bims-mikwok Biomed News
on Mitochondrial quality control
Issue of 2022‒05‒15
sixty-six papers selected by
Avinash N. Mukkala
University of Toronto

  1. FASEB J. 2022 May;36 Suppl 1
      Mitochondria are important organelle which regulate adenosine triphosphate (ATP) production, intracellular calcium buffering, cell survival and apoptosis. They are known to deliver the potential therapeutic role in injured cells through transcellular transfer via extracellular vesicles (EVs), gap junctions, and tunneling nanotubes (TNTs). Astrocytes secrete numerous factors that promote neuron survival, synapse formation, and plasticity. Recent studies have demonstrated that astrocytes transfer mitochondria into damaged neurons to enhance cell viability and recovery. In this study, we observed that treatment of isolated mitochondria from rat primary astrocytes enhance cell viability and ameliorate H2 O2 -damaged neurons. Interestingly, the isolated astrocytic mitochondria increased cell number in damaged neurons but not normal neurons, even though the mitochondrial transfer efficiency was no difference between them. Furthermore, this effect showed in astrocytic mitochondrial transplantation to rat middle cerebral artery occlusion (MCAO) models. These findings suggest that mitochondrial transfer therapy can be used to acute ischemic stroke and other diseases treatment.
  2. J Clin Transl Hepatol. 2022 Apr 28. 10(2): 321-328
      Defects in mitochondria are responsible for various genetic and acquired diseases. Mitochondrial transplantation, a method that involves introduction of healthy donor mitochondria into cells with dysfunctional mitochondria, could offer a novel approach to treat such diseases. Some studies have demonstrated the therapeutic benefit of mitochondrial transplantation and targeted delivery in vivo and in vitro within hepatocytes and the liver. This review discusses the issues regarding isolation and delivery of mitochondria to hepatocytes and the liver, and examines the existing literature in order to elucidate the utility and practicality of mitochondrial transplantation in the treatment of liver disease. Studies reviewed demonstrate that mitochondrial uptake could specifically target hepatocytes, address the challenge of non-specific localization of donor mitochondria, and provide evidence of changes in liver function following injection of mitochondria into mouse and rat disease models. While potential benefits and advantages of mitochondrial transplantation are evident, more research is needed to determine the practicality of mitochondrial transplantation for the treatment of genetic and acquired liver diseases.
    Keywords:  Hepatocytes; In vitro techniques; Liver; Mitochondria; Transplantation
  3. Cell Rep. 2022 May 10. pii: S2211-1247(22)00564-2. [Epub ahead of print]39(6): 110797
      The protein TRIM5α has multiple roles in antiretroviral defense, but the mechanisms underlying TRIM5α action are unclear. Here, we employ APEX2-based proteomics to identify TRIM5α-interacting partners. Our proteomics results connect TRIM5 to other proteins with actions in antiviral defense. Additionally, they link TRIM5 to mitophagy, an autophagy-based mode of mitochondrial quality control that is compromised in several human diseases. We find that TRIM5 is required for Parkin-dependent and -independent mitophagy pathways where TRIM5 recruits upstream autophagy regulators to damaged mitochondria. Expression of a TRIM5 mutant lacking ubiquitin ligase activity is unable to rescue mitophagy in TRIM5 knockout cells. Cells lacking TRIM5 show reduced mitochondrial function under basal conditions and are more susceptible to immune activation and death in response to mitochondrial damage than are wild-type cells. Taken together, our studies identify a homeostatic role for a protein previously recognized exclusively for its antiviral actions.
    Keywords:  APEX2; CP: Cell biology; CP: Immunology; ER-mitochondria contact site; HIV-1; TRIM5α; ULK1 complex; autophagy; inflammation; mitochondrial metabolism; proteomics; tripartite motif
  4. Autophagy. 2022 May 09. 1-2
      The unique cellular organization and metabolic demands of neurons pose a challenge in the maintenance of neuronal homeostasis. A critical element in maintaining neuronal health and homeostasis is mitochondrial quality control via replacement and rejuvenation at the axon. Dysregulation of mitochondrial quality control mechanisms such as mitophagy has been implicated in neurodegenerative diseases including Parkinson disease and amyotrophic lateral sclerosis. To sustain mitophagy at the axon, a continuous supply of PINK1 is required; however, how do neurons maintain a steady supply of this protein at the distal axons? In the study highlighted here, Harbauer et al. show that axonal mitophagy is supported by local translation of Pink1 mRNA that is co-transported with mitochondria to the distal ends of the neuron. This neuronal-specific pathway provides a continuous supply of PINK1 to sustain mitophagy.
    Keywords:  Autophagy; mitochondria; neurodegeneration; neuron; stress
  5. Cells. 2022 Apr 27. pii: 1469. [Epub ahead of print]11(9):
      Myeloid cell leukemia-1 (Mcl-1) is a unique antiapoptotic Bcl-2 member that is critical for mitochondrial homeostasis. Recent studies have demonstrated that Mcl-1's functions extend beyond its traditional role in preventing apoptotic cell death. Specifically, data suggest that Mcl-1 plays a regulatory role in autophagy, an essential degradation pathway involved in recycling and eliminating dysfunctional organelles. Here, we investigated whether Mcl-1 regulates autophagy in the heart. We found that cardiac-specific overexpression of Mcl-1 had little effect on baseline autophagic activity but strongly suppressed starvation-induced autophagy. In contrast, Mcl-1 did not inhibit activation of autophagy during myocardial infarction or mitochondrial depolarization. Instead, overexpression of Mcl-1 increased the clearance of depolarized mitochondria by mitophagy independent of Parkin. The increase in mitophagy was partially mediated via Mcl-1's LC3-interacting regions and mutation of these sites significantly reduced Mcl-1-mediated mitochondrial clearance. We also found that Mcl-1 interacted with the mitophagy receptor Bnip3 and that the interaction was increased in response to mitochondrial stress. Overall, these findings suggest that Mcl-1 suppresses nonselective autophagy during nutrient limiting conditions, whereas it enhances selective autophagy of dysfunctional mitochondria by functioning as a mitophagy receptor.
    Keywords:  Bnip3; Mcl-1; autophagy; heart; mitochondria; mitophagy
  6. FASEB J. 2022 May;36 Suppl 1
      The liver is the metabolic hub, and is responsible for the myriad of processes including the nutrient homeostasis and detoxification. Mitochondria of liver are critical for these functions. The detoxification process in liver, when severe, often results in liver damage through causing oxidative stress. Mitochondria are the main source of ROS and are also vulnerable to oxidant damage. Therefore, mitochondrial dysfunction is one of the prominent causes for drug-induced liver injury. Mitochondrial fission and fusion, the main processes of mitochondrial dynamics, determine mitochondrial shape, and are important for functional maintenance of mitochondria. However, the role of mitochondrial dynamics in drug-induced liver injury is poorly understood. In the current study, we examined the role of the optic atrophy 1 (OPA1) protein in drug-induced liver injury. OPA1 is associated with mitochondrial inner membrane (IM) and mediates IM fusion. OPA1 also regulates cristate structure, and is required for proper electron transport and ATP production. To investigate the OPA1's role, we used liver-specific OPA1-knockout (OPA1-LKO) mice with acetaminophen (APAP) administration as a model for drug-induced liver injury. We generated OPA1-LKO mice by crossing OPA1 flox mice with mice carrying the Cre recombinase under the albumin promoter. Whereas whole body KO of OPA1 causes embryonic lethality, OPA1-LKO mice appeared healthy and showed normal growth and behavior. Although the OPA1 gene in the liver was disrupted, OPA1-LKO mice showed approximately 30% of OPA1 remaining in the liver, presumably due to less efficient albumin promoter-mediated Cre expression and from other cell types of the liver. Liver histology revealed that OPA1-KO livers have disorganized hepatic cords with enlarged hepatocytes. Despite a reduced OPA1 level, mitochondria in OPA1-KO liver show near intact cristae structure and respiration, suggesting that a low level of OPA1 would support mitochondrial function in liver. We then tested the effect of OPA1 LKO on liver function under APAP stress. In APAP overdose, excess APAP metabolite depletes GSH in hepatocytes, which causes mitochondrial oxidative stress, leading to mitochondrial permeability transition, mitochondrial dysfunction, ATP depletion, and ultimately necrotic cell death. Upon administration of excess APAP, we found that OPA1-LKO mice were more sensitive to APAP-induced liver injury compared with the control mice. Histological analyses showed significantly more expanded focal centrilobular necrosis with vacuolization, cell swelling, and nuclear disintegration in OPA1-KO livers. Alanine aminotransferase levels, as a clinical chemistry parameter, were higher in OPA1-LKO mice than in control mice with APAP overdose. Furthermore, phospho-JNK levels were higher in OPA1-KO livers, indicating increased initial oxidative stress. However, depletion of hepatic glutathione (GSH) contents and reduction of GSH/GSSG ratio upon APAP treatment were similar between the LKO and control liver. Together, our experimental results indicate that although liver is tolerant to a reduced level of OPA1, OPA1 depletion lowers the stress threshold and makes hepatocytes more sensitive to APAP-induced liver injury.
  7. FASEB J. 2022 May;36 Suppl 1
      Mitochondria are dynamic powerhouses of cells and their fission and fusion must be tightly regulated for normal cell function. Mitochondrial fission is mediated by the GTPase, Drp1, and mis-regulation of Drp1 leads to mitochondrial hyper-fragmentation, a known marker of disease. It has been reported that Drp1 is modified by SUMO1 and SUMO2/3, however, the mechanisms of Drp1 regulation by individual SUMO paralogs remain to be fully understood. Here, we have used CRISPR/Cas9 derived SUMO1 and SUMO2 knockout (KO) cell lines, to perform a systematic investigation of paralog-specific effects on mitochondrial maintenance and function. In contrast to expectations, we observed multiple mitochondrial defects specifically in SUMO2 KO, but not SUMO1 KO, cells. SUMO2 KO cells had reduced mitochondrial activity based on results of MTT assays. By immunofluorescence microscopy, we also observed an increase in mitochondrial fragmentation in SUMO2 KO cells. Paradoxically, increased fragmentation occurred despite reduced levels of Drp1 protein expression and sequestration of Drp1 in large cytosolic foci. Taken together, our findings indicate that SUMO2 plays a non-redundant, paralog-specific role in regulating mitochondrial function, in part through effects on Drp1. We anticipate that a more detailed understanding of the molecular effects of SUMO2 on Drp1 may lead to novel therapeutic approaches to treat or prevent ischemic injury, neurodegeneration, and other mitochondrial-related maladies.
  8. FASEB J. 2022 May;36 Suppl 1
      Mitochondria are membrane-bound organelles composed of two membranes, the inner mitochondrial membrane (IMM) and the outer mitochondrial membrane (OMM), with the intermembrane space (IMS) localized between them. The IMM is divided into two subcompartments, the inner boundary membrane (IBM) that lies parallel to the OMM, and the cristae membrane (CM), which are IMM invaginations folded into cristae. The IBM and CM are connected at terminals forming circular-like openings known as cristae junctions (CJs). The structural integrity of the mitochondrial CJs is maintained by the mitochondrial contact site and cristae organizing system (MICOS), a specific protein complex containing several structural and regulatory proteins. The MICOS proteins control the IMM architecture through direct membrane shaping, formation of contact sites, and biogenesis of proteins and lipids. In addition, the optic atrophy 1 (OPA1), a mitochondrial fusion protein, localized also in the CM, has been shown to participate in IMM remodeling which mediates the fusion of the IMM. The structural organization of the IMM is regulated by changes in the matrix volume of mitochondria; excessive matrix swelling in response to energetic and oxidative stress induced by pathological stimuli such as cardiac ischemia-reperfusion (IR) impairs the integrity of CJs and thereby, alters mitochondrial function. The main goal of this study is to investigate the effects of cardiac IR-induced swelling on OPA1 and MICOS proteins. Hearts were isolated from male Sprague Dawley rats and perfused with Krebs-Henseleit solution (KHS) using the Langendorff-mode technique at a constant flow rate (10-12 ml/min). The animals were randomly assigned to the following groups: i) perfusion (no ischemia) for 55 min (C-55 group), ii) ischemia (I group) for 25 min, iii) ischemia in the presence of sanglifehrin A (SfA), an inhibitor of the permeability transition pore (IS), iv) perfusion (no ischemia) for 95 min (C-95), v) ischemia for 25-min followed by 40-min reperfusion (IR group), and vi) IR in the presence of SfA (IRS). SfA (0.5 µM) was present 10 min before ischemia (IS group) and throughout the entire period of reperfusion (IRS group). LDH activity was determined in the coronary effluent as a marker of cell death. At the end of reperfusion, mitochondria were isolated from the hearts by differential centrifugation for analysis of oxygen consumption rates, mitochondrial swelling, and protein levels of long and short forms of OPA1, and MICOS proteins. Our results show that ischemia diminished post-ischemic recovery of the hearts as evidenced by impaired cardiac contractility, increased LDH activity in the coronary effluent, and reduced mitochondrial respiration. Ischemia alone and IR differently affected the expression of both, OPA1 and mitofilin (Mic-60, a core MICOS protein) as well as other MICOS components. In conclusion, our data suggest that OPA1 and MICOS proteins are affected differently by ischemia alone and IR.
  9. Cell Death Dis. 2022 May 09. 13(5): 444
      Mitochondria are highly dynamic organelles that participate in ATP generation and involve calcium homeostasis, oxidative stress response, and apoptosis. Dysfunctional or damaged mitochondria could cause serious consequences even lead to cell death. Therefore, maintaining the homeostasis of mitochondria is critical for cellular functions. Mitophagy is a process of selectively degrading damaged mitochondria under mitochondrial toxicity conditions, which plays an essential role in mitochondrial quality control. The abnormal mitophagy that aggravates mitochondrial dysfunction is closely related to the pathogenesis of many diseases. As the myocardium is a highly oxidative metabolic tissue, mitochondria play a central role in maintaining optimal performance of the heart. Dysfunctional mitochondria accumulation is involved in the pathophysiology of cardiovascular diseases, such as myocardial infarction, cardiomyopathy and heart failure. This review discusses the most recent progress on mitophagy and its role in cardiovascular disease.
  10. FASEB J. 2022 May;36 Suppl 1
      Doxorubicin (DOX), an extremely effective and wide-spectrum antineoplastic anthracycline, has been known for its notorious adverse effect of dose-dependent dilated cardiotoxicity that culminates in heart failure. The current approach for reducing DOX-induced cardiotoxicity is to limit the overall cumulative dose of the drug, but at the expense of narrowing the therapeutic window for cancer treatment. Therefore, it is imperative to identify new strategies to protect against DOX-induced heart damage without compromising its antineoplastic activity. DOX cardiotoxicity is closely associated with mitochondrial injury which is characterized by an early loss of mitochondrial membrane potential followed by dysregulation of mitochondrial quality control mechanisms including mitophagy, a process through which injured mitochondria are degraded by the autophagic pathway. Mitophagy is generally believed to play protective roles under various normal and disease conditions. However, evidence also suggests that mitophagy can become detrimental, leading to cell death under certain conditions. The effect of DOX on mitophagy has been assessed previously, but neither mitophagy activity nor its functional role in DOX cardiotoxicity has been clearly defined. Parkin and FUNDC1 are two well-established positive regulators of mitophagy. Knockdown of Parkin diminished DOX-induced cell death, while overexpression of Parkin had the opposite effects, suggesting that DOX cardiotoxicity was mediated, at least in part, by accelerated mitophagy through a Parkin-dependent pathway. However, the role of FUNDC1-mediated mitophagy in DOX cardiotoxicity remains unclear. In this study, we investigated the functional role of FUNDC1 in DOX-induced cardiotoxicity using both FUNDC1 knockout (KO) and transgenic (TG) mice. We hypothesized that knockout of FUNDC1 would alleviate DOX-induced cardiotoxicity while overexpression of FUNDC1 would exacerbate DOX cardiotoxicity. DOX-induced cardiac injury was examined and compared with WT and FUNDC1 KO (FKO) or FUNDC1 transgenic (FTG) mice. The Fractional Shortening (FS) was measured by echocardiography. Serum LDH activity and cardiac troponin-I (cTnI) levels were measured and used as indicators of cardiac tissue damage. For the mitophagy flux assay, mice were treated with lysosomal proteasome inhibitors (25mg/kg pepA and 5mg/kg E64d) for 4 hours and the heart tissues were collected for Western blot analyses. Knockout of FUNDC1 reduced mitophagy in the mouse heart, while overexpression of FUNDC1 accelerated mitophagy flux, confirming FUNDC1 as a positive regulator of mitophagy. Knockout of FUNDC1 attenuated DOX-induced cardiac functional impairment as shown by improved fractional shortening (FS), supporting the conclusion that FUNDC1-dependent mitophagy may mediate DOX cardiotoxicity. Surprisingly, however, overexpression of FUNDC1 also alleviated DOX-induced cardiac injury, suggesting that FUNDC1 may function independently of mitophagy in the maintenance of cardiac function.
  11. FASEB J. 2022 May;36 Suppl 1
      BACKGROUND: Many patients that suffer from anterior cruciate ligament (ACL) injury have persistent quadriceps atrophy even after considerable rehabilitation. Our previous finding has revealed that mitochondrial dysfunction and redox disturbances are causal events in the initiation of muscle atrophy and central to maintenance of a healthy mitochondria is the removal of damaged mitochondria through mitophagy. However, the extent to which mitophagy play a key role in quadriceps muscle atrophy after ACL injury has yet to be explored. If mitophagy is found to play a central role in directing muscle atrophy after ACL injury than it may be an attractive therapeutic target.PURPOSE: Using a pre-clinical non-invasive ACL injury model, our objective was to use a time course study to investigate the potential role of mitophagy in quadriceps muscle atrophy after ACL injury.
    METHODS: 48 Long Evans rats (n=8 per group; 4m/4f) underwent non-invasive rupture of the right ACL and were euthanized at 7, 14, 28, 56 days post-injury. 8 rats (4m/4f) served as healthy controls (HC). Mitophagy-related cellular components of the vastus lateralis were analyzed by Western Blot analysis. One-way ANOVAs with LSD post-hoc were used to determine differences between groups (P < 0.05).
    RESULTS: Dynamin-related protein 1 (DRP1), a protein regulating mitochondrial fission, was increased significantly at 56 days post-injury [HC: n=8; 1.294 ± 0.364, 56D: n=7; 2.093 ± 1.519 (A.U), P<0.05]. Lysosomes are the terminal step in mitophagy and one of the lysosomal markers, Lysosomal-associated membrane protein 1 (LAMP-1) expression was significantly elevated at 56 days post-injury [HC: n=8; 1.270 ± 0.840, 56D: n=8; 2.266 ± 1.137 (A.U), P<0.05]. Upstream autophagy marker, Beclin-1 expression was also significantly increased at 56 days post-injury (HC: n=8; 1.117 ± 0.339, 56D: n=6; 2.158 ± 1.274, P<0.05).
    CONCLUSION: Collectively, these results imply that long-term ACL injury dysregulates mitochondrial quality control including key mitophagy markers, which contributes to ACL injury-induced quadriceps atrophy. Therefore, targeting mitophagy may be the one of the potential therapeutic interventions to prevent muscle atrophy in patients with an ACL injury.
  12. FASEB J. 2022 May;36 Suppl 1
      Mitochondria undergo coordinated rounds of fusion and fission that are critical for maintaining the functional integrity of this essential organelle. While a growing number of proteins have been identified as important regulators of mitochondrial dynamics, the direct role of membrane lipid composition during the fusion and fission processes is poorly understood. To address these shortcomings, we devised a protein-engineering platform that allows for the acute remodeling of structural phospholipids within the outer mitochondrial membrane (OMM) of intact cells. Specifically, we modified a bacterial phospholipase C (Bacillus cereus (Bc)PI-PLC) to initiate the rapid hydrolysis of phosphatidylinositol (PI) and locally generate diacylglycerol (DAG); an important intracellular signaling molecule and metabolic precursor that is used in diverse lipid biosynthetic pathways. Spatial restriction of enzyme activity was achieved using a chemically inducible system consisting of a rapamycin-dependent dimerization module (FKBP-BcPI-PLC) along with an OMM targeting sequence tagged with the FKBP-rapamycin binding domain (OMM-FRB). Using these unique molecular tools, we show that recruitment of FKBP-BcPI-PLC to the OMM not only causes the expected local accumulation of DAG, but also initiates the rapid and uniform fragmentation of the mitochondrial network. Mitochondrial fission induced by FKBP-BcPI-PLC is accompanied by profound swelling of the mitochondrial matrix along with vesiculation of the inner mitochondrial membrane (IMM) and a general loss of cristae, which all occur within minutes of tethering FKBP-BcPI-PLC to the OMM. Expression of dominant-negative constructs targeting essential GTPases known to regulate OMM fission suggest that both dynamin-related protein 1 (Drp1) and dynamin 2 (Dnm2) work together to drive efficient BcPI-PLC-induced mitochondrial division. However, results using a validated Drp1 knockout cell line show that the loss of Drp1 alone is sufficient to prevent the mitochondrial fragmentation initiated by FKBP-BcPI-PLC recruitment, indicating that Drp1 likely functions upstream or independent of Dnm2 in this context. Interestingly, unlike the induced OMM fission, removal of Drp1 from cells does not prevent the matrix swelling or OMM constrictions observed in response to acute generation of DAG within the OMM. Ongoing experiments are now focused on characterizing new methods to sequentially metabolize the DAG generated within the OMM as well as investigate how local lipid composition influences the binding and oligomerization of membrane-shaping proteins that may function in concert with Drp1 to regulate mitochondrial remodeling. Overall, these studies establish a direct relationship between lipid metabolism within the OMM and clinically relevant morphological changes that are known to manifest in mitochondrial-associated diseases.
  13. FASEB J. 2022 May;36 Suppl 1
      ATF4 is a transcriptional regulator that is selectively induced in response to cellular stress conditions, such as exercise, through the activation of the integrated stress response (ISR). Specifically, in the context of mitochondrial-specific stress, ATF4 is induced as a key component of the mitochondrial unfolded protein response (UPRmt ) and is suggested to upregulate various organellar chaperones and proteases that both preserve, and promote, mitochondrial function. In response to the stress brought about by contractile activity, ATF4 has been implicated in regulating skeletal muscle health by mediating the various signaling events associated with mitochondrial quality control (MQC), including i) mitochondrial biogenesis (expansion), ii) the mitophagy-lysosomal clearance of damaged and thus potentially harmful organelles, or iii) by activating the mitochondrial unfolded protein response (UPRmt ) as an intermediate response to acute cellular stress. However, it remains to be determined whether ATF4 is necessary for mitochondrial adaptations in skeletal muscle. Therefore, our aim was to determine whether ATF4 is required for the maintenance of mitochondrial function and adaptation following an acute 3h bout of contractile activity (ACA), or after repeated bouts (4 days; CCA) in C2C12 myotubes in which ATF4 was either overexpressed (OE) or knocked down (KD) via lentiviral transduction of plasmids containing the ATF4 open reading frame, or siRNA, respectively. Knockdown of ATF4 promoted elongated myotube formation following 5 days of differentiation, whereas ATF4 OE contributed to the opposite effect in which shorter myotubes were observed relative to the control condition. Induction of PGC-1α mRNA following ACA in ATF4 KD myotubes was attenuated, suggesting diminished drive for mitochondrial biogenesis in the absence of ATF4. The mRNA expression of ATF5, a downstream target of ATF4, was induced both in response to ACA as well as with ATF4 OE, and was reduced in the absence of ATF4. Mitophagy flux, measured by mitochondrial-localized LC3-II, was upregulated in ATF4 OE and KD myotubes basally, and was augmented in both control and ATF4 OE cells following ACA, but not in the absence of ATF4. Furthermore, ATF4 OE revealed decrements in mitochondrial content indicated by 20%, and 40%, reductions in VDAC and COX I protein expression relative to control, while 1.2-1.5-fold increases in these markers were observed when ATF4 was knocked down. However, following CCA, COX I and VDAC protein content were increased 3-4-fold in ATF4 OE cells, while there was no observable increase in mitochondrial content in ATF4 KD myotubes. Together, these data highlight a potential role of ATF4 in regulating basal mitochondrial content and further suggest that ATF4 may be required for contractile activity-induced increases in mitochondrial content.
  14. FASEB J. 2022 May;36 Suppl 1
      Mitochondria, which are often regarded as the "powerhouse of the cell", are labile organelles that regulate cellular metabolism, determine cell fate, and act as important signaling hubs. A phospholipid that is exclusively found within mitochondria that serves numerous roles is cardiolipin (CL). In healthy mitochondria, CL is predominately localized in the inner mitochondrial membrane (IMM) and binds to several membrane proteins including those in the electron transport chain (ETC) and in the protein import machinery (PIM). Similarly, CL has also been shown to regulate cell death and mitophagy in dysfunctional mitochondria. Following the biosynthesis of nascent CL in the inner membrane, it is remodeled into its mature form by a transacylase, tafazzin (Taz). The absence of this enzyme is associated with Barth Syndrome, a disease characterized by cardiomyopathy and skeletal myopathy. Interestingly, little is known about the role of mature CL in skeletal muscle. Therefore, the objective of this study was to identify whether Taz deficiency diminishes mitochondrial function in the presence or absence of changes in organelle volume and function. We hypothesized that a reduction in Taz will attenuate mitochondrial function, while lysosomal and mitochondrial content will be elevated. To test this hypothesis, C2C12 myotubes were transfected on day 3 of differentiation with either a scrambled or Taz siRNA vector. Cells were harvested for analysis on day 7 of differentiation. Organelle volume was assessed using immunohistochemistry and Western blot techniques, whereas Seahorse technology was used to measure mitochondrial respiration. In comparison to C2C12 myotubes that were treated with a scrambled vector, Taz protein content was reduced by 85% (P<0.05) in siRNA-treated cells. Maximal oxygen consumption and ATP production were attenuated in Taz-deficient cells by 18% and 14%, respectively, while basal respiration remained unaffected. Lysosome and mitochondrial content were increased 20% and 24%, respectively, in Taz-deficient cells. Similarly, myotubes lacking Taz exhibited a 7% increase (P=0.056) in Complex IV protein content, but lysosomal-associated membrane protein 1 (LAMP1) decreased 33% (P<0.05). Myosin heavy chain (MHC) IIX was also elevated by 32% in mature CL depleted myotubes. CL-deficient myotubes were 32% smaller in diameter and 10% longer when compared to scrambled treated myotubes. These data suggest that a reduction in mature CL impairs skeletal muscle mitochondrial function. Moreover, although lysosomal content was elevated, lysosomes appear to have a reduced capacity to fuse with autophagosomes, which may in part explain why there is an increase in dysfunctional mitochondria. Future research should evaluate mitophagy flux and mitochondrial membrane potential in the presence of reduced mature CL in C2C12 myotubes, and whether exercise has the ability to rescue mitochondrial function.
  15. FASEB J. 2022 May;36 Suppl 1
      P21-activated serine/threonine Kinase 1 (PAK1) plays a critical role in cardiomyocyte survival under numerous stressful conditions. Autophagy is a cellular process that promotes homeostasis by removing and replacing antiquated or damaged cellular components. Mitophagy is a form of selective autophagy that eliminates damaged mitochondria to maintain a pool of healthy mitochondria. We have previously demonstrated that PAK1 is essential for maintaining autophagy and mitophagy activities. However, the downstream mediators have not yet been discovered. In this study, we explored the mechanisms of PAK1-dependent autophagy and mitophagy by determining the protein expression levels of the major regulators of autophagy and mitophagy. H9c2 cardiac myoblasts were treated with siRNA to knockdown the expression of PAK1. Western blot analysis showed that PAK1 knockdown substantially reduced the protein expression levels of ATG5-12 complex, an essential promotor of autophagosome formation, TFEB, a master regulator of lysosome biogenesis and autophagy, and p62, an autophagy receptor for ubiquitinated cargos. All these changes are expected to reduce autophagy activity. In addition, PAK1 knockdown also reduced the expression of mitophagy receptor FUNDC1 and diminished the association of p62 with mitochondria, which coincided with reduced mitophagy activity. Collectively, these results suggest that the ability of PAK1 knockdown to inhibit autophagy and mitophagy is mediated by reduced expression levels of several important regulators of autophagy and/or mitophagy pathways. Future research is warranted to determine whether restoring the expression levels of these target genes can overcome the inhibition of autophagy and mitophagy by PAK1 deficiency.
  16. FASEB J. 2022 May;36 Suppl 1
      Mitochondria and peroxisomes are both dynamic signaling organelles that constantly undergo fission. While mitochondrial fission and fusion are known to coordinate cellular metabolism, proliferation, and apoptosis, the physiological relevance of peroxisome dynamics and the implications for cell fate are not fully understood. DRP1 (dynamin-related protein 1) is an essential GTPase that executes both mitochondrial and peroxisomal fission. Patients with de novo heterozygous missense mutations in the gene that encodes DRP1, DNM1L, present with encephalopathy due to mitochondrial and peroxisomal elongation (EMPF). EMPF is a devastating neurodevelopmental disease with no effective treatment. To interrogate the molecular mechanisms by which DRP1 mutations cause developmental defects, we are using patient-derived fibroblasts and iPSC-derived models from patients with mutations in different domains of DRP1 who present with clinically disparate conditions. Using super resolution imaging, we find that patient cells, in addition to displaying elongated mitochondrial and peroxisomal morphology, present with aberrant cristae structure. Given the direct link between cristae morphology and oxidative phosphorylation efficiency, we explored the impact of these mutations on cellular energy production. Patient cells display a lower coupling efficiency of the electron transport chain, increased proton leak, and Complex III deficiency. In addition to these metabolic abnormalities, mitochondrial hyperfusion results in hyperpolarized mitochondrial membrane potential. Intriguingly, human fibroblasts are capable of cellular reprogramming into iPSCs and appear to display peroxisome-mediated mitochondrial adaptations that could help sustain these cell fate transitions. Understanding the mechanism by which DRP1 mutations cause cellular dysfunction will give insight into the role of mitochondrial and peroxisome dynamics in neurodevelopment.
  17. FASEB J. 2022 May;36 Suppl 1
      Phosphorylation has long been appreciated to influence mitochondrial metabolism via the regulation of pyruvate dehydrogenase. However, the extent to which phosphorylation broadly influences mitochondrial function remains unclear, despite the presence of multiple protein phosphatases within the organelle. We recently demonstrated that deletion of the mitochondrial matrix phosphatase Pptc7 unexpectedly caused perinatal lethality in mice, suggesting that the regulation of mitochondrial phosphorylation is essential in mammalian development. Pptc7-/- mice exhibit severe metabolic deficiencies, including hypoglycemia and lactic acidosis, and die within one day of birth. Biochemical and proteomic approaches revealed that Pptc7-/- tissues have decreased mitochondrial function concomitant with a post-transcriptional downregulation of mitochondrial proteins. Multiple elevated mitochondrial protein phosphorylation sites in Pptc7-/- tissues suggest novel functional connections between Pptc7-mediated dephosphorylation and these observed metabolic consequences. Interestingly, these modifications occur on components of the import machinery of the mitochondria and within the mitochondrial targeting sequences of select nuclear-encoded precursor proteins. Collectively, our data reveal an unappreciated role for a matrix-localized phosphatase in the post-translational regulation of the mitochondrial proteome and organismal metabolic homeostasis.
  18. FASEB J. 2022 May;36 Suppl 1
      Spinal cord transection (ST) inactivates the neuromuscular complex and triggers progressive muscular atrophy of the affected skeletal muscles. Disruption in neural input also leads to a reduction in mitochondrial volume. The mitochondria lifecycle can be generally divided into processes of biogenesis, expression of OXPHOS proteins, fusion, fission, and mito/autophagy; specifically, the balance between biogenesis vs. mitophagy dictates the rate of formation and destruction, respectively. Here, we examined the expression profiles of the markers associated with the mitochondrial lifecycle up to one month following ST in phenotypically slow (soleus; SOL) and mixed (plantaris; PLT) skeletal muscle. We hypothesized that ST would induce a reduction in mitochondrial DNA (mtDNA) copy number, decrease expression in markers associated with biogenesis, and increased proteins that regulate mito/autophagy. Adult female Sprague Dawley rats were randomly divided into control (CON; n=6), 1 day (1dST; n=5), 8 day (8dST; n=8), and 28 day post-ST (28dST; n=8). Compared to CON relative SOL and PLT muscle masses (absolute mass / body mass) were 98, 65, and 62% and 85, 60, and 73% at 1, 8 and 28d post-ST, respectively (p<0.05). Compared to CON, no significant changes were observed in mtDNA copy number in ST muscles following amplification using standard end-point PCR protocols for mtDNA (ND1, COX1, or ATP6) and nuclear (β2M) genes (p>0.05). Total protein was isolated from SOL and PLT, separated using standard SDS-PAGE protocol, and probed for markers associated with various stages of the mitochondrial lifecycle using standard western blot protocols. Upstream markers known to influence mitochondrial lifecycle signaling (Rev-Erbα, AMPK) showed a varied temporal and muscle-specific response: in 1dST SOL muscles, Rev-Erbα increased 74% from CON then decreased 43% at 28dST, whereas 8dST PLT muscle exhibited a 44% increase (p<0.05). In 8dST SOL and PLT muscles, the pAMPK:AMPK ratio was elevated 366 and 85% from CON, respectively (p<0.05). Markers for mitochondrial biogenesis (PGC-1α, NRF1, NRF2) in SOL and PLT muscles were largely unchanged, although a ~50% decrease in Tfam protein expression was observed at each ST time point compared to CON (p<0.05). Mitofusion2, a marker for mitochondrial fusion, was unchanged (p>0.05). However, for markers detecting mitochondrial fission (MFF, Drp1, Fis1), we observed a ~60% increase in MFF expression in 1dST SOL and PLT muscles (p<0.05). As expected markers for mitophagy (Parkin, PINK, p62, LC3 I/II) were significantly elevated: Parkin increased ~70% at 28dST (p<0.05), whereas LC3 I/II increased 65% and 112% at 1dST in in SOL and PLT muscles, respectively (p<0.05), and remained significantly elevated in PLT muscles at 8dST (27%) and 28d (37%) (p<0.05). Taken together, these data suggest that mitochondrial volume and expression of markers regulating mitochondrial biogenesis and fusion are not influenced by acute ST despite a decrease in muscle mass. However, selective markers for fission and mitophagy are significantly elevated in both SOL and PLT muscles, suggesting that mitochondrial destruction mechanism are activated acutely following ST injury. Lastly, selective proteins of the OXPHOS protein complex significantly increased in 1dST SOL and PLT muscles suggesting possible activity-dependent regulation of mitochondrial gene expression.
  19. FASEB J. 2022 May;36 Suppl 1
      INTRODUCTION: The Ca2+ -activated Cl- channel Anoctamin-1 (Ano1) regulates multiple cell functions including cell proliferation, survival, and migration. We previously reported that overexpression of Ano1 is associated with hyperproliferation of pulmonary artery endothelial cells isolated from patients with idiopathic pulmonary arterial hypertension. We also showed that Ano1 is expressed not only in the plasma membrane (PM), but also in the mitochondria. However, the physiological and pathological roles of mitochondrial Ano1 have not been fully investigated.AIM: To investigate the role of mitochondria-localized Ano1 on the regulation of mitochondrial dynamics and reactive oxygen species (ROS) generation.
    METHODS: Stable overexpression and knockdown of Ano1 were employed in HEK293T cells and mouse embryonic fibroblasts (MEFs), respectively. Cells were used for biochemical and cell biological assays.
    RESULTS: First, mitochondrial localization of Ano1 in addition to the PM was confirmed by live cell imaging of HEK293T cells expressing GFP-tagged Ano1 and immunostaining of endogenous Ano1 in MEFs. Next, a co-immunoprecipitation assay showed that overexpressed Ano1 in HEK293T cells was associated with optic atrophy 1 (OPA1) which is an inner mitochondrial membrane (IMM) protein and a critical regulator of mitochondrial fusion, cristae formation, and bioenergetics. We also confirmed the interaction between endogenous Ano1 and OPA1 using wild-type and OPA1-knockout MEFs. Overexpression of wild-type Ano1 in HEK293T cells facilitated proliferation, but a pore-dead mutant (Ano1-R621E) did not. Importantly, Ano1 overexpressing HEK293T cells have higher cell death rates in response to oxidative stress compared to control cells. To further understand the role of endogenous Ano1 expression on mitochondrial functions, we next employed MEFs stably overexpressing shRNA targeting Ano1. Quantifying mitochondrial morphology from live MEFs expressing mitochondria-matrix targeted GFP revealed that Ano1-knockdown (Ano1-KD) MEFs contained more elongated mitochondria compared to cells stably expressing control shRNA. Importantly, we also found higher expression of mitofusin 2 and long-form OPA1 in Ano1-KD MEFs compared to control cells. The expression levels of other fission/fusion proteins were not altered by Ano1-KD. Lastly, significantly lower levels of mitochondrial superoxide in Ano1-KD cells and a tendency towards lower cellular oxidative levels in Ano1-KD cells compared to controls were observed by live cell imaging with mitochondrial superoxide-sensitive dye MitoSOX Red and protein carbonyl quantification in the whole cell lysates, respectively.
    SUMMARY: Ano1, expressed in both the IMM and the PM, is involved in the maintenance of mitochondrial morphology and ROS, possibly by modulating fusion protein expression levels. Future studies will include the development of genetically engineered mitochondria-targeted Ano1 to more precisely dissect the roles of IMM-localized Ano1 in mitochondrial functions.
  20. Commun Biol. 2022 May 12. 5(1): 453
      Humans are frequently exposed to time-varying and static weak magnetic fields (WMF). However, the effects of faint magnetic fields, weaker than the geomagnetic field, have been scarcely reported. Here we show that extremely low-frequency (ELF)-WMF, comprised of serial pulses of 10 µT intensity at 1-8 Hz, which is three or more times weaker than the geomagnetic field, reduces mitochondrial mass to 70% and the mitochondrial electron transport chain (ETC) complex II activity to 88%. Chemical inhibition of electron flux through the mitochondrial ETC complex II nullifies the effect of ELF-WMF. Suppression of ETC complex II subsequently induces mitophagy by translocating parkin and PINK1 to the mitochondria and by recruiting LC3-II. Thereafter, mitophagy induces PGC-1α-mediated mitochondrial biogenesis to rejuvenate mitochondria. The lack of PINK1 negates the effect of ELF-WMF. Thus, ELF-WMF may be applicable for the treatment of human diseases that exhibit compromised mitochondrial homeostasis, such as Parkinson's disease.
  21. FASEB J. 2022 May;36 Suppl 1
      INTRODUCTION: In aging post-menopausal women, Coronary Microvascular Disease (CMD) leads to hyperconstricted tone, reduced perfusion and chronic micro-ischemia with angina. We previously showed an age-related increase in coronary microvascular ROS alongside increased prooxidant gene and protein expression associated with blunted vasodilation. Adipose Stromal Vascular Fraction (SVF) is a heterogenous cell population that reduces vascular ROS to improve vasodilation. Oxidative stress with aging may be mediated by mitochondrial dysfunction, including fission/fusion imbalance. Therefore, we hypothesize aging leads to mitochondrial hyperfission and reduced fusion gene and protein expression, reversed by SVF therapy.METHODS: Coronary microvessels from young (YC, 4 months), old (OC, 24 months), or old + SVF (OSVF, tail vein injection 4 weeks prior to sacrifice) from female Fischer 344 rats were assessed via RNA sequencing (n=3) and immunofluorescence imaging of fission/fusion proteins Dynamin Related Protein 1 (DRP-1) and Mitofusin 1 & 2 (MFN1 & 2). Morphometric analysis was accomplished using ImageJ and the MiNA Image-J tool to analyze 60x confocal images of microvessels stained with antibodies against mitochondrial TOM20. Statistics were preformed using One-Way ANOVA with Bonferroni post hoc analysis.
    RESULTS: Gene expression of DRP-1 was significantly reduced, and MFN-1 significantly enhanced in OSVF vs. OC (p = 0.035, 0.019). There was no significant difference in gene expression of fission or fusion mediators Fis-1, MFN-2 or Opa-1. Protein expression of DRP-1 was enhanced with aging, reduced to youthful levels by SVF (p = 0.007, 0.033) (Fig.1a). There were no differences in MFN1 or MFN2 protein expression with aging, however, protein expression of MFN-1 was significantly increased after SVF (YC vs. OSVF p = 0.003, OC vs. OSVF p < .001). Mitochondrial aspect ratio and form factor were reduced in aging (p = 0.024, < 0.001), restored to youthful levels by SVF (p = < 0.001, < 0.001) (Fig. 1b). Mitochondrial fission factor was elevated with aging (p = < 0.001), reversed by SVF therapy (p = < 0.001) (Fig. 1c). Mitochondrial area was reduced with aging (p = 0.002). Mitochondrial branch length and numbers of branches within a network were greatest in youth, significantly attenuated with aging (p < 0.001, < 0.001), and significantly increased (albeit not to youthful levels) with SVF therapy (OC vs. OSVF: p = 0.017, 0.011, YC vs. OSVF: p = 0.004, 0.003) (Fig. 1d).
    CONCLUSIONS: During youth, mitochondrial morphology is more rod-like and interconnected, vs. punctate and isolated in aging. Injection of SVF reversed mitochondrial circularity and partially restored mitochondrial network density in aging. These findings were complimented by enhanced expression of DRP-1 with aging with reduced DRP-1 and enhanced MFN-1 expression with SVF. SVF is a plausible therapy for CMD by reversing mitochondrial dysfunction including alleviating oxidative stress, attenuating mitochondrial fission, and rejuvenating mitochondrial fusion.
  22. FASEB J. 2022 May;36 Suppl 1
      CD4 T cell differentiation to pro-inflammatory and immunosuppressive subsets requires distinct metabolic pathways. Pro-inflammatory CD4 subsets rely on glycolysis, while immunosuppressive (Treg cells) subsets, require functional mitochondria for their differentiation and function. Previous studies have shown that binge alcohol (ethanol, EtOH) administration increased Tbet-expressing (Th1) and decreased FOXP3-expressing (Treg) CD4 T cells in the colons of mice. We tested the hypothesis that EtOH dysregulates normal CD4 T cell differentiation, after stimulation, by impairing mitochondrial homeostasis. Human naïve CD4 T cells were isolated from buffy coats from blood bank donors (N = 6) using MACS sorting. Cells were stimulated using anti-CD3-coated dishes in the presence of anti-CD28 and IL-12, and exposed to EtOH (0 and 50 mM) for 3 days. Mitochondrial content was measured with Mitotracker Deep Red. Gene expression indicative of: autophagosome formation (ATG5, ATG7, ATG13, MAP1LC3B, BECN1, BNIP3L, ULK1), mitophagy (PINK1, PRKN),mitochondrial fusion (MFN1, MFN2, OPA1), mitochondrial fission (MFFand FIS1), and mitochondrial biogenesis (PPARC1A, PPARC1B, TFAM) was determined by RT2 profiler arrays. EtOH-treated CD4 T cells had increased mitochondrial content (p = 0.0008) with Tregs accounting for the greatest increase in mitochondria (p = 0.04). There was a main effect of stimulation (p < 0.05) to increase ATG5, ATG13, MAP1LC3B, BECN1, BNIP3L, ULK1, MFF, PPARC1B, and TFAM, and a main effect of EtOH (p < 0.05) to increase PINK1 and decrease ATG7. There was a main effect of both EtOH and stimulation (p < 0.05) to increase MFN2, and OPA1.Taken together, these results indicate that EtOH increases mitochondrial content in Treg cells and dysregulates mitochondrial gene expression important for mitochondrial repair and mitophagy. These EtOH-mediated alterations in gene expression could result in an inability of CD4 T cells to maintain mitochondrial homeostasis and remove damaged mitochondria that is required for normal differentiation and function of anti-inflammatory Treg cells.
  23. FASEB J. 2022 May;36 Suppl 1
      Mitochondria undergo permeability transition (PT) resulting in the opening of the non-selective PT pores (PTPs) in the inner mitochondrial membrane in response to energy and oxidative stresses associated with Ca2+ overload and ROS accumulation. The mitochondrial PTPs are permeable to ions and solutes with a molecular mass <1.5 kD that increases the colloidal osmotic pressure in the matrix leading to mitochondrial swelling. Calcium retention capacity (CRC) reflects the maximum amount of Ca2+ mitochondria can uptake to provoke the PTP opening. Quantification of CRC is important to study the effects of various pathological stimuli and the efficacy of pharmacological agents on the metabolism and function of mitochondria. Here, we performed a comparative analysis of CRC in mitochondria isolated from H9c2 cardioblasts, and in permeabilized H9c2 cells in situ to highlight the advantages/disadvantages of the fluorescent technique in isolated mitochondria vs. permeabilized cells. The cells were permeabilized using digitonin or saponin, and the CRC was assessed using the Ca2+ -sensitive fluorescence probe Calcium Green-5N. Results demonstrated the interference of dye-associated fluorescence signals with saponin and the adverse effects of digitonin on mitochondria at high concentrations. The CRC of saponin-permeabilized cells was higher than the CRC of digitonin-permeabilized cells. In addition, the mitochondrial CRC of saponin-permeabilized cells was higher than isolated mitochondria using the same number of cells. In conclusion, this study demonstrates that the fluorescent technique for CRC analysis in saponin-permeabilized cells has more advantages than isolated mitochondria.
  24. FASEB J. 2022 May;36 Suppl 1
      Mesenchymal stem cells (MSC) have been shown to improve heart function after myocardial infarction, but the exact mechanisms are not completely understood. Studies have shown that stem cells are able to transfer mitochondria through cytosol extensions to change the abilities and programming of surrounding cells. The purpose of this study was to determine whether mitochondrial transfer takes place between MSCs and cardiac H9c2 cells, and the effect of hypoxic conditions on this process. Mouse bone marrow MSC were cultured in Mesencult + 10% mouse supplement (Stem Cell Technologies) + 10% FBS and H9c2 cells were cultured in DMEM + 10% FBS. MSC mitochondria were stained using MitoTracker Red CMX Ros (Invitrogen), while H9C2 cells were stained using CellTracker Green CMFDA (Invitrogen) according to the manufacturer's instructions. Following staining, the cells were co-cultured for 24 hours in Fluorobrite DMEM (Gibco) + 10% FBS in 4-well glass culture slides. After washing with PBS and mounting with ProLong Live Antifade Reagent (Invitrogen), cells were observed using an Olympus BH2 fluorescent microscope. Our results showed close interactions between MSC and H9c2 cells with mitochondria in long filamentous extensions that made contact with H9c2. There was some evidence that mitochondria were transferred from MSC to H9c2 cells. Experiments are underway to determine whether mitochondria transfer from H9c2 cells to MSC, and the effect of hypoxia. These results continue to suggest that mitochondrial transfer may be one mechanism used by MSC to improve heart function after myocardial infarction.
  25. FASEB J. 2022 May;36 Suppl 1
      Metabolic dysfunction and mitochondria defect are implicated in several age-associated diseases including age-related macular degeneration (AMD), a leading cause of blindness in the elderly. In AMD, mitochondrial oxidative stress in the retinal pigment epithelium (RPE) drives disease progression and growth of atrophic lesions. Mitochondria release and uptake has been recently identified as a novel mechanism of intercellular communication but its implication in ocular diseases such as AMD has never been investigated. Here, we examined the role of mitochondrial transfer as a new mechanism of metabolic crosstalk between RPE cells. Diseased mitochondria were purified from RPE cells treated with the AMD-associated cytokine TNFα (10 ng/mL), administered to host RPE cells and the effects of MitoTNFA compared to MitoCtrl , isolated from control RPE, or exposure to exogenous TNFα. We showed that treatment of healthy RPE with MitoTNFA , and not MitoCtrl , triggers mitochondrial network fragmentation and transcriptional upregulation of inflammatory factors RelB, IL6, IL8, and repression of PGC1α, mirroring the effect of direct TNFα treatment. ELISA assay confirmed that the effects observed were not caused by presence of soluble TNFα within the mitochondrial fraction. Metabolic profiling using the Seahorse XFe96 bioanalyzer further validated the ability of MitoTNFA to phenocopy TNFα-induced metabolic reprograming in RPE. Finally, we demonstrated that transfer of MitoCtrl improved the bioenergetic functions of both healthy and diseased RPE with significant increases in basal, maximal, and spare respiratory functions. Our results showed that mitochondria transfer recapitulates the context-specific phenotype from donor to host cells in RPE. This new paradigm in RPE biology may not only explain the centrifugal expansion of RPE lesions in AMD but also presents a promising therapeutic avenue for mitochondria-driven disorders such as AMD.
  26. FASEB J. 2022 May;36 Suppl 1
      OBJECTIVE: . To elucidate mechanisms underlying alveolar-capillary crosstalk in the context of lipopolysaccharide (LPS)-induced Acute Lung Injury (ALI), which causes The Acute Respiratory Distress Syndrome (ARDS).HYPOTHESIS: Oxidative crosstalk between alveolar epithelium and adjacent endothelium causes mitochondrial injury and barrier failure in Acute Lung Injury METHODS: . We instilled LPS (5mg/kg) by the intranasal (i.n.) route in anesthetized mice. We evaluated microvascular endothelia of isolated blood-perfused mouse lungs by confocal microscopy, and ALI by bronchoalveolar lavage (BAL) 48h later. For microscopy, we gave the potentiometric mitochondrial dye MTDR, and the reactive oxygen species (ROS) indicator DCF by intravascular infusion. We determined the role of endothelial uncoupling protein 2 (UCP2) by (1) UCP2 knockdown in endothelia by vascular siRNA injection, and (2) endothelial-specific knockout of UCP2. We transfected alveolar epithelium by i.n. instillation of catalase-expressing plasmid, or depleted neutrophils by intraperitoneal injection of anti-Gr1 antibody, 24 hours before i.n. LPS. We quantified barrier permeability through BAL protein content.
    RESULTS: . Confocal imaging indicated that baseline fluorescence intensity of both endothelial MTDR and DCF was steady, indicating that endothelial mitochondrial potential and endothelial ROS were stable. However, 48 hours after LPS instillation, MTDR fluorescence decreased in venular capillaries more than in alveolar capillaries (n=4, p<0.05), indicating endothelial mitochondrial depolarization. Simultaneously, DCF fluorescence increased in venular capillaries more than in alveolar capillaries (n=4, p<0.05), indicating an increase in endothelial ROS. LPS instillation increased BAL protein (n=4, p<0.05). Knockdown of endothelial UCP2 expression, or expression of transfected catalase in the alveolar epithelium blocked LPS-induced endothelial mitochondrial depolarization and BAL protein increase (p<0.05). Endothelial-specific knockout of UCP2, but not neutrophil depletion, blocked LPS-induced BAL protein increase (p<0.05).
    CONCLUSIONS: . We show here for the first time that the hyper-permeable responses of LPS-induced ALI result from mitochondrial depolarization in venular capillaries. Transfer of H2 O2 from alveoli, not neutrophils activated UCP2, causing proton influx into the mitochondrial matrix. Our findings reveal a novel mechanism of LPS-induced ALI, implicating mitochondria-oxidant coupling in barrier failure. Thus, UCP2 presents a potential therapeutic target for ARDS.
  27. FASEB J. 2022 May;36 Suppl 1
      OBJECTIVE: Matrix metalloproteinase-2 (MMP-2) is a ubiquitous protease that cleaves several extracellular and intracellular proteins. MMP-2 is activated intracellularly in response to enhanced oxidative stress resulting from myocardial ischemia/reperfusion (I/R) injury. Oxidative stress impairs mitochondrial function which is regulated by different proteins including mitofusin-2 (Mfn-2) and dynamin-related protein-1 (Drp-1) which control mitochondrial dynamics involving fusion and fission, respectively. As both proteins are localized at the mitochondrial outer membrane and MMP-2 is localized at the mitochondrial-endoplasmic reticulum associated membrane we hypothesized that MMP-2 may proteolyze Mfn-2 and Drp-1. We therefore investigated whether inhibition of MMP-2 could protect against the loss of mitochondrial dynamics proteins during I/R injury.METHODS: Hearts isolated from 3 month old C57BL/6J mice were perfused according to Langendorff at constant pressure and subjected to I/R injury (30 min ischemia and 40 min reperfusion) in absence or presence of MMP-2 preferring inhibitors ARP-100 (10 µM) or ONO-4817 (50 µM) or their vehicle (n=5 hearts per group). Effects on mechanical function were recorded. At the end of reperfusion, the hearts were homogenized and subcellular fractions were prepared. The degradation of troponin I (TnI), an intracellular MMP-2 target, was measured in the cytosolic fraction as a marker of MMP-2 activity. Levels of Mfn-2 and Drp-1 in the mitochondrial fraction were also measured using western blot. In silico analysis of potential MMP-2 cleavage sites was performed using Procleave.
    RESULTS: ARP-100 or ONO-4817 significantly increased left ventricular developed pressure compared to vehicle-treated I/R hearts (% of pre-ischemic baseline value at R40: IR+vehicle 25.5±3.2, IR+ARP 50.1±3.2, IR+ONO 57.3±1.6%, p<0.05). Similarly, both inhibitors significantly improved the rates of contraction and relaxation (+/-dP/dt). TnI loss was increased in I/R hearts as shown by a significant reduction in TnI (~30 kDa) and the appearance of an ~22 kDa band. ARP-100 or ONO-4817 attenuated TnI loss, indicating MMP-2 activation. Levels of Mfn-2 and Drp-1 in the mitochondrial fraction were significantly reduced in the I/R group and ARP-100 or ONO-4817 attenuated this reduction (p<0.05). Similar results were obtained using a different Mfn-2 antibody that binds to its C-terminus. In silico analysis of both mouse Mfn-2 and Drp-1 sequences showed several potential sites that could be targeted by MMP-2.
    CONCLUSIONS: During myocardial I/R injury, MMP-2 may affect mitochondrial dynamics by proteolysis of Mfn-2 and Drp-1. Inhibition of MMP-2 activity could protect against cardiac contractile dysfunction in part by preserving these proteins affecting mitochondrial fusion and fission.
  28. FASEB J. 2022 May;36 Suppl 1
      Prolonged muscle disuse is accompanied by a phenotypic shift characterized by declines in mitochondrial content and function within skeletal muscle. This loss in mitochondrial content can be partially attributed to elevations in various catabolic processes that occur with atrophy. One of these is termed mitophagy, a selective form of cellular recycling (autophagy) whereby dysfunctional mitochondria are degraded via lysosomes. TFEB and TFE3 are key transcription factors that regulate lysosomes by activating the expression of various lysosomal genes. Loss of TFE3 has been associated with depressed levels of autophagy and with mitochondrial dysfunction. It is speculated that these functional impairments are due to diminished clearance via the lysosomes, however to date this has not been examined. We hypothesized that the loss of TFE3 would amplify the mitochondrial dysfunction associated with muscle disuse, while paradoxically preserving muscle mass and mitochondrial content. Using a severe model of muscle disuse, sciatic denervation, we observed a 10% loss in hindlimb muscle mass within 7 days of denervation. In the absence of TFE3, a trend for muscle preservation was observed, as these animals lost 20% less muscle mass than WT counterparts. Reduced rates of respiration supported by complex I and II were observed irrespective of genotype, concurrent with elevated ROS emissions, suggesting an impaired oxidative capacity following 7 days of denervation. Surprisingly, TFE3 KO animals did exhibit lower levels of oxidative stress basally, but this too increased following 7 days of denervation, and taken relative to baseline the fold induction of ROS emission was 3 times greater than WT animals. Higher levels of mitophagy flux were observed in the absence of TFE3 basally, which supports the observed 35% decline in mitochondrial content as measured by COX activity, as well as the lower levels of ROS emissions. Following only 1 day of denervation, increases in mitophagy flux were observed in WT animals, while the response in KO animals was clearly attenuated. In the WT animals, denervation led to a 30% reduction in mitochondrial content, however no change was observed in the absence of TFE3. Finally, increases in a number of autophagy-related markers such Beclin-1 and ATG7 were observed following denervation irrespective of genotype. However, the mature form of Cathepsin B, a hydrolytic enzyme, was markedly reduced by 55% in the absence of TFE3 and did not increase to the same extent as WT following 7 days of denervation suggesting an impairment in lysosomal function. Together, our results suggest that TFE3 exerts multiple roles in skeletal muscle plasticity, as a partial mediator of muscle mass, and in the control of lysosomal function and mitophagy in response to the acute stress of muscle disuse.
  29. FASEB J. 2022 May;36 Suppl 1
      AIMS: Sirtuin 3 (SIRT3) has been shown to contribute to the mitochondrial cardiomyopathy of Friedreich's Ataxia (FRDA), which is characterized by a deficiency in mitochondrial frataxin along with mitochondrial hyperacetylation. Using a cardiomyocyte-specific SIRT3 knockout (SIRT3cKO) mouse model, we addressed two key questions: (1) whether SIRT3-induced acetylation of proteins is specific to the mitochondria; and (2) if mitochondrial SIRT3 is a key regulator of mitochondrial frataxin which impacts mitochondrial iron homeostasis and ferroptosis.METHODS AND RESULTS: The mitochondrial and cytosolic fractions were isolated from the ventricles of the hearts of SIRT3cKO mice. The mitochondrial and cytosolic proteins and mitochondrial iron levels were analyzed by comparison to SIRT3-Loxp wild-type (WT) control mice. Cardiac function study showed that SIRT3cKO mice developed heart failure as evidenced by reduction of ejection fraction (EF) and fraction shortening (FS) and increased isovolumic relaxation time (IVRT) and myocardial performance index (MPI) when compared to WT controls. Comparison of the mitochondrial and cytosolic fractions of the SIRT3cKO model to those of the WT control shows that, upon loss of SIRT3, mitochondrial, but not cytosolic, total lysine acetylation was significantly increased in the heart. Similarly, acetylated p53 (p53ace) was significantly upregulated only in the mitochondria, while levels of p53 were not altered in either compartment. These data demonstrate that SIRT3 is the primary mitochondrial deacetylase, while acetylation in the cytosol is independent of SIRT3 in cardiomyocytes. Most importantly, loss of SIRT3 in the mitochondria resulted in significant reduction of the protein frataxin and the Iron (II) export protein ferroportin (FPN). Furthermore, levels of glutathione peroxidase 4 (GPX4) were also downregulated in the mitochondria. This was accompanied by a significant increase in levels of 4-hydroxynonenal (4-HNE), an indicator of lipid peroxidation, and suggestive of upregulated mitochondrial ferroptosis in SIRT3cKO mouse hearts. Additionally, mitophagy marker beclin-1 expression was significantly reduced in the mitochondria of SIRT3cKO mice. Levels of glucose transporter-1 (GLUT1), which functions to deliver the antioxidant Vitamin C to the mitochondria and reduce ROS production, were also diminished in the mitochondria. Mechanistically, mitochondrial levels of hypoxia inducible factor-2α (HIF-2α) were downregulated, while those of HIF-1α remained unchanged in SIRT3cKO mouse hearts. Treatment with ferroptosis inhibitor ferrostatin-1 for 14 days significantly reduced 4-HNE levels and rescued preexisting impaired cardiac function in SIRT3cKO mice.
    CONCLUSIONS: For the first time, we have demonstrated that cardiomyocyte SIRT3 deficiency causes mitochondrion-specific acetylation and impairment of frataxin and ferroportin potentially via downregulation of HIF-2α. Our results suggest that the SIRT3-ferroptosis pathway may be a novel target for the mitochondrial cardiomyopathy of FRDA.
  30. FASEB J. 2022 May;36 Suppl 1
      Endothelial cell (EC) inflammation is a major pathogenic feature of many diseases including acute lung injury (ALI). However, the role of the dynamin-related protein 1 (Drp1), a mitochondrial fission factor, in regulating EC inflammation is poorly understood. We show here that Drp1 is a critical mediator of EC inflammation via its ability to control NF-κB activation. When Drp1 is disabled, expression of adhesion molecules (ICAM-1, VCAM-1) and cytokines/chemokines (IL-6, MCP-1) is markedly reduced in cells stimulated with thrombin. Drp1-depleted cells were also impaired in their ability to activate NF-κB by thrombin. Mechanistic study revealed that thrombin-induced phosphorylation and nuclear DNA binding of RelAp65 was inhibited in Drp1-depleted cells. Surprisingly, the thrombin-induced degradation of IκBα in the cytosol remained unaffected in Drp1-depleted cells, suggesting a defect in the translocation of released RelA/p65 to the nucleus in these cells. Indeed, nuclear translocation of RelA/p65 caused by thrombin was inhibited in Drp1-depleted cells. Together, these data uncover a novel function of Drp1 in mediating EC inflammation via regulation of phosphorylation and nuclear translocation of RelA/p65.
  31. FASEB J. 2022 May;36 Suppl 1
      STUDY OBJECTIVE: Hyperoxia-induced acute lung injury (HALI) leads to respiratory failure and mitochondrial dysfunction is a critical mediator of HALI. We previously reported Toll-Like Receptor (TLR)4 prevents oxidant-induced ALI by maintaining mitochondrial bioenergetics in mammalian lung endothelium (Ec). Translocase of Outer Mitochondrial Membrane (TOM) have recently been identified to play important roles in mitochondrial quality control, primarily in yeast [1]. TOM70 is an essential subunit of TOM and mediates uptake of newly synthesized proteins to mitochondria as well has putative anti-oxidant and anti-apoptotic properties. TOM70 appeared to parallel many of the cytoprotective signaling properties of TLR4 [2-4]. Therefore, our hypothesis is TLR4 regulates TOM70 expression and TOM70 has mitochondrial and lung protective functions in HALI.METHODS: C57BL/6J mice (6-8 wks, both sex) were purchased from Jackson Laboratory, and Tlr4-/- mice were bred at Duke Animal facilities (IACUC A160-19-07) [5]. For hyperoxia exposure, mice were placed in a Plexiglas chamber with 5L/min of 100% oxygen continuous flow with food and water ad libitum. We previously reported 72 h of continuous hyperoxia as a time of maximal lung injury [6], which we used as a representative time point. Bodyweight and survival were monitored daily. Bronchoalveolar lavage (BAL) were collected and BAL protein were determined by a BCA assay. Protein and RNA were extracted from lung tissues and expression was determined by western blot and qPCR, respectively. Mouse lung Ec (MLEC) were isolated from lung tissues and treated with control or Tom70siRNA for 48 h. Statistical differences were analyzed by a Student-t test.
    RESULTS: Tlr4-/- MLEC decreased TOM70 and increased Parkin, PINK1, and MFN2 protein expression compared to WT. Tom70 siRNA effectively silenced Tom70 with >75% efficiency at 48 h after transfection. Tom70 siRNA-treated MLEC showed increased Mfn1, Mff, and Fis1mRNA expression, which are markers of mitochondrial fusion-fission. Hyperoxia-exposed mice showed marked weight loss and higher levels of BAL proteins compared to control, indicating increased permeability of the alveolar-capillary membrane. Moreover, TOM70 protein expression was significantly decreased by hyperoxia in both male and female mice.
    CONCLUSION: TLR4 deficiency decreased TOM70 expression and induced mitophagy and mitochondrial fusion, indicating that TOM70 is regulated by TLR4. Silencing TOM70 resulted in increased mitochondrial fusion and fission in MLEC, suggesting that TOM70 impacts mitochondrial quality control in mammalian, primary cells (MLEC) that are critical for lung airspace barrier function. Hyperoxia decreased TOM70 protein expression in mouse lungs, which may be a potential mechanism whereby hyperoxia leads to respiratory failure. Given that TOM70 maintains mitochondrial health, our data suggest that TOM70 may play a key role in the pathological mechanisms of HALI. 1. Liu et al. (2021) bioRxiv, 2. Zhang et al. (2019) Antioxid Redox Signal, 3. Zhang et al. (2016) Antioxid Redox Signal, 4. Takyar et al. (2015) FASEB J. 5. Kim et al. (2019) Aging Cell. 6. Zhang et al. (2021) AJP Lung Cell Mol Physiol.
  32. FASEB J. 2022 May;36 Suppl 1
      Mitochondria play an important role in energy production and cellular metabolism. Mitochondria contain their own DNA (mtDNA), which encodes 13 subunits necessary for oxidative phosphorylation. Over 1500 other mitochondrial proteins, including the 77 remaining subunits required for oxidative phosphorylation and the machinery required for transcription and translation of mtDNA, are encoded by the nuclear genome. Thus, the nuclear and mitochondrial genomes must communicate to respond to the energetic needs of the cell. The mechanism of this communication is unclear. The mitochondrial proteome, including the transcriptional machinery, is subject to post-translational modifications (PTMs) such as phosphorylation of serine, threonine, and tyrosine and acylation of lysine. We hypothesize that PTMs of the mitochondrial transcriptional machinery regulate mitochondrial gene expression, akin to mechanisms controlling nuclear gene expression. Transcription of mtDNA requires three nuclear-encoded proteins: mitochondrial transcription factor A (TFAM), transcription factor B2 (TFB2M), and mitochondrial RNA polymerase (POLRMT). An accessory factor, mitochondrial ribosomal protein L12 (MRPL12), is thought to stabilize POLRMT and may promote transcription. Prior experiments in our lab show phosphorylation mimics of TFB2M have significantly reduced binding affinity for mtDNA and exhibit transcription initiation defects in vitro. Using mass spectrometry POLRMT was previously shown to be acetylated at one lysine and phosphorylated at nine amino acids, while MRPL12 contains five acetylated lysines and one phosphorylated threonine. The biochemical function of these modifications is unknown. PTMs were studied by using site-directed mutagenesis to replace the amino acid of interest to mimic acetylation (lysine to glutamine) or phosphorylation (threonine to glutamate) of POLRMT and MRPL12. Mutated proteins were purified and their mtDNA promoter binding affinity was determined by fluorescence anisotropy experiments. Fluorescence anisotropy revealed that POLRMT PTM mimics had little effect on mtDNA binding. However, when WT MRPL12 was co-incubated with WT POLRMT mtDNA binding affinity was enhanced by 30%. Increased binding affinity was lost with MRPL12 PTM mimics. WT and PTM mimics of MRPL12 were also overexpressed in mammalian cell lines. mtDNA transcript levels and mtDNA content were measured using quantitative PCR. mtDNA content was largely unchanged while some transcript-dependent effects were observed in the presence of MRPL12 protein mutants.
  33. FASEB J. 2022 May;36 Suppl 1
      Mitochondria produce over 90% of cellular ATP and actively participate in maintaining ion homeostasis, redox status, lipid metabolism, and cell growth. Changes in the matrix volume of mitochondria affect their functional and structural integrity. Modest volume increases associated with modulation of the inner mitochondrial membrane can activate electron transfer chain and oxidative phosphorylation, whereas excessive swelling impairs structural organization of the membrane and initiate mitochondria-mediated cell death mechanisms. Therefore, clarifying the precise mechanisms of excessive mitochondrial swelling is important for regulation of mitochondria-mediated cell death pathways in response to energy and oxidative stresses. Opening of non-selective mitochondrial permeability transition pores (mPTP) is the primary cause of excessive matrix swelling. The molecular identity remains unknown and recent studies suggest the existence of two or more types of mPTP that can be composed of different protein(s). The adenine nucleotide translocator (ANT) and FO F1 -ATP synthase were proposed to be potential mPTP core components that can act together or independently each other. Here, we elucidated the role of ANT in mPTP opening by applying both experimental and computational approaches. mPTP opening was evaluated in cardiac mitochondria that were exposed to moderate and high Ca2+ concentrations in the absence and presence of respiratory substrates and ADP. We developed a detailed model of the ANT transport mechanism including the matrix (ANTM ), cytosolic (ANTC ), and pore (ANTP ) states of the transporter that was able to simulate our experimental data. In addition, we corroborated and simulated our ANT model based on previous ANT kinetics data. The model was successful not only in simulating ANT pore state transition, but also explained the potential role of ANT in mPTP opening in cardiac mitochondria.
  34. FASEB J. 2022 May;36 Suppl 1
      Nonalcoholic fatty liver disease (NAFLD) is a hepatic manifestation of metabolic syndrome that affects approximately 25% of adults globally. Although several pharmacological therapies have been reported to improve NAFLD, the therapeutic ranges of the drugs are limited. Mitochondrial dysfunction could be a key factor involved in NAFLD progression. However, the association between mitochondrial dynamics, which involves mitochondrial fission and fusion, and NAFLD development has not been fully characterized to date. Herein, we examined the effects of alterations of mitochondrial dynamics in hepatocytes and liver sinusoidal endothelial cells (LSECs), the most abundant non-parenchymal cells in the liver, on NAFLD development and hypothesized that rebalancing of mitochondrial dynamics in these cells could attenuate NAFLD progression. Mice were fed a choline-deficient, L-amino acid-defined diet (CDAA) for 1 or 4 weeks to recapitulate human NAFLD development. Malformed mitochondria, such as megamitochondria and mitochondria with deformed cristae, were substantially increased in both, hepatocytes and LSECs, of mice that were fed CDAA diet, and were associated with NAFLD development. In addition, decreased mitochondrial content and increased mitochondrial number, suggesting excessive mitochondrial fission, were induced in hepatocytes, but not in LSECs, after four weeks of CDAA feeding. These unfavorable changes in the mitochondria of hepatocytes, but not of LSECs, were attenuated by mdivi1, a chemical mitochondrial fission inhibitor. Liver inflammation and fibrosis were also attenuated by mdivi1, as assessed by serum biochemical and histochemical analyses. The expression of several inflammatory and fibrogenic genes (Tnfα, Il6, and Col3α1) tended to decrease with mdivi1 treatment. The expression of genes related to mitochondrial biogenesis, fission, fusion, and mitophagy was also significantly reduced by mdivi1 (p<0.05), suggesting rebalanced mitochondrial dynamics. Ultrastructural analyses revealed that endoplasmic reticulum stress and autophagy in hepatocytes were mitigated by mdivi1 administration. Taken together, these findings suggest that excessive mitochondrial fission in hepatocytes induces alterations in mitochondrial dynamics and other organellar injuries, and subsequently, liver inflammation and fibrogenesis. Therefore, rebalancing mitochondrial dynamics in hepatocytes by inhibiting excessive fission is a promising therapeutic strategy for NAFLD.
  35. FASEB J. 2022 May;36 Suppl 1
      BACKGROUND: . Acute kidney injury (AKI) is an independent risk factor for mortality and morbidity. Inflammation is now believed to play a major role in the pathophysiology of AKI. Receptor-interacting protein kinase 3 (RIP3) is a member of the receptor-interacting protein (RIP) family of serine/threonine protein kinases. RIP3 is a regulator of both programmed necrosis/necroptosis, an inflammatory form of cell death observed in pathogen-induced and sterile inflammation, and TNFα-induced apoptosis. In the induction of necroptosis, RIP3 is a major component of the tumor necrosis factor (TNF) receptor-I signaling complex which through additional interactions with (RIP1) and pseudokinase mixed lineage kinase domain-like protein (MLKL) forms the necrosome. More recently, RIP3 activity was found to promote sepsis-induced AKI via mitochondrial dysfunction. Since I/R injury triggers numerous pathological changes, including apoptosis, oxidative stress, and inflammation, suggesting a role of mitochondrial function in RIP3-dependent I/R injury.OBJECTIVE: We investigated whether RIP3 translocates into mitochondria in response to ischemia/reperfusion (I/R) to interact with Mitofilin and promote mitochondria damage that facilitates mtDNA release into the cytosol. We postulated that release of mtDNA activates cGAS/STING pathway leading to increased nuclear transcription of pro-inflammatory markers that exacerbates renal I/R injury.
    MATERIAL AND METHODS: . C57/6N and RIP3-/- mice as well as HK2 cells were used. Monolateral kidneys were subjected to 30 min of ischemia followed by either 12, 24, or 48 h of reperfusion. Protein levels of RIP3, Mitofilin, cGAS, STING, and p-p65 were measured using Western and immunofluorescence analysis, while IL-6, TNF-α, and ICAM-1 expressions as well as mtDNA release were assessed by qRT-PCR, ELISA. Kidney function was measured by blood urea nitrogen and creatinine kits, and the interaction between RIP3 and Mitofilin was measured by Co-Immunoprecipitation. In WT, RESULTS: . We found that renal I/R increased RIP3 levels, and its translocation into mitochondria. We observed that RIP3 interacts with Mitofilin likely promoting its degradation. While renal I/R associated with mitochondria damage, increased mtDNA release, activation of cGAS/STING/p65 pathway and increased transcription of pro-inflammatory markers including IL-6, TNF-α and ICAM-1, all these effects were decreased in RIP3-/- mice. In HK-2, RIP3 overexpression or Mitofilin knockdown increased cell death by activating the cGAS-STING/p65 pathway.
    CONCLUSION: . We demonstrated that kidney I/R increases RIP3 levels in the cytosol and it translocates into mitochondria, where it interacts with and promote Mitofilin degradation leading to increased mitochondrial structures damage and dysfunction. The subsequent increase in ROS production in mitochondria is postulated to facilitate mtDNA damage and release into the cytosol where it activates the cGAS/STING/p-p65 pathway leading to amplified nuclear transcription of pro-inflammatory markers that subsequently increase renal I/R injury. Together, this study point to an important role of RIP3 in the initiation and development of renal I/R injury.
  36. FASEB J. 2022 May;36 Suppl 1
      Mitochondrial dysfunction is a feature of heart failure with preserve ejection fraction (HFpEF). Central infusion of Ang II causes sympatho-excitation and hypertension with consequent HFpEF. The UPRmt activation is a retrograde mitochondrial stress response that promote recovery of defective mitochondria. UPRmt is activated by misfolded accumulation of nuclear-encoded mitochondrial proteins in cytosol and mitochondria. Despite this information, the mechanisms of UPRmt and mitochondrial dysfunction are largely unknown in the HFpEF heart. We hypothesized that concomitant sympathoexcitation with hypertension downregulates cardiac UPRmt leads to cardiac remodeling in HFpEF. Male Sprague-Drawly rats (250-300g) were subjected to central infusion of either Ang II (at 20 ng/min, 0.5 μl/h, ICV) or isotonic saline (0.5 μl/h, ICV, control) through osmotic mini-pumps for 14 days. This leads to concomitant sympathetic overstimulation and systemic hypertension with myriad features of HFpEF. Transthoracic echocardiography and haemodynamic recordings were performed at day 14 post ICV infusion. UPRmt , mitochondrial injury, and cardiac remodeling were assessed using, whole-tissue, cytosolic, and isolated mitochondrial protein Western immunoblots, cytochemistry, and histology. Sympatho-excitatory effect on UPRmt was examined in vitro using norepinephrine (NE) and H9c2 cardiomyocytes. The HFpEF rats showed significant diastolic dysfunction indicated by reduced E/A (HFpEF: 1.2 ± 0.1 vs Con: 1.5 ± 0.2) with preserved left ventricular ejection fraction (HFpEF: 75 ± 3% vs Con: 77 ± 4%). Histological evaluation showed increased cardiomyocyte hypertrophy (HFpEF: 46.3 ± 5 vs Con: 36.9 ± 6) and cardiac fibrosis (HFpEF: 4.3 ± 0.2 vs Con: 2.1 ± 0.3). Measurement of ATF5 (Activating Transcription Factor 5), a key UPRmt activation marker was decreased in HFpEF cytosol and mitochondria, while mitochondrial chaperonin HSP60 (heat-shock protein 60) was decreased in cytosol but increased in HFpEF mitochondria. Simultaneously, there was increased accumulation of oxidative phosphorylation (OXPH) Complex I, IV, and V misfolded subunits in the mitochondrial matrix. Furthermore, YME1L1, a mitochondrial matrix metalloprotease of misfolded protein degradation is reduced in HFpEF mitochondria. Concomitantly, there was increased mitochondrial ROS, reduced Mn-SOD, and increased autophagy markers p62-SQSTM1 and LC3B-II in HFpEF hearts. Notably, a reduced mitochondrial biogenesis and fusion, but increased fission, indicates a lack of UPRmt stress activated mitochondrial recovery in HFpEF heart. Our in vitro data corroborated a reduced UPRmt in response to NE treatment associated with mitochondrial depolarization and increased mitochondrial ROS level. In conclusion, concomitant sympatho-excitation and systemic hypertension contributes to reduced UPRmt , which is a unique feature of HFpEF heart. A lack of UPRmt activation fails to execute defective mitochondrial recovery in HFpEF heart conceivably driving HFpEF cardiac remodeling. This study identifies the key intermediary links of UPRmt downregulation in a potential HFpEF rat model. Therefore, boosting UPRmt can potentially have a therapeutic benefit for clinical HFpEF.
  37. FASEB J. 2022 May;36 Suppl 1
      Barth syndrome (BTHS) is a rare, X-linked disorder of mitochondrial phospholipid metabolism caused by variants in the gene TAFAZZIN.TAFAZZIN is a transacylase involved in the remodeling of cardiolipin (CL), a dimeric phospholipid localized to the inner mitochondrial membrane. Lack of TAFAZZIN-based remodeling results in irregular cardiolipin content, characterized by increased unremodeled CL, increased monoloysocardiolipin (the CL remodeling intermediate), and a decrease in remodeled CL, which is enriched in polyunsaturated fatty acyl chains that are tissue-specific in composition. BTHS is clinically characterized by cardiomyopathy, neutropenia, and myopathy, with a high morbidity and mortality. There are no approved disease-specific therapies. To investigate the cellular pathology and to identify new areas of potential therapeutic intervention, we developed two CRISPR-edited cell lines: TAFAZZIN-knockout (KO) HEK293 cells and iPSCs with which to perform broad-based discovery experiments and to study tissue-specific disease effects, respectively. A combined multi-omics approach including proteomics, lipidomics, and metabolomics in TAZ-KO HEK293 cells revealed diverse mitochondrial abnormalities, including defects in complex I of the respiratory chain, abnormal PDK2 expression, and dysregulation of proteins involved in mitochondrial quality control including PARL and PGAM5. Importantly, we discovered that molecules that bind to cardiolipin (SS-31) or inhibit nascent cardiolipin deacylation (bromoenol lactone), partially remediate these mitochondrial defects. We next explored cell-type specific dysfunction in iPSC-derived TAZ-KO and wild-type cardiomyocytes and neurons via lipidomics, RNA-seq, and functional studies. We identified disturbances in cellular lipid content including an expected increase in the monolysocardiolipin content and a reduction that exhibited cell-type specificity. RNAseq identified dysregulation in pathways regulated by PARL and PGAM5 including Wnt signaling, apoptosis, and autophagy in the undifferentiated state, with differentiated cell types highlighting pathways such as glucose metabolism and response to cellular stimuli. Oxygen consumption studies show impaired maximal respiratory capacity in TAZ-KO cardiomyocytes and neurons. Ongoing investigations aim to address cell type specific mitochondrial dysfunction by characterizing PARL abundance, PGAM5 cleavage, and mitochondrial morphology in TAZ-KO iPSC derived cell types. Additionally, we are currently targeting cardiolipin metabolism in differentiated TAZ-deficient cells with the goal of remediating cellular lipids, mitochondrial gene expression, and oxygen consumption abnormalities.
  38. Sci Rep. 2022 May 10. 12(1): 7652
      Autophagy is an essential cellular pathway that ensures degradation of a wide range of substrates including damaged organelles or large protein aggregates. Understanding how this proteolytic pathway is regulated would increase our comprehension on its role in cellular physiology and contribute to identify biomarkers or potential drug targets to develop more specific treatments for disease in which autophagy is dysregulated. Here, we report the development of molecular traps based in the tandem disposition of LC3-interacting regions (LIR). The estimated affinity of LC3-traps for distinct recombinant LC3/GABARAP proteins is in the low nanomolar range and allows the capture of these proteins from distinct mammalian cell lines, S. cerevisiae and C. elegans. LC3-traps show preferences for GABARAP/LGG1 or LC3/LGG2 and pull-down substrates targeted to proteaphagy and mitophagy. Therefore, LC3-traps are versatile tools that can be adapted to multiple applications to monitor selective autophagy events in distinct physiologic and pathologic circumstances.
  39. FASEB J. 2022 May;36 Suppl 1
      Spinal muscular atrophy (SMA) is a debilitating neuromuscular disorder caused by a mutation in the survival motor neuron 1 (SMN1) gene and it is the leading genetic cause of infant mortality. Recently approved genetic therapies designed to augment full length SMN protein in the central nervous system fail to ameliorate abnormal skeletal muscle features, which strongly suggests an important role for SMN protein in skeletal muscle homeostasis. This study aimed to characterize key modifiers of skeletal muscle mitochondrial turnover and dynamics during disease progression in a pre-clinical murine model of SMA in vivo. Additionally, we investigated the effects of a single dose of exercise on these mitochondrial biology-regulating pathways in the skeletal muscle of SMA mice. Muscle samples were collected from wild-type (WT) and Smn2B/- (SMA) mice at postnatal day 9 (P9), P13, and P21 to examine skeletal muscle-specific disease progression. Mitochondrial content did not differ between genotypes at all timepoints as evident by similar levels of mitochondrial oxidative phosphorylation proteins, succinate dehydrogenase staining, and citrate synthase content. However, mRNA content of key genes responsible for regulating mitochondrial quality such as nuclear respiratory factor 2, mitochondrial transcription factor A, and p53 were significantly higher in SMA mice versus WT at P21. Additionally, we observed elevated mitochondrial fission activity as indicated by a 2-fold increase (p < 0.05) in dynamin related protein 1 (DRP1) activation. Concomitantly, the expression of several mitophagy proteins including BLC2 interacting protein 3, parkin, and PTEN-induced kinase 1 were significantly increased by 2.9-, 2.7-, and 2-fold, respectively, compared to WT animals at P21. Interestingly, we observed blunted (p < 0.05) inclusion of optic atrophy 1 exon 4b, a key exon in mitochondrial biogenesis, between WT and SMA mice at P21. Acute exercise significantly increased the inhibition of DRP1-mediated mitochondrial fission activity in the muscles of SMA mice relative to sedentary SMA mice. However, a single exercise dose failed to alter the expression of mitophagy proteins up to three hours after running. Our data demonstrate that skeletal muscle mitochondrial health is compromised in SMA mice due to elevated fission and mitophagy processes. Furthermore, SMA mice display aberrant alternative splicing of Opa1 transcripts that may in turn hinder mitochondrial biogenesis in later disease stages. We also highlight that an acute bout of treadmill running elicits pro-fusion signaling to potentially improve the mitochondrial reticulum. Collectively, this study is the first to reveal mitochondrial dysfunction in SMA skeletal muscle and outlines signalling associated with mitochondrial plasticity following a single dose of exercise.
  40. FASEB J. 2022 May;36 Suppl 1
      Dynamin-related protein 1 (Drp1) is a key regulator of mitochondrial fission. Excessive Drp1-mediated mitochondrial fission in skeletal muscle from humans with severe obesity is associated with impaired insulin action. However, it remains unclear whether specific inhibition of Drp1 in skeletal muscle cells alleviates insulin resistance in obesity. Therefore, this study aims to determine the direct role of Drp1 on regulating insulin action in human skeletal muscle cells derived from humans with severe obesity. Human skeletal muscle cells from six lean, insulin-sensitive (LN, BMI = 22.7 ± 1.2 kg/m2 ,HOMA-IR = 1.9 ± 0.4) and six severely obese, insulin-resistant (OB, BMI = 47.3 ± 2.8 kg/m2 , HOMA-IR = 3.4 ± 0.4) subjects were pooled together, respectively. At 90% confluency, myoblasts were transfected using polyethyleneimine with a Drp1 shRNA (shDrp1) or scramble shRNA constructs (shCtrl). After 48 h, the medium was replaced with differentiation media with puromycin. On day 7 of differentiation, the mitochondrial network, reactive oxygen species (ROS), insulin signaling, glucose uptake, and protein markers of mitochondrial dynamics and mitochondrial content were assessed. RNA sequencing was also performed on OB-shCtrl and OB-shDrp1 myotubes. Differentially regulated genes were identified, and a gene set enrichment was used to determine pathway modulations. Drp1 protein expression was reduced in OB-shDrp1 myotubes compared to LN-shCtrl and OB-shCtrl (72% and 78%, respectively, P<0.05). OB-shCtrl myotubes exhibited fragmented mitochondrial networks with an increase in the number of non-networked individual mitochondria compared to the LN-shCtrl (P<0.05). The loss of Drp1 in OB-shDrp1 myotubes restored the mitochondrial network structure with the reduced number of non-networked mitochondria compared to OB-shCtrl (P<0.05), and is not different from LN-shCtrl. There were no differences in protein expression of markers of mitochondrial dynamics and content. Regarding insulin action, insulin-stimulated Akt Ser473 phosphorylation and glucose uptake (over basal condition) were both reduced in OB-shCtrl myotubes compared to LN-shCtrl (P<0.05). Importantly, OB-shDrp1 myotubes significantly increased insulin-stimulated Akt Ser473 phosphorylation and glucose uptake compared to OB-shCtrl (P<0.05). In addition, ROS was elevated in OB-shCtrl myotubes when compared to LN-shCtrl (P<0.05) but was reduced in OB-shDrp1 myotubes (P<0.05). Lastly, the loss of Drp1 revealed an upregulation in genes responsible for fatty acid oxidation and downregulation in genes for glycolysis in OB-shDrp1 myotubes compared to OB-shCtrl. These results demonstrate that the loss of Drp1 improves mitochondrial morphology and enhances insulin action, which may, at least partially, be due to reduced ROS production and improved fat oxidation in skeletal muscle cells from humans with severe obesity. Our data suggest that Drp1 may serve as an important regulator of skeletal muscle insulin action.
  41. FASEB J. 2022 May;36 Suppl 1
      INTRODUCTION: Cardiac fibrosis, characterized by wall stiffening, reduced contractility, and impaired overall heart performance, is a self-reinforcing process in response to injury (e.g., myocardial infarction) and pressure overload (e.g., systemic hypertension and pulmonary arterial hypertension [PAH]). The severity of cardiac fibrosis is associated with cardiac dysfunction, increased risk of arrhythmia, and mortality. However, there is a lack of effective therapies designed to inhibit or reverse cardiac fibrosis. Hyperproliferation of cardiac fibroblasts (CFs) and their differentiation into myofibroblasts are the key contributors to cardiac fibrosis. We previously showed that mitochondrial reactive oxygen species (mROS) are one of the regulators for activating proliferative signaling in rat neonatal CFs. Moreover, we reported that phosphorylation of dynamin-related protein 1 (DRP1) by a stress-responsive protein kinase D (PKD) at the outer mitochondrial membrane (OMM) promotes mROS generation via increased mitochondrial fission in H9c2 cardiac myoblasts.HYPOTHESIS: Mitochondrial PKD-DRP1 signaling increases mitochondrial fission leading to increased mROS levels, which activates proliferative signaling.
    METHODS: Primary adult CFs from human ventricles and the right ventricles (RVs) of a SU5415/hypoxia-induced rat PAH model were used. Mitochondrial morphology and mROS levels were measured by confocal microscopy.
    RESULTS: Overexpression of PKD1 increased DRP1 phosphorylation at Ser637, and activates proliferative signaling pathways including ERK1/2 and p38 in human CFs, but did not promote their differentiation into myofibroblasts, assessed by the amount of a-smooth muscle actin. To specifically identify the involvement of OMM-localized PKD activity in these processes, we generated an OMM-targeted dominant-negative PKD1 (termed mt-PKD-DN) by adding an OMM-target sequence (amino acid 1-33 from human TOM20) at the N-terminus of PKD1-K612W. Importantly, mt-PKD-DN was able to inhibit mitochondrial fission, decrease mROS, hyperpolarize mitochondrial membrane potential, and ultimately decrease proliferation in human CFs. To further understand the role of PKD-DRP1 signaling in vivo, we employed a preclinical PAH animal model. Immunofluorescence staining of RVs from PAH rats revealed that PKD activity and DRP1 phosphorylation increased only in CFs, but not in ventricular myocytes. RV-CFs isolated from PAH rats exhibited increased mitochondrial fission, mROS levels, and proliferative signaling compared to RV-CFs from control rats, and these changes were abolished by adenoviral expression of mt-PKD-DN.
    CONCLUSION: Mitochondrial mROS elevation via PKD-DRP1-dependent mitochondrial fission promotes CF proliferation. Targeting mitochondrial PKD may be a potential therapeutic strategy to reduce cardiac fibrosis under pathological conditions such as PAH.
  42. FASEB J. 2022 May;36 Suppl 1
      Caloric restriction (CR) in laboratory rodents prevents obesity, promotes healthy aging, and increases resilience against several pathological stimuli. At the mitochondrial level, protection promoted by CR in the brain and liver is related to higher calcium uptake rates and retention capacity, preventing Ca2+ -induced mitochondrial permeability transition. Dietary restriction has been demonstrated to increase kidney resistance against damaging stimuli such as ischemia/reperfusion, but if these effects are related to similar mitochondrial adaptations had not yet been uncovered. Here, we characterized changes in mitochondrial function in response to six months of CR in rats, measuring oxidative phosphorylation, redox balance and calcium homeostasis. CR promoted an increase in mitochondrial oxygen consumption rates under non-phosphorylating (state 4) and uncoupled (state 3U) conditions. While CR prevents mitochondrial reactive oxygen species production in many tissues, in kidney we found that mitochondrial H2 O2 release was enhanced in CR rats, although levels of carbonylated proteins and methionine sulfoxide were unchanged. Surprisingly, and opposite to the effects observed in brain and liver, mitochondria from CR animals are more prone to Ca2+ -induced mitochondrial permeability transition. CR mitochondria also displayed higher calcium uptake rates, which were not accompanied by changes in calcium efflux rates, nor related to altered inner mitochondrial membrane potentials, or the amounts of the Mitochondrial Calcium Uniporter (MCU). Instead, increased mitochondrial calcium uptake rates correlate with a loss of Mitochondrial Calcium Uptake 2 (MICU2), an MCU modulator, in CR kidneys. Interestingly, MICU2 is also modulated by CR in liver, suggesting it has a broader diet-sensitive regulatory role determining mitochondrial calcium homeostasis. Together, our results remark the highly organ-specific bioenergetic, redox, and ionic transport effects of CR. Specifically, we describe the regulation of the expression of MICU2, and its effects on mitochondrial calcium transport, as a novel and interesting aspect of the metabolic responses to dietary interventions.
  43. FASEB J. 2022 May;36 Suppl 1
      In the kidney, 95% of O2 consumed through mitochondrial respiration is used for Na+ transport. The circadian clock regulates Na+ transport with a necessary diurnal variation for optimal health. The circadian clock transcription factor BMAL1 is proposed to regulate mitochondrial bioenergetic function; however, this has not been extensively investigated in the kidney. Therefore, we tested the hypothesis that renal mitochondrial respiration may be impaired or altered in a novel Bmal1 clock gene knock-out rat model and is associated with loss of diurnal control of Na+ excretion. Male and female global Bmal1+/+ and Bmal1-/- rats at 12-14 weeks of age maintained in a regular 12-hour light/dark cycle was used to measure renal mitochondrial O2 consumption. Outer renal medullary tissue was dissected and prepared for respiration measurements at either day (ZT2-4) or night (ZT14-16) periods to correspond with minimum and maximum whole-body energy consumption as well as peak and trough BMAL1 protein expression, respectively (n = 10, Bmal1+/+ ; n = 22, Bmal1-/- ). Respiration was assessed using permeabilized tissue in the Oroboros Oxygraph and analyzed using Data Lab 2 software (O2K, Oroboros Instruments GmbH). Mitochondrial gene expression analysis was assessed using digital drop PCR. Data from both sexes were combined for each genotype at the two time points and analyzed by two-way ANOVA. Mitochondrial state 3 O2 consumption was significantly higher (main effect of genotype p = 0.0310) in Bmal1-/- (118 ± 156 pmol/s*mg) compared to BMAL1+/+ rats (75 ± 15 pmol/s*mg; Tukey post-hoc p = 0.0303). We also observed a significant increase in complex IV activity in Bmal1-/- rats during the dark vs. light period (164 ± 40 vs. 306 ± 40 pmol/s*mg; p=0.0031, t-test). Mitochondrial-associated genes, optic atrophy 1 (Opal1) and mitofusion 1 (Mfn1) show as significant day-night difference in mRNA expression in kidneys from BMAL1+/+ control rats that is lost in Bmal1-/- rats consistent with disrupted mitochondrial fusion and fission processes. Our lab previously reported that male Bmal1-/- rats do not have the typical night-day difference in Na+ excretion, which is consistent with our new data showing an alteration in mitochondrial function in kidneys from Bmal1-/- rats. Therefore, our findings demonstrate that BMAL1 may play a role in maintaining the coupling of mitochondrial respiration and ATP generation in the kidney and support the hypothesis that diurnal Na+ handling is regulated in part by BMAL1-dependent mitochondrial function.
  44. FASEB J. 2022 May;36 Suppl 1
      Autophagy is an essential cellular process regulated by intracellular calcium signals, which play an important role in autophagic activation during metabolic changes. Calcium transporters are present in mitochondria, an organelle able to uptake and release calcium ions, thus participating in cellular calcium signaling. Uptake by mitochondria is mediated by the mitochondrial calcium uniporter (MCU), while extrusion occurs through the mitochondrial sodium/lithium/calcium exchanger (NCLX). The aim of this work was to investigate how MCU and NCLX affect autophagy. To do so, we evaluated makers of autophagic activity in murine Aml-12 hepatic cells transfected with siRNAs targeting either MCU or NCLX expression, leading to genetic knockdown (KD). Additionally, we investigated the autophagic response to serum/amino acid starvation and rapamycin (mTORC1 inhibitor) treatment in Aml-12 cells with NCLX KD or pharmacological inhibition by CGP37157 (CGP). Using a cell line stably expressing the autophagic probe LC3-GFP-mCherry, we observed that NCLX KD leads to impaired autophagosome formation under basal conditions. Curiously, this effect was associated with a significant decrease in mRNA expression of LC3A and LC3B genes, while the expression of other autophagy-related genes, such as TFEB, ATG5, ATG12, and ATG7, was upregulated or unchanged. Conversely, MCU KD led to an apparent increase in autophagosome and autolysosome numbers, indicating enhanced autophagic activity. Interestingly, MCU KD also led to decreased expression of LC3A, but not LC3B. The expression of TFEB, ATG12, ATG5, and ATG7 were decreased or unchanged by MCU KD. After autophagic stimulation by serum/amino acid starvation, the levels of LC3 II were lower in NCLX KD cells compared to negative control in the presence or absence of bafilomycin A1, which indicates a reduction of autophagic flux. The levels of LC3 I were significantly lower in NCLX KD cells under basal and stimulated conditions, corroborating the decreased LC3 mRNA levels observed. Importantly, these effects were also observed using CGP to inhibit NCLX. The reduction of autophagic flux by NCLX KD or CGP was not observed in cells treated with rapamycin. We also measured the levels of phosphorylated 4E-BP1 as an indication of mTORC1 activity. As expected, serum/amino acid starvation and rapamycin decreased the levels of p-4E-BP1; however, this decrease was modulated by CGP only in starved cells, indicating that NCLX may affect mTORC1 activity in an upstream pathway. In conclusion, we show that mitochondrial calcium transporters are novel autophagy-regulating pathways: MCU modulates autophagic activation under basal conditions, while NCLX maintains autophagic activity under basal and stimulated conditions.
  45. FASEB J. 2022 May;36 Suppl 1
      It has long been thought that erythropoietin (Epo) is exclusively involved in erythropoiesis; now, it is known that EPO in mammal's brain plays key roles in the development, maintenance, protection, and repair of the nervous system. Also, EPO in mammals contributes to the efficient use of oxygen through the regulation of mitochondrial bioenergetic. Remarkably, a similar neuroprotective impact of recombinant human EPO (rhEPO) has been found in the brain of grasshoppers, raising questions about the evolutive origin of the EPO and its generic molecular function. The objective of this study is to show that the neuroprotective effect of rhEPO in insects involves the regulation of mitochondrial functions. The experiments were performed in crickets (Acheta domesticus). These insects were exposed under normoxia and hypoxia (5 days; 6% O2) conditions. Before experimentation, the animals were treated with EPO (30 IU/ml - intra-lymphatic injection) or PBS, as a control. The brains of the crickets were removed, and then we determined the mitochondrial respiration and production of mitochondrial ROS using our system oxygraphy - 2K (ORORBOROS). Our results show that compares to normoxia; hypoxia significantly reduces mitochondrial respiration of complexes 1 and 1&2. On the other hand, the treatment of EPO in hypoxia, despite significantly increasing these parameters, does not recover the levels of mitochondrial respiration under normoxic conditions. In addition, the activity of complex IV (an indicator of the number of mitochondria) does not vary significantly between any of the treatments. Furthermore, we observed that while hypoxia did not significantly affect H2 O2 production, the treatment with EPO increased ROS production under normoxic but not hypoxic conditions. Our data suggest that rhEPO regulates in some way the mitochondrial respiration and ROS production in the brain of crickets. Considering that insects appeared during a geological period (Cambrian explosion) in which the atmospheric O2 was increasing, which could cause great oxidative stress due to the change in the metabolism of these animals, this molecule would have appeared as a regulator of mitochondrial functions.
  46. Oxid Med Cell Longev. 2022 ;2022 9771743
      Cerebral ischemia reperfusion injury (IRI) induced by hemorrhagic shock and reperfusion (HSR) is the main cause of death following trauma. Previous studies indicated the neuroprotective effect of sevoflurane postconditioning (SP) in cerebral IRI. However, the mechanisms still remain elusive. Cerebral IRI models with SP were established by using HSR with C57BL/6 mice (male, 3-month-old) in vivo and by using oxygen glucose deprivation and reoxygenation (OGD/R) with HT22 cells in vitro. Postoperative cognition was evaluated by the Morris water maze, novel object recognition, and elevated plus maze tests. The role of SIRT1 was determined by using siRNA, a sensitive inhibitor (EX527), or an overexpression shRNA-GFP lentivirus. IRI caused significant disabilities of spatial learning and memory associated with enhanced cerebral infarct and neuronal apoptosis, which were effectively attenuated by SP. IRI also made a significant decrease of SIRT1 accompanied by oxidative stress, mitochondria dysfunction, and inactivated autophagy. SP or genetically overexpressing SIRT1 significantly suppressed defective autophagy, mitochondrial oxidative injury, and neuronal death caused by HSR or OGD/R. However, genetic suppression or pharmacological inhibition of SIRT1 significantly reversed the impact of SP treatment on mitochondrial DNA transcription ability and autophagy. Our results demonstrate that the loss of SIRT1 causes a sequential chain of mitochondrial dysfunction, defective autophagy, and neuronal apoptosis after IRI in the preclinical stroke models. Sevoflurane postconditioning treatment could effectively attenuate pathophysiological signatures induced by noxious stimuli, which maybe mediated by SIRT1.
  47. FASEB J. 2022 May;36 Suppl 1
      Arteries and veins are lined by non-proliferating endothelial cells that play a critical role in regulating blood flow. Endothelial cells also regulate tissue perfusion, metabolite exchange, and thrombosis. It is thought that endothelial cells rely on ATP generated via glycolysis to fuel each of these energy-demanding processes. However, endothelial metabolism has mainly been studied in the context of proliferative cells in angiogenesis, and little is known about energy production in endothelial cells within the fully-formed vascular wall. Using intact arteries isolated from rats and mice, we show that inhibiting mitochondrial oxidative phosphorylation at mitochondrial complex V disrupts calcium-dependent, nitric oxide-mediated endothelial cell control of vascular tone. Basal, mechanically-activated, and agonist-evoked calcium activity in intact artery endothelial cells are each prevented by inhibiting mitochondrial ATP synthesis. This effect is mimicked by blocking the transport of pyruvate, the master fuel for mitochondrial energy production, through the mitochondrial pyruvate carrier. The role for endothelial cell energy production is independent of species, sex, or vascular bed. These data show that mitochondrial ATP is necessary for the obligatory role of endothelial cells in the control of blood vessel diameter, and validate the idea of targeting endothelial cell metabolism to treat endothelial cell dysfunction in cardiovascular disease.
  48. Nat Commun. 2022 May 13. 13(1): 2673
      The folded mitochondria inner membrane-cristae is the structural foundation for oxidative phosphorylation (OXPHOS) and energy production. By mechanically simulating mitochondria morphogenesis, we speculate that efficient sculpting of the cristae is organelle non-autonomous. It has long been inferred that folding requires buckling in living systems. However, the tethering force for cristae formation and regulation has not been identified. Combining electron tomography, proteomics strategies, super resolution live cell imaging and mathematical modeling, we reveal that the mitochondria localized actin motor-myosin 19 (Myo19) is critical for maintaining cristae structure, by associating with the SAM-MICOS super complex. We discover that depletion of Myo19 or disruption of its motor activity leads to altered mitochondria membrane potential and decreased OXPHOS. We propose that Myo19 may act as a mechanical tether for effective ridging of the mitochondria cristae, thus sustaining the energy homeostasis essential for various cellular functions.
  49. FASEB J. 2022 May;36 Suppl 1
      Traumatic brain injury (TBI) is defined as an impact to the head by an external force that causes brain alterations and subsequent long-term functional deficits. TBI contributes to an economic burden of $17 billion USD annually and is a leading cause of death and disability for individuals under 45. The severity of TBI varies from mild to severe with repetitive and mild (rm) TBI, and accounts for the highest percentage of TBI-cases, leading to long-term cognitive impairment. There are no current treatment(s) for repetitive and mild TBI, therefore, we sought to identify novel signaling molecules/pathways that could contribute to TBI. We employed a clinically relevant (non-surgical) closed-head impact model of engineered rotational acceleration (CHIMERA) that allows free rotation of the head upon impact generated from an air-compressed piston. Our data suggest a novel role for PRMT7 (protein arginine methyltransferases) in the disease progression as indicated by the temporal decrease in protein expression post-rmTBI. Our central hypothesis is that the loss of PRMT7, due to repetitive and mild TBI, mediates excitotoxicity, increased cellular death, disturbed mitochondrial dynamics and contributes to behavioral deficits. PRMTs are novel targets that catalyze the methylation of arginine residues (a constitutive post-translational modification) involved in transcription, translation, receptor trafficking, and protein stability. There are currently 11 known PRMT isoforms (PRMT1-11), with PRMT7 gene deletion in human patients causing neurological deficits such as intellectual disability, microcephaly, and brachydactyly, along with hyperexcitability and impaired social behaviors in murine in vivo models. We assessed diffuse axonal injury in our model of mild and repetitive TBI (via CHIMERA) to suggest enhanced silver deposition (dark stained regions) throughout the brain, similar to human pathology. Next, we measured PRMT7 protein levels that were decreased in the cortex and hippocampus 7-days post-rmTBI. Relative PRMT7 mRNA (via real-time qPCR) was enhanced in the cortex 1-day post-rmTBI. Using LC-MS, we measured excitatory neurotransmitters to suggest that glutamate was enhanced in the hippocampus 3-day post-rmTBI. In addition, mitochondrial fission and fusion was assessed by measuring DRP1 and OPA1 and our results indicated increased polarization towards fission as indicated by significant increase of DRP1 protein expression 1,3,7 days post rmTBI. Furthermore, mitochondrial oxygen consumption rates were analyzed via Seahorse XF analyzer and indicated dynamic changes in ATP-linked respiration and maximal respiration 1 and 3 days post-rmTBI. Finally, learning, working memory, and locomotor skills were significantly impaired as indicated by decreased alternation ratios via T-maze, novel object recognition, and rotarod assessment. Overall, our results suggest that PRMT7 can mediate neuronal hyperexcitability, altered mitochondrial dynamics and can affect functional outcomes post-rmTBI.
  50. FASEB J. 2022 May;36 Suppl 1
      PURPOSE: Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) is an essential molecule in mitophagy process and known to act as a cytoprotective protein in mammalian cells. Mutation or deficiency of PINK1 has been closely related to several disease conditions. The purpose of this study was to determine PINK1 expression levels and subcellular localization under exercise-mimic laminar shear stress (LSS) condition in human aortic endothelial cells (HAECs) or in exercising mice, and its implication on endothelial homeostasis and cardiovascular disease (CVD) prevention.METHODS & RESULTS: First, we measured full-length PINK1 (FL-PINK1) mRNA and protein expression levels in HAECs under unidirectional laminar shear stress (LSS) at 15 dyne/cm2 for 48h. LSS significantly elevated both FL-PINK1 mRNA and protein expressions in HAECs compared to static control. Mitochondrial fractionation assays showed a decrease in FL-PINK1 accumulation in the mitochondria with a compensatory increase in the cytosolic FL-PINK1 level under LSS. Confocal microscopic analysis confirmed these subcellular localization patterns suggesting downregulation of mitophagy induction. Also, mitophagy flux was decreased under LSS, determined by a mtKeima probe. Mitochondrial morphometric analysis and mitochondrial membrane potential (JC-1) showed mitochondrial elongation and increased mitochondrial membrane potential, respectively, suggesting that an elevation of cytosolic PINK1 is not related to an immediate induction of mitophagy. Based on this observation, we further hypothesized that the elevation of the cytosolic pool of FL-PINK1 would increase the sensitivity to mitophagic signals in pathological conditions. Preconditioned HAECs with LSS showed lower mtDNA lesions under angiotensin II stimulation (100 nM, 6h). Moreover, LSS-preconditioned ECs showed rapid Parkin recruitment and mitophagy induction in response to mitochondrial toxin (i.e. CCCP) treatment compared to the control. We measured PINK1 expression at ECs of the thoracic aorta in exercised mice, a physiological LSS-enhanced model, which was significantly elevated compared to sedentary animals. In addition, exercise-preconditioned mice were more protective to angiotensin II-induced mtDNA lesion formation in the mouse abdominal aorta than sedentary mice, suggesting a potential protective mechanism of exercise in a PINK1-dependent manner.
    CONCLUSION: LSS increases a cytosolic pool of FL-PINK1, which may elevate the mitophagic sensitivity toward dysfunctional mitochondria or activate other cytoprotective mechanisms in endothelial cells. Our data suggest that exercise may support mitochondrial homeostasis in vascular endothelial cells by enhancing PINK1-dependent cell protection mechanisms.
  51. Elife. 2022 May 13. pii: e74552. [Epub ahead of print]11
      Proliferating cells undergo metabolic changes in synchrony with cell cycle progression and cell division. Mitochondria provide fuel, metabolites, and ATP during different phases of the cell cycle, however it is not completely understood how mitochondrial function and the cell cycle are coordinated. CLUH is a post-transcriptional regulator of mRNAs encoding mitochondrial proteins involved in oxidative phosphorylation and several metabolic pathways. Here, we show a role of CLUH in regulating the expression of astrin, which is involved in metaphase to anaphase progression, centrosome integrity, and mTORC1 inhibition. We find that CLUH binds both the SPAG5 mRNA and its product astrin, and controls the synthesis and the stability of the full-length astrin-1 isoform. We show that CLUH interacts with astrin-1 specifically during interphase. Astrin-depleted cells show mTORC1 hyperactivation and enhanced anabolism. On the other hand, cells lacking CLUH show decreased astrin levels and increased mTORC1 signaling, but cannot sustain anaplerotic and anabolic pathways. In absence of CLUH, cells fail to grow during G1, and progress faster through the cell cycle, indicating dysregulated matching of growth, metabolism and cell cycling. Our data reveal a role of CLUH in coupling growth signaling pathways and mitochondrial metabolism with cell cycle progression.
    Keywords:  cell biology; human
  52. Sci Adv. 2022 May 13. 8(19): eabl8716
      Several subunits in the matrix domain of mitochondrial complex I (CI) have been posited to be redox sensors for CI, but how elevated levels of reactive oxygen species (ROS) impinge on CI assembly is unknown. We report that genetic disruption of the mitochondrial NADPH-generating enzyme, isocitrate dehydrogenase 2 (IDH2), in Drosophila flight muscles results in elevated ROS levels and impairment of assembly of the oxidative phosphorylation system (OXPHOS). Mechanistically, this begins with an inhibition of biosynthesis of the matrix domain of CI and progresses to involve multiple OXPHOS complexes. Despite activation of multiple compensatory mechanisms, including enhanced coenzyme Q biosynthesis and the mitochondrial unfolded protein response, ferroptotic cell death ensues. Disruption of enzymes that eliminate hydrogen peroxide, but not those that eliminate the superoxide radical, recapitulates the phenotype, thereby implicating hydrogen peroxide as the signaling molecule involved. Thus, IDH2 modulates the assembly of the matrix domain of CI and ultimately that of the entire OXPHOS.
  53. Oxid Med Cell Longev. 2022 ;2022 9148246
      Current evidences indicate that both inflammation and oxidative stress contribute to the pathogenesis of sepsis-associated skeletal muscle atrophy. However, the interaction between inflammation and oxidative stress has not been completely understood in sepsis-associated skeletal muscle atrophy. Here in the present study, a murine model of sepsis has been established by cecal ligation and puncture (CLP) with wild-type and interleukin- (IL-) 6 knockout (KO) mice. Our results suggested that IL-6 KO largely attenuated skeletal muscle atrophy as reflected by reduced protein degradation, increased cross-sectional area (CSA) of myofibers, and improved muscle contractile function (all P < 0.05). In addition, we observed that IL-6 KO promoted the expression of peroxisome proliferator-activated receptor γ coactivator-1alpha (PGC-1α) and inhibited CLP-induced mitochondrial reactive oxygen species (ROS) production in skeletal muscles (all P < 0.05). However, the knockdown of PGC-1α abolished the protective effects of IL-6 KO in CLP-induced skeletal muscle atrophy and reversed the changes in mitochondrial ROS production (all P < 0.05). Ex vivo experiments found that exogenous IL-6 inhibited PGC-1α expression, promoted mitochondrial ROS production, and induced proteolysis in C2C12 cells (all P < 0.05). Together, these results suggested that IL-6 deficiency attenuated skeletal muscle atrophy by inhibiting mitochondrial ROS production through the upregulation of PGC-1α expression in septic mice.
  54. FASEB J. 2022 May;36 Suppl 1
      The heart is featured by high mitochondrial volume, by which continuous contractile activity can be maintained. However, it is unclear how mitochondrial structures are matured during postnatal development. Using FIB-SEM, we thus sought to identify 3D mitochondrial structural characteristics in the heart of mice at postnatal (P) day 1, 7, 14, and 42, respectively. Here, matured mitochondria (P14-42) were shown to have larger and more spherical structures and were highly connected with adjacent mitochondria as compared to early postnatal days (P1-7). Nevertheless, the immature mitochondria appeared to have more intense interaction with other subcellular organelles including lipid droplets and sarcotubular structures (i.e., SR/T-tubules). Thus, our study suggests that initial mitochondrial development can be augmented by crosstalk with adjacent mitochondria as well as other subcellular components and that it would be important for the further mitochondrial maturation process in the heart muscle.
  55. Cell Death Dis. 2022 May 12. 13(5): 452
      Osteoblast differentiation is critically reduced in various bone-related pathogenesis, including arthritis and osteoporosis. For future development of effective regenerative therapeutics, herein, we reveal the involved molecular mechanisms of a phytoestrogen, ferutinin-induced initiation of osteoblast differentiation from dental pulp-derived stem cell (DPSC). We demonstrate the significantly increased expression level of a transcription factor, Kruppel-like factor 2 (KLF2) along with autophagy-related molecules in DPSCs after induction with ferutinin. The loss-of-function and the gain-of-function approaches of KLF2 confirmed that the ferutinin-induced KLF2 modulated autophagic and OB differentiation-related molecules. Further, knockdown of the autophagic molecule (ATG7 or BECN1) from DPSC resulted not only in a decreased level of KLF2 but also in the reduced levels of OB differentiation-related molecules. Moreover, mitochondrial membrane potential-related molecules were increased and induction of mitophagy was observed in DPSCs after the addition of ferutinin. The reduction of mitochondrial as well as total ROS generations; and induction of intracellular Ca2+ production were also observed in ferutinin-treated DPSCs. To test the mitochondrial respiration in DPSCs, we found that the cells treated with ferutinin showed a reduced extracellular acidification rate (ECAR) than that of their vehicle-treated counterparts. Furthermore, mechanistically, chromatin immunoprecipitation (ChIP) analysis revealed that the addition of ferutinin in DPSCs not only induced the level of KLF2, but also induced the transcriptionally active epigenetic marks (H3K27Ac and H3K4me3) on the promoter region of the autophagic molecule ATG7. These results provide strong evidence that ferutinin stimulates OB differentiation via induction of KLF2-mediated autophagy/mitophagy.
  56. FASEB J. 2022 May;36 Suppl 1
      Tumor necrosis factor α (TNFα) contributes to the pathophysiology of several inflammatory airway diseases such as asthma, chronic obstructive pulmonary disease (COPD), chronic bronchitis, and COVID-19. Previously, we showed that acute (24-h) exposure to TNFα promotes mitochondrial fragmentation in human airway smooth muscle (hASM) cells, which is associated with an increased level of cytosolic GTPase dynamin-related protein 1 (DRP1), a major protein involved in mitochondrial fragmentation. Phosphorylation of DRP1 at Ser616 (S616) promotes its translocation and binding to the outer mitochondrial membrane and induces mitochondrial fragmentation; however, phosphorylation of DRP1 at the Ser637 (S637) residue reverses the total process and diminishes fragmentation. The balance between S616 and S637 phosphorylation of DRP1 regulates mitochondrial dynamics. In hASM cells, exposure to TNFα triggers protein unfolding and selectively activates the endoplasmic reticulum (ER) stress pathway involving the phosphorylation of inositol-requiring enzyme 1α (pIRE1α) and subsequent splicing of X-box binding protein 1 (XBP1s) that acts as a potent transcriptional activator for several downstream genes. We hypothesized that TNFα mediated activation of the pIRE1α/ XBP1 ER stress pathway transcriptionally activates genes that code for proteins involved in DRP1 phosphorylation at S616 leading to mitochondrial fragmentation. hASM cells were dissociated from bronchial biopsies from patients without a history of respiratory diseases and exposed to TNFα (20 ng/ ml for 6 h). Cells were also treated with Mdivi1 (50 μM for 6 h), a small-molecule inhibitor of mitochondrial fragmentation that targets the GTPase activity of DRP1. Bioinformatic analysis revealed the presence of putative binding sites for XBP1 on the promoter region of genes that translate into kinases (cyclin dependent kinases CDK1 and CDK5, cyclin B1), which are reported to phosphorylate DRP1 at S616. Western blot and/or immunocytochemistry were employed to quantify the expression and phosphorylation status of IRE1α, DRP1, XBP1 splicing, CDKs and cyclin B1. Mitochondrial morphology was assessed by 3-D confocal microscopy using MitoTracker green to label mitochondria. Chromatin immunoprecipitation (ChIP) and quantitative real time PCR was done to validate XBP1 target genes. Spliced XBP1 transcriptionally activates expression of CDK1, CDK5 and cyclin B1 and subsequent phosphorylation at S616 of DRP1 without significant alteration in S637 phosphorylation. As a result, TNFα induces an increase in S616/S637 phosphorylation ratio that promotes the translocation of DRP1 from cytosol to mitochondria and mediates mitochondrial fragmentation. Inhibition of DRP1 GTPase activity by Mdivi1 ameliorates DRP1 phosphorylation at S616 and significantly reduces mitochondrial fragmentation. These results reveal the mechanisms that underlie TNFα induced ER stress and downstream mitochondrial fragmentation.
  57. FASEB J. 2022 May;36 Suppl 1
      RATIONALE: Reactive oxygen species (ROS; e.g. O2 · - and H2 O2 ) play important roles in both physiological and pathophysiological processes. ROS in low concentrations contribute to physiological processes, such as cellular redox signaling and phagocytosis, whereas ROS in high concentrations are toxic to the cell causing tissue injury contributing to the pathogenesis of cardiovascular and chronic renal diseases, including salt-sensitive hypertension. Mitochondria, which produce ROS as byproducts of aerobic respiration via both forward electron transfer (FET) and reverse electron transfer (RET) are known to be one of the major cellular sources of ROS. Although it is recognized that the RET mechanism in which electrons flow back from complex II to complex I contribute significantly to ROS production in cardiac mitochondria, the mechanisms of ROS production and the role of RET in kidney mitochondria has remained poorly understood.METHOD: We evaluated the relative contributions of FET and RET towards overall ROS production in mitochondria isolated from the heart and kidney cortex and outer medulla (OM) of adult Sprague-Dawley rats. H2 O2 emission was measured by a spectrofluorometer in isolated mitochondria in the presence of either Succinate (Suc) simulating RET or succinate+rotenone (Suc+Rot) simulating FET. Furthermore, we measured mitochondrial rates of H2 O2 production along with respiration and membrane potential under three respiratory states namely (i) leak state (state 2; after substrate addition), (ii) oxidative phosphorylation (OxPhos) state (state 3; after ADP addition), and (iii) maximum respiratory state (state 5; after the addition of the uncoupler FCCP).
    RESULTS: It was found that mitochondria isolated from the heart and kidney cortex produced the least and the most ROS, respectively. The rate of ROS production in the presence of Suc+Rot compared to Suc alone decreased significantly in the heart and to a lesser extent in OM, indicating significant contribution of RET to overall ROS production in the heart and slightly in the OM. In contrast, there was not significant difference in ROS production rates in the presence of Suc and Suc +Rot in mitochondria from kidney cortex, showing that RET is not predominant in the kidney cortex. Also, we observed significant reduction in the ROS production rate in state 4 compared to state 2 in the heart mitochondria compared to kidney cortex and OM. A possible explanation for these differential results is that oxaloacetate (OAA), produced by the tricarboxylic acid cycle, accumulates resulting in succinate dehydrogenase (SDH) inhibition more rapidly in the heart than in the kidney affecting mitochondrial ROS production, respiration, and bioenergetics.
    CONCLUSION: RET mechanism contributes to mitochondrial ROS production significantly in the heart and slightly in the kidney OM, but not in the kidney cortex. OAA accumulation contributes to SDH inhibition significantly in the heart than in the kidney.
    Keywords:  Forward electron transfer; Mitochondrial bioenergetics; Oxidative stress; ROS production; Reverse electron transfer
  58. FASEB J. 2022 May;36 Suppl 1
      The insular cortex (IC) has been described as part of the central network implicated in the behavioral and physiological responses to emotional stress. Besides, it has been reported a site-specific control of physiological functions along the rostrocaudal axis of the IC. Nevertheless, evaluation of a functional topography of the IC in the regulation of the responses to stressful stimuli has never been documented. Therefore, this study aimed to investigate the impact of acute restraint stress in neuronal activation at different sites along the rostrocaudal axis of the IC in rats. Furthermore, we evaluated the involvement of IC rostrocaudal subregions in the cardiovascular responses to acute restraint stress. For this, male Wistar rats (60-days-old) were used. The restraint stress was performed by placing the animals in a cylindrical plastic tube for 60 minutes. For the cardiovascular study, different set of animals had cannula-guide bilaterally implanted into the anterior, rostral posterior of caudal posterior subregions of the IC; and the non-selective synaptic inhibitor CoCl2 (1mM/100nL) was microinjected 10 min before the restraint onset. We observed that acute restraint stress increased the number of Fos-immunoreactive cells in the rostral posterior region of the IC (t=2.95, df=14, P=0.0106), while fewer activated cells were identified in the anterior (t=2.44, df=14, P= 0.0276) and caudal posterior (t=2.27, df=14, P=0.0388) regions. Treatment of the anterior region of the IC with CoCl2 did not affect the blood pressure (F(1,15) =0.90, P>0.05) and heart rate (F(1,15) =0.3, P>0.05) increases and the sympathetically-mediated cutaneous vasoconstriction (F(1,15) =2.23, P>0.05) evoked by restraint stress. However, synaptic ablation of the rostral posterior IC decreased the restraint-evoked arterial pressure increase (F(1,21) =5.3, P=0.0313), but without affecting the tachycardia (F(1,21) =4.1, P=0.0561) and the reduction in tail skin temperature (F(1,21) =0.05, P>0.05). Besides, bilateral microinjection of CoCl2 into the caudal posterior IC decreased the tachycardia to restraint stress (F(1,17) =11.3, P=0.0036), but without affecting the blood pressure (F(1,17) = 0.2, P>0.05) and tail skin temperature (F(1,17) = 1.4, P>0.05) responses. Taken together, these findings indicate a site-specific regulation of stress response along the rostrocaudal axis of the IC.
  59. FASEB J. 2022 May;36 Suppl 1
      The endo-lysosomal pathway plays an important role in pathogen clearance and both bacteria and viruses have evolved complex mechanisms to evade this host system. Here, we describe a novel aspect of coronaviral infection, whereby the master transcriptional regulator of lysosome biogenesis - TFEB - is targeted for proteasomal-mediated degradation upon viral infection. Through mass spectrometry analysis and an unbiased siRNA screen, we identify that TFEB protein stability is coordinately regulated by the E3 ubiquitin ligase subunit DCAF7 and the PAK2 kinase. In particular, viral infection triggers marked PAK2 activation, which in turn, phosphorylates and primes TFEB for ubiquitin-mediated protein degradation. Deletion of either DCAF7 or PAK2 blocks viral-mediated TFEB degradation and protects against viral-induced cytopathic effects. We further derive a series of small molecules that interfere with the DCAF7-TFEB interaction. These agents inhibit viral-triggered TFEB degradation and demonstrate broad anti-viral activities including attenuating in vivo SARS-CoV-2 infection. Together, these results delineate a viral-triggered pathway that disables the endogenous cellular system that maintains lysosomal function and suggest that small molecule inhibitors of the E3 ubiquitin ligase DCAF7 represent a novel class of endo-lysosomal, host-directed, anti-viral therapies.
  60. FASEB J. 2022 May;36 Suppl 1
      Autophagy is a tightly controlled cellular recycling process that requires a host of autophagy machinery to form a double membraned vesicle called the autophagosome. This process is most understood in the context of stress-induced autophagy, with little known about autophagosome biogenesis in basal (nutrient replete) conditions. To understand the regulation of basal autophagy, our work has focused on the poorly understood protein ATG9A, a multi-pass transmembrane lipid scramblase that is essential for basal autophagy. To broadly understand the role ATG9A plays in basal autophagy, we utilized a quantitative proteome-level MS/MS approach to measure how ATG9A affects protein flux. We show that loss of ATG9A in basal conditions impairs the degradation of autophagy adaptors, particularly p62/SQSTM1. Using a panel of ATG knock-out cells, we demonstrate that the lipid transferase proteins ATG2A, ATG2B, and ATG9A promote the basal autophagic turnover of p62 and TAX1BP1 over other autophagy adaptors and do so independently of the LC3-lipidation machinery. Furthermore, we demonstrate that ATG2A and ATG9A lipid transferase activity regulates the rate of p62 condensate degradation. Finally, we show in CRISPR knock-in cell lines that ubiquitin is required for recruiting ATG9A to p62 condensates. Taken together, our data suggest that the lipid transferase activity of ATG9A and ATG2A is vital to basal autophagic regulation of protein homeostasis, and that ubiquitination is an apical signal that initiates recruitment of ATG9A to p62 condensates.
  61. Sci Rep. 2022 May 11. 12(1): 7704
      Aging of sensory organs is associated with a decline in mitochondrial function and the accumulation of dysfunctional mitochondria. Impaired mitophagy blocks the turnover of dysfunctional mitochondria and leads to their accumulation. Urolithin A (UA) induces mitophagy in various mammalian cells. This study was aimed at investigating the effect of the mitophagy activator, UA, on premature senescent auditory cells. The levels of cellular senescence-associated p53 and p21 significantly increased in H2O2-induced senescent House Ear Institute-Organ of Corti 1 (HEI-OC1) cells and cochlear explants. However, the levels of mitophagy-related molecules significantly decreased. UA significantly decreased the expression of senescence-associated p53 and p21, and increased the expression of mitophagy-related proteins, in H2O2-induced senescent cells and cochlear explants. The percentage of β-galactosidase-stained senescent cells also reduced in H2O2-treated cells and cochlear explants upon UA pre-treatment. The formation of mitophagosomes and mitophagolysosomes was restored upon UA pre-treatment of H2O2-induced senescent cells. The knockdown of mitophagy-related genes (Parkin and Bnip3) resulted in annulment of UA-induced anti-senescent activity. UA significantly increased the ATP content, mitochondrial DNA (mtDNA) integrity, and mitochondrial membrane potential in senescent HEI-OC1 cells. These findings indicate that UA counteracted mitophagy decline and prevented premature senescence in auditory cells. Hence, UA administration might be a promising strategy for preventing mitochondrial dysfunction in patients with age-related hearing loss.
  62. FASEB J. 2022 May;36 Suppl 1
      While it is well established that endurance exercise improves mitochondrial health in skeletal muscle, optimizing nutraceutical agents to mimic these metabolic achievements in the cell remains a challenge. Nonetheless, the application of exercise mimetics is a fruitful direction to pursue, as they may target and activate the same mechanisms that are upregulated with exercise administration alone. This is particularly useful under conditions where contractile activity is compromised due to muscle disuse, disease, or aging. The agents Sulforaphane (SFN) and Urolithin A (UroA) represent our preliminary candidates for antioxidation and mitophagy, respectively, for maintaining mitochondrial turnover and homeostasis. SFN is a powerful inducer of the Nrf-2-ARE pathway, a mechanism that upregulates cellular defences against oxidative stress. On the other hand, the metabolite, UroA, has developed a reputable role in regulating mitochondrial turnover via mitophagy. The purpose of this ongoing study is to characterize these nutraceutical agents both in their time- and dose-dependent capacities to induce changes in protein content relative to the antioxidant and mitophagy pathways, in C2C12 myotubes. Differentiated muscle cells were treated with two concentrations of each nutraceutical for 4 h, 24 h, or 48 h. Immunoblot analysis was conducted to measure changes in protein content. SFN treatment after 4 h rendered a marked increase in the master regulator for antioxidation, and transcription factor, Nrf-2, with no apparent changes in its negative regulator Keap-1 at any given time point. Nuclear-cytoplasmic fractions confirmed Nrf-2 translocation to the nucleus following 4 h of treatment, likely representing the potent activation of antioxidant genes. This aligns with the upregulation of the downstream antioxidant markers HO-1 and NQO1, showing 4-5-fold increases as early as 4 h and 24 h, respectively, and maintained after 48 h. Despite modest effects with Urolithin A, some significant changes took place under basal conditions. The upstream kinase phospho-AMPK was activated by 2-fold as early as 4 hr of treatment with UroA, which subsided by 24 and 48 h. Previous reports observed changes with the agent on autophagy markers, which includes the modest 1.3-fold increases in p62 and LC3-II after 48 h of treatment. However, no observable changes took place with respect to the mitophagy markers phospho-ULK1, Parkin, or PINK1. Nonetheless, our preliminary results suggest that these agents may be suitable candidates as exercise mimetics. These data also set the stage for an examination of the synergistic effect of these nutraceuticals, in combination with contractile activity, on mitochondrial turnover and function.
  63. FASEB J. 2022 May;36 Suppl 1
      Maintenance of the mitochondrial protein folding environment is essential for organellar and cellular homeostasis. Over 99% of mitochondrial proteins require import into mitochondria, followed by their folding and intraorganellar sorting. Mitochondrial stress can result in the accretion of misfolded proteins, establishing a requirement for mitochondrial protein quality control (MQC) strategies. The Mitochondrial Unfolded Protein Response (UPRmt ) is a compartment-specific MQC mechanism that increases the expression of protective enzymes by Activating Transcription Factor 5 (ATF5) to restore mitochondrial function. Contractile activity during acute exercise is a stressor that has the potential to temporarily disrupt organellar protein homeostasis. However, the roles of ATF5 and the UPRmt in basal mitochondrial maintenance and exercise-induced UPRmt signaling in skeletal muscle are not known. To investigate this, we subjected WT and whole-body ATF5 KO mice to a bout of acute exercise and collected skeletal muscle tissues immediately after. ATF5 KO animals exhibited 2-fold increases in phosphorylated JNK protein levels, indicative of enhanced stress signaling. Interestingly, in KO muscle, PGC-1a protein was enhanced by 50% and 40% in nuclear and cytosolic compartments, respectively, suggesting an increased drive toward mitochondrial biogenesis in the absence of ATF5. Muscle from these animals also displayed a more abundant, but dysfunctional, mitochondrial pool, with a 20% increase in mitochondrial content, 30-40% reductions in respiration, and a 20% increase in ROS emissions, corresponding with no changes in exercise performance. The UPRmt proteins mtHSP70 and LONP were upregulated 20-30% in KO muscle, while ATF4 mRNA was upregulated 2.5-3.7-fold, along with an 8% increase in its nuclear localization. Furthermore, KO muscle showed an impaired UPRmt mRNA response to acute exercise, suggesting a regulatory role for ATF5 in the maintenance of a high-quality mitochondrial pool, and in mediating the transcription of UPRmt genes during exercise.
  64. FASEB J. 2022 May;36 Suppl 1
      Mitochondria-early endosome (EE) interactions have been shown to facilitate the translocation of iron into mitochondria. Here we show that Divalent Metal Transporter 1 (DMT1) modulates iron exit from endosomes and transport into mitochondria via regulation of EE-mitochondria interactions. In cancer cells, mitochondria are the ultimate cellular iron sink, where iron can be either stored or used for example to shift cellular metabolism towards glycolysis (Warburg effect), a key adaptive mechanism of cancer cells. Moreover, a gene signature associated with reduced intracellular iron content, including low transferrin receptor (TfR) (anti-import) and high ferroportin (FPN) (pro-export) expression levels, has been related to favorable breast cancer prognosis. Similarly, reduced DMT1 expression associates with improved breast cancer patient survival. We evaluated the role of DMT1 in two distinct breast cancer cell lines: estrogen receptor positive T47D and triple-negative MDAMB231. In both cell lines, we demonstrate colocalization between EE, DMT1 and mitochondria. Interestingly, DMT1 is localized to the surface contact area between endosomes and mitochondria. To demonstrate that DMT1 plays a role in endosome-mitochondria interactions and Mitochondrial Iron Translocation (MIT), we have generated MDAMB231 as well as T47D CRISPR/Cas9 based DMT1 knockout (KO) stable cell lines. Several lines of evidence show that DMT1 regulates MIT and labile iron pool (LIP) levels via modulation of EE-mitochondria interactions in MDAMB231 cells. MIT decrease via DMT1 silencing was partially rescued by re-expression of DMT1 in MDAMB231, but not in T47D cells. MDAMB231 DMT1 KO cells showed increased Ferro-Orange staining, indicating higher LIP levels, as well as decreased TfR and increased FPN protein levels. Importantly, DMT1 silencing significantly reduced EE-mitochondria interactions and EE speed in MDAMB231 but not in T47D. Thus, DMT1 regulates MIT and LIP levels via EE-mitochondria interactions in MDAMB231. These results are in agreement with previous results showing that MDAMB231 display a delay in iron release in comparison to T47D, making them more sensitive to disruptions in MIT. Since mitophagy has been shown to act as a tumor suppressor in breast cancer, we tested whether it could be modulated by DMT1-mediated MIT. We found that DMT1 silencing increases mitochondrial ferritin, global autophagy marker LC3B and PINK1/Parkin-dependent mitophagy markers in MDAMB231; levels of all proteins evaluated were rescued to basal levels upon re-expression of DMT1 in DMT KO cells. Moreover, DMT1 silencing decreases Tom 20 (outer mitochondrial membrane marker) with PMPCB, a known DMT1 interactor that is required for PINK1 turnover. Concurring with the role of DMT1 in mitophagy and iron metabolism, both mitochondrial metabolism and invasive cell migration are significantly impaired by DMT1 silencing and are partially rescued by re-expression of DMT1. Overall, our results implicate DMT1 in the regulation of EE dynamics and EE-mitochondria interactions to support higher MIT/lower LIP levels, which are necessary for sustaining mitochondrial bioenergetics and invasive cell migration. Thus, we propose DMT1 as a key player associated with aggressive phenotypes in breast cancer.
  65. FASEB J. 2022 May;36 Suppl 1
      INTRODUCTION: Pyruvate kinase (PKM1) directs pyruvate to the Krebs cycle for oxidative metabolism in the healthy heart. Our lab described a hypoxia-mediated switch to the alternatively spliced isoform PKM2, enhancing pyruvate to lactate conversion. Recently, we have also found that Pkm2 knockout (KO) mice had profound depletion of basal glucose in the heart compared to control mice. Pkm2 has also been shown to reduce oxidative damage and promote cardiomyocyte cell proliferation after myocardial infarction (MI). We hypothesize that upregulation of PKM2 can alter metabolic pathways by promoting glycolysis, and that after injury, this can preserve ATP production, protecting the heart from the stresses of hypoxia and injury.METHODS: Global Pkm2 KO mice were subjected to permanent ligation of the left anterior descending coronary artery to mimic an MI. RNA-seq analysis of left ventricles from control (n=8) and Pkm2 KO mice (n=8) before and 3 days after sham or MI surgery was performed. Semi-quantitative real-time PCR was used to confirm changes in selected genes of interest.
    RESULTS: Loss of Pkm2 moderately altered gene expression at baseline (q<0.05, FDR<0.05). Notably, the mitochondrial gene COX3 was downregulated in Pkm2 KO hearts. 68 genes were differentially expressed in Pkm2 KO hearts after MI, not observed in control MI hearts. MI of Pkm2 KO hearts resulted in considerable reduction of transcripts of enzymes in the insulin signaling pathway, mitochondrial oxidative phosphorylation, mitochondrial uncoupling, fatty acid metabolism, and increase in transcripts encoding enzymes in the pentose phosphate pathway, response to oxidative stress, and apoptotic signaling. Semi-quantitative PCR of selected genes involved in glucose metabolism confirmed RNA-seq results.
    CONCLUSIONS: RNA-seq analysis of Pkm2 KO hearts demonstrated that loss of Pkm2 altered gene expression of metabolic and mitochondrial enzymes. Pkm2 KO hearts also showed increased abundance of pro-apoptotic markers which may be a result of increased oxidative stress.
  66. FASEB J. 2022 May;36 Suppl 1
      ATP-dependent AAA+ proteases are critical regulators of mitochondrial functions, playing crucial roles in the mitochondrial quality control response system. The past years have provided much structural insight into the molecular mechanisms associated with degradation of substrates by these proteolytic machines. Recent cryo-electron microscopy (cryo-EM) studies have provided critical insights into a conserved, AAA+-mediated hand-over-hand substrate translocation mechanism required to processively engage, unfold, and degrade proteolytic substrates. However, the underlying mechanisms regulating their various activities are not well understood. Numerous prior studies suggest that AAA+ protease have evolved numerous layers of regulation to control or tune proteolytic activity to meet cellular needs. Herein, we present compelling biochemical and structural data that support a long-range allosteric model linking substrate binding to proteolytic activity.