bims-mikwok Biomed News
on Mitochondrial quality control
Issue of 2020‒10‒18
twenty papers selected by
Avinash N. Mukkala
University of Toronto

  1. Shock. 2020 Oct 14.
    Zhang Z, Yan C, Miao J, Pu K, Ma H, Wang Q.
      BACKGROUND: Mitochondrial transplantation is a promising strategy for the treatment of several diseases. However, the effects of mitochondrial transplantation on the outcome of polymicrobial sepsis remain unclear.METHODS: The distribution of transplanted mitochondria in cecal ligation and puncture (CLP)-operated mice was detected at 2 and 12 h after intravenous injection in the tail (n = 3). Then, the effects of mitochondrial transplantation on bacterial clearance (n = 7), systemic inflammation (n = 10), organ injury (n = 8), and mortality (n = 19) during CLP-induced sepsis were explored. Microarray analysis (n = 3) was used to testify the molecular changes associated with decreased systemic inflammation and multiorgan dysfunction in sepsis.
    RESULTS: The extraneous mitochondria were distributed in the lung, liver, kidney, and brain of CLP-operated mice at 2 and 12 h after intravenous injection in the tail. Mitochondrial transplantation increased the survival rate of septic mice, which was associated with decreased bacterial burden, systemic inflammation, and organ injury. Spleen samples were utilized for microarray analysis. Pathway analysis revealed that in polymicrobial sepsis, gene expression was significantly changed in processes related to inflammatory response, complement and coagulation cascades, and rejection reaction.
    CONCLUSIONS: These data displayed that mitochondrial replenishment reduces systemic inflammation and organ injury, enhances bacterial clearance, and improves the survival rate in sepsis. Thus, extraneous mitochondrial replenishment may be an effective adjunctive treatment to reduce sepsis-related mortality.
  2. Front Physiol. 2020 ;11 533683
    Natarajan V, Mah T, Peishi C, Tan SY, Chawla R, Arumugam TV, Ramasamy A, Mallilankaraman K.
      Endothelial dysfunction, referring to a disturbance in the vascular homeostasis, has been implicated in many disease conditions including ischemic/reperfusion injury and atherosclerosis. Endothelial mitochondria have been increasingly recognized as a regulator of calcium homeostasis which has implications in the execution of diverse cellular events and energy production. The mitochondrial calcium uniporter complex through which calcium enters the mitochondria is composed of several proteins, including the pore-forming subunit MCU and its regulators MCUR1, MICU1, and MICU2. Mitochondrial calcium overload leads to opening of MPTP (mitochondrial permeability transition pore) and results in apoptotic cell death. Whereas, blockage of calcium entry into the mitochondria results in reduced ATP production thereby activates AMPK-mediated pro-survival autophagy. Here, we investigated the expression of mitochondrial calcium uniporter complex components (MCU, MCUR1, MICU1, and MICU2), induction of autophagy and apoptotic cell death in endothelial cells in response to oxygen-glucose deprivation. Human pulmonary microvascular endothelial cells (HPMVECs) were subjected to oxygen-glucose deprivation (OGD) at 3-h timepoints up to 12 h. Interestingly, except MCUR1 which was significantly downregulated, all other components of the uniporter (MCU, MICU1, and MICU2) remained unchanged. MCUR1 downregulation has been shown to activate AMPK mediated pro-survival autophagy. Similarly, MCUR1 downregulation in response to OGD resulted in AMPK phosphorylation and LC3 processing indicating the activation of pro-survival autophagy. Despite the activation of autophagy, OGD induced Caspase-mediated apoptotic cell death. Blockade of autophagy did not reduce OGD-induced apoptotic cell death whereas serum starvation conferred enough cellular and functional protection. In conclusion, the autophagic flux induced by MCUR1 downregulation in response to OGD is insufficient in protecting endothelial cells from undergoing apoptotic cell death and requires enhancement of autophagic flux by additional means such as serum starvation.
    Keywords:  MCUR1; apoptotic cell death; autophagy; endothelial dysfunction; oxygen-glucose deprivation
  3. Cell Biol Toxicol. 2020 Oct 17.
    De R, Mazumder S, Bandyopadhyay U.
      Mitochondria are double membrane-bound cellular work-horses constantly functioning to regulate vital aspects of cellular metabolism, bioenergetics, proliferation and death. Biogenesis, homeostasis and regulated turnover of mitochondria are stringently regulated to meet the bioenergetic requirements. Diverse external and internal stimuli including oxidative stress, diseases, xenobiotics and even age profoundly affect mitochondrial integrity. Damaged mitochondria need immediate segregation and selective culling to maintain physiological homeostasis. Mitophagy is a specialised form of macroautophagy that constantly checks mitochondrial quality followed by elimination of rogue mitochondria by lysosomal targeting through multiple pathways tightly regulated and activated in context-specific manners. Mitophagy is implicated in diverse oxidative stress-associated metabolic, proliferating and degenerative disorders owing to the centrality of mitopathology in diseases as well as the common mandate to eliminate damaged mitochondria for restoring physiological homeostasis. With improved health care and growing demand for precision medicine, specifically targeting the keystone factors in pathogenesis, more exploratory studies are focused on mitochondrial quality control as underlying guardian of cellular pathophysiology. In this context, mitophagy emerged as a promising area to focus biomedical research for identifying novel therapeutic targets against diseases linked with physiological redox perturbation. The present review provides a comprehensive account of the recent developments on mitophagy along with precise discussion on its impact on major diseases and possibilities of therapeutic modulation.
    Keywords:  Autophagy; Mitochondrial apoptosis; Mitochondrial disease; Mitochondrial quality control; Mitophagy; Oxidative stress
  4. Cell Transplant. 2020 Jan-Dec;29:29 963689720954140
    Lin KC, Yeh JN, Chen YL, Chiang JY, Sung PH, Lee FY, Guo J, Yip HK.
      This study tested the hypothesis that both allogenic adipose-derived mesenchymal stem cells (ADMSCs) and human inducible pluripotent stem cell-derived MSCs (iPS-MSCs) offered a comparable effect for protecting the lung against ischemia-reperfusion (IR) injury in rodent through downregulating the inflammatory, oxidative stress, and autophagic signaling pathways. Adult male Sprague-Dawley rats (n = 32) were categorized into group 1 (sham-operated control), group 2 (IRI), group 3 [IRI + ADMSCs (1.0 × 106 cells)/tail-vein administration at 0.5/18/36 h after IR], and group 4 [IRI + iPS-MSCs (1.0 × 106 cells)/tail-vein administration at 0.5/18/36 h after IR], and lungs were harvested at 72 h after IR procedure. In vitro study demonstrated that protein expressions of three signaling pathways in inflammation (TLR4/MyD88/TAK1/IKK/I-κB/NF-κB/Cox-2/TNF-α/IL-1ß), mitochondrial damage/cell apoptosis (cytochrome C/cyclophilin D/DRP1/ASK1/APAF-1/mitochondrial-Bax/caspase3/8/9), and autophagy/cell death (ULK1/beclin-1/Atg5,7,12, ratio of LCB3-II/LC3B-I, p-AKT/m-TOR) were significantly higher in lung epithelial cells + 6h hypoxia as compared with the control, and those were significantly reversed by iPS-MSC treatment (all P < 0.001). Flow cytometric analysis revealed that percentages of the inflammatory cells in bronchioalveolar lavage fluid and circulation, and immune cells in circulation/spleen as well as circulatory early and late apoptotic cells were highest in group 2, lowest in group 1, and significantly higher in group 3 than in group 4 (all P < 0.0001). Microscopy showed the lung injury score and numbers of inflammatory cells and Western blot analysis showed the signaling pathways of inflammation, mitochondrial damage/cell apoptosis, autophagy, and oxidative stress exhibited an identical pattern of flow cytometric results among the four groups (all P < 0.0001). Both xenogeneic and allogenic MSCs protected the lung against IRI via suppressing the inflammatory, oxidative stress, and autophagic signaling.
    Keywords:  acute lung ischemia-reperfusion injury; apoptosis; autophagy; inflammation; oxidative stress; xenogeneic and allogenic MSCs
  5. Inflammation. 2020 Oct 16.
    Qi Y, Zhang K, Li P, Wu Z.
      Downregulating miR-217-5p could protect cardiomyocytes against ischemia/reperfusion (I/R) injury, but its role in restoring mitochondrial function of I/R-injured cardiomyocytes remained unclear. H9C2 cardiomyocyte-derived cell line with I/R injury was established in vitro on the basis of hypoxia/reperfusion (H/R) model. Cell viability and apoptosis were respectively detected by MTT assay and flow cytometry. Contents of lactate dehydrogenase (LDH) and adenosine triphosphate (ATP) were determined. Flow cytometry was performed to measure the production of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP). Target gene and potential binding sites between miR-217-5p and Sirtuin1 (SIRT1) were predicted by TargetScan and confirmed by dual-luciferase reporter assay. Relative SIRT1 and expressions of autophagy-related and apoptosis-related genes were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. After I/R treatment, the viability of H9C2 cardiomyocyte-derived cell line and ATP contents were reduced, but LDH and ROS contents were increased, at the same time, cell apoptosis and the expressions of miR-217-5p, p62 and cleaved caspase-3 were increased, whereas the expressions of SIRT1, LC3 (light chain 3), PINK1 (PTEN-induced kinase 1), Parkin, Bcl-2, and c-IAP (inhibitor of apoptosis protein) were reduced. However, downregulating miR-217-5p expression reversed the effects of I/R. SIRT1 was predicted and verified to be the target of miR-217-5p, and silencing SIRT1 reversed the effects of downregulating miR-217-5p on I/R-injured cells. Downregulating miR-217-5p could help restore mitochondrial function via targeting SIRT1, so as to protect cardiomyocytes against I/R-induced injury.
    Keywords:  Ischemia/reperfusion injury; Mitochondrial function; miR-217-5p; sirtuin1
  6. J Biol Chem. 2020 10 15. pii: jbc.REV120.015101. [Epub ahead of print]
    Prole DL, Chinnery PF, Jones NS.
      Mitochondrial DNA (mtDNA) encodes proteins and RNAs that support the functions of mitochondria and thereby numerous physiological processes. Mutations of mtDNA can cause mitochondrial diseases and are implicated in ageing. The mtDNA within cells is organized into nucleoids within the mitochondrial matrix, but how mtDNA nucleoids are formed and regulated within cells remains incompletely resolved. Visualization of mtDNA within cells is a powerful means by which mechanistic insight can be gained. Manipulation of the amount, and sequence of, mtDNA within cells is important experimentally and for developing therapeutic interventions to treat mitochondrial disease. This review details recent developments and opportunities for improvements in the experimental tools and techniques that can be used to visualize, quantify and manipulate the properties of mtDNA within cells.
    Keywords:  aging; gene editing; microscopy; mitochondria; mitochondrial DNA (mtDNA); mitochondrial disease; mitophagy
  7. J Mol Cell Cardiol. 2020 Oct 07. pii: S0022-2828(20)30299-6. [Epub ahead of print]
    Lyra-Leite DM, Petersen AP, Ariyasinghe NR, Cho N, McCain ML.
      Contraction of cardiac myocytes depends on energy generated by the mitochondria. During cardiac development and disease, the structure and function of the mitochondrial network in cardiac myocytes is known to remodel in concert with many other factors, including changes in nutrient availability, hemodynamic load, extracellular matrix (ECM) rigidity, cell shape, and maturation of other intracellular structures. However, the independent role of each of these factors on mitochondrial network architecture is poorly understood. In this study, we tested the hypothesis that cell aspect ratio (AR) and ECM rigidity regulate the architecture of the mitochondrial network in cardiac myocytes. To do this, we spin-coated glass coverslips with a soft, moderate, or stiff polymer. Next, we microcontact printed cell-sized rectangles of fibronectin with AR matching cardiac myocytes at various developmental or disease states onto the polymer surface. We then cultured neonatal rat ventricular myocytes on the patterned surfaces and used confocal microscopy and image processing techniques to quantify sarcomeric α-actinin volume, nucleus volume, and mitochondrial volume, surface area, and size distribution. On some substrates, α-actinin volume increased with cell AR but was not affected by ECM rigidity. Nucleus volume was mostly uniform across all conditions. In contrast, mitochondrial volume increased with cell AR on all substrates. Furthermore, mitochondrial surface area to volume ratio decreased as AR increased on all substrates. Large mitochondria were also more prevalent in cardiac myocytes with higher AR. For select AR, mitochondria were also smaller as ECM rigidity increased. Collectively, these results suggest that mitochondrial architecture in cardiac myocytes is strongly influenced by cell shape and moderately influenced by ECM rigidity. These data have important implications for understanding the factors that impact metabolic performance during heart development and disease.
    Keywords:  Cytoskeleton; Fission; Fusion; Mechanotransduction; Micropatterning; PDMS
  8. J Cell Biol. 2020 Nov 02. pii: e202003024. [Epub ahead of print]219(11):
    Tirrell PS, Nguyen KN, Luby-Phelps K, Friedman JR.
      MICOS is a conserved multisubunit complex that localizes to mitochondrial cristae junctions and organizes cristae positioning within the organelle. MICOS is organized into two independent subcomplexes; however, the mechanisms that dictate the assembly and spatial positioning of each MICOS subcomplex are poorly understood. Here, we determine that MICOS subcomplexes target independently of one another to sites on the inner mitochondrial membrane that are in proximity to contact sites between mitochondria and the ER. One subcomplex, composed of Mic27/Mic26/Mic10/Mic12, requires ERMES complex function for its assembly. In contrast, the principal MICOS component, Mic60, self-assembles and localizes in close proximity to the ER through an independent mechanism. We also find that Mic60 can uniquely redistribute adjacent to forced mitochondria-vacuole contact sites. Our data suggest that nonoverlapping properties of interorganelle contact sites provide spatial cues that enable MICOS assembly and ultimately lead to proper physical and functional organization of mitochondria.
  9. Sci Rep. 2020 Oct 12. 10(1): 16751
    Koushi M, Aoyama Y, Kamei Y, Asakai R.
      Bisindolylpyrrole at 0.1 μM is cytoprotective in 2% FBS that is counteracted by cyclosporin-A (CsA), an inhibitor of cyclophilin-D (CypD). We hypothesized that the cytoprotective effect might be due to transient mitochondrial permeability transition (tPT). This study tested the hypothesis that bisindolylpyrrole can trigger tPT extensively, thereby leading to cell death under certain conditions. Indeed, CsA-sensitive tPT-mediated apoptosis could be induced by bisindolylpyrrole at > 5 μM in HeLa cells cultured in 0.1% FBS, depending on CypD and VDAC1/2, as shown by siRNA knockdown experiments. Rat liver mitochondria also underwent swelling in response to bisindolylpyrrole, which proceeded at a slower rate than Ca2+-induced swelling, and which was blocked by the VDAC inhibitor tubulin and the ANT inhibitor bongkrekate, indicating the involvement of the ANT-associated, smaller pore. We examined why 0.1% FBS is a prerequisite for apoptosis and found that apoptosis is blocked by PKC activation, which is counteracted by the overexpressed defective PKCε. In mitochondrial suspensions, bisindolylpyrrole triggered CsA-sensitive swelling, which was suppressed selectively by pretreatment with PKCε, but not in the co-presence of tubulin. These data suggest that upon PKC inactivation the cytoprotective compound bisindolylpyrrole can induce prolonged tPT causing apoptosis in a CypD-dependent manner through the VDAC1/2-regulated ANT-associated pore.
  10. Redox Biol. 2020 Oct 07. pii: S2213-2317(20)30955-1. [Epub ahead of print]37 101750
    Chi TF, Khoder-Agha F, Mennerich D, Kellokumpu S, Miinalainen I, Kietzmann T, Dimova EY.
      The upstream stimulatory factor 2 (USF2) is a transcription factor implicated in several cellular processes and among them, tumor development seems to stand out. However, the data with respect to the role of USF2 in tumor development are conflicting suggesting that it acts either as tumor promoter or suppressor. Here we show that absence of USF2 promotes proliferation and migration. Thereby, we reveal a previously unknown function of USF2 in mitochondrial homeostasis. Mechanistically, we demonstrate that deficiency of USF2 promotes survival by inducing mitophagy in a ROS-sensitive manner by activating both ERK1/2 and AKT. Altogether, this study supports USF2's function as tumor suppressor and highlights its novel role for mitochondrial function and energy homeostasis thereby linking USF2 to conditions such as insulin resistance, type-2 diabetes mellitus, and the metabolic syndrome.
    Keywords:  Compromised mitochondria; Migration; Mitophagy; Proliferation; Upstream stimulatory factor 2 (USF2)
  11. J Cell Sci. 2020 Oct 16. pii: jcs.249045. [Epub ahead of print]
    Harada T, Sada R, Osugi Y, Matsumoto S, Matsuda T, Hayashi-Nishino M, Nagai T, Harada A, Kikuchi A.
      Cytoskeleton-associated protein 4 (CKAP4) is palmitoylated type II transmembrane protein localized to the endoplasmic reticulum (ER). Knockout (KO) of CKAP4 in HeLaS3 cells induced the alterations of mitochondrial structures and increased the number of ER-mitochondria contact sites. To understand the involvement of CKAP4 in mitochondrial functions, the binding proteins of CKAP4 were explored, enabling identification of the mitochondrial porin voltage-dependent anion-selective channel protein 2 (VDAC2), which is localized to the outer mitochondrial membrane. Palmitoylation at Cys100 of CKAP4 was required for the binding of CKAP4 and VDAC2. In CKAP4 KO cells, the binding of inositol trisphosphate receptor (IP3R) and VDAC2 was enhanced, the intramitochondrial Ca2+ concentration increased, and the mitochondrial membrane potential decreased. In addition, CKAP4 KO decreased the oxidative consumption rate, in vitro cancer cell proliferation under low-glucose conditions, and in vivo xenograft tumor formation. The phenotypes were not rescued by a palmitoylation-deficient CKAP4 mutant. These results suggest that CKAP4 plays a role in maintaining mitochondrial functions through the binding to VDAC2 at ER-mitochondria contact sites and that palmitoylation is required for this novel function of CKAP4.
    Keywords:  CKAP4; ER; MAM; Mitochondria; Palmitoylation; VDAC2
  12. Redox Biol. 2020 Sep 30. pii: S2213-2317(20)30945-9. [Epub ahead of print]37 101740
    Sharma S, Bhattarai S, Ara H, Sun G, St Clair DK, Bhuiyan MS, Kevil C, Watts MN, Dominic P, Shimizu T, McCarthy KJ, Sun H, Panchatcharam M, Miriyala S.
      Electrophilic aldehyde (4-hydroxynonenal; 4-HNE), formed after lipid peroxidation, is a mediator of mitochondrial dysfunction and implicated in both the pathogenesis and the progression of cardiovascular disease. Manganese superoxide dismutase (MnSOD), a nuclear-encoded antioxidant enzyme, catalyzes the dismutation of superoxide radicals (O2•-) in mitochondria. To study the role of MnSOD in the myocardium, we generated a cardiomyocyte-specific SOD2 (SOD2Δ) deficient mouse strain. Unlike global SOD2 knockout mice, SOD2Δ mice reached adolescence; however, they die at ~4 months of age due to heart failure. Ultrastructural analysis of SOD2Δ hearts revealed altered mitochondrial architecture, with prominent disruption of the cristae and vacuole formation. Noninvasive echocardiographic measurements in SOD2Δ mice showed dilated cardiomyopathic features such as decreased ejection fraction and fractional shortening along with increased left ventricular internal diameter. An increased incidence of ventricular tachycardia was observed during electrophysiological studies of the heart in SOD2Δ mice. Oxidative phosphorylation (OXPHOS) measurement using a Seahorse XF analyzer in SOD2Δ neonatal cardiomyocytes and adult cardiac mitochondria displayed reduced O2 consumption, particularly during basal conditions and after the addition of FCCP (H+ ionophore/uncoupler), compared to that in SOD2fl hearts. Measurement of extracellular acidification (ECAR) to examine glycolysis in these cells showed a pattern precisely opposite that of the oxygen consumption rate (OCR) among SOD2Δ mice compared to their SOD2fl littermates. Analysis of the activity of the electron transport chain complex identified a reduction in Complex I and Complex V activity in SOD2Δ compared to SOD2fl mice. We demonstrated that a deficiency of SOD2 increases reactive oxygen species (ROS), leading to subsequent overproduction of 4-HNE inside mitochondria. Mechanistically, proteins in the mitochondrial respiratory chain complex and TCA cycle (NDUFS2, SDHA, ATP5B, and DLD) were the target of 4-HNE adduction in SOD2Δ hearts. Our findings suggest that the SOD2 mediated 4-HNE signaling nexus may play an important role in cardiomyopathy.
    Keywords:  Heart failure; Manganese superoxide dismutase; Superoxide radicals
  13. Am J Transl Res. 2020 ;12(9): 5131-5150
    Hu C, He Y, Fang S, Tian N, Gong M, Xu X, Zhao L, Wang Y, He T, Zhang Y, Bi Y.
      Urine-derived stem cells (USCs) are autologous stem cells that exhibit self-renewal ability and multi-lineage differentiation potential. These characteristics make USCs an ideal cell source for hepatocellular transplantation. Here, we investigated the biological characteristics of USCs and their potential use for the treatment of chronic liver injury. We characterized the cell-surface marker profile of USCs by flow cytometry and determined the osteogenic, adipogenic, and hepatic differentiation capacities of USCs using histology. We established a chronic liver-injury model by intraperitoneally injecting carbon tetrachloride into nude mice. USCs were then transplanted via tail vein injection. To determine liver function and histopathology following chronic liver injury, we calculated the liver index, measured serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and performed histological staining. USCs were small, adherent cells expressing mesenchymal but not hematopoietic stem-cell markers. Some induced USCs underwent osteogenic and adipogenic differentiation. When co-cultured with hepatic progenitor cells, about 10% of USCs underwent hepatic differentiation. The ALT and AST levels of the USC-transplanted group were lower than that of the chronic liver-injury model group, and there were no significant differences between the two USC-transplanted groups. However, hepatocyte degeneration and liver fibrosis substantially improved in the hypoxia-pretreated USC-transplanted group compared with the normoxia USC-transplanted group. Taken together, USCs display desirable proliferation and differentiation characteristics, and USC transplantation partially improves abnormal liver function and pathology associated with chronic liver injury. Furthermore, hypoxia pretreatment promotes cell proliferation, migration, and colony formation by inducing autophagy, leading to USC-elicited liver tissue recovery following injury in vivo.
    Keywords:  Urine-derived stem cells; autophagy; cell transplantation; chronic liver injury; hypoxia
  14. Int J Biol Sci. 2020 ;16(15): 2788-2802
    He M, Ma Y, Wang R, Zhang J, Jing L, Li PA.
      Deletion of mitochondrial uncoupling protein 2 (UCP2) has been shown to aggravate ischemic damage in the brain. However, the underlying mechanisms are not fully understood. The objective of this study is to explore the impact of homozygous UCP2 deletion (UCP2-/-) on mitochondrial fission and fusion dynamic balance in ischemic mice under normo- and hyperglycemic conditions. UCP2-/- and wildtype mice were subjected to a 60 min middle cerebral artery occlusion (MCAO) and allowed reperfusion for 6h, 24h and 72h. Our results demonstrated that deletion of UCP2 enlarged infarct volumes and increased numbers of cell death in both normo- and hyperglycemic ischemic mice compared with their wildtype counterparts subjected to the same duration of ischemia and reperfusion. The detrimental effects of UCP deletion were associated with increased ROS production, elevated mitochondrial fission markers Drp1 and Fis1 and suppressed fusion markers Opa1 and Mfn2 in UCP2-/- mice. Electron microscopic study demonstrated a marked mitochondrial swolling after 6h of reperfusion in UCP2-/- mice, contrasting to a mild mitochondrial swolling in wildtype ischemic animals. It is concluded that the exacerbating effects of UCP2-/- on ischemic outcome in both normo- and hyperglycemic animals are associated with increased ROS production, disturbed mitochondrial dynamic balance towards fission and early damage to mitochondrial ultrastructure.
    Keywords:  ROS.; Uncoupling protein 2; cerebral ischemia; hyperglycemia; mitochondrial dynamics; mitochondrial fission; mitochondrial ultrastructure
  15. Front Physiol. 2020 ;11 510600
    Natarajan GK, Glait L, Mishra J, Stowe DF, Camara AKS, Kwok WM.
      Mitochondrial Ca2+ handling is accomplished by balancing Ca2+ uptake, primarily via the Ru360-sensitive mitochondrial calcium uniporter (MCU), Ca2+ buffering in the matrix and Ca2+ efflux mainly via Ca2+ ion exchangers, such as the Na+/Ca2+ exchanger (NCLX) and the Ca2+/H+ exchanger (CHE). The mechanism of CHE in cardiac mitochondria is not well-understood and its contribution to matrix Ca2+ regulation is thought to be negligible, despite higher expression of the putative CHE protein, LETM1, compared to hepatic mitochondria. In this study, Ca2+ efflux via the CHE was investigated in isolated rat cardiac mitochondria and permeabilized H9c2 cells. Mitochondria were exposed to (a) increasing matrix Ca2+ load via repetitive application of a finite CaCl2 bolus to the external medium and (b) change in the pH gradient across the inner mitochondrial membrane (IMM). Ca2+ efflux at different matrix Ca2+ loads was revealed by inhibiting Ca2+ uptake or reuptake with Ru360 after increasing number of CaCl2 boluses. In Na+-free experimental buffer and with Ca2+ uptake inhibited, the rate of Ca2+ efflux and steady-state free matrix Ca2+ [mCa2+]ss increased as the number of administered CaCl2 boluses increased. ADP and cyclosporine A (CsA), which are known to increase Ca2+ buffering while maintaining a constant [mCa2+]ss, decreased the rate of Ca2+ efflux via the CHE, with a significantly greater decrease in the presence of ADP. ADP also increased Ca2+ buffering rate and decreased [mCa2+]ss. A change in the pH of the external medium to a more acidic value from 7.15 to 6.8∼6.9 caused a twofold increase in the Ca2+ efflux rate, while an alkaline change in pH from 7.15 to 7.4∼7.5 did not change the Ca2+ efflux rate. In addition, CHE activation was associated with membrane depolarization. Targeted transient knockdown of LETM1 in permeabilized H9c2 cells modulated Ca2+ efflux. The results indicate that Ca2+ efflux via the CHE in cardiac mitochondria is modulated by acidic buffer pH and by total matrix Ca2+. A mechanism is proposed whereby activation of CHE is sensitive to changes in both the matrix Ca2+ buffering system and the matrix free Ca2+ concentration.
    Keywords:  CHE; Ca2+ efflux; Ca2+/H+ exchanger; LETM1; calcium retention capacity; cardiac mitochondria; pH gradient; total matrix Ca2+
  16. Autophagy. 2020 Oct 12. 1-16
    da Silva Rosa SC, Martens MD, Field JT, Nguyen L, Kereliuk SM, Hai Y, Chapman D, Diehl-Jones W, Aliani M, West AR, Thliveris J, Ghavami S, Rampitsch C, Dolinsky VW, Gordon JW.
      Lipotoxicity is a form of cellular stress caused by the accumulation of lipids resulting in mitochondrial dysfunction and insulin resistance in muscle. Previously, we demonstrated that the mitophagy receptor BNIP3L/Nix is responsive to lipotoxicity and accumulates in response to a high-fat (HF) feeding. To provide a better understanding of this observation, we undertook gene expression array and shot-gun metabolomics studies in soleus muscle from rodents on an HF diet. Interestingly, we observed a modest reduction in several autophagy-related genes. Moreover, we observed alterations in the fatty acyl composition of cardiolipins and phosphatidic acids. Given the reported roles of these phospholipids and BNIP3L in mitochondrial dynamics, we investigated aberrant mitochondrial turnover as a mechanism of impaired myocyte insulin signaling. In a series of gain-of-function and loss-of-function experiments in rodent and human myotubes, we demonstrate that BNIP3L accumulation triggers mitochondrial depolarization, calcium-dependent activation of DNM1L/DRP1, and mitophagy. In addition, BNIP3L can inhibit insulin signaling through activation of MTOR-RPS6KB/p70S6 kinase inhibition of IRS1, which is contingent on phosphatidic acids and RHEB. Finally, we demonstrate that BNIP3L-induced mitophagy and impaired glucose uptake can be reversed by direct phosphorylation of BNIP3L by PRKA/PKA, leading to the translocation of BNIP3L from the mitochondria and sarcoplasmic reticulum to the cytosol. These findings provide insight into the role of BNIP3L, mitochondrial turnover, and impaired myocyte insulin signaling during an overfed state when overall autophagy-related gene expression is reduced. Furthermore, our data suggest a mechanism by which exercise or pharmacological activation of PRKA may overcome myocyte insulin resistance. Abbreviations: BCL2: B cell leukemia/lymphoma 2; BNIP3L/Nix: BCL2/adenovirus E1B interacting protein 3-like; DNM1L/DRP1: dynamin 1-like; FUNDC1: FUN14 domain containing 1; IRS1: insulin receptor substrate 1; MAP1LC3A/LC3: microtubule-associated protein 1 light chain 3 alpha; MFN1: mitofusin 1; MFN2: mitofusin 2; MTOR: mechanistic target of rapamycin kinase; OPA1: OPA1 mitochondrial dynamin like GTPase; PDE4i: phosphodiesterase 4 inhibitor; PLD1: phospholipase D1; PLD6: phospholipase D family member 6; PRKA/PKA: protein kinase, AMP-activated; PRKCD/PKCδ: protein kinase C, delta; PRKCQ/PKCθ: protein kinase C, theta; RHEB: Ras homolog enriched in brain; RPS6KB/p70S6K: ribosomal protein S6 kinase; SQSTM1/p62: sequestosome 1; YWHAB/14-3-3β: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein beta.
    Keywords:  Insulin signaling; MTOR; Nix; PKA; mitochondria; mitophagy; muscle
  17. Am J Transl Res. 2020 ;12(9): 5151-5169
    Miao Y, Ding Z, Zou Z, Yang Y, Yang M, Zhang X, Li Z, Zhou L, Zhang L, Zhang X, Du D, Jiang F, Zhou P.
      Cardiomyocytes, macrophages, and fibroblasts play important roles in inflammation and repair during myocardial ischemia/reperfusion injury (MIRI). Myeloid differentiation primary response 88 (MyD88) is upregulated in immunocytes, cardiomyocytes, and fibroblasts during MIRI. MyD88 induces the secretion of proinflammatory cytokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α), while fibroblasts are recruited and activated to mediate cardiac remodeling. The aim of this study was to assess the anti-MIRI effect and mode of action of the novel MyD88 inhibitor TJ-M2010-5. We synthesized TJ-M2010-5 and identified its target by co-immunoprecipitation, after which we established a murine MIRI model and tested the protective effect of TJ-M2010-5 by immunohistochemistry, flow cytometry, real-time PCR, and western blotting. Neonatal rat cardiomyocytes subjected to anoxia/reoxygenation were also isolated and their supernatants used to stimulate cardiac macrophagocytes and fibroblasts in vitro. MyD88 was found upregulated during the early and late phases after MIRI. The MyD88 inhibitor considerably improved cardiac function, reduced cardiomyocyte apoptosis, reduced IL-1β, IL-6, and TNF-α secretion, and inhibited CD80+CD86+MHCII+ macrophage and fibroblast migration. Moreover, TJ-M2010-5 markedly inhibited Toll-like receptor/MyD88 signaling in vivo and in vitro. Thus, our findings highlight TJ-M2010-5 as a potential therapeutic agent for MIRI treatment.
    Keywords:  Anoxia/reoxygenation; TJ-M2010-5; myeloid differentiation factor 88; myocardial ischemia and reperfusion injury; remodeling
  18. Curr Cardiol Rev. 2020 Oct 14.
    Krylatov A, Maslov L, Tsibulnikov SY, Voronkov N, Boshchenko A, Downey J, Mentzer R.
      There is considerable evidence in the heart that autophagy in cardiomyocytes is activated by hypoxia/reoxygenation (H/R) or in hearts by ischemia/reperfusion (I/R). Depending upon the experimental model and duration of ischemia, increases in autophagy in this setting maybe beneficial (cardioprotective) or deleterious (exacerbate I/R injury). Aside from the conundrum as to whether or not autophagy is an adaptive process, it is clearly regulated by a number of diverse molecules including reactive oxygen species (ROS), various kinases, hydrogen sulfide (H2S) and nitric oxide (NO). The purpose this review is to address briefly the controversy regarding the role of autophagy in this setting and to examine a variety of disparate molecules that are involved in its regulation.
    Keywords:  Autophagy; H2S; heart; ischemia; kinases; nitric oxide. ; reperfusion
  19. Pharmacol Res. 2020 Oct 13. pii: S1043-6618(20)31556-5. [Epub ahead of print] 105248
    Kodroń A, Mussulini BH, Pilecka I, Chacińska A.
      The ubiquitin-proteasome system constitutes a major pathway for protein degradation in the cell. Therefore the crosstalk of this pathway with mitochondria is a major topic with direct relevance to many mitochondrial diseases. Proteasome dysfunction triggers not only protein toxicity, but also mitochondrial dysfunction. The involvement of proteasomes in the regulation of protein transport into mitochondria contributes to an increase in mitochondrial function defects. On the other hand, mitochondrial impairment stimulates reactive oxygen species production, which increases protein damage, and protein misfolding and aggregation leading to proteasome overload. Concurrently, mitochondrial dysfunction compromises cellular ATP production leading to reduced protein ubiquitination and proteasome activity. In this review we discuss the complex relationship and interdependence of the ubiquitin-proteasome system and mitochondria. Furthermore, we describe pharmacological inhibition of proteasome activity as a novel strategy to treat a group of mitochondrial diseases.
    Keywords:  mitochondria; mitochondrial diseases; mitochondrial toxicity; proteasome; proteasome inhibitors; protein homeostasis
  20. Biochim Biophys Acta Bioenerg. 2020 Oct 13. pii: S0005-2728(20)30175-4. [Epub ahead of print] 148325
    Rai Y, Anita , Kumari N, Singh S, Kalra N, Soni R, Bhatt AN.
      Ionizing radiation (IR) induced mitochondrial dysfunction is associated with enhanced radiation stimulated metabolic oxidative stress that interacts randomly with intracellular bio-macromolecules causing lethal cellular injury and cell death. Since mild mitochondrial uncoupling emerged as a valuable therapeutic approach by regulating oxidative stress in most prevalent human diseases including ageing, ischemic reperfusion injury, and neurodegeneration with comparable features of IR inflicted mitochondrial damage. Therefore, we explored whether mitochondrial uncoupling could also protect from IR induced cytotoxic insult. Our results showed that DNP, BHT, FCCP, and BAM15 are safe to cells at different concentrations range depending on their respective mitochondrial uncoupling potential. Pre-incubation of murine fibroblast (NIH/3T3) cells with the safe concentration of these uncouplers followed by gamma (γ)-radiation showed significant cell growth recovery, reduced ROS generation, and apoptosis, compared to IR treatment alone. We observed that DNP pre-treatment increased the surviving fraction of IR exposed HEK-293, Raw 264.7 and NIH/3T3 cells. Additionally, DNP pre-treatment followed by IR leads to reduced total and mitochondrial oxidative stress (mos), regulated calcium (Ca2+) homeostasis, and mitochondrial bioenergetics in NIH/3T3 cells. It also significantly reduced macromolecular oxidation, correlated with the regulated ROS generation and antioxidant defence system. Moreover, DNP facilitated DNA repair kinetics evidenced by reducing the number of γ-H2AX foci formation and fragmented nuclei with time. DNP pre-incubation restrained the radiation induced pro-apoptotic factors and inhibits apoptosis. Our findings raise the possibility that mild mitochondrial uncoupling with DNP could be a potential therapeutic approach for radiation induced cytotoxic insult associated with an altered mitochondrial function.
    Keywords:  DNP; Ionizing radiation; Mitochondrial uncoupler; Oxidative stress; ROS; Radioresistance