bims-mikwok 2023-06-18 papers

bims-mikwok

Biomed News

on Mitochondrial quality control

Issue of 2023‒06‒18
eighteen papers selected by
Avinash N. Mukkala, University of Toronto

A mitochondrial iron-responsive pathway regulated by DELE1.
Inner mitochondrial membrane fission protein, MTP18, serves as a mitophagy receptor to prevent apoptosis in oral cancer.
Energy substrate metabolism, mitochondrial structure and oxidative stress after cardiac ischemia-reperfusion in mice lacking UCP3.
Mitochondria: at the crossroads between mechanobiology and cell metabolism.
Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes.
PERK signaling promotes mitochondrial elongation by remodeling membrane phosphatidic acid.
An Epstein-Barr virus protein interaction map reveals NLRP3 inflammasome evasion via MAVS UFMylation.
Hexokinase dissociation from mitochondria promotes oligomerization of VDAC that facilitates NLRP3 inflammasome assembly and activation.
A novel mitochondrial complex I ROS inhibitor partially improves muscle regeneration in adult but not old mice.
Time-resolved proteomic analyses of senescence highlight metabolic rewiring of mitochondria.
Mitochondrial Transfer to Host Cells from Ex Vivo Expanded Donor Hematopoietic Stem Cells.
The mitochondrial calcium uniporter (MCU) activates mitochondrial respiration and enhances mobility by regulating mitochondrial redox state.
Moderate exercise induces trained immunity in macrophages.
Investigation of Properties of the Mitochondrial Permeability Transition Pore Using Whole-Mitoplast Patch-Clamp Technique.
Ex vivo immunocapture and functional characterization of cell-type-specific mitochondria using MitoTag mice.
Reduced acetylation of TFAM promotes bioenergetic dysfunction in the failing heart.
The C-Terminal Tail of Mitochondrial Transcription Factor A Is Dispensable for Mitochondrial DNA Replication and Transcription In Situ.
Loss of PI3Kα Mediates Protection From Myocardial Ischemia-Reperfusion Injury Linked to Preserved Mitochondrial Function.

Mol Cell. 2023 Jun 15. pii: S1097-2765(23)00413-6. [Epub ahead of print]83(12): 2059-2076.e6

A mitochondrial iron-responsive pathway regulated by DELE1.

Yusuke Sekine, Ryan Houston, Eva-Maria Eckl, Evelyn Fessler, Derek P Narendra, Lucas T Jae, Shiori Sekine.

  The heme-regulated kinase HRI is activated under heme/iron deficient conditions; however, the underlying molecular mechanism is incompletely understood. Here, we show that iron-deficiency-induced HRI activation requires the mitochondrial protein DELE1. Notably, mitochondrial import of DELE1 and its subsequent protein stability are regulated by iron availability. Under steady-state conditions, DELE1 is degraded by the mitochondrial matrix-resident protease LONP1 soon after mitochondrial import. Upon iron chelation, DELE1 import is arrested, thereby stabilizing DELE1 on the mitochondrial surface to activate the HRI-mediated integrated stress response (ISR). Ablation of this DELE1-HRI-ISR pathway in an erythroid cell model enhances cell death under iron-limited conditions, suggesting a cell-protective role for this pathway in iron-demanding cell lineages. Our findings highlight mitochondrial import regulation of DELE1 as the core component of a previously unrecognized mitochondrial iron responsive pathway that elicits stress signaling following perturbation of iron homeostasis.

Keywords:  DELE1; HRI; LONP1; erythroid cells; integrated stress response; iron; mitochondria; mitochondrial import; mitochondrial proteostasis

DOI:  https://doi.org/10.1016/j.molcel.2023.05.031
J Cell Sci. 2023 Jun 14. pii: jcs.259986. [Epub ahead of print]

Inner mitochondrial membrane fission protein, MTP18, serves as a mitophagy receptor to prevent apoptosis in oral cancer.

Debasna P Panigrahi, Prakash P Praharaj, Bishnu P Behera, Srimanta Patra, Shankargouda Patil, Birija Sankar Patro, Sujit K Bhutia.

  MTP18, an inner mitochondrial membrane protein, plays a vital role in maintaining mitochondrial morphology. Furthermore, MTP18 induces mitochondrial fission with subsequent mitophagy, functioning as a mitophagy receptor that targets dysfunctional mitochondria into autophagosomes for elimination. Interestingly, MTP18 interacts with LC3 through its LC3 interacting region (LIR) to induce mitochondrial autophagy. Mutation in the LIR motif (mLIR) inhibits that interaction, thus suppressing mitophagy. Moreover, Parkin/PINK1 deficiency abrogates mitophagy in MTP18-overexpressing FaDu cells. Upon exposure to CCCP, MTP18[mLIR]-FaDu cells show decreased TOM20 expression without affecting COX IV expression. Conversely, loss of Parkin/PINK1 results in inhibition of TOM20 and COX IV degradation in MTP18[mLIR]-FaDu cells exposed to CCCP, establishing Parkin-mediated proteasomal degradation of outer mitochondrial membrane as essential for effective mitophagy. We found that MTP18 provides a survival advantage to oral cancer cells exposed to cellular stress and that inhibition of MTP18-dependent mitophagy induced cell death in oral cancer cells. The findings demonstrate that MTP18 is a novel mitophagy receptor and that MTP18-dependent mitophagy has pathophysiologic implications for oral cancer progression, indicating inhibition of MTP18-mitophagy could thus be a promising cancer therapy strategy.

Keywords:  Apoptosis; MTP18; Mitochondrial fission; Mitophagy; Parkin

DOI:  https://doi.org/10.1242/jcs.259986
Free Radic Biol Med. 2023 Jun 07. pii: S0891-5849(23)00430-6. [Epub ahead of print]

Energy substrate metabolism, mitochondrial structure and oxidative stress after cardiac ischemia-reperfusion in mice lacking UCP3.

Patricia Sánchez-Pérez, Ana Mata, May-Kristin Torp, Elia López-Bernardo, Christina M Heiestad, Jan Magnus Aronsen, Antonio Molina-Iracheta, Luis J Jiménez-Borreguero, Pablo García-Roves, Ana S H Costa, Christian Frezza, Michael P Murphy, Kåre-Olav Stenslokken, Susana Cadenas.

  Myocardial ischemia-reperfusion (IR) injury may result in cardiomyocyte dysfunction. Mitochondria play a critical role in cardiomyocyte recovery after IR injury. The mitochondrial uncoupling protein 3 (UCP3) has been proposed to reduce mitochondrial reactive oxygen species (ROS) production and to facilitate fatty acid oxidation. As both mechanisms might be protective following IR injury, we investigated functional, mitochondrial structural, and metabolic cardiac remodeling in wild-type mice and in mice lacking UCP3 (UCP3-KO) after IR. Results showed that infarct size in isolated perfused hearts subjected to IR ex vivo was larger in adult and old UCP3-KO mice than in equivalent wild-type mice, and was accompanied by higher levels of creatine kinase in the effluent and by more pronounced mitochondrial structural changes. The greater myocardial damage in UCP3-KO hearts was confirmed in vivo after coronary artery occlusion followed by reperfusion. S1QEL, a suppressor of superoxide generation from site IQ in complex I, limited infarct size in UCP3-KO hearts, pointing to exacerbated superoxide production as a possible cause of the damage. Metabolomics analysis of isolated perfused hearts confirmed the reported accumulation of succinate, xanthine and hypoxanthine during ischemia, and a shift to anaerobic glucose utilization, which all recovered upon reoxygenation. The metabolic response to ischemia and IR was similar in UCP3-KO and wild-type hearts, being lipid and energy metabolism the most affected pathways. Fatty acid oxidation and complex I (but not complex II) activity were equally impaired after IR. Overall, our results indicate that UCP3 deficiency promotes enhanced superoxide generation and mitochondrial structural changes that increase the vulnerability of the myocardium to IR injury.

Keywords:  Energy metabolism; Ischemia-reperfusion injury; Mitochondrial respiration; Mitochondrial structure; Oxidative stress; UCP3 (uncoupling protein 3)

DOI:  https://doi.org/10.1016/j.freeradbiomed.2023.05.014
Biol Cell. 2023 Jun 16.

Mitochondria: at the crossroads between mechanobiology and cell metabolism.

Émilie Su, Catherine Villard, Jean-Baptiste Manneville.

  Metabolism and mechanics are two key facets of structural and functional processes in cells, such as growth, proliferation, homeostasis and regeneration. Their reciprocal regulation has been increasingly acknowledged in recent years: external physical and mechanical cues entail metabolic changes, which in return regulate cell mechanosensing and mechanotransduction. Since mitochondria are pivotal regulators of metabolism, we review here the reciprocal links between mitochondrial morphodynamics, mechanics and metabolism. Mitochondria are highly dynamic organelles which sense and integrate mechanical, physical and metabolic cues to adapt their morphology, the organization of their network and their metabolic functions. While some of the links between mitochondrial morphodynamics, mechanics and metabolism are already well established, others are still poorly documented and open new fields of research. First, cell metabolism is known to correlate with mitochondrial morphodynamics. For instance, mitochondrial fission, fusion and cristae remodeling allow the cell to fine-tune its energy production through the contribution of mitochondrial oxidative phosphorylation and cytosolic glycolysis. Second, mechanical cues and alterations in mitochondrial mechanical properties reshape and reorganize the mitochondrial network. Mitochondrial membrane tension emerges as a decisive physical property which regulates mitochondrial morphodynamics. However, the converse link hypothesizing a contribution of morphodynamics to mitochondria mechanics and/or mechanosensitivity has not yet been demonstrated. Third, we highlight that mitochondrial mechanics and metabolism are reciprocally regulated, although little is known about the mechanical adaptation of mitochondria in response to metabolic cues. Deciphering the links between mitochondrial morphodynamics, mechanics and metabolism still presents significant technical and conceptual challenges but is crucial both for a better understanding of mechanobiology and for potential novel therapeutic approaches in diseases such as cancer. This article is protected by copyright. All rights reserved.

Keywords:  Mitochondrial morphodynamics; cancer; cytoskeleton; glycolysis; mechanotransduction; membrane tension; oxidative phosphorylation

DOI:  https://doi.org/10.1111/boc.202300010
J Vis Exp. 2023 May 19.

Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes.

Vijigisha Srivastava, Einav Gross.

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, regulated cell death, and the generation of reactive oxygen species (ROS). ROS are vital for key biological processes. However, when uncontrolled, they can lead to oxidative injury, including mitochondrial damage. Damaged mitochondria release more ROS, thereby intensifying cellular injury and the disease state. A homeostatic process named mitochondrial autophagy (mitophagy) selectively removes damaged mitochondria, which are then replaced by new ones. There are multiple mitophagy pathways, with the common endpoint being the breakdown of the damaged mitochondria in lysosomes. Several methodologies, including genetic sensors, antibody immunofluorescence, and electron microscopy, use this endpoint to quantify mitophagy. Each method for examining mitophagy has its advantages, such as specific tissue/cell targeting (with genetic sensors) and great detail (with electron microscopy). However, these methods often require expensive resources, trained personnel, and a lengthy preparation time before the actual experiment, such as for creating transgenic animals. Here, we present a cost-effective alternative for measuring mitophagy using commercially available fluorescent dyes targeting mitochondria and lysosomes. This method effectively measures mitophagy in the nematode Caenorhabditis elegans and human liver cells, which indicates its potential efficiency in other model systems.

DOI: https://doi.org/10.3791/65337
EMBO J. 2023 Jun 12. e113908

PERK signaling promotes mitochondrial elongation by remodeling membrane phosphatidic acid.

Valerie Perea, Christian Cole, Justine Lebeau, Vivian Dolina, Kelsey R Baron, Aparajita Madhavan, Jeffery W Kelly, Danielle A Grotjahn, R Luke Wiseman.

  Endoplasmic reticulum (ER) stress and mitochondrial dysfunction are linked in the onset and pathogenesis of numerous diseases. This has led to considerable interest in defining the mechanisms responsible for regulating mitochondria during ER stress. The PERK signaling arm of the unfolded protein response (UPR) has emerged as a prominent ER stress-responsive signaling pathway that regulates diverse aspects of mitochondrial biology. Here, we show that PERK activity promotes adaptive remodeling of mitochondrial membrane phosphatidic acid (PA) to induce protective mitochondrial elongation during acute ER stress. We find that PERK activity is required for ER stress-dependent increases in both cellular PA and YME1L-dependent degradation of the intramitochondrial PA transporter PRELID1. These two processes lead to the accumulation of PA on the outer mitochondrial membrane where it can induce mitochondrial elongation by inhibiting mitochondrial fission. Our results establish a new role for PERK in the adaptive remodeling of mitochondrial phospholipids and demonstrate that PERK-dependent PA regulation adapts organellar shape in response to ER stress.

Keywords:  endoplasmic reticulum (ER) stress; mitochondrial morphology; phosphatidic acid; unfolded protein response (UPR)

DOI:  https://doi.org/10.15252/embj.2023113908
Mol Cell. 2023 Jun 08. pii: S1097-2765(23)00374-X. [Epub ahead of print]

An Epstein-Barr virus protein interaction map reveals NLRP3 inflammasome evasion via MAVS UFMylation.

Stephanie Pei Tung Yiu, Cassie Zerbe, David Vanderwall, Edward L Huttlin, Michael P Weekes, Benjamin E Gewurz.

  Epstein-Barr virus (EBV) causes infectious mononucleosis, triggers multiple sclerosis, and is associated with 200,000 cancers/year. EBV colonizes the human B cell compartment and periodically reactivates, inducing expression of 80 viral proteins. However, much remains unknown about how EBV remodels host cells and dismantles key antiviral responses. We therefore created a map of EBV-host and EBV-EBV interactions in B cells undergoing EBV replication, uncovering conserved herpesvirus versus EBV-specific host cell targets. The EBV-encoded G-protein-coupled receptor BILF1 associated with MAVS and the UFM1 E3 ligase UFL1. Although UFMylation of 14-3-3 proteins drives RIG-I/MAVS signaling, BILF1-directed MAVS UFMylation instead triggered MAVS packaging into mitochondrial-derived vesicles and lysosomal proteolysis. In the absence of BILF1, EBV replication activated the NLRP3 inflammasome, which impaired viral replication and triggered pyroptosis. Our results provide a viral protein interaction network resource, reveal a UFM1-dependent pathway for selective degradation of mitochondrial cargo, and highlight BILF1 as a novel therapeutic target.

Keywords:  MAVS; NLRP3 inflammasome; UFMylation; antiviral defense; gamma-herpesvirus; herpesvirus; interactome; mitochondrial-derived vesicles; viral evasion; virus/host interaction

DOI:  https://doi.org/10.1016/j.molcel.2023.05.018
Sci Immunol. 2023 Jun 23. 8(84): eade7652

Hexokinase dissociation from mitochondria promotes oligomerization of VDAC that facilitates NLRP3 inflammasome assembly and activation.

Sung Hoon Baik, V Krishnan Ramanujan, Courtney Becker, Sarah Fett, David M Underhill, Andrea J Wolf.

NLRP3 inflammasome activation is a highly regulated process for controlling secretion of the potent inflammatory cytokines IL-1β and IL-18 that are essential during bacterial infection, sterile inflammation, and disease, including colitis, diabetes, Alzheimer's disease, and atherosclerosis. Diverse stimuli activate the NLRP3 inflammasome, and unifying upstream signals has been challenging to identify. Here, we report that a common upstream step in NLRP3 inflammasome activation is the dissociation of the glycolytic enzyme hexokinase 2 from the voltage-dependent anion channel (VDAC) in the outer membrane of mitochondria. Hexokinase 2 dissociation from VDAC triggers activation of inositol triphosphate receptors, leading to release of calcium from the ER, which is taken up by mitochondria. This influx of calcium into mitochondria leads to oligomerization of VDAC, which is known to form a macromolecule-sized pore in the outer membranes of mitochondria that allows proteins and mitochondrial DNA (mtDNA), often associated with apoptosis and inflammation, respectively, to exit the mitochondria. We observe that VDAC oligomers aggregate with NLRP3 during initial assembly of the multiprotein oligomeric NLRP3 inflammasome complex. We also find that mtDNA is necessary for NLRP3 association with VDAC oligomers. These data, together with other recent work, help to paint a more complete picture of the pathway leading to NLRP3 inflammasome activation.

DOI: https://doi.org/10.1126/sciimmunol.ade7652
Redox Biol. 2023 Jun 02. pii: S2213-2317(23)00171-4. [Epub ahead of print]64 102770

A novel mitochondrial complex I ROS inhibitor partially improves muscle regeneration in adult but not old mice.

Gavin Pharaoh, Ethan L Ostrom, Rudy Stuppard, Matthew Campbell, Jens Markus Borghardt, Michael Franti, Antonio Filareto, David J Marcinek.

  It is unclear whether mitochondrial dysfunction and redox stress contribute to impaired age-related muscle regenerative capacity. Here we characterized a novel compound, BI4500, that inhibits the release of reactive oxygen species (ROS) from the quinone site in mitochondrial complex I (site IQ). We tested the hypothesis that ROS release from site IQ contributes to impaired regenerative capacity in aging muscle. Electron transfer system site-specific ROS production was measured in adult and aged mouse isolated muscle mitochondria and permeabilized gastrocnemius fibers. BI4500 inhibited ROS production from site IQ in a concentration-dependent manner (IC50 = ∼985 nM) by inhibiting ROS release without impairing complex I-linked respiration. In vivo BI4500 treatment decreased ROS production from site IQ. Muscle injury and sham injury were induced using barium chloride or vehicle injection to the tibialis anterior (TA) muscle in adult and aged male mice. On the same day as injury, mice began a daily gavage of 30 mg/kg BI4500 (BI) or placebo (PLA). Muscle regeneration (H&E, Sirius Red, Pax7) was measured at 5 and 35 days after injury. Muscle injury increased centrally nucleated fibers (CNFs) and fibrosis with no treatment or age effect. There was a significant age by treatment interaction for CNFs at 5- and 35-days post injury with significantly more CNFs in BI adults compared to PLA adults. Muscle fiber cross-sectional area (CSA) recovered significantly more in adult BI mice (-89 ± 365 μm2) compared to old PLA (-599 ± 153 μm2) and old BI (-535 ± 222 μm2, mean ± SD). In situ TA force recovery was measured 35 days after injury and was not significantly different by age or treatment. Inhibition of site IQ ROS partially improves muscle regeneration in adult but not old muscle demonstrating a role for CI ROS in the response to muscle injury. Site IQ ROS does not contribute to impaired regenerative capacity in aging.

Keywords:  Barium chloride injury; Mitochondrial reactive oxygen species (ROS); Muscle injury and regeneration; Reverse electron transport (RET); Sarcopenia

DOI:  https://doi.org/10.1016/j.redox.2023.102770
Life Sci Alliance. 2023 Sep;pii: e202302127. [Epub ahead of print]6(9):

Time-resolved proteomic analyses of senescence highlight metabolic rewiring of mitochondria.

Jun Yong Kim, Ilian Atanassov, Frederik Dethloff, Lara Kroczek, Thomas Langer.

Mitochondrial dysfunction and cellular senescence are hallmarks of aging. However, the relationship between these two phenomena remains incompletely understood. In this study, we investigated the rewiring of mitochondria upon development of the senescent state in human IMR90 fibroblasts. Determining the bioenergetic activities and abundance of mitochondria, we demonstrate that senescent cells accumulate mitochondria with reduced OXPHOS activity, resulting in an overall increase of mitochondrial activities in senescent cells. Time-resolved proteomic analyses revealed extensive reprogramming of the mitochondrial proteome upon senescence development and allowed the identification of metabolic pathways that are rewired with different kinetics upon establishment of the senescent state. Among the early responding pathways, the degradation of branched-chain amino acid was increased, whereas the one carbon folate metabolism was decreased. Late-responding pathways include lipid metabolism and mitochondrial translation. These signatures were confirmed by metabolic flux analyses, highlighting metabolic rewiring as a central feature of mitochondria in cellular senescence. Together, our data provide a comprehensive view on the changes in mitochondrial proteome in senescent cells and reveal how the mitochondrial metabolism is rewired in senescent cells.

DOI: https://doi.org/10.26508/lsa.202302127
Cells. 2023 May 25. pii: 1473. [Epub ahead of print]12(11):

Mitochondrial Transfer to Host Cells from Ex Vivo Expanded Donor Hematopoietic Stem Cells.

Hiroki Kawano, Yuko Kawano, Chen Yu, Mark W LaMere, Matthew J McArthur, Michael W Becker, Scott W Ballinger, Satoshi Gojo, Roman A Eliseev, Laura M Calvi.

  Mitochondrial dysfunction is observed in various conditions, from metabolic syndromes to mitochondrial diseases. Moreover, mitochondrial DNA (mtDNA) transfer is an emerging mechanism that enables the restoration of mitochondrial function in damaged cells. Hence, developing a technology that facilitates the transfer of mtDNA can be a promising strategy for the treatment of these conditions. Here, we utilized an ex vivo culture of mouse hematopoietic stem cells (HSCs) and succeeded in expanding the HSCs efficiently. Upon transplantation, sufficient donor HSC engraftment was attained in-host. To assess the mitochondrial transfer via donor HSCs, we used mitochondrial-nuclear exchange (MNX) mice with nuclei from C57BL/6J and mitochondria from the C3H/HeN strain. Cells from MNX mice have C57BL/6J immunophenotype and C3H/HeN mtDNA, which is known to confer a higher stress resistance to mitochondria. Ex vivo expanded MNX HSCs were transplanted into irradiated C57BL/6J mice and the analyses were performed at six weeks post transplantation. We observed high engraftment of the donor cells in the bone marrow. We also found that HSCs from the MNX mice could transfer mtDNA to the host cells. This work highlights the utility of ex vivo expanded HSC to achieve the mitochondrial transfer from donor to host in the transplant setting.

Keywords:  MNX mouse, 5; ex vivo HSC expansion 3; in vivo mitochondrial transfer; mitochondrial DNA 2

DOI:  https://doi.org/10.3390/cells12111473
Redox Biol. 2023 Jun 04. pii: S2213-2317(23)00160-X. [Epub ahead of print]64 102759

The mitochondrial calcium uniporter (MCU) activates mitochondrial respiration and enhances mobility by regulating mitochondrial redox state.

Anna Weiser, Aurélie Hermant, Flavien Bermont, Federico Sizzano, Sonia Karaz, Pilar Alvarez-Illera, Jaime Santo-Domingo, Vincenzo Sorrentino, Jerome N Feige, Umberto De Marchi.

  Regulation of mitochondrial redox balance is emerging as a key event for cell signaling in both physiological and pathological conditions. However, the link between the mitochondrial redox state and the modulation of these conditions remains poorly defined. Here, we discovered that activation of the evolutionary conserved mitochondrial calcium uniporter (MCU) modulates mitochondrial redox state. By using mitochondria-targeted redox and calcium sensors and genetic MCU-ablated models, we provide evidence of the causality between MCU activation and net reduction of mitochondrial (but not cytosolic) redox state. Redox modulation of redox-sensitive groups via MCU stimulation is required for maintaining respiratory capacity in primary human myotubes and C. elegans, and boosts mobility in worms. The same benefits are obtained bypassing MCU via direct pharmacological reduction of mitochondrial proteins. Collectively, our results demonstrate that MCU regulates mitochondria redox balance and that this process is required to promote the MCU-dependent effects on mitochondrial respiration and mobility.

Keywords:  C. elegans; Calcium signaling; MCU; Mitochondria; Redox biology; Skeletal muscle

DOI:  https://doi.org/10.1016/j.redox.2023.102759
Am J Physiol Cell Physiol. 2023 Jun 12.

Moderate exercise induces trained immunity in macrophages.

Mayoorey Murugathasan, Ardavan Jafari, Amandeep Amandeep, Syed A Hassan, Matthew Chihata, Ali A Abdul-Sater.

  Despite its importance in protecting the host from infections and injury, excessive inflammation may lead to serious human diseases including autoimmune disorders, cardiovascular diseases, diabetes, and cancer. Exercise is a known immunomodulator; however, whether exercise causes long term changes in inflammatory responses and how these changes occur are lacking. Here, we show that chronic moderate intensity training of mice leads to persistent metabolic rewiring and changes to chromatin accessibility in bone marrow derived macrophages (BMDMs), which, in turn, tempers their inflammatory responses. We show that BMDMs from exercised mice exhibited a decrease in lipopolysaccharide (LPS) induced NF-kB activation and pro-inflammatory gene expression along with an increase in M2-like associated genes when compared to BMDMs from sedentary mice. This was associated with improved mitochondrial quality and increased reliance on oxidative phosphorylation accompanied with reduced mitochondrial ROS production. Mechanistically, ATAC-seq analysis showed changes in chromatin accessibility of genes associated with inflammatory and metabolic pathways. Overall, our data suggest that chronic moderate exercise can influence the inflammatory responses of macrophages by reprogramming their metabolic and epigenetic landscape.

Keywords:  Exercise; Inflammation; Macrophages; Mitochondria; epigenetic

DOI:  https://doi.org/10.1152/ajpcell.00130.2023
DNA Cell Biol. 2023 Jun 13.

Investigation of Properties of the Mitochondrial Permeability Transition Pore Using Whole-Mitoplast Patch-Clamp Technique.

Maria A Neginskaya, Evgeny V Pavlov.

  The mitochondrial permeability transition pore (mPTP) is a channel in the mitochondrial inner membrane that is activated by excessive calcium uptake. In this study, we used a whole-mitoplast patch-clamp approach to investigate the ionic currents associated with mPTP at the level of the whole single mitochondrion. The whole-mitoplast conductance was at the level of 5 to 7 nS, which is consistent with the presence of three to six single mPTP channels per mitochondrion. We found that mPTP currents are voltage dependent and inactivate at negative potential. The currents were inhibited by cyclosporine A and adenosine diphosphate. When mPTP was induced by oxidative stress, currents were partially blocked by the adenine nucleotide translocase inhibitor bongkrekic acid. Our data suggest that the whole-mitoplast patch-clamp approach is a useful method for investigating the biophysical properties and regulation of the mPTP.

Keywords:  calcium; cyclosporine A; mitochondria; mitochondrial permeability transition; whole-mitoplast patch clamp

DOI:  https://doi.org/10.1089/dna.2023.0171
Nat Protoc. 2023 Jun 16.

Ex vivo immunocapture and functional characterization of cell-type-specific mitochondria using MitoTag mice.

Natalia Prudente de Mello, Caroline Fecher, Adrian Marti Pastor, Fabiana Perocchi, Thomas Misgeld.

Mitochondria are key bioenergetic organelles involved in many biosynthetic and signaling pathways. However, their differential contribution to specific functions of cells within complex tissues is difficult to dissect with current methods. The present protocol addresses this need by enabling the ex vivo immunocapture of cell-type-specific mitochondria directly from their tissue context through a MitoTag reporter mouse. While other available methods were developed for bulk mitochondria isolation or more abundant cell-type-specific mitochondria, this protocol was optimized for the selective isolation of functional mitochondria from medium-to-low-abundant cell types in a heterogeneous tissue, such as the central nervous system. The protocol has three major parts: First, mitochondria of a cell type of interest are tagged via an outer mitochondrial membrane eGFP by crossing MitoTag mice to a cell-type-specific Cre-driver line or by delivery of viral vectors for Cre expression. Second, homogenates are prepared from relevant tissues by nitrogen cavitation, from which tagged organelles are immunocaptured using magnetic microbeads. Third, immunocaptured mitochondria are used for downstream assays, e.g., to probe respiratory capacity or calcium handling, revealing cell-type-specific mitochondrial diversity in molecular composition and function. The MitoTag approach enables the identification of marker proteins to label cell-type-specific organelle populations in situ, elucidates cell-type-enriched mitochondrial metabolic and signaling pathways, and reveals functional mitochondrial diversity between adjacent cell types in complex tissues, such as the brain. Apart from establishing the mouse colony (6-8 weeks without import), the immunocapture protocol takes 2 h and functional assays require 1-2 h.

DOI: https://doi.org/10.1038/s41596-023-00831-w
iScience. 2023 Jun 16. 26(6): 106942

Reduced acetylation of TFAM promotes bioenergetic dysfunction in the failing heart.

Manling Zhang, Ning Feng, Zishan Peng, Dharendra Thapa, Michael W Stoner, Janet R Manning, Charles F McTiernan, Xue Yang, Michael J Jurczak, Danielle Guimaraes, Krithika Rao, Sruti Shiva, Brett A Kaufman, Michael N Sack, Iain Scott.

  General control of amino acid synthesis 5-like 1 (GCN5L1) was previously identified as a key regulator of protein lysine acetylation in mitochondria. Subsequent studies demonstrated that GCN5L1 regulates the acetylation status and activity of mitochondrial fuel substrate metabolism enzymes. However, the role of GCN5L1 in response to chronic hemodynamic stress is largely unknown. Here, we show that cardiomyocyte-specific GCN5L1 knockout mice (cGCN5L1 KO) display exacerbated heart failure progression following transaortic constriction (TAC). Mitochondrial DNA and protein levels were decreased in cGCN5L1 KO hearts after TAC, and isolated neonatal cardiomyocytes with reduced GCN5L1 expression had lower bioenergetic output in response to hypertrophic stress. Loss of GCN5L1 expression led to a decrease in the acetylation status of mitochondrial transcription factor A (TFAM) after TAC in vivo, which was linked to a reduction in mtDNA levels in vitro. Together, these data suggest that GCN5L1 may protect from hemodynamic stress by maintaining mitochondrial bioenergetic output.

Keywords:  Hematology; Molecular biology; Physiology

DOI:  https://doi.org/10.1016/j.isci.2023.106942
Int J Mol Sci. 2023 May 29. pii: 9430. [Epub ahead of print]24(11):

The C-Terminal Tail of Mitochondrial Transcription Factor A Is Dispensable for Mitochondrial DNA Replication and Transcription In Situ.

Natalya Kozhukhar, Mikhail F Alexeyev.

  Mitochondrial transcription factor A (TFAM) is one of the widely studied but still incompletely understood mitochondrial protein, which plays a crucial role in the maintenance and transcription of mitochondrial DNA (mtDNA). The available experimental evidence is often contradictory in assigning the same function to various TFAM domains, partly owing to the limitations of those experimental systems. Recently, we developed the GeneSwap approach, which enables in situ reverse genetic analysis of mtDNA replication and transcription and is devoid of many of the limitations of the previously used techniques. Here, we utilized this approach to analyze the contributions of the TFAM C-terminal (tail) domain to mtDNA transcription and replication. We determined, at a single amino acid (aa) resolution, the TFAM tail requirements for in situ mtDNA replication in murine cells and established that tail-less TFAM supports both mtDNA replication and transcription. Unexpectedly, in cells expressing either C-terminally truncated murine TFAM or DNA-bending human TFAM mutant L6, HSP1 transcription was impaired to a greater extent than LSP transcription. Our findings are incompatible with the prevailing model of mtDNA transcription and thus suggest the need for further refinement.

Keywords:  GeneSwap approach; TFAM; mtDNA instability; mtDNA replication; mtDNA transcription

DOI:  https://doi.org/10.3390/ijms24119430
J Am Heart Assoc. 2023 Jun 15. e022352

Loss of PI3Kα Mediates Protection From Myocardial Ischemia-Reperfusion Injury Linked to Preserved Mitochondrial Function.

Pavel Zhabyeyev, Brent McLean, Wesam Bassiouni, Robert Valencia, Manish Paul, Ahmed M Darwesh, John M Seubert, Saugata Hazra, Gavin Y Oudit.

  Background Identifying new therapeutic targets for preventing the myocardial ischemia-reperfusion injury would have profound implications in cardiovascular medicine. Myocardial ischemia-reperfusion injury remains a major clinical burden in patients with coronary artery disease. Methods and Results We studied several key mechanistic pathways known to mediate cardioprotection in myocardial ischemia-reperfusion in 2 independent genetic models with reduced cardiac phosphoinositide 3-kinase-α (PI3Kα) activity. P3Kα-deficient genetic models (PI3KαDN and PI3Kα-Mer-Cre-Mer) showed profound resistance to myocardial ischemia-reperfusion injury. In an ex vivo reperfusion protocol, PI3Kα-deficient hearts had an 80% recovery of function compared with ≈10% recovery in the wild-type. Using an in vivo reperfusion protocol, PI3Kα-deficient hearts showed a 40% reduction in infarct size compared with wild-type hearts. Lack of PI3Kα increased late Na+ current, generating an influx of Na+, facilitating the lowering of mitochondrial Ca2+, thereby maintaining mitochondrial membrane potential and oxidative phosphorylation. Consistent with these functional differences, mitochondrial structure in PI3Kα-deficient hearts was preserved following ischemia-reperfusion injury. Computer modeling predicted that PIP3, the product of PI3Kα action, can interact with the murine and human NaV1.5 channels binding to the hydrophobic pocket below the selectivity filter and occluding the channel. Conclusions Loss of PI3Kα protects from global ischemic-reperfusion injury linked to improved mitochondrial structure and function associated with increased late Na+ current. Our results strongly support enhancement of mitochondrial function as a therapeutic strategy to minimize ischemia-reperfusion injury.

Keywords:  NaV1.5; PI3Kα; ischemia–reperfusion; mitochondria

DOI:  https://doi.org/10.1161/JAHA.122.022352