Mcl-1-mediated mitochondrial fission protects against stress but impairs cardiac adaptation to exercise.

Myeloid cell leukemia-1 (Mcl-1) is a structurally and functionally unique anti-apoptotic Bcl-2 protein. While elevated levels of Mcl-1 contribute to tumor cell survival and drug resistance, loss of Mcl-1 in cardiac myocytes leads to rapid mitochondrial dysfunction and heart failure development. Although Mcl-1 is an anti-apoptotic protein, previous studies indicate that its functions extend beyond regulating apoptosis. Mcl-1 is localized to both the mitochondrial outer membrane and matrix. Here, we have identified that Mcl-1 in the outer mitochondrial membrane mediates mitochondrial fission, which is independent of its anti-apoptotic function. We demonstrate that Mcl-1 interacts with Drp1 to promote mitochondrial fission in response to various challenges known to perturb mitochondria morphology. Induction of fission by Mcl-1 reduces nutrient deprivation-induced cell death and the protection is independent of its BH3 domain. Finally, cardiac-specific overexpression of Mcl-1OM, but not Mcl-1Matrix, contributes to a shift in the balance towards fission and leads to reduced exercise capacity, suggesting that a pre-existing fragmented mitochondrial network leads to decreased ability to adapt to an acute increase in workload and energy demand. Overall, these findings highlight the importance of Mcl-1 in maintaining mitochondrial health in cells.

Decline in cellular function of aged mouse c-kit+ cardiac progenitor cells.

KEY POINTS: While autologous stem cell-based therapies are currently being tested on elderly patients, there are limited data on the function of aged stem cells and in particular c-kit+ cardiac progenitor cells (CPCs). We isolated c-kit+ cells from young (3 months) and aged (24 months) C57BL/6 mice to compare their biological properties. Aged CPCs have increased senescence, decreased stemness and reduced capacity to proliferate or to differentiate following dexamethasone (Dex) treatment in vitro, as evidenced by lack of cardiac lineage gene upregulation. Aged CPCs fail to activate mitochondrial biogenesis and increase proteins involved in mitochondrial oxidative phosphorylation in response to Dex. Aged CPCs fail to upregulate paracrine factors that are potentially important for proliferation, survival and angiogenesis in response to Dex. The results highlight marked differences between young and aged CPCs, which may impact future design of autologous stem cell-based therapies. ABSTRACT: Therapeutic use of c-kit+ cardiac progenitor cells (CPCs) is being evaluated for regenerative therapy in older patients with ischaemic heart failure. Our understanding of the biology of these CPCs has, however, largely come from studies of young cells and animal models. In the present study we examined characteristics of CPCs isolated from young (3 months) and aged (24 months) mice that could underlie the diverse outcomes reported for CPC-based therapeutics. We observed morphological differences and altered senescence indicated by increased senescence-associated markers ß-galactosidase and p16 mRNA in aged CPCs. The aged CPCs also proliferated more slowly than their young counterparts and expressed lower levels of the stemness marker LIN28. We subsequently treated the cells with dexamethasone (Dex), routinely used to induce commitment in CPCs, for 7 days and analysed expression of cardiac lineage marker genes. While MEF2C, GATA4, GATA6 and PECAM mRNAs were significantly upregulated in response to Dex treatment in young CPCs, their expression was not increased in aged CPCs. Interestingly, Dex treatment of aged CPCs also failed to increase mitochondrial biogenesis and expression of the mitochondrial proteins Complex III and IV, consistent with a defect in mitochondria complex assembly in the aged CPCs. Dex-treated aged CPCs also had impaired ability to upregulate expression of paracrine factor genes and the conditioned media from these cells had reduced ability to induce angiogenesis in vitro. These findings could impact the design of future CPC-based therapeutic approaches for the treatment of older patients suffering from cardiac injury.

Protocol to separate small and large extracellular vesicles from mouse and human cardiac tissues.

Extracellular vesicles (EVs) are secreted by cells under various conditions and can contribute to the disease progression in tissues. Here, we present a protocol to separate small and large EVs from mouse hearts and cardiac tissues collected from patients. We describe steps for utilizing enzymatic digestion for release of EVs from interstitial space followed by differential centrifugation and immunoaffinity purification. The isolated EVs can be used for various experiments to gain insight into their in vivo functions. For complete details on the use and execution of this protocol, please refer to Liang et al. (2023).1.

The Small GTPase Rab7 Regulates Release of Mitochondria in Extracellular Vesicles in Response to Lysosomal Dysfunction.

Mitochondrial quality control is critical for cardiac homeostasis as these organelles are responsible for generating most of the energy needed to sustain contraction. Dysfunctional mitochondria are normally degraded via intracellular degradation pathways that converge on the lysosome. Here, we identified an alternative mechanism to eliminate mitochondria when lysosomal function is compromised. We show that lysosomal inhibition leads to increased secretion of mitochondria in large extracellular vesicles (EVs). The EVs are produced in multivesicular bodies, and their release is independent of autophagy. Deletion of the small GTPase Rab7 in cells or adult mouse heart leads to increased secretion of EVs containing ubiquitinated cargos, including intact mitochondria. The secreted EVs are captured by macrophages without activating inflammation. Hearts from aged mice or Danon disease patients have increased levels of secreted EVs containing mitochondria indicating activation of vesicular release during cardiac pathophysiology. Overall, these findings establish that mitochondria are eliminated in large EVs through the endosomal pathway when lysosomal degradation is inhibited.

Deciphering functional roles and interplay between Beclin1 and Beclin2 in autophagosome formation and mitophagy.

The degradation of macromolecules and organelles by the process of autophagy is critical for cellular homeostasis and is often compromised during aging and disease. Beclin1 and Beclin2 are implicated in autophagy induction, and these homologs share a high degree of amino acid sequence similarity but have divergent N-terminal regions. Here, we investigated the functions of the Beclin homologs in regulating autophagy and mitophagy, a specialized form of autophagy that targets mitochondria. Both Beclin homologs contributed to autophagosome formation, but a mechanism of autophagosome formation independent of either Beclin homolog occurred in response to starvation or mitochondrial damage. Mitophagy was compromised only in Beclin1-deficient HeLa cells and mouse embryonic fibroblasts because of defective autophagosomal engulfment of mitochondria, and the function of Beclin1 in mitophagy required the phosphorylation of the conserved Ser15 residue by the kinase Ulk1. Mitochondria-ER-associated membranes (MAMs) are important sites of autophagosome formation during mitophagy, and Beclin1, but not Beclin2 or a Beclin1 mutant that could not be phosphorylated at Ser15, localized to MAMs during mitophagy. Our findings establish a regulatory role for Beclin1 in selective mitophagy by initiating autophagosome formation adjacent to mitochondria, a function facilitated by Ulk1-mediated phosphorylation of Ser15 in its distinct N-terminal region.

Mitochondria are secreted in extracellular vesicles when lysosomal function is impaired.

Mitochondrial quality control is critical for cardiac homeostasis as these organelles are responsible for generating most of the energy needed to sustain contraction. Dysfunctional mitochondria are normally degraded via intracellular degradation pathways that converge on the lysosome. Here, we identified an alternative mechanism to eliminate mitochondria when lysosomal function is compromised. We show that lysosomal inhibition leads to increased secretion of mitochondria in large extracellular vesicles (EVs). The EVs are produced in multivesicular bodies, and their release is independent of autophagy. Deletion of the small GTPase Rab7 in cells or adult mouse heart leads to increased secretion of EVs containing ubiquitinated cargos, including intact mitochondria. The secreted EVs are captured by macrophages without activating inflammation. Hearts from aged mice or Danon disease patients have increased levels of secreted EVs containing mitochondria indicating activation of vesicular release during cardiac pathophysiology. Overall, these findings establish that mitochondria are eliminated in large EVs through the endosomal pathway when lysosomal degradation is inhibited.

Mcl-1 Differentially Regulates Autophagy in Response to Changes in Energy Status and Mitochondrial Damage.

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.

Aging is associated with a decline in Atg9b-mediated autophagosome formation and appearance of enlarged mitochondria in the heart.

Advancing age is a major risk factor for developing heart disease, and the biological processes contributing to aging are currently under intense investigation. Autophagy is an important cellular quality control mechanism that is reduced in tissues with age but the molecular mechanisms underlying the age-associated defects in autophagy remain poorly characterized. Here, we have investigated how the autophagic process is altered in aged mouse hearts. We report that autophagic activity is reduced in aged hearts due to a reduction in autophagosome formation. Gene expression profile analysis to evaluate changes in autophagy regulators uncovered a reduction in Atg9b transcript and protein levels. Atg9 proteins are critical in delivering membrane to the growing autophagosome, and siRNA knockdown of Atg9b in cells confirmed a reduction in autophagosome formation. Autophagy is also the main pathway involved in eliminating dysfunctional mitochondria via a process known as mitophagy. The E3 ubiquitin ligase Parkin plays a key role in labeling mitochondria for mitophagy. We also found increased levels of Parkin-positive mitochondria in the aged hearts, an indication that they have been labeled for mitophagy. In contrast, Nrf1, a major transcriptional regulator of mitochondrial biogenesis, was significantly reduced in aged hearts. Additionally, our data showed reduced Drp1-mediated mitochondrial fission and formation of enlarged mitochondria in the aged heart. Overall, our findings suggest that cardiac aging is associated with reduced autophagosome number, decreased mitochondrial turnover, and formation of megamitochondria.

Nuclear Parkin Activates the ERRα Transcriptional Program and Drives Widespread Changes in Gene Expression Following Hypoxia.

Parkin is an E3 ubiquitin ligase well-known for facilitating clearance of damaged mitochondria by ubiquitinating proteins on the outer mitochondrial membrane. However, knowledge of Parkin's functions beyond mitophagy is still limited. Here, we demonstrate that Parkin has functions in the nucleus and that Parkinson's disease-associated Parkin mutants, ParkinR42P and ParkinG430D, are selectively excluded from the nucleus. Further, Parkin translocates to the nucleus in response to hypoxia which correlates with increased ubiquitination of nuclear proteins. The serine-threonine kinase PINK1 is responsible for recruiting Parkin to mitochondria, but translocation of Parkin to the nucleus occurs independently of PINK1. Transcriptomic analyses of HeLa cells overexpressing wild type or a nuclear-targeted Parkin revealed that during hypoxia, Parkin contributes to both increased and decreased transcription of genes involved in regulating multiple metabolic pathways. Furthermore, a proteomics screen comparing ubiquitinated proteins in hearts from Parkin-/- and Parkin transgenic mice identified the transcription factor estrogen-related receptor α (ERRα) as a potential Parkin target. Co-immunoprecipitation confirmed that nuclear-targeted Parkin interacts with and ubiquitinates ERRα. Further analysis uncovered that nuclear Parkin increases the transcriptional activity of ERRα. Overall, our study supports diverse roles for Parkin and demonstrates that nuclear Parkin regulates transcription of genes involved in multiple metabolic pathways.

BNIP3L/NIX and FUNDC1-mediated mitophagy is required for mitochondrial network remodeling during cardiac progenitor cell differentiation.

Cell-based therapies represent a very promising strategy to repair and regenerate the injured heart to prevent progression to heart failure. To date, these therapies have had limited success due to a lack of survival and retention of the infused cells. Therefore, it is important to increase our understanding of the biology of these cells and utilize this information to enhance their survival and function in the injured heart. Mitochondria are critical for progenitor cell function and survival. Here, we demonstrate the importance of mitochondrial autophagy, or mitophagy, in the differentiation process in adult cardiac progenitor cells (CPCs). We found that mitophagy was rapidly induced upon initiation of differentiation in CPCs. We also found that mitophagy was mediated by mitophagy receptors, rather than the PINK1-PRKN/PARKIN pathway. Mitophagy mediated by BNIP3L/NIX and FUNDC1 was not involved in regulating progenitor cell fate determination, mitochondrial biogenesis, or reprogramming. Instead, mitophagy facilitated the CPCs to undergo proper mitochondrial network reorganization during differentiation. Abrogating BNIP3L- and FUNDC1-mediated mitophagy during differentiation led to sustained mitochondrial fission and formation of donut-shaped impaired mitochondria. It also resulted in increased susceptibility to cell death and failure to survive the infarcted heart. Finally, aging is associated with accumulation of mitochondrial DNA (mtDNA) damage in cells and we found that acquiring mtDNA mutations selectively disrupted the differentiation-activated mitophagy program in CPCs. These findings demonstrate the importance of BNIP3L- and FUNDC1-mediated mitophagy as a critical regulator of mitochondrial network formation during differentiation, as well as the consequences of accumulating mtDNA mutations. Abbreviations: Baf: bafilomycin A1; BCL2L13: BCL2 like 13; BNIP3: BCL2 interacting protein 3; BNIP3L: BCL2 interacting protein 3 like; CPCs: cardiac progenitor cells; DM: differentiation media; DNM1L: dynamin 1 like; EPCs: endothelial progenitor cells; FCCP: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; FUNDC1: FUN14 domain containing 1; HSCs: hematopoietic stem cells; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MFN1/2: mitofusin 1/2; MSCs: mesenchymal stem cells; mtDNA: mitochondrial DNA; OXPHOS: oxidative phosphorylation; PPARGC1A: PPARG coactivator 1 alpha; PHB2: prohibitin 2; POLG: DNA polymerase gamma, catalytic subunit; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TMRM: tetramethylrhodamine methyl ester.

Parkin does not prevent accelerated cardiac aging in mitochondrial DNA mutator mice.

The E3 ubiquitin ligase Parkin plays an important role in regulating clearance of dysfunctional or unwanted mitochondria in tissues, including the heart. However, whether Parkin also functions to prevent cardiac aging by maintaining a healthy population of mitochondria is still unclear. Here, we have examined the role of Parkin in the context of mtDNA damage and myocardial aging using a mouse model carrying a proofreading defective mitochondrial DNA polymerase gamma (POLG). We observed both decreased Parkin protein levels and development of cardiac hypertrophy in POLG hearts with age; however, cardiac hypertrophy in POLG mice was neither rescued, nor worsened by cardiac specific overexpression or global deletion of Parkin, respectively. Unexpectedly, mitochondrial fitness did not substantially decline with age in POLG mice when compared to WT. We found that baseline mitophagy receptor-mediated mitochondrial turnover and biogenesis were enhanced in aged POLG hearts. We also observed the presence of megamitochondria in aged POLG hearts. Thus, these processes may limit the accumulation of dysfunctional mitochondria as well as the degree of cardiac functional impairment in the aging POLG heart. Overall, our results demonstrate that Parkin is dispensable for constitutive mitochondrial quality control in a mtDNA mutation model of cardiac aging.

A Rab5 endosomal pathway mediates Parkin-dependent mitochondrial clearance.

Damaged mitochondria pose a lethal threat to cells that necessitates their prompt removal. The currently recognized mechanism for disposal of mitochondria is autophagy, where damaged organelles are marked for disposal via ubiquitylation by Parkin. Here we report a novel pathway for mitochondrial elimination, in which these organelles undergo Parkin-dependent sequestration into Rab5-positive early endosomes via the ESCRT machinery. Following maturation, these endosomes deliver mitochondria to lysosomes for degradation. Although this endosomal pathway is activated by stressors that also activate mitochondrial autophagy, endosomal-mediated mitochondrial clearance is initiated before autophagy. The autophagy protein Beclin1 regulates activation of Rab5 and endosomal-mediated degradation of mitochondria, suggesting cross-talk between these two pathways. Abrogation of Rab5 function and the endosomal pathway results in the accumulation of stressed mitochondria and increases susceptibility to cell death in embryonic fibroblasts and cardiac myocytes. These data reveal a new mechanism for mitochondrial quality control mediated by Rab5 and early endosomes.

PINK1 Is Dispensable for Mitochondrial Recruitment of Parkin and Activation of Mitophagy in Cardiac Myocytes.

Myocyte function and survival relies on the maintenance of a healthy population of mitochondria. The PINK1/Parkin pathway plays an important role in clearing defective mitochondria via autophagy in cells. However, how the PINK1/Parkin pathway regulates mitochondrial quality control and whether it coordinates with other mitophagy pathways are still unclear. Therefore, the objective of this study was to investigate the effect of PINK1-deficiency on mitochondrial quality control in myocytes. Using PINK1-deficient (PINK1-/-) mice, we found that Parkin is recruited to damaged cardiac mitochondria in hearts after treatment with the mitochondrial uncoupler FCCP or after a myocardial infarction even in the absence of PINK1. Parkin recruitment to depolarized mitochondria correlates with increased ubiquitination of mitochondrial proteins and activation of mitophagy in PINK1-/- myocytes. In addition, induction of mitophagy by the atypical BH3-only protein BNIP3 is unaffected by lack of PINK1. Overall, these data suggest that Parkin recruitment to depolarized cardiac mitochondria and subsequent activation of mitophagy is independent of PINK1. Moreover, alternative mechanisms of Parkin activation and pathways of mitophagy remain functional in PINK1-/- myocytes and could compensate for the PINK1 deficiency.