Mitochondrial quality control in the myocardium: cooperation between protein degradation and mitophagy.

Mitochondria are critical for cardiomyocyte survival and maintenance of normal cardiac function. However, changes in the extra- or intracellular environments during stress can cause excessive damage to mitochondria and lead to activation of cell death. In fact, there is evidence that mitochondrial dysfunction is an important contributor to both development of heart failure and the aging process. To counteract the adverse effects resulting from mitochondrial damage, cells have evolved mitochondrial quality control pathways that act at both the protein and organelle levels. Quality control of proteins in the outer mitochondrial membrane is monitored by the ubiquitin-protease system, whereas chaperones and proteases act in the various compartments of the mitochondria. When the damage is too excessive and the degradation machinery is overwhelmed, the entire mitochondrion is eliminated by an autophagosome. Together, these pathways ensure that myocytes maintain a functional network of mitochondria which provides ATP for contraction. Unfortunately, chronic stress and aging can negatively affect proteins that are involved in the mitochondrial quality control pathways which leads to accumulation of dysfunctional mitochondria and loss of myocytes. In this review, we provide an overview of the proteins and pathways that regulate mitochondrial quality control in the cell with an emphasis on pathways involved in maintaining protein and organelle homeostasis. We also delve into the effects of reduced mitochondrial quality control on aging and cardiovascular disease.

Isolation of Rab5-positive endosomes reveals a new mitochondrial degradation pathway utilized by BNIP3 and Parkin.

Degradation of mitochondria is an important cellular quality control mechanism mediated by two distinct pathways: one involving Parkin-mediated ubiquitination and the other dependent on mitophagy receptors. It is known that mitochondria are degraded by the autophagy pathway; however, we recently reported that the small GTPase Rab5 and early endosomes also participate in Parkin-mediated mitochondrial clearance. Here, we have developed a protocol to isolate Rab5-positive vesicles from cells for proteomics analysis and provide additional data confirming that mitophagy regulators and mitochondrial proteins are present in these vesicles. We also demonstrate that the mitophagy receptor BNIP3 utilizes the Rab5-endosomal pathway to clear mitochondria in cells. These findings indicate that a redundancy exists in the downstream degradation pathways to ensure efficient mitochondrial clearance.

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.

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.

The effect of glycomimetic functionalized collagen on peripheral nerve repair.

Increasing evidence suggests that the improper synaptic reconnection of regenerating axons is a significant cause of incomplete functional recovery following peripheral nerve injury. In this study, we evaluate the use of collagen hydrogels functionalized with two peptide glycomimetics of naturally occurring carbohydrates-polysialic acid (PSA) and human natural killer cell epitope epitope (HNK-1)-that have been independently shown to encourage nerve regeneration and axonal targeting. Our novel biomaterial was used to bridge a critical gap size (5 mm) in a mouse femoral nerve injury model. Functional recovery was assessed using gait and hind limb extension, and was significantly better in all glycomimetic peptide-coupled collagen conditions versus non-functional scrambled peptide-coupled collagen, native collagen, and saline controls. Analysis of cross-sections of the regenerated nerve demonstrated that hydrogels coupled with the PSA glycomimetic, but not HNK, had significant increases in the number of myelinated axons over controls. Conversely, hydrogels coupled with HNK, but not PSA, showed improvement in myelination. Additionally, significantly more correctly projecting motoneurons were observed in groups containing coupled HNK-1 mimicking peptide, but not PSA mimicking peptide. Given the distinct morphological outcomes between the two glycomimetics, our study indicates that the enhancement of recovery following peripheral nerve injury induced by PSA- and HNK-functionalized collagen hydrogels likely occurs through distinct mechanisms.