Cell based therapies represent a very promising strategy to repair and regenerate the injured heart to prevent progression to heart failure. To date, cell based 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 unfavorable environment of 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 is rapidly induced upon initiation of differentiation in CPCs. We also found that mitophagy was mediated by the mitophagy receptors pathway, rather than the PINK1/Parkin pathway. Mitophagy receptors Nix and Fundc1, but not Bnip3, were upregulated during differentiation. Mitophagy mediated by 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 Nix/Fundc1‐mediated mitophagy during differentiation led to mitochondrial fragmentation and failure to form an interconnected mitochondrial network. It also led to increased susceptibility to hydrogen peroxide mediated cell death during differentiation. Finally, aging is associated with accumulation of mtDNA mutations in cells and we found that acquiring mtDNA mutations selectively disrupted the differentiation‐activated mitophagy program in CPCs. These findings demonstrate the importance of Nix/Fundc1‐mediated mitophagy as a critical regulator of mitochondrial network formation in differentiating progenitor cells, as well as the consequences of accumulating mtDNA mutations. Support or Funding Information A.B. Gustafsson is supported by an AHA Established Investigator Award, and by NIH R21AG052280, R01HL087023, R01HL132300 and P01HL085577. M.A. Lampert is supported by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology grant T32GM007752. A.M. Orogo is supported in part by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology through an institutional training grant from the National Institute of General Medical Sciences T32GM007752, and National Institutes of Health NRSA Predoctoral Fellowship F31HL123309. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

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.