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 β-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.

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.

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.