Hypoxia Prevents Mitochondrial Dysfunction and Senescence in Human c-Kit+ Cardiac Progenitor Cells

Abstract

Senescence-associated dysfunction deleteriously affects biological activities of human c-Kit + cardiac progenitor cells (hCPCs), particularly under conditions of in vitro culture. In comparison, preservation of self-renewal and decreases in mitochondrial reactive oxygen species (ROS) are characteristics of murine CPCs in vivo that reside within hypoxic niches. Recapitulating hypoxic niche oxygen tension conditions of $1% O 2 in vitro for expansion of hCPCs rather than typical normoxic cell culture conditions (21% O 2 ) could provide significant improvement of functional and biological activities of hCPCs. hCPCs were isolated and expanded under permanent hypoxic (hCPC-1%) or normoxic (hCPC-21%) conditions from left ventricular tissue explants collected during left ventricular assist device implantation. hCPC-1% exhibit increased self-renewal and suppression of senescence characteristics relative to hCPC-21%. Oxidative stress contributed to higher susceptibility to apoptosis, as well as decreased mitochondrial function in hCPC-21%. Hypoxia prevented accumulation of dysfunctional mitochondria, supporting higher oxygen consumption rates and mitochondrial membrane potential. Mitochondrial ROS was an upstream mediator of senescence since treatment of hCPC-1% with mitochondrial inhibitor antimycin A recapitulated mitochondrial dysfunction and senescence observed in hCPC-21%. NAD + /NADH ratio and autophagic flux, which are key factors for mitochondrial function, were higher in hCPC-1%, but hCPC-21% were highly dependent on BNIP3/NIX-mediated mitophagy to maintain mitochondrial function. Overall, results demonstrate that supraphysiological oxygen tension during in vitro expansion initiates a downward spiral of oxidative stress, mitochondrial dysfunction, and cellular energy imbalance culminating in early proliferation arrest of hCPCs. Senescence is inhibited by preventing ROS through hypoxic culture of hCPCs. STEM CELLS 2019;37:555-567 SIGNIFICANCE STATEMENTHuman c-Kit + cardiac progenitor cell (hCPC) biological function is hampered by limited expansion potential and early replicative senescence. Permanent hypoxia (1% O 2 ) preserved clonogenicity and mitochondrial function of hCPCs derived from heart failure patients while maintaining high levels of intracellular NAD + /NADH ratio and suppressing reactive oxygen species and oxidative stress. Figure 7. Long-term hypoxic culture preserves human c-Kit + cardiac progenitor cell (hCPC) mitochondrial function and inhibits senescence. (A): Schematic of feed-forward cycle of oxidative damage on hCPC-21% that leads to mitochondrial dysfunction and senescence in normoxic culture. The cycle is rescued by hypoxic culture of hCPCs. (B): Illustration of hypoxic and normoxic isolation and expansion strategies and respective outcomes on either hCPC-1% or hCPC-21%.

Misoprostol Attenuates Cardiomyocyte Proliferation in the Neonatal Heart through Bnip3 and Perinuclear Calcium Signaling

“…Interestingly, maintaining a capacity for proliferation after birth appears to depend on the metabolic status of the cardiomyocyte, where the switch from glycolysis to oxidative phosphorylation and the resultant accumulation of cytotoxic free radicals is thought to result in cardiomyocyte senescence [11]. Moreover, recent evidence further suggests that preventing this metabolic shift is key to maintaining c-kit + cardiac progenitor cells in culture [48].…”

Adaptation Within Embryonic and Neonatal Heart Environment Reveals Alternative Fates for Adult c-Kit+Cardiac Interstitial Cells

“…Delivery to fetal myocardium yielded cCIC perivascular 16 localization with fibroblast-like phenotype, similar to cCICs introduced to postnatal P3 17 heart with persistent cell cycle activity for up to 4 weeks. Fibroblast-like phenotype of 18 exogenously transferred cCICs in fetal and postnatal cardiogenic environments is 19 consistent with inability to contribute directly toward cardiogenesis and lack of functional 20 integration with host myocardium. In contrast, cCICs incorporation into extra-embryonic 21 membranes is consistent with fate of polyploid cells in blastocysts.…”

“…Moreover, a plethora of selected subpopulations of 46 in vitro expanded CICs have been intensively studied for cardioprotective and reparative 47 potential upon reintroduction into pathologically injured myocardium for over a decade 48 10,15,16 , but consequences of cell culture environment upon CIC properties in terms of 49 reshaping population characteristics or individual cellular functional capabilities remains 50 6 relatively unstudied and poorly understood. Typically, such cultures involve two 51 dimensional (2D) monolayer growth and serial passaging to obtain sufficient numbers of 52 cells for treatments [17][18][19][20] shown by confocal microscopy immunolabeling ( Fig. S1e).…”

Cardiac interstitial tetraploid cells that escape replicative senescence are found in rodents but not large mammals

“…The impact of myocardial infarction injury upon CICs, including that of c-kit+ cells, has been extensively studied to exquisite detail for decades as summarized in many research studies and reviews [23][24][25][26] . Based upon our extensive experience studying c-kit+ cells for over a decade 21,22,[27][28][29][30] , we chose to focus our initial investigation upon furthering understanding of ploidy in this subset of the CIC population. Functional consequences of polyploidy in these interstitial cells may correlate with transcriptional changes leading to genetic diversity, adaptation to injury, and improved survival in stress response.…”