Abstract 468: Mitochondrial Dysfunction and Senescence of Human Cardiac Progenitor Cells Are Prevented by Hypoxic Culture

Abstract

Rationale: Therapeutic potential of c-Kit + cardiac progenitor cells (CPCs) is modest in autologous cell therapy clinical trials and could benefit from optimization to maximize functional activity. Early proliferation arrest limits expansion potential of human CPCs in vitro . In contrast, self-renewal is preserved and mitochondrial reactive oxidative species (mtROS) minimized within the CPC hypoxic niche in vivo . Expansion under oxygen tension comparable to the hypoxic niche may therefore improve CPC reparative function. Hypothesis: Senescence of CPCs derived from heart failure patients is suppressed by dampening mtROS through long-term hypoxic culture. Methods and Results: Environmental oxygen was maintained with constant hypoxia (1% O 2 ; H-CPCs) or atmospheric oxygen tension (20 - 21% O 2 ; N-CPCs) for isolation and expansion of CPCs from patients undergoing left ventricular assist device implantation. A 4-fold increase in clonogenesis and 73% fewer senescence-associated β-Gal-positive cells were found in H-CPCs. Oxidative stress and DNA damage were also reduced with 80% lower malondialdehyde content and significantly fewer nuclear γ-H2AX foci in H-CPCs. Accumulation of dysfunctional mitochondria was revealed in N-CPCs through functional metabolic analyses. Basal (-59%), maximal (-50%), and ATP production-coupled (-62%) oxygen consumption rates were significantly lower in N-CPCs, despite upregulation of electron transport chain subunits and 72% greater mitochondrial DNA content than H-CPCs. MtROS was elevated, and large mitochondria with swollen morphology were found by TEM in N-CPCs, confirming mitochondrial damage. Key indicators of mitochondrial function including NAD + /NADH ratio and autophagic flux were preserved in H-CPCs. Conclusions: Development of mitochondrial dysfunction and senescence are consequences of normoxic CPC expansion. CPC senescence is delayed by preserving mitochondrial health and limiting oxidative damage through hypoxic culture.