Non-apoptotic role of caspase enzymes in satellite cells from human skeletal muscle

Satellite cells (SCs) are essential for skeletal muscle regeneration, but their function declines with aging, often associated with increased pro-apoptotic signaling. This study investigated the impact of in vitro serum starvation-as a model of acute microenvironmental and nutrient stress-on the apoptosis and differentiation potential of human SCs from young and aged donors. SCs were isolated from the Vastus Lateralis of young and aged subjects and cultured in serum-free medium for up to 72 h. We assessed apoptosis through Annexin V/PI staining, TUNEL assays, and caspase activity measurements, while transcriptional profiles were analyzed via RT-PCR. Aged SCs displayed a significantly higher susceptibility to stress-induced apoptosis compared to young controls, marked by the early upregulation of CASP9 and FOXO1. While typical nucleosomal DNA fragmentation was absent, we observed the activation of caspase-3 after 72 h of starvation. In aged cells, activated caspase-3 co-localized with myogenin and extranuclear DNA at sites of nuclear remodeling. Notably, treatment with a pan-caspase inhibitor (z-VAD-fmk) prevented the formation of micronuclei and myotubes, further highlighting a non-apoptotic role for these enzymes. Aged SCs also showed a distinct cell cycle profile characterized by an enlarged G0/G1 phase and altered expression of CDK and CCNB1 genes. Our findings suggest that in human aged SCs, caspase enzymes serve a dual role: mediating a heightened stress response and facilitating the nuclear remodeling necessary for myogenic differentiation. These results clarify how intrinsic aging shapes the response of muscle stem cells under severe environmental and metabolic resource deprivation.