Revitalizing Muscle Regeneration: Cocoa Polyphenols Shield Mitochondrial Integrity and Boost Myogenesis Under Oxidative Stress

In this study, we describe the effect of cocoa polyphenol extract (CPE, from flavanols-rich cocoa) on myogenic differentiation in murine myoblasts (C2C12 cells) exposed to H2O2. The myogenic program was monitored using morphological, ultrastructural, and molecular approaches. Treatment with 100μM of H2O2 for 1h decreased cell viability. C2C12 (D1) exposed to H2O2 shows more apoptotic and necrotic cells, and mitochondria appear emptied, with cristae heavily damaged. To evaluate the effect of CPE on myoblast viability and myotube formation, 10μg/mL of CPE were added 24h prior to H2O2 treatment and cells were supplemented with fresh CPE every 24h during differentiation. Supplementation with CPE protected C2C12 myoblasts from H2O2-induced oxidative damage both at early (D1) and late (D6) phases of differentiation, preventing cell death and mitochon- drial damage. The number of mitochondria (per area of cell surface) increased 2-fold in both control and in CPE-supplemented and mitochondria in myotubes D6 showed a greater extension of mitochondrial cristae than mitochondria in D1. At D1 and D6 the monolayers showed surface and inner cell features relatively comparable to the untreated control suggesting that CPE supplementation significantly mitigated the effect of H2O2. Preliminary data obtained by the myogenic index (Giemsa staining) suggested that CPE-supplemented cells were partially protected from H2O2-induced myogenesis inhibition. The CPE supple- mentation seems to preserve the mitochondrial integrity and the myogenic differentiation ability of oxidatively injured C2C12 ensuing further nutraceutical perspectives.