SIRT1 pathway is involved in GS-enhanced mitochondrial function in cardiomyocytes
To further confirm that GS regulate mitochondrial function by the activation of SIRT1, we tested whether mitochondrial respiration, ATP production, and the NAD+-dependent SIRT1 pathway were affected by a SIRT1 inhibitor, nicotinamiede (NAM) combined with GS. Consistent with the results above, we observed increased basal or maximal mitochondrial respiration in H9c2 cells pretreated with GS for 48 h, which was significantly blocked by NAM (Fig. 7A-7B) . Similarly, GS-mediated increase of ATP content in H9c2 cells was inhibited by NAM( Fig. 7C) . For respiratory chain complexes, higher expression of three complexes, CIII-UQCRC2, CIV-MTCO1, and CII-SDHB, induced by GS was inhibited by the combination of GS with NAM (Fig. 7D-7E) . Importantly, GS combined with NAM significantly reduced GS-induced NAD+ level in H9c2 cells (Fig. 7F) . Then, to further determine whether increased mitochondrial function by GS is mediated by the NAD+-dependent SIRT1 pathway, we measured protein levels of SIRT1 and its targets in control, GS-, NAM-, or GS+NAM-treated H9c2 cells by Western blot analysis. As expected, GS-mediated activation of SIRT1 and its targets, PGC-1α, Nrf1, and Nrf2, were completely abrogated by the pretreatment of GS and NAM, which was similar to the control or NAM group (Fig. 7G-7H) . These findings suggest that the SIRT1 pathway is involved in GS-enhanced mitochondrial function in cardiomyocytes