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