T3 peptide, an active fragment of tumstatin, inhibits H2O2-induced apoptosis in H9c2 cardiomyoblasts.

Tumstatin, a cleaved fragment of α3 chain of type IV collagen, is an endogenous anti-angiogenetic peptide. Although the expression level of tumstatin changes in the heart tissues of certain experimental cardiac disease models, its effect on cardiomyocytes has not been clarified. In this study, we examined the effects of T3 peptide, an active subfragment of tumstatin, on hydrogen peroxide (H2O2)-induced cell death in H9c2 cardiomyoblasts. Cell viability was examined by a cell counting assay. Staining using 4', 6-diamidino-2-phenylindole was performed to observe nuclear morphology. Western blotting was performed to examine cleaved caspase-3 expression. Mitochondrial membrane potential and morphology were detected by a Mito Tracker Red staining. Intracellular reactive oxygen species production was examined by 2', 7'-dichlorodihydrofluorescein diacetate staining. T3 peptide (300, 1000ng/ml) suppressed H2O2 (1mM)-induced cell death, apoptotic changes of nuclei and cleaved caspas-3 expression in a concentration-dependent manner. T3 peptide also inhibited H2O2-induced loss of mitochondrial membrane potential, mitochondrial fission and reactive oxygen species production. Cilengitide, an integrin αvβ3/αvβ5 inhibitor, prevents the inhibitory effect of T3 peptide on H2O2-induced reactive oxygen species production. In conclusion, T3 peptide inhibits H2O2-induced apoptosis at least partly via the inhibition of intracellular reactive oxygen species production through the action on integrin.