T3 peptide, an active fragment of tumstatin, inhibits HO-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 (HO)-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 HO (1mM)-induced cell death, apoptotic changes of nuclei and cleaved caspas-3 expression in a concentration-dependent manner. T3 peptide also inhibited HO-induced loss of mitochondrial membrane potential, mitochondrial fission and reactive oxygen species production. Cilengitide, an integrin αβ/αβ inhibitor, prevents the inhibitory effect of T3 peptide on HO-induced reactive oxygen species production. In conclusion, T3 peptide inhibits HO-induced apoptosis at least partly via the inhibition of intracellular reactive oxygen species production through the action on integrin.