Helix B Surface Peptide Protects Cardiomyocytes Against Hypoxia/Reoxygenation-induced Apoptosis Through Mitochondrial Pathways.

Abstract

Helix B surface peptide (HBSP), a newly developed tissue-protective erythropoietin derivative, has beneficial effects on myocardial ischemia. This study aimed to investigate the cardio-protective effects of HBSP against hypoxia/reoxygenation (H/R) injury and its possible mechanism. A rat-derived cardiomyocyte cell line (H9C2 cells) were established and pretreated with HBSP. The pretreated primary cultures were subjected to H/R and monitored for cell viability using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Intracellular reactive oxygen species (ROS) levels, apoptosis, and mitochondrial membrane potential (ΔΨm) were detected by flow cytometry. The expression of cytochrome C and Bcl-2 family proteins, as well as the activities of caspases 3 and 9 were determined by Western blot analysis and a colorimetric method, respectively. HBSP reduced apoptotic cells in cardiomyocytes subjected to H/R. In HBSP-treated cardiomyocytes, the H/R-induced mitochondrial ROS production, ΔΨm collapse, and cytochrome C release from mitochondria to the cytosol significantly decreased. Moreover, HBSP inhibited the activation of caspases 9 and 3, as well as the alteration of Bcl-2 family proteins, which were induced by H/R. These results indicated that HBSP has protective effects against H/R-induced apoptosis by regulating the mitochondrial pathway. This mechanism involves inhibiting mitochondrial ROS generation, inhibiting caspase-3 activity, reducing ΔΨm collapse, reducing cytochrome release, and balancing anti and proapoptotic Bcl-2 family proteins.