High glucose-induced Ca2+ overload and oxidative stress contribute to apoptosis of cardiac cells through mitochondrial dependent and independent pathways

Background: Cardiac cell apoptosis is the initiating factor of cardiac complications especially diabetic cardiomyopathy. Mitochondria are susceptible to the damaging effects of elevated glucose condition. Calcium overload and oxidative insult are the two mutually non-exclusive phenomena suggested to cause cardiac dysfunction. Here, we examined the effect of high-glucose induced calcium overload in calpain-1 mediated cardiac apoptosis in an in vitro setting. Methods: H9c2, rat ventricular myoblast cell line was treated with elevated glucose condition and the cellular consequences were studied. Intracellular calcium trafficking. ROS generation, calpain-1 activation and caspase-12 and caspase-9 pathway were studied using flow cytometry, confocal microscopy and Western blot analysis. Results: High-glucose treatment resulted in increased intracellular calcium ([Ca2+]i) which was mobilized to the mitochondria. Concomitant intra-mitochondrial calcium ([Ca2+]m) increase resulted in enhanced reactive oxygen and nitrogen species generation. These events led to mitochondrial dysfunction and apoptosis. Cardiomyocyte death exhibited several classical markers of apoptosis, including activation of caspases, appearance of annexin V on the outer plasma membrane, increased population of cells with sub-G(0)/G(1) DNA content and nuclear condensation. Key findings include elucidation of cell signaling mechanism of high-glucose induced calcium-dependent cysteine protease calpain-1 activation, which triggers non-conventional caspases as alternate mode of cell death. Conclusion: This information increases the understanding of cardiac cell death under hyperglycemic condition and can possibly be extended for designing new therapeutic strategies for diabetic cardiomyopathy. General significance: The novel findings of the study reveal that high glucose induces apoptosis by both mitochondria-dependent and independent pathways via concomitant rise in intracellular calcium. (C) 2012 Elsevier B.V. All rights reserved.