A Non-Apoptotic Pattern of Caspase-9/Caspase-3 Activation During Differentiation of Human Embryonic Stem Cells into Cardiomyocytes

In vitro studies have demonstrated that the differentiation of embryonic stem cells (ESCs) into cardiomyocytes requires activation of caspases through the mitochondrial pathway. These studies have relied on synthetic substrates for activity measurements, which can be misleading due to potential none-specific hydrolysis of these substrates by proteases other than caspases. Hence, caspase-9 and caspase-3 activation are investigated during the differentiation of human ESCs (hESCs) by directly assessing caspase-9 and -3 cleavage. Western blot reveals the presence of the cleaved caspase-9 prior to and during the differentiation of human ESCs (hESCs) into cardiomyocytes at early stages, which diminishes as the differentiation progresses, without cleavage and activation of endogenous procaspase-3. Activation of exogenous procaspase-3 by endogenous caspase-9 and subsequent cleavage of chromogenic caspase-3 substrate i.e. DEVD-pNA during the course of differentiation confirmes that endogenous caspase-9 has the potency to recognize and activate procaspase-3, but for reasons that are unknown to us fails to do so. These observations suggest the existence of distinct mechanisms of caspase regulation in differentiation as compared to apoptosis. Bioinformatics analysis suggests the presence of caspase-9 regulators, which may influence proteolytic function under specific conditions. To investigate caspase-9/-3 activation pattern during cardiomyocytes generation, hESCs underwent small molecules-directed differentiation, with sampling at days 0, 1, 2, 4, and 12. Differentiation verification is done via immunofluorescence and real-time qRT-PCR, while caspases activity is measured using western blotting and spectrometric detection. Bioinformatic results suggested caspase-9 regulators, involved in cellular development and differentiation, which require further study. image