Cardiomyocyte Hypertrophy Induction Protocol

Mouse hearts were subjected to cardiomyocyte isolation based on a protocol described previously [19 (link), 20 (link)]. The mouse cardiomyocytes were cultured in DMEM (Gibco, #C11965500BT) supplemented with 10% FBS (Gibco, #A3840001). BrdU (Sigma, #B5002) was used to repress fibroblast proliferation. Before hypertrophy induction, the cardiomyocytes were cultured in FBS-free DMEM for 24 h. Cardiomyocyte hypertrophy was induced by treatment with Angiotensin II (Ang II; MCE, #HY-13948) for 48 h in FBS-free DMEM. For KLK11 overexpression or knockdown, cardiomyocytes were infected with adenovirus or transfected with siRNA 24 h before serum starvation and Ang II treatment. Cardiomyocytes were stained with an anti-a-actinin antibody (Sigma, #A7188) for size analysis, and cell size was measured with Image J software. Fifty cardiomyocytes of each group were analyzed, and the mean cardiomyocyte size of independent experiments was used to further statistical analysis. For mTOR inhibition, 100 nM rapamycin (MCE, #HY-10219) was used.