Abstract
This study aimed to investigate the protective effect and molecular mechanism of spironolactone in isoproterenol-induced cardiomyocyte hypertrophy. In this study, primary cardiomyocytes were extracted from the heart of neonatal rats. After stable culture, they were processed with isoproterenol alone or isoproterenol (10 μM) combined with different doses (low dose of 10 μM and high dose of 50 μM), and the cellular activity was determined by MTT experiment. The volume of cells was measured with an inverted microscope and CIAS-1000 cell image analysis system. The mRNA expression levels of ANP and BNP in cells were explored by RT-qPCR. The levels of ANP and BNP proteins and NFATc3 phosphorylation in the nucleus were detected by western blot. The extracellular Ca2+ concentration and CaN activity were measured by colorimetry with the kit. Isoproterenol significantly enlarged the volume of cardiomyocytes (p < 0.001), upregulated mRNA and expression levels of ANP and BNP proteins (p < 0.001), increased extracellular Ca2+ concentration and CaN activity (p < 0.001), and upregulated NFATc3 phosphorylation in the nucleus (p < 0.001). The volume of cells treated with isoproterenol combined with different doses of spironolactone significantly decreased compared with those treated with isoproterenol alone (p < 0.001). mRNA and expression levels of ANP and BNP proteins downregulated significantly (p < 0.001). The extracellular Ca2+ (p < 0.01) concentration and CaN activity (p < 0.001) decreased significantly, and NFATc3 phosphorylation in the nucleus downregulated significantly (p < 0.001). There was no significant difference in cell volume (p=0.999), ANP and BNP mRNA (p=0.695), expression levels of proteins, CaN activity (0.154), and NFATc3 phosphorylation in the nucleus between the cells treated with isoproterenol combined with high-dose spironolactone and those in the control group. In conclusion, spironolactone can reverse isoproterenol-induced cardiomyocyte hypertrophy by inhibiting the Ca2+/CaN/NFATc3 pathway.