Adiponectin Inhibited Calcification In Cultured Rat Neonatal Cardiomyocytes Via Reducing Endoplasmic Reticulum Stress-mediated Apoptosis

Aim:To investigate the effects of adiponectin on calcification in cultured cardiomyocytes via reducing endoplasmic reticulum stress-mediated apoptosis in vitro.Methods:Primary cultured cardiomyocytes were obtained from neonatal rats by collagenase Ⅱ digestion method. The a-actin expression as molecular marker of the cardiomyocytes was observed by immunocytochemistry. The cells cultured for72h were used in the experiment and divided into groups randomly:control group, calcified myocardial cells (CMC) group, adiponectin+CMC (3.10,20,30mg/L) groups. The morphological changes of the cardiomyocytes were observed under phase contracted microscope. CMC was prepared by incubation with β-glycerophosphate (BGP). Calcification was confirmed by Von Kossa staining and Alizarin Red S. Calcium content and alkaline phosphatases (ALP). Alkaline phosphatase was measured by spectrophotometric measurement of p-nitorphenol release. Osteocalcin was detected by radioimmunoassay. The content of lactate dehydrogenase (LDH) was measured. The cardiomyocyte apoptosis was detected by flow cytometry. The expression levels of glucose regulated protein78(GRP78) and cysteine-containing aspartate-specific proteases-12(Caspase-12) mRNA were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Western-blot observes the expression of proteins GRP78and Caspase-12.Results:The immunocytochemical stain for smooth muscle a-actin confirmed cardiomyocytes phenotype and the multicellular nodules spontaneously appeared in cardiomyocytes culture. ALP activity and the content of calcium and osteocalcin in calcified myocardial cells was increased by138%and136.9%(all P<0.05), respectively compared with control. The apoptotic rate and the levels of LDH was also significantly increased. Compared to untreated cells, the mRNA and protein levels of GRP78and Caspase-12increased in CMC. Adiponectin suppressed ALP activity, the content of calcium, osteocalcin secretion and decreased mRNA and protein levels of GRP78and Caspase-12in a dose-dependent manner.Conclusion:1.Adiponectin plays a protective role by extenuation of calcification in cultured rat neonatal cardiomyocytes induced by (3-glycerophosphate.2. Adiponectin inhibits calcification in a dose dependent. The concentration of30mg/L adiponectin inhibits calcification significantly.3.The role of adiponectin in inhibiting calcification may be related to reducing endoplasmic reticulum stress-induced apoptosis.