Calcium-sensing receptors induce apoptosis in cultured neonatal rat ventricular cardiomyocytes during simulated ischemia/reperfusion.
Calcium-sensing receptors (CaSRs) are G-protein coupled receptors which regulate systemic calcium homeostasis and also participate in cell proliferation, differentiation and apoptosis. We have previously shown that CaSR can induce apoptosis in isolated rat adult hearts and in normal rat neonatal cardiomyocytes. However, no knowledge exists concerning the role of CaSR in apoptosis induced by ischemia and reperfusion in neonatal cardiac myocytes. Therefore, in the present study, we incubated primary neonatal rat ventricular cardiomyocytes in ischemia-mimetic solution for 2h, then re-incubated them in a normal culture medium for 24h to establish a model of simulated ischemia/reperfusion (I/R). We assayed the apoptotic ratio of the cardiomyocytes by flow cytometry; observed morphological alterations by transmission electron microscope; analyzed the expression of caspase-3, Bcl-2, CaSR, extracellular signal-regulated protein kinase (ERK), and Fas/Fas ligand (FasL) by Western blotting; and measured the concentration of intracellular calcium by Laser Confocal Scanning Microscopy. The results showed that simulated I/R increased the expression of CaSR and cardiomyocyte apoptosis. GdCl3, a specific activator of CaSR, further enhanced CaSR expression, along with increases in intracellular calcium and apoptosis in cardiomyocytes during I/R. Activation of CaSR down-regulated Bcl-2 expression, up-regulated caspase-3 and Fas/FasL expression and stimulated ERK1/2 phosphorylation. In summary, CaSR is involved in I/R injury and apoptosis of neonatal rat ventricular cardiomyocytes by inhibiting Bcl-2, inducing calcium overload and activating the Fas/FasL death receptor pathway.
Jiang
Chun-Ming
CM
Department of Pathophysiology, Harbin Medical University, Baojian Street, Harbin 150086, China. jiangchunming1975@163.com
Han
Li-Ping
LP
Li
Hong-Zhu
HZ
Qu
Ying-Bo
YB
Zhang
Zhuo-Ran
ZR
Wang
Rui
R
Xu
Chang-Qing
CQ
Li
Wei-min
WM
Li
Wei-Ming
WM
D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004195:Disease Models, Animal; D005419:Flavonoids / Q000494:pharmacology; D005682:Gadolinium / Q000494:pharmacology; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D015428:Myocardial Reperfusion Injury / Q000378:metabolism* / Q000473:pathology; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D044169:Receptors, Calcium-Sensing / Q000378:metabolism*
D051017:Apoptosis Regulatory Proteins; D005419:Flavonoids; D044169:Receptors, Calcium-Sensing; D005682:Gadolinium; C038958:gadolinium chloride; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D002118:Calcium