Expression Of Calcium Sensing Receptor During Hypoxia/reoxygenation Rat Myocardial Damage And Relationship Between Casr And Cardiomyocyte Apoptosis

Background Despite the significant advances in pharmacological and interventional therapies for acute coronary syndrome, ischemic heart disease remains the leading cause of death in the world^[1].Myocardial salvage by reperfusion of ischemic tissue is the primary strategy to prevent heart failure^[2],which has to be delivered concomitant with acute reperfusion injury. Ischemia-reperfusion (I/R) injury is a complicated pathophysiological process, which is involved in calcium overload, free radical production, metabolic abnormalities, and inflammatory reaction.Ca²⁺ influx commonly by three means: voltage-gated calcium channels, Na⁺ / Ca²⁺ exchanger and a receptor-mediated pathway. It is well known that calcium is an important second messenger in the cardiovascular system. However, recent studies suggest that, in addition to as an intracellular messenger, Ca²⁺ may also act as an extracellular first messenger through the calcium-sensing receptor (CaSR)^[3]. CaSR is a member of the superfamily of G protein-coupled receptors .Brown et al. ^[4]first reported the presence of CaSR from bovine parathyroid. Since then, the CaSR had been detected in thyroid ^[5], kidney ^[6], bone ^[7], and gastrointestinal tract tissues[8].The receptor is not only important in maintaining and regulating systemic calcium homeostasis, but also modulates various cellular functions, including secretion of peptides, ion channel/transporter activity, gene expression, proliferation, differentiation, apoptosis and chemotaxis. CaSR was first reported in the cardiovascular system and increases cardiomyocyte apoptosis through the PLC-inositol 1,4,5-triphosphate (IP3) pathway in adult rat heart by Wang and Xu ^[9]. Furthermore, a recent report demonstrated^[10] that the CaSR is present in endothelial cells from human aorta and that it stimulates production of nitric oxide in these cells. However, little is known about the function of CaSR in the hypoxia/reoxygenation (H/R) injury. The present study aimed to investigate whether CaSR plays a role in regulating apoptosis in cardiomyocytes and to demonstrate that pretreatment of hepatocyte growth factor (HGF) has potent cardioprotective effects in dose-dependent manner against H/R injury.Objective To investigate expression of CaSR in the Sprague-Dawley rat neonatal cardiomyocytes induced by H/R injury and its relationship with apoptosis, and to demonstrate whether HGF can protects the heart from H/R injury .Methods After myocardial hypoxia/reoxygenation model was established by culturing primary myocardial cell of neonatal rat in vivo, the myocardial cells were divided into seven groups, the control group,H/R group,calcimimetic group(Ldcl3 group),inhibitor group(LdCl₃ + NiCl₂+ CdCl₂),LdCl₃ + small-(10ng/ml),middle-(20ng/ml )and large-dose(40ng/ml)of HGF group. LdCl₃ is CaSR activator,NiCl2 completely block Na+ / Ca²⁺ exchanger,CdCl2 completely block L-type voltage-gated calcium channels. The apoptotic ratio of rat neonatal cardiomyocytes was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, the level of serum lactate dehydrogenase(LDH) was measured by LDH kit, the expression of TNF-α,IL-1βand CaSR mRNA was detected by real reverse transcriptase polymerase chain reaction(RT-PCR). Results In H/R group , there was significant increases in the apoptotic ratio, the levels of LDH and the expression of TNF-α,CaSR and IL-1βcompared to the control group(P<0.01). Significant increase of those except IL-1βwere observed in Ldcl3 group in comparison to H/R group(P <0.01). IL-1βwas decreased in Ldcl3 group (P<0.01) and there was no obviously expression in the inhibitor group and the HGF groups. The apoptotic ratio, LDH and CaSR except TNF-αwere decreased slightly in inhibitor group compared to Ldcl3 group, but they have no significant difference(P >0.05). More over, compared to H/R group the apoptotic ratio, LDH, CaSR and TNF-αwere significant increase (P <0.05).The four index were significantly decreased in HGF groups compared to inhibitor group. Only the expression of CaSR was dose-depended increased in HGF groups, and there was significant in large-dose HGF group compared to the small -dose HGF(P<0.05).The other three were dose-depended decreased in HGF groups(P <0.05).Conclusion In conclusion, these results suggest that the CaSR was expressed in the atria and ventricle of the rat heart, CaSR activation may be partly associated with myocardial H/R injury through releasing the level of LDH, promoting the expression of inflammatory factor like TNF-a and causing intracellular calcium overload, which can accelerate apoptosis. Also, we found that CaSR activation could increase apoptosis in neonatal rat cardiomyocytes. These results did not change markedly when CdCl2 and NiCl2 were used to pretreat myocytes, which indicating that the Na⁺/Ca²⁺ exchanger or voltage-dependent calcium channels were not responsible for apoptosis. So we presumed that the role of CaSR was overwhelming in increase of intracellular calcium release. Furthermore, pretreatment of HGF inhibited increased free intracellular calcium, suppressed the release of TNF-α, decreased the level of LDH, resulted in reduced apoptosis to protect cardiomyocyte against H/R injury in a dose-effect manner.