The Calcium-sensing Receptor And Hepatocyte Growth Factor Against Myocardial Cell Apoptosis Related Research

Objective To examine whether anti-apoptotic role of HGF in preventing cardiomyocytes injury induced by ischemia/reperfusion is associated with downregulation of calcium sensing receptor (CaSR) mRNA expression and Phosphoinositide-3 Kinase(PI3K) phosphorylation signaling pathway.Methods Neonatal rat cardiomyocytes were isolated and randomly divided into 7 groups: control group; ischemia/reperfusion group (I/R group); GdCl₃ group; GdCl₃+ NiCl₂+ CdCl₂ group; GdCl₃+LY294002 group; GdCl₃+ HGF group; GdCl₃+ HGF+ LY294002 group. We incubated primary neonatal rat ventricular cardiomyocytes in ischemia-mimetic solution for 2h, then reincubated them in normal culture medium for 24h to establish a model of simulated ischemia/reperfusion(I/R). Cardiomyocyte apoptosis was detected by TUNEL. The expression of CaSR mRNA was detected by reverse transcriptase polymerase chain reaction(RT-PCR). In addition, we analyzed the expression of Caspase-3, Bcl-2 and Phosphoinositide-3 Kinase(PI3K) by Western blotting.Results The simulated I/R enhanced the expression of CaSR mRNA [I/R: (2.62±0.41); control: (1.00±0.31), P<0.01] and cardiomyocyte apoptosis[I/R: (15.32±2.54)%; control: (2.90±1.45)%, P<0.01]. GdCl₃, further increased the expression of CaSR mRNA[GdCl₃: (4.46±0.62); I/R: (2.62±0.41), P<0.01] and Cardiomyocyte apoptosis[GdCl₃: (25.36±2.60)%; I/R: (15.32±2.54)%, P<0.01], along with upregulation of Caspase-3[GdCl₃: (1.93±0.28); I/R: (1.50±0.21), P<0.01], downregulation of Bcl-2[GdCl₃: (0.82±0.18); I/R: (1.71±0.30), P<0.01] and inhibiting PI3K phosphorylation[I/R: (0.87±0.08); GdCl₃: (0.61±0.07); P<0.01]. Combination of GdCl₃ with LY294002 increased Cardiomyocytes apoptosis[GdCl₃+LY294002: (32.6±3.42)%; GdCl₃: (25.36±2.60)%, P<0.01] but did not increased CaSR mRNA expression[GdCl₃+ LY294002: (4.27±0.56); GdCl₃: (4.46±0.62), P﹥0.05].Treatment of HGF decreased I/R- and GdCl₃-induced apoptosis[GdCl₃+HGF: (11.8±1.89)%; GdCl₃: (25.36±2.60)%, P<0.05] by suppressing Caspase-3[GdCl₃+ HGF: (1.12±0.23); GdCl₃:(1.93±0.28), P<0.05;GdCl₃+ HGF+LY294002: (1.87±0.31); GdCl₃ + LY294002: (3.86±0.47), P<0.05] and promoting Bcl-2[GdCl₃+ HGF: (2.56±0.54); GdCl₃: (0.82±0.18), P<0.05;GdCl₃+ HGF+LY294002: (1.68±0.28); GdCl₃ + LY294002: (0.68±0.13), P<0.05] and PI3K phosphoration expression[GdCl₃+ HGF: (2.87±0.21); GdCl₃: (0.61±0.07), P<0.05; GdCl₃+ HGF+LY294002: (2.01±0.14); GdCl₃ + LY294002: (0.44±0.10), P<0.05] in accordance with downregulation of CaSR mRNA expression[GdCl₃+ HGF: (1.46±0.37); GdCl₃: (4.46±0.62), P<0.01].Conclusion HGF exerts protective role in I/R-induced apoptosis at least in part by inhibiting CaSR mRNA expression along with promoting Bcl-2, suppressing Caspase-3 expression and stimulating PI3K phosphorylation signaling pathway.