Effects And Mechanisms Of Substance P And Calcitonin Gene Related Peptide On Norepinephrine-induced Apoptosis In Cultured Rat Cardiomyocytes

Objective The aim of the study was to investigate the effects and mechanisms of substance P(SP) and calcitonin gene related peptide(CGRP) on the apoptosis induced by norepinepherine(NE) in cultured neonatal rat cardiomyocytes by examining the changes of apoptosis rate, SP, CGRP and apoptosis related protein, including Bcl-2, Bax and αB-crystallin.Methods The neonatal cardiomyocytes from the new-born (1-3days after birth) Sprague-Dawley rats were isolated and cultured. The cardiomyocytes were identified with morphology and immunocytochemical(IC) assay under a reverse microscope. The cardiomyocytes incubated for72hours were used for further experiments. Thirty wells of the cultured cardiomyocytes were randomly divided into5groups (6wells in each group):control group (incubated with the culture only) and four NE (10-5mol·L-1) groups according to the incubating time of the presence of NE in the culture, which were incubated with NE for15min,30min,60min and180min, respectively. Apoptosis of the cardiomyocytes were examined as scheduled using TUNEL(n=3) assay and flowcytometry (FCM, n=3). The dose-apoptosis relationship for NE was examined. Eighteen wells of rat cardiomyocytes were randomly divided into6groups (3wells in each group): control group and five NE groups according to the concentration of NE in the culture, which were10-8mol·L-1,10-7mol·L-1,10-6mol·L-1,10-5mol·L-1and10-4mol·L-1. The apoptosis of the cultured cardiomyocytes was examined using FCM at3hours after incubation of the myocytes with NE. Effects of NE at a variaty of concentration (from10-8mol·L-1to10-4mol·L-1) on the expression of SP, CGRP, Bcl-2, Bax and aB-crystallin in cultured cardiomyocytes were analyzed by enzyme-linked immunosorbent assay and western blot. Effects of SP and CGRP pretreatment on NE-induced apoptosis and the expressions of the protein and coding mRNA of Bcl-2, Bax and αB-crystallin in cultured cardiomyocytes were investigated. The protein expression of Bcl-2, Bax and αB-crystallin of the cardiomyocytes were detected by western blot. The coding mRNA of Bcl-2, Bax and αB-crystallin were detected by quantitative real-time PCR(qRT-PCR).Results The apoptosis ratio of the cardiomyocytes treated with NE (at10-5mol·L-1) for15min,30min,1h and3h groups were significantly higher than that in control group, obtained by TUNEL and FCM respectiely (all P<0.05). The dose-apoptosis test showed that NE at the concentrations of10-8,10-7,10-6,10-5,10-4mol·L-1caused significantly higher ratio of apoptosis in the cardiomyocytes, when compared with the control (P<0.05). However, the expressions of Bcl-2and aB-crystallin were markedly decreased (P<0.05) while Bax was significantly increased (P<0.05) by most of the concentrations of NE. Compared with control group, the content of SP in the cardiomyocytes were decreased in the NE groups while CGRP was increased (P <0.05) in higher concentration groups of NE (at10-5,10-4mol·L-1). Compared with control group, greater levels (P<0.05) of the mRNA and protein of Bax were observed in the cardiomyocytes treated with10-5mol·L-1NE group. The mRNA and protein of Bcl-2and αB-crystallin, the ratio of Bcl-2to Bax were decreased (P<0.05) in the NE group. SP could reverse the effect of NE on the mRNA and protein of Bcl-2and αB-crystallin, the ratio of Bcl-2to Bax, the mRNA of Bax. But no significant effect on the level of the protein of Bax was found. D-SP, a specific antagonist of neurokinin1receptor, reversed the effect of SP. CGRP could inhibit the effect of NE on the mRNA and protein of Bcl-2and αB-crystallin, the ratio of Bcl-2to Bax. But no significant effects of NE on the mRNA and the protein expression of Bax were found. CGRP8-37, a selective antagonist of CGRP receptor, abolished the effect of CGRP on NE.Conclusions NE could induce the increase of apoptosis and the content of Bax, but decrease of the level of Bcl-2, aB-crystallin and the ratio of Bcl-2to Bax in cultured cardiomyocytes. NE could change the protein contents of SP and CGRP in cultured cardiomyocytes. Both SP and CGRP could inhibit NE-induced apoptosis, which may be associated with the increase of the coding mRNA and protein levels of Bcl-2, aB-crystallin and the ratio of Bcl-2to Bax.