The Effect Of Calcitonin Gene-Related Peptide(CGRP) On Noradrenaline Induced Neonatal Rat Cardiomyocytes Apoptosis And Its Mechanism

Background:Recent studies suggest that Calcitonin Gene-Related Peptide, noradrenaline levels were significantly increased in the local myocytes, when acute ischemia and infarction. Adrenergic nerves system and sensory nerves system are involved in myocardial ischemic injury and protection, but its mechanism has not yet clarified. In this study, we investigated its roles and its mechanism on the cellular level.Objective:The aim of this study was to investigate the effect of calcitonin gene-related peptide on norepinephrine induced apoptosis on cultured neonatal rat myocytes and whether this effect was mediated by the PKA, PKC signal pathways.Methods:The study was carried out in a series of experiments:(1) Culture and identification of cardiomyocytes.The model of primary cultured cardiomyocytes of Sprague-Dawley(SD) rats was established and observed. The neonatal rat cardiomyocytes were identified with morphology and immunocytochemical(IC) assay method under a reverse microscope. The cardiomyocytes obtained from 1-3 day old SD rats were cultured for 72 hours for experimental study.(2) The effect of Calcitonin Gene-Related Peptide(CGRP) on noradrenaline induced neonatal rat myocytes apoptosis. Cultured myocytes were given NE coupled with PKA inhibitor (H89), CGRP, CGRP receptor antagonist CGRP8-37, forming the following 5 groups:control group(C group), NE group(10'5M), NH group(NE 10-5M+H89 3×10-5 M), NC group (NE 10-5M+CGRP 10-8M), NC8 group(NE 10-5M+CGRP 10-8M+CGRP8-37 10-7M). Each group were cultuled for 15 minutes, then, apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated nick end-labeling(TUNEL)(3) The effect of Calcitonin Gene-Related Peptide(CGRP) on the PKA activity of noradrenaline treated neonatal rat myocytes. Cultured myocytes were randomly divided into 5 groups as before. The PKA activity of extracted myocardial cell protein was measured by using a non-radioactive method.(4) The effect of Calcitonin Gene-Related Peptide(CGRP) on the PKC activity of noradrenaline treated neonatal rat myocytes. Cultured myocytes were randomly divided into 4 groups as before:C group, NE group, NC group, NC8 group. The PKC activity of extracted myocardial cell protein was measured by using a non-radioactive method.Results:(1) The model of primary cultured cardiomyocytes of SD rats was successfully established.(2)Compared with control group(C group), the apoptosis ratio of NE group, NC8 group was significantly increased (P<0.05), Compared with NE group, the apoptosis ratio of NH group, NC group was significantly decreased (P<0.05)(3)Compared with control group(C group), the PKA activity of NE group, NC group was significantly increased (P<0.05), while the PKA activity of NH group was decreased(P<0.05). The PKA activity of NE group showed no significant difference compared with NC group or NC8 group(P>0.05). There was significant difference between NC group and NC8 group(P< 0.05).(4)Compared with control group(C group), the activity of PKC was increased in NE group, NC group and NC8 group (P<0.05), but there were no significant difference between them(P> 0.05).Conclusions:(1) Norepinephrine induced apoptosis on cultured neonatal rat myocytes was maybe mediated by PKA signal pathway.(2) CGRP plays an important role as an inhibitory effect on the myocytes apoptosis induced by norepinephrine, and this effect could be blocked by CGRP receptor 1 antagonist (CGRP8-37).(3) CGRP inhibited the NE induced cardiomyocytes apoptosis was not by regulating the PKA or PKC signaling pathways. Its mechanism remains to be explored.