Effects Of Norepinephrine And Calcitonin Gene-related Peptide On αB-crystallin Expression In Neonatal Rat Cardiomyocytes

Objective The aim of this study was to investigate the expression of aB-crystallin induced by norepinephrine(NE) and calcitonin gene-related peptide(CGRP) individually in neonatal rat cardiomyocytes, and explore the effects of NE combined with CGRP on aB-crystallin expression.Methods (1) Culture and identification of cardiomyocytes. The cardiomyocytes obtained from1-3day old Wistar rats were cultured for72hours for experimental study.(2) Effects of NE on the expression of aB-crystallin in neonatal rat cardiomyocytes.16wells of neonatal rat cardiomyocytes were randomly divided into4groups(4wells in each group), C group(control group). NE8group(10-8mol/L), NE7group(10-7mol/L), NE6group(10-6mol/E). Each group was exposed to NE for1hour and then the expression of αB-erystallin was detected and analyzed by Western blot.3wells was collected for immunolluorescence to observe the distribution of αB-crystallin in neonatal rat cardiomyocytes.(3) Effects of CGRP on the expression of αB-crystallin in neonatal rat cardiomyocytes.24wells of neonatal rat cardiomyocytes were randomly divided into6groups(4wells in each group). C group(contiol group), CGRP11group(10-11mol/L), CGRP10group(10-10mol/L), CGRP9group(10-9mol/E), CGRP8group(10-8mol/L), CGRP7group(10-7mol/L). Each group was exposed to CGRP for1hour and then Western blot was used to detect and analyze the expression of aB-crystallin in neonatal rat cardiomyocytes.(4) Effects of NE combined with CGRP on the expression of aB-crystallin in neonatal rat cardiomyocytes.16wells of neonatal rat cardiomyocytes were randomly divided into4groups(4wells in each group), C group(control group), NE group(10-6mol/L), NC group(10-6mol/L NE and10-10mol/L CGRP), NC8group(10-6mol/L NE,10-10mol/L CGRP and10-9mol/L CGRP8-37). Each group was exposed to the agent or the agents for1hour and then Western blot was used to detect and analyze the expression of aB-crystallin in neonatal rat cardiomyocytes.(5) Effects of NE combined with CGRP on the expression of aB-crystallin mRNA in neonatal rat cardiomyocytes.15wells of neonatal rat cardiomyocytes were randomly divided into5groups(3wells in each group), C group(control group), NE group(10-6mol/L), NC group(10-6mol/L NE and10-10mol/L CGRP), NC8group(10-6mol/L NE,10-10mol/L CGRP and10-9mol/L CGRP8-37), CGRP group(10-10mol/L). Each group was was exposed to the agent or the agents for1hour and then quantitative reverse transcription-PCR(RT-PCR) was used to detect and analyze the expression of αB-crystallin mRNA in neonatal rat cardiomyocytes.Results (1) The model of primary cultured cardiomyocytes of Wistar rats was successfully established, presenting more than90%of the cultured cells were cardiomyocytes and they were alive at3days of incubation.(2) The results showed the αB-crystallin of cardiomyocytes was distributed widely but mainly around cell nucleus.(3) Compared with control group, the expression of aB-crystallin of NE8group, NE7group and NE6group was significantly decreased with pecked drop in the expression of aB-crystallin in NE6group (P<0.01). (4) Compared with control group, the expression of aB-crystallin of CGRP11group, CGRP10group, CGRP9group, CGRP8group and CGRP7group was significantly increased (P<0.01). It was the highest in the expression of aB-crystallin in CGRP10group (P<0.01).(5) Compared with control group, the expression of aB-crystallin of NE group was significantly decreased (P<0.01); Compared with NE group, the expression of aB-crystallin of NC group was increased greatly (P<0.05); Compared with NC group, the expression of aB-crystallin of NC8group was decreased. But there is no significant difference (P>0.05).(6) Compared with control group, the expression of aB-crystallin mRNA of NE group, NC group, NC8group and CGRP group was significantly decreased. It was the lowest in the expression of aB-crystallin mRNA in NE and NC group (P<0.05). Compared with NE group, the expression of aB-crystallin mRNA of NC group was increased. But there is no significant difference (P>0.05). The expression of aB-crystallin mRNA showed no significant difference between NC group and NC8group (P>0.05).Conclusions (1) NE could inhibit the expression of aB-crystallin in neonatal rat cardiomyocytes.(2) CGRP could up-regulate the expression of aB-crystallin in neonatal rat cardiomyocytes.(3) CGRP could block NE-induced down-regulation of expression of αB-crystallin in neonatal rat cardiomyocytes.