Effect Of Different Glucose Concentrations On Autophagy In Neonatal Rat Cardiomyocyte Culture

Objective: To investigate the effects of different glucose concentrations on autophagy in neonatal rat cardiomyocyte culture.Methods: Neonatal rat myocytes were isolated from newborn Sprague-Dawley rat heart ventricles by 0.125% trypsin. Then use differential attachment method and the 5 - bromodeoxyuridine (Brdu) to purify myocardial cells. After culture for 24 hours, dividing into four groups as follows: L: means control group, cardiomyocytes were exposed to 5.5mmol/L glucose; H: means high glucose: cardiomyocytes were exposed to 25mmol/L glucose; LR: means cardiomyocytes were exposed to 5.5mmol/L glucose and inducer of autophagy (rapamycin 100nm/L ); HR: means cardiomyocytes were exposed to 25mmol /L glucose and inducer of autophagy (rapamycin 100nm /L ). Cells were visualized with an inverted microscopy. Cardiomyocyte cells surface area was determined using Mocha Software. RT-PCR was performed to identified mRNA expression of hypertrophy-related ANP(atrial natriuretic peptid) andβ-MHC(β-myosin heavy chain) gene and of autophagy-related Beclin-1gene. Western blotting was performed to identified protein expression of autophagy-related marker microtubule-associated protein 3 (LC-3). Bandscan 5.0 were used to measure optical density of PCR bands, Sigma Scan Pro 5 software were used to measure optical density of Western blotting bands. All data were analyzed by SSPS17.0 statistical software, P <0.05 was considered statistically significant.Results: Detection of myocardial cell area, RT-PCR and Western blotting results were as follows:1, Cardiac hypertrophy index (myocardial cell area, ANP andβ-MHC gene expression) in high glucose group was increased significantly than the control group (P <0.05) ; autophagy -related indicators (beclin-1 gene expression and the expression of LC-3) in high glucose was reduced significantly than the control group (P <0.05). 2, Indicators of myocardial hypertrophy in high glucose + inducer of autophagy group was increased than in the control + autophagy inducer group, but the difference was not statistically significant; Indicators of autophagy in high glucose+ inducer of autophagy group was reduced than the control + inducer of autophagy group , but the difference was not statistically significant.3, Indicators of cardiac hypertrophy in high glucose + inducer of autophagy group was reduced than the high glucose group, the difference was statistically significant; indicators of autophagy in high glucose + inducer of autophagy group was increased than high glucose group, the difference was significance.4, Indicators of cardiac hypertrophy in control + inducer of autophagy group was reduced than the control group, but the difference was not statistically significant; indicators of autophagy in control glucose + inducer of autophagy group was increased than control group, but the difference was not statistically Significance.Conclusions:1. Cultured with high glucose levels can lead to cardiac hypertrophy. The level of autophagy in myocardial cells is decreased .2. In high glucose medium , autophagy inducers could increase the level of autophagy in myocardial cells, and can inhibit of myocardial hypertrophy caused by high glucose effect.3. Cultured with high glucose levels lead to myocardial hypertrophy and these effects may be associated with the level of autophagy.4. With the findings of this study, we hypothesized that autophagy play a role in diabetic cardiomyopathy. The mechanism of preventting cardiac hypertrophy and diabetic cardiomyopathy by autophagy requires further study, this may provide new targets for clinical treatment of diabetic cardiomyopathy.