Autophagy Mediates The Transition Of Cardiac Hypertrophy To Cardiac Dysfunction In Hsp27 Transgenic Mice

Background and Aim:Activation of autophagy has been shown in the process of cardiac hypertrophy. Autophagy is a highly conserved progress in the organism, it is formed by parcel damaged proteins or organelles, and then autophagy and lysosome are combined into autophagy-lysosome, degrade the contents of autophagy body, to maintain cell stable environment. Study has demonstrated that, autophagy levels abnormal may be involved cardiac hypertrophy occurrence and development, but whether autophagy involved the transition of myocardial hypertrophy compensatory to decompensation is unclear. In this study, by using our previously established cardiac hypertrophy model in transgenic mice with high expression of heat shock protein 27 (heat shock protein 27, Hsp27), we investigate that the role of autophagy in the transition of cardiac hjypertrophy to heart failure.Methods:1. Animals:High expression of Hsp27 transgenic mice (Hsp27 transgenic mice, Hsp27 Tg) was constructed by Nanjing university institute of animal models and our research group, they were bred in the SPF animals houses. According to myocardial Hsp27 expression level, in our study, high expression of Hsp27 transgenic mice was named Tg10 transgenic strains(highly expressed transgenic mice, hTg), low expression of Hsp27 transgenic mice was named Tg85 transgenic strains.2. Detect the expression levels of Hsp27 in the Tg10 and Tg85 transgenic strains by western blot. WT mice as the control group, hTg mice as the experimental group.3. Cardiac hypertrophy detection:weigh the hearts and calculate the ratios of heart weight/body weight (HW/BW) and heart weight/tibia length (HW/TL), observe the heart and cardiomyocytes morphology by biopsy.4. Detect cardiac function and heart structure: 1) Left ventricular systolic function:measure left ventricular ejection fraction (EF%), fractional shortening (FS%) and Stroke volume (SV) by echocardiographic. 2)left ventricular internal diameter at diastolic/systolic phase (LVIDd/LVIDs).5. Analysis the expression levels of the ATP energy synthesis related genes mRNAs by real time-PCR6. Histopathology and western blot analysis1) Detection myocardial histology:HE staining and immunohistochemistry (IHC)2) Observe the myocardial ultrastructure by electron microscopy3) Detect myocardial protein aggregation by western blot7. Detection myocardial autophagy level1) Observe myocardial autophagy body number by electron microscopy2) Test the expressions of LC3â…¡/â… ,p62 and hVps34 by western blot8. Autophagy inhibition.4 weeks hTg mice was induced by intraperitoneally injection of autophagy inhibitors wortmannin (WM), daily administration of 1, for 3 weeks, solvent (vehicle) injection hTg mice as control group. After the experiment, detect cardiac function by echocardiographic, measure and calculate HW/BW and HW/TL, observe the myocardial ultrastructure and autophagy body quantity change, dectect the histologic change by HE and IHC, dectect the protein aggregation and autophagy related protein expression levels by western blot.Results:1. HTg mice appeared myocardial hypertrophy (heart size, the ratios of HW/Bw and HW/TL increased significantly compared with the WT group) in 2 weeks of age2. HTg mice cardiac function significant reduced in 4 weeks of age, ATP synthesis related genes mRNA level dropped significantly3. HTg mice in 3 weeks of age, myocardial autophagy level increased significantly, compared with WT group, autophagy body number, myocardial ratio of LC3 â…¡/ â…  and the expression of hVps34 protein significantly increased in hTg group, but p62 level significantly decreased4. Compared with solvent injection hTg rat, WM treatment hTg mice has the following changes:1) Cardiac function improved significantly, characterized by a significant rise in EF% and FS%2) Myocardial ultrastructure integrity improved significantly3) Mitochondrial energy synthetic gene expression increased significantly4) Autophagy were significantly reduced, the number of cardiocytes autophagy body decreased, the ratio of LC3 â…¡/â…  and hVps34 level decreased, p62 level significantly increased5. WM treatment on hTg mice ratios of HW/BW and HW/TL, histological change had no significant effectConclusion:Autophagy plays a key role in autophagy in the transition of cardiac hjypertrophy to heart failure caused by the Hsp27 high erpression. Autophagy level over up-regulation may nonselective phagocytose normal protein and cell organelle, lead to myocardial cell and tissue damage, eventually cause cardiac insufficiency, the occurrence of heart failure.