Study Of The Role And Mechanisms Of HSP75 Mediates Cardiac Hypertrophy

Background:Cardiac hypertrophy is a common response of the heart to a variety of pathological stimuli, such as hypertension, ischemia, pressure overload, and sarcomeric gene mutation, and it is frequently characterized by myocyte hypertrophy, disarray, and interstitial fibrosis. Although pathological hypertrophy of the myocardium is temporarily compensatory, prolonged hypertrophy may result in ventricular arrhythmias, heart failure, and subsequent cardiovascular mortality. Therefore, it is very important to elucidate the underlying mechanism of cardiac hypertrophy. A growing body of evidence indicates that heat shock proteins (HSPs) are involved in cardiac hypertrophy. HSPs are a family of proteins that become active following increases in temperature and other environmental stresses, and they are well known to play roles in signal transduction, protein folding, translocation, degradation, and the assembly of intracellular proteins, which may protect against various environmental challenges. The activities of several members of the HSP family, such as HSP56, HSP70, HSP 90, and HSP20, have been shown to be induced by hypertrophic stimuli and regulate cardiac hypertrophy via different mechanisms. For example, the cardioprotective effect of the anti-HSP70 antibody was indicated by attenuating pro- hypertrophy signal the mitogen-activated protein kinases (MAPK) P38 and ERK. Cardiac hypertrophy and fibrosis are dependent on many signaling pathways. These important signaling pathways include the MAPK pathway and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway.HSP75, which is also called tumor necrosis factor-associated protein 1 (TRAP-1), is a mitochondrially localized member of the HSP90 family. HSP75 is induced by various environmental stresses, such as glucose deprivation, oxidative injury, and ultraviolet A irradiation, and acts as a mitochondrial chaperone involved in maintaining mitochondrial function and regulating cell apoptosis. The overexpression of HSP75 has been shown to protect against mitochondrial dysfunction, oxidative stress, and ischemic injury-induced apoptosis in brain cells and cardiac myocytes; however, the effects of HSP75 on cardiac hypertrophy and the related signaling mechanisms therein still remain unclear. In the present study, we used HSP75-transgenic mice to investigate the role of HSP75 in pressure overload-induced cardiac hypertrophy and its related molecular mechanisms.Methods:Part one:Wild type C57BL/6 male mice,whose body weight from 23.5～26g and age from 8 to 10 weeks, were divided into sham and Aortic banding (AB)-1day,1 week, 2 week,4 week and 8 week group. Western blot were used to detect the expression of HSP75 protein in different group.Part two:A human HSP75 cDNA construct that contained full-length human HSP75 cDNA was cloned downstream of the human cardiac a-myosin heavy chain (α-MHC) promoter. Transgenic mice were produced by microinjecting theα-MHC-HSP75 construct into fertilized mouse embryos. Transgenic mice were identified by PCR and western blot.Wild type C57BL/6 and cardiac specific HSP75 transgenic male mice, whose body weight from 23.5-26g and age from 8 to 10 weeks, were divided into sham and AB group. After 4 weeks, echocardiography and hemodynamics was performed in order to test the morphology and function of the mice. Then the hearts and lungs of the sacrificed mice were dissected and weighed to compare the heart weight/body weight (HW/BW, mg/g), lung weight/body weight (LW/BW, mg/g), and heart weight/tibia length (HW/TL, mg/cm) ratios. HE and SPR staining were used to detect the myocyte area and collagen deposition. Real-Time PCR was used to detect the expression of mRNA of hypertrophic and fibrosis markers in different groups.Part three:Wild type C57BL/6 and cardiac specific HSP75 transgenic male mice, whose body weight from 23.5-27.5g and age from 8 to 10 weeks, were divided into sham and AB group. After 4 weeks, Western blot were used to detect the expression of TAK, ERK1/2, JNK1/2, p38 and AKT protein in different groups. Results:Part one:HSP75 expression is low in normal mice, but HSP75 levels were gradually elevated from 1 day to 4 weeks after the AB operation; however, HSP75 expression was markedly decreased 8 weeks after the AB operation.Part two:Four independent transgenic lines were established and studied. HSP75-TG mice that were subjected to AB exhibited fewer indications of cardiac dysfunction (including increases in FS, EF, dp/dt max, and dp/dt min and decreases in LVEDD, LVESD, IVSD, and LVPWD) than the WT mice that were subjected to AB. The TG mice that had been subjected to AB exhibited an intriguing diminution in cardiac hypertrophy, as measured by the HW/BW, LW/BW, and HW/TL ratios. The augmentation in cardiac myocyte size and fibrosis area observed after HE and PSR staining in the TG mice was also markedly attenuated in comparison to that in WT mice 4 weeks postoperation. In comparison to WT mice, the overexpression of HSP75 attenuated the pressure overload-induced changes in the expression of hypertrophic markers ANP, Myh7, CTGF, TGF-β, and collagensⅠandⅢand the induction of hypertrophic markers Myh6 was increased in TG mice response to AB compared to that in WT; however, the sham-operated TG mice exhibited no significant changes in the expression levels of the ANP, Myh6, Myh7, CTGF, TGF-β, collagenⅠandⅢgenes in comparison to the control mice.Part three:AKT, TAK, ERK1/2, JNK1/2, and p38 were significantly phosphorylated in mice that had been subjected to AB; however, the phosphorylations of AKT, TAK, JNK1/2, and P38 were almost completely blocked in the TG mice after AB, whereas ERK1/2 was not significantly affected.ConclusionWe show that HSP75 protein levels markedly changed in the mice after AB,and HSP75 functions as a novel anti-hypertrophic regulator by blocking the TAK/P38, JNK, and AKT pathways.