Study On Calsarcin-1 And Its Signaling Pathway In Remodeling Myocardium

Backgroud:Heart failure(HF)is the final common end of many cardiovascular diseases,including sustained pressure overload(eg, hypertension),genetical heart disease(eg,HCM or part of DCM)and ischemia/infarction(eg,coronar heart disease).Regardless of the etiology, these disorders initiate ventricular remodeling,an adaptive,compensatory process which becomes progressively maladaptive and the cause of functional and clinical deterioration and,eventually,HF.The molecular and cellular mechanisms for myocardial remodeling are complecated and less clear. Pathological hypertrophy is a hallmark of structural remodeling,which was gauged by the extent of increase in ventricular size,mass and sphericity of the left ventricle and "cellular or structural" as manifested by changes in the individual myocyte,interstitium,signal transduction pathways,sub-cellular apparatus and nucleus are essentially synonymous.Long term of sustained chronic activation of neuroendocrine system prompts the myocardial remodeling and deteriorates the cardiac function,which in turn aggravate the neuroendocrine system activation.Therefore,understanding the cellular and molecular mechanisms of myocardial remodeling is important in elucidating potential therapeutic targets,and using these insights to develop HF therapies designed to alter maladaptive myocardial remodeling could transform HF into a potentially curable and/or nonfatal condition.Calcineurin is the calcium-calmodulin-activated protein phosphatase 2B (PP2B),which is a serine-/threonine-specific phosphatase that is uniquely activated by sustained elevation in[Ca²⁺].When activated,calcineurin directly binds to NFAT transcription factors in the cytoplasm,resulting in their dephosphorylation and subsequent translocation into the nucleus.And the calcineurin and its downstream transcriptional effector NFAT now have been implicated as critical transducers of the hypertrophic response that uniquely link alterations in intracellular calcium handling in a myocyte to the hypertrophic growth response.Calsarcin-1 belongs to a family of musclespecific sarcomeric proteins that appear to function as a bridge between calcineurin and a-actinin at the z-line of cardiac and skeletal muscle.In addition to serving as a potential docking site for calcineurin on the z-line, calsarcins could act as inhibitors or activators of the enzyme,or they could be substrates for calcineurin dephosphorylation.Preliminary studies indicate calsarcin-1 participates in coupling of signaling events at the sarcomere with downstream signals that control cardiac gene expression and myocardial remodeling.Calsarcin-1-deficient hearts further showed exaggerated cardiomyopathy and super-induction of hypertrophic genes in response to pressure overload,suggesting that calsarcin-1 is a negative regulator of stress-induced signaling in vivo.A novel mutation,cTnI Arginine145→tryptophan linking with hypertrophic cardiomyopathy among Chinese population was first discovered by our institute.And the animal models carried with mutant cTnI(cTnI R146W in the mouse sequence)were established successfully.So those animal models together with 2K1C animal model were used to investigate the underlying mechanisms of calsarcin-1 and calcineurin signal transduction pathway in the process of ventricular remodeling.Objective:To investigate the proteins related to the pathological myocardial remodeling,2K1C rat model was use to recapitulate the hypertension induced myocardium remodeling.And 2D combined with MS techneqiues was applied to compare the differential expressed proteins between sham and remodeling myocardium.After then,the differential expressed proteins related to myocardial remodeling were studied in mice carried with cTn I R146W which was set by our institute.In addition,the signaling pathway and its regulation involved in the process of myocardial remodeling in this HCM mice model was also studied in vivo and in vitro.Material and methods:1.The rats were anesthetized,and their left renal arteries were isolated and clipped by 0.2mm silver clamp for 8 weeks,their cardiac structural and function were evaluated by Doppler Ultrasound,and HE& mason dye were used to confirm the changes of remodeling myocardiummyocardial remodeling.Left ventricular of those rats were dissected,the total protein were extracted and purified by 2D cleanup kit.2.By 2D combined with MS methods,the differential expressed proteins of sham operated and 2K1C rats were identified and categorized,their functions were further discussed.3.The differential expression of calsarcin-1 identified by proteomics methods in these two groups was further confirmed by Western and RT-PCR methods.In addition,the calcineurin signaling transduction pathway which related to calsarcin-1 were further investigated in the remodeling myocardium.4.The model of balb/c mice carried with wild type or mutant cardiac troponin I R146W was set up by intraperitoneal injection with the recombinant adenovirus,which was assessed by Doppler Ultrasond and H&E staining. The expression of its downstream molecular of beta-MyHC were further investigated by RT-PCR methods.5.The cTnI R146W transgenic mice set by our lab were identified by RT-PCR methods,and their cardiac structure and functions were assessed by Doppler Ultrasound.6.The expression of calsarcin-1 in genotype(+)and genotype(-)mice were investigated by western and RT-PCR methods.The calcineurin enzyme activity and nuclear NFATc1 binding activity were studied in transgenic mice,and the significance of this changed calcineurin signaling pathway was further discussed.7.The full length of rat calsarcin-1 gene was amplified and cloned into EGFP-N1 plasmid and named as EGFP-N1-Cal.After transfected into Hek293T cells,the expression of calsarcin-1 was observed under fluorescence microscope,and confirmed by Western blotting.8.Neonate rat ventricular myocytes(NRVM)were cultured and transfected by EGFP-N1-Cal.The expression of calsarcin-1 was also observed under fluorescence microscope,and confirmed by Western blotting.9.The recombinant adenovirus containing wild or mutant(R146W)cTnI gene was transfected into the NRVM,and its influence on NRVM was evaluated.10.EGFP-N1-Cal and the recombinant adenovirus containing wild or mutant (R146W)cTnI gene were co-transfected into NRVM,their action on calsarcin-1/calcineurin signaling pathway was further studies.11.Statistics:All data are presented as mean±SD.The independent samples T test,One way ANOVA and chi-square test were used to analyze differences and statistical tests were performed with the use of SPSS 13.0 statistical software.Statistical significance was accepted at P＜0.05.Results:1 After clipped for 8 weeks,higher blood pressure was observed in 2K1C group than in sham operated rats,which was 183±9 and 107±5mmHg (p＜0.01),respectively.The ratio of whole heart weight and left ventricular weight to body weight was also significant greater in 2K1C rats.2.M-mode echocardiographic data showed that LVPWd and IVSd were slightly increased in 2K1C rats,while the IVDd of 2K1C rats was decreased (0.62±0.03 vs.0.65±0.02 cm p＜0.01).The cardiac systolic function evaluated by color Doppler showed no significant difference in these two groups.However,transmitral pulsed Doppler and tissue Doppler measurements showed that compared with the Genotype(-)mice,there was a significant decrease in Peak E,Ea and increased E/Ea in 2K1C rats (P＜0.01).The histological analysis from hematoxylin and eosin in left ventricular showed that 2K1C rats exhibited hypertrophied myocytes with large hyperchromatic nuclei and nuclei hyperplasy.And histological results from Masson's Trichrome also showed that 2K1C rats exhibited serious interstitial fibrosis.3.The extraction and purification methods of left ventricular protein were successful established.4.With 2D and MS techniques,16 differential expressed proteins between 2K1C and sham operated rats were identified,which includes energy metabolism proteins,cellular skeleton related proteins,signaling related proteins and heat shock protein.Furthermore,the expression of calsarcin-1 in the two groups was studied by RT-PCR and Western blotting.Compared with the sham group,2K1C rats showed increased calcineurin activity, which was correlated with the decreased expression of calsarcin-1.5.Although no significant different was found in the morphological and echological characteristic among the three group of balb/c mice or the balb/c mice carried with wild type or mutant cTnI R146W,the pathological sections revealed significant difference in the mice carried with mutant cardiac troponin I,which was demonstrated as disarray of cardiac myocytes with increased einterstitial fibrosis.Furthermore,calsarcin-1 was found to be down-regulated in the mice with mutant cardiac troponin I as well as significant upregulatedβ-MyHC expression.6.Echocardiography results revealed that the cTnI R146W transgenic mice had abnormal indicators for diastolic function.7.Calsarcin-1 was significantly down-regulated in the left ventricular of Genotype(+)mice.However,the expression ofβ-MyHC was up-regulated in Genotype(+)mice when compared to the Genotype(-) mice.8.The activity of calcineurin was significantly increased in Genotype(+)mice (6.39±0.95 vs.Genotype(-)9.09±1.27 pmol Pi/mg protein/min p＜0.01) as well as the nuclear binding activity of NFATc1 in 2K1C rats(0.13±0.01 vs.0.21±0.03 p＜0.01).9.The full length of rat calsarcin-1 was successful amplified and transcripted into cDNA,which was cloned into EGFP-N1 and confirmed by directly sequencing.10.Recombinant pEGFP-N1-Cal was successfully transfected into Hek293 cells using M-PEI that resulted 60%to 70%transfection efficiency and significantly increasing of calsarcin-1 was confirmed by Western blotting.11.The cultured NRVM were transduced with recombinant adenovirus at a titer of 50 MOI.After transduction for 48h,bright green fluorescence was observed And the proteins of EGFP expressed by recombinant viruses was identified by its specific monoclonal antibodies.12.Recombinant mutant cTn I adenovirus increased the calcineurin enzyme activity and nuclear NFATc1 binding activity of NVRM,which could be inhibited by co-transfection with recombinant EGFP-N1-Cal.Discussion and conclusion:1.2K1C for 8 weeks in rats lead to significant myocardial remodeling,which was manifested as concentric hypertrophy,hypertrophied myocytes with large hyperchromatic nuclei,interstitial fibrosis and cardiac diastolic dysfunction.2.Proteomics techniques could be used to compare the differential expressed proteins in normal and remodeling hearts.With this technique,energy metabolism proteins,cellular skeleton related proteins,signaling related proteins and heat shock protein were found to be involved in remodeling myocardium.Especially,calsarcin-1/Calcineurin signaling transduction pathway were investigated,which was found to be involved in the hypertension induced ventricular remodeling.3.By transgenic method,cTnI R146W was found to be related to the myocardial remodeling process.In addition,the abnormality in Calsarcin-1/Calcineurin signaling transduction pathway may also involved in the cTnI R146W mutation induced myocardial remodeling.4.Recombinant calsarcin-1 can reverse the cTnI R146W mutation induced abnormality in Calsarcin-1/Calcineurin signialing transduction pathway. However,whether overexpression of calsarcin-1 can relieve the cTnI R146W induced myocardium remodeling still need to be investigated in further studies.