Study Of The Role And Mechanisms Of Lmcd1 Mediates Cardiac Hypertrophy

Background:Cardiac hypertrophy is common development of cardiovascular diseases such as hypertension, myocardial infarct and arrhythmia. And cardiac hypertrophy is a response of the myocardium to increased workload, characterized by increase of myocardial mass and accumulation of extracellular matrix. Although initially a beneficial adaptive response, prolonged hypertrophy may result in dilated cardiomyopathy, heart failure and even sudden death which is increasing in prevalence to 6-10 times.Thus cardiac hypertrophy represents an independent risk factor for cardiovascular diseases with high rates of mortality and morbidity. Therefore, it is necessary to clarify the mechanisms of the cardiac hypertrophy and find the effective target for the treatment of pathological hypertrophy.Cardiac hypertrophy is dynamic process regulated by complicate multiple factors, and a chronic compensatory mechanism to long-term workload. The pathology appearance of cardiac hypertrophy shows hypertrophy of myocytes, proliferation of mesenchymal and fibrosis of interstitium, which impaired systolic function and cardiac compliance. It shows increased heart weight and vascular capacitance. It is also reported that inflammation and angio neogenesis also participate the development of cardiac hypertrophy. Cardiac hypertrophy was caused by many factors through various mechanisms. For example, mechanical irritation caused the cardiac hypertrophy through promote protein synthesis or deplete protein degradation. And the neurohumor factor such as AngiotensinⅡ, endothelin, catecholamine, Insulin-Like factor, transforming growth factor-βand interleukin-1 act as external stimulus signal which induced intranuclear gene expression alteration, then promote the hypertrophy or proliferation of cells.LIM and cysteine-rich domains 1 (Lmcd1) was identified by screening of the expressed sequence tag database (dbEST) with partially overlapping EST clones, which encodes a family of LIM domain proteins. The predicted Lmcd1 protein contains a novel cysteine-rich domain residing in the amino-terminal region and two LIM domains with regular spacing in the carboxy-terminal region. Northern blot analysis indicated expression of the 1.7-kb transcript in many tissues, and it was found to be highly expressed in both cardiac and skeletal muscle. LIM domain proteins are important regulators in cell growth, cell fate determination, cell differentiation and remodeling of the cell cytoskeleton by their interaction with various structural proteins, kinases and transcriptional regulators. Rath, et al. reported that LIM domain proteins act as co-activators of GATA function in both hematopoietic and cardiovascular tissues. However, the potentially significant role of Lmcd1 in cardiac hypertrophy hasn’t been reported yet. Therefore, the purpose of this study is to determine whether Lmcd1 can regulate cardiac hypertrophy in the condition of pathology and physiology, and to elucidate the molecular mechanisms of Lmcd1 on cardiac hypertrophy in vitro and in vivo.Methods:Part one:Wild type C57BL/6 male mice ranging in age from 8 to 10 weeks, body weight from 24-26g. We used a model of aortic banding (AB)-induced cardiac hypertrophy in male mice. Mice were divided into sham and AB-1week,4week and 8week group. Western Blot Analysis were used to detect the expression of Lmcd1 In wild-type (WT) mice in different group.Part two:Wild type C57BL/6 mice and cardiac specific Lmcd1 transgenic mice ranging in age from 8 to 10 weeks, body weight from 24-26g. We used a model of aortic banding (AB)-induced cardiac hypertrophy in male mice. Mice were divided into sham and AB group. Echocardiography and hemodynamics was performed to analysis the cardiac function. HE and WGA staining were used to detect the myocyte area. Real-Time PCR and Western Blot were used to analysis the expression of transcripts of hypertrophic and fibrosis markers. Cardiomyocytes were infected with AdLmcd1 and Ad-GFP as well as Ad-shLmcd1 and Ad-shRNA, subsequently stimulated with 100 nM AngⅡfor the indicated times. [3H]Leucine-incorporation were used to observe the protein synthesis, and Western Blot were used to analysis the expression of ANP and BNP. Part three:The mice were treated with calcineurin inhibitor cyclosporin A, and echocardiography and hemodynamics were performed to analysis the cardiac function. Real-Time PCR was used to analysis the expression of transcripts of hypertrophic and fibrosis markers. Western Blot was used to analysis the expression of NFAT and MCIP in vivo and in vitro.Results:Lmcdl protein levels in the LV were markedly increased compared to basal levels after AB 4weeks, especially 8 weeks (P<0.01).In wild-type (WT) mice subjected to the AB, cardiac hypertrophy developed after 8 weeks. In cardiac specific Lmcdl transgenic mice, however, cardiac hypertrophy was significantly greater than in WT mice as estimated by HW/BW, cardiomyocyte area, echocardiographic measurements as well as cardiac mRNA and protein level of ANP、BNP、β-MHC and TGFβ1、Col1α1、Col3α1、CTGF. [3H]Leucine-incorporation and the expression of ANP and BNP were significantly increased in cardiomyocytes which were infected with AdLmcdl (P<0.01).The HW/BW, echocardiographic measurements as well as cardiac mRNA level of ANP、BNP、β-MHC and Col1agenⅠ、CollagenⅣ、CTGF were significant downregulated in the cardiac specific Lmcdl transgenic mice which were treated with calcineurin inhibitor cyclosporin A. And overexpression of Lmcdl enhanced the expression of NFAT and MCIP in in vivo and in vitro.Conclusion:Our results suggest that Lmcdl protein levels markedly increased in the mice after AB, and Lmcdl plays a critical role in the development of cardiac hypertrophy via activation of calcineurin/NFAT signaling pathway.