| Part one Effects of the ubiquitin proteasome inhibitor on left ventricular hypertrophy and ion channelsBackground:Left ventricular hypertrophy (LVH) is an adaptive response of the myocardium to an increased load with increased myocyte size. LVH is mostly associated with various disease states including hypertension, myocardial infarction, congenital and valvular heart diseases. LVH is a critical pathogenetic process leading to heart failure. Multiple electrophysiological abnormalities are also associated with LVH, which has been shown to be an independent risk factor for arrhythmias. LVH is usually associated with prolongation of ventricular action potentials and alterations in the dispersion of repolarization, which will cause electric instability and increase the incidence of life-threatening arrhythmia. Atrial and ventricular arrhythmias are common complications in patients with LVH. The mechanism of arrhythmia induced by LVH is poorly understood. Some data showed a significant change in mRNA or protein expression level of ion channels in LVH animal models, which suggest ion channel remodeling in the LVH is associated with mechanism of arrhythmia. The ubiquitin-proteasome system (UPS) is mainly composed of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), ubiquitin-ligating enzyme (E3) and 26S proteasome. Up to 80-90% of all intracellular proteins are degraded via the UPS. UPS is not only the major pathway for intracellular degradation of injured proteins, but also plays a major role in regulating many cellular processes including inflammation, apoptosis, cell proliferation, and DNA repair. Previous researchs have proved that the UPS is closely related to tumor and autoimmune diseases. Over the past decade, the role of the UPS has been the subject of many studies to elucidate its role in cardiovascular disease. Researchers have found that the UPS is related to coronary heart disease, cardiomyopathy and heart failure, but the role of the UPS on LVH and arrhysmia are poorly understood.Objective:To establish LVH model by transverse aortic constriction (TAC 2W,4W) and Ang II infusion (2000ng/kg/min,3W) in Wild-type (WT) C57BL/6 mice; To explore changes of protein and mRNA expression level of calcium and potassium ion channels in LVH animal models; To observe the effects of ubiquitin-protease inhibitors (PYR-41, PR-957) on LVH and ion channels.Methods:TAC (2W,4W) and Ang II infusion (2000ng/kg/min,3W) were performed in Wild-type (WT) C57BL/6 mice. Blood pressure was measured by the non-invasive tail-cuff system. Echocardiography was performed using a Vevo 2100 High-Resolution Imaging System to measure the left ventricular anterior wall thickness, the left ventricular posterior wall thickness, the left ventricular systolic and end-diastolic volume, ejection fraction (EF) and fractional shortening (FS). Heart sections were stained with H&E to detect the infiltration of inflammatory cells in the left ventricular tissues, left ventricular sections were stained with Masson trichrome, the mRNA levels of collagen I and collagen III in the left ventricular tissues were measured by using qPCR analysis to detect the cardiac fibrosis. Heart sections were stained with wheat germ agglutinin (WGA) to evaluate cross-section area of cardiomyocytes, the mRNA levels of brain natriuretic protein (BNP) and P-myosin heavy chain (β-MHC) in the left ventricular tissues were measured by using qPCR analysis to evaluate cardiac hypertrophy. The protein levels of calcium and potassium ion channels were examined by Western blot analysis, the mRNA levels of calcium and potassium ion channels in left ventricular tissues were measured by using qPCR analysis to explore changes in expression level of ion channels in LVH animal models. WT C57BL/6 mice were randomly divided into groups according to experimental design, mice were injected with saline or Ang II (2000ng/kg/min) using osmotic mini-pumps for 3 weeks, separately added with PYR-41 or PR-957 according to experimental design. Echocardiography, histopathological staining of myocardial samples, western blot analysis were performed to observe the effects of the ubiquiti-protease inhibitor (PYR-41 and PR-957) on LVH and ion channels.Results:1. Compared with sham-operated mice, the ratio of heart weight and body weight (HW/BW), the left ventricular anterior wall thickness, the left ventricular posterior wall thickness, cross-sectional area of cardiomyocyte were markedly increased in both TAC 2W and 4W mice with significant differences. Compared with sham-operated mice, ejection fraction (EF) and fractional shortening (FS) were significantly decreased in TAC 4W mice. Western blot analysis showed that compared with sham-operated mice, the protein levels of SERCA2, CASQ2, KIR3.4, KIR2.1 were decreased in both TAC 2W and 4W mice, and the protein levels of CACNA1C were increased in both TAC 2W and 4W mice. SERCA2 and KIR3.4 expression was decreased in TAC 4W mice with significant differences, CACNA1C expression was increased in TAC 2W and 4W mice with significant difference.2. Compared with saline-treated mice, the blood pressure, the ratio of heart weight and body weight (HW/BW), the left ventricular anterior wall thickness, the left ventricular posterior wall thickness, cross-section area of cardiomyocytes were markedly increased in Ang II-infused mice with significant differences. Compared with saline-treated mice, ejection fraction (EF) and fractional shortening (FS) were slightly increased in Ang II-infused mice without significant differences. The qRT-PCR data showed that compared with saline-treated mice, there is a significant increase in mRNA expression of brain natriuretic protein (BNP) and β-myosin heavy chain (ββ-MHC) in the left ventricular tissues in Ang Ⅱ-infused mice. Western blot analysis and qRT-PCR data showed that compared with sham-operated mice, the protein and mRNA levels of SERCA2, CASQ2, RyR2, CACNA1C, KCNQ1 and KIR3.4 were decreased in Ang Ⅱ-infused mice, and the protein and mRNA levels of KIR2.1 were increased in Ang Ⅱ-infused infusion mice. SERCA2, CASQ2 and KIR3.4 expression was decreased in Ang Ⅱ-infused mice with significant differences.3. The ratio of heart weight and body weight (HW/BW), the left ventricular anterior wall thickness, the left ventricular posterior wall thickness were significantly smaller (P＜0.05) in Ang Ⅱ+PYR-41 group compared with those in Ang Ⅱ+DMSO group. Western blot analysis showed that compared with Ang Ⅱ+DMSO group mice, the protein levels of SERCA2 and KIR3.4 were increased in Ang Ⅱ+PYR-41 group, but no significant differences were observed between two groups.4. The left ventricular anterior wall thickness were significantly smaller in Ang Ⅱ+ PR-957 group compared with that in Ang Ⅱ+DMSO group (P＜0.05). The ratio of heart weight and body weight (HW/BW), the left ventricular posterior wall thickness were smaller in Ang Ⅱ+PR-957 group compared with those in Ang Ⅱ+DMSO group without significant differences. Western blot analysis showed that compared with Ang Ⅱ+DMSO group mice, the protein levels of KIR3.4 were increased in Ang Ⅱ+PR-957 group without significant difference, and the protein expression of SERCA2 was unaffected in AngⅡ+PR-957 group compared with that in Ang Ⅱ+DMSO group.Conclusion:LVH model in Wild-type (WT) C57BL/6 mice can successfully established by transverse aortic constriction (TAC2W,4W) or Ang Ⅱ infusion (2000ng/kg/min,3W) with change in expression level of calcium and potassium ion channels. SERCA2 and KIR3.4 protein expression were markedly decreased in LVH model established by TAC or Ang Ⅱ infusion with significant differences. PYR-41 produced a significantly regression of LVH induced by Ang Ⅱ infusion. PYR-41 produced a partial regression of SERCA2 and KIR3.4 protein expression change in LVH induced by Ang II infusion, but no significant differences were observed. PR-957 was not as significant as PYR-41 to prevent LVH and protein expression change of ion channels induced by Ang II infusion. The effect of ubiquitin proteasome inhibitor on cardiac diseases and ion channels needs further experimental research and observation.Part two Lack of association between rs3807989 in CAV1 and atrial fibrillation in Chinese Han populationBackground:Atrial fibrillation (AF) is one of the most common cardiac arrhythmias, it affects 1-2% of the general population. The prevalence of AF increases substantially with aging, from 1% in young adults to 10% in people older than 80 years. AF has been shown to be an independent risk factor for ischemic stroke (IS), one of main causes of mortality and morbidity. AF is mostly associated with cardiac risk factors, including hypertension, congenital heart disease, coronary artery disease and valvular heart disease. Nearly 30% of AF cases, which have no predisposing factors, are therefore classified as lone AF. Accumulating studies have indicated that genetic factors play an important role in the etiology of AF, in particular lone AF. In the last decade, a number of AF associated genes and loci, such as KCNQ1, KCNH2, SCN5A, KCNE2, KCNJ2, NUP155, NPPA, SCN3B,4q25, ZFHX3 and KCNN3, have been identified with genetic linkage and genome-wide association studies (GWAS). Caveolin-1 gene (CAV1) encodes a caveolae protein which is expressed in almost all cell types. CAV1 is highly enriched in epithelial cells, adipocytes and endothelial cells, it regulates various cell functions such as vesicle transport, signal transduction and so on. Scholars have found that CAV1 is also expressed in atrial myocytes. In two independent GWAS studies, CAV1 has been reported to be associated with AF. More recently, a SNP in CAV1 gene, rs3807989, has been associated with early-onset lone AF before the age of 40 years. However, these study results need to be independently confirmed in other ethnic populations.Objective:To investigated the association of rs3807989, a tag SNP in CAV1, with AF in Chinese Han population.Methods:The present study is a case-control association. The study subjects were enrolled from multiple hospitals in Dalian, Wuhan, Suizhou, Xiangfan and Shiyan cities and all were Chinese Han people. The study cohort consisted of 839 AF patients and 1215 healthy controls. AF was diagnosed by a panel of cardiologists, according to standard diagnostic criteria. We recruited patients of younger than 80 years old with AF. Lone AF was defined as AF patients without history of hypertension, coronary artery disease (CAD), congenital heart disease, congestive heart failure, ischemic stroke or diabetes. Peripheral venous blood samples were drawn from all participants. Genomic DNA was extracted from leucocytes following standard protocols of the Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA). The SNP rs3807989 was genotyped on a Rotor-GeneTM6000 High Resolution Melt (HRM) system (QIAGEN, Hilden, Germany), with a reaction of 25μL polymerase chain reaction (PCR), containing 1μL of LC Green dye,5pmol of each primer,25ng of genomic DNA,2.5μL of 10×PCR buffer with 1.5mmol/L MgCl2,5mmol deoxynucleotide triphosphates, and 1U of Taq polymerase. Two positive controls for each genotype (A/A, A/G, and G/G) were included in each run. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using the x2 test (PLINK). Multivariate regression analysis was performed by incorporating age, sex, hypertension and CAD (for AF dataset) as covariates using multivariate logistic regression (PLINK) to calculate genotypic association of the SNP rs3807989 with AF in three inheritance models (dominant, recessive and additive models).Results:No significant association was detected between rs3807989 and AF in Chinese Han population after logistic regression with multiple covariates (allelic P-adj=0.828 with OR=1.02; genotypic P-adj=0.815,0.405,0.760 with a dominant model, recessive model, additive model). When the AF cases were further divided into lone AF (31.5%) and other types of AF (68.5%), no significant association was found between rs3807989 and lone AF (P-adj=0.929 with OR=0.990) and other types of AF (P-adj=0.597 with OR=1.060).Conclusion:The SNP rs3807989 in CAV1 gene was not associated with lone AF or other AF group in our studies, which suggested that the SNP rs3807989 in CAV1 may not be a risk factor for AF in Chinese Han population. |
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