Study of reciprocal effects of cardiac myosin and tropomyosin isoforms on actin-myosin interaction with in vitro motility assay

Interaction of myosin with actin in striated muscle is controlled by Ca²⁺ via thin filament associated proteins: troponin and tropomyosin. In cardiac muscle there is a whole pattern of myosin and tropomyosin isoforms. The aim of the current work is to study regulatory effect of tropomyosin on sliding velocity of actin filaments in the in vitro motility assay over cardiac isomyosins. It was found that tropomyosins of different content of α- and β-chains being added to actin filament effects the sliding velocity of filaments in different ways. On the other hand the velocity of filaments with the same tropomyosins depends on both heavy and light chains isoforms of cardiac myosin.     

MiR‐451 is decreased in hypertrophic cardiomyopathy and regulates autophagy by targeting TSC1

The molecular mechanisms that drive the development of cardiac hypertrophy in hypertrophic cardiomyopathy (HCM) remain elusive. Accumulated evidence suggests that microRNAs are essential regulators of cardiac remodelling. We have been suggested that microRNAs could play a role in the process of HCM. To uncover which microRNAs were changed in their expression, microRNA microarrays were performed on heart tissue from HCM patients (n = 7) and from healthy donors (n = 5). Among the 13 microRNAs that were differentially expressed in HCM, miR‐451 was the most down‐regulated. Ectopic overexpression of miR‐451 in neonatal rat cardiomyocytes (NRCM) decreased the cell size, whereas knockdown of endogenous miR‐451 increased the cell surface area. Luciferase reporter assay analyses demonstrated that tuberous sclerosis complex 1 (TSC1) was a direct target of miR‐451. Overexpression of miR‐451 in both HeLa cells and NRCM suppressed the expression of TSC1. Furthermore, TSC1 was significantly up‐regulated in HCM myocardia, which correlated with the decreased levels of miR‐451. As TSC1 is a known positive regulator of autophagy, we examined the role of miR‐451 in the regulation of autophagy. Overexpression of miR‐451 in vitro inhibited the formation of the autophagosome. Conversely, miR‐451 knockdown accelerated autophagosome formation. Consistently, an increased number of autophagosomes was observed in HCM myocardia, accompanied by up‐regulated autophagy markers, and the lipidated form of LC3 and Beclin‐1. Taken together, our findings indicate that miR‐451 regulates cardiac hypertrophy and cardiac autophagy by targeting TSC1. The down‐regulation of miR‐451 may contribute to the development of HCM and may be a potential therapeutic target for this disease.     

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.     

Modulatory effects of connexin-43 expression on gap junction intercellular communications with mast cells and fibroblasts

The influence of mast cells upon aberrant wound repair and excessive fibrosis has supportive evidence, but the mechanism for these mast cell activities is unclear. It is proposed that heterocellular gap junction intercellular communication (GJIC) between fibroblasts and mast cells directs some fibroblast activities. An in vitro model was used employing a rodent derived peritoneal mast cell line (RMC-1) and human dermal derived fibroblasts. The influence of the expression of the gap junction channel structural protein, connexin 43 (Cx-43) on heterocellular GJIC, the expression of microtubule β-tubulin and microfilament α smooth muscle actin (SMA) were investigated. The knockdown of Cx-43 by siRNA in RMC-1 cells completely blocked GJIC between RMC-1 cells. SiRNA knockdown of Cx-43 within fibroblasts only dampened GJIC between fibroblasts. It appears Cx-43 is the only expressed connexin (Cx) in RMC-1 cells. Fibroblasts express other Cxs that participate in GJIC between fibroblasts in the absence of Cx-43 expression. Heterocellular GJIC between RMC-1 cells and fibroblasts transformed fibroblasts into myofibroblasts, expressing α SMA within cytoplasmic stress fibers. The knockdown of Cx-43 in RMC-1 cells increased β-tubulin expression, but its knockdown in fibroblasts reduced β-tubulin expression. Knocking down the expression of Cx-43 in fibroblasts limited αSMA expression. Cx-43 participation is critical for heterocellular GJIC between mast cells and fibroblasts, which may herald a novel direction for controlling fibrosis.     

A variant of unknown significance in the GLA gene causing diagnostic uncertainty in a young female with isolated hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is genetically heterogeneous, and largely caused by mutations in genes encoding sarcomere proteins. However, GLA mutations causing Fabry disease, an X-linked lysosomal storage disorder, may also present with isolated HCM. As HCM genetic testing panels are increasingly being used clinically, variants of unknown significance (VUS) are encountered, leading to challenges in interpretation. We present an illustrative case: a 10-year-old girl with isolated HCM who, on testing with a HCM multi-gene panel, was found to carry a maternally inherited p.W24R variant in GLA. Attempts to evaluate the significance of this variant, by direct biochemical testing of patient specimens, gave inconclusive results. Subsequent in vitro protein expression studies suggested that the variant is unlikely to be pathogenic. This case highlights diagnostic dilemmas that can be provoked by VUS in general, and specifically raises a question whether GLA sequencing should be included in first-line diagnostic testing for female children with isolated hypertrophic cardiomyopathy.     

心尖肥厚型心肌病的临床特征及治疗

目的:总结心尖肥厚型心肌病的临床特征及治疗方法.方法:回顾性分析我院2008年6月至2012年2月期间经心脏超声或左室造影证实的33例心尖肥厚型心肌病患者的临床特征及治疗情况.结果:患者的临床症状表现为以胸闷、气短为主者20例;心前区不适4例;心前区疼痛5例;乏力2例;心悸l例;发现心电图异常前来就诊l例.32例患者行左室造影诊断为心尖肥厚型心肌病.l例因肾功能不全,未行左室造影,但经心脏超声诊断为心尖肥厚型心肌病.27例患者服用β-受体阻滞剂,2例患者服用钙离子拮抗剂,4例患者因心率慢,未服用药物,临床随访1～36(平均16.7± 4.1)个月,临床症状均明显缓解.结论:β-受体阻滞剂和钙离子拮抗剂均适用于治疗心尖肥厚型心肌病.