Dystrophin deficiency markedly increases enterovirus-induced cardiomyopathy: a genetic predisposition to viral heart disease

Both enteroviral infection of the heart and mutations in the dystrophin gene can cause cardiomyopathy. Little is known, however, about the interaction between genetic and acquired forms of cardiomyopathy. We previously demonstrated that the enteroviral protease 2A cleaves dystrophin; therefore, we hypothesized that dystrophin deficiency would predispose to enterovirus-induced cardiomyopathy. We observed more severe cardiomyopathy, worsening over time, and greater viral replication in dystrophin-deficient mice infected with enterovirus than in infected wild-type mice. This difference appears to be a result of more efficient release of the virus from dystrophin-deficient myocytes. In addition, we found that expression of wild-type dystrophin in cultured cells decreased the cytopathic effect of enteroviral infection and the release of virus from the cell. We also found that expression of a cleavage-resistant mutant dystrophin further inhibited the virally mediated cytopathic effect and viral release. These results indicate that viral infection can influence the severity and penetrance of the cardiomyopathy that occurs in the hearts of dystrophin-deficient individuals.     

Enteroviral protease 2A directly cleaves dystrophin and is inhibited by a dystrophin-based substrate analogue

Enteroviruses such as Coxsackievirus B3 can cause dilated cardiomyopathy through unknown pathological mechanism(s). Dystrophin is a large extrasarcomeric cytoskeletal protein whose genetic deficiency causes hereditary dilated cardiomyopathy. In addition, we have recently shown that dystrophin is proteolytically cleaved by the Coxsackievirus protease 2A leading to functional impairment and morphological disruption. However, the mechanism of dystrophin cleavage and the exact cleavage site remained to be identified. Antibody epitope mapping of endogenous dystrophin indicated protease 2A-mediated cleavage at the site in the hinge 3 region predicted by a neural network algorithm (human, amino acid 2434; mouse, amino acid 2427). Using site-directed mutagenesis, peptide sequencing, and fluorescence resonance energy transfer assays with recombinant dystrophin, we demonstrate that this putative site in mouse and human dystrophin is a direct substrate for the Coxsackieviral protease 2A both in vitro and in vivo. The substrate analogue protease inhibitor z-LSTT-fmk was designed based on the dystrophin sequence that interacts with the protease 2A and was found to have an IC(50) of 550 nM in vitro. Dystrophin is the first cellular substrate of the enteroviral protease 2A that was identified using by a bioinformatic approach and for which the cleavage site was molecularly mapped within living cells.     

Dysferlin deficiency confers increased susceptibility to coxsackievirus‐induced cardiomyopathy

Coxsackievirus infection can lead to viral myocarditis and its sequela, dilated cardiomyopathy, which represent major causes of cardiovascular mortality worldwide in children. Yet, the host genetic susceptible factors and the underlying mechanisms by which viral infection damages cardiac function remain to be fully resolved. Dysferlin is a transmembrane protein highly expressed in skeletal and cardiac muscles. In humans, mutations in the dysferlin gene can cause limb‐girdle muscular dystrophy type 2B and Miyoshi myopathy. Dysferlin deficiency has also been linked to cardiomyopathy. Defective muscle membrane repair has been suggested to be an important mechanism responsible for muscle degeneration in dysferlin‐deficient patients and animals. Using both naturally occurring and genetically engineered dysferlin‐deficient mice, we demonstrated that loss of dysferlin confers increased susceptibility to coxsackievirus infection and myocardial damage. More interestingly, we found that dysferlin is cleaved following coxsackieviral infection through the proteolytic activity of virally encoded proteinases, suggesting an important mechanism underlying virus‐induced cardiac dysfunction. Our results in this study not only identify dysferlin deficiency as a novel host risk factor for viral myocarditis but also reveal a key mechanism by which coxsackievirus infection impairs cardiac function, leading to the development of dilated cardiomyopathy.     

The tyrosine kinase p56lck is essential in coxsackievirus B3-mediated heart disease

Infections are thought to be important in the pathogenesis of many heart diseases. Coxsackievirus B3 (CVB3) has been linked to chronic dilated cardiomyopathy, a common cause of progressive heart disease, heart failure and sudden death. We show here that the sarcoma (Src) family kinase Lck (p56lck) is required for efficient CVB3 replication in T-cell lines and for viral replication and persistence in vivo. Whereas infection of wild-type mice with human pathogenic CVB3 caused acute and very severe myocarditis, meningitis, hepatitis, pancreatitis and dilated cardiomyopathy, mice lacking the p56lck gene were completely protected from CVB3-induced acute pathogenicity and chronic heart disease. These data identify a previously unknown function of Src family kinases and indicate that p56lck is the essential host factor that controls the replication and pathogenicity of CVB3.     

Ultrastructural and Functional Alterations of EC Coupling Elements in mdx Cardiomyocytes: An Analysis from Membrane Surface to Depth

A dilated cardiomyopathy (DCM) is associated with Duchenne muscular dystrophy (DMD). The loss of dystrophin leads to membrane instability and calcium dysregulation in skeletal muscle but effects of such a loss are not elucidated at cardiomyocytes level. We sought to examine whether membrane and transverse tubules damages occur in ventricular myocytes from mdx mouse model of DMD and how they impact the function of single excitation–contraction coupling elements. Scanning ion conductance microscopy (SICM) was used to characterize the integrity loss of living mdx cardiomyocytes surface. 2D Fourier transform analysis of labeled internal networks (transverse tubules, alpha-actinin, dihydropyridine receptors, ryanodine receptors) was performed to evaluate internal alterations. During calcium measurements, “smart microperfusions” of depolarizing solutions were applied through SICM nanopipette, stimulating single tubules elements. These approaches revealed structural membrane surface (39 % decrease for Z-groove ratio) and transverse tubules disorganization (21 % transverse tubules ratio decrease) in mdx as compared to control. These disruptions were associated with functional alterations (sixfold increase of calcium signal duration and twofold increase of sparks frequency). In DCM associated with DMD, myocytes display evident membrane alterations at the surface level but also in the cell depth with a disruption of transverse tubules network as observed in other cases of heart failure. These ultrastructural changes are associated with changes in the function of some coupling elements. Thus, these profound disruptions may play a role in calcium dysregulation through excitation–contraction coupling elements perturbation and suggest a transverse tubules stabilizing role for dystrophin.     

H9c2细胞对B组柯萨奇病毒3型重组株的易感性

H9c2细胞是来源于大鼠胚胎心脏组织的成肌细胞系,B组柯萨奇病毒(group B Coxsackievirus,CVB)是心肌炎和扩张型心肌病的主要病原.本研究观察了CVB3在H9c2细胞中的感染性,探讨H9c2细胞是否可用于CVB致心肌疾病的实验研究.用整合了增强型绿色荧光蛋白(EGFP)或海肾荧光素酶(RLuc)的...     

A novel scheme of dystrophin disruption for the progression of advanced heart failure

The precise mechanism of the progression of advanced heart failure is unknown. We assessed a new scheme in two heart failure models: (I) congenital dilated cardiomyopathy (DCM) in TO-2 strain hamsters lacking delta-sarcoglycan (SG) gene and (II) administration of a high-dose of isoproterenol, as an acute heart failure in normal rats. In TO-2 hamsters, we followed the time course of the histological, physiological and metabolic the progressions of heart failure to the end stage. Dystrophin localization detected by immunostaining age-dependently to the myoplasm and the in situ sarcolemma fragility evaluated by Evans blue entry was increased in the same cardiomyocytes. Western blotting revealed a limited cleavage of the dystrophin protein at the rod domain, strongly suggesting a contribution of endogenous protease(s). We found a remarkable up-regulation of the amount of calpain-1 and -2, and no change of their counterpart, calpastatin. After supplementing TO-2 hearts with the normal delta-SG gene in vivo, these pathological alterations and the animals' survival improved. Furthermore, dystrophin but not delta-SG was disrupted by a high dose of isoproterenol, translocated from the sarcolemma to the myoplasm and fragmented. These results of heart failure, irrespective of the hereditary or acquired origin, indicate a vicious cycle formed by the increased sarcolemma permeability, preferential activation of calpain over calpastatin, and translocation and cleavage of dystrophin would commonly lead to advanced heart failure.     

免疫系统在病毒性心肌炎发病中的作用

心肌炎通常由病毒感染引起,有证据表明心肌炎最终发展成扩张性心肌病,是发达国家主要致死的原因,越来越多的人认为细胞因子在心肌炎和心肌病发病中起重要作用,心力衰竭病人血循环中细胞因子水平较正常人高。已证明多种细胞因子能在体内外抑制心肌收缩,细胞因子由活化的免疫细胞产生,它可诱生NO合酶,继而产生NO,已证明NO既有利又有害,关键在于产生NO量的多少,NO能抑制病毒复制,而保护心脏抗柯萨奇B病毒感染,无论是病毒感染对心脏的直接作用,还是免疫应答的利弊平衡,对此两者分子机制的了解都将是掌握人类心肌炎发病的关键。     

Structure and dynamics of coxsackievirus B4 2A proteinase, an enyzme involved in the etiology of heart disease

The 2A proteinases (2A(pro)) from the picornavirus family are multifunctional cysteine proteinases that perform essential roles during viral replication, involving viral polyprotein self-processing and shutting down host cell protein synthesis through cleavage of the eukaryotic initiation factor 4G (eIF4G) proteins. Coxsackievirus B4 (CVB4) 2A(pro) also cleaves heart muscle dystrophin, leading to cytoskeletal dysfunction and the symptoms of human acquired dilated cardiomyopathy. We have determined the solution structure of CVB4 2A(pro) (extending in an N-terminal direction to include the C-terminal eight residues of CVB4 VP1, which completes the VP1-2A(pro) substrate region). In terms of overall fold, it is similar to the crystal structure of the mature human rhinovirus serotype 2 (HRV2) 2A(pro), but the relatively low level (40%) of sequence identity leads to a substantially different surface. We show that differences in the cI-to-eI2 loop between HRV2 and CVB4 2A(pro) translate to differences in the mechanism of eIF4GI recognition. Additionally, the nuclear magnetic resonance relaxation properties of CVB4 2A(pro), particularly of residues G1 to S7, F64 to S67, and P107 to G111, reveal that the substrate region is exchanging in and out of a conformation in which it occupies the active site with association and dissociation rates in the range of 100 to 1,000 s(-1). This exchange influences the conformation of the active site and points to a mechanism for how self-processing can occur efficiently while product inhibition is avoided.     

A Role for Toll-like Receptor 3 Variants in Host Susceptibility to Enteroviral Myocarditis and Dilated Cardiomyopathy

The innate antiviral response is mediated, at least in part, by Toll-like receptors (TLRs). TLR3 signaling is activated in response to viral infection, and the absence of TLR3 in mice significantly increases mortality after infection with enteroviruses that cause myocarditis and/or dilated cardiomyopathy. We screened TLR3 in patients diagnosed with enteroviral myocarditis/cardiomyopathy and identified a rare variant in one patient as well as a significantly increased occurrence of a common polymorphism compared with controls. Expression of either variant resulted in significantly reduced TLR3-mediated signaling after stimulation with synthetic double-stranded RNA. Furthermore, Coxsackievirus B3 infection of cell lines expressing mutated TLR3 abrogated activation of the type I interferon pathway, leading to increased viral replication. TLR3-mediated type I interferon signaling required cellular autophagy and was suppressed by 3-methyladenine and bafilomycin A1, by inhibitors of lysosomal proteolysis, and by reduced expression of Beclin 1, Atg5, or microtubule-associated protein 1 light chain 3β (MAP1LC3β). However, TLR3-mediated signaling was restored upon exogenous expression of Beclin 1 or a variant MAP1LC3β fusion protein refractory to RNA interference. These data suggest that individuals harboring these variants may have a blunted innate immune response to enteroviral infection, leading to reduced viral clearance and an increased risk of cardiac pathology.     

Molecular genetics of left ventricular dysfunction

The left ventricle (LV) plays a central role in the maintenance of health of children and adults due to its role as the major pump of the heart. In cases of LV dysfunction, a significant percentage of affected individuals develop signs and symptoms of congestive heart failure (CHF), leading to the need for therapeutic intervention. Therapy for these patients include anticongestive medications and, in some, placement of devices such as aortic balloon pump or left ventricular assist device (LVAD), or cardiac transplantation. In the majority of patients the etiology is unknown, leading to the term idiopathic dilated cardiomyopathy (IDC). During the past decade, the basis of LV dysfunction has begun to unravel. In approximately 30-40% of cases, the disorder is inherited; autosomal dominant inheritance is most common (although X-linked, autosomal recessive and mitochondrial inheritance occurs). In the remaining patients, the disorder is presumed to be acquired, with inflammatory heart disease playing an important role. In the case of familial dilated cardiomyopathy (FDCM), the genetic basis is beginning to unfold. To date, two genes for X-linked FDCM (dystrophin, G4.5) have been identified and four genes for the autosomal dominant form (actin, desmin, lamin A/C, delta-sarcoglycan) have been described. In one form of inflammatory heart disease, coxsackievirus myocarditis, inflammatory mediators and dystrophin cleavage play a role in the development of LV dysfunction. In this review, we will describe the molecular genetics of LV dysfunction and provide evidence for a "final common pathway" responsible for the phenotype.     

Ex vivo stretch reveals altered mechanical properties of isolated dystrophin-deficient hearts

Duchenne muscular dystrophy (DMD) is a progressive and fatal disease of muscle wasting caused by loss of the cytoskeletal protein dystrophin. In the heart, DMD results in progressive cardiomyopathy and dilation of the left ventricle through mechanisms that are not fully understood. Previous reports have shown that loss of dystrophin causes sarcolemmal instability and reduced mechanical compliance of isolated cardiac myocytes. To expand upon these findings, here we have subjected the left ventricles of dystrophin-deficient mdx hearts to mechanical stretch. Unexpectedly, isolated mdx hearts showed increased left ventricular (LV) compliance compared to controls during stretch as LV volume was increased above normal end diastolic volume. During LV chamber distention, sarcomere lengths increased similarly in mdx and WT hearts despite greater excursions in volume of mdx hearts. This suggests that the mechanical properties of the intact heart cannot be modeled as a simple extrapolation of findings in single cardiac myocytes. To explain these findings, a model is proposed in which disruption of the dystrophin-glycoprotein complex perturbs cell-extracellular matrix contacts and promotes the apparent slippage of myocytes past each other during LV distension. In comparison, similar increases in LV compliance were obtained in isolated hearts from β-sarcoglycan-null and laminin-α(2) mutant mice, but not in dysferlin-null mice, suggesting that increased whole-organ compliance in mdx mice is a specific effect of disrupted cell-extracellular matrix contacts and not a general consequence of cardiomyopathy via membrane defect processes. Collectively, these findings suggest a novel and cell-death independent mechanism for the progressive pathological LV dilation that occurs in DMD.     

Early dystrophin disruption in the pathogenesis of experimental chronic Chagas cardiomyopathy

Chronic Chagas cardiomyopathy evolves over a long period of time after initial infection by Trypanosoma cruzi. Similarly, a cardiomyopathy appears later in life in muscular dystrophies. This study tested the hypothesis that dystrophin levels are decreased in the early stage of T. cruzi-infected mice that precedes the later development of a cardiomyopathy. CD1 mice were infected with T. cruzi (Brazil strain), killed at 30 and 100 days post infection (dpi), and the intensity of inflammation, percentage of interstitial fibrosis, and dystrophin levels evaluated. Echocardiography and magnetic resonance imaging data were evaluated from 15 to 100 dpi. At 30 dpi an intense acute myocarditis with ruptured or intact intracellular parasite nests was observed. At 100 dpi a mild chronic fibrosing myocarditis was detected without parasites in the myocardium. Dystrophin was focally reduced or completely lost in cardiomyocytes at 30 dpi, with the reduction maintained up to 100 dpi. Concurrently, ejection fraction was reduced and the right ventricle was dilated. These findings support the hypothesis that the initial parasitic infection-induced myocardial dystrophin reduction/loss, maintained over time, might be essential to the late development of a cardiomyopathy in mice.     

Subcellular Localization of Dystrophin Isoforms in Cardiomyocytes and Phenotypic Analysis of Dystrophin-deficient Mice Reveal Cardiac Myopathy is Predominantly Caused by a Deficiency in Full-length Dystrophin

Duchenne muscular dystrophy (DMD) is an X-linked recessive progressive muscle degenerative disorder that causes dilated cardiomyopathy in the second decade of life in affected males. Dystrophin, the gene responsible for DMD, encodes full-length dystrophin and various short dystrophin isoforms. In the mouse heart, full-length dystrophin Dp427 and a short dystrophin isoform, Dp71, are expressed. In this study, we intended to clarify the functions of these dystrophin isoforms in DMD-related cardiomyopathy. We used two strains of mice: mdx mice, in which Dp427 was absent but Dp71 was present, and DMD-null mice, in which both were absent. By immunohistochemical staining and density-gradient centrifugation, we found that Dp427 was located in the cardiac sarcolemma and also at the T-tubules, whereas Dp71 was specifically located at the T-tubules. In order to determine whether T tubule-associated Dp71 was involved in DMD-related cardiac disruption, we compared the cardiac phenotypes between DMD-null mice and mdx mice. Both DMD-null mice and mdx mice exhibited severe necrosis, which was followed by fibrosis in cardiac muscle. However, we could not detect a significant difference in myocardial fibrosis between mdx mice and DMD-null mice. Based on the present results, we have shown that cardiac myopathy is caused predominantly by a deficiency of full-length dystrophin Dp427.     

Hypocalcemic cardiomyopathy due to untreated hypoparathyroidism

Hypocalcemic cardiomyopathy due to hypoparathyroidism is a very rare condition which is usually refractory to conventional treatment for cardiac failure but which responds favorably to restoration of normocalcemia. A 55-year-old man and a 46-year-old woman with a history of postoperative hypoparathyroidism presented with symptoms of cardiac failure and hypocalcemia. A presumptive diagnosis of dilated cardiomyopathy was considered by echocardiography and endomyocardial biopsies were consistent with cardiomyopathy. The coronary angiograms were normal and there was no apparent cause for dilated cardiomyopathy in these patients. The history of the patients and partial recovery of cardiac function after restoration of normocalcemia suggest that hypocalcemia was the cause of dilated cardiomyopathy.     

Decreased Levels of BAG3 in a Family With a Rare Variant and in Idiopathic Dilated Cardiomyopathy

The most common cause of dilated cardiomyopathy and heart failure (HF) is ischemic heart disease; however, in a third of all patients the cause remains undefined and patients are diagnosed as having idiopathic dilated cardiomyopathy (IDC). Recent studies suggest that many patients with IDC have a family history of HF and rare genetic variants in over 35 genes have been shown to be causative of disease. We employed whole‐exome sequencing to identify the causative variant in a large family with autosomal dominant transmission of dilated cardiomyopathy. Sequencing and subsequent informatics revealed a novel 10‐nucleotide deletion in the BCL2‐associated athanogene 3 (BAG3) gene (Ch10:del 121436332_12143641: del. 1266_1275 [NM 004281]) that segregated with all affected individuals. The deletion predicted a shift in the reading frame with the resultant deletion of 135 amino acids from the C‐terminal end of the protein. Consistent with genetic variants in genes encoding other sarcomeric proteins there was a considerable amount of genetic heterogeneity in the affected family members. Interestingly, we also found that the levels of BAG3 protein were significantly reduced in the hearts from unrelated patients with end‐stage HF undergoing cardiac transplantation when compared with non‐failing controls. Diminished levels of BAG3 protein may be associated with both familial and non‐familial forms of dilated cardiomyopathy. J. Cell. Physiol. 229: 1697–1702, 2014. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.