Unexpectedly low mutation rates in beta-myosin heavy chain and cardiac myosin binding protein genes in Italian patients with hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disease. Fourteen sarcomeric and sarcomere‐related genes have been implicated in HCM etiology, those encoding β‐myosin heavy chain (MYH7) and cardiac myosin binding protein C (MYBPC3) reported as the most frequently mutated: in fact, these account for around 50% of all cases related to sarcomeric gene mutations, which are collectively responsible for approximately 70% of all HCM cases. Here, we used denaturing high‐performance liquid chromatography followed by bidirectional sequencing to screen the coding regions of MYH7 and MYBPC3 in a cohort (n = 125) of Italian patients presenting with HCM. We found 6 MHY7 mutations in 9/125 patients and 18 MYBPC3 mutations in 19/125 patients. Of the three novel MYH7 mutations found, two were missense, and one was a silent mutation; of the eight novel MYBPC3 mutations, one was a substitution, three were stop codons, and four were missense mutations. Thus, our cohort of Italian HCM patients did not harbor the high frequency of mutations usually found in MYH7 and MYBPC3. This finding, coupled to the clinical diversity of our cohort, emphasizes the complexity of HCM and the need for more inclusive investigative approaches in order to fully understand the pathogenesis of this disease. J. Cell. Physiol. 226: 2894–2900, 2011. © 2011 Wiley‐Liss, Inc.     

Impact of beta-myosin heavy chain isoform expression on cross-bridge cycling kinetics

Myosin heavy chain (MHC) isoforms alpha and beta have intrinsically different ATP hydrolysis activities (ATPase) and therefore cross-bridge cycling rates in solution. There is considerable evidence of altered MHC expression in rodent cardiac disease models; however, the effect of incremental beta-MHC expression over a wide range on the rate of high-strain, isometric cross-bridge cycling is yet to be ascertained. We treated male rats with 6-propyl-2-thiouracil (PTU; 0.8 g/l in drinking water) for short intervals (6, 11, 16, and 21 days) to generate cardiac MHC patterns in transition from predominantly alpha-MHC to predominantly beta-MHC. Steady-state calcium-dependent tension development and tension-dependent ATP consumption (tension cost; proportional to cross-bridge cycling) were measured in chemically permeabilized (skinned) right ventricular muscles at 20 degrees C. To assess dynamic cross-bridge cycling kinetics, the rate of force redevelopment (ktr) was determined after rapid release-restretch of fully activated muscles. MHC isoform content in each experimental muscle was measured by SDS-PAGE and densitometry. alpha-MHC content decreased significantly and progressively with length of PTU treatment [68 +/- 5%, 58 +/- 4%, 37 +/- 4%, and 27 +/- 6% for 6, 11, 16, and 21 days, respectively; P < 0.001 (ANOVA)]. Tension cost decreased, linearly, with decreased alpha-MHC content [6.7 +/- 0.4, 5.6 +/- 0.5, 4.0 +/- 0.4, and 3.9 +/- 0.3 ATPase/tension for 6, 11, 16, and 21 days, respectively; P < 0.001 (ANOVA)]. Likewise, ktr was significantly and progressively depressed with length of PTU treatment [11.1 +/- 0.6, 9.1 +/- 0.5, 8.2 +/- 0.7, and 6.2 +/- 0.3 s(-1) for 6, 11, 16, and 21 days, respectively; P < 0.05 (ANOVA)] Thus cross-bridge cycling, under high strain, for alpha-MHC is three times higher than for beta-MHC. Furthermore, under isometric conditions, alpha-MHC and beta-MHC cross bridges hydrolyze ATP independently of one another.     

HLA haplotypes and susceptibility to Graves' disease.

Graves' disease is a polygenic disease in which the HLA cluster could play a role. The purpose of our study is to identify HLA haplotypes in a family with closely related susceptibility to Graves' disease and foresee the risk of disease in the youngest daughter. The family studied had included the father (47 years), mother (46 years) and 3 daughters (18, 17 and 13 years). The mother and 2 eldest daughters were affected by Graves' disease. HLA-A, -B, -C, -DR and -DQ were performed with standard microlymphotoxicity techniques. A mother's role in passing susceptibility to Graves' disease to daughters is undisputed; it seems to be due to the B35 HLA allele. Also, the third daughter (at 15 years) has an HLA B35 allele, and actually has an incipient humoral hyperthyroidism.     

Functional effects of the hypertrophic cardiomyopathy R403Q mutation are different in an alpha- or beta-myosin heavy chain backbone

The R403Q mutation in the beta-myosin heavy chain (MHC) was the first mutation to be linked to familial hypertrophic cardiomyopathy (FHC), a primary disease of heart muscle. The initial studies with R403Q myosin, isolated from biopsies of patients, showed a large decrease in myosin motor function, leading to the hypothesis that hypertrophy was a compensatory response. The introduction of the mouse model for FHC (the mouse expresses predominantly alpha-MHC as opposed to the beta-isoform in larger mammals) created a new paradigm for FHC based on finding enhanced motor function for R403Q alpha-MHC. To help resolve these conflicting mechanisms, we used a transgenic mouse model in which the endogenous alpha-MHC was largely replaced with transgenically encoded beta-MHC. A His(6) tag was cloned at the N terminus of the alpha-and beta-MHC to facilitate protein isolation by Ni(2+)-chelating chromatography. Characterization of the R403Q alpha-MHC by the in vitro motility assay showed a 30-40% increase in actin filament velocity compared with wild type, consistent with published studies. In contrast, the R403Q mutation in a beta-MHC backbone showed no enhancement in velocity. Cleavage of the His-tagged myosin by chymotrypsin made it possible to isolate homogeneous myosin subfragment 1 (S1), uncontaminated by endogenous myosin. We find that the actin-activated MgATPase activity for R403Q alpha-S1 is approximately 30% higher than for wild type, whereas the enzymatic activity for R403Q beta-S1 is reduced by approximately 10%. Thus, the functional consequences of the mutation are fundamentally changed depending upon the context of the cardiac MHC isoform.     

Low sequence variation in the gene encoding the human beta-myosin heavy chain

Over 40 different mutations in the cardiac myosin heavy chain gene (MYH7) have been associated with familial hypertrophic cardiomyopathy (FHC), but no study has analyzed variation at this locus within the normal human population. Here we determine the extent and distribution of nucleotide variation in the 5808-bp MYH7 coding sequence in 25 normal individuals without FHC. We identified six single-nucleotide polymorphisms, none of which changes the encoded amino acid. At one of these sites, the frequencies of both alleles are equal; at the other five sites, the frequency of the rarer allele varies from 0.02 to 0.08. The nucleotide diversity (pi) calculated from these data is 1.73x10(-4)+/-0.49x10(-4), which is lower than the nucleotide diversity found in most other human autosomal genes. Substitution analysis of homologous genes between human and rodent also indicates that the MYH7 sequence has evolved at a very slow rate. The rate of both synonymous and nonsynonymous substitutions, especially in the portion of the sequence that encodes the alpha-helical myosin rod, is extremely low. The low level of even silent sequence variation in MYH7 in comparisons between human sequences and between human and rodent sequences may be a consequence of strong selective pressure against mutations that cause cardiomyopathy.     

Immunopathology of cardiomyopathy in the experimental Chagas disease

The mechanisms by which Trypanosoma cruzi causes cardiomyopathy and induces neuronal destruction are discussed in this paper. The results suggest that autoimmunity in the chronic phase is the main cause of the progressive cardiac destruction, and that autoreactivity is restricted to the CD4+ T cell compartment. During the acute phase, the neuronal and cardiac fiber destruction occurs when ruptured parasite nests release T. cruzi antigens that bind to the cell surface in the vicinity which become targets for the cellular and humoral immune response against T. cruzi. The various factors involved in the genesis of autoimmunity in chronic T. cruzi infection include molecular mimicry, presentation of self-antigens and imbalance of immune regulation.     

TNF blockade aggravates experimental chronic Chagas disease cardiomyopathy

Chronic Chagas disease cardiomyopathy (CCC), caused by Trypanosoma cruzi, is an inflammatory dilated cardiomyopathy associated with increased circulating levels of TNF-alpha. We investigate whether TNF blockade with Etanercept during the chronic phase of T. cruzi infection could attenuate experimental CCC development. The effect of Etanercept was evaluated after 11 months of T. cruzi infection on survival, parasitism, left ventricular function, intensity of myocarditis, fibrosis, and left ventricular mRNA expression of cytokines and TNF-alpha-induced genes. Left ventricular function was significantly reduced in treated animals as compared to infected untreated animals. Blood and cardiac parasitism as well as survival rate were not altered with Etanercept treatment. Inflammatory infiltrates were located predominantly in the subendocardic region in treated animals, whereas in untreated animals inflammation was scattered throughout the myocardium. Left ventricular mRNA IL-10 expression was significantly higher, and iNOS, significantly lower in treated than in untreated animals. mRNA expression of TNF-alpha, IFN-gamma, TGF-beta, A20 and ANP was similar in both groups. Our results suggest that TNF-alpha/LT-alpha blockade with Etanercept enhances left ventricular dysfunction in T. cruzi-induced chronic cardiomyopathy and the absence of TNF signaling may be deleterious to the failing heart in Chagas disease cardiomyopathy.     

The human beta-myosin heavy chain gene: sequence diversity and functional characteristics of the protein

The beta-myosin heavy chain gene (MYH7) encodes the motor protein that drives myocardial contraction. It has been proven to be a disease gene for hypertrophic cardiomyopathy (HCM). We analyzed the DNA sequence variation of MYH7 (about 16 kb) of eight individuals: six patients with HCM and two healthy controls. The overall DNA sequence identity was up to 97.2% compared to Jaenicke and coworkers (Jaenicke et al. [1990] Genomics 8:194-206), while the corresponding amino acid sequences revealed 100% identity. In HCM patients, eleven nucleotide substitutions were identified but no causative disease mutation was found: six were detected in coding, four in intronic, and one in 5' regulatory regions. The average nucleotide diversity across this locus was 0.015% with an average of 0.02% in the coding and 0.012% in the noncoding sequence. Analysis of the kinetic behaviour of beta-MHC in the intact contractile structure of normal individuals and HCM patients revealed apparent rate constants of tension development ranging between 1.58 s(-1) and 1.48 s(-1).     

Trypanosoma cruzi benznidazole susceptibility in vitro does not predict the therapeutic outcome of human Chagas disease

Therapeutic failure of benznidazole (BZ) is widely documented in Chagas disease and has been primarily associated with variations in the drug susceptibility of Trypanosoma cruzi strains. In humans, therapeutic success has been assessed by the negativation of anti-T. cruzi antibodies, a process that may take up to 10 years. A protocol for early screening of the drug resistance of infective strains would be valuable for orienting physicians towards alternative therapies, with a combination of existing drugs or new anti-T. cruzi agents. We developed a procedure that couples the isolation of parasites by haemoculture with quantification of BZ susceptibility in the resultant epimastigote forms. BZ activity was standardized with reference strains, which showed IC?? to BZ between 7.6-32 μM. The assay was then applied to isolates from seven chronic patients prior to administration of BZ therapy. The IC?? of the strains varied from 15.6 ± 3-51.4 ± 1 μM. Comparison of BZ susceptibility of the pre-treatment isolates of patients considered cured by several criteria and of non-cured patients indicates that the assay does not predict therapeutic outcome. A two-fold increase in BZ resistance in the post-treatment isolates of two patients was verified. Based on the profile of nine microsatellite loci, sub-population selection in non-cured patients was ruled out.     

High fat diet aggravates cardiomyopathy in murine chronic Chagas disease

Infection with Trypanosoma cruzi, the etiologic agent in Chagas disease, may result in heart disease. Over the last decades, Chagas disease endemic areas in Latin America have seen a dietary transition from the traditional regional diet to a Western style, fat rich diet. Previously, we demonstrated that during acute infection high fat diet (HFD) protects mice from the consequences of infection-induced myocardial damage through effects on adipogenesis in adipose tissue and reduced cardiac lipidopathy. However, the effect of HFD on the subsequent stages of infection – the indeterminate and chronic stages – has not been investigated. To address this gap in knowledge, we studied the effect of HFD during indeterminate and chronic stages of Chagas disease in the mouse model. We report, for the first time, the effect of HFD on myocardial inflammation, vasculopathy, and other types of dysfunction observed during chronic T. cruzi infection. Our results show that HFD perturbs lipid metabolism and induces oxidative stress to exacerbate late chronic Chagas disease cardiac pathology.