Gap junction protein beta 4 plays an important role in cardiac function in humans, rodents, and zebrafish.

AIMS: GJB4 encodes a transmembrane connexin protein (Cx30.3) that is a component of gap junctions. This study investigated whether GJB4 plays an important role in human heart disease and function. METHODS AND RESULTS: We examined a patient and her older brother who both presented with complicated severe hypertrophic cardiomyopathy (HCM) and whose parents are healthy married cousins. The gene exome analysis showed 340 single nucleotide polymorphisms (SNPs) that caused amino acid changes for which the patient was homozygous and both parents were heterozygous. After excluding all known common (>10%) SNP gene mutations, the gene for GJB4 was the only identified gene that is possibly associated with cardiac muscle. The resultant E204A substitution exists in the 4th transmembrane domain. GJB4-E204A impaired the binding with gap junction protein A1 (GJA1) compared with GJB4-WT. The expression of GJB4 was induced in rat disease models of left and right ventricle hypertrophy and mouse disease models of adriamycin-induced cardiomyopathy and myocardial infarction, while it was not detected at all in control. An immunohistochemical study was performed for autopsied human hearts and the explanted heart of the patient. GJB4 was expressed and colocalized with GJA1 in intercalated discs in human diseased hearts, which was extensively enhanced in the explanted heart of the patient. The abnormal expression and localization of GJB4 were observed in beating spheres of patient's induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs). We generated knockout zebrafish of GJB4 by CRISPR/Cas9 and the endodiastolic volume and the ventricular ejection fraction were significantly lower in GJB4-deficient than in wild-type zebrafish at five days post-fertilization. CONCLUSIONS: These results indicate both that GJB4 is defined as a new connexin in diseased hearts, of which mutation can cause a familial form of HCM, and that GJB4 may be a new target for the treatment of cardiac hypertrophy and dysfunction.

The Fabry disease-causing mutation, GLA IVS4+919G>A, originated in Mainland China more than 800 years ago.

The Fabry disease-causing mutation, the GLA IVS4+919G>A (designated GLA IVS4), is very prevalent in patients with hypertrophic cardiomyopathy in Taiwan. This X-linked mutation has also been found in patients in Kyushu, Japan and Southeast Asia. To investigate the age and the possible ancestral origin of this mutation, a total of 33 male patients with the GLA IVS4+919G>A mutation, born in Taiwan, Japan, Singapore, Malaysia, Vietnam, and the Fujian and Guangdong provinces of China, were studied. Peripheral bloods were collected, and the Ilumina Infinium CoreExome-24 microarray was used for dense genotyping. A mutation-carrying haplotype was discovered which was shared by all 33 patients. This haplotype does not exist in 15 healthy persons without the mutation. Rather, a wide diversity of haplotypes was found in the vicinity of the mutation site, supporting the existence of a single founder of the GLA IVS4 mutation. The age of the founder mutation was estimated by the lengths of the mutation-carrying haplotypes based on the linkage-disequilibrium decay theory. The first, second, and third quartile of the age estimates are 800.7, 922.6, and 1068.4 years, respectively. We concluded that the GLA IVS4+919G>A mutation originated from a single mutational event that occurred in a Chinese chromosome more than 800 years ago.

Hypertrophic cardiomyopathy masked by pericarditis

Hypertrophic cardiomyopathy used to be regarded as a rare untreatable cause of sudden death in young male athletes. This report is the case of a middle-aged female patient with hereditary hypertrophic cardiomyopathy masked by superimposed pericarditis and revealed by autopsy. This case report illustrates how co-morbidity can hide a crucial diagnosis. This case report also illustrates the value of autopsy disclosing a familial disease that is increasingly recognized and dramatically more treatable than a few decades ago. Sudden death due to hypertrophic cardiomyopathy has become preventable, if the diagnosis is made soon enough. The lessons for patient care from this case include the importance of not missing the diagnosis of hypertrophic cardiomyopathy in female patients.

Novel α-Actin Gene Mutation p.(Ala21Val) Causing Familial Hypertrophic Cardiomyopathy, Myocardial Noncompaction, and Transmural Crypts. Clinical-Pathologic Correlation.

BACKGROUND: Mutations of α-actin gene (ACTC1) have been phenotypically related to various cardiac anomalies, including hypertrophic cardiomyopathy and dilated cardiomyopathy and left ventricular (LV) myocardial noncompaction. A novel ACTC mutation is reported as cosegregating for familial hypertrophic cardiomyopathy and LV myocardial noncompaction with transmural crypts. METHODS AND RESULTS: In an Italian family of 7 subjects, 4 aged 10 (II-1), 14 (II-2), 43 (I-4) and 46 years (I-5), presenting abnormal ECG changes, dyspnea and palpitation (II-2, I-4, and I-5), and recurrent cerebral ischemic attack (I-5), underwent 2-dimensional echo, cardiac magnetic resonance, Holter monitoring, and next-generation sequencing gene analysis. Patients II-2 and I-5 with ventricular tachycardia underwent a cardiac invasive study, including coronary with LV angiography and endomyocardial biopsy. In all the affected members, ECG showed right bundle branch block and left anterior hemiblock with age-related prolongation of QRS duration. Two-dimensional echo and cardiac magnetic resonance documented LV myocardial noncompaction in all and in I-4, I-5, and II-2 a progressive LV hypertrophy up to 22-mm maximal wall thickness. Coronary arteries were normal. LV angiography showed transmural crypts progressing to spongeous myocardial transformation with LV dilatation and dysfunction in the oldest subject. At histology and electron microscopy detachment of myocardiocytes were associated with cell and myofibrillar disarray and degradation of intercalated discs causing disanchorage of myofilaments to cell membrane. Next-generation sequencing showed in affected members an unreported p.(Ala21Val) mutation of ACTC. CONCLUSIONS: Novel p.(Ala21Val) mutation of ACTC1 causes myofibrillar and intercalated disc alteration leading to familial hypertrophic cardiomyopathy and LV myocardial noncompaction with transmural crypts.

Surgical pathology of subaortic septal myectomy: histology skips over clinical diagnosis.

BACKGROUND: Subaortic septal myectomy is usually performed to mitigate obstruction in patients with the obstructive form of hypertrophic cardiomyopathy (HCM) or in those with congenital subaortic stenosis. Moreover, it is combined with aortic valve replacement in patients with severe aortic valve stenosis (SAS) and asymmetrical septal hypertrophy causing concomitant left ventricular outflow tract obstruction. When both conditions coexist, it is conceptually difficult to identify a cardiomyopathy beyond an adaptive myocardial hypertrophy, strictly related to pressure overload. Myectomy histopathology might be useful to enlighten the cause of the obstruction and establish the diagnosis. AIM: The aim was to describe the pathological findings of surgical septal myectomy specimens obtained from a group of patients with diverse clinical diagnosis, including HCM, severe aortic stenosis, and asymmetrical septal hypertrophy. METHODS: This was a retrospective study of 56 patients undergoing septal myectomy along a 10-year period at a tertiary cardiac surgical center. Clinical, interventional, and anatomopathological findings between patients with and without a preoperative diagnosis of HCM were analyzed and compared. RESULTS: Mean age at intervention was 67.5±20.5 years; 37 (66.1%) were female Preoperative diagnosis of sarcomeric obstructive HCM was assumed in 23 (41.1%) patients. All the other patients (58.9%) were referred for surgery with preoperative diagnosis of asymmetric septal hypertrophy, mainly in the context of severe aortic stenosis (24 patients). Twenty-seven (48.2%) patients had a greater than 30 mmHg intraventricular gradient at rest. Patients with presumed HCM were significantly younger (56.5±15.8 vs. 70.2±13.3 years, P<.001), had higher prevalence of significant intraventricular obstruction at rest [20 (87.0%) vs. 8 (34.8%), P<.001], and more frequently had moderate or severe mitral regurgitation [9 (39.1%) vs. 5(15.1%), P=.043]. All patients with aortic valve stenosis underwent both aortic valve replacement and septal myectomy. Twelve (52.1%) of the patients with obstructive HCM had isolated septal myectomy, while in the remaining 11, the procedure was combined with intervention on the mitral valve. Histopathological final diagnosis was of nonspecific reactive myocardial hypertrophy in all but 4 (92.2%) patients. In those, 2 (3.6%) had the final diagnosis of HCM and 2 (3.6%) the diagnosis of congenital subaortic membranous stenosis with reactive myocardial hypertrophy. Different grades of subendocardial fibroelastosis and myocardial fibrosis, mainly interstitial, were present [27 (48.2%) and 18 (32%) patients, respectively]. When microscopic data were compared between patients with or without a preoperative clinical diagnosis of HCM, no significant differences were found. CONCLUSION: In patients submitted to surgical septal myectomy, histology was mostly indistinctive among different clinical entities. Since different myocardial hypertrophy etiologies may share similar pathological expression, there is a need for detailed clinical assessment when trying to define the best strategy for clinical management.

α-Galactosidase A Genotype N215S Induces a Specific Cardiac Variant of Fabry Disease.

BACKGROUND: Hypertrophic cardiomyopathy is the most common type of cardiomyopathy, but many patients lack sarcomeric/myofilament mutations. We studied whether cardio-specific α-galactosidase A gene variants are misinterpreted as hypertrophic cardiomyopathy because of the lack of extracardiac organ involvement. METHODS AND RESULTS: All subjects who tested positive for the N215S genotype (n=26, 13 females, mean age 49±17 [range, 14-74] years) were characterized in this prospective monocentric longitudinal cohort study to determine genotype-specific clinical characteristics of the N215S (c.644A>G [p.Asn215Ser]) α-galactosidase A gene variant. All subjects were initially referred with suspicion of genetically determined hypertrophic cardiomyopathy. Cardiac hypertrophy (interventricular septum, 12±4 [7-23] mm; left ventricular posterior wall, 11±4 [7-21] mm; left ventricular mass, 86±41 [46-195] g/m2) was progressive, systolic function mainly preserved (cardiac index 2.8±0.6 [1.9-3.9] L/min per m2), and diastolic function mildly abnormal. Cardiac magnetic resonance imaging revealed replacement fibrosis in loco typico (18/26, 69%), particularly in subjects >50 years. Elderly subjects had advanced heart failure, and 6 (23%) were suggested for implantable cardioverter-defibrillator therapy. Leukocyte α-galactosidase A enzyme activity was mildly reduced in 19 subjects and lyso-globotriaosylceramide slightly elevated (median, 4.9; interquartile range, 1.3-9.1 ng/mL). Neurological and renal impairments (serum creatinine, 0.87±0.20; median, 0.80; interquartile range, 0.70-1.01 mg/dL; glomerular filtration rate, 102±23; median, 106; interquartile range, 84-113 mL/min) were discreet. Only 2 subjects developed clinically relevant proteinuria. CONCLUSIONS: α-Galactosidase A genotype N215S does not lead to the development of a classical Fabry phenotype but induces a specific cardiac variant of Fabry disease mimicking nonobstructive hypertrophic cardiomyopathy. The lack of prominent noncardiac impairment leads to a significant delay in diagnosis and Fabry-specific therapy.

Sex dimorphisms of crossbridge cycling kinetics in transgenic hypertrophic cardiomyopathy mice.

Familial hypertrophic cardiomyopathy (HCM) is a disease of the sarcomere and may lead to hypertrophic, dilated, restrictive, and/or arrhythmogenic cardiomyopathy, congestive heart failure, or sudden cardiac death. We hypothesized that hearts from transgenic HCM mice harboring a mutant myosin heavy chain increase the energetic cost of contraction in a sex-specific manner. To do this, we assessed Ca(2+) sensitivity of tension and crossbridge kinetics in demembranated cardiac trabeculas from male and female wild-type (WT) and HCM hearts at an early time point (2 mo of age). We found a significant effect of sex on Ca(2+) sensitivity such that male, but not female, HCM mice displayed a decrease in Ca(2+) sensitivity compared with WT counterparts. The HCM transgene and sex significantly impacted the rate of force redevelopment by a rapid release-restretch protocol and tension cost by the ATPase-tension relationship. In each of these measures, HCM male trabeculas displayed a gain-of-function when compared with WT counterparts. In addition, cardiac remodeling measured by echocardiography, histology, morphometry, and posttranslational modifications demonstrated sex- and HCM-specific effects. In conclusion, female and male HCM mice display sex dimorphic crossbridge kinetics accompanied by sex- and HCM-dependent cardiac remodeling at the morphometric, histological, and cellular level.

A small-molecule inhibitor of sarcomere contractility suppresses hypertrophic cardiomyopathy in mice.

Hypertrophic cardiomyopathy (HCM) is an inherited disease of heart muscle that can be caused by mutations in sarcomere proteins. Clinical diagnosis depends on an abnormal thickening of the heart, but the earliest signs of disease are hyperdynamic contraction and impaired relaxation. Whereas some in vitro studies of power generation by mutant and wild-type sarcomere proteins are consistent with mutant sarcomeres exhibiting enhanced contractile power, others are not. We identified a small molecule, MYK-461, that reduces contractility by decreasing the adenosine triphosphatase activity of the cardiac myosin heavy chain. Here we demonstrate that early, chronic administration of MYK-461 suppresses the development of ventricular hypertrophy, cardiomyocyte disarray, and myocardial fibrosis and attenuates hypertrophic and profibrotic gene expression in mice harboring heterozygous human mutations in the myosin heavy chain. These data indicate that hyperdynamic contraction is essential for HCM pathobiology and that inhibitors of sarcomere contraction may be a valuable therapeutic approach for HCM.

The structural and functional effects of the familial hypertrophic cardiomyopathy-linked cardiac troponin C mutation, L29Q.

Familial hypertrophic cardiomyopathy (FHC) is characterized by severe abnormal cardiac muscle growth. The traditional view of disease progression in FHC is that an increase in the Ca(2+)-sensitivity of cardiac muscle contraction ultimately leads to pathogenic myocardial remodeling, though recent studies suggest this may be an oversimplification. For example, FHC may be developed through altered signaling that prevents downstream regulation of contraction. The mutation L29Q, found in the Ca(2+)-binding regulatory protein in heart muscle, cardiac troponin C (cTnC), has been linked to cardiac hypertrophy. However, reports on the functional effects of this mutation are conflicting, and our goal was to combine in vitro and in situ structural and functional data to elucidate its mechanism of action. We used nuclear magnetic resonance and circular dichroism to solve the structure and characterize the backbone dynamics and stability of the regulatory domain of cTnC with the L29Q mutation. The overall structure and dynamics of cTnC were unperturbed, although a slight rearrangement of site 1, an increase in backbone flexibility, and a small decrease in protein stability were observed. The structure and function of cTnC was also assessed in demembranated ventricular trabeculae using fluorescence for in situ structure. L29Q reduced the cooperativity of the Ca(2+)-dependent structural change in cTnC in trabeculae under basal conditions and abolished the effect of force-generating myosin cross-bridges on this structural change. These effects could contribute to the pathogenesis of this mutation.

ß-Myosin heavy chain variant Val606Met causes very mild hypertrophic cardiomyopathy in mice, but exacerbates HCM phenotypes in mice carrying other HCM mutations.

RATIONALE: Approximately 40% of hypertrophic cardiomyopathy (HCM) is caused by heterozygous missense mutations in ß-cardiac myosin heavy chain (ß-MHC). Associating disease phenotype with mutation is confounded by extensive background genetic and lifestyle/environmental differences between subjects even from the same family. OBJECTIVE: To characterize disease caused by ß-cardiac myosin heavy chain Val606Met substitution (VM) that has been identified in several HCM families with wide variation of clinical outcomes, in mice. METHODS AND RESULTS: Unlike 2 mouse lines bearing the malignant myosin mutations Arg453Cys (RC/+) or Arg719Trp (RW/+), VM/+ mice with an identical inbred genetic background lacked hallmarks of HCM such as left ventricular hypertrophy, disarray of myofibers, and interstitial fibrosis. Even homozygous VM/VM mice were indistinguishable from wild-type animals, whereas RC/RC- and RW/RW-mutant mice died within 9 days after birth. However, hypertrophic effects of the VM mutation were observed both in mice treated with cyclosporine, a known stimulator of the HCM response, and compound VM/RC heterozygous mice, which developed a severe HCM phenotype. In contrast to all heterozygous mutants, both systolic and diastolic function of VM/RC hearts was severely impaired already before the onset of cardiac remodeling. CONCLUSIONS: The VM mutation per se causes mild HCM-related phenotypes; however, in combination with other HCM activators it exacerbates the HCM phenotype. Double-mutant mice are suitable for assessing the severity of benign mutations.

Human-induced pluripotent stem cell models of inherited cardiomyopathies.

PURPOSE OF REVIEW: This article provides an overview of the latest advances in in-vitro modeling of inherited cardiomyopathies using human-induced pluripotent stem cells (iPSCs). RECENT FINDINGS: Inherited cardiomyopathies have been recently modeled by generating iPSCs from patients harboring mutations in genes associated with the pathogenesis of hypertrophic cardiomyopathy, dilated cardiomyopathy, and arrhythmogenic right ventricular cardiomyopathy/dysplasia. SUMMARY: Patient-specific iPSCs and their differentiated cardiomyocytes (induced pluripotent stem cell-derived cardiomyocytes) now provide a novel model to study the underlying molecular mechanism of the pathogenesis of familial cardiomyopathies as well as for in-vitro drug screening and drug discovery.

Post-mortem genetic testing in a family with long-QT syndrome and hypertrophic cardiomyopathy.

Pediatric sudden unexplained deaths are rare and tragic events that should be evaluated with all the tools available to the medical community. The current state of genetic testing is an excellent resource that improves our ability to diagnose cardiovascular disorders that can lead to sudden cardiac arrest. Post-mortem genetic testing is not typically a covered benefit of health insurance and may not be offered to families in the setting of a negative autopsy. This unusual case includes two separate cardiovascular disorders that highlight the use of genetic testing and its role in diagnosis, screening, and risk stratification. The insurance company's decision to cover post-mortem testing demonstrated both compassion as well as an understanding of the long-term cost effectiveness.

Confirmation of cause and manner of death via a comprehensive cardiac autopsy including whole exome next-generation sequencing.

Annually, the sudden death of thousands of young people remains inadequately explained despite medicolegal investigation. Postmortem genetic testing for channelopathies/cardiomyopathies may illuminate a potential cardiac mechanism and establish a more accurate cause and manner of death and provide an actionable genetic marker to test surviving family members who may be at risk for a fatal arrhythmia. Whole exome sequencing allows for simultaneous genetic interrogation of an individual's entire estimated library of approximately 30000 genes. Following an inconclusive autopsy, whole exome sequencing and gene-specific surveillance of all known major cardiac channelopathy/cardiomyopathy genes (90 total) were performed on autopsy blood-derived genomic DNA from a previously healthy 16-year-old adolescent female found deceased in her bedroom. Whole exome sequencing analysis revealed a R249Q-MYH7 mutation associated previously with familial hypertrophic cardiomyopathy, sudden death, and impaired ß-myosin heavy chain (MHC-ß) actin-translocating and actin-activated ATPase (adenosine triphosphatase) activity. Whole exome sequencing may be an efficient and cost-effective approach to incorporate molecular studies into the conventional postmortem examination.

Japanese patients with Fabry disease predominantly showing cardiac and neurological manifestation with novel missense mutation: R220P.

BACKGROUND: Fabry disease, an X-linked lysosomal sphingolipid storage disorder caused by mutation of the α-galactosidase A (GLA) gene, results in systemic organ damage. However, the age of onset of clinical manifestations and course of the disease are variable even within the same family. OBJECTIVE: In this study, we evaluated the clinical phenotype and the molecular lesions associated with the GLA gene in a Japanese family with Fabry disease that predominantly showed cardiac and neurological manifestations. METHODS: A genetic analysis of the GLA gene using conventional genomic sequencing was performed in all seven members of this family, including four hemizygous males and three heterozygous females. Endomyocardial biopsy was performed in two patients with severe left ventricular (LV) hypertrophy. RESULTS: A novel missense mutation was identified at codon 220 in exon 5, thus resulting in an arginine to proline substitution (R220P) in all seven family members. The three adult hemizygous males had LV hypertrophy and developed neurological manifestations in their 50s. One of the adult hemizygotes developed complete atrioventricular block. On the other hand, we could not find any organ damage in a young hemizygous male or the three heterozygous females. CONCLUSION: We identified a novel missense mutation in a Japanese family with Fabry disease showing cardiac and neurological manifestations. In patients with Fabry disease, advanced organ damage in the heart and brain can be life-threatening, even if renal failure is lacking.

Cardiac and skeletal muscle expression of mutant ß-myosin heavy chains, degree of functional impairment and phenotypic heterogeneity in hypertrophic cardiomyopathy.

Several mutations in distinct genes, all coding for sarcomeric proteins, have been reported in unrelated kindreds with familial hypertrophic cardiomyopathy (FHC). We have identified nine individuals from three families harboring two distinct mutations in one copy of the ß-myosin heavy chain (ß-MHC) gene. In this study, the expression of the mutant ß-myosin protein isoform, isolated from slow-twitch fibers of skeletal muscle, was demonstrated by Northern and Western blot analysis; this myosin showed a decreased in vitro motility activity and produced a lower actin-activated ATPase activity. Isometric tension, measured in single slow-twitch fibers isolated from the affected individuals, also showed a significant decrease. The degree of impairment of ß-myosin function, as well as the loss in isometric tension development, were strictly dependent on the amount of the isoform transcribed from the mutated allele. Interestingly, a strong correlation was also demonstrated between mutant ß-myosin content and clinical features of FHC. On the other hand, we were unable to detect any correlation between mutant ß-myosin expression and degree of cardiac hypertrophy, thereby strengthening the hypothesis that hypertrophy, one of the hallmarks of FHC, might not necessarily be related to the clinical evolution of this disease. These findings lend support to the notion that additional factors rather than the mutated gene may play a pathogenetic role in cardiac wall thickening, whereas the prognosis appears to be strongly related to the amount of mutant protein.

Generation and functional characterization of knock-in mice harboring the cardiac troponin I-R21C mutation associated with hypertrophic cardiomyopathy.

The R21C substitution in cardiac troponin I (cTnI) is the only identified mutation within its unique N-terminal extension that is associated with hypertrophic cardiomyopathy (HCM) in man. Particularly, this mutation is located in the consensus sequence for ß-adrenergic-activated protein kinase A (PKA)-mediated phosphorylation. The mechanisms by which this mutation leads to heart disease are still unclear. Therefore, we generated cTnI knock-in mouse models carrying an R21C mutation to evaluate the resultant functional consequences. Measuring the in vivo levels of incorporated mutant and WT cTnI, and their basal phosphorylation levels by top-down mass spectrometry demonstrated: 1) a dominant-negative effect such that, the R21C+/- hearts incorporated 24.9% of the mutant cTnI within the myofilament; and 2) the R21C mutation abolished the in vivo phosphorylation of Ser(23)/Ser(24) in the mutant cTnI. Adult heterozygous (R21C+/-) and homozygous (R21C+/+) mutant mice activated the fetal gene program and developed a remarkable degree of cardiac hypertrophy and fibrosis. Investigation of cardiac skinned fibers isolated from WT and heterozygous mice revealed that the WT cTnI was completely phosphorylated at Ser(23)/Ser(24) unless the mice were pre-treated with propranolol. After propranolol treatment (-PKA), the pCa-tension relationships of all three mice (i.e. WT, R21C+/-, and R21C+/+) were essentially the same. However, after treatment with propranolol and PKA, the R21C cTnI mutation reduced (R21C+/-) or abolished (R21C+/+) the well known decrease in the Ca(2+) sensitivity of tension that accompanies Ser(23)/Ser(24) cTnI phosphorylation. Altogether, the combined effects of the R21C mutation appear to contribute toward the development of HCM and suggest that another physiological role for the phosphorylation of Ser(23)/Ser(24) in cTnI is to prevent cardiac hypertrophy.

Cardiac myosin binding protein C phosphorylation in cardiac disease.

Perturbations in sarcomeric function may in part underlie systolic and diastolic dysfunction of the failing heart. Sarcomeric dysfunction has been ascribed to changes in phosphorylation status of sarcomeric proteins caused by an altered balance between intracellular kinases and phosphatases during the development of cardiac disease. In the present review we discuss changes in phosphorylation of the thick filament protein myosin binding protein C (cMyBP-C) reported in failing myocardium, with emphasis on phosphorylation changes observed in familial hypertrophic cardiomyopathy caused by mutations in MYBPC3. Moreover, we will discuss assays which allow to distinguish between functional consequences of mutant sarcomeric proteins and (mal)adaptive changes in sarcomeric protein phosphorylation.

Myosin cross-bridges do not form precise rigor bonds in hypertrophic heart muscle carrying troponin T mutations.

Distribution of orientations of myosin was examined in ex-vivo myofibrils from hearts of transgenic (Tg) mice expressing Familial Hypertrophic Cardiomyopathy (FHC) troponin T (TnT) mutations I79N, F110I and R278C. Humans are heterozygous for sarcomeric FHC mutations and so hypertrophic myocardium contains a mixture of the wild-type (WT) and mutated (MUT) TnT. If mutations are expressed at a low level there may not be a significant change in the global properties of heart muscle. In contrast, measurements from a few molecules avoid averaging inherent in the global measurements. It is thus important to examine the properties of only a few molecules of muscle. To this end, the lever arm of one out of every 60,000 myosin molecules was labeled with a fluorescent dye and a small volume within the A-band (~1 fL) was observed by confocal microscopy. This volume contained on average 5 fluorescent myosin molecules. The lever arm assumes different orientations reflecting different stages of acto-myosin enzymatic cycle. We measured the distribution of these orientations by recording polarization of fluorescent light emitted by myosin-bound fluorophore during rigor and contraction. The distribution of orientations of rigor WT and MUT myofibrils was significantly different. There was a large difference in the width and of skewness and kurtosis of rigor distributions. These findings suggest that the hypertrophic phenotype associated with the TnT mutations can be characterized by a significant increase in disorder of rigor cross-bridges.

Mutations in the beta-myosin heavy chain gene in southern Chinese families with hypertrophic cardiomyopathy.

In this study, 14 unrelated hypertrophic cardiomyopathy (HCM) probands were scanned by polymerase chain reaction-single-strand conformation polymorphism analysis and DNA sequencing. Three mis-sense mutations of the beta-myosin heavy chain gene, MYH7, were found: valine (Val) 606 methionine (Met), arginine (Arg) 694 leucine (Leu), and Arg 723 glycine (Gly). All are reported here for the first time in Chinese subjects. The results showed that: Val606Met is an intermediate malignancy mutation; Arg694Leu is a novel mutation with a benign phenotype; and the Arg723Gly mutation is linked to malignancy - it can lead not only to HCM but also to dilated cardiomyopathy at various ages. The clinical symptoms associated with Arg723Gly emerged early and caused more severe clinical manifestation and poorer prognosis in females than in males. Mis-sense mutations were not detected in the myosin binding protein C, cardiac, cardiac troponin T type 2, or cardiac troponin I type 3 genes. The MYH7 gene may be an HCM mutation hotspot in the Chinese and have unique features in this study population.