Familial Hypertrophic Cardiomyopathy Research Articles

Pseudo-Phosphorylation Mediated Rescue of R58Q-Linked Familial Hypertrophic Cardiomyopathy Phenotype

Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease characterized by ventricular hypertrophy, myofibrillar disarray and often caused by mutations in genes encoding for major sarcomeric proteins. This study is focused on Arginine to Glutamine substitution (R58Q) in the ventricular myosin regulatory light chain (RLC) resulting in a malignant hypertrophic phenotype. We previously showed a mutation-induced decrease in the endogenous level of cardiac RLC phosphorylation in R58Q mice, resulting in compromised communication between the functional regions of the RLC and affecting the interaction of myosin and actin and cardiac muscle contraction. In this report we explored the therapeutic potential of pseudo-phosphorylation, in which Aspartic Acid is replaced for phosphorylatable Serine-15 (S15D) in the R58Q background. In vitro actin-activated myosin ATPase activity was assessed using S15D-R58Q and R58Q vs. WT reconstituted porcine cardiac myosin. R58Q largely decreased maximal ATPase activity, Vmax (∼30% lower) compared to WT. S15D-R58Q rescued the maximal ATPase activity to the WT level. The Michaelis–Menten constant (in μM) for R58Q (Km=4.04±0.45) was significantly increased compared to WT (Km=1.68±0.19), suggesting that a higher concentration of actin is needed to activate cross-bridges. S15D-R58Q-RLC showed a small but significant increase in Km (2.56±0.32), compared with WT. Steady-state acto-myosin binding was studied using fluorescently labelled actin to quantify the dissociation constants (Kd) and stoichiometry of binding (n) between myosin and actin in the absence of nucleotide. Binding of WT-RLC to actin was strong (Kd value =5.5 nM, n=0.89). A large decrease in binding (∼67-fold increase in Kd) was observed for R58Q-RLC. Kd of S15D-R58Q-RLC was only slightly increased (∼3-fold) compared with WT indicating a rescue in binding affinity by pseudo-phosphorylated RLC mutant. Our findings on S15D-R58Q support the idea that myosin phosphorylation may have important translational applications for the treatment of RLC induced hypertrophic cardiomyopathy. Supported by NIH-HL123255 (DSC).

Abnormal Cardiac Cross-Bridge Kinetics in a Troponin T ILE79ASN Transgenic Mouse Model

The missense mutation Ile79Asn in human cardiac troponin T (HcTnT-I79N) has been associated with phenotypic outcomes of familial hypertrophic cardiomyopathy, arrhythmias, and sudden cardiac death. Previous characterization of a transgenic mouse model bearing the HcTnT-I79N mutation, which expressed 52% HcTnT-I79N of total troponin T, demonstrated increased Ca2+ sensitivity of isometric force. However, little is known about the changes in cross-bridge kinetics, if any, that result from the mutation. This study investigates isometric force, sinusoidal stiffness and rate of tension redevelopment (kTR) of skinned papillary muscle fiber bundles at two sarcomere lengths (1.9 µm and 2.1 µm); cardiac muscle was isolated from the left ventricle of non-transgenic wild-type (WT) mice or transgenic mice bearing the HcTnT-I79N mutation. Sarcomere length (SL) was set at pCa8 using HeNe laser diffraction after the attachment points of each skinned fiber bundle were fixed with glutaraldehyde to minimize end compliance. Sinusoidal stiffness and kTR were obtained after recording steady-state isometric tension by oscillating the fiber bundle 0.2% of its relaxed length or by applying a ramped shortening followed by a quick re-stretch to original fiber bundle length, respectively. As previously reported, the HcTnT-I79N mutation resulted in increased Ca2+-sensitivity of isometric force although no difference in maximum force per cross-sectional area was observed. Length-dependent activation at the longer SL resulted in increased Ca2+-sensitivity of isometric force and sinusoidal stiffness, and decreased kTR for both WT and HcTnT-I79N. However, HcTnT-I79N exhibited faster kTR and greater sinusoidal stiffness at sub-maximum pCa (-log[Ca2+]) solutions compared to WT regardless of SL. These data indicate that HcTnT-I79N mutation, and SL, markedly influence not only the dynamics of individual regulatory units, but also the kinetics of cross-bridge cycling.

Early remodeling of repolarizing K+ currents in the αMHC403/+ mouse model of familial hypertrophic cardiomyopathy

Familial hypertrophic cardiomyopathy (HCM), linked to mutations in myosin, myosin-binding proteins and other sarcolemmal proteins, is associated with increased risk of life threatening ventricular arrhythmias, and a number of animal models have been developed to facilitate analysis of disease progression and mechanisms. In the experiments here, we use the αMHC403/+ mouse line in which one αMHC allele harbors a common HCM mutation (in βMHC, Arg403 Gln). Here, we demonstrate marked prolongation of QT intervals in young adult (10–12week) male αMHC403/+ mice, well in advance of the onset of measurable left ventricular hypertrophy. Electrophysiological recordings from myocytes isolated from the interventricular septum of these animals revealed significantly (P<0.001) lower peak repolarizing voltage-gated K+ (Kv) current (IK,peak) amplitudes, compared with cells isolated from wild type (WT) littermate controls. Analysis of Kv current waveforms revealed that the amplitudes of the inactivating components of the total outward Kv current, Ito,f, Ito,s and IK,slow, were significantly lower in αMHC403/+, compared with WT, septum cells, whereas Iss amplitudes were similar. The amplitudes/densities of IK,peak and IK,slow were also lower in αMHC403/+, compared with WT, LV wall and LV apex myocytes, whereas Ito,f was attenuated in αMHC403/+ LV wall, but not LV apex, cells. These regional differences in the remodeling of repolarizing Kv currents in the αMHC403/+ mice would be expected to increase the dispersion of ventricular repolarization and be proarrhythmic. Quantitative RT-PCR analysis revealed reductions in the expression of transcripts encoding several K+ channel subunits in the interventricular septum, LV free wall and LV apex of (10–12week) αMHC403/+ mice, although this transcriptional remodeling was not correlated with the observed decreases in K+ current amplitudes.

Altered myocyte contractility and calcium homeostasis in alpha-myosin heavy chain point mutations linked to familial dilated cardiomyopathy

Mutations in the human cardiac motor protein beta-myosin heavy chain (βMHC) have been long recognized as a cause of familial hypertrophic cardiomyopathy. Recently, mutations (P830L and A1004S) in the less abundant but faster isoform alpha-myosin heavy chain (αMHC) have been linked to dilated cardiomyopathy (DCM). In this study, we sought to determine the cellular contractile phenotype associated with these point mutations. Ventricular myocytes were isolated from 2 month male Sprague Dawley rats. Cells were cultured in M199 media and infected with recombinant adenovirus containing the P830L or the A1004S mutant human αMHC at a MOI of 500 for 18 h. Uninfected cells (UI), human βMHC (MOI 500, 18 h), and human αMHC (MOI 500, 18 h) were used as controls. Cells were loaded with fura-2 (1 μM, 15 min) after 48 h. Sarcomere shortening and calcium transients were recorded in CO2 buffered M199 media (36°±1 C) with and without 10 nM isoproterenol (Iso). The A1004S mutation resulted in decreased peak sarcomere shortening while P830L demonstrated near normal shortening kinetics at baseline. In the presence of Iso, the A1004S sarcomere shortening was identical to the βMHC shortening while the P830L was identical to the αMHC control. All experimental groups had identical calcium transients. Despite a shared association with DCM, the P830L and A1004S αMHC mutations alter myocyte contractility in completely different ways while at the same preserving peak intracellular calcium.

Epidemiology of cardiomyopathy – A Clinical and Genetic Study of Restrictive Cardiomyopathy: The EPOCH-R Study

Background: Hypertrophic cardiomyopathy (HCM) is a genetic disorder with the prevalence of 1 in 500 globally. HCM is clinically characterized by thickening of the wall of the heart, predominantly left ventricle (LV), and interventricular septum (IVS). Our study aims to report the demographical, clinical and genetic profile of Indian HCM patients. Methods: HCM patients were recruited on the basis of WHO criteria. The clinical phenotypes were analyzed using electrocardiography, two-dimensional electrocardiography, and hotspot region of the MYH7 gene was sequenced for all patients as well as for controls. Results: There were 59 patients with a clinical diagnosis of HCM with a preponderance of disease in males with a ratio (men, women) of 5.5:1. Average age of onset of the disease was late 30 s (39.2 ± 14.5) with familial HCM accounting for 18% (n = 9) for total HCM families (n = 50). Nonobstructive kind of HCM was more prevalent as compared to obstructive HCM (66.1% vs. 33.9%). Average posterior wall LV thickness of the HCM patients was 16 ± 4.8 mm and IVS thickness was 21 ± 8.3 mm with familial patients having greater wall thickness as compared to sporadic patients. Sequencing of hotspot region of MYH7 identified three mutations in three different patients. Two mutations were found to be segregating in familial cases. Conclusion: HCM is more prevalent in males with a predominance of hypertrophic nonobstructive cardiomyopathy form. Eighteen percent of cases were familial and showed an early onset of the disease and worse prognosis as compared to sporadic cases. Hotspot sequencing of MYH7 only explains 6% of its genetic basis. More of the candidate genes need to be screened through advanced techniques like next generation sequencing to identify the causal genes which could make us understand the mechanistic pathways.

Analysis of cardiac troponin C gene TNNC1 c. G175C mutation in a Chinese pedigree with familial hypertrophic cardiomyopathy and the correlation between genotype and phenotype

Objective: To investigate the genotype-phenotype correlation in Chinese familial hypertrophic cardiomyopathy (HCM )focusing on the cardiac troponin C gene TNNC1 c. G175C mutation. Methods: All family members of a Chinese pedigree with hypertrophic cardiomyopathy admitted in Third People's Hospital of Qingdao in February 2005 and 200 healthy volunteers were included in this study. The coding exons of 30 hypertrophic cardiomyopathy associated genes were identified by whole exons amplification and high-throughput sequencing in the proband, and the identified mutation were further detected through bi-directional Sanger sequencing in all family members and 200 healthy volunteers. Pedigree analysis included clinical manifestation, physical examination, ECG and echocardiogram. Results: A missense mutation c. G175C was identified in the TNNC1 gene in 2 family members, which resulted in a glutamic acid (E) to glutamine (Q) exchange at amino acid residue 59. A mutation c. A1319G was identified in the MYLK2 gene in 1 family member, which resulted in a lysine (K) to arginine (R) exchange at amino acid residue 440. These mutations were absent in 200 healthy controls. The proband carried the two kinds of mutations and expressed various clinical manifestations of heart failure and had history of ventricular tachycardia, paraxial atrial fibrillation, pacemaker implantation, electrocardiogram showed right bundle branch block and echocardiography examination evidenced thickened interventricular septum (23.3 mm) and apex and reduced wall motion of these segments. The daughter of the proband carried the TNNC1 c. G175C mutation and was also diagnosed with asymptomatic HCM by echocardiography with thickened interventricular septum (19 mm) and apex (15 mm). Conclusion: The novel missense mutation of TNNC1 c. G175C might be the disease-causing gene mutation in this Chinese pedigree with familiar HCM.

Amino Acid Changes at Arginine 204 of Troponin I Result in Increased Calcium Sensitivity of Force Development.

Mutations in human cardiac troponin I (cTnI) have been associated with restrictive, dilated, and hypertrophic cardiomyopathies. The most commonly occurring residue on cTnI associated with familial hypertrophic cardiomyopathy (FHC) is arginine (R), which is also the most common residue at which multiple mutations occur. Two FHC mutations are known to occur at cTnI arginine 204, R204C and R204H, and both are associated with poor clinical prognosis. The R204H mutation has also been associated with restrictive cardiomyopathy (RCM). To characterize the effects of different mutations at the same residue (R204) on the physiological function of cTnI, six mutations at R204 (C, G, H, P, Q, W) were investigated in skinned fiber studies. Skinned fiber studies showed that all tested mutations at R204 caused significant increases in Ca2+ sensitivity of force development (ΔpCa50 = 0.22–0.35) when compared to wild-type (WT) cTnI. Investigation of the interactions between the cTnI mutants and WT cardiac troponin C (cTnC) or WT cardiac troponin T (cTnT) showed that all the mutations investigated, except R204G, affected either or both cTnI:cTnT and cTnI:cTnC interactions. The R204H mutation affected both cTnI:cTnT and cTnI:cTnC interactions while the R204C mutation affected only the cTnI:cTnC interaction. These results suggest that different mutations at the same site on cTnI could have varying effects on thin filament interactions. A mutation in fast skeletal TnI (R174Q, homologous to cTnI R204Q) also significantly increased Ca2+ sensitivity of force development (ΔpCa50 = 0.16). Our studies indicate that known cTnI mutations associated with poor prognosis (R204C and R204H) exhibit large increases in Ca2+ sensitivity of force development. Therefore, other R204 mutations that cause similar increases in Ca2+ sensitivity are also likely to have poor prognoses.

Abstract 12739: Gap Junction Protein Beta 4 Plays an Important Role In Cardiac Hypertrophy and Cardiac Function in Human and Zebrafish

Introduction: Gap junction protein beta 4 (GJB4) encodes a transmembrane connexin protein (Cx30.3) that is a component of gap junctions. Mutations in this gene remain unknown to be associated with hypertrophic cardiomyopathy (HCM) and cardiac dysfunction. Hypothesis: Our hypothesis is that mutations in GJB4 can cause familial HCM, which can progresses into dilated-phase HCM (d-HCM). Methods: We have previously examined a patient and her elder brother who both presented with complicated severe HCM and whose parents are married cousins. The elder brother had died suddenly as a child and the autopsy demonstrated the development of HCM. The patient was diagnosed with HCM, which progressed to d-HCM during the 20-year follow-up. The patient has recently undergone cardiac transplantation. The parents are healthy. We hypothesized that an autosomal recessive mutation caused their familial HCM. The whole-exome sequencing was performed from each of the three participants. We generated knockout zebrafish of GJB4 and ELP2 by CRISPER/Cas9 system and analysis the volume and function of ventricle at five days post fertilization (dpf). Results: The gene exome analysis showed 34 single nucleotide polymorphisms (SNPs) that caused amino acid change for which the patient was homozygous and both parents were heterozygous after excluding all known common (&gt;10%) SNP gene mutations in the dbSNP. Among them, GJB4 and ELP2 were the identified genes that are possibly associated with cardiac muscle. The PolyPhen-2 scores of mutations in GJB4 and ELP2 were 1.000 and 0.001 respectively. The identified missense mutation was a 981 A&gt;C nucleotide transition in GJB4. The resultant E204A substitution replaces a polar carboxylic glutamate with a nonpolar alanine. The endodiastolic volume was significantly lower in GJB4-KO (94pl, p&lt;0.01) and ELP2-KO (95pl, p&lt;0.01) than in wild type (WT, 145pl) zebrafish at 5 dpf. Furthermore the ventricular ejection fraction was decreased in GJB4 KO (0.66, p&lt;0.01) and unchanged in ELP2-KO (0.80) respectively compared with WT zebrafish (0.82). Conclusions: These results indicate both that mutations in GJB4 and ELP2 can cause a familial form of HCM and that GJB4 especially may be a new target for treatment of cardiac function and hypertrophy.

Abstract 11235: Improvement of Defective Channel Function on RyR2 Mediated by CaMKII Activation Suppresses Arrhythmogenesis in TnT-related Hypertrophic Cardiomyopathy

Backgrounds: In Familial Hypertrophic Cardiomyopathy (FHC) caused by cardiac troponin T (cTnT) mutations, Ca 2+ sensitivity in myofilaments is increased, results in leading to sudden cardiac death. The activation of calcium/calmodulin-dependent protein kinase II (CaMKII) is known to be one of the key elements for the alteration of calcium handling in heart failure, presumably mediated through defective channel function of cardiac ryanodine receptor (RyR2). However, how cTnT mutations lead to lethal arrhythmias and abnormal calcium handling through defective RyR2 remains elusive. Here, we investigated the pathogenic role of CaMKII on aberrant Ca 2+ release through RyR2 for arrhythmogenesis in FHC hearts and therapeutic effects of dantrolene, which was found to correct inter-domain interactions between N-terminal and central domain of RyR2 in FHC-related cTnT-mutated transgenic mouse (TG; cTnT-delta160E). TG have been reported myocardial disarray in histology and increased Ca 2+ sensitivity in myofilaments. Methods and results: On confocal Ca 2+ imaging by fluo4-AM exposed to isoproterenol (ISO; 10 nmol/L), the Ca 2+ spark frequency (SpF:s-1·100μm-1) was much higher in TG cardiomyocytes (ISO-TG: n=11: 7.8±0.6; p&lt;0.05) than in non-TG (n=8: 3.4±0.4). It was largely reversed by CaMKII inhibitor {KN-93(1μM), n=7: 5.2±0.5; p&lt;0.05}, but not by PKA inhibitor (PKI){H-89(1μM), n=7: 7.3±0.4}. The significant increase of spontaneous Ca 2+ transient (sCaT), which was measured after sequential pacing at 5 Hz was appeared in ISO-TG (42.1%; p&lt;0.05 vs ISO-treated non-TG 14.0%), whereas it was again attenuated by CaMKII inhibitor (15.8%), but not by PKI (33.0%). These abnormal events in ISO-TG were reproduced by adding EGTA-AM into ISO-treated non-TG, suggesting that increased Ca 2+ buffering capacity, causing an increase in diastolic [Ca 2+ ], predisposes to aberrant Ca 2+ release events. Moreover, after the treatment of dantrolene (1μM), ISO-induced SpF (n=6: 5.2±0.3; p&lt;0.05 vs ISO-TG) and sCaT (25.9%) were decreased in TG. Conclusions: CaMKII activation may play a role in the development of abnormal calcium handling in FHC-linked cTnT mutated hearts, possibly through defective inter-domain interaction between N-terminal and central domain of RyR2.

Hypertrophic Cardiomyopathy: Cell-to-Cell Imbalance in Gene Expression and Contraction Force as Trigger for Disease Phenotype Development.

Hypertrophic cardiomyopathy (HCM) is an inherited cardiac disease with an incidence of about 1 in 500 individuals.1 It is characterized by asymmetrical hypertrophy of the left ventricle in the absence of other causes for hypertrophy. HCM can vary from essentially asymptomatic to highly malignant up to end-stage heart failure or cause life-threatening arrhythmias with sudden cardiac death particularly in young adults. In most familial HCM cases, heterozygous mutations in sarcomeric proteins have been identified as underlying cause. About one third of the patients are heterozygous for mutations in the β-cardiac myosin heavy chain gene MYH7 , another third in cardiac myosin-binding protein C (cMyBPC).2 Few mutations were found in nonsarcomeric proteins. Cardiomyocyte and myofibrillar disarray with interstitial fibrosis and hypertrophy are hallmarks of HCM.3 The degree of myocardial disarray correlates with risk factors for sudden cardiac death,4 and it was suggested that myocyte disarray directly results from functional changes induced by the HCM-related mutations at the sarcomeric level.5 From studies mostly based on animal models or recombinant contractile proteins, it was postulated that HCM-associated mutations generally enhance contractility of the myocardium, whereas mutations with reduced contractility would lead to dilated cardiomyopathy.6–8 Accordingly, the different HCM mutations are thought to lead to the HCM phenotype by increasing calcium sensitivity, contractility, and ATPase activity of the cardiomyocytes.5 Results from our group and others, however, were inconsistent with this hypothesis. Instead, contractility and calcium sensitivity can be enhanced or decreased in HCM.6,9–12 Thus, it is still unclear what triggers development of HCM. On the other hand, trials with new therapeutic approaches for HCM would greatly benefit from detailed knowledge of the disease-causing mechanisms. Here, we propose an entirely new concept of how different mutations in sarcomeric proteins may lead …

CRISPR (Clustered Regularly Interspaced Palindromic Repeat)/Cas9 System: A Revolutionary Disease-Modifying Technology.

As clinicians, we are equipped with an ever-expanding armamentarium of drug and device therapies that have extended the lifespan of countless patients with cardiovascular disease. Nevertheless, it is likely that future therapies aimed at the root cause of disease, rather than secondary effects, will improve clinical outcomes in our patients yet further. Imagine if we could restore expression of a critical protein that is reduced as a consequence of remodeling associated with heart failure? Alternatively, what if a mutated gene in one of your patients with familial hypertrophic cardiomyopathy could actually be corrected such that they were essentially cured of their disease? Although these scenarios would have been inconceivable just a few years ago, molecular genetic tools now exist in principle to modify genomes and essentially fix a variety of diseases that affect the heart and blood vessels. To achieve this seemingly impossible objective, several research groups have swiftly repurposed a bacterial system for adaptive immunity, called the CRISPR (Clustered Regularly Interspaced Palindromic Repeat)/Cas9 System, as a revolutionary genome editing tool in mammalian tissues.1 For a genome modification technology to be translatable to human disease, it must be able to navigate the complex milieu of the human genome and precisely cut the DNA only at the …

P.156 - Exome sequencing identifies a novel MYH7 mutation in a patient with nemaline myopathy and cardiomyopathy

Nemaline myopathies (NM) are rare and severe muscle diseases are characterised by the presence of nemaline bodies (rods) in the muscle biopsy. Eleven genes have been implicated in the aetiology of NM to date. Myosin 7 gene (MYH7), encoding the low/beta-cardiac myosin heavy chain (MHC-b) expressed both in skeletal muscle and the heart, has been found mutated in Laing Distal Myopathy (LDM), Myosin Storage myopathy, (MSM) and Familial Hypertrophic Cardiomyopathy (FHCM). Here we describe a 60-year-old French woman presenting with cardiomyopathy revealed by tachycardic episodes at 32 years, and mild slowly progressive muscular weakness involving pelvic girdle and hamstring muscles from her forties. A deltoid muscle biopsy at 44 years showed the presence of clusters of nemaline bodies in 20% of fibres, multiple and poorly defined irregularities of the intermyofibrillar network, and type 1 fibre disproportion. Whole-body MRI at 55 years confirmed the presence of mild global muscle involvement predominating on spine erectors, gluteus maximus, and thigh muscles. Exome sequencing disclosed a novel c.4828G > A, p.Glu1610Lys affecting the MYH7 mid-rod domain. The p.Glu1610Lys, predicted to be damaging, affects a conserved amino acid located in the MYH7 rod-tail domain, a region rarely mutated in patients with a combined cardiac and skeletal muscle involvement. By contrast these clinical and histological presentations have never been described before in association with MYH7 defects. Our study significantly broadens the clinical and pathological spectrum of MYH7-related disorders and suggests screening MYH7 gene in adult-onset nemaline myopathies with cardiomyopathy.

In vivo definition of cardiac myosin-binding protein C's critical interactions with myosin.

Cardiac myosin-binding protein C (cMyBP-C) is an integral part of the sarcomeric machinery in cardiac muscle that enables normal function. cMyBP-C regulates normal cardiac contraction by functioning as a brake through interactions with the sarcomere's thick, thin, and titin filaments. cMyBP-C's precise effects as it binds to the different filament systems remain obscure, particularly as it impacts on the myosin heavy chain's head domain, contained within the subfragment 2 (S2) region. This portion of the myosin heavy chain also contains the ATPase activity critical for myosin's function. Mutations in myosin's head, as well as in cMyBP-C, are a frequent cause of familial hypertrophic cardiomyopathy (FHC). We generated transgenic lines in which endogenous cMyBP-C was replaced by protein lacking the residues necessary for binding to S2 (cMyBP-C(S2-)). We found, surprisingly, that cMyBP-C lacking the S2 binding site is incorporated normally into the sarcomere, although systolic function is compromised. We show for the first time the acute and chronic in vivo consequences of ablating a filament-specific interaction of cMyBP-C. This work probes the functional consequences, in the whole animal, of modifying a critical structure-function relationship, the protein's ability to bind to a region of the critical enzyme responsible for muscle contraction, the subfragment 2 domain of the myosin heavy chain. We show that the binding is not critical for the protein's correct insertion into the sarcomere's architecture, but is essential for long-term, normal function in the physiological context of the heart.

Delineation of Molecular Pathways Involved in Cardiomyopathies Caused by Troponin T Mutations

Familial hypertrophic cardiomyopathy (FHC) is associated with mild to severe cardiac problems and is the leading cause of sudden death in young people and athletes. Although the genetic basis for FHC is well-established, the molecular mechanisms that ultimately lead to cardiac dysfunction are not well understood. To obtain important insights into the molecular mechanism(s) involved in FHC, hearts from two FHC troponin T models (Ile79Asn [I79N] and Arg278Cys [R278C]) were investigated using label-free proteomics and metabolomics. Mutations in troponin T are the third most common cause of FHC, and the I79N mutation is associated with a high risk of sudden cardiac death. Most FHC-causing mutations, including I79N, increase the Ca(2+) sensitivity of the myofilament; however, the R278C mutation does not alter Ca(2+) sensitivity and is associated with a better prognosis than most FHC mutations. Out of more than 1200 identified proteins, 53 and 76 proteins were differentially expressed in I79N and R278C hearts, respectively, when compared with wild-type hearts. Interestingly, more than 400 proteins were differentially expressed when the I79N and R278C hearts were directly compared. The three major pathways affected in I79N hearts relative to R278C and wild-type hearts were the ubiquitin-proteasome system, antioxidant systems, and energy production pathways. Further investigation of the proteasome system using Western blotting and activity assays showed that proteasome dysfunction occurs in I79N hearts. Metabolomic results corroborate the proteomic data and suggest the glycolytic, citric acid, and electron transport chain pathways are important pathways that are altered in I79N hearts relative to R278C or wild-type hearts. Our findings suggest that impaired energy production and protein degradation dysfunction are important mechanisms in FHCs associated with poor prognosis and that cardiac hypertrophy is not likely needed for a switch from fatty acid to glucose metabolism.

Familial hypertrophic cardiomyopathy caused by a de novo Gly716Arg mutation of the β-myosin heavy chain.

The present study was performed to identify the genotype of a hypertrophic cardiomyopathy family and investigate the clinicopathogenic characteristics and prognostic features of relevant genetic abnormalities. Target sequence capture sequencing was performed to screen for pathogenic alleles in a 32-year-old female patient (proband). Sanger sequencing was carried out to verify the results. Sanger sequencing was also performed on other family members to identify allele carriers. A survival analysis was carried out using published literature and our findings. We found that the proband and her son harboured a Gly716Arg sequence variant of the β-myosin heavy chain. Neither the proband's father nor the mother were carriers of this sequence variant; thus, the mutation was classified as "de novo". Further survival analysis revealed that female patients appear to have a longer life expectancy compared with males. Our study may provide an effective approach for the genetic diagnosis of hypertrophic cardiomyopathy.

Galliera Genetic Bank: A DNA and Cell Line Biobank from Patients Affected by Genetic Diseases

The Galliera Genetic Bank is part of the Laboratory of Human Genetics of Galliera Hospital in Genoa and has collected samples from patients affected by genetic diseases since 1983. Presently, it stores 10,259 biospecimens and associated data from about 200 genetic disorders. The most representative disorders are chromosome disorders (Down s., Ring chromosome 14 s., Cri du chat s. and Isodicentric 15 chromosome), neurological diseases (Fragile X s., Mowat Wilson s. and Dravet s.), rare bone diseases (Crouzon s., Achondroplasia, Grieg s. and Thanathophoric dwarfism), overgrowth syndromes (Sotos s.), familial hypertrophic cardiomyopathy and other rare disease such as IPEX and Aarskog s. The biobank has been supported by Italian Telethon grants since 1993 and since 2008 is partner of the Telethon Network of Genetic Biobanks. It operates according to Italian and international regulations. Since 2008 the biobank is certified ISO 9001, and in 2010 it was officially authorized by the Liguria Region to operate as a facility in support of diagnosis and research on genetic diseases. Since its inception, the biobank has offered the following services to the biomedical community: (i) access to sample and data collection; (ii) sample processing (e.g., cell lines establishment, DNA/RNA extraction, etc); (iii) preservation of biological specimens and related data (repository service), garnering more than 110 acknowledgements in scientific articles.

Identification of novel mutations including a double mutation in patients with inherited cardiomyopathy by a targeted sequencing approach using the Ion Torrent PGM system.

Inherited cardiomyopathy is the major cause of sudden cardiac death (SCD) and heart failure (HF). The disease is associated with extensive genetic heterogeneity; pathogenic mutations in cardiac sarcomere protein genes, cytoskeletal protein genes and nuclear envelope protein genes have been linked to its etiology. Early diagnosis is conducive to clinical monitoring and allows for presymptomatic interventions as needed. In the present study, the entire coding sequences and flanking regions of 12 major disease (cardiomyopathy)-related genes [namely myosin, heavy chain 7, cardiac muscle, β (MYH7); myosin binding protein C, cardiac (MYBPC3); lamin A/C (LMNA); troponin I type 3 (cardiac) (TNNI3); troponin T type 2 (cardiac) (TNNT2); actin, α, cardiac muscle 1 (ACTC1); tropomyosin 1 (α) (TPM1); sodium channel, voltage gated, type V alpha subunit (SCN5A); myosin, light chain 2, regulatory, cardiac, slow (MYL2); myosin, heavy chain 6, cardiac muscle, α (MYH6); myosin, light chain 3, alkali, ventricular, skeletal, slow (MYL3); and protein kinase, AMP-activated, gamma 2 non-catalytic subunit (PRKAG2)] in 8 patients with dilated cardiomyopathy (DCM) and in 8 patients with hypertrophic cardiomyopathy (HCM) were amplified and then sequenced using the Ion Torrent Personal Genome Machine (PGM) system. As a result, a novel heterozygous mutation (MYH7, p.Asn885Thr) and a variant of uncertain significance (TNNT2, p.Arg296His) were identified in 2 patients with HCM. These 2 missense mutations, which were absent in the samples obtained from the 200 healthy control subjects, altered the amino acid that was evolutionarily conserved among a number of vertebrate species; this illustrates that these 2 non-synonymous mutations play a role in the pathogenesis of HCM. Moreover, a double heterozygous mutation (PRKAG2, p.Gly100Ser plus MYH7, p.Arg719Trp) was identified in a patient with severe familial HCM, for the first time to the best of our knowledge. This patient provided us with more information regarding the genotype-phenotype correlation between mutations of MYH7 and PRKAG2. Taken together, these findings provide insight into the molecular mechanisms underlying inherited cardiomyopathy. The mutations identified in this study may be further investigated in the future in order to improve the diagnosis and treatment of patients with inherited cardiomyopathy. Furthermore, our findings indicated that sequencing using the Ion Torrent PGM system is a useful approach for the identification of pathogenic mutations associated with inherited cardiomyopathy, and it may be used for the risk evaluation of individuals with a possible susceptibility to inherited cardiomyopathy.

Calcium‐dependent sliding of actin thin filaments isolated from hearts with obstructive hypertrophic cardiomyopathy and donor controls does not differ

Cardiac contraction and relaxation results from the calcium‐dependent sliding of actin‐thin filaments along myosin‐thick filaments. Nearly 1 in 500 individuals are affected by familial hypertrophic cardiomyopathy (HCM) which impairs relaxation. Genetic mutations to myosin‐binding protein C (MyBP‐C) are commonly found in patients with HCM, but this disease is also associated with non‐sarcomeric etiologies. To investigate whether increases in thin filament calcium sensitivity impair relaxation in HCM from differing origins, we examined the calcium‐dependent motion of thin filaments isolated from human hearts with MyBP‐C mutations, non‐sarcomeric etiologies and donor controls. Thin filaments containing their calcium regulator proteins, troponin‐tropomyosin, were isolated from flash frozen myocardium by mechanochemical dispersion and fluorescently labeled. Myosin molecules were isolated from a single control heart and thin filament sliding on myosin was visualized using epifluorescence microscopy. At low calcium levels (&lt;100 nM free Ca2+), 95% of the thin filaments were immobilized on the surface in all 3 groups. Addition of calcium (1 nM – 0.1 mM) resulted in sigmoidal increases in the fraction of filaments moving and their sliding velocities. Neither calcium sensitivity (EC50 0.25 μM) nor maximal sliding velocities (1.2 μm/sec) differed between the groups (1 mM ATP and 30°C). These results suggest that changes in thin filament calcium sensitivity do not contribute to impaired relaxation.Support or Funding InformationFunded by APS UGSRF.

0178 : Global and regional echocardiographic strain to assess early phase of hypertrophic cardiomyopathy due to sarcomeric mutations

Hypertrophic cardiomyopathy (HCM) is a genetic disease with delayed cardiac expression. Our objective was to characterize global and regional LV myocardial strain by two-dimensional imaging in sarcomeric HCM families and hypothesized that early systolic dysfunction, before hypertrophic stage, may be diagnosed by this technique. We analyzed 79 adults: HCM patients with LV hypertrophy (LVH+, n=38), mutation carriers without LV hypertrophy (LVH-/G+, n=20), and normal control subjects (n=21). We calculated global longitudinal strain (GLS) and regional peak longitudinal strain. Age, sex ratio and body surface area were not significantly different between groups. Maximal 2D wall thickness of left ventricle was 10.1±1.6 mm in LVH-/G+ and not different from controls (9.9 ±1.2 mm). We observed that LV GLS was not different in LVH-/G+ as compared to controls (–20.6% IQ:–24.2/– 8.3 vs –22.9% IQ:-26.8/-20.9) but was reduced in HCM patients (–14.1% IQ:–18.5/–11.8) although a normal ejection fraction. Interestingly, regional peak longitudinal strain was similar in LVH-/G+ and controls except in four segments: basal anteroseptal (BAS) wall (–16% vs –19%, p=0.018), basal inferoseptal wall (–16.0% vs –19.0%, p=0.047), basal inferior wall (–21.0% vs –24.0%, p=0.006) and mid anteroseptal wall (–21.0% vs –22.5%, p=0.022). Regional BAS strain <-16.5% yielded a sensitivity of 57% and a specificity of 90% to differentiate LVH-/G+ patients from controls. We also evaluated accuracy of ratio between normal and abnormal segments. Septo-basal/Latero-basal strain ratio <0.76 yielded a sensitivity of 73% and a specificity of 92% to differentiate LVH-/G+ patients from controls. Regional longitudinal strain but not global strain, was significantly reduced at early stage of HCM before development of LVH. This suggests the integration of myocardial deformation in the evaluation of HCM relatives to identify better mutation carriers and early LV abnormalities. The author hereby declares no conflict of interest

Calcium Ions in Inherited Cardiomyopathies.

Inherited cardiomyopathies are a known cause of heart failure, although the pathways and mechanisms leading from mutation to the heart failure phenotype have not been elucidated. There is strong evidence that this transition is mediated, at least in part, by abnormal intracellular Ca(2+) handling, a key ion in ventricular excitation, contraction and relaxation. Studies in human myocytes, animal models and in vitro reconstituted contractile protein complexes have shown consistent correlations between Ca(2+) sensitivity and cardiomyopathy phenotype, irrespective of the causal mutation. In this review we present the available data about the connection between mutations linked to familial hypertrophic (HCM), dilated (DCM) and restrictive (RCM) cardiomyopathy, right ventricular arrhythmogenic cardiomyopathy/dysplasia (ARVC/D) as well as left ventricular non-compaction and the increase or decrease in Ca(2+) sensitivity, together with the results of attempts to reverse the manifestation of heart failure by manipulating Ca(2+) homeostasis.