ENerGetIcs in hypertrophic cardiomyopathy: traNslation between MRI, PET and cardiac myofilament function (ENGINE study)

Introduction

Hypertrophic cardiomyopathy (HCM) is an autosomal dominant heart disease mostly due to mutations in genes encoding sarcomeric proteins. HCM is characterised by asymmetric hypertrophy of the left ventricle (LV) in the absence of another cardiac or systemic disease. At present it lacks specific treatment to prevent or reverse cardiac dysfunction and hypertrophy in mutation carriers and HCM patients. Previous studies have indicated that sarcomere mutations increase energetic costs of cardiac contraction and cause myocardial dysfunction and hypertrophy. By using a translational approach, we aim to determine to what extent disturbances of myocardial energy metabolism underlie disease progression in HCM.

Methods

Hypertrophic obstructive cardiomyopathy (HOCM) patients and aortic valve stenosis (AVS) patients will undergo a positron emission tomography (PET) with acetate and cardiovascular magnetic resonance imaging (CMR) with tissue tagging before and 4 months after myectomy surgery or aortic valve replacement + septal biopsy. Myectomy tissue or septal biopsy will be used to determine efficiency of sarcomere contraction in-vitro, and results will be compared with in-vivo cardiac performance. Healthy subjects and non-hypertrophic HCM mutation carriers will serve as a control group.

Endpoints

Our study will reveal whether perturbations in cardiac energetics deteriorate during disease progression in HCM and whether these changes are attributed to cardiac remodelling or the presence of a sarcomere mutation per se. In-vitro studies in hypertrophied cardiac muscle from HOCM and AVS patients will establish whether sarcomere mutations increase ATP consumption of sarcomeres in human myocardium. Our follow-up imaging study in HOCM and AVS patients will reveal whether impaired cardiac energetics are restored by cardiac surgery.     

Hypertrophic cardiomyopathy：from gene defect to clinical disease

Major advances have been made over the last decade in our understanding of the molecular basis of several cardiac conditions.Hypertrophic cardiomyopathy(HCM)was the first cardiac disorder in which a genetic basis was identified and as such,has acted as a paradigm for the study of an inherited cardiac disorder.HCM can result in clinical symptoms ranging from no symptoms to severe heart failure and premature sudden death.HCM is the commonest cause of sudden death in those aged less than 35 years, including competitive athletes.At least ten genes have now been identified,defects in which cause HCM.All of these genes encode proteins which comprise the basic contractile unit of the heart,i.e.the sarcomere.While much is now known about which genes cause disease and the various clinical presentations,very little is known about how these gene defects cause disease,and what factors modify the expression of the mutant genes.Studies in both cell culture and animal models of HCM are now beginning to shed light on the signalling pathways involved in HCM,and the role of both environmental and genetic modifying factors.Understanding these mechanisms will ultimately improve our knowledge of the basic biology of heart muscle function,and will therefore provide new avenues for treating cardiovascular disease in man.     

Hypertrophic cardiornyopathy: from gene defect to clinical disease

Major advances have been made over the last decade in our understanding of the molecular basis of several cardiac conditions. Hypertrophic cardiomyopathy (HCM) was the first cardiac disorder in which a genetic basis was identified and as such, has acted as a paradigm for the study of an inherited cardiac disorder. HCM can result in clinical symptoms ranging from no symptoms to severe heart failure and premature sudden death. HCM is the commonest cause of sudden death in those aged less than 35 years, including competitive athletes. At least ten genes have now been identified, defects in which cause HCM. All of these genes encode proteins which comprise the basic contractile unit of the heart, i.e. the sarcomere. While much is now known about which genes cause disease and the various clinical presentations, very little is known about how these gene defects cause disease, and what factors modify the expression of the mutant genes. Studies in both cell culture and animal models of HCM are now beginning to     

Life-long tailoring of management for patients with hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease, characterised by complex pathophysiology and extensive genetic and clinical heterogeneity. In most patients, HCM is caused by mutations in cardiac sarcomere protein genes and inherited as an autosomal dominant trait. The clinical phenotype ranges from severe presentations at a young age to lack of left ventricular hypertrophy in genotype-positive individuals. No preventative treatment is available as the sequence and causality of the pathomechanisms that initiate and exacerbate HCM are unknown. Sudden cardiac death and end-stage heart failure are devastating expressions of this disease. Contemporary management including surgical myectomy and implantable cardiac defibrillators has shown significant impact on long-term prognosis. However, timely recognition of specific scenarios – including transition to the end-stage phase – may be challenging due to limited awareness of the progression patterns of HCM. This in turn may lead to missed therapeutic opportunities. To illustrate these difficulties, we describe two HCM patients who progressed from the typical hyperdynamic stage of asymmetric septal thickening to end-stage heart failure with severely reduced ejection fraction. We highlight the different stages of this complex inherited cardiomyopathy based on the clinical staging proposed by Olivotto and colleagues. In this way, we aim to provide a practical guide for clinicians and hope to increase awareness for this common form of cardiac disease.     

Beyond the cardiac myofilament: hypertrophic cardiomyopathy- associated mutations in genes that encode calcium-handling proteins

Traditionally regarded as a genetic disease of the cardiac sarcomere, hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disease and a significant cause of sudden cardiac death. While the most common etiologies of this phenotypically diverse disease lie in a handful of genes encoding critical contractile myofilament proteins, approximately 50% of patients diagnosed with HCM worldwide do not host sarcomeric gene mutations. Recently, mutations in genes encoding calcium-sensitive and calcium-handling proteins have been implicated in the pathogenesis of HCM. Among these are mutations in TNNC1- encoded cardiac troponin C, PLN-encoded phospholamban, and JPH2-encoded junctophilin 2 which have each been associated with HCM in multiple studies. In addition, mutations in RYR2-encoded ryanodine receptor 2, CASQ2-encoded calsequestrin 2, CALR3-encoded calreticulin 3, and SRI-encoded sorcin have been associated with HCM, although more studies are required to validate initial findings. While a relatively uncommon cause of HCM, mutations in genes that encode calcium-handling proteins represent an emerging genetic subset of HCM. Furthermore, these naturally occurring disease-associated mutations have provided useful molecular tools for uncovering novel mechanisms of disease pathogenesis, increasing our understanding of basic cardiac physiology, and dissecting important structure-function relationships within these proteins.     

Mechanical dysfunction of the sarcomere induced by a pathogenic mutation in troponin T drives cellular adaptation

Familial hypertrophic cardiomyopathy (HCM), a leading cause of sudden cardiac death, is primarily caused by mutations in sarcomeric proteins. The pathogenesis of HCM is complex, with functional changes that span scales, from molecules to tissues. This makes it challenging to deconvolve the biophysical molecular defect that drives the disease pathogenesis from downstream changes in cellular function. In this study, we examine an HCM mutation in troponin T, R92Q, for which several models explaining its effects in disease have been put forward. We demonstrate that the primary molecular insult driving disease pathogenesis is mutation-induced alterations in tropomyosin positioning, which causes increased molecular and cellular force generation during calcium-based activation. Computational modeling shows that the increased cellular force is consistent with the molecular mechanism. These changes in cellular contractility cause downstream alterations in gene expression, calcium handling, and electrophysiology. Taken together, our results demonstrate that molecularly driven changes in mechanical tension drive the early disease pathogenesis of familial HCM, leading to activation of adaptive mechanobiological signaling pathways.     

Hypertrophic cardiomyopathy in daily practice: an introduction on diagnosis,prognosis and treatment

Hypertrophic cardiomyopathy (HCM) is a complex, inherited cardiac disease that has been subject to intense investigation since it was first described in 1957. Over the past 40 years, understanding has evolved regarding the diagnosis, prognosis and treatment of HCM. Analyses of HCM populations from nonreferral centres have refined the insights into the natural history and the occurrence of sudden cardiac death, which is the most devastating component of its natural history. Therapeutic strategies are diverse and may vary during the course of the disease. Optimal therapy depends on symptoms, haemodynamic findings and the presence of risk factors for sudden cardiac death. At present, invasive therapy for patients with obstructive HCM and drug-refractory symptoms includes surgery or percutaneous transluminal septal myocardial ablation.This report summarises the diagnostic criteria, clinical course and therapeutic management of HCM. Attention is also paid to certain issues of special interest in this disease.     

Structural analysis of obscurin gene in hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a cardiac disease characterized by left ventricular hypertrophy with diastolic dysfunction. Molecular genetic studies have revealed that HCM is caused by mutations in genes for sarcomere/Z-band components including titin/connectin and its associate proteins. However, disease-causing mutations can be found in about half of the patients, suggesting that other disease-causing genes remain to be identified. To explore a novel disease gene, we searched for obscurin gene (OBSCN) mutations in HCM patients, because obscurin interacts with titin/connectin. Two linked variants, Arg4344Gln and Ala4484Thr, were identified in a patient and functional analyses demonstrated that Arg4344Gln affected binding of obscurin to Z9-Z10 domains of titin/connectin, whereas Ala4484Thr did not. Myc-tagged obscurin showed that Arg4344Gln impaired obscurin localization to Z-band. These observations suggest that the obscurin abnormality may be involved in the pathogenesis of HCM.     

Mutations in NEXN, a Z-disc gene, are associated with hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM), the most common inherited cardiac disorder, is characterized by increased ventricular wall thickness that cannot be explained by underlying conditions, cadiomyocyte hypertrophy and disarray, and increased myocardial fibrosis. In as many as 50% of HCM cases, the genetic cause remains unknown, suggesting that more genes may be involved. Nexilin, encoded by NEXN, is a cardiac Z-disc protein recently identified as a crucial protein that functions to protect cardiac Z-discs from forces generated within the sarcomere. We screened NEXN in 121 unrelated HCM patients who did not carry any mutation in eight genes commonly mutated in myofilament disease. Two missense mutations, c.391C>G (p.Q131E) and c.835C>T (p.R279C), were identified in exons 5 and 8 of NEXN, respectively, in two probands. Each of the two mutations segregated with the HCM phenotype in the family and was absent in 384 control chromosomes. In silico analysis revealed that both of the mutations affect highly conserved amino acid residues, which are predicted to be functionally deleterious. Cellular transfection studies showed that the two mutations resulted in local accumulations of nexilin and that the expressed fragment of actin-binding domain containing p.Q131E completely lost the ability to bind F-actin in C2C12 cells. Coimmunoprecipitation assay indicated that the p.Q131E mutation decreased the binding of full-length NEXN to α-actin and abolished the interaction between the fragment of actin-binding domain and α-actin. Therefore, the mutations in NEXN that we describe here may further expand the knowledge of Z-disc genes in the pathogenesis of HCM.     

Mice expressing mutant myosin heavy chains are a model for familial hypertrophic cardiomyopathy.

BACKGROUND: Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease characterized by ventricular hypertrophy, myocellular disarray, arrhythmias, and sudden death. Mutations in several contractile proteins, including cardiac myosin heavy chains, have been described in families with this disease, leading to the hypothesis that HCM is a disease of the sarcomere. MATERIALS AND METHODS: A mutation in the myosin heavy chain (Myh) predicted to interfere strongly with myosin's binding to actin was designed and used to create an animal model for HCM. Five independent lines of transgenic mice were produced with cardiac-specific expression of the mutant Myh. RESULTS: Although the mutant Myh represents a small proportion (1-12%) of the heart's myosin, the mice exhibit the cardiac histopathology seen in HCM patients. Histopathology is absent from the atria and primarily restricted to the left ventricle. The line exhibiting the highest level of mutant Myh expression demonstrates ventricular hypertrophy by 12 weeks of age, but the further course of the disease is strongly affected by the sex of the animal. Hypertrophy increases with age in female animals while the hearts of male show severe dilation by 8 months of age, in the absence of increased mass. CONCLUSIONS: The low levels of the transgene protein in the presence of the phenotypic features of HCM suggest that the mutant protein acts as a dominant negative. In addition, the distinct phenotypes developed by aging male or female transgenic mice suggest that extragenic factors strongly influence the development of the disease phenotype.     

Familial screening and genetic counselling in hypertrophic cardiomyopathy: the Rotterdam experience

Hypertrophic cardiomyopathy (HCM) is a disease characterised by unexplained left ventricular hypertrophy (LVH) (i.e. LVH in the absence of another cardiac or systemic disease that could produce a similar degree of hypertrophy), electrical instability and sudden death (SD). Germline mutations in genes encoding for sarcomere proteins are found in more than half of the cases of unexplained LVH. The autosomal dominant inherited forms of HCM are characterised by incomplete penetrance and variability in clinical and echocardiographic features, prognosis and therapeutic modalities. The identification of the genetic defect in one of the HCM genes allows accurate presymptomatic detection of mutation carriers in a family. Cardiac evaluation of at-risk relatives enables early diagnosis and identification of those patients at high risk for SD, which can be the first manifestation of the disease in asymptomatic persons. In this article we present our experience with genetic testing and cardiac screening in our HCM population and give an overview of the current literature available on this subject. (Neth Heart J 2007;15:184-9.)     

心肌肌钙蛋白T基因突变在心肌病发病机制中的研究进展

肥厚型和扩张型心肌病中,基因缺陷分别占发病的50％和35％,其病理生理机制,主要包括肌小节蛋白基因突变引起的收缩力产生缺陷,细胞骨架蛋白基因突变引起的收缩力传递缺陷等。心肌肌钙蛋白T将肌钙蛋白C和肌钙蛋白I连接到肌动蛋白和原肌球蛋白上,在心肌细胞收缩和舒张过程中发挥重要作用。在肥厚型和扩张型心肌病中发现了多种心肌肌钙蛋白T的基因突变,围绕心肌肌钙蛋白T的研究有助于阐明心肌病的发病机制。本文总结了心肌肌钙蛋白T基因突变在心肌病发病机制中的研究情况。     

A Murine Hypertrophic Cardiomyopathy Model: The DBA/2J Strain

Familial hypertrophic cardiomyopathy (HCM) is attributed to mutations in genes that encode for the sarcomere proteins, especially Mybpc3 and Myh7. Genotype-phenotype correlation studies show significant variability in HCM phenotypes among affected individuals with identical causal mutations. Morphological changes and clinical expression of HCM are the result of interactions with modifier genes. With the exceptions of angiotensin converting enzyme, these modifiers have not been identified. Although mouse models have been used to investigate the genetics of many complex diseases, natural murine models for HCM are still lacking. In this study we show that the DBA/2J (D2) strain of mouse has sequence variants in Mybpc3 and Myh7, relative to widely used C57BL/6J (B6) reference strain and the key features of human HCM. Four-month-old of male D2 mice exhibit hallmarks of HCM including increased heart weight and cardiomyocyte size relative to B6 mice, as well as elevated markers for cardiac hypertrophy including β-myosin heavy chain (MHC), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and skeletal muscle alpha actin (α1-actin). Furthermore, cardiac interstitial fibrosis, another feature of HCM, is also evident in the D2 strain, and is accompanied by up-regulation of type I collagen and α-smooth muscle actin (SMA)-markers of fibrosis. Of great interest, blood pressure and cardiac function are within the normal range in the D2 strain, demonstrating that cardiac hypertrophy and fibrosis are not secondary to hypertension, myocardial infarction, or heart failure. Because D2 and B6 strains have been used to generate a large family of recombinant inbred strains, the BXD cohort, the D2 model can be effectively exploited for in-depth genetic analysis of HCM susceptibility and modifier screens.     

Mitochondrial Haplogroups Modify the Risk of Developing Hypertrophic Cardiomyopathy in a Danish Population

Hypertrophic cardiomyopathy (HCM) is a genetic disorder caused by mutations in genes coding for proteins involved in sarcomere function. The disease is associated with mitochondrial dysfunction. Evolutionarily developed variation in mitochondrial DNA (mtDNA), defining mtDNA haplogroups and haplogroup clusters, is associated with functional differences in mitochondrial function and susceptibility to various diseases, including ischemic cardiomyopathy. We hypothesized that mtDNA haplogroups, in particular H, J and K, might modify disease susceptibility to HCM. Mitochondrial DNA, isolated from blood, was sequenced and haplogroups identified in 91 probands with HCM. The association with HCM was ascertained using two Danish control populations. Haplogroup H was more prevalent in HCM patients, 60% versus 46% (p = 0.006) and 41% (p = 0.003), in the two control populations. Haplogroup J was less prevalent, 3% vs. 12.4% (p = 0.017) and 9.1%, (p = 0.06). Likewise, the UK haplogroup cluster was less prevalent in HCM, 11% vs. 22.1% (p = 0.02) and 22.8% (p = 0.04). These results indicate that haplogroup H constitutes a susceptibility factor and that haplogroup J and haplogroup cluster UK are protective factors in the development of HCM. Thus, constitutive differences in mitochondrial function may influence the occurrence and clinical presentation of HCM. This could explain some of the phenotypic variability in HCM. The fact that haplogroup H and J are also modifying factors in ischemic cardiomyopathy suggests that mtDNA haplotypes may be of significance in determining whether a physiological hypertrophy develops into myopathy. mtDNA haplotypes may have the potential of becoming significant biomarkers in cardiomyopathy.     

A Mutation in TNNC1-encoded Cardiac Troponin C, TNNC1-A31S, Predisposes to Hypertrophic Cardiomyopathy and Ventricular Fibrillation

Defined as clinically unexplained hypertrophy of the left ventricle, hypertrophic cardiomyopathy (HCM) is traditionally understood as a disease of the cardiac sarcomere. Mutations in TNNC1-encoded cardiac troponin C (cTnC) are a relatively rare cause of HCM. Here, we report clinical and functional characterization of a novel TNNC1 mutation, A31S, identified in a pediatric HCM proband with multiple episodes of ventricular fibrillation and aborted sudden cardiac death. Diagnosed at age 5, the proband is family history-negative for HCM or sudden cardiac death, suggesting a de novo mutation. TnC-extracted cardiac skinned fibers were reconstituted with the cTnC-A31S mutant, which increased Ca(2+) sensitivity with no effect on the maximal contractile force generation. Reconstituted actomyosin ATPase assays with 50% cTnC-A31S:50% cTnC-WT demonstrated Ca(2+) sensitivity that was intermediate between 100% cTnC-A31S and 100% cTnC-WT, whereas the mutant increased the activation of the actomyosin ATPase without affecting the inhibitory qualities of the ATPase. The secondary structure of the cTnC mutant was evaluated by circular dichroism, which did not indicate global changes in structure. Fluorescence studies demonstrated increased Ca(2+) affinity in isolated cTnC, the troponin complex, thin filament, and to a lesser degree, thin filament with myosin subfragment 1. These results suggest that this mutation has a direct effect on the Ca(2+) sensitivity of the myofilament, which may alter Ca(2+) handling and contribute to the arrhythmogenesis observed in the proband. In summary, we report a novel mutation in the TNNC1 gene that is associated with HCM pathogenesis and may predispose to the pathogenesis of a fatal arrhythmogenic subtype of HCM.     

Blocking cardiac growth in hypertrophic cardiomyopathy induces cardiac dysfunction and decreased survival only in males

Mutations in myosin heavy chain (MyHC) can cause hypertrophic cardiomyopathy (HCM) that is characterized by hypertrophy, histopathology, contractile dysfunction, and sudden death. The signaling pathways involved in the pathology of HCM have not been elucidated, and an unresolved question is whether blocking hypertrophic growth in HCM may be maladaptive or beneficial. To address these questions, a mouse model of HCM was crossed with an antihypertrophic mouse model of constitutive activated glycogen synthase kinase-3beta (caGSK-3beta). Active GSK-3beta blocked cardiac hypertrophy in both male and female HCM mice. However, doubly transgenic males (HCM/GSK-3beta) demonstrated depressed contractile function, reduced sarcoplasmic (endo) reticulum Ca(2+)-ATPase (SERCA) expression, elevated atrial natriuretic factor (ANF) expression, and premature death. In contrast, female HCM/GSK-3beta double transgenic mice exhibited similar cardiac histology, function, and survival to their female HCM littermates. Remarkably, dietary modification from a soy-based diet to a casein-based diet significantly improved survival in HCM/GSK-3beta males. These findings indicate that activation of GSK-3beta is sufficient to limit cardiac growth in this HCM model and the consequence of caGSK-3beta was sexually dimorphic. Furthermore, these results show that blocking hypertrophy by active GSK-3beta in this HCM model is not therapeutic.     

Cardiolipin remodeling in Barth syndrome and other hereditary cardiomyopathies

Mitochondria play a prominent role in cardiac energy metabolism, and their function is critically dependent on the integrity of mitochondrial membranes. Disorders characterized by mitochondrial dysfunction are commonly associated with cardiac disease. The mitochondrial phospholipid cardiolipin directly interacts with a number of essential protein complexes in the mitochondrial membranes including the respiratory chain, mitochondrial metabolite carriers, and proteins critical for mitochondrial morphology. Barth syndrome is an X-linked disorder caused by an inherited defect in the biogenesis of the mitochondrial phospholipid cardiolipin. How cardiolipin deficiency impacts on mitochondrial function and how mitochondrial dysfunction causes cardiomyopathy has been intensively studied in cellular and animal models of Barth syndrome. These findings may also have implications for the molecular mechanisms underlying other inherited disorders associated with defects in cardiolipin, such as Sengers syndrome and dilated cardiomyopathy with ataxia (DCMA).     

Four and a half LIM domain protein signaling and cardiomyopathy

Four and a half LIM domain (FHL) protein family members, FHL1 and FHL2, are multifunctional proteins that are enriched in cardiac muscle. Although they both localize within the cardiomyocyte sarcomere (titin N2B), they have been shown to have important yet unique functions within the context of cardiac hypertrophy and disease. Studies in FHL1-deficient mice have primarily uncovered mitogen-activated protein kinase (MAPK) scaffolding functions for FHL1 as part of a novel biomechanical stretch sensor within the cardiomyocyte sarcomere, which acts as a positive regulator of pressure overload-mediated cardiac hypertrophy. New data have highlighted a novel role for the serine/threonine protein phosphatase (PP5) as a deactivator of the FHL1-based biomechanical stretch sensor, which has implications in not only cardiac hypertrophy but also heart failure. In contrast, studies in FHL2-deficient mice have primarily uncovered an opposing role for FHL2 as a negative regulator of adrenergic-mediated signaling and cardiac hypertrophy, further suggesting unique functions targeted by FHL proteins in the “stressed” cardiomyocyte. In this review, we provide current knowledge of the role of FHL1 and FHL2 in cardiac muscle as it relates to their actions in cardiac hypertrophy and cardiomyopathy. A specific focus will be to dissect the pathways and protein-protein interactions that underlie FHLs’ signaling role in cardiac hypertrophy as well as provide a comprehensive list of FHL mutations linked to cardiac disease, using evidence gained from genetic mouse models and human genetic studies.     

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

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