Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C). The mechanisms leading from gene mutations to the HCM phenotype remain incompletely understood, partially because current mouse models of HCM do not faithfully reflect the human situation and early hypertrophy confounds the interpretation of functional alterations. The goal of this study was to evaluate whether myofilament Ca2+ sensitization and diastolic dysfunction are associated or precede the development of left ventricular hypertrophy (LVH) in HCM. We evaluated the function of skinned and intact cardiac myocytes, as well as the intact heart in a recently developed Mybpc3-targeted knock-in mouse model carrying a point mutation frequently associated with HCM. Compared to wild-type, 10-week old homozygous knock-in mice exhibited i) higher myofilament Ca2+ sensitivity in skinned ventricular trabeculae, ii) lower diastolic sarcomere length, and faster Ca2+ transient decay in intact myocytes, and iii) LVH, reduced fractional shortening, lower E/A and E′/A′, and higher E/E′ ratios by echocardiography and Doppler analysis, suggesting systolic and diastolic dysfunction. In contrast, heterozygous knock-in mice, which mimic the human HCM situation, did not exhibit LVH or systolic dysfunction, but exhibited higher myofilament Ca2+ sensitivity, faster Ca2+ transient decay, and diastolic dysfunction. These data demonstrate that myofilament Ca2+ sensitization and diastolic dysfunction are early phenotypic consequences of Mybpc3 mutations independent of LVH. The accelerated Ca2+ transients point to compensatory mechanisms directed towards normalization of relaxation. We propose that HCM is a model for diastolic heart failure and this mouse model could be valuable in studying mechanisms and treatment modalities.
► Absence of left ventricular hypertrophy in heterozygous Mybpc3-targeted knock-in mice. ► Myofilament Ca2+ sensitization in heterozygous Mybpc3-targeted knock-in mice. ► Diastolic dysfunction independent of left ventricular hypertrophy. ► Hypertrophic cardiomyopathy as a model of diastolic heart failure.
cMyBP-C, cardiac myosin-binding protein C; cTnI, cardiac troponin I; CSQ, calsequestrin; HCM, hypertrophic cardiomyopathy; Het, heterozygous Mybpc3-targeted knock-in mice; KI, homozygous Mybpc3-targeted knock-in mice; KO, homozygous Mybpc3-targeted knock-out mice; LVH, left ventricular hypertrophy; max F, maximal Ca2+-activated force; MYBPC3, human cardiac myosin-binding protein C gene; Mybpc3, mouse cardiac myosin-binding protein C gene; NCX, Na+/Ca2+ exchanger; nH, Hill coefficient; pCa50, log of [Ca2+] required for 50% of maximal activation; PKA, cAMP-dependent protein kinase A; PLB, phospholamban; SERCA2, SR-Ca2+ ATPase; SL, sarcomere length; SR, sarcoplasmic reticulum; Ca2+ sensitivity; Ca2+ transient; Diastolic dysfunction; Hypertrophy; Mouse model
High-myofilament Ca2+-sensitivity has been proposed as trigger of disease pathogenesis in familial hypertrophic cardiomyopathy (HCM) based on in vitro and transgenic mice studies. However, myofilament Ca2+-sensitivity depends on protein phosphorylation and muscle length, and at present, data in human are scarce.
To investigate whether high-myofilament Ca2+-sensitivity and perturbed length-dependent activation are characteristics for human HCM with mutations in thick- and thin-filament proteins.
Methods and Results
Cardiac samples from patients with HCM harboring mutations in genes encoding thick (MYH7, MYBPC3) and thin (TNNT2, TNNI3, TPM1) filament proteins were compared with sarcomere mutation-negative HCM and nonfailing donors. Cardiomyocyte force measurements showed higher myofilament Ca2+-sensitivity in all HCM samples and low phosphorylation of protein kinase A (PKA)-targets compared with donors. After exogenous PKA treatment, myofilament Ca2+-sensitivity was either similar (MYBPC3mut, TPM1mut, sarcomere mutation-negative HCM), higher (MYH7mut, TNNT2mut), or even significantly lower (TNNI3mut) compared with donors. Length-dependent activation was significantly smaller in all HCM than in donor samples. PKA treatment increased phosphorylation of PKA-targets in HCM myocardium and normalized length-dependent activation to donor values in sarcomere mutation-negative HCM and HCM with truncating MYBPC3 mutations, but not in HCM with missense mutations. Replacement of mutant by wild-type troponin in TNNT2mut and TNNI3mut corrected length-dependent activation to donor values.
High-myofilament Ca2+-sensitivity is a common characteristic of human HCM and partly reflects hypophosphorylation of PKA-targets compared with donors. Length-dependent sarcomere activation is perturbed by missense mutations, possibly via post-translational modifications other than PKA-hypophosphorylation or altered protein–protein interactions, and represents a common pathomechanism in HCM.
calcium; cardiomyopathy; contractility; hypertrophy; myocardium
Mutations in sarcomere protein genes can cause hypertrophic cardiomyopathy (HCM), a disorder characterized by myocyte enlargement, fibrosis, and impaired ventricular relaxation. Here, we demonstrate that sarcomere protein gene mutations activate proliferative and profibrotic signals in non-myocyte cells to produce pathologic remodeling in HCM. Gene expression analyses of non-myocyte cells isolated from HCM mouse hearts showed increased levels of RNAs encoding cell-cycle proteins, Tgf-β, periostin, and other profibrotic proteins. Markedly increased BrdU labeling, Ki67 antigen expression, and periostin immunohistochemistry in the fibrotic regions of HCM hearts confirmed the transcriptional profiling data. Genetic ablation of periostin in HCM mice reduced but did not extinguish non-myocyte proliferation and fibrosis. In contrast, administration of Tgf-β–neutralizing antibodies abrogated non-myocyte proliferation and fibrosis. Chronic administration of the angiotensin II type 1 receptor antagonist losartan to mutation-positive, hypertrophy-negative (prehypertrophic) mice prevented the emergence of hypertrophy, non-myocyte proliferation, and fibrosis. Losartan treatment did not reverse pathologic remodeling of established HCM but did reduce non-myocyte proliferation. These data define non-myocyte activation of Tgf-β signaling as a pivotal mechanism for increased fibrosis in HCM and a potentially important factor contributing to diastolic dysfunction and heart failure. Preemptive pharmacologic inhibition of Tgf-β signals warrants study in human patients with sarcomere gene mutations.
We sought to assess the indexes of myocardial activation delay, using Doppler myocardial imaging (DMI), as potential diagnostic tools and predictors of cardiac events in patients with hypertrophic cardiomyopathy (HCM) compared with power athletes.
the distribution and magnitude of left ventricular (LV) hypertrophy are not uniform in patients with HCM, which results in heterogeneity of regional LV systolic function.
The study population comprised 70 young patients with HCM (mean (SD) age 29.4 (5.9) years) with mild septal hypertrophy (15–19 mm) and 85 age and sex matched athletes with septal thickness >12 mm, followed up for 44.4 (10.8) months. Using pulsed DMI, myocardial peak velocities, systolic time intervals, and myocardial intraventricular and interventricular systolic delays were measured in six different basal myocardial segments.
DMI analysis showed in HCM lower myocardial both systolic and early diastolic peak velocities of all the segments. Patients with HCM also showed significant interventricular and intraventricular delay (p<0.0001), whereas athletes showed homogeneous systolic activation of the ventricular walls. During the follow up, seven sudden deaths occurred in the HCM group, while no cardiovascular event was observed in the group of athletes. In patients with HCM, intraventricular delay on DMI was the most powerful independent predictor of sudden cardiac death (p<0.0001). An intraventricular delay >45 ms identified with high sensitivity and specificity patients with HCM at higher risk of ventricular tachycardia and cardiac events (test accuracy 90.6%).
DMI may be a valid supporting tool for the differential diagnosis between HCM and “athlete's heart”. In patients with HCM, DMI indexes of intraventricular delay may provide additional information for selecting subgroups of patients with HCM at increased risk of ventricular arrhythmias and sudden cardiac death at follow up. Accordingly, such patients may benefit from early intensive treatment and survey.
Doppler myocardial imaging may represent a valid supporting tool for the differential diagnosis between mild hypertrophic cardiomyopathy (HCM) and “athlete's heart”. In patients with HCM, DMI indexes of intraventricular delay may provide additional information for selecting subgroups of patients with HCM at increased risk of ventricular arrhythmias and sudden cardiac death at follow up.
Doppler myocardial imaging; hypertrophic cardiomyopathy athlete; intra‐ventricular delay; arrhythmias; prognosis; sudden cardiac death
Based on evidence that FHL2 (four and a half LIM domains protein 2) negatively regulates cardiac hypertrophy we tested whether FHL2 altered expression or variants could be associated with hypertrophic cardiomyopathy (HCM). HCM is a myocardial disease characterized by left ventricular hypertrophy, diastolic dysfunction and increased interstitial fibrosis and is mainly caused by mutations in genes coding for sarcomeric proteins. FHL2 mRNA level, FHL2 protein level and I-band-binding density were lower in HCM patients than control individuals. Screening of 121 HCM patients without mutations in established disease genes identified 2 novel (T171M, V187L) and 4 known (R177Q, N226N, D268D, P273P) FHL2 variants in unrelated HCM families. We assessed the structural and functional consequences of the nonsynonymous substitutions after adeno-associated viral-mediated gene transfer in cardiac myocytes and in 3D-engineered heart tissue (EHT). Overexpression of FHL2 wild type or nonsynonymous substitutions in cardiac myocytes markedly down-regulated α-skeletal actin and partially blunted hypertrophy induced by phenylephrine or endothelin-1. After gene transfer in EHTs, force and velocity of both contraction and relaxation were higher with T171M and V187L FHL2 variants than wild type under basal conditions. Finally, chronic phenylephrine stimulation depressed EHT function in all groups, but to a lower extent in T171M-transduced EHTs. These data suggest that (1) FHL2 is down-regulated in HCM, (2) both FHL2 wild type and variants partially protected phenylephrine- or endothelin-1-induced hypertrophy in cardiac myocytes, and (3) FHL2 T171M and V187L nonsynonymous variants induced altered EHT contractility. These findings provide evidence that the 2 novel FHL2 variants could increase cardiac function in HCM.
Electronic supplementary material
The online version of this article (doi:10.1007/s00395-014-0451-8) contains supplementary material, which is available to authorized users.
Hypertrophic cardiomyopathy; Hypertrophy; FHL2; Engineered heart tissue; Hypercontractility
To determine whether 3.0-T magnetic resonance imaging (MRI) could assess right ventricular (RV) function in patients with hypertrophic cardiomyopathy (HCM), and if this assessment is correlated with the New York Heart Function Assessment (NYHA) classification.
Materials and Methods
Forty-six patients with HCM and 23 normal individuals were recruited. Left and right ventricular function parameters including end-diastolic and end-systolic volumes (EDV, ESV), stroke volume (SV) and ejection fraction (EF) and dimensions were measured and compared using 3.0-T MRI. RV function parameters between HCM patients and controls were compared using independent sample t tests. A one way ANOVA test with Bonferroni correction was used to determine significant differences among different NYHA groups. Receiver operating characteristic analyses calculated the sensitivity and specificity of RV dysfunction on MRI for the prediction of HCM severity.
Statistical analysis revealed significant differences of left ventricular (LV) and RV volumetric values and masses between the HCM patients and controls (all p<0.05). Within the HCM group, the simultaneously decreased maximum RVEDD correlated well with the LVEDD (r = 0.53; p<0.001). The function and dimension parameters among Class I to III were not determined to be significantly different (all p>0.05). However, significant differences between the Class IV and I-III groups (all P<0.0167) indicated that the diastolic and systolic function in both the RV and LV were impaired in Class IV patients. ROC analyses identified the EDV, ESV and EDD of both the LV and RV with a high sensitivity cutoff value to predict the HCM patients with severe heart failure (Class IV) with high sensitivity and specificity.
RV involvements were comparable to those of LV global function impairments in patients with HCM. The presence of RV dysfunction and decreased dimension on the MRI helped to predict the severe symptomatic HCM with high sensitivity and specificity.
Restrictive cardiomyopathy (RCM) is a debilitating disease characterized by impaired ventricular filling, reduced ventricular volumes, and severe diastolic dysfunction. Hypertrophic cardiomyopathy (HCM) is characterized by ventricular hypertrophy and heightened risk of premature sudden cardiac death. These cardiomyopathies can result from mutations in the same gene that encodes for cardiac troponin I (cTnI). Acute genetic engineering of adult rat cardiac myocytes was used to ascertain whether primary physiologic outcomes could distinguish between RCM and HCM alleles at the cellular level. Co-transduction of cardiac myocytes with wild-type (WT) cTnI and RCM/HCM linked mutants in cTnI’s inhibitory region (IR) demonstrated that WT cTnI preferentially incorporates into the sarcomere over IR mutants. The cTnI IR mutants exhibited minor effects in single, rodent, myocyte Ca2+-activated tension assays yet prolonged relaxation and Ca2+ decay. RCM cTnI mutants in the helix-4/C-terminal region demonstrated a) hyper-sensitivity to Ca2+ under loaded conditions, b) slower myocyte mechanical relaxation and Ca2+ transient decay, c) frequency-dependent Ca2+-independent diastolic tone, d) heightened myofilament incorporation and e) irreversible cellular contractile defects with acute diltiazem administration. For species comparison, a subset of cTnI mutants were tested in isolated adult rabbit cardiac myocytes. Here, RCM and HCM mutant cTnIs exerted similar effects of slowed sarcomere length relaxation and Ca2+ transient decay but did not show variable phenotypes by cTnI region. This study highlights cellular contractile defects by cardiomyopathy mutant cTnIs that are allele and species dependent. The species dependent results in particular raise important issues toward elucidating a unifying mechanistic pathway underling the inherited cardiomyopathies.
inherited cardiomyopathy; cardiac troponin I; myocyte; contraction
Hypertrophic cardiomyopathy (HCM) has been recently recognized as the most common inherited cardiovascular disorder, affecting 1 in 500 adults worldwide. HCM is characterized by myocyte hypertrophy resulting in thickening of the ventricular wall, myocyte disarray, interstitial and/or replacement fibrosis, decreased ventricular cavity volume and diastolic dysfunction. HCM is also the most common cause of sudden death in the young. A large proportion of patients diagnosed with HCM have mutations in sarcomeric proteins. However, it is unclear how these mutations lead to the cardiac phenotype, which is variable even in patients carrying the same causal mutation. Abnormalities in calcium cycling, oxidative stress, mitochondrial dysfunction and energetic deficiency have been described constituting the basis of therapies in experimental models of HCM and HCM patients. This review focuses on evidence supporting the role of cellular metabolism and mitochondria in HCM.
hypertrophic cardiomyopathy; mitochondria; calcium handling; bioenergetic deficit; induced pluripotent stem cells (iPSCs)
We tested the hypothesis that the apical myocardial mechanics differ from those of other ventricular segments in hypertensive patients with and without apical hypertrophic cardiomyopathy (ApHCM).
We retrospectively studied hypertensive patients with and without ApHCM. Left ventricular longitudinal, circumferential, and radial strains were examined by two-dimensional speckle-tracking echocardiography at the basal, middle, and apical walls of the parasternal short-axis and apical 2-, 3- and 4-chamber views.
Fourteen consecutive patients with hypertension and ApHCM and 14 patients with hypertension without ApHCM were studied. Lower mitral annular peak systolic velocity and greater diastolic dysfunction were present in hypertensive patients with ApHCM than in hypertensive patients without ApHCM. Compared with hypertensive patients without ApHCM, hypertensive patients with ApHCM had significantly lower apical longitudinal (−13.9% vs −21.9%, p = 0.010) and radial strains (4.4% vs 11.5%, p = 0.017) without the base-to-apex gradient. The global longitudinal (−15.6% vs −18.8%, p = 0.027) and circumferential strains (−16.1% vs −19.2%, p = 0.019) were significantly lower in hypertensive patients with ApHCM than in hypertensive patients without ApHCM. Among systolic parameters, the global longitudinal strain was independently associated with hypertension with ApHCM (odds ratio, 1.457; 95% confidence interval, 1.002–2.119; p = 0.049).
Reduced apical longitudinal and radial strains without a base-to-apex gradient were present in hypertensive patients with ApHCM. The global longitudinal strain was independently associated with ApHCM in hypertensive patients.
Hypertrophic cardiomyopathy; Echocardiography; Left ventricular function
Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) are classic forms of systolic and diastolic heart failure, respectively. Mutations in genes encoding sarcomere and cytoskeletal proteins are major causes of HCM and DCM. MURC, encoding muscle-restricted coiled-coil, a Z line protein, regulates cardiac function in mice. We investigated potential causal role of MURC in human cardiomyopathies.
Methods and Results
We sequenced MURC in 1,199 individuals including 383 probands with DCM, 307 with HCM and 509 healthy controls. We found six heterozygous DCM-specific missense variants (p.N128K, p.R140W, p.L153P, p.S307T, p.P324L and p.S364L) in eight unrelated probands. Variants p.N128K and p.S307T segregated with inheritance of DCM in small families (χ2=8.5, p=0.003). Variants p.N128K, p.R140W, p.L153P and p.S364L were considered probably or possibly damaging. Variant p.P324L recurred in three independent probands, including one proband with a TPM1 mutation (p.M245T). A deletion variant (p.L232-R238del) was present in three unrelated HCM probands but it did not segregate with HCM in a family who also had a MYH7 mutation (p.L970V). The phenotype in mutation carriers was notable for progressive heart failure leading to heart transplantation in four patients, conduction defects and atrial arrhythmias. Expression of mutant MURC proteins in neonatal rat cardiac myocytes transduced with recombinant adenoviruses was associated with reduced RhoA activity, lower mRNA levels of hypertrophic markers and smaller myocyte size as compared to wild type MURC.
MURC mutations impart loss-of-function effects on MURC functions and are likely causal variants in human DCM. The causal role of a deletion mutation in HCM is uncertain.
heart failure; genetics; cardiomyopathy; mutation; RhoA
Hypertrophic Cardiomyopathy (HCM) is a common primary cardiac disorder defined by a hypertrophied left ventricle, is one of the main causes of sudden death in young athletes and has been associated with mutations in most sarcomeric proteins (tropomyosin, Troponin T and I, and actin, etc.). Many of these mutations appear to affect the functional properties of cardiac troponin C (cTnC), i.e., by increasing the Ca2+-sensitivity of contraction, a hallmark of HCM, and surprisingly, prior to this report, cTnC had not been classified as a HCM susceptibility gene. In this study, we show that mutations occurring in the human cTnC (HcTnC) gene (TNNC1) have the same prevalence (~0.4%) as well established HCM-susceptibility genes that encode other sarcomeric proteins. Comprehensive open reading frame/splice site mutation analysis of TNNC1 performed on 1025 unrelated HCM patients over the last 10 years revealed novel missense mutations in TNNC1: A8V, C84Y, E134D, and D145E. Functional studies with these recombinant HcTnC HCM mutations showed increased Ca2+ sensitivity of force development (A8V, C84Y and D145E) and force recovery (A8V and D145E). These results are consistent with the HCM functional phenotypes seen with other sarcomeric HCM mutations (E134D showed no changes in these parameters). This is the largest cohort analysis of TNNC1 in HCM that details the discovery of at least three novel HCM-associated mutations and more strongly links TNNC1 to HCM along with functional evidence that supports a central role for its involvement in the disease. These types of studies may help to further define TNNC1 as an HCM-susceptibility gene that has already been established for the other members of the Troponin complex.
troponin C; TnC; hypertrophic cardiomyopathy; HCM; mutation; calcium; genetics
Hypertrophic cardiomyopathy (HCM) is diagnosed clinically by the presence of left ventricular hypertrophy (LVH). However, LVH is absent in a significant number of genotype-positive patients. Because myocyte dysfunction and disarray are the primary abnormalities in HCM, we reasoned that tissue Doppler imaging could identify contraction and relaxation abnormalities, irrespective of hypertrophy, in a transgenic rabbit model of human HCM.
Methods and Results
M-mode, 2D, Doppler echocardiography and tissue Doppler imaging were performed in nontransgenic (n=24), wild-type β-myosin heavy chain-arginine403 (n=14), and mutant β-myosin heavy chain-glutamic acid403 (n=24) transgenic rabbits. Mean septal thicknesses were 2.0±0.3, 2.0±0.25, and 2.75±0.3 mm in the 3 groups, respectively (P=0.001). LVH was absent in 9 of the 24 mutant rabbits. Left ventricular dimensions, systolic function, heart rate, mitral inflow velocities, and time intervals were similar in the groups. However, the difference between atrial reversal and transmitral A wave duration was increased in the mutant rabbits (P<0.001). More importantly, systolic and early diastolic tissue Doppler velocities were significantly lower in all mutant rabbits (7.45±2.2 versus 10.8±2.3 cm/s in nontransgenic and 9.0±0.76 cm/s in wild-type; P<0.001), including the 9 without LVH. A systolic velocity <8.5 cm/s had an 86% sensitivity and 100% specificity in identifying the mutant transgenic rabbits.
Myocardial contraction and relaxation were reduced in the mutant β-myosin heavy chain-glutamic acid403 transgenic rabbit model of human HCM, irrespective of the presence or absence of LVH. In addition, tissue Doppler imaging is more sensitive than conventional echocardiography for HCM screening.
cardiomyopathy; hypertrophy; genetics; echocardiography; imaging
Hypertrophic cardiomyopathy (HCM), the most common cause of sudden cardiac death in the young, is characterized by a diverse array of cardiac phenotypes evolving over several decades. We have developed transgenic rabbits that fully recapitulate the phenotype of human HCM and provide for the opportunity to delineate the sequence of evolution of cardiac phenotypes, and thus, the pathogenesis of HCM.
We determined evolution of biochemical, molecular, histological, structural and functional phenotypes at 4 age-periods in 47 β-myosin heavy chain-glutamine (MyHC-Q)-403 transgenic rabbits. Ca+2 sensitivity of myofibrillar ATPase activity was reduced very early and in the absence of other discernible phenotypes. Myocyte disarray also occurred early, prior to, and independent of hypertrophy and fibrosis. The latter phenotypes evolved predominantly during puberty in conjunction with activation of stress-related signaling kinases. Myocardial contraction and relaxation velocities were decreased early despite normal global cardiac function and in the absence of histological phenotype. Global cardiac function declined with aging, while left atrial size was increased along with Doppler indices of left ventricular filling pressure.
Thus, Ca+2 sensitivity of myofibrillar ATPase activity is a primary phenotype expressed early and independent of the ensuing phenotypes. Pathogenesis of myocyte disarray, which exhibits age-independent penetrance, differs from those of hypertrophy and fibrosis, which show age-dependent expression. Myocardial dysfunction is an early marker that predicts subsequent development of hypertrophy. These findings in an animal model that recapitulates the phenotype of human HCM, implicate involvement of multiple independent mechanisms in the pathogenesis of cardiac phenotypes in HCM.
Transgenic animal models; Cardiomyopathy; Hypertrophy; Genetics; ATPase; Echocardiography; Tissue Doppler
Background: Hypertrophic cardiomyopathy (HCM), an auto-somal dominant disorder due to mutation of genes encoding sarcomeric proteins, leads to left ventricular diastolic dysfunction. Recently, the research in this area suggests that systolic dysfunction exists in the patients with HCM even though traditional measures of systolic dysfunction are normal. So, we carried out this study to determine global systolic dysfunction in patients with HCM.
Materials and Methods: A total of 18 patients, diagnosed with HCM according to echocardiography parameters, that is thickness of interventricular septum/posterior wall thickness >1.3 or hypertrophy involving apex only with or without left ventricular outflow tract obstruction, were included in the study and were compared with normal age-matched controls. We measured torsion and strain imaging by 2-dimensional echocardiography as well as strain imaging by tissue Doppler echocardiography.
Result: The results of the study showed that there was considerable increased torsion in patients with HCM as compared to normal subjects (16.61±7.43 vs. 10.42±4.73, p=0.006). Tissue Doppler indices—systolic annular velocity (7.7±0.7 vs. 8.7±1.00, p=0.012) and lateral wall E/E’ (12.52±5.27 vs. 6.66±1.67, p<0.001) were significantly different in patients with HCM and normal subjects. The average systolic strain and strain rate as well as diastolic strain rate were significantly different in both the groups when strain imaging was performed by tissue Doppler echocardiography. We also observed significantly reduced global longitudinal, circumferential and radial strain in patients with HCM when strain analysis was carried out with 2-dimensional speckle tracking echocardiography.
Conclusion: The global subtle systolic dysfunction, as measured by left ventricular torsion and strain imaging, is present in patients with HCM even though traditional measure of systolic dysfunction is normal.
Global systolic dysfunction; Hypertrophic cardiomyopathy; Left-ventricular torsion; Strain imaging
Advances in molecular genetics of hypertrophic cardiomyopathy (HCM) have led to identification of mutations in 11 genes coding for sarcomeric proteins. In addition, mutations in gene coding for the γ subunit of AMP-activated protein kinase and triplet-repeat syndromes, as well as in mitochondrial DNA have been identified in patients with HCM. Mutations in genes coding for the β-myosin heavy chain, myosin binding protein-C, and cardiac troponin T account for approximately 2/3 of all HCM cases. Accordingly, HCM is considered a disease of contractile sarcomeric proteins. Genotype-phenotype correlation studies show mutations and the genetic background affect the phenotypic expression of HCM. The final phenotype is the result of interactions between the causal genes, genetic background (modifier genes), and probably the environmental factors. The molecular pathogenesis of HCM is not completely understood. The initial defects caused by the mutant proteins are diverse. However, despite their diversity, they converge into common final pathway of impaired cardiac myocyte function. The latter leads to an increased myocyte stress and subsequent activation of stress-responsive signaling kinases and trophic factors, which activate the transcriptional machinery inducing cardiac hypertrophy, interstitial fibrosis and myocyte disarray, the pathological characteristics of HCM. Studies in transgenic animal models show that cardiac hypertrophy, interstitial fibrosis, and myocyte disarray are potentially reversible. These findings raise the possibility of reversal of evolving phenotype or prevention of phenotypes in human patients with HCM. Elucidation of the molecular genetic basis and the pathogenesis of HCM could provide the opportunity for genetic based diagnosis, risk stratification, and implementation of preventive and therapeutic measures in those who have inherited the causal mutations for HCM.
Cardiomyopathy; hypertrophic; genetics - Genes - Mutation - Death; sudden; cardiac
Cardiac hypertrophy, the clinical hallmark of hypertrophic cardiomyopathy (HCM), is a major determinant of morbidity and mortality not only in HCM but also in a number of cardiovascular diseases. There is no effective therapy for HCM and generally for cardiac hypertrophy. Myocardial oxidative stress and thiol-sensitive signaling molecules are implicated in pathogenesis of hypertrophy and fibrosis. We posit that treatment with N-acetylcysteine, a precursor of glutathione, the largest intracellular thiol pool against oxidative stress, could reverse cardiac hypertrophy and fibrosis in HCM.
Methods and Results
We treated 2-year-old β-myosin heavy-chain Q403 transgenic rabbits with established cardiac hypertrophy and preserved systolic function with N-acetylcysteine or a placebo for 12 months (n = 10 per group). Transgenic rabbits in the placebo group had cardiac hypertrophy, fibrosis, systolic dysfunction, increased oxidized to total glutathione ratio, higher levels of activated thiol-sensitive active protein kinase G, dephosphorylated nuclear factor of activated T cells (NFATc1) and phospho-p38, and reduced levels of glutathiolated cardiac α-actin. Treatment with N-acetylcysteine restored oxidized to total glutathione ratio, normalized levels of glutathiolated cardiac α-actin, reversed cardiac and myocyte hypertrophy and interstitial fibrosis, reduced the propensity for ventricular arrhythmias, prevented cardiac dysfunction, restored myocardial levels of active protein kinase G, and dephosphorylated NFATc1 and phospho-p38.
Treatment with N-acetylcysteine, a safe prodrug against oxidation, reversed established cardiac phenotype in a transgenic rabbit model of human HCM. Because there is no effective pharmacological therapy for HCM and given that hypertrophy, fibrosis, and cardiac dysfunction are common and major predictors of clinical outcomes, the findings could have implications in various cardiovascular disorders.
antioxidants; cardiomyopathy; fibrosis; genetics; hypertrophy
Mutations in myofilament proteins, most commonly MYBPC3-encoded myosin binding protein C and MYH7-encoded β-myosin heavy chain, can cause hypertrophic cardiomyopathy (HCM). Despite significant advances in structure-function relationships pertaining to the cardiac sarcomere, there is limited knowledge of how a mutation leads to clinical HCM. We therefore set out to study expression and localization of myofilament proteins in left ventricular tissue of patients with HCM.
Methods and Results
Frozen surgical myectomy specimens from 47 patients with HCM were examined and genotyped for mutations involving 8 myofilament-encoding genes. Myofilament protein levels were quantified by western blot with localization graded from immunohistochemical staining of tissue sections. Overall, 25/47 (53%) patients had myofilament-HCM including 12 with MYBPC3-HCM and 9 with MYH7-HCM. Compared to healthy heart tissue, levels of myofilament proteins were increased in patients manifesting a mutation in either gene. Patients with a frameshift mutation predicted to truncate MYBPC3 exhibited marked disturbances in protein localization as compared to missense mutations in either MYBPC3 or MYH7.
In this first expression study in human HCM tissue, increased myofilament protein levels in patients with either MYBPC3 or MYH7-mediated HCM suggest a poison peptide mechanism. Specifically, the mechanism of dysfunction may vary according to the genetic subgroup suggested by a distinctly abnormal distribution of myofilament proteins in patients manifesting a truncation mutation in MYBPC3.
Cardiomyopathy; Hypertrophy; Genetics; Protein
To investigate the role of inflammation in the phenotypic expression of myocardial fibrosis in hypertrophic cardiomyopathy (HCM).
Kuopio University Hospital and University of Eastern Finland, Kuopio, Finland.
Twenty-four patients with a single HCM-causing mutation D175N in the α-tropomyosin gene and 17 control subjects.
Main outcome measures
Endomyocardial biopsy samples taken from the patients with HCM were compared with matched myocardial autopsy specimens. Levels of high-sensitivity C-reactive protein (hsCRP) and proinflammatory cytokines were measured in patients and controls. Myocardial late gadolinium enhancement (LGE) in cardiac MRI (CMRI) was detected.
Endomyocardial samples in patients with HCM showed variable myocyte hypertrophy and size heterogeneity, myofibre disarray, fibrosis, inflammatory cell infiltration and nuclear factor kappa B (NF-κB) activation. Levels of hsCRP and interleukins (IL-1β, IL-1RA, IL-6, IL-10) were significantly higher in patients with HCM than in control subjects. In patients with HCM, there was a significant association between the degree of myocardial inflammatory cell infiltration, fibrosis in histopathological samples and myocardial LGE in CMRI. Levels of hsCRP were significantly associated with histopathological myocardial fibrosis. hsCRP, tumour necrosis factor α and IL-1RA levels had significant correlations with LGE in CMRI.
A variable myocardial and systemic inflammatory response was demonstrated in patients with HCM attributable to an identified sarcometric mutation. Inflammatory response was associated with myocardial fibrosis, suggesting that myocardial fibrosis in HCM is an active process modified by an inflammatory response.
Hypertrophic cardiomyopathy; inflammation; fibrosis; genetics; late gadolinium enhancement; coronary angioplasty; aortic stenosis; invasive cardiology; coronary artery disease; cardiomyopathy hypertrophic; tissue characters; HCM; MRI; myocardial function; myocardial perfusion; myocardial ischaemia; myocardial infarction; arrhythmias; endocrinology
Mild hypertrophy but increased arrhythmic risk characterizes the stereotypic phenotype proposed for hypertrophic cardiomyopathy (HCM) caused by thin-filament mutations. However, whether such clinical profile is different from more prevalent thick-filament–associated disease is unresolved.
This study aimed to assess clinical features and outcomes in a large cohort of patients with HCM associated with thin-filament mutations compared with thick-filament HCM.
Adult HCM patients (age >18 years), 80 with thin-filament and 150 with thick-filament mutations, were followed for an average of 4.5 years.
Compared with thick-filament HCM, patients with thin-filament mutations showed: 1) milder and atypically distributed left ventricular (LV) hypertrophy (maximal wall thickness 18 ± 5 mm vs. 24 ± 6 mm; p < 0.001) and less prevalent outflow tract obstruction (19% vs. 34%; p = 0.015); 2) higher rate of progression to New York Heart Association functional class III or IV (15% vs. 5%; p = 0.013); 3) higher prevalence of systolic dysfunction or restrictive LV filling at last evaluation (20% vs. 9%; p = 0.038); 4) 2.4-fold increase in prevalence of triphasic LV filling pattern (26% vs. 11%; p = 0.002); and 5) similar rates of malignant ventricular arrhythmias and sudden cardiac death (p = 0.593).
In adult HCM patients, thin-filament mutations are associated with increased likelihood of advanced LV dysfunction and heart failure compared with thick-filament disease, whereas arrhythmic risk in both subsets is comparable. Triphasic LV filling is particularly common in thin-filament HCM, reflecting profound diastolic dysfunction.
diastolic function; end-stage; genotype to phenotype correlation; triphasic filling; troponin; ACTC, cardiac α-actin gene; AF, atrial fibrillation; CMR, cardiac magnetic resonance; ECG, electrocardiography; HCM, hypertrophic cardiomyopathy; HR, hazard ratio; ICD, implantable cardioverter-defibrillator; LGE, late gadolinium enhancement; LV, left ventricular; LVH, left ventricular hypertrophy; MYBPC3, myosin binding protein C; MYH7, myosin heavy chain; NSVT, nonsustained ventricular tachycardia; NYHA, New York Heart Association; SCD, sudden cardiac death; TNNT2, cardiac troponin T gene; TNNI3, cardiac troponin I gene; TPM1, cardiac α-tropomyosin gene
To compare the extent and distribution of focal fibrosis by gadolinium contrast‐enhanced magnetic resonance imaging (MRI; delayed hyperenhancement) in severe left ventricular (LV) hypertrophy in patients with pressure overload caused by aortic stenosis (AS) and with genetically determined hypertrophic cardiomyopathy (HCM).
44 patients with symptomatic valvular AS (n = 22) and HCM (n = 22) were studied. Cine images were acquired with fast imaging with steady‐state precession (trueFISP) on a 1.5 T scanner (Sonata, Siemens Medical Solutions). Gadolinium contrast‐enhanced MRI was performed with a segmented inversion–recovery sequence. The location, extent and enhancement pattern of hyperenhanced myocardium was analysed in a 12‐segment model.
Mean LV mass was 238.6 (SD 75.3) g in AS and 205.4 (SD 80.5) g in HCM (p = 0.17). Hyperenhancement was observed in 27% of patients with AS and in 73% of patients with HCM (p < 0.01). In AS, hyperenhancement was observed in 60% of patients with a maximum diastolic wall thickness ⩾ 18 mm, whereas no patient with a maximum diastolic wall thickness < 18 mm had hyperenhancement (p < 0.05). Patients with hyperenhancement had more severe AS than patients without hyperenhancement (aortic valve area 0.80 (0.09) cm2v 0.99 (0.3) cm2, p < 0.05; maximum gradient 98 (22) mm Hg v 74 (24) mm Hg, p < 0.05). In HCM, hyperenhancement was predominant in the anteroseptal regions and patients with hyperenhancement had higher end diastolic (125.4 (36.9) ml v 98.8 (16.9) ml, p < 0.05) and end systolic volumes (38.9 (18.2) ml v 25.2 (1.7) ml, p < 0.05). The volume of hyperenhancement (percentage of total LV myocardium), where present, was lower in AS than in HCM (4.3 (1.9)% v 8.6 (7.4)%, p< 0.05). Hyperenhancement was observed in 4.5 (3.1) and 4.6 (2.7) segments in AS and HCM, respectively (p = 0.93), and the enhancement pattern was mostly patchy with multiple foci.
Focal scarring can be observed in severe LV hypertrophy caused by AS and HCM, and correlates with the severity of LV remodelling. However, focal scarring is significantly less prevalent in adaptive LV hypertrophy caused by AS than in genetically determined HCM.
aortic stenosis; focal fibrosis; hypertrophic cardiomyopathy; magnetic resonance imaging
Patient: Male, 2
Final Diagnosis: Obstructive hypertrophic cardiomyopathy
Symptoms: Congestive heart failure
Clinical Procedure: Left ventricular septal myectomy • repair of congenital heart disease
Hypertrophic cardiomyopathy (HCM) is uncommon in Down syndrome (DS). When combined with congenital heart disease (CHD) both morbidity and mortality may be greater compared to CHD alone. Whether HCM in DS patients is related to having trisomy 21 versus a second site mutation is unknown.
We report a case of severe HCM in an infant with DS in combination with double outlet right ventricle (DORV) who required surgery for relive of sub-aortic obstruction and congestive heart failure. We predicted that this infant would have a second site mutation involving either a sarcomeric protein or metabolic disorder as a cause for his HCM. Using current genetic and metabolic testing as well as histologic assessment of excised cardiac tissue we sought to further characterize the nature of the HCM. A successful resection of sub-aortic stenosis and DORV repair was performed. Genetic and metabolic testing was negative for gene defects and/or syndromes commonly associated with familial HCM. Excised cardiac tissue from the ventricular septum exhibited myocyte hypertrophy and sub-endocardial fibrosis but no sarcomeric disarray, myocyte fibrosis or glycogen storage. Metabolic testing for common forms of mitochondrial disease was negative. Post-operative echocardiograms show persistent, non-obstructive septal hypertrophy.
Unlike prior reports, this child required a surgical intervention to relieve his sub-aortic obstruction. Thus, HCM in this population can be more serious that previously suspected. Although testing did not reveal the cause of his HCM, we still suggest screening for known causes of HSC until the etiology of the HCM in DS is well understood.
conal-truncal defect; hypertrophic cardiomyopathy; Down syndrome
We sought to evaluate the relation between atrial fibrillation (AF) and the extent of myocardial scarring together with left ventricular (LV) and atrial parameters assessed by late gadolinium-enhancement (LGE) cardiovascular magnetic resonance (CMR) in patients with hypertrophic cardiomyopathy (HCM).
AF is the most common arrhythmia in HCM. Myocardial scarring is also identified frequently in HCM. However, the impact of myocardial scarring assessed by LGE CMR on the presence of AF has not been evaluated yet.
87 HCM patients underwent LGE CMR, echocardiography and regular ECG recordings. LV function, volumes, myocardial thickness, left atrial (LA) volume and the extent of LGE, were assessed using CMR and correlated to AF. Additionally, the presence of diastolic dysfunction and mitral regurgitation were obtained by echocardiography and also correlated to AF.
Episodes of AF were documented in 37 patients (42%). Indexed LV volumes and mass were comparable between HCM patients with and without AF. However, indexed LA volume was significantly higher in HCM patients with AF than in HCM patients without AF (68 ± 24 ml·m-2 versus 46 ± 18 ml·m-2, p = 0.0002, respectively). The mean extent of LGE was higher in HCM patients with AF than those without AF (12.4 ± 14.5% versus 6.0 ± 8.6%, p = 0.02). When adjusting for age, gender and LV mass, LGE and indexed LA volume significantly correlated to AF (r = 0.34, p = 0.02 and r = 0.42, p < 0.001 respectively). By echocardiographic examination, LV diastolic dysfunction was evident in 35 (40%) patients. Mitral regurgitation greater than II was observed in 12 patients (14%). Multivariate analysis demonstrated that LA volume and presence of diastolic dysfunction were the only independent determinant of AF in HCM patients (p = 0.006, p = 0.01 respectively). Receiver operating characteristic curve analysis indicated good predictive performance of LA volume and LGE (AUC = 0.74 and 0.64 respectively) with respect to AF.
HCM patients with AF display significantly more LGE than HCM patients without AF. However, the extent of LGE is inferior to the LA size for predicting AF prevalence. LA dilation is the strongest determinant of AF in HCM patients, and is related to the extent of LGE in the LV, irrespective of LV mass.
RNA trans-splicing has been explored as a therapeutic option for a variety of genetic diseases, but not for cardiac genetic disease. Hypertrophic cardiomyopathy (HCM) is an autosomal-dominant disease, characterized by left ventricular hypertrophy (LVH) and diastolic dysfunction. MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C) is frequently mutated. We evaluated the 5′-trans-splicing strategy in a mouse model of HCM carrying a Mybpc3 mutation. 5′-trans-splicing was induced between two independently transcribed molecules, the mutant endogenous Mypbc3 pre-mRNA and an engineered pre-trans-splicing molecule (PTM) carrying a FLAG-tagged wild-type (WT) Mybpc3 cDNA sequence. PTMs were packaged into adeno-associated virus (AAV) for transduction of cultured cardiac myocytes and the heart in vivo. Full-length repaired Mybpc3 mRNA represented up to 66% of total Mybpc3 transcripts in cardiac myocytes and 0.14% in the heart. Repaired cMyBP-C protein was detected by immunoprecipitation in cells and in vivo and exhibited correct incorporation into the sarcomere in cardiac myocytes. This study provides (i) the first evidence of successful 5′-trans-splicing in vivo and (ii) proof-of-concept of mRNA repair in the most prevalent cardiac genetic disease. Since current therapeutic options for HCM only alleviate symptoms, these findings open new horizons for causal therapy of the severe forms of the disease.
hypertrophic cardiomyopathy; Mybpc3; RNA-based therapy; trans-splicing
Cardiac diffusion tensor imaging (cDTI) measures the magnitudes and directions of intramyocardial water diffusion. Assuming the cross-myocyte components to be constrained by the laminar microstructures of myocardium, we hypothesized that cDTI at two cardiac phases might identify any abnormalities of laminar orientation and mobility in hypertrophic cardiomyopathy (HCM).
We performed cDTI in vivo at 3 Tesla at end-systole and late diastole in 11 healthy controls and 11 patients with HCM, as well as late gadolinium enhancement (LGE) for detection of regional fibrosis.
Voxel-wise analysis of diffusion tensors relative to left ventricular coordinates showed expected transmural changes of myocardial helix-angle, with no significant differences between phases or between HCM and control groups. In controls, the angle of the second eigenvector of diffusion (E2A) relative to the local wall tangent plane was larger in systole than diastole, in accord with previously reported changes of laminar orientation. HCM hearts showed higher than normal global E2A in systole (63.9° vs 56.4° controls, p = 0.026) and markedly raised E2A in diastole (46.8° vs 24.0° controls, p < 0.001). In hypertrophic regions, E2A retained a high, systole-like angulation even in diastole, independent of LGE, while regions of normal wall thickness did not (LGE present 57.8°, p = 0.0028, LGE absent 54.8°, p = 0.0022 vs normal thickness 38.1°).
In healthy controls, the angles of cross-myocyte components of diffusion were consistent with previously reported transmural orientations of laminar microstructures and their changes with contraction. In HCM, especially in hypertrophic regions, they were consistent with hypercontraction in systole and failure of relaxation in diastole. Further investigation of this finding is required as previously postulated effects of strain might be a confounding factor.
Electronic supplementary material
The online version of this article (doi:10.1186/s12968-014-0087-8) contains supplementary material, which is available to authorized users.
Diffusion tensor imaging; Hypertrophic cardiomyopathy; Cardiovascular magnetic resonance; Myocardial architecture; Laminar structure; Sheet and shear layers; Diastolic dysfunction
Hypertrophic cardiomyopathy (HCM) is the most common genetic disease of the heart. HCM is characterized by a wide range of clinical expression, ranging from asymptomatic mutation carriers to sudden cardiac death as the first manifestation of the disease. Over 1000 mutations have been identified, classically in genes encoding sarcomeric proteins. Noninvasive imaging is central to the diagnosis of HCM and cardiovascular magnetic resonance (CMR) is increasingly used to characterize morphologic, functional and tissue abnormalities associated with HCM. The purpose of this review is to provide an overview of the clinical, pathological and imaging features relevant to understanding the diagnosis of HCM. The early and overt phenotypic expression of disease that may be identified by CMR is reviewed. Diastolic dysfunction may be an early marker of the disease, present in mutation carriers prior to the development of left ventricular hypertrophy (LVH). Late gadolinium enhancement by CMR is present in approximately 60% of HCM patients with LVH and may provide novel information regarding risk stratification in HCM. It is likely that integrating genetic advances with enhanced phenotypic characterization of HCM with novel CMR techniques will importantly improve our understanding of this complex disease.