A Tension-Based Model Distinguishes Hypertrophic versus Dilated Cardiomyopathy.

The heart either hypertrophies or dilates in response to familial mutations in genes encoding sarcomeric proteins, which are responsible for contraction and pumping. These mutations typically alter calcium-dependent tension generation within the sarcomeres, but how this translates into the spectrum of hypertrophic versus dilated cardiomyopathy is unknown. By generating a series of cardiac-specific mouse models that permit the systematic tuning of sarcomeric tension generation and calcium fluxing, we identify a significant relationship between the magnitude of tension developed over time and heart growth. When formulated into a computational model, the integral of myofilament tension development predicts hypertrophic and dilated cardiomyopathies in mice associated with essentially any sarcomeric gene mutations, but also accurately predicts human cardiac phenotypes from data generated in induced-pluripotent-stem-cell-derived myocytes from familial cardiomyopathy patients. This tension-based model also has the potential to inform pharmacologic treatment options in cardiomyopathy patients.

Telomere shortening is a hallmark of genetic cardiomyopathies.

This study demonstrates that significantly shortened telomeres are a hallmark of cardiomyocytes (CMs) from individuals with end-stage hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy (DCM) as a result of heritable defects in cardiac proteins critical to contractile function. Positioned at the ends of chromosomes, telomeres are DNA repeats that serve as protective caps that shorten with each cell division, a marker of aging. CMs are a known exception in which telomeres remain relatively stable throughout life in healthy individuals. We found that, relative to healthy controls, telomeres are significantly shorter in CMs of genetic HCM and DCM patient tissues harboring pathogenic mutations: TNNI3, MYBPC3, MYH7, DMD, TNNT2, and TTN Quantitative FISH (Q-FISH) of single cells revealed that telomeres were significantly reduced by 26% in HCM and 40% in DCM patient CMs in fixed tissue sections compared with CMs from age- and sex-matched healthy controls. In the cardiac tissues of the same patients, telomere shortening was not evident in vascular smooth muscle cells that do not express or require the contractile proteins, an important control. Telomere shortening was recapitulated in DCM and HCM CMs differentiated from patient-derived human-induced pluripotent stem cells (hiPSCs) measured by two independent assays. This study reveals telomere shortening as a hallmark of genetic HCM and DCM and demonstrates that this shortening can be modeled in vitro by using the hiPSC platform, enabling drug discovery.

Changes in the dynamics of the cardiac troponin C molecule explain the effects of Ca2+-sensitizing mutations.

Cardiac troponin C (cTnC) is the regulatory protein that initiates cardiac contraction in response to Ca2+ TnC binding Ca2+ initiates a cascade of protein-protein interactions that begins with the opening of the N-terminal domain of cTnC, followed by cTnC binding the troponin I switch peptide (TnISW). We have evaluated, through isothermal titration calorimetry and molecular-dynamics simulation, the effect of several clinically relevant mutations (A8V, L29Q, A31S, L48Q, Q50R, and C84Y) on the Ca2+ affinity, structural dynamics, and calculated interaction strengths between cTnC and each of Ca2+ and TnISW Surprisingly the Ca2+ affinity measured by isothermal titration calorimetry was only significantly affected by half of these mutations including L48Q, which had a 10-fold higher affinity than WT, and the Q50R and C84Y mutants, each of which had affinities 3-fold higher than wild type. This suggests that Ca2+ affinity of the N-terminal domain of cTnC in isolation is insufficient to explain the pathogenicity of these mutations. Molecular-dynamics simulation was used to evaluate the effects of these mutations on Ca2+ binding, structural dynamics, and TnI interaction independently. Many of the mutations had a pronounced effect on the balance between the open and closed conformations of the TnC molecule, which provides an indirect mechanism for their pathogenic properties. Our data demonstrate that the structural dynamics of the cTnC molecule are key in determining myofilament Ca2+ sensitivity. Our data further suggest that modulation of the structural dynamics is the underlying molecular mechanism for many disease mutations that are far from the regulatory Ca2+-binding site of cTnC.

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.

iASPP, a previously unidentified regulator of desmosomes, prevents arrhythmogenic right ventricular cardiomyopathy (ARVC)-induced sudden death.

Desmosomes are anchoring junctions that exist in cells that endure physical stress such as cardiac myocytes. The importance of desmosomes in maintaining the homeostasis of the myocardium is underscored by frequent mutations of desmosome components found in human patients and animal models. Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a phenotype caused by mutations in desmosomal components in ∼ 50% of patients, however, the causes in the remaining 50% of patients still remain unknown. A deficiency of inhibitor of apoptosis-stimulating protein of p53 (iASPP), an evolutionarily conserved inhibitor of p53, caused by spontaneous mutation recently has been associated with a lethal autosomal recessive cardiomyopathy in Poll Hereford calves and Wa3 mice. However, the molecular mechanisms that mediate this putative function of iASPP are completely unknown. Here, we show that iASPP is expressed at intercalated discs in human and mouse postmitotic cardiomyocytes. iASPP interacts with desmoplakin and desmin in cardiomyocytes to maintain the integrity of desmosomes and intermediate filament networks in vitro and in vivo. iASPP deficiency specifically induces right ventricular dilatation in mouse embryos at embryonic day 16.5. iASPP-deficient mice with exon 8 deletion (Ppp1r13l(Δ8/Δ8)) die of sudden cardiac death, displaying features of ARVC. Intercalated discs in cardiomyocytes from four of six human ARVC cases show reduced or loss of iASPP. ARVC-derived desmoplakin mutants DSP-1-V30M and DSP-1-S299R exhibit weaker binding to iASPP. These data demonstrate that by interacting with desmoplakin and desmin, iASPP is an important regulator of desmosomal function both in vitro and in vivo. This newly identified property of iASPP may provide new molecular insight into the pathogenesis of ARVC.

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 ßMHC, 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.

Clinically Divergent Mutation Effects on the Structure and Function of the Human Cardiac Tropomyosin Overlap.

The progression of genetically inherited cardiomyopathies from an altered protein structure to clinical presentation of disease is not well understood. One of the main roadblocks to mechanistic insight remains a lack of high-resolution structural information about multiprotein complexes within the cardiac sarcomere. One example is the tropomyosin (Tm) overlap region of the thin filament that is crucial for the function of the cardiac sarcomere. To address this central question, we devised coupled experimental and computational modalities to characterize the baseline function and structure of the Tm overlap, as well as the effects of mutations causing divergent patterns of ventricular remodeling on both structure and function. Because the Tm overlap contributes to the cooperativity of myofilament activation, we hypothesized that mutations that enhance the interactions between overlap proteins result in more cooperativity, and conversely, those that weaken interaction between these elements lower cooperativity. Our results suggest that the Tm overlap region is affected differentially by dilated cardiomyopathy-associated Tm D230N and hypertrophic cardiomyopathy-associated human cardiac troponin T (cTnT) R92L. The Tm D230N mutation compacts the Tm overlap region, increasing the cooperativity of the Tm filament, contributing to a dilated cardiomyopathy phenotype. The cTnT R92L mutation causes weakened interactions closer to the N-terminal end of the overlap, resulting in decreased cooperativity. These studies demonstrate that mutations with differential phenotypes exert opposite effects on the Tm-Tn overlap, and that these effects can be directly correlated to a molecular level understanding of the structure and dynamics of the component proteins.

Familial hypertrophic cardiomyopathy caused by a de novo Gly716Arg mutation of the ß-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 ß-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.

Mutation in the γ2-subunit of AMP-activated protein kinase stimulates cardiomyocyte proliferation and hypertrophy independent of glycogen storage.

RATIONALE: AMP-activated protein kinase is a master regulator of cell metabolism and an attractive drug target for cancer and metabolic and cardiovascular diseases. Point mutations in the regulatory γ2-subunit of AMP-activated protein kinase (encoded by Prkag2 gene) caused a unique form of human cardiomyopathy characterized by cardiac hypertrophy, ventricular preexcitation, and glycogen storage. Understanding the disease mechanisms of Prkag2 cardiomyopathy is not only beneficial for the patients but also critical to the use of AMP-activated protein kinase as a drug target. OBJECTIVE: We sought to identify the pro-growth-signaling pathway(s) triggered by Prkag2 mutation and to distinguish it from the secondary response to glycogen storage. METHODS AND RESULTS: In a mouse model of N488I mutation of the Prkag2 gene (R2M), we rescued the glycogen storage phenotype by genetic inhibition of glucose-6-phosphate-stimulated glycogen synthase activity. Ablation of glycogen storage eliminated the ventricular preexcitation but did not affect the excessive cardiac growth in R2M mice. The progrowth effect in R2M hearts was mediated via increased insulin sensitivity and hyperactivity of Akt, resulting in activation of mammalian target of rapamycin and inactivation of forkhead box O transcription factor-signaling pathways. Consequently, cardiac myocyte proliferation during the postnatal period was enhanced in R2M hearts followed by hypertrophic growth in adult hearts. Inhibition of mammalian target of rapamycin activity by rapamycin or restoration of forkhead box O transcription factor activity by overexpressing forkhead box O transcription factor 1 rescued the abnormal cardiac growth. CONCLUSIONS: Our study reveals a novel mechanism for Prkag2 cardiomyopathy, independent of glycogen storage. The role of γ2-AMP-activated protein kinase in cell growth also has broad implications in cardiac development, growth, and regeneration.

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

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

Effect of a myosin regulatory light chain mutation K104E on actin-myosin interactions.

Familial hypertrophic cardiomyopathy (FHC) is the most common cause of sudden cardiac death in young individuals. Molecular mechanisms underlying this disorder are largely unknown; this study aims at revealing how disruptions in actin-myosin interactions can play a role in this disorder. Cross-bridge (XB) kinetics and the degree of order were examined in contracting myofibrils from the ex vivo left ventricles of transgenic (Tg) mice expressing FHC regulatory light chain (RLC) mutation K104E. Because the degree of order and the kinetics are best studied when an individual XB makes a significant contribution to the overall signal, the number of observed XBs in an ex vivo ventricle was minimized to ∼20. Autofluorescence and photobleaching were minimized by labeling the myosin lever arm with a relatively long-lived red-emitting dye containing a chromophore system encapsulated in a cyclic macromolecule. Mutated XBs were significantly better ordered during steady-state contraction and during rigor, but the mutation had no effect on the degree of order in relaxed myofibrils. The K104E mutation increased the rate of XB binding to thin filaments and the rate of execution of the power stroke. The stopped-flow experiments revealed a significantly faster observed dissociation rate in Tg-K104E vs. Tg-wild-type (WT) myosin and a smaller second-order ATP-binding rate for the K104E compared with WT myosin. Collectively, our data indicate that the mutation-induced changes in the interaction of myosin with actin during the contraction-relaxation cycle may contribute to altered contractility and the development of FHC.

N-acetylcysteine reverses diastolic dysfunction and hypertrophy in familial hypertrophic cardiomyopathy.

S-glutathionylation of cardiac myosin-binding protein C (cMyBP-C) induces Ca(2+) sensitization and a slowing of cross-bridge kinetics as a result of increased oxidative signaling. Although there is evidence for a role of oxidative stress in disorders associated with hypertrophic cardiomyopathy (HCM), this mechanism is not well understood. We investigated whether oxidative myofilament modifications may be in part responsible for diastolic dysfunction in HCM. We administered N-acetylcysteine (NAC) for 30 days to 1-mo-old wild-type mice and to transgenic mice expressing a mutant tropomyosin (Tm-E180G) and nontransgenic littermates. Tm-E180G hearts demonstrate a phenotype similar to human HCM. After NAC administration, the morphology and diastolic function of Tm-E180G mice was not significantly different from controls, indicating that NAC had reversed baseline diastolic dysfunction and hypertrophy in our model. NAC administration also increased sarco(endo)plasmic reticulum Ca(2+) ATPase protein expression, reduced extracellular signal-related kinase 1/2 phosphorylation, and normalized phosphorylation of phospholamban, as assessed by Western blot. Detergent-extracted fiber bundles from NAC-administered Tm-E180G mice showed nearly nontransgenic (NTG) myofilament Ca(2+) sensitivity. Additionally, we found that NAC increased tension cost and rate of cross-bridge reattachment. Tm-E180G myofilaments were found to have a significant increase in S-glutathionylation of cMyBP-C, which was returned to NTG levels upon NAC administration. Taken together, our results indicate that oxidative myofilament modifications are an important mediator in diastolic function, and by relieving this modification we were able to reverse established diastolic dysfunction and hypertrophy in HCM.

Decreasing tropomyosin phosphorylation rescues tropomyosin-induced familial hypertrophic cardiomyopathy.

Studies indicate that tropomyosin (Tm) phosphorylation status varies in different mouse models of cardiac disease. Investigation of basal and acute cardiac function utilizing a mouse model expressing an α-Tm protein that cannot be phosphorylated (S283A) shows a compensated hypertrophic phenotype with significant increases in SERCA2a expression and phosphorylation of phospholamban Ser-16 (Schulz, E. M., Correll, R. N., Sheikh, H. N., Lofrano-Alves, M. S., Engel, P. L., Newman, G., Schultz Jel, J., Molkentin, J. D., Wolska, B. M., Solaro, R. J., and Wieczorek, D. F. (2012) J. Biol. Chem. 287, 44478-44489). With these results, we hypothesized that decreasing α-Tm phosphorylation may be beneficial in the context of a chronic, intrinsic stressor. To test this hypothesis, we utilized the familial hypertrophic cardiomyopathy (FHC) α-Tm E180G model (Prabhakar, R., Boivin, G. P., Grupp, I. L., Hoit, B., Arteaga, G., Solaro, R. J., and Wieczorek, D. F. (2001) J. Mol. Cell. Cardiol. 33, 1815-1828). These FHC hearts are characterized by increased heart:body weight ratios, fibrosis, increased myofilament Ca(2+) sensitivity, and contractile defects. The FHC mice die by 6-8 months of age. We generated mice expressing both the E180G and S283A mutations and found that the hypertrophic phenotype was rescued in the α-Tm E180G/S283A double mutant transgenic animals; these mice exhibited no signs of cardiac hypertrophy and displayed improved cardiac function. These double mutant transgenic hearts showed increased phosphorylation of phospholamban Ser-16 and Thr-17 compared with the α-Tm E180G mice. This is the first study to demonstrate that decreasing phosphorylation of tropomyosin can rescue a hypertrophic cardiomyopathic phenotype.

In vitro rescue study of a malignant familial hypertrophic cardiomyopathy phenotype by pseudo-phosphorylation of myosin regulatory light chain.

Pseudo-phosphorylation of cardiac myosin regulatory light chain (RLC) has never been examined as a rescue method to alleviate a cardiomyopathy phenotype brought about by a disease causing mutation in the myosin RLC. This study focuses on the aspartic acid to valine substitution (D166V) in the myosin RLC shown to be associated with a malignant phenotype of familial hypertrophic cardiomyopathy (FHC). The mutation has also been demonstrated to cause severe functional abnormalities in transgenic mice expressing D166V in the heart. To explore this novel rescue strategy, pseudo-phosphorylation of D166V was used to determine whether the D166V-induced detrimental phenotype could be brought back to the level of wild-type (WT) RLC. The S15D substitution at the phosphorylation site of RLC was inserted into the recombinant WT and D166V mutant to mimic constitutively phosphorylated RLC proteins. Non-phosphorylatable (S15A) constructs were used as controls. A multi-faceted approach was taken to determine the effect of pseudo-phosphorylation on the ability of myosin to generate force and motion. Using mutant reconstituted porcine cardiac muscle preparations, we showed an S15D-induced rescue of both the enzymatic and binding properties of D166V-myosin to actin. A significant increase in force production capacity was noted in the in vitro motility assays for S15D-D166V vs. D166V reconstituted myosin. A similar pseudo-phosphorylation induced effect was observed on the D166V-elicited abnormal Ca(2+) sensitivity of force in porcine papillary muscle strips reconstituted with phosphomimic recombinant RLCs. Results from this study demonstrate a novel in vitro rescue strategy that could be utilized in vivo to ameliorate a malignant cardiomyopathic phenotype. We show for the first time that pseudo-RLC phosphorylation can reverse the majority of the mutation-induced phenotypes highlighting the importance of RLC phosphorylation in combating cardiac disease.

Ca(2+)-regulatory function of the inhibitory peptide region of cardiac troponin I is aided by the C-terminus of cardiac troponin T: Effects of familial hypertrophic cardiomyopathy mutations cTnI R145G and cTnT R278C, alone and in combination, on filament sliding.

Investigations of cardiomyopathy mutations in Ca(2+) regulatory proteins troponin and tropomyosin provide crucial information about cardiac disease mechanisms, and also provide insights into functional domains in the affected polypeptides. Hypertrophic cardiomyopathy-associated mutations TnI R145G, located within the inhibitory peptide (Ip) of human cardiac troponin I (hcTnI), and TnT R278C, located immediately C-terminal to the IT arm in human cardiac troponin T (hcTnT), share some remarkable features: structurally, biochemically, and pathologically. Using bioinformatics, we find compelling evidence that TnI and TnT, and more specifically the affected regions of hcTnI and hcTnT, may be related not just structurally but also evolutionarily. To test for functional interactions of these mutations on Ca(2+)-regulation, we generated and characterized Tn complexes containing either mutation alone, or both mutations simultaneously. The most important results from in vitro motility assays (varying [Ca(2+)], temperature or HMM density) show that the TnT mutant "rescued" some deleterious effects of the TnI mutant at high Ca(2+), but exacerbated the loss of function, i.e., switching off the actomyosin interaction, at low Ca(2+). Taken together, our experimental results suggest that the C-terminus of cTnT aids Ca(2+)-regulatory function of cTnI Ip within the troponin complex.

[Renin-angiotensin-aldosterone system in hypertrophic cardiomyopathy].

Keeping in mind an important role of renin-angiotensin aldosterone system (RAS) in developing of cardiac remodeling and fibrosis, genetic polymorphisms coding its components could have influence with clinical variants of the course. Biomarkers could appear predictors of adverse. To examine the contribution of the RAS to developing of different hypertrophic cardiomyopathy (HCM) clinical variants of the course we studied 58 patients with HCM and controls comparable by age and gender. All patients were genotyped of gene polymorphisms CMA1 A(-1903)G rs1800875, AGTM235T rs699, AGTR1 A1166C rs5186, CYP11B2-344 T/C rs1799998. Angiotensin-converting enzyme (ACE) and angiotensin II (AII) levels were measured in 40 patients with HCM and 39 controls. We found out that AII were significantly decreased in patients with HCM than in healthy controls. The positive correlation between AII and left ventricle posterior wall (LVPW) were detected. Severity of heart hypertrophy were associated with pejorative genotype of AGT M235T polymorphism and CMA1 A(-1903) polymorphism. Significant association between the AG genotype of CMA1 A(-1903) polymorphism and angina class II-III and ventricular extrasystole of high gradation was observed. Our data not only support the hypothesis that RAAS polymorphisms may influence phenotype, but also allow for create new approaches to possible predicting adverse outcomes.

Generation of induced pluripotent stem cell lines from two unrelated individuals with familial hypertrophic cardiomyopathy carrying the MYBPC3 missense c.1484G>A mutation.

Familial hypertrophic cardiomyopathy (HCM) is the most common inherited heart condition. HCM patients show left ventricle hypertrophy without any associated loading conditions, being at risk for heart failure and sudden cardiac death. Two induced pluripotent stem cell (iPSC) lines were generated from peripheral blood mononuclear cells obtained from two unrelated individuals, a 54-year-old male (F81) and a 44-year-old female (F93), both carrying the MYBPC3 c.1484G>A HCM mutation. iPSCs show expression of pluripotency markers, trilineage differentiation capacity and a normal karyotype. This resource enables further assessment of the pathophysiological development of HCM.

Generation of induced pluripotent stem cell lines from two unrelated individuals with familial hypertrophic cardiomyopathy carrying MYBPC3 nonsense mutations.

Familial hypertrophic cardiomyopathy (HCM) stands as a predominant heart condition, characterised by left ventricle hypertrophy in the absence of any associated loading conditions, with affected individuals having an increased risk of developing heart failure and sudden cardiac death (SCD). Two induced pluripotent stem cell (iPSC) lines were derived from peripheral blood mononuclear cells obtained from two unrelated individuals with previously reported nonsense mutations in the MYBPC3 gene. The first individual is a 48-year-old male (F26) with the MYBPC3 c.1731G > A HCM mutation, whereas the second individual is a 43-year-old female (F82) carrying the MYBPC3 c.2670G > A HCM mutation. The generated iPSCs exhibit appropriate expression of pluripotency markers, trilineage differentiation capacity and a normal karyotype. This resource contributes to gaining deeper insights into the pathophysiological mechanisms that underlie HCM.

Correlation of molecular and functional effects of mutations in cardiac troponin T linked to familial hypertrophic cardiomyopathy: an integrative in silico/in vitro approach.

Nearly 70% of all of the known cTnT mutations that cause familial hypertrophic cardiomyopathy fall within the TNT1 region that is critical to cTn-Tm binding. The high resolution structure of this domain has not been determined, and this lack of information has hindered structure-function analysis. In the current study, a coupled computational experimental approach was employed to correlate changes in cTnT dynamics to basic function using the regulated in vitro motility assay (R-IVM). An in silico approach to calculate forces in terms of a bending coordinate was used to precisely identify decreases in bending forces at residues 105 and 106 within the proposed cTnT "hinge" region. Significant functional changes were observed in multiple functional properties, including a decrease in the cooperativity of calcium activation, the calcium sensitivity of sliding speed, and maximum sliding speed. Correlation of the computational and experimental findings revealed an association between TNT1 flexibility and the cooperativity of thin filament calcium activation where an increase in flexibility led to a decrease in cooperativity. Further analysis of the primary sequence of the TNT1 region revealed a unique pattern of conserved charged TNT1 residues altered by the R92W and R92L mutations and may represent the underlying "structure" modulating this central functional domain. These data provide a framework for further integrated in silico/in vitro approaches that may be extended into a high-throughput predictive screen to overcome the current structural limitations in linking molecular phenotype to genotype in thin filament cardiomyopathies.

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

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