Mutations In Cardiac Troponin Research Articles

Mouse Models of Cardiomyopathies Caused by Mutations in Troponin C.

Cardiac muscle contraction is regulated via Ca2+ exchange with the hetero-trimeric troponin complex located on the thin filament. Binding of Ca2+ to cardiac troponin C, a Ca2+ sensing subunit within the troponin complex, results in a series of conformational re-arrangements among the thin filament components, leading to an increase in the formation of actomyosin cross-bridges and muscle contraction. Ultimately, a decline in intracellular Ca2+ leads to the dissociation of Ca2+ from troponin C, inhibiting cross-bridge cycling and initiating muscle relaxation. Therefore, troponin C plays a crucial role in the regulation of cardiac muscle contraction and relaxation. Naturally occurring and engineered mutations in troponin C can lead to altered interactions among components of the thin filament and to aberrant Ca2+ binding and exchange with the thin filament. Mutations in troponin C have been associated with various forms of cardiac disease, including hypertrophic, restrictive, dilated, and left ventricular noncompaction cardiomyopathies. Despite progress made to date, more information from human studies, biophysical characterizations, and animal models is required for a clearer understanding of disease drivers that lead to cardiomyopathies. The unique use of engineered cardiac troponin C with the L48Q mutation that had been thoroughly characterized and genetically introduced into mouse myocardium clearly demonstrates that Ca2+ sensitization in and of itself should not necessarily be considered a disease driver. This opens the door for small molecule and protein engineering strategies to help boost impaired systolic function. On the other hand, the engineered troponin C mutants (I61Q and D73N), genetically introduced into mouse myocardium, demonstrate that Ca2+ desensitization under basal conditions may be a driving factor for dilated cardiomyopathy. In addition to enhancing our knowledge of molecular mechanisms that trigger hypertrophy, dilation, morbidity, and mortality, these cardiomyopathy mouse models could be used to test novel treatment strategies for cardiovascular diseases. In this review, we will discuss (1) the various ways mutations in cardiac troponin C might lead to disease; (2) relevant data on mutations in cardiac troponin C linked to human disease, and (3) all currently existing mouse models containing cardiac troponin C mutations (disease-associated and engineered).

Computational Exploration and Characterization of Potential Calcium Sensitizing Mutations in Cardiac Troponin C.

Calcium-dependent heart muscle contraction is regulated by the cardiac troponin protein complex (cTn) and specifically by the N-terminal domain of its calcium binding subunit (cNTnC). cNTnC contains one calcium binding site (site II), and altered calcium binding in this site has been studied for decades. It has been previously shown that cNTnC mutants, which increase calcium sensitization may have therapeutic benefits, such as restoring cardiac muscle contractility and functionality post-myocardial infarction events. Here, we computationally characterized eight mutations for their potential effects on calcium binding affinity in site II of cNTnC. We utilized two distinct methods to estimate calcium binding: adaptive steered molecular dynamics (ASMD) and thermodynamic integration (TI). We observed a sensitizing trend for all mutations based on the employed ASMD methodology. The TI results showed excellent agreement with experimentally known calcium binding affinities in wild-type cNTnC. Based on the TI results, five mutants were predicted to increase calcium sensitivity in site II. This study presents an interesting comparison of the two computational methods, which have both been shown to be valuable tools in characterizing the impacts of calcium sensitivity in mutant cNTnC systems.

Increased cytosolic calcium buffering contributes to a cellular arrhythmogenic substrate in iPSC-cardiomyocytes from patients with dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a major risk factor for heart failure and is associated with the development of life-threatening cardiac arrhythmias. Using a patient-specific induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model harbouring a mutation in cardiac troponin T (R173W), we aim to examine the cellular basis of arrhythmogenesis in DCM patients with this mutation. iPSC from control (Ctrl) and DCM-TnT-R173W donors from the same family were differentiated into iPSC-CM and analysed through optical action potential (AP) recordings, simultaneous measurement of cytosolic calcium concentration ([Ca2+]i) and membrane currents and separately assayed using field stimulation to detect the threshold for AP- and [Ca2+]i-alternans development. AP duration was unaltered in TnT-R173W iPSC-CM. Nevertheless, TnT-R173W iPSC-CM showed a strikingly low stimulation threshold for AP- and [Ca2+]i-alternans. Myofilaments are known to play a role as intracellular Ca2+ buffers and here we show increased Ca2+ affinity of intracellular buffers in TnT-R173W cells, indicating increased myofilament sensitivity to Ca2+. Similarly, EMD57033, a myofilament Ca2+ sensitiser, replicated the abnormal [Ca2+]i dynamics observed in TnT-R173W samples and lowered the threshold for alternans development. In contrast, application of a Ca2+ desensitiser (blebbistatin) to TnT-R173W iPSC-CM was able to phenotypically rescue Ca2+ dynamics, normalising Ca2+ transient profile and minimising the occurrence of Ca2+ alternans at physiological frequencies. This finding suggests that increased Ca2+ buffering likely plays a major arrhythmogenic role in patients with DCM, specifically in those with mutations in cardiac troponin T. In addition, we propose that modulation of myofilament Ca2+ sensitivity could be an effective anti-arrhythmic target for pharmacological management of this disease.

Allele-specific silencing by RNAi of R92Q and R173W mutations in cardiac troponin T

Autosomal dominant mutations in sarcomere proteins such as the cardiac troponin T (TNNT2) are the main genetic causes of human hypertrophic cardiomyopathy and dilated cardiomyopathy. Allele-specific silencing by RNA interference (ASP-RNAi) holds promise as a therapeutic strategy for downregulating a single mutant allele with minimal suppression of the corresponding wild-type allele. Here, we propose ASP-RNAi as a possible strategy to specifically knockdown mutant alleles coding for R92Q and R173W mutant TNNT2 proteins, identified in hypertrophic and dilated cardiomyopathy, respectively. Different siRNAs were designed and validated by luciferase reporter assay and following analysis in HEK293T cells expressing either the wild-type or mutant TNNT2 alleles. This study is the first exploration of ASP-RNAi on TNNT2-R173W and TNNT2-R92Q mutations in vitro and gives a base for further application of allele silencing as a therapeutic treatment for TNNT2-mutation-associated cardiomyopathies.

Exosomally derived Y RNA fragment alleviates hypertrophic cardiomyopathy in transgenic mice

Cardiosphere-derived cell exosomes (CDCexo) and YF1, a CDCexo-derived non-coding RNA, elicit therapeutic bioactivity in models of myocardial infarction and hypertensive hypertrophy. Here we tested the hypothesis that YF1, a 56-nucleotide Y RNA fragment, could alleviate cardiomyocyte hypertrophy, inflammation, and fibrosis associated with hypertrophic cardiomyopathy (HCM) in transgenic mice harboring a clinically relevant mutation in cardiac troponin I (cTnIGly146). By quantitative PCR, YF1 was detectable in bone marrow, spleen, liver, and heart 30 min after intravenous (i.v.) infusion. For efficacy studies, mice were randomly allocated to receive i.v. YF1 or vehicle, monitored for ambulatory and cardiac function, and sacrificed at 4 weeks. YF1 (but not vehicle) improved ambulation and reduced cardiac hypertrophy and fibrosis. In parallel, peripheral mobilization of neutrophils and proinflammatory monocytes was decreased, and fewer macrophages infiltrated the heart. RNA-sequencing of macrophages revealed that YF1 confers substantive and broad changes in gene expression, modulating pathways associated with immunological disease and inflammatory responses. Together, these data demonstrate that YF1 can reverse hypertrophic and fibrotic signaling pathways associated with HCM, while improving function, raising the prospect that YF1 may be a viable novel therapeutic candidate for HCM.

The effect of variable troponin C mutation thin filament incorporation on cardiac muscle twitch contractions

One of the complexities of understanding the pathology of familial forms of cardiac diseases is the level of mutation incorporation in sarcomeres. Computational models of the sarcomere that are spatially explicit offer an approach to study aspects of mutational incorporation into myofilaments that are more challenging to get at experimentally. We studied two well characterized mutations of cardiac TnC, L48Q and I61Q, that decrease or increase the release rate of Ca2+ from cTnC, k−Ca, resulting in HCM and DCM respectively [1]. Expression of these mutations in transgenic mice was used to provide experimental data for incorporation of 30 and 50% (respectively) into sarcomeres. Here we demonstrate that fixed length twitch contractions of trabeculae from mice containing mutant differ from WT; L48Q trabeculae have slower relaxation while I61Q trabeculae have markedly reduced peak tension. Using our multiscale modelling approach [2] we were able to describe the tension transients of WT mouse myocardium. Tension transients for the mutant cTnCs were simulated with changes in k−Ca, measured experimentally for each cTnC mutant in whole troponin complex, a change in the affinity of cTnC for cTnI, and a reduction in the number of detached crossbridges available for binding. A major advantage of the multiscale explicit 3-D model is that it predicts the effects of variable mutation incorporation, and the effects of variations in mutation distribution within thin filaments in sarcomeres. Such effects are currently impossible to explore experimentally. We explored random and clustered distributions of mutant cTnCs in thin filaments, as well as distributions of individual thin filaments with only WT or mutant cTnCs present. The effects of variable amounts of incorporation and non-random distribution of mutant cTnCs are more marked for I61Q than L48Q cTnC. We conclude that this approach can be effective for study on mutations in multiple proteins of the sarcomere. SummaryA challenge in experimental studies of diseases is accounting for the effect of variable mutation incorporation into myofilaments. Here we use a spatially explicit computational approach, informed by experimental data from transgenic mice expressing one of two mutations in cardiac Troponin C that increase or decrease calcium sensitivity. We demonstrate that the model can accurately describe twitch contractions for the data and go on to explore the effect of variable mutant incorporation and localization on simulated cardiac muscle twitches.

NRSF-GNAO1-CaMK2 axis exacerbates cardiac remodeling and progresses heart failure by impairing Ca2+ homeostasis

Abstract Background In the development of heart failure, pathological intracellular signaling reactivates fetal cardiac genes, which leads to maladaptive remodeling and cardiac dysfunction. We previously reported that a transcriptional repressor, neuron restrictive silencer factor (NRSF) represses fetal cardiac genes and maintains normal cardiac function under normal conditions, while hypertrophic stimuli de-repress this NRSF mediated repression via activation of CaMKII. Molecular mechanisms by which NRSF maintains cardiac systolic function remains to be determined, however. Purpose To elucidate how NRSF maintains normal cardiac homeostasis and identify the novel therapeutic targets for heart failure. Methods and results We generated cardiac-specific NRSF knockout mice (NRSF cKO), and found that these NRSF cKO showed cardiac dysfunction and premature deaths accompanied with lethal arrhythmias, as was observed in our previously reported cardiac-specific dominant-negative mutant of NRSF transgenic mice (dnNRSF-Tg). By cDNA microarray analysis of dnNRSF-Tg and NRSF-cKO, we identified that expression of Gnao1 gene encoding Gαo, a member of inhibitory G proteins, was commonly increased in ventricles of both types of mice. ChIP-seq analysis, reporter assay and electrophoretic mobility shift assay identified that NRSF transcriptionally regulates Gnao1 gene expression. Genetic Knockdown of Gαo in dnNRSF-Tg and NRSF-cKO by crossing these mice with Gnao1 knockout mice ameliorated the reduced systolic function, increased arrhythmogenicity and reduced survival rates. Transgenic mice expressing a human GNAO1 in their hearts (GNAO1-Tg) showed progressive cardiac dysfunction with cardiac dilation. Ventricles obtained from GNAO1-Tg have increased phosphorylation level of CaMKII and increased expression level of endogenous mouse Gnao1 gene. These data suggest that increased cardiac expression of Gαo is sufficient to induce pathological Ca2+-dependent signaling and cardiac dysfunction, and that Gαo forms a positive regulatory circuit with CaMKII and NRSF. Electrophysiological analysis in ventricular myocytes of dnNRSF-Tg revealed that impaired Ca2+ handling via alterations in localized L-type calcium channel (LTCC) activities; decreased T-tubular and increased surface sarcolemmal LTCC activities, underlies Gαo-mediated cardiac dysfunction. Furthermore, we also identified increased expression of Gαo in ventricles of two different heart failure mice models, mice with transverse aortic constriction and mice carrying a mutant cardiac troponin T, and confirmed that genetic reduction of Gαo prevented the progression of cardiac dysfunction in both types of mice. Conclusions Increased expression of Gαo, induced by attenuation of NRSF-mediated repression forms a pathological circuit via activation of CaMKII. This circuit exacerbates cardiac remodeling and progresses heart failure by impairing Ca2+ homeostasis. Gαo is a potential therapeutic target for heart failure. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Grants-in –Aid for Scientific Research from the Japan Society for the Promotion of Science

095 Characterising the Phenotypic Switch in a Murine Model of Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) occurs due to mutations in sarcomeric proteins. At the cellular level, HCM characteristics include increased protein synthesis, increased mitochondrial number, altered cell metabolism and cardiomyocyte disorganisation. HCM has variable phenotypic expression and clinical characteristics often present later in life. Using a murine model of human HCM, we have shown that a mutation in cardiac troponin I (cTnI-G203S) is associated with cytoskeletal disorganisation and a hypermetabolic mitochondrial state. Alterations in metabolic activity precede manifestation of the HCM phenotype. However, little is known about the role of signalling pathways in disease progression. The mTOR signalling pathway is a key regulator of protein synthesis and cell metabolism, and may be involved in the development of HCM. Here we examined myocardial ultrastructure and cardiac mTOR signalling in cTnI-G203S mice. Studies were performed in mice before and after the development of HCM (pre- and post-HCM) using TEM and immunoblot methodologies. Pre-HCM cTnI-G203S hearts demonstrated an increasing trend in total and activated mTOR), and a significant increase in sarcomere area (p<0.001, n=2), versus age-matched wt hearts. Post-HCM cTnI-G203S hearts demonstrated (i) significantly higher total and activated mTOR (ptotal<0.05, n=3; pactivated: p<0.05, n=3), and (ii) a significant decrease in sarcomere area (p<0.001, n=2) versus age-matched wt hearts. We conclude that alterations in the mTOR signalling pathway may be involved in the progression of the HCM phenotype. Myocardial ultrastructure appears to alter as the phenotype develops from a pre- to post-cardiomyopathic state.

Dilated cardiomyopathy mutation (R174W) in troponin T attenuates the length-mediated increase in cross-bridge recruitment and myofilament Ca2+ sensitivity.

Alterations in length-dependent activation (LDA) may constitute a mechanism by which cardiomyopathy mutations lead to deleterious phenotypes and compromised heart function, because LDA underlies the molecular basis by which the heart tunes myocardial force production on a beat-to-beat basis (Frank-Starling mechanism). In this study, we investigated the effect of DCM-linked mutation (R173W) in human cardiac troponin T (TnT) on myofilament LDA. R173W mutation is associated with left ventricular dilatation and systolic dysfunction and is found in multiple families. R173W mutation is in the central region (residues 80-180) of TnT, which is known to be important for myofilament cooperativity and cross-bridge (XB) recruitment. Steady-state and dynamic contractile parameters were measured in detergent-skinned guinea pig left ventricular muscle fibers reconstituted with recombinant guinea pig wild-type TnT (TnTWT) or mutant TnT (TnTR174W; guinea pig analog of human R173W mutation) at two different sarcomere lengths (SL): short (1.9 µm) and long (2.3 µm). TnTR174W decreased pCa50 (-log [Ca2+]free required for half-maximal activation) to a greater extent at long than at short SL; for example, pCa50 decreased by 0.12 pCa units at long SL and by 0.06 pCa units at short SL. Differential changes in pCa50 at short and long SL attenuated the SL-dependent increase in myofilament Ca2+ sensitivity (ΔpCa50) in TnTR174W fibers; ΔpCa50 was 0.10 units in TnTWT fibers but only 0.04 units in TnTR174W fibers. Furthermore, TnTR174W blunted the SL-dependent increase in the magnitude of XB recruitment. Our observations suggest that the R173W mutation in human cardiac TnT may impair Frank-Starling mechanism.NEW & NOTEWORTHY This work characterizes the effect of dilated cardiomyopathy mutation in cardiac troponin T (TnTR174W) on myofilament length-dependent activation. TnTR174W attenuates the length-dependent increase in cross-bridge recruitment and myofilament Ca2+ sensitivity.

A case report of recessive restrictive cardiomyopathy caused by a novel mutation in cardiac troponin I (TNNI3)

BackgroundRestrictive cardiomyopathy is a rare cardiac disease, for which several genes including TNNT2, MYPN, FLNC and TNNI3 have been associated with its familial form.Case presentationHere we describe a female proband with a severely manifested restrictive phenotype leading to heart transplantation at the age of 41, who was found homozygous for the novel TNNI3 mutation: NM_000363.4:c.586G > C, p.(Asp196His). Her parents were third-degree cousins originating from a small village and although they were found heterozygous for the same variant they displayed no symptoms of the disease. Her older sister who was also found heterozygous was asymptomatic. Her twin sister and her brother who were homozygous for the same variant displayed a restrictive and a hypertrophic phenotype, respectively. Their children are all carriers of the mutation and remain asymptomatic until the age of 21.ConclusionThese observations point to a recessive mode of inheritance reported for the first time for this combination of gene/disease.

The Mechanistic Role of Tropomyosin Overlap Dysregulation in Early Cardiomyopathic Disease Progression

An oft-noted component of sarcomeric dilated (DCM) and hypertrophic (HCM) cardiomyopathies is that primary mutations can exhibit significant phenotypic variability. This variability extends even to families carrying the same mutation suggesting that independently segregating genetic modifiers exist that differentially affect susceptible individuals. In addition, recent clinical studies have demonstrated the importance of early stage treatment, emphasizing the mechanistic role of the primary biophysical disruption “domain” on pathogenesis. One possible site of primary biophysical disruption is the crucial tropomyosin (Tm)-overlap which provides the stability and flexibility required during cross bridge cycling. We hypothesize that the cardiac thin filament (CTF) has an innate but finite capacity to tolerate changes in these parameters, beyond which cardiomyopathies result. Highly penetrant mutations throughout the CTF were studied using differential scanning calorimetry to assess changes in thermodynamic stability and flexibility of fully reconstituted CTF's. N-terminus proximal Tm mutations (E62Q, D84N) decreased Tm-cTn stability while increasing flexibility, where C-terminus proximal Tm mutations (D219N, D230N) do the opposite. Strikingly these proximal-terminal dependent effects occurred independent of late-stage pathology. Mutations in cardiac troponin T (cTnT), however, only increased thermal stability and flexibility when directly adjacent to the overlap. Furthermore, we investigated the role of developmental cTnT isoform switching on the Tm-overlap to assess its capacity as an independent modifier. Fetal-cTnT alone had little effect, however, when expressed with D230N-Tm, filaments exhibited an additive decrease in flexibility of the Tm-overlap. Fetal-cTnT did not cause additive changes when expressed with D84N-Tm and R92L-cTnT. Similarly, mice expressing D230N-Tm and fetal-cTnT exhibited additively reduced % fractional shortening whereas R92L-cTnT did not display additive changes, suggesting fetal-cTnT may be a mutation-specific modifier. Together these data suggest that the Tm overlap represents a sensitive “domain” of early biophysical dysregulation in cardiomyopathic disease progression.

Computational and Experimental Investigation of Cardiac Troponin T R173Q, R173W and Δ160E Mutation Specific Correlates to Disease

Mutations in cardiac troponin T (cTnT), a component of the cardiac thin filament (cTF), are known to cause changes in protein structure and dynamics leading to pathological cardiac remodeling observed in patients with hypertrophic (HCM) and dilated (DCM) cardiomyopathies. In this complex system, mutations in cTnT separated by only a few amino acids can cause divergent cardiomyopathies, highlighting the importance of primary structural and dynamic “triggers” that lead to disease. To begin to correlate genotype to phenotype, we investigated three cardiomyopathy-linked mutations in the cTnT linker region: DCM-associated mutations R173W and R173Q, and HCM-associated mutation Δ160E. We coupled computational predictions with in-vitro biophysical measurementsto better understand the divergent disease mechanisms. We hypothesize that R173Q/W and Δ160E cause differential structural changes in the cTF linker domain leading to atomic and molecular level changes that initiate pathogenic remodeling. Differential scanning calorimetry on all 3 mutations showed an increased thermal stability required to unfold the Tm:Tn complex from actin, suggesting altered protein interactions. To obtain further resolution, molecular dynamics (MD) simulations and time-resolved fluorescence resonance energy transfer (TR-FRET) studies were employed. Previous studies showed that Δ160E caused a decreased distance between the linker region of cTnT and the tropomyosin C-terminus, resulting in decreased linker flexibility. In contrast, MD simulations of the TF containing R173W/Q show an increase distance between the linker region of cTnT and the C-terminus of tropomyosin and actin 374. Further clarification of these data will be provided by ongoing TR-FRET studies to confirm structural distance changes in the liker region in fully reconstituted cTF. This iterative process will allow us to build predictive tools to test cTF mutations associated with these cardiomyopathies and eventually to design and implement more precise therapeutic interventions.

Characterization of the L29Q Hypertrophic Cardiomyopathy Mutation in Cardiac Troponin C by Paramagnetic Relaxation Enhancement Nuclear Magnetic Resonance.

The key events in regulating muscle contraction involve the troponin (Tn) heterotrimeric protein complex in which the binding to and release of Ca2+ from the highly conserved troponin C (TnC) subunit trigger a series of structural changes within Tn, and the other thin filament proteins, to result in contraction. In the heart, the control of contraction and relaxation events can be altered by many single-point mutations that may result in cardiomyopathy and sometimes sudden cardiac death. Here we have examined the structural effects of one hypertrophic cardiomyopathy mutation, L29Q, on Ca2+-induced structural transitions within whole TnC. This mutation is of particular interest as several physiological and structural studies have indicated that the response of TnC to Ca2+ binding is altered in the presence of the L29Q mutation, but the structural nature of these changes continues to be debated. In addition, little is known about the effect of this mutation in the Ca2+ free state. Here we have used paramagnetic relaxation enhancement nuclear magnetic resonance (PRE-NMR) to assess the structural effects arising from the L29Q mutation. PRE-NMR distances obtained from a nitroxide spin-label at Cys84 showed that the L29Q mutation perturbs the structure of the TnC N-domain in the presence and absence of Ca2+, with a more "open" TnC N-domain observed in the apo form. In addition, binding of Ca2+ to the TnC-L29Q construct triggers a change in the orientation between the two domains of TnC. Together, these structural perturbations, revealed by PRE-NMR, provide insight into the pathogenesis of this mutation.

The homozygous K280N troponin T mutation alters cross-bridge kinetics and energetics in human HCM.

Hypertrophic cardiomyopathy (HCM) is a genetic form of left ventricular hypertrophy, primarily caused by mutations in sarcomere proteins. The cardiac remodeling that occurs as the disease develops can mask the pathogenic impact of the mutation. Here, to discriminate between mutation-induced and disease-related changes in myofilament function, we investigate the pathogenic mechanisms underlying HCM in a patient carrying a homozygous mutation (K280N) in the cardiac troponin T gene (TNNT2), which results in 100% mutant cardiac troponin T. We examine sarcomere mechanics and energetics in K280N-isolated myofibrils and demembranated muscle strips, before and after replacement of the endogenous troponin. We also compare these data to those of control preparations from donor hearts, aortic stenosis patients (LVHao), and HCM patients negative for sarcomeric protein mutations (HCMsmn). The rate constant of tension generation following maximal Ca2+ activation (k ACT) and the rate constant of isometric relaxation (slow k REL) are markedly faster in K280N myofibrils than in all control groups. Simultaneous measurements of maximal isometric ATPase activity and Ca2+-activated tension in demembranated muscle strips also demonstrate that the energy cost of tension generation is higher in the K280N than in all controls. Replacement of mutant protein by exchange with wild-type troponin in the K280N preparations reduces k ACT, slow k REL, and tension cost close to control values. In donor myofibrils and HCMsmn demembranated strips, replacement of endogenous troponin with troponin containing the K280N mutant increases k ACT, slow k REL, and tension cost. The K280N TNNT2 mutation directly alters the apparent cross-bridge kinetics and impairs sarcomere energetics. This result supports the hypothesis that inefficient ATP utilization by myofilaments plays a central role in the pathogenesis of the disease.

Pathogenic troponin T mutants with opposing effects on myofilament Ca2+ sensitivity attenuate cardiomyopathy phenotypes in mice

Mutations in cardiac troponin T (TnT) associated with hypertrophic cardiomyopathy generally lead to an increase in the Ca2+ sensitivity of contraction and susceptibility to arrhythmias. In contrast, TnT mutations linked to dilated cardiomyopathy decrease the Ca2+ sensitivity of contraction. Here we tested the hypothesis that two TnT disease mutations with opposite effects on myofilament Ca2+ sensitivity can attenuate each other's phenotype. We crossed transgenic mice expressing the HCM TnT-I79N mutation (I79N) with a DCM knock-in mouse model carrying the heterozygous TnT-R141W mutation (HET). The results of the Ca2+ sensitivity in skinned cardiac muscle preparations ranked from highest to lowest were as follow: I79N > I79N/HET > NTg > HET. Echocardiographic measurements revealed an improvement in hemodynamic parameters in I79N/HET compared to I79N and normalization of left ventricular dimensions and volumes compared to both I79N and HET. Ex vivo testing showed that the I79N/HET mouse hearts had reduced arrhythmia susceptibility compared to I79N mice. These results suggest that two disease mutations in TnT that have opposite effects on the myofilament Ca2+ sensitivity can paradoxically ameliorate each other's disease phenotype. Normalizing myofilament Ca2+ sensitivity may be a promising new treatment approach for a variety of diseases.

Hypertrophic cardiomyopathy mutation in cardiac troponin T (R95H) attenuates length-dependent activation in guinea pig cardiac muscle fibers.

The central region of cardiac troponin T (TnT) is important for modulating the dynamics of muscle length-mediated cross-bridge recruitment. Therefore, hypertrophic cardiomyopathy mutations in the central region may affect cross-bridge recruitment dynamics to alter myofilament Ca2+ sensitivity and length-dependent activation of cardiac myofilaments. Given the importance of the central region of TnT for cardiac contractile dynamics, we studied if hypertrophic cardiomyopathy-linked mutation (TnTR94H)-induced effects on contractile function would be differently modulated by sarcomere length (SL). Recombinant wild-type TnT (TnTWT) and the guinea pig analog of the human R94H mutation (TnTR95H) were reconstituted into detergent-skinned cardiac muscle fibers from guinea pigs. Steady-state and dynamic contractile measurements were made at short and long SLs (1.9 and 2.3 µm, respectively). Our results demonstrated that TnTR95H increased pCa50 (-log of free Ca2+ concentration) to a greater extent at short SL; TnTR95H increased pCa50 by 0.11 pCa units at short SL and 0.07 pCa units at long SL. The increase in pCa50 associated with an increase in SL from 1.9 to 2.3 µm (ΔpCa50) was attenuated nearly twofold in TnTR95H fibers; ΔpCa50 was 0.09 pCa units for TnTWT fibers but only 0.05 pCa units for TnTR95H fibers. The SL dependency of rate constants of cross-bridge distortion dynamics and tension redevelopment was also blunted by TnTR95H Collectively, our observations on the SL dependency of pCa50 and rate constants of cross-bridge distortion dynamics and tension redevelopment suggest that mechanisms underlying the length-dependent activation cardiac myofilaments are attenuated by TnTR95HNEW & NOTEWORTHY Mutant cardiac troponin T (TnTR95H) differently affects myofilament Ca2+ sensitivity at short and long sarcomere length, indicating that mechanisms underlying length-dependent activation are altered by TnTR95H TnTR95H enhances myofilament Ca2+ sensitivity to a greater extent at short sarcomere length, thus attenuating the length-dependent increase in myofilament Ca2+ sensitivity.

Abstract 383: Modulating Calcium Dysregulation in Troponin T Linked Hypertrophic Cardiomyopathy

Mutations in cardiac troponin T (cTnT) account for approximately 5-10% of all Hypertrophic Cardiomyopathy (HCM). Residue Arg92 of cTnT represents a HCM-mutational hotspot, including Arg92Leu (R92L) and Arg92Trp (R92W). These mutations, although differing by only a single amino acid, present with varying degrees of cardiac remodeling and disease severity in the human population. Studying the differential effects of the R92 mutations on Ca 2+ homeostasis revealed an age-dependent disruption in down-stream calcium handling in R92W mice, including differential diastolic calcium levels and SERCA2a uptake activity. To determine the effects of treating this differential calcium handling disruption, R92L and R92W mice were treated with diltiazem hydrochloride, an L-type Ca 2+ channel blocker. While R92L mice showed a progressive worsening in diastolic function and remodeling throughout treatment, R92W mice showed improvement in diastolic function but no change in cardiac remodeling, suggesting an improvement but not amelioration of disease with diltiazem treatment. Of note, previous studies also suggested an increase in phospholamban (PLB) phosphorylation at Thr17 (CaMK-II-mediated) over time in R92W mice only. Thus, we hypothesized a mutation-specific role of CaMK-II in cTnT-linked HCM progression. We tested this via genetic partial inhibition of CaMK-II (AC3-I peptide) in both R92W and R92L transgenic mice. Both R92W and AC3-IxRW hearts exhibited a normalization of SERCA2a activity to non-transgenic levels over time. This improved Ca 2+ re-uptake was coupled with a decrease in atrial mass, improved ventricular relaxation, and normalized diastolic function only in AC3-IxRW animals, consistent with blunting of disease progression in vivo . In contrast, AC3-IxRL mice showed depressed SERCA2a activity, increased atrial mass and ventricular thickening, and diastolic dysfunction as compared to R92L alone mice, indicating a CaMK-II independent progression to HCM. These results suggest that closely related primary mutations can result in specific ventricular remodeling and targeting these discrete molecular secondary effects represent a novel targeted treatment approach in HCM.

Thin Filament-Mediated Modulation of Mouse Cardiac Cross-Bridge Kinetics by Ca2+-Sensitizing Mutation CTNC-A8V or Bepridil

Recent genetic and functional data with the Ala8Val missense mutation in cardiac troponin C (cTnC-A8V) suggest its pathogenicity for the development of hypertrophic cardiomyopathy (HCM) in humans and mice. Previous functional studies performed using cTnC-A8V knock-in mice revealed increased Ca2+-sensitivity of contraction and impaired kinetics of cardiac muscle relaxation of skinned and intact cardiomyocytes. This study aims to further understand the molecular mechanisms that underlie the development of HCM caused by the cTnC-A8V mutation by measuring the kinetics of tension redevelopment (kTR) and sinusoidal stiffness at two distinct sarcomere lengths (SL). Papillary muscles were isolated from the left ventricle of cTnC-A8V knock-in homozygote and WT mice. SL of skinned preparations was set to 1.9µm or 2.1µm using HeNe laser diffraction after the ends were chemically fixed with glutaraldehyde to minimize end-compliance. SL dependent changes in mechanical properties were similar in WT and cTnC-A8V preparations: increased pCa50, with increased stiffness and decreased kTR at any given activation level. However, regardless of SL, cTnC-A8V preparations exhibited an increased pCa50, kTR and sinusoidal stiffness at submaximum Ca2+ levels relative to WT. Maximum sinusoidal stiffness increased 2x in WT preparations stretched from SL 1.9µm to 2.1µm, while for the cTnC-A8V preparations the SL-dependent increase in maximum sinusoidal stiffness was blunted. To determine whether the difference in cross-bridge kinetics at SL 2.1µm between cTnC-A8V and WT were due to the increased Ca2+-sensitivity of cTnC, WT preparations were treated with 300μM Bepridil, a Ca2+-sensitizer. As seen with the cTnC-A8V, sensitizing the thin filament with Bepridil resulted in an increased pCa50 and kTR. These data suggest an important role of the thin filament controlling not only the number of cross-bridges during Ca2+-activation, but may also influence cross-bridge kinetics.

Identifying the Therapeutic Window for Treatment of Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) affects 1:200 of the population, and is associated with altered metabolic activity and hypercontractile function. We have previously demonstrated that the cardiac L-type Ca2+ channel (LTCC) can regulate mitochondrial function due to an association via the cytoskeletal network. We have also shown that human HCM causing mutations in cardiac troponin I or β-myosin heavy chain lead to a faster LTCC inactivation rate, and impaired functional communication between LTCC and mitochondria in mouse models of human disease (cTnI-G203S and αMHC403/+ respectively). This results in a “hypermetabolic” mitochondrial state, which precedes development of HCM. Application of a peptide derived against the LTCC alpha-interacting domain (AID-TAT) slows LTCC inactivation rate and decreases metabolic function in wt cardiomyocytes. We examined the efficacy of in vivo exposure of cTnI-G203S and αMHC403/+ mice to AID-TAT (10 μM 3x/wk, 5wk) on mitochondrial function by assessing alterations in mitochondrial membrane potential (Ψm, JC-1 fluorescence) and mitochondrial metabolic activity (MMA, flavoprotein autofluorescence). Treatment of pre-cardiomyopathic cTnI-G203S mice normalised Ψm (AID-TAT: 20.1 ± 2.2% increase, n = 18 vs AID(S)-TAT: 30.3 ± 1.8% increase, n = 18; p < 0.05), and MMA (AID-TAT: 17.8 ± 2.1% increase, n = 29 vs AID(S)-TAT: 42.3 ± 5.0% increase, n = 26; p < 0.05). AID-TAT also prevented hypertrophy as evidenced by changes on echocardiography. Treatment of post-cardiomyopathic αMHC403/+ mice normalised Ψm (AID-TAT: 20.6 ± 3.4% increase, n = 6 vs AID(S)-TAT: 30.3 ± 1.7% increase, n = 24; p < 0.05), but not MMA (AID-TAT: 29.2 ± 2.8% increase, n = 14 vs AID(S)-TAT: 40.8 ± 6.6% increase, n = 13; p = NS). Treatment did not appear to reverse hypertrophy. We conclude that early-intervention AID-TAT treatment may be more effective in restoring mitochondrial function and preventing development of HCM.

Prognostic predictive value of gene mutations in Japanese patients with hypertrophic cardiomyopathy.

Although some studies have attempted to find useful prognostic factors in hypertrophic cardiomyopathy (HCM), those results are not fully helpful for use in actual clinical practice. Furthermore, several genetic abnormalities associated with HCM have been identified. However, the genotype-phenotype correlation in HCM remains to be elucidated. Here, we attempted to assess patients with different types of gene mutations causing HCM and investigate the prognosis. A total of 140 patients with HCM underwent a screening test for myofilament gene mutations by direct sequencing of eight sarcomeric genes. Patients with a single mutation in cardiac troponin T, cardiac troponin I, α-tropomyosin, and regulatory and essential light chains were excluded from the study because the number of cases was too small. The clinical presentations and outcomes of the remaining 127 patients with HCM, 31 β-myosin heavy chain (MYH7) mutation carriers, 19 cardiac myosin-binding protein C (MYBPC3) mutation carriers, and 77 mutation non-carriers were analyzed retrospectively. MYBPC3 mutation carriers had a high frequency of ventricular arrhythmia and syncope. Kaplan-Meier curves revealed no significant difference in prognosis among the three groups, but a lack of family history of sudden death (SD) and a past history of syncope were significantly related to poor prognosis. An absence of family history of SD and past history of syncope are useful prognostic factors in patients with HCM. MYH7 and MYBPC3 mutations did not significantly influence prognosis compared to non-carriers. However, patients with the MYBPC3 mutation should be closely followed for the possibility of SD.