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A Heterozygous Mutation in Cardiac Troponin T Promotes Ca²⁺ Dysregulation and Adult Cardiomyopathy in Zebrafish.

ABSTRACT: Cardiomyopathies are a group of heterogeneous diseases that affect the muscles of the heart, leading to early morbidity and mortality in young and adults. Genetic forms of cardiomyopathy are caused predominantly by mutations in structural components of the cardiomyocyte sarcomeres, the contractile units of the heart, which includes cardiac Troponin T (TnT). Here, we generated mutations with CRISPR/Cas9 technology in the zebrafish tnnt2a gene, encoding cardiac TnT, at a mutational "hotspot" site to establish a zebrafish model for genetic cardiomyopathies. We found that a heterozygous tnnt2a mutation deleting Arginine at position 94 and Lysine at position 95 of TnT causes progressive cardiac structural changes resulting in heart failure. The cardiac remodeling is presented by an enlarged atrium, decreased ventricle size, increased myocardial stress as well as increased fibrosis. As early as five days post fertilization, larvae carrying the TnT RK94del mutation display diastolic dysfunction and impaired calcium dynamics related to increased Ca²⁺ sensitivity. In conclusion, adult zebrafish with a heterozygous TnT-RK94del mutation develop cardiomyopathy as seen in patients with TnT mutations and therefore represent a promising model to study disease mechanisms and to screen for putative therapeutic compounds.

SUBMITTER: Kamel SM

PROVIDER: S-EPMC8072640 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Cardiomyopathy mutation (F88L) in troponin T abolishes length dependency of myofilament Ca2+ sensitivity.

Project description:Recent clinical studies have revealed a new hypertrophic cardiomyopathy-associated mutation (F87L) in the central region of human cardiac troponin T (TnT). However, despite its implication in several incidences of sudden cardiac death in young and old adults, whether F87L is associated with cardiac contractile dysfunction is unknown. Because the central region of TnT is important for modulating the muscle length-mediated recruitment of new force-bearing cross-bridges (XBs), we hypothesize that the F87L mutation causes molecular changes that are linked to the length-dependent activation of cardiac myofilaments. Length-dependent activation is important because it contributes significantly to the Frank-Starling mechanism, which enables the heart to vary stroke volume as a function of changes in venous return. We measured steady-state and dynamic contractile parameters in detergent-skinned guinea pig cardiac muscle fibers reconstituted with recombinant guinea pig wild-type TnT (TnTWT) or the guinea pig analogue (TnTF88L) of the human mutation at two different sarcomere lengths (SLs): short (1.9 µm) and long (2.3 µm). TnTF88L increases pCa50 (-log [Ca2+]free required for half-maximal activation) to a greater extent at short SL than at long SL; for example, pCa50 increases by 0.25 pCa units at short SL and 0.17 pCa units at long SL. The greater increase in pCa50 at short SL leads to the abolishment of the SL-dependent increase in myofilament Ca2+ sensitivity (ΔpCa50) in TnTF88L fibers, ΔpCa50 being 0.10 units in TnTWT fibers but only 0.02 units in TnTF88L fibers. Furthermore, at short SL, TnTF88L attenuates the negative impact of strained XBs on force-bearing XBs and augments the magnitude of muscle length-mediated recruitment of new force-bearing XBs. Our findings suggest that the TnTF88L-mediated effects on cardiac thin filaments may lead to a negative impact on the Frank-Starling mechanism.

| S-EPMC5987878 | biostudies-literature

Late onset sporadic dilated cardiomyopathy caused by a cardiac troponin T mutation.

Project description:Mutations in TNNT2, encoding cardiac troponin T, commonly shows early onset, aggressive dilated cardiomyopathy (DCM). This observation may influence the decision of whether to undertake clinical genetic testing for TNNT2 in later onset DCM. Further, the trigger for late onset DCM remains enigmatic. A 70-year-old woman, previously healthy with a left ventricular ejection fraction of 50%-55% at age 69, presented with DCM of unknown cause and a 4-month history progressive heart failure requiring cardiac transplantation. Clinical genetic testing revealed a novel TNNT2 R139H mutation but no relevant variants in 18 other DCM genes. Her explanted heart showed partial fatty replacement in the right ventricle. Sequencing for five arrhythmogenic right ventricular dysplasia genes was negative. Functional studies in porcine cardiac skinned fibers reconstituted with the mutant R139H troponin T protein showed decreased Ca(2+) sensitivity at pH 7, characteristic of DCM. Because fatty infiltration may acidify the myocellular environment, maximal force development examined at pH 6.5 was diminished, suggesting a possible environmental trigger. We conclude that the TNNT2 R139H mutation was likely to be disease causing. Further, later age of onset may not be relevant to exclude genetic testing for TNNT2 mutations.

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Founder Mutation in N Terminus of Cardiac Troponin I Causes Malignant Hypertrophic Cardiomyopathy.

Project description:BackgroundCardiac troponin I (TNNI3) gene mutations account for 3% of hypertrophic cardiomyopathy and carriers have a heterogeneous phenotype, with increased risk of sudden cardiac death (SCD). Only one mutation (p.Arg21Cys) has been reported in the N terminus of the protein. In model organisms, it impairs PKA (protein kinase A) phosphorylation, increases calcium sensitivity, and causes diastolic dysfunction. The phenotype of this unique mutation in patients with hypertrophic cardiomyopathy remains unknown.MethodsWe sequenced 29 families with hypertrophic cardiomyopathy enriched for pediatric-onset disease and identified 5 families with the TNNI3 p.Arg21Cys mutation. Using cascade screening, we studied the clinical phenotype of 57 individuals from the 5 families with TNNI3 p.Arg21Cys-related cardiomyopathy. We performed survival analysis investigating the age at first SCD in carriers of the mutation.ResultsAll 5 families with TNNI3 p.Arg21Cys were from South Lebanon. TNNI3 p.Arg21Cys-related cardiomyopathy manifested a malignant phenotype-SCD occurred in 30 (53%) of 57 affected individuals at a median age of 22.5 years. In select carriers without left ventricular hypertrophy on echocardiogram, SCD occurred, myocyte disarray was found on autopsy heart, and tissue Doppler and cardiac magnetic resonance imaging identified subclinical disease features such as diastolic dysfunction and late gadolinium enhancement.ConclusionsThe TNNI3 p.Arg21Cys mutation has a founder effect in South Lebanon and causes malignant hypertrophic cardiomyopathy with early SCD even in the absence of hypertrophy. Genetic diagnosis with this mutation may be sufficient for risk stratification for SCD.

| S-EPMC7676616 | biostudies-literature

Green Tea Catechin Normalizes the Enhanced Ca2+ Sensitivity of Myofilaments Regulated by a Hypertrophic Cardiomyopathy-Associated Mutation in Human Cardiac Troponin I (K206I).

Project description:BackgroundHypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disease characterized by thickening of ventricular walls and decreased left ventricular chamber volume. The majority of HCM-associated mutations are found in genes encoding sarcomere proteins. Herein, we set out to functionally characterize a novel HCM-associated mutation (K206I-TNNI3) and elucidate the mechanism of dysfunction at the level of myofilament proteins.Methods and resultsThe male index case was diagnosed with HCM after an out-of-hospital cardiac arrest, which was followed by comprehensive clinical evaluation, transthoracic echocardiography, and clinical genetic testing. To determine molecular mechanism(s) of the mutant human cardiac troponin I (K206I), we tested the Ca(2+) dependence of thin filament-activated myosin-S1-ATPase activity in a reconstituted, regulated, actomyosin system comparing wild-type human troponin complex, 50% mix of K206I/wildtype, or 100% K206I. We also exchanged native troponin detergent extracted fibers with reconstituted troponin containing either wildtype or a 65% mix of K206I/wildtype and measured force generation. The Ca(2+) sensitivity of the myofilaments containing the K206I variant was significantly increased, and when treated with 20 µmol/L (-)-epigallocatechin gallate (green tea) was restored back to wild-type levels in ATPase and force measurements. The K206I mutation impairs the ability of the troponin I to inhibit ATPase activity in the absence of calcium-bound human cardiac troponin C. The ability of calcium-bound human cardiac troponin C to neutralize the inhibition of K206I was greater than with wild-type TnI.ConclusionsCompromised interactions of K206I with actin and hcTnC may lead to impaired relaxation and HCM.

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Enhanced troponin I binding explains the functional changes produced by the hypertrophic cardiomyopathy mutation A8V of cardiac troponin C.

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Familial hypertrophic cardiomyopathy related cardiac troponin C L29Q mutation alters length-dependent activation and functional effects of phosphomimetic troponin I*.

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Fetal cardiac troponin isoforms rescue the increased Ca2+ sensitivity produced by a novel double deletion in cardiac troponin T linked to restrictive cardiomyopathy: a clinical, genetic, and functional approach.

Project description:A novel double deletion in cardiac troponin T (cTnT) of two highly conserved amino acids (Asn-100 and Glu-101) was found in a restrictive cardiomyopathic (RCM) pediatric patient. Clinical evaluation revealed the presence of left atrial enlargement and marked left ventricle diastolic dysfunction. The explanted heart examined by electron microscopy revealed myofibrillar disarray and mild fibrosis. Pedigree analysis established that this mutation arose de novo. The patient tested negative for six other sarcomeric genes. The single and double recombinant cTnT mutants were generated, and their functional consequences were analyzed in porcine skinned cardiac muscle. In the adult Tn environment (cTnT3 + cardiac troponin I), the single cTnT3-?N100 and cTnT3-?E101 mutations had opposing effects on the Ca(2+) sensitivity of force development compared with WT, whereas the double deletion cTnT3-?N100/?E101 increased the Ca(2+) sensitivity + 0.19 pCa units. In addition, cTnT3-?N100/?E101 decreased the cooperativity of force development, suggesting alterations in intrafilament protein-protein interactions. In the fetal Tn environment, (cTnT1 + slow skeletal troponin I), the single (cTnT1-?N110) and double (cTnT1-?N110/?E111) deletions did not change the Ca(2+) sensitivity compared with control. To recreate the patient's heterozygous genotype, we performed a reconstituted ATPase activity assay. Thin filaments containing 50:50 cTnT3-?N100/?E101:cTnT3-WT also increased the myofilament Ca(2+) sensitivity compared with WT. Co-sedimentation of thin filament proteins indicated that no significant changes occurred in the binding of Tn containing the RCM cTnT mutation to actin-Tm. This report reveals the protective role of Tn fetal isoforms as they rescue the increased Ca(2+) sensitivity produced by a cTnT-RCM mutation and may account for the lack of lethality during gestation.

| S-EPMC3121488 | biostudies-literature

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

Project description: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.

| S-EPMC4640586 | biostudies-literature

A mutation in TNNC1-encoded cardiac troponin C, TNNC1-A31S, predisposes to hypertrophic cardiomyopathy and ventricular fibrillation.

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

| S-EPMC3442518 | biostudies-literature

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

Project description:BACKGROUND:Restrictive cardiomyopathy is a rare cardiac disease, for which several genes including TNNT2, MYPN, FLNC and TNNI3 have been associated with its familial form. CASE PRESENTATION:Here 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. CONCLUSION:These observations point to a recessive mode of inheritance reported for the first time for this combination of gene/disease.

| S-EPMC6451262 | biostudies-literature