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Variant R94C in TNNT2-Encoded Troponin T Predisposes to Pediatric Restrictive Cardiomyopathy and Sudden Death Through Impaired Thin Filament Relaxation Resulting in Myocardial Diastolic Dysfunction.

ABSTRACT: Background Pediatric-onset restrictive cardiomyopathy (RCM) is associated with high mortality, but underlying mechanisms of disease are under investigated. RCM-associated diastolic dysfunction secondary to variants in TNNT2-encoded cardiac troponin T (TNNT2) is poorly described. Methods and Results Genetic analysis of a proband and kindred with RCM identified TNNT2-R94C, which cosegregated in a family with 2 generations of RCM, ventricular arrhythmias, and sudden death. TNNT2-R94C was absent among large, population-based cohorts Genome Aggregation Database (gnomAD) and predicted to be pathologic by in silico modeling. Biophysical experiments using recombinant human TNNT2-R94C demonstrated impaired cardiac regulation at the molecular level attributed to reduced calcium-dependent blocking of myosin's interaction with the thin filament. Computational modeling predicted a shift in the force-calcium curve for the R94C mutant toward submaximal calcium activation compared within the wild type, suggesting low levels of muscle activation even at resting calcium concentrations and hypercontractility following activation by calcium. Conclusions The pathogenic TNNT2-R94C variant activates thin-filament-mediated sarcomeric contraction at submaximal calcium concentrations, likely resulting in increased muscle tension during diastole and hypercontractility during systole. This describes the proximal biophysical mechanism for development of RCM in this family.

SUBMITTER: Ezekian JE

PROVIDER: S-EPMC7335540 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Variant R94C in <i>TNNT2</i>-Encoded Troponin T Predisposes to Pediatric Restrictive Cardiomyopathy and Sudden Death Through Impaired Thin Filament Relaxation Resulting in Myocardial Diastolic Dysfunction.

Ezekian Jordan E JE Clippinger Sarah R SR Garcia Jaquelin M JM Yang Qixin Q Denfield Susan S Jeewa Aamir A Dreyer William J WJ Zou Wenxin W Fan Yuxin Y Allen Hugh D HD Kim Jeffrey J JJ Greenberg Michael J MJ Landstrom Andrew P AP

Journal of the American Heart Association 20200226 5

Background Pediatric-onset restrictive cardiomyopathy (RCM) is associated with high mortality, but underlying mechanisms of disease are under investigated. RCM-associated diastolic dysfunction secondary to variants in <i>TNNT2</i>-encoded cardiac troponin T (TNNT2) is poorly described. Methods and Results Genetic analysis of a proband and kindred with RCM identified TNNT2-R94C, which cosegregated in a family with 2 generations of RCM, ventricular arrhythmias, and sudden death. TNNT2-R94C was abs ...[more]

PMID: 32098556

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Project description:Troponin (Tn) is the calcium-sensing protein of the thin filament. Although cardiac troponin (cTn) and skeletal troponin (sTn) accomplish the same function, their subunit interactions within Tn and with actin-tropomyosin are different. To further characterize these differences, myofibril ATPase activity as a function of pCa and labeled Tn exchange in rigor myofibrils was used to estimate Tn dissociation rates from the nonoverlap and overlap region as a function of pCa. Measurement of ATPase activity showed that skeletal myofibrils containing >96% cTn had a higher pCa 9 ATPase activity than, but similar pCa 4 activity to, sTn-containing myofibrils. Analysis of the pCa-ATPase activity relation showed that cTn myofibrils were more calcium sensitive but less cooperative (pCa50 = 6.14, nH = 1.46) than sTn myofibrils (pCa50= 5.90, nH = 3.36). The time course of labeled Tn exchange at pCa 9 and 4 were quite different between cTn and sTn. The apparent cTn dissociation rates were approximately 2-10-fold faster than sTn under all the conditions studied. The apparent dissociation rates for cTn were 5 x 10(-3) min(-1), 150 x 10(-3) min(-1), and 260 x 10(-3) min(-1), whereas for sTn they were 0.6 x 10(-3) min(-1), 88 x 10(-3) min(-1), and 68 x 10(-3) min(-1) for the nonoverlap region at pCa 9, nonoverlap region at pCa 4, and overlap region at pCa 4, respectively. Normalization of the apparent dissociation rates gives 1:30:50 for cTn compared with 1:150:110 for sTn (nonoverlap at pCa 9:nonoverlap at pCa 4:overlap at pCa 4) suggesting that calcium has a smaller influence, whereas strong cross-bridges have a larger influence on cTn dissociation compared with sTn. The higher cTn dissociation rate in the nonoverlap region and ATPase activity at pCa 9 suggest that it gives a less off or inactive thin filament. Analysis of the intensity ratio (after a short time of exchange) as a function of pCa showed that cTn had greater calcium sensitivity but lower cooperativity than sTn. In addition, the magnitude of the change in intensity ratio going from pCa 9 to 4 was less for cTn than sTn. These data suggest that the influence of calcium on cTn exchange is less than sTn even though calcium can activate ATPase activity to a similar extent in cTn compared with sTn myofibrils. This may be explained partially by cTn being less off or inactive at pCa 9. Modeling of the intensity profiles obtained after Tn exchange at pCa 5.8 suggest that the profiles are best explained by a model that includes a long-range cross-bridge effect that grades with distance from the rigor cross-bridge for both cTn and sTn.

Project description:Tropomyosin and troponin regulate muscle contraction by participating in a macromolecular scale steric-mechanism to control myosin-crossbridge - actin interactions and consequently contraction. At low-Ca2+, the C-terminal 30% of troponin subunit-I (TnI) is proposed to trap tropomyosin in a position on thin filaments that sterically interferes with myosin-binding, thus causing muscle relaxation. In contrast, at high-Ca2+, inhibition is released after the C-terminal domains dissociate from F-actin-tropomyosin as its component switch-peptide domain binds to the N-lobe of troponin-C (TnC). Recent, paradigm-shifting, cryo-EM reconstructions by the Namba group have revealed density attributed to TnI along cardiac muscle thin filaments at both low- and high-Ca2+ concentration. Modeling the reconstructions showed expected high-Ca2+ hydrophobic interactions of the TnI switch-peptide and TnC. However, under low-Ca2+ conditions, sparse interactions of TnI and tropomyosin, and in particular juxtaposition of non-polar switch-peptide residues and charged tropomyosin amino acids in the published model seem difficult to reconcile with an expected steric-blocking conformation. This anomaly is likely due to inaccurate fitting of tropomyosin into the cryo-EM volume. In the current study, the low-Ca2+ cryo-EM volume was fitted with a more accurate tropomyosin model and representation of cardiac TnI. Our results show that at low-Ca2+ a cluster of hydrophobic residues at the TnI switch-peptide and adjacent H4 helix (Ala149, Ala151, Met 154, Leu159, Gly160, Ala161, Ala163, Leu167, Leu169, Ala171, Leu173) draw-in tropomyosin surface residues (Ile143, Ile146, Ala151, Ile154), presumably attracting the entire tropomyosin cable to its myosin-blocking position on actin. The modeling confirms that neighboring TnI "inhibitory domain" residues (Arg145, Arg148) bind to thin filaments at actin residue Asp25, as previously suggested. ClusPro docking of TnI residues 137-184 to actin-tropomyosin, including the TnI inhibitory-domain, switch-peptide and Helix H4, verified the modeled configuration. Our residue-to-residue contact-mapping of the TnI-tropomyosin association lends itself to experimental validation and functional localization of disease-bearing mutations.

Project description: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. SUMMARY: A 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.

Dataset Information

Variant R94C in TNNT2-Encoded Troponin T Predisposes to Pediatric Restrictive Cardiomyopathy and Sudden Death Through Impaired Thin Filament Relaxation Resulting in Myocardial Diastolic Dysfunction.

Publications

Variant R94C in <i>TNNT2</i>-Encoded Troponin T Predisposes to Pediatric Restrictive Cardiomyopathy and Sudden Death Through Impaired Thin Filament Relaxation Resulting in Myocardial Diastolic Dysfunction.

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