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Mechanisms of Pathogenicity of Hypertrophic Cardiomyopathy-Associated Troponin T (TNNT2) Variant R278C^+/- During Development.

ABSTRACT: Hypertrophic cardiomyopathy (HCM) is one of the most common heritable cardiovascular diseases and variants of TNNT2 (cardiac troponin T) are linked to increased risk of sudden cardiac arrest despite causing limited hypertrophy. In this study, a TNNT2 variant, R278C^+/-, was generated in both human cardiac recombinant/reconstituted thin filaments (hcRTF) and human- induced pluripotent stem cells (hiPSCs) to investigate the mechanisms by which the R278C^+/- variant affects cardiomyocytes at the proteomic and functional levels. The results of proteomics analysis showed a significant upregulation of markers of cardiac hypertrophy and remodeling in R278C^+/- vs. the isogenic control. Functional measurements showed that R278C^+/- variant enhances the myofilament sensitivity to Ca²⁺, increases the kinetics of contraction, and causes arrhythmia at frequencies >75 bpm. This study uniquely shows the profound impact of the TNNT2 R278C^+/- variant on the cardiomyocyte proteomic profile, cardiac electrical and contractile function in the early stages of cardiac development.

SUBMITTER: Shafaattalab S

PROVIDER: S-EPMC10441323 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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Mechanisms of Pathogenicity of Hypertrophic Cardiomyopathy-Associated Troponin T (TNNT2) Variant R278C+/- During Development.

Shafaattalab Sanam S Li Alison Y AY Jayousi Farah F Maaref Yasaman Y Dababneh Saif S Hamledari Homa H Baygi Dina Hosseini DH Barszczewski Tiffany T Ruprai Balwinder B Jannati Shayan S Nagalingam Raghu R Cool Austin M AM Langa Paulina P Chiao Mu M Roston Thomas T Solaro R John RJ Sanatani Shubhayan S Toepfer Christopher C Lindert Steffen S Lange Philipp P Tibbits Glen F GF

bioRxiv : the preprint server for biology 20230813

Hypertrophic cardiomyopathy (HCM) is one of the most common heritable cardiovascular diseases and variants of TNNT2 (cardiac troponin T) are linked to increased risk of sudden cardiac arrest despite causing limited hypertrophy. In this study, a TNNT2 variant, R278C+/-, was generated in both human cardiac recombinant/reconstituted thin filaments (hcRTF) and human- induced pluripotent stem cells (hiPSCs) to investigate the mechanisms by which the R278C+/- varian ...[more]

PMID: 37609317

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Project description:Hypertrophic cardiomyopathy (HCM) is the most common heritable cardiovascular disease and often results in cardiac remodeling and an increased incidence of sudden cardiac arrest (SCA) and death, especially in youth and young adults. Among thousands of different variants found in HCM patients, variants of TNNT2 (cardiac troponin T-TNNT2) are linked to increased risk of ventricular arrhythmogenesis and sudden death despite causing little to no cardiac hypertrophy. Therefore, studying the effect of TNNT2 variants on cardiac propensity for arrhythmogenesis can pave the way for characterizing HCM in susceptible patients before sudden cardiac arrest occurs. In this study, a TNNT2 variant, I79N, was generated in human cardiac recombinant/reconstituted thin filaments (hcRTF) to investigate the effect of the mutation on myofilament Ca2+ sensitivity and Ca2+ dissociation rate using steady-state and stopped-flow fluorescence techniques. The results revealed that the I79N variant significantly increases myofilament Ca2+ sensitivity and decreases the Ca2+ off-rate constant (k off). To investigate further, a heterozygous I79N+/- TNNT2 variant was introduced into human-induced pluripotent stem cells using CRISPR/Cas9 and subsequently differentiated into ventricular cardiomyocytes (hiPSC-CMs). To study the arrhythmogenic properties, monolayers of I79N+/- hiPSC-CMs were studied in comparison to their isogenic controls. Arrhythmogenesis was investigated by measuring voltage (V m) and cytosolic Ca2+ transients over a range of stimulation frequencies. An increasing stimulation frequency was applied to the cells, from 55 to 75 bpm. The results of this protocol showed that the TnT-I79N cells had reduced intracellular Ca2+ transients due to the enhanced cytosolic Ca2+ buffering. These changes in Ca2+ handling resulted in beat-to-beat instability and triangulation of the cardiac action potential, which are predictors of arrhythmia risk. While wild-type (WT) hiPSC-CMs were accurately entrained to frequencies of at least 150 bpm, the I79N hiPSC-CMs demonstrated clear patterns of alternans for both V m and Ca2+ transients at frequencies >75 bpm. Lastly, a transcriptomic analysis was conducted on WT vs. I79N+/- TNNT2 hiPSC-CMs using a custom NanoString codeset. The results showed a significant upregulation of NPPA (atrial natriuretic peptide), NPPB (brain natriuretic peptide), Notch signaling pathway components, and other extracellular matrix (ECM) remodeling components in I79N+/- vs. the isogenic control. This significant shift demonstrates that this missense in the TNNT2 transcript likely causes a biophysical trigger, which initiates this significant alteration in the transcriptome. This TnT-I79N hiPSC-CM model not only reproduces key cellular features of HCM-linked mutations but also suggests that this variant causes uncharted pro-arrhythmic changes to the human action potential and gene expression.

Dataset Information

Mechanisms of Pathogenicity of Hypertrophic Cardiomyopathy-Associated Troponin T (TNNT2) Variant R278C^+/- During Development.

Publications

Mechanisms of Pathogenicity of Hypertrophic Cardiomyopathy-Associated Troponin T (TNNT2) Variant R278C<sup>+/-</sup> During Development.

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