Abstract
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.
Highlights
Hypertrophic cardiomyopathy (HCM) is a genetic disorder that presents clinically as ventricular and/or septal hypertrophy, myocyte disarray, and increased myocardial fibrosis (Maron et al, 1995; Maron et al, 2006)
Despite over 20 years of studies conducted on HCM-causing TNNT2 variants, there is an unmet need to understand the connection between biophysical triggers and disease progression
We focused on the missense TNNT2 mutation I79N+/−, which has been associated with a high rate of sudden cardiac death. human cardiac reconstituted thin filament (hcRTF) and hiPSC-CMs were used to assess the effects of the I79N variant on Ca2+ dissociation rate [koff (Ca2)+ dissociation kinetics, regulation of excitation/relaxation, and the changes in transcriptome
Summary
Hypertrophic cardiomyopathy (HCM) is a genetic disorder that presents clinically as ventricular and/or septal hypertrophy, myocyte disarray, and increased myocardial fibrosis (Maron et al, 1995; Maron et al, 2006). Mice expressing the I79N variant were susceptible to ventricular tachycardia and arrhythmias that was coincident with an increased myofibrillar Ca2+ sensitivity, action potential triangulation, and increased dispersion of ventricular conduction velocities at fast heart rates in the absence of structural abnormalities (Baudenbacher et al, 2008; Schober et al, 2012) These results with I79N TNNT2 in transgenic mice were subsequently corroborated by the same group using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) (Wang et al, 2018). The mRNA expression profile strongly indicates that this TNNT2 variant induces a major remodeling response even with the cells not being fully mature, a finding which deserves further exploration
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