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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 Ca 2+  sensitivity and Ca 2+  dissociation rate using steady-state and stopped-flow fluorescence techniques. The results revealed that the I79N variant significantly increases myofilament Ca 2+  sensitivity and decreases the Ca 2+  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 Ca 2+  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 Ca 2+  transients due to the enhanced cytosolic Ca 2+  buffering. These changes in Ca 2+  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 Ca 2+  transients at frequencies &amp;gt;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.

Mechanisms of Arrhythmogenicity of Hypertrophic Cardiomyopathy-Associated Troponin T (TNNT2) Variant I79N