Generation and Characterization of a Human iPSC Cardiomyocyte Model of Troponin T I79N Linked Hypertrophic Cardiomyopathy

Abstract

Objective: The aim of this study was to examine the functional consequences of a troponin T mutation (I79N) associated with hypertrophic cardiomyopathy (HCM) in cardiomyocytes derived from human induced pluripotent stem cells (hiPSC). Method and Results: We generated a TnT-I79N hiPSC HCM model by inserting the 236T>A mutation into the TNNT2 gene of one of the alleles of hiPSC derived from a healthy donor using CRISPR/Cas9. There was no off-target cleavage produced by CRISPR/Cas9. Nano-liquid chromatography mass spectrometry showed that 43% of TnT protein was mutant in cardiomyocytes (CM) derived from TnT-I79N hiPSCs. In 2D monolayers, the impedance amplitude (a measure of contractility) was increased and the Ca-contractility relationship leftward shifted in TnT-I79N hiPSC-CMs compared to isogenic control hiPSC-CM. Results were confirmed using single hiPSC-CM cultured on a matrigel matress: The TnT-I79N mutation altered cytosolic Ca buffering properties by lowering the apparent dissociation constant Kd (0.40±0.04 μM in TnT-I79N, n=12 vs. 0.72±0.10 μM in control, n=16, P<0.05) without changing the maximal binding capacity Bmax, indicating that TnT-I79N mutation enhanced the Ca-binding affinity of myofilaments. Likely as a result of the increased cytosolic Ca-binding affinity, I79N hiPSC-CM paced at 0.2 Hz exhibited significantly reduced intracellular Ca-transients (ΔF/Fratio 0.85±0.03 in I79N vs. 1.01±0.06 in control, P<0.05) and reduced caffeine transient (ΔF/Fratio 0.92±0.06 in I79N vs. 1.10±0.06 in control, P<0.05), whereas Ca-decay kinetics where unchanged. Despite the reduced Ca-transient amplitude, TnT-I79N mutation enhanced contractility but delayed the relaxation in single hiPSC-CMs, indicating an increased sensitivity to Ca. Conclusion: The I79N hiPSC-CM model reproduces key findings from I79N transgenic mice: increased myofilament Ca-sensitivity, enhances contractility, impaired relaxation and enhanced cytosolic Ca-binding affinity. Human iPSC-CM could be a promising tool to model HCM in vitro.