Translational investigation of electrophysiology in hypertrophic cardiomyopathy

Abstract

Hypertrophic cardiomyopathy (HCM) patients are at increased risk of ventricular arrhythmias and sudden cardiac death, which can occur even in the absence of structural changes of the heart. HCM mouse models suggest mutations in myofilament components to affect Ca 2+ homeostasis and thereby favor arrhythmia development. Additionally, some of them show indications of pro-arrhythmic changes in cardiac electrophysiology. In this study, we explored arrhythmia mechanisms in mice carrying a HCM mutation in Mybpc3 ( Mybpc3 -KI) and tested the translatability of our findings in human engineered heart tissues (EHTs) derived from CRISPR/Cas9-generated homozygous MYBPC3 mutant ( MYBPC3 hom) in induced pluripotent stem cells (iPSC) and to left ventricular septum samples obtained from HCM patients. We observed higher arrhythmia susceptibility in contractility measurements of field-stimulated intact cardiomyocytes and ventricular muscle strips as well as in electromyogram recordings of Langendorff-perfused hearts from adult Mybpc3 -KI mice than in wild-type (WT) controls. The latter only occurred in homozygous (Hom-KI) but not in heterozygous (Het-KI) mouse hearts. Both Het- and Hom-KI are known to display pro-arrhythmic increased Ca 2+ myofilament sensitivity as a direct consequence of the mutation. In the electrophysiological characterization of the model, we observed smaller repolarizing K + currents in single cell patch clamp, longer ventricular action potentials in sharp microelectrode recordings and longer ventricular refractory periods in Langendorff-perfused hearts in Hom-KI, but not Het-KI. Interestingly, reduced K + channel subunit transcript levels and prolonged action potentials were already detectable in newborn, pre-hypertrophic Hom-KI mice. Human iPSC-derived MYBPC3 hom EHTs, which genetically mimicked the Hom-KI mice, did exhibit lower mutant mRNA and protein levels, lower force, beating frequency and relaxation time, but no significant alteration of the force-Ca 2+ relation in skinned EHTs. Furthermore, MYBPC3 hom EHTs did show higher spontaneous arrhythmic behavior, whereas action potentials measured by sharp microelectrode did not differ to isogenic controls. Action potentials measured in septal myectomy samples did not differ between patients with HCM and patients with aortic stenosis, except for the only sample with a MYBPC3 mutation. The data demonstrate that increased myofilament Ca 2+ sensitivity is not sufficient to induce arrhythmias in the Mybpc3 -KI mouse model and suggest that reduced K + currents can be a pro-arrhythmic trigger in Hom-KI mice, probably already in early disease stages. However, neither data fro...

Ion Channel Impairment and Myofilament Ca2+ Sensitization: Two Parallel Mechanisms Underlying Arrhythmogenesis in Hypertrophic Cardiomyopathy

“…The same pattern of decreased K + channel expression was observed in several transgenic mouse models of HCM carrying different mutations [28][29][30]56]. Toib and colleagues, in a HCM transgenic rodent model with MyBPC knockout (KO), demonstrated that a reduction of K + currents resulted in a significant prolongation of the QT interval at the ECG evaluation and premature ventricular contractions (PVCs) evidenced by telemetry recordings [28].…”

“…Measurements with sharp microelectrodes in the LV endocardium revealed that hearts from homozygous mutant mice were characterized by significantly longer APs compared to heterozygous mutant mice and WT mice, both in postnatal day 1 and 30-week-old mice, confirming that: 1) AP prolongation represents one of the main EP abnormalities of HCM and 2) the electrical impairment typical of HCM occurs early during the pathological phenotype development. Interestingly, all outward currents were reduced in aged homozygous mice compared to WT animals and this decrease was particularly evident for I to and I Ks [29]. The reliability of these measurements was confirmed by incorporating all the data related to K + and Ca 2+ currents in a computational model of a mouse ventricular AP: the "in silico" representation resulted in a similar prolongation of APD in computed homozygous MYBPC3 mutant cells, as compared to WT myocytes [29].…”

“…Interestingly, all outward currents were reduced in aged homozygous mice compared to WT animals and this decrease was particularly evident for I to and I Ks [29]. The reliability of these measurements was confirmed by incorporating all the data related to K + and Ca 2+ currents in a computational model of a mouse ventricular AP: the "in silico" representation resulted in a similar prolongation of APD in computed homozygous MYBPC3 mutant cells, as compared to WT myocytes [29]. Together with MYBPC3, mutations in β-cardiac MHC (myosin heavy chain) are about 80% of pathology-related mutations in human HCM.…”

“…To satisfy this need, many studies have been drawn up through the years to gain deeper insight into the cellular and molecular mechanisms underpinning the pathogenesis of life-threatening arrhythmias in HCM patients with mild hypertrophic phenotype and no major fibrotic scars, using various tools and models to study the underlying electrical mechanisms. Different tools have been used in the recent years to model HCM [26][27][28][29][30][31][32][33][34] in order to unveil the different pathomechanisms contributing to the arrhythmogenic substrate in HCM. Notwithstanding that the aforementioned histological abnormalities represent well-established mechanisms contributing to the arrhythmogenic substrate in advanced HCM, at earlier stages of the disease, when the classical structural abnormalities are much less prominent, different arrhythmic mechanisms originating within the affected cardiomyocytes must be the main cause of ventricular tachycardia in these patients.…”

Human Engineered Heart Tissue Models for Disease Modeling and Drug Discovery

“…After demonstrating the efficacy with diltiazem in EHTs, the drug improved the electrical phenotype of HCM-affected family members. Flenner et al generated hiPSCs with an HCM mutation (homozygous MYBPC3 mutant; MYBPC3hom) and the hiPSC-CM-derived EHTs harboring this mutation exhibited lower K + current protein levels, force, beating frequency, and relaxation time, highlighting the application of not only force measurements but also arrhythmia susceptibility ( Flenner et al, 2021 ). Tsan et al generated micron-scale cardiac muscle bundles from hiPSC-CMs with a deletion of the MYBPC3 promoter, and demonstrated that contractile velocities were higher while relaxation velocities were lower, recapitulating the kinetics of HCM ( Tsan et al, 2021 ).…”