Translational investigation of electrophysiology in hypertrophic cardiomyopathy

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 Ca2+ 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 (MYBPC3hom) 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 Ca2+ 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 MYBPC3hom 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-Ca2+ relation in skinned EHTs. Furthermore, MYBPC3hom 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 Ca2+ 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 from EHTs nor from left ventricular samples indicate relevant reduction of K+ currents in human HCM. Therefore, our study highlights the species difference between mouse and human and emphasizes the importance of research in human samples and human-like models.publishedVersionPeer reviewe

Impact of AT2 Receptor Deficiency on Postnatal Cardiovascular Development

<div><h3>Background</h3><p>The angiotensin II receptor subtype 2 (AT2 receptor) is ubiquitously and highly expressed in early postnatal life. However, its role in postnatal cardiac development remained unclear.</p> <h3>Methodology/Principal Findings</h3><p>Hearts from 1, 7, 14 and 56 days old wild-type (WT) and AT2 receptor-deficient (KO) mice were extracted for histomorphometrical analysis as well as analysis of cardiac signaling and gene expression. Furthermore, heart and body weights of examined animals were recorded and echocardiographic analysis of cardiac function as well as telemetric blood pressure measurements were performed. Moreover, gene expression, sarcomere shortening and calcium transients were examined in ventricular cardiomyocytes isolated from both genotypes. KO mice exhibited an accelerated body weight gain and a reduced heart to body weight ratio as compared to WT mice in the postnatal period. However, in adult KO mice the heart to body weight ratio was significantly increased most likely due to elevated systemic blood pressure. At postnatal day 7 ventricular capillarization index and the density of α-smooth muscle cell actin-positive blood vessels were higher in KO mice as compared to WT mice but normalized during adolescence. Echocardiographic assessment of cardiac systolic function at postnatal day 7 revealed decreased contractility of KO hearts in response to beta-adrenergic stimulation. Moreover, cardiomyocytes from KO mice showed a decreased sarcomere shortening and an increased peak Ca²⁺ transient in response to isoprenaline when stimulated concomitantly with angiotensin II.</p> <h3>Conclusion</h3><p>The AT2 receptor affects postnatal cardiac growth possibly via reducing body weight gain and systemic blood pressure. Moreover, it moderately attenuates postnatal vascularization of the heart and modulates the beta adrenergic response of the neonatal heart. These AT2 receptor-mediated effects may be implicated in the physiological maturation process of the heart.</p> </div

AT2 receptor deficiency is associated with reduced cardiac contractility in the neonatal organism.

<p>A) Echocardiographic assessment of cardiac function in 7-day-old KO and WT mice. A significantly reduced fractional shortening (FS) after application of dobutamine (5 µg/g body weight) was observed in KO as compared to WT mice. # <i>P</i><0.01 vs. KO/WT (n = 8/10). Data are shown as mean ± SEM. B) and C) Isoprenaline- and calcium-induced inotropic response of isolated ventricular stripes derived from 7-day-old KO and WT mice (n = 8/6). A non-significant trend towards reduced calcium- and isoprenaline-mediated stimulation of ventricular contractility in KO mice was observed, respectively.</p

Angiotensin II-induced ANP mRNA expression in neonatal cardiomycytes derived from KO and WT mice.

<p>Angiotensin II (Ang II, 10⁻⁷ mol/L)-stimulated cardiomyocytes from KO mice show a similar increase in ANP mRNA expression as compared to WT mice, but have higher basal ANP mRNA levels. Angiotensin II-induced ANP mRNA expression is significantly reduced by AT1 receptor antagonist losartan, but not by AT2 receptor antagonist PD123,319 (10⁻⁷ mol/L each). * <i>P</i><0.05 vs. untreated WT or KO cardiomyocytes, respectively, § <i>P</i><0.05 vs. WT control, $ <i>P</i><0.05 vs. Ang II. Data are shown as mean ± SD; n = 6–8.</p

Continuous recording of systolic and diastolic blood pressure in adult AT2 receptor-deficient and wild-type mice (A) and analysis of the average mean blood pressure at daytime or at night (B,C).

<p>Measurements show an increased systemic blood pressure in AT2 receptor-deficient mice. Similar activity (D) and heart rates (E,F) were observed in knockout as compared with wild-type mice throughout the observational period. The data are from five wild-type mice and six knockout mice.</p

Body weight (A) and heart weight gain (B) as well as heart to body weight ratio (C) of AT2 receptor-deficient (KO) and wild-type (WT) mice during development.

<p>Accelerated cardiac and body weight gain in postnatal KO as compared to WT mice resulting in a significantly reduced heart to body weight ratio of neonatal and adolescent KO mice. In contrast, the heart to body weight ratio of adult eight week-old KO mice is significantly increased. * <i>P</i><0.001 vs. WT (n = 43–233/36–210). Data are shown as mean ± SEM.</p

Expression of angiotensin II receptor subtypes (AT1a and AT2), atrial natriuretic peptide (ANP) and Bax in the postnatal heart.

<p>A) In WT mice cardiac AT2 receptor mRNA expression peaks at postnatal day 7 (NN7) and declines thereafter. B) Cardiac AT1a receptor mRNA expression also peaked at NN7 and a significantly higher AT1a receptor mRNA expression was then observed in WT mice as compared to that observed in KO mice. C) Cardiac ANP mRNA expression declined in both WT and KO mice during cardiac development in the postnatal period, but ANP mRNA levels remained higher in KO as compared with WT mice from postnatal day 14 on. D) mRNA expression of pro-apoptotic protein Bax is decreased in cardiac tissue of neonatal AT2 receptor knockout mice. * <i>P</i><0.05 vs. WT (n = 10/10). Data are shown as mean ± SD.</p

Effects of angiotensin II (Ang II, 10⁻⁷ mol/L), isoprenaline (ISO, 10⁻⁶ mol/L), or the combination of both on sarcomere shortening (A) and calcium transients (B) of isolated cardiomyocytes from WT and KO mice (n = 10–36 cardiomyocytes from both three KO and WT hearts were analysed, respectively).

<p>** <i>P</i><0.05 vs. basal sarcomere shortening of WT or KO cardiomyocytes, # <i>P</i><0.05 vs. sarcomere shortening of WT cardiomyocytes treated with isoprenaline in the presence of angiotensin II, * <i>P</i><0.05 vs. basal WT calcium % peak height, $ <i>P</i><0.05 vs. basal KO calcium % peak height, # P<0.05 vs. calcium % peak height of WT cardiomyocytes treated with isoprenaline in the presence of angiotensin II. Data are shown as mean ± SEM. C) Representative recordings of sarcomere shortening (black/grey lines) and calcium transients (pink/red lines) in cardiomyocytes derived from KO and WT mice.</p