MYBPC3-c.772G>A mutation results in haploinsufficiency and altered myosin cycling kinetics in a patient induced stem cell derived cardiomyocyte model of hypertrophic cardiomyopathy.

Approximately 40% of hypertrophic cardiomyopathy (HCM) mutations are linked to the sarcomere protein cardiac myosin binding protein-C (cMyBP-C). These mutations are either classified as missense mutations or truncation mutations. One mutation whose nature has been inconsistently reported in the literature is the MYBPC3-c.772G > A mutation. Using patient-derived human induced pluripotent stem cells differentiated to cardiomyocytes (hiPSC-CMs), we have performed a mechanistic study of the structure-function relationship for this MYBPC3-c.772G > A mutation versus a mutation corrected, isogenic cell line. Our results confirm that this mutation leads to exon skipping and mRNA truncation that ultimately suggests ∼20% less cMyBP-C protein (i.e., haploinsufficiency). This, in turn, results in increased myosin recruitment and accelerated myofibril cycling kinetics. Our mechanistic studies suggest that faster ADP release from myosin is a primary cause of accelerated myofibril cross-bridge cycling due to this mutation. Additionally, the reduction in force generating heads expected from faster ADP release during isometric contractions is outweighed by a cMyBP-C phosphorylation mediated increase in myosin recruitment that leads to a net increase of myofibril force, primarily at submaximal calcium activations. These results match well with our previous report on contractile properties from myectomy samples of the patients from whom the hiPSC-CMs were generated, demonstrating that these cell lines are a good model to study this pathological mutation and extends our understanding of the mechanisms of altered contractile properties of this HCM MYBPC3-c.772G > A mutation.

IPSC derived cardiac fibroblasts of DMD patients show compromised actin microfilaments, metabolic shift and pro-fibrotic phenotype.

Duchenne muscular dystrophy (DMD) is a severe form of muscular dystrophy caused by mutations in the dystrophin gene. We characterized which isoforms of dystrophin were expressed by human induced pluripotent stem cell (hiPSC)-derived cardiac fibroblasts obtained from control and DMD patients. Distinct dystrophin isoforms were observed; however, highest molecular weight isoform was absent in DMD patients carrying exon deletions or mutations in the dystrophin gene. The loss of the full-length dystrophin isoform in hiPSC-derived cardiac fibroblasts from DMD patients resulted in deficient formation of actin microfilaments and a metabolic switch from mitochondrial oxidation to glycolysis. The DMD hiPSC-derived cardiac fibroblasts exhibited a dysregulated mitochondria network and reduced mitochondrial respiration, with enhanced compensatory glycolysis to sustain cellular ATP production. This metabolic remodeling was associated with an exacerbated myofibroblast phenotype and increased fibroblast activation in response to pro fibrotic challenges. As cardiac fibrosis is a critical pathological feature of the DMD heart, the myofibroblast phenotype induced by the absence of dystrophin may contribute to deterioration in cardiac function. Our study highlights the relationship between cytoskeletal dynamics, metabolism of the cell and myofibroblast differentiation and provides a new mechanism by which inactivation of dystrophin in non-cardiomyocyte cells may increase the severity of cardiopathy.

Corrigendum: Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: the impact of full-length dystrophin deficiency.

[This corrects the article DOI: 10.3389/fphys.2022.1030920.].

Slower Calcium Handling Balances Faster Cross-Bridge Cycling in Human MYBPC3 HCM.

BACKGROUND: The pathogenesis of MYBPC3-associated hypertrophic cardiomyopathy (HCM) is still unresolved. In our HCM patient cohort, a large and well-characterized population carrying the MYBPC3:c772G>A variant (p.Glu258Lys, E258K) provides the unique opportunity to study the basic mechanisms of MYBPC3-HCM with a comprehensive translational approach. METHODS: We collected clinical and genetic data from 93 HCM patients carrying the MYBPC3:c772G>A variant. Functional perturbations were investigated using different biophysical techniques in left ventricular samples from 4 patients who underwent myectomy for refractory outflow obstruction, compared with samples from non-failing non-hypertrophic surgical patients and healthy donors. Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and engineered heart tissues (EHTs) were also investigated. RESULTS: Haplotype analysis revealed MYBPC3:c772G>A as a founder mutation in Tuscany. In ventricular myocardium, the mutation leads to reduced cMyBP-C (cardiac myosin binding protein-C) expression, supporting haploinsufficiency as the main primary disease mechanism. Mechanical studies in single myofibrils and permeabilized muscle strips highlighted faster cross-bridge cycling, and higher energy cost of tension generation. A novel approach based on tissue clearing and advanced optical microscopy supported the idea that the sarcomere energetics dysfunction is intrinsically related with the reduction in cMyBP-C. Studies in single cardiomyocytes (native and hiPSC-derived), intact trabeculae and hiPSC-EHTs revealed prolonged action potentials, slower Ca2+ transients and preserved twitch duration, suggesting that the slower excitation-contraction coupling counterbalanced the faster sarcomere kinetics. This conclusion was strengthened by in silico simulations. CONCLUSIONS: HCM-related MYBPC3:c772G>A mutation invariably impairs sarcomere energetics and cross-bridge cycling. Compensatory electrophysiological changes (eg, reduced potassium channel expression) appear to preserve twitch contraction parameters, but may expose patients to greater arrhythmic propensity and disease progression. Therapeutic approaches correcting the primary sarcomeric defects may prevent secondary cardiomyocyte remodeling.

Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: The impact of full-length dystrophin deficiency.

Cardiomyocytes differentiated from human induced Pluripotent Stem Cells (hiPSC- CMs) are a unique source for modelling inherited cardiomyopathies. In particular, the possibility of observing maturation processes in a simple culture dish opens novel perspectives in the study of early-disease defects caused by genetic mutations before the onset of clinical manifestations. For instance, calcium handling abnormalities are considered as a leading cause of cardiomyocyte dysfunction in several genetic-based dilated cardiomyopathies, including rare types such as Duchenne Muscular Dystrophy (DMD)-associated cardiomyopathy. To better define the maturation of calcium handling we simultaneously measured action potential and calcium transients (Ca-Ts) using fluorescent indicators at specific time points. We combined micropatterned substrates with long-term cultures to improve maturation of hiPSC-CMs (60, 75 or 90 days post-differentiation). Control-(hiPSC)-CMs displayed increased maturation over time (90 vs 60 days), with longer action potential duration (APD), increased Ca-T amplitude, faster Ca-T rise (time to peak) and Ca-T decay (RT50). The progressively increased contribution of the SR to Ca release (estimated by post-rest potentiation or Caffeine-induced Ca-Ts) appeared as the main determinant of the progressive rise of Ca-T amplitude during maturation. As an example of severe cardiomyopathy with early onset, we compared hiPSC-CMs generated from a DMD patient (DMD-ΔExon50) and a CRISPR-Cas9 genome edited cell line isogenic to the healthy control with deletion of a G base at position 263 of the DMD gene (c.263delG-CMs). In DMD-hiPSC-CMs, changes of Ca-Ts during maturation were less pronounced: indeed, DMD cells at 90 days showed reduced Ca-T amplitude and faster Ca-T rise and RT50, as compared with control hiPSC-CMs. Caffeine-Ca-T was reduced in amplitude and had a slower time course, suggesting lower SR calcium content and NCX function in DMD vs control cells. Nonetheless, the inotropic and lusitropic responses to forskolin were preserved. CRISPR-induced c.263delG-CM line recapitulated the same developmental calcium handling alterations observed in DMD-CMs. We then tested the effects of micropatterned substrates with higher stiffness. In control hiPSC-CMs, higher stiffness leads to higher amplitude of Ca-T with faster decay kinetics. In hiPSC-CMs lacking full-length dystrophin, however, stiffer substrates did not modify Ca-Ts but only led to higher SR Ca content. These findings highlighted the inability of dystrophin-deficient cardiomyocytes to adjust their calcium homeostasis in response to increases of extracellular matrix stiffness, which suggests a mechanism occurring during the physiological and pathological development (i.e. fibrosis).

Paradoxical prolongation of QT interval during exercise in patients with hypertrophic cardiomyopathy: cellular mechanisms and implications for diastolic function.

Aims: Ventricular cardiomyocytes from hypertrophic cardiomyopathy (HCM) patient hearts show prolonged action potential duration (APD), impaired intracellular Ca2+ homeostasis and abnormal electrical response to beta -adrenergic stimulation. We sought to determine whether this behaviour is associated with abnormal changes of repolarization during exercise and worsening of diastolic function, ultimately explaining the intolerance to exercise experienced by some patients without obstruction. Methods and results: Non-obstructive HCM patients (178) and control subjects (81) underwent standard exercise testing, including exercise echocardiography. Ventricular myocytes were isolated from myocardial samples of 23 HCM and eight non-failing non-hypertrophic surgical patients. The APD shortening in response to high frequencies was maintained in HCM myocytes, while ß-adrenergic stimulation unexpectedly prolonged APDs, ultimately leading to a lesser shortening of APDs in response to exercise. In HCM vs. control subjects, we observed a lesser shortening of QT interval at peak exercise (QTc: +27 ± 52 ms in HCM, -4 ± 50 ms in controls, P < 0.0001). In patients showing a marked QTc prolongation (>30 ms), the excessive shortening of the electrical diastolic period was linked with a limited increase of heart-rate and deterioration of diastolic function at peak effort. Conclusions: Abnormal balance of Ca2+- and K+-currents in HCM cardiomyocytes determines insufficient APD and Ca2+-transient shortening with exercise. In HCM patients, exercise-induced QTc prolongation was associated with impaired diastolic reserve, contributing to the reduced exercise tolerance. Our results support the idea that severe electrical cardiomyocyte abnormalities underlie exercise intolerance in a subgroup of HCM patients without obstruction.

Genotype-Driven Pathogenesis of Atrial Fibrillation in Hypertrophic Cardiomyopathy: The Case of Different TNNT2 Mutations.

Atrial dilation and atrial fibrillation (AF) are common in Hypertrophic CardioMyopathy (HCM) patients and associated with a worsening of prognosis. The pathogenesis of atrial myopathy in HCM remains poorly investigated and no specific association with genotype has been identified. By re-analysis of our cohort of thin-filament HCM patients (Coppini et al. 2014) AF was identified in 10% of patients with sporadic mutations in the cardiac Troponin T gene (TNNT2), while AF occurrence was much higher (25-75%) in patients carrying specific "hot-spot" TNNT2 mutations. To determine the molecular basis of arrhythmia occurrence, two HCM mouse models expressing human TNNT2 variants (a "hot-spot" one, R92Q, and a "sporadic" one, E163R) were selected according to the different pathophysiological pathways previously demonstrated in ventricular tissue. Echocardiography studies showed a significant left atrial dilation in both models, but more pronounced in the R92Q. In E163R atrial trabeculae, in line with what previously observed in ventricular preparations, the energy cost of tension generation was markedly increased. However, no changes of twitch amplitude and kinetics were observed, and there was no atrial arrhythmic propensity. R92Q atrial trabeculae, instead, displayed normal ATP consumption but markedly increased myofilament calcium sensitivity, as previously observed in ventricular preparations. This was associated with reduced inotropic reserve and slower kinetics of twitch contractions and, importantly, with an increased occurrence of spontaneous beats and triggered contractions that represent an intrinsic arrhythmogenic mechanism promoting AF. The association of specific TNNT2 mutations with AF occurrence depends on the mutation-driven pathomechanism (i.e., increased atrial myofilament calcium sensitivity rather than increased myofilament tension cost) and may influence the individual response to treatment.

The harder the climb the better the view: The impact of substrate stiffness on cardiomyocyte fate.

The quest for novel methods to mature human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for cardiac regeneration, modelling and drug testing has emphasized a need to create microenvironments with physiological features. Many studies have reported on how cardiomyocytes sense substrate stiffness and adapt their morphological and functional properties. However, these observations have raised new biological questions and a shared vision to translate it into a tissue or organ context is still elusive. In this review, we will focus on the relevance of substrates mimicking cardiac extracellular matrix (cECM) rigidity for the understanding of the biomechanical crosstalk between the extracellular and intracellular environment. The ability to opportunely modulate these pathways could be a key to regulate in vitro hiPSC-CM maturation. Therefore, both hiPSC-CM models and substrate stiffness appear as intriguing tools for the investigation of cECM-cell interactions. More understanding of these mechanisms may provide novel insights on how cECM affects cardiac cell function in the context of genetic cardiomyopathies.

Mavacamten has a differential impact on force generation in myofibrils from rabbit psoas and human cardiac muscle.

Mavacamten (MYK-461) is a small-molecule allosteric inhibitor of sarcomeric myosins being used in preclinical/clinical trials for hypertrophic cardiomyopathy treatment. A better understanding of its impact on force generation in intact or skinned striated muscle preparations, especially for human cardiac muscle, has been hindered by diffusional barriers. These limitations have been overcome by mechanical experiments using myofibrils subject to perturbations of the contractile environment by sudden solution changes. Here, we characterize the action of mavacamten in human ventricular myofibrils compared with fast skeletal myofibrils from rabbit psoas. Mavacamten had a fast, fully reversible, and dose-dependent negative effect on maximal Ca2+-activated isometric force at 15°C, which can be explained by a sudden decrease in the number of heads functionally available for interaction with actin. It also decreased the kinetics of force development in fast skeletal myofibrils, while it had no effect in human ventricular myofibrils. For both myofibril types, the effects of mavacamten were independent from phosphate in the low-concentration range. Mavacamten did not alter force relaxation of fast skeletal myofibrils, but it significantly accelerated the relaxation of human ventricular myofibrils. Lastly, mavacamten had no effect on resting tension but inhibited the ADP-stimulated force in the absence of Ca2+. Altogether, these effects outline a motor isoform-specific dependence of the inhibitory effect of mavacamten on force generation, which is mediated by a reduction in the availability of strongly actin-binding heads. Mavacamten may thus alter the interplay between thick and thin filament regulation mechanisms of contraction in association with the widely documented drug effect of stabilizing myosin motor heads into autoinhibited states.

The relation between sarcomere energetics and the rate of isometric tension relaxation in healthy and diseased cardiac muscle.

Full muscle relaxation happens when [Ca2+] falls below the threshold for force activation. Several experimental models, from whole muscle organs and intact muscle down to skinned fibers, have been used to explore the cascade of kinetic events leading to mechanical relaxation. The use of single myofibrils together with fast solution switching techniques, has provided new information about the role of cross-bridge (CB) dissociation in the time course of isometric force decay. Myofibril's relaxation is biphasic starting with a slow seemingly linear phase, with a rate constant, slow kREL, followed by a fast mono-exponential phase. Sarcomeres remain isometric during the slow force decay that reflects CB detachment under isometric conditions while the final fast relaxation phase begins with a sudden give of few sarcomeres and is then dominated by intersarcomere dynamics. Based on a simple two-state model of the CB cycle, myofibril slow kREL represents the apparent forward rate with which CBs leave force generating states (gapp) under isometric conditions and correlates with the energy cost of tension generation (ATPase/tension ratio); in short slow kREL ~ gapp ~ tension cost. The validation of this relationship is obtained by simultaneously measuring maximal isometric force and ATP consumption in skinned myocardial strips that provide an unambiguous determination of the relation between contractile and energetic properties of the sarcomere. Thus, combining kinetic experiments in isolated myofibrils and mechanical and energetic measurements in multicellular cardiac strips, we are able to provide direct evidence for a positive linear correlation between myofibril isometric relaxation kinetics (slow kREL) and the energy cost of force production both measured in preparations from the same cardiac sample. This correlation remains true among different types of muscles with different ATPase activities and also when CB kinetics are altered by cardiomyopathy-related mutations. Sarcomeric mutations associated to hypertrophic cardiomyopathy (HCM), a primary cardiac disorder caused by mutations in genes encoding sarcomeric proteins, have been often found to accelerate CB turnover rate and increase the energy cost of myocardial contraction. Here we review data showing that faster CB detachment results in a proportional increase in the energetic cost of tension generation in heart samples from both HCM patients and mouse models of the disease.

Advances in Stem Cell Modeling of Dystrophin-Associated Disease: Implications for the Wider World of Dilated Cardiomyopathy.

Familial dilated cardiomyopathy (DCM) is mostly caused by mutations in genes encoding cytoskeletal and sarcomeric proteins. In the pediatric population, DCM is the predominant type of primitive myocardial disease. A severe form of DCM is associated with mutations in the DMD gene encoding dystrophin, which are the cause of Duchenne Muscular Dystrophy (DMD). DMD-associated cardiomyopathy is still poorly understood and orphan of a specific therapy. In the last 5 years, a rise of interest in disease models using human induced pluripotent stem cells (hiPSCs) has led to more than 50 original studies on DCM models. In this review paper, we provide a comprehensive overview on the advances in DMD cardiomyopathy disease modeling and highlight the most remarkable findings obtained from cardiomyocytes differentiated from hiPSCs of DMD patients. We will also describe how hiPSCs based studies have contributed to the identification of specific myocardial disease mechanisms that may be relevant in the pathogenesis of DCM, representing novel potential therapeutic targets.

Optical Investigation of Action Potential and Calcium Handling Maturation of hiPSC-Cardiomyocytes on Biomimetic Substrates.

Cardiomyocytes from human induced pluripotent stem cells (hiPSC-CMs) are the most promising human source with preserved genetic background of healthy individuals or patients. This study aimed to establish a systematic procedure for exploring development of hiPSC-CM functional output to predict genetic cardiomyopathy outcomes and identify molecular targets for therapy. Biomimetic substrates with microtopography and physiological stiffness can overcome the immaturity of hiPSC-CM function. We have developed a custom-made apparatus for simultaneous optical measurements of hiPSC-CM action potential and calcium transients to correlate these parameters at specific time points (day 60, 75 and 90 post differentiation) and under inotropic interventions. In later-stages, single hiPSC-CMs revealed prolonged action potential duration, increased calcium transient amplitude and shorter duration that closely resembled those of human adult cardiomyocytes from fresh ventricular tissue of patients. Thus, the major contribution of sarcoplasmic reticulum and positive inotropic response to ß-adrenergic stimulation are time-dependent events underlying excitation contraction coupling (ECC) maturation of hiPSC-CM; biomimetic substrates can promote calcium-handling regulation towards adult-like kinetics. Simultaneous optical recordings of long-term cultured hiPSC-CMs on biomimetic substrates favor high-throughput electrophysiological analysis aimed at testing (mechanistic hypothesis on) disease progression and pharmacological interventions in patient-derived hiPSC-CMs.

A Novel Method of Isolating Myofibrils From Primary Cardiomyocyte Culture Suitable for Myofibril Mechanical Study.

Myofibril based mechanical studies allow evaluation of sarcomeric protein function. We describe a novel method of obtaining myofibrils from primary cardiomyocyte culture. Adult rat ventricular myocytes (ARVMs) were obtained by enzymatic digestion and maintained in serum free condition. ARVMs were homogenized in relaxing solution (pCa 9.0) with 20% sucrose, and myofibril suspension was made. Myofibrils were Ca2+-activated and relaxed at 15°C. Results from ARVM myofibrils were compared to myofibrils obtained from ventricular tissue skinned with Triton X-100. At maximal Ca2+-activation (pCa 4.5) myofibril mechanical parameters from ARVMs were 6.8 ± 0.9 mN/mm2 (resting tension), 146.8 ± 13.8 mN/mm2 (maximal active tension, P0), 5.4 ± 0.22 s-1 (rate of force activation), 53.4 ± 4.4 ms (linear relaxation duration), 0.69 ± 0.36 s-1 (linear relaxation rate), and 10.8 ± 1.3 s-1 (exponential relaxation rate). Force-pCa curves were constructed from Triton skinned tissue, ARVM culture day 1, and ARVM culture day 3 myofibrils, and pCa50 were 5.79 ± 0.01, 5.69 ± 0.01, and 5.71 ± 0.01, respectively. Mechanical parameters from myofibrils isolated from ARVMs treated with phenylephrine were compared to myofibrils isolated from time-matched non-treated ARVMs. Phenylephrine treatment did not change the kinetics of activation or relaxation but decreased the pCa50 to 5.56 ± 0.03 (vehicle treated control: 5.67 ± 0.03). For determination of protein expression and post-translational modifications, myofibril slurry was re-suspended and resolved for immunoblotting and protein staining. Troponin I phosphorylation was significantly increased at serine 23/24 in phenylephrine treated group. Myofibrils obtained from ARVMs are a viable method to study myofibril mechanics. Phenylephrine treatment led to significant decrease in Ca2+-sensitivity that is due to increased phosphorylation of TnI at serine 23/24. This culture based approach to obtaining myofibrils will allow pharmacological and genetic manipulation of the cardiomyocytes to correlate biochemical and biophysical properties.

Electrophysiological and Contractile Effects of Disopyramide in Patients With Obstructive Hypertrophic Cardiomyopathy: A Translational Study.

Disopyramide is effective and safe in patients with obstructive hypertrophic cardiomyopathy. However, its cellular and molecular mechanisms of action are unknown. We tested disopyramide in cardiomyocytes from the septum of surgical myectomy patients: disopyramide inhibits multiple ion channels, leading to lower Ca transients and force, and shortens action potentials, thus reducing cellular arrhythmias. The electrophysiological profile of disopyramide explains the efficient reduction of outflow gradients but also the limited prolongation of the QT interval and the absence of arrhythmic side effects observed in 39 disopyramide-treated patients. In conclusion, our results support the idea that disopyramide is safe for outpatient use in obstructive patients.

Late sodium current inhibitors to treat exercise-induced obstruction in hypertrophic cardiomyopathy: an in vitro study in human myocardium.

BACKGROUND AND PURPOSE: In 30-40% of hypertrophic cardiomyopathy (HCM) patients, symptomatic left ventricular (LV) outflow gradients develop only during exercise due to catecholamine-induced LV hypercontractility (inducible obstruction). Negative inotropic pharmacological options are limited to ß-blockers or disopyramide, with low efficacy and tolerability. We assessed the potential of late sodium current (INaL )-inhibitors to treat inducible obstruction in HCM. EXPERIMENTAL APPROACH: The electrophysiological and mechanical responses to ß-adrenoceptor stimulation were studied in human myocardium from HCM and control patients. Effects of INaL -inhibitors (ranolazine and GS-967) in HCM samples were investigated under conditions simulating rest and exercise. KEY RESULTS: In cardiomyocytes and trabeculae from 18 surgical septal samples of patients with obstruction, the selective INaL -inhibitor GS-967 (0.5 µM) hastened twitch kinetics, decreased diastolic [Ca2+ ] and shortened action potentials, matching the effects of ranolazine (10µM). Mechanical responses to isoprenaline (inotropic and lusitropic) were comparable in HCM and control myocardium. However, isoprenaline prolonged action potentials in HCM myocardium, while it shortened them in controls. Unlike disopyramide, neither GS-967 nor ranolazine reduced force at rest. However, in the presence of isoprenaline, they reduced Ca2+ -transient amplitude and twitch tension, while the acceleration of relaxation was maintained. INaL -inhibitors were more effective than disopyramide in reducing contractility during exercise. Finally, INaL -inhibitors abolished arrhythmias induced by isoprenaline. CONCLUSIONS AND IMPLICATIONS: Ranolazine and GS-967 reduced septal myocardium tension during simulated exercise in vitro and therefore have the potential to ameliorate symptoms caused by inducible obstruction in HCM patients, with some advantages over disopyramide and ß-blockers.

Pathogenesis of Hypertrophic Cardiomyopathy is Mutation Rather Than Disease Specific: A Comparison of the Cardiac Troponin T E163R and R92Q Mouse Models.

BACKGROUND: In cardiomyocytes from patients with hypertrophic cardiomyopathy, mechanical dysfunction and arrhythmogenicity are caused by mutation-driven changes in myofilament function combined with excitation-contraction (E-C) coupling abnormalities related to adverse remodeling. Whether myofilament or E-C coupling alterations are more relevant in disease development is unknown. Here, we aim to investigate whether the relative roles of myofilament dysfunction and E-C coupling remodeling in determining the hypertrophic cardiomyopathy phenotype are mutation specific. METHODS AND RESULTS: Two hypertrophic cardiomyopathy mouse models carrying the R92Q and the E163R TNNT2 mutations were investigated. Echocardiography showed left ventricular hypertrophy, enhanced contractility, and diastolic dysfunction in both models; however, these phenotypes were more pronounced in the R92Q mice. Both E163R and R92Q trabeculae showed prolonged twitch relaxation and increased occurrence of premature beats. In E163R ventricular myofibrils or skinned trabeculae, relaxation following Ca2+ removal was prolonged; resting tension and resting ATPase were higher; and isometric ATPase at maximal Ca2+ activation, the energy cost of tension generation, and myofilament Ca2+ sensitivity were increased compared with that in wild-type mice. No sarcomeric changes were observed in R92Q versus wild-type mice, except for a large increase in myofilament Ca2+ sensitivity. In R92Q myocardium, we found a blunted response to inotropic interventions, slower decay of Ca2+ transients, reduced SERCA function, and increased Ca2+/calmodulin kinase II activity. Contrarily, secondary alterations of E-C coupling and signaling were minimal in E163R myocardium. CONCLUSIONS: In E163R models, mutation-driven myofilament abnormalities directly cause myocardial dysfunction. In R92Q, diastolic dysfunction and arrhythmogenicity are mediated by profound cardiomyocyte signaling and E-C coupling changes. Similar hypertrophic cardiomyopathy phenotypes can be generated through different pathways, implying different strategies for a precision medicine approach to treatment.

Ranolazine Prevents Phenotype Development in a Mouse Model of Hypertrophic Cardiomyopathy.

BACKGROUND: Current therapies are ineffective in preventing the development of cardiac phenotype in young carriers of mutations associated with hypertrophic cardiomyopathy (HCM). Ranolazine, a late Na+ current blocker, reduced the electromechanical dysfunction of human HCM myocardium in vitro. METHODS AND RESULTS: To test whether long-term treatment prevents cardiomyopathy in vivo, transgenic mice harboring the R92Q troponin-T mutation and wild-type littermates received an oral lifelong treatment with ranolazine and were compared with age-matched vehicle-treated animals. In 12-months-old male R92Q mice, ranolazine at therapeutic plasma concentrations prevented the development of HCM-related cardiac phenotype, including thickening of the interventricular septum, left ventricular volume reduction, left ventricular hypercontractility, diastolic dysfunction, left-atrial enlargement and left ventricular fibrosis, as evaluated in vivo using echocardiography and magnetic resonance. Left ventricular cardiomyocytes from vehicle-treated R92Q mice showed marked excitation-contraction coupling abnormalities, including increased diastolic [Ca2+] and Ca2+ waves, whereas cells from treated mutants were undistinguishable from those from wild-type mice. Intact trabeculae from vehicle-treated mutants displayed inotropic insufficiency, increased diastolic tension, and premature contractions; ranolazine treatment counteracted the development of myocardial mechanical abnormalities. In mutant myocytes, ranolazine inhibited the enhanced late Na+ current and reduced intracellular [Na+] and diastolic [Ca2+], ultimately preventing the pathological increase of calmodulin kinase activity in treated mice. CONCLUSIONS: Owing to the sustained reduction of intracellular Ca2+ and calmodulin kinase activity, ranolazine prevented the development of morphological and functional cardiac phenotype in mice carrying a clinically relevant HCM-related mutation. Pharmacological inhibitors of late Na+ current are promising candidates for an early preventive therapy in young phenotype-negative subjects carrying high-risk HCM-related mutations.

Isolation and Mechanical Measurements of Myofibrils from Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Tension production and contractile properties are poorly characterized aspects of excitation-contraction coupling of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Previous approaches have been limited due to the small size and structural immaturity of early-stage hiPSC-CMs. We developed a substrate nanopatterning approach to produce hiPSC-CMs in culture with adult-like dimensions, T-tubule-like structures, and aligned myofibrils. We then isolated myofibrils from hiPSC-CMs and measured the tension and kinetics of activation and relaxation using a custom-built apparatus with fast solution switching. The contractile properties and ultrastructure of myofibrils more closely resembled human fetal myofibrils of similar gestational age than adult preparations. We also demonstrated the ability to study the development of contractile dysfunction of myofibrils from a patient-derived hiPSC-CM cell line carrying the familial cardiomyopathy MYH7 mutation (E848G). These methods can bring new insights to understanding cardiomyocyte maturation and developmental mechanical dysfunction of hiPSC-CMs with cardiomyopathic mutations.