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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 Ca 2+ 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.

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 β-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 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.

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.

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.

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.

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 β-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 β-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 β-blockers.

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.

Rationale: Despite major advances in cardiovascular medicine, heart disease remains a leading cause of death worldwide. However, the field of tissue engineering has been growing exponentially in the last decade and restoring heart functionality is now an affordable target; yet, new materials are still needed for effectively provide rapid and long-lasting interventions. Liquid crystalline elastomers (LCEs) are biocompatible polymers able to reversibly change shape in response to a given stimulus and generate movement. Once stimulated, LCEs can produce tension or movement like a muscle. However, so far their application in biology was limited by slow response times and a modest possibility to modulate tension levels during activation. Objective: To develop suitable LCE-based materials to assist cardiac contraction. Methods and Results: Thanks to a quick, simple, and versatile synthetic approach, a palette of biocompatible acrylate-based light-responsive LCEs with different molecular composition was prepared and mechanically characterized. Out of this, the more compliant one was selected. This material was able to contract for some weeks when activated with very low light intensity within a physiological environment. Its contraction was modulated in terms of light intensity, stimulation frequency, and t on /t off ratio to fit different contraction amplitude/time courses, including those of the human heart. Finally, LCE strips were mounted in parallel with cardiac trabeculae, and we demonstrated their ability to improve muscular systolic function, with no impact on diastolic properties. Conclusions: Our results indicated LCEs are promising in assisting cardiac mechanical function and developing a new generation of contraction assist devices.

Abnormalities of cardiomyocyte Ca2 + homeostasis and excitation–contraction (E–C) coupling are early events in the pathogenesis of hypertrophic cardiomyopathy (HCM) and concomitant determinants of the diastolic dysfunction and arrhythmias typical of the disease. T-tubule remodelling has been reported to occur in HCM but little is known about its role in the E–C coupling alterations of HCM. Here, the role of T-tubule remodelling in the electro-mechanical dysfunction associated to HCM is investigated in the Δ160E cTnT mouse model that expresses a clinically-relevant HCM mutation. Contractile function of intact ventricular trabeculae is assessed in Δ160E mice and wild-type siblings. As compared with wild-type, Δ160E trabeculae show prolonged kinetics of force development and relaxation, blunted force-frequency response with reduced active tension at high stimulation frequency, and increased occurrence of spontaneous contractions. Consistently, prolonged Ca2 + transient in terms of rise and duration are also observed in Δ160E trabeculae and isolated cardiomyocytes. Confocal imaging in cells isolated from Δ160E mice reveals significant, though modest, remodelling of T-tubular architecture. A two-photon random access microscope is employed to dissect the spatio-temporal relationship between T-tubular electrical activity and local Ca2 + release in isolated cardiomyocytes. In Δ160E cardiomyocytes, a significant number of T-tubules (> 20%) fails to propagate action potentials, with consequent delay of local Ca2 + release. At variance with wild-type, we also observe significantly increased variability of local Ca2 + transient rise as well as higher Ca2 +-spark frequency. Although T-tubule structural remodelling in Δ160E myocytes is modest, T-tubule functional defects determine non-homogeneous Ca2 + release and delayed myofilament activation that significantly contribute to mechanical dysfunction., Highlights • Contraction and Ca2 + transient kinetics are impaired in myocardial preparations from mice carrying the cardiac troponin T ∆ 160E mutation. • T-tubules architecture is mildly altered in ∆160E cardiomyocytes. • 20% of T-tubules fail to propagate action potential and produce delay of local Ca2 + rise. • Higher spatio-temporal variability of local Ca2 + rise and increased Ca2 + sparks frequency are found in ∆160E cardiomyocytes.

A ryanodine receptor 2 mutation associated with catecholaminergic polymorphic ventricular tachycardia renders cardiomyocytes incapable of mediating a positive inotropic response., Ryanodine receptor (RyR2) is the major Ca2+ channel of the cardiac sarcoplasmic reticulum (SR) and plays a crucial role in the generation of myocardial force. Changes in RyR2 gating properties and resulting increases in its open probability (Po) are associated with Ca2+ leakage from the SR and arrhythmias; however, the effects of RyR2 dysfunction on myocardial contractility are unknown. Here, we investigated the possibility that a RyR2 mutation associated with catecholaminergic polymorphic ventricular tachycardia, R4496C, affects the contractile function of atrial and ventricular myocardium. We measured isometric twitch tension in left ventricular and atrial trabeculae from wild-type mice and heterozygous transgenic mice carrying the R4496C RyR2 mutation and found that twitch force was comparable under baseline conditions (30°C, 2 mM [Ca2+]o, 1 Hz). However, the positive inotropic responses to high stimulation frequency, 0.1 µM isoproterenol, and 5 mM [Ca2+]o were decreased in R4496C trabeculae, as was post-rest potentiation. We investigated the mechanisms underlying inotropic insufficiency in R4496C muscles in single ventricular myocytes. Under baseline conditions, the amplitude of the Ca2+ transient was normal, despite the reduced SR Ca2+ content. Under inotropic challenge, however, R4496C myocytes were unable to boost the amplitude of Ca2+ transients because they are incapable of properly increasing the amount of Ca2+ stored in the SR because of a larger SR Ca2+ leakage. Recovery of force in response to premature stimuli was faster in R4496C myocardium, despite the unchanged rates of recovery of L-type Ca2+ channel current (ICa-L) and SR Ca2+ content in single myocytes. A faster recovery from inactivation of the mutant R4496C channels could explain this behavior. In conclusion, changes in RyR2 channel gating associated with the R4496C mutation could be directly responsible for the alterations in both ventricular and atrial contractility. The increased RyR2 Po and fractional Ca2+ release from the SR induced by the R4496C mutation preserves baseline contractility despite a slight decrease in SR Ca2+ content, but cannot compensate for the inability to increase SR Ca2+ content during inotropic challenge.

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 β‐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 β‐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 β‐blockers.

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 [Ca 2+ ] and Ca 2+ 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 [Ca 2+ ], ultimately preventing the pathological increase of calmodulin kinase activity in treated mice. Conclusions— Owing to the sustained reduction of intracellular Ca 2+ 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.

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 Ca 2+ removal was prolonged; resting tension and resting ATPase were higher; and isometric ATPase at maximal Ca 2+ activation, the energy cost of tension generation, and myofilament Ca 2+ 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 Ca 2+ sensitivity. In R92Q myocardium, we found a blunted response to inotropic interventions, slower decay of Ca 2+ transients, reduced SERCA function, and increased Ca 2+ /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.

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 &beta, 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.

Summary 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., Highlights • The contractile properties of hiPSC-CM myofibrils have not been previously studied • hiPSC-CMs cultured on nanopatterned surfaces develop elongated, aligned myofibrils • hiPSC-CMs myofibrils have contractile properties similar to human fetal myofibrils • hiPSC-CMs can be used to study development of genetically based cardiac diseases, In this article, Pioner and colleagues reported contractile properties of isolated myofibrils from hiPSC-CMs with highly mature morphology. This approach permits quantitative assessment of maturation and contractile properties of hiPSC-CMs and can be used to study the development of contractile dysfunction in genetically based cardiac diseases. The authors present a patient-derived cell line carrying a novel familial cardiomyopathy MYH7 mutation (E848G).

Duchenne Muscular Dystrophy (DMD) is a wasting disease of striated muscle resulting from membrane fragility. We modeled DMD cardiac disease using urine-derived cells from a patient, reprogrammed to induced pluripotent stem cells (hiPSCs) and differentiated into cardiomyocytes (DMD-hiPSC-CMs). DMD-hiPSC-CMs were dystrophin-deficient (exon 50 deletion) and manifested physiological consequences of the disease such as calcium-handling abnormalities. Here we report, for the first time, isolation and functional characterization of myofibrils from hiPSC-CMs to study DMD cardiomyopathy.At day 20 post-differentiation, DMD-hiPSC-CMs and control hiPSC-CMs (from a healthy volunteer) were replated onto fibronectin-coated nanopatterned coverslides and cultured until day 80. Both DMD- and controls-CMs exhibited more mature morphology with aligned myofibrils and clearly defined Z-bands, cell lengths of 100-150µm and widths of 40µm. For mechanical measurements, cells were harvested and skinned in a rigor solution containing Triton 1% for 5 minutes.Control hiPSC-CM myofibrils had mechanical and kinetic proporties more similar to human fetal skeletal (Racca, 2013) or cardiac myofibrils than human adult cardiac myofibrils. Preliminary data for DMD-hiPSC-CMs showed lower force development, prolonged duration of early, slow phase relaxation kinetics (tREL slow), but no differences in the other kinetic properties. Preliminary data from intact DMD-hiPSC-CMs paced at 1Hz showed no difference in the rate of cell shortening, but depressed magnitude and prolonged relaxation (t50). We previously reported (Xuan G, 2014) prolongation of Ca2+ transient decay.Together these data suggest DMD-hiPSC-CMs have slower relaxation due to both myofibril properties and Ca2+ sequestration, compared with control hiPSC-CMs, and both cell lines have myofibril mechanic and kinetic properties more similar to fetal than adult myofibrils.In conclusion, we demonstrated that isolated functional myofibrils from hiPSC-CMs can be obtained after growing on nano-patterned surfaces in culture to study cardiac diseases in vitro.

In hypertrophic cardiomyopathy (HCM) atrial dilatation (AD) and fibrillation (AF) are very common and associated with worse outcome. The cellular and molecular basis of atrial remodeling in HCM remain undefined. We previously characterized (Coppini et al. ABS Biophysical Journal 2015) the changes in sarcomere function and E-C coupling that occur in ventricular myocardium of two HCM mouse models carrying different mutations in cTnT (R92Q and E163R). Both models exhibited diastolic dysfunction that was, however, related to different mechanisms i.e. E-C coupling abnormalities in R92Q and sarcomere changes in E163R. Here we employ these mouse models to study whether atrial remodeling is a consequence of diastolic dysfunction or is also influenced by the specific underlying mutation.Echocardiographic measurements of left atrial (LA) dimensions showed that LA area was severely increased in R92Q hearts while it was only mildly increased in E163R (in mm2 : 6.73±0.5 in R92Q, 4.82±0.16 in E163R vs 3.97±0.26 in WT). Left atrial trabeculae were dissected and mounted isometrically to record twitch tension. We studied the steady-state force-frequency relationship and the response to positive inotropic stimuli such as Isoproterenol 10-7 mM (ISO) and 8 mM extracellular [Ca2+]. Compared to WT, R92Q atrial trabeculae showed: (i) slower kinetics of both force development and relaxation (e.g. at 1 Hz, 50% relaxation was prolonged by 35%), (ii) impaired twitch amplitude at high pacing rates (50% reduction), (iii) depressed rested-state contractions and (iv) blunted increase of twitch tension in ISO and high [Ca2+]. None of these changes were observed in intact E163R atrial trabeculae. These findings suggest that atrial remodeling in R92Q is more pronounced compared to E163R, and related to E-C coupling alterations. Supported by the Italian Ministry of Health (WFR GR-2011-02350583).

The communication reports the use of liquid crystalline networks (LCNs) for engineering tissue cultures with human cells. Their ability as cell scaffolds for different cell lines is demonstrated. Preliminary assessments of the material biocompatibility are performed on human dermal fibroblasts and murine muscle cells (C2C12), demonstrating that coatings or other treatments are not needed to use the acrylate-based materials as support. Moreover, it is found that adherent C2C12 cells undergo differentiation, forming multinucleated myotubes, which show the typical elongated shape, and contain bundles of stress fibers. Once biocompatibility is demonstrated, the same LCN films are used as a substrate for culturing human induced pluripotent stem cell-derived cardiomyocites (hiPSC-CMs) proving that LCNs are capable to develop adult-like dimensions and a more mature cell function in a short period of culture in respect to standard supports. The demonstrated biocompatibility together with the extraordinary features of LCNs opens to preparation of complex cell scaffolds, both patterned and stimulated, for dynamic cell culturing. The ability of these materials to improve cell maturation and differentiation will be developed toward engineered heart and skeletal muscular tissues exploring regenerative medicine toward bioartificial muscles for injured sites replacement.

Changes in myofilament function related to HCM-associated mutations contribute to the diastolic dysfunction observed in the in vivo patient heart and in intact ventricular preparations from patient samples. HCM mutations that are ubiquitously expressed in the heart (e.g. cMyBP-C or cTnT) could also affect atrial function. Here we investigate whether HCM-associated atrial myopathy is a consequence of mutation-driven sarcomere dysfunction or results from atrial remodeling due to the diastolic dysfunction and increased LV filling pressures. In one HCM patient carrying the Lys814del cMyB-C mutation, changes in sarcomere function (increased myofilament Ca2+ sensitivity and increased cross bridges detachment rate under isometric conditions) were similar in atrial and ventricular myofibrils compared to donor preparations. However, isometric twitch mechanics and kinetics of intact trabeculae from the right atrium of 4 cMyB-C-mutant patients were unaffected as compared to trabeculae from non-HCM patients (N=8), or mutation negative HCM patients (N=3), or HCM patients carrying mutations in beta-myosin (N=2). We extended the study to HCM mouse models carrying mutations in cTnT. In the E163R mouse, atrial and ventricular sarcomere kinetics and energetics were similarly altered compared to WT mice. Isometric ATPase, both at rest and at maximal Ca2+-activation and the energy cost of tension generation were increased in both atrial and ventricular preparations of E163R vs WT. However, isometric twitch kinetics were prolonged in intact ventricular trabeculae of E163R mice vs WT while they were unaffected in atrial trabeculae. In the R92Q mouse model, that is associated with a much more severe degree of LV diastolic dysfunction and left atrial dilatation compared to the E163R, left atrial trabeculae showed prolonged twitch contractions, increased spontaneous activity and a number of E-C coupling alterations that resemble those observed in ventricular preparations. HCM-mutations in cMyBP-C and cTnT induce similar alterations in both atrial and ventricular sarcomeres. However, likely due to the different working conditions of the two chambers, sarcomere dysfunction can significantly alter the mechanics and kinetics of the intact myocardium only in the ventricles. Atrial muscle dysfunction in HCM is induced by remodeling processes that depend on the increased filling pressures.

Ca2+ handling abnormalities are an early-onset pathogenic element in HCM. Here we characterize pro-arrhythmogenic changes in E-C coupling that occur in intact trabeculae and cardiomyocytes from cTnT mutant mouse models of HCM (R92Q, E163R and Δ160E) and test the effects of specific pharmacological interventions. Compared to WT, R92Q trabeculae ([Ca2+]o 2 mM, 30°C) showed (i) preserved peak isometric twitch tension and prolonged relaxation kinetics associated to decreased SERCA levels, (ii) faster mechanical restitution, further accelerated by isoproterenol (Iso) 100nM, (iii) decreased Ca2+-recirculation fraction markedly increased by Iso (iv) frequent after-contractions or regular spontaneous beats during stimulation pauses that increased in response to Iso. Compared to WT, R92Q cardiomyocytesshowed (i) prolonged action potentials associated with ionic current remodeling, (ii) slower rate of Ca2+transient decay, (iii) elevated diastolic [Ca2+]i, (iv) spontaneous Ca2+ waves during stimulation pauses. In R92Q preparations, the late-Na+ current blocker Ranolazine (Ran 10 μM) (i) reduced the rate of spontaneous beats and spontaneous Ca2+ waves, (ii) hastened Ca2+ transient kinetics and reduced diastolic Ca2+, (iii) reduced and reversed the acceleration of mechanical restitution and the increase in Ca2+ recirculation fraction induced by Iso.Compared to R92Q, occurrence of spontaneous contractions was similar in E163R but less pronounced in Δ160E. Iso and Ran showed similar effects in all three mouse models, in spite of some quantitative differences. The results are consistent with those recently reported in human HCM myocytes (Coppini et al, Circulation 2013) and suggest that remodeling and dysfunction of NCX and RyR2 contribute to the pro-arrhythmogenic E-C coupling abnormalities observed in HCM.

Background: Rest or exercise obstruction is present in two thirds of patients with hypertrophic cardiomyopathy (HCM) and is a major determinant of symptoms and disability. Hypercontractile upper septum is the main pathophysiological determinant of obstruction, thus negative inotropic interventions such as dysopiramide or beta-blockers are the only available options to pharmacologically treat obstruction, commonly with partial efficacy. We have previously demonstrated that ranolazine ameliorates diastolic function in trabeculae from septal samples of obstructive HCM patients undergoing myectomy (Coppini et al, Circulation 2013).Methods: Patch clamp studies and intracellular Ca2+ recordings were performed in isolated myocytes from myectomy samples of obstructive HCM patients; intact trabeculae were used for mechanical measurements. Myocardial specimens from non-failing non-hypertrophic patients or patients with secondary hypertrophy were used as controls.Results: Dysopiramide (Dys) reduced twitch tension in a dose dependent manner and 5µM Dys accelerated contraction kinetics in HCM trabeculae. Isoproterenol 10−7 mol/L (Iso) determined a significant potentiation of twitch amplitude and an accelleration of contraction kinetics (both time to peak and relaxation). Changes induced by Iso in control trabeculae were similar. Interestingly, Iso caused APD prolongation in HCM cardiomyocytes instead of the shortening observed in control cells. This was likely related to the unbalance between depolarizing and repolarizing currents, including increased Late-Na+ current (INaL). The INaL blocker Ranolazine 10 µM (Ran) applied on top of Iso (Iso+Ran) markedly reduced isometric twitch tension of HCM trabeculae, while Ran alone showed no negative inotropic effect. Contraction kinetics in Iso+Ran were still significantly faster compared to baseline.Conclusions: Beta adrenergic stimulation may enhance septal contractility and determine obstruction in HCM. Ranolazine, by reducing septal tension at peak exercise but not at rest, may represent a safe therapeutic option for obstruction.

Introduction: Ca2+ handling abnormalities are a main determinant of electro-mechanical dysfunction in human hypertrophic cardiomyopathy (HCM), leading to a pro-arrhythmogenic cellular phenotype. The late-Na+ current blocker ranolazine reduced the rate of arrhythmogenic events in HCM human myocardium via shortening of action potentials and reduction of intracellular Na+ and Ca2+, with increased forward NCX function (Circulation. 2013 Feb 5;127(5):575-84). Methods: Here we characterize pro-arrhythmogenic changes in E-C coupling that occur in cardiomyocytes and intact trabeculae from cTnT mutant mouse models of HCM (R92Q and E163R) and test the effects of ranolazine. Results: Compared to wild type (WT), R92Q cardiomyocytes showed (i) prolonged action potentials associated with ionic current remodeling; (ii) lower Ca2+transient amplitude with slower rate of decay; (iii) elevated diastolic [Ca2+]i; (iv) spontaneous Ca2+ waves and premature Ca2+ transients during pauses. Compared to WT, R92Q trabeculae showed (i) prolonged relaxation kinetics associated with reduced SERCA function and (ii) Frequent after-contractions or regular spontaneous beats during stimulation pauses. In E163R vs. WT trabeculae and cells, the kinetics of force development and relaxation was prolonged, despite Ca2+ transient kinetics and SERCA function were not slower. E163R vs. WT myocardium showed increased rate of spontaneous arrhythmogenic activity. In R92Q preparations, ranolazine (10μM): (i) hastened Ca2+ transient kinetics and reduced diastolic Ca2+; (ii) increased forward NCX function and (iii) reduced the rate of spontaneous beats and Ca2+ waves. In E163R trabeculae and cells ranolazine did not affect the kinetics of Ca2+ transients. Nonetheless, the drug prolonged RyR2 refractoriness and reduced the occurrence of spontaneous Ca2+ and contractile activity. None of these effects of ranolazine was observed in WT. Discussion: As in human HCM, the beneficial effects of ranolazine on R92Q myocardium are likely mediated by the consequences of late Na+ current inhibition. In E163R preparations, myocardial arrhythmogeneicity is not accompanied by remodeling of ion currents or changes of SERCA/NCX function, and thus appear to be a direct consequence of increased myofilaments Ca2+ sensitivity or anomalies of RyR2 function. Therefore the beneficial effects of ranolazine on Ca2+ mediated spontaneous activity in E163R myocardium supports the hypothesis that the anti-arrhythmic effect of ranolazine can also be mediated by mechanisms other that INaL inhibition (i.e. reduction of myofilaments Ca2+ sensitivity or RyR2 inhibition).

Introduction: Many of cTnT mutations linked to cardiomyopathies fall the TNT1 domain/N terminal tail region of unresolved high definition structure. This region (∼94-170) of cTnT is critical to Tm binding and contraction regulation. Here, the impact of the E163R mutation in cTnT-TNT1 on contractile function and tension cost was investigated using intact and skinned preparations from WT and transgenic mouse hearts.Methods: Left and right ventricular trabeculae were dissected from non-transgenic wild type (WT) and heterozygous (Δ160E or E163R) mouse hearts and mounted isometrically to record twitch tension or, when skinned, Ca2+ activated force. Myofibrillar ATPase activity was measured by fluorimetric enzyme coupled assay (de Tombe and Stienen, 1995).Results: Myocardium of E163R mice shows: (i) no change of myosin isoform expression (ii) maintained peak isomentric twitch tension at all stimulation frequencies, (iii) prolonged time to peak and time to 50% relaxation, with preserved rate-adaptation of twitch duration, (iv) changes of the short-term interval force relationship and increased occurrence of spontaneous contractions. No significant differences were found in maximum Ca2+ activated tension of E163R and WT skinned trabeculae. However, Ca2+ sensitivity of tension was significantly increased in E163R skinned trabeculae when compared with WT. As to the economy of force maintenance, preliminary experiments suggest an increase of tension cost in trabeculae from E163R hearts. Resting ATPase activity also tended to be higher in E163R preparations. Kinetics of force development and relaxation will be assessed on single myofibrils, isolated from the same hearts.Conclusions: Both primary sarcomeric changes and secondary E-C coupling alterations contribute to mechanical impairment in E163R cTnT mutant myocardium. Supported by: EC Grant n. 241577 (BIG-Heart)

Myosin heavy chain 7 (MYH7) mutations are associated with familial cardiomyopathies (FCM) and result in a high rate of sudden cardiac death. Human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) have recently shown promise as a model for studying FCM. We identified a cohort with familial cardiomyopathy (FCM) associated with a MYH7 mutation (E848G) and middle-age onset of systolic dysfunction and arrhythmias. hiPSC-CMs from patient affected (FCM-CMs) and non-affected (WT-CMs) individuals were generated from skin fibroblasts. Here we report, for the first time, contractile properties of isolated myofibrils from these cultured hiPSC-CMs for comparison using cultured cells and 3D engineered cardiac tissue (3D-ECT) constructs. Isolated myofibrils were obtained from differentiation day 20 hiPSC-CMs that were replated onto fibronectin-coated nanopatterned cover slides and matured in culture for an additional 60 days to obtain elongated and aligned myofibrils. This procedure produced hiPSC-CMs that were usually > 100+ µm in length. hiPSC- FCM-CMs and WT-CMs were harvested and skinned in a rigor solution containing 1% Triton and contractile properties of single or small bundles of myofibrils were measured in a custom built apparatus with rapid solution switching capabilities. During maximal calcium activation FCM-CM myofibrils produced approximately half the amount of force of WT-CM myofibrils, but preliminary data suggests no differences in the kinetics of force development or relaxation. This compares well with 50 day cardiomyocytes plated on nano-patterned surfaces or seeded into 3D-ECT constructs, where shortening and force (respectively) of FCM-CMs was much less than for WT-CMs, with no difference in calcium transient amplitudes. We speculate this early stage contractile deficit may contribute to disease development and conclude hiPSC-FCM-CMs can be a viable model for mechanical studies of cardiomyopathies in vitro.