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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).

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.

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.