At the roots of genetic cardiomyopathies: from 2D to 3D models based on human iPSC-derived cardiomyocytes

Human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful model for studying the mechanisms underlying inherited cardiomyopathies. Since hiPSCs preserve the entire individual genetic profile, they can help to identify the most appropriate pharmacological interventions to correct specific functional alterations. HiPSC-CMs have been used to study various pathological mechanisms underlying inherited genetic heart disease, particularly as a model to study dilated (DCM) and hypertrophic cardiomyopathies (HCM). However, the most consistent limitation of hiPSC-CMs as a model of cardiomyopathies is their immature cellular features after differentiation; therefore, new methods to promote their maturation have been investigated in the recent years. This work describes approaches to mature hiPSC-CMs, such as biomaterial-based micropatterned substrates (2D-system) and engineered cardiac tissues (EHTs, 3D-system); both can promote cell alignment and elongation, and overall enhancing cardiac cell maturation. In the first part of this work, we demonstrated that micropatterned surfaces have a strong impact on the cardiomyocytes regulation of calcium homeostasis and cellular electrophysiology. We developed different (polyethylene- and diethyleneglycol-based) substrates with different stiffness that were applied to Duchenne Muscular Dystrophy (DMD) hiPSC models. These results provided understanding on the lack of full-length dystrophin in cardiomyocytes and the possible role of cardiac cells with the extracellular environment interaction. A simultaneous measurement of the time-course of action potentials and calcium transients revealed that abnormal calcium handling in DMD-hiPSC-CMs is mostly related to defects in SR calcium accumulation (likely due to RyR leakage) and reduced ability to remove intracellular calcium during diastole. These mechanisms were exacerbated on stiffer substrates. Furthermore, hiPSC-CMs maturation can be enhanced by cardiac tissue engineering approaches by organizing the cells in a 3D environment that more closely resembles the physiological cardiac tissue. In the second part of this work, hiPSC-CMs were used to EHTs, which can be used for in vitro disease modeling and potentially developing precise therapies based on genotype-driven pathogenesis. EHTs were used for contractile force recordings and a direct comparison with cardiac samples from patients. In particular, we focused on the c.772G>A variant, present in the MYBPC3 gene, that causes hypertrophic cardiomyopathy (HCM). To better understand the pathogenetic mechanisms driven by this variant frequent in the florentine patient cohort, myectomy samples were collected from HCM patients carrying this mutation, and PBMCs were obtained from the same patients to be reprogrammed into hiPSCs. We observed that the c.772G>A mutation impairs sarcomere energetics and cross bridge cycling leading to a reduction of cMyBP-C expression in myectomy samples and c.772G>A -hiPSC-CMs. In addition, myocardial samples showed prolonged APs and Ca-T duration and preserved twitch duration. The same electrophysiological changes were observed in patient hiPSC-CMs and -EHTs, suggesting an early adaptive response to primary sarcomeric changes. In the last part of the thesis, EHTs were used as a model to test the long-term effect of Mavacamten, a novel first-in-class allosteric myosin inhibitor developed to reduce contractility and improve myocardial energy in HCM patients. After chronic treatment with Mavacamten (0.3µM and 0.75µM) for 20 days, the HCM-EHTs showed reduced contractile force development under isometric conditions in drug-treated EHTs compared with untreated EHTs, with mild reduction of the twitch duration. Overall, this work provides an overview on the advantage and limitations of using both 2D and 3D approaches for modeling genetic cardiomyopathies and drug testing using hiPSC models

DataSheet1_Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: The impact of full-length dystrophin deficiency.pdf

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).</p