Biotechnological methods for the study of cardiomyopathies and the evaluation of new pharmacological therapies based on patient-specific in vitro models

Inherited cardiomyopathies are a heterogeneous group of cardiac disease characterized by functional and structural alteration of myocardium, representing the major cause of morbidity and mortality over the past two decades. Recently, extensive genetic screenings have highlighted the importance to expand our knowledge regarding the effects that specific mutations cardiomyopathies-associated have on contractile functionality of the heart. Therefore, focused and specific pharmacological treatments for cardiomyopathies are missing, considering all the different clinical manifestations observed in the various forms of cardiomyopathies, including hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM), the two most widely represented forms of these cardiac diseases. In this context, human pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) are a powerful model of study to investigate the pathological mechanisms underlying these diseases, allowing to preserve the genetic heritage of patient and then model satisfactorily inherited cardiomyopathies. Moreover, hiPSC-CMs enable pharmacological in vitro testing and functional investigations, identifying specific drug interventions to address the pathological alterations caused from specific associated mutations. Despite this potentiality, electrophysiological and mechanical immaturity of hiPSC-CMs represent a limitation on their use. In recent years, new methods to induce their maturation were developed, including micropatterned substrates and engineered heart tissues (EHTs), to promote cellular elongation and maturation. In particular, up of 60% of HCM cases are caused from mutation occurring in β-myosin heavy chain (β-MHC) and myosin-binding protein C (MYBPC3). Then, in this work we used hiPSC-CMs to model HCM end DCM, by using different hiPSC lines derived from patients carrying β-MHC and MYBPC3 mutations, to investigate the main pathological characteristic associated to this disease. Also, we used four different hiPSCs line derived from patients affected from dilated cardiomyopathy associated to Duchenne Muscular Dystrophy (DMD), resulting in total absence of dystrophin. In the first part of this work, we elucidated that specific micropatterned substrates with different stiffness could strongly affect calcium handling and electrophysiological features of DMD cardiomyocytes in comparison to control. In fact, the absence of full-length dystrophin in DMD patients induce pathological modifications due the lack of interaction between intracellular environment and extracellular matrix (ECM), resulting in calcium handling abnormalities and alterations in sarcoplasmic reticulum (SR) re-uptake. Moreover, a deficit of diastolic calcium mobilization occurs in dystrophin-deficient cardiomyocytes. Based on this evidence, EHTs were derived from HCM and DMD lines, as previously described, to more closely mimic the physiological myocardium. More in detail, we performed contractile force recording and action potential evaluation by using a confocal microscope using HCM lines carrying c.772G>A variant in MYBPC3 gene, highlighting an effect of this mutation on cross bridge cycling end contractile properties of HCM-EHTs. In addition, we performed chronic treatment with Mavacamten, an allosteric myosin inhibitor, on HCM-EHTs showing a significant decrease of contractile force development in HCM-EHTs treated compared to basal. Moreover, we performed mechanical evaluation, via calcium transient measurements and In Vitro Motility Assay (IVMA) on myosin isolated from hiPSC-CMs carrying R403Q mutation, resulting in a particularly severe form of HCM, showing calcium handling modification and impaired contractility in mutated cardiomyocytes compared to control. Therefore, in the second part of this work, we evaluated DMD-EHTs derived from four patients, carrying mutation that result in total absence of full-length dystrophin (Δexon 46 48, Δexon 50, Δexon 51, Δexon 49). Also, contractile force measurements were performed on DMD EHTs, demonstrated a reduction of active tension exerted from DMD tissues in association to kinetics preserved. Finally, Dapagliflozin, a novel type 2 sodium glucose cotransporter inhibitor, that act “off label” bringing various beneficial effects at cardiovascular level, through effects still unclear. For these reasons, we performed acute treatment with Dapagliflozin (1 and 5 µM) on DMD-EHTs andcardiomyocytes isolated from human surgical samples, highlighting an increase of active tension in DMD-tissues treated compared to untreated. Moreover, a reduction in action potential duration (APD) occur in HCM cardiomyocytes compared to basal conditions. Overall, these results provide evidence that, although there is different phenotypical manifestation in different inherited cardiomyopathies, specific pathological mechanisms could be in common, including calcium handling and electrophysiological alterations. These mechanisms can be exploited to performed specialized patient-specific medicine, to improve the knowledge on these genetic disorders, establishing the basis for ever more innovative pharmacological treatments.