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

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

Generation of human induced pluripotent stem cells (EURACi001-A, EURACi002-A, EURACi003-A) from peripheral blood mononuclear cells of three patients carrying mutations in the CAV3 gene

Caveolinopathies are a heterogeneous family of genetic pathologies arising from alterations of the caveolin-3 gene (CAV3), encoding for the isoform specifically constituting muscle caveolae. Here, by reprogramming peripheral blood mononuclear cells, we report the generation of induced pluripotent stem cells (iPSCs) from three patients carrying the ΔYTT deletion, T78K and W101C missense mutations in caveolin-3. iPSCs displayed normal karyotypes and all the features of pluripotent stem cells in terms of morphology, specific marker expression and ability to differentiate in vitro into the three germ layers. These lines thus represent a human cellular model to study the molecular basis of caveolinopathies.Resource tableImage 1Unique stem cell lines identifierEURACi001-AEURACi002-AEURACi003-AAlternative names of stem cell linesB2CAV3 (EURACi001-A)L1CAV3 (EURACi002-A)N1CAV3 (EURACi003-A)InstitutionInstitute for Biomedicine, Eurac ResearchContact information of distributorAlessandra Rossini ([email protected])Type of cell linesiPSCsOriginHumanCell sourcePeripheral blood mononuclear cells (PBMCs)Method of reprogrammingElectroporation of episomal vectors (pCXLE hOCT3/4-shp53-F, pCXLE-hSK, and pCXLE-hUL)Multiline rationaleNon-isogenic cell lines obtained from patients with mutations in the same gene (CAV3)Gene modificationNOType of modificationSpontaneous mutationsAssociated diseaseCaveolinopathiesGene/locusHeterozygous CAV3 c.Δ184–192 (EURACi001-A)Heterozygous CAV3 c.303 TGG > TGC (EURACi002-A)Heterozygous CAV3 c.233 ACG > AAG (EURACi003-A)Method of modificationN/AName of transgene or resistanceN/AInducible/constitutive systemN/ADate archived/stock dateJanuary 2016 (EURACi001-A)September 2016 (EURACi002-A)May 2016 (EURACi003-A)Cell line repository/bankN/AEthical approvalPeripheral blood was collected from patients after signing the informed consent provided by Cell Line and DNA Biobank from Patients Affected by Genetic Diseases, member of the Telethon Network of Genetic Biobanks, Istituto G. Gaslini, Genoa, Italy. The generation and use of iPSCs was reviewed and approved by Ethical Committee at Universita’ degli Studi di Milano (03.06.15, number 29/15)

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