Combinatorial maturation strategy for disease modeling and phenotypic drug screening of Duchenne muscular dystrophy cardiomyopathy

Thesis (Ph.D.)--University of Washington, 2017-08Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) offer great promise for regenerative medicine, preclinical drug screening, and cardiac disease modeling applications. One of the most significant hurdles towards adoption of hPSC-CM technologies, however, is cardiomyocyte developmental immaturity. Current differentiation methods produce hPSC-CMs with structural and functional characteristics most closely resembling fetal cardiomyocytes, which significantly hinders our ability to predict patient drug responses or model adult-onset cardiomyopathies. The following dissertation addresses this challenge with the goal of engineering structurally and functionally mature cardiac tissues from hPSC-CMs for in vitro disease modeling and drug screening applications. Here, we present the development of a bio-inspired, combinatorial method for enhancing the maturation of hPSC-CMs that incorporates distinct physical, biochemical, and genetics cues. We began by investigating the role of surface nanotopography on hPSC-CM development and found that, similar to primary cardiomyocytes, hPSC-CMs exhibited a nanotopographic size-dependent phenotype. Utilizing the optimal nanotopographic surfaces dimensions for promoting maturation, we tested whether this maturation cue alone could improve our ability to model the cardiomyopathy associated with Duchenne Muscular Dystrophy (DMD). Although we were able to measure a blunted cytoskeletal response to the nanotopography in dystrophin-null hPSC-CMs, this difference was mild and we were unable to detect a functional disease phenotype. We therefore explored more comprehensive methods for inducing hPSC-CM maturation and developed our combinatorial maturation (ComboMat) protocol. The ComboMat protocol incorporates biomimetic nanotopography, thyroid hormone T3, and Let7i microRNA overexpression to produce hPSC-CMs with enhanced sarcomere development, improved electrophysiological and contractile function, improved mitochondrial respiratory capacity, and a transcriptome upregulated for metabolic and muscle development. When the ComboMat protocol is applied to a CRISPR-edited dystrophin knockout (KO) model of DMD cardiomyopathy, a distinctive, endogenously occurring disease phenotype emerges. Mature dystrophin KO hPSC-CMs exhibit greater propensity for arrhythmia with a higher resting cytosolic calcium content compared to Normal hPSC-CM controls. A phenotypic drug screen of dystrophin KO hPSC-CMs using the ComboMat protocol identified compounds that mitigated arrhythmogenic behavior. The ComboMat protocol can be applied to other cardiac disease models, cardiotoxicity studies, or cardiac tissue engineering applications. In vitro screening assays must predict the response of the human heart with high fidelity in order to be adopted. Taken together, this research demonstrates the utility of bioengineering strategies to mature hPSC-CMs in order to develop more biomimetic, adult-like cardiac tissues for preclinical screening applications

Cell and Myofibril Contractile Properties of hiPSC-Derived Cardiomyocytes from a Patient with a MYH7 Mutation Associated with Familial Cardiomyopathy

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

Absence of full-length dystrophin impairs normal maturation and contraction of cardiomyocytes derived from human-induced pluripotent stem cells.

AIMS: Heart failure invariably affects patients with various forms of muscular dystrophy (MD), but the onset and molecular sequelae of altered structure and function resulting from full-length dystrophin (Dp427) deficiency in MD heart tissue are poorly understood. To better understand the role of dystrophin in cardiomyocyte development and the earliest phase of Duchenne muscular dystrophy (DMD) cardiomyopathy, we studied human cardiomyocytes differentiated from induced pluripotent stem cells (hiPSC-CMs) obtained from the urine of a DMD patient. METHODS AND RESULTS: The contractile properties of patient-specific hiPSC-CMs, with no detectable dystrophin (DMD-CMs with a deletion of exon 50), were compared to CMs containing a CRISPR-Cas9 mediated deletion of a single G base at position 263 of the dystrophin gene (c.263delG-CMs) isogenic to the parental line of hiPSC-CMs from a healthy individual. We hypothesized that the absence of a dystrophin-actin linkage would adversely affect myofibril and cardiomyocyte structure and function. Cardiomyocyte maturation was driven by culturing long-term (80-100 days) on a nanopatterned surface, which resulted in hiPSC-CMs with adult-like dimensions and aligned myofibrils. CONCLUSIONS: Our data demonstrate that lack of Dp427 results in reduced myofibril contractile tension, slower relaxation kinetics, and to Ca2+ handling abnormalities, similar to DMD cells, suggesting either retarded or altered maturation of cardiomyocyte structures associated with these functions. This study offers new insights into the functional consequences of Dp427 deficiency at an early stage of cardiomyocyte development in both patient-derived and CRISPR-generated models of dystrophin deficiency

TFPa/HADHA is required for fatty acid beta-oxidation and cardiolipin re-modeling in human cardiomyocytes.

Mitochondrial trifunctional protein deficiency, due to mutations in hydratase subunit A (HADHA), results in sudden infant death syndrome with no cure. To reveal the disease etiology, we generated stem cell-derived cardiomyocytes from HADHA-deficient hiPSCs and accelerated their maturation via an engineered microRNA maturation cocktail that upregulated the epigenetic regulator, HOPX.  Here we report, matured HADHA mutant cardiomyocytes treated with an endogenous mixture of fatty acids manifest the disease phenotype: defective calcium dynamics and repolarization kinetics which results in a pro-arrhythmic state. Single cell RNA-seq reveals a cardiomyocyte developmental intermediate, based on metabolic gene expression. This intermediate gives rise to mature-like cardiomyocytes in control cells but, mutant cells transition to a pathological state with reduced fatty acid beta-oxidation, reduced mitochondrial proton gradient, disrupted cristae structure and defective cardiolipin remodeling. This study reveals that HADHA (tri-functional protein alpha), a monolysocardiolipin acyltransferase-like enzyme, is required for fatty acid beta-oxidation and cardiolipin remodeling, essential for functional mitochondria in human cardiomyocytes