טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentEisen Binyamin
SubjectFunctional Properties of Induced Pluripotent Stem
Cell-Derived Cardiomyocytes Generated from
Duchenne Muscular Dystrophy Patients
DepartmentDepartment of Medicine
Supervisor Professor Emeritus Ofer Binah


Abstract

Duchenne muscular dystrophy (DMD), an X-linked progressive muscle degenerative disease, results in death by the third decade of life due to respiratory or cardiac failure. DMD is caused by mutations in the dystrophin gene leading to loss of protein, destabilization of dystrophin-glycoprotein complex (DGC), and sarcolemma instability. Eventually, degenerated muscle is replaced by fibrous tissue, leading to loss of function. Dilated cardiomyopathy (DCM) is a major cause of morbidity and mortality in DMD patients. To investigate the cardiac cellular manifestation of DMD, we generated induced pluripotent stem cells (iPSCs) and iPSC-derived cardiomyocytes (iPSC-CMs) from two DMD patients: a male and female manifesting carrier.

Dystrophin mRNA and protein expression were analyzed by qRT-PCR, RNAseq, Western blot (WB) and immunofluorescence staining. For comprehensive electrophysiological analysis, current and voltage clamp were used to record transmembrane action potentials and ion currents, respectively. Microelectrode array was used to record extracellular electrograms. [Ca2]i transients and contractions were recorded using Fura-2 fluorescent dye and video edge detector.

X-inactive specific transcript (XIST) and dystrophin expression analyses revealed that female iPSCs underwent X chromosome reactivation (XCR) or erosion of X chromosome inactivation (XCI), which was maintained in female iPSC-CM population displaying mixed X chromosome expression of wild type (WT) and mutated alleles. Both DMD female and male iPSC-CMs presented low spontaneous firing rate, delayed afterdepolarizations (DADs) and prolonged action potential duration (APD). DMD iPSC-CMs exhibited decreased If density relatively to HCN channel expression likely accounting for the observed low firing rate. In addition, DMD female and male iPSC-CMs showed increased ICa,L density as well as increased expression of the respective channel (CACNA1C), which possibly contribute to APD prolongation. DMD iPSC-CMs displayed increased beat rate variability (BRV) compared to control iPSC-CMs. Notably, DMD female iPSC-CM population exhibited a significant BRV increase compared to the male DMD iPSC-CM population as well, which may be attributed to the heterogenous nature of the population expressing both the WT and mutated allele. Under β-adrenergic stimulation, DMD male iPSC-CMs showed a greater chronotropic increase compared to control. A similar increase was observed in If density of DMD male iPSC-CMs exposed to β-adrenergic stimulation, which likely leads to the observed chronotropic increase. Both DMD female and male iPSC-CMs displayed a lower inotropic and lusitropic increase measured by means of Fura-2 fluorescent dye and video edge detector. The altered responses to β-adrenergic stimulation are consistent with the clinical manifestation of DMD in which patients display elevated heart rate decreased inotropy and lusitrpy.

Our findings demonstrate cellular mechanisms underlying functional abnormalities that play a role in the cardiac manifestation of DMD, thus enabling better understanding of the disease pathophysiology and supporting the validity of our experimental model. Furthermore, our discoveries may serve as means for further research and development of novel therapeutic approaches for DMD.