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PhD seminar- Functional properties of induced pluripotent stem cell-derived cardiomyocytes generated from Duchenne muscular dystrophy patients

February 10 @ 10:00 - 12:00

Duchenne muscular dystrophy (DMD), an X-linked progressive muscle degenerative disease, results in death by the 3rd 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-CMs displaying mixed X chromosome expression of wild type (WT) and mutated alleles. Both DMD female and male iPSC-CMs presented low spontaneous firing rate, arrhythmias and prolonged action potential duration (APD). DMD male iPSC-CMs manifested decreased If density, and DMD female and male iPSC-CMs showed increased ICa,L density. DMD female iPSC-CMs displayed increased beat rate variability (BRV). Under β-adrenergic stimulation, DMD male iPSC-CMs showed a greater relative chronotropic increase compared to control and both DMD female and male iPSC-CMs displayed a lower relative inotropic and lusitropic increase.

Our findings demonstrate cellular mechanisms underlying functional abnormalities which 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.


February 10
10:00 - 12:00
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