Functional Abnormalities in Induced Pluripotent Stem Cell-Derived Cardiomyocytes Generate from a Duchenne Muscular Dystrophy Patient and a Female Manifesting Carrier

Binyamin Eisen Ruth and Bruce Rappaport Faculty of Medicine, Technion – Israel Institute of Technology, Haifa, Israel Ronen Ben Jehuda Ruth and Bruce Rappaport Faculty of Medicine, Technion – Israel Institute of Technology, Haifa, Israel Department of Biotechnology, Technion – Israel Institute of Technology, Haifa, Israel Ashley J. Cuttitta Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan, USA Lucy N. Mekies Ruth and Bruce Rappaport Faculty of Medicine, Technion – Israel Institute of Technology, Haifa, Israel Lubna Willi Ruth and Bruce Rappaport Faculty of Medicine, Technion – Israel Institute of Technology, Haifa, Israel Yuval Shemer Ruth and Bruce Rappaport Faculty of Medicine, Technion – Israel Institute of Technology, Haifa, Israel Irina Reiter Ruth and Bruce Rappaport Faculty of Medicine, Technion – Israel Institute of Technology, Haifa, Israel Ifat Abramovich Ruth and Bruce Rappaport Faculty of Medicine, Technion – Israel Institute of Technology, Haifa, Israel Dov Freimark Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Leviev Heart Center, Sheba Medical Center, Ramat Gan, Israel Mihaela Gherghiceanu Department of Pathology, Victor Babes National Institute of Pathology, Bucharest, Romania Lorenzo Monserrat Clinical Department, Health in Code, A Coruña, Spain Daniel E. Michele Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan, USA Eyal Gottlieb Ruth and Bruce Rappaport Faculty of Medicine, Technion – Israel Institute of Technology, Haifa, Israel Michael Arad Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Leviev Heart Center, Sheba Medical Center, Ramat Gan, Israel Ofer Binah Ruth and Bruce Rappaport Faculty of Medicine, Technion – Israel Institute of Technology, Haifa, Israel

Background

Duchenne muscular dystrophy (DMD), an X-linked muscle degenerative fatal disease, is caused by dystrophin mutations. Dilated cardiomyopathy (DCM) is a major cause of morbidity and mortality in DMD patients.

Objective

We hypothesized that DMD patients induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) manifest key aspects of the disease.

Methods

We generated iPSC-CMs from a DMD male patient and a female manifesting carrier, and investigated their molecular and functional properties using NGS, patch clamp, RT-PCR, Ca²⁺ imaging and metabolic indices.

Results

NGS demonstrated DMD female iPSCs underwent X chromosome reactivation, and iPSC-CMs displayed mosaic X chromosome expression of the WT and mutated alleles. WT dystrophin allele levels were higher in female iPSC-CMs compared to control. DMD iPSC-CMs exhibited low automaticity, prolonged action potential duration, and presented delayed afterdepolarizations. Beat rate variability (BRV) analysis demonstrated DMD female iPSC-CMs displayed significantly increased BRV.

DMD iPSC-CMs displayed: (a) increased expression of hyperpolarization-activated cyclic nucleotide-gated channel – isoform 4 (HCN4); (b) increased L-type Ca²⁺ current (I_Ca,L) density; (c) increased expression of the Ca²⁺ voltage-gated channel subunit alpha1 C (CACNA1C). Additionally, DMD male iPSC-CMs presented smaller pacemaker current (I_f) density.

Under basal conditions, [Ca²⁺]_i and contraction parameters were similar in DMD and control cardiomyocytes, but mutated iPSC-CMs manifested attenuated positive inotropic response to β-adrenergic stimulation with isoproterenol.

Seahorse XF analyzer demonstrated decreased oxidative phosphorylation accompanied by a correlated increase in glycolysis in DMD iPSC-CMs. Accordingly, mass spectrometry analysis showed a dramatic fall in phosphocreatine levels in DMD iPSC-CMs as well.

Conclusions

DMD iPSC-CMs exhibit electrophysiological abnormalities, attenuated response to β-adrenergic stimulation, metabolic deficits and reduced energy stores. Importantly, mosaic dystrophin expression in DMD female iPSC-CMs contributes to the observed abnormalities. These features render this model as a valuable tool for investigating DCM in DMD.