Depressed β-adrenergic Inotropic Responsiveness and Intracellular Calcium Handling Abnormalities in Duchenne Muscular Dystrophy Patients’ Induced Pluripotent Stem Cell-derived Cardiomyocytes

Introduction: Duchenne Muscular Dystrophy (DMD) caused by mutations in the dystrophin gene, is an X-linked disease affecting male and rarely adult heterozygous females. DMD is characterized by progressive muscle degeneration and weakness, loss of ambulation and death by the late 20`s.

Hypothesis: Our goal was to test the hypothesis that DMD patients` iPSC-derived cardiomyocytes (iPSC-CMs) exhibit functional abnormalities contributing to the in vivo cardiac pathology.

Methods: Dystrophin‐mutated iPSC-CMs were generated from male and female DMD patients. To test the hypothesis, [Ca²⁺]_i transients and contractions were recorded from control and DMD cardiomyocytes. To investigate the molecular mechanisms underlying Ca²⁺ handling in DMD iPSC-CMs, phosphorylation patterns in the CREB signaling pathway were detected using the CREB signaling phospho antibody array.

Results: While in control cardiomyocytes isoproterenol caused a concentration-dependent positive inotropic and lusitropic effects, DMD iPSC-CMs displayed a markedly depressed response. To determine whether the reduced response was due to dysfunctional β-adrenergic cascade or impaired downstream elements mediating common positive inotropic interventions, we investigated the effect of elevating [Ca²⁺]_out.

Like isoproterenol, in healthy iPSC-CMs, elevated [Ca²⁺]_out caused positive inotropic and lusitropic effects, while DMD iPSC-CMs were unresponsive. Next, we tested the functionality of the SR by measuring caffeine-induced Ca²⁺ release. In healthy iPSC-CMs caffeine caused an abrupt increase in [Ca²⁺]_i followed by a gradual decline in [Ca²⁺]_i level. In contrast, DMD iPSC-CMs exhibited a reduced caffeine-induced Ca²⁺ signal amplitude and recovery time. To decipher the molecular mechanisms underlying the DMD cardiomyocytes dysfunction, we employed an enzyme-linked immunosorbent assay-based CREB antibody microarray to determine the phosphorylation patterns in the CREB signaling pathway. Briefly, in DMD male and female iPSC-CMs, MEK2 was more phosphorylated at Thr394 by at least 2-fold and PKC theta was less phosphorylated at Thr538 by at least 2.5-fold respectively.

Conclusion: DMD iPSC-CMs exhibit intracellular Ca²⁺ handling, mechanical and molecular abnormalities.