Albano C. Meli

Background Duchenne muscular dystrophy (DMD) is an X‐linked disorder characterized by progressive muscle weakness due to the absence of functional dystrophin. DMD patients also develop dilated cardiomyopathy (DCM). We have previously shown that DMD ( mdx ) mice and a canine DMD model (GRMD) exhibit abnormal intracellular calcium (Ca 2+ ) cycling related to early‐stage pathological remodelling of the ryanodine receptor intracellular calcium release channel (RyR2) on the sarcoplasmic reticulum (SR) contributing to age‐dependent DCM. Methods Here, we used hiPSC‐CMs from DMD patients selected by Speckle‐tracking echocardiography and canine DMD cardiac biopsies to assess key early‐stage Duchenne DCM features. Results Dystrophin deficiency was associated with RyR2 remodelling and SR Ca 2+ leak (RyR2 Po of 0.03 ± 0.01 for HC vs. 0.16 ± 0.01 for DMD, P < 0.01), which led to early‐stage defects including senescence. We observed higher levels of senescence markers including p15 (2.03 ± 0.75 for HC vs. 13.67 ± 5.49 for DMD, P < 0.05) and p16 (1.86 ± 0.83 for HC vs. 10.71 ± 3.00 for DMD, P < 0.01) in DMD hiPSC‐CMs and in the canine DMD model. The fibrosis was increased in DMD hiPSC‐CMs. We observed cardiac hypocontractility in DMD hiPSC‐CMs. Stabilizing RyR2 pharmacologically by S107 prevented most of these pathological features, including the rescue of the contraction amplitude (1.65 ± 0.06 μm for DMD vs. 2.26 ± 0.08 μm for DMD + S107, P < 0.01). These data were confirmed by proteomic analyses, in particular ECM remodelling and fibrosis. Conclusions We identified key cellular damages that are established earlier than cardiac clinical pathology in DMD patients, with major perturbation of the cardiac ECC. Our results demonstrated that cardiac fibrosis and premature senescence are induced by RyR2 mediated SR Ca 2+ leak in DMD cardiomyocytes. We revealed that RyR2 is an early biomarker of DMD‐associated cardiac damages in DMD patients. The progressive and later DCM onset could be linked with the RyR2‐mediated increased fibrosis and premature senescence, eventually causing cell death and further cardiac fibrosis in a vicious cycle leading to further hypocontractility as a major feature of DCM. The present study provides a novel understanding of the pathophysiological mechanisms of the DMD‐induced DCM. By targeting RyR2 channels, it provides a potential pharmacological treatment.

Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes raise the possibility of generating pluripotent stem cells from a wide range of human diseases. In the cardiology field, hiPSCs have been used to address the mechanistic bases of primary arrhythmias and in investigations of drug safety. These studies have been focused primarily on atrial and ventricular pathologies. Consequently, many hiPSC-based cardiac differentiation protocols have been developed to differentiate between atrial- or ventricular-like cardiomyocytes. Few protocols have successfully proposed ways to obtain hiPSC-derived cardiac pacemaker cells, despite the very limited availability of human tissues from the sinoatrial node. Providing an in vitro source of pacemaker-like cells would be of paramount importance in terms of furthering our understanding of the mechanisms underlying sinoatrial node pathophysiology and testing innovative clinical strategies against sinoatrial node dysfunction (i.e., biological pacemakers and genetic- and pharmacological- based therapy). Here, we summarize and detail the currently available protocols used to obtain patient-derived pacemaker-like cells.