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Abstract：
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 (Ca2+) 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 Ca2+ 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 Ca2+ 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.
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 Ca2+ 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 Ca2+ 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.
摘要：
背景：杜氏肌营养不良症（DMD）是一种X连锁疾病，其特征是由于缺乏功能性肌营养不良蛋白而导致的进行性肌肉无力。DMD患者也发展为扩张型心肌病(DCM)。我们先前已经表明，DMD（mdx）小鼠和犬DMD模型（GRMD）表现出异常的细胞内钙（Ca2）循环，与肌浆网（SR）上的ryanodine受体细胞内钙释放通道（RyR2）的早期病理重塑有关。
方法：这里，我们使用斑点追踪超声心动图和犬DMD心脏活检选择的DMD患者的hiPSC-CM来评估关键的早期DuchenneDCM特征.
结果：肌营养不良蛋白缺乏与RyR2重塑和SRCa2渗漏相关（对于HC，RyR2Po为0.03±0.01DMD为0.16±0.01，P<0.01),导致包括衰老在内的早期缺陷。我们观察到较高水平的衰老标志物，包括p15（HC的2.03±0.75与13.67±5.49对于DMD，P<0.05）和p16（HCvs.10.71±3.00对于DMD，在DMDhiPSC-CM和犬DMD模型中P<0.01）。纤维化在DMDhiPSC-CM中增加。我们观察到DMDhiPSC-CM中的心脏收缩不足。通过S107稳定RyR2药理学阻止了大多数这些病理特征，包括收缩幅度的抢救(DMD与1.65±0.06μmDMD+S107为2.26±0.08μm,P<0.01)。这些数据通过蛋白质组学分析得到证实，特别是ECM重塑和纤维化。
结论：我们在DMD患者中确定了比心脏临床病理更早建立的关键细胞损伤，心脏ECC的主要扰动。我们的结果表明，RyR2介导的SRCa2泄漏在DMD心肌细胞中诱导了心脏纤维化和过早衰老。我们发现RyR2是DMD患者中DMD相关心脏损害的早期生物标志物。进行性和较晚的DCM发病可能与RyR2介导的纤维化增加和过早衰老有关。最终导致细胞死亡和进一步的心脏纤维化的恶性循环，导致进一步的收缩功能低下是DCM的主要特征。本研究提供了对DMD诱导的DCM的病理生理机制的新理解。通过瞄准RyR2通道,它提供了潜在的药物治疗。
方法：这里，我们使用斑点追踪超声心动图和犬DMD心脏活检选择的DMD患者的hiPSC-CM来评估关键的早期DuchenneDCM特征.
结果：肌营养不良蛋白缺乏与RyR2重塑和SRCa2渗漏相关（对于HC，RyR2Po为0.03±0.01DMD为0.16±0.01，P<0.01),导致包括衰老在内的早期缺陷。我们观察到较高水平的衰老标志物，包括p15（HC的2.03±0.75与13.67±5.49对于DMD，P<0.05）和p16（HCvs.10.71±3.00对于DMD，在DMDhiPSC-CM和犬DMD模型中P<0.01）。纤维化在DMDhiPSC-CM中增加。我们观察到DMDhiPSC-CM中的心脏收缩不足。通过S107稳定RyR2药理学阻止了大多数这些病理特征，包括收缩幅度的抢救(DMD与1.65±0.06μmDMD+S107为2.26±0.08μm,P<0.01)。这些数据通过蛋白质组学分析得到证实，特别是ECM重塑和纤维化。
结论：我们在DMD患者中确定了比心脏临床病理更早建立的关键细胞损伤，心脏ECC的主要扰动。我们的结果表明，RyR2介导的SRCa2泄漏在DMD心肌细胞中诱导了心脏纤维化和过早衰老。我们发现RyR2是DMD患者中DMD相关心脏损害的早期生物标志物。进行性和较晚的DCM发病可能与RyR2介导的纤维化增加和过早衰老有关。最终导致细胞死亡和进一步的心脏纤维化的恶性循环，导致进一步的收缩功能低下是DCM的主要特征。本研究提供了对DMD诱导的DCM的病理生理机制的新理解。通过瞄准RyR2通道,它提供了潜在的药物治疗。
