Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Fondazione Intesa San Paolo Introduction Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive degeneration of striated muscles; in addition to skeletal muscle impairment, DMD is also characterized by progressive myocardial disfunction. The low translational value of animal models and the low availability of human samples make DMD hard to investigate; induced pluripotent stem cells (iPSCs) represent a novel tool to model this disease, preserving the genetic heritage of the patient, including the pathogenic mutation causing dystrophy. Aim Our aim is to characterize cardiomyocytes differentiated from iPSCs (iPSC-CMs) derived from healthy donors (CTRL) and DMD patients, to identify the pathophysiological mechanisms of DMD-related cardiomyopathy. Materials and Methods Cardiomyocytes are differentiated from IPSCs obtained by reprogramming isolated mononucleated blood cells from healthy donors and DMD patients. IPSC-CMs are cultured until day 60, 75 or 90 post-differentiation after plating on nanostructured substrates with two different stiffness levels: PEG-substrates, with lower rigidity, mimicking healthy extracellular tissue, and DEG-substrates, with greater rigidity, that mimic the presence of myocardial fibrosis. Through imaging techniques, we evaluated calcium handling and action potentials (AP) on DMD and CTRL iPSC-CMs by using specific fluorescent dyes for Ca2+ (CAL630) and membrane voltage (Fluovolt). Cells were stimulated at different pacing rates. Results The calcium transient amplitude of CTRL-iPSC-CMs became larger during maturation. This adaptation did not occur in DMD lines, showing a deficit calcium release due to poor maturation of the sarcoplasmic reticulum (SR). AP duration was shorter in the DMD line at d75 but at d90 we observed no differences when compared with the CTRL line. CTRL iPSC-CMs showed a marked ability to adapt to different substrate stiffnesses. Indeed, the calcium transient amplitude was larger and its kinetics faster when cells were grown on the rigid DEG substrates rather than on PEG plates. In the DMD line, however, no differences were observed between the substrates. Conclusions Our results highlight a scarce ability of DMD iPSC-CM to adapt to different substrate stiffness, resulting in mechanical and electrical impairment, especially in the presence of stiffer substrates. This might explain why cardiac impairment is usually absent in the early stages of DMD, when cardiac structural changes are still absent. However, the electrophysiological and mechanical impairment of DMD hearts may precipitate rapidly when extracellular stiffness starts to increase due to development of cardiac fibrosis.

中文翻译：

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细胞外僵硬度作为杜氏肌营养不良症心功能不全的决定因素：人类 iPSC 衍生心肌细胞的研究

资金致谢 资金来源类型：基金会。主要资金来源： Fondazione Intesa San Paolo 除了骨骼肌损伤外，DMD 的特征还在于进行性心肌功能障碍。动物模型的低转化价值和人类样本的低可用性使得 DMD 难以研究；诱导多能干细胞 (iPSCs) 代表了一种模拟这种疾病的新工具，它保留了患者的遗传遗产，包括导致营养不良的致病突变。目标 我们的目标是表征从健康供体 (CTRL) 和 DMD 患者衍生的 iPSC (iPSC-CM) 分化的心肌细胞，确定 DMD 相关心肌病的病理生理机制。材料和方法 心肌细胞从通过重新编程来自健康供体和 DMD 患者的分离的单核血细胞获得的 IPSC 中分化出来。IPSC-CM 在具有两种不同刚度水平的纳米结构基底上电镀后培养至分化后第 60、75 或 90 天：具有较低刚度的 PEG 基底，模拟健康的细胞外组织，以及具有更大刚度的 DEG 基底，模拟心肌纤维化的存在。通过成像技术，我们通过使用用于 Ca2+ (CAL630) 和膜电压 (Fluovolt) 的特定荧光染料来评估 DMD 和 CTRL iPSC-CM 上的钙处理和动作电位 (AP)。以不同的起搏速率刺激细胞。结果CTRL-iPSC-CMs的钙瞬变振幅在成熟过程中变大。这种适应在 DMD 系中没有发生，表明由于肌质网 (SR) 的成熟不良导致钙释放不足。在 d75 时，DMD 线中的 AP 持续时间较短，但在 d90 时，与 CTRL 线相比，我们没有观察到差异。CTRL iPSC-CMs 显示出适应不同基板刚度的显着能力。事实上，当细胞在刚性 DEG 基板上而不是在 PEG 板上生长时，钙瞬态幅度更大，其动力学更快。然而，在 DMD 线中，在基板之间没有观察到差异。结论 我们的结果突出了 DMD iPSC-CM 适应不同基板刚度的稀缺能力，导致机械和电气损伤，特别是在较硬的基材存在的情况下。这可以解释为什么在 DMD 的早期阶段通常不存在心脏损伤，此时心脏结构变化仍然不存在。然而，当细胞外僵硬由于心脏纤维化的发展而开始增加时，DMD 心脏的电生理和机械损伤可能会迅速出现。