Opto-Electrophysiological Method to Study Human Skeletal Muscle Channelopathies

摘要 肌病源于离子通道或调节其前-RNA的蛋白质编码基因的突变 剪接分别称为通道病和剪接病。不正常的表情、结构或功能 这些突变导致的离子通道的改变导致肌肉电生理和兴奋- 收缩耦合。这些遗传性疾病目前还无法治愈，严重影响患者的生活质量 并跨越罕见疾病的流行范围(例如，低钾和高钾性周期性瘫痪，1：100000) 对于最常见的肌病(如营养不良性肌强直，1-5：10000)。虽然他们的遗传原因很容易 可识别，了解通道病和剪接病的潜在机制，并设计 合理的治疗策略，需要对所有患者进行详细的电生理研究 有意义的人类细胞环境。这些研究虽然可行，但由于普遍缺乏实用的、 可用于人体肌肉的高通量方法。用动物模型来绕过这一点 缺点往往不能概括大多数疾病，也不能再现人类对治疗药物的反应。 我们打算通过设计和测试一种新的、实用的电生理学方法来克服这些限制 很容易与从活组织切片中分离的人类肌肉纤维一起使用。我们将结合一项革命性的实验 用两种不同的电生理方法进行技术量化，状态为 ART，在接近理想的条件下对长度为50-400μm的纤维段进行电生理学研究。与之前的不同 方法，这种新方法易于实现、用户友好，并提供了必要的高吞吐量 具有统计学意义的研究。我们的方法将允许逐个病例的电生理研究和筛查 作用强烈的药物，能够设计针对患者的治疗方案，符合当前的趋势 在当代精准医学中。我们预计，我们的方法将对人类产生变革性的影响 肌肉生理学和病理生理学。

ABSTRACT Myopathies stemming from mutations in genes coding for ion channels or proteins that regulate their pre-RNA splicing are called, respectively, channelopathies and spliceopathies. Abnormal expression, structure or function of ion channels resulting from those mutations leads to altered muscle electrophysiology and excitation- contraction coupling. These genetic disorders have currently no cure, severely affect the quality of life of patients and span the prevalence spectrum from rare diseases (e.g. hypo- and hyperkalemic periodic paralysis, 1:100000) to the most common myopathies (e.g. dystrophic myotonia, 1-5:10000). While their genetic cause is readily identifiable, understanding the mechanisms underlying channelopathies and spliceopathies, and designing sound therapeutic strategies for them, demands detailed electrophysiological studies performed in the all- meaningful human cellular context. These studies, though feasible, are impeded by a pervasive lack of practical, high throughput methods amenable for use in human muscles. Animal models used to circumvent this shortcoming often fail to recapitulate most diseases or to reproduce the human response to therapeutic drugs. We intend to overcome these limitations by designing and testing a novel, practical electrophysiological method facilely used with human muscle fibers dissected from biopsies. We will combine a revolutionary experimental chamber with technologies from two different electrophysiological methods to perform quantitative, state of the art, electrophysiological studies in segments of fibers 50-400μm long in near-ideal conditions. Unlike previous methods, this new method is readily implemented, user-friendly, and affords the requisite high throughput for statistically significant studies. Our method will allow case-by-case electrophysiological studies and screening of acutely acting drugs, enabling the design of patient specific treatment schemes, coinciding with current trends in contemporary precision medicine. We expect, then, our method will have a transformative impact in human muscle physiology and pathophysiology.