Mitochondrial respiratory chain (RC) disease is a heterogeneous and highly morbid group of energy deficiency disorders for which no proven effective therapies exist. Robust vertebrate animal models of primary RC dysfunction are needed to explore the effects of variation in RC disease subtypes, tissue-specific manifestations, and major pathogenic factors contributing to each disorder, as well as their pre-clinical response to therapeutic candidates. We have developed a series of zebrafish (Danio rerio) models that inhibit, to variable degrees, distinct aspects of RC function, and enable quantification of animal development, survival, behaviors, and organ-level treatment effects as well as effects on mitochondrial biochemistry and physiology. Here, we characterize four pharmacologic inhibitor models of mitochondrial RC dysfunction in early larval zebrafish, including rotenone (complex I inhibitor), azide (complex IV inhibitor), oligomycin (complex V inhibitor), and chloramphenicol (mitochondrial translation inhibitor that leads to multiple RC complex dysfunction). A range of concentrations and exposure times of each RC inhibitor were systematically evaluated on early larval development, animal survival, integrated behaviors (touch and startle responses), organ physiology (brain death, neurologic tone, heart rate), and fluorescence-based analyses of mitochondrial physiology in zebrafish skeletal muscle. Pharmacologic RC inhibitor effects were validated by spectrophotometric analysis of Complex I, II and IV enzyme activities, or relative quantitation of ATP levels in larvae. Outcomes were prioritized that utilize in vivo animal imaging and quantitative behavioral assessments, as may optimally inform the translational potential of pre-clinical drug screens for future clinical study in human mitochondrial disease subjects. The RC complex inhibitors each delayed early embryo development, with short-term exposures of these three agents or chloramphenicol from 5 to 7 days post fertilization also causing reduced larval survival and organ-specific defects ranging from brain death, behavioral and neurologic alterations, reduced mitochondrial membrane potential in skeletal muscle (rotenone), and/or cardiac edema with visible blood pooling (oligomycin). Remarkably, we demonstrate that treating animals with probucol, a nutrient-sensing signaling network modulating drug that has been shown to yield therapeutic effects in a range of other RC disease cellular and animal models, both prevented acute rotenone-induced brain death in zebrafish larvae, and significantly rescued early embryo developmental delay from either rotenone or oligomycin exposure. Overall, these zebrafish pharmacologic RC function inhibition models offer a unique opportunity to gain novel insights into diverse developmental, survival, organ-level, and behavioral defects of varying severity, as well as their individual response to candidate therapies, in a highly tractable and cost-effective vertebrate animal model system.

线粒体呼吸链 (RC) 疾病是一组异质性且发病率很高的能量缺乏性疾病，尚无经过证实的有效治疗方法。需要建立原发性 RC 功能障碍的稳健脊椎动物模型来探索 RC 疾病亚型、组织特异性表现和导致每种疾病的主要致病因素的变异的影响，以及它们对候选治疗方案的临床前反应。我们开发了一系列斑马鱼（斑马鱼）模型，这些模型可以在不同程度上抑制 RC 功能的不同方面，并能够量化动物发育、生存、行为和器官水平治疗效果以及对线粒体生化和线粒体功能的影响。生理。在这里，我们描述了早期幼虫斑马鱼线粒体 RC 功能障碍的四种药理学抑制剂模型，包括鱼藤酮（复合物 I 抑制剂）、叠氮化物（复合物 IV 抑制剂）、寡霉素（复合物 V 抑制剂）和氯霉素（导致多种 RC 的线粒体翻译抑制剂）复杂的功能障碍）。对每种 RC 抑制剂的一系列浓度和暴露时间进行了系统评估，包括早期幼虫发育、动物存活、综合行为（触摸和惊吓反应）、器官生理学（脑死亡、神经张力、心率）和基于荧光的分析。斑马鱼骨骼肌的线粒体生理学。通过复合物 I、II 和 IV 酶活性的分光光度分析或幼虫中 ATP 水平的相对定量来验证 RC 抑制剂的药理学作用。 优先考虑利用体内动物成像和定量行为评估的结果，因为可以最好地了解临床前药物筛选的转化潜力，以用于人类线粒体疾病受试者的未来临床研究。 RC 复合物抑制剂均延迟了早期胚胎发育，受精后 5 至 7 天短期暴露于这三种药物或氯霉素也会导致幼虫存活率降低和器官特异性缺陷，包括脑死亡、行为和神经系统改变、线粒体减少等。骨骼肌膜电位（鱼藤酮）和/或可见血池的心脏水肿（寡霉素）。值得注意的是，我们证明用普罗布考治疗动物，普罗布考是一种营养感应信号网络调节药物，已被证明在一系列其他 RC 疾病细胞和动物模型中产生治疗效果，都可以预防斑马鱼幼虫急性鱼藤酮诱导的脑死亡，并显着挽救了因鱼藤酮或寡霉素暴露而导致的早期胚胎发育迟缓。总体而言，这些斑马鱼药理 RC 功能抑制模型提供了一个独特的机会，以高度易于处理和成本低廉的方式获得对不同严重程度的各种发育、生存、器官水平和行为缺陷的新见解，以及它们对候选疗法的个体反应。 -有效的脊椎动物模型系统。