Aims: Multiple mitochondrial dysfunction (MMD) can lead to complex damage of mitochondrial structure and function, which then lead to the serious damage of various metabolic pathways including cerebral abnormalities. However, the effects of MMD on heart, a highly mitochondria-dependent tissue, are still unclear. In this study, we use iron-sulfur cluster assembly 1 (Isca1), which has been shown to cause MMD syndromes type 5 (MMDS5), to verify the above scientific question. Main methods: We generated myocardium-specific Isca1 knockout rat (Isca1(flox/flox)/alpha-MHC-Cre) using CRISPR-Cas9 technology. Echocardiography, magnetic resonance imaging (MRI), histopathological examinations and molecular markers detection demonstrated phenotypic characteristics of our model. Immunoprecipitation, immunofluorescence co-location, mitochondrial activity, ATP generation and iron ions detection were used to verify the molecular mechanism. Key findings: This study was the first to verify the effects of Isca1 deficiency on cardiac development in vivo, that is cardiomyocytes suffer from mitochondria damage and iron metabolism disorder, which leads to myocardial oncosis and eventually heart failure and body death in rat. Furthermore, forward and reverse validation experiments demonstrated that six-transmembrane epithelial antigen of prostate 3 (STEAP3), a new interacting molecule for ISCA1, plays an important role in iron metabolism and energy generation impairment induced by ISCA1 deficiency. Significance: This result provides theoretical basis for understanding of MMDS pathogenesis, especially on heart development and the pathological process of heart diseases, and finally provides new clues for searching clinical therapeutic targets of MMDS.

目的 心肌肥厚是一种缓慢发展的有效代偿功能,是对血流动力学或心肌损伤的适应性反应,是导致心衰的独立风险因素.已有报道显示,经异丙肾上腺素(isoproterenol,ISO)诱导的心肌肥厚大鼠模型,其给药途径和诱导剂量差异很大,模型动物病理表型发展进程不均一.方法 综合以往方法的先进性、模型表型的均一性、方法稳定性及复制的难易性等因素,本文利用皮下植入渗透泵的方式,采用低剂量长时程给予ISO(4 mg/kg,持续28 d)诱导构建心肌肥厚大鼠模型,并利用超声影像、病理组织学染色、免疫荧光染色及Real-time PCR等技术对模型表型进行评估.结果 模型大鼠病理表型典型.整体表型,包括增大的心脏体积、心室壁厚度及心重指数;细胞表型,包括增大的心肌细胞,严重的心肌纤维化,心肌纤维断裂、溶解,线粒体脊消失、空泡化,心肌Z线、M线以及闰盘扭曲模糊等;分子表型,包括心肌肥厚标志物心钠肽(atrial natriuretic peptide,ANP)及脑钠肽(brain natriuretic peptide,BNP)显著升高.结论 本文建立的心肌肥厚大鼠模型,方法稳定易复制,表型特征典型,更适于基因功能分析及相关药物筛选等科学研究.

The effects of multiple mitochondrial dysfunction (MMD) on heart, a highly mitochondria-dependent tissue, is still unclear. This study was the first to verify the effect of ISCA1 gene deficiency, which has been shown to cause multiple mitochondrial dysfunction syndromes type 5 (MMDS5), on cardiac development in vivo , that is cardiomyocytes suffer from energy shortage due to abnormal metabolism of iron ion, which leads to oncosis and eventually HF and body death. Subsequently, we determine a new interacting molecule for ISCA1, six-transmembrane epithelial antigen of prostate 3 (STEAP3), which acts as a reductase in the reduction of Fe 3+ to Fe 2+ . Forward and reverse validation experiments demonstrated that STEAP3 plays an important role in iron metabolism and energy generation impairment induced by ISCA1 deficiency. This result provides theoretical basis for understanding of MMDS pathogenesis, especially on heart development and the pathological process of heart diseases, and finally provides new clues for searching of clinical therapeutic targets.

目的 构建Prpf40b基因敲除大鼠,为该基因的生物学研究建立工具动物,同时初步探究该基因缺失后对心脏发育的影响.方法 利用CRISPR/Cas9技术构建Prpf40b基因敲除大鼠,测序及PCR技术鉴定敲除大鼠构建成功及仔代大鼠基因型.通过超声影像技术分析敲除大鼠心脏结构形态和功能改变,随后通过病理组织学观察分析该基因敲除后对大鼠心肌显微形态的组织学影响.结果 经PCR鉴定和测序比对,确认Prpf40b基因敲除大鼠构建成功.经超声影像学分析,与同窝阴性大鼠相比2月龄敲除大鼠心脏结构形态未见明显异常,而12月龄敲除大鼠收缩期及舒张期时心室腔内径及容积显著减小,同时射血分数显著减小.经病理组织学分析,与同窝阴性大鼠相比12月龄敲除大鼠心肌出现排列不齐,心肌纤维粗细不均及肌浆网扩张等现象.结论 Prpf40b基因缺失可诱发大鼠心脏整体结构形态改变及心肌组织学异常.

心肌肥厚是一种缓慢发展的有效代偿功能,主要发生在长期压力负荷的情况下,是对血流动力学或心肌损伤的适应性反应,心肌肥厚失代偿最终会增加心力衰竭和猝死的发生率,目前尚无有效的治愈方法.本文就心肌肥厚动物模型,包括小鼠、大鼠及大型动物的制备方法及各自特点,进行总结和比较.同时对心肌肥厚经典分子信号机制,包括有丝分裂蛋白激酶(MAPKs)信号通路及Ca2+介导的信号通路等,以及心肌肥厚引发的失代偿分子机制,包括儿茶酚胺及心肌细胞凋亡等信号通路进行的总结和比较.