背景：多发性线粒体功能障碍综合征（multiple mitochondrial dysfunctions syndrome，MMDS）是一类与线粒体铁硫簇（iron-sulfur cluster，ISC）合成相关的细胞核基因（nuclear DNA，nDNA）突变密切相关的线粒体疾病。MMDS可造成线粒体结构和功能的严重破坏，从而损害多种代谢途径，临床上主要呈现神经系统损伤、脑损伤、发育不良、肌肉张力下降和呼吸功能不全等。线粒体功能障碍会累及使用ATP的所有组织，关于MMDS的研究目前主要集中在神经系统和脑组织。然而，近年来已有MMDS患者累及心脏的报道，但是相关机制研究很少，尤其是ISC相关基因突变或表达异常引起的MMDS对心脏的影响和机制尚不清楚。ISCA1是线粒体铁硫簇组装系统的重要组成部分，我们前期研究发现，在心肌病和心力衰竭状态下，ISCA1的表达显著增加，提示该类基因的表达调控可能参与心肌病及心衰的发展，因此对ISC相关基因参与心肌病等心血管疾病的作用研究具有重要的意义。

目的：建立心肌组织特异性过表达Isca1大鼠模型，与课题组已建立的心肌组织特异性Isca1敲除大鼠模型形成功能获得和功能缺失的对比模型。对比研究Isca1对大鼠心脏发育、心脏病理进程的影响及可能的分子机制。为由Isca1基因异常诱发的MMDS并累及心脏的研究提供动物模型。

方法：（1）利用基因工程技术建立心肌组织特异性过表达Isca1大鼠。利用Isca1条件敲除大鼠与α-MHC-Cre工具大鼠杂交培育心肌组织特异性Isca1敲除大鼠。

（2）利用基因组PCR方法鉴定心肌特异的Isca1敲除大鼠和心肌特异的过表达Isca1大鼠的基因型，并进行生存状况观察。

（3）超声影像学检测大鼠在体心脏结构和功能变化；

（4）免疫荧光观察ISCA1与STEAP3于心肌细胞中的定位，观察大鼠心肌组织和H9c2细胞的结构以及胀亡情况，观察大鼠心肌组织和H9c2细胞的膜和骨架的形态学变化。

（5）透射电子显微镜观察大鼠心肌细胞超微结构的形态变化。

（6）Real-Time PCR检测大鼠心脏组织和H9c2细胞的分子标记物表达水平。

（7）Western blot检测ISCA1及线粒体呼吸链复合物相关分子的表达水平。

（8）筛选并建立Isca1过表达和Steap3表达敲低的H9c2细胞系。

（9）免疫沉淀和质谱法分析ISCA1互作蛋白分子。

（10）比色法检测大鼠心肌组织和H9c2细胞的ATP浓度和铁离子浓度。

结果：（1）Western blot结果显示ISCA1在野生小鼠心肌中的表达随年龄增加而下调，但在同龄cTnT^R141W心肌病小鼠模型心肌中表达下调速度减缓。ISCA1主要表达于心肌线粒体。

（2）通过杂交繁育，得到了心肌组织特异性Isca1敲除纯合子大鼠（简称：Isca1^-/-）、心肌组织特异性Isca1敲除杂合子大鼠（简称：Isca1^-/+）和心肌组织特异性过表达Isca1大鼠（简称：Isca1-Tg）三种模型，可用于不同的研究目的。ISCA1在Isca1^-/-大鼠心肌中的敲除效率为86%，Isca1^-/+大鼠Isca1表达降低46%，而Isca1-Tg大鼠Isca1表达增加253%。

（3）Isca1^-/-大鼠出生后10天内死亡。大体解剖可观察到Isca1^-/-大鼠心室壁塌陷，心脏整体显明显增大。Isca1^-/-大鼠心脏重量与体重比（HW/BW）增加了20%（P<0.01）。超声影像结果表明Isca1^-/-大鼠表现出典型的心衰表型，包括心腔变大、室壁变薄、心脏收缩功能迅速下降。病理组织学结果显示Isca1^-/-大鼠心肌纤维结构紊乱，大面积的心肌断裂溶解，线粒体肿胀，膜结构受损。伊文斯蓝（Evans blue）、WGA和dystrophin共染色显示Isca1^-/-大鼠心肌细胞膜结构受损严重。Isca1^-/-大鼠心脏组织中肥大标志物心钠肽（nppa）和脑钠肽（nppb）均显著增加（分别为309%和329%，P<0.001）。整体、组织和分子三个水平的观察结果均证实Isca1的缺失会诱发心肌细胞发生胀亡。

（4）Isca1^-/-大鼠心肌中Fe²⁺水平降低，Fe³⁺水平升高，Isca1缺失诱发心肌组织铁离子代谢失衡。免疫沉淀和质谱法分析结果证实，STEAP3是ISCA1的互作蛋白分子，二者共定位于线粒体。体外实验结果显示，Steap3表达敲低后，ISCA1结合的Fe²⁺浓度亦显著降低（P<0.05），而Isca1过表达可部分逆转Steap3表达敲低后诱导的细胞死亡、ATP减少和心肌肥大标志物的表达，并改善细胞的胀亡表型。证实ISCA1与STEAP3相互作用，共同参与调节铁离子的代谢过程。

结论:（1）Isca1^-/-大鼠表现为严重的心衰和心肌细胞胀亡。

（2）Isca1参与调节心肌组织中铁离子代谢平衡，Isca1敲除导致Fe²⁺水平降低，进而引起线粒体呼吸链和能量代谢异常，即为Isca1缺失后诱发心肌细胞胀亡的可能机制。

（3）STEAP3是ISCA1的互作蛋白分子，ISCA1与STEAP3相互作用，共同调节铁离子的代谢过程。

（4）Isca1^-/+大鼠存在半合子效应，生理情况下表现为心脏扩张，心功能下降，并呈渐进性发展的特点，可作为MMDS累及心脏的大鼠模型。

Background: Multiple mitochondrial dysfunctions syndrome (MMDS) is a group of mitochondrial diseases caused by mutations in genes related to mitochondrial iron-sulfur cluster synthesis (ISC). MMDS is characterized as serious damages of mitochondrial structure and function, and thus impair various metabolic pathways. The main clinical manifestations of MMDS are nervous system injury, encephalopathy, hypoplasia, hypotonia, and respiratory dysfunction. It was supposed that mitochondrial dysfunction would affect all tissue, which needed ATP to maintain its function. At present, the research on MMDS mainly focuses on the nervous system and brain tissue. However, the studies on MMDS related heart diseases are limited, especially the heart disease of MMDS caused by mutations of ISC related genes. ISCA1, an important component of the mitochondrial iron-sulfur cluster assembly machinery. In our previous study, expression of ISCA1 was found to be increased significantly in the heart tissues with hypertrophic cardiomyopathy (HCM) and heart failure (HF) and suggesting Isca1 might be involved in cardiomyopathy and heart failure. Thus, illustration of Isca1 function in hearts would be beneficial to understand the role of ISC related genes in cardiovascular diseases such as cardiomyopathy.

Objective: Three rat models were built to investigate Isca1 function in hearts, including Isca1 transgenic rats, heterozygous and homozygous Isca1 knockout rats. The homozygous Isca1 knockout rats were used to investigate the effects of Isca1 on cardiac development and pathological processes as well as molecular mechanisms. The heterozygous Isca1 knockout rats were phenotyped to generated an MMDS related heart disease model.

Methods: (1) Isca1 transgenic rats were generated by insertion of the floxed Isca1 in Rosa26 loci, which over-expressed ISCA1 protein specifically in the heart induced by α-MHC-Cre. The heterozygous and homozygous Isca1 heart conditional knockout rats were obtained by cross Isca1 gene floxed rats with α-MHC-Cre rats.

(2) The genotypes of the rats were identified by PCR. The survival rate of homozygous Isca1 knockout rats was compared with wild-type rats.

(3) Echocardiography was used to analyze the structure and function of hearts.

(4) Immunofluorescence was applied to observe the structure, integrity, and oncosis of myocardial tissue and the localization of ISCA1 and STEAP3 in cardiomyocytes.

(5) The ultrastructure of heart tissues was observed by transmission electron microscopy (TEM).

(6) Real-time qPCR was used to detect the levels of cardiac hypertrophy molecular markers of rat heart and H9c2 cells.

(7) The level of ISCA1 and mitochondrial respiratory chain complex related molecules were determined by Western blot.

(8) The Isca1 over-expression and Steap3 knockdown H9c2 were screened and established.

(9) ISCA1 interacting protein molecules were identified by immunoprecipitation assays and mass spectrometry.

(10) ATP concentration and iron concentration in heart tissue and H9c2 cells were detected by colorimetry.

Results: (1) Western blot results showed that the expression of ISCA1 in the hearts of wild mice decreased with age, but decreased slowly in the cardiomyopathy cTnT^R141W model mice. ISCA1 was mainly located in myocardial mitochondria.

(2) Three models of homozygous Isca1 heart conditional knockout rat (referred to as Isca1^-/-), heterozygous Isca1 heart conditional knockout rat (referred to as Isca1^-/+), and Isca1 transgenic rat (referred to as Isca1-Tg) were obtained by transgenic and cross-breeding, which can be used for different purposes. The knockout efficiency of ISCA1 was 86% in the myocardium of Isca1^-/- rat. The expression of ISCA1 in Isca1^-/- rats was decreased by 46%. The expression of ISCA1 in Isca1-Tg rats was increased by 253%.

(3) The Isca1^-/- rats showed an enlarged heart and collapsed ventricular wall and died within 10 days after birth. The heart weight/body weight ratio (HW/BW) of Isca1^-/- rats increased by 20% (P<0.01). Echocardiography showed that Isca1^-/- rats exhibited a typical heart failure phenotype with enlarged cardiac chambers, thinner ventricular wall, and decreased cardiac systolic function. Histopathological results showed that poorly organized myocardium, large area of myocardiolysis, swollen mitochondria with damaged membrane structure in heart tissues of Isca1^-/- rats. Evans blue, WGA, and dystrophin staining showed that the deletion of Isca1 caused severe damage of myocardial cell membrane and increased significantly the expression of hypertrophic markers, nppa (309%, P<0.001) and nppb (329%, P<0.001). The rusults of three levels (whole, tissue and molecular) showed that Isca1 deletion induced cardiomyocytes oncosis.

(4) The level of Fe²⁺ decreased and Fe³⁺ increased in Isca1^-/- rats, suggesting that deletion of Isca1 induced iron ion metabolic imbalance in the myocardium. STEAP3 was indicated to be a new interacting protein molecule of ISCA1, and co-localization in mitochondria. The results of in vitro experiment showed that knockdown of the expression of Steap3 in H9c2 cells resulted in the decrease of 30% of the ISCA1-binding Fe²⁺ significantly (P<0.05). Knockdown of the expression of Steap3 was resulted in the decrease of ATP production, increased expression of hypertrophic markers, and oncosis and those features could be partially ameliorated by overexpression of Isca1 in H9c2 cells. These results suggesting that the interaction between ISCA1 and STEAP3 regulated the metabolism of iron ions.

Conclusion: (1) Isca1 deletion caused typical heart failure with cardiomyocytes oncosis.

(2) Isca1 is involved in the regulation of the balance of iron ions metabolism in the myocardium. Isca1 deletion caused the decrease of Fe²⁺ level and subsequently impaired mitochondrial respiratory chain and energy metabolism, which was the mechanism of cardiomyocytes oncosis induced by Isca1 deletion.

(3) STEAP3 is an interacting protein molecule of ISCA1. The interaction between ISCA1 and STEAP3 regulates the metabolism of iron ions.

(4) Isca1^-/+ rats had a hemizygous effect and showed cardiac dilatation, dysfunctions, and progressive development in normal physiological conditions which could be used as a rat model of MMDS cumulative heart.