MTDNA AND CARDIAC FUNCTION

描述(改编自申请人的摘要)：突变 线粒体DNA有可能导致心肌病和猝死，因为 见于某些罕见的遗传性综合征(Kain-Sayres和其他)。 相对于功能完整的线粒体DNA的突变形式的积累 线粒体基因组也与心脏收缩有关 与更常见的疾病相关的功能障碍，包括： 高龄、特发性扩张型心肌病、肥厚型 心肌病、慢性缺血性心脏病和药物毒性(AZT 或阿霉素)。然而，在后一种情况下，直接证据 线粒体DNA缺陷与心脏疾病之间的因果关系 功能障碍一直欠缺。到目前为止，实验性的操纵 在动物身上检测线粒体DNA是不可能的。在此应用程序中， 申请者提出了一系列实验来确定这种关系 DNA拷贝数、呼吸功能和收缩功能之间的关系 表现在完好无损的心里。首先，调查员将完成 小鼠线粒体DNA聚合酶(MtPOL)的分子克隆 以及终止线粒体DNA复制的一个因素(MtTER)。他假设 在体内改变这些蛋白质的表达将有两种情况之一 后遗症：正常线粒体DNA基因组拷贝数的变化或 线粒体DNA缺失频率增加。为了检验这一假设， 申请者将操纵这些基因在培养的骨骼中的表达 肌管(使用不可磨灭的过表达和反义寡核苷酸) 并评估其对线粒体DNA拷贝数和缺失丰度的影响 线粒体DNA的形式。随后，他预计将调整mtDNA拷贝数 并在转基因动物的完整心脏中产生缺失形式的线粒体DNA 使用心脏特异性mtPOL基因敲除的小鼠和常规 分别敲除了一个必需的mtTER亚单位基因。在……里面 此外，研究人员建议培养肌源性细胞系和 线粒体DNA生理性显著点突变的转基因小鼠 通过3‘-5’中mtPOL缺陷突变变体的强制表达 核酸外切酶(校对)功能。心率与左心室 将在这些动物身上测量dp/dtmax，以及心脏呼吸 将使用31P核磁共振波谱评估离体心的功能 在常氧和缺血条件下。申请者希望这些 实验将定量地确定收缩的严重程度。 以及由于线粒体DNA复制的特定减少而导致的代谢功能障碍 数量，或来自给定程度的异质性，用于定义的缺失或 线粒体DNA的点突变。

DESCRIPTION (adapted from the applicant's abstract): Mutations with mtDNA have the potential to cause cardiomyopathy and sudden death, as seen in certain rare, inherited syndromes (Kearn-Sayres and others). An accumulation of mutated forms of mtDNA relative to functional, intact mitochondrial genomes also has been implicated in cardiac contractile dysfunction associated with much more common disorders, including: advanced age, idiopathic dilated cardiomyopathy, hypertrophic cardiomyopathy, chronic ischemic heart disease, and drug toxicity (AZT or doxorubicin). In these latter conditions, however, direct evidence for a causal relationship between defects in mtDNA and cardiac dysfunction has been lacking. Up to now, experimental manipulation of mtDNA in animals has not been possible. In this application, the applicant proposes a set of experiments to determine the relationship between DNA copy number, respiratory function, and contractile performance in the intact heart. First, the investigator will complete the molecular cloning of the mouse mitochondrial DNA polymerase (mtPOL) and a factor that terminates mtDNA replication (mtTER). He hypothesizes that altering expression of these proteins in vivo will have one of two sequelae: changes in the copy number of normal mtDNA genomes or increased frequency of mtDNA deletions. To test this hypothesis, the applicant will manipulate expression of these genes in cultured skeletal myotubes (using indelible overexpression and antisense oligonucleotides) and assess the effects on mtDNA copy number and the abundance of deleted forms of mtDNA. Subsequently, he expects to modulate mtDNA copy number and generate deleted forms of mtDNA in the intact heart of transgenic mice using cardiac-specific knock-out of the mtPOL gene and conventional knock-out of an essential mtTER subunit gene, respectively. In addition, the investigator proposes to generate myogenic cell lines and transgenic mice with physiologically significant point mutations in mtDNA by forced expression of a mutated variant of mtPOL defective in 3'-5' exonuclease (proofreading) function. Heart rate and left ventricular dP/dtmax will be measured in these animals, and cardiac respiratory function will be assessed in isolated hearts using 31P NMR spectroscopy under normoxic and ischemic conditions. The applicant hopes these experiments will determine, quantitatively, the severity of contractile and metabolic dysfunction resulting from a given reduction in mtDNA copy number, or from a given degree of heteroplasmy for defined deletions or point mutations in mtDNA.