MTDNA AND CARDIAC FUNCTION

描述（改编自申请人的摘要）： 线粒体DNA有可能导致心肌病和猝死， 见于某些罕见的遗传性综合征（Kearn-Sayres等人）。 相对于功能完整的线粒体DNA， 线粒体基因组也与心肌收缩有关， 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缺陷与心脏病之间的因果关系 功能缺失。到目前为止，实验操作 mtDNA在动物中是不可能的。在本申请中， 申请人提出了一组实验来确定 DNA拷贝数、呼吸功能和收缩功能之间 在完整心脏中的表现。首先，调查人员将完成 小鼠线粒体DNA聚合酶（mtPOL）的分子克隆 和终止mtDNA复制的因子（mtTER）。他假设 改变这些蛋白质在体内的表达， 后遗症：正常mtDNA基因组拷贝数的变化，或 mtDNA缺失频率增加。为了验证这一假设， 申请人将在培养的骨骼肌细胞中操纵这些基因的表达， 肌管（使用不可消除的过表达和反义寡核苷酸） 并评估对mtDNA拷贝数和缺失的丰度的影响。 mtDNA的形式。随后，他希望调节mtDNA拷贝数， 并在转基因小鼠的完整心脏中产生缺失形式的mtDNA， 使用mtPOL基因的心脏特异性敲除的小鼠和常规的 敲除必需mtTER亚基基因。在 此外，研究者建议产生肌原性细胞系， 线粒体DNA生理学显著点突变转基因小鼠 通过强制表达在3 '-5'端缺陷的mtPOL突变变体， 外切核酸酶（校对）功能。心率和左心室 将在这些动物中测量dP/dtmax，并测量心脏呼吸 将使用31 P NMR光谱法在离体心脏中评估功能 在常氧和缺血条件下。申请人希望这些 实验将定量地确定收缩的严重程度， 以及线粒体DNA拷贝减少导致的代谢功能障碍 数量，或从给定程度的异质性定义的缺失，或 mtDNA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.