Nucleoside analogs, mitochondria and AIDS cardiomyopathy

描述（由申请人提供）： 本研究旨在探讨艾滋病高效抗逆转录病毒治疗（HAART）中使用的核苷类逆转录酶抑制剂（NRTIs）引起线粒体功能障碍和心肌病（CM）的机制。自从引入包括NRTI如齐多夫定（3 '-叠氮基-2'，3 '-脱氧胸苷; AZT）的HAART组合以来，艾滋病生存率有所改善，但HAART的线粒体毒性机制尚未完全了解。“DNA聚合γ假说”提供了一个框架来解释HAART CM中线粒体亚细胞病理生理机制。 它强调了NRTI细胞内和线粒体内磷酸化的重要性，通过细胞激酶（活性部分），抑制DNA聚合酶γ的NRTI三磷酸盐，和组织靶点中的mtDNA消耗。线粒体DNA突变和线粒体氧化应激（活性氧和抗氧化剂之间的不平衡）的贡献也得到了解决。 靶向转基因小鼠（TG）是检验“DNA聚合酶γ假说”（基于DNA聚合酶γ复制mtDNA的功能）的活体系统。靶向心脏TG（由α肌球蛋白重链启动子驱动）是探索HAART CM机制的活系统。TG：（t）表达HIV反式激活蛋白（达特）或（2）过表达人TK 2（线粒体胸苷激酶）以用于HAART方案。 该假说指出：心脏线粒体NRTI单磷酸化（通过TK 2）和HIV达特在病理生理学上有助于HAART CM。AZT抑制线粒体DNA复制，消耗线粒体DNA，改变线粒体超微结构，导致CM。 过表达心脏TK 2的靶向TG增加AZT的线粒体单磷酸化（含AZT的HAART方案）。这导致线粒体内AZTTP增加、DNA pol-gamma聚合酶功能抑制、mtDNA耗竭和突变以及CM。将达特靶向TG表达至心肌细胞抑制心脏GSH合成，消耗GSH，并引起导致CM的氧化应激。达特TG治疗CM。 目标1：利用mtDNA和mtDNA丰度从生化上确定HAART所致CM的线粒体生物发生，并通过8-OHdG（mtDNA中）丰度、GSH/GSSG比值和顺乌头酸酶失活来确定氧化应激。 目的2：应用形态计量学方法从显微镜和超微结构上对HAART引起的CM进行定义. 目的3：通过超声心动图和Langendorff制剂确定HAART治疗CM的心脏功能。

DESCRIPTION (provided by applicant): This project characterizes mechanisms of mitochondrial dysfunction and cardiomyopathy (CM) from nucleoside reverse transcriptase inhibitors (NRTIs) used in highly active antiretroviral therapy (HAART) for AIDS. Survival with AIDS improved since the introduction of HAART combinations that include NRTIs like zidovudine (3'-azido-2',3'-deoxythymidine; AZT) but mechanisms of mitochondrial toxicity from HAART are incompletely understood. The "DNA pol gamma hypothesis" offers a framework to explain mitochondrial subcellular pathophysiological mechanisms in HAART CM. It underscores the importance of NRTI intracellular and intramitochondrial phosphorylation by cellular kinases (to active moieties), inhibition of DNA pol gamma by NRTI triphosphates, and mtDNA depletion in tissue targets. Contributions of mtDNA mutations and mitochondrial oxidative stress (imbalance between reactive oxygen species and antioxidants) are also addressed. Targeted transgenic mice (TG) are living systems to test the "DNA pol gamma hypothesis" (based on the function of DNA pol gamma that replicates mtDNA). Targeted cardiac TGs (driven by the alpha myosin heavy chain promoter) are living systems to explore mechanisms of CM from HAART. The TGs: (t) express HIV transactivator protein (Tat) or (2) over-express human TK2 (the mitochondrial thymidine kinase) to be used with HAART protocols. The HYPOTHESIS states: Cardiac mitochondrial NRTI monophosphorylation (by TK2) and HIV Tat contribute pathophysiologically to HAART CM. AZT inhibits mtDNA replication, depletes mtDNA, alters mitochondrial ultrastructure, and causes CM. Targeted TGs that overexpress cardiac TK2 increase mitochondrial monophosphorylation of AZT (with AZT-containing HAART regimens). This leads to increased intramitochondrial AZTTP, inhibition of DNA pol-gamma polymerase function, mtDNA depletion and mutations, and CM. Targeted TG expression of Tat to cardiac myocytes inhibits cardiac GSH synthesis, depletes GSH, and causes oxidative stress that results in CM. HAART treatment of Tat TGs worsens CM. AIM1: to define mitochondrial biogenesis biochemically in CM from HAART using mtDNA and mtRNA abundance, and to define oxidative stress by abundance of 8-OHdG (in mtDNA), GSH/GSSG ratios and aconitase inactivation. AIM 2: to define CM from HAART microscopically and ultrastructurally using morphometric methods. AIM 3: to define cardiac performance in CM from HAART echocardiographically and using Langendorff preparations.