Nucleoside analogs, mitochondria and AIDS cardiomyopathy

描述(由申请人提供)： 该项目描述了用于艾滋病高效抗逆转录病毒疗法(HAART)的核苷类逆转录酶抑制剂(NRTI)导致线粒体功能障碍和心肌病(CM)的机制。自从引入包括齐多夫定(3‘-叠氮-2’，3‘-脱氧胸苷；AZT)等NRTI的HAART组合以来，艾滋病患者的存活率有所改善，但HAART的线粒体毒性机制尚不完全清楚。DNA polGamma假说为解释HAART CM的线粒体亚细胞病理生理学机制提供了一个框架。它强调了NRTI在细胞内和线粒体内的磷酸化(对活性部分)的重要性，NRTI三磷酸对DNA Pol伽马的抑制，以及组织靶标中mtDNA的耗尽。线粒体DNA突变和线粒体氧化应激(活性氧物种和抗氧化剂之间的失衡)的贡献也被提及。靶向转基因小鼠(Tg)是一种活体系统，用来检验“DNA Pol-Gamma假说”(基于复制mtDNA的DNA Pol-Gamma的功能)。靶向心肌TGS(由α肌球蛋白重链启动子驱动)是从HAART中探索CM机制的活体系统。TGS：(T)表达HIV反式激活蛋白(TAT)或(2)过表达人类TK2(线粒体胸苷激酶)，用于HAART方案。假设：心肌线粒体NRTI单磷酸化(通过TK2)和HIV Tat在HAART CM的病理生理学中起作用。AZT抑制线粒体DNA复制，耗尽线粒体DNA，改变线粒体超微结构，导致CM。靶向过表达心脏TK2的TGS增加AZT的线粒体单磷酸化(使用含AZT的HAART方案)。这会导致线粒体内AZTTP增加，DNA聚合酶功能抑制，线粒体DNA枯竭和突变，以及CM。TAT靶向心肌细胞的TG表达抑制了心脏GSH的合成，消耗了GSH，并导致氧化应激，从而导致CM。HAART治疗TAT TGS加重CM。目的：利用线粒体DNA和mtRNA丰度，从生物化学的角度确定HAART的线粒体生物发生，并通过线粒体DNA中8-OHdG的丰度、GSH/GSSG比值和乌头酸酶失活来确定氧化应激。目的：用形态计量学方法从显微和超微结构上确定HAART的CM。目的3：通过HAART超声心动图和使用朗宁多夫制剂确定心肌梗死患者的心功能。

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.