Mechanisms of Heart Failure in HIV/AIDS: Nucleotides and NRTIs

描述（由申请人提供）：该项目定义了核苷逆转录酶抑制剂（NRTIs）对HIV/AIDS的代谢如何导致线粒体（mt-） DNA复制缺陷、氧化应激和心肌病（CM，心脏肌肉无力，泵功能差）。除了增加艾滋病毒/艾滋病患者的生存率外，包括nrti在内的组合还会带来毁灭性的线粒体副作用，包括CM。线粒体（mt-） DNA缺失和突变以及氧化应激是CM和CHF的关键机制，也是NRTI基于mtDNA复制缺陷的线粒体毒性副作用的发展。工作假设是：在心肌细胞中，nrti与控制核苷酸生物合成和挽救mtDNA复制的蛋白质相互作用。核糖核苷酸还原酶（RNR）、SAM结构域和含HD结构域的蛋白1 （SAMHD1）改变dNTPs的丰度，从而促进mtDNA的复制。RNR抑制或SAMHD1活性增加会耗尽原生dNTP池，增加nrti - tp的丰度（或两者兼而有之）。mtDNA缺失和突变、氧化应激和CM是表型特征。本项目将通过基因工程小鼠的临床相关实验，剖析HIV/AIDS患者CM肌细胞的肌浆和线粒体中的核苷酸池失衡。氧化应激和CM是通过保护心肌线粒体与锰卟啉催化抗氧化剂，线粒体定位改善。目的1：确定RNR活性和抑制在HIV/AIDS CM中调节心肌细胞质和线粒体核苷酸池中的作用。目前在实验室中已经有过表达RNR的转基因小鼠。在心脏细胞中，RNR过表达（通过转基因）或其抑制（如外源性白藜芦醇）改变核苷酸池，使其磷酸化并结合到mtDNA中。核苷酸池是通过dNTP检测确定的，并与心脏生理、病理和药理学研究相关。目的2：确定SAMHD1活性（和缺失）在调节HIV/AIDS CM中心肌细胞质和线粒体核苷酸池中的作用。SAMHD1缺失小鼠是增加心脏细胞dNTPs池的强大生物工具。相反，心脏靶向转基因SAMHD1过表达增加dN库。NRTI处理产生抑制pol ？创建CM。通过dNTP检测和相关的生理、病理和药理学参数来确定心脏核苷酸池。目的3：通过改善线粒体氧化应激的“替代”来预防和治疗HIV/AIDS中的CM。“功能替代”是通过AIM 1和AIM 2中的TGs来完成的，它们是锰卟啉催化抗氧化剂（提供SOD2和过氧化氢酶样活性，增强抗氧化防御），可以预防或减弱RNR-或samhd1诱导的心脏变化，并减弱CM病理生理变化。

DESCRIPTION (provided by applicant): This project defines how metabolism of nucleoside reverse transcriptase inhibitors (NRTIs) for HIV/AIDS causes defective mitochondrial (mt-) DNA replication, oxidative stress and cardiomyopathy (CM, a weakness of the heart muscle with poor pump function). Along with the benefit of increased survival in HIV/AIDS, combinations that include NRTIs carry devastating mitochondrial side effects, including CM. Mitochondrial (mt-) DNA depletion and mutation, and oxidative stress are key mechanisms in CM and CHF, and for development of NRTI toxic mitochondrial side effects based of defective mtDNA replication. The working hypothesis states: In myocardial cells, NRTIs interact with proteins that control the nucleotide biosynthesis and salvage for mtDNA replication. Ribonucleotide reductase (RNR) and SAM domain and HD domain-containing protein 1 (SAMHD1) alter abundance of dNTPs for mtDNA replication. RNR inhibition or increased activity of SAMHD1 depletes native dNTP pools, increases abundance of NRTI-TPs (or both). mtDNA depletion and mutation, oxidative stress, and CM are the phenotypic signature. Through clinically relevant experiments using genetically engineered mice, the project will dissect nucleotide pool imbalance in the sarcoplasm and mitochondria of CM myocytes in HIV/AIDS. Oxidative stress and CM are ameliorated by protecting cardiac mitochondria with manganese porphyrin catalytic antioxidants that localize to mitochondria. AIM 1: To define the role of RNR activity and inhibition in modulating cardiac cytoplasmic and mitochondrial nucleotide pools in the CM of HIV/AIDS. Transgenic mice (TGs) that overexpress RNR are in the lab presently. In heart cells, RNR overexpression (by transgenesis) or its inhibition (e.g., exogenous resveratrol) alters nucleotide pools for phosphorylation and incorporation into mtDNA. Nucleotide pools are defined with dNTP assays and correlated with cardiac physiological, pathological and pharmacological studies. AIM 2: To define the role of SAMHD1 activity (and absence) in modulating the myocardial cytoplasmic and mitochondrial nucleotide pools in the CM of HIV/AIDS. SAMHD1 null mice are powerful biological tools to increase the pool of dNTPs in heart cells. Conversely, cardiac targeted transgenic overexpression of SAMHD1 increases dN pools. NRTI treatment generates NRTI-TPs that inhibit pol ? and create CM. Cardiac nucleotide pools are determined with dNTP assays and correlative physiological, pathological and pharmacological parameters. AIM 3: To prevent and treat CM in HIV/AIDS with "replacement" by ameliorating mitochondrial oxidative stress. "Replacement of function" is accomplished using TGs from AIM 1 and 2 that are co- administered manganese porphyrin catalytic antioxidants (to provide SOD2- and catalase-like activity that bolsters antioxidant defense) and prevent or attenuate RNR- or SAMHD1-induced cardiac changes and attenuate CM pathophysiological changes.