Age Related Change in Mitochondrial Angiotensin System and Mitochondrial Decline

描述（申请人提供）：衰老与线粒体功能下降密切相关。线粒体功能障碍的关键信号包括活性氧生成增加，每消耗O2产生的ATP分子减少，细胞凋亡增加。ATP的减少转化为细胞维持过程的较低能量，包括有丝分裂和更新。我们最近报道了功能性线粒体血管紧张素系统（MAS）的鉴定，并发现衰老影响线粒体中关键血管紧张素1型和2型受体（AT1R和AT2R）的比例，AT1R阻断剂可能部分逆转这些变化并改善线粒体功能。重要的是，AT1R-/-小鼠的寿命延长了25%，部分原因是线粒体数量的增加。衰老对线粒体（mt） AT1R和mtAT2R表达的影响及其在线粒体功能障碍中与年龄相关的变化中的作用尚未得到研究。新的初步证据表明，老年小鼠的AT1R阻断可能通过改变p53诱导蛋白（MIEAP）的表达来恢复与年龄相关的线粒体能量产生的下降，并提高线粒体自噬效率。我们假设年龄相关的mtAT1R/AT2R比率增加通过增加活性氧（ROS）的产生和受损线粒体的消除受损介导线粒体能量代谢的下降。为了验证这一假设，我们提出在对照和氯沙坦（AT1R阻滞剂）治疗的年轻和老年（38月龄）C57BL/6、AT1-/-和AT2-/-小鼠中对mtAT1R和mtAT2R进行全面研究。将以年龄相关心肌生物能量衰竭为模型，研究mtAT1R和mtAT2R变化的影响，提出具体目的：采用Q-PCR、western blot、共聚焦和电镜技术，研究外周血管紧张素系统（PAS）和MAS与年龄相关的变化及其对活性氮/活性氧ROS/RNS生成的影响。我们将使用特定的荧光探针在所有动物组分离的心肌细胞和线粒体中量化ROS/RNS的产生。2. 通过磁共振波谱和成像，在基线和安慰剂或氯沙坦治疗四周后的活体动物心脏组织中，指定PAS和MAS的年龄相关变化对生物能量功能障碍的贡献。MAS对生物能量学变化的贡献将在所有动物群体的分离心脏线粒体中进行研究。3. 为了研究PAS和MAS在线粒体生物发生、修复和消除中的作用，我们利用带有P53活性报告基因的心脏细胞系（H9C2）来测量过表达AT1R和/或AT2R对MIEAP、氧化应激、mtDNA损伤和线粒体自噬的影响。还将比较对照组和LOS治疗小鼠、年轻和老年（38月龄）C57BL/6、AT1-/-和AT2-/-小鼠之间的类似结果。

DESCRIPTION (provided by applicant): Aging and decline in mitochondrial function are closely linked. Key signs of mitochondrial dysfunction include increased generation of reactive oxygen species, fewer ATP molecules produced per O2 consumed, and increased apoptosis. The reduction in ATP translates to lower energy for cellular maintenance processes including mitobiogenesis and turnover. We recently reported the identification of a functional Mitochondrial Angiotensin System (MAS), and found that aging impacts the ratio of key angiotensin type 1 and type 2 receptors in mitochondria (AT1R and AT2R), that AT1R blocking agents may partially reverse these changes and improve mitochondrial function. Importantly, AT1R-/- mice have an enhanced life span by 25%, in part through an increase in mitochondrial numbers. The effects of aging on the expression of mitochondrial (mt) AT1R and mtAT2R and their contribution to age-related changes in mitochondrial dysfunction have not been previously studied. New preliminary evidence suggests that AT1R blockade in old mice may restore the age- related decline in mitochondrial energy production and improve mitophagy efficiency via alterations in p53- inducible protein (MIEAP) expression. We hypothesize that an age-related increase in mtAT1R/AT2R ratio mediates declines in mitochondrial energy metabolism via increased reactive oxygen species (ROS) production and impaired elimination of damaged mitochondria. In order to test this hypothesis we propose a comprehensive study of mtAT1R and mtAT2R, in control and losartan (AT1R blocker) treated, young and aged (38-month old) C57BL/6, AT1-/- and AT2-/- mice. Will use age related cardiac muscle bioenergetics failure as a model to study the impact of changes in mtAT1R and mtAT2R following proposed specific aims: 1. To identify age-related changes in peripheral angiotensin system (PAS) and MAS and their contribution to altered reactive nitrogen/oxygen species ROS/RNS generation, using Q-PCR, western blot, confocal and electron microscopy. Generation of ROS/RNS will be quantified in isolated cardiomyocytes and mitochondria from all our animal groups using specific fluorescent probes. 2. To specify the contribution of age-related changes in PAS and MAS to bioenergetic dysfunction by magnetic resonance spectroscopy and imaging in the cardiac tissue of living animals at baseline and after four weeks of placebo or losartan treatment. MAS contributions to the changes in bioenergetics will studied in isolated cardiac mitochondria from all animal groups. 3. To investigate the role of the PAS and MAS on mitochondrial biogenesis, repair, and elimination by utilizing a cardiac cell line (H9C2) instrumented with a P53 activity reporter to measure the effects of over-expressing AT1R and/or AT2R on MIEAP, oxidative stress, mtDNA damage and mitophagy. Similar outcomes will also be compared between control and LOS treated, young and aged (38-month old) C57BL/6, AT1-/- and AT2-/- mice.