Investigating the role of SIRT3 in metabolic flexibility and proteostasis in the aging heart

研究 SIRT3 在衰老心脏代谢灵活性和蛋白质稳态中的作用

• 批准号: 10625412
• 负责人: Kenneth M Humphries
• 金额: $ 21.85万
• 依托单位: OKLAHOMA MEDICAL RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10625412
• 关键词: Acceleration; Acetylation; Adult; Affect; Age; Aging; Carbon; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cause of Death; Cell Culture Techniques; Data; Deacetylase; Deuterium Oxide; Enzymes; Event; Exhibits; Fibrosis; Future; Global Change; Glucose; Goals; Half-Life; Heart; Heart Hypertrophy; Homeostasis; Hypertrophy; Individual; Intervention; Knockout Mice; Label; Longevity; Mass Spectrum Analysis; Measures; Metabolic; Metabolism; Mitochondria; Mitochondrial Proteins; Modeling; Modification; Mus; Pathology; Phenotype; Post-Translational Protein Processing; Predisposition; Process; Protein Acetylation; Protein Biosynthesis; Proteins; Proteomics; Role; Sirtuins; Stress; Structure; Testing; Tissues; United States; age related; aged; coronary fibrosis; experimental study; flexibility; healthspan; heart function; heart metabolism; improved; in vivo; mitochondrial dysfunction; model organism; novel; premature; prevent; protein degradation; proteostasis; restoration

Cardiovascular disease is the leading cause of death in the United States and its occurrence dramatically increases with age. Preventing or delaying cardiac aging can therefore have a significant effect on longevity and healthspan. A central goal of this proposal is to explore a novel mechanism by which mitochondria decline with age and contribute to loss of cardiac function. We are focusing on the post-translational modification, acetylation, because it increases with age and the enzyme that reverses this modification, sirtuin-3 (SIRT3), is a known longevity factor that decreases with age. However, if mitochondrial acetylation accelerates cardiac aging remains unresolved and controversial. Our overarching hypothesis is that an increase in acetylation has two effects in the aged heart. First, it causes metabolic inflexibility by directly affecting the activity of key regulatory enzymes. Second, the increase in acetylation affects proteostasis (protein synthesis and degradation homeostasis). Dysfunctional proteostasis in turn contributes to metabolic inflexibility by causing improper synthesis and degradation of modified mitochondrial metabolic enzymes. For this proposal, we generated a cardiomyocyte (CM) specific SIRT3 knockout mice (SIRT3CM-/-). Our preliminary data show that SIRT3CM-/- mice exhibit dramatic hypertrophy, loss of contractile function, fibrosis, metabolic abnormalities, and dysfunctional proteostasis by 10- months. This accelerated model of aging and hyperacetylation will be used to test our hypothesis through the following aims. Aim 1 will determine if the increase in acetylation caused by the loss of SIRT3 affects metabolic flexibility with age. We hypothesize that the loss of SIRT3 causes metabolic inflexibility by affecting the activity of discrete mitochondrial regulatory enzymes that result in the increased reliance on glucose. This aim will determine if the loss of SIRT3 affects metabolic flexibility by measuring cardiac and mitochondrial functions, enzymatic activities, and metabolic flexibility longitudinally in SIRT3CM-/- mice and isolated adult cardiomyocytes. Global acetylation and the acetylation of specific metabolic enzymes will be measured by mass spectrometry. The results of these studies will determine if there is a direct consequence of hyperacetylation on cardiac pathology in the absence of SIRT3. Aim 2 will determine how the loss of SIRT3 affects mitochondrial proteostasis. Acetylation of proteins can affect structure and function, yet little is known regarding its role in global changes in mitochondrial protein quality and turnover. This aim will use deuterium oxide (D2O) labeling and proteomics to determine if the loss of SIRT3 affects mitochondrial protein synthesis and the relative turnover rates of specific metabolic regulatory enzymes. Rescue experiments will be performed by AAV delivery of SIRT3. Mechanistic studies in cell culture will be performed to demonstrate how acetylation affects proteostasis. These will be the first studies to employ in vivo labeling to determine how acetylation affects protein synthesis and turnover. The results will provide an impetus for future projects further defining how acetylation and other carbon stresses affect cardiac aging.

心血管疾病是美国死亡的主要原因，其发生急剧发生 随着年龄的增长而增加。因此，防止或延迟心脏衰老可能会对寿命产生重大影响和 HealthSpan。该提议的一个核心目标是探索一种新型机制，线粒体通过该机制而下降。 年龄并导致心脏功能的丧失。我们专注于翻译后修饰，乙酰化， 因为它随着年龄的增长而增加，并且逆转这种修饰的酶是Sirtuin-3（SIRT3），是已知的 寿命因素随着年龄的增长而降低。但是，如果线粒体乙酰化会加速心脏老化 未解决和有争议。我们的总体假设是，乙酰化的增加在 老化的心。首先，它通过直接影响关键调节酶的活性而导致代谢僵化。 其次，乙酰化的增加会影响蛋白质抑制（蛋白质合成和降解稳态）。 功能失调的蛋白质症反过 修饰的线粒体代谢酶的降解。对于此建议，我们产生了一个心肌细胞 （CM）特定的SIRT3敲除小鼠（SIRT3CM - / - ）。我们的初步数据显示SIRT3CM - / - 鼠标表现出戏剧性 10- 月份。这种加速的衰老和高乙酰化模型将用于通过 以下目标。 AIM 1将确定由SIRT3损失引起的乙酰化增加是否影响代谢 随着年龄的增长。我们假设SIRT3的损失通过影响活动而导致代谢僵化 导致对葡萄糖的依赖增加的离散线粒体调节酶。这个目标 确定SIRT3的丢失是否通过测量心脏和线粒体功能来影响代谢柔韧性， 酶促活性和代谢柔韧性在SIRT3CM - / - 小鼠和孤立的成年心肌细胞中纵向纵向。 特定代谢酶的全球乙酰化和乙酰化将通过质谱法测量。 这些研究的结果将确定心脏过度乙酰化是否直接结果 在没有SIRT3的情况下病理。 AIM 2将决定SIRT3的损失如何影响线粒体 蛋白毒酸。蛋白质的乙酰化会影响结构和功能，但关于其在 线粒体蛋白质质量和周转率的全球变化。此目标将使用氧化氘（D2O）标签 和蛋白质组学确定SIRT3的丢失是否影响线粒体蛋白质合成和相对周转 特定代谢调节酶的速率。救援实验将通过SIRT3的AAV交付进行。 将进行细胞培养的机械研究，以证明乙酰化如何影响蛋白质。这些 将是首次采用体内标记的研究来确定乙酰化如何影响蛋白质的合成和 周转。结果将为未来的项目提供进一步定义乙酰化和其他碳的动力 压力会影响心脏衰老。