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

基本信息

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
项目类别：
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

项目摘要

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.

心血管疾病是美国的首要死因，其发病率急剧上升随着年龄的增长而增加。因此，预防或延缓心脏衰老可以对寿命和寿命产生重大影响。健康寿命。该提案的一个中心目标是探索一种新的机制，通过该机制，线粒体会随年龄增长并导致心脏功能丧失。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，而逆转这种修饰的酶 Sirtuin-3 (SIRT3) 是一种已知的酶长寿系数随着年龄的增长而降低。然而，如果线粒体乙酰化加速心脏衰老仍然存在尚未解决且有争议。我们的总体假设是乙酰化的增加有两个影响：年迈的心。首先，它通过直接影响关键调节酶的活性而导致代谢不灵活。其次，乙酰化的增加影响蛋白质稳态（蛋白质合成和降解稳态）。功能失调的蛋白质稳态反过来会导致合成不当和代谢不灵活。修饰的线粒体代谢酶的降解。对于这个提案，我们生成了一个心肌细胞 (CM) 特异性 SIRT3 敲除小鼠 (SIRT3CM-/-)。我们的初步数据表明 SIRT3CM-/- 小鼠表现出显着的肥大、收缩功能丧失、纤维化、代谢异常和蛋白质稳态功能失调 10- 几个月。这种衰老和过度乙酰化的加速模型将用于通过以下方法来检验我们的假设：以下目标。目标 1 将确定 SIRT3 缺失引起的乙酰化增加是否会影响代谢随着年龄的增长而变得灵活。我们假设 SIRT3 的缺失通过影响活性而导致代谢不灵活离散的线粒体调节酶导致对葡萄糖的依赖增加。这一目标将通过测量心脏和线粒体功能来确定 SIRT3 的缺失是否会影响代谢灵活性， SIRT3CM-/- 小鼠和分离的成年心肌细胞的酶活性和纵向代谢灵活性。整体乙酰化和特定代谢酶的乙酰化将通过质谱法测量。这些研究的结果将确定高乙酰化是否对心脏有直接影响。缺乏 SIRT3 时的病理学。目标 2 将确定 SIRT3 的缺失如何影响线粒体蛋白质稳态。蛋白质的乙酰化可以影响结构和功能，但对其在蛋白质中的作用知之甚少。线粒体蛋白质质量和周转率的全球变化。该目标将使用氧化氘 (D2O) 标记和蛋白质组学，以确定 SIRT3 的缺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 AAV 递送 SIRT3 进行。将进行细胞培养的机制研究以证明乙酰化如何影响蛋白质稳态。这些将是第一个采用体内标记来确定乙酰化如何影响蛋白质合成和周转。研究结果将为未来的项目提供动力，进一步定义乙酰化和其他碳压力会影响心脏老化。