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

心血管疾病是美国主要的死亡原因，其发病率极高 随年龄增长。因此，预防或延缓心脏衰老可以显著延长寿命和 健康跨度。这项提议的一个中心目标是探索一种新的机制，线粒体通过这种机制 年龄增长，并导致心脏功能丧失。我们的重点是翻译后修饰，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(sirtuin-3)，是一种已知的 随年龄增长而减少的长寿系数。然而，如果线粒体乙酰化加速心脏衰老， 悬而未决且备受争议。我们最重要的假设是，乙酰化程度的增加在 那颗衰老的心。首先，它通过直接影响关键调节酶的活性而导致代谢缺乏灵活性。 其次，乙酰化的增加影响蛋白质平衡(蛋白质合成和降解平衡)。 功能失调的蛋白平衡进而导致代谢不灵活，导致不适当的合成和 修饰的线粒体代谢酶的降解。对于这个提议，我们制造了一个心肌细胞 (Cm)特异性SIRT3基因敲除小鼠(SIRT3CM-/-)。我们的初步数据显示，SIRT3CM-/-小鼠表现出戏剧性 肥大、收缩功能丧失、纤维化、代谢异常和蛋白平衡失调 月份。这一加速的衰老和高乙酰化模型将被用来通过 遵循目标。目标1将确定SIRT3缺失引起的乙酰化增加是否影响新陈代谢 随年龄增长的灵活性。我们假设SIRT3的缺失通过影响活动而导致代谢不灵活。 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的缺失是否影响代谢的灵活性， SIRT3CM-/-小鼠和分离的成人心肌细胞的纵向酶活性和代谢灵活性。 整体乙酰化和特定代谢酶的乙酰化将通过质谱学进行测量。 这些研究的结果将确定高乙酰化是否对心脏有直接影响 在缺乏SIRT3的情况下的病理学。目标2将确定SIRT3的缺失如何影响线粒体 蛋白质平衡。蛋白质的乙酰化可以影响结构和功能，但对其在 线粒体蛋白质质量和周转的全球变化。这一目标将使用氧化氢(D2O)标记 和蛋白质组学来确定SIRT3的缺失是否影响线粒体蛋白质的合成和相对周转 特定代谢调节酶的速率。救援实验将通过AAV交付SIRT3进行。 将在细胞培养中进行机制研究，以证明乙酰化如何影响蛋白平衡。这些 将是第一个使用体内标记来确定乙酰化如何影响蛋白质合成和 营业额。这一结果将为未来的项目进一步定义乙酰化和其他碳 压力会影响心脏老化。

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.