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

在美国，心血管疾病是导致死亡的主要原因， 随着年龄增长。因此，预防或延缓心脏衰老可以对长寿和 healthspan.这项提议的一个中心目标是探索一种新的机制， 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（SIRT 3），是一种已知的 长寿因素随着年龄的增长而减少。然而，如果线粒体乙酰化加速心脏衰老， 未解决的和有争议的。我们的总体假设是，乙酰化的增加对 老的心。首先，它通过直接影响关键调节酶的活性而引起代谢紊乱。 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又通过引起不适当的合成和代谢而导致代谢不稳定。 修饰的线粒体代谢酶的降解。为了这个提议，我们产生了一个心肌细胞， (CM)特异性SIRT 3敲除小鼠（SIRT 3CM-/-）。我们的初步数据显示SIRT 3CM-/-小鼠表现出显著的 肥大、收缩功能丧失、纤维化、代谢异常和功能失调的蛋白质稳态， 个月这个加速老化和超乙酰化的模型将被用来测试我们的假设， 的目标。目的1将确定SIRT 3缺失引起的乙酰化增加是否影响代谢 年龄的灵活性。我们假设SIRT 3的缺失通过影响代谢酶的活性而导致代谢的不稳定性。 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SIRT 3的缺失是否影响代谢灵活性， 酶活性和代谢灵活性纵向SIRT 3CM-/-小鼠和分离的成年心肌细胞。 将通过质谱法测量总体乙酰化和特定代谢酶的乙酰化。 这些研究的结果将确定是否有一个直接后果，高乙酰化对心脏 在没有SIRT 3的情况下的病理学。目的2将确定SIRT 3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，但关于其在蛋白质合成中的作用知之甚少。 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氘（D2 O）标记 和蛋白质组学，以确定SIRT 3的缺失是否影响线粒体蛋白质合成和相对营业额， 特定代谢调节酶的速率。拯救实验将通过SIRT 3的AAV递送进行。 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质稳态。这些 这将是第一项采用体内标记来确定乙酰化如何影响蛋白质合成的研究， 周转这些结果将为未来的项目提供动力，进一步确定乙酰化和其他碳 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.