Enhancing Beneficial Autophagy to Prevent Heart Failure

自噬是一种进化上保守的细胞内溶酶体降解途径， 清除受损的细胞器和蛋白质以确保心肌细胞存活。快速 自噬的上调对于在应激下维持心肌细胞活力是必需的，例如在应激期间， 饥饿和缺血，通过产生营养物质作为能量来源， 蛋白质和细胞器。自噬对于去除异常结蛋白聚集体也是至关重要的， 伴伴侣蛋白B晶体蛋白R120 G突变的心肌病。因此，自相矛盾的是， 自噬的上调与再灌注损伤中引起心肌细胞死亡有关， 造成了缺血性损伤再灌注后，我们观察到自噬体受损， 处理，由活性氧产生的爆发触发，作为自噬体的原因 积累;这意味着通过自噬的通量随着缺血-再灌注损伤而受损。的确， 我们已经证明，心肌细胞中自噬体的形成是由BNIP 3的表达诱导的， 一种由缺氧转录诱导并介导缺血心肌细胞死亡的促死亡蛋白， 再灌注损伤;但自噬体加工受损，因为自噬-溶酶体机制变得 不堪重负，包裹受损线粒体的自噬体积累。的外源表达 转录因子EB（TFEB），自噬-溶酶体途径生物发生的主要调节因子，重新建立 自噬体加工，去除BNIP 3损伤的线粒体和减弱的心肌细胞 死亡我们还观察到，饥饿是心肌细胞自噬的有效诱导剂， 小鼠心脏中自噬-溶酶体机制的多个组分的转录上调; 和通过间歇性禁食的重复饥饿（每隔一天剥夺小鼠食物24小时，持续6 周）导致针对缺血再灌注（IR）损伤的保护， 与ad-lib喂养的小鼠相比。这与快速禁食诱导的TFEB核转位有关 从细胞质中，这表明TFEB对自噬-溶酶体机制的充分启动 促进足够的自噬，这在缺血-再灌注损伤的情况下是有益的。在这 建议，我们将测试的假设，间歇性禁食和外源性表达TFEB保护 抗缺血-再灌注诱导的心肌细胞死亡和蛋白聚集诱导的心肌病 和心力衰竭的治疗。在具体目标1中，将进行研究，以确定 自噬在间歇性禁食诱导的心肌保护中的作用， 使LAMP 2缺陷的小鼠（其也是自噬缺陷的，因为LAMP 2对于 自噬体-溶酶体融合）对IR损伤的影响，随后进行系列超声心动图和终末侵入性 压力-容积环研究，以评估左心室大小和功能的变化以及心脏发育 失败，作为LAMP 2表达的函数。在具体目标2中，具有条件性成年发作心肌细胞的小鼠 TFEB的特异性过表达将受到IR损伤，以确定外源性TFEB是否赋予IR损伤。 通过增强自噬，防止细胞死亡、梗死后重塑和心力衰竭。在特定 目的3，将具有心肌细胞特异性表达突变晶体蛋白的小鼠进行间歇性禁食 或与TFEB过表达者杂交，以确定自噬的诱导是否阻止蛋白- 聚集诱发心力衰竭。这些研究将评估转录诱导的范式， 自噬-溶酶体机制增强应激诱导的自噬， 缺血-再灌注损伤和结蛋白诱导的心肌病中的死亡和心力衰竭。

Autophagy, an evolutionarily conserved intracellular lysosomal degradative pathway, is constitutively active in the myocardium, and removes damaged organelles and proteins to ensure cardiomyocyte survival. Rapid upregulation of autophagy is essential for maintaining cardiomyocyte viability under stress, such as during starvation and ischemia, by generating nutrients as a source of energy and removing potentially deleterious proteins and organelles. Autophagy is also critical for removal of abnormal desmin aggregates that cause cardiomyopathy with the R120G mutation in chaperone protein, ¿-B crystallin. It is therefore paradoxical that an upregulation of autophagy has been implicated in causing cardiomyocyte death in reperfusion injury, following an ischemic insult. With reperfusion, we have observed an impairment in autophagosome processing, triggered by a burst in reactive oxygen species generation, as a cause of autophagosome accumulation; which implies that flux through autophagy is impaired with ischemia-reperfusion injury. Indeed, we have demonstrated that autophagosome formation is induced in cardiomyocytes by expression of BNIP3, a pro-death protein that is transcriptionally induced by hypoxia and mediates cardiomyocyte death in ischemia- reperfusion injury; but autophagosome processing is impaired as the autophagy-lysosome machinery gets overwhelmed and autophagosomes enclosing damaged mitochondria accumulate. Exogenous expression of transcription factor EB (TFEB), a master regulator of autophagy-lysosome pathway biogenesis, re-established autophagosome processing, with removal of BNIP3-damaged mitochondria and attenuated cardiomyocyte death. We have also observed that starvation, a potent inducer of cardiomyocyte autophagy, provokes transcriptional upregulation of multiple components of the autophagy-lysosome machinery in the mouse heart; and repetitive starvation by intermittently fasting (depriving mice of food for 24 hours every other day for 6 weeks) results in protection against ischemia-reperfusion (IR) injury, with >50% reduction in infarct size as compared with ad-lib fed mice. This is associated with a rapid fasting-induced nuclear translocation of TFEB from the cytoplasm, suggesting that adequate priming of the autophagy-lysosome machinery by TFEB promotes sufficient autophagy, which is then beneficial in the setting of ischemia-reperfusion injury. In this proposal, we will test the hypothesis that intermittent fasting and exogenous expression of TFEB protect against ischemia-reperfusion induced cardiomyocyte death and protein-aggregate-induced cardiomyopathy and heart failure, by enhancing beneficial autophagy. In specific aim 1, studies will be performed to determine the role of autophagy in intermittent fasting-induced cardioprotection against post-infarction remodeling, by subjecting mice deficient for LAMP2, (which are also autophagy-deficient as LAMP2 is critical for autophagosome-lysosome fusion) to IR injury followed by serial echocardiography and terminal invasive pressure-volume loop studies, to assess changes in left ventricular size and function and development of heart failure, as a function of LAMP2 expression. In specific aim 2, mice with conditional adult onset cardiomyocyte specific overexpression of TFEB will be subjected to IR injury to determine whether exogenous TFEB confers protection against cell death, post-infarction remodeling and heart failure, by enhancing autophagy. In specific aim 3, mice with cardiomyocyte-specific expression of mutant crystallin will be subjected to intermittent fasting or crossed with TFEB overexpressors, to determine whether induction of autophagy prevents protein- aggregate induced heart failure. These studies will evaluate the paradigm that transcriptional induction of autophagy-lysosome machinery enhances stress-induced autophagy, and protects against cardiomyocyte death and heart failure in ischemia-reperfusion injury and desmin-induced cardiomyopathy.