MOLECULAR ORCHESTRATION OF MITOCHONDRIAL FITNESS VIA REPLACEMENT OR REPAIR

 描述(申请人提供)：线粒体是大多数ATP的基本来源，它为心脏的兴奋-收缩偶联提供燃料。它们也是有毒的活性氧(ROS)的主要来源。为了维持线粒体和新陈代谢的整体健康，细胞依赖于监测、先发制人的隔离和有针对性地移除受损的细胞器，同时保留健康的线粒体。以这种方式选择性的线粒体剔除利用了细胞自噬装置，因此被称为“有丝分裂吞噬”。识别、隔离和去除受损线粒体的完整过程通常被称为“线粒体质量控制”。最好理解的线粒体质量控制的细胞机制依赖于帕金森氏病因子Parkin对线粒体的定位和蛋白质的泛素化。这一提议背后的新概念是，“线粒体质量”不是一种特定的条件。由于代谢需求和底物利用率根据心脏发育和病理生理状态而波动，因此在一种情况下最高质量的线粒体在另一种情况下可能是次优的或有害的。线粒体可塑性的例子包括正常的围产期从糖酵解到脂肪酸代谢的转变，以及患病成人心脏这种代谢转变的病理逆转。传统观点认为，线粒体在这些代谢过程中被重新编程，但我们认为这过于简单化了。我们同时进行的实验数据表明，必须首先以Parkin依赖的方式移除原有的线粒体，然后生物发生和线粒体融合才能完成代谢上不同的继任者的替换。因此，我们假设Parkin有丝分裂途径在心脏中起着两种不同的作用：一种是选择和移除单个受损线粒体的典型功能，另一种是在代谢过渡期间引发广泛的线粒体更新的功能，这是生物性线粒体替换所必需的。我们的研究工作提供了一种全新的方法，通过表达主要激活或抑制的线粒体外膜Parkin受体(PINK1-磷酸化的Mfn2)来调节其线粒体分子界面上的Parkin信号。我们将使用这种方法在活体小鼠心脏中操纵Parkin信号，并在TAC/De-TAC研究中评估在正常围产期向脂肪酸代谢过渡期间、手术诱导心脏压力超负荷(TAC)后以及肥厚逆转重塑期间对线粒体质量和代谢重塑的影响。如果我们的假设是正确的，那么增强Parkin信号将有助于早期剔除受损的 而阻断Parkin介导的有丝分裂则会导致受损细胞器的早期积累，但会延迟非适应性向胎儿样代谢的恢复。因此，我们将确定与疾病进展相关的条件增强或抑制Parkin介导的有丝分裂的最佳时间，以最好地补充不同病理生理状态下的线粒体功能和代谢。总之，我们的研究将严格评估这一概念，即在线粒体受体上操纵Parkin信号(这种相互作用是药理学上有针对性的)既可以促进对受损细胞器的剔除，也可以纠正线粒体代谢偏好和心肌需求之间的不匹配。

 DESCRIPTION (provided by applicant): Mitochondria are the essential sources of most ATP that fuels excitation-contraction coupling in the heart. They are also major sources of toxic reactive oxygen species (ROS). To maintain overall mitochondrial and metabolic health, cells rely upon surveillance, pre-emptive sequestration, and targeted removal of damaged organelles while retaining healthy mitochondria. Selective mitochondrial culling in this manner utilizes the cellular autophagy apparatus, and is therefore designated "mitophagy." The integrated process of identification, sequestration, and mitophagic removal of damaged mitochondria is commonly referred to as "mitochondrial quality control." The best understood cellular mechanism for mitochondrial quality control depends upon mitochondrial localization of, and protein ubiquitination by, the Parkinson's disease factor Parkin. The novel concept underlying this proposal is that "mitochondrial quality" is not a specific condition. Because metabolic demands and substrate availability fluctuate based on cardiac developmental and pathophysiological status, the highest quality mitochondria in one condition may be sub-optimal or detrimental in another. Examples of mitochondrial plasticity include the normal perinatal transition from glycolytic to fatty acid metabolism, and the pathological reversal of this metabolic transition in diseased adult hearts. Conventional wisdom is that mitochondria are genetically "reprogrammed" during these metabolic transformations, but we believe this to be overly simplistic. Our intercurrent experimental data reveal that pre-existing mitochondria must first be removed in a Parkin-dependent manner before biogenesis and mitochondrial fusion can accomplish their replacement by metabolically distinct successors. Thus, we hypothesize that the Parkin mitophagy pathway serves two distinct roles in hearts: the canonical function of selecting and removing individual damaged mitochondria, and a previously undescribed function evoking generalized mitochondrial turnover essential to biogenic mitochondrial replacement during metabolic transitions. Our research efforts have produced a completely novel approach to modulating Parkin signaling specifically at its mitochondrial molecular interface by expressing dominantly active or inhibitory mitochondrial outer membrane Parkin receptors (PINK1-phosphorylated Mfn2). We will use this approach to manipulate Parkin signaling in the in vivo mouse heart and evaluate the consequences on mitochondrial quality and metabolic remodeling during the normal perinatal transition to fatty acid metabolism, after surgical induction of cardia pressure overload (TAC), and during hypertrophy reverse remodeling in TAC/de-TAC studies. If our hypothesis is correct, then enhancing Parkin signaling will facilitate early culling of damaged organelles at the cost of accelerating delayed metabolic remodeling back to the glycolytic fetal phenotype, whereas interrupting Parkin-mediated mitophagy will cause early accumulation of damaged organelles, but delay the maladaptive reversion to fetal-like metabolism. Thus, we will establish the optimal times, relative to disease progression, for conditional enhancement or suppression of Parkin-mediated mitophagy to best complement mitochondrial function and metabolism in different pathophysiological states. Together, our studies will rigorously evaluate the concept that manipulating Parkin signaling at its mitochondrial receptor (an interaction that i pharmacologically targetable) can both promote culling of damaged organelles and correct mismatches between mitochondrial metabolic preference and myocardial demand.