Investigating the effects of calorie restriction on mitochondrial biology in adult and aged skeletal muscles

随着年龄的增长，骨骼肌逐渐失去质量和功能，这一过程被称为肌肉减少症。先前的研究，包括我们的研究，已经表明肌肉减少症涉及线粒体功能障碍，细胞内细胞器在肌肉生物学中起关键作用。这些与衰老相关的功能障碍包括(i)线粒体体内生物能量学受损，在细胞水平上由线粒体感知和响应能量需求的能力下降或“ADP亲和力”引起；（ii）由增敏的线粒体通透性过渡孔（mPTP）引起的线粒体介导的细胞凋亡增加。该建议建立在这些最新发现的基础上，并使用最有效的干预措施之一-卡路里限制(CR) -来研究线粒体在肌肉减少症中的作用。CR，包括限制每天的食物摄入量，被认为是通过影响线粒体功能来调节其保护性的“抗衰老”效果。然而，CR对线粒体生物学的许多具体影响，特别是在衰老肌肉中，仍然不清楚或未知。特别是，线粒体生物学的三个基本方面对我们对肌肉减少症的理解具有重要意义，需要进一步的研究。首先，CR是否影响线粒体对成人骨骼肌ADP和mPTP功能的亲和力，从而防止这些参数的衰老相关损伤尚不清楚。其次，线粒体形成一个动态网络，能够经历融合和裂变事件，其中线粒体形态的变化影响线粒体功能，反之亦然。迄今为止，衰老对线粒体形态和动力学的影响尚不清楚，CR的影响尚未探索。第三，新出现的证据表明，骨骼肌衰老可能与通过正常的线粒体自噬过程去除受损线粒体的能力受损有关，从而导致功能失调线粒体的积累。CR对老年骨骼肌有丝分裂过程的影响目前从未被研究过。**目前的研究计划旨在通过(i)确定CR对成人和老年骨骼肌线粒体功能关键指标的影响，（ii）确定衰老和CR对线粒体功能的影响是否与线粒体形态/动力学的变化有关，以及（iii）确定CR对肌肉和线粒体功能的保护作用是否涉及线粒体自噬过程的优化。将使用的方法包括非破坏性方法，其中肌肉细胞渗透，使我们能够在细胞内环境中获得形态完整的线粒体以进行功能测量；一种定量电子显微镜方法，能够在三维中精确量化线粒体网络；详细的肌纤维类型分析，以加强我们的解释和背景我们的结果；动态通量评估量化有丝分裂。**拟议的研究计划将产生关于衰老如何影响线粒体生物学未被研究的方面的新的基础知识。此外，它将揭示CR影响骨骼肌线粒体生物学的机制，从而在肌肉减少症的背景下发挥其保护作用。因此，我的研究计划将加强我们目前对肌肉减少症机制的理解。它还将提供关于骨骼肌线粒体生物学调控的新知识-连接功能，形态和质量控制过程。最终，新知识可以在预防骨骼肌减少症和其他影响骨骼肌的不良事件中找到应用。

With aging, skeletal muscle progressively loose mass and function, a process termed sarcopenia. Previous investigations, including ours, have shown that sarcopenia involves dysfunction of mitochondria, intracellular organelles playing key roles in muscle biology. These aging-related dysfunctions include (i) an impaired mitochondrial bioenergetics in vivo, caused at the cellular level by a decreased mitochondrial capacity to sense and respond to energy demand, or "affinity for ADP"; and (ii) an increase in mitochondrial-mediated cell death via apoptosis, caused by a sensitized mitochondrial permeability transition pore (mPTP). This proposal builds upon these recent discoveries, and uses one of the most efficient interventions to attenuate sarcopenia - calorie restriction (CR) - to investigate the role of mitochondria in sarcopenia. CR, which consists in limiting food intake per day, is believed to mediate its protective "anti-aging" effects by impacting mitochondrial function. However, many of the specific effects of CR on mitochondrial biology, especially in aged muscle, remain unclear or unknown. **In particular, three fundamental aspects of mitochondrial biology that have important implications for our understanding of sarcopenia require further work. First, whether CR affects mitochondrial affinity for ADP and mPTP function in adult skeletal muscles, and thus prevents the aging-related impairment in these parameters is unknown. Second, it is now well established that mitochondria form a dynamic network able to undergo fusion and fission events, where changes in mitochondrial morphology impact mitochondrial function, and vice versa. To date, the effects of aging on mitochondrial morphology and dynamics remain unclear, and those of CR are unexplored. Third, emerging evidence indicates that skeletal muscle aging might be associated with an impaired capacity to remove damaged mitochondria through the normal process of mitophagy, causing the accumulation of dysfunctional mitochondria. The effects of CR on mitophagic processes in aged skeletal muscles have currently never been investigated. **The present research program was designed to address these issues by (i) defining the effects of CR on crucial indices of mitochondrial function in adult and aged skeletal muscles, (ii) determining if the impact of aging and CR on mitochondrial function is linked to changes mitochondrial morphology/dynamics and (iii) defining if the protective effects of CR on muscle and mitochondrial function involve optimization of mitophagic processes. Methods that will be used include a non-disruptive approach where muscle cells are permeabilized, giving us access to morphologically intact mitochondria in their intracellular environment for functional measures; a quantitative electron microscopy approach enabling precise quantification of the mitochondrial network in 3-dimension; detailed muscle fiber-type analysis to reinforce our interpretation and contextualize our results; and dynamic flux assessments for quantifying mitophagy.**The proposed research program will generate new fundamental knowledge on how aging affects understudied aspects of mitochondrial biology. In addition, it will shed new light on the mechanisms by which CR impacts mitochondrial biology in skeletal muscle, and thereby exerts its protective effects in the context of sarcopenia. My research program will therefore enhance our current understanding of the mechanisms underlying sarcopenia. It will also provide novel knowledge on the regulation of mitochondrial biology - linking function, morphology and quality control processes - in skeletal muscle. Ultimately, the novel knowledge could find applications in the prevention of sarcopenia and other adverse events affecting skeletal muscle.