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，这一过程称为骨质疏松症。以前的研究，包括我们的研究，已经表明石棺减少与线粒体功能障碍有关，线粒体是细胞内细胞器在肌肉生物学中发挥关键作用。这些与衰老相关的功能障碍包括：(1)体内线粒体生物能量学受损，原因是线粒体对能量需求的感知和反应能力降低，即对ADP的亲和力降低；(2)由于敏化的线粒体通透性转换孔(MPTP)，线粒体通过细胞凋亡介导的细胞死亡增加。这项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)-来研究线粒体在石棺减少症中的作用。CR，即限制每天的食物摄入量，被认为通过影响线粒体功能来调节其保护性的“抗衰老”效应。然而，CR对线粒体生物学的许多具体影响，特别是在衰老的肌肉中，仍然不清楚或不清楚。特别是，线粒体生物学的三个基本方面对我们理解石棺减少症具有重要意义，需要进一步研究。首先，CR是否影响成年骨骼肌线粒体对ADP和MPTP功能的亲和力，从而防止这些参数中与衰老相关的损害尚不清楚。其次，现在已经确定，线粒体形成了一个能够经历融合和裂变事件的动态网络，其中线粒体形态的变化影响线粒体的功能，反之亦然。到目前为止，衰老对线粒体形态和动力学的影响尚不清楚，CR的影响也不是很清楚。第三，新出现的证据表明，骨骼肌衰老可能与通过正常吞噬线粒体过程移除受损线粒体的能力受损有关，导致功能障碍的线粒体积累。CR对老年骨骼肌有丝分裂过程的影响目前从未被研究过。本研究计划旨在通过以下方式解决这些问题：(I)确定CR对成年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吞噬过程的优化。将使用的方法包括：肌肉细胞通透性的非破坏性方法，使我们能够接触到细胞内环境中形态完整的线粒体进行功能测量；定量电子显微镜方法，能够在3维空间精确量化线粒体网络；详细的肌肉纤维类型分析，以加强我们的解释并将我们的结果与背景联系起来；以及动态通量评估，以量化有丝分裂。拟议的研究计划将产生关于衰老如何影响线粒体生物学中未被研究的方面的新的基础知识。此外，它将阐明CR影响骨骼肌线粒体生物学的机制，从而在骨骼肌减少的情况下发挥其保护作用。因此，我的研究计划将加强我们目前对石棺减少的机制的理解。它还将提供有关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，这项新知识可以应用于预防骨骼肌减少症和其他影响骨骼肌的不良事件。

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.