Reduced oxidative capacity of skeletal muscle mitochondria IS a fundamental consequence of adult ageing

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Mitochondria were mechanistically linked to ageing over 80 years ago by Denham Harman (Harman, 1972) and studies have been examining the relationship between in vivo oxidative capacity and ageing skeletal muscle for over three decades. Yet, whether mitochondrial energetics and, specifically, in vivo oxidative capacity decline with ageing and contribute to the decline of muscle health and physical function remains controversial. Mitochondria are often defined as the powerhouse of the cell due to their central role in producing ATP to meet cellular demand. This production of ATP by oxidative phosphorylation is particularly important in high-energy demand tissues such as skeletal muscle, heart and the brain. Not surprisingly, mitochondrial energy production has been a major focus of ageing research particularly in relation to ageing skeletal muscle and mobility. However, it is now clear that mitochondria play a central role in cellular physiology and sit at the nexus integrating environmental and cell stressors with cell signalling and energetics that ultimately lead to adaptive or pathological responses (Monzel et al., 2023). ATP production and redox homeostasis have received the most attention of the many mechanisms that mitochondria use to communicate with the cytoplasm, nucleus and the other cells to affect cellular health and contribute to cellular resilience and prevention of damage accumulation with ageing. Mitochondria also process several metabolites that play an important role in regulating diverse cellular processes. For example, the tricarboxylic acid (TCA) cycle intermediates NAD+, acetyl CoA and alpha-ketoglutarate play key roles regulating gene expression and cell signalling through stress response and epigenetic mechanisms (Martinez-Reyes & Chandel, 2020; Martinez-Reyes et al., 2016). Under healthy conditions, mitochondrial health and energetic efficiency is maintained by multiple quality control mechanisms such as the mitochondrial unfolded protein response (mUPR) and mitophagy, mitochondrial targeted autophagy (Sligar et al., 2022; Wang et al., 2023). The breakdown of efficient mitochondrial quality control with age can lead to a reduced ability to meet energy demands, increased redox stress, inflammation and impaired cell signalling. Furthermore, improperly degraded mitochondria in aged tissues can release mitochondrial components into the cytoplasm and circulation as damage-associated molecular patterns (DAMPs). Mitochondrial DNA (mtDNA) and other macromolecules released into the cytosol stimulate the release of proinflammatory cytokines through activation of the NLRP3 inflammasome or the stimulator of interferon genes (STING) pathways that induce chronic inflammation (Chen et al., 2021; Lin et al., 2022; Masumoto et al., 2021; Qiu et al., 2022; Walker et al., 2022; Zhang et al., 2019). Thus, changes in mitochondria with age can alter cell and tissue health and contribute to age-related pathology through multiple complex and interrelated mechanisms beyond ATP production. There is wide consensus that the derangement of mitochondrial integrity and physiological functions probably contribute to ageing skeletal muscle through a multitude of mechanisms. However, mitochondrial ATP production or oxidative capacity are still the unique focus of most research in the field. In large part, this is due to the fact that measuring mitochondrial physiological functions other than oxidative phosphorylation is challenging and substantially imprecise. Mitochondrial oxidative capacity can be assessed in vivo in skeletal muscle using 31phosphorous magnetic resonace spectroscopy (31P MRS). In muscle tissue, the energy produced by mitochondria is accumulated in the form of phosphocreatine (PCr) that functions as a short-term energy supply and buffers ATP levels. In 31P MRS, an individual performs a short exercise bout to reduce PCr, which is split into inorganic P and Cr, followed by a period of recovery, during with PCr recovers back to resting levels using the ATP produced by mitochondria (Amara et al., 2008; Blei et al., 1993). The recovery constant (τ) provides a measure of how fast mitochondria can recharge PCr and is a proxy measure of mitochondrial capacity for ATP production (Layec et al., 2016; Paganini et al., 1997). Mitochondrial capacity can also be measured ‘in vitro’ from biopsy specimens assessing maximum respiration rate in permeabilized muscle fibres (PMFs), also considered a proxy for maximum ATP production (Kuznetsov et al., 2008). Age-related changes in in vivo oxidative capacity (OxCap) and PMF respiration typically track one another and we will primarily focus on in vivo OxCap for this discussion. Many studies have described significant relationships between OxCap and phenotypes of physical function that typically decline with ageing, including V ̇ O 2 peak V̇_O₂peak , leg strength and power (Coen et al., 2013; Mau et al., 2022). Skeletal muscle OxCap is also significantly associated with and predicts future declines of gait speed and mobility, even after adjusting for confounders such as muscle strength, sex and comorbidities (Coen et al., 2013; Tian et al., 2022). Lower OxCap is also associated with other physiological parameters important for healthy ageing, including cognitive function and insulin resistance (Fabbri et al., 2017; Tian et al., 2023). These declines in OxCap with age and its association with impaired mobility are supported by studies that have found reduced mitochondrial protein expression in aged skeletal muscle (Adelnia et al., 2020). Multiple studies support the hypothesis that ageing is associated with reduced in vivo OxCap in skeletal muscle (Conley et al., 2000; Grevendonk et al., 2021; Taylor et al., 1997). However, the relationship between physical activity and OxCap and the decline in physical activity is complex since the stronger preditor of skeletal muscle mitochondrial volume and function is the level of physical activity. Thus, not surprisingly, studies that matched older and younger subjects for physical activity level reported no difference in in vivo OxCap (Hart et al., 2014; Kent-Braun & Ng, 2000; Zhang et al., 2021). However, a very small minority of older individuals are able to maintain the level of physical activity necessary for mitochondria preservation. Understanding the effect of ageing on mitochondria is also further complicated by the many factors that affect OxCap. Factors such as muscle fibre type, muscle use, comorbidities (e.g. diabetes, kidney disease), genetic background and life history all can effect skeletal muscle mitochondrial energetics. The resulting heterogeneity of mitochondria changes with age probably contributes to the conflicting results reported by many studies, which were often obtained from relatively small studies that compared younger to older individuals while accounting for potential confounders. To shed light on the question of whether skeletal muscle mitochondrial OxCap declines with ageing, independent of physical activity, we analysed large cross-sectional publicly available datasets from two ongoing large cohort studies with which we are involved (Fig. 1). Using data from participants (ages 22–97 years) of the Baltimore Longitudinal Study of Aging (BLSA) (https://www.blsa.nih.gov/) we found that in vivo OxCap (expressed as τ, a higher value represents a lower OxCap) was significantly lower at older ages (r = 0.42, P < 0.0001; n = 737) after adjusting for sex, body mass index (BMI) and self-reported level of physical activity. Interestingly, in this analysis sex and BMI were not significant predictors of oxidative capacity, while physical activity was a strong and highly significant positive predictor (r = −0.29, P < 0.0001). We replicated these findings using the baseline data from the the Study of Muscle, Mobility and Aging (SOMMA, age >70 years; SOMMA Jr age 30–69 years) (https://sommaonline.ucsf.edu) (Cummings et al., 2023). There was a significant negative correlation between OxCap and age (r = −0.167, P < 0.0001; n = 883). This negative correlation persisted even after adjusting the analysis for physical activity using actigraphy measures of time spent in moderate, vigorous or very vigorous activity (r = −0.123, P = 0.0004; n = 824) and for physical activity, sex, height, weight and diabetes diagnosis (r = −0.101, P = 0.0038; n = 820). Thus, the significant association with declining OxCap and age persists even after adjusting for physical activity using both self-reported and actigraphy measurements. Despite the fact that physical activity remains an important driver of mitochondrial energetics into older ages, these results provide strong support for the conclusion that reduced mitochondrial OxCap is an intrinsic aspect of ageing skeletal muscle and is not solely due to reduced physical activity in older populations. Understanding the relationship between physical activity, mobility performance and in vivo OxCap in older adults is complex. Some literature suggests that ageing and associated reduced physical activity leads to reduced mechanical efficiency (Layec et al., 2015; Venturelli et al., 2024). In addition, ageing is also associated with reduced mitochondrial efficiency, lower ATP produced per oxygen consumed by the mitochondria (P/O) in skeletal muscle (Conley et al., 2000, 2007). These factors may contribute to lower exercise efficiency and impaired lower extremity performance (Distefano et al., 2018; Lewsey et al., 2020). Lower efficiency would result in a greater energetic stress experienced by the muscle, while improved efficiency would reduce the energy stress, for a given activity level, thereby affecting the relationship between mitochondrial function and lower extremity performance. Given the important role for energy stress in signalling mitochondrial biogenesis and adaptation with physical activity, older and younger subjects matched for physical activity may in fact not represent matching for the underlying molecular signals determining mitochondrial biogenesis and mitophagy. Furthermore, in vivo OxCap represents an integrative measure of ATP production that incorporates mitochondrial capacity as well as delivery of oxygen and substrates to the muscle through the circulation. Indeed, studies in the BLSA found evidence that in older persons lower extremity muscle OxCap is negatively affected by even minor reduction of perfusion assessed by the ankle–brachial index within the normal range (Oberdier et al., 2019). These data suggest that the decline of muscle mitochondrial function may be of partial vascular origin. In summary, ongoing analyses of large cohort studies of ageing support the conclusion that a decline in mitochondrial OxCap is an intrinsic part of skeletal muscle ageing independently of reduced physical activity as we age. Of course, mitochondrial energetic function is also affected by many factors other than ageing, such as physical activity, comorbidities, perfusion, as well as cellular level changes in mitochondrial quality control and adaptive cell signalling that also change with age. These multiple age-related stressors combine to impair ATP production and other physiological roles of mitochondria in aged tissues, including skeletal muscle, and contribute to disruptions in energetic, metabolite and redox homeostasis critical for healthy function. This loss of energy reserve and cell homeostasis then becomes an important contributor to the loss of resilience at the cell, tissue and organismal level with ageing. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. No competing interests declared. D.M.: Conception or design of the work; Drafting the work or revising it critically for important intellectual content; Final approval of the version to be published; Agreement to be accountable for all aspects of the work. L.F.: Conception or design of the work; Drafting the work or revising it critically for important intellectual content; Final approval of the version to be published; Agreement to be accountable for all aspects of the work. NIA: David J. Marcinek, R01 AG059416; NIA: David J. Marcinek, R01 AG078279; NIA: David J. Marcinek, P30 AG024827. Luigi Ferrucci, Intramural Research Program at the National Institute on Aging. This work was supported by Study of Muscle Mobility and Aging (SOMMA) R01 AG059416 and NIA R01AG078279 and the Intramural Research Program at the National Institute on Aging, NIH, in Baltimore, MD, USA. Infractructure support was provided by the NIA Claude D. Pepper Older American Independence Centers at the University of Pittsburgh, P30AG024827, and Wake Forest University, P30AG021332, and the Clinical and Translational Science Institutes, funded by the National Center for Advancing Translational Science, at Wake Forest University, UL1 0TR001420.