Aging is a complex biological phenomenon that results in functional decline across tissues and organ systems (López-Otín et al., 2013). Age is one of the most significant contributors to disease risk (Kennedy et al., 2014), and the aging process is influenced by a variety of genetic and environmental factors at the tissue and organismal level (Tian et al., 2023; Nie et al., 2022; Kuo et al., 2022). The application of omics technologies in recent years has led to unprecedented insights into the complex biology of aging (Rutledge et al., 2022). Bulk and single-cell transcriptomic analyses of mouse tissues across the lifespan have shown that aging tempo and trajectory, as indicated by tissue transcriptomic signatures, varies widely across tissues (Schaum et al., 2020; Zhang et al., 2021; Almanzar et al., 2020). Remarkably, many of the transcriptomic signatures of aging across tissues can be significantly reversed by caloric restriction or rejuvenated by the transfusion of young blood (Pálovics et al., 2022; Ma et al., 2020). Large-scale proteomic analyses of human plasma also reveal distinct waves of changes across the lifespan, which are associated with distinct biological pathways and affect age-related phenotypic traits and diseases (Lehallier et al., 2019; Oh et al., 2023).

Despite enormous progress, there is no consensus regarding the mechanisms underlying aging at the cellular or tissue level. Although many non-mutually exclusive hypotheses have been put forth to explain the root cause of aging - oxidative damage, genomic instability, epigenetic changes, loss of proteostasis, mitochondrial dysfunction, DNA damage, telomere shortening, cellular senescence, stem cell exhaustion - it remains a major challenge to distinguish between the driver and passenger mechanisms of aging (de Magalhães, 2024). Nevertheless, efforts to understand the proximal and ultimate cause of aging will facilitate development of therapeutics aimed to improve healthy aging (Kaeberlein et al., 2015; Campisi et al., 2019).

In the present study, we focused on aging from a mitochondrial perspective, as this organelle is known to play an important role in the aging process (Jang et al., 2018; Lima et al., 2022; Lanza and Nair, 2010; Sun et al., 2016). Mitochondria supply the bulk of the energy needed to maintain tissue health and repair tissue damage, and their function tends to decline with age. Over time, damage accumulates in mitochondrial DNA, proteins, and lipids, which compromises their functional integrity and leads to dysregulated metabolism and increased oxidative stress. Recent transcriptomic analyses have highlighted major reductions in electron transport chain gene expression across the lifespan (Schaum et al., 2020), and these changes can be significantly reversed by the transfusion of young blood into older mice (Pálovics et al., 2022). Aging-associated reduction in mitochondrial OXPHOS gene expression appears to be conserved between human, mouse, fly, and worm (Zahn et al., 2007). Accordingly, mitochondrial dysfunction has been implicated in various age-related diseases, including neurodegenerative disorders, cardiovascular diseases, and metabolic syndromes (Haas, 2019). Boosting mitochondrial health has been shown to delay age-related decline in organ function (Foote et al., 2018; Lima et al., 2023; Chiao et al., 2020; Nilsson and Tarnopolsky, 2019).

Given the central role of mitochondria in tissue health, we aimed to address the extent and magnitude of aging-induced changes in mitochondrial function across tissues and organ systems. Although many studies have examined mitochondrial respiratory capacity in various tissues, the scale was limited in that only a very small number of tissues could be interrogated at the same time. This is largely due to the inherent low-throughput method of assessing respiration which requires freshly isolated mitochondria or cells from tissues (Salabei et al., 2014). Consequently, it was not previously feasible to have a comprehensive and systems-level analysis of mitochondrial function across many tissues and the lifespan.

This barrier, however, has been recently overcome. An innovative method by Acin-Perez and coworkers has made it possible to now assess mitochondrial function in previously frozen tissues (Acin-Perez et al., 2020). This new method circumvents the need to isolate mitochondria at the time of tissue harvest, allowing many tissues to be collected, frozen, and assayed at a later time. We adopted the new method in a standardized workflow to profile mitochondrial activity in 33 tissues from young and old mice of both sexes. The dataset consists of a total of 1320 tissue samples from 40 mice and 3960 high-resolution respirometry assays encompassing three technical replicates. Our study represents the largest and the most comprehensive tissue respirometry analysis to date. Our data provide an unprecedented view on the variations and changes in mitochondrial functional capacity across tissues, sex, and age, thus informing ongoing studies on the causes and consequences of aging.

Here, we provide the most comprehensive catalog of sex- and age-associated mitochondrial respiration signatures across diverse tissues and organs system. One important aspect of our study to emphasize is the experimental uniformity. All the tissue samples were collected from the same set of mice, and processed and analyzed in a uniform and standardized manner on the same respirometry platform in a single laboratory. Combined with the 10 biological replicates, our experimental setup and workflow markedly enhanced the rigor and reduced the variations in data quality. From the mitochondrial respiration compendium, we can clearly see that mitochondrial function varies widely across tissues. This is perhaps not surprising given the highly variable energetic demands of different tissues (Rolfe and Brown, 1997) and the known differences in the composition of mitochondrial proteomes across tissues (Mootha et al., 2003; Pagliarini et al., 2008). Importantly, our data provide evidence that aging has a disproportionately larger effect than sex on mitochondrial activity across tissues.

The transcriptomic dataset generated by the Tabula Muris Consortium clearly indicates organ-specific temporal signatures across the lifespan (Schaum et al., 2020; Zhang et al., 2021; Almanzar et al., 2020), and this is assumed to track closely with tissue function even though no equivalent systematic interrogation of tissue function across the lifespan has been performed. In this regard, our mitochondrial activity atlas aligned and corroborated with the conclusion from large-scale transcriptomic analyses that the aging process and trajectory can vary substantially across tissues (Schaum et al., 2020; Zhang et al., 2021). This reinforces the notion that while an organism ages, the aging-associated decline in organ function is not uniform across tissues and organ systems. Given that genetics and environmental factors all contribute to tissue health and their functional breakdown with age, this further exemplifies the heterogenous process of aging across a population.

Although sex is a significant biological variable influencing many aspects of physiology and pathophysiology (Mauvais-Jarvis et al., 2020; Austad and Fischer, 2016), and sex hormones are known to affect mitochondrial function (Klinge, 2020; Gaignard et al., 2017; Sultanova et al., 2020), we were surprised that sex has a relatively modest impact on respiratory capacity in mitochondria across tissues and age. The modest impact of sex on mitochondrial respiration across tissues is also concordant with the relatively minor impact of sex on tissue transcriptome across the lifespan (Schaum et al., 2020; Ma et al., 2020). The caveat, however, is that our study is based on a single strain of mice with a uniform genetic background. The contribution of biological sex may be more pronounced in an outbred population where sex hormones interact with other genetic and environmental determinants to influence mitochondrial activity across tissues and age.

Aging is a dynamic process that unfolds across the lifespan, which involves changes at the molecular, cellular, biochemical, and tissue level. Although the magnitude of change may vary between individuals, with increasing age, humans generally experience a gradual and progressive decline in cognitive function, visual acuity, skin elasticity, motor control, digestive function, fertility, and metabolism (e.g. increased adiposity) (López-Otín et al., 2013; Tian et al., 2023). Interestingly, in our dataset, we also observed an aging-associated decline in mitochondrial respiration in most brain regions, eyes, skin, fallopian tubes, different fat depots and BAT, pancreas, and the digestive tracts in male and/or female mice. This age-dependent reduction in mitochondrial function likely contributes, at least in part, to the functional decline across different organ systems. Depending on the tissue, the magnitude of change in mitochondrial respiration in old mice, when compared to young mice, can be substantial or relatively modest. This variability is consistent with the heterogenous rate of aging across tissues (Tian et al., 2023).

The underlying cause of reduced mitochondrial respiration in aging is not well understood and it may involve multiple mechanisms. Transcriptomic analyses have consistently shown that aging induces a reduction in mitochondrial OXPHOS gene expression (Schaum et al., 2020; Zhang et al., 2021), and this likely is one of the mechanisms that contributes to the reduced mitochondrial activity we observed across many tissues in old mice. The contributing factors which drive an age-dependent decrease in mitochondrial OXPHOS gene expression across tissues, however, is largely unknown. Other potential mechanisms may involve age-dependent accumulation of oxidative damage in mitochondrial DNA, proteins, and lipids that adversely affect mitochondrial function (Sun et al., 2016; Sastre et al., 2000). The damage to mitochondrial integrity in aging is frequently attributed to reactive oxygen species (ROS) that are naturally generated as byproducts of electron transfer through the mitochondrial OXPHOS complexes (Orrenius et al., 2007), or to inadequate stress response pathways (e.g. proteostasis and mitophagy) (Lima et al., 2022). While cells have mechanisms to guard against oxidative stress due to mitochondrial activity, these antioxidant defense mechanisms are known to decline with age despite an age-dependent increase in ROS production (Jones et al., 2002; Kozakiewicz et al., 2019). Similarly, the integrated stress response pathways also tend to diminished with age (Lima et al., 2022). Thus, it is often thought the gradual accumulation of ROS with age, coupled with a decline in mitochondria’s ability to handle proteotoxic stress, progressively compromises mitochondrial function.

It should be noted that changes in cell, tissue, and organ function are often counteracted by homeostatic and compensatory responses. While many tissues showed a significant reduction in mitochondrial function, some tissues also showed a concomitant increase in mitochondrial activity with age. For example, we observed an increase in mitochondrial respiration in the heart, different skeletal muscle groups, and mesenteric fat in old male and/or female mice. Increased mitochondrial respiration is seen in both the oxidative (diaphragm, soleus, tongue) and glycolytic (plantaris) muscle fibers. This increase in mitochondrial activity is not attributed to differences in mitochondrial content since the OCR is normalized to mitochondrial content.

Skeletal muscle mass is known to decrease with age (Wilkinson et al., 2018), and it is frequently but not always accompanied by a reduction in skeletal muscle mitochondrial function in vivo (Gouspillou et al., 2014; Short et al., 2005; Conley et al., 2000; Kent-Braun and Ng, 2000; Chilibeck et al., 1998) and in vitro (Short et al., 2005; Mansouri et al., 2006; Kumaran et al., 2005; Tonkonogi et al., 2003; Drew et al., 2003; Picard et al., 2010). Some studies, however, have also suggested that the energetic efficiency of mitochondria in skeletal muscle - located beneath the sarcolemmal membrane or between the myofibrils - appears to increase with age (Crescenzo et al., 2015; Crescenzo et al., 2014). While we observed an increase in the maximal uncoupled respiration in skeletal muscle lysates of aged mice, our results cannot be directly compared to prior studies that examine coupled respiration in intact tissue or isolated mitochondria. It has been shown that skeletal muscle mitochondrial number increases, whereas mitochondrial size decreases, with age (Del Campo et al., 2018). Additionally, the ultrastructure of mitochondria may also change with age (Brandt et al., 2017). Whether these parameters correlate with and contribute to age-dependent changes in mitochondrial respiration remains to be established.

Similar to skeletal muscle, the heart also experiences functional decline with age, and this is thought to be attributed to multiple mechanisms that include age-dependent changes in mitochondrial content, morphology, lipid composition (e.g. cardiolipin) and dynamics (e.g. fusion and fission), activity of the electron transport chain, ROS generation, and susceptibility to mitochondrial permeability transition pore opening (Lesnefsky et al., 2016; Boengler et al., 2017). Additional contributing factors to cardiac functional decline include age-related oxidative stress and chronic low-grade inflammation, which further impact mitochondrial integrity and function (de Almeida et al., 2020). With regard to impaired mitochondrial OXPHOS in the aging heart, it appears the subpopulation of mitochondria located between the myofibrils, the interfibrillar mitochondria (IFM), are affected to a much greater extent than the population of mitochondria located in the subsarcolemmal membrane (Suh et al., 2003; Fannin et al., 1999; Lesnefsky et al., 2001). This suggests that IFM, being more closely associated with energy-demanding myofibrils, might be more vulnerable to age-related dysfunction. In contrast to studies using isolated mitochondria from the heart, mitochondrial function was found to be largely preserved in permeabilized intact cardiomyocyte bundles from senescent rats, in which all intact mitochondria are represented (Picard et al., 2012).

In our assay, however, maximal mitochondrial respiration in the heart ventricles increases with age. Since the contractile property of the heart declines and the stiffness of the extracellular matrix increases with age due to fibrosis (Biernacka and Frangogiannis, 2011; Travers et al., 2016), the heart tissue becomes less efficient and requires more energy to carry out its normal pumping function. Thus, we speculate that reduced efficiency of the aging heart may drive a compensatory response to increase mitochondrial capacity. An increase in mitochondrial respiration, however, may come at the cost of generating greater amounts of reactive oxygen species, which are thought to be detrimental to the aging heart. Thus, with the general observation that organ function declines with age, our data suggests that some tissues such as the heart and skeletal muscle may increase their mitochondrial respiration to meet the energetic demands resulting from reduced mass (e.g. sarcopenia) and/or declining efficiency of organ function. This hypothesis awaits future experimental verification.

It is worth noting that our respirometry analysis assesses NADH-dependent respiration through CI, succinate-dependent respiration through CII, and TMPD/ascorbate-dependent respiration at CIV. Regardless of whether electrons enter the respiratory chain through CI or CII, or at CIV, oxygen (the final electron acceptor) is only consumed at CIV. Since the OCR is normalized to mitochondrial content, the variations we observed in OCR within any given tissue through CI or CII, or at CIV, likely reflect how tightly coupled respiratory complex chains consisting of CI+CIII2+CIV and CII+CIII2+CIV are relative to CIV alone. Although our data suggest that sex and age affect the flow of electrons through respiratory complex CI+CIII2+CIV and CII+CIII2+CIV across tissues, this remains speculative given that our assays were not performed on intact mitochondria.

Every respirometry method has its advantages and limitations (Salabei et al., 2014). Unlike the mitochondrial respiration analysis in intact cells or isolated mitochondria, our respirometry analyses were performed in mitochondria-enriched lysates derived from frozen tissues (Acin-Perez et al., 2020). In an intact mitochondrion, the rate of respiration is regulated by both substrate supply across the mitochondrial membrane and the rate of NADH and FADH production via the TCA cycle and fatty acid β-oxidation. In the absence of an intact mitochondrial membrane, our assays measure maximal mitochondrial respiration through CI, CII, or CIV. Thus, our mitochondrial activity reflects maximal respiration across tissues. In an in vivo milieu, mitochondrial activity is likely regulated and may not reach its maximal capacity as reflected in our assay. Other relevant and informative parameters concerning mitochondrial functions - e.g., P/O ratios, basal respiration, and ATP-coupled respiration - could not be obtained from our current approach, which relied on non-intact mitochondria. However, despite these limitations, the major advantage of the frozen-tissue method is that it is now feasible to carry out large-scale respirometry analyses across tissues, thus providing a broader understanding of systemic bioenergetic health that outweighs its limitations.

It should be emphasized that our mitochondrial respiration represents the average values for each tissue, even though all tissues consist of multiple distinct cell types that may vary in their cellular composition and mitochondrial activity across sex and age. While individual cell types can be isolated prior to respirometry analysis, this labor-intensive method is not suited for assessing mitochondrial respiration at scale (i.e. across a large number of tissues from the same animal). In the future, this challenge may be overcome with the development of new technologies that allow for high-resolution mitochondrial respirometry at cell-type or single-cell resolution in situ.

In summary, our mitochondria functional signatures demonstrate similar and divergent responses to aging across tissues. Whether changes in tissue mitochondrial respiration reflect the cause, consequence, or both, of aging remains to be determined. We anticipate that the integration of this knowledge and approach with other large-scale omics data will help uncover potential genetic, epigenetic, and biochemical mechanisms linking mitochondrial health and organismal aging.

All data generated or analyzed during this study are included in the manuscript and supporting files; source data files have been provided for Figure 1.

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Author details

1. Dylan C Sarver

   1. Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, United States
   2. Center for Metabolism and Obesity Research, Johns Hopkins University School of Medicine, Baltimore, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Writing – original draft, Writing - review and editing

   Competing interests

   No competing interests declared

2. Muzna Saqib

   1. Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, United States
   2. Center for Metabolism and Obesity Research, Johns Hopkins University School of Medicine, Baltimore, United States

   Contribution

   Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

3. Fangluo Chen

   1. Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, United States
   2. Center for Metabolism and Obesity Research, Johns Hopkins University School of Medicine, Baltimore, United States

   Contribution

   Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

4. G William Wong

   1. Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, United States
   2. Center for Metabolism and Obesity Research, Johns Hopkins University School of Medicine, Baltimore, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Investigation, Writing – original draft, Project administration

   For correspondence

   gwwong@jhmi.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5286-6506

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