A practical perspective on how to develop, implement, execute, and reproduce high-resolution respirometry experiments: The physiologist's guide to an Oroboros O2k.

The development of high-resolution respirometry (HRR) has greatly expanded the analytical scope to study mitochondrial respiratory control relative to specific tissue/cell types across various metabolic states. Specifically, the Oroboros Oxygraph 2000 (O2k) is a common tool for measuring rates of mitochondrial respiration and is the focus of this perspective. The O2k platform is amenable for answering numerous bioenergetic questions. However, inherent variability with HRR-derived data, both within and amongst users, can impede progress in bioenergetics research. Therefore, we advocate for several vital considerations when planning and conducting O2k experiments to ultimately enhance transparency and reproducibility across laboratories. In this perspective, we offer guidance for best practices of mitochondrial preparation, protocol selection, and measures to increase reproducibility. The goal of this perspective is to propagate the use of the O2k, enhance reliability and validity for both new and experienced O2k users, and provide a reference for peer reviewers.

Phytochemical compound PB125 attenuates skeletal muscle mitochondrial dysfunction and impaired proteostasis in a model of musculoskeletal decline.

Impaired mitochondrial function and disrupted proteostasis contribute to musculoskeletal dysfunction. However, few interventions simultaneously target these two drivers to prevent musculoskeletal decline. Nuclear factor erythroid 2-related factor 2 (Nrf2) activates a transcriptional programme promoting cytoprotection, metabolism, and proteostasis. We hypothesized daily treatment with a purported Nrf2 activator, PB125, in Hartley guinea pigs, a model of musculoskeletal decline, would attenuate the progression of skeletal muscle mitochondrial dysfunction and impaired proteostasis and preserve musculoskeletal function. We treated 2- and 5-month-old male and female Hartley guinea pigs for 3 and 10 months, respectively, with the phytochemical compound PB125. Longitudinal assessments of voluntary mobility were measured using Any-MazeTM open-field enclosure monitoring. Cumulative skeletal muscle protein synthesis rates were measured using deuterium oxide over the final 30 days of treatment. Mitochondrial oxygen consumption in soleus muscles was measured using high resolution respirometry. In both sexes, PB125 (1) increased electron transfer system capacity; (2) attenuated the disease/age-related decline in coupled and uncoupled mitochondrial respiration; and (3) attenuated declines in protein synthesis in the myofibrillar, mitochondrial and cytosolic subfractions of the soleus. These effects were not associated with statistically significant prolonged maintenance of voluntary mobility in guinea pigs. Collectively, treatment with PB125 contributed to maintenance of skeletal muscle mitochondrial respiration and proteostasis in a pre-clinical model of musculoskeletal decline. Further investigation is necessary to determine if these documented effects of PB125 are also accompanied by slowed progression of other aspects of musculoskeletal dysfunction. KEY POINTS: Aside from exercise, there are no effective interventions for musculoskeletal decline, which begins in the fifth decade of life and contributes to disability and cardiometabolic diseases. Targeting both mitochondrial dysfunction and impaired protein homeostasis (proteostasis), which contribute to the age and disease process, may mitigate the progressive decline in overall musculoskeletal function (e.g. gait, strength). A potential intervention to target disease drivers is to stimulate nuclear factor erythroid 2-related factor 2 (Nrf2) activation, which leads to the transcription of genes responsible for redox homeostasis, proteome maintenance and mitochondrial energetics. Here, we tested a purported phytochemical Nrf2 activator, PB125, to improve mitochondrial function and proteostasis in male and female Hartley guinea pigs, which are a model for musculoskeletal ageing. PB125 improved mitochondrial respiration and attenuated disease- and age-related declines in skeletal muscle protein synthesis, a component of proteostasis, in both male and female Hartley guinea pigs.

Targeting mitochondrial function and proteostasis to mitigate dynapenia.

Traditionally, interventions to treat skeletal muscle aging have largely targeted sarcopenia-the age-related loss of skeletal muscle mass. Dynapenia refers to the age-related loss in skeletal muscle function due to factors outside of muscle mass, which helps to inform treatment strategies for aging skeletal muscle. There is evidence that mechanisms to maintain protein homeostasis and proteostasis, deteriorate with age. One key mechanism to maintain proteostasis is protein turnover, which is an energetically costly process. When there is a mismatch between cellular energy demands and energy provision, inelastic processes related to metabolism are maintained, but there is competition for the remaining energy between the elastic processes of somatic maintenance and growth. With aging, mitochondrial dysfunction reduces ATP generation capacity, constraining the instantaneous supply of energy, thus compromising growth and somatic maintenance processes. Further, with age the need for somatic maintenance increases because of the accumulation of protein damage. In this review, we highlight the significant role mitochondria have in maintaining skeletal muscle proteostasis through increased energy provision, protein turnover, and substrate flux. In addition, we provide evidence that improving mitochondrial function could promote a cellular environment that is conducive to somatic maintenance, and consequently for mitigating dynapenia. Finally, we highlight interventions, such as aerobic exercise, that could be used to improve mitochondrial function and improve outcomes related to dynapenia.

Running Economy Changes Alter Predicted Running Speed and Performance in Collegiate Runners.

The goal of the study was to determine the effect of altering running strategy on predicted running performance in distance runners through application of a novel prediction model. Fifteen male (n = 10; Age: 22.2 ± 4.9 years; Height: 177.7 ± 7.4 cm; Mass: 68.6 ± 6.7 kg) and female (n = 5; Age: 21.8 ± 4.1 years; Height: 167.4 ± 7.8 cm; Mass: 59.3 ± 8.1 kg) long distance runners were recruited to participate in the study. Participants' oxygen consumption (VO2) and carbon dioxide (VCO2) were measured by a metabolic cart using a face mask. After a brief warm-up, participants rested for the initial five minutes then ran at their preferred speed for five minutes. Participants rested for another five minutes while their oxygen consumption returned to baseline measurements and ran for five minutes while increasing step rate by 7.5%. There was no significant difference between conditions for VO2 measurements and energetic cost (p > 0.05). There was also no significant difference in the baseline, self-selected speed and predicted speed resulting from the increase in step rate (p > 0.05). Increasing stride rate 7.5% resulted in an average decrease in predicted running speed of 1%. While statistically insignificant, small decrements in running speed can accrue over time and negatively impact running performance.

Three-dimensional imaging studies in mice identify cellular dynamics of skeletal muscle regeneration.

The function of many organs, including skeletal muscle, depends on their three-dimensional structure. Muscle regeneration therefore requires not only reestablishment of myofibers but also restoration of tissue architecture. Resident muscle stem cells (SCs) are essential for regeneration, but how SCs regenerate muscle architecture is largely unknown. We address this problem using genetic labeling of mouse SCs and whole-mount imaging to reconstruct, in three dimensions, muscle regeneration. Unexpectedly, we found that myofibers form via two distinct phases of fusion and the residual basement membrane of necrotic myofibers is critical for promoting fusion and orienting regenerated myofibers. Furthermore, the centralized myonuclei characteristic of regenerated myofibers are associated with myofibrillogenesis and endure months post injury. Finally, we elucidate two cellular mechanisms for the formation of branched myofibers, a pathology characteristic of diseased muscle. We provide a synthesis of the cellular events of regeneration and show that these differ from those used during development.

Age-induced changes in skeletal muscle mitochondrial DNA synthesis, quantity, and quality in genetically unique rats.

Mitochondrial genomic integrity is a key element of physiological processes and health. Changes in the half-life of the mitochondrial genome are implicated in the generation and accumulation of age-induced mitochondrial DNA (mtDNA) mutations, which are implicated in skeletal muscle aging and sarcopenia. There are conflicting data on the half-life of mtDNA, and there is limited information on how aging affects half-life in skeletal muscle. We hypothesized that skeletal muscle mtDNA synthesis rates would decrease with age in both female and male rats concomitant with changes in mtDNA integrity reflected in mtDNA copy number and mutation frequency. We measured mitochondrial genome half-life using stable isotope labeling over a period of 14 days and assessed mtDNA copy number and deletion mutation frequency using digital PCR in the quadriceps muscle of 9-month-old and 26-month-old male and female OKC-HET rats. We found a significant age-related increase in mtDNA half-life, from 132 days at 9 months to 216 days at 26 months of age in OKC-HET quadriceps. Concomitant with the increase in mtDNA half-life, we found an age-related increase in mtDNA deletion mutation frequency in both male and female rats. Notably, 26-month-old female rats had a lower mutation frequency than male rats, and there were no changes in mtDNA copy number with sex, age, or mitochondrial genotype. These data reveal several key findings: (1) mtDNA turnover in rat skeletal muscle decreases with age, (2) mtDNA half-lives in skeletal muscle are approximately an order of magnitude longer than what is reported for other tissues, and (3) muscle mtDNA turnover differs significantly from the turnover of other mitochondrial macromolecules including components of the mitochondrial nucleoid. These findings provide insight into the factors driving age-induced mtDNA mutation accumulation, which contribute to losses of mitochondrial genomic integrity and may play a role in skeletal muscle dysfunction.

Non-transgenic guinea pig strains exhibit divergent age-related changes in hippocampal mitochondrial respiration.

AIM: Alzheimer's disease (AD) is the most common form of dementia. However, while 150+ animal models of AD exist, drug translation from preclinical models to humans for treatment usually fails. One factor contributing to low translation is likely the absence of neurodegenerative models that also encompass the multi-morbidities of human aging. We previously demonstrated that, in comparison to the PigmEnTed (PET) guinea pig strain which models "typical" brain aging, the Hartley strain develops hallmarks of AD like aging humans. Hartleys also exhibit age-related impairments in cartilage and skeletal muscle. Impaired mitochondrial respiration is one driver of both cellular aging and AD. In humans with cognitive decline, diminished skeletal muscle and brain respiratory control occurs in parallel. We previously reported age-related declines in skeletal muscle mitochondrial respiration in Hartleys. It is unknown if there is concomitant mitochondrial dysfunction in the brain. METHODS: Therefore, we assessed hippocampal mitochondrial respiration in 5- and 12-month Hartley and PET guinea pigs using high-resolution respirometry. RESULTS: At 12 months, PETs had higher complex I supported mitochondrial respiration paralleling their increase in body mass compared to 5 months PETs. Hartleys were also heavier at 12 months compared to 5 months but did not have higher complex I respiration. Compared to 5 months Hartleys, 12 months Hartleys had lower complex I mitochondrial efficiency and compensatory increases in mitochondrial proteins collectively suggesting mitochondrial dysfunction with age. CONCLUSIONS: Therefore, Hartleys might be a relevant model to test promising therapies targeting mitochondria to slow brain aging and AD progression.

Nontransgenic Guinea Pig Strains Exhibit Hallmarks of Human Brain Aging and Alzheimer's Disease.

Older age is the primary risk factor for most chronic diseases, including Alzheimer's disease (AD). Current preclinical models to study brain aging and AD are mainly transgenic and harbor mutations intended to mirror brain pathologies associated with human brain aging/AD (eg, by increasing production of the amyloid precursor protein, amyloid beta [Aß], and/or phosphorylated tau, all of which are key pathological mediators of AD). Although these models may provide insight on pathophysiological processes in AD, none completely recapitulate the disease and its strong age-dependence, and there has been limited success in translating preclinical results and treatments to humans. Here, we describe 2 nontransgenic guinea pig (GP) models, a standard PigmEnTed (PET) strain, and lesser-studied Dunkin-Hartley (DH) strain, that may naturally mimic key features of brain aging and AD in humans. We show that brain aging in PET GP is transcriptomically similar to human brain aging, whereas older DH brains are transcriptomically more similar to human AD. Both strains/models also exhibit increased neurofilament light chain (NFL, a marker of neuronal damage) with aging, and DH animals display greater S100 calcium-binding protein B (S100ß), ionized calcium-binding adapter molecule 1 (Iba1), and Aß and phosphorylated tau-which are all important markers of neuroinflammation-associated AD. Collectively, our results suggest that both the PET and DH GP may be useful, nontransgenic models to study brain aging and AD, respectively.

Sex differences in changes of protein synthesis with rapamycin treatment are minimized when metformin is added to rapamycin.

Loss of protein homeostasis is a hallmark of the aging process. We and others have previously shown that maintenance of proteostasis is a shared characteristic of slowed-aging models. Rapamycin (Rap) exerts sex-specific effects on murine lifespan, but the combination of Rap with the anti-hyperglycemic drug metformin (Rap + Met) equally increases male and female mouse median lifespan. In the current investigation, we compare the effects of short-term (8 weeks) Rap and Rap + Met treatments on bulk and individual protein synthesis in two key metabolic organs (the liver and skeletal muscle) of young genetically heterogeneous mice using deuterium oxide. We report for the first time distinct effects of Rap and Rap + Met treatments on bulk and individual protein synthesis in young mice. Although there were decreases in protein synthesis as assessed by bulk measurements, individual protein synthesis analyses demonstrate there were nearly as many proteins that increased synthesis as decreased synthesis rates. While we observed the established sex- and tissue-specific effects of Rap on protein synthesis, adding Met yielded more uniform effects between tissue and sex. These data offer mechanistic insight as to how Rap + Met may extend lifespan in both sexes while Rap does not.

Differential Effects of Rapamycin and Metformin in Combination With Rapamycin on Mechanisms of Proteostasis in Cultured Skeletal Myotubes.

mTOR inhibition extends life span in multiple organisms. In mice, when metformin treatment (Met) is added to the mTOR inhibitor rapamycin (Rap), median and maximal life span is extended to a greater degree than with Rap or Met alone. Treatments that extend life span often maintain proteostasis. However, it is less clear how individual tissues, such as skeletal muscle, maintain proteostasis with life span-extending treatments. In C2C12 myotubes, we used deuterium oxide (D2O) to directly measure two primary determinants of proteostasis, protein synthesis, and degradation rates, with Rap or Met+Rap treatments. We accounted for the independent effects of cell growth and loss, and isolated the contribution of autophagy and mitochondrial fission to obtain a comprehensive assessment of protein turnover. Compared with control, both Rap and Met+Rap treatments lowered mitochondrial protein synthesis rates (p < .001) and slowed cellular proliferation (p < .01). These changes resulted in greater activation of mechanisms promoting proteostasis for Rap, but not Met+Rap. Compared with control, both Rap and Met+Rap slowed protein breakdown. Autophagy and mitochondrial fission differentially influenced the proteostatic effects of Rap and Met+Rap in C2C12 myotubes. In conclusion, we demonstrate that Met+Rap did not increase protein turnover and that these treatments do not seem to promote proteostasis through increased autophagy.

The Dunkin Hartley Guinea Pig Is a Model of Primary Osteoarthritis That Also Exhibits Early Onset Myofiber Remodeling That Resembles Human Musculoskeletal Aging.

Skeletal muscle dysfunction, articular cartilage degeneration, and bone loss occur essentially in parallel during aging. Mechanisms contributing to this systemic musculoskeletal decline remain incompletely understood, limiting progress toward developing effective therapeutics. Because the progression of human musculoskeletal aging is slow, researchers rely on rodent models to identify mechanisms and test interventions. The Dunkin Hartley guinea pig is an outbred strain that begins developing primary osteoarthritis by 4 months of age with a progression and pathology similar to aging humans. The purpose of this study was to determine if skeletal muscle remodeling during the progression of osteoarthritis in these guinea pigs resembles musculoskeletal aging in humans. We compared Dunkin Hartley guinea pigs to Strain 13 guinea pigs, which develop osteoarthritis much later in the lifespan. We measured myofiber type and size, muscle density, and long-term fractional protein synthesis rates of the gastrocnemius and soleus muscles in 5, 9, and 15-month-old guinea pigs. There was an age-related decline in skeletal muscle density, a greater proportion of smaller myofibers, and a decline in type II concomitant with a rise in type I myofibers in the gastrocnemius muscles from Dunkin Hartley guinea pigs only. These changes were accompanied by age-related declines in myofibrillar and mitochondrial protein synthesis in the gastrocnemius and soleus. Collectively, these findings suggest Dunkin Hartley guinea pigs experience myofiber remodeling alongside the progression of osteoarthritis, consistent with human musculoskeletal aging. Thus, Dunkin Hartley guinea pigs may be a model to advance discovery and therapeutic development for human musculoskeletal aging.

Exercise-Induced Mitohormesis for the Maintenance of Skeletal Muscle and Healthspan Extension.

Oxidative damage is one mechanism linking aging with chronic diseases including the progressive loss of skeletal muscle mass and function called sarcopenia. Thus, mitigating oxidative damage is a potential avenue to prevent or delay the onset of chronic disease and/or extend healthspan. Mitochondrial hormesis (mitohormesis) occurs when acute exposure to stress stimulates adaptive mitochondrial responses that improve mitochondrial function and resistance to stress. For example, an acute oxidative stress via mitochondrial superoxide production stimulates the activation of endogenous antioxidant gene transcription regulated by the redox sensitive transcription factor Nrf2, resulting in an adaptive hormetic response. In addition, acute stresses such as aerobic exercise stimulate the expansion of skeletal muscle mitochondria (i.e., mitochondrial biogenesis), constituting a mitohormetic response that protects from sarcopenia through a variety of mechanisms. This review summarized the effects of age-related declines in mitochondrial and redox homeostasis on skeletal muscle protein homeostasis and highlights the mitohormetic mechanisms by which aerobic exercise mitigates these age-related declines and maintains function. We discussed the potential efficacy of targeting the Nrf2 signaling pathway, which partially mediates adaptation to aerobic exercise, to restore mitochondrial and skeletal muscle function. Finally, we highlight knowledge gaps related to improving redox signaling and make recommendations for future research.

Muscle-specific changes in protein synthesis with aging and reloading after disuse atrophy.

BACKGROUND: Successful strategies to halt or reverse sarcopenia require a basic understanding of the factors that cause muscle loss with age. Acute periods of muscle loss in older individuals have an incomplete recovery of muscle mass and strength, thus accelerating sarcopenic progression. The purpose of the current study was to further understand the mechanisms underlying the failure of old animals to completely recover muscle mass and function after a period of hindlimb unloading. METHODS: Hindlimb unloading was used to induce muscle atrophy in Fischer 344-Brown Norway (F344BN F1) rats at 24, 28, and 30 months of age. Rats were hindlimb unloaded for 14 days and then reloaded at 24 months (Reloaded 24), 28 months (Reloaded 28), and 24 and 28 months (Reloaded 24/28) of age. Isometric torque was determined at 24 months of age (24 months), at 28 months of age (28 months), immediately after 14 days of reloading, and at 30 months of age (30 months). During control or reloaded conditions, rats were labelled with deuterium oxide (D2 O) to determine rates of muscle protein synthesis and RNA synthesis. RESULTS: After 14 days of reloading, in vivo isometric torque returned to baseline in Reloaded 24, but not Reloaded 28 and Reloaded 24/28. Despite the failure of Reloaded 28 and Reloaded 24/28 to regain peak force, all groups were equally depressed in peak force generation at 30 months. Increased age did not decrease muscle protein synthesis rates, and in fact, increased resting rates of protein synthesis were measured in the myofibrillar fraction (Fractional synthesis rate (FSR): %/day) of the plantaris (24 months: 2.53 ± 0.17; 30 months: 3.29 ± 0.17), and in the myofibrillar (24 months: 2.29 ± 0.07; 30 months: 3.34 ± 0.11), collagen (24 months: 1.11 ± 0.07; 30 months: 1.55 ± 0.14), and mitochondrial (24 months: 2.38 ± 0.16; 30 months: 3.20 ± 0.10) fractions of the tibialis anterior (TA). All muscles increased myofibrillar protein synthesis (%/day) in Reloaded 24 (soleus: 3.36 ± 0.11, 5.23 ± 0.19; plantaris: 2.53 ± 0.17, 3.66 ± 0.07; TA: 2.29 ± 0.14, 3.15 ± 0.12); however, in Reloaded 28, only the soleus had myofibrillar protein synthesis rates (%/day) >28 months (28 months: 3.80 ± 0.10; Reloaded 28: 4.86 ± 0.19). Across the muscles, rates of protein synthesis were correlated with RNA synthesis (all muscles combined, R2 = 0.807, P < 0.0001). CONCLUSIONS: These data add to the growing body of literature that indicate that changes with age, including following disuse atrophy, differ by muscle. In addition, our findings lead to additional questions of the underlying mechanisms by which some muscles are maintained with age while others are not.

Six Weeks of Low-Load Blood Flow Restricted and High-Load Resistance Exercise Training Produce Similar Increases in Cumulative Myofibrillar Protein Synthesis and Ribosomal Biogenesis in Healthy Males.

Purpose: High-load resistance exercise contributes to maintenance of muscle mass, muscle protein quality, and contractile function by stimulation of muscle protein synthesis (MPS), hypertrophy, and strength gains. However, high loading may not be feasible in several clinical populations. Low-load blood flow restricted resistance exercise (BFRRE) may provide an alternative approach. However, the long-term protein synthetic response to BFRRE is unknown and the myocellular adaptations to prolonged BFRRE are not well described. Methods: To investigate this, 34 healthy young subjects were randomized to 6 weeks of low-load BFRRE, HLRE, or non-exercise control (CON). Deuterium oxide (D2O) was orally administered throughout the intervention period. Muscle biopsies from m. vastus lateralis were collected before and after the 6-week intervention period to assess long-term myofibrillar MPS and RNA synthesis as well as muscle fiber-type-specific cross-sectional area (CSA), satellite cell content, and myonuclei content. Muscle biopsies were also collected in the immediate hours following single-bout exercise to assess signaling for muscle protein degradation. Isometric and dynamic quadriceps muscle strength was evaluated before and after the intervention. Results: Myofibrillar MPS was higher in BFRRE (1.34%/day, p < 0.01) and HLRE (1.12%/day, p < 0.05) compared to CON (0.96%/day) with no significant differences between exercise groups. Muscle RNA synthesis was higher in BFRRE (0.65%/day, p < 0.001) and HLRE (0.55%/day, p < 0.01) compared to CON (0.38%/day) and both training groups increased RNA content, indicating ribosomal biogenesis in response to exercise. BFRRE and HLRE both activated muscle degradation signaling. Muscle strength increased 6-10% in BFRRE (p < 0.05) and 13-23% in HLRE (p < 0.01). Dynamic muscle strength increased to a greater extent in HLRE (p < 0.05). No changes in type I and type II muscle fiber-type-specific CSA, satellite cell content, or myonuclei content were observed. Conclusions: These results demonstrate that BFRRE increases long-term muscle protein turnover, ribosomal biogenesis, and muscle strength to a similar degree as HLRE. These findings emphasize the potential application of low-load BFRRE to stimulate muscle protein turnover and increase muscle function in clinical populations where high loading is untenable.

Metformin inhibits mitochondrial adaptations to aerobic exercise training in older adults.

Metformin and exercise independently improve insulin sensitivity and decrease the risk of diabetes. Metformin was also recently proposed as a potential therapy to slow aging. However, recent evidence indicates that adding metformin to exercise antagonizes the exercise-induced improvement in insulin sensitivity and cardiorespiratory fitness. The purpose of this study was to test the hypothesis that metformin diminishes the improvement in insulin sensitivity and cardiorespiratory fitness after aerobic exercise training (AET) by inhibiting skeletal muscle mitochondrial respiration and protein synthesis in older adults (62 ± 1 years). In a double-blinded fashion, participants were randomized to placebo (n = 26) or metformin (n = 27) treatment during 12 weeks of AET. Independent of treatment, AET decreased fat mass, HbA1c, fasting plasma insulin, 24-hr ambulant mean glucose, and glycemic variability. However, metformin attenuated the increase in whole-body insulin sensitivity and VO2 max after AET. In the metformin group, there was no overall change in whole-body insulin sensitivity after AET due to positive and negative responders. Metformin also abrogated the exercise-mediated increase in skeletal muscle mitochondrial respiration. The change in whole-body insulin sensitivity was correlated to the change in mitochondrial respiration. Mitochondrial protein synthesis rates assessed during AET were not different between treatments. The influence of metformin on AET-induced improvements in physiological function was highly variable and associated with the effect of metformin on the mitochondria. These data suggest that prior to prescribing metformin to slow aging, additional studies are needed to understand the mechanisms that elicit positive and negative responses to metformin with and without exercise.

Mitochondria as a Target for Mitigating Sarcopenia.

Sarcopenia is the loss of muscle mass, strength, and physical function that is characteristic of aging. The progression of sarcopenia is gradual but may be accelerated by periods of muscle loss during physical inactivity secondary to illness or injury. The loss of mobility and independence and increased comorbidities associated with sarcopenia represent a major healthcare challenge for older adults. Mitochondrial dysfunction and impaired proteostatic mechanisms are important contributors to the complex etiology of sarcopenia. As such, interventions that target improving mitochondrial function and proteostatic maintenance could mitigate or treat sarcopenia. Exercise is currently the only effective option to treat sarcopenia and does so, in part, by improving mitochondrial energetics and protein turnover. Exercise interventions also serve as a discovery tool to identify molecular targets for development of alternative therapies to treat sarcopenia. In summary, we review the evidence linking mitochondria and proteostatic maintenance to sarcopenia and discuss the therapeutic potential of interventions addressing these two factors to mitigate sarcopenia.

Skeletal Muscle Mitochondrial Protein Synthesis and Respiration Increase With Low-Load Blood Flow Restricted as Well as High-Load Resistance Training.

Purpose: It is well established that high-load resistance exercise (HLRE) can stimulate myofibrillar accretion. Additionally, recent studies suggest that HLRE can also stimulate mitochondrial biogenesis and respiratory function. However, in several clinical situations, the use of resistance exercise with high loading may not constitute a viable approach. Low-load blood flow restricted resistance exercise (BFRRE) has emerged as a time-effective low-load alternative to stimulate myofibrillar accretion. It is unknown if BFRRE can also stimulate mitochondrial biogenesis and respiratory function. If so, BFRRE could provide a feasible strategy to stimulate muscle metabolic health. Methods: To study this, 34 healthy previously untrained individuals (24 ± 3 years) participated in BFRRE, HLRE, or non-exercise control intervention (CON) 3 times per week for 6 weeks. Skeletal muscle biopsies were collected; (1) before and after the 6-week intervention period to assess mitochondrial biogenesis and respiratory function and; (2) during recovery from single-bout exercise to assess myocellular signaling events involved in transcriptional regulation of mitochondrial biogenesis. During the 6-week intervention period, deuterium oxide (D2O) was continuously administered to the participants to label newly synthesized skeletal muscle mitochondrial proteins. Mitochondrial respiratory function was assessed in permeabilized muscle fibers with high-resolution respirometry. Mitochondrial content was assessed with a citrate synthase activity assay. Myocellular signaling was assessed with immunoblotting. Results: Mitochondrial protein synthesis rate was higher with BFRRE (1.19%/day) and HLRE (1.15%/day) compared to CON (0.92%/day) (P < 0.05) but similar between exercise groups. Mitochondrial respiratory function increased to similar degree with both exercise regimens and did not change with CON. For instance, coupled respiration supported by convergent electron flow from complex I and II increased 38% with BFRRE and 24% with HLRE (P < 0.01). Training did not alter citrate synthase activity compared to CON. BFRRE and HLRE elicited similar myocellular signaling responses. Conclusion: These results support recent findings that resistance exercise can stimulate mitochondrial biogenesis and respiratory function to support healthy skeletal muscle and whole-body metabolism. Intriquingly, BFRRE produces similar mitochondrial adaptations at a markedly lower load, which entail great clinical perspective for populations in whom exercise with high loading is untenable.

Skeletal muscle mitochondrial protein synthesis and respiration in response to the energetic stress of an ultra-endurance race.

The 2016 Colorado Trail Race (CTR) was an ultra-endurance mountain bike race in which competitors cycled for up to 24 h/day between altitudes of 1,675 and 4,025 m to complete 800 km and 21,000 m of elevation gain. In one athlete, we had the unique opportunity to characterize skeletal muscle protein synthesis and mitochondrial respiration in response to a normal activity control period (CON) and the CTR. We hypothesized that mitochondrial protein synthesis would be elevated and mitochondrial respiration would be maintained during the extreme stresses of the CTR. Titrated and bolus doses of ADP were provided to determine substrate-specific oxidative phosphorylation (OXPHOS) and electron transport system (ETS) capacities in permeabilized muscle fibers via high-resolution respirometry. Protein synthetic rates were determined by daily oral consumption of deuterium oxide (2H2O). The endurance athlete had OXPHOS (226 pmol·s-1·mg tissue-1) and ETS (231 pmol·s-1·mg tissue-1) capacities that rank among the highest published to date in humans. Mitochondrial (3.2-fold), cytoplasmic (2.3-fold), and myofibrillar (1.5-fold) protein synthesis rates were greater during CTR compared with CON. With titrated ADP doses, the apparent Km of ADP, OXPHOS, and ETS increased after the CTR. With provision of ADP boluses after the CTR, the addition of fatty acids (-12 and -14%) mitigated the decline in OXPHOS and ETS capacity during carbohydrate-supported respiration (-26 and -31%). In the face of extreme stresses during the CTR, elevated rates of mitochondrial protein synthesis may contribute to rapid adaptations in mitochondrial bioenergetics. NEW & NOTEWORTHY The mechanisms that maintain skeletal muscle function during extreme stresses remain incompletely understood. In the current study, greater rates of mitochondrial protein synthesis during the energetic demands of ultra-endurance exercise may contribute to rapid adaptations in mitochondrial bioenergetics. The endurance athlete herein achieved mitochondrial respiratory capacities among the highest published for humans. Greater mitochondrial protein synthesis during ultra-endurance exercise may contribute to improved mitochondrial respiration and serve as a mechanism to resist cellular energetic stresses.

Influence of Nrf2 activators on subcellular skeletal muscle protein and DNA synthesis rates after 6 weeks of milk protein feeding in older adults.

In older adults, chronic oxidative and inflammatory stresses are associated with an impaired increase in skeletal muscle protein synthesis after acute anabolic stimuli. Conjugated linoleic acid (CLA) and Protandim have been shown to activate nuclear factor erythroid-derived 2-like 2 (Nrf2), a transcription factor for the antioxidant response element and anti-inflammatory pathways. This study tested the hypothesis that compared to a placebo control (CON), CLA and Protandim would increase skeletal muscle subcellular protein (myofibrillar, mitochondrial, cytoplasmic) and DNA synthesis in older adults after 6 weeks of milk protein feeding. CLA decreased oxidative stress and skeletal muscle oxidative damage with a trend to increase messenger RNA (mRNA) expression of a Nrf2 target, NAD(P)H dehydrogenase quinone 1 (NQO1). However, CLA did not influence other Nrf2 targets (heme oxygenase-1 (HO-1), glutathione peroxidase 1 (Gpx1)) or protein or DNA synthesis. Conversely, Protandim increased HO-1 protein content but not the mRNA expression of downstream Nrf2 targets, oxidative stress, or skeletal muscle oxidative damage. Rates of myofibrillar protein synthesis were maintained despite lower mitochondrial and cytoplasmic protein syntheses after Protandim versus CON. Similarly, DNA synthesis was non-significantly lower after Protandim compared to CON. After Protandim, the ratio of protein to DNA synthesis tended to be greater in the myofibrillar fraction and maintained in the mitochondrial and cytoplasmic fractions, emphasizing the importance of measuring both protein and DNA synthesis to gain insight into proteostasis. Overall, these data suggest that Protandim may enhance proteostatic mechanisms of skeletal muscle contractile proteins after 6 weeks of milk protein feeding in older adults.