High-Resolution Fluorespirometry to Assess Dynamic Changes in Mitochondrial Membrane Potential in Human Immune Cells.

Peripheral mononuclear cells (PBMCs) exhibit robust changes in mitochondrial respiratory capacity in response to health and disease. While these changes do not always reflect what occurs in other tissues, such as skeletal muscle, these cells are an accessible and valuable source of viable mitochondria from human subjects. PBMCs are exposed to systemic signals that impact their bioenergetic state. Thus, expanding our tools to interrogate mitochondrial metabolism in this population will elucidate mechanisms related to disease progression. Functional assays of mitochondria are often limited to using respiratory outputs following maximal substrate, inhibitor, and uncoupler concentrations to determine the full range of respiratory capacity, which may not be achievable in vivo. The conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) by ATP-synthase results in a decrease in mitochondrial membrane potential (mMP) and an increase in oxygen consumption. To provide a more integrated analysis of mitochondrial dynamics, this article describes the use of high-resolution fluorespirometry to measure the simultaneous response of oxygen consumption and mitochondrial membrane potential (mMP) to physiologically relevant concentrations of ADP. This technique uses tetramethylrhodamine methylester (TMRM) to measure mMP polarization in response to ADP titrations following maximal hyperpolarization with complex I and II substrates. This technique can be used to quantify how changes in health status, such as aging and metabolic disease, affect the sensitivity of mitochondrial response to energy demand in PBMCs, T-cells, and monocytes from human subjects.

Time-restricted feeding improves aortic endothelial relaxation by enhancing mitochondrial function and attenuating oxidative stress in aged mice.

Age-related endothelial dysfunction is a pivotal factor in the development of cardiovascular diseases, stemming, at least in part, from mitochondrial dysfunction and a consequential increase in oxidative stress. These alterations are central to the decline in vascular health seen with aging, underscoring the urgent need for interventions capable of restoring endothelial function for preventing cardiovascular diseases. Dietary interventions, notably time-restricted feeding (TRF), have been identified for their anti-aging effects on mitochondria, offering protection against age-associated declines in skeletal muscle and other organs. Motivated by these findings, our study aimed to investigate whether TRF could similarly exert protective effects on endothelial health in the vasculature, enhancing mitochondrial function and reducing oxidative stress. To explore this, 12-month-old C57BL/6 mice were placed on a TRF diet, with food access limited to a 6-h window daily for 12 months. For comparison, we included groups of young mice and age-matched controls with unrestricted feeding. We evaluated the impact of TRF on endothelial function by measuring acetylcholine-induced vasorelaxation of the aorta. Mitochondrial health was assessed using fluororespirometry, and vascular reactive oxygen species (ROS) production was quantified with the redox-sensitive dye dihydroethidium. We also quantified 4-hydroxynonenal (4-HNE) levels, a stable marker of lipid peroxidation, in the aorta using ELISA. Our findings demonstrated that aged mice on a standard diet exhibited significant impairments in aortic endothelial relaxation and mitochondrial function, associated with elevated vascular oxidative stress. Remarkably, the TRF regimen led to substantial improvements in these parameters, indicating enhanced endothelial vasorelaxation, better mitochondrial function, and reduced oxidative stress in the aortas of aged mice. This investigation establishes a vital foundation, paving the way for subsequent clinical research aimed at exploring the cardiovascular protective benefits of intermittent fasting.

Role of Cardiorespiratory Fitness and Mitochondrial Oxidative Capacity in Reduced Walk Speed of Older Adults with Diabetes.

Cardiorespiratory fitness and mitochondrial oxidative capacity are associated with reduced walking speed in older adults. The impact of cardiorespiratory fitness and mitochondrial oxidative capacity on walking speed in older adults with diabetes has not been clearly defined. We examined differences in cardiorespiratory fitness and skeletal muscle mitochondrial oxidative capacity between older adults with and without diabetes as well as determine their relative contribution to slower walking speed in older adults with diabetes. Participants with diabetes (n=159) had lower cardiorespiratory fitness and mitochondrial respiration in permeabilized fiber bundles when compared to those without diabetes (n=717), following adjustments for covariates including BMI, chronic comorbid health conditions, and physical activity. 4-m and 400-m walking speeds were slower in those with diabetes. Mitochondrial oxidative capacity alone or combined with cardiorespiratory fitness mediated ∼20-70% of the difference in walk speed between older adults with and without diabetes. Additional adjustments with BMI and co-morbidities further explained the group differences in walk speed. Cardiorespiratory fitness and skeletal muscle mitochondrial oxidative capacity contribute to slower walking speeds in older adults with diabetes.

Childhood adverse life events and skeletal muscle mitochondrial function.

Social stress experienced in childhood is associated with adverse health later in life. Mitochondrial function has been implicated as a mechanism for how stressful life events "get under the skin" to influence physical well-being. Using data from the Study of Muscle, Mobility, and Aging (n = 879, 59% women), linear models examined whether adverse childhood events (i.e., physical abuse) were associated with two measures of skeletal muscle mitochondrial energetics in older adults: (i) maximal adenosine triphosphate production (ATPmax) and (ii) maximal state 3 respiration (Max OXPHOS). Forty-five percent of the sample reported experiencing one or more adverse childhood events. After adjustment, each additional event was associated with -0.08 SD (95% confidence interval = -0.13, -0.02) lower ATPmax. No association was observed with Max OXPHOS. Adverse childhood events are associated with lower ATP production in later life. Findings indicate that mitochondrial function may be a mechanism for understanding how early social stress influences health in later life.

Signatures of cysteine oxidation on muscle structural and contractile proteins are associated with physical performance and muscle function in older adults: Study of Muscle, Mobility and Aging (SOMMA).

Oxidative stress is considered a contributor to declining muscle function and mobility during aging; however, the underlying molecular mechanisms remain poorly described. We hypothesized that greater levels of cysteine (Cys) oxidation on muscle proteins are associated with decreased measures of mobility. Herein, we applied a novel redox proteomics approach to measure reversible protein Cys oxidation in vastus lateralis muscle biopsies collected from 56 subjects in the Study of Muscle, Mobility and Aging (SOMMA), a community-based cohort study of individuals aged 70 years and older. We tested whether levels of Cys oxidation on key muscle proteins involved in muscle structure and contraction were associated with muscle function (leg power and strength), walking speed, and fitness (VO2 peak on cardiopulmonary exercise testing) using linear regression models adjusted for age, sex, and body weight. Higher oxidation levels of select nebulin Cys sites were associated with lower VO2 peak, while greater oxidation of myomesin-1, myomesin-2, and nebulin Cys sites was associated with slower walking speed. Higher oxidation of Cys sites in key proteins such as myomesin-2, alpha-actinin-2, and skeletal muscle alpha-actin were associated with lower leg power and strength. We also observed an unexpected correlation (R = 0.48) between a higher oxidation level of eight Cys sites in alpha-actinin-3 and stronger leg power. Despite this observation, the results generally support the hypothesis that Cys oxidation of muscle proteins impairs muscle power and strength, walking speed, and cardiopulmonary fitness with aging.

Energetics and clinical factors for the time required to walk 400 m: The Study of Muscle, Mobility and Aging (SOMMA).

BACKGROUND: Walking slows with aging often leading to mobility disability. Mitochondrial energetics has been found to be associated with gait speed over short distances. Additionally, walking is a complex activity but few clinical factors that may be associated with walk time have been studied. METHODS: We examined 879 participants ≥70 years and measured the time to walk 400 m. We tested the hypothesis that decreased mitochondrial energetics by respirometry in muscle biopsies and magnetic resonance spectroscopy in the thigh and is associated with longer time to walk 400 m. We also used cardiopulmonary exercise testing to assess the energetic costs of walking: maximum oxygen consumption (VO2peak) and energy cost-capacity (the ratio of VO2, at a slow speed to VO2peak). In addition, we tested the hypothesis that selected clinical factors would also be associated with 400-m walk time. RESULTS: Lower Max OXPHOS was associated with longer walk time, and the association was explained by the energetic costs of walking, leg power, and weight. Additionally, a multivariate model revealed that longer walk time was also significantly associated with lower VO2peak, greater cost-capacity ratio, weaker leg power, heavier weight, hip and knee stiffness, peripheral neuropathy, greater perceived exertion while walking slowly, greater physical fatigability, less moderate-to-vigorous exercise, less sedentary time, and anemia. Significant associations between age, sex, muscle mass, and peripheral artery disease with 400-m walk time were explained by other clinical and physiologic factors. CONCLUSIONS: Lower mitochondrial energetics is associated with needing more time to walk 400 m. This supports the value of developing interventions to improve mitochondrial energetics. Additionally, doing more moderate-to-vigorous exercise, increasing leg power, reducing weight, treating hip and knee stiffness, and screening for and treating anemia may reduce the time required to walk 400 m and reduce the risk of mobility disability.

Mitochondrial Targeted Interventions for Aging.

Changes in mitochondrial function play a critical role in the basic biology of aging and age-related disease. Mitochondria are typically thought of in the context of ATP production and oxidant production. However, it is clear that the mitochondria sit at a nexus of cell signaling where they affect metabolite, redox, and energy status, which influence many factors that contribute to the biology of aging, including stress responses, proteostasis, epigenetics, and inflammation. This has led to growing interest in identifying mitochondrial targeted interventions to delay or reverse age-related decline in function and promote healthy aging. In this review, we discuss the diverse roles of mitochondria in the cell. We then highlight some of the most promising strategies and compounds to target aging mitochondria in preclinical testing. Finally, we review the strategies and compounds that have advanced to clinical trials to test their ability to improve health in older adults.

Lower muscle mitochondrial energetics is associated with greater phenotypic frailty in older women and men: the Study of Muscle, Mobility and Aging.

BACKGROUND: Phenotypic frailty syndrome identifies older adults at greater risk for adverse health outcomes. Despite the critical role of mitochondria in maintaining cellular function, including energy production, the associations between muscle mitochondrial energetics and frailty have not been widely explored in a large, well-phenotyped, older population. METHODS: The Study of Muscle, Mobility and Aging (SOMMA) assessed muscle energetics in older adults (N = 879, mean age = 76.3 years, 59.2% women). 31Phosporous magnetic resonance spectroscopy measured maximal production of adenosine triphosphate (ATPmax) in vivo, while ex vivo high-resolution respirometry of permeabilized muscle fibers from the vastus lateralis measured maximal oxygen consumption supported by fatty acids and complex I- and II-linked carbohydrates (e.g., Max OXPHOSCI+CII). Five frailty criteria, shrinking, weakness, exhaustion, slowness, and low activity, were used to classify participants as robust (0, N = 397), intermediate (1-2, N = 410), or frail (≥ 3, N = 66). We estimated the proportional odds ratio (POR) for greater frailty, adjusted for multiple potential confounders. RESULTS: One-SD decrements of most respirometry measures (e.g., Max OXPHOSCI+CII, adjusted POR = 1.5, 95%CI [1.2,1.8], p = 0.0001) were significantly associated with greater frailty classification. The associations of ATPmax with frailty were weaker than those between Max OXPHOSCI+CII and frailty. Muscle energetics was most strongly associated with slowness and low physical activity components. CONCLUSIONS: Our data suggest that deficits in muscle mitochondrial energetics may be a biological driver of frailty in older adults. On the other hand, we did observe differential relationships between measures of muscle mitochondrial energetics and the individual components of frailty.

Skeletal Muscle Energetics Explain the Sex Disparity in Mobility Impairment in the Study of Muscle, Mobility and Aging.

The age-related decline in muscle mitochondrial energetics contributes to the loss of mobility in older adults. Women experience a higher prevalence of mobility impairment compared to men, but it is unknown whether sex-specific differences in muscle energetics underlie this disparity. In the Study of Muscle, Mobility and Aging (SOMMA), muscle energetics were characterized using in vivo phosphorus-31 magnetic resonance spectroscopy and high-resolution respirometry of vastus lateralis biopsies in 773 participants (56.4% women, age 70-94 years). A Short Physical Performance Battery (SPPB) score ≤8 was used to define lower-extremity mobility impairment. Muscle mitochondrial energetics were lower in women compared to men (eg, Maximal Complex I&II OXPHOS: Women = 55.06 ± 15.95; Men = 65.80 ± 19.74; p < .001) and in individuals with mobility impairment compared to those without (eg, Maximal Complex I&II OXPHOS in women: SPPB ≥ 9 = 56.59 ± 16.22; SPPB ≤ 8 = 47.37 ± 11.85; p < .001). Muscle energetics were negatively associated with age only in men (eg, Maximal ETS capacity: R = -0.15, p = .02; age/sex interaction, p = .04), resulting in muscle energetics measures that were significantly lower in women than men in the 70-79 age group but not the 80+ age group. Similarly, the odds of mobility impairment were greater in women than men only in the 70-79 age group (70-79 age group, odds ratio [OR]age-adjusted = 1.78, 95% confidence interval [CI] = 1.03, 3.08, p = .038; 80+ age group, ORage-adjusted = 1.05, 95% CI = 0.52, 2.15, p = .89). Accounting for muscle energetics attenuated up to 75% of the greater odds of mobility impairment in women. Women had lower muscle mitochondrial energetics compared to men, which largely explain their greater odds of lower-extremity mobility impairment.

Signatures of Cysteine Oxidation on Muscle Structural and Contractile Proteins Are Associated with Physical Performance and Muscle Function in Older Adults: Study of Muscle, Mobility and Aging (SOMMA).

Oxidative stress is considered a contributor to declining muscle function and mobility during aging; however, the underlying molecular mechanisms remain poorly described. We hypothesized that greater levels of cysteine (Cys) oxidation on muscle proteins are associated with decreased measures of mobility. Herein, we applied a novel redox proteomics approach to measure reversible protein Cys oxidation in vastus lateralis muscle biopsies collected from 56 subjects in the Study of Muscle, Mobility and Aging (SOMMA), a community-based cohort study of individuals aged 70 years and older. We tested whether levels of Cys oxidation on key muscle proteins involved in muscle structure and contraction were associated with muscle function (leg power and strength), walking speed, and fitness (VO2 peak on cardiopulmonary exercise testing) using linear regression models adjusted for age, sex, and body weight. Higher oxidation levels of select nebulin Cys sites were associated with lower VO2 peak, while greater oxidation of myomesin-1, myomesin-2, and nebulin Cys sites was associated with slower walking speed. Higher oxidation of Cys sites in key proteins such as myomesin-2, alpha-actinin-2, and skeletal muscle alpha-actin were associated with lower leg power and strength. We also observed an unexpected correlation (r = 0.48) between a higher oxidation level of 8 Cys sites in alpha-actinin-3 and stronger leg power. Despite this observation, the results generally support the hypothesis that Cys oxidation of muscle proteins impair muscle power and strength, walking speed, and cardiopulmonary fitness with aging.

Role of Cardiorespiratory Fitness and Mitochondrial Energetics in Reduced Walk Speed of Older Adults with Diabetes in the Study of Muscle, Mobility and Aging (SOMMA).

Rationale: Cardiorespiratory fitness and mitochondrial energetics are associated with reduced walking speed in older adults. The impact of cardiorespiratory fitness and mitochondrial energetics on walking speed in older adults with diabetes has not been clearly defined. Objective: To examine differences in cardiorespiratory fitness and skeletal muscle mitochondrial energetics between older adults with and without diabetes. We also assessed the contribution of cardiorespiratory fitness and skeletal muscle mitochondrial energetics to slower walking speed in older adults with diabetes. Findings: Participants with diabetes had lower cardiorespiratory fitness and mitochondrial energetics when compared to those without diabetes, following adjustments for covariates including BMI, chronic comorbid health conditions, and physical activity. 4-m and 400-m walking speeds were slower in those with diabetes. Mitochondrial oxidative capacity alone or combined with cardiorespiratory fitness mediated ∼20-70% of the difference in walk speed between older adults with and without diabetes. Further adjustments of BMI and co-morbidities further explained the group differences in walk speed. Conclusions: Skeletal muscle mitochondrial energetics and cardiorespiratory fitness contribute to slower walking speeds in older adults with diabetes. Cardiorespiratory fitness and mitochondrial energetics may be therapeutic targets to maintain or improve mobility in older adults with diabetes. ARTICLE HIGHLIGHTS: Why did we undertake this study? To determine if mitochondrial energetics and cardiorespiratory fitness contribute to slower walking speed in older adults with diabetes. What is the specific question(s) we wanted to answer? Are mitochondrial energetics and cardiorespiratory fitness in older adults with diabetes lower than those without diabetes? How does mitochondrial energetics and cardiorespiratory fitness impact walking speed in older adults with diabetes? What did we find? Mitochondrial energetics and cardiorespiratory fitness were lower in older adults with diabetes compared to those without diabetes, and energetics, and cardiorespiratory fitness, contributed to slower walking speed in those with diabetes. What are the implications of our findings? Cardiorespiratory fitness and mitochondrial energetics may be key therapeutic targets to maintain or improve mobility in older adults with diabetes.

Energetics and Clinical Factors for the Time Required to Walk 400 Meters The Study of Muscle, Mobility and Aging (SOMMA).

Background: Walking slows with aging often leading to mobility disability. Mitochondrial energetics has been found to influence gait speed over short distances. Additionally, walking is a complex activity but few clinical factors that may influence walk time have been studied. Methods: We examined 879 participants ≥70 years and measured the time to walk 400m. We tested the hypothesis that decreased mitochondrial energetics by respirometry in muscle biopsies and magnetic resonance spectroscopy in the thigh, is associated with longer time to walk 400m. We also used cardiopulmonary exercise testing to assess the energetic costs of walking: maximum oxygen consumption (VO 2 peak) and energy cost-capacity (the ratio of VO2, at a slow speed to VO 2 peak). In addition, we tested the hypothesis that selected clinical factors would also be associated with 400m walk time. Results: Lower Max OXPHOS was associated with longer walk time and the association was explained by the energetics costs of walking, leg power and weight. Additionally, a multivariate model revealed that longer walk time was also significantly associated with lower VO 2 peak, greater cost-capacity ratio, weaker leg power, heavier weight, hip and knee stiffness, peripheral neuropathy, greater perceived exertion while walking slowly, greater physical fatigability, less moderate-to-vigorous exercise, less sedentary time and anemia. Significant associations between age, sex, muscle mass, and peripheral artery disease with 400m walk time were explained by other clinical and physiologic factors. Conclusions: Lower mitochondrial energetics is associated with needing more time to walk 400m. This supports the value of developing interventions to improve mitochondrial energetics. Additionally, doing more moderate-to-vigorous exercise, increasing leg power, reducing weight, treating hip and knee stiffness, and screening for and treating anemia may reduce the time required to walk 400m and reduce the risk of mobility disability.

Childhood adverse life events and skeletal muscle mitochondrial function.

Social stress experienced in childhood is associated with adverse health later in life. Mitochondrial function has been implicated as a mechanism for how stressful life events "get under the skin" to influence physical wellbeing. Using data from the Study of Muscle, Mobility and Aging (n=879, 59% women), linear models examined whether adverse childhood events (i.e., physical abuse) were associated with two measures of skeletal muscle mitochondrial energetics in older adults: (1) maximal adenosine triphosphate production (ATP max ) and (2) maximal state 3 respiration (Max OXPHOS). Forty-five percent of the sample reported experiencing 1+ adverse childhood event. After adjustment, each additional event was associated with -0.07 SD (95% CI= - 0.12, -0.01) lower ATP max . No association was observed with Max OXPHOS. Adverse childhood events are associated with lower ATP production in later life. Findings indicate that mitochondrial function may be a mechanism in understanding how early social stress influences health in later life.

Skeletal muscle energetics explain the sex disparity in mobility impairment in the Study of Muscle, Mobility and Aging (SOMMA).

The age-related decline in muscle mitochondrial energetics contributes to the loss of mobility in older adults. Women experience a higher prevalence of mobility impairment compared to men, but it is unknown whether sex-specific differences in muscle energetics underlie this disparity. In the Study of Muscle, Mobility and Aging (SOMMA), muscle energetics were characterized using in vivo phosphorus-31 magnetic resonance spectroscopy and high-resolution respirometry of vastus lateralis biopsies in 773 participants (56.4% women, age 70-94 years). A Short Physical Performance Battery score ≤ 8 was used to define lower-extremity mobility impairment. Muscle mitochondrial energetics were lower in women compared to men (e.g. Maximal Complex I&II OXPHOS: Women=55.06 +/- 15.95; Men=65.80 +/- 19.74; p<0.001) and in individuals with mobility impairment compared to those without (e.g., Maximal Complex I&II OXPHOS in women: SPPB≥9=56.59 +/- 16.22; SPPB≤8=47.37 +/- 11.85; p<0.001). Muscle energetics were negatively associated with age only in men (e.g., Maximal ETS capacity: R=-0.15, p=0.02; age/sex interaction, p=0.04), resulting in muscle energetics measures that were significantly lower in women than men in the 70-79 age group but not the 80+ age group. Similarly, the odds of mobility impairment were greater in women than men only in the 70-79 age group (70-79 age group, OR age-adjusted =1.78, 95% CI=1.03, 3.08, p=0.038; 80+ age group, OR age-adjusted =1.05, 95% CI=0.52, 2.15, p=0.89). Accounting for muscle energetics attenuated up to 75% of the greater odds of mobility impairment in women. Women had lower muscle mitochondrial energetics compared to men, which largely explain their greater odds of lower-extremity mobility impairment.

Age-related changes of skeletal muscle metabolic response to contraction are also sex-dependent.

Mitochondria adapt to increased energy demands during muscle contraction by acutely altering metabolite fluxes and substrate oxidation. With age, an impaired mitochondrial metabolic response may contribute to reduced exercise tolerance and decreased skeletal muscle mass, specific force, increased overall fatty depositions in the skeletal muscle, frailty and depressed energy maintenance. We hypothesized that elevated energy stress in mitochondria with age alters the capacity of mitochondria to utilize different substrates following muscle contraction. To test this hypothesis, we used in vivo electrical stimulation to simulate high-intensity intervals (HII) or low intensity steady-state (LISS) exercise in young (5-7 months) and aged (27-29 months) male and female mice to characterize effects of age and sex on mitochondrial substrate utilization in skeletal muscle following contraction. Mitochondrial respiration using glutamate decreased in aged males following HII and glutamate oxidation was inhibited following HII in both the contracted and non-stimulated muscle of aged female muscle. Analyses of the muscle metabolome of female mice indicated that changes in metabolic pathways induced by HII and LISS contractions in young muscle are absent in aged muscle. To test improved mitochondrial function on substrate utilization following HII, we treated aged females with elamipretide (ELAM), a mitochondrially-targeted peptide shown to improve mitochondrial bioenergetics and restore redox status in aged muscle. ELAM removed inhibition of glutamate oxidation and showed increased metabolic pathway changes following HII, suggesting rescuing redox status and improving bioenergetic function in mitochondria from aged muscle increases glutamate utilization and enhances the metabolic response to muscle contraction in aged muscle. KEY POINTS: Acute local contraction of gastrocnemius can systemically alter mitochondrial respiration in non-stimulated muscle. Age-related changes in mitochondrial respiration using glutamate or palmitoyl carnitine following contraction are sex-dependent. Respiration using glutamate after high-intensity contraction is inhibited in aged female muscle. Metabolite level and pathway changes following muscle contraction decrease with age in female mice. Treatment with the mitochondrially-targeted peptide elamipretide can partially rescue metabolite response to muscle contraction.

The mitochondrially targeted peptide elamipretide (SS-31) improves ADP sensitivity in aged mitochondria by increasing uptake through the adenine nucleotide translocator (ANT).

Aging muscle experiences functional decline in part mediated by impaired mitochondrial ADP sensitivity. Elamipretide (ELAM) rapidly improves physiological and mitochondrial function in aging and binds directly to the mitochondrial ADP transporter ANT. We hypothesized that ELAM improves ADP sensitivity in aging leading to rescued physiological function. We measured the response to ADP stimulation in young and old muscle mitochondria with ELAM treatment, in vivo heart and muscle function, and compared protein abundance, phosphorylation, and S-glutathionylation of ADP/ATP pathway proteins. ELAM treatment increased ADP sensitivity in old muscle mitochondria by increasing uptake of ADP through the ANT and rescued muscle force and heart systolic function. Protein abundance in the ADP/ATP transport and synthesis pathway was unchanged, but ELAM treatment decreased protein s-glutathionylation incuding of ANT. Mitochondrial ADP sensitivity is rapidly modifiable. This research supports the hypothesis that ELAM improves ANT function in aging and links mitochondrial ADP sensitivity to physiological function. ELAM binds directly to ANT and ATP synthase and ELAM treatment improves ADP sensitivity, increases ATP production, and improves physiological function in old muscles. ADP (adenosine diphosphate), ATP (adenosine triphosphate), VDAC (voltage-dependent anion channel), ANT (adenine nucleotide translocator), H+ (proton), ROS (reactive oxygen species), NADH (nicotinamide adenine dinucleotide), FADH2 (flavin adenine dinucleotide), O2 (oxygen), ELAM (elamipretide), -SH (free thiol), -SSG (glutathionylated protein).

A novel mitochondrial complex I ROS inhibitor partially improves muscle regeneration in adult but not old mice.

It is unclear whether mitochondrial dysfunction and redox stress contribute to impaired age-related muscle regenerative capacity. Here we characterized a novel compound, BI4500, that inhibits the release of reactive oxygen species (ROS) from the quinone site in mitochondrial complex I (site IQ). We tested the hypothesis that ROS release from site IQ contributes to impaired regenerative capacity in aging muscle. Electron transfer system site-specific ROS production was measured in adult and aged mouse isolated muscle mitochondria and permeabilized gastrocnemius fibers. BI4500 inhibited ROS production from site IQ in a concentration-dependent manner (IC50 = âˆ¼985 nM) by inhibiting ROS release without impairing complex I-linked respiration. In vivo BI4500 treatment decreased ROS production from site IQ. Muscle injury and sham injury were induced using barium chloride or vehicle injection to the tibialis anterior (TA) muscle in adult and aged male mice. On the same day as injury, mice began a daily gavage of 30 mg/kg BI4500 (BI) or placebo (PLA). Muscle regeneration (H&E, Sirius Red, Pax7) was measured at 5 and 35 days after injury. Muscle injury increased centrally nucleated fibers (CNFs) and fibrosis with no treatment or age effect. There was a significant age by treatment interaction for CNFs at 5- and 35-days post injury with significantly more CNFs in BI adults compared to PLA adults. Muscle fiber cross-sectional area (CSA) recovered significantly more in adult BI mice (-89 ± 365 µm2) compared to old PLA (-599 ± 153 µm2) and old BI (-535 ± 222 µm2, mean ± SD). In situ TA force recovery was measured 35 days after injury and was not significantly different by age or treatment. Inhibition of site IQ ROS partially improves muscle regeneration in adult but not old muscle demonstrating a role for CI ROS in the response to muscle injury. Site IQ ROS does not contribute to impaired regenerative capacity in aging.

Randomized crossover clinical trial of coenzyme Q10 and nicotinamide riboside in chronic kidney disease.

BackgroundCurrent studies suggest mitochondrial dysfunction is a major contributor to impaired physical performance and exercise intolerance in chronic kidney disease (CKD). We conducted a clinical trial of coenzyme Q10 (CoQ10) and nicotinamide riboside (NR) to determine their impact on exercise tolerance and metabolic profile in patients with CKD.MethodsWe conducted a randomized, placebo-controlled, double-blind, crossover trial comparing CoQ10, NR, and placebo in 25 patients with an estimated glomerular filtration rate (eGFR) of less than 60mL/min/1.73 m2. Participants received NR (1,000 mg/day), CoQ10 (1,200 mg/day), or placebo for 6 weeks each. The primary outcomes were aerobic capacity measured by peak rate of oxygen consumption (VO2 peak) and work efficiency measured using graded cycle ergometry testing. We performed semitargeted plasma metabolomics and lipidomics.ResultsParticipant mean age was 61.0 ± 11.6 years and mean eGFR was 36.9 ± 9.2 mL/min/1.73 m2. Compared with placebo, we found no differences in VO2 peak (P = 0.30, 0.17), total work (P = 0.47, 0.77), and total work efficiency (P = 0.46, 0.55) after NR or CoQ10 supplementation. NR decreased submaximal VO2 at 30 W (P = 0.03) and VO2 at 60 W (P = 0.07) compared with placebo. No changes in eGFR were observed after NR or CoQ10 treatment (P = 0.14, 0.88). CoQ10 increased free fatty acids and decreased complex medium- and long-chain triglycerides. NR supplementation significantly altered TCA cycle intermediates and glutamate that were involved in reactions that exclusively use NAD+ and NADP+ as cofactors. NR decreased a broad range of lipid groups including triglycerides and ceramides.ConclusionsSix weeks of treatment with NR or CoQ10 improved markers of systemic mitochondrial metabolism and lipid profiles but did not improve VO2 peak or total work efficiency.Trial registrationClinicalTrials.gov NCT03579693.FundingNational Institutes of Diabetes and Digestive and Kidney Diseases (grants R01 DK101509, R03 DK114502, R01 DK125794, and R01 DK101509).

Age and Sex as Determinants of Acute Domoic Acid Toxicity in a Mouse Model.

The excitatory neurotoxin domoic acid (DA) consistently contaminates food webs in coastal regions around the world. Acute exposure to the toxin causes Amnesic Shellfish Poisoning, a potentially lethal syndrome of gastrointestinal- and seizure-related outcomes. Both advanced age and male sex have been suggested to contribute to interindividual DA susceptibility. To test this, we administered DA doses between 0.5 and 2.5 mg/kg body weight to female and male C57Bl/6 mice at adult (7-9-month-old) and aged (25-28-month-old) life stages and observed seizure-related activity for 90 min, at which point we euthanized the mice and collected serum, cortical, and kidney samples. We observed severe clonic-tonic convulsions in some aged individuals, but not in younger adults. We also saw an association between advanced age and the incidence of a moderately severe seizure-related outcome, hindlimb tremors, and between advanced age and overall symptom severity and persistence. Surprisingly, we additionally report that female mice, particularly aged female mice, demonstrated more severe neurotoxic symptoms following acute exposure to DA than males. Both age and sex patterns were reflected in tissue DA concentrations as well: aged mice and females had generally higher concentrations of DA in their tissues at 90 min post-exposure. This study contributes to the body of work that can inform intelligent, evidence-based public health protections for communities threatened by more frequent and extensive DA-producing algal blooms.

Skeletal muscle mitochondrial interactome remodeling is linked to functional decline in aged female mice.

Genomic, transcriptomic and proteomic approaches have been used to gain insight into molecular underpinnings of aging in laboratory animals and in humans. However, protein function in biological systems is under complex regulation and includes factors besides abundance levels, such as modifications, localization, conformation and protein-protein interactions. By making use of quantitative chemical cross-linking technologies, we show that changes in the muscle mitochondrial interactome contribute to mitochondrial functional decline in aging in female mice. Specifically, we identify age-related changes in protein cross-links relating to assembly of electron transport system complexes I and IV, activity of glutamate dehydrogenase, and coenzyme-A binding in fatty acid ß-oxidation and tricarboxylic acid cycle enzymes. These changes show a remarkable correlation with complex I respiration differences within the same young-old animal pairs. Each observed cross-link can serve as a protein conformational or protein-protein interaction probe in future studies, which will provide further molecular insights into commonly observed age-related phenotypic differences. Therefore, this data set could become a valuable resource for additional in-depth molecular studies that are needed to better understand complex age-related molecular changes.