dATP elevation induces myocardial metabolic remodeling to support improved cardiac function.

Hallmark features of systolic heart failure are reduced contractility and impaired metabolic flexibility of the myocardium. Cardiomyocytes (CMs) with elevated deoxy ATP (dATP) via overexpression of ribonucleotide reductase (RNR) enzyme robustly improve contractility. However, the effect of dATP elevation on cardiac metabolism is unknown. Here, we developed proteolysis-resistant versions of RNR and demonstrate that elevation of dATP/ATP to ∼1% in CMs in a transgenic mouse (TgRRB) resulted in robust improvement of cardiac function. Pharmacological approaches showed that CMs with elevated dATP have greater basal respiratory rates by shifting myosin states to more active forms, independent of its isoform, in relaxed CMs. Targeted metabolomic profiling revealed a significant reprogramming towards oxidative phosphorylation in TgRRB-CMs. Higher cristae density and activity in the mitochondria of TgRRB-CMs improved respiratory capacity. Our results revealed a critical property of dATP to modulate myosin states to enhance contractility and induce metabolic flexibility to support improved function in CMs.

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.

A replication-linked mutational gradient drives somatic mutation accumulation and influences germline polymorphisms and genome composition in mitochondrial DNA.

Mutations in mitochondrial DNA (mtDNA) cause maternally inherited diseases, while somatic mutations are linked to common diseases of aging. Although mtDNA mutations impact health, the processes that give rise to them are under considerable debate. To investigate the mechanism by which de novo mutations arise, we analyzed the distribution of naturally occurring somatic mutations across the mouse and human mtDNA obtained by Duplex Sequencing. We observe distinct mutational gradients in G→A and T→C transitions delimited by the light-strand origin and the mitochondrial Control Region (mCR). The gradient increases unequally across the mtDNA with age and is lost in the absence of DNA polymerase γ proofreading activity. In addition, high-resolution analysis of the mCR shows that important regulatory elements exhibit considerable variability in mutation frequency, consistent with them being mutational 'hot-spots' or 'cold-spots'. Collectively, these patterns support genome replication via a deamination prone asymmetric strand-displacement mechanism as the fundamental driver of mutagenesis in mammalian DNA. Moreover, the distribution of mtDNA single nucleotide polymorphisms in humans and the distribution of bases in the mtDNA across vertebrate species mirror this gradient, indicating that replication-linked mutations are likely the primary source of inherited polymorphisms that, over evolutionary timescales, influences genome composition during speciation.

Mitochondrial protein interaction landscape of SS-31.

Mitochondrial dysfunction underlies the etiology of a broad spectrum of diseases including heart disease, cancer, neurodegenerative diseases, and the general aging process. Therapeutics that restore healthy mitochondrial function hold promise for treatment of these conditions. The synthetic tetrapeptide, elamipretide (SS-31), improves mitochondrial function, but mechanistic details of its pharmacological effects are unknown. Reportedly, SS-31 primarily interacts with the phospholipid cardiolipin in the inner mitochondrial membrane. Here we utilize chemical cross-linking with mass spectrometry to identify protein interactors of SS-31 in mitochondria. The SS-31-interacting proteins, all known cardiolipin binders, fall into two groups, those involved in ATP production through the oxidative phosphorylation pathway and those involved in 2-oxoglutarate metabolic processes. Residues cross-linked with SS-31 reveal binding regions that in many cases, are proximal to cardiolipin-protein interacting regions. These results offer a glimpse of the protein interaction landscape of SS-31 and provide mechanistic insight relevant to SS-31 mitochondrial therapy.

Ice seals as sentinels for algal toxin presence in the Pacific Arctic and subarctic marine ecosystems.

Domoic acid (DA) and saxitoxin (STX)-producing algae are present in Alaskan seas, presenting exposure risks to marine mammals that may be increasing due to climate change. To investigate potential increases in exposure risks to four pagophilic ice seal species (Erignathus barbatus, bearded seals; Pusa hispida, ringed seals; Phoca largha, spotted seals; and Histriophoca fasciata, ribbon seals), this study analyzed samples from 998 seals harvested for subsistence purposes in western and northern Alaska during 2005-2019 for DA and STX. Both toxins were detected in bearded, ringed, and spotted seals, though no clinical signs of acute neurotoxicity were reported in harvested seals. Bearded seals had the highest prevalence of each toxin, followed by ringed seals. Bearded seal stomach content samples from the Bering Sea showed a significant increase in DA prevalence with time (logistic regression, p = .004). These findings are consistent with predicted northward expansion of DA-producing algae. A comparison of paired samples taken from the stomachs and colons of 15 seals found that colon content consistently had higher concentrations of both toxins. Collectively, these results suggest that ice seals, particularly bearded seals (benthic foraging specialists), are suitable sentinels for monitoring HAB prevalence in the Pacific Arctic and subarctic.

Mitochondrial protein interactome elucidated by chemical cross-linking mass spectrometry.

Mitochondrial protein interactions and complexes facilitate mitochondrial function. These complexes range from simple dimers to the respirasome supercomplex consisting of oxidative phosphorylation complexes I, III, and IV. To improve understanding of mitochondrial function, we used chemical cross-linking mass spectrometry to identify 2,427 cross-linked peptide pairs from 327 mitochondrial proteins in whole, respiring murine mitochondria. In situ interactions were observed in proteins throughout the electron transport chain membrane complexes, ATP synthase, and the mitochondrial contact site and cristae organizing system (MICOS) complex. Cross-linked sites showed excellent agreement with empirical protein structures and delivered complementary constraints for in silico protein docking. These data established direct physical evidence of the assembly of the complex I-III respirasome and enabled prediction of in situ interfacial regions of the complexes. Finally, we established a database and tools to harness the cross-linked interactions we observed as molecular probes, allowing quantification of conformation-dependent protein interfaces and dynamic protein complex assembly.

Evaluation of in vivo mitochondrial bioenergetics in skeletal muscle using NMR and optical methods.

It is now clear that mitochondria are involved as either a cause or consequence of many chronic diseases. This central role of the mitochondria is due to their position in the cell as important integrators of cellular energetics and signaling. Mitochondrial function affects many aspects of the cellular environment such as redox homeostasis and calcium signaling, which then also exert control over mitochondrial function. This complex dynamic between mitochondrial function and the cellular environment highlights the value of examining mitochondria in vivo in the intact physiological environment. This review discusses NMR and optical approaches used to measure mitochondria ATP and oxygen fluxes that provide in vivo measures of mitochondrial capacity and quality in animal and human models. Combining these in vivo measurements with more traditional ex vivo analyses can lead to new insights into the importance of the cellular environment in controlling mitochondrial function under pathological conditions. Interpretation and underlying assumptions for each technique are discussed with the goal of providing an overview of some of the most common approaches used to measure in vivo mitochondrial function encountered in the literature.

The mitochondrial-targeted peptide, SS-31, improves glomerular architecture in mice of advanced age.

Although age-associated changes in kidney glomerular architecture have been described in mice and man, the mechanisms are unknown. It is unclear if these changes can be prevented or even reversed by systemic therapies administered at advanced age. Using light microscopy and transmission electron microscopy, our results showed glomerulosclerosis with injury to mitochondria in glomerular epithelial cells in mice aged 26 months (equivalent to a 79-year-old human). To test the hypothesis that reducing mitochondrial damage in late age would result in lowered glomerulosclerosis, we administered the mitochondrial targeted peptide, SS-31, to aged mice. Baseline (24-month-old) mice were randomized to receive 8 weeks of SS-31, or saline, and killed at 26 months of age. SS-31 treatment improved age-related mitochondrial morphology and glomerulosclerosis. Assessment of glomeruli revealed that SS-31 reduced senescence (p16, senescence-associated-ß-Gal) and increased the density of parietal epithelial cells. However, SS-31 treatment reduced markers of parietal epithelial cell activation (Collagen IV, pERK1/2, and α-smooth muscle actin). SS-31 did not impact podocyte density, but it reduced markers of podocyte injury (desmin) and improved cytoskeletal integrity (synaptopodin). This was accompanied by higher glomerular endothelial cell density (CD31). Thus, despite initiating therapy in late-age mice, a short course of SS-31 has protective benefits on glomerular mitochondria, accompanied by temporal changes to the glomerular architecture. This systemic pharmacological intervention in old-aged animals limits glomerulosclerosis and senescence, reduces parietal epithelial cell activation, and improves podocyte and endothelial cell integrity.

Skeletal muscle bioenergetics in aging and heart failure.

Changes in mitochondrial capacity and quality play a critical role in skeletal and cardiac muscle dysfunction. In vivo measurements of mitochondrial capacity provide a clear link between physical activity and mitochondrial function in aging and heart failure, although the cause and effect relationship remains unclear. Age-related decline in mitochondrial quality leads to mitochondrial defects that affect redox, calcium, and energy-sensitive signaling by altering the cellular environment that can result in skeletal muscle dysfunction independent of reduced mitochondrial capacity. This reduced mitochondrial quality with age is also likely to sensitize skeletal muscle mitochondria to elevated angiotensin or beta-adrenergic signaling associated with heart failure. This synergy between aging and heart failure could further disrupt cell energy and redox homeostasis and contribute to exercise intolerance in this patient population. Therefore, the interaction between aging and heart failure, particularly with respect to mitochondrial dysfunction, should be a consideration when developing strategies to improve quality of life in heart failure patients. Given the central role of the mitochondria in skeletal and cardiac muscle dysfunction, mitochondrial quality may provide a common link for targeted interventions in these populations.

Respiratory chain protein turnover rates in mice are highly heterogeneous but strikingly conserved across tissues, ages, and treatments.

The mitochondrial respiratory chain (RC) produces most of the cellular ATP and requires strict quality-control mechanisms. To examine RC subunit proteostasis in vivo, we measured RC protein half-lives (HLs) in mice by liquid chromatography-tandem mass spectrometry with metabolic [(2)H3]-leucine heavy isotope labeling under divergent conditions. We studied 7 tissues/fractions of young and old mice on control diet or one of 2 diet regimens (caloric restriction or rapamycin) that altered protein turnover (42 conditions in total). We observed a 6.5-fold difference in mean HL across tissues and an 11.5-fold difference across all conditions. Normalization to the mean HL of each condition showed that relative HLs were conserved across conditions (Spearman's ρ = 0.57; P < 10(-4)), but were highly heterogeneous between subunits, with a 7.3-fold mean range overall, and a 2.2- to 4.6-fold range within each complex. To identify factors regulating this conserved distribution, we performed statistical analyses to study the correlation of HLs to the properties of the subunits. HLs significantly correlated with localization within the mitochondria, evolutionary origin, location of protein-encoding, and ubiquitination levels. These findings challenge the notion that all subunits in a complex turnover at comparable rates and suggest that there are common rules governing the differential proteolysis of RC protein subunits under divergent cellular conditions.

Defects in mitochondrial localization and ATP synthesis in the mdx mouse model of Duchenne muscular dystrophy are not alleviated by PDE5 inhibition.

Given the crucial roles for mitochondria in ATP energy supply, Ca(2+) handling and cell death, mitochondrial dysfunction has long been suspected to be an important pathogenic feature in Duchenne muscular dystrophy (DMD). Despite this foresight, mitochondrial function in dystrophin-deficient muscles has remained poorly defined and unknown in vivo. Here, we used the mdx mouse model of DMD and non-invasive spectroscopy to determine the impact of dystrophin-deficiency on skeletal muscle mitochondrial localization and oxidative phosphorylation function in vivo. Mdx mitochondria exhibited significant uncoupling of oxidative phosphorylation (reduced P/O) and a reduction in maximal ATP synthesis capacity that together decreased intramuscular ATP levels. Uncoupling was not driven by increased UCP3 or ANT1 expression. Dystrophin was required to maintain subsarcolemmal mitochondria (SSM) pool density, implicating it in the spatial control of mitochondrial localization. Given that nitric oxide-cGMP pathways regulate mitochondria and that sildenafil-mediated phosphodiesterase 5 inhibition ameliorates dystrophic pathology, we tested whether sildenafil's benefits result from decreased mitochondrial dysfunction in mdx mice. Unexpectedly, sildenafil treatment did not affect mitochondrial content or oxidative phosphorylation defects in mdx mice. Rather, PDE5 inhibition decreased resting levels of ATP, phosphocreatine and myoglobin, suggesting that sildenafil improves dystrophic pathology through other mechanisms. Overall, these data indicate that dystrophin-deficiency disrupts SSM localization, promotes mitochondrial inefficiency and restricts maximal mitochondrial ATP-generating capacity. Together these defects decrease intramuscular ATP and the ability of mdx muscle mitochondria to meet ATP demand. These findings further understanding of how mitochondrial bioenergetic dysfunction contributes to disease pathogenesis in dystrophin-deficient skeletal muscle in vivo.

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.

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.

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.

Associations of Skeletal Muscle Mass, Muscle Fat Infiltration, Mitochondrial Energetics, and Cardiorespiratory Fitness With Liver Fat Among Older Adults.

BACKGROUND: Muscle mass loss may be associated with liver fat accumulation, yet scientific consensus is lacking and evidence in older adults is scant. It is unclear which muscle characteristics might contribute to this association in older adults. METHODS: We associated comprehensive muscle-related phenotypes including muscle mass normalized to body weight (D3-creatine dilution), muscle fat infiltration (magnetic resonance imaging), carbohydrate-supported muscle mitochondrial maximal oxidative phosphorylation (respirometry), and cardiorespiratory fitness (VO2 peak) with liver fat among older adults. Linear regression models adjusted for age, gender, technician (respirometry only), daily minutes of moderate-to-vigorous physical activity, and prediabetes/diabetes status tested main effects and interactions of each independent variable with waist circumference (high: women-≥88 cm, men-≥102 cm) and gender. RESULTS: Among older adults aged 75 (interquartile range: 73, 79 years; 59.8% women), muscle mass and liver fat were not associated overall (N = 362) but were positively associated among participants with a high waist circumference (ß: 25.2; 95% confidence intervals [95% CI]: 11.7, 40.4; p = .0002; N = 160). Muscle fat infiltration and liver fat were positively associated (ß: 15.2; 95% CI: 6.8, 24.3; p = .0003; N = 378). Carbohydrate-supported maximum oxidative phosphorylation (before adjustment) and VO2 peak (after adjustment; ß: -12.9; 95% CI: -20.3, -4.8; p = .003; N = 361) were inversely associated with liver fat; adjustment attenuated the estimate for maximum oxidative phosphorylation although the point estimate remained negative (ß: -4.0; 95% CI: -11.6, 4.2; p = .32; N = 321). CONCLUSIONS: Skeletal muscle-related characteristics are metabolically relevant factors linked to liver fat in older adults. Future research should confirm our results to determine whether trials targeting mechanisms common to liver and muscle fat accumulation are warranted.

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.

Randomized Crossover Clinical Trial of Nicotinamide Riboside and Coenzyme Q10 on Metabolic Health and Mitochondrial Bioenergetics in CKD.

Background: Mitochondria-driven oxidative/redox stress and inflammation play a major role in chronic kidney disease (CKD) pathophysiology. Compounds targeting mitochondrial metabolism may improve mitochondrial function, inflammation, and redox stress; however, there is limited evidence of their efficacy in CKD. Methods: We conducted a randomized, double-blind, placebo-controlled crossover trial comparing the effects of 1200 mg/day of coenzyme Q10 (CoQ10) or 1000 mg/day of nicotinamide riboside (NR) supplementation to placebo in 25 people with moderate-to-severe CKD (eGFR <60mL/min/1.73 m2). We assessed changes in the blood transcriptome using 3'-Tag-Seq gene expression profiling and changes in pre-specified secondary outcomes of inflammatory and oxidative stress biomarkers. For a subsample of participants (n=14), we assessed lymphocyte and monocyte bioenergetics using an extracellular flux analyzer. Results: The (mean±SD) age, eGFR, and BMI of the participants were 61±11 years, 37±9 mL/min/1.73m2, and 28±5 kg/m2 respectively. Of the participants, 16% had diabetes and 40% were female. Compared to placebo, NR-mediated transcriptomic changes were enriched in gene ontology (GO) terms associated with carbohydrate/lipid metabolism and immune signaling while, CoQ10 changes were enriched in immune/stress response and lipid metabolism GO terms. NR increased plasma IL-2 (estimated difference, 0.32, 95% CI of 0.14 to 0.49 pg/mL), and CoQ10 decreased both IL-13 (estimated difference, -0.12, 95% CI of -0.24 to -0.01 pg/mL) and CRP (estimated difference, -0.11, 95% CI of -0.22 to 0.00 mg/dL) compared to placebo. Both NR and CoQ10 reduced 5 series F2-Isoprostanes (estimated difference, -0.16 and -0.11 pg/mL, respectively; P<0.05 for both). NR, but not CoQ10, increased the bioenergetic health index (BHI) (estimated difference, 0.29, 95% CI of 0.06 to 0.53) and spare respiratory capacity (estimated difference, 3.52, 95% CI of 0.04 to 7 pmol/min/10,000 cells) in monocytes. Conclusion: Six weeks of NR and CoQ10 improved in oxidative stress, inflammation, and cell bioenergetics in persons with moderate to severe CKD.