Muscle Mitochondrial Function Research Articles

Activation of innate immunity selectively compromises mitochondrial complex I, proline oxidation, and flight activity in the major arbovirus vector Aedes aegypti.

Aedes aegypti females are natural vectors of important arboviruses such as dengue, zika, and yellow fever. Mosquitoes activate innate immune response signaling pathways upon infection, as a resistance mechanism to fight pathogens and limit their propagation. Despite the beneficial effects of immune activation for insect vectors, phenotypic costs ultimately affect their fitness. However, the underlying mechanisms that mediate these fitness costs remain poorly understood. Given the high energy required to mount a proper immune response, we hypothesized that systemic activation of innate immunity would impair flight muscle mitochondrial function, compromising tissue energy demand and flight activity. Here, we investigated the dynamic effects of activation of innate immunity by intra-thoracic zymosan injection on A. aegypti flight muscle mitochondrial metabolism. Zymosan injection significantly increased defensin A expression in fat bodies in a time-dependent manner that compromised flight activity. Although oxidant levels in flight muscle were hardly altered, ATP-linked respiratory rates driven by mitochondrial pyruvate+proline oxidation were significantly reduced at 24 h upon zymosan injection. Oxidative phosphorylation coupling was preserved regardless of innate immune response activation along 24 h. Importantly, rotenone-sensitive respiration and complex I-III activity were specifically reduced 24 h upon zymosan injection. Also, loss of complex I activity compromised ATP-linked and maximal respiratory rates mediated by mitochondrial proline oxidation. Finally, the magnitude of innate immune response activation negatively correlated with respiratory rates, regardless of the metabolic states. Collectively, we demonstrate that activation of innate immunity is strongly associated with reduced flight muscle complex I activity with direct consequences to mitochondrial proline oxidation and flight activity. Remarkably, our results indicate a trade-off between dispersal and immunity exists in an insect vector, underscoring the potential consequences of disrupted flight muscle mitochondrial energy metabolism to arbovirus transmission.

104 Effects of providing vitamin and mineral supplementation throughout gestation on subsequent F1 replacement heifer liver and muscle oxygen consumption and mitochondrial function throughout pregnancy

Abstract The study aimed to assess the impact of maternal vitamin/mineral supplementation during gestation on the energy utilization in liver and muscle tissues of F1 heifer offspring. We hypothesized that VTM supplementation during gestation would mitigate mitochondrial energy loss and uncoupling of liver and muscle tissue in pregnant offspring. At breeding, crossbred Angus heifers [n = 31; initial body weight (BW) = 412.5 ± 53.68 kg] were randomly assigned to either a basal diet targeting BW gains of 0.45 kg•heifer-1•d-1(CON; n = 14) or the basal diet plus 113 g•heifer-1•d-1of a vitamin/mineral supplement (VTM; n = 17). Heifers remained on their respective dietary treatments until calving. After calving, all F1 heifer calves were raised in a single management group with their dams until weaning (BW = 209 ± 4.82 kg; VTM, n = 17; CON, n = 12) and fed a common diet throughout postnatal development. At breeding and throughout gestation, F1 heifers (n = 9, VTM; n = 7, CON) were allotted daily feed intakes of 1.5% of BW on a dry-matter basis as a total mixed ration and used to evaluate cellular energy metabolism throughout pregnancy. Liver and Longissimus dorsi muscle biopsies were collected on d 179 and d 247 +/- 3 of gestation and mitochondrial function was assessed via high resolution respirometry (Oroboros Instruments, Innsbruck, Austria). The following respiratory states were evaluated: LEAK respiration (L), OXPHOS capacity (P), NADH-linked OXPHOS (PI), and electron transfer capacity (E). Data were analyzed using the MIXED procedure of SAS with repeated measures where appropriate. No treatment × trimester interaction (P ≥ 0.13) was observed for any of the respiratory states in liver and muscle. Hepatic oxygen consumption was not influenced by maternal gestational VTM supplementation (P ≥ 0.68), or stage of gestation (P ≥ 0.27). In muscle, L respiration was the only state affected by maternal dietary treatments, being greater (P = 0.05) in CON than VTM heifers. Additionally, L respiration was greater (P = 0.02) during the third trimester compared with the second trimester. This observation in CON heifers is important because increased L respiration (proton leak) results in decreased ATP production. Protons that escape the intermembrane space, are dissipated as heat rather than be used for ATP synthesis; thus, oxygen consumption increases to compensate for the reduction of protons entering ATP turnover. Results show increased muscle mitochondria inefficiency in offspring with dams lacking supplementation during pregnancy. The increased incidence of proton leak results in potentially less efficient cattle as protons escape the electron transport chain rather than be transformed for energy utilization need of the animal, thus VTM supplementation throughout pregnancy impacts fetal development with potential effects in live offspring 18 to 22 mo post-parturition.

Heat therapy preconditioning improves the skeletal muscle contractile and mitochondrial function in a mouse model of access-related hand dysfunction

Introduction: Hemodialysis via arteriovenous fistula (AVF) is a life-line treatment for patients with end-stage kidney disease. However, hemodialysis access-related hand dysfunction (ARHD) including ischemia, muscle weakness and alterations in mitochondrial function is prevalent following AVF placement. We tested the hypothesis that two-weeks of daily heat therapy prior to AVF surgery would improve hindlimb function in a mouse model of ARHD. Methods: Adult male C57BL/6J mice (N=9-11/group) were fed a 0.2% adenine diet to induce chronic kidney disease. Following two weeks of the initial adenine diet, animals were randomly allocated to heat therapy (HT) or control (CON) groups. Animals assigned to HT were exposed to environmental heat at 40°C in a climate chamber for 30 min/day, six days/week, for two weeks while the control mice were exposed to room temperature (~22°C). The AVF was created using the left common iliac artery and vein 24-48 hours after the last treatment session. Laser Doppler perfusion to the ventral paw was assessed post-operatively (POD) and AVF patency was verified using a high-frequency duplex ultrasound system three days prior to euthanasia. Following 14 days of recovery, in situ tibialis anterior (TA) muscle contractility and gastrocnemius muscle mitochondrial function were assessed. RNA sequencing analysis of the hindlimb skeletal muscle (N=4/group) was performed. Results: Compared to baseline, the level of blood urea nitrogen was similarly elevated ( P<0.05) in both groups following four weeks of adenine diet. In both groups, blood perfusion to the paw was decreased by ~50% immediately after AVF creation and progressively recovered to ~80% at POD13 without a treatment effect ( P=0.4467). No differences in body weight and muscle mass in the TA, extensor digitorum longus, and soleus muscles were observed (all P>0.05). Notably, however, the absolute ( P=0.0250) and specific force ( P=0.0101) frequency relationship of the TA muscle were significantly improved in the animals exposed to HT. In addition, compared with control animals, HT mice had significant improvement in oxidative phosphorylation (OXPHOS) in both pyruvate ( P=0.0145) and octanoylcarnitine ( P=0.0196) fueled conditions. RNA sequencing results revealed that genes involved in nervous system development and metabolism were significantly different between HT and CON groups. Conclusion: Daily application of heat therapy for two weeks prior to the AVF surgery has beneficial effects on both skeletal muscle contractile and mitochondrial function suggesting this pre-habilitation strategy may mitigate the risk of ARHD in patients undergoing AVF surgery. Funding Support: National Institutes of Health grant R01HL148597 and American Heart Association grant POST903198. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inhibiting Mitochondrial Fission Protein Drp1 Enhances Mitochondrial Structure, Attenuates Lipid Peroxidation, and Improves Muscle Strength in D2-mdx Mice

Duchenne Muscular Dystrophy (DMD) is the most severe muscular dystrophy manifested by progressive muscle wasting and weakness. Mitochondrial dysfunction and reactive oxygen species (ROS) accumulation are early pathological features of DMD and precede myopathy. A fine balance of mitochondrial dynamics (fission and fusion) is crucial to maintain mitochondrial function and skeletal muscle health. We recently reported excessive activation of Dynamin-Related Protein 1 (Drp1)-mediated mitochondrial fission in two animal models of DMD. Mitochondrial fission inhibitor‐1 (Mdivi‐1) is an effective pharmacological inhibitor of Drp1-mediated mitochondrial fission and has been shown benefits in numerous diseases. The objective of the study was to test whether Mdivi-1 could improve skeletal muscle mitochondrial structure, function and muscle strength in D2- mdx mouse model of DMD. Methods: 9-10-week-old male D2- mdx mice were treated with either Mdivi-1 (I.P., 40 mg/kg BW, mdx/Mdivi-1) or vehicle (2%DMSO in PBS, mdx/VEH) for 5 weeks. D2-wildtype mice treated with vehicle (WT/VEH) were used as the control (n=8/group). Muscle strength was measured using a grip strength meter. Mitochondria were isolated from quad muscles collected 24 hours after the last injection to measure mitochondrial respiration. Transmission Electron Microscopy was used to image mitochondrial structure. Gastrocnemius muscles were collected for immunoblot analysis. Results: Grip strength was significantly lower in muscles from D2- mdx mice compared to WT controls (P = 0.01) but was improved in mdx+Mdivi-1 mice (P = 0.05). Mitochondrial fission protein markers, Drp1(Ser616) phosphorylation and Fis1 were markedly higher in skeletal muscles from mdx/VEH than WT/VEH (P= 0.009 and 0.001) but were attenuated in mdx/Mdivi-1 mice (P = 0.041 and 0.1), indicating a rebalance of mitochondrial dynamics. This rebalance subsequently led to improvement in skeletal muscle mitochondrial structure in mdx/Mdivi-1 mice, with evidence of lower numbers of damaged mitochondria and reduced circularity (P = 0.005 and 0.038). Although not statistically significant, ADP and FCCP-stimulated mitochondrial respiration were enhanced by 93.8% and 92.4% in mdx+Mdivi-1 mice compared to mdx/VEH. In addition, lipid peroxidation marker, 4-HNE was significantly higher in mdx/VEH group in comparison to WT/VEH group (P = <0.0001), but was attenuated in mdx/Mdivi-1 mice (P = 0.08). Conclusion: These data demonstrate that inhibition of Drp1-mediated mitochondrial fission is effective in improving muscular strength in D2- mdx mice, which is at least partly due to enhanced mitochondrial dynamics, structure and attenuated lipid peroxidation. Mdivi-1 may be a viable treatment for DMD. National Institute of Health (R15DK131512). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Deletion of the Aryl Hydrocarbon Receptor in Endothelial Cells Improves Ischemic Angiogenesis in Chronic Kidney Disease

Background: Chronic kidney disease (CKD) is a strong yet underappreciated risk factor for peripheral arterial disease (PAD) that exacerbates disease progression and increases risk for limb amputation and cardiovascular mortality. An estimated 25-35% of CKD patients develop PAD, but mechanistic data linking these two pathologies is limited. Several tryptophan-derived uremic metabolites are retained in the blood and tissues as a consequence of CKD and are endogenous ligands of the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor that has been shown to regulate angiogenesis. Hypothesis: We tested the hypothesis that endothelial cell-specific conditional deletion of the AHR would enhance ischemic limb outcomes in mice with CKD and femoral artery ligation (FAL). Methods: Twelve-week-old male and female endothelium-specific AHR knockout mice (AHRecKO) and AHR floxed mice (AHRfl/fl, Cre-negative control) were fed 0.2% adenine to induce CKD or casein control diet (n=10/group/sex) prior to undergoing FAL to induce limb ischemia. Laser Doppler flowmetry was used to measure limb perfusion. Isometric contraction of the tibialis anterior in-situ via nerve stimulus was performed to measure muscle function. Skeletal muscle mitochondrial respiratory function and H2O2 production were evaluated. Primary endothelial cells were isolated from male and female wildtype mice and treated with known AHR ligand indoxyl sulfate (IS) under hypoxic conditions to uncover sex-dependent differences in AHR activating potential. Results: In male mice with CKD, endothelium-specific ablation of the AHR resulted in significant increases in percentage of perfused capillaries within the ischemic limb (AHRecKO 73.41% ± 4.80% vs AHRfl/fl 64.75% ± 8.325%; p= 0.0074), improved laser Doppler perfusion recovery in the paw and gastrocnemius muscle, and larger myofiber cross-sectional area ( p=0.0258) compared to AHRfl/fl mice. However, these improvements did not translate to improved muscle contractile function. In contrast, deletion of the AHR in female mice with CKD had no significant impact on major outcome measures. Primary cells from male mice treated with IS had significantly higher expression of the AHR controlled gene Cyp1a1 ( p=0.026) compared to cells from female mice. Conclusion: Endothelium-specific deletion of the AHR improved ischemic angiogenesis in male, but not female, mice with CKD. These data reveal sex-dependent differences in AHR activating potential within endothelial cells that exist in the absence of sex hormones. This study was supported by National Institutes of Health (NIH) grant R01-HL149704 (T.E.R.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Peripheral artery disease patients with chronic kidney disease have lower muscle strength and mitochondrial function compared to those with normal kidney function

Introduction: Chronic kidney disease is a significant risk factor for both the development of peripheral artery disease (PAD) and major adverse limb events. However, little is known about how the coalescence of these two conditions impacts limb muscle pathophysiology. The objective of the current study was to determine the impact of CKD on skeletal muscle strength and mitochondrial health in patients with and without PAD. Methods: A cross-sectional prospective study design was employed to assess plantar flexor muscle strength, muscle fiber cross-sectional area (CSA), and mitochondrial health in four groups of patients (61 males and 29 females): PAD only (Toe brachial index, TBI<0.5 and eGFR>60 mL/min), CKD only (TBI>0.75 and eGFR:15-45 mL/min), PAD with CKD (TBI<0.5 and eGFR:15-45 mL/min), and adult controls without PAD and CKD (TBI>0.75 and eGFR>60 mL/min). Results: Plantar flexor muscle strength was lowest in PAD patients with CKD compared to adult controls ( P<0.0001), PAD only ( P=0.001), and CKD only ( P=0.003). No differences in the mean muscle fiber CSA were detected. The percentage of COX+ fibers was significantly lower in PAD patients with CKD compared to adult controls ( P=0.03). High resolution respirometry analysis of mitochondrial oxidative phosphorylation (OXPHOS) function demonstrated that CKD has a significant negative impact on muscle bioenergetics. PAD patients with CKD had lower OXPHOS function than adult controls ( P=0.0016) and PAD patients without CKD ( P=0.0015). Notably, CKD patients without PAD also had lower OXPHOS function than adult controls ( P=0.0054) and PAD patients without CKD ( P=0.005). Conclusion: The current results demonstrate a profound negative impact of CKD on the associated myopathy of PAD patients. This study was supported by National Institutes of Health (NIH) grant R01-HL149704 (T.E.R.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 1151: Concurrent Obesity And Chronic Alcohol Consumption Exacerbates Skeletal Muscle Mitochondrial Dysfunction In A Murine Model Of Peripheral Artery Disease (PAD).

PAD causes ischemic mitochondrial and consequent muscle damage in the legs, amplifying patient morbidity and mortality. While obesity and alcoholism are common comorbidities, their impact on PAD myopathy is unclear. We sought to determine the effect of concurrent hindlimb ischemia (HLI), obesity and chronic alcohol consumption on muscle oxidative stress, and mitochondrial and limb function. Methods: Twenty-four male, 24 female, 8 month old C57BL/6 mice received high-fat-sucrose (HFS) or low-fat-sucrose (LFS) diets for 16 weeks, followed by either 20% ethanol (EtOH) added to the drinking water / continued water access for another 12 weeks ( n =12 mice/4 groups). The left femoral artery was then ligated to induce HLI, and mice continued their assigned diets for another 4 weeks. The quadriceps from ischemic and non-ischemic limbs were excised at euthanasia. High-resolution respirometry measured muscle mitochondrial respiration and reactive oxygen species (H 2 O 2 ) output. Muscle oxidative stress and mitochondrial quality control (mitophagy) were assessed enzymatically and by western blot. Results: Compared to LFS animals with/without EtOH access, two-way ANOVA showed post-HLI limb function was lower in HFS+water and HFS+EtOH animals (p<0.05). Mitochondrial respiration was lower in LFS+EtOH, HFS+water, and HFS+EtOH ischemic muscle during electron transport chain Complex I, state 2 (I.2), Complex I, state 3 (I.3) and Complex III versus LFS+water controls (p<0.05), and was further reduced during Complex II and IV in LFS and HFS-ischemic muscle exposed to EtOH (p<0.01). Mitochondrial H 2 O 2 yield was increased in HFS+water and HFS+EtOH ischemic muscle at complexes I.2, I.3, II, III and IV (p<0.05), but not in LFS+EtOH muscle. Oxidative damage (carbonyl accumulation) (p<0.0001) and reduced antioxidant levels (superoxide dismutase (p<0.05) and PGC-1alpha (p<0.001)) was only evident in HFS ischemic muscle with/without EtOH exposure. However, the mitochondrial detoxifying enzyme, aldehyde dehydrogenase-2 (p=0.03), and mitophagy markers LC3II:I ratio (p=0.002) and mitofusins 1 (p=0.02) and 2 (p=0.002), were only reduced in HFS+EtOH muscle. Conclusion: Obesity and chronic alcohol consumption exacerbates muscle mitochondriopathy in mice with HLI.

Loss of FAM210A in Satellite Cell Drives Mitochondrial Dysfunction and Muscle Weakness in Experimental Peripheral Artery Disease

Introduction: Peripheral artery disease (PAD) affects approximately 200 million people globally. Chronic limb-threatening ischemia (CLTI) represents the most advanced clinical phenotype of PAD. Mitochondrial metabolism defects are apparent in the satellite cells of CLTI patients, and the muscle regeneration process demands increased energy, with satellite cell mitochondrial metabolism and dynamics playing a crucial role. However, our current understanding of the role of satellite cells and muscle regeneration in PAD pathology is notably inadequate. Recent studies have shown that the absence of FAM210A, a newly identified mitochondrial protein, can trigger morphological and energetic abnormalities in mitochondria, resulting in muscle function deterioration and promoting muscle wasting. Given the impaired mitochondrial function in satellite cells in CLTI patients and the heightened energy demand regulated by mitochondrial dynamics during muscle regeneration in satellite cell, it is therefore essential to exploring the role of FAM210A in satellite cells in CLTI limb pathology. The objective of this study was to investigate the impact of the loss of FAM210A in satellite cells in mice with experimental PAD. Methods: Male and female conditional FAM210A satellite cell knock out (FAM210ASCKO) and FAM210A floxed (FAM210Afl/fl) mice (N=3-5) were used for this project. Femoral artery ligation surgery was performed to induce PAD. Tamoxifen was administered for 5 days intraperitoneally one week prior to the surgery. Mice were euthanized twenty-eight days after surgery. Outcome measures included limb perfusion assessment, muscle weight, muscle force production, and mitochondrial respiration. Results: FAM210ASCKO mice had significantly decreased muscle mass of the tibialis anterior and extensor digitorum longus ( p<0.0001 and p=0.0004 respectively). More importantly, maximal muscle force with in-situ contraction in tibialis anterior was significantly lower in FAM210ASCKO mice compared to FAM210Afl/fl mice (FAM210Afl/fl mice: 1085.16 ± 331.08 mN; FAM210ASCKO mice: 430.77 ± 467.91 mN; p=0.0139). Furthermore, analysis of mitochondrial oxidative phosphorylation (OXPHOS) revealed that FAM210ASCKO mice had a significant reduction compared to FAM210Afl/fl mice (48%) in skeletal muscle mitochondrial bioenergetic function at various levels of energy demand mimicking from resting to maximal exercise when carbohydrate was used to energize mitochondria ( p=0.034). Importantly, the analysis of mitochondrial membrane potential consistently reveals a substantial reduction in FAM210ASCKO mice compared to FAM210Afl/fl mice, which can partially explain the diminished mitochondrial bioenergetic function observed in ischemic FAM210ASCKO mice. Conclusion: The results of this study indicate that the loss of FAM210A in satellite cells exacerbates ischemic muscle pathology evidenced by muscle weakness and mitochondrial failure in FAM210ASCKO mice with PAD. Future work is needed to elucidate the mechanisms by which FAM210A regulate ischemic satellite cell function and the therapeutic potential of preventing ischemia-induced loss of FAM210A in PAD. This study was supported by National Institutes of Health (NIH) grant R01-HL149704 (to Dr Ryan). Mr Thome was supported by NIH grant F31-DK128920. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mitochondrial and Microvascular Impairments in a Novel Swine Model of Peripheral Artery Disease

Introduction: Impaired skeletal muscle mitochondria and microvascular function have been reported in human peripheral artery disease (PAD). However, no large animal models of PAD have mimicked these impairments. Purpose: The purpose of this study was to examine the skeletal muscle microvasculature and mitochondria in a novel swine model of PAD. Hypothesis: We hypothesized that hindlimb ischemia leads to dysfunction of skeletal muscle microvessels and mitochondria. Methods: Ossabaw swine (n=5) with metabolic syndrome underwent endovascular coil occlusion of the right external iliac artery to generate hindlimb ischemia (ISC). The left leg served as a non-ischemic control (CON). After 4-weeks, biopsies were collected from both legs (lateral gastrocnemius). Skeletal muscle arterioles were assessed using video microscopy. Microvascular vasodilatory function was assessed in response to flow (Shear stress), acetylcholine (ACh), and sodium nitroprusside (SNP). Skeletal muscle mitochondrial function was assessed using high-resolution respirometry. Substrates were administered as follows: malate (M) & glutamate (G), adenosine diphosphate (ADP), succinate (S), oligomycin, and ascorbate (Asc) & N,N,N,N-tetramethyl-phenylenediamine (TMPD). Complex I state 2 (CI-S2) was assessed as M+G, complex I state 3 (CI-S3) was assessed as (M+G+ADP)-(M+G), complex II state 3 (CII-S3) was assessed as (M+G+ADP+S)-(M+G+ADP), complex I&II state 3 (CI&II S3) was assessed as (M+G+ADP+S)-(M+G), state 4 (S4) was assessed after oligomycin, and complex IV (CIV) respiration was assessed after Asc+TMPD. The respiratory control ratio (RCR) was calculated as CI&II-S3/S4. Results: Vasodilation in response to flow was reduced in ISC vs. CON (Δ-13.1±13.7%, p=0.01). but not in response to ACh (Δ-6.2±17.7%, p=0.23) or SNP (Δ1.7±11.0%, p=0.53). Additionally, CI-S2 (Δ-0.3±3.7 pmolO2·s−1·mg−1, p=0.65), S4 (Δ-1.7±4.4 pmolO2·s−1·mg−1, p=0.14), and CIV (Δ-2.8±20.9 pmolO2·s−1·mg−1, p=0.65) were not different between ISC and CON. However, CI-S3 (Δ-4.7±5.0 pmolO2·s−1·mg−1, p<0.01), CII-S3 (Δ-3.5±5.0 pmolO2·s−1·mg−1, p=0.02), CI&II-S3 (Δ-8.5±8.7 pmolO2·s−1·mg−1, p<0.01), and RCR (Δ-0.8±1.3 pmolO2·s−1·mg−1, p=0.04) were all reduced in ISC vs. CON. Conclusions: We found that skeletal muscle microvessels and mitochondria were impaired in our swine model. Based on the present findings, we conclude that our novel porcine model closely replicates the pathophysiology PAD, making it a promising large animal model for studying human PAD. This research was supported by the National Institutes of Health (R01HD106911, R01AG062198, R01AG0778031) and the Great Plains IdeA-CTR (U54 GM115458). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Skeletal Muscle Mitochondrial Function and Mitochondria-related Protein Expression in Men and Women During Acute Exercise-Heat Stress and Acclimation

INTRODUCTION: Military personnel can exhibit improved performance and reduced physiological strain in hot environments with the induction of heat adaptations. Mild heat exposure at 40°C heat serves therapeutic purposes, enhancing metabolic flexibility, and promoting positive muscle adaptations. Research indicates that heat acclimation (HA) leads to increased mitochondrial biogenesis and reduced reactive oxygen species in skeletal muscle, improving thermal tolerance. However, understanding sex differences in mitochondrial function and related protein expression affected by heat acclimation remain limited. Therefore, this study aimed to explore mitochondrial respiration and the expression of mitochondria-related proteins to better understand the impact of heat stress on mitochondrial adaptations in skeletal muscle in both males and females. METHODS: Healthy men (M:n = 7, 22±2yo, VO2max 52.86±2.35ml·kg−1·min−1) and women (F:n = 7, 24±1yo, VO2max 45.97±2.57 ml·kg−1·min−1) participated in a 5-day heat acclimation (HA) at 40°C and 40% relative humidity. Before and after HA, participants completed a heat tolerance test (HTT) measuring time to reach a rectal temperature of 39.5°C or volitional failure. Biopsies from the vastus lateralis were taken immediately post-HTT for musculoskeletal analysis. Mitochondrial respiratory function was assessed using Oroboros O2K, employing high-resolution respirometry. Paired sample t-tests were used to determine statistical significance between groups (p<0.05). Proteomics data were mined for Gene Ontology (GO) terms related to mitochondrial function (STRING V.12.0) (adjusted p<0.05). RESULTS: Both men and women exhibited significant enhancements in complex I-mediated respiration and complex I+II maximal coupled respiration after HA (PRE-HA: M:P=0.009, P=0.026; F:P=0.016, P=0.011). No sex differences in mitochondria respiration were observed before or after HA. Before HA, skeletal muscle from women showed higher expression in 4 proteins (ND61, NDUFB1, COX1, ATP5MF) associated with 9 GO terms related to mitochondrial function compared to men. However, after HA, men exhibited higher expression in 14 proteins (MTX2, APOOL, DNAJC11, TIMM13, NDUFAB1, DNAJC11, MAVS, CHCHD4, NDUFAF4, ATP5PF, NDUFAS, UQCRH, SSBP1, ND3) associated with 21 mitochondria-related GO terms compared to women. CONCLUSION: Acclimation to heat stress is associated with positive mitochondrial adaptation in both men and women. Although enhancements in mitochondrial respiratory function in both men and women after HA, there were different skeletal muscle responses between men and women regarding the regulation of mitochondria-related proteins after a bout of exercise-heat stress. Funding: DoD BA200299. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Role of Endothelial Cell Specific Nrf2 on Skeletal Muscle Mitochondrial Function

Introduction: Cardiovascular disease (CVD) is a leading cause of death worldwide. Excessive reactive oxygen species (ROS) in the vasculature has been suggested as one of the major players in CVD. In addition, CVDs such as peripheral arterial disease (PAD) have elevated oxidative stress (OS) damage, stemming from an imbalance between antioxidants and ROS in both the vasculature and skeletal muscles. Although a vascular disease, the most common symptom of PAD is leg pain due to skeletal muscle ischemia and OS damage. The Nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway is a redox transcription factor and is likely the master regulator of endogenous antioxidants. Modulation of Nrf2 expression may be a potential therapeutic target in CVDs. However, the role of endothelial cell (EC) Nrf2 on skeletal muscle mitochondrial function is currently unknown. Therefore, the purpose of this study was to examine the role of endothelial specific Nrf2 in skeletal muscle mitochondrial function.Hypothesis: We hypothesized that 1) EC-specific Nrf2-knockout (Tie2-Cre-Nrf2 Floxed) would result in blunted skeletal muscle mitochondrial function and 2) EC specific Keap1 knockout (Tie2-Cre-Keap1 Floxed) would improve skeletal muscle mitochondrial function. Methods: Gastrocnemius muscles were harvested from 6-month-old C57BL/6 mice (WT, n = 11), EC specific Nrf2-KO mice (n = 15) and EC specific Keap1-KO mice (n = 7). Muscle fibers were permeabilized and placed in a high-resolution respirometer to assess mitochondrial respiratory complex function (Oroboros Instruments). Results: Complex I state 3 respiration was blunted in Nrf2-KO compared to both Keap1-KO and WT (6.5±1.7 vs 13.6±3.5 vs 18.5±3.3 pmols−1･mg−1, respectively; p<0.05). Complex I+II state 3 respiration was attenuated in Nrf2-KO compared to Keap1-KO (13.63±1.5 vs 27.8±4.2 pmols−1･mg−1; p<0.05). Complex II state 3 respiration was increased in the Keap1-KO compared to both Nrf2-KO and WT (23.9±2.8 vs 7.1±1.1 vs 5.6±0.7 pmols−1･mg−1, respectively; p<0.05) and Nrf2-KO exhibited reduced Complex IV respiration compared to both Keap1-KO and WT (19.5±6.1 vs 57.7±13.2 vs 76.6±12.4 pmols−1･mg−1, respectively; p<0.05).Conclusions: This study provides novel findings showing skeletal muscle mitochondrial dysfunction is present in EC specific Nrf2-KO mice. Yet, EC specific Keap1-KO mice had increased mitochondrial respiration. To our knowledge, this is the first study to demonstrate a substantial impact that EC specific Nrf2 contributes to skeletal muscle mitochondrial function. Our findings provide evidence that the vascular EC Nrf2 pathway can be a potential therapeutic target for skeletal muscle dysfunction in CVDs such as PAD. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Myokine Secretion following an Aerobic Exercise Intervention in Individuals with Type 2 Diabetes with or without Exercise Resistance.

Type 2 diabetes (T2D) is characterized by muscle metabolic dysfunction that exercise can minimize, but some patients do not respond to an exercise intervention. Myokine secretion is intrinsically altered in patients with T2D, but the role of myokines in exercise resistance in this patient population has never been studied. We sought to determine if changes in myokine secretion were linked to the response to an exercise intervention in patients with T2D. The participants followed a 10-week aerobic exercise training intervention, and patients with T2D were grouped based on muscle mitochondrial function improvement (responders versus non-responders). We measured myokines in serum and cell-culture medium of myotubes derived from participants pre- and post-intervention and in response to an in vitro model of muscle contraction. We also quantified the expression of genes related to inflammation in the myotubes pre- and post-intervention. No significant differences were detected depending on T2D status or response to exercise in the biological markers measured, with the exception of modest differences in expression patterns for certain myokines (IL-1β, IL-8, IL-10, and IL-15). Further investigation into the molecular mechanisms involving myokines may explain exercise resistance with T2D; however, the role in metabolic adaptations to exercise in T2D requires further investigation.

Local and systemic responses to repeated gluteal muscle microbiopsies in mature sedentary horses

We aimed to test the hypothesis that repeated muscle collections would impact mitochondrial function, antioxidant status, and markers of inflammation and muscle damage. Twenty-six horses (8 geldings, 18 mares; mean ± SD 9.5 ± 3.5 y) had gluteus medius muscle biopsy samples collected at: 0 and 24h (n=7); 0 and 6h (n = 6); 0, 6, and 12h (n=7); or 0, 6, 12, and 24h (n=6). Blood was collected from all horses every 6h for 72h, starting 24h prior to the 0h muscle collection. Data were analyzed using mixed linear models. Muscle integrative (per mg tissue) electron transfer capacity of complex II decreased (P=0.004) and intrinsic (relative to citrate synthase (CS) activity) LEAK increased (P<0.03) from 0 to 6h but both returned to 0h levels by 12h. Activity of CS was greater at 0 than 12 and 24h (P≤0.02). Serum creatine kinase (CK) activity was similar from -24 through 0h but increased in all horses at 6h and remained elevated through 48h (P<0.05) though not above reference ranges. Whole blood superoxide dismutase activity fluctuated throughout the 72-h collection period (P=0.03) and serum cortisol concentration displayed a circadian pattern (P<0.0001) but neither were altered by muscle collections. No other variable, including muscle mitochondrial capacities and function, blood and muscle antioxidant status and concentrations of select cytokines, and serum amyloid A, differed by time or muscle collection. Repeated gluteal collections had limited short-term or no effect on physiological markers in unstressed, mature horses except serum CK activity, which should be interpreted with caution during repeated tissue collections.

Effects of manipulating Kingfish (Seriola lalandi) routine oxygen demand and supply on ventricular and skeletal muscle mitochondrial function

SummaryYellowtail kingfish (Seriola lalandi) are a highly active marine species, with increasing value in commercial aquaculture. While practices which manipulate oxygen (O2) dynamics through metabolic demand and environmental O2 supply have been shown to impact juvenile growth rate in fish development, there has been no work exploring the effects of O2 at the subcellular level. In addition, manipulation of O2 supply or demand via changes in environmental factors such as O2 levels or salinity, may impact whole animal performance. Still, there has been little work exploring the physiological effects at a subcellular level. Mitochondria (mt) consume O2 and are pivotal in central metabolism, directly impacting growth, and dictating whole animal performance. Here we explore the effects of manipulating O2 demand and supply on mt in yellowtail kingfish ventricle and skeletal muscle by exposure to chronic hyperoxia (200% [O2], 35 ppt) and to hypoosmotic conditions (100% [O2], 17.5 ppt). Chronic exposure to hypoosmotic conditions resulted in greater overall biomass, while it lowered relative ventricular mass compared to control fish. Mitochondrial respiratory flux for most measures remained consistent with control fish, with the exception of cytochrome c oxidase (COX) respiration. Chronic hyperoxia had no impact on overall morphology, and no significant effect on ventricle mt respiration, yet increased COX activity in skeletal muscle. These data indicate that lowering routine mt O2 demands in S. lalandi with hypoosmotic conditions has more impact than increasing O2 availability, and that this may enhance biomass output while maintaining mt integrity.

Migration increases mitochondrial oxidative capacity without increasing reactive oxygen species emission in a songbird.

Birds remodel their flight muscle metabolism prior to migration to meet the physiological demands of migratory flight, including increases in both oxidative capacity and defence against reactive oxygen species. The degree of plasticity mediated by changes in these mitochondrial properties is poorly understood but may be explained by two non-mutually exclusive hypotheses: variation in mitochondrial quantity or in individual mitochondrial function. We tested these hypotheses using yellow-rumped warblers (Setophaga coronata), a Nearctic songbird which biannually migrates 2000-5000 km. We predicted higher flight muscle mitochondrial abundance and substrate oxidative capacity, and decreased reactive oxygen species emission in migratory warblers captured during autumn migration compared with a short-day photoperiod-induced non-migratory phenotype. We assessed mitochondrial abundance via citrate synthase activity and assessed isolated mitochondrial function using high-resolution fluororespirometry. We found 60% higher tissue citrate synthase activity in the migratory phenotype, indicating higher mitochondrial abundance. We also found 70% higher State 3 respiration (expressed per unit citrate synthase) in mitochondria from migratory warblers when oxidizing palmitoylcarnitine, but similar H2O2 emission rates between phenotypes. By contrast, non-phosphorylating respiration was higher and H2O2 emission rates were lower in the migratory phenotype. However, flux through electron transport system complexes I-IV, II-IV and IV was similar between phenotypes. In support of our hypotheses, these data suggest that flight muscle mitochondrial abundance and function are seasonally remodelled in migratory songbirds to increase tissue oxidative capacity without increasing reactive oxygen species formation.

Skeletal muscle mitochondrial correlates of critical power and W' in healthy active individuals.

The asymptote (critical power; CP) and curvature constant (W') of the hyperbolic power-duration relationship can predict performance within the severe-intensity exercise domain. However, the extent to which these parameters relate to skeletal muscle mitochondrial content and respiratory function is not known. Fifteen males (peak O2 uptake, 52.2±8.7mLkg-1min-1; peak work rate, 366±40W; and gas exchange threshold, 162±41W) performed three to five constant-load tests to task failure for the determination of CP (246±44W) and W' (18.6±4.1kJ). Skeletal muscle biopsies were obtained from the vastus lateralis to determine citrate synthase (CS) activity, as a marker of mitochondrial content, and the ADP-stimulated respiration (P) and maximal electron transfer (E) through mitochondrial complexes (C) I-IV. The CP was positively correlated with CS activity (absolute CP, r=0.881, P<0.001; relative CP, r=0.751, P=0.001). The W' was not correlated with CS activity (P>0.05). Relative CP was positively correlated with mass-corrected CI+IIE (r=0.659, P=0.038), with absolute CP being inversely correlated with CS activity-corrected CIVE (r=-0.701, P=0.024). Relative W' was positively correlated with CS activity-corrected CI+IIP (r=0.713, P=0.021) and the phosphorylation control ratio (r=0.661, P=0.038). There were no further correlations between CP or W' and mitochondrial respiratory variables. These findings support the assertion that skeletal muscle mitochondrial oxidative capacity is positively associated with CP and that this relationship is strongly determined by mitochondrial content.

Reduced exercise capacity occurs before intrinsic skeletal muscle dysfunction in experimental rat models of pulmonary hypertension.

Reduced exercise capacity in pulmonary hypertension (PH) significantly impacts quality of life. However, the cause of reduced exercise capacity in PH remains unclear. The objective of this study was to investigate whether intrinsic skeletal muscle changes are causative in reduced exercise capacity in PH using preclinical PH rat models with different PH severity. PH was induced in adult Sprague-Dawley (SD) or Fischer (CDF) rats with one dose of SU5416 (20 mg/kg) injection, followed by 3 weeks of hypoxia and additional 0-4 weeks of normoxia exposure. Control s rats were injected with vehicle and housed in normoxia. Echocardiography was performed to assess cardiac function. Exercise capacity was assessed by VO2 max. Skeletal muscle structural changes (atrophy, fiber type switching, and capillary density), mitochondrial function, isometric force, and fatigue profile were assessed. In SD rats, right ventricular systolic dysfunction is associated with reduced exercise capacity in PH rats at 7-weektimepoint in comparison to control rats, while no changes were observed in skeletal muscle structure, mitochondrial function, isometric force, or fatigue profile. CDF rats at 4-weektimepoint developed a more severe PH and, in addition to right ventricular dysfunction, the reduced exercise capacity in these rats is associated with skeletal muscle atrophy; however, mitochondrial function, isometric force, and fatigue profile in skeletal muscle remain unchanged. Our data suggest that cardiopulmonary impairments in PH are the primary cause of reduced exercise capacity, which occurs before intrinsic skeletal muscle dysfunction.

Plasma metabolomic markers underlying skeletal muscle mitochondrial function relationships with cognition and motor function.

Lower skeletal muscle mitochondrial function is associated with future cognitive impairment and mobility decline, but the biological underpinnings for these associations are unclear. We examined metabolomic markers underlying skeletal muscle mitochondrial function, cognition and motor function. We analysed data from 560 participants from the Baltimore Longitudinal Study of Aging (mean age: 68.4years, 56% women, 28% Black) who had data on skeletal muscle oxidative capacity (post-exercise recovery rate of phosphocreatine, kPCr) via 31P magnetic resonance spectroscopy and targeted plasma metabolomics using LASSO model. We then examined which kPCr-related markers were also associated with cognition and motor function in a larger sample (n = 918, mean age: 69.4, 55% women, 27% Black). The LASSO model revealed 24 metabolites significantly predicting kPCr, with the top 5 being asymmetric dimethylarginine, lactic acid, lysophosphatidylcholine a C18:1, indoleacetic acid and triacylglyceride (17:1_34:3), also significant in multivariable linear regression. The kPCr metabolite score was associated with cognitive or motor function, with 2.5-minute usual gait speed showing the strongest association (r = 0.182). Five lipids (lysophosphatidylcholine a C18:1, phosphatidylcholine ae C42:3, cholesteryl ester 18:1, sphingomyelin C26:0, octadecenoic acid) and 2 amino acids (leucine, cystine) were associated with both cognitive and motor function measures. Our findings add evidence to the hypothesis that mitochondrial function is implicated in the pathogenesis of cognitive and physical decline with aging and suggest that targeting specific metabolites may prevent cognitive and mobility decline through their effects on mitochondria. Future omics studies are warranted to confirm these findings and explore mechanisms underlying mitochondrial dysfunction in aging phenotypes.

Association between dietary phosphate intake and skeletal muscle energetics in adults without cardiovascular disease.

Highly bioavailable inorganic phosphate (Pi) is present in large quantities in the typical Western diet and represents a large fraction of total phosphate intake. Dietary Pi excess induces exercise intolerance and skeletal muscle mitochondrial dysfunction in normal mice. However, the relevance of this to humans remains unknown. The study was conducted on 13 individuals without a history of cardiopulmonary disease (46% female, 15% Black participants) enrolled in the pilot-phase of the Dallas Heart and Mind Study. Total dietary phosphate was estimated from 24-h dietary recall (ASA24). Muscle ATP synthesis was measured at rest, and phosphocreatinine (PCr) dynamics was measured during plantar flexion exercise using 7-T 31P magnetic resonance (MR) spectroscopy in the calf muscle. Correlation was assessed between dietary phosphate intake normalized to total caloric intake, resting ATP synthesis, and PCr depletion during exercise. Higher dietary phosphate intake was associated with lower resting ATP synthesis (r = -0.62, P = 0.03), and with higher levels of PCr depletion during plantar flexion exercise relative to the resting period (r = -0.72; P = 0.004). These associations remain significant after adjustment for age and estimated glomerular filtration rate (both P < 0.05). High dietary phosphate intake was also associated with lower serum Klotho levels, and Klotho levels are in turn associated with PCr depletion and higher ADP accumulation post exercise. Our study suggests that higher dietary phosphate is associated with reduced skeletal muscle mitochondrial function at rest and exercise in humans providing new insight into potential mechanisms linking the Western diet to impaired energy metabolism.NEW & NOTEWORTHY This is the first translational research study directly demonstrating the adverse effects of dietary phosphate on muscle energy metabolism in humans. Importantly, our data show that dietary phosphate is associated with impaired muscle ATP synthesis at rest and during exercise, independent of age and renal function. This is a new biologic paradigm with significant clinical dietary implications.

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.