The use of peripheral blood mononuclear cells (PBMCs) in cardiovascular research is increasingly common. However, little is known regarding potential age-related changes in mitochondrial bioenergetics and oxidative stress in PBMCs, or whether such changes relate to endothelial function. We assessed mitochondrial bioenergetics and antioxidant buffering capacity (AoxBC) capacity in PBMCs from young (n = 18; 21 ± 2 yr) and older (n = 17, 66 ± 4 yr) adults. High-resolution respirometry and fluorometry measured mitochondrial respiration rate (JO2) and membrane potential (Δψm), respectively, in response to substrate provision (pyruvate, glutamate, malate, and succinate; PGMS) and a bioenergetic creatine kinase (CK) clamp at physiological ATP:ADP ratios (PCr1, PCr2, and PCr3). MtROS emission was measured as hydrogen peroxide (H2O2) emission, and H2O2 production was quantified using inhibitors of glutathione reductase and thioredoxin/peroxiredoxin. AoxBC was calculated as the percentage of H2O2 produced but not emitted. Endothelial function was assessed through flow-mediated dilation (FMD). JO2 was similar between groups at baseline (P = 0.08) and lower energetic states (PCr2, PCr3; P ≥ 0.09), but was lower in older adults at higher energetic states (PCr1: 14.05 ± 2.11 vs. 12.03 ± 2.98 pmol·s-1·106 cells-1, P = 0.03; PGMS: 20.61 ± 2.11 vs. 16.58 ± 3.56 pmol·s-1·106 cells-1; P = 0.0009). Δψm was hypopolarized in older compared with young adults at all energetic states (P ≤ 0.003). Although there were no statistical differences in H2O2 emission (P = 0.43) or production (P = 0.18), AoxBC was lower in older adults (52.59 ± 15.44% vs. 63.49 ± 10.30%; P = 0.03). Age-related changes in JO2 (PGMS, P = 0.02) and Δψm (PGMS, P = 0.0008; PCr2, P = 0.04; PCr3, P = 0.02) were related to FMD. These data demonstrate associations between altered PBMC mitochondrial bioenergetics and age-related vascular endothelial dysfunction.NEW & NOTEWORTHY The use of peripheral blood mononuclear cells (PBMCs) is increasingly common in cardiovascular research. However, relatively little is known regarding potential age-related changes in PBMC mitochondrial bioenergetics and oxidative stress, or whether age-related changes are related to endothelial (dys)function. We demonstrate altered mitochondrial bioenergetics (oxygen consumption rates and membrane potential) and antioxidant buffering capacity in PBMCs from older compared with young adults. We additionally demonstrate associations between mitochondrial bioenergetics and endothelial function (brachial-artery flow-mediated dilation).

The objective of this study was to investigate the role of 17β-estradiol (E2) in modulating mitochondrial bioenergetics and reactive oxygen species (ROS) homeostasis following acute and chronic volumetric muscle loss (VML) injury in female mice. The central hypothesis was that the loss of ovarian hormones would lessen mitochondrial bioenergetic efficiency causing greater ROS production and that E2 replacement would rescue this phenotype in the context of VML. Female C57BL/6J mice were divided into three groups: VML+Sham surgery (VML), VML+Ovariectomized (OVX)+Placebo pellets (VML+OVX+Placebo), and VML+OVX+Estradiol (E2) pellets (VML+OVX+E2) and skeletal muscle was analyzed at 3-, 7-, and 60-day post-injury (dpi). At 12 weeks of age, sham or OVX surgery was performed, followed by either placebo or E2 pellet implantation in OVX cohorts. Two weeks after surgery, unilateral VML surgery was conducted on the hindlimb plantar flexors (gastrocnemius, soleus, plantaris muscles). At 3- and 7-dpi, antioxidant buffering capacity (AoxBC, balance between ROS production and ROS emission), and mitochondrial transcriptomics were evaluated. At 60-dpi, mitochondrial respiration (JO 2 ) and AoxBC were measured. The loss of ovarian hormones led to lower mitochondrial AoxBC when either carb- or fat-substrates were supplied (p≤0.02) at 3- and 7-dpi. A hallmark gene set enrichment analysis (GSEA) following bulk RNA sequencing revealed that oxidative phosphorylation was among the top-3 pathways modulated by the loss of ovarian hormones and E2 replacement. A focused mitochondrial-pathway GSEA showed a robust and temporal regulation of the mitochondrial transcriptome at 3- and 7-dpi for both VML+OVX+Placebo and VML+OVX+E2. At 60-dpi, both carb- and fat-mediated JO 2 were 30% and 44% lower in the VML+OVX+Placebo group (p≤0.008) compared to VML; however, both carb- and fat-mediated JO 2 was 49% and 92% greater in VML+OVX+E2 muscle fibers compared to the VML+OVX+Placebo fibers (p≤0.001). Both carb- and fat-mediated AoxBC were 27% and 40% less, respectively, in the VML+OVX+Placebo compared to the VML mice (p≤0.001); however, they were 40% and 61% greater in the VML+OVX+E2 muscle fiber bundles relative to the VML+OVX+Placebo fibers (p≤0.001). Our findings demonstrated that VML injury led to significant impairments in mitochondrial bioenergetics, increased ROS production, and diminished antioxidant defenses. OVX exacerbates VML-induced decrease in antioxidant defense, whereas E2 replacement mitigated these disruptions, improving mitochondrial bioenergetics and AoxBC under ovarian hormone-deficient conditions following VML injury. RNA sequencing and gene set enrichment analysis revealed key shifts in mitochondrial maintenance, oxidative phosphorylation, and metabolic pathways, highlighting the crucial role of E2 in mitochondrial adaptation following traumatic injury in female mice. These findings suggest the therapeutic potential of hormone therapy in restoring metabolic homeostasis and redox balance following traumatic muscle injury, particularly in naturally or surgically estrogen-deficient conditions. Funding: NIH R01 AR078903. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Volumetric muscle loss (VML) is characterized by an irrecoverable loss of skeletal muscle mass, persistent functional deficits, and metabolic dysfunction. A disrupted cellular redox homeostasis is one attribute of this metabolic dysfunction and can lead to excessive reactive oxygen species (ROS) emissions and chronic oxidative stress. The primary objective of this study was to define the role of ovarian hormones, specifically 17β-estradiol (17β-E2), in driving mitochondrial bioenergetic and redox balance after VML injury. Female C57BL/6J mice were randomized into experimental and control groups (VML-sham OVX, VML-OVX, and VML-OVX-E2). A time course of ROS emissions and antioxidant buffering capacity (AoxBC) for VML-injured muscles was established across the first 60 days post injury (dpi) in ovary-intact females. Ovariectomy (OVX) was performed before injury to deplete ovarian hormones, and 17β-E2 was administered via continuous-release pellets to investigate the effects of hormone loss and repletion on ROS emissions and mitochondrial bioenergetics. The long-term effects of 17β-E2 were evaluated to determine whether restoring redox led to sustained redox balance in the long term. Transcriptomic analyses were conducted to explore molecular mechanisms of 17β-E2 benefit after VML. In intact females, ROS emissions were greater during the first 14-dpi, but AoxBC recovered more rapidly than previously observed in males. OVX exacerbated VML-induced metabolic dysfunction, resulting in less AoxBC, greater ROS emissions, and an early suppression of mitochondrial gene networks. 17β-E2 repletion attenuated ROS emissions and improved AoxBC at 7-dpi, and led to greater mitochondrial respiratory capacity, conductance, and bioenergetic efficiency out to 60-dpi. Chronic 17β-E2 depletion resulted in impaired glucose tolerance and greater adiposity, which were mitigated by 17β-E2 treatment. Transcriptomic analyses suggest that 17β-E2 contributes to resolving inflammation and enforcing a temporal decoupling of cellular expansion and mitochondrial maturation after VML injury.NEW & NOTEWORTHY Female mice exhibit accelerated recovery of mitochondrial redox balance after volumetric muscle loss (VML) compared with males. This study demonstrates that 17β-estradiol (17β-E2) drives this resilience. Following VML, ovariectomy induced an early transcriptional arrest and asynchronous repair signaling. 17β-E2 replacement restored regenerative coordination by temporally decoupling early cellular expansion from mitochondrial biogenesis. This precise transcriptional regulation translated to long-term functional resilience, restoring mitochondrial bioenergetic efficiency and resolving oxidative stress.

The sympathetic nervous system (SNS) is recognized for its role in the physiological regulation of organs, such as heart, vasculature and lungs, and has emerged as a potential player in skeletal muscle metabolic and neuromuscular junction (NMJ) health. However, the mechanism through which SNS signaling influences skeletal muscle function and adaptation to exercise remains unclear. Using molecular, electrophysiological, immunohistochemical, and high-resolution respirometry techniques, we tested the role of sympathetic innervation to skeletal muscle in response to exercise. Our findings reveal that sympathetic denervation disrupts the NMJ, reducing motor and sympathetic receptor expression, with concomitant deficits in skeletal muscle function. Mechanistically, these deficits are linked to diminished CPT1 enzyme activity, which impairs long-chain fatty acid-mediated oxidation in skeletal muscle mitochondria. These findings reveal a key role for sympathetic innervation in maintaining mitochondrial metabolic function and by extension, skeletal muscle performance, offering novel insight into the interplay between the SNS, exercise, and muscle mitochondria.

The purpose of this study was to investigate a potential mechanism of mitochondrial dysfunction after volumetric muscle loss (VML) injury by assessing oxidative stress and protein post-translational oxidation (PTO). We hypothesized VML injury would result in greater redox imbalance and mitochondrial protein PTO at 7-days post-injury (dpi). Male C57BL/6J mice underwent VML injury to the hindlimb plantarflexors (gastrocnemius, soleus, plantaris muscles), and at 7-dpi mitochondrial reactive oxygen species (ROS) were assessed along with complementary total proteomic and PTO analysis of the remaining skeletal muscle. Statistical comparisons were made to uninjured muscles from age-matched naïve male mice. ROS emissions from VML-injured permeabilized myofibers were greater for both fat- (1.4-fold, p < 0.001) and carbohydrate-substrates (3.4-fold, p< 0.0001) compared to naive. Furthermore, the remaining muscle’s antioxidant buffering capacity, the ability to neutralize ROS, was significantly less in VML compared to naïve under both substrates (carb -44% and fat -29%, p < 0.01). Proteomic analysis was performed on enriched mitochondrial fractions at 7-dpi. There were 214 differentially expressed proteins at 7-dpi compared to naïve (|log2 fold-change| > 1 and FDR < 0.05). Of these differentially expressed proteins, only 14 were down-regulated while the remaining 197 were up-regulated; however, a mitochondrial pathway enrichment analysis revealed that there were 29 significant pathways (|NES| > 1.5 and FDR < 0.05), all of which were down-regulated compared to naïve. This robust down-regulation of mitochondrial protein pathways is consistent with prior transcriptomic studies. Finally, a PTO analysis was performed on the mitochondrial enriched fractions. This form of proteomics distinctly examines both reversible and irreversible PTO events (e.g. sulfenic acid and sulfonic acid). At the peptide level, there were distinct differences in the number of oxidized peptides detected between VML-injured and naïve muscles particularly evident in reversible oxidation events. The largest differences were seen in mono-oxidation events of cysteine residues (46% greater, VML = 5814 vs. naïve = 3979), followed by the reversible oxidation of methionine (40% greater, VML = 2166 vs. naïve = 1548). Irreversible tri-oxidation of cysteine was 20% greater in VML (VML = 145 vs. naïve = 121). Finally, irreversible di-oxidation of cysteine was 13% greater in VML (VML = 35 vs. naïve = 31). Of the 17,925 peptides examined for PTO after VML injury, 20% (3,511) were found to be significant (|Log2 Fold-Change| > 1 and coefficient of variance < 30%) after normalizing by protein abundance compared to naïve. Of the significant peptides, 88% (3,056) were up-regulated and 30% (1,056) are associated with the mitochondria (MitoCarta 3.0). In terms of types of oxidation events, the majority (61%) of significant peptides did not contain any oxidation event. While 28% (984) contained at least one mono-oxidation of cysteine, followed by methionine oxidation with 13% (447), and lastly tri-oxidation with 1% (35). Funding: NIH R01AR078903 (SMG and JAC). This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Neurometabolism is increasingly recognized as a pathogenic contributor to neurodegenerative disease. However, commonly reported mitochondrial functional outcomes (e.g., respiration) often lack specificity with respect to energetic demand, carbon substrate utilization, and key bioenergetic parameters such as mitochondrial membrane potential. To address this limitation, the present study sought to determine whether oxidative phosphorylation conductance differs across brain regions and as a function of carbon substrate. Oxidative phosphorylation conductance was investigated in permeabilized frontal cortex and hippocampus of female and male C57BL/6J mice using pyruvate/malate substrate (PM, supporting complex I) or succinate with rotenone complex-I inhibition (SR, supporting complex II). Both mitochondrial volume (multiphoton microscopy) and abundance (flow cytometry) assessments showed no regional differences (P > 0.05 in both sexes). Mitochondria's ability to titer respiration to clamped energetic demands was lower with SR compared to PM in both sexes, regardless of brain region (P < 0.001). The production of ATP-to-respiration ratio (P/O ratio) was less at low energetic demands with SR compared to PM in males (P < 0.001) and less regardless of energetic demand with SR compared to PM in females (P < 0.05). This study, utilizing otherwise healthy, young brain tissue, demonstrates the necessity for greater precision in mitochondrial bioenergetic approaches to rigorously advance understanding of neurometabolism.NEW & NOTEWORTHY By integrating multi-modal imaging and high-resolution respirometry, this study reveals a critical divergence in regional brain mitochondrial bioenergetics. Although succinate-supported (Complex II) respiration yields higher absolute flux, it exhibits significantly lower oxidative phosphorylation conductance and enzymatic activity than pyruvate/malate-supported (Complex I) states. This substrate-specific inefficiency provides a novel mechanistic basis for mitochondrial failure in neurodegenerative diseases where Complex I is dysfunctional, highlighting the necessity for precise bioenergetic profiling in brain health.

Volumetric muscle loss (VML) injury results in the unrecoverable loss of muscle mass and contractility. Oral delivery of formoterol, a β2-adrenergic receptor agonist, produces a modest recovery of muscle mass and contractility in VML-injured mice. The objective of this study was to determine if a regenerative rehabilitation paradigm or a regenerative medicine paradigm could enhance the recovery of VML-injured muscle. Regenerative rehabilitation involved oral formoterol delivery combined with voluntary wheel running. Regenerative medicine involved direct delivery of formoterol to VML-injured muscle using a non-biodegradable poly(ethylene glycol) biomaterial. To determine if the regenerative rehabilitation or regenerative medicine approaches were effective at 8 weeks post-injury, muscle mass, contractile function, metabolic function, and histological evaluations were used. One model of regenerative rehabilitation, in which rehabilitation was delayed until 1 month post-injury, resulted in greater muscle mass, muscle contractility, and permeabilized muscle fiber mitochondrial respiration compared to untreated VML-injured mice. Histologically, these mice had evidence of greater total muscle fiber number and oxidative fibers; however, they also had a greater percentage of densely packed collagen. The regenerative medicine model produced greater permeabilized muscle fiber mitochondrial respiration compared to untreated VML-injured mice; however, the non-biodegradable biomaterial was associated with fewer total muscle fibers and lower muscle quality (i.e. lower muscle mass-normalized contractility). The conclusions reached from this study are: (i) regenerative rehabilitation and regenerative medicine strategies utilizing formoterol require further optimization but showed promising outcomes; and (ii) in general, β-adrenergic receptor agonism continues to be a physiologically supportive intervention to improve muscle contractile and metabolic function after VML injury.

Cottonseed oil (CSO) is a seed oil with a unique fatty acid composition and the ability to reduce lipid levels in humans and mice. The present study aimed to characterize the effects of dihydrosterculic acid (DHSA), a cyclopropyl fatty acid found in CSO, on lipid metabolism. First, male wild-type mice were fed CSO- or isocaloric oil-enriched diets (lacking DHSA) for 6 weeks. Tissues were analyzed via RNA-sequencing which identified 45 differentially expressed genes within the CSO group, the majority of which are associated with lipid metabolic processes. Despite being a moderate-fat diet, no changes in hepatic or plasma triglyceride were observed in the CSO group. Confirmational tissue analysis showed an increase in hepatic peroxisome proliferator-activated receptor alpha (PPAR alpha) and PPAR alpha target gene expression in the CSO group compared to control groups, suggesting that DHSA effects may be mediated through increased PPAR alpha transcriptional activity and fatty acid oxidation (FAO). To test this hypothesis, female PPAR alpha knockout mice were fed a CSO-enriched diet. In the absence of PPAR alpha, the lipid-lowering effect of the CSO diet was lost. Next, FAO was assessed in DHSA-treated HepG2 cells by measuring mitochondrial respiration with long-chain fatty acids and adenosine diphosphate substrates. Compared to the control, DHSA-treated cells demonstrated a higher capacity to utilize FAO for energy production. Lastly, CSO-fed mice exhibited significantly lower respiratory exchange ratio with an elevated energy expenditure (EE) compared to SFO-fed mice. In total, these data suggest that the effects of CSO are the result of a DHSA-dependent increase in EE via PPAR alpha induction of FAO pathways.Key points Previous studies with cottonseed oil- (CSO) enriched diets showed reductions in hepatic and plasma lipids; however, it is unclear whether linoleic acid or dihydrosterculic acid (DHSA), a cyclopropyl fatty acid found in CSO, is responsible for these phenotypic changes. This study utilized a unique diet design in which mice were fed either a CSO-enriched diet (DHSA + linoleic acid) or an isocaloric oil-enriched diet (containing linoleic acid but lacking DHSA). RNA-sequencing analysis indicated CSO-fed mice demonstrated increased expression of genes associated with fatty acid oxidation (FAO) in addition to increases in the transcription factor and FAO regulator peroxisome proliferator-activated receptor alpha (PPAR alpha) and its oxidative target genes. Knockout of PPAR alpha confirmed this transcription factor is required for the lipid-lowering phenotype seen following CSO-enriched diets. CSO-fed mice demonstrated significantly lower respiratory exchange ratio and higher energy expenditure compared to chow- and SFO-fed mice, indicative of elevated FAO exclusive to the CSO group.

Volumetric muscle loss (VML) is characterized by contractile weakness, dysfunctional mitochondrial bioenergetics, and poor rehabilitation plasticity. A hyperpolarized mitochondrial membrane potential is one attribute of the dysfunction bioenergetics and can lead to excessive reactive oxygen species (ROS) emissions. The primary objective of this study was to define the role of acute ROS emissions after VML injury. Male C57BL/6J mice were randomized into experimental and control groups. A time course of ROS emissions and antioxidant buffering capacity (AoxBC) for VML-injured muscles was established across the first 60 days postinjury (dpi). SS-31, a mitochondrial-targeted peptide, was administered subcutaneously (8 mg/kg/day) for upto 14 dpi, and specific electron transport chain complex ROS emissions and mitochondrial bioenergetics were investigated. SS-31 and wheel running were combined in a regenerative rehabilitation model to determine whether attenuating acute ROS emissions improved adaptive capability of the remaining muscle. Lipidomic and proteomic analyses were conducted to explore mechanisms of SS-31 benefit after VML. ROS emissions were greater and AoxBC was less during the first 14 dpi and this was associated with dysfunctional mitochondrial bioenergetics regardless of carbohydrate or fat fuel substrate. Complexes I, II, and III were identified as the primary sources of ROS emissions. SS-31 attenuated ROS emissions at both 7 and 14dpi and led to greater mitochondrial respiratory conductance and efficiency out to 30 dpi. Regenerative rehabilitation did not produce greater contractile adaptations, but there was modest evidence of greater metabolic adaptations compared with rehabilitation alone. Lipidomic and proteomic analyses suggest that SS-31 contributes to redox protein abundance alterations after VML injury.NEW & NOTEWORTHY Volumetric muscle loss (VML) impairs mitochondrial bioenergetics, causing hyperpolarization, reduced respiratory conductance, and elevated reactive oxygen species (ROS). A mitochondrial-targeted peptide, SS-31, improved mitochondrial efficiency, lowered ROS, and boosted antioxidant buffering in VML-injured muscle. Combining SS-31 with rehabilitation slightly enhanced metabolism but not contractile function. This suggests oxidative stress is not the sole factor in contractile dysfunction after VML injury and underscores the need for multifaceted therapies to restore muscle after VML.

Macrophages are crucial to the innate immune system and their dysfunction has been implicated in many diseases, including autoimmunity, cancer, and obesity. Activated macrophages can alter their oxidative phosphorylation, reactive oxygen species (ROS) generation, and antioxidant production to elicit their diverse functions. Tumor progression locus 2 (TPL2, MAP3K8, or COT) is a serine-threonine kinase involved in macrophage activation and its ablation induces high type I interferon (IFN) production upon stimulation. Previous research has implicated type I IFNs in metabolic regulation, but their exact mechanism remains unclear in pro-inflammatory macrophages. We hypothesize that TPL2 promotes oxidative phosphorylation in LPS-stimulated macrophages by suppressing type I IFN signaling. LPS-stimulated Tpl2-/- bone marrow-derived macrophages had decreased oxygen consumption compared to LPS-stimulated wild type and Tpl2-/-IFNAR-/- macrophages. Tpl2-/- macrophages had reduced mitochondrial complex II activity and ROS production suggesting high type I IFNs decreased macrophage mitochondrial function. This resulted in LPS-stimulated Tpl2-/- macrophages having reduced SOD2, HMOX-1, and PRDX5 antioxidant gene expression relative to wild type, IFNAR-/-, and Tpl2-/-IFNAR-/- macrophages. These data indicate that Tpl2-/- macrophages are metabolically defective and their elevated type I IFN production contributes to their impaired mitochondria in a pro-inflammatory environment. Supported in part by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1TR002378 and TL1TR002382. Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

The brain is a non-uniform organ comprised of discrete structures of varying function and cell composition. The differing function of brain regions ostensibly would necessitate disparate energetic demands, thereby suggesting brain bioenergetics would be different among brain regions. This study aimed to address whether brain regions indeed have different bioenergetics by evaluating substrate preference, fuel-specific mitochondrial function at physiologically relevant energy demands as well as associated membrane potential and electron conductance using high-resolution respirometry (Oroboros O2k) in the frontal cortex, hypothalamus, and hippocampus. We hypothesize there will be different bioenergetics among brain regions. Substrate preference was established using a substrate-uncoupler-inhibitor-titration (SUIT) with additions, in order: ADP, Pyruvate/Malate, UK5099, Octanoylcarnitine, Rotenone, Succinate, and Antimycin A. There was no statistically significant interaction between region and substrate (p=0.15) nor a main effect of region (p=0.91), however, there was a main effect of substrate in which pyruvate/malate (+275%) and succinate/rotenone (+170%) had significantly greater respiration than octanoylcarnitine, independent of region (p<0.001). Given the results of the SUIT test, pyruvate/malate was used for subsequent tests. The SUIT method relies upon saturating ADP levels and non-physiological ATP:ADP concentration ratios, the extent to which this test obscures the interpretation of brain region respiration is unclear. Instead, an alternative approach that leverages the enzymatic reaction of creatine kinase and phosphocreatine to assess mitochondrial respiration and membrane potential at clamped physiological ATP:ADP ratios, “CK Clamp,” was used. Function of mitochondria with a pyruvate fuel was then evaluated in all three brain regions in which energetic demand was clamped at ΔGATP = -12.87 (high demand), -14.08, -14.5, and -14.85 (low demand). There was a statistically significant interaction between brain region and clamped ΔGATP values (p = 0.02). Frontal cortex displayed higher respiration than hippocampus at -12.87 ΔGATP (33%). There was no statistically significant interaction between region and clamp for mitochondrial membrane potential assessed during the CK clamp (p=0.16), however there was a significant main effect of clamped ΔGATP (p<0.001) in which membrane potential increased (i.e., hyperpolarized) as energetic demand decreased. This agrees with the bioenergetic model that a decrease in energetic demand increases the proton motive force (aka, hyperpolarization), thereby applying a backpressure on the electron transport chain, slowing respiration. Electron conductance was then assessed by calculating the slope of respiration versus clamped ΔGATP. Electron conductance was greater in the frontal cortex than hypothalamus (+70%, p = 0.015). The conclusion of this study is there are differences in mitochondrial bioenergetics among brain regions and the CK clamp was more sensitive at detecting these differences than the SUIT method. None. 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.

This study's objective was to investigate the extent to which two different levels of low-intensity vibration training (0.6 g or 1.0 g) affected musculoskeletal structure and function after a volumetric muscle loss (VML) injury in male C57BL/6J mice. All mice received a unilateral VML injury to the posterior plantar flexors. Mice were randomized into a control group (no vibration; VML-noTX), or one of two experimental groups. The two experimental groups received vibration training for 15-min/day, 5-days/week for 8 weeks at either 0.6 g (VML-0.6 g) or 1.0 g (VML-1.0 g) beginning 3-days after induction of VML. Muscles were analyzed for contractile and metabolic adaptations. Tibial bone mechanical properties and geometric structure were assessed by a three-point bending test and microcomputed tomography (µCT). Body mass-normalized peak isometric-torque was 18% less in VML-0.6 g mice compared with VML-noTx mice (p = 0.030). There were no statistically significant differences of vibration intervention on contractile power or muscle oxygen consumption (p ≥ 0.191). Bone ultimate load, but not stiffness, was ~16% greater in tibias of VML-1.0 g mice compared with those from VML-noTx mice (p = 0.048). Cortical bone volume was ~12% greater in tibias of both vibration groups compared with VML-noTx mice (p = 0.003). Importantly, cross-section moment of inertia, the primary determinant of bone ultimate load, was 44% larger in tibias of VML-0.6 g mice compared with VML-noTx mice (p = 0.006). These changes indicate that following VML, bones are more responsive to the selected vibration training parameters than muscle. Vibration training represents a possible adjuvant intervention to address bone deficits following VML.

Volumetric muscle loss (VML) injury results in the non-recoverable loss of muscle mass and function. Our previous studies indicate mitochondrial dysfunction in the remaining muscle fibers after VML injury, marked by changes in mitochondrial respiration, membrane potential, and enzyme activities. In particular, mitochondrial membrane potential is hyperpolarized during the first 14-days post-injury, a condition that could lead to the production of reactive oxygen species (ROS). When ROS emissions exceed the antioxidant buffering capacity, cellular damage can ensue. The primary objective of this study was to determine ROS production, ROS emission, and antioxidant buffering capacity of remaining muscle fibers after VML injury in the first month after injury. The secondary objective of this study was to determine if a mitochondrial antioxidant (SS-31) could mitigate mitochondrial dysfunction after VML injury. We hypothesized that VML injury will alter the redox status of the remaining muscle. Study 1: Male C57BL/6J mice (n=40) were randomized to Uninjured or VML-injured cohorts. ROS (H2O2) emission/production and endogenous antioxidant buffering capacity were analyzed fluorometrically during live muscle fiber respiration (Oroboros O2k) at 3-, 7-, 14-, 21-, and 30-days post-injury. Study 2: Male C57BL/6J mice (n=32) were randomized into two groups: VML and VML+SS-31. SS-31 is a mitochondrial-targeted peptide with ROS-scavenging properties. VML+SS-31 received SS-31 (8mg/kg/day) for 14 days, and the VML received equal volume saline injections. At 14- and 28-days post-VML, mitochondrial respiration and electron conductance, ROS emission, and antioxidant buffering capacity were assessed in permeabilized gastrocnemius muscle fibers from the muscle remaining after the initial injury. Group differences were detected through ANOVA and unpaired t-test. ROS emission and antioxidant buffering capacity were different between VML-injured and Uninjured mice through 14-days post-injury, specifically at 3-, 7-, and 14-days, mitochondrial ROS emission was significantly higher (+28%, p<0.001), and antioxidant buffering capacity was less (-28%, p=0.002) in VML mice compared to Uninjured mice. Two weeks of SS-31 treatment lead to greater muscle fiber respiration and electron conductance (p≤0.008) at both 14- and 28-days post-VML. Importantly, at 14-days post-injury, mitochondrial ROS emission was 71% lower in VML+SS-31 than VML. At both 14- and 28-days post-injury, antioxidant buffering capacity was 12% greater in VML+SS-31 (p≤0.003) compared to VML. Oxidative stress is a feature of early VML pathophysiology marked by greater ROS emission and less antioxidant buffering capacity. Attenuating oxidative stress during the first two-weeks post-VML injury improves mitochondrial respiration. W81XWH-20-1-0885 and R01AR078903 to SG and JC. 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.

This study's objective was to investigate how contractile strength loss associated with a volumetric muscle loss (VML) injury affects the adjacent tibial bone structural and functional properties in male C57BL/6J mice. Mice were randomized into one of two experimental groups: VML-injured mice that were injured at age 12 weeks and aged to 20 weeks (8 weeks postinjury, VML) and 20-week-old age-matched uninjured mice (Uninjured-20). Tibial bone strength, mid-diaphysis cortical geometry, intrinsic material properties, and metaphyseal trabecular bone structure were assessed by three-point bending and microcomputed tomography (& mu;CT). The plantar flexor muscle group (gastrocnemius, soleus, plantaris) was analyzed for its functional capacities, that is, peak-isometric torque and peak-isokinetic power. VML-injured limbs had 25% less peak-isometric torque and 31% less peak-isokinetic power compared to those of Uninjured-20 mice (p < 0.001). Ultimate load, but not stiffness, was significantly less (10%) in tibias of VML-injured limbs compared to those from Uninjured-20 (p = 0.014). & mu;CT analyses showed cortical bone thickness was 6% less in tibias of VML-injured limbs compared to Uninjured-20 (p = 0.001). Importantly, tibial bone cross-section moment of inertia, the primary determinant of bone ultimate load, was 16% smaller in bones of VML-injured limbs compared to bones from Uninjured-20 (p = 0.046). Metaphyseal trabecular bone structure was also altered up to 23% in tibias of VML-injured limbs (p < 0.010). These changes in tibial bone structure and function after a VML injury occur during a natural maturation phase between the age of 12 and 20 weeks, as evidenced by Uninjured-20 mice having greater tibial bone size and strength compared to uninjured-aged 12-week mice.

Volumetric muscle loss (VML) injuries are characterized by non-recoverable loss of tissue resulting in contractile and metabolic dysfunction. The characterization of metabolic dysfunction in volumetric muscle loss-injured muscle has been interpreted from permeabilized myofiber respiration experiments involving saturating ADP levels and non-physiologic ATP:ADP concentration ratios. The extent to which this testing condition obscures the analysis of mitochondrial (dys) function after volumetric muscle loss injury is unclear. An alternative approach is described that leverages the enzymatic reaction of creatine kinase and phosphocreatine to assess mitochondrial respiration and membrane potential at clamped physiologic ATP:ADP ratios, “CK Clamp.” The objective of this study was to validate the CK Clamp in volumetric muscle loss-injured muscle and to detect differences that may exist between volumetric muscle loss-injured and uninjured muscles at 1, 3, 5, 7, 10, and 14 days post-injury. Volumetric muscle loss-injured muscle maintains bioenergetic features of the CK Clamp approach, i.e., mitochondrial respiration rate (JO2) titters down and mitochondrial membrane potential is more polarized with increasing ATP:ADP ratios. Pyruvate/malate/succinate-supported JO2 was significantly less in volumetric muscle loss-injured muscle at all timepoints compared to uninjured controls (−26% to −84%, p < 0.001) and electron conductance was less at day 1 (−60%), 5 (−52%), 7 (−35%), 10 (−59%), and 14 (−41%) (p < 0.001). Palmitoyl-carnitine/malate-supported JO2 and electron conductance were less affected following volumetric muscle loss injury. volumetric muscle loss-injury also corresponded with a more polarized mitochondrial membrane potential across the clamped ATP:ADP ratios at day 1 and 10 (pyruvate and palmitoyl-carnitine, respectively) (+5%, p < 0.001). This study supports previous characterizations of metabolic dysfunction and validates the CK Clamp as a tool to investigate bioenergetics in traumatically-injured muscle.

Exercise benefits many organ systems, including having a panacea-like effect on the brain. For example, aerobic exercise improves cognition and attention and reduces the risk of brain-related diseases, such as dementia, stress, and depression. Recent advances suggest that endocrine signaling from peripheral systems, such as skeletal muscle, mediates the effects of exercise on the brain. Consequently, it has been proposed that factors secreted by all organs in response to physical exercise should be more broadly termed the "exerkines." Accumulating findings suggest that exerkines derived from skeletal muscle, liver, and adipose tissues directly impact brain mitochondrial function. Mitochondria play a pivotal role in regulating neuronal energy metabolism, neurotransmission, cell repair, and maintenance in the brain, and therefore exerkines may act via impacting brain mitochondria to improve brain function and disease resistance. Therefore, herein we review studies investigating the impact of muscle-, liver-, and adipose tissue-derived exerkines on brain cognitive and metabolic function via modulating mitochondrial bioenergetics, content, and dynamics under healthy and/or disease conditions.

PURPOSE: With the widespread of mitochondrial function, the need to accurately quantify and comprehend mitochondrial O2 respiration has gained prominence within the physiological and pathological spheres. Various in situ, in vitro, and in vivo approaches have been available to investigate numerous aspects of mitochondrial O2 consumption. This review aims to provide a specific method for researchers to consider in examining mitochondrial O2 respiration using permeabilized or homogenized tissues. These techniques allow direct access to mitochondrial O2 respiration, facilitating an exploration of mitochondrial bioenergetics under well-defined substrate conditions.METHODS: Here, we present a method for measuring mitochondrial O2 consumption in permeabilized or homogenized tissues using high-resolution respirometry (Oroboros Oxygraph-2K, Oroboros Instrument).RESULTS: A high-resolution respirometry provides the most direct way to assess mitochondrial O2 respiration with interpretations based on specific modeling approaches.CONCLUSIONS: As many diseases are closely associated with mitochondrial dysfunction, ongoing advancements in this methodology may provide researchers with exciting and novel opportunities for finding the potential role of mitochondria in the etiology and treatment of various diseases.

Skeletal muscles and bones are structurally and functionally linked, such that bone strength is primarily determined the frequency and magnitude of the mechanical strain derived from muscular contractions. Volumetric muscle loss (VML) injury results in a significant loss of muscle tissue and a non-recoverable loss of muscle strength. However, the extent to which limbs that sustain a VML injury have associated changes to bone structure and functional capacity is unknown. This study’s objective was to investigate whether VML injury affects the adjacent tibial bone structure and functional capacity in adult male C57BL/6J mice. At 12 weeks of age, mice (n=14) underwent a unilateral VML injury (4mm diameter muscle biopsy) to the left hindlimb plantar flexor muscles, while the right limbs served as uninjured controls. At 20 weeks of age (2 months post-VML injury), mice were bilaterally tested for in vivo peak-isometric plantarflexion torque. Post-mortem analyses of tibial bone structure and mechanical properties was assessed via μCT and 3-point bending tests, respectively, at the mid-diaphysis. Statistical analyses were performed between injured and uninjured limbs via paired t-test, with an α-level of 0.05. At 2 months post-injury, VML-injured limbs had significantly less gastrocnemius muscle mass (28%), and peak-isometric torque (35%) as compared to uninjured limbs (p<0.001). Tibial bone strength, as measured by ultimate load, demonstrated a trend in VML-injured limbs to be 3% lower than that in uninjured limbs (p=0.055). Most notably, the cross-sectional moment of inertia (CSMI), the principal structural determinant of the bone’s ultimate load was 9% smaller in VML-injured limbs as compared to uninjured limbs (p=0.035). Additional structural changes in cortical bone were detected: notably, less cortical bone thickness (4.5%), volume (6.6%), and cross-sectional area (4.9%) in VML-injured limbs as compared to the uninjured limb (p≤ 0.025). Bone-to-muscle functional ratio (i.e., ultimate load:peak-isometric torque) was 38% greater in VML-injured limbs compared to uninjured limbs (p<0.001) indicating the deficits in muscle far outweigh that of bone at 2 months post-injury. Future research directions include determining if more time post-VML injury has a greater effect on bone ultimate load and exploring the extent to which the uninjured limbs in VML-injured mice experience compensatory or related changes to bone structure and function. W81XWH-20-1-0885 to JAC and SMG This is the full abstract presented at the American Physiology Summit 2023 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.