Skeletal Muscle Mitochondrial Function Research Articles

Vgll2 as an integrative regulator of mitochondrial function and contractility specific to skeletal muscle.

During skeletal muscle adaptation to physiological or pathophysiological signals, contractile apparatus and mitochondrial function are coordinated to alter muscle fiber type. Although recent studies have identified various factors involved in modifying contractile proteins and mitochondrial function, the molecular mechanisms coordinating contractile and metabolic functions during muscle fiber transition are not fully understood. Using a gene-deficient mouse approach, our previous studies uncovered that vestigial-like family member 2 (Vgll2), a skeletal muscle-specific transcription cofactor activated by exercise, is essential for fast-to-slow adaptation of skeletal muscle. The current study provides evidence that Vgll2 plays a role in increasing muscle mitochondrial mass and oxidative capacity. Transgenic Vgll2 overexpression in mice altered muscle fiber composition toward the slow type and enhanced exercise endurance, which contradicted the outcomes observed with Vgll2 deficiency. Vgll2 expression was positively correlated with the expression of genes related to mitochondrial function in skeletal muscle, mitochondrial DNA content, and protein abundance of oxidative phosphorylation complexes. Additionally, Vgll2 overexpression significantly increased the maximal respiration of isolated muscle fibers and enhanced the suppressive effects of endurance training on weight gain. Notably, no additional alteration in expression of myosin heavy chain genes was observed after exercise, suggesting that Vgll2 plays a direct role in regulating mitochondrial function, independent of its effect on contractile components. The observed increase in exercise endurance and metabolic efficiency may be attributed to the acute upregulation of genes promoting fatty acid utilization as a direct consequence of Vgll2 activation facilitated by endurance exercise. Thus, the current study establishes that Vgll2 is an integrative regulator of mitochondrial function and contractility in skeletal muscle.

Hypomethylation in promoters of PGC-1α involved in exercise-driven skeletal muscular alterations in old age.

Exercise training can significantly improve skeletal muscle mitochondrial function and has been proven to be highly relevant to alterations in skeletal muscle DNA methylation. However, it remains unclear whether late-in-life exercise has an effect on promoter methylation of PGC-1α, a key regulator of mitochondrial biogenesis. Here we employed two distinct exercise modalities, constant medium intensity exercise training (CMIT) and high-intensity interval exercise training (HIIT), to investigate their impacts on PGC-1α expression and methylation regulation in skeletal muscle of aged mice. The results revealed a notable decrease in PGC-1α expression in skeletal muscle of aged mice, accompanied by elevated methylation levels of the PGC-1α promoter, and increased DNA methyltransferase (DNMT) protein expressions. However, both forms of exercise training significantly corrected PGC-1α epigenetic changes, increased PGC-1α expression, and ameliorated skeletal muscle reduction. Furthermore, exercise training led to elevated expression of proteins related to mitochondrial biogenesis and energy metabolism in skeletal muscle, improving mitochondrial structure and function. In conclusion, late-in-life exercise improved skeletal muscle function, morphology, and mitochondria biogenesis, which may be associated with hypomethylation in promoters of PGC-1α and increased content of skeletal muscle PGC-1α. Notably, there was no clear difference between HIIT and CMIT in PGC-1α expression and skeletal muscle function.

Impact of sprint interval training on post-fatigue mitochondrial rate in professional boxers.

Professional boxing is a sport that requires a high aerobic capacity to prevent fatigue and allow athletes to perform over 4-12 rounds. Typically, athletes will go into a heavy training period in a pre-bout camp lasting 6 to 9weeks. This study investigates the impact of 3weeks of repeated Wingate sprint interval training, performed on standard gym ergometer bikes, on skeletal muscle endurance and mitochondrial function. Ten male professional boxers (age: 26 ± 4years, height: 175 ± 5cm, weight: 70 ± 5kg) participated in the study. Baseline testing involved a NIRS monitor attached to the rectus femoris muscle prior to an incremental time to exhaustion test on a treadmill. After the treadmill test participants underwent a series of arterial occlusions to determine mitochondrial function post-volitional exhaustion. Participants then continued their own training for 3weeks and then repeated baseline testing. After the second testing session, participants undertook three weekly sprint sessions consisting of 3 × 30s maximal sprints with 60s recovery. Testing was repeated 3weeks later. The time to exhaustion increased by > 6% after 3weeks of sprint interval training as compared to baseline and control (p < 0.05). Skeletal muscle oxygen saturation (SmO2) at exhaustion was increased by 5.5% after 3weeks of sprint interval training as compared to baseline and control (p = 0.008). Skeletal muscle mitochondrial rate post exhaustion was increased by 160% after 3weeks of sprint interval training as compared to baseline and control (p < 0.001). The study demonstrated that SIT led to increased incremental time to exhaustion, higher SmO2 levels at volitional exhaustion and increased mitochondrial rates in professional boxers. These findings suggest that SIT should be an integral part of a boxe's conditioning regimen to improve performance and safety within the ring.

Akt1 deficiency does not affect fiber type composition or mitochondrial protein expression in skeletal muscle of male mice.

Insulin-like growth factor-1-induced activation of ATP citrate lyase (ACLY) improves muscle mitochondrial function through an Akt-dependent mechanism. In this study, we examined whether Akt1 deficiency alters skeletal muscle fiber type and mitochondrial function by regulating ACLY-dependent signaling in male Akt1 knockout (KO) mice (12-16 weeks old). Akt1 KO mice exhibited decreased body weight and muscle wet weight, with reduced cross-sectional areas of slow- and fast-type muscle fibers. Loss of Akt1 did not affect the phosphorylation status of ACLY in skeletal muscle. The skeletal muscle fiber type and expression of mitochondrial oxidative phosphorylation complex proteins were unchanged in Akt1 KO mice compared with the wild-type control. These observations indicate that Akt1 is important for the regulation of skeletal muscle fiber size, whereas the regulation of muscle fiber type and muscle mitochondrial content occurs independently of Akt1 activity.

PSIV-10 Thyroid hormone does not protect skeletal muscle mitochondrial function from heat stress in pigs

PSIV-10 Thyroid hormone does not protect skeletal muscle mitochondrial function from heat stress in pigs

Cadmium exposure induces skeletal muscle insulin resistance through the reactive oxygen species-mediated PINK1/Parkin pathway

Epidemiological studies have suggested a positive association between environmental cadmium (Cd) exposure and type 2 diabetes mellitus (T2DM). Skeletal muscle insulin resistance (IR) plays a critical role in the pathogenesis of T2DM. This study aimed to investigate the effects of chronic low-level Cd exposure on skeletal muscle IR and its potential mechanism. Rats were exposed to drinking water containing 2 or 10 mg/L Cd for 24 weeks. Differentiated L6 myotubes were treated with Cd for 72 h. Immunofluorescence, flow cytometry assay, RNA-sequencing, and Seahorse analysis were conducted to determine the effects of Cd and its underlying mechanism on relevant parameters, including insulin sensitivity, glucose uptake, oxidative stress, mitophagy, and mitochondrial function in skeletal muscle and L6 myotubes. N-acetyl-cysteine (NAC), a scavenger of reactive oxygen species (ROS), and mitophagy inhibitor Cyclosporin A (CsA) were used to confirm the role of oxidative stress in mitophagy and mitochondrial dysfunction caused by Cd. We found that rats exposed to 10 mg/L Cd exhibited hyperglycemia and skeletal muscle IR. Cd markedly increased IRS-1 phosphorylation at Ser612, while decreased levels of phosphorylated PI3K, Akt, AS160, inhibited GLUT4 translocation and glucose uptake. Mechanistically, Cd increased the intracellular ROS, hydrogen peroxide, and malondialdehyde levels and decreased antioxidase activity in L6 myotubes. Furthermore, Cd upregulated the mRNA and protein levels of LC3II/I, PINK1, and Parkin. In addition, Cd induced the formation of mitophagosomes, reduced the mitochondrial membrane potential, decreased the adenosine triphosphate content, and impaired the mitochondrial respiratory capacity. Strikingly, NAC ameliorated oxidative stress, excessive mitophagy, and the associated reduction in myotube insulin sensitivity, while inhibition of mitophagy by CsA alleviated skeletal muscle IR. In conclusion, this study reveals a previously unrecognized mechanism that chronic low-level Cd exposure may induce mitophagy by activating the PINK1/Parkin signal pathway by increasing ROS, thus causing skeletal muscle IR and elevated blood glucose.

Effects of Dietary Linoleic Acid on Mitochondrial Function in Skeletal Muscles Isolated From a Mouse Model of Cancer Cachexia

Effects of Dietary Linoleic Acid on Mitochondrial Function in Skeletal Muscles Isolated From a Mouse Model of Cancer Cachexia

Embryo thermal manipulation enhances mitochondrial function in the skeletal muscle of heat-stressed broilers by regulating transient receptor potential V2 expression

Heat stress induces mitochondrial dysfunction, thereby impeding skeletal muscle development and significantly impacting the economic efficiency of poultry production. This study aimed to investigate the effects of embryo thermal manipulation (TM, 41.5°C, 65% RH, 3 h/d during 16-18th embryonic age) on the mitochondrial function of the pectoralis major (PM) in broiler chickens exposed to thermoneutral (24 ± 1°C, 60% RH) or cyclic heat stress (35 ± 1°C, 60% RH, 12 h/d) from day 22 to 28, and to explore potential mechanisms involving transient receptor potential V2 (TRPV2). Additionally, in vitro experiments were conducted to assess the regulatory effects of TRPV2 pharmacological activation and inhibition on mitochondrial function in primary myotubes. The results revealed that TM had no discernible effect on the body weight and feed intake of broiler chickens under heat stress conditions (P > 0.05). However, it did delay the increase in rectal temperature and accelerate the decrease in serum T3 levels (P < 0.05). Furthermore, TM promoted the development of PM muscle fibers, significantly increasing myofiber diameter and cross-sectional area (P < 0.05). Under heat stress conditions, TM significantly upregulated the expression of mitochondrial electron transport chain (ETC) genes and TRPV2 in broiler PM muscle (P < 0.05), with a clear positive correlation observed between the two (P < 0.05). In vitro, pharmacological activation of TRPV2 not only increased its own expression but also enhanced mitochondrial ETC genes expression and oxidative phosphorylation function by upregulating intracellular calcium ion levels (P < 0.05). Conversely, TRPV2 inhibition had the opposite effect. Overall, this study underscores the potential of prenatal thermal manipulation in regulating postnatal broiler skeletal muscle development and mitochondrial function through the modulation of TRPV2 expression.

Adiposity in mares induces insulin dysregulation and mitochondrial dysfunction which can be mitigated by nutritional intervention

Obesity is a complex disease associated with augmented risk of metabolic disorder development and cellular dysfunction in various species. The goal of the present study was to investigate the impacts of obesity on the metabolic health of old mares as well as test the ability of diet supplementation with either a complex blend of nutrients designed to improve equine metabolism and gastrointestinal health or L-carnitine alone to mitigate negative effects of obesity. Mares (n = 19, 17.9 ± 3.7 years) were placed into one of three group: normal-weight (NW, n = 6), obese (OB, n = 7) or obese fed a complex diet supplement for 12 weeks (OBD, n = 6). After 12 weeks and completion of sample collections, OB mares received L-carnitine alone for an additional 6 weeks. Obesity in mares was significantly associated with insulin dysregulation, reduced muscle mitochondrial function, and decreased skeletal muscle oxidative capacity with greater ROS production when compared to NW. Obese mares fed the complex diet supplement had better insulin sensivity, greater cell lipid metabolism, and higher muscle oxidative capacity with reduced ROS production than OB. L-carnitine supplementation alone did not significantly alter insulin signaling, but improved lipid metabolism and muscle oxidative capacity with reduced ROS. In conclusion, obesity is associated with insulin dysregulation and altered skeletal muscle metabolism in older mares. However, dietary interventions are an effective strategy to improve metabolic status and skeletal muscle mitochondrial function in older mares.

Skeletal muscle mitochondrial dysfunction mediated by Pseudomonas aeruginosa quorum-sensing transcription factor MvfR: reversing effects with anti-MvfR and mitochondrial-targeted compounds.

Skeletal muscle, pivotal for many functions in the human body, including breathing and protecting internal organs, contains abundant mitochondria essential for maintaining cellular homeostasis during infection. The effect of Pseudomonas aeruginosa (PA) infections on skeletal muscle remains poorly understood. Our study delves into the role of a central quorum-sensing transcription factor, multiple virulence factor regulator (MvfR), that controls the expression of multiple acute and chronic virulence functions that contribute to the pathogenicity of PA. The significance of our study lies in the role of MvfR in the metabolic perturbances linked to mitochondrial functions in skeletal muscle and the effectiveness of the novel MvfR inhibitor and the mitochondrial-targeted peptide SS-31 in alleviating the mitochondrial disturbances caused by PA in skeletal muscle. Inhibiting MvfR or interfering with its effects can be a potential therapeutic strategy to curb PA virulence.

Arginine Regulates Skeletal Muscle Fiber Type Formation via mTOR Signaling Pathway.

The composition of skeletal muscle fiber types affects the quality of livestock meat and human athletic performance and health. L-arginine (Arg), a semi-essential amino acid, has been observed to promote the formation of slow-twitch muscle fibers in animal models. However, the precise molecular mechanisms are still unclear. This study investigates the role of Arg in skeletal muscle fiber composition and mitochondrial function through the mTOR signaling pathway. In vivo, 4-week C56BL/6J male mice were divided into three treatment groups and fed a basal diet supplemented with different concentrations of Arg in their drinking water. The trial lasted 7 weeks. The results show that Arg supplementation significantly improved endurance exercise performance, along with increased SDH enzyme activity and upregulated expression of the MyHC I, MyHC IIA, PGC-1α, and NRF1 genes in the gastrocnemius (GAS) and quadriceps (QUA) muscles compared to the control group. In addition, Arg activated the mTOR signaling pathway in the skeletal muscle of mice. In vitro experiments using cultured C2C12 myotubes demonstrated that Arg elevated the expression of slow-fiber genes (MyHC I and Tnnt1) as well as mitochondrial genes (PGC-1α, TFAM, MEF2C, and NRF1), whereas the effects of Arg were inhibited by the mTOR inhibitor rapamycin. In conclusion, these findings suggest that Arg modulates skeletal muscle fiber type towards slow-twitch fibers and enhances mitochondrial functions by upregulating gene expression through the mTOR signaling pathway.

Shenqi Fuzheng injection facilitates skeletal muscle mitophagy mediated by the ubiquitination of HIF-1α to ameliorate cancer-associated fatigue.

Cancer-related fatigue (CRF) significantly impacts the quality of life of cancer patients. This study investigates the therapeutic potential of Shenqi Fuzheng injection (SFI) in managing CRF, focusing on its mechanistic action in skeletal muscle. We utilized a CRF mouse model to examine the effects of SFI on physical endurance, monitoring activity levels, swimming times and rest periods. Proteomic analysis of the gastrocnemius muscle was performed using isobaric tags and liquid chromatography-tandem mass spectrometry to map the muscle proteome changes post-SFI treatment. Mitochondrial function in skeletal muscle was assessed via ATP bioluminescence assay. Furthermore, the regulatory role of the hypoxia inducible factor 1 subunit alpha (HIF-1α) signalling pathway in mediating SFI's effects was explored through western blotting. In CRF-induced C2C12 myoblasts, we evaluated cell viability (CCK-8 assay), apoptosis (flow cytometry) and mitophagy (electron microscopy). The study also employed pulldown, luciferase and chromatin immunoprecipitation assays to elucidate the molecular mechanisms underlying SFI's action, particularly focusing on the transcriptional regulation of PINK1 through HIF-1α binding at the PINK1 promoter region. Our findings reveal that SFI enhances physical mobility, reduces fatigue symptoms and exerts protective effects on skeletal muscles by mitigating mitochondrial damage and augmenting antioxidative responses. SFI promotes cell viability and induces mitophagy while decreasing apoptosis, primarily through the modulation of HIF-1α, PINK1 and p62 proteins. These results underscore SFI's efficacy in enhancing mitochondrial autophagy, thereby offering a promising approach for ameliorating CRF. The study not only provides insight into SFI's potential therapeutic mechanisms but also establishes a foundation for further exploration of SFI interventions in CRF management.

CISD3/MiNT is required for complex I function, mitochondrial integrity, and skeletal muscle maintenance

Mitochondria play a central role in muscle metabolism and function. A unique family of iron-sulfur proteins, termed CDGSH Iron Sulfur Domain-containing (CISD/NEET) proteins, support mitochondrial function in skeletal muscles. The abundance of these proteins declines during aging leading to muscle degeneration. Although the function of the outer mitochondrial CISD/NEET proteins, CISD1/mitoNEET and CISD2/NAF-1, has been defined in skeletal muscle cells, the role of the inner mitochondrial CISD protein, CISD3/MiNT, is currently unknown. Here, we show that CISD3 deficiency in mice results in muscle atrophy that shares proteomic features with Duchenne muscular dystrophy. We further reveal that CISD3 deficiency impairs the function and structure of skeletal muscles, as well as their mitochondria, and that CISD3 interacts with, and donates its [2Fe-2S] clusters to, complex I respiratory chain subunit NADH Ubiquinone Oxidoreductase Core Subunit V2 (NDUFV2). Using coevolutionary and structural computational tools, we model a CISD3-NDUFV2 complex with proximal coevolving residue interactions conducive of [2Fe-2S] cluster transfer reactions, placing the clusters of the two proteins 10 to 16 Å apart. Taken together, our findings reveal that CISD3/MiNT is important for supporting the biogenesis and function of complex I, essential for muscle maintenance and function. Interventions that target CISD3 could therefore impact different muscle degeneration syndromes, aging, and related conditions.

Ablation of Sam50 is associated with fragmentation and alterations in metabolism in murine and human myotubes.

The sorting and assembly machinery (SAM) Complex is responsible for assembling β-barrel proteins in the mitochondrial membrane. Comprising three subunits, Sam35, Sam37, and Sam50, the SAM complex connects the inner and outer mitochondrial membranes by interacting with the mitochondrial contact site and cristae organizing systemcomplex. Sam50, in particular, stabilizes the mitochondrial intermembrane space bridging (MIB) complex, which is crucial for protein transport, respiratory chain complex assembly, and regulation of cristae integrity. While the role of Sam50 in mitochondrial structure and metabolism in skeletal muscle remains unclear, this study aims to investigate its impact. Serial block-face-scanning electron microscopyand computer-assisted 3D renderings were employed to compare mitochondrial structure and networking in Sam50-deficient myotubes from mice and humans with wild-type (WT) myotubes. Furthermore, autophagosome 3D structure was assessed in human myotubes. Mitochondrial metabolic phenotypes were assessed using Gas Chromatography-Mass Spectrometry-based metabolomics to explore differential changes in WT and Sam50-deficient myotubes. The results revealed increased mitochondrial fragmentation and autophagosome formation in Sam50-deficient myotubes compared to controls. Metabolomic analysis indicated elevated metabolism of propanoate and several amino acids, including ß-Alanine, phenylalanine, and tyrosine, along with increased amino acid and fatty acid metabolism in Sam50-deficient myotubes. Furthermore, impairment of oxidative capacity was observed upon Sam50 ablation in both murine and human myotubes, as measured with the XF24 Seahorse Analyzer. Collectively, these findings support the critical role of Sam50 in establishing and maintaining mitochondrial integrity, cristae structure, and mitochondrial metabolism. By elucidating the impact of Sam50-deficiency, this study enhances our understanding of mitochondrial function in skeletal muscle.

Differential impact of sex on regulation of skeletal muscle mitochondrial function and protein homeostasis by hypoxia-inducible factor-1α in normoxia.

Hypoxia-inducible factor (HIF)-1α is continuously synthesized and degraded in normoxia. During hypoxia, HIF1α stabilization restricts cellular/mitochondrial oxygen utilization. Cellular stressors can stabilize HIF1α even during normoxia. However, less is known about HIF1α function(s) and sex-specific effects during normoxia in the basal state. Since skeletal muscle is the largest protein store in mammals and protein homeostasis has high energy demands, we determined HIF1α function at baseline during normoxia in skeletal muscle. Untargeted multiomics data analyses were followed by experimental validation in differentiated murine myotubes with loss/gain of function and skeletal muscle from mice without/with post-natal muscle-specific Hif1a deletion (Hif1amsd). Mitochondrial oxygen consumption studies using substrate, uncoupler, inhibitor, titration protocols; targeted metabolite quantification by gas chromatography-mass spectrometry; and post-mitotic senescence markers using biochemical assays were performed. Multiomics analyses showed enrichment in mitochondrial and cell cycle regulatory pathways in Hif1a deleted cells/tissue. Experimentally, mitochondrial oxidative functions and ATP content were higher with less mitochondrial free radical generation with Hif1a deletion. Deletion of Hif1a also resulted in higher concentrations of TCA cycle intermediates and HIF2α proteins in myotubes. Overall responses to Hif1amsd were similar in male and female mice, but changes in complex II function, maximum respiration, Sirt3 and HIF1β protein expression and muscle fibre diameter were sex-dependent. Adaptive responses to hypoxia are mediated by stabilization of constantly synthesized HIF1α. Despite rapid degradation, the presence of HIF1α during normoxia contributes to lower mitochondrial oxidative efficiency and greater post-mitotic senescence in skeletal muscle. In vivo responses to HIF1α in skeletal muscle were differentially impacted by sex. KEY POINTS: Hypoxia-inducible factor -1α (HIF1α), a critical transcription factor, undergoes continuous synthesis and proteolysis, enabling rapid adaptive responses to hypoxia by reducing mitochondrial oxygen consumption. In mammals, skeletal muscle is the largest protein store which is determined by a balance between protein synthesis and breakdown and is sensitive to mitochondrial oxidative function. To investigate the functional consequences of transient HIF1α expression during normoxia in the basal state, myotubes and skeletal muscle from male and female mice with HIF1α knockout were studied using complementary multiomics, biochemical and metabolite assays. HIF1α knockout altered the electron transport chain, mitochondrial oxidative function, signalling molecules for protein homeostasis, and post-mitotic senescence markers, some of which were differentially impacted by sex. The cost of rapid adaptive responses mediated by HIF1α is lower mitochondrial oxidative efficiency and post-mitotic senescence during normoxia.

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&lt;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&lt;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&lt;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&lt;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.

Mitochondrial NAD Profile in a Rat Model of Attention-Deficit/Hyperactivity Disorder

Mitochondria require nicotinamide adenine dinucleotide (NAD) to satisfy cellular energy needs. Mitochondrial stress has been reported in skeletal muscle tissue of humans with attention-deficit/hyperactivity disorder. NAD deficiency may indicate mitochondrial stress that can be assessed through NAD concentration assays and Western blotting of Nampt and Sirtuin 3 (SIRT3) proteins. Nampt is the rate-limiting enzyme in intracellular NAD recycling, while SIRT3 is an NAD-dependent protein deacetylase that regulates mitochondrial mass and activity. The objective of this study was to investigate skeletal muscle and liver tissues of rat strains that exhibit behaviors associated with ADHD. We hypothesized that tissues from rat models with ADHD-like behavior (Lewis and F344 strains) would have lower NAD concentrations and reduced Nampt and SIRT3 protein expression relative to the control strain (Sprague Dawley). To examine this hypothesis, we performed fluorescence tissue NAD concentration assays. We found a significant difference in liver NAD between Sprague Dawley and F344 rats (Sprague Dawley, 0.14 ± 0.07 mM/mg [mean ± standard deviation], F344, 0.22 ± 0.11; p &lt; 0.05). Western blot analysis for Nampt and SIRT3 proteins revealed a significant difference between the F344 and Sprague Dawley rats for Nampt protein in the extensor digitorum longus (EDL) muscle (Sprague Dawley, 1043930 ± 303677 arbitrary units [AU], F344, 1729014 ± 553568 AU; p &lt; 0.05). Our results indicate minor differences in tissue NAD concentration and Nampt protein expression in rat models with ADHD-like behaviors. This could be indicative of differential mitochondrial function in skeletal muscle or liver tissues. Further studies will examine the implications of increasing nicotinamide riboside in the skeletal muscles to observe the effects of NAD functioning. This work was supported by funds provided by the Cross-Disciplinary Science Institute at Gettysburg College (X-SIG) (HMS, JB) and a Gettysburg College Professional Development Grant (JB). 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 Extracellular Vesicles Enhance Mitochondrial Movement and have a Dose-dependent Effect on Increasing Mitochondrial Respiration

Chronic exercise leads to systemic health benefits across tissues, including an increase in mitochondrial function in skeletal muscle. One potential mechanism is via the release of extracellular vesicles (EVs), which can transfer functional cargo to recipient cells. Previously, we have shown that using an in vitro model of exercise, chronic contractile activity (CCA)-derived EVs (CCA-EV) increased mitochondrial biogenesis in healthy myoblasts. Here, we hypothesize that CCA-EVs will have a concentration-dependent effect on mitochondrial respiration, and also regulate mitochondrial dynamics in myoblasts. C2C12 myoblasts were differentiated into myotubes, and electrically paced (3hrs/day, 4 days, 14V, C-PACE EM, IonOptix). EVs from control and CCA-stimulated myotubes were isolated from conditioned media using differential ultracentrifugation and biophysically characterized by size and concentration using TRPS (Izon). C2C12 myoblasts were treated daily for 4 days using three different concentrations: 1.0E4; 2.0E4 and 4.0E4 EVs/cell (N=4-5). Basal and maximal oxygen consumption rates (OCR) were measured using the Seahorse XFe24 (Agilent). We used live-cell fluorescence microscopy and Mitometer software to access mitochondrial movement: area/volume, displacement, distance, length, perimeter, speed and velocity. Average size was unchanged between control-EVs (123±10.8 nm) and CCA-EVs (123±11.9 nm) (p=0.9137, N=3), and 2.3-fold more CCA-EVs were released vs. control-EVs (p=0.2072, N=3). Lowest EV concentration (1.0E+4 EVs/cell, N=5) had no effect on basal (p=0.4226) and maximal (p=0.1835) OCR. Intermediate dosage of CCA-EVs (2.0E+4 EVs/cell, N=5) increased basal OCR by 20% (p=0.0037) and maximal OCR by 18% (p=0.0095), while the highest concentration (4.0E+4 EVs/cell, N=4) decreased basal OCR by 9% (p=0.0001) and maximal OCR by 29% (p=0.0009), compared to control-EVs. Mitochondrial motility (N=484-976) in myoblasts treated daily after each day of CCA showed enhanced mitochondrial dynamics with CCA-EVs: increased area/volume (p&lt;0.0001), displacement (p&lt;0.0001), distance (p&lt;0.0001), mean intensity (p&lt;0.0001), perimeter/surface area (p=0.0137) and speed (p&lt;0.0001), compared to control-EVs. In summary, CCA-EVs induced concentration-dependent improvements in mitochondrial respiration in healthy myoblasts, concomitant with enhanced mitochondrial movement. These results open exciting avenues for the therapeutic potential of CCA-EVs in rescuing mitochondrial dysfunction. TFGS is funded by a Research Manitoba Postdoctoral Fellowship. Grants from CHRIM, CHF, DREAM and NSERC to AS funded the research. 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.

Unraveling the Interplay of p21, Autophagy, and Mitophagy in Mitigating Skeletal Muscle Dysfunction during Aging and Obesity

Introduction: The accumulation of senescent cells accelerates aging, a process further exacerbated by obesity. The decline of muscle function is a hallmark of this progression due to muscle atrophy stemming from dysregulated autophagy and mitochondrial function in skeletal muscles. p21 is one of the major regulators, which accumulates in tissues with aging and obesity. Previous studies have demonstrated that the elimination of p21-highly-expressing ( p21high) cells improves muscle function. However, the connection between p21 and its influence on muscle function remains unclear. One possible explanation is associated with alternations in autophagy and mitophagy through mitochondria dysfunction and increased reactive oxygen species. Therefore, this study investigated the link between autophagy, mitophagy, and p21 in the context of aging and obesity. Methods: Four 18-month-old male p21-Cre/+; +/+ (P) and p21-Cre/+; DTA/+ (PD) mice, fed by high-fat diet for 15 months were administered with tamoxifen for twice. We previously observed the P group with accumulation of p21high cells, which were effectively eliminated in PD group. Soleus muscles were collected for subsequent immunoblotting analysis, employing primary antibodies targeting key proteins involved in autophagy and mitophagy cellular processes. Results: Our study elucidates dynamic changes driven by eliminating p21high cells in autophagy and mitophagy marker levels within the soleus muscles of mice exposed to a high-fat diet for 15 months. There was no significant difference in the body weight of P and PD mice (65.1±2.58 g vs. 64.3±2.22 g). In P mice, an increase in the upstream autophagy initiator, Beclin1, was observed, along with elevated levels of ATG4A, ATG7, and the ATG12-ATG5 complex, indicating elongation of autophagosome formation. Despite increased autophagosome marker p62, the LC3-II/LC3-I ratio remained unchanged. Lysosomal markers, LAMP1 and LAMP2, were both elevated in P mice. Conversely, PD mice exhibited heightened mitochondrial damage elimination markers, Parkin, VDAC1, and DRP1. Although PINK1 levels were not significantly changed, they showed an increasing trend. Conclusion: Our results suggest that eliminating p21high cells may be the potential therapeutic approach to enhance skeletal muscle function in the context of obesity and aging, possibly through autophagy and mitophagy. 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.

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&lt;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&gt;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.