Constitutive activation of estrogen receptor α signaling in muscle prolongs exercise endurance in mice.

Estrogen is a female hormone that plays a role in various tissues, although the mechanism in skeletal muscle has not been fully clarified. We previously showed that systemic administration of estrogen for 10 weeks ameliorated decreased exercise endurance in ovariectomized mice. To assess whether a long-term and muscle-specific activation of estrogen signaling modulates muscle function, we constructed an expression plasmid for a constitutively active estrogen receptor α (caERα) under the control of muscle creatine kinase (Mck) gene promoter/enhancer. In C2C12 mouse myoblastic cells, transfection of the Mck-caERα plasmid elevated the estrogen response element-driven transcription in a ligand-independent manner. Using this construct, we generated Mck-caERα transgenic mice, in which caERα is predominantly expressed in muscle. Treadmill running test revealed that female Mck-caERα mice exhibit a prolonged running time and distance compared with the wild-type mice. Moreover, microarray expression analysis revealed that the genes related to lipid metabolism, insulin signaling, and growth factor signaling were particularly upregulated in the quadriceps femoris muscle of Mck-caERα mice. These results suggest that estrogen signaling potentiates exercise endurance in skeletal muscle through modulating the expression of metabolism-associated genes.

Hydrogen Rich Water Consumption Positively Affects Muscle Performance, Lactate Response, and Alleviates Delayed Onset of Muscle Soreness After Resistance Training.

ABSTRACT: Botek, M, Krejcí, J, McKune, A, Valenta, M, and Sládecková, B. Hydrogen rich water consumption positively affects muscle performance, lactate response, and alleviates delayed onset of muscle soreness after resistance training. J Strength Cond Res 36(10): 2792-2799, 2022-Positive outcomes of hydrogen rich water (HRW) supplementation on endurance performance have been shown, but the effects of HRW in resistance training are unclear. The aim of this study was to assess the effects of 1,260 ml of HRW intake on physiological, perceptual, and performance responses to a resistance training and after 24 hours of recovery. This randomized, double-blinded placebo-controlled cross-over study included 12 men aged 23.8 ± 1.9 years. Subjects performed a half squat, knee flexion, and extension exercises with the load set at 70% of 1 repetition maximum for 3 sets (10 reps/set). Lunges were performed with a load of 30% of body mass for 3 sets (20 reps/set). Time of each set, lactate, and ratings of perceived exertion were assessed mid-way through exercise and immediately after the exercise. Creatine kinase, muscle soreness visual analog scale ratings, countermovement jump, and heart rate variability were evaluated before the training and at 30 minutes, 6, and 24 hours of recovery. Lunges were performed faster with HRW compared with placebo ( p < 0.001). Hydrogen rich water reduced lactate at mid-way and immediately after the exercise (HRW: 5.3 ± 2.1 and 5.1 ± 2.2, placebo: 6.5 ± 1.8 and 6.3 ± 2.2 mmol·L -1 , p ≤ 0.008). Visual analog scale ratings were significantly lower with HRW (26 ± 11 vs. 41 ± 20 mm, p = 0.002) after 24 hours of recovery. In conclusion, an acute intermittent HRW hydration improved muscle function, reduced the lactate response, and alleviated delayed onset of muscle soreness.

Naturally Occurring Mutations to Muscle-Type Creatine Kinase Impact Its Canonical and Pharmacological Activities in a Substrate-Dependent Manner In Vitro.

Tenofovir (TFV) is a key component of human immunodeficiency virus (HIV) pre-exposure prophylaxis (PrEP). TFV is a nucleotide analog reverse-transcriptase inhibitor prodrug that requires two separate phosphorylation reactions by intracellular kinases to form the active metabolite tenofovir-diphosphate (TFV-DP). Muscle-type creatine kinase (CKM) has previously been demonstrated to be the kinase most responsible for the phosphorylation of tenofovir-monophosphate (TFV-MP) to the active metabolite in colon tissue. Because of the importance of CKM in TFV activation, genetic variation in CKM may contribute to interindividual variability in TFV-DP levels. In the present study, we report 10 naturally occurring CKM mutations that reduced TFV-MP phosphorylation in vitro: T35I, R43Q, I92M, H97Y, R130H, R132C, F169L, Y173C, W211R, V280L, and N286I. Interestingly, of these 10, only 4-R130H, R132C, W211R, and N286I-reduced both canonical CKM activities: ADP phosphorylation and ATP dephosphorylation. Although positions 130, 132, and 286 are located in the active site, the other mutations that resulted in decreased TFV-MP phosphorylation occur elsewhere in the protein structure. Four of these eight mutations-T35I, R43Q, I92M, and W211R-were found to decrease the thermal stability of the protein. Additionally, the W211R mutation was found to impact protein structure both locally and at a distance. These data suggest a substrate-specific effect such that certain mutations are tolerated for canonical activities while being deleterious toward the pharmacological activity of TFV activation, which could influence PrEP outcomes. SIGNIFICANCE STATEMENT: Muscle-type creatine kinase (CKM) is important to the activation of tenofovir, a key component of HIV prophylaxis. This study demonstrates that naturally occurring CKM mutations impact enzyme function in a substrate-dependent manner such that some mutations that do not reduce canonical activities lead to reductions in the pharmacologically relevant activity. This finding at the intersection of drug metabolism and energy metabolism is important to the perspective on pharmacology of other drugs acted on by atypical drug-metabolizing enzymes.

Quantification of creatine kinase reaction rate in mouse hindlimb using phosphorus-31 magnetic resonance spectroscopic fingerprinting.

The goal of this study was to evaluate the accuracy, reproducibility, and efficiency of a 31 P magnetic resonance spectroscopic fingerprinting (31 P-MRSF) method for fast quantification of the forward rate constant of creatine kinase (CK) in mouse hindlimb. The 31 P-MRSF method acquired spectroscopic fingerprints using interleaved acquisition of phosphocreatine (PCr) and γATP with ramped flip angles and a saturation scheme sensitive to chemical exchange between PCr and γATP. Parameter estimation was performed by matching the acquired fingerprints to a dictionary of simulated fingerprints generated from the Bloch-McConnell model. The accuracy of 31 P-MRSF measurements was compared with the magnetization transfer (MT-MRS) method in mouse hindlimb at 9.4 T (n = 8). The reproducibility of 31 P-MRSF was also assessed by repeated measurements. Estimation of the CK rate constant using 31 P-MRSF (0.39 ± 0.03 s-1 ) showed a strong agreement with that using MT-MRS measurements (0.40 ± 0.05 s-1 ). Variations less than 10% were achieved with 2 min acquisition of 31 P-MRSF data. Application of the 31 P-MRSF method to mice subjected to an electrical stimulation protocol detected an increase in CK rate constant in response to stimulation-induced muscle contraction. These results demonstrated the potential of the 31 P-MRSF framework for rapid, accurate, and reproducible quantification of the chemical exchange rate of CK in vivo.

Kinetic analysis of the transphosphorylation with creatine kinase by pressure-assisted capillary electrophoresis/dynamic frontal analysis.

Kinetic reactions of the transphosphorylation with creatine kinase (CK) were individually investigated between creatine (Cr) and creatine phosphate (CrP) by pressure-assisted capillary electrophoresis/dynamic frontal analysis (pCE/DFA). The transphosphorylations are reversible between Cr and CrP, and reverse reactions inevitably accompany in general batch analyses. In pCE/DFA, the kinetic reaction proceeds in a separation capillary and the product is continuously resolved from the substrate zone. Therefore, the formation rate is kept constant at the substrate zone without the reverse reaction, and the product is detected as a plateau signal. This study demonstrates the direct and individual analyses of both the forward and the backward kinetic reactions with CK by pCE/DFA. A plateau signal was detected in the pCE/DFA with ADP or ATP as one of the products on either the forward or the backward reactions. The Michaelis-Menten constants of Km,ATP (from Cr to CrP) and Km,ADP (from CrP to Cr) were successfully determined through the plateau signal. Determined values of Km,ATP and Km,ADP by pCE/DFA were smaller than the ones obtained by the pre-capillary batch analyses. The results agree with the fact that the reverse reaction is excluded in the analysis of the kinetic reactions. The proposed pCE/DFA is useful on individual analyses of both forward and backward kinetic reactions without any interference from the reverse reaction.

Alkylation of rabbit muscle creatine kinase surface methionine residues inhibits enzyme activity in vitro.

Creatine kinase (CK) catalyzes the formation of phosphocreatine from adenosine triphosphate (ATP) and creatine. The highly reactive free cysteine residue in the active site of the enzyme (Cys283) is considered essential for the enzymatic activity. In previous studies we demonstrated that Cys283 is targeted by the alkylating chemical warfare agent sulfur mustard (SM) yielding a thioether with a hydroxyethylthioethyl (HETE)-moiety. In the present study, the effect of SM on rabbit muscle CK (rmCK) activity was investigated with special focus on the alkylation of Cys283 and of reactive methionine (Met) residues. For investigation of SM-alkylated amino acids in rmCK, micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry measurements were performed using the Orbitrap technology. The treatment of rmCK with SM resulted in a decrease of enzyme activity. However, this decrease did only weakly correlate to the modification of Cys283 but was conclusive for the formation of Met70-HETE and Met179-HETE. In contrast, the activity of mutants of rmCK produced by side-directed mutagenesis that contained substitutions of the respective Met residues (Met70Ala, Met179Leu, and Met70Ala/Met179Leu) was highly resistant against SM. Our results point to a critical role of the surface exposed Met70 and Met179 residues for CK activity.

The Effects of Marine Algal Polyphenols, Phlorotannins, on Skeletal Muscle Growth in C2C12 Muscle Cells via Smad and IGF-1 Signaling Pathways.

Skeletal muscle is an important tissue in energy metabolism and athletic performance. The use of effective synthetic supplements and drugs to promote muscle growth is limited by various side effects. Moreover, their use is prohibited by anti-doping agencies; hence, natural alternatives are needed. Therefore, we evaluated the muscle growth effect of substances that can act like synthetic supplements from edible marine algae. First, we isolated six marine algal polyphenols belonging to the phlorotannin class, namely dieckol (DK), 2,7″-phloroglucinol-6,6'-bieckol (PHB), phlorofucofuroeckol A (PFFA), 6,6'-bieckol (6,6-BK), pyrogallol-phloroglucinol-6,6'-bieckol (PPB), and phloroglucinol (PG) from an edible brown alga, Ecklonia cava and evaluated their effects on C2C12 myoblasts proliferation and differentiation. Of the six phlorotannin isolates evaluated, DK and PHB induced the highest degree of C2C12 myoblast proliferation. In addition, DK and PHB regulates myogenesis by down-regulating the Smad signaling, a negative regulator, and up-regulating the insulin-like growth factor-1 (IGF-1) signaling, a positive regulator. Interestingly, DK and PHB bind strongly to myostatin, which is an inhibitor of myoblast proliferation, while also binding to IGF-1 receptors. Moreover, they bind to IGF-1 receptor. These results suggest that DK and PHB are potential natural muscle building supplements and could be a safer alternative to synthetic drugs.

Prevalence of work-related musculoskeletal disorders among Egyptian printing workers evidenced by using serum biomarkers of inflammation, oxidative stress, muscle injury, and collagen type I turnover.

OBJECTIVE: Printing workers experience a high rate of musculoskeletal disorders (MSDs). This study aims to determine the prevalence of MSDs, estimate serum biomarkers denoting musculoskeletal tissue changes, and determine some individual risk factors for MSDs among Egyptian printing workers. METHODS: Eighty-five male printing workers and 90 male administrative employees (control group) were recruited from a printing press in Giza. A validated version of the standardized Nordic questionnaire was used. Serum biomarkers of inflammation (interleukin (IL)-1α, IL-1ß, IL-6, tumor necrosis factor (TNF)-α, and C-reactive protein (CRP)), cell stress or injury (malondialdehyde (MDA) and creatine kinase skeletal muscle (CK-MM)), and collagen metabolism (collagen-I carboxy-terminal propeptide (PICP) and type-I collagen cross-linked C-telopeptide (CTx)) were measured for all participants. RESULTS: This study showed a significant (p < 0.001) prevalence of the musculoskeletal symptoms (76.5%) and significant (p < 0.001) elevation in the levels of all measured biomarkers among the printing workers (means ± SD: IL-1α = 1.55 ± 0.9, IL-1ß = 1.53 ± 0.87, IL-6 = 1.55 ± 0.85, TNF-α = 4.9 ± 2.25, CRP = 6.78 ± 3.07, MDA = 3.41 ± 1.29, CK-MM = 132.47 ± 69.01, PICP = 103.48 ± 36.44, and CTx = 0.47 ± 0.16) when compared with their controls (prevalence: 34.4%; means ± SD: IL-1α = 0.88 ± 0.61, IL-1ß = 0.96 ± 0.72, IL-6 = 1.03 ± 0.75, TNF-α = 2.56 ± 1.99, CRP = 2.36 ± 1.1, MDA = 0.85 ± 0.21, CK-MM = 53.48 ± 33.05, PICP = 56.49 ± 9.05, and CTx = 0.31 ± 0.06). Also, significant (p < 0.001) positive strong associations were observed between age, body mass index (BMI), and the duration of employment with all measured biomarkers, where all correlation coefficients were >0.7. CONCLUSION: Printing workers suffer a high prevalence of work-related MSDs that might be related to some individual factors (age, BMI, and duration of employment). Consequently, preventive ergonomic interventions should be applied. Further studies should be done to elucidate the link between tissue changes and detected biomarkers to follow the initiation and progression of MSDs and study the effectiveness of curative interventions.

The potential of the novel NAD+ supplementing agent, SNH6, as a therapeutic strategy for the treatment of Friedreich's ataxia.

Friedreich's ataxia (FA) is due to deficiency of the mitochondrial protein, frataxin, which results in multiple pathologies including a deadly, hypertrophic cardiomyopathy. Frataxin loss leads to deleterious accumulations of redox-active, mitochondrial iron, and suppressed mitochondrial bioenergetics. Hence, there is an urgent need to develop innovative pharmaceuticals. Herein, the activity of the novel compound, 6-methoxy-2-salicylaldehyde nicotinoyl hydrazone (SNH6), was assessed in vivo using the well-characterized muscle creatine kinase (MCK) conditional frataxin knockout (KO) mouse model of FA. The design of SNH6 incorporated a dual-mechanism mediating: (1) NAD+-supplementation to restore cardiac bioenergetics; and (2) iron chelation to remove toxic mitochondrial iron. In these studies, MCK wild-type (WT) and KO mice were treated for 4-weeks from the asymptomatic age of 4.5-weeks to 8.5-weeks of age, where the mouse displays an overt cardiomyopathy. SNH6-treatment significantly elevated NAD+ and markedly increased NAD+ consumption in WT and KO hearts. In SNH6-treated KO mice, nuclear Sirt1 activity was also significantly increased together with the NAD+-metabolic product, nicotinamide (NAM). Therefore, NAD+-supplementation by SNH6 aided mitochondrial function and cardiac bioenergetics. SNH6 also chelated iron in cultured cardiac cells and also removed iron-loading in vivo from the MCK KO heart. Despite its dual beneficial properties of supplementing NAD+ and chelating iron, SNH6 did not mitigate cardiomyopathy development in the MCK KO mouse. Collectively, SNH6 is an innovative therapeutic with marked pharmacological efficacy, which successfully enhanced cardiac NAD+ and nuclear Sirt1 activity and reduced cardiac iron-loading in MCK KO mice. No other pharmaceutical yet designed exhibits both these effective pharmacological properties.

Structure-activity relationship analysis of dammarane-type natural products as muscle-type creatine kinase activators.

Muscle-type creatine kinase (CK-MM) is the target protein of ginsenosides in skeletal muscle. 20(S)-protopanaxadiol [20(S)-PPD] is an activator of CK-MM and exerts an anti-fatigue effect. In this study, twelve dammarane-type compounds were used for structure-activity relationship analysis in terms of enzyme activity, intermolecular interaction, and molecular docking. Enzyme activity analysis showed that 20(S)-PPD, 20(R)-PPD, 20(S)-protopanaxatriol [20(S)-PPT], 25-OH-PPD, 24-COOH-PPD, panaxadiol (PD), and ginsenoside Rh2 significantly increased CK-MM activity. Panaxatriol (PT), ocotillol, ginsenoside Rg1, and ginsenoside Rd had no significant influence on CK-MM activity, while jujubogenin inhibited its activity. Biolayer Interferometry (BLI) assay produced the same results as those on enzyme activity. The interaction intensity between dammarane-type compounds and CK-MM was linearly related to the compounds' maximum increment rate of enzyme activity. Molecular docking showed the following sequence of docking scores: Rd > Rg1 > Rh2 > 24-COOH-PPD > 20(S)-PPD > 20(S)-PPT > 25-OH-PPD > 20(R)-PPD > ocotillol > PT > PD > jujubogenin. We demonstrated that 20(S)-PPD was the best activator of CK-MM among the 12 dammarane-type compounds. The cyclization of the dammarane side chain, the hydroxyl group at position C6, and the glycosylation of C3, C6, and C20 reduced the ability to activate CK-MM. These findings can help in the development of enhanced CK-MM activators through structural modification.

Association between Muscle Damage and Head Impacts in High School American Football.

Subconcussive head impacts (SHI), defined as impacts to the cranium that do not result in concussion symptoms, are gaining traction as a major public health concern. The contribution of physiological factors such as physical exertion and muscle damage to SHI-dependent changes in neurological measures remains unknown. A prospective longitudinal study examined the association between physiological factors and SHI kinematics in 15 high school American football players over one season. Players wore a sensor-installed mouthguard for all practices and games, recording frequency and magnitude of all head impacts. Serum samples were collected at 12 time points (pre-season, pre- and post-game for five in-season games, and post-season) and were assessed for an isoenzyme of creatine kinase (CK-MM) primarily found in skeletal muscle. Physical exertion was estimated in the form of excess post-exercise oxygen consumption (EPOC) from heart rate data captured during the five games. Mixed-effect regression models indicated that head impact kinematics were significantly and positively associated with change in CK-MM but not EPOC. There was a significant and positive association between CK-MM and EPOC. These data suggest that when examining SHI, effects of skeletal muscle damage should be considered when using outcome measures that may have an interaction with muscle damage.

Antipsychotic-induced disorders: Reported cases and prospective study on muscle biomarkers after high exposure to haloperidol.

Antipsychotic drugs are known to induce neuromuscular effects. In this study, we review 13 years (2002-2014) of antipsychotic intoxications reported by the anti-poisoning center of Algiers (APCA). The most recorded symptoms were neuromuscular/muscular disorders, of which haloperidol was the most inducer among all antipsychotics. A prospective study was conducted between December 2012 and January 2017 to evaluate muscle effects generated after intentional or accidental ingestion of haloperidol. Fifty-one patients admitted in different emergency departments in Algiers were included in this study. Urine and blood samples were collected from each patient for biological and toxicological monitoring and a group of healthy volunteers was assessed for comparison purpose. There was no significant difference in plasma lactate dehydrogenase (LDH) activity between healthy volunteers and exposed patients even when high levels of haloperidol were recorded. In contrast, selenium concentration and creatine kinase (CK) activity in plasma samples were significantly higher in patients exposed to high levels of haloperidol compared to healthy volunteers. Large percentage of patients exposed to high levels of haloperidol presented a significant elevated CK activity and high selenium concentration regarding the physiological thresholds. Additionally, CK activity and selenium concentration correlated positively with plasma content of haloperidol suggesting a dose-dependent relationship. In conclusion, some biomarkers (CK and selenium) may reflect muscle adverse effects of high haloperidol exposure that result possibly from muscle rigidity.

IL-10 prevents aging-associated inflammation and insulin resistance in skeletal muscle.

Altered energy balance and insulin resistance are important characteristics of aging. Skeletal muscle is a major site of glucose disposal, and the role of aging-associated inflammation in skeletal muscle insulin resistance remains unclear. To investigate, we examined glucose metabolism in 18-mo-old transgenic mice with muscle-specific overexpression of IL-10 (MIL10) and in wild-type mice during hyperinsulinemic-euglycemic clamping. Despite similar fat mass and energy balance, MIL10 mice were protected from aging-associated insulin resistance with significant increases in glucose infusion rates, whole-body glucose turnover, and skeletal muscle glucose uptake (∼60%; P < 0.05), as compared to age-matched WT mice. This protective effect was associated with decreased muscle inflammation, but no changes in adipose tissue inflammation in aging MIL10 mice. These results demonstrate the importance of skeletal muscle inflammation in aging-mediated insulin resistance, and our findings further implicate a potential therapeutic role of anti-inflammatory cytokine in the treatment of aging-mediated insulin resistance.-Dagdeviren, S., Jung, D. Y., Friedline, R. H., Noh, H. L., Kim, J. H., Patel, P. R., Tsitsilianos, N., Inashima, K., Tran, D. A., Hu, X., Loubato, M. M., Craige, S. M., Kwon, J. Y., Lee, K. W., Kim, J. K. IL-10 prevents aging-associated inflammation and insulin resistance in skeletal muscle.

Proteomic analysis of cardiac ventricles: baso-apical differences.

The heart is characterized by a remarkable degree of heterogeneity. Since different cardiac pathologies affect different cardiac regions, it is important to understand molecular mechanisms by which these parts respond to pathological stimuli. In addition to already described left ventricular (LV)/right ventricular (RV) and transmural differences, possible baso-apical heterogeneity has to be taken into consideration. The aim of our study has been, therefore, to compare proteomes in the apical and basal parts of the rat RV and LV. Two-dimensional electrophoresis was used for the proteomic analysis. The major result of this study has revealed for the first time significant baso-apical differences in concentration of several proteins, both in the LV and RV. As far as the LV is concerned, five proteins had higher concentration in the apical compared to basal part of the ventricle. Three of them are mitochondrial and belong to the "metabolism and energy pathways" (myofibrillar creatine kinase M-type, L-lactate dehydrogenase, dihydrolipoamide dehydrogenase). Myosin light chain 3 is a contractile protein and HSP60 belongs to heat shock proteins. In the RV, higher concentration in the apical part was observed in two mitochondrial proteins (creatine kinase S-type and proton pumping NADH:ubiquinone oxidoreductase). The described changes were more pronounced in the LV, which is subjected to higher workload. However, in both chambers was the concentration of proteins markedly higher in the apical than that in basal part, which corresponds to the higher energetic demand and contractile activity of these segments of both ventricles.

Rhabdomyolysis in a Tertiary PICU: A 10-Year Study.

OBJECTIVES: Rhabdomyolysis is a disorder of muscle breakdown. The aim of this study was to describe the epidemiology of rhabdomyolysis in children admitted to a PICU and to assess the relationship between peak creatinine kinase and mortality. DESIGN: Retrospective cohort study in children admitted to the PICU with rhabdomyolysis between January 1, 2005, and December 31, 2014. Demographic, clinical, and outcome data were recorded. Outcomes were analyzed by level of peak creatinine kinase value (0-10,000, 10,001-50,000, > 50,000IU/L). Long-term renal outcomes were reported for PICU survivors. SETTING: A single-centre academic tertiary PICU. PATIENTS: Children admitted to the PICU with serum creatinine kinase level greater than 1,000 IU/L. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: There were 182 children with rhabdomyolysis. The median peak creatinine kinase value was 3,583 IU/L (1,554-9,608). The primary diagnostic categories included sepsis, trauma, and cardiac arrest. Mortality for peak creatinine kinase values 0-10,000, 10,001-50,000, and > 50,000 IU/L were 24/138 (17%), 6/28 (21%), and 3/16 (19%), respectively (p = 0.87). Children with a peak creatinine kinase greater than 10,000 IU/L had a longer duration of mechanical ventilation and ICU length of stay than children with peak creatinine kinase less than 10,000. Renal replacement therapy was administered in 29/182 (16%). There was longer duration of mechanical ventilation (273 [141-548] vs. 73 [17-206] hr [p < 0.001]) and ICU length of stay (334 [147-618] vs. 100 [37-232] hr (p < 0.001)] in children receiving renal replacement therapy. Continuous veno-venous hemofiltration was the most common modality 23/29 (79%). Only one child required renal replacement therapy postintensive care stay, and adverse long-term renal outcomes were uncommon. CONCLUSIONS: In children with rhabdomyolysis requiring intensive care, peak creatinine kinase was not associated with mortality but is associated with greater use of intensive care resources. Chronic kidney disease is an uncommon sequelae of rhabdomyolysis in children requiring intensive care.

Exercise Inducible Lactate Dehydrogenase B Regulates Mitochondrial Function in Skeletal Muscle.

Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate, which are critical fuel metabolites of skeletal muscle particularly during exercise. However, the physiological relevance of LDH remains poorly understood. Here we show that Ldhb expression is induced by exercise in human muscle and negatively correlated with changes in intramuscular pH levels, a marker of lactate production, during isometric exercise. We found that the expression of Ldhb is regulated by exercise-induced peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α). Ldhb gene promoter reporter studies demonstrated that PGC-1α activates Ldhb gene expression through multiple conserved estrogen-related receptor (ERR) and myocyte enhancer factor 2 (MEF2) binding sites. Transgenic mice overexpressing Ldhb in muscle (muscle creatine kinase (MCK)-Ldhb) exhibited increased exercise performance and enhanced oxygen consumption during exercise. MCK-Ldhb muscle was shown to have enhanced mitochondrial enzyme activity and increased mitochondrial gene expression, suggesting an adaptive oxidative muscle transformation. In addition, mitochondrial respiration capacity was increased and lactate production decreased in MCK-Ldhb skeletal myotubes in culture. Together, these results identified a previously unrecognized Ldhb-driven alteration in muscle mitochondrial function and suggested a mechanism for the adaptive metabolic response induced by exercise training.

Top-Down Proteomics of Large Proteins up to 223 kDa Enabled by Serial Size Exclusion Chromatography Strategy.

Mass spectrometry (MS)-based top-down proteomics is a powerful method for the comprehensive analysis of proteoforms that arise from genetic variations and post-translational modifications (PTMs). However, top-down MS analysis of high molecular weight (MW) proteins remains challenging mainly due to the exponential decay of signal-to-noise ratio with increasing MW. Size exclusion chromatography (SEC) is a favored method for size-based separation of biomacromolecules but typically suffers from low resolution. Herein, we developed a serial size exclusion chromatography (sSEC) strategy to enable high-resolution size-based fractionation of intact proteins (10-223 kDa) from complex protein mixtures. The sSEC fractions could be further separated by reverse phase chromatography (RPC) coupled online with high-resolution MS. We have shown that two-dimensional (2D) sSEC-RPC allowed for the identification of 4044 more unique proteoforms and a 15-fold increase in the detection of proteins above 60 kDa, compared to one-dimensional (1D) RPC. Notably, effective sSEC-RPC separation of proteins significantly enhanced the detection of high MW proteins up to 223 kDa and also revealed low abundance proteoforms that are post-translationally modified. This sSEC method is MS-friendly, robust, and reproducible and, thus, can be applied to both high-efficiency protein purification and large-scale proteomics analysis of cell or tissue lysate for enhanced proteome coverage, particularly for low abundance and high MW proteoforms.

Hyperhomocysteinemia associated skeletal muscle weakness involves mitochondrial dysfunction and epigenetic modifications.

HHcy has been implicated in elderly frailty, but the underlying mechanisms are poorly understood. Using C57 and CBS+/- mice and C2C12 cell line, we investigated mechanisms behind HHcy induced skeletal muscle weakness and fatigability. Possible alterations in metabolic capacity (levels of LDH, CS, MM-CK and COX-IV), in structural proteins (levels of dystrophin) and in mitochondrial function (ATP production) were examined. An exercise regimen was employed to reverse HHcy induced changes. CBS+/- mice exhibited more fatigability, and generated less contraction force. No significant changes in muscle morphology were observed. However, there is a corresponding reduction in large muscle fiber number in CBS+/- mice. Excess fatigability was not due to changes in key enzymes involved in metabolism, but was due to reduced ATP levels. A marginal reduction in dystrophin levels along with a decrease in mitochondrial transcription factor A (mtTFA) were observed. There was also an increase in the mir-31, and mir-494 quantities that were implicated in dystrophin and mtTFA regulation respectively. The molecular changes elevated during HHcy, with the exception of dystrophin levels, were reversed after exercise. In addition, the amount of NRF-1, one of the transcriptional regulators of mtTFA, was significantly decreased. Furthermore, there was enhancement in mir-494 levels and a concomitant decline in mtTFA protein quantity in homocysteine treated cells. These changes in C2C12 cells were also accompanied by an increase in DNMT3a and DNMT3b proteins and global DNA methylation levels. Together, these results suggest that HHcy plays a causal role in enhanced fatigability through mitochondrial dysfunction which involves epigenetic changes.

Proteome-wide muscle protein fractional synthesis rates predict muscle mass gain in response to a selective androgen receptor modulator in rats.

Biomarkers of muscle protein synthesis rate could provide early data demonstrating anabolic efficacy for treating muscle-wasting conditions. Androgenic therapies have been shown to increase muscle mass primarily by increasing the rate of muscle protein synthesis. We hypothesized that the synthesis rate of large numbers of individual muscle proteins could serve as early response biomarkers and potentially treatment-specific signaling for predicting the effect of anabolic treatments on muscle mass. Utilizing selective androgen receptor modulator (SARM) treatment in the ovariectomized (OVX) rat, we applied an unbiased, dynamic proteomics approach to measure the fractional synthesis rates (FSR) of 167-201 individual skeletal muscle proteins in triceps, EDL, and soleus. OVX rats treated with a SARM molecule (GSK212A at 0.1, 0.3, or 1 mg/kg) for 10 or 28 days showed significant, dose-related increases in body weight, lean body mass, and individual triceps but not EDL or soleus weights. Thirty-four out of the 94 proteins measured from the triceps of all rats exhibited a significant, dose-related increase in FSR after 10 days of SARM treatment. For several cytoplasmic proteins, including carbonic anhydrase 3, creatine kinase M-type (CK-M), pyruvate kinase, and aldolase-A, a change in 10-day FSR was strongly correlated (r(2) = 0.90-0.99) to the 28-day change in lean body mass and triceps weight gains, suggesting a noninvasive measurement of SARM effects. In summary, FSR of multiple muscle proteins measured by dynamics of moderate- to high-abundance proteins provides early biomarkers of the anabolic response of skeletal muscle to SARM.

Cellular compartmentation of energy metabolism: creatine kinase microcompartments and recruitment of B-type creatine kinase to specific subcellular sites.

There is an increasing body of evidence for local circuits of ATP generation and consumption that are largely independent of global cellular ATP levels. These are mostly based on the formation of multiprotein(-lipid) complexes and diffusion limitations existing in cells at different levels of organization, e.g., due to the viscosity of the cytosolic medium, macromolecular crowding, multiple and bulky intracellular structures, or controlled permeability across membranes. Enzymes generating ATP or GTP are found associated with ATPases and GTPases enabling the direct fueling of these energy-dependent processes, and thereby implying that it is the local and not the global concentration of high-energy metabolites that is functionally relevant. A paradigm for such microcompartmentation is creatine kinase (CK). Cytosolic and mitochondrial isoforms of CK constitute a well established energy buffering and shuttling system whose functions are very much based on local association of CK isoforms with ATP-providing and ATP-consuming processes. Here we review current knowledge on the subcellular localization and direct protein and lipid interactions of CK isoforms, in particular about cytosolic brain-type CK (BCK) much less is known compared to muscle-type CK (MCK). We further present novel data on BCK, based on three different experimental approaches: (1) co-purification experiments, suggesting association of BCK with membrane structures such as synaptic vesicles and mitochondria, involving hydrophobic and electrostatic interactions, respectively; (2) yeast-two-hybrid analysis using cytosolic split-protein assays and the identifying membrane proteins VAMP2, VAMP3 and JWA as putative BCK interaction partners; and (3) phosphorylation experiments, showing that the cellular energy sensor AMP-activated protein kinase (AMPK) is able to phosphorylate BCK at serine 6 to trigger BCK localization at the ER, in close vicinity of the highly energy-demanding Ca(2+) ATPase pump. Thus, membrane localization of BCK seems to be an important and regulated feature for the fueling of membrane-located, ATP-dependent processes, stressing again the importance of local rather than global ATP concentrations.