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.

A randomized placebo-controlled trial of nicotinamide riboside and pterostilbene supplementation in experimental muscle injury in elderly individuals.

BACKGROUNDDuring aging, there is a functional decline in the pool of muscle stem cells (MuSCs) that influences the functional and regenerative capacity of skeletal muscle. Preclinical evidence has suggested that nicotinamide riboside (NR) and pterostilbene (PT) can improve muscle regeneration, e.g., by increasing MuSC function. The objective of this study was to investigate if supplementation with NR and PT (NRPT) promotes skeletal muscle regeneration after muscle injury in elderly individuals by improved recruitment of MuSCs.METHODSThirty-two elderly individuals (55-80 years of age) were randomized to daily supplementation with either NRPT (1,000 mg NR and 200 mg PT) or matched placebo. Two weeks after initiation of supplementation, skeletal muscle injury was induced by electrically induced eccentric muscle work. Skeletal muscle biopsies were obtained before, 2 hours after, and 2, 8, and 30 days after injury.RESULTSA substantial skeletal muscle injury was induced by the protocol and associated with release of myoglobin and creatine kinase, muscle soreness, tissue edema, and a decrease in muscle strength. MuSC content, proliferation, and cell size revealed a large demand for recruitment after injury, but this was not affected by NRPT. Furthermore, histological analyses of muscle fiber area, central nuclei, and embryonic myosin heavy chain showed no NRPT supplementation effect.CONCLUSIONDaily supplementation with 1,000 mg NR and 200 mg PT is safe but does not improve recruitment of the MuSC pool or other measures of muscle recovery in response to injury or subsequent regeneration in elderly individuals.TRIAL REGISTRATIONClinicalTrials.gov NCT03754842.FUNDINGNovo Nordisk Foundation (NNF17OC0027242) and Novo Nordisk Foundation CBMR.

scFv-oligopeptide chaperoning system-assisted on-column refolding and purification of human muscle creatine kinase from inclusion bodies.

The formation of inclusion bodies in bacterial hosts poses a major challenge for the large-scale recovery of bioactive proteins. The process of obtaining bioactive protein from inclusion bodies is labor intensive, and the yields of recombinant protein are often low. Here, we describe a novel method for the renaturation and purification of inclusion bodies. This method combines a scFv-oligopeptide chaperoning system and an on-column refolding system to help refold human muscle creatine kinase (HCK) inclusion bodies. This method could significantly increase the activity recovery of denatured HCK inclusion bodies and provides an effective method for the production of bioactive proteins from inclusion bodies.

Simultaneous loss of TSC1 and DEPDC5 in skeletal and cardiac muscles produces early-onset myopathy and cardiac dysfunction associated with oxidative damage and SQSTM1/p62 accumulation.

By promoting anabolism, MTORC1 is critical for muscle growth and maintenance. However, genetic MTORC1 upregulation promotes muscle aging and produces age-associated myopathy. Whether MTORC1 activation is sufficient to produce myopathy or indirectly promotes it by accelerating tissue aging is elusive. Here we examined the effects of muscular MTORC1 hyperactivation, produced by simultaneous depletion of TSC1 and DEPDC5 (CKM-TD). CKM-TD mice produced myopathy, associated with loss of skeletal muscle mass and force, as well as cardiac failure and bradypnea. These pathologies were manifested at eight weeks of age, leading to a highly penetrant fatality at around twelve weeks of age. Transcriptome analysis indicated that genes mediating proteasomal and macroautophagic/autophagic pathways were highly upregulated in CKM-TD skeletal muscle, in addition to inflammation, oxidative stress, and DNA damage signaling pathways. In CKM-TD muscle, autophagosome levels were increased, and the AMPK and ULK1 pathways were activated; in addition, autophagy induction was not completely blocked in CKM-TD myotubes. Despite the upregulation of autolysosomal markers, CKM-TD myofibers exhibited accumulation of autophagy substrates, such as SQSTM1/p62 and ubiquitinated proteins, suggesting that the autophagic activities were insufficient. Administration of a superoxide scavenger, tempol, normalized most of these molecular pathologies and subsequently restored muscle histology and force generation. However, CKM-TD autophagy alterations were not normalized by rapamycin or tempol, suggesting that they may involve non-canonical targets other than MTORC1. These results collectively indicate that the concomitant muscle deficiency of TSC1 and DEPDC5 can produce early-onset myopathy through accumulation of oxidative stress, which dysregulates myocellular homeostasis.Abbreviations: AMPK: AMP-activated protein kinase; CKM: creatine kinase, M-type; COX: cytochrome oxidase; DEPDC5: DEP domain containing 5, GATOR1 subcomplex subunit; DHE: dihydroethidium; EDL: extensor digitorum longus; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; GAP: GTPase-activating protein; GTN: gastrocnemius; MTORC1: mechanistic target of rapamycin kinase complex 1; PLA: plantaris; QUAD: quadriceps; RPS6KB/S6K: ribosomal protein S6 kinase beta; SDH: succinate dehydrogenase; SOL: soleus; SQSTM1: sequestosome 1; TA: tibialis anterior; TSC1: TSC complex subunit 1; ULK1: unc-51 like autophagy activating kinase 1.

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.

CKM Gene rs8111989 Polymorphism and Power Athlete Status.

Multiple genetic variants are known to influence athletic performance. These include polymorphisms of the muscle-specific creatine kinase (CKM) gene, which have been associated with endurance and/or power phenotypes. However, independent replication is required to support those findings. The aim of the present study was to determine whether the CKM (rs8111989, c.*800A>G) polymorphism is associated with power athlete status in professional Russian and Lithuanian competitors. Genomic DNA was collected from 693 national and international standard athletes from Russia (n = 458) and Lithuania (n = 235), and 500 healthy non-athlete subjects from Russia (n = 291) and Lithuania (n = 209). Genotyping for the CKM rs8111989 (A/G) polymorphism was performed using PCR or micro-array analysis. Genotype and allele frequencies were compared between all athletes and non-athletes, and between non-athletes and athletes, segregated according to population and sporting discipline (from anaerobic-type events). No statistically significant differences in genotype or allele frequencies were observed between non-athletes and power athletes (strength-, sprint- and speed/strength-oriented) athletes. The present study reports the non-association of the CKM rs8111989 with elite status in athletes from sports in which anaerobic energy pathways determine success.

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.

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.

Consumption of New Zealand Blackcurrant Extract Improves Recovery from Exercise-Induced Muscle Damage in Non-Resistance Trained Men and Women: A Double-Blind Randomised Trial.

BACKGROUND: Blackcurrant is rich in anthocyanins that may protect against exercise-induced muscle damage (EIMD) and facilitate a faster recovery of muscle function. We examined the effects of New Zealand blackcurrant (NZBC) extract on indices of muscle damage and recovery following a bout of strenuous isokinetic resistance exercise. METHODS: Using a double-blind, randomised, placebo controlled, parallel design, twenty-seven healthy participants received either a 3 g·day-1 NZBC extract (n = 14) or the placebo (PLA) (n = 13) for 8 days prior to and 4 days following 60 strenuous concentric and eccentric contractions of the biceps brachii muscle on an isokinetic dynamometer. Muscle soreness (using a visual analogue scale), maximal voluntary contraction (MVC), range of motion (ROM) and blood creatine kinase (CK) were assessed before (0 h) and after (24, 48, 72 and 96 h) exercise. RESULTS: Consumption of NZBC extract resulted in faster recovery of baseline MVC (p = 0.04), attenuated muscle soreness at 24 h (NZBC: 21 ± 10 mm vs. PLA: 40 ± 23 mm, p = 0.02) and 48 h (NZBC: 22 ± 17 vs. PLA: 44 ± 26 mm, p = 0.03) and serum CK concentration at 96 h (NZBC: 635 ± 921 UL vs. PLA: 4021 ± 4319 UL, p = 0.04) following EIMD. CONCLUSIONS: Consumption of NZBC extract prior to and following a bout of eccentric exercise attenuates muscle damage and improves functional recovery. These findings are of practical importance in recreationally active and potentially athletic populations, who may benefit from accelerated recovery following EIMD.

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.

Acetylation of muscle creatine kinase negatively impacts high-energy phosphotransfer in heart failure.

A hallmark of impaired myocardial energetics in failing hearts is the downregulation of the creatine kinase (CK) system. In heart failure patients and animal models, myocardial phosphocreatine content and the flux of the CK reaction are negatively correlated with the outcome of heart failure. While decreased CK activity is highly reproducible in failing hearts, the underlying mechanisms remains elusive. Here, we report an inverse relationship between the activity and acetylation of CK muscle form (CKM) in human and mouse failing hearts. Hyperacetylation of recombinant CKM disrupted MM homodimer formation and reduced enzymatic activity, which could be reversed by sirtuin 2 treatment. Mass spectrometry analysis identified multiple lysine residues on the MM dimer interface, which were hyperacetylated in the failing hearts. Molecular modeling of CK MM homodimer suggested that hyperacetylation prevented dimer formation through interfering salt bridges within and between the 2 monomers. Deacetylation by sirtuin 2 reduced acetylation of the critical lysine residues, improved dimer formation, and restored CKM activity from failing heart tissue. These findings reveal a potentially novel mechanism in the regulation of CK activity and provide a potential target for improving high-energy phosphoryl transfer in heart failure.

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.

Ancestral contribution of the muscle-specific creatine kinase (CKM) polymorphism rs4884 in the knee osteoarthritis risk: a preliminary study.

INTRODUCTION/OBJECTIVES: Articular cartilage and periarticular muscle tissues are strongly affected during knee osteoarthritis (OA). Creatine kinase (CK) is an enzyme expressed in several tissues, but the isoform CK-MM is specific of skeletal muscle, and its serum concentration is used as a biomarker of muscle damage. Genetic variants of the CKM gene have been associated with various pathologies, but to date, there are no reports of association with OA. Due to the rs4884 polymorphism being well represented in the Mexican population, it is used as an ancestry informative marker; thus, the goal of this preliminary report was to evaluate the association of this polymorphism in primary knee OA Mexican patients. METHOD: Eighty-seven patients with primary knee OA were compared with 107 healthy controls. Serum CK-MM was determined using the dot blot system, and genotyping was performed using the OpenArray system. Logistic regression models were used to assess the association between the rs4884 polymorphism and OA susceptibility adjusting by gender, age, and body mass index. RESULTS: There were no significant differences in serum CK-MM values between patients and controls. The GG genotype and the G allele had a higher frequency in the control group compared with the OA group (24.3% vs. 12.6%, OR = 0.34, 95% CI = 0.14-0.84, P = 0.019; and 40.2% vs. 28.2%, OR = 0.51, 95% CI = 0.32-0.82, P = 0.005, respectively). CONCLUSIONS: Our results suggest a protection role of the rs4884 polymorphism against knee OA development; further studies are required to confirm it. Key Points • CK-MM enzyme catalyzes the conversion of creatine and ATP to create phosphocreatine and ADP; this reaction is reversible. • In tissues that consume ATP rapidly, such as skeletal muscle, the phosphocreatine serves as an important energy reservoir. • During knee OA, peripheral muscle tissues of the joint may be affected. • The rs4884 polymorphism of the CKM gene may participate as a protective factor in the development of OA.

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.

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.

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.

Genetic Code Expansion, Protein Expression, and Protein Functionalization in Bacillus subtilis.

The site-specific chemical modification of proteins through incorporation of noncanonical amino acids enables diverse applications, such as imaging, probing, and expanding protein functions, as well as to precisely engineer therapeutics. Here we report a general strategy that allows the incorporation of noncanonical amino acids into target proteins using the amber suppression method and their efficient secretion in the biotechnological relevant expression host Bacillus subtilis. This facilitates efficient purification of target proteins directly from the supernatant, followed by their functionalization using click chemistry. We used this strategy to site-specifically introduce norbornene lysine into a single chain antibody and functionalize it with fluorophores for the detection of human target proteins.

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.

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.

The Effect of High-Dose Vitamin C on Biochemical Markers of Myocardial Injury in Coronary Artery Bypass Surgery.

OBJECTIVE: To evaluate the effect of high-dose vitamin C on cardiac reperfusion injury and plasma levels of creatine kinase-muscle/brain (CK-MB), troponin I, and lactate dehydrogenase (LDH) in patients undergoing coronary artery bypass grafting (CABG). METHODS: This is a double-blind randomized clinical trial study. Fifty patients (50-80 years old) who had CABG surgery were selected. The intervention group received 5 g of intravenous vitamin C before anesthesia induction and 5 g of vitamin C in cardioplegic solution. The control group received the same amount of placebo (normal saline). Arterial blood samples were taken to determine the serum levels of CK-MB, troponin I, and LDH enzymes. Left ventricular ejection fraction was measured and hemodynamic parameters were recorded at intervals. RESULTS: High doses of vitamin C in the treatment group led to improvement of ventricular function (ejection fraction [EF]) and low Intensive Care Unit (ICU) stay. The cardiac enzymes level in the vitamin C group was lower than in the control group. These changes were not significant between the groups in different time intervals (anesthesia induction, end of bypass, 6 h after surgery, and 24 h after surgery) for CK-MB, LDH, and troponin I. Hemodynamic parameters, hematocrit, potassium, urinary output, blood transfusion, arrhythmia, and inotropic support showed no significant difference between the groups. CONCLUSION: Vitamin C has significantly improved the patients' ventricular function (EF) 72 h after surgery and reduced the length of ICU stay. No significant changes in cardiac biomarkers, including CK-MB, troponin I, and LDH, were seen over time in each group. IRCT CODE: IRCT2016053019470N33.