Muscle-specific Creatine Kinase Research Articles

Acute responses of strength-related gene expressions to maximum strength and force sense acuity.

Although high muscle strength worsens the sense of force, it is unknown whether there is a relationship between this deterioration and the underlying molecular mechanisms. This study examined the relationship between decreased force sense (FS) acuity and strength-related gene expressions. Maximal voluntary isometric contraction (MVIC) and FS (50% MVIC) tests were performed on the knee joints of twenty-two subjects. The expression analyses were evaluated by qRT-PCR in blood samples taken before, after MVIC, after 50% MVIC, and 15 min after the test. MVIC and FS error values were significantly correlated with each other (r = .659, p = .001). The qRT-PCR analyses demonstrated that the expressed mRNAs of the interleukin 6 (IL-6), alpha-actinin 3 (ACTN3), angiotensin-converting enzyme (ACE), brain-derived neurotrophic factor (BDNF), and ciliary neurotrophic factor receptor (CNTFR) genes dramatically increased until 50% MVIC and subsequently decreased 15 min after the exercise (p < .05). The muscle-specific creatine kinase (CKMM), myosin light chain kinase (MLCK), and G-protein β3 subunit (GNB3) genes reached their peak expression levels 30 min after MVIC (p < .05). ACE and ACTN3 gene expression increased significantly in parallel with the increased FS error (p < .05). These gene expression fluctuations observed at 50% MVIC and after the rest could be related to changes in cellular metabolism leading to fatigue. The time points of gene expression levels during exercise need to be considered. The force acuity of those whose maximal force develops too much may deteriorate.

한국인 투척 선수군에서 CKMM 유전자의 rs8111989(NcoI, A/G) 다형성에 대한 분포 양상

In order to explore the useful genetic marker(s) influencing the athletic performance of throwers, we performed a kind of case-control study genetic association study using the rs8111989(NcoI, A/G) polymorphism in the muscle-specific creatine kinase(CKMM) gene as a candidate gene. A total of 160 unrelated subjects including controls(n=70) and thrower (n=90) were recruited in this study, and total genomic DNA isolated from their saliva. The analysis of A/G polymorphisms in CKMM gene from their total genomic DNA was performed by using SNaPshot method in automated machine. By the result of genotype analysis, there was the significant differences in both genotype and allele frequencies of the A/G polymorphism in CKMM gene between controls and throwers, respectively(p.05). Especially, the frequencies of GG genotype and G allele in this genetic polymorphism showed the significantly higher frequencies in throwers than in controls, respectively. Thus, this genetic polymorphism may be one of useful genetic markers influencing the athletic performance in throwers. However, because athletic performance indicate the polygenic and multifactorial trait, the further studies using other genetic polymorphisms in more candidate genes will be required.

Etiology, risk factors and complications of exercise induced muscle injury

Healthy lifestyle includes physical activity as the basic component however, exercises with eccentricity have been associated to a higher risk of muscle injury and slower recovery. The development of exercise-induced muscle injury is a phenomenon that results from atypical or unaccustomed activity; the intensity of the pain and the extent of the damage steadily deteriorate over time, and when the exercise session contains an eccentric component, they may last for several days. Increasing levels of muscle-specific circulating protein are present, along with increased levels of muscular pain and decreased muscle strength. The extent of severity of the symptoms following eccentric exercise varies greatly. Exercise-induced muscle injury is influenced by a number of factors including intensity, duration, mode, muscle group, age, gender, genetics, and dietary state. Numerous scientific studies illustrate the circumstances under which exercise causes muscle injury. It is widely acknowledged that exercise that is unfamiliar, particularly exercise that involves eccentric contractions, causes damage. exercise-induced muscle injury is accompanied by an inflammatory response involving numerous mediators, including the production of muscle-specific creatine kinase and interleukins and interleukins receptor antagonist and acute phase proteins. Complications of exercise-induced muscle injury include temporary muscle inflammation, loss of strength, muscle discomfort, restricted range of motion, elevated passive tension, discomfort, and a momentary decline in insulin sensitivity and can also lead to rhabdomyolysis. The purpose of this research is to review the available information about an etiology, risk factors and complications of exercise-induced muscle injury.

A pilot study of newborn screening for Duchenne muscular dystrophy in Guangzhou

BackgroundTo estimate the overall situation of Duchenne muscular dystrophy (DMD) screening in newborns in Guangzhou, China. MethodA total of 62553 newborns including 44268 males and 18285 females were screened for DMD by measuring muscle specific creatine kinase isoform (CK-MM) concentrations using the GSP® Neonatal CK-MM kit based on time-resolved immunofluorescence. We recalled positive cases and recollected dried blood spots (DBS) for retest of CK-MM. The newborns with retest positive result were recalled again for serum creatine kinase (CK) and multiplex ligation-dependent probe amplification (MLPA) test. Whole exon sequencing was performed when MLPA test was negative. ResultsFour males were diagnosed with DMD. The incidence of males was 1/11067. No DMD patient was found in female. There were significant differences of CK-MM concentration between male and female newborns. Among gestational age (GA), birth weight (BW) and age at sampling, linear regression analysis showed that CK-MM concentration was much more closely correlated with GA and age at sampling. ConclusionsCK-MM concentration is affected by gender, GA, BW and age at sampling. The efficiency of DMD screening might be improved by adjusting a multitier cut-off value according to GA and age at sampling. DMD newborn screening should be male priority.

TAZ exhibits phase separation properties and interacts with Smad7 and β-catenin to repress skeletal myogenesis.

Hippo signaling in Drosophila and mammals is prominent in regulating cell proliferation, death and differentiation. Hippo signaling effectors (YAP and TAZ; also known as YAP1 and WWTR1, respectively) exhibit crosstalk with transforming growth factor-β (TGF-β)-Smad and Wnt/β-catenin pathways. Previously, we implicated Smad7 and β-catenin in mammalian myogenesis. Therefore, we assessed a potential role of TAZ on the Smad7-β-catenin complex in muscle cells. Here, we document functional interactions between Smad7, TAZ and β-catenin in mouse myogenic cells. Ectopic TAZ expression resulted in repression of the muscle-specific creatine kinase muscle (Ckm) gene promoter and its corresponding protein level. Depletion of endogenous TAZ enhanced Ckm promoter activation. Ectopic TAZ, while potently active on a TEAD reporter (HIP-HOP), repressed myogenin (Myog) and Myod1 enhancer regions and myogenin protein level. Additionally, a Wnt/β-catenin readout (TOP flash) demonstrated TAZ-mediated inhibition of β-catenin activity. In myoblasts, TAZ was predominantly localized in nuclear speckles, while in differentiation conditions TAZ was hyperphosphorylated at Ser89, leading to enhanced cytoplasmic sequestration. Finally, live-cell imaging indicated that TAZ exhibits properties of liquid-liquid phase separation (LLPS). These observations indicate that TAZ, as an effector of Hippo signaling, suppresses the myogenic differentiation machinery.

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.

Systemic delivery of AAVrh74.tMCK.hCAPN3 rescues the phenotype in a mouse model for LGMD2A/R1

Limb girdle muscular dystrophy (LGMD) 2A/R1, caused by mutations in the CAPN3 gene and CAPN3 loss of function, is known to play a role in disease pathogenicity. In this study, AAVrh74.tMCK.CAPN3 was delivered systemically to two different age groups of CAPN3 knockout (KO) mice; each group included two treatment cohorts receiving low (1.17 × 1014 vg/kg) and high (2.35 × 1014 vg/kg) doses of the vector and untreated controls. Treatment efficacy was tested 20 weeks after gene delivery using functional (treadmill), physiological (in vivo muscle contractility assay), and histopathological outcomes. AAV.CAPN3 gene therapy resulted in significant, robust improvements in functional outcomes and muscle physiology at low and high doses in both age groups. Histological analyses of skeletal muscle showed remodeling of muscle, a switch to fatigue-resistant oxidative fibers in females, and fiber size increases in both sexes. Safety studies revealed no organ tissue abnormalities; specifically, there was no histopathological evidence of cardiotoxicity. These results show that CAPN3 gene replacement therapy improved the phenotype in the CAPN3 KO mouse model at both doses independent of age at the time of vector administration. The improvements were supported by an absence of cardiotoxicity, showing the efficacy and safety of the AAV.CAPN3 vector as a potential gene therapy for LGMDR1.

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

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. 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. 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). 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.

Extracellular xenogeneic hemoglobin suppresses the capacity for C2C12 myoblast myogenic differentiation

Functional characteristics of satellite cells (SCs) that act as myogenesis initiators and have emerged as a promising target for cell therapy, are dependent on their microenvironment. The aim of this study was to investigate the effect of cell-free hemoglobin, as a part of the microenvironment of SCs, on their functional characteristics. The C2C12 cell line served as the experimental model of SCs; hemoglobin isolated from porcine (PHb) and bovine (BHb) slaughterhouse blood served as the experimental model for extracellular hemoglobin. The proliferation rate of C2C12 cells was assessed by the MTT test, migration capacity by the scratch assay, and myogenic differentiation capacity by histochemical staining and RT-PCR analysis of the expression of genes specific for myogenic lineage. The effect of hemoglobin on the proliferation and migration of C2C12 cells was dependent on its concentration and the animal species it was isolated from, but the effect of BHb was more prominent. Both PHb and BHb decreased the expression levels of myogenin and muscle specific creatine kinase at a 10 ?M concentration. While PHb had no effect on the morphometric parameters of C2C12 myotubes, BHb modified the area and length of C2C12 myotubes cultivated in DMEM/2% horse serum and DMEM/10% fetal calf serum. While PHb and BHb had no effect on heme oxygenase 1 (Hmox1) expression, they stimulated the expression of hypoxia-inducible factor 1-alpha (Hif1?) at a concentration of 10 ?M. The mainly inhibitory effect of cell-free hemoglobin on myogenic differentiation suggests that it could be a relevant factor in the outcome of cell therapy of muscle injury.

Raumenų adaptaciją prie fizinių krūvių lemiančių „CKM“ ir „AMPD1“ genetinių variantų analizė Lietuvos sportininkų grupėje

[full article and abstract in Lithuanian; abstract in English] Energy homeostasis and different muscle contraction properties demands for interactive processes of diverse specialty enzymes, including important reactions of enzymes-regulators – adenosine monophosphate deaminase 1 (encoded by AMPD1 gene) and muscle-specific creatine kinase (encoded by CKM gene), in the human body during the physical activity. This research aims to analyse the effect of CKM (c.*800A >G, rs8111989) and AMPD1 (c.133C >T, p.Gln45Ter, rs17602729) gene polymorphisms on physical capacities of Lithuanian elite athletes. The study involved 150 Lithuanian elite athletes (60 females and 90 males, Olympic candidates, World’ and European Olympic medallists) and 150 controls (60 females and 90 males, healthy unrelated citizens of Lithuania without any competitive sport experience). The athletes were prospectively stratified into three groups according to the event duration and distance, spanning a spectrum from the endurance-oriented to the power-oriented athletes: ‘endurance group’ (n = 63), ‘sprint/power group’ (n = 65) and “mixed” (n = 22), sports utilized anaerobic and aerobic energy production). Physical (anthropometric) measurements (height, weight, muscle mass and fat mass, body mass index (BMI)) and indices of functional capacity (anaerobic muscle strength based on right and left hands grip strength, short-term explosive muscle power (STEMP), anaerobic alactic muscle power (AAMP) and aerobic capacity based on maximum oxygen uptake (VO2max)) were determined for athletes. For molecular genetic analysis, DNA was extracted from blood leukocytes using standard phenol-chloroform method. Two different strategies were used for CKM and AMPD1 polymorphisms genotyping: CKM polymorphism genotyping was performedby allelic discrimination technology based real-time polymerase chain reaction and AMPD1 polymorphism – by restriction fragment length polymorphism method. Phenotypic analysis showed that mean values of physical measurements and functional capacity indices (except height, weight, muscle and fat mass) were significantly different between sport groups and were specific to each group (p G) a ndAMPD1 (c.133C>T) genetic variants have different effect on male and female physical capacity. CKM AA and AG genotype confers ability to achieve better muscle efficiency in short-term, maximum-effort requiring physical activity and AMPD1 CC genotype athletes have better aerobic capacity. Our study approves the opinion of many sports and genetic scientists that sprint and power qualities are more effected by inheritance than the endurance qualities. Keywords: creatine kinase, myoadenylate deaminase, gene polymorphisms. DOI: https://doi.org/10.15823/sm.2019.95.7

Detection of muscle-specific creatine kinase expression as physiological indicator for Atlantic salmon (Salmo salar L) skeletal muscle damage

Plasma creatine kinase (CK) release is a marker of damage in fish; their expression can indicate drug/contaminant exposure. Three CK isoforms have been described in fish: muscle CK (M-CK), brain CK (B-CK) and mitochondrial sarcomeric, and the release of any of them could be detected in the serum. Nevertheless, the expression and properties of M-CK in Atlantic salmon are still unknown. In this study, we evaluated differential M-CK expression in Atlantic salmon (Salmo salar L) skeletal muscle and serum at 0, 5, 10, and 15 days post-infection (dpi) with the infectious salmon anaemia virus, a primary aquaculture pathogen in Chile. The M-CK isoform was assessed using a Western blot-validated antibody. Results for qPCR (+10.8 AU) and ELISA (1.24, p < 0.05) showed increased skeletal muscle M-CK at 10 dpi (r = 0.9817, p < 0.05). Serum M-CK significantly increased at 5 and 10 dpi (r = 1.26 and 1.17, p < 0.05). In situ M-CK detection in the myofibrils showed co-localization with the pro-inflammatory cytokine IL-1β, particularly at 10 dpi. In summary, serum M-CK detection apparently serves as a non-invasive, functional indicator for Atlantic salmon skeletal muscle damage, which could indicate disease or environmental stress.

Protein Turnover Measurements in Human Serum by Serial Immunoaffinity LC-MS/MS.

The half-life of target proteins is frequently an important parameter in mechanistic pharmacokinetic and pharmacodynamic (PK/PD) modeling of biotherapeutics. Clinical studies for accurate measurement of physiologically relevant protein turnover can reduce the uncertainty in PK/PD model-based predictions, for example, of the therapeutic dose and dosing regimen in first-in-human clinical trials. We used a targeted mass spectrometry work flow based on serial immunoaffinity enrichment ofmultiple human serum proteins from a [5,5,5-2H3]-L-leucine tracer pulse-chase study in healthy volunteers. To confirm the reproducibility of turnover measurements from serial immunoaffinity enrichment, multiple aliquots from the same sample set were subjected to protein turnover analysis in varying order. Tracer incorporation was measured by multiple-reaction-monitoring mass spectrometry and target turnover was calculated using a four-compartment pharmacokinetic model. Five proteins of clinical or therapeutic relevance including soluble tumor necrosis factor receptor superfamily member 12A, tissue factor pathway inhibitor, soluble interleukin 1 receptor like 1, soluble mucosal addressin cell adhesion molecule 1, and muscle-specific creatine kinase were sequentially subjected to turnover analysis from the same human serum sample. Calculated half-lives ranged from 5-15 h; however, no tracer incorporation was observed for mucosal addressin cell adhesion molecule 1. The utility of clinical pulse-chase studies to investigate protein turnover can be extended by serial immunoaffinity enrichment of target proteins. Turnover analysis from serum and subsequently from remaining supernatants provided analytical sensitivity and reproducibility for multiple human target proteins in the same sample set, irrespective of the order of analysis.

Comparison of Muscle-Specific Creatine Kinase (CK-MM) Gene Polymorphism (rs8111989) Among Professional, Amateur Athletes and Non-athlete Karatekas

Background: Identification of genetic markers is one of the priority trends to perform in athletes for evaluation of their efficacy. Previous studies have revealed that CK-MM may be used as a valuable marker to reflect the magnitude of skeletal muscle destruction in response to exercise. Objectives: In this study we analyzed the frequency of rs8111989CK-MM polymorphism in karatekas to find whether there is a difference among professional, amateur athletes and non- karatekas. Methods: Distribution of allele and genotype frequencies of the muscle specific creatine kinase (CK-MM) gene A/G polymorphism was assessed in a survey among 275 athletes residing in state of Isfahan (86 professional karatekas (43 male and 43 female) and 86 amateur karatekas (50 male and 36 female) and 103 non-athlete individuals (50 male and 53 female). Blood samples were taken and genotyping was performed by restriction fragment length polymorphism (RFLP) approach. Statistical analyses carried out using SPSS software and data with P < 0.05 were considered to be significant. Results: The CK-MM AG genotype frequency was significantly higher in professional and amateur karate athletes and control subjects (AG genotype: 52.4% vs. AA genotype: 33.4%; GG genotype: 14.1%; χ2 = 16.79, P < 0.05). Furthermore, the CK-MM genotype in the professional group (AA:31.3%, AG:56.9, GG:11.6) was different of those observed for amateur karatekas (AA:32.5%, AG:43.0, GG:24.4)(χ2 = 11.39; P = 0.003) and CK-MM genotype in the amateur group was also different from non-athletes (AA:36%, AG: 56.3, GG: 7.7)(χ2 = 11.39; P = 0.003). Conclusions: The CK-MM gene A/G polymorphism is associated with the physical performance levels of karatekas.

A meta-analysis of the association of CKM gene rs8111989 polymorphism with sport performance.

The muscle-specific creatine kinase (CKM) A/G variants (rs8111989) have been associated with skeletal muscle performance in humans; they are correlated with physical performance and contribute to differences in the maximum oxygen uptake (VO2max) responses during power or endurance training. However, there is not enough definitive evidence to demonstrate whether the A and G allelic variants of the CKM gene rs8111989 are indeed genetic factors that can influence human physical performance. In our study, we identified 9 articles on CKM in a literature search, and conducted two meta-analyses on the CKM rs8111989 A/G allele or genotype differences between power or endurance athletes and general controls. We found that the power athletes had a significantly higher frequency of the G allele (OR, 1.14; 95% CI, 1.02-1.28, P=0.03) and GG genotype (OR, 1.54; 95% CI, 1.24-1.91, P<0.0001) compared to controls, but there was no significant difference for the endurance athletes (G allele, OR, 0.95, 95%CI, 0.85-1.06, P=0.34; GG genotype, OR, 1.00, 95%CI, 0.78-1.27, P=1.00). The results provide additional evidence to support the notion that human physical performance might be influenced by genetic profiles, especially in power sports.

CK-MM Polymorphism is Associated With Physical Fitness Test Scores in Military Recruits.

Muscle-specific creatine kinase is thought to play an integral role in maintaining energy homeostasis by providing a supply of creatine phosphate. The genetic variant, rs8111989, contributes to individual differences in physical performance, and thus the purpose of this study was to determine if rs8111989 variant is predictive of Physical Fitness Test (PFT) scores in male, military infantry recruits. DNA was extracted from whole blood, and genotyping was performed in 176 Marines. Relationships between PFT measures (run, sit-ups, and pull-ups) and genotype were determined. Participants with 2 copies of the T allele for rs8111989 variant had higher PFT scores for run time, pull-ups, and total PFT score. Specifically, participants with 2 copies of the TT allele (variant) (n = 97) demonstrated an overall higher total PFT score as compared with those with one copy of the C allele (n = 79) (TT: 250 ± 31 vs. 238 ± 31; p = 0.02), run score (TT: 82 ± 10 vs. 78 ± 11; p = 0.04) and pull-up score (TT: 78 ± 11 vs. 65 ± 21; p = 0.04) or those with the CC/CT genotype. These results demonstrate an association between physical performance measures and genetic variation in the muscle-specific creatine kinase gene (rs8111989).

CKM gene polymorphism in Russian and Polish rowers.

Muscle-specific creatine kinase (CKMM) plays a vital role in the energy homeostasis of muscle cells. The A/G variation (rs8111989) located in the 3'-untranslated region of the CKM gene has been found to be the most relevant in terms of genetic testing in sport. The aim of the presented study was to test the hypothesis that the G allele might represent a genetic element that contributes to the improvement of endurance performance in Polish and Russian rowers. The distribution of the CKM genotypes was examined in a group of Polish and Russian athletes in comparison with non-athlete controls. There were no statistical differences between the rowers and the control groups across the CKM genotypes when Polish or Russian participants were analyzed. Based on the obtained results, it may be speculated that the CKM A/G polymorphism is not an important determinant of endurance performance level in Polish and Russian rowers. However, these results should be interpreted with caution as they can be limited by many factors.

Arginylation of Myosin Heavy Chain Regulates Skeletal Muscle Strength

Protein arginylation is a posttranslational modification with an emerging global role in the regulation of actin cytoskeleton. To test the role of arginylation in the skeletal muscle, we generated a mouse model with Ate1 deletion driven by the skeletal muscle-specific creatine kinase (Ckmm) promoter. Ckmm-Ate1 mice were viable and outwardly normal; however, their skeletal muscle strength was significantly reduced in comparison to controls. Mass spectrometry of isolated skeletal myofibrils showed a limited set of proteins, including myosin heavy chain, arginylated on specific sites. Atomic force microscopy measurements of contractile strength in individual myofibrils and isolated myosin filaments from these mice showed a significant reduction of contractile forces, which, in the case of myosin filaments, could be fully rescued by rearginylation with purified Ate1. Our results demonstrate that arginylation regulates force production in muscle and exerts a direct effect on muscle strength through arginylation of myosin.

The activity of carp muscle-specific creatine kinase at low temperature is enhanced by decreased hydrophobicity of residue 268.

Abstract The muscle-specific forms of creatine kinase in rabbit (RM-CK) and carp (M1-CK) exhibit different temperature-dependent functional properties. Replacing the glycine at residue 268 of RM-CK with asparagine increases the enzyme's activity at 10°C. In this study, we investigated how hydrophobicity of residue 268 affects the biochemical properties of RM-CK and M1-CK at low temperature. We generated three mutants of both RM-CK and M1-CK: Asp268, Lys268, and Leu268. The secondary structures of these mutants were similar, as revealed by their circular dichroism spectra. Similar to the Asn268 mutants, the Asp268 and Lys268 mutants of RM-CK and M1-CK exhibited higher specific activities at 10°C and pH 8.0. However, no such effect was observed for the RM-CK and M1-CK Leu268 mutants. While in the presence of cryoprotectant (sucrose or trehalose), the activities of wild-type RM-CK and M1-CK mutant enzymes with a hydrophobic residue at 268 were higher, and the effect was more profound at pH 8.0. It may be inferred that water molecules affect protein conformation around residue 268, thereby influencing protein stability at low temperature.

Disturbed energy metabolism and muscular dystrophy caused by pure creatine deficiency are reversible by creatine intake

Creatine (Cr) plays an important role in muscle energy homeostasis by its participation in the ATP-phosphocreatine phosphoryl exchange reaction mediated by creatine kinase. Given that the consequences of Cr depletion are incompletely understood, we assessed the morphological, metabolic and functional consequences of systemic depletion on skeletal muscle in a mouse model with deficiency of l-arginine:glycine amidinotransferase (AGAT(-/-)), which catalyses the first step of Cr biosynthesis. In vivo magnetic resonance spectroscopy showed a near-complete absence of Cr and phosphocreatine in resting hindlimb muscle of AGAT(-/-) mice. Compared with wild-type, the inorganic phosphate/β-ATP ratio was increased fourfold, while ATP levels were reduced by nearly half. Activities of proton-pumping respiratory chain enzymes were reduced, whereas F(1)F(0)-ATPase activity and overall mitochondrial content were increased. The Cr-deficient AGAT(-/-) mice had a reduced grip strength and suffered from severe muscle atrophy. Electron microscopy revealed increased amounts of intramyocellular lipid droplets and crystal formation within mitochondria of AGAT(-/-) muscle fibres. Ischaemia resulted in exacerbation of the decrease of pH and increased glycolytic ATP synthesis. Oral Cr administration led to rapid accumulation in skeletal muscle (faster than in brain) and reversed all the muscle abnormalities, revealing that the condition of the AGAT(-/-) mice can be switched between Cr deficient and normal simply by dietary manipulation. Systemic creatine depletion results in mitochondrial dysfunction and intracellular energy deficiency, as well as structural and physiological abnormalities. The consequences of AGAT deficiency are more pronounced than those of muscle-specific creatine kinase deficiency, which suggests a multifaceted involvement of creatine in muscle energy homeostasis in addition to its role in the phosphocreatine-creatine kinase system.

Sustained Reduction of Hyperbilirubinemia in Gunn Rats After Adeno-Associated Virus-Mediated Gene Transfer of Bilirubin UDP-Glucuronosyltransferase Isozyme 1A1 to Skeletal Muscle

Crigler-Najjar syndrome is an autosomal recessive disorder with severe unconjugated hyperbilirubinemia due to deficiency of bilirubin UDP-glucuronosyltransferase isozyme 1A1 (UGT1A1) encoded by the UGT1A1 gene. Current therapy relies on phototherapy to prevent life-threatening elevations of serum bilirubin levels, but liver transplantation is the only permanent treatment. Muscle-directed gene therapy has several advantages, including easy and safe access through simple intramuscular injections, and has been investigated in human clinical trials. In this study, we have investigated the efficacy of adeno-associated viral (AAV) vector-mediated muscle-directed gene therapy in the preclinical animal model of Crigler-Najjar syndrome, that is the Gunn rat. Serotype 1 AAV vector expressing rat UGT1A1 under the control of muscle-specific creatine kinase promoter was injected at a dose of 3×10(12) genome copies/kg into the muscles of Gunn rats and resulted in expression of UGT1A1 protein and functionally active enzyme in injected muscles. AAV-injected Gunn rats showed an approximately 50% reduction in serum bilirubin levels as compared with saline-treated controls, and this reduction was sustained for at least 1 year postinjection. Increased excretion of alkali-labile metabolites of bilirubin in bile and urine was detected in AAV-injected animals. High-performance liquid chromatography analysis of bile from AAV-injected Gunn rats showed a metabolite with retention time close to that of bilirubin diglucuronide. Taken together, these data show that clinically relevant and sustained reduction of serum bilirubin levels can be achieved by simple and safe intramuscular injections in Gunn rats. AAV-mediated muscle directed gene therapy has potential for the treatment of patients with Crigler-Najjar syndrome type 1.