Creatine Kinase Gene Research Articles

The muscle tissue transcriptome of idiopathic inflammatory myopathy reflects the muscle damage process by monocytes and presence of skin lesions.

To investigate transcriptomic and immunophenotypic features of muscle specimens from patients with idiopathic inflammatory myopathy (IIM). Bulk RNA-sequencing was performed on muscle biopsy samples from 16 patients with dermatomyositis (DM) and 9 patients with polymyositis (PM). Seven tested positive for anti-aminoacyl t-RNA synthetase antibodies in the DM patients (ARS-DM). We conducted weighted gene coexpression network analysis (WGCNA), differentially expressed gene (DEG) analysis, and gene set variation analysis (GSVA) to assess contributions of specific pathways. Cell proportions in muscle specimens were estimated using a deconvolution approach. WGCNA revealed significant positive correlations between serum creatine kinase (CK) levels and gene modules involved in cellular respiration, phagocytosis, and oxidative phosphorylation (OXPHOS). Significant positive correlations were also observed between CK levels and proportions of CD16-positive and -negative monocytes and myeloid dendritic cells. Notably, DM patients demonstrated enrichment of complement and interferon-α and -γ pathway genes compared to those with PM. Furthermore, ARS-DM demonstrated a higher proportion of Th1 cells and DEGs related to OXPHOS. Additionally, serum Krebs von den Lungen-6 levels correlated with gene modules associated with extracellular matrix and transforming growth factor-β signaling pathway. Our study highlights a significant involvement of monocytes in muscle damage and delineates pathological differences among IIM subtypes. DM was characterized by complement, interferon-α and -γ signaling, whilst ARS-DM was associated with OXPHOS. Distinctive gene expression variations in muscle specimens suggest that different pathologic mechanisms underlie muscle damage in each IIM phenotype.

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.

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.

Creatine Kinase Is Decreased in Childhood Asthma.

Rationale: The identification of novel molecules associated with asthma may provide insights into the mechanisms of disease and their potential clinical implications. Objectives: To conduct a screening of circulating proteins in childhood asthma and to study proteins that emerged from human studies in a mouse model of asthma. Methods: We included 2,264 children from eight birth cohorts from the Mechanisms of the Development of ALLergy project and the Tucson Children's Respiratory Study. In cross-sectional analyses, we tested 46 circulating proteins for association with asthma in the selection stage and carried significant signals forward to a validation and replication stage. As CK (creatine kinase) was the only protein consistently associated with asthma, we also compared whole blood CK gene expression between subjects with and without asthma (n = 249) and used a house dust mite (HDM)-challenged mouse model to gain insights into CK lung expression and its role in the resolution of asthma phenotypes. Measurements and Main Results: As compared with the lowest CK tertile, children in the highest tertile had significantly lower odds for asthma in selection (adjusted odds ratio, 95% confidence interval: 0.31; 0.15-0.65; P = 0.002), validation (0.63; 0.42-0.95; P = 0.03), and replication (0.40; 0.16-0.97; P = 0.04) stages. Both cytosolic CK forms (CKM and CKB) were underexpressed in blood from asthmatics compared with control subjects (P = 0.01 and 0.006, respectively). In the lungs of HDM-challenged mice, Ckb expression was reduced, and after the HDM challenge, a CKB inhibitor blocked the resolution of airway hyperresponsiveness and reduction of airway mucin. Conclusions: Circulating concentrations and gene expression of CK are inversely associated with childhood asthma. Mouse models support a possible direct involvement of CK in asthma protection via inhibition of airway hyperresponsiveness and reduction of airway mucin.

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.

Serum Creatine Kinase and Other Profile of Duchenne Muscular Dystrophy and Becker Muscular Dystrophy

Background: Serum creatine kinase (CK) level is increased muscular dystrophy (MD) and may be used as a clue to identify MDs. Objective: The objective is to compare CK levels between Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), to correlate value of serum CK with number of deletions, duration of illness and to establish a cut off value of CK for screening. Materials and Methods: A cross-sectional survey was carried out in a tertiary care institute of Kolkata. Clinically diagnosed patients of 139 DMDs and 50 BMDs along with 69 age-matched individuals suffering from diseases other than MDs was included. Estimation of serum CK levels and gene analysis were done for all. Results: DMD victims were found to be younger with low age of onset and lesser disease duration but higher serum CK level compared to those having BMD. Most of the genetic deletions were happened in distal region of dystrophin gene and a significant difference was revealed to exist between DMD and BMD neither in regard to proportion of overall deletion nor deletions in proximal and distal region. However, gene deletion was found absent in 31% and 42% of DMD and BMD cases. Serum CK level of 511.5 unit/L was seemed to be a reliable cut-off for detection of DMD and BMD with 97.3% sensitivity, 100% specificity, and area under the curve 0.989 with a P = 0.000. Conclusion: In case of nonavailability of genetic test facility as well as negative genetic test serum CK may be tried for identifying MD.

Genetically determined variations of selenoprotein P are associated with antioxidant, muscular, and lipid biomarkers in response to Brazil nut consumption by patients using statins

Several single nucleotide polymorphisms (SNPs) could indirectly, as well directly, influence metabolic parameters related to health effects in response to selenium (Se)supplementation. This study aimed to investigate whether the selenoprotein SNPs were associated with the response of Se status biomarkers to the Brazil nut consumption in patients using statins and if the variation in Se homoeostasis could affect antioxidant protection, lipid profile, muscle homoeostasis and selenoproteins mRNA. The study was performed in the Ribeirão Preto Medical School University Hospital. Thirty-two patients using statins received one unit of Brazil nut daily for 3 months. Body composition, blood Se concentrations, erythrocyte glutathione peroxidase (GPX) activity, total cholesterol, low-density lipoprotein(LDL), high-density lipoprotein (HDL), triacylglycerol (TAG), creatine kinase (CK) activity and gene expression of GPX1 and selenoprotein P (SELENOP) were evaluated before and after Brazil nut consumption. The volunteers were genotyped for SNP in GPX1 (rs1050450) and SELENOP (rs3877899 and rs7579). SNPs in selenoproteins were not associated with plasma and erythrocyte Se, but SNPs in SELENOP influenced the response of erythrocyte GPX activity and CK activity, TAG and LDL after Brazil nut consumption. Also, Brazil nut consumption increased GPX1 mRNA expression only in subjects with rs1050450 CC genotype. SELENOP mRNA expression was significantly lower in subjects with rs7579 GG genotype before and after the intervention. Thus, SNP in SELENOP could be associated with interindividual differences in Se homeostasis after Brazil nut consumption, emphasising the involvement of genetic variability in response to Se consumption towards health maintenance and disease prevention.

Phylogeny, Characterisation and Identification of Creatine Kinase Genes (ckma and ckmb) in Zebrafish (Danio rerio)

Creatine kinase genes (ckma and ckmb) in zebrafish (Danio rerio), an aquatic model organism, have been characterized and identified. However, the gene structure is designed using exons, introns, amino acids produced by the exons. TATA boxex, poly A tails and 5' UTR and 3' UTR regions of zebrafish ckma and ckma genes are showed at the gene structure. In addition, chromosomal regions of ckma and ckmb genes were determined. The other genes which are placed in the same region with ckma and ckmb genes were found in medaka and human which are the orthologs of zebrafish, and conserved gene syntheny was designed manually according to these regions. In addition, phylogenetic relationship was determined between zebrafish and it’s some orthologs using ckma and ckmb gene sequences. Genetic affinity between zebrafish and its orthologs was calculated as similarity-identity % rate and given as a table. For all these studies, bioinformatics databases (NCBI database, Ensembl genomic database, Expasy, Reverse Complementary) and programs (MEGA6 program, BLOSUM62 matrix program and BioEdit software) were used. In this study, characterization and identification of ckma and ckmb genes in zebrafish (D. rerio) was completed using bioinformatics tools and some data to be used in the future studies on molecular stress response were presented.

Structure and function of resistance arteries from BB-creatine kinase and ubiquitous Mt-creatine kinase double knockout mice

Increasing evidence indicates that the enzyme creatine kinase (CK) is intimately involved in microvascular contractility. The mitochondrial isoenzyme catalyses phosphocreatine synthesis from ATP, while cytoplasmic CK, predominantly the BB isoenzyme in vascular tissue, is tightly bound near myosin ATPase, where it favours ATP production from phosphocreatine to metabolically support vascular contractility. However, the effect of CK gene inactivation on microvascular function is hitherto unknown. We studied functional and structural parameters of mesenteric resistance arteries isolated from 5 adult male mice lacking cytoplasmic BB-CK and ubiquitous mitochondrial CK (CK-/-) vs 6 sex/age-matched controls. Using a Mulvany Halpern myograph, we assessed the acute maximum contractile force with 125mMK+ and 10-5M norepinephrine, and the effect of two inhibitors, dinitrofluorobenzene, which inhibits phosphotransfer enzymes (0.1μM), and the specific adenylate kinase inhibitor P1, P5-di(adenosine 5') pentaphosphate (10-6 to 10-5M). WT and CK-/- did not significantly differ in media thickness, vascular elasticity parameters, or acute maximum contractile force. CK-/- arteries displayed greater reduction in contractility after dinitrofluorobenzene 38%; vs 14% in WT; and after AK inhibition, 14% vs 5.5% in WT, and displayed abnormal mitochondria, with a partial loss of the inner membrane. Thus, CK-/- mice display a surprisingly mild phenotype in vascular dysfunction. However, the mitochondrial abnormalities and greater effect of inhibitors on contractility may reflect a compromised energy metabolism. In CK-/- mice, compensatory mechanisms salvage energy metabolism, as described for other CK knock-out models.

Creatine kinase, energy reserve, and hypertension: from bench to bedside.

We hypothesized that human variation in the activity of the ATP regenerating enzyme creatine kinase (CK) activity affects hypertension and cardiovascular disease risk. CK is tightly bound close to ATP-utilizing enzymes including Ca2+-ATPase, myosin ATPase, and Na+/K+-ATPase, where it rapidly regenerates ATP from ADP, H+, and phosphocreatine. Thus, relatively high CK was thought to enhance ATP-demanding processes including resistance artery contractility and sodium retention, and reduce ADP-dependent functions. In a series of studies of our group and others, CK was linked to hypertension and bleeding risk. Plasma CK after rest, used as a surrogate measure for tissue CK, was associated with high blood pressure and failure of antihypertensive therapy in case-control and population studies. Importantly, high tissue CK preceded hypertension in animal models and in humans, and human vascular tissue CK gene expression was strongly associated with clinical blood pressure. In line with this, CK inhibition substantially reduced the contractility of human resistance arteries ex vivo. We also presented evidence that plasma CK reduced ADP-dependent platelet aggregation. In subsequent intervention studies, the oral competitive CK inhibitor beta-guanidinopropionic acid (GPA) reduced blood pressure in spontaneously hypertensive rats (SHRs), and a 1-week trial of sub-therapeutic dose GPA in healthy men was uneventful. Thus, based on theoretical concepts, evidence was gathered in laboratory, case-control, and population studies that high CK is associated with hypertension and with bleeding risk, potentially leading to a new mode of cardiovascular risk reduction with CK inhibition.

Research of the heart protective effect of exercise precondition mediated by calcitonin gene related peptide on acute exhaustion rats

To observe the heart protective effect of exercise preconditioning (EP) in the acute exhaustion exercise (EE) rats, and explore its action mechanism further. Eighty healthy male Sprague-Dawley (SD) rats were divided into control group (C group), EP group, EE group, and EP+EE group randomly, with 20 rats in each group. The rats in EP and EP+EE groups were trained for 3 weeks according to the daily swimming for 60 minutes (swimming 15 minutes, resting 5 minutes, repeating 3 times) with 6 days each week. The rats in EE and EP+EE groups on the last 1 day after 3 weeks, 3% weight heavy weight was carried once for swimming EE. Two hours after the last EE, abdominal aortic blood and heart was harvested, the levels of serum MB isoenzyme of creatine kinase (CK-MB) and calcitonin gene related peptide (CGRP) were determined by enzyme linked immunosorbent assay (ELISA); the ultrastructure of myocardium was observed by optical microscopy; the levels of myocardial malondialdehyde (MDA) and superoxide dismutase (SOD) were determined by ELISA, the mRNA expression of myocardial CGRP was assayed by reverse transcription-polymerase chain reaction (RT-PCR), and the protein expression of myocardial CGRP was assayed by Western Blot. Compared with C group, the levels of serum CK-MB and myocardial MDA were significantly increased, serum CGRP content, myocardial SOD activity, and mRNA and protein expressions of myocardial CGRP were significantly decreased in EE group and EP+EE group. Compared with EE group, the levels of serum CK-MB and myocardial MDA in EP+EE group were decreased [CK-MB (U/L): 13.11±0.77 vs. 15.55±0.90, MDA (μmol/L): 389.57±49.60 vs. 709.08±160.49], the level of serum CGRP, and mRNA and protein expressions of myocardium CGRP were increased [serum CGRP (ng/L): 120.41±9.07 vs. 97.97±9.05, CGRP mRNA (2 -ΔΔCT): 0.45±0.09 vs. 0.14±0.02, CGRP protein (gray value): 0.78±0.08 vs. 0.41±0.04, all P < 0.05], the degree of myocardial injury was obviously alleviated. There was no significant difference in the indexes between the EP group and C group. EP has the heart protective effect for the acute EE rats, and the mechanism is closely related to the endogenous protective substance CGRP.

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.

Omega-3 fatty acid EPA improves regenerative capacity of mouse skeletal muscle cells exposed to saturated fat and inflammation

Sarcopenic obesity is characterised by high fat mass, low muscle mass and an elevated inflammatory environmental milieu. We therefore investigated the effects of elevated inflammatory cytokine TNF-α (aging/obesity) and saturated fatty acid, palmitate (obesity) on skeletal muscle cells in the presence/absence of EPA, a-3 polyunsaturated fatty acid with proposed anti-inflammatory, anti-obesity activities. In the present study we show that palmitate was lipotoxic, inducing high levels of cell death and blocking myotube formation. Cell death under these conditions was associated with increased caspase activity, suppression of differentiation, reductions in both creatine kinase activity and gene expression of myogenic factors; IGF-II, IGFBP-5, MyoD and myogenin. However, inhibition of caspase activity via administration of Z-VDVAD-FMK (caspase-2), Z-DEVD-FMK (caspase-3) and ZIETD-KMK (caspase 8) was without effect on cell death. By contrast, lipotoxicity associated with elevated palmitate was reduced with the MEK inhibitor PD98059, indicating palmitate induced cell death was MAPK mediated. These lipotoxic conditions were further exacerbated in the presence of inflammation via TNF-α co-administration. Addition of EPA under cytotoxic stress (TNF-α) was shown to partially rescue differentiation with enhanced myotube formation being associated with increased MyoD, myogenin, IGF-II and IGFBP-5 expression. EPA had little impact on the cell death phenotype observed in lipotoxic conditions but did show benefit in restoring differentiation under lipotoxic plus cytotoxic conditions. Under these conditions Id3 (inhibitor of differentiation) gene expression was inversely linked with survival rates, potentially indicating a novel role of EPA and Id3 in the regulation of apoptosis in lipotoxic/cytotoxic conditions. Additionally, signalling studies indicated the combination of lipo- and cyto-toxic effects on the muscle cells acted through ceramide, JNK and MAPK pathways and blocking these pathways using PD98059 (MEK inhibitor) and Fumonisin B1 (ceramide inhibitor) significantly reduced levels of cell death. These findings highlight novel pathways associated with in vitro models of lipotoxicity (palmitate-mediated) and cytotoxicity (inflammatory cytokine mediated) in the potential targeting of molecular modulators of sarcopenic obesity.

Arginine kinase from Myzostoma cirriferum, a basal member of annelids.

We assembled a phosphagen kinase gene from the Expressed Sequence Tags database of Myzostoma cirriferum, a basal member of annelids. The assembled gene sequence was synthesized using an overlap extension polymerase chain reaction method and was expressed in Escherichia coli. The recombinant enzyme (355 residues) exhibited monomeric behavior on a gel filtration column and showed strong activity only for l-arginine. Thus, the enzyme was identified as arginine kinase (AK). The two-substrate kinetic parameters were obtained and compared with other AKs. Phylogenetic analysis of amino acid sequences of phosphagen kinases indicated that the Myzostoma AK gene lineage differed from that of the polychaete Sabellastarte spectabilis AK, which is a dimer of creatine kinase (CK) origin. It is likely that the Myzostoma AK gene lineage was lost at an early stage of annelid evolution and that Sabellastarte AK evolved secondarily from the CK gene. This work contributes to our understanding of the evolution of phosphagen kinases of annelids with marked diversity.

Effects of Exposure to Two Fragrances on the Gene Expression of Ckm and Ckmt2 and Total CK Activity in the Hearts of Wistar Rats

Background: Some of the most commercially used compounds in fragrances have been associated with various adverse effects in various experimental in vivo and in vitro models and are still being used promiscuously in perfumes and as additives in other household products. Objectives: This study sought to determine the effects of exposing wistar rats to two locally made Nigerian perfumes on some cardiac performance enzyme and genes. Materials and Methods: In this experimental study, 18 animals were allocated into three groups (A, B and C) of six each. Groups B and C animals were exposed (by inhalation) to the first and second perfumes (designated F1 and F2) respectively for 77 days, while animals in group A were unexposed control. The rats were sacrificed at the end of the exposure period after which heart tissue was excised for creatine kinase enzyme assay and formalin fixed, paraffin embedded heart tissues were processed for RNA extraction and analyzed by quantitative real time polymerase chain reaction for the mRNA expression of creatine kinase genes Ckm and Ckmt2. Results: The results showed that animals in both exposure groups demonstrated significantly (P < 0.05) increased expression of striated muscle associated creatine kinase and sarcomeric mitochondria Ck genes as well as the increased release of the cardiomyocyte enzyme CK in the hearts of Wistar rats. Conclusions: These results suggest that exposure to these two locally made fragrances contributes to cardiomyocyte stress.

Common Creatine Kinase gene mutation results in falsely reassuring CK levels in muscle disorders.

Learning point for clinicians The mean allele frequency of this SNP is 0.02 (∼2.5 million people in the UK are carriers). Therefore, greater awareness of this CKM SNP is needed as there may be instances where the diagnosis of rhabdomyolysis, statin-induced myopathy, poly-/dermatomyositis and other muscle conditions that result in raised CK’s, is missed in these patients. Creatine Kinase (CK) is the enzyme responsible for catalysing the exchange of phosphates in the creatine/phosphocreatine shuttle.1 Skeletal muscle CK is made …

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).

Association of lamin A/C with muscle gene-specific promoters in myoblasts

The A-type and B-type lamins form a filamentous meshwork underneath the inner nuclear membrane called the nuclear lamina, which is an important component of nuclear architecture in metazoan cells. The lamina interacts with large, mostly repressive chromatin domains at the nuclear periphery. In addition, genome–lamina interactions also involve dynamic association of lamin A/C with gene promoters in adipocytes. Mutations in the human lamin A gene cause a spectrum of hereditary diseases called the laminopathies which affect muscle, cardiac and adipose tissues. Since most mutations in lamin A/C affect skeletal muscle, we investigated lamin–chromatin interactions at promoters of muscle specific genes in both muscle and non-muscle cell lines by ChIP-qPCR. We observed that lamin A/C was specifically associated with promoter regions of muscle genes in myoblasts but not in fibroblasts. Lamin A/C dissociated from the promoter regions of the differentiation specific MyoD, myogenin and muscle creatine kinase genes when myoblasts were induced to differentiate. In the promoter regions of the myogenin and MyoD genes, the binding of lamin A/C in myoblasts inversely correlated with the active histone mark, H3K4me3. Lamin A/C binding on muscle genes was reduced and differentiation potential was enhanced on treatment of myoblasts with a histone deacetylase inhibitor. These findings suggest a role for lamina–chromatin interactions in muscle differentiation and have important implications for the pathological mechanisms of striated muscle associated laminopathies.

Arginine kinases from the marine feather star Tropiometra afra macrodiscus: The first finding of a prenylation signal sequence in metazoan phosphagen kinases.

Two arginine kinase cDNAs (AK1 and AK2) were isolated from the marine feather star Tropiometra afra macrodiscus, and the gene structure (exon/intron organization) of AK1 was determined. The cDNA-derived amino acid sequences and the exon/intron organization of the Tropiometra AK1 gene were homologous to those of a human creatine kinase (CK) as well as the AK of the sea cucumber Stichopus. Phylogenetic analysis also supports the close relationship between human CKs and echinoderm AKs, indicating that the latter AKs evolved from an ancestral CK gene. We observed that the Tropiometra AK1 gene has a novel C-terminal extension (approximately 50 amino acid residues) encoded by a unique exon. Moreover, a typical prenylation signal sequence (CSLL) was found at the C-terminal end of this extension, suggesting that AK1 is anchored to a membrane. AK2 had no such C-terminal extension. This is the first finding of a prenylation signal in metazoan phosphagen kinases. Recombinant Tropiometra AK1 and AK2 enzymes were successfully expressed in Escherichia coli, and their kinetic constants were determined. Both enzymes showed activity comparable to that of typical invertebrate AKs.

Contrasting roles for MyoD in organizing myogenic promoter structures during embryonic skeletal muscle development.

Among the complexities of skeletal muscle differentiation is a temporal distinction in the onset of expression of different lineage-specific genes. The lineage-determining factor MyoD is bound to myogenic genes at the onset of differentiation whether gene activation is immediate or delayed. How temporal regulation of differentiation-specific genes is established remains unclear. Using embryonic tissue, we addressed the molecular differences in the organization of the myogenin and muscle creatine kinase (MCK) gene promoters by examining regulatory factor binding as a function of both time and spatial organization during somitogenesis. At the myogenin promoter, binding of the homeodomain factor Pbx1 coincided with H3 hyperacetylation and was followed by binding of co-activators that modulate chromatin structure. MyoD and myogenin binding occurred subsequently, demonstrating that Pbx1 facilitates chromatin remodeling and modification before myogenic regulatory factor binding. At the same time, the MCK promoter was bound by HDAC2 and MyoD, and activating histone marks were largely absent. The association of HDAC2 and MyoD was confirmed by co-immunoprecipitation, proximity ligation assay (PLA), and sequential ChIP. MyoD differentially promotes activated and repressed chromatin structures at myogenic genes early after the onset of skeletal muscle differentiation in the developing mouse embryo.