The presence of serum creatine kinase 2 (MB) in hypokalemic familial periodic paralysis.

We recently found 17 U/l of isoenzyme creatine kinase (CK) 2 (MB), or 3.2% of total 533 U/l CK activity, in a patient with hypokalemic familial periodic paralysis who did not show clinical or EKG evidence of acute myocardial necrosis. The myopathy associated with hypokalemic familial periodic paralysis is thus another cause for the presence of CK 2 (MB). CK 2 (MB) is not a specific isoenzyme for myocardial damage since it may be identified in the serum of patients with skeletal muscle conditions.     

Stem cell biotherapy: A new remedy for Trichinella spiralis-induced inflammatory myopathy

Trichinella spiralis (T. spiralis)-induced myopathy is an inflammatory myopathy that is difficult to treat unless the parasite is combated in its early intestinal phase before it reaches the muscles. This study aimed to evaluate the effect of local mesenchymal stem cell (MSC) therapy on T. spiralis-induced inflammatory myopathy in rats. Rats were divided into four groups: Group 1 (non-infected non-treated group); Group 2 (infected non-treated group); Group 3 (infected albendazole (ABZ)-treated group); and Group 4 (infected MSC-treated group). Their muscle status was assessed physiologically with the righting reflex and electromyography (EMG), parasitologically with the total muscle larval count, histopathologically with hematoxylin and eosin and Mallory's trichrome stains, as well as immunohistochemically for myogenin as a marker of muscle regeneration. Additionally, serum muscle enzymes creatine kinase (CK) and lactate dehydrogenase (LDH), as well as muscle matrix metalloproteinases MMP1 and MMP9, were assayed. Finally, the immunological response was assessed by measuring the levels of the muscle inflammatory cytokines tumor necrosis factor-alpha (TNF-α), interferon-gamma (INF-γ), and interleukin-4 (IL-4). Our findings revealed that MSC therapy markedly improved muscle EMG and righting reflex, as well as the histopathological appearance of the muscles, reduced inflammatory cellular infiltrates, and increased myogenin immunostaining. It also reduced serum CK and LDH levels, as well as muscle INF-γ, TNF-α, IL-4, MMP1, and MMP9 levels. However, it had no effect on the total muscle larval count. Accordingly, due to its anti-inflammatory properties and muscle-regenerative effect, MSC therapy could be a promising new remedy for T. spiralis-induced myopathy.     

Elimination by necrosis,not apoptosis,of embryonic extraocular muscles in the<Emphasis Type="Italic"> muscular dysgenesis</Emphasis> mutant of the mouse

Muscular dysgenesis (mdg) in the mouse is a loss-of-function mutation of the skeletal muscle isoform of the voltage-sensor Ca²⁺ channel of skeletal muscle (DHP receptor alpha1 subunit, Cchl1a3, Chr1), which is essential for excitation-contraction coupling. Affected individuals (genotype mdg/mdg, phenotype MDG) are unable to breathe and die perinatally. We introduce here extraocular muscles in the study of MDG myopathy and show that, despite their developmental origin from head placodes, they are affected like trunk and limb muscles. MDG myotubes in situ are eliminated by necrosis, not apoptosis.The study was supported by the Deutsche Forschungsgemeinschaft, SFB 223 C03, E02     

Acute and remote biochemical and physiological effects of exhaustive weightlifting exercise

The goal of the work was a study of exhaustive weightlifting exercise effect on prolonged changes in physiological and biochemical variables characterized functional status of skeletal muscles. An exercise gave rise to significant blood lactate concentration increase that was indicative of an anaerobic metabolism to be a predominant mechanism of muscle contraction energy supply. A reduction of m. rectus femoris EMG activity (amplitude and frequency), tonus of tension and an increase in tonus of relaxation were found immediately after exercise. Both EMG amplitude and frequency were increased 1 day post-exercise. However, after 3 days of recovery, EMG amplitude and frequency were decreased again and, in parallel, blood serum creatine kinase (CK) activity was significantly increased. After 9 recovery days, all measured variables with the exception of CK were normalized. A significant reverse correlation was found between blood serum lactate concentration and m. rectus femoris EMG activity at the same time points. Blood serum CK activity and m. rectus femoris EMG and tonus variables were observed to be significantly reversely correlated on the 3rd post-exercise day. Presented data demonstrate that exhaustive exercise-induced muscle injury resulted in phase alterations in electrical activity and tonus which correlated with lactate concentration and CK activity in blood serum.     

Loss of tafazzin results in decreased myoblast differentiation in C2C12 cells: A myoblast model of Barth syndrome and cardiolipin deficiency

Barth syndrome (BTHS) is an X-linked genetic disorder resulting from mutations in the tafazzin gene (TAZ), which encodes the transacylase that remodels the mitochondrial phospholipid cardiolipin (CL). While most BTHS patients exhibit pronounced skeletal myopathy, the mechanisms linking defective CL remodeling and skeletal myopathy have not been determined. In this study, we constructed a CRISPR-generated stable tafazzin knockout (TAZ-KO) C2C12 myoblast cell line. TAZ-KO cells exhibit mitochondrial deficits consistent with other models of BTHS, including accumulation of monolyso-CL (MLCL), decreased mitochondrial respiration, and increased mitochondrial ROS production. Additionally, tafazzin deficiency was associated with impairment of myocyte differentiation. Future studies should determine whether alterations in myogenic determination contribute to the skeletal myopathy observed in BTHS patients. The BTHS myoblast model will enable studies to elucidate mechanisms by which defective CL remodeling interferes with normal myocyte differentiation and skeletal muscle ontogenesis.     

Characterization of creatine kinase isoforms in herring (Clupea harengus) skeletal muscle

It is known that mitochondrial creatine kinase (MtCK) in mammals is always expressed in conjunction with one of the cytosolic forms of creatine kinase (CK), either muscle-type (MM-CK) or brain-type (BB-CK) in tissues of high, sudden energy demand. The two creatine kinase (CK) isoforms were detected in herring (Clupea harengus) skeletal muscle: cytosolic CK and mitochondrial CK (MtCK) that displayed the different electrophoretic mobility. These isoforms differ in molecular weight and some biochemical properties. Isolation and purification procedures allowed to obtain purified enzymes with specific activity of the 206 μmol/min/mg for cytosolic CK and 240 μmol/min/mg for MtCK. Native M_rs of the cytosolic CK and MtCK determined by gel permeation chromatography were 86.000 and 345.000, respectively. The results indicate that one of isoforms found in herring skeletal muscle is a cytosolic dimer and the other one, is a mitochondrial octamer. Octamerization of MtCK is not an advanced feature and also exists in fish. These values correspond well with published values for MtCKs and cytosolic CK isoforms from higher vertebrate classes and even from lower invertebrates.     

模拟氮沉降对天山云杉细根分解及其养分释放的影响

采用野外模拟试验,设计4种氮处理——对照(不施氮,CK)、低氮(施氮5kg·hm-2·a-1,LN)、中氮(施氮10kg·hm-2·a-1,MN)、高氮(施氮15kg·hm-2·a-1,HN),研究氮沉降对天山云杉细根分解及养分释放的影响。结果表明:(1)不同氮处理分解2年后天山云杉细根残留率依次为74.044%(HN)、71.967%(MN)、68.156%(CK)、61.933%(LN),且差异显著。(2)天山云杉的细根月分解速率在试验前期不同氮处理下规律不明显;而在试验后期呈现为对照中氮低氮高氮。(3)4种氮处理下天山云杉细根分解50%需要的时间依次为3.31年(LN)、3.67年(CK)、4.28年(MN)、4.64年(HN),分解95%需要的时间依次为14.39年(LN)、15.93年(CK)、18.58年(MN)和20.17年(HN)。(4)天山云杉细根C元素迁移模式总体表现为直接释放,N元氮为富集-释放模式,残留率呈现波动式下降趋势。(5)不同氮处理下天山云杉细根分解率与C元素浓度间均呈线性负相关关系;对照和低氮处理下,天山云杉细根分解率与N元素浓度间均为线性负相关关系,中氮和高氮处理下,细根分解率随N元素浓度的增加呈先增加后降低的趋势。     

Stable reference genes for expression studies in breast muscle of normal and white striping-affected chickens

The normalization with proper reference genes is a crucial step to obtain accurate mRNA expression levels in quantitative PCR (qPCR) studies. Therefore, in this study, 10 reference candidate genes were evaluated to determine their stability in normal pectoralis major muscle of broilers and those counterparts affected with White Striping (WS) myopathy at 42 days age. Four different tools were used for ranking the most stable genes: GeNorm, NormFinder, BestKeeper and Comparative Ct (ΔCt), and a general ranking was performed using the RankAggreg tool to select the best reference genes among all tools. From the 10 genes evaluated in the breast muscle of broilers, 8 were amplified. Most of the algorithms/tools indicated the same two genes, RPL30 and RPL5, as the most stable in the broilers breast muscle. In addition, there was agreement among the tools for the least stable genes: MRPS27, GAPDH and RPLP1 in the broilers breast muscle. Therefore, it is interesting to note that even with different tools for evaluating gene expression, there was consensus on the most and least stable genes. These results indicate that the Ribosomal protein L30 (RPL30) and Ribosomal protein L5 (RPL5) can be recommended for accurate normalization in qPCR studies with chicken pectoralis major muscle affected with White Striping and other myopathies.

     

Defective collagen VI α6 chain expression in the skeletal muscle of patients with collagen VI-related myopathies

Collagen VI is a non-fibrillar collagen present in the extracellular matrix (ECM) as a complex polymer; the mainly expressed form is composed of α1, α2 and α3 chains; mutations in genes encoding these chains cause myopathies known as Ullrich congenital muscular dystrophy (UCMD), Bethlem myopathy (BM) and myosclerosis myopathy (MM). The collagen VI α6 chain is a recently identified component of the ECM of the human skeletal muscle. Here we report that the α6 chain was dramatically reduced in skeletal muscle and muscle cell cultures of genetically characterized UCMD, BM and MM patients, independently of the clinical phenotype, the gene involved and the effect of the mutation on the expression of the “classical” α1α2α3 heterotrimer. By contrast, the collagen VI α6 chain was normally expressed or increased in the muscle of patients affected by other forms of muscular dystrophy, the overexpression matching with areas of increased fibrosis. In vitro treatment with TGF-β1, a potent collagen inducer, promoted the collagen VI α6 chain deposition in the ECM of normal muscle cells, whereas, in cultures derived from collagen VI-related myopathy patients, the collagen VI α6 chain failed to develop a network outside the cells and accumulated in the endoplasmic reticulum. The defect of the α6 chain points to a contribution to the pathogenesis of collagen VI-related disorders.     

Leucocytosis as a marker of organ damage induced by chronic strenuous physical exercise

The effects of strenuous physical exercise on the serial changes in the haematological, biochemical and hormonal markers were investigated. A group of 14 soldiers, aged 24–36 years, took part in a military training course for about 13 weeks. After severe exercise stress, an increase (90%) in the number of peripheral blood leucocytes was observed. The degree of leucocytosis showed a close correlation with the values of some serum parameters, such as concentrations of aspartate aminotransferase (AST;r = 0.747), lactate dehydrogenase (LD;r = 0.748), blood urea nitrogen (r = 0.756), creatine kinase (CK;r = 0.637), manganese-superoxide dismutase (Mn-SOD;r = 0.508), alanine aminotransferase (ALT;r = 0.542) and uric acid (r = 0.538), and concentrations of urinary parameters, such as vanilmandelic acid (r = 0.429) and free cortisol (r = 0.437). The subjects showing prominent leucocytosis over 9500 cells · l^–1 exhibited a lower concentration of serum cholinesterase than those who showed milder leucocytosis. The serum Mn-SOD concentration was closely correlated with the serial changes in serum concentrations of AST, ALT, LD and CK, indicating exercise-induced muscle and liver damage. The change in peripheral leucocyte number was assumed to be diagnostically informative and may be a prognostic marker, reflecting organ damage and restoration after strenuous physical exercise.     

Autophagy induction rescues muscular dystrophy

Collagen VI is an extracellular matrix protein forming a microfibrillar network in the endomysium of skeletal muscles. In humans, mutations in any of the three genes coding for collagen VI cause several skeletal muscle diseases, including Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD). Collagen VI null (Col6a1^–/–) mice display a myopathic phenotype resembling that of BM and UCMD patients. Muscles lacking collagen VI are characterized by the presence of dilated sarcoplasmic reticulum and dysfunctional mitochondria, which triggers apoptosis and leads to muscle wasting. We have found that accumulation of abnormal organelles is due to an impairment of autophagy. Reactivation of the autophagic flux by either nutritional approaches or by pharmacological and genetics tools removes dysfunctional organelles and greatly ameliorates the dystrophic phenotype.     

Identification of valid reference genes during the differentiation of human myoblasts

Background  

Analysis of RNA expression using real-time PCR (qRT-PCR) traditionally includes reference genes (RG) as an internal control. This practice is being questioned as it becomes increasingly clear that RG may vary considerably under certain experimental conditions. Thus, the validity of a particular RG must be determined for each experimental setting. We used qRT-PCR to measure the levels of six RG, which have been reported in the literature to be invariant. The RG were analyzed in human myoblast cultures under differentiation conditions. We examined the expression by qRT-PCR of mRNA encoding Beta-actin (ACTB), Beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), peptidylprolyl isomerase A (PPIA), TATA box binding protein (TBP) and ribosomal protein, large, P0 (RPLPO). The mRNA expression of the following genes of interest (GOI) were analyzed: skeletal muscle alpha 1 actin (ACTA1), myogenin/myogenic factor 4 (MYOG), embryonic skeletal muscle myosin heavy chain 3 (MYH3) and the activity of creatine phosphokinase (CK). The geNorm, NormFinder and BestKeeper software programs were used to ascertain the most suitable RG to normalize the RNA input.     

Cardiac and skeletal muscle troponin I isoforms are encoded by a dispersed gene family on mouse Chromosomes 1 and 7

We mapped the locations of the genes encoding the slow skeletal muscle, fast skeletal muscle, and cardiac isoforms of troponin I (Tnni) in the mouse genome by interspecific hybrid backcross analysis of species-specific (C57BL/6 vs Mus spretus) restriction fragment length polymorphisms (RFLPs). The slow skeletal muscle troponin I locus (Tnni1) mapped to Chromosome (Chr) 1. The fast skeletal muscle troponin I locus (Tnni2), mapped to Chr 7, approximately 70 cM from the centromere. The cardiac troponin I locus (Tnni3) also mapped to Chr 7, approximately 5–10 cM from the centromere and unlinked to the fast skeletal muscle troponin I locus. Thus, the troponin I gene family is dispersed in the mouse genome. Received: 10 May 1995 / Accepted: 1 September 1995     

Subclinical myopathy in a child with neutral lipid storage disease and mutations in the PNPLA2 gene

We report a 14-year-old-boy with markedly elevated serum creatine kinase (CK) levels, in whom massive triglyceride storage was found in peripheral blood leukocytes and in muscle biopsy. Sequencing PNPLA2, the gene encoding the adipose triglyceride lipase (ATGL) and responsible for the neutral lipid storage disease with myopathy (NLSDM), we identified two heterozygous mutations, including a previously reported nonsense and a novel missense mutation in the patatin domain of the gene.Lipid storage myopathy can be clinically silent in childhood and presenting only with hyperCKemia.     

Creatine kinase regulation by reversible phosphorylation in frog muscle

Creatine kinase (CK) was analyzed from skeletal muscle of wood frogs, Rana sylvatica, a species that survives natural whole body freezing during the winter months. Muscle CK activity increased by 35% and apparent K_m creatine decreased by 29% when frogs froze. Immunoblotting analysis showed that this activity increase was not due to a change in total CK protein. Frog muscle CK was regulated by reversible protein phosphorylation; in vitro incubations with ³²P-ATP under conditions that facilitated the actions of various protein kinases (PKA, PKG, PKC, CaMK or AMPK) resulted in immunoprecipitation of ³²P-labeled CK. Furthermore, incubations that stimulated CaMK or AMPK altered CK kinetics. Incubation under conditions that facilitated protein phosphatases (PP2B or PP2C) reversed these effects. Phosphorylation of CK increased activity, whereas dephosphorylation decreased activity. Ion-exchange chromatography revealed that two forms of CK with different phosphorylation states were present in muscle; low versus high phosphate forms dominated in muscle of control versus frozen frogs, respectively. However, CK from control versus frozen frogs showed no differences in susceptibility to urea denaturation or sensitivity to limited proteolysis by thermolysin. The increased activity, increased substrate affinity and altered phosphorylation state of CK in skeletal muscle from frozen frogs argues for altered regulation of CK under energy stress in ischemic frozen muscle.     

Role of Autophagy in Glycogen Breakdown and Its Relevance to Chloroquine Myopathy

Several myopathies are associated with defects in autophagic and lysosomal degradation of glycogen, but it remains unclear how glycogen is targeted to the lysosome and what significance this process has for muscle cells. We have established a Drosophila melanogaster model to study glycogen autophagy in skeletal muscles, using chloroquine (CQ) to simulate a vacuolar myopathy that is completely dependent on the core autophagy genes. We show that autophagy is required for the most efficient degradation of glycogen in response to starvation. Furthermore, we show that CQ-induced myopathy can be improved by reduction of either autophagy or glycogen synthesis, the latter possibly due to a direct role of Glycogen Synthase in regulating autophagy through its interaction with Atg8.