Novel isoform of myotonin protein kinase: gene product of myotonic dystrophy is localized in the sarcoplasmic reticulum of skeletal muscle.

It is quite important to know the exact localization and function of myotonin protein kinase (MtPK), identified as the gene product of myotonic dystrophy, the most prevalent disease with multisystem disorders among muscular dystrophies. To investigate the localization of MtPK, we raised a polyclonal antibody against a synthetic peptide chosen within the deduced sequence of MtPK. This antibody detected both a membrane-bound 70-kd protein and a soluble 55-kd protein on Western blots of human muscles. By using this antibody for immunohistochemical studies of both biopsied human skeletal muscle fibers and mature innervated cultured muscle fibers, we can now demonstrate by confocal laser scanning microscopy that MtPK is localized mainly in the I-band. By immunoelectron microscopy, it was determined that MtPK is a membrane-bound protein localized mainly in the terminal cisternae of the sarcoplasmic reticulum. To our knowledge, this is the first documentation of the ultrastructural localization of MtPK. This finding is quite important for clarifying the pathophysiological basis of myotonic dystrophy, which might be due to a dysregulation of calcium metabolism.     

抗阻训练衰老小鼠骨骼肌p53与bax基因的表达

背景：抗阻训练能够缓解肌肉衰减征的发生并能够弱化骨骼肌细胞凋亡。 目的：观察年龄和抗阻训练对SAMP8小鼠骨骼肌p53,bax基因表达的影响。 方法：分别取3月龄(青年组)和6月龄(老年组)的SAMP8小鼠进行8周爬梯运动,每周3次,以不参加运动的同龄小鼠作为对照。 结果与结论：Real-time PCR结果显示,与青年组比较,老年组小鼠骨骼肌纤维p53,bax mRNA的表达量显著上升(P < 0.05),而与相同月龄对照组比较,抗阻训练的小鼠骨骼肌纤维中p53,bax mRNA的表达量明显降低(P < 0.05)。提示抗阻训练能抑制衰老小鼠骨骼肌p53,bax基因的表达。     

Differential expression and regulation of the PKA signalling pathway in fast and slow skeletal muscle

To identify intracellular signalling pathways that transduce muscle electrical activity, we have investigated the Protein Kinase A (PKA) pathway in fast and slow skeletal muscle. The slow soleus muscle (SOL) displayed approximately twice as much PKA catalytic activity and cAMP-binding compared to the fast Extensor Digitorum Longus (EDL) muscle. These results were confirmed by Western blot analysis using antibodies directed against the catalytic or regulatory subunits of PKA. PKA subunits were concentrated at the neuromuscular junction in innervated and denervated muscle fibers demonstrating that PKA is expressed post-synaptically. In addition, we also detected PKA subunits outside the junctional area, suggesting that PKA functions outside of the synaptic regions. Following denervation, levels of cyclic AMP, PKA C activity, R cAMP-binding and RI alpha protein levels increased significantly in the SOL, in contrast to the EDL where only elevated levels of RI alpha protein were observed. These observations demonstrate that PKA levels in skeletal muscle are subject to control at several levels and suggest that some of the differences may be in the pattern of electrical activity that motoneurons impose on the SOL and EDL.     

Studies of gene expression and activity of hexokinase, phosphofructokinase and glycogen synthase in human skeletal muscle in states of altered insulin-stimulated glucose metabolism

When whole body insulin-stimulated glucose disposal rate is measured in man applying the euglycaemic, hyperinsulinaemic clamp technique it has been shown that approximately 75% of glucose is taken up by skeletal muscle. After the initial transport step, glucose is rapidly phosphorylated to glucose-6-phosphate and routed into the major pathways of either glucose storage as glycogen or the glycolytic/tricarboxylic acid pathway. Glucose uptake in skeletal muscle involves-the activity of specific glucose transporters and hexokinases, whereas, phosphofructokinase and glycogen synthase hold critical roles in glucose oxidation/glycolysis and glucose storage, respectively. Glucose transporters and glycogen synthase activities are directly and acutely stimulated by insulin whereas the activities of hexokinases and phosphofructokinase may primarily be allosterically regulated. The aim of the review is to discuss our present knowledge of the activities and gene expression of hexokinase II (HKII), phosphofructokinase (PFK) and glycogen synthase (GS) in human skeletal muscle in states of altered insulin-stimulated glucose metabolism. My own experimental studies have comprised patients with disorders characterized by insulin resistance like non-insulin-dependent diabetes mellitus (NIDDM) and insulin-dependent diabetes mellitus (IDDM) before and after therapeutic interventions, patients with microvascular angina and patients with severe insulin resistant diabetes mellitus and congenital muscle fiber type disproportion myopathy as well as athletes who are in a state of improved insulin sensitivity. By applying the glucose insulin clamp method in combination with nuclear magnetic resonance 31P spectroscopy to normoglycaemic or hyperglycaemic insulin resistant subjects impairment of insulin-stimulated glucose transport and/or phosphorylation in skeletal muscle has been shown. In states characterized by insulin resistance but normoglycaemia, the activity of HKII measured in needle revealed any genetic variability that contributes to explain the decreased muscle levels of GS mRNA or the decreased activity and activation of muscle GS in NIDDM patients and their glucose tolerant but insulin resistant relatives. Thus, the causes of impaired insulin-stimulated glycogen synthesis of skeletal muscle in normoglycaemic insulin resistant subjects are likely to be found in the insulin signalling network proximal to the GS protein. In insulin resistant diabetic patients the impact of these yet unknown abnormalities may be accentuated by the prevailing hyperglycaemia and hyperlipidaemia. Endurance training in young healthy subjects results in improved insulin-stimulated glucose disposal rates, predominantly due to an increased glycogen synthesis rate in muscle, which is paralleled by an increased total GS activity, increased GS mRNA levels and enhanced insulin-stimulated activation of GS. These changes are probably due to local contraction-dependent mechanisms. Likewise, one-legged exercise training has been reported to increase the basal concentration of muscle GS mRNA in NIDDM patients to a level similar to that seen in control subjects although insulin-stimulated glucose disposal rates remain reduced in NIDDM patients. In the insulin resistant states examined so far, basal and insulin-stimulated glucose oxidation rate at the whole body level and PFK activity in muscle are normal. In parallel, no changes have been found in skeletal muscle levels of PFK mRNA and immunoreactive protein in NIDDM or IDDM patients. In endurance trained subjects insulin-stimulated whole body glucose oxidation rate is often increased. However, depending on the intensity and frequency, physical exercise may induce an increased, a decreased or an unaltered level of muscle PFK activity. In athletes the muscle PFK mRNA is similar to what is found in sedentary subjects whereas the immunoreactive PFK protein concentration is decreased.     

De-phosphorylation of MyoD is linking nerve-evoked activity to fast myosin heavy chain expression in rodent adult skeletal muscle

Elucidating the molecular pathways linking electrical activity to gene expression is necessary for understanding the effects of exercise on muscle. Fast muscles express higher levels of MyoD and lower levels of myogenin than slow muscles, and we have previously linked myogenin to expression of oxidative enzymes. We here report that in slow muscles, compared with fast, 6 times as much of the MyoD is in an inactive form phosphorylated at T115. In fast muscles, 10 h of slow electrical stimulation had no effect on the total MyoD protein level, but the fraction of phosphorylated MyoD was increased 4-fold. Longer stimulation also decreased the total level of MyoD mRNA and protein, while the level of myogenin protein was increased. Fast patterned stimulation did not have any of these effects. Overexpression of wild type MyoD had variable effects in active slow muscles, but increased expression of fast myosin heavy chain in denervated muscles. In normally active soleus muscles, MyoD mutated at T115 (but not at S200) increased the number of fibres containing fast myosin from 50% to 85% in mice and from 13% to 62% in rats. These data establish de-phosphorylated active MyoD as a link between the pattern of electrical activity and fast fibre type in adult muscles.     

Changes in muscle fibre type, muscle mass and IGF-I gene expression in rabbit skeletal muscle subjected to stretch

The relationship between IGF-I and changes in muscle fibre phenotype in response to 6 d of stretch or disuse of the lower limb muscles of the rabbit was studied by combining in situ hybridisation and immunohistochemistry procedures. Passive stretch by plaster cast immobilisation of the muscle in its lengthened position not only induced an increase in IGF-I mRNA expression within the individual muscle fibres but also an increase in the percentage of fibres expressing neonatal and slow myosin. This change in phenotype was also found to be accompanied by a rapid and marked increase of muscle mass, total RNA content as well as IGF-I gene expression. In contrast, IGF-I appears not to be involved in muscle atrophy induced by immobilisation in the shortened position and the inactivity which results from this procedure. The level of increase in expression of IGF-I mRNA varied from fibre to fibre. By using adjacent serial sections, the fibres which expressed IGF-I mRNA at the highest levels were identified as expressing neonatal and the slow type 1 myosin. These data suggest that the expression of IGF-I within individual muscle fibres is correlated not only with hypertrophy but also with the muscle phenotypic adaptation that results from stretch and overload.     

Expression of the Na(+)/H(+) exchanger isoform NHE1 in rat skeletal muscle and effect of training

The expression of the Na(+)/H(+) exchanger isoform NHE1 was quantified in homogenates of various rat skeletal muscles by means of immunoblotting, and the effect of 3 weeks of treadmill training on NHE1 expression was determined in a red (oxidative) as well as a white (glycolytic)-muscle preparation. The NHE1 antibodies recognized a glycosylated protein at 101-111 kDa. There was a positive correlation between the NHE1 expression in the muscle and percent type IIB fibres and percent type IID/X fibres, whereas the NHE1 expressions were negatively correlated to percent type I fibres and percent type I + IIA fibres. Thus the highest NHE1 expression was evident in the most glycolytic fibres. Treadmill training increased (P < 0.05) the NHE1 content by 29 and 36% in oxidative and glycolytic fibres, respectively, suggesting that training enhanced the NHE1 content of all muscle-fibre types. Therefore training may improve the capacity for pH regulation in skeletal muscle.     

Expression of the Na+/H+ exchanger isoform NHE1 in rat skeletal muscle and effect of training

The expression of the Na⁺/H⁺ exchanger isoform NHE1 was quantified in homogenates of various rat skeletal muscles by means of immunoblotting, and the effect of 3 weeks of treadmill training on NHE1 expression was determined in a red (oxidative) as well as a white (glycolytic)‐muscle preparation. The NHE1 antibodies recognized a glycosylated protein at 101–111 kDa. There was a positive correlation between the NHE1 expression in the muscle and percent type IIB fibres and percent type IID/X fibres, whereas the NHE1 expressions were negatively correlated to percent type I fibres and percent type I + IIA fibres. Thus the highest NHE1 expression was evident in the most glycolytic fibres. Treadmill training increased (P < 0.05) the NHE1 content by 29 and 36% in oxidative and glycolytic fibres, respectively, suggesting that training enhanced the NHE1 content of all muscle‐fibre types. Therefore training may improve the capacity for pH regulation in skeletal muscle.