Identification of secondary effects of hyperexcitability by proteomic profiling of myotonic mouse muscle

Myotonia is a symptom of various genetic and acquired skeletal muscular disorders and is characterized by hyperexcitability of the sarcolemma. Here, we have performed a comparative proteomic study of the genetic mouse models ADR, MTO and MTO*5J of human congenital myotonia in order to determine myotonia-specific changes in the global protein complement of gastrocnemius muscle. Proteomic analyses of myotonia in the mouse, which is caused by mutations in the gene encoding the muscular chloride channel Clc1, revealed a generally perturbed protein expression pattern in severely affected ADR and MTO muscle, but less pronounced alterations in mildly diseased MTO*5J mice. Alterations were found in major metabolic pathways, the contractile machinery, ion handling elements, the cellular stress response and cell signaling mechanisms, clearly confirming a glycolytic-to-oxidative transformation process in myotonic fast muscle. In the long-term, a detailed biomarker signature of myotonia will improve our understanding of the pathobiochemical processes underlying this disorder and be helpful in determining how a single mutation in a tissue-specific gene can trigger severe downstream effects on the expression levels of a very large number of genes in contractile tissues.     

Multiple phosphorylation of mouse muscle glycogen synthase

Glycogen synthase was isolated from extracts of mouse diaphragm muscle by immunoprecipitation with specific antibodies raised against the rabbit muscle enzyme. A procedure was developed which permitted phosphorylation of the immunoprecipitated enzyme by several purified protein kinases. Peptide mapping techniques (including reverse-phase HPLC and thin-layer electrophoresis and chromatography) were used to compare tryptic phosphopeptides of the rabbit and mouse muscle enzymes. The results demonstrated a high degree of similarity in the chemical properties of these peptides, suggesting significant homology around the phosphorylation sites in these proteins. Thus, mouse peptides corresponding to the rabbit muscle peptides containing sites 1a, 1b, 2, 3, and 5 were identified, with protein kinase recognition specificities identical to those of the rabbit enzyme. The study indicates significant conservation in the muscle isozymes of glycogen synthase between mouse and rabbit as well as a similar distribution of phosphorylation sites throughout the enzyme subunit.     

Isolation of neuronal parvalbumin by high-performance liquid chromatography. Characterization and comparison with muscle parvalbumin

Neuronal parvalbumin has been isolated from rat brain and purified to homogeneity by high-performance liquid chromatography (HPLC) on reverse-phase supports. This procedure includes four consecutive chromatographic steps with an overall protein recovery of 74% and a 26 400-fold purification. The concentration of parvalbumin was found to be approximately 10 mg/kg wet weight in brain tissue, which is about 100 times lower than that in rat muscle. The physical properties of brain parvalbumin are described and compared with those of the muscle counterpart. These proteins were identical in their molecular weights (12 000), isoelectric points (4.9), retention times on C-18 reverse-phase HPLC columns, Ca2+ content (two per molecule), amino acid compositions, and immunological properties. A comparison of the tryptic peptide maps of brain and muscle parvalbumin by analytical HPLC also revealed identity and showed that the isolation method described here did not alter the chemical structure of the protein.     

Inhibition of smooth muscle 5'-nucleotidase by imidazole and its reversal by magnesium

Imidazole, commonly used as an effective pH-buffering reagent in aqueous media maintained at pH 7-8, was found to depress the 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) activity of microsomal membrane fraction isolated from rat vas deferens smooth muscle in a dose-dependent manner in the absence of added Mg2+. Such an inhibitory effect of imidazole on the smooth muscle 5'-nucleotidase was not dependent upon the purity or integrity of the membrane fractions used and could be fully reversed by the inclusion of 5-10 mM Mg2+ in the assay medium. Of the five different pH-buffering reagents tested, imidazole was specific in exerting inhibitory effect on the 5'-nucleotidase in the absence of Mg2+ and this inhibition could not be accounted for by the impurities present in the imidazole. Differential effects of chelating reagents and other divalent metal ions on the 5'-nucleotidase activity were also observed in imidazole and Tris buffer solutions. The 5'-nucleotidase activity was not affected if the membranes were preincubated and washed with a large volume of 50 mM imidazole and subsequently assayed in 50 mM Tris in the absence of Mg2+. Similar findings were obtained with EDTA treated membrane. These results suggest that imidazole does not act by removal of the activating metal ion but rather interacts directly with 5'-nucleotidase and alters the metal-enzyme interactions.     

The myosin cross-bridge cycle and its control by twitchin phosphorylation in catch muscle

The anterior byssus retractor muscle of Mytilus edulis was used to characterize the myosin cross-bridge during catch, a state of tonic force maintenance with a very low rate of energy utilization. Addition of MgATP to permeabilized muscles in high force rigor at pCa > 8 results in a rapid loss of some force followed by a very slow rate of relaxation that is characteristic of catch. The fast component is slowed 3-4-fold in the presence of 1 mM MgADP, but the distribution between the fast and slow (catch) components is not dependent on [MgADP]. Phosphorylation of twitchin results in loss of the catch component. Fewer than 4% of the myosin heads have ADP bound in rigor, and the time course (0.2-10 s) of ADP formation following release of ATP from caged ATP is similar whether or not twitchin is phosphorylated. This suggests that MgATP binding to the cross-bridge and subsequent splitting are independent of twitchin phosphorylation, but detachment occurs only if twitchin is phosphorylated. A similar dependence of detachment on twitchin phosphorylation is seen with AMP-PNP and ATPgammaS. Single turnover experiments on bound ADP suggest an increase in the rate of release of ADP from the cross-bridge when catch is released by phosphorylation of twitchin. Low [Ca(2+)] and unphosphorylated twitchin appear to cause catch by 1) markedly slowing ADP release from attached cross-bridges and 2) preventing detachment following ATP binding to the rigor cross-bridge.     

Sarcomere matrix of striated muscle: in vivo phosphorylation of titin and nebulin in mouse diaphragm muscle

Titin and nebulin are two major protein components of a cytoskeletal matrix that coexists with thick and thin filaments within the sarcomere of a wide range of striated muscles. Purified titin and nebulin from mouse diaphragm muscle are similar in size, in relative abundance, and in amino acid composition to analogous proteins from other mammals or avians. Phosphate analysis of these nucleic-acid-free proteins indicated that both proteins contain substantial amounts of protein-bound phosphate: about 12 mol of phosphate per mole of titin subunit and 11 mol of phosphate per mole of nebulin subunit. Incubation of intact, excised mouse diaphragm with radioactive inorganic phosphate resulted in significant incorporation of radiophosphate into titin and nebulin. The identification of titin and nebulin phosphorylation was facilitated by a simple salt fractionation and nuclease digestion procedure that effectively separated titin and nebulin from radiolabeled nucleic acids. Such in vivo phosphorylation studies indicated that approximately 2 mol of phosphate per titin subunit and 5 to 7 mol of phosphate per nebulin subunit were incorporated within 5 h of incubation. The incorporation nearly doubled when the beta-adrenergic agonist, isoproterenol, or a phosphodiesterase inhibitor, theophylline, was present in the medium. For both proteins, phosphorylation occurred mainly on serine residues. Nebulin also appears to possess a smaller number of threonine sites. Taken together, our data indicate that a small proportion (20 to 40%) of the steady-state titin phosphates are rapidly turning over. In contrast, most of the nebulin phosphates (50 to 100%) are readily exchanged. The modulation of turnover by external stimuli that increase cytosolic cAMP raises the possibility that at least a portion of the multiple phosphorylation sites of titin and nebulin may be involved in the functional regulation of the sarcomere matrix.     

Inhibition of denuded mouse oocyte meiotic maturation by tumor-promoting phorbol esters and its reversal by retinoids

Two tumor-promoting phorbol esters, phorbol 12,13-dibutyrate (PDBu) and phorbol 12-myristate 13-acetate (PMA), when added to the culture medium of denuded mouse oocytes prevent their spontaneous meiotic maturation, whereas phorbol 13-acetate, which is inactive as a tumor promoter, does not inhibit this process. Retinoids appear to antagonize this inhibitory action of tumor promoters. However, the inhibitory effect of forskolin on meiotic maturation is not prevented, but is potentiated by retinal. These data indirectly suggest a role for calcium and/or phospholipids in the regulation of meiotic maturation. They also suggest that forskolin and phorbol esters mediate their effects through different pathways.     

Downhill exercise alters immunoproteasome content in mouse skeletal muscle

Content of the immunoproteasome, the inducible form of the standard proteasome, increases in atrophic muscle suggesting it may be associated with skeletal muscle remodeling. However, it remains unknown if the immunoproteasome responds to stressful situations that do not promote large perturbations in skeletal muscle proteolysis. The purpose of this study was to determine how an acute bout of muscular stress influences immunoproteasome content. To accomplish this, wild-type (WT) and immunoproteasome knockout lmp7 ^?/? /mecl1 ^?/? (L7M1) mice were run downhill on a motorized treadmill. Soleus muscles were excised 1 and 3 days post-exercise and compared to unexercised muscle (control). Ex vivo physiology, histology and biochemical analyses were used to assess the effects of immunoproteasome knockout and unaccustomed exercise. Besides L7M1 muscle being LMP7/MECL1 deficient, no other major biochemical, histological or functional differences were observed between the control muscles. In both strains, the downhill run shifted the force-frequency curve to the right and reduced twitch force; however, it did not alter tetanic force or inflammatory markers. In the days post-exercise, several of the proteasome’s catalytic subunits were upregulated. Specifically, WT muscle increased LMP7 while L7M1 muscle instead increased β5. These findings indicate that running mice downhill results in subtle contractile characteristics that correspond to skeletal muscle injury, yet it does not appear to induce a significant inflammatory response. Interestingly, this minor stress activated the production of specific immunoproteasome subunits that if knocked out were replaced by components of the standard proteasome. These data suggest that the immunoproteasome may be involved in maintaining cellular homeostasis.     

Length-dependence of isometric twitch tension potentiation and myosin phosphorylation in mouse skeletal muscle

The effect of changes in muscle length on post-tetanic isometric twitch tension potentiation and myosin P-light chain phosphorylation-was studied at 23°C in the mouse extensor digitorum longus muscle. The length-tension relationship was determined for the same muscles after a 30 min period of quiescence and between 30 s and 3 min after a 1.5 s tetanus at L₀. Isometric twitch tension is increased at all muscle lengths after the tetanus; however, the fractional increase in twitch tension rises from 0.2 at L₀ to a maximum of 0.3 at 1.2 L_0. The fractional increase in twitch tension measured at any fixed muscle length is constant between 30 s and 3 min post-tetanus. P-light chain phosphorylation remains constant between 30 s and 3 min post-tetanus followed by a slow decline to basal values. Under fixed length conditions, there is linear relationship between the relative magnitude of the twitch tension and the extent of P-light chain phosphor-ylation. Net myosin phosphorylalion measured after a 1.5 s tetanus at 1.23 L₀ is 35% less than that obtained under the same conditions at L_0. Thus, contraction-induced phosphorylation of P-light chain decreases with increased muscle length and post-tetanic potentiation at a constant level of P-light chain phosphorylation increases with increasing muscle length. These observations may be consistent with alterations in the sarcoplasmic Ca²⁺ ion transient as the muscle is lengthened.     

Regulation of Ca2+ handling by phosphorylation status in mouse fast- and slow-twitch skeletal muscle fibers

The effects ofphosphorylation status on Ca²⁺release and Ca²⁺ removal werestudied in fast-twitch flexor digitorum brevis and slow-twitch soleusskeletal muscle fibers enzymatically isolated from wild-type andphospholamban knockout (PLBko) mice. In all fibers the adenosine3',5'-cyclic monophosphate-dependent protein kinase (PKA)inhibitor H-89 decreased the peak amplitude of the intracellularCa²⁺ concentration([Ca²⁺]) transient fora single action potential, and the PKA activator dibutyryl adenosine3',5'-cyclic monophosphate (DBcAMP) reversed this effect,indicating modulation of Ca²⁺release by phosphorylation status in all fibers. H-89 decreased thedecay rate constant of the[Ca²⁺] transient andDBcAMP reversed this effect only in phospholamban-expressing fibers(wild-type soleus), indicating modulation ofCa²⁺ removal only in the presenceof phospholamban. A high basal level of PKA phosphorylation in soleusfibers maintained under our control conditions was indicated bythe lack of effect of direct application of DBcAMP onCa²⁺ release orCa²⁺ removal in wild-type or PLBkosoleus fibers and was confirmed by analysis of phospholamban fromwild-type soleus fibers.

     

Proteolytic cleavage sites in smooth muscle myosin-light-chain kinase and their relation to structural and regulatory domains

Proteolysis of the smooth muscle myosin-light-chain kinase with either thermolysin or endoproteinase Lys-C cleaves the enzyme towards the amino-terminus between the first and second unc domains, unc-II-1 and unc-II-2, and in the calmodulin-binding domain. The thermolytic fragment extends 532 residues from Ser275 to Ala806 and is resistant to further digestion. It is catalytically inactive and does not bind calmodulin. Further proteolysis of the thermolytic fragment with trypsin generates a constitutively active fragment. Digestion with endoproteinase Lys-C initially results in an inactive fragment of 516 residues, Ala287 to Lys802. Further digestion with Lys-C endoproteinase results in a constitutively active 474-residue fragment with the same amino-terminus, but a carboxyl-terminus at Lys760, near Arg762, the last conserved residue of protein kinase catalytic domains. There is no cleavage in the acidic-residue-rich connecting peptide between the amino-terminus of the catalytic domain and the unc-I domain, nor within the unc-II or unc-I domains or between the adjacent unc-II-2 and unc-I domains. The pattern of cleavages by these proteases reflects well the predicted domain structure of the myosin-light-chain kinase and further delineates the regulatory pseudosubstrate region. A synthetic peptide corresponding to the pseudosubstrate sequence, MLCK(787-807) was a more potent inhibitor by three orders of magnitude than the overlapping peptide MLCK(777-793) proposed by Ikebe et al. (1989) [Ikebe, M., Maruta, S. & Reardon, S. (1989) J. Biol. Chem. 264, 6967-6971] to be important in autoregulation of the myosin-light-chain kinase.     

Impaired mitochondrial oxidative phosphorylation in skeletal muscle of the dystrophin-deficient mdx mouse

The mdx mouse, an animal model of the Duchenne muscular dystrophy, was used for the investigation of changes in mitochondrial function associated with dystrophin deficiency. Enzymatic analysis of skeletal muscle showed an approximately 50% decrease in the activity of all respiratory chain-linked enzymes in musculus quadriceps of adult mdx mice as compared with controls, while in cardiac muscle no difference was observed. The activities of cytosolic and mitochondrial matrix enzymes were not significantly different from the control values in both cardiac and skeletal muscles. In saponin-permeabilized skeletal muscle fibers of mdx mice the maximal rates of mitochondrial respiration were about two times lower than those of controls. These changes were also demonstrated on the level of isolated mitochondria. Mdx muscle mitochondria had only 60% of maximal respiration activities of control mice skeletal muscle mitochondria and contained only about 60% of hemoproteins of mitochondrial inner membrane. Similar findings were observed in a skeletal muscle biopsy of a Duchenne muscular dystrophy patient. These data strongly suggest that a specific decrease in the amount of all mitochondrial inner membrane enzymes, most probably as result of Ca²⁺ overload of muscle fibers, is the reason for the bioenergetic deficits in dystrophin-deficient skeletal muscle.     

Inhibition by interferon (alpha + beta) of mouse liver regeneration and its reversal by putrescine

Mouse interferon (alpha + beta) given to mice by intraperitoneal injection suppressed both the accumulation of putrescine and stimulation of DNA synthesis in liver caused by partial hepatectomy. The suppression of DNA synthesis was completely reversed by exogenous putrescine. The same results were obtained when core 2',5'-oligoadenylate instead of interferon was given to partially hepatectomized mice. These results suggest that interferon inhibits putrescine formation through elevating the 2',5'-oligoadenylate level and thus inhibits DNA synthesis in the regenerating liver.     

Pachytene exit controlled by reversal of Mek1-dependent phosphorylation

During yeast meiosis, a checkpoint prevents exit from pachytene in response to defects in meiotic recombination and chromosome synapsis. This pachytene checkpoint requires two meiotic chromosomal proteins, Red1 and Mek1; Mek1 is a kinase that phosphorylates Red1. In mutants that undergo checkpoint-mediated pachytene arrest, Mek1 is active and Red1 remains phosphorylated. Activation of Mek1 requires the initiation of meiotic recombination and certain DNA damage checkpoint proteins. Mek1 kinase activity and checkpoint-induced pachytene arrest are counteracted by protein phosphatase type 1 (Glc7). Glc7 coimmunoprecipitates with Red1, colocalizes with Red1 on chromosomes, and dephosphorylates Red1 in vitro. We speculate that phosphorylated Red1 prevents exit from pachytene and that completion of meiotic recombination triggers Glc7-dependent dephosphorylation of Red1.