Endurance training-induced changes in alkali light chain patterns in type IIB fibers of the rat.

The effects of endurance training on the expression of myosin were electrophoretically analyzed in the deep portion of vastus lateralis muscle from the rat. A 10-wk running program led to increases (P < 0.01) in myosin heavy chain (MHC) 2a and 2d with a decrease (P < 0.01) in MHC(2b). Training also evoked a rearrangement of the isomyosin pattern with decreases in fast isomyosin (FM) 1 (P < 0.01) and FM2 (P < 0.05) and a rise in intermediate isomyosin (P < 0.01). These changes were accompanied by a 61% decrease (P < 0.01) in myosin light chain (MLC) 3F (11.8 +/- 2.7 vs. 4.6 +/- 4.2%). Two-dimensional electrophoresis made it possible to separate the triplet of isomyosins (FMb) consisting of MHC(2b). Training elicited a 26% decrease (P < 0.05) in the FM1b fraction within FMb, i.e., FM1b/(FM1b + FM2b + FM3b) (24.2 +/- 5.5 vs. 18.0 +/- 4.3%). These changes resulted in a 10% decrease (P < 0.05) in the MLC(3F) fraction, i.e., MLC(3F)/(MLC(1F) + MLC(3F)), in FMb (44.9 +/- 4.5 vs. 40.3 +/- 3.2%). These results suggest that endurance training may exert the depressive effect on the contractile velocity of type IIB fibers and that a training-induced decrease in the contractile velocity of whole muscle may be caused by alterations in fast alkali MLC complements within a given fiber type as well as by transitions in MHC-based fiber populations.     

Oxidative stress of myosin contributes to skeletal muscle dysfunction in rats with chronic heart failure

Intrinsic muscle abnormalities affecting skeletal muscle are often reported during chronic heart failure (CHF). Because myosin is the molecular motor of force generation, we sought to determine whether its dysfunction contributes to skeletal muscle weakness in CHF and, if so, to identify the underlying causative factors. Severe CHF was induced in rats by aortic stenosis. In diaphragm and soleus muscles, we investigated in vitro mechanical performance, myosin-based actin filament motility, myosin heavy (MHC) and light (MLC) chain isoform compositions, MLC integrity, caspase-3 activation, and oxidative damage. Diaphragm and soleus muscles from CHF exhibited depressed mechanical performance. Myosin sliding velocities were 16 and 20% slower in CHF than in sham in diaphragm (1.9 +/- 0.1 vs. 1.6 +/- 0.1 microm/s) and soleus (0.6 +/- 0.1 vs. 0.5 +/- 0.1 microm/s), respectively (each P < 0.05). The ratio of slow-to-fast myosin isoform did not differ between sham and CHF. Immunoblots with anti-MLC antibodies did not detect the presence of protein fragments, and no activation of caspase-3 was evidenced. Immunolabeling revealed oxidative damage in CHF muscles, and MHC was the main oxidized protein. Lipid peroxidation and expression of oxidized MHC were significantly higher in CHF than in shams. In vitro myosin exposure to increasing ONOO(-) concentrations was associated with an increasing amount of oxidized MHC and a reduced myosin velocity. These data provide experimental evidence that intrinsic myosin dysfunction occurs in CHF and may be related to oxidative damage to myosin.     

Paradoxical effects of endurance training and chronic hypoxia on myofibrillar ATPase activity

This study aimed to determine the changes in soleus myofibrillar ATPase (m-ATPase) activity and myosin heavy chain (MHC) isoform expression after endurance training and/or chronic hypoxic exposure. Dark Agouti rats were randomly divided into four groups: control, normoxic sedentary (N; n = 14), normoxic endurance trained (NT; n = 14), hypoxic sedentary (H; n = 10), and hypoxic endurance trained (HT; n = 14). Rats lived and trained in normoxia at 760 mmHg (N and NT) or hypobaric hypoxia at 550 mmHg (approximately 2,800 m) (H and HT). m-ATPase activity was measured by rapid flow quench technique; myosin subunits were analyzed with mono- and two-dimensional gel electrophoresis. Endurance training significantly increased m-ATPase (P < 0.01), although an increase in MHC-I content occurred (P < 0.01). In spite of slow-to-fast transitions in MHC isoform distribution in chronic hypoxia (P < 0.05) no increase in m-ATPase was observed. The rate constants of m-ATPase were 0.0350 +/- 0.0023 s(-1) and 0.047 +/- 0.0050 s(-1) for N and NT and 0.033 +/- 0.0021 s(-1) and 0.038 +/- 0.0032 s(-1) for H and HT. Thus, dissociation between variations in m-ATPase and changes in MHC isoform expression was observed. Changes in fraction of active myosin heads, in myosin light chain isoform (MLC) distribution or in MLC phosphorylation, could not explain the variations in m-ATPase. Myosin posttranslational modifications or changes in other myofibrillar proteins may therefore be responsible for the observed variations in m-ATPase activity.     

Regulation of myosin heavy-chain gene expression during skeletal-muscle hypertrophy.

Changes in the myosin phenotype of differentiated muscle are a prominent feature of the adaptation of the tissue to a variety of physiological stimuli. In the present study the molecular basis of changes in the proportion of myosin isoenzymes in rat skeletal muscle which occur during compensatory hypertrophy caused by the combined removal of synergist muscles and spontaneous running exercise was investigated. The relative amounts of sarcomeric myosin heavy (MHC)- and light (MLC)-chain mRNAs in the plantaris (fast) and soleus (slow) muscles from rats was assessed with cDNA probes specific for different MHC and MLC genes. Changes in the proportion of specific MHC mRNA levels were in the same direction as, and of similar magnitude to, changes in the proportion of myosin isoenzymes encoded for by the mRNAs. No significant changes in the proportion of MLC proteins or mRNA were detected. However, high levels of MLC3 mRNA were measured in both normal and hypertrophied soleus muscles which contained only trace amounts of MLC3 protein. Small amounts of embryonic and neonatal MHC mRNAs were induced in both muscles during hypertrophy. We conclude that the change in the pattern of myosin isoenzymes during skeletal-muscle adaptation to work overload is a consequence of changes in specific MHC mRNA levels.     

Myosin subunits and contractile properties of single fibers from hypokinetic rat muscles

The effects of prolonged hypokinesia on the contractile properties and myosin isozymes of single fibers from the synergistic fast-twitch plantaris (PL) and slow-twitch soleus (SOL) skeletal muscles of adult rats were studied after 28 days of hindlimb suspension. There was a 31% increase in the mean maximal velocity of unloaded shortening (Vmax) among fibers from SOL with no change in the mean Vmax of fibers from PL after suspension. The myosin heavy and light chain (MHC and MLC) composition of bundles and the MHC composition of single fibers from control and suspended muscles were examined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was a marked increase in the relative amount of fast-type MHC's in hypokinetic SOL and a smaller increase in the amount of fast-type MHC's in the PL. Relatively minor changes occurred in the MLC's during hypokinesia. As Vmax increased among individual fibers from control and suspended muscles, the relative amount of fast-type MHC's increased. The results demonstrate that the myosin isozyme composition of skeletal muscle, especially the heavy chains, is altered during hypokinesia, and this finding provides an explanation for changes in Vmax of rat single muscle fibers under the same conditions.     

Evidence for distinct phosphorylatable myosin light chains in avian heart and slow skeletal muscle

In mammalian organisms the regulatory or phosphorylatable myosin light chains in heart and slow skeletal muscle have been shown to be identical and presumable constitute the product of a single gene. We analyzed the expression of the avian cardiac myosin light chain (MLC) 2-A in heart and slow skeletal muscle by a combination of experimental approaches, e.g., two-dimensional gel electrophoresis of the protein and hybridization of mRNA to specific MLC 2-A sequences cloned from chicken. The investigations have indicated that, unlike in mammals, in avian organisms the phosphorylatable myosin light chains from heart and slow skeletal muscle are distinct proteins and therefore products of different genes. The expression of MLC 2-A is restricted to the myocardium and no evidence was found that it is shared with slow skeletal muscle.     

Transition of myosin isozymes during development of human masseter muscle. Persistence of developmental isoforms during postnatal stage

Previous results have shown that the adult human masseter muscle contains myosin isoforms that are specific to early stages of development in trunk and limb muscles, i.e. embryonic and fetal (neonatal) myosin heavy chains (MHC) and embryonic myosin light chain (MLC1emb). We wanted to know if this specific pattern is the result of a late maturation or of a distinct evolution during development. We show here that the embryonic and the fetal MHC and the MLC1emb are expressed throughout perinatal and postnatal masseter development. Our results also demonstrate that MLC1emb accumulation increases considerably during the postnatal period. In addition, both the slow MLCs and the slow isoform of tropomyosin are expressed later in the masseter than quadriceps and the fast skeletal muscle isoform MLC3 is not detected during fetal and early postnatal development in the masseter whereas it is expressed throughout fetal development in the quadriceps. Our results thus confirm previous histochemical data and demonstrate that the masseter muscle displays a pattern of myosin and tropomyosin isoform transitions different to that previously described in trunk and limb muscles. This suggests that control of masseter muscle development involves mechanisms distinct from other body muscles, possibly as a result of either its craniofacial innervation or of a possibly different embryonic origin.     

Progressive resistance training reduces myosin heavy chain coexpression in single muscle fibers from older men.

The purpose of this study was to examine myosin heavy chain (MHC) and myosin light chain (MLC) isoforms following 12 wk of progressive resistance training (PRT). A needle biopsy was taken from the vastus lateralis to determine fiber-type expression [ATPase (pH 4.54) and MHC/MLC] in seven healthy men (age = 74.0 +/- 1.8 yr). Subjects were also tested for 1-repetition maximum (1-RM), pre- and posttraining. The progressive knee extensor protocol consisted of three sets at 80% of 1-RM 3 days/wk for 12 wk. Freeze-dried, single muscle fibers were dissected for MHC and MLC analysis and then subjected to SDS-PAGE and silver staining, pre- and posttraining. MHC expression increased in the I (10.4%; P < 0.05) and decreased in I/IIa (9.0%; P < 0.05), I/IIa/x (0.9%; P < 0.05), and IIa/x (8.9%; P < 0.05) isoforms, with no change in the IIa and IIx isoforms, pre- vs. posttraining (total fibers = 3,059). The MLC(3f)-to-MLC(2) ratio did not change with the PRT in either the MHC I or MHC IIa isoforms (total fibers = 902), pre- to posttraining. ATPase fiber distribution did not significantly differ following training (I: 50. 4 +/- 6.7 vs. 51.9 +/- 7.9, IIa: 36.8 +/- 5.3 vs. 41.1 +/- 7.0, IIb: 12.8 +/- 5.6 vs. 7.0 +/- 4.0%; pre- vs. posttraining, respectively). 1-RM increased (51.9%; P < 0.05) from pre- to posttraining. The PRT provide a stimulus for alterations in MHC isoforms, which demonstrated a decrease in all hybrid isoforms and an increase in MHC I expression (not found in the ATPase results), unlike the MLC ratio (3:2), which was not altered with training.     

Smooth muscle myosin light chain kinase: rapid purification by anion exchange high-performance liquid chromatography

A rapid procedure for the purification of myosin light chain kinase present in chicken gizzard smooth muscle using anion exchange high-performance liquid chromatography is described. The procedure allows preparation of microgram amounts of the protein directly from the extract of gizzard myofibrils and then is suitable for the study of myosin light chain kinase in small muscles. The protein was judged to be greater than 95% pure by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme retains its activity since it catalyzes the calcium-calmodulin-dependent phosphorylation of the 20,000-Da myosin light chain.     

Purification of native myosin filaments from muscle

Analysis of the structure and function of native thick (myosin-containing) filaments of muscle has been hampered in the past by the difficulty of obtaining a pure preparation. We have developed a simple method for purifying native myosin filaments from muscle filament suspensions. The method involves severing thin (actin-containing) filaments into short segments using a Ca(2+)-insensitive fragment of gelsolin, followed by differential centrifugation to purify the thick filaments. By gel electrophoresis, the purified thick filaments show myosin heavy and light chains together with nonmyosin thick filament components. Contamination with actin is below 3.5%. Electron microscopy demonstrates intact thick filaments, with helical cross-bridge order preserved, and essentially complete removal of thin filaments. The method has been developed for striated muscles but can also be used in a modified form to remove contaminating thin filaments from native smooth muscle myofibrils. Such preparations should be useful for thick filament structural and biochemical studies.     

Gene duplication and conversion events shaped three homologous, differentially expressed myosin regulatory light chain (MLC2) genes

Myosin II is a hexameric protein complex consisting of two myosin heavy chains, two myosin essential light chains and two myosin regulatory light chains. Multiple subunit isoforms exist, allowing great diversity in myosin II composition which likely impacts on its contractile properties. Little is known about the evolutionary origin, expression pattern and function of myosin regulatory light chain (MLC2) isoforms. We analysed the evolutionary relationship between smooth muscle (sm), nonmuscle (nm) and nonmuscle-like (nml) MLC2 genes, which encode three homologous proteins expressed in nonmuscle cells. The three genes arose by successive gene duplication events. The high sequence similarity between the tandemly arranged nm- and nml-MLC2 genes is best explained by gene conversion. Urea/glycerol-polyacrylamide gel electrophoresis and RNA analysis were employed to monitor expression of sm-, nm- and nml-MLC2 in human and mouse cell lines. Conspicuous differences between transformed and non-transformed cells were observed, with sm-MLC2 being suppressed in Ras-transformed cells. Our findings shed light on the evolutionary history of three homologous MLC2 proteins and point to isoform-specific cell growth-related roles in nonmuscle cell myosin II contractility.     

Induction of endogenous myosin light chain 1 and cardiac alpha-actin expression in L6E9 cells by MyoD1.

Rat L6E9 muscle cells commit to terminal differentiation by forming a large muscle syncitia complete with the expression of a large number of muscle-specific contractile protein genes. To determine whether these cells, which fail to synthesize MLC (myosin light chain) 1 and cardiac alpha-actin, exhibit a deficiency in the expression of muscle determination genes, we measured expression of MyoD1, myogenin, Myf-5, and MRF-4. Results show these cells do not synthesize MyoD1, yet express the other myogenic determination genes. Transient expression of exogenous MyoD1 in these cells is sufficient to activate endogenous MLC 1 and cardiac alpha-actin mRNA synthesis during muscle differentiation. Previously undetected myosin heavy chain (MHC) isoforms (beta-MHC and perinatal MHC) are also transcribed at low levels in L6E9 muscle cells, and in MyoD1-transfected L6E9 cells no change occurs in their expression. Furthermore, treatment with the demethylating agent 5-azacytidine activates expression of the endogenous MyoD1 gene in L6E9 cells and, subsequently, rescues deficiencies in their myogenic biochemical program. These results demonstrate that the endogenous MyoD1 gene in L6E9 cells is not defective and can be functionally activated. Also, the MyoD1 protein plays an essential role, which cannot be compensated by other known muscle determination proteins, in the induction of MLC 1 and cardiac alpha-actin expression.     

Comparative studies on the structure and aggregative properties of the myosin molecule. I. The structure of the lobster myosin molecule.

Myosin purified from the abdominal flexor muscle of the lobster, Homarus americanus, has a number average length of 1559 +/- 218 A, a rod like tail 1335 A long and a globular head 225 X 45 A as determined from electron microscopic observations on platinum shadowed preparations. The mass of the molecule was determined to be ca. 486,000 daltons from high speed equilibrium centrifugation studies at neutral and alkaline pH, and by SDS-acrylamide gel electrophoresis. Both sedimentation equilibrium centrifuge studies at alkaline pH and SDS-acrylamide gel electrophoresis experiments, indicate that the molecule contains a heavy chain core (two polypeptide chains weighing ca. 210,000 daltons each) and ca. four light chains of two weight classes (ca. 16,000 and 20,000 daltons). The amino acid composition of the myosin was determined. The specific activities of the Mg2+ -activated, K+/EDTA-activated, and Ca2+ -activated ATPases of the myosin were determined. Kinetic analysis of the digestion of lobster myosin with trypsin suggests that lobster myosin contains three classes of lysine and arginine residues; slowly split (k = 2.07 +/- 0.31 X 10(-2) moles/min2), rapidly split (k = 11.0 +/- 1.83 X 10(-2) moles/min2) and trypsin insensitive. There are 187 +/- 22 slowly split residues, 280 +/- 35 rapidly split residues, and 144 +/- 41 trypsin insensitive bonds per molecule. Comparison of these molecular parameters with those for the vertebrate skeletal muscle myosin indicates that the two myosins are similar in terms of mass, shape and overall polypeptide chain composition but may be considerably different in terms of local polypeptide chain conformation or composition.     

Regulation of Limulus skeletal muscle contraction

Skeletal muscle contraction of Limulus polyphemus, the horseshoe crab, seemed to be regulated in a dual manner, namely Ca2+ binding to the troponin complex as well phosphorylation of the myosin light chains (MLC) by a Ca2+/calmodulin-dependent myosin light chain kinase. We investigated muscle contraction in Limulus skinned fibers in the presence of Ca2+ and of Ca2+/calmodulin to find out which of the two mechanisms prevails in Limulus skeletal muscle contraction. Although skinned fibers revealed high basal MLC mono- and biphosphorylation levels (0.48 mol phosphate/mol 31 kDa MLC; 0.52 mol phosphate/mol 21 kDa MLC), the muscle fibers were fully relaxed at pCa 8. Upon C2+ or Ca2+/calmodulin activation, the fibers developed force (357+/-78.7 mN/mm2; 338+/-69.7 mN/mm2, respectively) while the MLC phosphorylation remained essentially unchanged. We conclude that Ca2+ activation is the dominant regulatory mechanism in Limulus skeletal muscle contraction.     

Changes in myosin ATPase activity in skeletal muscles of rat during cold stress

Three skeletal muscles viz., gastrocnemius, pectoralis and diaphragm from rats acclimated to a low temperature (4 +/- 1 degrees C; 16 hr daily; maximum for 8 weeks) exhibit an increased myosin ATPase activity. An analysis of native myosin from these muscles under non-dissociating conditions reveals two myosin isozymes instead of a single isozyme expressed in control muscles. Isoelectric focusing (IEF) coupled with two dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE) confirms an increased phosphorylation of myosin light chain 2 (MLC2) in muscles from cold acclimated rats.     

Production of glycerinated models of muscle fibers with phosphorylated myosin light chains

A simple method for obtaining glycerinated muscle fibres of m. psoas of rabbit containing regulatory myosin light chains (LC2) of different levels of phosphorylation. The glycerination conditions stimulated endogenic kinase LC2 or phosphatase LC2. Glycerinated muscle fibres contained phosphorylated and dephosphorylated (levels of phosphorylation are 95 +/- 5%, and 5 +/- 5%, respectively) LC2 of myosin. To determine the level of phosphorylation the method of polyacrylamide gel electrophoresis in 8 M urea was modified.     

Human skeletal muscle fiber type specific protein content

The aim of this project was to develop a method to assess fiber type specific protein content across the continuum of human skeletal muscle fibers. Individual vastus lateralis muscle fibers (n = 264) were clipped into two portions: one for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) fiber typing and one for Western blot protein identification. Following fiber type determination, fiber segments were combined into fiber type specific pools (～20 fibers/pool) and measured for total protein quantity, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), citrate synthase (CS), and total p38 content. GAPDH content was 64, 54, 160, and 138% more abundant in myosin heavy chain (MHC) I/IIa, MHC IIa, MHC IIa/IIx, and MHC IIx fibers, respectively, when compared with MHC I. Inversely, CS content was 528, 472, 242, and 47% more abundant in MHC I, MHC I/IIa, MHC IIa, and MHC IIa/IIx fibers, respectively, when compared with MHC IIx. Total p38 content was 87% greater in MHC IIa versus MHC I fibers. These data and this approach establish a reliable method for human skeletal muscle fiber type specific protein analysis. Initial results show that particular proteins exist in a hierarchal fashion throughout the continuum of human skeletal muscle fiber types, further highlighting the necessity of fiber type specific analysis.     

Regenerated rat fast muscle transplanted to the slow muscle bed and innervated by the slow nerve,exhibits an identical myosin heavy chain repertoire to that of the slow muscle

 The hypothesis that the limited adaptive range observed in fast rat muscles in regard to expression of the slow myosin is due to intrinsic properties of their myogenic stem cells was tested by examining myosin heavy chain (MHC) expression in regenerated rat extensor digitorum longus (EDL) and soleus (SOL) muscles. The muscles were injured by bupivacaine, transplanted to the SOL muscle bed and innervated by the SOL nerve. Three months later, muscle fibre types were determined. MHC expression in muscle fibres was demonstrated immunohistochemically and analysed by SDS-glycerol gel electrophoresis. Regenerated EDL transplants became very similar to the control SOL muscles and indistinguishable from the SOL transplants. Slow type 1 fibres predominated and the slow MHC-1 isoform was present in more than 90% of all muscle fibres. It contributed more than 80% of total MHC content in the EDL transplants. About 7% of fibres exhibited MHC-2a and about 7% of fibres coexpressed MHC-1 and MHC-2a. MHC-2x/d contributed about 5–10% of the whole MHCs in regenerated EDL and SOL transplants. The restricted adaptive range of adult rat EDL muscle in regard to the synthesis of MHC-1 is not rooted in muscle progenitor cells; it is probably due to an irreversible maturation-related change switching off the gene for the slow MHC isoform. Accepted: 11 June 1996