A rapid method for preparation of sarcolemma from frog skeletal muscle

A rapid method for the preparation of sarcolema from frog skeletal muscle has been described. The purified cell segments were transparent and devoid of contractile material. The Na⁺, K⁺ -ATPase and 5'-nucleotidase activities in sarcolemma purified by this method were comparable to those reported for sarcolemmal preparations purified by density gradient centrifugation. The preparation also possessed acid phosphatase, alkaline phosphatase and K⁺ -activated, ouabain-sensitive p-nitrophenyl phosphatase activities. The cholesterol to phospholipid ratio of the sarcolemma was 0.33, indicating its high purity; further, the preparation was free from mitochondria and contractile proteins.     

Isolation of plasma membrane vesicles,derived from transverse tubules,by selective homogenization of subcellular fractions of frog skeletal muscle in isotonic media

A new technique for isolating fragmented plasma membranes from skeletal muscle has been developed that is based on gentle mechanical disruption of selected homogenate fractions. $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-stimulated}$, Mg²⁺-dependent ATPase was used as an enzymatic marker for the plasma membrane, Ca²⁺-stimulated, Mg²⁺-dependent ATPase as a marker for sarcoplasmic reticulum, and succinate dehydrogenase for mitochondria. Cell Cell segments in an amber low-speed ($\text{800 × g}$) pellet of a frog muscle homogenate were disrupted by repeated gentle shearing with a Polytron homogenizer. Sarcoplasmic reticulum was released into the low-speed supernatant, whereas most of the plasma membrane marker remained in a white, fluffy layer of the sediment, which contained sarcolemma and myofibrils. Additional gentle shearing of the white low-speed sediment extracted plasma membranes in a form that required centrifugation at $\text{100 000 × g}$ for pelleting. This pellet, the fragmented plasma membrane fraction, had a relatively high specific activity of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-stimulated}$ ATPase compared with the other fractions, but it had essentially no Ca²⁺-stimulated ATPase activity and only a small percentage of the succinate dehydrogenase activity of the homogenate.Experimental evidence suggests that the fragmented plasma membrane fraction is derived from delicate transverse tubules rather than from the thicker, basement membrane-coated sarcolemmal sheath of muscle cells. Electron microscopy showed small vesicles lined by a single thin membrane. Hydroxyproline, a characteristic constituent of collagen and basement membrane, could not be detected in this fraction.     

A novel cholesterol transfer protein in cardiac sarcolemma. Purification and initial characterization

Summary In contrast to several sterol carrier proteins isolated from soluble cytosolic fractions, a cholesterol transfer protein (CHTP) with an apparent molecular weight of 73,000 was isolated from a cardiac sarcolemmal fraction by detergent solubilization, column chromatography, and preparative electrophoresis using nondissociating polyacrylamide gels. This protein must be reconstituted into an artificial membrane in order to mediate cholesterol transfer activity. For the expression of its full activity, CHTP must also be present in the membrane in a multimeric form, since the monomer was shown not to be active. We believe this novel protein might represent an important molecule in the regulation of the homeostasis of cholesterol in cardiac sarcolemma.     

Quantitative analysis of regional variability in the distribution of transverse tubules in rabbit myocardium

Summary The goal of the present investigation was to compare quantitatively the distribution of T-tubules between regions of the myocardium. The volume fraction and surface density of T-tubules in rabbit right atrial free wall, left atrial free wall, right ventricular free wall, left ventricular free wall, right ventricular papillary muscle, and left ventricular papillary muscle were measured using established, electron-microscopic, morphometric techniques. T-tubules were delineated using wheat-germ agglutinin conjugated to horseradish peroxidase as a tracer. No significant differences were found in the morphometric parameters between any two ventricular samples or between atrial samples. Furthermore, little difference between T-tubule volume fraction or surface density was found between individual animals for any given site. Both volume fraction and surface density of ventricular T-tubules were more than ten-times their values in atrial tissue (volume fraction: 3.43%±0.35 vs. 0.20±0.09; surface density: 2.46 m²/m³±0.11 vs 0.10±0.04). Measurements show that there is greater variation of T-tubule volume fraction and surface density within atrial samples than within ventricular samples. This suggests greater inhomogeneity in T-tubule distribution in atrial myocardium than in ventricular myocardium. Morphometric data also indicate that the mean diameter of atrial T-tubules is greater than that of ventricular T-tubules while qualitative observations show that atrial T-tubules are distributed less regularly and have a larger longitudinal component to their organization than those in the ventricular myocardium.     

Isolation and characterization of a 37,000-dalton protein associated with the erythrocyte membrane

We have purified a 37,000-dalton polypeptide (p37) from the red cell membrane that was found in previous studies to undergo a lineage-specific alteration in its membrane association. Our data suggest that p37 associates with the red cell membrane through electrostatic interactions that are resistant to 0.5 M NaCl or 10 mM EDTA. Conditions found to elute p37 from red cell ghosts include H2O at pH 12, 0.1 N NaOH + 1 mM ethanol and 1.0% Triton X-100. p37 was purified substantially from ghosts by Triton X-100 solubilization followed by sequential DEAE-Sephadex and CM-Sephadex chromatography. When p37 was analyzed by two-dimensional gel electrophoresis, a family of isoelectric focusing variants was detected ranging in pI from 7.0 to 7.8. All of the isoelectric focusing variants showed homology to one another when compared serologically with anti-p37 antibodies or by limited peptide mapping using Staphylococcus aureus V8 protease. The isoelectric focusing variants appear to represent distinct, yet related polypeptides rather than degrees of post-translational modifications to a single species, inasmuch as all of the variants are present in anti-p37 immunoprecipitates prepared from in vitro translations programmed with p37 mRNA.     

Electron microscopy and 3D reconstruction of F-actin decorated with cardiac myosin-binding protein C (cMyBP-C)

Myosin-binding protein C (MyBP-C) is an ∼ 130-kDa rod-shaped protein of the thick (myosin containing) filaments of vertebrate striated muscle. It is composed of 10 or 11 globular 10-kDa domains from the immunoglobulin and fibronectin type III families and an additional MyBP-C-specific motif. The cardiac isoform cMyBP-C plays a key role in the phosphorylation-dependent enhancement of cardiac function that occurs upon β-adrenergic stimulation, and mutations in MyBP-C cause skeletal muscle and heart diseases. In addition to binding to myosin, MyBP-C can also bind to actin via its N-terminal end, potentially modulating contraction in a novel way via this thick-thin filament bridge. To understand the structural basis of actin binding, we have used negative stain electron microscopy and three-dimensional reconstruction to study the structure of F-actin decorated with bacterially expressed N-terminal cMyBP-C fragments. Clear decoration was obtained under a variety of salt conditions varying from 25 to 180 mM KCl concentration. Three-dimensional helical reconstructions, carried out at the 180-mM KCl level to minimize nonspecific binding, showed MyBP-C density over a broad portion of the periphery of subdomain 1 of actin and extending tangentially from its surface in the direction of actin's pointed end. Molecular fitting with an atomic structure of a MyBP-C Ig domain suggested that most of the N-terminal domains may be well ordered on actin. The location of binding was such that it could modulate tropomyosin position and would interfere with myosin head binding to actin.     

Selective loss of a plasma membrane protein associated with contraction of skeletal muscle

Muscle proteins were labeled by incubating isolated frog sartorius muscles with [³H]- or [¹⁴C]phenylalanine. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of plasma membrane fractions revealed a major protein band with an apparent molecular weight of approx. 96 000. Radioactivity in this band showed a clearly delineated decrease, relative to other bands, when previously labeled muscles were induced to contract either by electrical stimulation or by increasing the influx of Ca²⁺ from the incubation medium. It is postulated that a Ca²⁺-activated neutral protease may account for this decrease in labeled membrane protein.     

The role of GDF11 in aging and skeletal muscle,cardiac and bone homeostasis

GDF11 is a secreted factor in the TGFß family of cytokines. Its nearest neighbor evolutionarily is myostatin, a factor discovered as being a negative regulator of skeletal muscle growth. High profile studies several years ago suggested that GDF11 declines with age, and that restoration of systemic GDF11 to ‘youthful’ levels is beneficial for several age-related conditions. Particularly surprising was a report that supplementation of GDF11 aided skeletal muscle regeneration, as its homolog, myostatin, has the opposite role. Given this apparent contradiction in functionality, multiple independent labs sought to discern differences between the two factors and better elucidate age-related changes in circulating GDF11, with most failing to reproduce the initial finding of declining GDF11 levels, and, importantly, all subsequent studies examining the effects of GDF11 on skeletal muscle described an inhibitory effect on regeneration – and that higher doses induce skeletal muscle atrophy and cachexia. There have also been several studies examining the effect of GDF11 and/or the downstream ActRII pathway on cardiac function, along with several interesting reports on bone. A review of the GDF11 literature, as it relates in particular to aging and skeletal muscle, cardiac and bone biology, is presented.     

Inhibitors of protein synthesis,puromycin and emetine,inhibit prostaglandin E2 release by skeletal muscle

Addition of 1μM puromycin or 1 μM emetine to rat soleus muscle in vitro decreases muscle prostaglandin E₂ release by 51–77%. This inhibition appears to be caused by decreased availability of endogenous arachidonic acid for prostaglandin E₂ synthesis, because neither puromycin nor emetine inhibits muscle prostaglandin E₂ production from arachidonic acid added into the incubation medium.     

Role of nuclear Lamin A/C in cardiomyocyte functions

Lamin A/C is a structural protein of the nuclear envelope (NE) and cardiac involvement in Lamin A/C mutations was one of the first phenotypes to be reported in humans, suggesting a crucial role of this protein in the cardiomyocytes function. Mutations in LMNA gene cause a class of pathologies generically named ‘Lamanopathies’ mainly involving heart and skeletal muscles. Moreover, the well‐known disease called Hutchinson–Gilford Progeria Syndrome due to extensive mutations in LMNA gene, in addition to the systemic phenotype of premature aging, is characterised by the death of patients at around 13 typically for a heart attack or stroke, suggesting again the heart as the main site sensitive to Lamin A/C disfunction. Indeed, the identification of the roles of the Lamin A/C in cardiomyocytes function is a key area of exploration. One of the primary biological roles recently conferred to Lamin A/C is to affect contractile cells lineage determination and senescence. Then, in differentiated adult cardiomyocytes both the ‘structural’ and ‘gene expression hypothesis’ could explain the role of Lamin A in the function of cardiomyocytes. In fact, recent advances in the field propose that the structural weakness/stiffness of the NE, regulated by Lamin A/C amount in NE, can ‘consequently’ alter gene expression.     

Entrance of colloidal ThO2 tracer into the T tubules and longitudinal tubules of the guinea pig heart

Summary Colloidal ThO₂ particles (diameter of 60 Å) were used as electron-opaque markers to trace the intracellular compartments continuous with the bulk interstitial fluid of guinea pig ventricular muscle. Beating and quiescent hearts in a Langendorff preparation were perfused for 30 min with oxygenated Ringer solution containing 1% ThO₂. The hearts were immediately fixed by perfusing with glutaraldehyde solution. The colloidal ThO₂ particles entered into many of the T tubules and into longitudinallyrunning tubules. No differences in distribution of ThO₂ were observed in a heart which was not exposed to ThO₂ until after it was fixed. Tracer did not penetrate into the intercalated disk clefts in the guinea pig hearts and one frog heart used for comparison. Tubular profiles filled with ThO₂ were not seen in frog heart, an observation which confirms the absence of T tubules in this amphibian. It is concluded that, in mammalian cardiac muscle, the lumens of the longitudinal tubules are continuous with the lumens of the T tubules, forming an extensively interconnected T-L tubular system. Hence, every myofibril has close access to a fluid-filled space which is continuous with the interstitial fluid and which may be of similar cationic composition; such an arrangement should facilitate excitation-contraction coupling.Supported by grants from the American Heart Association and from the Public Health Service (HE-11155, HE-05815 and HE-10384). The authors wish to acknowledge the expert technical assistance of Mrs. Jan Redick and to thank Dr. James Smith of Marquette University for the supply of Thorotrast used in these studies.     

Aquaporin-4 water channel oligomers are associated with the transverse tubules of skeletal myofibers

Transverse (T) tubules comprise a tortuous network inside the skeletal myofibers enclosing a distinct osmotic environment. Here we have examined whether the T tubules contain aquaporin type 4 (AQP4) water channels to mediate rapid transmembrane water flow. Separation of T tubular and sarcolemmal membranes by sucrose density gradient centrifugation revealed that two main isoforms of AQP4, namely M23 and M1, were present in both membrane fractions. Compatible with this, expression of fluorescent Venus-AQP4.M23 in rat muscle showed the protein both in the T tubules and at the sarcolemma. Blue-Native polyacrylamide gel electrophoresis showed that higher order oligomers typical to the AQP4 water channel were present in both membrane compartments. Interestingly, α-syntrophin that mediates binding of AQP4 to the sarcolemmal dystrophin glycoprotein complex was also present in the T tubule fraction. Deletion of the syntrophin-binding sequence of AQP4 increased its mobile fraction at the sarcolemma but not in the T tubules. Taken together, our results strongly suggest that both the sarcolemma and the T tubules harbor higher order oligomers of the AQP4 water channel but the interactions with adjacent macromolecules are different.     

Both oxidative and nitrosative stress are associated with muscle wasting in tumour-bearing rats

Reactive oxygen and nitrogen species (ROS and RNS) have been proposed as mechanisms of cancer-induced cachexia. In this study, we assessed using Western blot analysis the levels of total protein carbonylation (2,4-dinitrophenylhydrazine assay), both malondialdehyde- (MDA-) and 2-hydroxy-4-nonenal- (HNE-) protein adducts, Mn-superoxide dismutase (Mn-SOD), catalase, heme oxygenase-1 (HO-1) and 3-nitrotyrosine formation in gastrocnemius muscles of rats bearing the Yoshida AH-130 hepatoma. In the muscles of the tumour-bearing animals, protein carbonylation as measured by total levels of carbonyl group formation and both HNE and MDA-protein adducts, and protein tyrosine nitration were significantly greater than in control muscles. Protein levels of the antioxidant enzymes Mn-SOD, catalase, and HO-1 were not significantly modified in the rat cachectic muscles compared to controls. The inefficiency of the antioxidant enzymes in neutralizing excessive ROS production may account for elevated markers of protein oxidation and be responsible for the development of both oxidative and nitrosative stress in cancer-induced cachexia.     

Purification and cell-surface marker characterization of quiescent satellite cells from murine skeletal muscle by a novel monoclonal antibody

A novel monoclonal antibody, SM/C-2.6, specific for mouse muscle satellite cells was established. SM/C-2.6 detects mononucleated cells beneath the basal lamina of skeletal muscle, and the cells co-express M-cadherin. Single fiber analyses revealed that M-cadherin+ mononucleated cells attaching to muscle fibers are stained with SM/C-2.6. SM/C-2.6+ cells, which were freshly purified by FACS from mouse skeletal muscle, became MyoD+ in vitro in proliferating medium, and the cells differentiated into desmin+ and nuclear-MyoD+ myofibers in vitro when placed under differentiation conditions. When the sorted cells were injected into mdx mouse muscles, donor cells differentiated into muscle fibers. Flow cytometric analyses of SM/C-2.6+ cells showed that the quiescent satellite cells were c-kit-, Sca-1-, CD34+, and CD45-. More, SM/C-2.6+ cells were barely included in the side population but in the main population of cells in Hoechst dye efflux assay. These results suggest that SM/C-2.6 identifies and enriches quiescent satellite cells from adult mouse muscle, and that the antibody will be useful as a powerful tool for the characterization of cellular and molecular mechanisms of satellite cell activation and proliferation.     

Proteomic analysis of fast and slow muscles from normal and kyphoscoliotic mice using protein arrays, 2-DE and MS

A proteomic strategy based upon the integrated use of SELDI-TOF/MS, 2-DE and MALDI-TOF/MS has been used to identify a panel of fast muscle protein markers: MLC1F, MLC3F, fast troponin C (STNC) and slow muscle markers: MLC1SB and MLC2v. MLC3F, MLC1F and STNC were virtually absent in the physiologically pure slow soleus muscle of kyphoscoliotic mutant mice compared to control BDmice, whereas MLC2v increased threefold. A SELDI-TOF/MS peak at 18,012 Da in spectra from strong anionic exchange protein array fractions of fast vastus muscle was confirmed as STNC by its specific depletion from crude extracts of vastus muscle using an anti-TNC mAb. SELDI-TOF/MS also identified MLC2F phosphorylation in crude muscle extracts after treatment with alkaline phosphatase. High probability protein identifications were achieved by SELDI-TOF/MS PMF based upon the resolution of large peptides formed by partial cleavage and high peptide coverage. When the pI from 2-D gels and molecular weight estimations from SELDI-TOF/MS were entered into the TagIdent algorithm, high probability protein identity predictions were obtained that were confirmed later by PMF. We confirm that SELDI-TOF/MS can be integrated with other proteomics techniques for the efficient analysis of protein expression changes and PTMs associated with physiological changes in skeletal muscle.     

The effects of selenium deficiency on differentiation, degradation, dand cell lysis of L8 rat skeletal muscle cells

We investigated the effects of selenium (Se) deficiency on differentiation, protein degradation, and cell lysis in cultured skeletal muscle cells, using L8 rat skeletal muscle cells cultured in serum-free (SF) medium to induce differentiation and to maintain myotubes. Creatine kinase activity was reduced (p < 0.05) by approximately 15% without Se supplementation for 96 h. Confluent myoblasts were treated with SF media with four different levels of vitamin E (0,10, 35, and 100 μM) in the absence and presence of Se (0 and 0.25 μM, respectively). After 96 h, vitamin E at a high dose (100 μM) was effective in the prevention of the decrease of differentiation caused by Se deficiency (p < 0.05). Following differentiation, the effects of three Se concentrations (0, 0.25, and 2.5 μM) on degradation of proteins as assessed by release of³H-labeled free amino acids secreted into the media were studied. Selenium supplementation did not affect (p > 0.05) total protein degradation. However, Se deficiency increased (p < 0.05) lactate dehydrogenase released from lyzed dead cells. The results indicate that Se is required to maintain an optimal rate of muscle cell differentiation and health of myotube cultures.     

Expression and localization of myotonic dystrophy protein kinase in human skeletal muscle cells determined with a novel antibody: possible role of the protein in cytoskeleton rearrangements during differentiation

Myotonic dystrophy is a multisystemic disorder, due to a CTG triplet expansion at the 3'UTR of the DM1 gene encoding for myotonic dystrophy protein kinase. Recent studies indicate that decreased DMPK levels could account for part of the symptoms suggesting a role of this protein in skeletal muscle differentiation. To investigate this aspect, polyclonal antibodies were raised against two peptides of the catalytic domain and against the human full-length DMPK (DMFL). In western blots, anti-hDMFL antibody was able to detect low amounts of purified human recombinant protein and recognized the splicing isoforms in heart and stomach of overexpressing mice. In human muscle extracts, this antibody specifically recognized a protein of apparent molecular weight of 85 kDa and it specifically stained neuromuscular junctions in skeletal muscle sections. In contrast, both anti-peptide antibodies demonstrated low specificity for either denatured or native DMPK, suggesting that these two epitopes are probably cryptic sites. Using anti-hDMFL, the expression and localization of DMPK was studied in human skeletal muscle cells (SkMC). Western blot analysis indicated that the antibody recognizes a main protein of apparent MW of 75 kDa, which appears to be expressed during differentiation into myotubes. Immunolocalization showed low levels of DMPK in the cytoplasm of undifferentiated cells; during differentiation the staining became more intense and was localized to the terminal part of the cells, suggesting that DMPK might have a role in cell elongation and fusion.     

The sulfhydryl groups of the 35,000-dalton C-terminal segment of band 3 are located in a 9000-dalton fragment produced by chymotrypsin treatment of red cell ghosts

Five sulfhydryl groups of band 3, the anion-transport protein of the red blood cell membrane, can be labeled byN-ethylmaleimide (NEM). Two of these are located in a 35,000-dalton, C-terminal segment produced by chymotrypsin treatment of cells. Extensive treatment of unsealed ghosts with chymotrypsin results in the disappearance of the 35,000-dalton segment, but its two NEM-binding sites are preserved in a 9000-dalton peptide. The latter must therefore be a proteolytic product of the larger segment. Labeling of sulfhydryl groups of band 3 by an impermeant analog of NEM occurs in inside-out, but not in right-side-out vesicles derived from red cell ghosts, supporting the conclusion that NEM-reactive sulfhydryl groups, including those in the 35,000- and 9000-dalton segments, are exposed at the cytoplasmic face of the membrane. These findings support the conclusion that the 35,000-dalton segment crosses the bilayer, and suggest that the 9000-dalton segment may be a membrane-crossing portion of the 35,000-dalton segment.     

Functional characterisation of the R2452W ryanodine receptor variant associated with malignant hyperthermia susceptibility

Malignant hyperthermia (MH) is a pharmacogenetic disorder that manifests in susceptible individuals exposed to volatile anaesthetics. Over 400 variants in the ryanodine receptor 1 (RYR1) have been reported but relatively few have been definitively associated with susceptibility to MH. This is largely due to the technical challenges of demonstrating abnormal Ca²⁺ release from the sarcoplasmic reticulum. This study focuses on the R2452W variant and its functional characterisation with the aim of classifying this variant as MH causative. HEK293 cells were transiently transfected with full-length human wildtype or R2452W mutant RYR1 cDNA. In addition, B-lymphoblastoid cells from blood and myoblasts propagated from in vitro contracture tests were extracted from patients positive for the R2452W variant. All cell lines generated were loaded with the ratiometric dye Fura-2 AM, stimulated with the RYR1-specific agonist 4-chloro-m-cresol and Ca²⁺ release from the sarcoplasmic/endoplasmic reticulum was monitored by fluorescence emission. All cells expressing the RYR1 R2452W variant show a significantly higher Ca²⁺ release in response to the agonist, 4-chloro-m-cresol, compared to cells expressing RYR1 WT. These results indicate that the R2452W variant results in a hypersensitive ryanodine receptor 1 and suggest that the R2452W variant in the ryanodine receptor 1 is likely to be causative of MH.