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At least 19 recordsLinked to original sources

PubMed2001

Na,K-ATPase in skeletal muscle: two populations of beta-spectrin control localization in the sarcolemma but not partitioning between the sarcolemma and the transverse tubules.

We used immunological approaches to study the factors controlling the distribution of the Na,K-ATPase in fast twitch skeletal muscle of the rat. Both alpha subunits of the Na,K-ATPase colocalize with beta-spectrin and ankyrin 3 in costameres, structures at the sarcolemma that lie over Z and M-lines and in longitudinal strands. In immunoprecipitates, the alpha1 and alpha2 subunits of the Na,K-ATPase as well as ankyrin 3 associate with beta-spectrin/alpha- fodrin heteromers and with a pool of beta-spectrin at the sarcolemma that does not contain alpha-fodrin. Myofibers of mutant mice lacking beta-spectrin (ja/ja) have a more uniform distribution of both the alpha1 and alpha2 subunits of the Na,K-ATPase in the sarcolemma, supporting the idea that the rectilinear sarcomeric pattern assumed by the Na,K-ATPase in wild-type muscle requires beta-spectrin. The Na,K-ATPase and beta-spectrin are distributed normally in muscle fibers of the nb/nb mouse, which lacks ankyrin 1, suggesting that this isoform of ankyrin is not necessary to link the Na,K-ATPase to the spectrin-based membrane skeleton. In immunofluorescence and subcellular fractionation experiments, the alpha2 but not the alpha1 subunit of the Na,K-ATPase is present in transverse (t-) tubules. The alpha1 subunit of the pump is not detected in increased amounts in the t-tubules of muscle from the ja/ja mouse, however. Our results suggest that the spectrin-based membrane skeleton, including ankyrin 3, concentrates both isoforms of the Na,K-ATPase in costameres, but that it does not play a significant role in restricting the entry of the alpha1 subunit into the t-tubules.

Animals↗

PubMed1985

alpha-Adrenergic stimulation of trans-sarcolemma electron efflux in perfused rat heart. Possible regulation of Ca2+-channels by a sarcolemma redox system.

The role of trans-sarcolemma membrane electron efflux in the alpha-adrenergic control of Ca2+ influx in perfused rat heart was examined. Electron efflux was measured by monitoring the rate of reduction of extracellular ferricyanide and compared with changes in contractility, as an indirect assessment of changes in cytoplasmic Ca2+ concentration. Methoxamine and phenylephrine each increased the rate of ferricyanide reduction from 80 to approx. 114 nmol/min per g wet wt. of heart, with half-maximal activation occurring at 10 microM for each agonist. Activation of the rate of ferricyanide reduction by both 10 microM methoxamine and 10 microM phenylephrine was blocked by the alpha-adrenergic antagonist, phenoxybenzamine, but not by the beta-antagonist, propranolol. Stimulation of the rate of ferricyanide reduction by the alpha-agonist coincided with the increase in contractility, each reaching maximum values at approx. 80 s. Removal of the alpha-agonists led to parallel decreases in contractility and the rate of reduction, each returning to pre-stimulation values in approx. 400 s. In addition, the relationship between Ca2+ and ferricyanide reduction was examined. Perfusion of the heart with medium containing 6 mM CaCl2 significantly increased contractility and the rate of ferricyanide reduction. Perfusion of the heart with low Ca2+ diminished contractility, did not affect the rate of ferricyanide reduction, but amplified the stimulatory effect of methoxamine on this rate. The increase in ferricyanide reduction by alpha-adrenergic agonists resulted from a change in the apparent Vmax, indicative of an increase in electron efflux sites in the plasma membrane. It is concluded that alpha-adrenergic control of electron efflux closely parallels changes in contractility and therefore changes in the cytoplasmic concentration of Ca2+. The data suggest that alpha-agonist-mediated changes in electron efflux may lead to Ca2+ influx.

Adrenergic alpha-Agonists↗

PubMed1979

Temperature effects on 1,8-anilinonaphthalene sulfonic acid fluorescence with sarcolemma vesicles.

Increased temperature produces a red shift and decreased fluorescence intensity of the emission peak of 1,8-anilinonaphthalene sulfonic acid (ANS) in suspensions of biomembrane vesicles. These changes have been attributed to a conjectured increase in polarity of the microenvironment of ANS. If the conjecture is correct, fluorescence lifetimes must be decreased with warming. We showed than ANS binds to both protein and lipid protein of sarcolemma, that there are two kinds of sarcolemma-lipid ANS-binding sites, and that there are three fluorescence lifetimes of excited sarcolemma-bound ANS. The three fluroescence lifetimes were unchanged on warming, or decreased too little to account for the observations. Fluorescence lifetime data were consistent with the notion that the effect of increasing temperature is to decrease the amount of ANS bound to sarcolemma. From studies of liposomes prepared from lipid extracts of sarcolemma, and of proteins from sarcolemma it was deduced that warming reducted the amount of ANS bound to both of these sarcolemma components, probably mainly by reducing binding capacity. There might also be a shift of affinities such that the ratio, KA sarcolemma lipid/KA sarcolemma protein, is larger at higher temperature. Except at very small concentration ratios of ANS/sarcolemma, more than twice as much ANS was bound to sarcolemma lipids as to proteins.

Anilino Naphthalenesulfonates↗

PubMed1988

Phospholipid composition and amphiphile content of isolated sarcolemma from normal and autolytic rat myocardium.

Sarcolemmal vesicles were purified to a similar extent, 50- to 60-fold on a protein basis, from normal rat hearts and hearts subjected to 30 or 60 min of autolysis at 37 degrees C (total ischemia in vitro). Electron microscopic examination of the autolytic hearts revealed sarcolemmal discontinuities and other morphological characteristics typical of irreversible cell injury. Total contents and percentage composition of phospholipid classes did not differ between normal and autolytic hearts or between sarcolemmal preparations from these hearts. There was no increase in lysophospholipid contents of whole hearts or of purified sarcolemma after autolysis. Long chain acyl-CoAs or acylcarnitines did not accumulate in autolytic hearts under our experimental conditions. The molar long chain acyl-CoA: phospholipid ratio in isolated sarcolemma was extremely low (1:100,000). It increased 3-fold after autolysis but the increase was most probably the result of an increase in mitochondrial contamination of the sarcolemmal preparations from autolytic hearts. The molar long chain acylcarnitine: phospholipid ratio of isolated sarcolemma was much larger (1:100), but it did not change after autolysis. Experiments, in which radioactive amphiphiles were incorporated in isolated sarcolemma that was subsequently repeatedly washed, indicated that the lysophospholipid and acylcarnitine contents of isolated sarcolemma reflect the contents of sarcolemma in situ, but that sarcolemmal acyl-CoA is used for re-acylation reactions during purification, explaining the low acyl-CoA content of isolated sarcolemma. Na/K-ATPase and Na/Ca-exchange activities were markedly depressed in isolated sarcolemma from autolytic hearts. Our results suggest that sarcolemmal phospholipid breakdown and sarcolemmal amphiphile accumulation are not responsible for the structural and functional defects of the sarcolemma after autolysis.

Acetylcarnitine↗

PubMed1991

Dystrophin-glycoprotein complex is highly enriched in isolated skeletal muscle sarcolemma.

mAbs specific for protein components of the surface membrane of rabbit skeletal muscle have been used as markers in the isolation and characterization of skeletal muscle sarcolemma membranes. Highly purified sarcolemma membranes from rabbit skeletal muscle were isolated from a crude surface membrane preparation by wheat germ agglutination. Immunoblot analysis of subcellular fractions from skeletal muscle revealed that dystrophin and its associated glycoproteins of 156 and 50 kD are greatly enriched in purified sarcolemma vesicles. The purified sarcolemma was also enriched in novel sarcolemma markers (SL45, SL/TS230) and Na+/K(+)-ATPase, whereas t-tubule markers (alpha 1 and alpha 2 subunits of dihydropyridine receptor, TS28) and sarcoplasmic reticulum markers (Ca2(+)-ATPase, ryanodine receptor) were greatly diminished in this preparation. Analysis of isolated sarcolemma by SDS-PAGE and densitometric scanning demonstrated that dystrophin made up 2% of the total protein in the rabbit sarcolemma preparation. Therefore, our results demonstrate that although dystrophin is a minor muscle protein it is a major constituent of the sarcolemma membrane in skeletal muscle. Thus the absence of dystrophin in Duchenne muscular dystrophy may result in a major disruption of the cytoskeletal network underlying the sarcolemma in dystrophic muscle.

Animals↗

PubMedDate not supplied

[ATPase systems of the skeletal muscle sarcolemma in E-avitaminotic muscular dystrophy].

The sarcolemma preparations isolated from the skeletal muscles of the normal rabbits and those with E-avitaminosis are characterized by electron microscopy according to the activity of 5-nucleotidase and different ATPase systems. As the data of electron microscopy evidence, the sarcolemma preparations of the normal and dystrophic muscles are deprived of admixtures of other subsellular structures. The 5-nucleotidase activity in the sarcolemma of dystrophic muscles is almost thrice as high as in the sarcolemma of normal muscles. On the basis of the results of studies in the kinetic parameters the optimal conditions are selected to investigate the Mg2+, Ca2+ and Na+, K+-ATPase activity. It is shown that under dystrophy the ATPase activity in the sarcolemma preparations in the presence of Ca2+ and Mg2+ does not change as compared to the norm. As to the Mg2+-dependent Na+, K+-ATPase system its activity in sarcolemma of muscles under dystrophy lowers noticeably. Ouabain-sensitive Na+, K+-ATPase of sarcolemma under dystrophy is considerably lower than at the normal level and is 4.1 and 12.1 mumol and phi n per 1 mg of protein for 1 h, respectively. Proceeding from the data on the changes in the lipid composition of sarcolemma of muscles with E-avitaminous dystrophy a problem is under discussion concerning dependence of the transport Na+, K+-ATPase activity on the structure of sarcolemma.

Adenosine Triphosphatases↗

PubMed1984

The effect of lipid intermediates on Ca2+ and Na+ permeability and (Na+ + K+)-ATPase of cardiac sarcolemma. A possible role in myocardial ischemia.

The effect of fatty acid and acylcarnitine on Ca2+ and Na+ transporting enzymes and carriers was studied in sealed cardiac sarcolemma vesicles of mixed polarity. Palmitoylcarnitine markedly reduced the Na+ gradient-induced Ca2+ uptake. Half-maximal reduction was obtained at 15 microM of the carnitine derivative. In a same concentration range palmitoylcarnitine caused a rapid release of accumulated Ca2+ when added to Ca2+-filled vesicles, which suggests that palmitoylcarnitine increases the permeability of the sarcolemma vesicles to Ca2+. A rapid release of Ca2+ was also observed if Ca2+ was taken up by action of the Ca2+ pump. The (Ca2+ + Mg2+)-ATPase, which most likely drives this active Ca2+ uptake, was 90% increased by 50 microM palmitoylcarnitine and evidence was presented that the acylcarnitine effect again was linked to an alteration of Ca2+ permeability of the vesicles. At the same concentration acylcarnitine was not able to unmask the latent protein kinase, so that probably the sarcolemma ATP permeability was not affected. Palmitoylcarnitine at 25 microM did not affect the ouabain-sensitive (Na+ + K+) -ATPase in native sarcolemma vesicles, however, it inhibited markedly if the enzyme was measured in SDS-treated vesicles. The effect of increased free fatty acid concentration on some of the sarcolemma transporting properties was tested by adding oleate-albumin complexes with different molar ratios to the sarcolemma vesicles. In contrast to molar ratios 1 and 5, the ratio of 7 was able to induce a rapid Ca2+ release and to inhibit (Na+ + K+)-ATPase in either native or SDS-treated vesicles markedly. 22Na release from 22Na-preloaded sarcolemma vesicles was shown to be stimulated by either palmitoylcarnitine (50 microM) or oleate-albumin complex (with a molar ratio of 7). The possible significance of the observed effects of lipid intermediates on ion permeability and (Na+ + K+)-ATPase activity in isolated sarcolemma vesicles for the derangement of cardiac cell function in ischemia is discussed.

Animals↗

PubMed1986

Characterization of the membrane-bound protein kinase C and its substrate proteins in canine cardiac sarcolemma.

Cardiac sarcolemma was purified from canine ventricles. Enrichment of the sarcolemmal membranes was demonstrated by the high (Na+ + K+)-ATPase activity of 28.0 +/- 1.5 mumol Pi/mg protein per h and the high concentration of muscarinic receptors with the Bmax of 8.2 +/- 2.5 pmol/mg protein as determined by [3H]QNB binding. The purified sarcolemma also contains significant levels of a membrane-bound Ca2+ and phospholipid-dependent protein kinase (protein kinase C). To elucidate the protein kinase C activity in sarcolemma, a prior incubation of the membranes with EGTA and Triton X-100 was necessary. The specific activity of protein kinase C was found to be 131.4 pmol Pi/mg per min, in the presence of 6.25 micrograms phosphatidylserine and 0.5 mM CaCl2. Treatment of sarcolemma with 12-O-tetradecanoylphorbol 13-acetate (TPA) and phorbol 12,13-dibutyrate (PBu2) resulted in a concentration-dependent activation of protein kinase C activity. The effect of TPA and PBu2 on protein kinase C in sarcolemma was independent of exogenous Ca2+ and phosphatidylserine. Polymyxin B inhibited phorbol-ester-induced activation of protein kinase C activity. The distribution of protein kinase C in the cytosolic fraction was also examined. The specific activity of the kinase in the cytosolic fraction was 59.7 pmol Pi/mg per min. However, the total protein kinase C activity in the cytosol was 213500 pmol Pi/min, compared to that of 1025 pmol Pi/min in the sarcolemma isolated from approx. 100 g of canine ventricular muscle. Several endogenous proteins in cardiac sarcolemma were phosphorylated in the presence of Ca2+ and phosphatidylserine. The major substrates for protein kinase C were proteins of Mr 94 000, 87 000, 78 000, 51 000, 46 000, 11 500 and 10 000. Most of these substrate proteins have not been identified before. Other proteins of Mr 38 000, 31 000 and 15 000 were markedly phosphorylated in the presence of Ca2+ only. Phosphorylation of phospholamban (Mr 27 000 and 11 000) was also stimulated in the presence of Ca2+ and phosphatidylserine, but the low Mr form of phospholamban was distinct from two other low Mr substrate proteins for protein kinase C. Polymyxin B was more selective in inhibiting the protein kinase C dependent phosphorylation. On the other hand, trifluoperazine selectively inhibited the phosphorylation of phospholamban and Mr 15 000 protein. Although the exact function of this kinase is unknown, based on these observations, we believe that protein kinase C in the cardiac sarcolemma may play an important role in the cell-surface-signal regulated cardiac function.

Animals↗

PubMed2000

Skeletal muscle fibres show NADPH diaphorase activity associated with mitochondria, the sarcoplasmic reticulum and the NOS-1-containing sarcolemma.

The subcellular appearance of NADPH diaphorase activity in different rat skeletal muscles has been analyzed. Both a sarcolemma-associated as well as a non-sarcolemma-associated NADPH diaphorase-dependent generation of formazan was observed. The sarcolemma-associated NADPH diaphorase staining appeared regularly in two manifestations: one observed in longitudinal sections as dotted costameres at the cell surface which accordingly appeared in transversal sections as rings surrounding the myofibre surface. At this site, nitric oxide synthase (NOS)-1 was located. The second sarcolemma-associated site of NADPH diaphorase staining was found as bundles of longitudinal-orientated stripes of hitherto unidentified origin. The non-sarcolemma-associated production of formazan was likewise manifested at two sites: the first was found regularly in longitudinal sections as intense sarcomere-like striations occurring parallel to the I-bands and indicating mitochondria. The second non-sarcolemma-associated NADPH diaphorase staining was realized as fine longitudinal filaments of variable occurrence connecting the mitochondria and presumably belonging to the sarcoplasmic reticulum. Attempts to identify single NADPH diaphorase(s) existing in skeletal muscles by incubation with specific inhibitors failed but showed the presence of two different subpopulations of NADPH diaphorases in myofibres: a urea-resistant fraction in the sarcolemma region containing NOS-1 and a non-sarcolemma-associated, urea-sensitive fraction depleted of NOS-1.

Animals↗

PubMed1979

Characteristics of saxitoxin binding to the sodium channel of sarcolemma isolated from rat skeletal muscle.

1. The characteristics of saxitoxin (STX) binding to the mammalian Na channel have been studied in purified sarcolemma isolated from rat skeletal muscle. 2. STX binds specifically to isolated sarcolemma with a Kd of 1.43 x 10(-9) M and Bmax of 7-8 p-mole STX bound/mg membrane protein at 0 degrees C in the presence of 140 mM-NaCl. In rat muscle homogenate under the same conditions the corresponding values are Kd = 1.53 x 10(-9) M and Bmax = 0.15-0.20 p-mole/mg protein (18-20 p-mole/g wet wt.). Membrane purification produced a fortyfold increase in STX binding site concentration per milligram protein. Calculated binding site density in isolated sarcolemma was about 30 sites/micron 2 of membrane surface. 3. Denervation (10-14 days) results in a 43% reduction in the density of high-affinity STX binding sites in purified sarcolemma, but the Kd for this class of sites is not changed. 4. In sarcolemma, the apparent Kd for STX binding is dependent on temperature pH and ionic strength. The Q10 for Kd between 0 and 40 degrees C is 1.3. Protonation of a group having a pK of 6.0 markedly raises Kd without affecting Bmax. Apparent Kd increases eightfold when ionic strength is raised from 20 to 600 mM. 5. Dissociation and association rate constants for STX binding are temperature dependent with Q10 of 2.6 and 1.9 respectively between 0 and 20 degrees C. 6. STX binding is competitively inhibited by monovalent and divalent cations under conditions of constant total ionic strength. An affinity sequence of Tl+ greater than Li+ greater than Na+ greater than K+ greater than Rb+ greater than Cs+ is seen for the monovalent cation-binding site. 7. The STX binding site is relatively stable to heat and to enzymic degradation. A specific modifier of carboxyl residues inactivates subsequent STX binding. This process can be prevented by the presence of STX during the reaction. 8. Characteristics of the STX binding site in isolated sarcolemma are compared to those reported for other isolated excitable membranes and for studies of whole muscle and muscle homogenate. Sarcolemma provides a potential source of enriched Na channels for further purification efforts in a mammalian system.

Animals↗

PubMed1987

The role of calcium at the sarcolemma in the control of myocardial contractility.

The importance of sarcolemmal-bound calcium (Ca) in the control of contraction in mammalian myocardium is indicated by the following results. The curve that relates [Ca]o (from 50 microM to 10 mM) to force development and that which relates [Ca]o to Ca bound to a highly purified sarcolemmal fraction are superimposable. The ability of a series of cations to uncouple excitation from contraction is the same as their relative ability to displace Ca from the sarcolemma. Dimethonium, which specifically displaces cation from the diffuse double layer of the cellular surface, has little effect on contractile force. This indicates that the Ca actually bound to the sarcolemma is the surface Ca important in contractile control. Polymyxin B, a highly charged cationic amphiphilic peptidolipid, specifically competes for Ca-binding sites on anionic and zwitterionic phospholipid. It is a potent displacer of Ca from myocardial cells and purified sarcolemma and a potent uncoupler. Phospholipase D cleaves the nitrogenous base from sarcolemmal phospholipid with production of anionic phosphatidic acid. Phospholipase D treatment increases Ca bound to cells and purified sarcolemma and increases force development of ventricular tissue from both neonatal rat and adult rabbit. Insertion of charged amphiphiles in the sarcolemma as phospholipid analogues modulate interaction of Ca with the sarcolemma, e.g., anionic dodecylsulfate increases Ca bound to sarcolemmal vesicles by more than 80% and increases force development in rabbit papillary muscle by 100%. The effect of pH variation on Ca binding to phospholipid extracted from sarcolemma indicates that phospholipid accounts for at least 75% of the binding. The current model proposes a two-site control of Ca binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

[Lipid levels in rabbit sarcolemma normally and during experimental muscular dystrophy].

The cholesterin content, total content and phsopholipid composition were studied in sarcolemma of normal rabbits and those with experimental muscular dystrophy. The total content of phospholipids in the sarcolemma of young normal rabbits is 4.8 mg of phosphorus per 1 mg of protein which is higher than that in the sarcolemma of adult rabbits 33%. With experimental muscular dystrophy the content of cholesterin is 2.7 as high as its content in the sarcolemma of the normal rabbits. It is established that the content of phosphatidilserine in the sarcolemma of young normal rabbits is by 61% higher than that in adult animals. The content of phosphatidilserine of lysophosphatidilcholine decreases by 40 and 39%, respectively, with dystrophy. The content of phosphatidilcholine increases by 16% as compared with sarcolemma extracted from the muscles of the young normal rabbits. Molar ratio of phospholipid/cholesterine in the sarcolemma of normal rabbits is 1.8 and decreases with the experimental muscular dystrophy to 0.75.

Age Factors↗

PubMed1978

Fluorescence of 1,8-anilinonaphthalene sulfonic acid bound to proteins and to lipids of sarcolemma.

Relative contributions to fluorescnece of 1,8-anilinonaphthalene sulfonic acid-sarcolemma by 1,8-anilinonaphthalene sulfonic acid bound to proteins and to lipid were assessed on the basis of fluorescence lifetime and steady-state emission spectra. The lifetime spectrum of lipid-poor proteins prepared from sarcolemma had a major time constant of 16 ns; that of lipids extracted from sarcolemma had a major time constant of 7 ns and a minor one of 4 ns. In sarcolemma, components having all three time constants were observed with weighting factors 0.33, 0.6 and 0.07, respectively, indicating the fraction of 1,8-anilinonaphthalene sulfonic acid bound to protein and to lipid. Steady-state emission spectra of 1,8-anilinonaphthalene sulfonic acid-sarcolemma were resolved into and resynthesized from contributions by 1,8-anilinonaphthalene sulfonic acid bound to proteins and to lipids. The latter was at least two thirds of total 1,8-anilinonaphthalene sulfonic acid bound. Results of analyses of spectra obtained with various concentrations of sarcolemma and 1,8-anilinonaphthalene sulfonic acid suggest that the proteins have a higher affinity, but only about half the binding capacity for 1,8-anilinonaphthalene sulfonic acid as do lipids.

Anilino Naphthalenesulfonates↗

PubMed1987

Skeletal muscle sarcolemma in malignant hyperthermia: evidence for a defect in calcium regulation.

Sarcolemmal properties implicated in the skeletal muscle disorder, malignant hyperthermia (MH), were examined using sarcolemma-membrane vesicles isolated from normal and MH-susceptible (MHS) porcine skeletal muscle. MHS and normal sarcolemma did not differ in the distribution of the major proteins, cholesterol or phospholipid content, vesicle size and sidedness, (Na+ + K+)-ATPase activity, ouabain binding, or adenylate cyclase activity (total and isoproterenol sensitivity). The regulation of the initial rates of MHS and normal sarcolemmal ATP-dependent calcium transport (calcium uptake after 1 min) by Ca2+ (K1/2 = 0.64-0.81 microM), calmodulin, and cAMP-dependent protein kinase were similar. However, when sarcolemmal calcium content was measured at either 2 or 20 min after the initiation of active calcium transport, a significant difference between MHS and normal sarcolemmal calcium uptake became apparent, with MHS sarcolemma accumulating approximately 25% less calcium than normal sarcolemma. Calcium transport by MHS and normal sarcolemma, at 2 or 20 min, had a similar calmodulin dependence (C1/2 = 150 nM), and was stimulated to a similar extent by cAMP-dependent protein kinase or calmodulin. Halothane inhibited MHS and normal sarcolemmal active calcium uptake in a similar fashion (half-maximal inhibition at 10 mM halothane), while dantrolene (30 microM) and nitrendipine (1 microM) had little effect on either MHS or normal sarcolemmal calcium transport. After 20 min of ATP-supported calcium uptake, 2 mM EGTA plus 10 microM sodium orthovanadate were added to initiate sarcolemmal calcium efflux. Following an initial rapid phase of calcium release, an extended slow phase of calcium efflux (k = 0.012 min-1) was similar for both MHS and normal sarcolemma vesicles. We conclude that although a number of sarcolemmal properties, including passive calcium permeability, are normal in MH, a small but significant defect in MHS sarcolemmal ATP-dependent calcium transport may contribute to the abnormal calcium homeostasis and altered contractile properties of MHS skeletal muscle.

Adenosine Triphosphate↗

PubMed1990

Glycoproteins of rat skeletal muscle sarcolemma: characterization by two-dimensional gel electrophoresis and effect of denervation.

Sarcolemmal membrane glycoproteins from rat mixed, fast, and slow muscles were characterized by concanavalin A (ConA) binding following two-dimensional polyacrylamide gel electrophoresis (PAGE). Analysis of electrophoretic profiles revealed that although sarcolemmal membranes prepared from these muscle types contained common glycoprotein species, each had a distinct glycoprotein pattern. In sarcolemma from mixed muscle, three major classes of ConA binding glycoproteins could be distinguished: (i) an acidic species of 110,000-120,000 Da, pI 5.0 to 5.3 (CG-1, ConA binding glycoproteins-1); (ii) a group of highly charged isomers, ranging from 75,000 to 80,000 Da pI 5.2 to 8.2 (CG-2); and (iii) a group of charged isomers of predominantly acidic nature of approximately 50,000 Da pI 5.2 to 5.8 (CG-3). ConA bound exclusively to CG-1 in sarcolemma from a fast muscle (extensor digitorum longus muscle, EDL). In soleus muscle sarcolemma (slow fiber type) both CG-1 and CG-3 were readily detected but CG-2 was markedly diminished. ConA binding to slow muscle sarcolemma revealed as well a glycoprotein species of 66,000-70,000 Da, pI 4.3-5.1 (CG-4), which was unique to this fiber type and as such may be a specific marker for slow fiber type. Denervation had no significant effect on the properties of ConA binding to mixed or slow muscle sarcolemma but dramatically altered the ConA binding to fast muscle sarcolemma, specifically increasing binding to CG-2. These findings demonstrate that denervation differentially affects the metabolism of ConA binding glycoproteins in these muscle types.

Animals↗

PubMed1987

Relationship between reestablishment of sarcolemma-glycocalyx ultrastructures and restoration of transmembrane potentials in cultured rat heart cells.

Simultaneous studies on sarcolemma-glycocalyx ultrastructures and electrophysiological properties of the trypsin-released rat heart cells were carried out in order to define the relationship between sarcolemmal membrane repair and transmembrane potential recovery in the long-term cultured heart cells. Based on electron microscopic observations of the trypsinized heart cells maintained in the long-term culture, serial alterations of sarcolemma-glycocalyx complex could be divided into three successive stages. A defective stage of the sarcolemma-glycocalyx complex was present in the cultured cells between day 3 and day 6 of incubation. A repaired stage of the sarcolemma-glycocalyx complex was observed in the cells from day 7 to day 9 of incubation. A well-organized stage of the sarcolemma-glycocalyx complex was seen in the cells after ten days of incubation. Sequential measurements of electrophysiological parameters of the cultured heart cells showed a diphasic evolution of maximum diastolic potential and action potential amplitude, with an initial decrease from day 3 to day 9 of incubation and a later return to normal range after ten days of incubation. There seemed a good correlation between electrophysiological properties and sarcolemma-glycocalyx ultrastructures. Thus, we conclude that restoration of the electrophysiological properties of the cultured cells is closely related to the reorganization of the defective sarcolemmal membrane.

Action Potentials↗

PubMed1999

Differential distribution of dystrophin and beta-spectrin at the sarcolemma of fast twitch skeletal muscle fibers.

We used double label immunofluorescence and confocal microscopy to examine the organization of beta-spectrin and dystrophin at the sarcolemma of fast twitch myofibers in the Extensor Digitorum Longus (EDL) of the rat. Both beta-spectrin and dystrophin are concentrated in costameres, a rectilinear sarcolemmal array composed of longitudinal strands and transverse elements overlying Z and M lines. In contrast, intercostameric regions, lying between these linear structures, contain significant levels of dystrophin but little detectable beta-spectrin. The dystrophin-associated proteins, syntrophin and beta-dystroglycan, are also concentrated at costameres but, like dystrophin, are present in intercostameric regions as well. Dystrophin is present at costameres and intercostameric regions in fast twitch muscles of the mouse but is absent from all regions of the sarcolemma in the mdx mouse, which lacks dystrophin. Areas of the sarcolemma near myonuclei also contain dystrophin without beta-spectrin, consistent with the idea that the distribution of dystrophin at the sarcolemma is not dependent on beta-spectrin. We conclude that dystrophin is present under all areas of the sarcolemma. The increased fragility of the sarcolemma in patients with Duchennes muscular dystrophy may be explained in part by the absence of dystrophin not only from costameres, but also from intercostameric regions.

Animals↗

PubMed2006

Computational evidence for protein-mediated fatty acid transport across the sarcolemma.

Long-chain fatty acids (FAs) are important substrates used by the heart to fulfil its energy requirements. Prior to mitochondrial oxidation, blood-borne FAs must pass through the cell membrane of the cardiac myocyte (sarcolemma). The mechanism underlying the sarcolemmal transport of FAs is incompletely understood. The aim of the present study was to estimate the trans-sarcolemmal FA uptake rate using a comprehensive computer model, in which the most relevant mechanisms proposed for cardiac FA uptake were incorporated. Our in silico findings show that diffusion of FA, present in its unbound form (uFA) in close proximity to the outer leaflet of the sarcolemma and serving as sole FA source, is insufficient to account for the physiological FA uptake rate. The inclusion of a hypothetical membrane-associated FA-TFPC (FA-transport-facilitating protein complex) in the model calculations substantially increased the FA uptake rate across the sarcolemma. The model requires that the biological properties of the FA-TFPC allow for increasing the rate of absorption of FA into the outer leaflet and the 'flip-flop' rate of FA from the outer to the inner leaflet of the sarcolemma. Experimental studies have identified various sarcolemma-associated proteins promoting cardiac FA uptake. It remains to be established whether these proteins possess the properties predicted by our model. Our findings also indicate that albumin receptors located on the outer leaflet of the sarcolemma facilitate the transfer of FA across the membrane to a significant extent. The outcomes of the computer simulations were verified with physiologically relevant FA uptake rates as assessed in the intact, beating heart in experimental studies.

Biological Transport, Active↗