Sarcolemmal Invaginations Research Articles

Centronuclear Myopathy Caused by Defective Membrane Remodelling of Dynamin 2 and BIN1 Variants.

Centronuclear myopathy (CNM) is a congenital myopathy characterised by centralised nuclei in skeletal myofibers. T-tubules, sarcolemmal invaginations required for excitation-contraction coupling, are disorganised in the skeletal muscles of CNM patients. Previous studies showed that various endocytic proteins are involved in T-tubule biogenesis and their dysfunction is tightly associated with CNM pathogenesis. DNM2 and BIN1 are two causative genes for CNM that encode essential membrane remodelling proteins in endocytosis, dynamin 2 and BIN1, respectively. In this review, we overview the functions of dynamin 2 and BIN1 in T-tubule biogenesis and discuss how their dysfunction in membrane remodelling leads to CNM pathogenesis.

Mutant BIN1-Dynamin 2 complexes dysregulate membrane remodeling in the pathogenesis of centronuclear myopathy

Membrane remodeling is required for dynamic cellular processes such as cell division, polarization, and motility. BAR domain proteins and dynamins are key molecules in membrane remodeling that work together for membrane deformation and fission. In striated muscles, sarcolemmal invaginations termed T-tubules are required for excitation–contraction coupling. BIN1 and DNM2, which encode a BAR domain protein BIN1 and dynamin 2, respectively, have been reported to be causative genes of centronuclear myopathy (CNM), a hereditary degenerative disease of skeletal muscle, and deformation of T-tubules is often observed in the CNM patients. However, it remains unclear how BIN1 and dynamin 2 are implicated in T-tubule biogenesis and how mutations in these molecules cause CNM to develop. Here, using an in cellulo reconstitution assay, we demonstrate that dynamin 2 is required for stabilization of membranous structures equivalent to T-tubules. GTPase activity of wild-type dynamin 2 is suppressed through interaction with BIN1, whereas that of the disease-associated mutant dynamin 2 remains active due to lack of the BIN1-mediated regulation, thus causing aberrant membrane remodeling. Finally, we show that in cellulo aberrant membrane remodeling by mutant dynamin 2 variants is correlated with their enhanced membrane fission activities, and the results can explain severity of the symptoms in patients. Thus, this study provides molecular insights into dysregulated membrane remodeling triggering the pathogenesis of DNM2-related CNM.

Quantitative assessment of passive electrical properties of the cardiac T-tubular system by FRAP microscopy

Well-coordinated activation of all cardiomyocytes must occur on every heartbeat. At the cell level, a complex network of sarcolemmal invaginations, called the transverse-axial tubular system (TATS), propagates membrane potential changes to the cell core, ensuring synchronous and uniform excitation-contraction coupling. Although myocardial conduction of excitation has been widely described, the electrical properties of the TATS remain mostly unknown. Here, we exploit the formal analogy between diffusion and electrical conductivity to link the latter with the diffusional properties of TATS. Fluorescence recovery after photobleaching (FRAP) microscopy is used to probe the diffusion properties of TATS in isolated rat cardiomyocytes: A fluorescent dextran inside TATS lumen is photobleached, and signal recovery by diffusion of unbleached dextran from the extracellular space is monitored. We designed a mathematical model to correlate the time constant of fluorescence recovery with the apparent diffusion coefficient of the fluorescent molecules. Then, apparent diffusion is linked to electrical conductivity and used to evaluate the efficiency of the passive spread of membrane depolarization along TATS. The method is first validated in cells where most TATS elements are acutely detached by osmotic shock and then applied to probe TATS electrical conductivity in failing heart cells. We find that acute and pathological tubular remodeling significantly affect TATS electrical conductivity. This may explain the occurrence of defects in action potential propagation at the level of single T-tubules, recently observed in diseased cardiomyocytes.

Exendin-4 ameliorates cardiac ischemia/reperfusion injury via caveolae and caveolins-3.

BackgroundExendin-4, an exogenous glucagon-like peptide-1 receptor (GLP-1R) agonist, protects the heart from ischemia/reperfusion injury. However, the mechanisms for this protection are poorly understood. Caveolae, sarcolemmal invaginations, and caveolins, scaffolding proteins in caveolae, localize molecules involved in cardiac protection. We tested the hypothesis that caveolae and caveolins are essential for exendin-4 induced cardiac protection using in vitro and in vivo studies in control and caveolin-3 (Cav-3) knockout mice (Cav-3 KO).MethodsMyocytes were treated with exendin-4 and then incubated with methyl-β-cyclodextrin (MβCD) to disrupt caveolae formation. This was then followed by simulated ischemia/reperfusion (SI/R). In addition, cardiac protection in vivo was assessed by measuring infarct size and cardiac troponin levels.ResultsExendin-4 protected cardiac myocytes (CM) from SI/R [35.6 ± 12.6% vs. 64.4 ± 18.0% cell death, P = 0.034] and apoptosis but this protection was abolished by MβCD (71.8 ± 10.8% cell death, P = 0.004). Furthermore, Cav-3/GLP-1R co-localization was observed and membrane fractionation by sucrose density gradient centrifugation of CM treated with MβCD + exendin-4 revealed that buoyant (caveolae enriched) fractions decreased Cav-3 compared to CM treated with exendin-4 exclusively. Furthermore, exendin-4 induced a reduction in infarct size and cardiac troponin relative to control (infarct size: 25.1 ± 8.2% vs. 41.4 ± 4.1%, P < 0.001; troponin: 36.9 ± 14.2 vs. 101.1 ± 22.3 ng/ml, P < 0.001). However, exendin-4 induced cardiac protection was abolished in Cav-3 KO mice (infarct size: 43.0 ± 6.4%, P < 0.001; troponin: 96.8 ± 26.6 ng/ml, P = 0.001).ConclusionsWe conclude that caveolae and caveolin-3 are critical for exendin-4 induced protection of the heart from ischemia/reperfusion injury.

Atomic force and electron microscopic-based study of sarcolemmal surface of living cardiomyocytes unveils unexpected mitochondrial shift in heart failure

Loss of T-tubules (TT), sarcolemmal invaginations of cardiomyocytes (CMs), was recently identified as a general heart failure (HF) hallmark. However, whether TT per se or the overall sarcolemma is altered during HF process is still unknown. In this study, we directly examined sarcolemmal surface topography and physical properties using Atomic Force Microscopy (AFM) in living CMs from healthy and failing mice hearts. We confirmed the presence of highly organized crests and hollows along myofilaments in isolated healthy CMs. Sarcolemma topography was tightly correlated with elasticity, with crests stiffer than hollows and related to the presence of few packed subsarcolemmal mitochondria (SSM) as evidenced by electron microscopy. Three days after myocardial infarction (MI), CMs already exhibit an overall sarcolemma disorganization with general loss of crests topography thus becoming smooth and correlating with a decreased elasticity while interfibrillar mitochondria (IFM), myofilaments alignment and TT network were unaltered. End-stage post-ischemic condition (15days post-MI) exacerbates overall sarcolemma disorganization with, in addition to general loss of crest/hollow periodicity, a significant increase of cell surface stiffness. Strikingly, electron microscopy revealed the total depletion of SSM while some IFM heaps could be visualized beneath the membrane. Accordingly, mitochondrial Ca2+ studies showed a heterogeneous pattern between SSM and IFM in healthy CMs which disappeared in HF. In vitro, formamide-induced sarcolemmal stress on healthy CMs phenocopied post-ischemic kinetics abnormalities and revealed initial SSM death and crest/hollow disorganization followed by IFM later disarray which moved toward the cell surface and structured heaps correlating with TT loss. This study demonstrates that the loss of crest/hollow organization of CM surface in HF occurs early and precedes disruption of the TT network. It also highlights a general stiffness increased of the CM surface most likely related to atypical IFM heaps while SSM died during HF process. Overall, these results indicate that initial sarcolemmal stress leading to SSM death could underlie subsequent TT disarray and HF setting.

Exendin-4 ameliorates cardiac ischemia/reperfusion injury via caveolae and caveolins-3

BackgroundExendin-4, an exogenous glucagon-like peptide-1 receptor (GLP-1R) agonist, protects the heart from ischemia/reperfusion injury. However, the mechanisms for this protection are poorly understood. Caveolae, sarcolemmal invaginations, and caveolins, scaffolding proteins in caveolae, localize molecules involved in cardiac protection. We tested the hypothesis that caveolae and caveolins are essential for exendin-4 induced cardiac protection using in vitro and in vivo studies in control and caveolin-3 (Cav-3) knockout mice (Cav-3 KO).MethodsMyocytes were treated with exendin-4 and then incubated with methyl-β-cyclodextrin (MβCD) to disrupt caveolae formation. This was then followed by simulated ischemia/reperfusion (SI/R). In addition, cardiac protection in vivo was assessed by measuring infarct size and cardiac troponin levels.ResultsExendin-4 protected cardiac myocytes (CM) from SI/R [35.6 ± 12.6% vs. 64.4 ± 18.0% cell death, P = 0.034] and apoptosis but this protection was abolished by MβCD (71.8 ± 10.8% cell death, P = 0.004). Furthermore, Cav-3/GLP-1R co-localization was observed and membrane fractionation by sucrose density gradient centrifugation of CM treated with MβCD + exendin-4 revealed that buoyant (caveolae enriched) fractions decreased Cav-3 compared to CM treated with exendin-4 exclusively. Furthermore, exendin-4 induced a reduction in infarct size and cardiac troponin relative to control (infarct size: 25.1 ± 8.2% vs. 41.4 ± 4.1%, P < 0.001; troponin: 36.9 ± 14.2 vs. 101.1 ± 22.3 ng/ml, P < 0.001). However, exendin-4 induced cardiac protection was abolished in Cav-3 KO mice (infarct size: 43.0 ± 6.4%, P < 0.001; troponin: 96.8 ± 26.6 ng/ml, P = 0.001).ConclusionsWe conclude that caveolae and caveolin-3 are critical for exendin-4 induced protection of the heart from ischemia/reperfusion injury.

Modeling Calcium Dynamics in Realistic Rabbit Ventricular Myocytes with Several Transverse Tubules

Calcium signaling in cardiomyocytes is strongly influenced by the topology of the sarcolemma (SL) and the distribution of sarcolemmal proteins, including the L-type calcium channel (LCC) and sodium-calcium exchanger (NCX). Peculiar to mammalian ventricular cardiomyocytes are sarcolemmal invaginations called transverse tubules (TT) exhibiting high densities of LCC clusters that trigger Ca2+ release from the sarcoplasmic reticulum (SR). Prior studies of pharmacologically-disabled SR release in rabbit ventricular myocytes have demonstrated that sub-micrometer resolution details of the SL geometry shape local Ca2+ dynamics and suggest a feedback between cytosolic [Ca2+] and SL ion channel and transporter activity. Here we investigate the hypothesis that the ordered spatial arrangement of TT and coupling between adjacent tubules leads to an organized Ca2+ transient. Moreover, we compare Ca2+ transients arising from clustered or continuously-distributed trigger fluxes for the propensity to produce Ca2+ waves. Our findings are that 1) the arrangement of TTs promotes a faster rise in [Ca2+] transversely relative to the longitudinal direction of cardiomyocytes and 2) clustering of SL transporters along the TT promotes an axially-uniform calcium transient. These results evidence contribution of structural detail at sub-micrometer resolution to excitation-contraction coupling and anomalous Ca2+ dynamics underlying cardiac arrhythmias.Supported by NBCR (NIH grant 2 P41 RR08605), NIH GM31749, NSF MCB-0506593, MCA93S013, Center for Theoretical Biological Physics, Howard Hughes Medical Institute, SDSC, W. M. Keck foundation, Richard A. and Nora Eccles Fund for Cardiovascular Research.

Caveolae compartmentalise β2-adrenoceptor signals by curtailing cAMP production and maintaining phosphatase activity in the sarcoplasmic reticulum of the adult ventricular myocyte

Inotropy and lusitropy in the ventricular myocyte can be efficiently induced by activation of β1-, but not β2-, adrenoceptors (ARs). Compartmentation of β2-AR-derived cAMP-dependent signalling underlies this functional discrepancy. Here we investigate the mechanism by which caveolae (specialised sarcolemmal invaginations rich in cholesterol and caveolin-3) contribute to compartmentation in the adult rat ventricular myocyte. Selective activation of β2-ARs (with zinterol/CGP20712A) produced little contractile response in control cells but pronounced inotropic and lusitropic responses in cells treated with the cholesterol-depleting agent methyl-β-cyclodextrin (MBCD). This was not linked to modulation of L-type Ca2+ current, but instead to a discrete PKA-mediated phosphorylation of phospholamban at Ser16. Application of a cell-permeable inhibitor of caveolin-3 scaffolding interactions mimicked the effect of MBCD on phosphorylated phospholamban (pPLB) during β2-AR stimulation, consistent with MBCD acting via caveolae. Biosensor experiments revealed β2-AR mobilisation of cAMP in PKA II signalling domains of intact cells only after MBCD treatment, providing a real-time demonstration of cAMP freed from caveolar constraint. Other proteins have roles in compartmentation, so the effects of phosphodiesterase (PDE), protein phosphatase (PP) and phosphoinositide-3-kinase (PI3K) inhibitors on pPLB and contraction were compared in control and MBCD treated cells. PP inhibition alone was conspicuous in showing robust de-compartmentation of β2-AR-derived signalling in control cells and a comparatively diminutive effect after cholesterol depletion. Collating all evidence, we promote the novel concept that caveolae limit β2-AR-cAMP signalling by providing a platform that not only attenuates production of cAMP but also prevents inhibitory modulation of PPs at the sarcoplasmic reticulum. This article is part of a Special Issue entitled “Local Signaling in Myocytes”.

Caveolin-3 expression and caveolae are required for isoflurane-induced cardiac protection from hypoxia and ischemia/reperfusion injury

Volatile anesthetics protect the heart from ischemia/reperfusion injury but the mechanisms for this protection are poorly understood. Caveolae, sarcolemmal invaginations, and caveolins, scaffolding proteins in caveolae, localize molecules involved in cardiac protection. We tested the hypothesis that caveolae and caveolins are essential for volatile anesthetic-induced cardiac protection using cardiac myocytes (CMs) from adult rats and in vivo studies in caveolin-3 knockout mice (Cav-3 −/−). We incubated CM with methyl-β-cyclodextrin (MβCD) or colchicine to disrupt caveolae formation, and then exposed the myocytes to the volatile anesthetic isoflurane (30 min, 1.4%), followed by simulated ischemia/reperfusion (SI/R). Isoflurane protected CM from SI/R [23.2 ± 1.6% vs. 71.0 ± 5.8% cell death (assessed by trypan blue exclusion), P < 0.001] but this protection was abolished by MβCD or colchicine (84.9 ± 5.5% and 64.5 ± 6.1% cell death, P < 0.001). Membrane fractionation by sucrose density gradient centrifugation of CM treated with MβCD or colchicine revealed that buoyant (caveolae-enriched) fractions had decreased phosphocaveolin-1 and caveolin-3 compared to control CM. Cardiac protection in vivo was assessed by measurement of infarct size relative to the area at risk and cardiac troponin levels. Isoflurane-induced a reduction in infarct size and cardiac troponin relative to control (infarct size: 26.5% ± 2.6% vs. 45.3% ± 5.4%, P < 0.01; troponin: 27.7 ± 4.4 vs. 77.7 ± 11.8 ng/ml, P < 0.05). Isoflurane-induced cardiac protection was abolished in Cav-3 −/− mice (infarct size: 53.4% ± 6.1% vs. 53.2% ± 3.5%, P < 0.01; troponin: 102.1 ± 22.3 vs. 105.9 ± 8.2 ng/ml, P < 0.01). Isoflurane-induced cardiac protection is thus dependent on the presence of caveolae and the expression of caveolin-3. We conclude that caveolae and caveolin-3 are critical for volatile anesthetic-induced protection of the heart from ischemia/reperfusion injury.

Spatiotemporal characteristics of SR Ca 2+ uptake and release in detubulated rat ventricular myocytes

In cardiac ventricular myocytes, sarcoplasmic reticulum (SR) Ca 2+ load is a key determinant of SR Ca 2+ release. This release normally occurs predominantly from SR junctions at sarcolemmal invaginations (t-tubules), ensuring synchronous SR Ca 2+ release throughout the cell. However under conditions of Ca 2+ overload, spontaneous SR Ca 2+ release and propagating Ca 2+ waves can occur, which are pro-arrhythmic. We used detubulated rat ventricular myocytes to determine the dependence of Ca 2+ wave propagation on SR Ca 2+ load, and the role of t-tubules in SR Ca 2+ uptake and spontaneous release. After SR Ca 2+ depletion, recovery of Ca 2+ transient amplitude (and SR Ca 2+ load) was slower in detubulated than control myocytes (half-maximal recovery: 9.9 ± 1.4 vs. 5.5 ± 0.7 beats). In detubulated myocytes the extent and velocity of Ca 2+ propagation from the cell periphery increased with each beat and depended steeply on SR Ca 2+ load. Isoproterenol (ISO) accelerated recovery, increased maximal propagation velocity and reduced the threshold SR Ca 2+ load for propagation. Ca 2+ spark frequency was uniform across control cell width and was similar at the periphery of detubulated cells. However, internal Ca 2+ spark frequency in detubulated cells was 75% lower (despite comparable local SR Ca 2+ load); this transverse spark frequency profile was similar to that in atrial myocytes. We conclude that: (1) t-tubule Ca 2+ fluxes normally control SR Ca 2+ refilling; (2) Ca 2+ wave propagation depends steeply on SR Ca 2+ content (3) SR-t-tubule junctions are important in initiating SR Ca 2+ release and (4) ISO enhances propagation of SR Ca release, but not the initiation of SR Ca release events (for given SR Ca 2+ loads).

Skeletal muscle fibre ends of the frog as revealed by scanning electron microscopy.

Muscle fibre ends at myotendinous junctions were examined by scanning electron microscopy after removal of tendon connective tissues by HCl-hydrolysis in the fourth extensor digitorum longus muscle and the extraocular muscle of the frog. The fibre ends in both muscles were characterized by the presence of small pit-like and short slit-like invaginations. The fibre ends in the extensor digitorum longus muscle took a spongy appearance with a great number of invaginations, while those on the extraocular muscle consisted of a number of pits and short slits arranged linearly toward the fibre tip. These findings confirm that muscle fibre ends in the frog consist of sarcolemmal invaginations to increase the surface area where collagen fibrils attach to the basal laminae of muscle fibres. The difference of the surface specializations between two kinds of muscle fibres may intimately reflect the functional properties of the muscle.

Fibronectin and laminin in transverse tubules of cardiac myocytes studied by laser confocal microscopy and immunocytochemistry.

The distribution of two non-collagenous glycoproteins of high molecular weight, fibronectin (FN) and laminin (LMN), was investigated in myocardial cells from the ventricle of rats, and from biopsies collected from the auricle of patients undergoing a coronary bypass operation. In order to elucidate the expression of FN and LMN across cells, non-invasive serial sectioning has been carried out by laser scanning confocal microscopy of frozen, immunostained tissue sections. In addition, immunoelectron microscopy was used to study the distribution of these antigens at higher magnifications. These studies show that FN is part of the basement membrane of the surface sarcolemma of both ventricular and atrial cells, in addition to being an abundant protein of the extracellular matrix (ECM). Along transverse tubular(TT)-membranes, FN was only detected in tubules exceeding 200 nm in diameter. Even here, the intensity of labelling varied greatly and was generally low. By contrast, a heavy investment of LMN was organized in the basal lamina along the surface sarcolemma and along ramifications of the entire TT-system in ventricular heart muscle cells. In this way, the network of TT-membrane systems of working heart muscle cells provides a supply of LMN to all depths of the myocardial fibre. In human atrial muscle cells, a regular TT-system appears to be absent. Instead occasional, deep sarcolemmal invaginations occur with diameters of 300-500 nm, the surfaces of which are also invested with LMN. The significance of the present findings has been discussed, with special reference to LMN as a possible component of a series of proteins involved in transmembrane communication between the ECM and the sarcoplasm.

Scanning Electron Microscopical Study of Skeletal Muscle Fiber Ends in Normal and Dystrophic Mice.

Muscle fiber ends at myotendinous junctions were examined by scanning electron microscopy after removal of tendon connective tissue components by HCl hydrolysis in the extensor digitorum longus muscle of 30-, 60- and 120-day-old normal and dystrophic (dy) mice. A remarkable morphological difference between normal and dystrophic mice was observed. In normal 30-day-old mice, muscle fiber ends had already assumed a complicated three-dimensional morphology with many thin cytoplasmic processes and lateral longitudinal clefts. On the other hand, in dystrophic animals at the 60th day, muscle fiber ends were characterized by a simple conical form with a rather smooth surface in which existed a number of pit-like sarcolemmal invaginations, and short and narrow longitudinal slits, possibly an indication of developmental immaturity. Thereafter, the slits increased in number and in length, although the linear elongation of the slits seems to be caused by the fusion of adjacent pits to one another and to existing slits. From these findings, the fiber ends in the adult (120-day-old) dystrophic mouse are suggested to retain a preceding state of their unique structural differentiation.

Studies of hamster cardiac myofibrillogenesis in vivo with Antibodies to spectrin, desmin, and alpha‐actinin

The spectrins are a family of cytoskeletal-membrane proteins that have generated much interest in the past decade. In the present study, we utilized immunohistochemical, morphological, and electrophoretic techniques to assess the possible function(s) of spectrin in mammalian cardiac tissue during development. Antibodies generated against alpha-actinin and desmin were also employed to identify myofibrils and intermediate filaments in relation to changes in the distribution of spectrin. Spectrin is localized along the sarcolemma of pre-myofibrillar hamster cardiac myocytes (day 8, postcoitum) and remains associated with the cell membrane throughout development. The staining pattern is somewhat diffuse at first, but eventually the cell margin becomes clearly defined by day 13 postcoitum. A second, more profound change in the distribution of spectrin occurs during the newborn stage, when spectrin begins to appear in the sarcoplasm. It appears as regularly spaced invaginations that are diffuse at first, eventually attaining a position around the Z-bands of adult muscle. The change in the distribution of spectrin coincides temporally with the appearance of T-tubules, which are sarcolemmal invaginations that reside at the Z-bands of adult heart. Thus, spectrin may act as a guidance mechanism for the proper positioning of T-tubules around the Z-discs of mammalian cardiac tissue. Although spectrin does not appear to interact directly with early myofibrils it may assist in the proper alignment of T-tubules and, in doing so, act to stabilize the entire contractile apparatus by enveloping it and attaching it to the sarcolemma.

In vitro attachment of skeletal muscle fibers to a collagen gel duplicates the structure of the myotendinous junction

The myotendinous junction (MTJ) and its associated cells and connective tissue are important structures involved in transmission of contractile force from skeletal muscle to tendon. A model culture system was developed to investigate the formation of the MTJ and its attachment to collagen fibers. Skeletal muscle cells were cultured in a well modeled from two layers of a native gel of type I collagen. Muscle cells cultured in this manner formed attachments to the collagen gel and developed into highly contractile multinucleated muscle fibers with the development of extensive terminal invaginations of the sarcolemma. In addition, the subsarcolemma at the ends of muscle fibers showed areas of increased electron density which corresponded well with the termini of myofibrils. The results indicate that the development of sarcolemmal invaginations at the end of a muscle fiber probably occurs intrinsically during muscle development in vivo. The direct association of collagen fibers with the basal lamina at the end of muscle fibers was only occasionally observed in culture, suggesting that other fibrils or proteins may also be involved in the attachment of collagen fibers to the basal lamina of muscle fibers at the MTJ.

The cell surface of isolated cardiac myocytes—A light microscope study with use of fluorochrome-coupled lectins

In cells isolated from guinea-pig or rat ventricular muscle occurrence and distribution of carbohydrate components of the surface coat were monitored using fluorochrome-coupled lectins. Fluorescence of membrane-bound lectins was assayed by an image analysis system. The lectins ConA † † Abbreviations: BSA, bovine serum albumin; ConA, Concanavalin A; DBA, Dolichos biflorus agglutinin; DMSO, dimethyl sulfoxide; EGTA, ethyleneglycol-bis-(β-aminoethyl ether)- N,N,N′,N′-tetraacetic acid; FITC, fluorescein isothiocyanate; Gal, d-galactose; GalNAc, N-acetyl- d-galactosamine; Glc, d-glucose; GlcNAc, N-acetyl- d-glucosamine; HEPES, N-(2-hydroxyethyl)piperazine- N′-(2-ethanesulfonic acid); LFA, Limax flavus agglutinin; Man, d-mannose; met-Glc, methyl- d-glucoside; met-Man, methyl- d-mannoside; NeuNAc, N-acetylneuraminic acid; NeuNGc, N-glycoloylneuraminic acid; PNA, peanut agglutinin; RCA-I, Ricinus communis agglutinin-I; SBA, soy bean agglutinin; TMA-DPH, 1-[4(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene; TRITC, tetramethylrhodamine isothiocyanate; UEA-I, Ulex europaeus agglutinin-I; WGA, wheat germ agglutinin; sWGA, succinylated WGA. , WGA, sWGA, LFA and RCA-I showed specific binding to the whole myocyte surface, indicating a homogeneous distribution of α-mannosyl, α-glycosyl, N-acetylglucosaminyl, N-acetylneuraminate and β-galactosyl residues. Binding of DBA and SBA, with specific affinity for N-acetylgalactosaminyl residues, to guinea-pig cardiac myocytes was mainly at the cell poles corresponding to intercalated discs in intact tissue. Both lectins failed to interact with rat myocytes. UEA-I, specific for α- l-fucose, bound slightly to rat and not to guinea-pig myocytes. Binding of PNA to guinea-pig myocytes was observed only after cleaving off sialic acids from cell surface, suggesting that sialic acids mask galactosyl-β(1,3)- N-acetylgalactosamine residues. Specifity of lectin-cell interaction was tested by an inhibition assay where free sugars were tested for their capacity to inhibit lectin binding to the myocytes. When comparing different isolation procedures based on different proteolytic enzymes, the myocytes' affinity to any lectin was found to be qualitatively unchanged. Investigation of lectin-decorated myocytes by means of confocal laser scan microscopy showed that lectin binding sites are not confined to the cell surface but are also present in sarcolemmal invaginations, i.e. transverse tubules. This suggests that the tubular system is lined with a carbohydrate layer similar to, and continuous with, that of the peripheral cell surface.

Two types of focal accumulations of acetylcholinesterase appear in noninnervated regenerating skeletal muscles of the rat.

Muscle fibers in the soleus muscle of the rat, injured by bupivacaine and free autografting, were allowed to regenerate within their old basal laminae. Histochemical and cytochemical analysis of newly synthesized acetylcholinesterase (AChE) revealed that two kinds of focal accumulations of AChE appeared in regenerating myotubes. First, AChE gets concentrated at the sites of the former motor endplates. Accumulation of AChE starts in places where a tight contact between the remnants of the old junctional basal lamina and the budding surface of the myotube engulf the extracellular material. Appearance of these AChE accumulations can be prevented by papain treatment of the soleus muscle before autografting but not by predenervating it for 1 month. Focalization of AChE is probably induced by a component of the junctional basal lamina, possibly a protein, the existence of which is not dependent upon continuous presence of the motor nerve and may be produced by the muscle. This view is corroborated by the fact that an additional kind of AChE accumulation appeared in regenerating muscles in regions remote from the sites where motor endplates were located in the muscles of origin. Although differing in localization, size, and appearance, both kinds of AChE accumulations ultrastructurally resemble the postsynaptic specialization of the motor endplate: they consist of tubelike sarcolemmal invaginations containing AChE. The extrajunctional AChE accumulations seem to arise spontaneously and are usually located more than 750 micron away from the junctional ones as if some local inhibitory mechanism prevents their formation in the immediate vicinity.

Is a guanine nucleotide-binding protein involved in excitation-contraction coupling in skeletal muscle?

Plasma membrane depolarization causes skeletal muscle contraction by triggering Ca2+ release from an intracellular membrane network, the sarcoplasmic reticulum. A specialized portion of the sarcoplasmic reticulum, the terminal cisternae, is junctionally associated with sarcolemmal invaginations called the transverse tubules, but the mechanism by which the action potential at the level of the transverse tubules is coupled to Ca2+ release from the terminal cisternae is still mysterious. Here we show that: (i) GTP gamma S, a non-hydrolyzable analog of GTP, elicits isometric force development in skinned muscle fibre; (ii) GTP gamma S is unable to release CA2+ from isolated sarcoplasmic reticulum fractions; (iii) the threshold for tension development is shifted to higher GTP gamma S concentrations by pre-incubation with pertussis toxin. These results suggest that a GTP-binding protein is involved in coupling the action potential of transverse tubules to Ca2+ release from the terminal cisternae.

Inositol 1,4,5-trisphosphate induces calcium release from sarcoplasmic reticulum of skeletal muscle

The sarcoplasmic reticulum of skeletal muscle is a specialized form of endoplasmic reticulum that controls myoplasmic calcium concentration and, therefore, the contraction-relaxation cycle. Ultrastructural studies have shown that the sarcoplasmic reticulum is a continuous but heterogeneous membranous network composed of longitudinal tubules that surround myofibrils and terminal cisternae. These cisternae are junctionally associated, via bridging structures called 'feet', with sarcolemmal invaginations (the transverse tubules) to form the triadic junction. Following transverse tubule depolarization, a signal, transmitted along the triadic junction, triggers Ca2+ release from terminal cisternae, but the mechanism of this coupling is still unknown. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) has recently been shown to mobilize Ca2+ from intracellular stores, referable to endoplasmic reticulum, in a variety of cell types (see ref. 8 for review), including smooth muscle cells of the porcine coronary artery and canine cardiac muscle cells. Here we show that Ins(1,4,5)P3 releases Ca2+ from isolated, purified sarcoplasmic reticulum fractions of rabbit fast-twitch skeletal muscle, the effect being more pronounced on a fraction of terminal cisternae that contains morphologically intact feet structures; and elicits isometric force development in chemically skinned muscle fibres.

Slow asymmetric currents and tubulo-reticular junction ultrastructure in crayfish muscle fibers

Asymmetric membrane currents in isolated muscle fibers of the crayfishAstacus fluviatilis were studied under voltage clamp conditions with controlled composition of the external and internal medium. Besides fast asymmetric currents which are probably associated with opening of calcium channels in the surface membrane, slow asymmetric currents with a time course almost an order of magnitude slower than in fast frog muscle fibers also are present in fibers of this type. The value of the charge transported across a single "foot" in the tubulo-reticular junction was calculated. The number of feet and their arrangement in each junction were studied by transmission electron microscopy. The number of charges transported across one foot agrees with the hypothesis that slow displacement currents are linked with movement of charge particles distributed over the whole area of the sarcolemmal and tubular invaginations, and not only in the feet.