Not Just a Plasma Membrane Protein: in Cardiac Muscle Cells Alpha-II Spectrin also Shows a Close Association with Myofibrils

Spectrin and its associated proteins are essential for the integrity of muscle cells and there is increasing evidence for their involvement in signalling pathways as well as having a structural function in mediating stress. Spectrin is a multigene family and it is essential to determine which isoforms are present and their location in the cell. In heart muscle, we have found that one spectrin isoform, alphaII-spectrin, is strongly represented and, using immunofluorescence, we show that it lies within the contractile fibres near the Z-disc as well as on the cardiomyocyte plasma membrane. Electron microscopy of immunogold-labelled cryosections reveals statistically significant clustering of gold particles near the Z-disc, within and close to the edge of myofibrils. betaII-spectrin and ankyrin-R and G are both known to occupy this region. We suggest that alphaIIbetaII spectrin tetramers with ankyrin organise and/or stabilise cardiac muscle cell membrane components relative to the contractile apparatus.     

Nebulin is a thin filament protein of the cardiac muscle of the agnathans

Nebulin is an integral protein of skeletal muscle thin filaments and probably acts as a ruler for the thin filament length. Cardiac muscles of higher vertebrates have been shown earlier to lack nebulin. Instead in human and chicken cardiac muscle the much smaller protein nebulette replaces nebulin. Since nebulette is confined to the Z-disc region of the sarcomere and does not span the whole thin filament length, it must have functions significantly different from those assumed for nebulin. We have investigated nebulin in skeletal and cardiac muscles of the agnathans (lamprey, hagfish), elasmobranchs (shark), chondrosts (sturgeon) and teleosts (trout, eel) by SDS-PAGE and immunodetection methods. Among these, lamprey and hagfish cardiac muscles are unique in that both contain full-length nebulin identical in molecular mass to the nebulin of the respective body muscle. Using immunofluorescence microscopy, lamprey cardiac nebulin was localised in the I-band of the sarcomere, the same as for nebulin in skeletal muscle. In contrast to this, all gnathostome species investigated lacked nebulin in cardiac muscles, while it was present in the respective skeletal muscles. This clearly shows that nebulin is not exclusively present in skeletal muscles of chordates. The findings also demonstrate a rare case of dramatic size reduction of a protein during evolution.     

The vertebrate muscle Z-disc: sarcomere anchor for structure and signalling

The Z-disc, appearing as a fine dense line forming sarcomere boundaries in striated muscles, when studied in detail reveals crosslinked filament arrays that transmit tension and house myriads of proteins with diverse functions. At the Z-disc the barbed ends of the antiparallel actin filaments from adjoining sarcomeres interdigitate and are crosslinked primarily by layers of α-actinin. The Z-disc is therefore the site of polarity reversal of the actin filaments, as needed to interact with the bipolar myosin filaments in successive sarcomeres. The layers of α-actinin determine the Z-disc width: fast fibres have narrow (~30–50 nm) Z-discs and slow and cardiac fibres have wide (~100 nm) Z-discs. Comprehensive reviews on the roles of the numerous proteins located at the Z-disc in signalling and disease have been published; the aim here is different, namely to review the advances in structural aspects of the Z-disc.     

Exercise training-induced improvements in insulin action

Individuals with insulin resistance are characterized by impaired insulin action on whole-body glucose uptake, in part due to impaired insulin-stimulated glucose uptake into skeletal muscle. A single bout of exercise increases skeletal muscle glucose uptake via an insulin-independent mechanism that bypasses the typical insulin signalling defects associated with these conditions. However, this 'insulin sensitizing' effect is short-lived and disappears after approximately 48 h. In contrast, repeated physical activity (i.e. exercise training) results in a persistent increase in insulin action in skeletal muscle from obese and insulin-resistant individuals. The molecular mechanism(s) for the enhanced glucose uptake with exercise training have been attributed to the increased expression and/or activity of key signalling proteins involved in the regulation of glucose uptake and metabolism in skeletal muscle. Evidence now suggests that the improvements in insulin sensitivity associated with exercise training are also related to changes in the expression and/or activity of proteins involved in insulin signal transduction in skeletal muscle such as the AMP-activated protein kinase (AMPK) and the protein kinase B (Akt) substrate AS160. In addition, increased lipid oxidation and/or turnover is likely to be another mechanism by which exercise improves insulin sensitivity: exercise training results in an increase in the oxidative capacity of skeletal muscle by up-regulating lipid oxidation and the expression of proteins involved in mitochondrial biogenesis. Determination of the underlying biological mechanisms that result from exercise training is essential in order to define the precise variations in physical activity that result in the most desired effects on targeted risk factors, and to aid in the development of such interventions.     

The central role of myostatin in skeletal muscle and whole body homeostasis

Myostatin is a powerful negative regulator of skeletal muscle mass in mammalian species. It plays a key role in skeletal muscle homeostasis and has now been well described since its discovery. Myostatin is capable of inducing muscle atrophy via its inhibition of myoblast proliferation, increasing ubiquitin-proteasomal activity and downregulating activity of the IGF-Akt pathway. These well-recognized effects are seen in multiple atrophy causing situations, including injury, diseases such as cachexia, disuse and space flight, demonstrating the importance of the myostatin signalling mechanism. Based on this central role, significant work has been pursued to inhibit myostatin's actions in vivo. Importantly, several new studies have uncovered roles for myostatin distinct from skeletal muscle size. Myostatin has been suggested to play a role in cardiomyocyte homeostasis, glucose metabolism and adipocyte proliferation, all of which are examined in detail below. Based on these effects, myostatin inhibition has potential to be widely utilized in many Western diseases such as chronic obstructive pulmonary disease, type II diabetes and obesity. However, if myostatin inhibitors are to successfully translate from bench-top to bedside in the near future, awareness must be raised on these non-traditional effects of myostatin away from skeletal muscle. Indeed, further research into these novel areas is required.     

Peroxisome proliferator‐activated receptors (PPARS): regulators of gene expression in heart and skeletal muscle

The peroxisome proliferator‐activated receptors (PPARs) are members of the nuclear hormone receptor superfamily. The three isoforms (PPARα, β/δ and γ) have been implicated in the regulation of the expression of genes involved in lipid metabolism. Although their prominent role in lipid homeostasis is well established, the way in which the activity of each of the PPAR isoforms is regulated under physiological and pathological conditions is still subject of intensive research. In skeletal as well as cardiac muscle cells it has been demonstrated that the expression of a large panel of proteins involved in the transport and metabolic conversion of fatty acids is under control of PPARs. The pivotal role of the PPARα isoform in cardiac fatty acid metabolism has been confirmed in PPARα‐null mice. The exact role of PPARβ/δ in the regulation of muscle metabolism is still a matter of debate. Whereas several studies provided evidence to support the notion that PPARα and PPARβ/δ have redundant roles, other studies suggest that PPARα activity is counteracted by PPARβ/δ. Marked effects of bona fide PPARγ ligands (the anti‐diabetic thiazolidinediones) on skeletal and cardiac muscle function and phenotype, have also been reported. However, next to activating PPARγ, the thiazolidinediones do affect other cellular processes as well. To date it is being realized that the control of the trans‐activating capacity of each of the PPAR isoforms is multi‐factorial and, in addition to ligand availability, depends on such factors as isoform‐specific phosphorylation and selective interaction with various proteins acting either as co‐activator or co‐repressor.     

Transgenic mice expressing the myotilin T57I mutation unite the pathology associated with LGMD1A and MFM

Myotilin is a muscle-specific Z-disc protein with putative roles in myofibril assembly and structural upkeep of the sarcomere. Several myotilin point mutations have been described in patients with limb-girdle muscular dystrophy type 1A (LGMD1A), myofibrillar myopathy (MFM), spheroid body myopathy (SBM), three similar adult-onset, progressive and autosomal dominant muscular dystrophies. To further investigate myotilin's role in the pathogenesis of these muscle diseases, we have characterized three independent lines of transgenic mice expressing mutant (T57I) myotilin under the control of the human skeletal actin promoter. Similar to LGMD1A and MFM patients, these mice develop progressive myofibrillar pathology that includes Z-disc streaming, excess myofibrillar vacuolization and plaque-like myofibrillar aggregation. These aggregates become progressively larger and more numerous with age. We show that the mutant myotilin protein properly localizes to the Z-disc and also heavily populates the aggregates, along with several other Z-disc associated proteins. Whole muscle physiological analysis reveals that the extensor digitorum longus muscle of transgenic mice exhibits significantly reduced maximum specific isometric force compared with littermate controls. Intriguingly, the soleus and diaphragm muscles are spared of any abnormal myopathology and show no reductions in maximum specific force. These data provide evidence that myotilin mutations promote aggregate-dependent contractile dysfunction. In sum, we have established a promising patho-physiological mouse model that unifies the phenotypes of LGMD1A, MFM and SBM.     

Novel regulators of RyR Ca2+ release channels: insight into molecular changes in genetically-linked myopathies

There are many mutations in the ryanodine receptor (RyR) Ca²⁺ release channel that are implicated in skeletal muscle disorders and cardiac arrhythmias. More than 80 mutations in the skeletal RyR1 have been identified and linked to malignant hyperthermia, central core disease or multi-minicore disease, while more than 40 mutations in the cardiac RyR2 lead to ventricular arrhythmias and sudden cardiac death in patients with structurally normal hearts. These RyR mutations cause diverse changes in RyR activity which either excessively activate or block the channel in a manner that disrupts Ca²⁺ signalling in the muscle fibres. In a different myopathy, myotonic dystrophy (DM), a juvenile isoform of the skeletal RyR is preferentially expressed in adults. There are two regions of RyR1 that are variably spiced and developmentally regulated (ASI and ASII). The juvenile isoform (ASI (−)) is less active than the adult isoform (ASI(+)) and its over-expression in adults with DM may contribute to functional changes. Finally, mutations in an important regulator of the RyR, the Ca²⁺ binding protein calsequestrin (CSQ), have been linked to a disruption of Ca²⁺ homeostasis in cardiac myocytes that results in arrhythmias. We discuss evidence supporting the hypothesis that mutations in each of these situations alter protein/protein interactions within the RyR complex or between the RyR and its associated proteins. The disruption of these protein–protein interactions can lead either to excess Ca²⁺ release or reduced Ca²⁺ release and thus to abnormal Ca²⁺ homeostasis. Much of the evidence for disruption of protein–protein interactions has been provided by the actions of a group of novel RyR regulators, domain peptides with sequences that correspond to sequences within the RyR and which compete with the endogenous residues for their interaction sites.     

Toll‐like receptor signalling in regenerative myogenesis: friend and foe

Skeletal muscle regeneration in normal and diseased muscle is regulated by multiple factors and cells present in the injured muscle micro‐environment. In addition to muscle progenitor cells, several immunocytes participate in the regenerative response. Among them, macrophages are one of the most important components of the immune response that governs the step‐wise progression of muscle regeneration. The initial role of macrophages is to phagocytose muscle cell debris and later, through their transition to an anti‐inflammatory phenotype, they promote regeneration. However, in several genetic muscle disorders, continuous muscle injury disrupts the balance between pro‐inflammatory and anti‐inflammatory macrophages, leading to an overall inflammatory milieu and inhibition of muscle regeneration. Accumulating evidence suggests that Toll‐like receptor (TLR)‐mediated signalling plays an important role in the regulation of macrophage phenotypes during regenerative myogenesis in response to both acute and chronic muscle injury. Here, we discuss the role of TLR signalling in regulating macrophage phenotypes and skeletal muscle regeneration in healthy and diseased muscle. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Evidence for immunological cross-reactivity between smooth and skeletal muscle.

In 1965 Johnson, Holborow & Glynn showed that some patients with chronic liver disease have antibodies which react with smooth muscle by indirect immunofluorescence. This work was rapidly confirmed and it was soon showed that the same serum may also react with renal glomeruli and other tissue components. Reactions with skeletal muscle were not demonstrated. In 1960 Strauss and co-workers deomonstrated that patients with myasthenia gravis may have antibodies which react with skeletal muscle. Subsequent work has suggested that these antibodies are specific for skeletal as opposed to smooth muscle. More recently smooth muscles antibodies (SMA) have been shown to react with a wide range of tissues and cells including neoplastic cells (Gabbiani et al,, 1973; Holborow et al., 1975). Throughout this work it has been assumed that such reactivity is due to the presence of smooth muscle contractile proteins. This conclusion was further supported by the demonstration that animals immunized with smooth muscle contractile proteins developed antibodies which produce similar patterns to those found with the naturally occurring antismooth muscle antibodies (Trenchev, Sneyd & Holborow, 1974). From these studies it appeared that there was very little if any cross reactivity between smooth and skeletal muscle, although Bray (1974) pointed to the need to re-examine this conclusion. In the course of screening human sera for autoantibodies we have been impressed by the occasional occurrence of sera which gave both smooth and skeletal muscle staining patterns. In this report we describe selected sera which react with antigens which appear specific for either smooth or striated muscle, and additional sera which react with antigens which appear common to both smooth and striated muscle. Immunization of rabbits with skeletal muscle contractile protein has provided further evidence for such cross reactivity,     

Heart failure: a model of cardiac and skeletal muscle energetic failure

Chronic heart failure (CHF), the new epidemic in cardiology, is characterized by energetic failure of both cardiac and skeletal muscles. The failing heart wastes energy due to anatomical changes that include cavity enlargement, altered geometry, tachycardia, mitral insufficiency and abnormal loading, while skeletal muscle undergoes atrophy. Cardiac and skeletal muscles also have altered high-energy phosphate production and handling in CHF. Nevertheless, there are differences in the phenotype of myocardial and skeletal muscle myopathy in CHF: cardiomyocytes have a lower mitochondrial oxidative capacity, abnormal substrate utilisation and intracellular signalling but a maintained oxidative profile; in skeletal muscle, by contrast, mitochondrial failure is less clear, and there is altered microvascular reactivity, fibre type shifts and abnormalities in the enzymatic systems involved in energy distribution. Underlying these phenotypic abnormalities are changes in gene regulation in both cardiac and skeletal muscle cells. Here, we review the latest advances in cardiac and skeletal muscle energetic research and argue that energetic failure could be taken as a unifying mechanism leading to contractile failure, ultimately resulting in skeletal muscle energetic failure, exertional fatigue and death.     

Insight towards the identification of cytosolic Ca2+‐binding sites in ryanodine receptors from skeletal and cardiac muscle

Ca²⁺ plays a critical role in several processes involved in skeletal and cardiac muscle contraction. One key step in cardiac excitation–contraction (E–C) coupling is the activation of the cardiac ryanodine receptor (RYR2) by cytosolic Ca²⁺ elevations. Although this process is not critical for skeletal E–C coupling, the activation and inhibition of the skeletal ryanodine receptor (RYR1) seem to be important for overall muscle function. The RYR1 and RYR2 channels fall within the large category of Ca²⁺‐binding proteins that harbour highly selective Ca²⁺ ‐binding sites to receive and translate the various Ca²⁺ signals into specific functional responses. However, little is known about the precise localization of these sites within the cytosolic assembly of both RYR isoforms, although several experimental lines of evidence have highlighted their EF‐hand nature. EF‐hand proteins share a common helix‐loop‐helix structural motif with highly conserved residues involved in Ca²⁺ coordination. The first step in predicting EF‐hand positive regions is to compare the primary protein structure with the EF‐hand motif by employing available bioinformatics tools. Although this simple method narrows down search regions, it does not provide solid evidence regarding which regions bind Ca²⁺ in both RYR isoforms. In this review, we seek to highlight the key findings and experimental approaches that should strengthen our future efforts to identify the cytosolic Ca²⁺‐binding sites responsible for activation and inhibition in the RYR1 channel, as much less work has been conducted on the RYR2 channel.     

Sarcoglycans in human skeletal muscle and human cardiac muscle: a confocal laser scanning microscope study

Sarcoglycans are a subcomplex of transmembrane proteins which are part of the dystrophin-glycoprotein complex. They are expressed in the skeletal, cardiac and smooth muscle. Although numerous studies have been conducted on the sarcoglycan subcomplex in skeletal and cardiac muscle, the manner of the distribution and localization of these proteins along the nonjunctional sarcolemma is not clear. We therefore carried out an indirect immunofluorescence study on surgical biopsies of normal human skeletal muscle and of healthy human atrial myocardium biopsies of patients affected by valvulopathy. Our results indicate that, in skeletal muscle, sarcoglycans have a costameric distribution and all colocalize with each other. Only in a few cases did the alpha-sarcoglycan not colocalize with other sarcoglycans. In addition, these glycoproteins can be localized in different fibers either in the regions of the sarcolemma over band I or band A. In cardiac muscle, our results show a costameric distribution of all proteins examined and, unlike in skeletal muscle, they show a constant colocalization of all sarcoglycans with each other, along with a consistent localization of these proteins in the region of the sarcolemma over band I. In our opinion, this situation seems to confirm the hypothesis of a correlation between the region of the sarcolemma occupied by costameric proteins and the metabolic type, fast or slow, of the muscular fibers. These data, besides opening a new line of research in understanding interactions between the sarcoglycans and other transmembrane proteins, could also be extended to skeletal and cardiac muscles affected by neuromuscular and cardiovascular pathologies to understand possible structural alterations.     

Intermediate filament proteins increase during chronic stimulation of skeletal muscle

Summary Chronic low-frequency electrical stimulation of rabbit fast-twitch skeletal muscle induces increased levels of two intermediate filament proteins, desmin and vimentin, during the first 3 weeks of stimulation. These increases occur over the same timecourse as reported shifts in -actinin expression and increased Z-disc width, but precede the fast-to-slow shifts in contractile proteins, which have been described by others. Desmin and vimentin levels increase during the first 2 weeks of stimulation, at which time the increase in desmin appears to plateau while vimentin continues to increase significantly through 3 weeks of stimulation. Absolute amounts of vimentin are lower than desmin at all time points, however increases in desmin and vimentin levels are strongly correlated during the stimulation period, suggesting that the two proteins are coordinately increased during the initial phases of muscle transformation. We suggest that rapid increases in the expression of intermediate filament proteins, which coincide with alterations in Z-disc structure, may indicate a fortification of the force-bearing ultrastructure of the muscle fibre in response to the increased activity that is induced by stimulation. The presence of vimentin and elevated levels of desmin expression suggest that mature skeletal muscle reverts toward a developmental program of intermediate filament protein expression during fast-to-slow transformation.     

Recruitment of bone-marrow-derived cells by skeletal and cardiac muscle in adult dystrophic mdx mice

 It is commonly accepted, that regenerative capacity of striated muscle is confined to skeletal muscle by activation of satellite cells that normally reside quiescent between the plasmalemma and the basement membrane of muscle fibers. Muscular dystrophies are characterized by repetitive cycles of de- and regeneration of skeletal muscle fibers and by the frequent involvement of the cardiac muscle. Since during the longstanding course of muscular dystrophies there is a permanent demand of myogenic progenitors we hypothesized that this may necessitate a recruitment of additional myogenic precursors from an undifferentiated, permanently renewed cell pool, such as bone marrow (BM) cells. To this end normal and dystrophic (mdx) female mice received bone marrow transplantation (BMT) from normal congenic male donor mice. After 70 days, histological sections of skeletal and cardiac muscle from BMT mice were probed for the donor-derived Y chromosomes. In normal BMT recipients, no Y chromosome-containing myonuclei were detected, either in skeletal or in cardiac muscle. However, in all samples from dystrophic mdx skeletal muscles Y chromosome-specific signals were detected within muscle fiber nuclei, which additionally were found to express the myoregulatory proteins myogenin and myf-5. Moreover, in the hearts of BMT-mdx mice single cardiomyocytes with donor derived nuclei were identified, indicating, that even cardiac muscle cells are able to regenerate by recruitment of circulating BM-derived progenitors. Our findings suggest that further characterization and identification of the BM cells capable of undergoing myogenic differentiation may have an outstanding impact on therapeutic strategies for diseases of skeletal and cardiac muscle. Accepted: 27 October 1998     

The Wnt/Frizzled pathway as a therapeutic target for cardiac hypertrophy: where do we stand?

Cardiac hypertrophy is an enlargement of the heart muscle in response to wall stress. This hypertrophic response often leads to heart failure. In recent years, several studies have shown the involvement of Wnt signalling in hypertrophic growth. In this review, the role of Wnt signalling and the possibilities for therapeutic interventions are discussed. In healthy adult heart tissue, Wnt signalling is very low. However, under pathological condition such as hypertension, Wnt signalling is activated. In recent years, it has become clear that both β-catenin-dependent signalling and β-catenin-independent signalling are involved in hypertrophic growth. Several studies, both in vitro and in vivo, have shown that genetic interventions in Wnt signalling at different levels resulted in an attenuated or diminished hypertrophic response. Therefore, inhibition of Wnt signalling could provide a new therapeutic strategy for cardiac hypertrophy, but further research on the Wnts and Frizzleds involved in the different forms of cardiac hypertrophy will be needed to achieve this goal.     

Developmental changes in the protein profiles of human cardiac and skeletal muscle

1. The use of SDS electrophoresis as a tool for the analysis of development processes in man has been evaluated. 2. The protein profiles of cardiac and skeletal muscle from foetal (10--24 weeks gestation) infant and adult specimens have been analysed and striking developmental changes were found which involved all the major proteins. 3. Before 20 weeks gestation the soluble protein profile of skeletal muscle appears to consist largely of extracellular proteins. 4. Myoglobin was found in foetal cardiac muscle from 20 weeks gestation but was not demonstrable in foetal (greater than 24 weeks) skeletal muscle. Foetal and adult myoglobin were indistinguishable. 5. A limited survey of the protein patterns of brain, liver and kidney was carried out. In general these tissues show less developmental change than skeletal or cardiac muscle.     

Desmin‐related myopathies in mice and man

Desmin, the main intermediate filament (IF) protein in skeletal and heart muscle cells, is of great importance as a part of the cytoskeleton. The IFs surround and interlink myofibrils, and connect the peripheral myofibrils with the sarcolemma. In myotendinous junctions and neuromuscular junctions of skeletal muscle fibres, desmin is enriched. In the heart, desmin is increased at intercalated discs, the attachment between cardiomyocytes, and it is the main component in Purkinje fibres of the conduction system. Desmin is the first muscle‐specific protein to appear during myogenesis. Nevertheless, lack of desmin, as shown from experiments with desmin knockout (K/O) mice, does not influence myogenesis or myofibrillogenesis. However, the desmin knock‐out mice postnatally develop a cardiomyopathy and a muscle dystrophy in highly used skeletal muscles. In other skeletal muscles the organization of myofibrils is remarkably unaffected. Thus, the main consequence of the lack of desmin is that the muscle fibres become more susceptible to damage. The loss of membrane integrity leads to a dystrophic process, with degeneration and fibrosis. In the heart cardiac failure develops, whereas in affected skeletal muscles regenerative attempts are seen. In humans, accumulations of desmin have been a hallmark for presumptive desmin myopathies. Recent investigations have shown that some families with such a myopathy have a defect in the gene coding for αB‐crystallin, whereas others have mutations in the desmin gene. Typical features of these patients are cardiac affections and muscle weakness. Thus, mutations in the desmin gene is pathogenic for a distinct type of muscle disorder.