Impact of capillary and sarcolemmal proximity on mitochondrial structure and energetic function in skeletal muscle.

Mitochondria within skeletal muscle cells are located either between the muscle contractile apparatus (interfibrillar mitochondria, IFM) or beneath the cell membrane (subsarcolemmal mitochondria, SSM), with several structural and functional differences reported between IFM and SSM. However, recent 3D imaging studies demonstrate that mitochondria are particularly concentrated in the proximity of capillaries embedded in sarcolemmal grooves rather than in proximity to the sarcolemma itself (paravascular mitochondria, PVM). To evaluate the impact of capillary vs. sarcolemmal proximity, we compared the structure and function of skeletal muscle mitochondria located either lateral to embedded capillaries (PVM), adjacent to the sarcolemma but not in PVM pools (SSM) or interspersed between sarcomeres (IFM). Mitochondrial morphology and interactions were assessed by 3D electron microscopy coupled with machine learning segmentation, whereas mitochondrial energy conversion was assessed by two-photon microscopy of mitochondrial membrane potential, content, calcium, NADH redox and flux in live, intact cells. Structurally, although PVM and SSM were similarly larger than IFM, PVM were larger, rounder and had more physical connections to neighbouring mitochondria compared to both IFM and SSM. Functionally, PVM had similar or greater basal NADH flux compared to SSM and IFM, respectively, despite a more oxidized NADH pool and a greater membrane potential, signifying a greater activation of the electron transport chain in PVM. Together, these data indicate that proximity to capillaries has a greater impact on resting mitochondrial energy conversion and distribution in skeletal muscle than the sarcolemma alone. KEY POINTS: Capillaries have a greater impact on mitochondrial energy conversion in skeletal muscle than the sarcolemma. Paravascular mitochondria are larger, and the outer mitochondrial membrane is more connected with neighbouring mitochondria. Interfibrillar mitochondria are longer and have greater contact sites with other organelles (i.e. sarcoplasmic reticulum and lipid droplets). Paravascular mitochondria have greater activation of oxidative phosphorylation than interfibrillar mitochondria at rest, although this is not regulated by calcium.

Cardiac T-Tubule Microanatomy and Function.

Unique to striated muscle cells, transverse tubules (t-tubules) are membrane organelles that consist of sarcolemma penetrating into the myocyte interior, forming a highly branched and interconnected network. Mature t-tubule networks are found in mammalian ventricular cardiomyocytes, with the transverse components of t-tubules occurring near sarcomeric z-discs. Cardiac t-tubules contain membrane microdomains enriched with ion channels and signaling molecules. The microdomains serve as key signaling hubs in regulation of cardiomyocyte function. Dyad microdomains formed at the junctional contact between t-tubule membrane and neighboring sarcoplasmic reticulum are critical in calcium signaling and excitation-contraction coupling necessary for beat-to-beat heart contraction. In this review, we provide an overview of the current knowledge in gross morphology and structure, membrane and protein composition, and function of the cardiac t-tubule network. We also review in detail current knowledge on the formation of functional membrane subdomains within t-tubules, with a particular focus on the cardiac dyad microdomain. Lastly, we discuss the dynamic nature of t-tubules including membrane turnover, trafficking of transmembrane proteins, and the life cycles of membrane subdomains such as the cardiac BIN1-microdomain, as well as t-tubule remodeling and alteration in diseased hearts. Understanding cardiac t-tubule biology in normal and failing hearts is providing novel diagnostic and therapeutic opportunities to better treat patients with failing hearts.

Beat-by-Beat Cardiomyocyte T-Tubule Deformation Drives Tubular Content Exchange.

RATIONALE: The sarcolemma of cardiomyocytes contains many proteins that are essential for electromechanical function in general, and excitation-contraction coupling in particular. The distribution of these proteins is nonuniform between the bulk sarcolemmal surface and membrane invaginations known as transverse tubules (TT). TT form an intricate network of fluid-filled conduits that support electromechanical synchronicity within cardiomyocytes. Although continuous with the extracellular space, the narrow lumen and the tortuous structure of TT can form domains of restricted diffusion. As a result of unequal ion fluxes across cell surface and TT membranes, limited diffusion may generate ion gradients within TT, especially deep within the TT network and at high pacing rates. OBJECTIVE: We postulate that there may be an advective component to TT content exchange, wherein cyclic deformation of TT during diastolic stretch and systolic shortening serves to mix TT luminal content and assists equilibration with bulk extracellular fluid. METHODS AND RESULTS: Using electron tomography, we explore the 3-dimensional nanostructure of TT in rabbit ventricular myocytes, preserved at different stages of the dynamic cycle of cell contraction and relaxation. We show that cellular deformation affects TT shape in a sarcomere length-dependent manner and on a beat-by-beat time-scale. Using fluorescence recovery after photobleaching microscopy, we show that apparent speed of diffusion is affected by the mechanical state of cardiomyocytes, and that cyclic contractile activity of cardiomyocytes accelerates TT diffusion dynamics. CONCLUSIONS: Our data confirm the existence of an advective component to TT content exchange. This points toward a novel mechanism of cardiac autoregulation, whereby the previously implied increased propensity for TT luminal concentration imbalances at high electrical stimulation rates would be countered by elevated advection-assisted diffusion at high mechanical beating rates. The relevance of this mechanism in health and during pathological remodeling (eg, cardiac hypertrophy or failure) forms an exciting target for further research.

Microutrophin expression in dystrophic mice displays myofiber type differences in therapeutic effects.

Gene therapy approaches for DMD using recombinant adeno-associated viral (rAAV) vectors to deliver miniaturized (or micro) dystrophin genes to striated muscles have shown significant progress. However, concerns remain about the potential for immune responses against dystrophin in some patients. Utrophin, a developmental paralogue of dystrophin, may provide a viable treatment option. Here we examine the functional capacity of an rAAV-mediated microutrophin (µUtrn) therapy in the mdx4cv mouse model of DMD. We found that rAAV-µUtrn led to improvement in dystrophic histopathology & mostly restored the architecture of the neuromuscular and myotendinous junctions. Physiological studies of tibialis anterior muscles indicated peak force maintenance, with partial improvement of specific force. A fundamental question for µUtrn therapeutics is not only can it replace critical functions of dystrophin, but whether full-length utrophin impacts the therapeutic efficacy of the smaller, highly expressed µUtrn. As such, we found that µUtrn significantly reduced the spacing of the costameric lattice relative to full-length utrophin. Further, immunostaining suggested the improvement in dystrophic pathophysiology was largely influenced by favored correction of fast 2b fibers. However, unlike µUtrn, µdystrophin (µDys) expression did not show this fiber type preference. Interestingly, µUtrn was better able to protect 2a and 2d fibers in mdx:utrn-/- mice than in mdx4cv mice where the endogenous full-length utrophin was most prevalent. Altogether, these data are consistent with the role of steric hindrance between full-length utrophin & µUtrn within the sarcolemma. Understanding the stoichiometry of this effect may be important for predicting clinical efficacy.

Zebrafish integrin a3b is required for cardiac contractility and cardiomyocyte proliferation.

In cardiac muscle cells, heterodimeric integrin transmembrane receptors are known to serve as mechanotransducers, translating mechanical force to biochemical signaling. However, the roles of many individual integrins have still not been delineated. In this report, we demonstrate that Itga3b is localized to the sarcolemma of cardiomyocytes from 24 to 96 hpf. We further show that heterozygous and homozygous itga3b/bdf mutant embryos display a cardiomyopathy phenotype, with decreased cardiac contractility and reduced cardiomyocyte number. Correspondingly, proliferation of ventricular and atrial cardiomyoctyes and ventricular epicardial cells is decreased in itga3b mutant hearts. The contractile dysfunction of itga3b mutants can be attributed to cardiomyocyte sarcomeric disorganization, including thin myofilaments with blurred and shortened Z-discs. Together, our results reveal that Itga3b localizes to the myocardium sarcolemma, and it is required for cardiac contractility and cardiomyocyte proliferation.

Multi-system neurological disorder associated with a CRYAB variant.

We report a multiplex family with extended multisystem neurological phenotype associated with a CRYAB variant. Two affected siblings were evaluated with whole exome sequencing, muscle biopsy, laser microdissection, and mass spectrometry-based proteomic analysis. Both patients and their mother manifested a combination of early-onset cataracts, cardiomyopathy, cerebellar ataxia, optic atrophy, cognitive impairment, and myopathy. Whole exome sequencing identified a heterozygous c.458C>T variant mapped to the C-terminal extension domain of the Alpha-crystallin B chain, disrupting its function as a molecular chaperone and its ability to suppress protein aggregation. In accordance with the molecular findings, muscle biopsies revealed subsarcolemmal deposits that appeared dark with H&E and trichrome staining were negative for the other routine histochemical staining and for amyloid with the Congo-red stain. Electron microscopy demonstrated that the deposits were composed of numerous parallel fibrils. Laser microdissection and mass spectrometry-based proteomic analysis revealed that the inclusions are almost exclusively composed of crystallized chaperones/heat shock proteins. Moreover,  a structural model suggests that Ser153 could be involved in monomer stabilization, dimer association, and possible binding of partner proteins. We propose that our report potentially expands the complex phenotypic spectrum of alpha B-crystallinopathies with possible effect of a CRYAB variant on the central nervous system.

How the central domain of dystrophin acts to bridge F-actin to sarcolemmal lipids.

Dystrophin is a large intracellular protein that prevents sarcolemmal ruptures by providing a mechanical link between the intracellular actin cytoskeleton and the transmembrane dystroglycan complex. Dystrophin deficiency leads to the severe muscle wasting disease Duchenne Muscular Dystrophy and the milder allelic variant, Becker Muscular Dystrophy (DMD and BMD). Previous work has shown that concomitant interaction of the actin binding domain 2 (ABD2) comprising spectrin like repeats 11 to 15 (R11-15) of the central domain of dystrophin, with both actin and membrane lipids, can greatly increase membrane stiffness. Based on a combination of SAXS and SANS measurements, mass spectrometry analysis of cross-linked complexes and interactive low-resolution simulations, we explored in vitro the molecular properties of dystrophin that allow the formation of ABD2-F-actin and ABD2-membrane model complexes. In dystrophin we identified two subdomains interacting with F-actin, one located in R11 and a neighbouring region in R12 and another one in R15, while a single lipid binding domain was identified at the C-terminal end of R12. Relative orientations of the dystrophin central domain with F-actin and a membrane model were obtained from docking simulation under experimental constraints. SAXS-based models were then built for an extended central subdomain from R4 to R19, including ABD2. Overall results are compatible with a potential F-actin/dystrophin/membrane lipids ternary complex. Our description of this selected part of the dystrophin associated complex bridging muscle cell membrane and cytoskeleton opens the way to a better understanding of how cell muscle scaffolding is maintained through this essential protein.

Live-imaging of revertant and therapeutically restored dystrophin in the DmdEGFP-mdx mouse model for Duchenne muscular dystrophy.

BACKGROUND: Dmdmdx , harbouring the c.2983C>T nonsense mutation in Dmd exon 23, is a mouse model for Duchenne muscular dystrophy (DMD), frequently used to test therapies aimed at dystrophin restoration. Current translational research is methodologically hampered by the lack of a reporter mouse model, which would allow direct visualization of dystrophin expression as well as longitudinal in vivo studies. METHODS: We generated a DmdEGFP-mdx reporter allele carrying in cis the mdx-23 mutation and a C-terminal EGFP-tag. This mouse model allows direct visualization of spontaneously and therapeutically restored dystrophin-EGFP fusion protein either after natural fibre reversion, or for example, after splice modulation using tricyclo-DNA to skip Dmd exon 23, or after gene editing using AAV-encoded CRISPR/Cas9 for Dmd exon 23 excision. RESULTS: Intravital microscopy in anaesthetized mice allowed live-imaging of sarcolemmal dystrophin-EGFP fusion protein of revertant fibres as well as following therapeutic restoration. Dystrophin-EGFP-fluorescence persisted ex vivo, allowing live-imaging of revertant and therapeutically restored dystrophin in isolated fibres ex vivo. Expression of the shorter dystrophin-EGFP isoforms Dp71 in the brain, Dp260 in the retina, and Dp116 in the peripheral nerve remained unabated by the mdx-23 mutation. CONCLUSION: Intravital imaging of DmdEGFP-mdx muscle permits novel experimental approaches such as the study of revertant and therapeutically restored dystrophin in vivo and ex vivo.

Inducible Fgf13 ablation enhances caveolae-mediated cardioprotection during cardiac pressure overload.

The fibroblast growth factor (FGF) homologous factor FGF13, a noncanonical FGF, has been best characterized as a voltage-gated Na+ channel auxiliary subunit. Other cellular functions have been suggested, but not explored. In inducible, cardiac-specific Fgf13 knockout mice, we found-even in the context of the expected reduction in Na+ channel current-an unanticipated protection from the maladaptive hypertrophic response to pressure overload. To uncover the underlying mechanisms, we searched for components of the FGF13 interactome in cardiomyocytes and discovered the complete set of the cavin family of caveolar coat proteins. Detailed biochemical investigations showed that FGF13 acts as a negative regulator of caveolae abundance in cardiomyocytes by controlling the relative distribution of cavin 1 between the sarcolemma and cytosol. In cardiac-specific Fgf13 knockout mice, cavin 1 redistribution to the sarcolemma stabilized the caveolar structural protein caveolin 3. The consequent increase in caveolae density afforded protection against pressure overload-induced cardiac dysfunction by two mechanisms: (i) enhancing cardioprotective signaling pathways enriched in caveolae, and (ii) increasing the caveolar membrane reserve available to buffer membrane tension. Thus, our results uncover unexpected roles for a FGF homologous factor and establish FGF13 as a regulator of caveolae-mediated mechanoprotection and adaptive hypertrophic signaling.

Suppression of ß1-Adrenoceptor Autoantibodies is Involved in the Antiarrhythmic Effects of Omega-3 Fatty Acids in Male and Female Hypertensive Rats.

The arrhythmogenic potential of ß1-adrenoceptor autoantibodies (ß1-AA), as well as antiarrhythmic properties of omega-3 in heart diseases, have been reported while underlying mechanisms are poorly understood. We aimed to test our hypothesis that omega-3 (eicosapentaenoic acid-EPA, docosahexaenoic acid-DHA) may inhibit matrix metalloproteinase (MMP-2) activity to prevent cleavage of ß1-AR and formation of ß1-AA resulting in attenuation of pro-arrhythmic connexin-43 (Cx43) and protein kinase C (PKC) signaling in the diseased heart. We have demonstrated that the appearance and increase of ß1-AA in blood serum of male and female 12-month-old spontaneously hypertensive rats (SHR) was associated with an increase of inducible ventricular fibrillation (VF) comparing to normotensive controls. In contrast, supplementation of hypertensive rats with omega-3 for two months suppressed ß1-AA levels and reduced incidence of VF. Suppression of ß1-AA was accompanied by a decrease of elevated myocardial MMP-2 activity, preservation of cardiac cell membrane integrity and Cx43 topology. Moreover, omega-3 abrogated decline in expression of total Cx43 as well as its phosphorylated forms at serine 368 along with PKC-ε, while decreased pro-fibrotic PKC-δ levels in hypertensive rat heart regardless the sex. The implication of MMP-2 in the action of omega-3 was also demonstrated in cultured cardiomyocytes in which desensitization of ß1-AR due to permanent activation of ß1-AR with isoproterenol was prevented by MMP-2 inhibitor or EPA. Collectively, these data support the notion that omega-3 via suppression of ß1-AA mechanistically controlled by MMP-2 may attenuate abnormal of Cx43 and PKC-ε signaling; thus, abolish arrhythmia substrate and protect rats with an advanced stage of hypertension from malignant arrhythmias.

Dysferlin mediates membrane tubulation and links T-tubule biogenesis to muscular dystrophy.

The multi-C2 domain protein dysferlin localizes to the plasma membrane and the T-tubule system in skeletal muscle; however, its physiological mode of action is unknown. Mutations in the DYSF gene lead to autosomal recessive limb-girdle muscular dystrophy type 2B and Miyoshi myopathy. Here, we show that dysferlin has membrane tubulating capacity and that it shapes the T-tubule system. Dysferlin tubulates liposomes, generates a T-tubule-like membrane system in non-muscle cells, and links the recruitment of phosphatidylinositol 4,5-bisphosphate to the biogenesis of the T-tubule system. Pathogenic mutant forms interfere with all of these functions, indicating that muscular wasting and dystrophy are caused by the dysferlin mutants' inability to form a functional T-tubule membrane system.

Quantification of lectin fluorescence in GNE myopathy muscle biopsies.

INTRODUCTION: GNE myopathy is an adult-onset muscle disorder characterized by impaired sialylation of (muscle) glycans, detectable by lectin histochemistry. We describe a standardized method to quantify (lectin-) fluorescence in muscle sections, applicable for diagnosis and response to therapy for GNE myopathy. METHODS: Muscle sections were fluorescently labeled with the sialic acid-binding Sambucus nigra agglutinin (SNA) lectin and antibodies to sarcolemma residence protein caveolin-3 (CAV-3). Entire tissue sections were imaged in tiles and fluorescence was quantified. RESULTS: SNA fluorescence co-localizing with CAV-3 was ∼50% decreased in GNE myopathy biopsies compared with muscle-matched controls, confirming previous qualitative results. DISCUSSION: This quantitative fluorescence method can accurately determine sialylation status of GNE myopathy muscle biopsies. This method is adaptable for expression of other membrane-associated muscle proteins, and may be of benefit for disorders in which therapeutic changes in expression are subtle and difficult to assess by other methods. Muscle Nerve 58: 286-292, 2018.

Intracellular Reorganization of Cardiomyocytes in Dyslipidemic Cardiomyopathies.

The study examined the myocardial ultrastructural alterations in rats maintained on various atherogenic diets. It revealed the complex ultrastructural alterations of cardiomyocytes and endotheliocytes (including the lytic and destructive changes of the intracellular organelles, upregulation of the autophagocytosis in the cardiomyocytes, and necrobiosis with apoptosis of endotheliocytes) reflecting the cytopathic features of circulating cholesterol and lipoproteins, whose elevation determined the intensity of destructive processes. The revealed peculiarities in the changes of lipid inclusions (their osmiophilic transformation) in cardiomyocytes can be provoked by entry of cholesterol into the cells and its further metabolic modifications. During moderate dyslipidemia, the cardiomyocytes demonstrated the ultrastructural signs of induction of intracellular regeneration (marked with the clusters of polysomes in the intermyofibrillar and subsarcolemmal spaces with appearance of neogenic myofilaments) and upregulated pinocytotic activity. In all cases, up-regulated autophagocytosis in cardiomyocytes was accompanied by accumulation of myelin- and vacuole-like structures in the intercellular spaces and capillary lumens paralleled with appearance of activated forms of macrophages and fibroblasts in the myocardium.

MG53 is dispensable for T-tubule maturation but critical for maintaining T-tubule integrity following cardiac stress.

The cardiac transverse (T)-tubule membrane system is the safeguard for cardiac function and undergoes dramatic remodeling in response to cardiac stress. However, the mechanism by which cardiomyocytes repair damaged T-tubule network remains unclear. In the present study, we tested the hypothesis that MG53, a muscle-specific membrane repair protein, antagonizes T-tubule damage to protect against maladaptive remodeling and thereby loss of excitation-contraction coupling and cardiac function. Using MG53-knockout (MG53-KO) mice, we first established that deficiency of MG53 had no impact on maturation of the T-tubule network in developing hearts. Additionally, MG53 ablation did not influence T-tubule integrity in unstressed adult hearts as late as 10months of age. Following left ventricular pressure overload-induced cardiac stress, MG53 protein levels were increased by approximately three-fold in wild-type mice, indicating that pathological stress induces a significant upregulation of MG53. MG53-deficient mice had worsened T-tubule disruption and pronounced dysregulation of Ca2+ handling properties, including decreased Ca2+ transient amplitude and prolonged time to peak and decay. Moreover, MG53 deficiency exacerbated cardiac hypertrophy and dysfunction and decreased survival following cardiac stress. Our data suggest MG53 is not required for T-tubule development and maintenance in normal physiology. However, MG53 is essential to preserve T-tubule integrity and thereby Ca2+ handling properties and cardiac function under pathological cardiac stress.

Ultra-structural time-course study in the C. elegans model for Duchenne muscular dystrophy highlights a crucial role for sarcomere-anchoring structures and sarcolemma integrity in the earliest steps of the muscle degeneration process.

Duchenne muscular dystrophy (DMD) is a genetic disease characterized by progressive muscle degeneration due to mutations in the dystrophin gene. In spite of great advances in the design of curative treatments, most patients currently receive palliative therapies with steroid molecules such as prednisone or deflazacort thought to act through their immunosuppressive properties. These molecules only slightly slow down the progression of the disease and lead to severe side effects. Fundamental research is still needed to reveal the mechanisms involved in the disease that could be exploited as therapeutic targets. By studying a Caenorhabditis elegans model for DMD, we show here that dystrophin-dependent muscle degeneration is likely to be cell autonomous and affects the muscle cells the most involved in locomotion. We demonstrate that muscle degeneration is dependent on exercise and force production. Exhaustive studies by electron microscopy allowed establishing for the first time the chronology of subcellular events occurring during the entire process of muscle degeneration. This chronology highlighted the crucial role for dystrophin in stabilizing sarcomeric anchoring structures and the sarcolemma. Our results suggest that the disruption of sarcomeric anchoring structures and sarcolemma integrity, observed at the onset of the muscle degeneration process, triggers subcellular consequences that lead to muscle cell death. An ultra-structural analysis of muscle biopsies from DMD patients suggested that the chronology of subcellular events established in C. elegans models the pathogenesis in human. Finally, we found that the loss of sarcolemma integrity was greatly reduced after prednisone treatment suggesting a role for this molecule in plasma membrane stabilization.

Novel insights on the relationship between T-tubular defects and contractile dysfunction in a mouse model of hypertrophic cardiomyopathy.

Abnormalities of cardiomyocyte Ca(2+) homeostasis and excitation-contraction (E-C) coupling are early events in the pathogenesis of hypertrophic cardiomyopathy (HCM) and concomitant determinants of the diastolic dysfunction and arrhythmias typical of the disease. T-tubule remodelling has been reported to occur in HCM but little is known about its role in the E-C coupling alterations of HCM. Here, the role of T-tubule remodelling in the electro-mechanical dysfunction associated to HCM is investigated in the Δ160E cTnT mouse model that expresses a clinically-relevant HCM mutation. Contractile function of intact ventricular trabeculae is assessed in Δ160E mice and wild-type siblings. As compared with wild-type, Δ160E trabeculae show prolonged kinetics of force development and relaxation, blunted force-frequency response with reduced active tension at high stimulation frequency, and increased occurrence of spontaneous contractions. Consistently, prolonged Ca(2+) transient in terms of rise and duration are also observed in Δ160E trabeculae and isolated cardiomyocytes. Confocal imaging in cells isolated from Δ160E mice reveals significant, though modest, remodelling of T-tubular architecture. A two-photon random access microscope is employed to dissect the spatio-temporal relationship between T-tubular electrical activity and local Ca(2+) release in isolated cardiomyocytes. In Δ160E cardiomyocytes, a significant number of T-tubules (>20%) fails to propagate action potentials, with consequent delay of local Ca(2+) release. At variance with wild-type, we also observe significantly increased variability of local Ca(2+) transient rise as well as higher Ca(2+)-spark frequency. Although T-tubule structural remodelling in Δ160E myocytes is modest, T-tubule functional defects determine non-homogeneous Ca(2+) release and delayed myofilament activation that significantly contribute to mechanical dysfunction.

Supramolecular organization of the human N-BAR domain in shaping the sarcolemma membrane.

The 30kDa N-BAR domain of the human Bin1 protein is essential for the generation of skeletal muscle T-tubules. By electron cryo-microscopy and electron cryo-tomography with a direct electron detector, we found that Bin1-N-BAR domains assemble into scaffolds of low long-range order that form flexible membrane tubules. The diameter of the tubules closely matches the curved shape of the N-BAR domain, which depends on the composition of the target membrane. These insights are fundamental to our understanding of T-tubule formation and function in human skeletal muscle.

Costamere proteins and their involvement in myopathic processes.

Muscle fibres are very specialised cells with a complex structure that requires a high level of organisation of the constituent proteins. For muscle contraction to function properly, there is a need for not only sarcomeres, the contractile structures of the muscle fibre, but also costameres. These are supramolecular structures associated with the sarcolemma that allow muscle adhesion to the extracellular matrix. They are composed of protein complexes that interact and whose functions include maintaining cell structure and signal transduction mediated by their constituent proteins. It is important to improve our understanding of these structures, as mutations in various genes that code for costamere proteins cause many types of muscular dystrophy. In this review, we provide a description of costameres detailing each of their constituent proteins, such as dystrophin, dystrobrevin, syntrophin, sarcoglycans, dystroglycans, vinculin, talin, integrins, desmin, plectin, etc. We describe as well the diseases associated with deficiency thereof, providing a general overview of their importance.

The human myotendinous junction: an ultrastructural and 3D analysis study.

The myotendinous junction (MTJ) is a specialized structure in the musculotendinous system, where force is transmitted from muscle to tendon. Animal models have shown that the MTJ takes form of tendon finger-like processes merging with muscle tissue. The human MTJ is largely unknown and has never been described in three dimensions (3D). The aim of this study was to describe the ultrastructure of the human MTJ and render 3D reconstructions. Fourteen subjects (age 25 ± 3 years) with isolated injury of the anterior cruciate ligament (ACL), scheduled for reconstruction with a semitendinosus/gracilis graft were included. Semitendinosus and gracilis tendons were stripped as grafts for the ACL reconstruction. The MTJ was isolated from the grafts and prepared for transmission electron microscopy (TEM) and focused ion beam/scanning electron microscopy. It was possible to isolate recognizable MTJ tissue from all 14 patients. TEM images displayed similarities to observations in animals: Sarcolemmal evaginations observed as finger-like processes from the tendon and endomysium surrounding the muscle fibers, with myofilaments extending from the final Z-line of the muscle fiber merging with the tendon tissue. The 3D reconstruction revealed that tendon made ridge-like protrusions, which interdigitiated with groove-like indentations in the muscle cell.

Complex sarcolemmal invaginations mimicking myotendinous junctions in a case of Laing early-onset distal myopathy.

Distal myopathies are a group of clinically and pathologically overlapping muscle diseases that are genetically complex and can represent a diagnostic challenge. Laing early-onset distal myopathy (MPD1) is a form of distal myopathy caused by mutations in the MYH7 gene, which encodes the beta myosin heavy chain protein expressed in type 1 skeletal muscle fibers and cardiac myocytes. Here, we present a case of genetically confirmed MPD1 with a typical clinical presentation but distinctive light microscopic and ultrastructural findings on muscle biopsy. A 39-year-old professional male cellist presented with a bilateral foot drop that developed by age 8; analysis of the family pedigree showed an autosomal dominant pattern of inheritance. The physical exam demonstrated bilateral weakness of ankle dorsiflexors, toe extensors and finger extensors; creatine kinase level was normal. Biopsy of the quadriceps femoris muscle showed predominance and hypotrophy of type 1 fibers, hybrid fibers with co-expression of slow and fast myosin proteins (both in highly atrophic and normal size range), moth-eaten fibers and mini-cores, lack of rimmed vacuoles and rare desmin-positive eosinophilic sarcoplasmic inclusions. In addition to these abnormalities often observed in MPD1, the biopsy demonstrated frequent clefted fibers with complex sarcolemmal invaginations; on ultrastructural examination, these structures closely mimicked myotendinous junctions but were present away from the tendon and were almost exclusively found in type 1 fibers. Sequencing analysis of the MYH7 gene in the index patient and other affected family members demonstrated a previously described heterozygous c.4522_4524delGAG (p.Glu1508del) mutation. This case widens the pathologic spectrum of MPD1 and highlights the pathologic and clinical variability that can accompany the same genetic mutation, suggesting a significant role for modifier genes in MPD1 pathogenesis.