Skeletal Muscle Sarcomere Research Articles

Modulation of Skeletal Muscle Contraction by Myosin Phosphorylation.

The striated muscle sarcomere is a highly organized and complex enzymatic and structural organelle. Evolutionary pressures have played a vital role in determining the structure-function relationship of each protein within the sarcomere. A key part of this multimeric assembly is the light chain-binding domain (LCBD) of the myosin II motor molecule. This elongated "beam" functions as a biological lever, amplifying small interdomain movements within the myosin head into piconewton forces and nanometer displacements against the thin filament during the cross-bridge cycle. The LCBD contains two subunits known as the essential and regulatory myosin light chains (ELC and RLC, respectively). Isoformic differences in these respective species provide molecular diversity and, in addition, sites for phosphorylation of serine residues, a highly conserved feature of striated muscle systems. Work on permeabilized skeletal fibers and thick filament systems shows that the skeletal myosin light chain kinase catalyzed phosphorylation of the RLC alters the "interacting head motif" of myosin motor heads on the thick filament surface, with myriad consequences for muscle biology. At rest, structure-function changes may upregulate actomyosin ATPase activity of phosphorylated cross-bridges. During activation, these same changes may increase the Ca2+ sensitivity of force development to enhance force, work, and power output, outcomes known as "potentiation." Thus, although other mechanisms may contribute, RLC phosphorylation may represent a form of thick filament activation that provides a "molecular memory" of contraction. The clinical significance of these RLC phosphorylation mediated alterations to contractile performance of various striated muscle systems are just beginning to be understood. © 2017 American Physiological Society. Compr Physiol 7:171-212, 2017.

Muscle contraction: Sliding filament history, sarcomere dynamics and the two Huxleys.

Despite having all the evidence needed to come to the right conclusions in the middle of the 1800s, it was not until the 1950s that it was realised by two unrelated Huxleys and their collaborators that striated muscle sarcomeres contain overlapping sets of filaments which do not change much in length and which slide past each other when the muscle sarcomere shortens. It then took quite a while to convince others that this was the case, but now the idea of sliding filaments is fundamental to our understanding of how any muscle works. Here a brief overview of the history of the discovery of sliding filaments and the factors that were missed in the 1800s is followed by an analysis of the more recent experiments which have added to the conviction that all muscles operate on the same guiding principles; two sets of sliding filaments, independent force generators and a mechanism of protein rowing that makes the filaments slide.

Cardiomyocyte-Specific Human Bcl2-Associated Anthanogene 3 P209L Expression Induces Mitochondrial Fragmentation, Bcl2-Associated Anthanogene 3 Haploinsufficiency, and Activates p38 Signaling

The Bcl2-associated anthanogene (BAG) 3 protein is a member of the BAG family of cochaperones, which supports multiple critical cellular processes, including critical structural roles supporting desmin and interactions with heat shock proteins and ubiquitin ligases intimately involved in protein quality control. The missense mutation P209L in exon 3 results in a primarily cardiac phenotype leading to skeletal muscle and cardiac complications. At least 10 other Bag3 mutations have been reported, nine resulting in a dilated cardiomyopathy for which no specific therapy is available. We generated αMHC-human Bag3 P209L transgenic mice and characterized the progressive cardiac phenotype invivo to investigate its utility in modeling human disease, understand the underlying molecular mechanisms, and identify potential therapeutic targets. We identified a progressive heart failure by echocardiography and Doppler analysis and the presence of pre-amyloid oligomers at 1 year. Paralleling the pathogenesis of neurodegenerative diseases (eg, Parkinson disease), pre-amyloid oligomers-associated alterations in cardiac mitochondrial dynamics, haploinsufficiency of wild-type BAG3, and activation of p38 signaling were identified. Unexpectedly, increased numbers of activated cardiac fibroblasts were identified in Bag3 P209L Tg+ hearts without increased fibrosis. Together, these findings point to a previously undescribed therapeutic target that may have application to mutation-induced myofibrillar myopathies as well as other common causes of heart failure that commonly harbor misfolded proteins.

Disorder profile of nebulin encodes a vernierlike position sensor for the sliding thin and thick filaments of the skeletal muscle sarcomere.

Nebulin is an about 1μm long intrinsically disordered scaffold for the thin filaments of skeletal muscle sarcomere. It is a multifunctional elastic protein that wraps around actin filament, stabilizes thin filaments, and regulates Ca-dependent actomyosin interactions. This study investigates whether the disorder profile of nebulin might encode guidelines for thin and thick filament interactions in the sarcomere of the skeletal muscle. The question was addressed computationally by analyzing the predicted disorder profile of human nebulin (6669 residues, ∼200 actin-binding repeats) by pondr and the periodicity of the A-band stripes (reflecting the locations of myosin-associated proteins) in the electron micrographs of the sarcomere. Using the detrended fluctuation analysis, a scale factor for the A-band stripe image data with respect to the nebulin disorder profile was determined to make the thin and thick filaments aligned to have maximum correlation. The empirical mode decomposition method was then applied to identify hidden periodicities in both the nebulin disorder profile and the rescaled A-band data. The decomposition reveals three characteristic length scales (45 nm, 100 nm, and 200 nm) that are relevant for correlational analysis. The dynamical cross-correlation analyses with moving windows at various sarcomere lengths depict a vernierlike design for both periodicities, thus enabling nebulin to sense position and fine tune sarcomere overlap. This shows that the disorder profile of scaffolding proteins may encode a guideline for cellular architecture.

Nebulin Deficiency in Adult Muscle Causes Sarcomeric Defects and Muscle-Type Dependent Changes in Trophicity -- Novel Insights in Nemaline Myopathy

Nebulin is a giant filamentous protein that is coextensive with the actin filaments of the skeletal muscle sarcomere. Nebulin mutations are the main cause of nemaline myopathy (NEM), with typical adult patients having low expression of nebulin, yet the roles of nebulin in adult muscle remain poorly understood. To establish nebulin's functional roles in adult muscle we studied a novel conditional nebulin KO (Neb cKO) mouse model in which nebulin deletion was driven by the muscle creatine kinase (MCK) promotor. Neb cKO mice are born with high nebulin levels in their skeletal muscles but within weeks after birth nebulin expression rapidly falls to barely detectable levels Surprisingly, a large fraction of the mice survive to adulthood with low nebulin levels (<5% of control), contain nemaline rods, and undergo fiber-type switching towards oxidative types. Nebulin deficiency causes a large deficit in specific-force and mechanistic studies provide evidence that a reduced fraction of force-generating crossbridges and shortened thin filaments contribute to the force deficit. Muscles rich in glycolytic fibers upregulate proteolysis pathways (MuRF-1, Fbxo30/MUSA1, Gadd45a) and undergo hypotrophy with smaller cross-sectional areas, worsening their force deficit. Muscles rich in oxidative fibers do not have smaller weights and can even hypertrophy, offsetting their specific-force deficit. These studies reveal nebulin as critically important for force development and trophicity in adult muscle. The Neb cKO phenocopies important aspects of nemaline myopathy (muscle weakness, oxidative fiber type predominance, variable trophicity effects, nemaline rods) and will be highly useful to test therapeutic approaches to ameliorate muscle weakness.

Decreased force enhancement in skeletal muscle sarcomeres with a deletion in titin.

In the cross-bridge theory, contractile force is produced by cross-bridges that form between actin and myosin filaments. However, when a contracting muscle is stretched, its active force vastly exceeds the force that can be attributed to cross-bridges. This unexplained, enhanced force has been thought to originate in the giant protein titin, which becomes stiffer in actively compared with passively stretched sarcomeres by an unknown mechanism. We investigated this mechanism using a genetic mutation (mdm) with a small but crucial deletion in the titin protein. Myofibrils from normal and mdm mice were stretched from sarcomere lengths of 2.5 to 6.0 μm. Actively stretched myofibrils from normal mice were stiffer and generated more force than passively stretched myofibrils at all sarcomere lengths. No increase in stiffness and just a small increase in force were observed in actively compared with passively stretched mdm myofibrils. These results are in agreement with the idea that titin force enhancement stiffens and stabilizes the sarcomere during contraction and that this mechanism is lost with the mdm mutation.

Nebulin deficiency in adult muscle causes sarcomere defects and muscle-type-dependent changes in trophicity: novel insights in nemaline myopathy.

Nebulin is a giant filamentous protein that is coextensive with the actin filaments of the skeletal muscle sarcomere. Nebulin mutations are the main cause of nemaline myopathy (NEM), with typical adult patients having low expression of nebulin, yet the roles of nebulin in adult muscle remain poorly understood. To establish nebulin's functional roles in adult muscle, we studied a novel conditional nebulin KO (Neb cKO) mouse model in which nebulin deletion was driven by the muscle creatine kinase (MCK) promotor. Neb cKO mice are born with high nebulin levels in their skeletal muscles, but within weeks after birth nebulin expression rapidly falls to barely detectable levels Surprisingly, a large fraction of the mice survive to adulthood with low nebulin levels (<5% of control), contain nemaline rods and undergo fiber-type switching toward oxidative types. Nebulin deficiency causes a large deficit in specific force, and mechanistic studies provide evidence that a reduced fraction of force-generating cross-bridges and shortened thin filaments contribute to the force deficit. Muscles rich in glycolytic fibers upregulate proteolysis pathways (MuRF-1, Fbxo30/MUSA1, Gadd45a) and undergo hypotrophy with smaller cross-sectional areas (CSAs), worsening their force deficit. Muscles rich in oxidative fibers do not have smaller weights and can even have hypertrophy, offsetting their specific-force deficit. These studies reveal nebulin as critically important for force development and trophicity in adult muscle. The Neb cKO phenocopies important aspects of NEM (muscle weakness, oxidative fiber-type predominance, variable trophicity effects, nemaline rods) and will be highly useful to test therapeutic approaches to ameliorate muscle weakness.

An "enigmatic" L-carnosine (β-alanyl-L-histidine)? Cell proliferative activity as a fundamental property of a natural dipeptide inherent to traditional antioxidant, anti-aging biological activities: balancing and a hormonally correct agent, novel patented oral therapy dosage formulation for mobility, skeletal muscle power and functional performance, hypothalamic-pituitary- brain relationship in health, aging and stress studies.

Hypothalamic releasing and inhibiting hormones are major neuroendocrine regulators of human body metabolism being driven directly to the anterior pituitary gland via hypothalamic-hypophyseal portal veins. The alternative physiological or therapeutic interventions utilizing the pharmaco-nutritional boost of imidazole-containing dipeptides (non-hydrolized oral form of carnosine, carcinine, N-acetylcarnosine lubricant eye drops) can maintain health, enhance physical exercise performance and prevent ageing. Carnosine (β-alanyl-L-histidine) is synthesized in mammalian skeletal muscle. There is an evidence that the release of carnosine from the skeletal muscle sarcomeres moieties during physical exercise affects autonomic neurotransmission and physiological functions. Carnosine released from skeletal muscle during exercise acts as a powerful afferent physiological signaling stimulus for hypothalamus, may be transported into the hypothalamic tuberomammillary nucleus (TMN), specifically to TMN-histamine neurons and hydrolyzed herewith via activities of carnosine-degrading enzyme (carnosinase 2) localized in situ. Through the colocalized enzymatic activity of Histidine decarboxylase in the histaminergic neurons, the resulting L-histidine may subsequently be converted into histamine, which could be responsible for the effects of carnosine on neurotransmission and physiological function. Carnosine and its imidazole-containing dipeptide derivatives are renowned for their anti-aging, antioxidant, membrane protective, metal ion chelating, buffering, anti-glycation/ transglycating activities used to prevent and treat a spectrum of age-related and metabolic diseases, such as neurodegenerative disease, sight threatening eye diseases, Diabetes mellitus and its complications, cancers and other disorders due to their wide spectrum biological activities. The precursor of carnosine (and related imidazole containing compounds) synthesis in skeletal muscles beta-alanine is used as the oral supplement by athletes to achieve the fine sporting art results due to the buffering activities of carnosine and its related imidazole- containing compounds which contribute to the maintenance of the acid-base balance in the acting muscles. This work originally emphasizes that overall data indicate the signaling activities of carnosine in skeletal and cardiac muscles switching on the mechanisms of exercise-induced telomere protection and point to the stress response and growth/cellular proliferation pathways as high-priority candidates for the ongoing studies and therapeutic concepts. The therapeutic interventions utilizing the specific oral formulation (Can-C Plus), timing dosing and pharmaco-nutritional boost of imidazolecontaining dipeptides can maintain health, enhance physical exercise performance and prevent aging. The patented therapeutic concept protects the existence of the interesting physiological major activities, better controls and therapeutic treatments for aging/age-related disorders (including age-related loss of muscle mass and muscle function) using carnosine dipeptide for cellular rejuvenation and manipulating telomeres and enzyme telomerase activity that may reduce some of the physiological declines that accompany aging.

Structural insight into the Phosphoinositide-Regulated Cellular Dynamics of Alpha-Actinin

Alpha-actinin2 is a fundamental component of striated muscle sarcomeres. It localizes to the Z-disk, the macromolecular assembly that connects two adjacent sarcomeres where it provides mechanical stability during contraction but also plays a pivotal role during Z-disk assembly. A regulatory role by phosphatidylinositol-bis-phosphate (PiP2) has been proposed for all 4 alpha-actinin isoforms but not validated at structural level. The X-ray structure of full-length alpha-actinin2 suggests the existence of two different conformational states. In the ground state, alpha-actinin exists in an intramolecular autoinhibited conformation, where the C-terminal EF-hands bind a titin-like pseudoligand at the connection between rod and actin-binding domains. PiP2 acts as a potential trigger for the transition to an open state capable of titin-binding. We hypothesize this mechanism plays a key role during Z-disk maturation, with the open-state allowing interaction with key binding partners. Super-resolution and correlative electron microscopy of mutant-transfected Neonatal Rat Cardiomyocytes (NRC) shed new light on the internal organization of the Z-disk and the regulatory mechanism of alpha-actinin ligand binding. Combining fluorescence confocal microscopy and live-cell imaging in NRC, we identified a crucial role for PiP2, which regulates the intracellular dynamics of alpha-actinin as seen in Fluorescence Recovery After Photobleaching experiments with a PiP2-binding deficient mutant. A second alpha-actinin mutant, in constitutively open-conformation, impaired overall sarcomere assembly by reduced intracellular dynamics, suggesting that PiP2 might not be limited to a positive regulation, but is also essential for the dynamic equilibrium of the protein.

Titin Hysteresis is Greater for Actively Lengthened Compared to Passively Lengthened Skeletal Muscle Sarcomeres

Titin is a giant protein found in skeletal muscle which behaves mechanically like a molecular spring. The extensible region of titin within the sarcomere contains spring-like domains arranged in series with stiffness and visco-elastic properties which come into play in an orderly fashion with increasing sarcomere length. When lengthened within the physiologically relevant range of sarcomere excursion, first the Ig domains align, followed by stretching of the PEVK region. Beyond the physiological range, Ig domains unfold, and this unfolding is thought to be highly visco-elastic.Active muscle lengthened from 1.8 to 4.0µm and to beyond myofilament overlap (> 4.0µm) will produce more force than passively lengthened muscle at the same sarcomere length [1]. Higher force beyond 4µm is expected to increase the hysteresis of titin by increasing Ig domain unfolding.Rabbit psoas myofibrils were used for these experiments. Single myofibrils with an initial mean sarcomere length (SL) of 2.8µm were activated with Ca+2 and ATP (no Ca+2 for passive) and lengthened to approximately 4.5µm and then immediately cycled (10x) ±0.25µm. Mean 1st peak force was higher in active (n=6) versus passive (n=5) at 338.9±137.2nN/µm2 versus 103.2±17.2nN/µm2, as expected. Mean peak force fell continuously from the 1st to the 10th cycle in active, while the peak force stabilized in passive tests by the 4th cycle. Hysteresis was greater in the active tests; mean 1st cycle hysteresis was 10.8±5.4nN·µm versus 1.3±0.9nN·µm. We conclude that the higher forces observed at all SL for active compared to passive lengthening results in increased visco-elastic behaviour in titin through increased Ig domain unfolding and this unfolding is not completed in active tests even after 10 repeated stretch-shortening cycles at long SL.[1] Leonard and Herzog, 2010, AJP-(Cell) 299:14-20.

Actin scaffolding by clathrin heavy chain is required for skeletal muscle sarcomere organization

Actin scaffolding by clathrin heavy chain is required for skeletal muscle sarcomere organization

Clathrin’s muscle-building regimen

The clathrin heavy chain forms a membrane scaffold that organizes skeletal muscle sarcomeres.

Actin scaffolding by clathrin heavy chain is required for skeletal muscle sarcomere organization.

The ubiquitous clathrin heavy chain (CHC), the main component of clathrin-coated vesicles, is well characterized for its role in intracellular membrane traffic and endocytosis from the plasma membrane (PM). Here, we demonstrate that in skeletal muscle CHC regulates the formation and maintenance of PM-sarcomere attachment sites also known as costameres. We show that clathrin forms large coated lattices associated with actin filaments and the muscle-specific isoform of α-actinin at the PM of differentiated myotubes. Depletion of CHC in myotubes induced a loss of actin and α-actinin sarcomeric organization, whereas CHC depletion in vivo induced a loss of contractile force due to the detachment of sarcomeres from the PM. Our results suggest that CHC contributes to the formation and maintenance of the contractile apparatus through interactions with costameric proteins and highlight an unconventional role for CHC in skeletal muscle that may be relevant to pathophysiology of neuromuscular disorders.

Localization and Binding Partners of SESTD1 in Skeletal Muscles

Myofibrillogenesis is a complex production system of the striated muscles, which are correctly built by a lot of structural and signaling proteins. Some insights about the molecular mechanism of myofibrillogenesis were elucidated but most of them are not uncovered. SESTD1 is a novel protein that consists of SEC14 domain, three spectrin repeats and unique sequences and is expressed in various tissues including striated muscles. One research suggests that SESTD1 is involved in the planar cell polarity pathway during mammalian embryonic development and another research suggests that SESTD1 regulates some transient receptor potential channels in smooth muscle. However, it remains unclear about the function of SESTD1 in striated muscles. In this research, we investigated localization and binding partner of SESTD1 in striated muscles. SESTD1 was detected in the whole extracts from skeletal but not cardiac muscles by western blot using anti-SESTD1 monoclonal antibody. Immunofluorescence microscopy using the antibody revealed that SESDT1 is localized at Z-line of sarcomere. To search the binding partners of SESTD1, we carried out yeast two-hybrid screening with skeletal muscle cDNA libraries. Several proteins located in Z-line were obtained as candidates for SESTD1 C-terminus binding proteins and alpha-actin was obtained as a SESTD1 N-terminus binding protein. These results indicate that SESTD1 is the novel Z-line protein of the sarcomere in skeletal muscle and may function through binding to both Z-line structural proteins and actin filaments. We are currently investigating the role of SESTD1 in Z-line formation and actin filament assembly.

Loss of Tropomodulin4 in the zebrafish mutant träge causes cytoplasmic rod formation and muscle weakness reminiscent of nemaline myopathy.

Nemaline myopathy is an inherited muscle disease that is mainly diagnosed by the presence of nemaline rods in muscle biopsies. Of the nine genes associated with the disease, five encode components of striated muscle sarcomeres. In a genetic zebrafish screen, the mutant träge (trg) was isolated based on its reduction in muscle birefringence, indicating muscle damage. Myofibres in trg appeared disorganised and showed inhomogeneous cytoplasmic eosin staining alongside malformed nuclei. Linkage analysis of trg combined with sequencing identified a nonsense mutation in tropomodulin4 (tmod4), a regulator of thin filament length and stability. Accordingly, although actin monomers polymerize to form thin filaments in the skeletal muscle of tmod4trg mutants, thin filaments often appeared to be dispersed throughout myofibres. Organised myofibrils with the typical striation rarely assemble, leading to severe muscle weakness, impaired locomotion and early death. Myofibrils of tmod4trg mutants often featured thin filaments of various lengths, widened Z-disks, undefined H-zones and electron-dense aggregations of various shapes and sizes. Importantly, Gomori trichrome staining and the lattice pattern of the detected cytoplasmic rods, together with the reactivity of rods with phalloidin and an antibody against actinin, is reminiscent of nemaline rods found in nemaline myopathy, suggesting that misregulation of thin filament length causes cytoplasmic rod formation in tmod4trg mutants. Although Tropomodulin4 has not been associated with myopathy, the results presented here implicateTMOD4 as a novel candidate for unresolved nemaline myopathies and suggest that the tmod4trg mutant will be a valuable tool to study human muscle disorders.

Interactive Properties of A-Band Titin

The giant protein titin (chain weight ∼3 MDa) has important roles in assembly and contractile function in vertebrate striated muscle sarcomeres. The molecule consists principally of ∼300 Ig and Fn3 domains in a chain of more than 1 μm long spanning half the sarcomere. The A-band part is attached to the thick (myosin) filament with six molecules in each half filament. The I-band part makes an elastic connection between the tip of the filament and the Z-disc. The central region of A-band titin (C-zone, ∼0.5 μm long) contains 11 copies of what is called the large super-repeat, Ig-Fn3-Fn3-Ig-Fn3-Fn3-Fn3-Ig-Fn3-Fn3-Fn3. The lengths of the large super-repeat (∼43 nm) and of its sub-periods (∼14 nm) correspond to the two main periodicities of the thick filament. C-zone titin is thought to make at least three distinct types of interactions: with itself, with myosin, and with myosin binding protein-C. We studied three recombinant 2-domain fragments from the three sub-periods of a large super-repeat. At physiological ionic strength all three constructs were soluble and mostly monomeric, whereas reduced salt resulted in self-association. Self-association was strongest in the case of the third sub-period construct leading to its complete sedimentation. Electron microscopy revealed large net-like oligomers suggesting regularity in the domains interactions. In the constructs from the first two sub-periods, side-by-side dimers were observed and oligomers were seen only rarely. Co-sedimentation and solid phase assays showed binding of the constructs to the light meromyosin part of myosin but did not reveal any significant differences in the interactions. Modelling of the proteins folds and electrostatics was consistent with the observed interactions being mainly electrostatic.

The titin A-band rod domain is dispensable for initial thick filament assembly in zebrafish

The sarcomeres of skeletal and cardiac muscle are highly structured protein arrays, consisting of thick and thin filaments aligned precisely to one another and to their surrounding matrix. The contractile mechanisms of sarcomeres are generally well understood, but how the patterning of sarcomeres is initiated during early skeletal muscle and cardiac development remains uncertain. Two of the most widely accepted hypotheses for this process include the “molecular ruler” model, in which the massive protein titin defines the length of the sarcomere and provides a scaffold along which the myosin thick filament is assembled, and the “premyofibril” model, which proposes that thick filament formation does not require titin, but that a “premyofibril” consisting of non-muscle myosin, α-actinin and cytoskeletal actin is used as a template. Each model posits a different order of necessity of the various components, but these have been difficult to test in vivo. Zebrafish motility mutants with developmental defects in sarcomere patterning are useful for the elucidation of such mechanisms, and here we report the analysis of the herzschlag mutant, which shows deficits in both cardiac and skeletal muscle. The herzschlag mutant produces a truncated titin protein, lacking the C-terminal rod domain that is proposed to act as a thick filament scaffold, yet muscle patterning is still initiated, with grossly normal thick and thin filament assembly. Only after embryonic muscle contraction begins is breakdown of sarcomeric myosin patterning observed, consistent with the previously noted role of titin in maintaining the contractile integrity of mature sarcomeres. This conflicts with the “molecular ruler” model of early sarcomere patterning and supports a titin-independent model of thick filament organization during sarcomerogenesis. These findings are also consistent with the symptoms of human titin myopathies that exhibit a late onset, such as tibial muscular dystrophy.

Engineered Human Muscle Tissue from Skeletal Muscle Derived Stem Cells and Induced Pluripotent Stem Cell Derived Cardiac Cells.

During development, cardiac and skeletal muscle share major transcription factors and sarcomere proteins which were generally regarded as specific to either cardiac or skeletal muscle but not both in terminally differentiated adult cardiac or skeletal muscle. Here, we investigated whether artificial muscle constructed from human skeletal muscle derived stem cells (MDSCs) recapitulates developmental similarities between cardiac and skeletal muscle. We constructed 3-dimensional collagen-based engineered muscle tissue (EMT) using MDSCs (MDSC-EMT) and compared the biochemical and contractile properties with EMT using induced pluripotent stem (iPS) cell-derived cardiac cells (iPS-EMT). Both MDSC-EMT and iPS-EMT expressed cardiac specific troponins, fast skeletal muscle myosin heavy chain, and connexin-43 mimicking developing cardiac or skeletal muscle. At the transcriptional level, MDSC-EMT and iPS-EMT upregulated both cardiac and skeletal muscle-specific genes and expressed Nkx2.5 and Myo-D proteins. MDSC-EMT displayed intracellular calcium ion transients and responses to isoproterenol. Contractile force measurements of MDSC-EMT demonstrated functional properties of immature cardiac and skeletal muscle in both tissues. Results suggest that the EMT from MDSCs mimics developing cardiac and skeletal muscle and can serve as a useful in vitro functioning striated muscle model for investigation of stem cell differentiation and therapeutic options of MDSCs for cardiac repair.

The Novel Desmin Mutant p.A120D Impairs Filament Formation, Prevents Intercalated Disk Localization, and Causes Sudden Cardiac Death

The intermediate filament protein desmin is encoded by the gene DES and contributes to the mechanical stabilization of the striated muscle sarcomere and cell contacts within the cardiac intercalated disk. DES mutations cause severe skeletal and cardiac muscle diseases with heterogeneous phenotypes. Recently, DES mutations were also found in patients with arrhythmogenic right ventricular cardiomyopathy. Currently, the cellular and molecular pathomechanisms of the DES mutations leading to this disease are not exactly known. We identified the 2 novel variants DES-p.A120D (c.359C>A) and DES-p.H326R (c.977A>G), which were characterized by cell culture experiments and atomic force microscopy. Family analysis indicated a broad spectrum of cardiomyopathies with a striking frequency of arrhythmias and sudden cardiac deaths. The in vitro experiments of desmin-p.A120D reveal a severe intrinsic filament formation defect causing cytoplasmic aggregates in cell lines and of the isolated recombinant protein. Model variants of codon 120 indicated that ionic interactions contribute to this filament formation defect. Ex vivo analysis of ventricular tissue slices revealed a loss of desmin staining within the intercalated disk and severe cytoplasmic aggregate formation, whereas z-band localization was not affected. The functional experiments of desmin-p.H326R did not demonstrate any differences from wild type. Because of the functional in vivo and in vitro characterization, DES-p.A120D has to be regarded as a pathogenic mutation and DES-p.H326R as a rare variant with unknown significance. Presumably, the loss of the desmin-p. A120D filament localization at the intercalated disk explains its clinical arrhythmogenic potential.

Determining the role of sarcomeric proteins in facioscapulohumeral muscular dystrophy: a study protocol

BackgroundAlthough muscle weakness is a hallmark of facioscapulohumeral muscular dystrophy (FSHD), the molecular mechanisms that lead to weakness in FSHD remain largely unknown. Recent studies suggest aberrant expression of genes involved in skeletal muscle development and sarcomere contractility, and activation of pathways involved in sarcomeric protein degradation. This study will investigate the contribution of sarcomeric protein dysfunction to the pathogenesis of muscle weakness in FSHD.Methods/DesignEvaluation of sarcomeric function using skinned single muscle fiber contractile studies and protein analysis in muscle biopsies (quadriceps femoris and tibialis anterior) from patients with FSHD and age- and gender-matched healthy controls. Patients with other forms of muscular dystrophy and inflammatory myopathy will be included as disease controls to assess whether results are due to changes specific for FSHD, or a consequence of muscle disease in general. A total of 56 participants will be included. Extensive clinical parameters will be measured using MRI, quantitative muscle studies and physical activity assessments.DiscussionThis study is the first to extensively investigate muscle fiber physiology in FSHD following an earlier pilot study suggesting sarcomeric dysfunction in FSHD. The results obtained in this study will increase the understanding of the pathophysiology of muscle weakness in FSHD, and possibly identify novel targets for therapeutic intervention.