The role of the M-band myomesin proteins in muscle integrity and cardiac disease.

Transversal structural elements in cross-striated muscles, such as the M-band or the Z-disc, anchor and mechanically stabilize the contractile apparatus and its minimal unit-the sarcomere. The ability of proteins to target and interact with these structural sarcomeric elements is an inevitable necessity for the correct assembly and functionality of the myofibrillar apparatus. Specifically, the M-band is a well-recognized mechanical and signaling hub dealing with active forces during contraction, while impairment of its function leads to disease and death. Research on the M-band architecture is focusing on the assembly and interactions of the three major filamentous proteins in the region, mainly the three myomesin proteins including their embryonic heart (EH) isoform, titin and obscurin. These proteins form the basic filamentous network of the M-band, interacting with each other as also with additional proteins in the region that are involved in signaling, energetic or mechanosensitive processes. While myomesin-1, titin and obscurin are found in every muscle, the expression levels of myomesin-2 (also known as M-protein) and myomesin-3 are tissue specific: myomesin-2 is mainly expressed in the cardiac and fast skeletal muscles, while myomesin-3 is mainly expressed in intermediate muscles and specific regions of the cardiac muscle. Furthermore, EH-myomesin apart from its role during embryonic stages, is present in adults with specific cardiac diseases. The current work in structural, molecular, and cellular biology as well as in animal models, provides important details about the assembly of myomesin-1, obscurin and titin, the information however about the myomesin-2 and -3, such as their interactions, localization and structural details remain very limited. Remarkably, an increasing number of reports is linking all three myomesin proteins and particularly myomesin-2 to serious cardiovascular diseases suggesting that this protein family could be more important than originally thought. In this review we will focus on the myomesin protein family, the myomesin interactions and structural differences between isoforms and we will provide the most recent evidence why the structurally and biophysically unexplored myomesin-2 and myomesin-3 are emerging as hot targets for understanding muscle function and disease.

Knockout of MYOM1 in human cardiomyocytes leads to myocardial atrophy via impairing calcium homeostasis.

Myomesin-1 (encoded by MYOM1 gene) is expressed in almost all cross-striated muscles, whose family (together with myomesin-2 and myomesin-3) helps to cross-link adjacent myosin to form the M-line in myofibrils. However, little is known about its biological function, causal relationship and mechanisms underlying the MYOM1-related myopathies (especially in the heart). Regrettably, there is no MYMO1 knockout model for its study so far. A better and further understanding of MYOM1 biology is urgently needed. Here, we used CRISPR/Cas9 gene-editing technology to establish an MYOM1 knockout human embryonic stem cell line (MYOM1-/- hESC), which was then differentiated into myomesin-1 deficient cardiomyocytes (MYOM1-/- hESC-CMs) in vitro. We found that myomesin-1 plays an important role in sarcomere assembly, contractility regulation and cardiomyocytes development. Moreover, myomesin-1-deficient hESC-CMs can recapitulate myocardial atrophy phenotype in vitro. Based on this model, not only the biological function of MYOM1, but also the aetiology, pathogenesis, and potential treatments of myocardial atrophy caused by myomesin-1 deficiency can be studied.

Assembly and Maintenance of Myofibrils in Striated Muscle.

In this chapter, we present the current knowledge on de novo assembly, growth, and dynamics of striated myofibrils, the functional architectural elements developed in skeletal and cardiac muscle. The data were obtained in studies of myofibrils formed in cultures of mouse skeletal and quail myotubes, in the somites of living zebrafish embryos, and in mouse neonatal and quail embryonic cardiac cells. The comparative view obtained revealed that the assembly of striated myofibrils is a three-step process progressing from premyofibrils to nascent myofibrils to mature myofibrils. This process is specified by the addition of new structural proteins, the arrangement of myofibrillar components like actin and myosin filaments with their companions into so-called sarcomeres, and in their precise alignment. Accompanying the formation of mature myofibrils is a decrease in the dynamic behavior of the assembling proteins. Proteins are most dynamic in the premyofibrils during the early phase and least dynamic in mature myofibrils in the final stage of myofibrillogenesis. This is probably due to increased interactions between proteins during the maturation process. The dynamic properties of myofibrillar proteins provide a mechanism for the exchange of older proteins or a change in isoforms to take place without disassembling the structural integrity needed for myofibril function. An important aspect of myofibril assembly is the role of actin-nucleating proteins in the formation, maintenance, and sarcomeric arrangement of the myofibrillar actin filaments. This is a very active field of research. We also report on several actin mutations that result in human muscle diseases.

The sarcomeric cytoskeleton: from molecules to motion.

Highly ordered organisation of striated muscle is the prerequisite for the fast and unidirectional development of force and motion during heart and skeletal muscle contraction. A group of proteins, summarised as the sarcomeric cytoskeleton, is essential for the ordered assembly of actin and myosin filaments into sarcomeres, by combining architectural, mechanical and signalling functions. This review discusses recent cell biological, biophysical and structural insight into the regulated assembly of sarcomeric cytoskeleton proteins and their roles in dissipating mechanical forces in order to maintain sarcomere integrity during passive extension and active contraction. α-Actinin crosslinks in the Z-disk show a pivot-and-rod structure that anchors both titin and actin filaments. In contrast, the myosin crosslinks formed by myomesin in the M-band are of a ball-and-spring type and may be crucial in providing stable yet elastic connections during active contractions, especially eccentric exercise.

Fibre typing of intrafusal fibres.

The first descriptions of muscle spindles with intrafusal fibres containing striated myofibrils and nervous elements were given approximately 150 years ago. It took, however, another 100 years to establish the presence of two types of intrafusal muscle fibres: nuclear bag and nuclear chain fibres. The present paper highlights primarily the contribution of Robert Banks in fibre typing of intrafusal fibres: the confirmation of the principle of two types of nuclear bag fibres in mammalian spindles and the variation in occurrence of a dense M-band along the fibres. Furthermore, this paper summarizes how studies from the Umeå University group (Laboratory of Muscle Biology in the Department of Integrative Medical Biology) on fibre typing and the structure and composition of M-bands have contributed to the current understanding of muscle spindle complexity in adult humans as well as to muscle spindle development and effects of ageing. The variable molecular composition of the intrafusal sarcomeres with respect to myosin heavy chains and M-band proteins gives new perspectives on the role of the intrafusal myofibrils as stretch-activated sensors influencing tension/stiffness and signalling to nuclei.

Tropomyosin is required for cardiac morphogenesis, myofibril assembly, and formation of adherens junctions in the developing mouse embryo.

BACKGROUND: We explored a function for tropomyosin (TM) in mammalian myofibril assembly and cardiac development by analyzing a deletion in the mouse TPM1 gene targeting αTM1, the major striated muscle TM isoform. RESULTS: Mice lacking αTM1 are embryonic lethal at E9.5 with enlarged, misshapen, and non-beating hearts characterized by an abnormally thin myocardium and reduced trabeculae. αTM1-deficient cardiomyocytes do not assemble striated myofibrils, instead displaying aberrant non-striated F-actin fibrils with α-actinin puncta dispersed irregularly along their lengths. αTM1's binding partner, tropomodulin1 (Tmod1), is also disorganized, and both myomesin-containing thick filaments as well as titin Z1Z2 fail to assemble in a striated pattern. Adherens junctions are reduced in size in αTM1-deficient cardiomyocytes, α-actinin/F-actin adherens belts fail to assemble at apical cell-cell contacts, and cell contours are highly irregular, resulting in abnormal cell shapes and a highly folded cardiac surface. In addition, Tmod1-deficient cardiomyocytes exhibit failure of α-actinin/F-actin adherens belt assembly. CONCLUSIONS: Absence of αTM1 resulting in unstable F-actin may preclude sarcomere formation and/or lead to degeneration of partially assembled sarcomeres due to unregulated actomyosin interactions. Our data also identify a novel αTM1/Tmod1-based pathway stabilizing F-actin at cell-cell junctions, which may be required for maintenance of cell shapes during embryonic cardiac morphogenesis.

An acute eccentric exercise increases circulating myomesin 3 fragments.

Discovery of blood biomarkers to evaluate exercise-induced muscle damage have attracted many researchers and coaches. This study aimed to determine changes in circulating myomesin 3 fragments as a novel biomarker for exercise-induced muscle damage. Nine healthy males performed 10 sets of 40 repetitions of one-leg calf-raise exercise by the load corresponding to the half of their body weight. Muscle symptoms were evaluated by a visual analog scale (VAS). Blood samples were collected before and 2, 4, 24, 48, 72, and 96 h post-exercise. Plasma myomesin 3 fragments levels were significantly increased at 96 h after the eccentric exercise. The myomesin 3 fragments levels were correlated with other biomarkers of muscle damage and the muscle symptoms. These results suggest that the circulating myomesin 3 fragments levels are potential biomarkers reflecting eccentric exercise-induced muscle damage.

The M-band: The underestimated part of the sarcomere.

The sarcomere is the basic unit of the myofibrils, which mediate skeletal and cardiac Muscle contraction. Two transverse structures, the Z-disc and the M-band, anchor the thin (actin and associated proteins) and thick (myosin and associated proteins) filaments to the elastic filament system composed of titin. A plethora of proteins are known to be integral or associated proteins of the Z-disc and its structural and signalling role in muscle is better understood, while the molecular constituents of the M-band and its function are less well defined. Evidence discussed here suggests that the M-band is important for managing force imbalances during active muscle contraction. Its molecular composition is fine-tuned, especially as far as the structural linkers encoded by members of the myomesin family are concerned and depends on the specific mechanical characteristics of each particular muscle fibre type. Muscle activity signals from the M-band to the nucleus and affects transcription of sarcomeric genes, especially via serum response factor (SRF). Due to its important role as shock absorber in contracting muscle, the M-band is also more and more recognised as a contributor to muscle disease.

Identification of MYOM2 as a candidate gene in hypertrophic cardiomyopathy and Tetralogy of Fallot, and its functional evaluation in the Drosophila heart.

The causal genetic underpinnings of congenital heart diseases, which are often complex and multigenic, are still far from understood. Moreover, there are also predominantly monogenic heart defects, such as cardiomyopathies, with known disease genes for the majority of cases. In this study, we identified mutations in myomesin 2 (MYOM2) in patients with Tetralogy of Fallot (TOF), the most common cyanotic heart malformation, as well as in patients with hypertrophic cardiomyopathy (HCM), who do not exhibit any mutations in the known disease genes. MYOM2 is a major component of the myofibrillar M-band of the sarcomere, and a hub gene within interactions of sarcomere genes. We show that patient-derived cardiomyocytes exhibit myofibrillar disarray and reduced passive force with increasing sarcomere lengths. Moreover, our comprehensive functional analyses in the Drosophila animal model reveal that the so far uncharacterized fly gene CG14964 [herein referred to as Drosophila myomesin and myosin binding protein (dMnM)] may be an ortholog of MYOM2, as well as other myosin binding proteins. Its partial loss of function or moderate cardiac knockdown results in cardiac dilation, whereas more severely reduced function causes a constricted phenotype and an increase in sarcomere myosin protein. Moreover, compound heterozygous combinations of CG14964 and the sarcomere gene Mhc (MYH6/7) exhibited synergistic genetic interactions. In summary, our results suggest that MYOM2 not only plays a critical role in maintaining robust heart function but may also be a candidate gene for heart diseases such as HCM and TOF, as it is clearly involved in the development of the heart.This article has an associated First Person interview with Emilie Auxerre-Plantié and Tanja Nielsen, joint first authors of the paper.

Cardiomyocytes have mosaic patterns of protein expression.

Skeletal myocytes have well-established fast and slow twitch fibers with unique gene and protein specific expression patterns. By immunohistochemical staining, these show a mosaic pattern across myocytes. We hypothesized cardiac myocytes may behave similarly where some proteins are differentially expressed between mature cardiomyocytes. We utilized the tool HPASubC on over 52,000 cardiac images of the Human Protein Atlas to identify differential protein expression patterns by immunohistochemistry across the cardiomyocytes. We matched identified proteins to open chromatin and gene expression data. We identified 143 putative proteins with mosaic patterns of expression across the cardiomyocytes. We validated four of these proteins (MYL3, MYL4, PAM, and MYOM1) and demonstrated unique atrial or ventricular patterns of expression for each. Acetylation of histone H3K27 at the promoters of these four genes were consistent with the atrial/ventricular expression patterns. Despite the generally accepted homogeneity of cardiomyocytes, a small subset of proteins varies between cardiomyocytes in a mosaic pattern. This fundamental process has been previously uncharacterized. These changes may inform on different functional and disease-related activities of proteins in individual cardiomyocytes.

Rare genetic mutations in Pakistani patients with dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a leading cause of heart failure, and heart transplantation globally. There is enlargement of left ventricle of the heart impairing the systolic function in this disorder. The involvement of genetic factors in the pathogenesis of DCM has been reported in up to 50% of the cases. However, due to the complexity and heterogeneity of the disease, the complete pathophysiology remains unclear. In this study, whole exomes of five unrelated patients of idiopathic DCM were sequenced to an average depth of 100× using Illumina HiSeq4000 system. The analysis of the data with in silico tools SIFT, Polyphen2, and CADD showed 494 rare (AF < 1.0%) missense SNVs predicted as deleterious. Detrimental variants in genes highly expressed in cardiac tissue included 3 rare allele frequency loss-of-function SNVs in C2orf40, MYOM3, and TMED4 genes, a homozygous frameshift insertion in RTKN2, and a splice site homozygous deletion in SLC6A6 in at least one of the patients. The stop-gained SNV rs143187236 of MYOM3 (myomesin 3) was found in perfect linkage disequilibrium (r2 = 1.0) with its neighboring missense SNV rs149105212 in two of the patients, representing the role of myomesin 3 in pathophysiology of DCM. Allele frequency comparison showed three variants rs375563861 (C2orf40), rs143187236 (MYOM3), and rs564181443 (RTKN2) having 3 fold or higher allele frequency in South Asians than in the global populations. The identified pathogenic variants can be used in risk assessment and precision therapy in DCM patients.

Zebrafish Embryonic Slow Muscle Is a Rapid System for Genetic Analysis of Sarcomere Organization by CRISPR/Cas9, but Not NgAgo.

Zebrafish embryonic slow muscle cells, with their superficial localization and clear sarcomere organization, provide a useful model system for genetic analysis of muscle cell differentiation and sarcomere assembly. To develop a quick assay for testing CRISPR-mediated gene editing in slow muscles of zebrafish embryos, we targeted a red fluorescence protein (RFP) reporter gene specifically expressed in slow muscles of myomesin-3-RFP (Myom3-RFP) zebrafish embryos. We demonstrated that microinjection of RFP-sgRNA with Cas9 protein or Cas9 mRNA resulted in a mosaic pattern in loss of RFP expression in slow muscle fibers of the injected zebrafish embryos. To uncover gene functions in sarcomere organization, we targeted two endogenous genes, slow myosin heavy chain-1 (smyhc1) and heat shock protein 90 α1 (hsp90α1), which are specifically expressed in zebrafish muscle cells. We demonstrated that injection of Cas9 protein or mRNA with respective sgRNAs targeted to smyhc1 or hsp90a1 resulted in a mosaic pattern of myosin thick filament disruption in slow myofibers of the injected zebrafish embryos. Moreover, Myom3-RFP expression and M-line localization were also abolished in these defective myofibers. Given that zebrafish embryonic slow muscles are a rapid in vivo system for testing genome editing and uncovering gene functions in muscle cell differentiation, we investigated whether microinjection of Natronobacterium gregoryi Argonaute (NgAgo) system could induce genetic mutations and muscle defects in zebrafish embryos. Single-strand guide DNAs targeted to RFP, Smyhc1, or Hsp90α1 were injected with NgAgo mRNA into Myom3-RFP zebrafish embryos. Myom3-RFP expression was analyzed in the injected embryos. The results showed that, in contrast to the CRISPR/Cas9 system, injection of the NgAgo-gDNA system did not affect Myom3-RFP expression and sarcomere organization in myofibers of the injected embryos. Sequence analysis failed to detect genetic mutations at the target genes. Together, our studies demonstrate that zebrafish embryonic slow muscle is a rapid model for testing gene editing technologies in vivo and uncovering gene functions in muscle cell differentiation.

Specific titin and myomesin domains stimulate myoblast proliferation.

Myofibrillar proteins titin and myomesin stimulated myoblast proliferation as determined by MTT-test and labelled thymidine incorporation in the DNA. Specific Fn type III and Ig-like domains of these proteins were able to exert mitogenic effects as well. Proliferative effect of Fn type III domains was highly sensitive to inhibition of Ca2+/calmodulin dependent protein kinase, whereas the effect of Ig-like domains showed greater sensitivity to the inhibition of adenylyl cyclase - cAMP - PKA pathway. IGF-1 autocrine signalling inhibition partially suppressed mitogenic effects revealed by both domain types.

Binding of Myomesin to Obscurin-Like-1 at the Muscle M-Band Provides a Strategy for Isoform-Specific Mechanical Protection.

The sarcomeric cytoskeleton is a network of modular proteins that integrate mechanical and signaling roles. Obscurin, or its homolog obscurin-like-1, bridges the giant ruler titin and the myosin crosslinker myomesin at the M-band. Yet, the molecular mechanisms underlying the physical obscurin(-like-1):myomesin connection, important for mechanical integrity of the M-band, remained elusive. Here, using a combination of structural, cellular, and single-molecule force spectroscopy techniques, we decode the architectural and functional determinants defining the obscurin(-like-1):myomesin complex. The crystal structure reveals a trans-complementation mechanism whereby an incomplete immunoglobulin-like domain assimilates an isoform-specific myomesin interdomain sequence. Crucially, this unconventional architecture provides mechanical stability up to forces of ∼135 pN. A cellular competition assay in neonatal rat cardiomyocytes validates the complex and provides the rationale for the isoform specificity of the interaction. Altogether, our results reveal a novel binding strategy in sarcomere assembly, which might have implications on muscle nanomechanics and overall M-band organization.

Induction of insulin-like growth factor 1 splice forms by subfragments of myofibrillar proteins.

Expression of insulin-like growth factor 1 (IGF-1) mRNAs splice forms was recently shown to be stimulated by myofibrillar proteins released from the damaged muscle. In this study, we report that individual subfragments of titin and myomesin composed of Fn type III and Ig-like domains can activate expression of two IGF-1 splice forms in cultured myoblasts, both at protein and mRNA levels. Competition studies showed that each of the domain-types interacts with its own receptor. Induction of IGF-1 expression caused by domains of different types showed dissimilar sensitivity to inhibitors of regulatory cascades. The effect of Fn type III domains was more sensitive to inhibition of Ca(2+)/calmodulin dependent protein kinase, whereas the effect of Ig-like domains showed greater sensitivity to the inhibition of the adenylyl cyclase-cAMP-PKA pathway.

Dysferlin interacts with calsequestrin-1, myomesin-2 and dynein in human skeletal muscle.

Dysferlinopathies are a group of progressive muscular dystrophies characterized by mutations in the gene DYSF. These mutations cause scarcity or complete absence of dysferlin, a protein that is expressed in skeletal muscle and plays a role in membrane repair. Our objective was to unravel the proteins that constitute the dysferlin complex and their interaction within the complex using immunoprecipitation assays (IP), blue native gel electrophoresis (BN) in healthy adult skeletal muscle and healthy cultured myotubes, and fluorescence lifetime imaging-fluorescence resonance energy transfer (FLIM-FRET) analysis in healthy myotubes. The combination of immunoprecipitations and blue native electrophoresis allowed us to identify previously reported partners of dysferlin - such as caveolin-3, AHNAK, annexins, or Trim72/MG53 - and new interacting partners. Fluorescence lifetime imaging showed a direct interaction of dysferlin with Trim72/MG53, AHNAK, cytoplasmic dynein, myomesin-2 and calsequestrin-1, but not with caveolin-3 or dystrophin. In conclusion, although IP and BN are useful tools to identify the proteins in a complex, techniques such as fluorescence lifetime imaging analysis are needed to determine the direct and indirect interactions of these proteins within the complex. This knowledge may help us to better understand the roles of dysferlin in muscle tissue and identify new genes involved in muscular dystrophies in which the responsible gene is unknown.