Journal of Molecular Biology
Volume 376, Issue 2, 15 February 2008, Pages 338-351
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Myomesin 3, a Novel Structural Component of the M-band in Striated Muscle

https://doi.org/10.1016/j.jmb.2007.11.048Get rights and content

Abstract

The M-band is the cytoskeletal structure that cross-links the myosin and titin filaments in the middle of the sarcomere. Apart from the myosin tails and the C-termini of titin, only two closely related structural proteins had been detected at the M-band so far, myomesin and M-protein. However, electron microscopy studies revealed structural features that do not correlate with the expression of these two proteins, indicating the presence of unknown constituents in the M-band.

Using comparative sequence analysis, we have identified a third member of this gene family, myomesin 3, and characterised its biological properties. Myomesin 3 is predicted to consist of a unique head domain followed by a conserved sequence of either fibronectin- or immunoglobulin-like domains, similarly to myomesin 3 and M-protein. While all three members of the myomesin family are localised to the M-band of the sarcomere, each member shows its specific expression pattern. In contrast to myomesin, which is ubiquitously expressed in all striated muscles, and M-protein, whose expression is restricted to adult heart and fast-twitch skeletal muscle, myomesin 3 can be detected mainly in intermediate speed fibers of skeletal muscle. In analogy to myomesin, myomesin 3 targets to the M-band region of the sarcomere via its N-terminal part and forms homodimers via its C-terminal domain. However, despite the high degree of homology, no heterodimer between distinct members of the myomesin gene family can be detected. We propose that each member of the myomesin family is a component of one of the distinct ultrastructures, the M-lines, which modulate the mechanical properties of the M-bands in different muscle types.

Introduction

The M-band, a prominent part of the sarcomeric cytoskeleton, is a complex protein network that ensures the proper interaction of contractile filaments in muscle. This structure is believed to stabilise the thick filament lattice in the sarcomere during the contraction cycle.1 Apart from myosin, the main structural components of the sarcomeric M-band identified so far are the C-terminal part of titin2 and two closely related proteins, myomesin† (gene nomenclature: Myom1)3, 4 and M-protein (gene nomenclature: Myom2),5 which consist of a unique N-terminal domain followed by 12 identically arranged repeats of immunoglobulin (Ig)-like and fibronectin (Fn) type III domains (two Igs–five Fns–five Igs; Fig. 1). Myomesin is found in all kinds of vertebrate cross-striated muscles studied so far,6, 7 whereas M-protein displays a more restricted expression pattern.8, 9

The molecular organisation of the M-band has been characterised by biochemical and biophysical methods. These studies have indicated that myomesin not only can bind to myosin and titin10 but also has the ability to form antiparallel dimers via its C-terminal domain.11 These findings were integrated into a novel three-dimensional model of the sarcomeric M-band in which myomesin is considered to be the principal thick filament cross-linking protein, a role analogous to α-actinin in the Z-disk.7 Myomesin-titin interaction10 might be crucial for the coupling of mechanical events in the M-band of the sarcomere and signalling pathways that are mediated by the stretch-activated Ser/Thr-kinase domain in the C-terminal portion of titin.12

Several muscle types express the EH-myomesin splice isoform generated by the inclusion of a unique EH segment (about 100 amino acids) in the centre of the myomesin molecule.6, 7, 13 Single-molecule manipulations have established that the EH segment has a disordered conformation and functions as an entropic spring, similar to the PEVK domain of titin.14

The structure of the M-band was extensively studied by electron microscopy (EM). Negative-contrasted pictures can resolve five major M-lines (designated M6, M4, M1, M4′ and M6′), 22 nm apart, in the central “bare” zone of the thick filament.15 The M4/M4′ lines show a similar appearance in all studied muscles, while the densities of the M1 line and the M6/M6′ lines depend on the physiological performance of a particular fiber and change during development. Data from immuno-EM suggest myomesin as an integral component of the M4 line,16 which is supported by the uniform expression of myomesin in all types of cross-striated muscle. The expression of the longer EH-myomesin isoform correlates with a fuzzy appearance of the M-band in the EM studies,7, 13 indicating that myomesin is involved in the longitudinal alignment of the myosin filaments.17 M-protein was proposed to be responsible for the appearance of the M1 line,16 which is confirmed by the simultaneous absence of M-protein and the M1 line in slow fibers.9 The molecular nature of the M6/M6′ lines has not been defined so far. However, the regular distances and similar appearance of all M-lines indicate the presence of hitherto unidentified proteins that might be closely related to the well-characterised myomesin and M-protein.

Intrigued by the existence of these unexplained structural features of the M-band, we screened the mouse genome for the presence of genes related to myomesin and M-protein. Using comparative sequence analysis, we have identified a novel gene that shares the same intron–exon and predicted domain arrangement compared with myomesin and M-protein. To study the expression of all three myomesin family genes in different types of adult and developing muscles, we performed RT–PCR analysis of mouse tissues. We show that the new member of the myomesin family, myomesin 3, is differentially expressed in various kinds of striated muscle, with high levels in newborn skeletal and adult slow muscles. Using specific antibodies, we show that myomesin 3 is a novel protein component of the sarcomeric M-band. Its expression in skeletal muscle is fiber type specific, with the highest expression level in IIA fibers of mouse hind limbs, while it is absent from the mouse heart at any stage during normal development. Furthermore, the N-terminal fragment of myomesin 3 is responsible for M-band targeting in transfected neonatal rat cardiomyocytes (NRCs), while the C-terminal domain homodimerises as shown by glutathione S-transferase (GST) pull-down assays.

Section snippets

The expression of myomesin, M-protein and myomesin 3 genes in mouse is tissue and developmental stage specific

To study the expression of the three myomesin gene family members, we performed semi-quantitative RT–PCR analysis on total RNA isolated from mouse (C57/BL6) tissues at different developmental stages (Fig. 2). Myomesin primers were derived from exons flanking the alternatively spliced EH segment to monitor both myomesin isoforms (EH-myomesin and myomesin lacking the EH segment; Fig. 2, first panel). The myomesin transcript can be detected in all types of striated muscle but not in smooth muscle

Discussion

In this work, we report the discovery and characterisation of a novel muscle protein, myomesin 3. It was exciting to find a third member of the myomesin family and a novel component of the sarcomeric M-band after more than 30 years of research in this field.3, 4, 5 The structure of the myomesin 3 gene is very similar to the structures of the myomesin and M-protein genes analysed earlier18, 19, since the intron positions and phases are essentially identical. According to the alignment of the

RT–PCR analysis

Total RNA was isolated from the heart (EH 10.5 p.c., 12.5 p.c., 16.5 p.c., 18.5 p.c. and adult heart), somites (10.5 and 12.5 p.c.), whole leg (12.5 p.c., 14.5 p.c. and newborn), m. tibialis anterior, m. soleus, diaphragm, extraocular muscle and stomach of adult mouse using the TRIZOL Reagent (Invitrogen AG, Basel, Switzerland). RT–PCR was carried out on approximately 1 μg of total RNA, and cDNA was produced with the ThermoScript RT–PCR System (Invitrogen) and amplified with Taq polymerase

Acknowledgements

This work was funded by grants from Swiss Foundation for Research on Muscle Diseases and Wolfermann-Nägeli Foundation to I.A. In addition, it was supported by grants from the Swiss National Science Foundation (to J.C.P. and I.A.), a grant from the Gebert-Rüf Stiftung, a grant from the Swiss Cardiovascular Teaching and Research Network. R.S. was supported by a predoctoral training grant from Swiss Federal Institute of Technology, Zurich.

We would like to thank Sereina Bodenmann for performing

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