Background— Muscle LIM protein (MLP) is an essential nuclear regulator of myogenic differentiation. Additionally, it may act as an integrator of protein assembly of the actin-based cytoskeleton. MLP-knockout mice develop a marked cardiac hypertrophy reaction and dilated cardiomyopathy (DCM). MLP is therefore a candidate gene for heritable forms of hypertrophic cardiomyopathy (HCM) and DCM in humans. Methods and Results— We analyzed 1100 unrelated individuals (400 patients with DCM, 200 patients with HCM, and 500 controls) for mutations in the human CRP3 gene that encodes MLP. We found 3 different missense mutations in 3 unrelated patients with familial HCM but detected no mutation in the DCM group or the controls. All mutations predicted an amino acid exchange at highly conserved residues in the functionally important LIM1 domain, which is responsible for interaction with α-actinin and with certain muscle-specific transcription factors. Protein-binding studies indicate that mutations in the CRP3 gene lead to a decreased binding activity of MLP to α-actinin. All 3 index patients were characterized by typical asymmetrical septal hypertrophy. Family studies revealed cosegregation of clinically affected individuals with the respective mutations in MLP. Conclusion— Here, we present evidence that mutations in the CRP3/MLP gene can cause HCM.

The M band of vertebrate cross-striated myofibrils has remained an enigmatic structure. In addition to myosin thick filaments, two major structural proteins, myomesin and M-protein, have been localized to the M band. Also, titin is expected to be anchored in this structure. To begin to understand the molecular layout of these three proteins, a panel of 16 polyclonal and monoclonal antibodies directed against unique epitopes of defined sequence was assembled, and immunoelectron microscopy was used to locate the position of the epitopes at the sarcomere level. The results allow the localization and orientation of defined domains of titin, myomesin, and M-protein at high resolution. The 250-kD carboxy-terminal region of titin clearly enters the M band with the kinase domain situated approximately 52 nm from the central M1-line. The positions of three additional epitopes are compatible with the view that the titin molecule reaches approximately 60 nm into the opposite sarcomere half. Myomesin also seems to bridge the central M1-line and is oriented parallel to the long axis of the myofibril. The neighboring molecules are oriented in an antiparallel and staggered fashion. The amino-terminal portion of the protein, known to contain a myosin binding site, seems to adopt a specific three-dimensional arrangement. While myomesin is present in both slow and fast fibers, M-protein is restricted to fast fibers. It appears to be organized in a fundamentally different manner: the central portion of the polypeptide is around the M1-line, while the terminal epitopes seem to be arranged along thick filaments. This orientation fits the conspicuously stronger M1-lines in fast twitch fibers. Obvious implications of this model are discussed.

γ-Filamin, also called ABP-L, is a filamin isoform that is specifically expressed in striated muscles, where it is predominantly localized in myofibrillar Z-discs. A minor fraction of the protein shows subsarcolemmal localization. Although γ-filamin has the same overall structure as the two other known isoforms, it is the only isoform that carries a unique insertion in its immunoglobulin (Ig)-like domain 20. Sequencing of the genomic region encoding this part of the molecule shows that this insert is encoded by an extra exon. Transient transfections of the insert-bearing domain in skeletal muscle cells and cardiomyocytes show that this single domain is sufficient for targeting to developing and mature Z-discs. The yeast two-hybrid method was used to identify possible binding partners for the insert-bearing Ig-like domain 20 of γ-filamin. The two Ig-like domains of the recently described α-actinin–binding Z-disc protein myotilin were found to interact directly with this filamin domain, indicating that the amino-terminal end of γ-filamin may be indirectly anchored to α-actinin in the Z-disc via myotilin. Since defects in the myotilin gene were recently reported to cause a form of autosomal dominant limb-girdle muscular dystrophy, our findings provide a further contribution to the molecular understanding of this disease.