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Human embryonic stem cells (hESCs) are a pluripotent cell type considered to have high potential for the treatment of cardiovascular disease by cell replacement therapies. Several groups have shown that hESCs can be differentiated in vitro into cardiac myocytes, which may be used to facilitate tissue regeneration by injection directly into damaged myocardium. However, several hurdles still need to be overcome before these cells can be used in clinical trials. In particular, because transplanted hESC-cardiac myocytes should integrate fully within the damaged heart, these cells must be functionally compatible with the host myocardium. To assess this aspect of hESC-cardiac myocytes, Brito-Martins et al. (2008) in this issue of the BJP, describe the responses of hESC-cardiac myocytes to beta-adrenoceptor stimulation, compared to those of myocytes from adult human hearts. Data obtained using specific beta-adrenoceptor agonists showed good compatibility of hESC-cardiac myocytes with adult human myocardium in terms of beta-adrenoceptor response. [ABSTRACT FROM AUTHOR]

Titin is a giant vertebrate striated muscle protein with critical importance for myofibril elasticity and structural integrity. We show here that the complete sequence of the human titin gene contains 363 exons, which together code for 38 138 residues (4200 kDa). In its central I-band region, 47 novel PEVK exons were found, which contribute to titin's extensible spring properties. Additionally, 3 unique I-band titin exons were identified (named novex-1 to -3). Novex-3 functions as an alternative titin C-terminus. The novex-3 titin isoform is approximately 700 kDa in size and spans from Z1-Z2 (titin's N-terminus) to novex-3 (C-terminal exon). Novex-3 titin specifically interacts with obscurin, a 721-kDa myofibrillar protein composed of 57 Ig/FN3 domains, followed by one IQ, SH3, DH, and a PH domain at its C-terminus. The obscurin domains Ig48/Ig49 bind to novex-3 titin and target to the Z-line region when expressed as a GFP fusion protein in live cardiac myocytes. Immunoelectron microscopy detected the C-terminal Ig48/Ig49 obscurin epitope near the Z-line edge. The distance from the Z-line varied with sarcomere length, suggesting that the novex-3 titin/obscurin complex forms an elastic Z-disc to I-band linking system. This system could link together calcium-dependent, SH3-, and GTPase-regulated signaling pathways in close proximity to the Z-disc, a structure increasingly implicated in the restructuring of sarcomeres during cardiomyopathies.

Ion-channel beta-subunits are ancillary proteins that co-assemble with alpha-subunits to modulate gating kinetics and enhance stability of multimeric channel complexes. They provide binding sites for other regulatory proteins and are medically important as the targets of many pharmacological compounds. MinK is the beta-subunit of the slow activating component of the delayed rectifier potassium current (I(Ks)) channel, and associates with the alpha-subunit, KvLQT1. We report here that minK specifically interacts with the sarcomeric Z-line component, T-cap (also called telethonin). In vitro interaction studies indicated that the cytoplasmic domain of minK specifically binds to the sixteen C-terminal residues of T-cap; these residues are sufficient for its interaction with minK. Consistent with our in vitro studies, immunofluorescence staining followed by confocal analysis revealed that both minK and T-cap are localized within the Z-line region in cardiac muscle. Striated staining of minK was observed in non-washed, membrane-intact cardiac myofibrils, but not in well-washed, membrane-removed cardiac myofibrils, suggesting that minK localizes on T-tubular membranes surrounding the Z-line in the inner ventricular myocardium. Together with our previous data on the colocalization and interaction of T-cap with the N-terminus of the giant protein titin in the periphery of the Z-line, these data suggest that T-cap functions as an adapter protein to link together myofibrillar components with the membranous beta-subunit of the I(Ks) channel. We speculate that this interaction may contribute to a stretch-dependent regulation of potassium flux in cardiac muscle, providing a "mechano-electrical feedback" system., (Copyright 2001 Academic Press.)

We describe here a novel sarcomeric 145-kD protein, myopalladin, which tethers together the COOH-terminal Src homology 3 domains of nebulin and nebulette with the EF hand motifs of alpha-actinin in vertebrate Z-lines. Myopalladin's nebulin/nebulette and alpha-actinin-binding sites are contained in two distinct regions within its COOH-terminal 90-kD domain. Both sites are highly homologous with those found in palladin, a protein described recently required for actin cytoskeletal assembly (Parast, M.M., and C.A. Otey. 2000. J. Cell Biol. 150:643-656). This suggests that palladin and myopalladin may have conserved roles in stress fiber and Z-line assembly. The NH(2)-terminal region of myopalladin specifically binds to the cardiac ankyrin repeat protein (CARP), a nuclear protein involved in control of muscle gene expression. Immunofluorescence and immunoelectron microscopy studies revealed that myopalladin also colocalized with CARP in the central I-band of striated muscle sarcomeres. Overexpression of myopalladin's NH(2)-terminal CARP-binding region in live cardiac myocytes resulted in severe disruption of all sarcomeric components studied, suggesting that the myopalladin-CARP complex in the central I-band may have an important regulatory role in maintaining sarcomeric integrity. Our data also suggest that myopalladin may link regulatory mechanisms involved in Z-line structure (via alpha-actinin and nebulin/nebulette) to those involved in muscle gene expression (via CARP).

The giant myofibrillar protein titin contains within its C-terminal region a serine-threonine kinase of unknown function. We have identified a novel muscle specific RING finger protein, referred to as MURF-1, that binds in vitro to the titin repeats A168/A169 adjacent to the titin kinase domain. In myofibrils, MURF-1 is present within the periphery of the M-line lattice in close proximity to titin's catalytic kinase domain, within the Z-line lattice, and also in soluble form within the cytoplasm. Yeast two-hybrid screens with MURF-1 as a bait identified two other highly homologous MURF proteins, MURF-2 and MURF-3. MURF-1,2,3 proteins are encoded by distinct genes, share highly conserved N-terminal RING domains and in vitro form dimers/heterodimers by shared coiled-coil motifs. Of the MURF family, only MURF-1 interacts with titin repeats A168/A169, whereas MURF-3 has been reported to affect microtubule stability. Association of MURF-1 with M-line titin may potentially modulate titin's kinase activity similar to other known kinase-associated proteins, whereas differential expression and heterodimerization of MURF1, 2 and 3 may link together titin kinase and microtubule-dependent signal pathways in striated muscles., (Copyright 2001 Academic Press.)

The congenital nemaline myopathies are rare hereditary muscle disorders characterized by the presence in the muscle fibers of nemaline bodies consisting of proteins derived from the Z disc and thin filament. In a single large Australian family with an autosomal dominant form of nemaline myopathy, the disease is caused by a mutation in the alpha-tropomyosin gene TPM3. The typical form of nemaline myopathy is inherited as an autosomal recessive trait, the locus of which we previously assigned to chromosome 2q21.2-q22. We show here that mutations in the nebulin gene located within this region are associated with the disease. The nebulin protein is a giant protein found in the thin filaments of striated muscle. A variety of nebulin isoforms are thought to contribute to the molecular diversity of Z discs. We have studied the 3' end of the 20. 8-kb cDNA encoding the Z disc part of the 800-kDa protein and describe six disease-associated mutations in patients from five families of different ethnic origins. In two families with consanguineous parents, the patients were homozygous for point mutations. In one family with nonconsanguineous parents, the affected siblings were compound heterozygotes for two different mutations, and in two further families with one detected mutation each, haplotypes are compatible with compound heterozygosity. Immunofluorescence studies with antibodies specific to the C-terminal region of nebulin indicate that the mutations may cause protein truncation possibly associated with loss of fiber-type diversity, which may be relevant to disease pathogenesis.