Z-disk Region Research Articles

Desmin intermediate filaments and tubulin detyrosination stabilize growing microtubules in the cardiomyocyte

In heart failure, an increased abundance of post-translationally detyrosinated microtubules stiffens the cardiomyocyte and impedes its contractile function. Detyrosination promotes interactions between microtubules, desmin intermediate filaments, and the sarcomere to increase cytoskeletal stiffness, yet the mechanism by which this occurs is unknown. We hypothesized that detyrosination may regulate the growth and shrinkage of dynamic microtubules to facilitate interactions with desmin and the sarcomere. Through a combination of biochemical assays and direct observation of growing microtubule plus-ends in adult cardiomyocytes, we find that desmin is required to stabilize growing microtubules at the level of the sarcomere Z-disk, where desmin also rescues shrinking microtubules from continued depolymerization. Further, reducing detyrosination (i.e. tyrosination) below basal levels promotes frequent depolymerization and less efficient growth of microtubules. This is concomitant with tyrosination promoting the interaction of microtubules with the depolymerizing protein complex of end-binding protein 1 (EB1) and CAP-Gly domain-containing linker protein 1 (CLIP1/CLIP170). The dynamic growth and shrinkage of tyrosinated microtubules reduce their opportunity for stabilizing interactions at the Z-disk region, coincident with tyrosination globally reducing microtubule stability. These data provide a model for how intermediate filaments and tubulin detyrosination establish long-lived and physically reinforced microtubules that stiffen the cardiomyocyte and inform both the mechanism of action and therapeutic index for strategies aimed at restoring tyrosination for the treatment of cardiac disease.

The Effect of Mutation of L345P in the Desmin Gene on the Process of Autophagy in Muscle Cells of the C2C12 Line

The process of autophagy plays an important role in the life of the cell, in particular, in the process of maintaining proteostasis. In muscle cells, the process of autophagy is highly dynamic due to the need for constant renewal of the degrading proteins of the Z-disk region during muscle contraction. In this case, the cytoskeleton and, in particular, intermediate filaments play a major role in maintaining the structural and functional integrity of the muscle cell. We hypothesize that disruption of the structure of intermediate filaments of muscle cells, in particular, desmin, leads to a change in the dynamics of the process of autophagy. We have demonstrated that, in C2C12 cells, a mutation of the desmin gene (DES L345P) leads to a significant increase in the baseline level of autophagy and the rate of degradation of cellular components due to the process of autophagy. It has been shown that, in cells with the mutation DES L345P, after stimulation of the process of autophagy by serum starvation for 2 and 4 h, the amount of LC3-II protein is halved relative to cells with wild-type desmin. The rate of conversion of LC3-I to LC3-II remains unchanged. This observation may be associated with an increase in the base level of the process of autophagy and the rate of degradation of autophagosomes for the removal of mutant aggregate forms of the desmin protein from the cell due to mutation L345P.

Deconstructing sarcomeric structure\u2013function relations in titin-BioID knock-in mice

Proximity proteomics has greatly advanced the analysis of native protein complexes and subcellular structures in culture, but has not been amenable to study development and disease in vivo. Here, we have generated a knock-in mouse with the biotin ligase (BioID) inserted at titin’s Z-disc region to identify protein networks that connect the sarcomere to signal transduction and metabolism. Our census of the sarcomeric proteome from neonatal to adult heart and quadriceps reveals how perinatal signaling, protein homeostasis and the shift to adult energy metabolism shape the properties of striated muscle cells. Mapping biotinylation sites to sarcomere structures refines our understanding of myofilament dynamics and supports the hypothesis that myosin filaments penetrate Z-discs to dampen contraction. Extending this proof of concept study to BioID fusion proteins generated with Crispr/CAS9 in animal models recapitulating human pathology will facilitate the future analysis of molecular machines and signaling hubs in physiological, pharmacological, and disease context.

Expressing a Z-disk nebulin fragment in nebulin-deficient mouse muscle: effects on muscle structure and function

BackgroundNebulin is a critical thin filament-binding protein that spans from the Z-disk of the skeletal muscle sarcomere to near the pointed end of the thin filament. Its massive size and actin-binding property allows it to provide the thin filaments with structural and regulatory support. When this protein is lost, nemaline myopathy occurs. Nemaline myopathy causes severe muscle weakness as well as structural defects on a sarcomeric level. There is no known cure for this disease.MethodsWe studied whether sarcomeric structure and function can be improved by introducing nebulin’s Z-disk region into a nebulin-deficient mouse model (Neb cKO) through adeno-associated viral (AAV) vector therapy. Following this treatment, the structural and functional characteristics of both vehicle-treated and AAV-treated Neb cKO and control muscles were studied.ResultsIntramuscular injection of this AAV construct resulted in a successful expression of the Z-disk fragment within the target muscles. This expression was significantly higher in Neb cKO mice than control mice. Analysis of protein expression revealed that the nebulin fragment was localized exclusively to the Z-disks and that Neb cKO expressed the nebulin fragment at levels comparable to the level of full-length nebulin in control mice. Additionally, the Z-disk fragment displaced full-length nebulin in control mice, resulting in nemaline rod body formation and a worsening of muscle function. Neb cKO mice experienced a slight functional benefit from the AAV treatment, with a small increase in force and fatigue resistance. Disease progression was also slowed as indicated by improved muscle structure and myosin isoform expression.ConclusionsThis study reveals that nebulin fragments are well-received by nebulin-deficient mouse muscles and that limited functional benefits are achievable.

Novex-3, the tiny titin of muscle.

The giant multi-functional striated muscle protein titin is the third most abundant muscle protein after myosin and actin. Titin plays a pivotal role in myocardial passive stiffness, structural integrity and stress-initiated signaling pathways. The complete sequence of the human titin gene contains three isoform-specific mutually exclusive exons [termed novel exons (novex)] coding for the I-band sequence, named novex-1 (exon 45), novex-2 (exon 46) and novex-3 (exon 48). Transcripts containing either the novex-1 or novex-2 exons code for the novex-1 and novex-2 titin isoforms. The novex-3 transcript contains a stop codon and polyA tail signal, resulting in an unusually small (∼700 kDa) isoform, referred to as novex-3 titin. This 'tiny titin' isoform extends from the Z-disc (N-terminus) to novex-3 (C-terminus) and is expressed in all striated muscles. Biochemical analysis of novex-3 titin in cardiomyocytes shows that obscurin, a vertebrate muscle protein, binds to novex-3 titin. The novex-3/obscurin complex localizes to the Z-disc region and may regulate calcium, and SH3- and GTPase-associated myofibrillar signaling pathways. Therefore, novex-3 titin could be involved in stress-initiated sarcomeric restructuring.

From Ribosome to Sarcomere - Titin Dynamics in Striated Muscle Cells

The giant sarcomeric protein titin (up to 3.7 MDa) spans the half-sarcomere and has been regarded as blueprint for sarcomere assembly. Only little is known about the kinetics of titin's integration into the myofilament and its mechanistic role in sarcomere assembly. We study sarcomere dynamics by recombining fluorophores into the titin filament. In cardiomyocytes derived from knock-in mice expressing a titin fusion protein with eGFP attached to M-Band exon 6, FRAP experiments show an unexpected level of mobility of M-band titin that is calcium dependent. To investigate titin's role in sarcomere assembly and disassembly we inserted dsRed into titin's Z-disc region. The insertion of the fluorescent proteins at the N- and C-terminus of titin does not interfere with normal cellular function and double heterozygous knock-in animals have no obvious phenotype. The animals survive, are fertile, and have normal body and heart weights. Their myocytes allow us to simultaneously visualize titin's N- and the C-terminus using live cell imaging. Towards elucidating titin's role in myofilament dynamics we follow titin expression as well as sarcomere assembly and disassembly in differentiated mouse ES-cells, embryonic and neonatal myocytes, and satellite cells derived from titin-eGFP and -dsRed double heterozygous mice. During myofibrillogenesis in cardiomyocytes and skeletal muscle cells, titin integrates simultaneously into the Z-disc and M-band of the sarcomere. Finally, we use our animal models to follow titin synthesis, transport and degradation in developing and mature myocytes. The combination of fluorescent proteins, mouse genetics, live imaging, and super resolution microscopy provides a deeper understanding of sarcomere assembly and dynamics.

257 THE EFFECT OF REMOTE ISCHAEMIC PRECONDITIONING (RIPC) ON CARDIAC PHOSPHOPROTEINS PROFILING USING TANDEM MASS TAGS AND LC-MS/MS

<h3>Introduction</h3> Remote ischaemic preconditioning (RIPC) is a powerful protective phenomenon in which brief ischaemia of one organ or tissue confers protection of another organ or tissue against a sustained ischaemia-reperfusion insult. The nature of the signal released from the remote organ and the timing of the triggered changes in the target organ (the myocardium) remians controversial. However, several studies have implicated changes in survival signalling seen after ischemia and reperfusion. In this study we characterised a mouse model of RIPC and investigated changes in the expression of phosphoproteins in the myocardium prior to sustained ischaemia. <h3>Methods</h3> RIPC was induced in anaesthetised male C57/Bl6 mice using four cycles of 5 min of ischaemia and 5 min of reperfusion of the hindlimb using blood pressure cuff inflated at 200mmHg. The effect of this applied pressure on blood flow was measured using Laser doppler Flowmetry. Ischaemia/reperfusion (I/R) injury was determined by measuring infarct size at the end of reperfusion using a Langendorff-perfused mouse heart model. Reperfusion injury was also determined by creatine kinase release and changes in diastolic tension. To monitor changes in phosphoproteins, hearts were excised either at the end of RIPC or from sham mice. Proteins were extracted for phoshpo-proteomic analysis using the TMT isobaric mass tagging for LTQ Orbitrap velos mass spectrometer. <h3>Results</h3> RIPC significantly reduces infarct size, CK release and diastolic tension following I/R. Phoshpo-proteomic analysis showed that RIPC increases the expression of several phosph-proteins localized in the Z-disk region of the sarcomere. Thesae include Myozenin 2 (Calsarcin1), a cardiac Calcineurin-adaptor protein localized at Z disks. <h3>Conclusion</h3> Our mouse model of RIPC is associated with significant cardioprotection against I/R. RIPC triggers changes in several cardiac phospho-proteins prior to sustained ischemia some of which may be involved in calcineurin signalling.

Thick-Filament Strain and Interfilament Spacing in Passive Muscle: Effect of Titin-Based Passive Tension

We studied the effect of titin-based passive tension on sarcomere structure by simultaneously measuring passive tension and low-angle x-ray diffraction patterns on passive fiber bundles from rabbit skinned psoas muscle. We used a stretch-hold-release protocol with measurement of x-ray diffraction patterns at various passive tension levels during the hold phase before and after passive stress relaxation. Measurements were performed in relaxing solution without and with dextran T-500 to compress the lattice toward physiological levels. The myofilament lattice spacing was measured in the A-band ( d 1,0) and Z-disk ( d Z) regions of the sarcomere. The axial spacing of the thick-filament backbone was determined from the sixth myosin meridional reflection (M6) and the equilibrium positions of myosin heads from the fourth myosin layer line peak position and the I 1,1/I 1,0 intensity ratio. Total passive tension was measured during the x-ray experiments, and a differential extraction technique was used to determine the relations between collagen- and titin-based passive tension and sarcomere length. Within the employed range of sarcomere lengths (∼2.2–3.4 μm), titin accounted for >80% of passive tension. X-ray results indicate that titin compresses both the A-band and Z-disk lattice spacing with viscoelastic behavior when fibers are swollen after skinning, and elastic behavior when the lattice is reduced with dextran. Titin also increases the axial thick-filament spacing, M6, in an elastic manner in both the presence and absence of dextran. No changes were detected in either I 1,1/I 1,0 or the position of peaks on the fourth myosin layer line during passive stress relaxation. Passive tension and M6 measurements were converted to thick-filament compliance, yielding a value of ∼85 m/N, which is several-fold larger than the thick-filament compliance determined by others during the tetanic tension plateau of activated intact muscle. This difference can be explained by the fact that thick filaments are more compliant at low tension (passive muscle) than at high tension (tetanic tension). The implications of our findings are discussed.

Titin is a Target of Matrix Metalloproteinase-2

Background— Titin is the largest mammalian (≈3000 to 4000 kDa) and myofilament protein that acts as a molecular spring in the cardiac sarcomere and determines systolic and diastolic function. Loss of titin in ischemic hearts has been reported, but the mechanism of titin degradation is not well understood. Matrix metalloproteinase-2 (MMP-2) is localized to the cardiac sarcomere and, on activation in ischemia/reperfusion injury, proteolyzes specific myofilament proteins. Here we determine whether titin is an intracellular substrate for MMP-2 and if its degradation during ischemia/reperfusion contributes to cardiac contractile dysfunction. Methods and Results— Immunohistochemistry and confocal microscopy in rat and human hearts showed discrete colocalization between MMP-2 and titin in the Z-disk region of titin and that MMP-2 is localized mainly to titin near the Z disk of the cardiac sarcomere. Both purified titin and titin in skinned cardiomyocytes were proteolyzed when incubated with MMP-2 in a concentration-dependent manner, and this was prevented by MMP inhibitors. Isolated rat hearts subjected to ischemia/reperfusion injury showed cleavage of titin in ventricular extracts by gel electrophoresis, which was confirmed by reduced titin immunostaining in tissue sections. Inhibition of MMP activity with ONO-4817 prevented ischemia/reperfusion-induced titin degradation and improved the recovery of myocardial contractile function. Titin degradation was also reduced in hearts from MMP-2 knockout mice subjected to ischemia/reperfusion in vivo compared with wild-type controls. Conclusion— MMP-2 localizes to titin at the Z-disk region of the cardiac sarcomere and contributes to titin degradation in myocardial ischemia/reperfusion injury.

Long term bed rest with and without vibration exercise countermeasures: Effects on human muscle protein dysregulation

The present investigation, the first in the field, was aimed at analyzing differentially, on individual samples, the effects of 55 days of horizontal bed rest, a model for microgravity, on myosin heavy and myosin light chain isoforms distribution (by SDS) and on the proteome (by 2-D DIGE and MS) in the vastus lateralis (VL), a mixed type II/I (∼50:50%) head of the quadriceps and in the calf soleus (SOL), a predominantly slow (∼35:65%) twitch muscle. Two separate studies were performed on six subjects without (BR) and six with resistive vibration exercise (RVE) countermeasures, respectively. Both VL and SOL underwent in BR decrements of ∼15% in cross-sectional area and of ∼22% in maximal torque that were prevented by RVE. Myosin heavy chain distribution showed increased type I and decreased type IIA in BR both in VL and in SOL, the opposite with RVE. A substantial downregulation of proteins involved in aerobic metabolism characterized both in SOL and VL in BR. RVE reversed the pattern more in VL than in SOL, whereas proteins involved in anaerobic glycolysis were upregulated. Proteins from the Z-disk region and from costamers were differently dysregulated during bed rest (both BR and RVE), particularly in VL.

Differential splicing of the large sarcomeric protein nebulin during skeletal muscle development.

We studied differential splicing of nebulin, a giant filamentous F-actin binding protein (M(r) approximately 700-800kDa) that is found in skeletal muscle. Nebulin spans the thin filament length, its C-terminus is anchored in the Z-disc, and its N-terminal region is located toward the thin filament pointed end. Various lines of evidence indicate that nebulin plays important roles in thin filament and Z-disc structure in skeletal muscle. In the present work we studied nebulin in a range of muscle types during postnatal development and performed transcript studies with a mouse nebulin exon microarray, developed by us, whose results were confirmed by RT-PCR. We also performed protein studies with high-resolution SDS-agarose gels and Western blots, and structural studies with electron microscopy. We found during postnatal development of the soleus muscle major changes in splicing in both the super-repeat region and the Z-disc region of nebulin; interestingly, these changes were absent in other muscle types. Three novel Z-disc exons, previously described in the mouse gene, were upregulated during postnatal development of soleus muscle and this was correlated with a significant increase in Z-disc width. These findings support the view that nebulin plays an important role in Z-disc width regulation. In summary, we discovered changes in both the super-repeat region and the Z-disc region of nebulin, that these changes are muscle-type specific, and that they correlate with differences in sarcomere structure.

Transversal Stiffness and Young's Modulus of Single Fibers from Rat Soleus Muscle Probed by Atomic Force Microscopy

The structural integrity of striated muscle is determined by extra-sarcomere cytoskeleton that includes structures that connect the Z-disks and M-bands of a sarcomere to sarcomeres of neighbor myofibrils or to sarcolemma. Mechanical properties of these structures are not well characterized. The surface structure and transversal stiffness of single fibers from soleus muscle of the rat were studied with atomic force microscopy in liquid. We identified surface regions that correspond to projections of the Z-disks, M-bands, and structures between them. Transversal stiffness of the fibers was measured in each of these three regions. The stiffness was higher in the Z-disk regions, minimal between the Z-disks and the M-bands, and intermediate in the M-band regions. The stiffness increased twofold when relaxed fibers were maximally activated with calcium and threefold when they were transferred to rigor (ATP-free) solution. Transversal stiffness of fibers heavily treated with Triton X-100 was about twice higher than that of the permeabilized ones, however, its regional difference and the dependence on physiological state of the fiber remained the same. The data may be useful for understanding mechanics of muscle fibers when it is subjected to both axial and transversal strain and stress.

Dynamic Strength of Titin's Z-Disk End

Titin is a giant filamentous protein traversing the half sarcomere of striated muscle with putative functions as diverse as providing structural template, generating elastic response, and sensing and relaying mechanical information. The Z-disk region of titin, which corresponds to the N-terminal end of the molecule, has been thought to be a hot spot for mechanosensing while also serving as anchorage for its sarcomeric attachment. Understanding the mechanics of titin's Z-disk region, particularly under the effect of binding proteins, is of great interest. Here we briefly review recent findings on the structure, molecular associations, and mechanics of titin's Z-disk region. In addition, we report experimental results on the dynamic strength of titin's Z1Z2 domains measured by nanomechanical manipulation of the chemical dimer of a recombinant protein fragment.

Direct Observation of Protein Complex Disassembly by Single Molecule Force Spectroscopy

In recent years, single molecule force spectroscopy has opened unique possibilities to investigate the mechanical properties of single protein molecules. So far, experiments have focused on the mechanical behavior of a protein's tertiary structure. For a large number of proteins, however, multiple folded protein molecules are arranged into a multi-subunit complex. How the quaternary structure of such a complex responds to force is not clear. Here, we present a toolkit to study the mechanical properties of dimeric and trimeric protein complexes by single molecule force spectroscopy. We apply these methods to two different model systems:The dimeric α-crystallin domain is the building block of Hsp 16.5 from Methanococcus janashii. We can directly observe that this dimer dissociates at ∼ 200 pN into two metastable monomeric subunits, which subsequently unfold independently.The two most N-terminal domains of human titin are assembled into an antiparallel complex by telethonin in the Z-disk region of the sarcomere. We show that the Ig domains of titin are stabilized in the presence of telethonin, and that this stabilization is optimized to provide a high level of mechanical strength in the sarcomere. The dissociation force of the titin-telethonin complex exceeds 600 pN, making it one of the highest rupture forces known to date.Single molecule force spectroscopy allows us not only to measure directly the dissociation forces of protein complexes, but also to observe the mechanical hierarchy of the involved building blocks. Building on these results, future experiments will attempt to observe the assembly of protein complexes using single molecule force spectroscopy.

Spatial distribution of the transverse stiffness of relaxed and activated rat soleus muscle fibers

The transverse stiffness of glycerinated and demembranated fibers from the soleus muscle of Wistar rats in different functional states was measured by atomic force microscopy. It was demonstrated that the transverse stiffness of relaxed fibers near the Z disk is approximately twofold higher as compared with the M-line region. However, the stiffness of glycerinated fibers in the Z-disk and M-line regions is considerably lower than that of demembranated fibers. The values of mechanical parameters of activated fibers are significantly higher as compared with the relaxed fibers. However, the stiffness of activated glycerinated fibers near the Z disk approximately doubled as compared with the relaxed state, whereas the stiffness of the Z-disk region in demembranated fibers increased more than fourfold. The stiffness of both glycerinated and demembranized fibers near the M-line increased approximately threefold.

Titin expression in human articular cartilage and cultured chondrocytes : A novel component in articular cartilage biomechanical sensing?

In striated muscle tissues, the giant protein titin acts as a biomechanically active filament system, coupling stress/strain to gene expression. The objective of the study is to show the existence of titin fragments in human articular cartilage, as in diarthodial joints, chondrocytes are also known to sense and respond to stretching. We have surveyed human cultured cartilage collected from adults with osteoarthritis (OA), without OA and from infants with a set of titin antibodies and primer pairs. Three different antibodies were used for immunolabelling, reacting with titin's N-terminal Z1–Z2 domains, its Novex III exon, and with its PEVK region. An antibody directed to a titin ligand was included, since in cardiac muscle, this has been shown to participate in the transmission of stretch dependent titin-based signals. Our results indicate that although at low levels, titin is expressed in cartilage. Primer pairs detected titin transcripts in cartilage, and consistent with this, antibodies directed to titin's Z-disc region and to its elastic region stained cartilage. Moreover, we also could detect transcription of the titin ligand CARP. Components of the stretch dependent signal machinery in muscle are also expressed in cartilage. Further studies are warranted to address if common stress/strain dependent signalling are conserved in muscle and cartilage tissues.

Evidence for a dimeric assembly of two titin/telethonin complexes induced by the telethonin C-terminus

The Z-disk region defines the lateral boundary of the sarcomere and requires a high level of mechanical strength to provide a stable framework for large filamentous muscle proteins. The level of complexity at this area is reflected by a large number of protein–protein interactions. Recently, we unraveled how the N-terminus of the longest filament component, the giant muscle protein titin, is assembled into an antiparallel (2:1) sandwich complex by the N-terminal titin-binding segment of the Z-disk ligand telethonin/T-cap [Zou, P., Pinotsis, N., Lange, S., Song, Y.H., Popov, A., Mavridis, I., Mayans, O.M., Gautel, M., Wilmanns, M., 2006. Palindromic assembly of the giant muscle protein titin in the sarcomeric Z-disk. Nature 439, 229–233]. In this contribution, we present structural data of a related complex of the titin N-terminus with full-length telethonin. The C-terminus of telethonin remains invisible, suggesting that it does not fold into a defined structure even in the presence of titin. In contrast to the structure with truncated telethonin, a dimer of two titin/telethonin complexes is formed within the crystal environment, potentially indicating the formation of higher oligomers. We further investigated the structure and dynamics of this assembly by small-angle X-ray scattering, circular dichroism, and in vivo complementation data. The data consistently indicate the involvement of the C-terminal part of telethonin into the assembly of two titin/telethonin complexes.

Mechanical stress-strain sensors embedded in cardiac cytoskeleton: Z disk, titin, and associated structures

Cardiac muscle is equipped with intricate intrinsic mechanisms to regulate adaptive remodeling. Recent and extensive experimental findings powered by novel strategies for screening protein-protein interactions, improved imaging technologies, and versatile transgenic mouse methodologies reveal that Z disks and titin filaments possess unexpectedly complicated sensory and modulatory mechanisms for signal reception and transduction. These mechanisms employ molecules such as muscle-enriched LIM domain proteins, PDZ-LIM domain proteins, myozenin gene family members, titin-associated ankyrin repeat family proteins, and muscle-specific ring finger proteins, which have been identified as potential molecular sensor components. Moreover, classic transmembrane signaling processes, including mitogen-activated kinase, protein kinase C, and calcium signaling, also involve novel interactions with the Z disk/titin network. This compartmentalization of signaling complexes permits alteration of receptor-dependent transcriptional regulation by direct sensing of intrinsic stress. Newly identified mechanical stress sensors are not limited to Z-disk region and to I-band and M-band regions of titin but are also embedded in muscle-specific membrane systems such as the costamere, intercalated disks, and caveolae-like microdomains. This review summarizes current knowledge of this rapidly developing area with focus on how the heart adjusts physiological remodeling process to meet with mechanical demands and how this process fails in cardiac pathologies.

Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly.

Vertebrate-striated muscle is assumed to owe its remarkable order to the molecular ruler functions of the giant modular signaling proteins, titin and nebulin. It was believed that these two proteins represented unique results of protein evolution in vertebrate muscle. In this paper we report the identification of a third giant protein from vertebrate muscle, obscurin, encoded on chromosome 1q42. Obscurin is ∼800 kD and is expressed specifically in skeletal and cardiac muscle. The complete cDNA sequence of obscurin reveals a modular architecture, consisting of >67 intracellular immunoglobulin (Ig)- or fibronectin-3–like domains with multiple splice variants. A large region of obscurin shows a modular architecture of tandem Ig domains reminiscent of the elastic region of titin. The COOH-terminal region of obscurin interacts via two specific Ig-like domains with the NH2-terminal Z-disk region of titin. Both proteins coassemble during myofibrillogenesis. During the progression of myofibrillogenesis, all obscurin epitopes become detectable at the M band. The presence of a calmodulin-binding IQ motif, and a Rho guanine nucleotide exchange factor domain in the COOH-terminal region suggest that obscurin is involved in Ca2+/calmodulin, as well as G protein–coupled signal transduction in the sarcomere.

ENH, Containing PDZ and LIM Domains, Heart/Skeletal Muscle-Specific Protein, Associates with Cytoskeletal Proteins through the PDZ Domain

The Enigma homologue protein (ENH), containing an N-terminal PDZ domain and three C-terminal LIM domains, is a heart and skeletal muscle-specific protein that has been shown to preferentially interact with protein kinase C β (PKCβ) through the LIM domains (Kuroda et al., J. Biol. Chem. 271, 31029–31032, 1996). We here demonstrate that ENH is colocalized with a cytoskeletal protein α-actinin in the Z-disk region of rat neonatal cardiomyocytes. Pull-down assays using the glutathione-S-transferase-fusion system also showed the interaction of the PDZ domain of ENH with actin and α-actinin. Furthermore, by combined use of the in silico and conventional cDNA cloning methods, we have isolated three ENH-related clones from a mouse heart-derived cDNA library: mENH1 (591 amino acid residues) corresponding to rat ENH, mENH2 (337 residues), and mENH3 (239 residues); the latter two containing only a single PDZ domain. Deciphering their cDNA sequences, these mENH1–3 mRNAs appear to be generated from a single mENH gene by alternative splicing. Northern blot analyses using human cancer cells and mouse embryos have shown expression of each mENH mRNA to vary considerably among the cell types and during the developmental stage. Together with a recent finding that PKCβ is markedly activated in the cardiac hypertrophic signaling, these results suggest that ENH1 plays an important role in the heart development by scaffolding PKCβ to the Z-disk region and that ENH2 and ENH3 negatively modulate the scaffolding activity of ENH1.