Myosin Type Research Articles

Molecular basis of skeletal muscle plasticity--from gene to form and function.

Skeletal muscle shows an enormous plasticity to adapt to stimuli such as contractile activity (endurance exercise, electrical stimulation, denervation), loading conditions (resistance training, microgravity), substrate supply (nutritional interventions) or environmental factors (hypoxia). The presented data show that adaptive structural events occur in both muscle fibres (myofibrils, mitochondria) and associated structures (motoneurons and capillaries). Functional adaptations appear to involve alterations in regulatory mechanisms (neuronal, endocrine and intracellular signalling), contractile properties and metabolic capacities. With the appropriate molecular techniques it has been demonstrated over the past 10 years that rapid changes in skeletal muscle mRNA expression occur with exercise in human and rodent species. Recently, gene expression profiling analysis has demonstrated that transcriptional adaptations in skeletal muscle due to changes in loading involve a broad range of genes and that mRNA changes often run parallel for genes in the same functional categories. These changes can be matched to the structural/functional adaptations known to occur with corresponding stimuli. Several signalling pathways involving cytoplasmic protein kinases and nuclear-encoded transcription factors are recognized as potential master regulators that transduce physiological stress into transcriptional adaptations of batteries of metabolic and contractile genes. Nuclear reprogramming is recognized as an important event in muscle plasticity and may be related to the adaptations in the myosin type, protein turnover, and the cytoplasma-to-myonucleus ratio. The accessibility of muscle tissue to biopsies in conjunction with the advent of high-throughput gene expression analysis technology points to skeletal muscle plasticity as a particularly useful paradigm for studying gene regulatory phenomena in humans.

KLF5/BTEB2, a Krüppel-like zinc-finger type transcription factor, mediates both smooth muscle cell activation and cardiac hypertrophy.

Cardiac and vascular biology need to be approached interactively because they share many common biological features as seen in activation of the local renin-angiotensin system, angiogenesis, and extracellular matrix production. We previously reported KLF5/BTEB2, a Krüppel-like zinc-finger type transcription factor, to activate various gene promoters that are activated in phenotypically modulated smooth muscle cells, such as a nonmuscle type myosin heavy chain gene SMemb, plasminogen activator inhibitor-1 (PAI-1), iNOS, PDGF-A, Egr-1 and VEGF receptors at least in vitro. KLF5/BTEB2 mRNA levels are downregulated with vascular development but upregulated in neointima that is produced in response to vascular injury. Mitogenic stimulation activates KLF5/BTEB2 gene expression through MEK1 and Egr-1. Chromatin immunoprecipitation assay showed KLF5/BTEB2 to be induced and to bind the promoter of the PDGF-A gene in response to angiotensin II stimulation. In order to define the role of KLF5/BTEB2 in cardiovascular remodeling, we targeted the KLF5/BTEB2 gene in mice. Homozygous mice resulted in early embryonic lethality whereas heterozygous mice were apparently normal. However, in response to external stress, arteries of heterozygotes exhibited diminished levels of smooth muscle and adventitial cell activation. Furthermore, cardiac fibrosis and hypertrophy induced by continuous angiotensin II infusion. We also found that RARa binds KLF5/BTEB2, and that Am80, a potent synthetic RAR agonist, inhibits angiotensin II-induced cardiac hypertrophy. These results indicate that KLF5/BTEB2 is an essential transcription factor that causes not only smooth muscle phenotypic modulation but also cardiac hypertrophy and fibrosis.

The expression of cytokeratin in hepatic stellate cells of the cod.

In the current study, we examined the cytoskeletal architecture of cod hepatic stellate cells. We found that the cod hepatic stellate cells contain abundant cytoplasmic filaments. Deep-etch electron microscopy showed that the major component of the cytoplasmic filaments was intermediate filaments, although microtubules and microfilaments were also found in the cytoplasmic filament bundles. Immunoelectron microscopy revealed the presence of beta-tubulin, alpha-smooth muscle actin, smooth muscle type myosin, desmin and cytokeratin but not vimentin or glial fibrillar acidic protein. These results demonstrate that the cytoplasmic filaments of cod hepatic stellate cells are composed of desmin and cytokeratin intermediate filaments, acto-myosin complexes and microtubules, suggesting that the cod hepatic stellate cells have both contractile and structural functions. The expression of cytokeratin in cod hepatic stellate cells indicates that they serve for mechanical support in the extremely soft liver tissues of cods with their abundant lipids.

The role of myosin in vesicle transport during bovine chromaffin cell secretion.

Bovine adrenomedullary cells in culture have been used to study the role of myosin in vesicle transport during exocytosis. Amperometric determination of calcium-dependent catecholamine release from individual digitonin-permeabilized cells treated with 3 microM wortmannin or 20 mM 2,3-butanedione monoxime (BDM) and stimulated by continuous as well as repetitive calcium pulses showed alteration of slow phases of secretion when compared with control untreated cells. The specificity of these drugs for myosin inhibition was further supported by the use of peptide-18, a potent peptide affecting myosin light-chain kinase activity. These results were supported also by studying the impact of these myosin inhibitors on chromaffin granule mobility using direct visualization by dynamic confocal microscopy. Wortmannin and BDM affect drastically vesicle transport throughout the cell cytoplasm, including the region beneath the plasma membrane. Immunocytochemical studies demonstrate the presence of myosin types II and V in the cell periphery. The capability of antibodies to myosin II in abrogating the secretory response from populations of digitonin-permeabilized cells compared with the modest effect caused by anti-myosin V suggests that myosin II plays a fundamental role in the active transport of vesicles occurring in the sub-plasmalemmal area during chromaffin cell secretory activity.

Regulation of arterial tone by smooth muscle myosin type II.

The initiation of contractile force in arterial smooth muscle (SM) is believed to be regulated by the intracellular Ca2+ concentration and SM myosin type II phosphorylation. We tested the hypothesis that SM myosin type II operates as a molecular motor protein in electromechanical, but not in protein kinase C (PKC)-induced, contraction of small resistance-sized cerebral arteries. We utilized a SM type II myosin heavy chain (MHC) knockout mouse model and measured arterial wall Ca2+ concentration ([Ca2+](i)) and the diameter of pressurized cerebral arteries (30-100 microm) by means of digital fluorescence video imaging. Intravasal pressure elevation caused a graded [Ca2+](i) increase and constricted cerebral arteries of neonatal wild-type mice by 20-30%. In contrast, intravasal pressure elevation caused a graded increase of [Ca2+](i) without constriction in (-/-) MHC-deficient arteries. KCl (60 mM) induced a further [Ca2+](i) increase but failed to induce vasoconstriction of (-/-) MHC-deficient cerebral arteries. Activation of PKC by phorbol ester (phorbol 12-myristate 13-acetate, 100 nM) induced a strong, sustained constriction of (-/-) MHC-deficient cerebral arteries without changing [Ca2+](i). These results demonstrate a major role for SM type II myosin in the development of myogenic tone and Ca2+ -dependent constriction of resistance-sized cerebral arteries. In contrast, the sustained contractile response did not depend on myosin and intracellular Ca2+ but instead depended on PKC. We suggest that SM myosin type II operates as a molecular motor protein in the development of myogenic tone but not in pharmacomechanical coupling by PKC in cerebral arteries. Thus PKC-dependent phosphorylation of cytoskeletal proteins may be responsible for sustained contraction in vascular SM.

Identification and phylogenetic analysis of Drosophila melanogaster myosins.

Myosins constitute a superfamily of motor proteins that convert energy from ATP hydrolysis into mechanical movement along the actin filaments. Phylogenetic analysis currently places myosins into 17 classes based on class-specific features of their conserved motor domain. Traditionally, the myosins have been divided into two classes depending on whether they form monomers or dimers. The conventional myosin of muscle and nonmuscle cells forms class II myosins. They are complex molecules of four light chains bound to two heavy chains that form bipolar filaments via interactions between their coiled-coil tails (type II). Class I myosins are smaller monomeric myosins referred to as unconventional myosins. Now, at least 15 other classes of unconventional myosins are known. How many myosins are needed to ensure the proper development and function of eukaryotic organisms? Thus far, three types of myosins were found in budding yeast, six in the nematode Caenorhabditis elegans, and at least 12 in human. Here, we report on the identification and classification of Drosophila melanogaster myosins. Analysis of the Drosophila genome sequence identified 13 myosin genes. Phylogenetic analysis based on the sequence comparison of the myosin motor domains, as well as the presence of the class-specific domains, suggests that Drosophila myosins can be divided into nine major classes. Myosins belonging to previously described classes I, II, III, V, VI, and VII are present. Molecular and phylogenetic analysis indicates that the fruitfly genome contains at least five new myosins. Three of them fall into previously described myosin classes I, VII, and XV. Another myosin is a homolog of the mouse and human PDZ-containing myosins, forming the recently defined class XVIII myosins. PDZ domains are named after the postsynaptic density, disc-large, ZO-1 proteins in which they were first described. The fifth myosin shows a unique domain composition and a low homology to any of the existing classes. We propose that this is classified when similar myosins are identified in other species.

Developmental expression of alpha4 protein phosphatase regulatory subunit in tissues affected by Opitz syndrome.

Mutations in the Mid1 gene are responsible for X-linked Opitz syndrome, characterized by midline defects of the brain, face, heart, and trachea. The regulatory subunit alpha4 binds protein phosphatase 2A to Mid1 and promotes Mid1 dephosphorylation, in opposition to MAP kinase. To examine the relationship between alpha4 expression and human defects in Opitz syndrome, developmental expression of alpha4 in embryonic day (E) 8-E16 mouse embryos was mapped by immunohistochemistry. At E10, alpha4 was first detected only in the heart. At E11, alpha4 was expressed in epithelium of the mandibular and maxillary arches and in specific subsets of mesenchymal cells within the arches. The fetal heart and brain also highly expressed alpha4. At E16, alpha4 expression broadened to include the heart, brain, skeletal muscle, and tracheal and esophageal epithelium but not smooth muscle. Consistent with immunolocalization in embryos, Western immunoblotting of adult rabbit tissues demonstrated high levels of alpha4 expression in brain, heart, lung, liver, and skeletal muscle with low expression in kidney, uterus, and intestine. Expression in slow type I skeletal muscle was much higher than in fast type II muscle. By using double immunohistochemical staining, alpha4 was coexpressed in mouse skeletal muscle cells containing slow type myosin. Expression of alpha4 overlaps with the tissue defects in Opitz syndrome. The alpha4 gene lies in the linkage interval for FG syndrome, characterized by skeletal muscle and brain defects that coincide closely to the expression pattern of alpha4.

Muscle-fiber characteristics in adult mouse-tongue muscles.

To clarify functions of the mouse-tongue muscles, proteins such as myocin heavy chain (MHC) 2a and MHC-2b, which are isoforms of the fast-twitch fiber type myosin heavy chain, in the lateral margin of the tongue were observed by reverse transcription polymerase chain reaction and immunohistochemical analyses. The main MHC isoform in the superior longitudinal muscle of the tongue was MHC-2b, with the fastest function and the main MHC isoform in the transverse muscle of the tongue was MHC-2a. These findings suggested that the fastest function is necessary for the superior longitudinal muscle of the tongue, which is useful for moving the tongue in and out of the mouth in the sagittal direction, showing different cellular biological properties of the myofibers from those of the transverse muscle of the tongue.

Orientation Changes of the Myosin Light Chain Domain During Filament Sliding in Active and Rigor Muscle

Structural changes in myosin power many types of cell motility including muscle contraction. Tilting of the myosin light chain domain (LCD) seems to be the final step in transducing the energy of ATP hydrolysis, amplifying small structural changes near the ATP binding site into nanometer-scale motions of the filaments. Here we used polarized fluorescence measurements from bifunctional rhodamine probes attached at known orientations in the LCD to describe the distribution of orientations of the LCD in active contraction and rigor. We applied rapid length steps to perturb the orientations of the population of myosin heads that are attached to actin, and thereby characterized the motions of these force-bearing myosin heads. During active contraction, this population is a small fraction of the total. When the filaments slide in the shortening direction in active contraction, the long axis of LCD tilts towards its nucleotide-free orientation with no significant twisting around this axis. In contrast, filament sliding in rigor produces coordinated tilting and twisting motions.

Hamy3, a novel type 100 kDa myosin from sunflower.

Hamy3, a novel type myosin heavy chain from sunflower is the smallest myosin described so far, with only 900 amino acid residues. One interesting finding in Hamy3 is the glycine to glutamine alteration at residue 741, which corresponds to chicken skeletal muscle myosin glycine 699 (G699). G699 is found in 125 out of 129 myosin sequences and is interpreted in terms of its role as a pivot point for motion in the myosin "lever arm hypothesis". Changes in this crucial part of myosin might indicate a role that is different from the generation of intracellular motility.

Differing ADP Release Rates from Myosin Heavy Chain Isoforms Define the Shortening Velocity of Skeletal Muscle Fibers

To understand mammalian skeletal myosin isoform diversity, pure myosin isoforms of the four major skeletal muscle myosin types (myosin heavy chains I, IIA, IIX, and IIB) were extracted from single rat muscle fibers. The extracted myosin (1-2 microg/15-mm length) was sufficient to define the actomyosin dissociation reaction in flash photolysis using caged-ATP (Weiss, S., Chizhov, I., and Geeves, M. A. (2000) J. Muscle Res. Cell Motil. 21, 423-432). The ADP inhibition of the dissociation reaction was also studied to give the ADP affinity for actomyosin (K(AD)). The apparent second order rate constant of actomyosin dissociation gets faster (K(1)k(+2) = 0.17 -0.26 microm(-1) x s(-1)), whereas the affinity for ADP is weakened (250-930 microm) in the isoform order I, IIA, IIX, IIB. Both sets of values correlate well with the measured maximum shortening velocity (V(0)) of the parent fibers. If the value of K(AD) is controlled largely by the rate constant of ADP release (k(-AD)), then the estimated value of k(-AD) is sufficiently low to limit V(0). In contrast, [ATP]K(1)k(+2) at a physiological concentration of 5 mm ATP would be 2.5-6 times faster than k(-AD).

The Tip of the Coiled-coil Rod Determines the Filament Formation of Smooth Muscle and Nonmuscle Myosin

Myosin II self-assembles to form thick filaments that are attributed to its long coiled-coil tail domain. The present study has determined a region critical for filament formation of vertebrate smooth muscle and nonmuscle myosin II. A monoclonal antibody recognizing the 28 residues from the C-terminal end of the coiled-coil domain of smooth muscle myosin II completely inhibited filament formation, whereas other antibodies recognizing other parts of the coiled-coil did not. To determine the importance of this region in the filament assembly in vivo, green fluorescent protein (GFP)-tagged smooth muscle myosin was expressed in COS-7 cells, and the filamentous localization of the GFP signal was monitored by fluorescence microscopy. Wild type GFP-tagged smooth muscle myosin colocalized with F-actin during interphase and was also recruited into the contractile ring during cytokinesis. Myosin with the nonhelical tail piece deleted showed similar behavior, whereas deletion of the 28 residues at the C-terminal end of the coiled-coil domain abolished this localization. Deletion of the corresponding region of GFP-tagged nonmuscle myosin IIA also abolished this localization. We conclude that the C-terminal end of the coiled-coil domain, but not the nonhelical tail piece, of myosin II is critical for myosin filament formation both in vitro and in vivo.

Reparative myogenesis in long-term denervated skeletal muscles of adult rats results in a reduction of the satellite cell population.

This study, conducted on 25-month denervated rat hindlimb muscles, was directed toward elucidating the basis for the poor regeneration that is observed in long-term denervated muscles. Despite a approximately 97.6% loss in mean cross-sectional area of muscle fibers, the muscles retained their fascicular arrangement, with the fascicles containing approximately 1.5 times more fibers than age-matched control muscles. At least three distinct types of muscle fibers were observed: degenerating, persisting (original), and newly formed (regenerated) fibers. A majority of newly formed fibers did not appear to undergo complete maturation, and morphologically they resembled myotubes. Sites of former motor end-plates remained identifiable in persisting muscle fibers. Nuclear death was seen in all types of muscle fibers, especially in degenerating fibers. Nevertheless, the severely atrophic skeletal muscles continued to express developmentally and functionally important proteins, such as MyoD, myogenin, adult and embryonic subunits of the nicotinic acetylcholine receptor, and neural-cell adhesion molecule. Despite the prolonged period of denervation, slow and fast types of myosin were found in surviving muscle fibers. The number of satellite cells was significantly reduced in long-term denervated muscles, as compared with age-matched control muscles. In 25-month denervated muscle, satellite cells were only attached to persisting muscle fibers, but were never seen on newly formed fibers. Our data suggest that the absence of satellite cells in a population of immature newly formed muscle fibers that has arisen as a result of continuous reparative myogenesis may be a crucial, although not necessarily the only, factor underlying the poor regenerative ability of long-term denervated muscle.

Changes in Ca2+ signaling and contractile protein isoforms in smooth muscle cells from guinea pig ileum during culture.

Single smooth muscle cells (SMCs) isolated from guinea pig ileum using collagenase and papain were cultured on laminin-coated dishes in MEM containing fetal calf serum. Temporal changes in intracellular calcium ion concentration in response to carbachol and to ATP were investigated using fluo-3/AM and fluorescence microscopy. It was observed that carbachol caused an increased intracellular calcium ion in freshly isolated single SMCs but a reduced or negative response of cultured SMCs before confluence. On the other hand, ATP was observed to cause an increase in the calcium ion content of SMCs throughout the culture. SDS-PAGE and Western blot analyses revealed changes in the expression of contractile proteins as follows. l-Caldesmon and non-muscle type myosin heavy chain (NMHC) (considered to be marker molecules for dedifferentiation in smooth muscle cells) and non-muscle type tropomyosin were not observed in freshly isolated single SMCs. l-Caldesmon and NMHC appeared in the cultured SMCs within 2 days and the tropomyosin isoform was observed 6 days following seeding. Simultaneously, smooth muscle type myosin heavy chain (SMHC) decreased strikingly and the 41 kDa tropomyosin monomer was lost. The content of alpha-actin decreased gradually to a minimum on day 6 when non-muscle type tropomyosin appeared, and the cells began to proliferate rapidly. These results suggest that the loss of contractility in cultured smooth muscle cells is more closely related to changes in contractile protein profiles than to receptor-mediated signal transduction and that in addition to NMHC and l-caldesmon, non-muscle type tropomyosin may be useful as a marker molecule for de-differentiation of smooth muscle cells.

Isolation of a cDNA encoding the motor domain of nonmuscle myosin which is specifically expressed in the mantle pallial cell layer of scallop (Patinopecten yessoensis).

It has been reported that catch and striated muscle myosin heavy chains of scallop are generated through alternative splicing from a single gene [Nyitray et al. (1994) Proc. Natl. Acad. Sci. USA 91, 12686-12690]. They suggested that the catch muscle type myosin was expressed in various tissues of scallop, including the gonad, heart, foot, and mantle. However, there have been no reports of the primary structure of myosin from tissues other than the adductor muscles. In this study, we isolated a cDNA encoding the motor domain of myosin from the mantle tissue of scallop (Patinopecten yessoensis), and determined its nucleotide sequence. Sequence analysis revealed that mantle myosin exhibited 65% identity with Drosophila non muscle myosin, 60% with chicken gizzard smooth muscle myosin, and 44% with scallop striated muscle myosin. The mantle myosin has inserted sequences in the 27 kDa domain of the head region, and has a longer loop 1 structure than those of scallop striated and catch muscle myosins. Phylogenetic analysis suggested that the mantle myosin is classified as a smooth/nonmuscle type myosin. Western blot analysis with antibodies produced against the N-terminal region of the mantle myosin revealed that this myosin was specifically expressed in the mantle pallial cell layer consisting of nonmuscle cells. Our results show that mantle myosin is classified as a nonmuscle type myosin in scallop.

Differences in molecular structure among the porcine myosin heavy chain-2a, -2x, and -2b isoforms

Full coding regions for fast type myosin heavy chain (MyHC) isoforms were sequenced from a porcine skeletal muscle to analyze sequence diversity relating to the contractile properties of muscle fibers. An approximately 6-kb fragment for each MyHC was amplified through RT-PCR using isoform type-specific primers, which were designed in the 5′ and 3′ non-coding regions of the porcine MyHCs. The lengths of deduced amino acid sequences were 1939, 1939, and 1937 for the porcine MyHC-2a,-2x, and-2b, respectively. The entire amino acid sequences were highly conserved among the three MyHCs, except for the 50/20 k junction region (loop 2) which would weakly bind actin molecules. The porcine MyHC-2b possessed different amino acids from MyHC-2a and-2x, in loop1 and ELC binding region. The sequence data suggested the diversity of contractile properties among the porcine MyHC isoforms.

Role of cortical tumour-suppressor proteins in asymmetric division of Drosophila neuroblast.

Cellular diversity during development arises in part from asymmetric divisions, which generate two distinct cells by transmitting localized determinants from a progenitor cell into one daughter cell. In Drosophila, neuroblasts undergo typical asymmetric divisions to produce another neuroblast and a ganglion mother cell. At mitosis, neural fate determinants, including Prospero and Numb, localize to the basal cortex, from which the ganglion mother cell buds off; Inscuteable and Bazooka, which regulate spindle orientation, localize apically. Here we show that a tumour-suppressor protein, Lethal giant larvae (Lgl), is essential for asymmetric cortical localization of all basal determinants in mitotic neuroblasts, and is therefore indispensable for neural fate decisions. Lgl, which itself is uniformly cortical, interacts with several types of Myosin to localize the determinants. Another tumour-suppressor protein, Lethal discs large (Dlg), participates in this process by regulating the localization of Lgl. The localization of the apical components is unaffected in lgl or dlg mutants. Thus, Lgl and Dlg act in a common process that differentially mediates cortical protein targeting in mitotic neuroblasts, and that creates intrinsic differences between daughter cells.

ATP-Induced Transconformation of Myosin Revealed by Determining Three-Dimensional Positions of Fluorophores from Fluorescence Energy Transfer Measurements

The method of fluorescence resonance energy transfer (FRET) is one of the most important techniques for measuring the distance between two fluorophores and for detecting the changes in protein structure under physiological conditions. The use of green fluorescent protein is also a powerful technology that has been used to elucidate dynamic molecular events. From these we have developed a novel method to determine the three-dimensional positions of fluorophores by combining the FRET data and other structural information available. Using this method, we could determine the ATP-induced changes of three-dimensional structure of truncated Dictyostelium myosin in solution. The myosin structure with ADP in solution was found to be similar to that of the crystal structure of MgADPBeFx-bound truncated Dictyostelium myosin (type I structure), whereas myosin with ATP in solution was similar to the crystal structure of MgAdPVi-bound one (type II structure). However, the crystal structure of MgADP-bound scallop myosin (type III structure) could not be explained by any of our FRET data under various conditions. This indicates that the type III crystal structure might represent a transient intermediate conformation that could not be detected using fluorescence energy transfer.

The Identification of Tryptophan Residues Responsible for ATP-Induced Increase in Intrinsic Fluorescence of Myosin Subfragment 1

ATP binding to myosin subfragment 1 (S1) induces an increase in tryptophan fluorescence. Chymotryptic rabbit skeletal S1 has 5 tryptophan residues (Trp113, 131, 440, 510 and 595), and therefore the identification of tryptophan residues perturbed by ATP is quite complex. To solve this problem we resolved the complex fluorescence spectra into log-normal and decay-associated components, and carried out the structural analysis of the microenvironment of each tryptophan in S1. The decomposition of fluorescence spectra of S1 and S1-ATP complex revealed 3 components with maxima at ca. 318, 331 and 339–342 nm. The comparison of structural parameters of microenvironment of 5 tryptophan residues with the same parameters of single- tryptophan-containing proteins with well identified fluorescence properties applying statistical method of cluster analysis, enabled us to assign Trp595 to 318 nm, Trp440 to 331 nm, and Trp113, 131 and 510 to 342 nm spectral components. ATP induced an almost equal increase in the intensities of the intermediate (331 nm) and long-wavelength (342 nm) components, and a small decrease in the short component (318 nm). The increase in the intermediate component fluorescence most likely results from an immobilization of some quenching groups (Met437, Met441 and/or Arg444) in the environment of Trp440. The increase in the intensity and a blue shift of the long component might be associated with conformational changes in the vicinity of Trp510. However, these conclusions can not be extended directly to the other types of myosins due to the diversity in the tryptophan content and their microenvironments.

Expression of the transcriptional factor Egr-1/BTEB2 in cardiac xenograft vascular remodeling

IT IS important to consider that phenotypic change of smooth muscle cells (SMCs) is one of the important factors involved in vascular remodeling in cardiovascular diseases such as atherosclerosis and restenosis after balloon angioplasty. Phenotypic change of SMCs induces fibrosis of vessels by collagen synthesis, degradation of vessels by depletion of extracellular matrix, or intimal thickening by proliferation and migration of SMCs. In xenograft rejection, phenotypic change of SMCs plays an important role in the process of forming vascular lesion formation, as it does in common other cardiovascular diseases. However, little is known about this mechanisms. Early growth response factor-1 (Egr-1) and basic transcriptional element binding protein-2 (BTEB2) regulate multiple genes involved in the remodeling of vessels in various vascular diseases. Egr-1 is a transcriptional factor of BTEB2, which is itself a transcriptional factor regulating nonmuscle type myosin heavy chain (SMemb), which is expressed in embryonic SMCs of fetal aorta and proliferating SMCs. Therefore, Egr-1 and BTEB2 play an important role in the dedifferentiation of SMCs. It would be useful to consider expression of Egr-1 and BTEB2 in cardiac xenografts to investigate the mechanisms of xenotransplant rejection.