黒尾 誠

We have previously shown that smooth muscle myosin heavy chain isoforms (SMs), including SM1, SM2, and SMemb, are differentially expressed during vascular development, and in vascular lesions, such as atherosclerosis. The SM1/2 gene is expressed exclusively in smooth muscle cells and generates SM1 and SM2 mRNAs by alternative splicing. Whereas SM1 is constitutively expressed from early development, SM2 appears only after birth. In this study, we have isolated and characterized the 5'-flanking region of the mouse SM1/2 gene. Transient transfection assays using a series of promoter-luciferase chimeric constructs demonstrated that tandem elements of the CCTCCC sequence, located at -89 and -61 bp relative to the transcription start site, were essential for transcriptional activity of the SM1/2 gene in primary cultured rabbit aortic smooth muscle cells and smooth muscle cell lines derived from the rabbit aorta but not in non-smooth muscle cells. Gel mobility shift assays indicated that CCTCCC was a binding site for nuclear proteins prepared from smooth muscle cells. Double-stranded oligonucleotides containing either the CACC box or the Sp1 consensus sequence efficiently competed with the CCTCCC elements for binding the nuclear extracts. Site-specific mutations of CCTCCC elements resulted in a significant reduction of the promoter activity. Moreover, CCTCCC elements are evolutionary conserved between mouse and rabbit. In conclusion, the results of this study indicate an important role for the interaction of the CCTCCC sequence with Sp1 or related factors in activating transcription from the SM1/2 gene promoter.

Recent investigations have revealed that an enhancement of Na+/H+ exchange activity is a frequently observed ion transport abnormality in hypertensive patients. To test the hypothesis that increased Na+/H+ exchange causes hypertension, we produced transgenic mice overexpressing Na+/H+ exchanger and analyzed their Na+ metabolism and blood pressure. Urinary excretion of water and Na+ was significantly decreased in transgenic mice and systolic blood pressure was elevated after salt loading. The impaired Na+ excretion suggested that the Na+/H+ exchanger overexpressed in the renal tubules increased Na+ reabsorption, which caused a blood pressure elevation by Na+ retention after excessive salt intake. Our results demonstrate that overexpression of Na+/H+ exchanger can be a genetic factor for salt-sensitive hypertension.

A smooth muscle layer exists in the parietal endocardium. Notwithstanding previous pioneer work, little has been known on the phenotypes of these smooth muscles. In humans as in animals, smooth muscles show two distinct phenotypes, the 'synthetic' and 'contractile' states, which can be differentiated on the basis of the expression of two types of myosin heavy chain isoforms, SM1 and SM2. In this study, using SM1- and SM2-specific monoclonal antibodies, we performed immunohistochemical examinations to determine the phenotype of the endocardial smooth muscles. We report here that the endocardial smooth muscles have the contractile phenotype.

Vascular smooth muscles contain at least three types of developmentally regulated myosin heavy-chain (MHC) isoforms; SM1, SM2, and SMemb. By investigating the expression of the three MHC isoforms, we previously demonstrated in rabbits that smooth muscles proliferating in the neointima of arterio- and atherosclerotic lesions regain an "embryonic" phenotype. In the present study, we examined if neointimal cells are morphologically similar to embryonal smooth muscles and if dedifferentiation of neointimal smooth muscles is a reversible process. Vascular injury was produced in rabbits either by endothelial cell denudation of the aorta or by poststenotic dilation of the carotid artery. We have demonstrated in this study that the proliferating neointimal cells expressed SM1 and SMemb, but not SM2, indicating smooth muscles of an "embryonic" phenotype. The dedifferentiation of neointimal smooth muscles was found to be reversible; at 4 to 8 weeks after injury, a majority of the cells reexpressed both SM1 and SM2, but not SMemb. By electron microscopy, we have revealed smooth-muscle phenotypes determined by MHC isoforms to correspond to the morphologic phenotypes as an increase in membranous organelles, and a decrease in myofilaments was associated with the reexpression of SMemb. Interestingly, we also found that in the medial wall at 4 to 8 weeks after ballooning injury, a number of SM1-negative cells proliferated rapidly, replacing normal smooth muscles. These cells were negative against SM1 and SM2 but positive for SMemb. These SM1-negative cells contained abundant membranous organelles and few myofilaments. These cells did not express SM1 or SM2 even after 8 weeks postinjury. We conclude from these results that the proliferating synthetic-type smooth muscles after vascular injury are composed of SM1-positive cells that are morphologically similar to embryonal smooth muscle and that maintain ability to redifferentiate, and SM1-negative cells that contain few myofilaments and remain dedifferentiated.

The contractility and distensibility of renal arterioles are important in the regulation of glomerular filtration. However, little is known regarding the characteristics of contractile proteins in these arterioles. Recently it was demonstrated that vascular smooth muscles contain two types of myosin heavy chain (MHC) isoforms, SM1 and SM2, which are unique molecular markers of smooth muscle cell phenotypes. SM1 is constitutively expressed in all types of smooth muscles, whereas SM2 exists only in mature smooth muscles. We characterized the expression of MHC isoforms as well as the ultrastructural myofilament assembly of renal arteriolar smooth muscles in human, rat and rabbit by immunohistochemical techniques. SM1 and alpha-smooth muscle actin were localized in both the preglomerular vessels (including the afferent arterioles) and efferent arterioles, whereas SM2 was present only in the preglomerular vessels. Renin-producing cells in the afferent arterioles (juxtaglomerular granular cells, JG cells) were positive for alpha-smooth muscle actin but negative for SM2. When renin synthesis was stimulated, the more proximal afferent arteriolar smooth muscles turned renin-positive and SM2 disappeared. Glomerular mesangial cells did not show immunoreactivities for SM1, SM2 or alpha-smooth muscle actin. The difference in MHC isoform expression in these arterioles was also reflected by ultrastructures; the afferent arteriolar smooth muscles contained abundant myofilaments including thick filaments, whereas the efferent arteriolar smooth muscles had a few myofilaments composed only of thin microfilaments. The JG cells displayed a myofilament assembly similar to that in the efferent arteriolar smooth muscles. We conclude from these observations that smooth muscles in pre-and postglomerular arterioles, the glomerular mesangial cells and JG cells differ in phenotypes, suggesting that they may have different contractile properties which may be critically involved in the regulation of glomerular filtration.

Smooth muscle is an important component of the vessel wall. Smooth muscle cell undergoes phenotypic modulation during development of vascular lesions, such as atherosclerosis and restenosis following percutaneous transluminal coronary angioplasty (PTCA). In order to understand the mechanism of vascular remodeling, it is important to identify the smooth muscle cell in the vascular lesion and identify its phenotype by using molecular markers specific to the smooth muscle cell. Three types of myosin heavy chain (MHC) isoforms (SM1, SM2 and SMemb) expressed in smooth muscles are suitable for this purpose. In this study we first demonstrated that the expression of smooth muscle specific MHCs, such as SM1 and SM2, is reduced in human coronary arteries after the fifth decade. On the other hand, rapidly proliferating smooth muscles in the restenotic lesion express abundant SMemb but less amount of SM2. These observations indicate that deranged vascular smooth muscle differentiation is involved the development of vascular lesion. We furthermore demonstrated that smooth muscle-specific MHC is released into serum from the arterial wall following vascular damage as in dissecting aneurysm. Circulating smooth muscle MHC level was elevated 5-10 times above normal at 24 hours after aortic dissection as determined using a sensitive ELISA. We conclude from these results that smooth muscle MHC isoforms are important molecular markers for vascular pathology as well as for biochemical diagnosis of vascular injuries.

The Na(+)-H+ antiporter is a unique transmembrane protein with multiple roles in cellular functions through intracellular alkalization. It participates in the regulation of intracellular pH, cell volume and intracellular signalling in response to various mitogenic stimuli. To clarify its role as a subcellular signal in cardiovascular remodeling like vascular hyperplasia or cardiac hypertrophy, we determined mRNA levels of the Na(+)-H+ antiporter isoform, NHE-1, in vascular smooth muscles and pressure-overloaded hearts in rabbits. The NHE-1 mRNA levels in rabbit aortas and hearts were developmentally regulated with high levels at embryonic and neonatal stages than in adults. In primary-cultured smooth muscle cells (SMC), the mRNA levels were increased during exponential growth, but decreased to initial levels at confluency. Growth of a mutant SMC line, C5, which is deficient in Na(+)-H+ antiporter activity, was markedly reduced in bicarbonate-free medium. However, when the activity was restored by transfecting cells with a full-length NHE-1 cDNA in an expression vector, the growth rate of C5 was accelerated again. After balloon injury to the vascular wall, the NHE-1 mRNA levels of the injured arteries were also increased, suggesting that Na(+)-H+ antiporter contributes to the network of the growth promoting systems in smooth muscle cells in vivo. Pressure-overload on the ventricle increased the NHE-1 mRNA levels in hearts approximately two-fold of sham-operated rabbits after 3 days and remained for at least two weeks (P < 0.05). We further demonstrated that 3-methylsulfonyl-4-piperidino-benzoyl guanidine mesylate (Hoe 694), a potent antagonist of Na(+)-H+ antiporter, partially inhibited stretch-induced activation of mitogen-activated kinase (MAP kinase) in the cultured cardiomyocytes. From these results, we conclude that activation of the Na(+)-H+ antiporter and its gene expression is involved in molecular mechanisms of both cardiac hypertrophy and vascular smooth muscle cell proliferation, indicating a potential target in developing new therapeutics for cardiovascular diseases.

Essential hypertension is one of the most common diseases that exacerbate the risk of cardiovascular or cerebrovascular attacks. Although the etiology of essential hypertension remains unclear, recent investigations have revealed that an enhancement of Na(+)-proton (Na(+)-H+) exchange activity is a frequently observed ion transport abnormality in hypertensive patients and animal models. To test the hypothesis that increased Na(+)-H+ exchange causes hypertension, we produced transgenic mice overexpressing Na(+)-H+ exchanger and analyzed their Na+ metabolism and blood pressure. Urinary excretion of water and Na+ was significantly decreased in transgenic mice, and systolic blood pressure was elevated after salt loading. The impaired urinary excretion of Na+ suggested that the Na(+)-H+ exchanger overexpressed in the renal tubules increased reabsorption of Na+, which caused a blood pressure elevation by Na+ retention after excessive salt intake. Our results demonstrate that overexpression of Na(+)-H+ exchanger can be a genetic factor that interacts with excessive salt intake and causes salt-sensitive blood pressure elevation.

Smooth muscle myosin heavy chains (MHCs) exist in multiple isoforms. Rabbit smooth muscles contain at least three types of MHC isoforms: SM1 (204 kD), SM2 (200 kD), and SMemb (200 kD). SM1 and SM2 are specific to smooth muscles, but SMemb is a nonmuscle-type MHC abundantly expressed in the embryonic aorta. We recently reported that these three MHC isoforms are differentially expressed in rabbit during normal vascular development and in experimental arteriosclerosis and atherosclerosis. The purpose of this study was to clarify whether expression of human smooth muscle MHC isoforms is regulated in developing arteries and in atherosclerotic lesions. To accomplish this, we have isolated and characterized three cDNA clones from human smooth muscle: SMHC94 (SM1), SMHC93 (SM2), and HSME6 (SMemb). The expression of SM2 mRNA in the fetal aorta was significantly lower as compared with SM1 mRNA, but the ratio of SM2 to SM1 mRNA was increased after birth. SMemb mRNA in the aorta was decreased after birth but appeared to be increased in the aged. To further examine the MHC expression at the histological level, we have developed three antibodies against human SM1, SM2, and SMemb using the isoform-specific sequences of the carboxyl terminal end. Immunohistologically, SM1 was constitutively positive from the fetal stage to adulthood in the apparently normal media of the aorta and coronary arteries, whereas SM2 was negative in fetal arteries of the early gestational stage. In human, unlike rabbit, aorta or coronary arteries, SMemb was detected even in the adult. However, smaller-sized arteries, like the vasa vasorum of the aorta or intramyocardial coronary arterioles, were negative for SMemb. Diffuse intimal thickening in the major coronary arteries was found to be composed of smooth muscles, reacting equally to three antibodies for MHC isoforms, but reactivities with anti-SM2 antibody were reduced with aging. With progression of atherosclerosis, intimal smooth muscles diminished the expression of not only SM2 but also SM1, whereas alpha-smooth muscle actin was well preserved. We conclude from these results that smooth muscle MHC isoforms are important molecular markers for studying human vascular smooth muscle cell differentiation as well as the cellular mechanisms of atherosclerosis.

The objective of this study was to examine the identity and characteristics of a spontaneously occurring murine retroperitoneal tumor of BALB/c mouse origin that selectively metastasized to the liver. From the primary tumor, a permanent cell line, termed LMFS (liver metastasis from sarcoma) was established in vivo and in vitro. After a subcutaneous injection of more than 1 x 10(5) cells in the side back of mice, the LMFS cells proliferated at the inoculation site (100% take) and induced metastatic nodules spontaneously in the liver, but not in the lung. By the limiting dilution technique, a cloned cell line, LMFS-1, was established in vitro. The LMFS-1 cell line had similar morphological characteristics to the LMFS cells both in vitro and in vivo. The doubling time of the LMFS-1 cell line was 10 h in passage 60. The number of chromosomes ranged from 71 to 108 and 93% of metaphases showed near-tetraploidy. In microscopic examination, no specific arrangement of the LMFS tumor cells was seen; the LMFS cell had medium- to large-sized atypical nuclei and clear and large cytoplasm. Electronmicroscopy showed that the cytoplasm of the LMFS cell had a moderate amount of rough-surfaced endoplasmic reticulum but no desmosomes or microvilli. Immunohistochemically, the LMFS cells were positive for vimentin, but showed no reaction for keratin or cytokeratin. Therefore, the LMFS tumor was considered to be an undifferentiated sarcoma. The LMFS cell line should be a useful tool not only for studies of metastasis, but also for experiments on the therapy of hepatic tumors.

BACKGROUND: The closure of the ductus arteriosus (DA) is one of the most striking cardiovascular events that occur at birth. It has been attributed to oxygenation and intrinsic prostaglandins. However, selective constriction of DA suggests the presence of highly specialized contractile mechanisms in DA. We previously reported that smooth muscle myosin heavy chain isoforms, SM1 and SM2, are molecular markers for smooth muscle differentiation because of their unique expression pattern during vascular development. SM1 and SM2 are generated from a single gene through developmentally regulated alternative RNA splicing; SM1 is expressed in almost all stages of differentiation of the vascular smooth muscles, but SM2 is found only after birth. METHODS AND RESULTS: Immunohistochemistry was performed to study the expression of the different types of myosin heavy chain isoforms in DA of fetal and neonatal rabbits. Electron microscopic examinations were also carried out to demonstrate ultrastructural characteristics of ductus muscles. We found that SM2 is expressed before birth in the medial layer of DA, indicating advanced differentiation of smooth muscle cells in DA. The exact location of immunoreactivity for SM2 was in the smooth muscle cell of the medial layer of DA. Immunoreactivity for SM1, however, was not different for DA and adjacent great arteries. Transmission electron microscopy demonstrated greater amounts of myofilaments in medial smooth muscles of DA than those of aorta. CONCLUSIONS: These results indicate that smooth muscles in DA are more differentiated than those in other arteries, which may be one of the cellular mechanisms responsible for the unique closure of DA at birth.

Recent investigations have raised a possibility that abnormal ion transportation through cell membranes may be involved in the pathogenesis of essential hypertension. In order to test the hypothesis that increased activity of Na+/H+ antiporter may cause hypertension, we developed transgenic mice overexpressing the Na+/H+ antiporter. We isolated a full-length cDNA clone encoding the rabbit Na+/H+ antiporter and constructed the transgene by ligating it with the human elongation factor 1 alpha promoter. We obtained three transgenic strains which express the transgene in various tissues such as kidney, heart and aorta. These transgenic mice may be useful for the analysis of pathogenesis of essential hypertension.

The various species of Japanese hagfish, namely, Eptatretus okinoseanus (types A and B), Eptatretus burgeri and Myxine garmani, are known to eliminate a fraction of their chromosomes during early embryogenesis. High molecular weight DNA from germ line cells and somatic cells of these hagfish species was isolated and digested with different restriction enzymes. The DNA fragments were separated by agarose gel electrophoresis. Digestion with BamHI and DraI generated two weak bands and one weak band, respectively, that were estimated to be about 90, and 180 bp and about 90 bp long and were limited to the germ line DNA in both types of E. okinoseanus. DNA filter hybridization experiments showed that the two BamHI fragments and the one DraI fragment were present almost exclusively in the germ line DNA of E. okinoseanus. Thus, these DNA fragments appear to be eliminated during embryogenesis. Moreover, evidence was obtained that these fragments are highly and tandemly repeated. Molecular cloning and sequence analysis revealed that the BamHI fragments are mainly composed of a family of closely related sequences that are 95 bp long (EEEo1, for Eliminated Element of E. okinoseanus 1), and the DraI fragment is composed of another family of closely related sequences that are 85 bp long (EEEo2). The two DNA families account for about 19% of the total eliminated DNA in E. okinoseanus type A. Fluorescence in situ hybridization experiments demonstrated that the two families of DNA are located on several C-band-positive, small chromosomes that are limited to germ cells in both types of E. okinoseanus.

Rabbit smooth muscles contain at least three types of myosin heavy chain (MHC) isoforms; SM1, SM2 and SMemb (NMHC-B), the expression of which is developmentally regulated. We have recently reported that smooth muscles with the embryonic phenotype accumulate in the neointimas produced by endothelial denudation or high-cholesterol feeding. In this study, we examined MHC isoform expression in the neointimas and the media of poststenotic dilatation of the rabbit carotid artery, and determined the phenotype of the smooth muscle cell in the dilated segment. We report here that neointimal cells in the dilated segment are smooth muscle cells with the embryonic phenotype as previously reported in our ballooning-injury study. The medial smooth muscles, however, are composed of heterogeneous population of smooth muscles which differ in stage of differentiation as determined by the MHC isoform expression. These results indicate that MHC isoforms are useful molecular markers to identify abnormally proliferating smooth muscles in diseased arteries and to understand the process of atherogenesis occurring following vascular injury.

PDGF-like peptides secreted from smooth muscles have been suggested to be responsible for the smooth muscle growth. In order to elucidate the nature of PDGF-like molecules expressed in vascular smooth muscles, we have isolated and characterized cDNA clones for PDGF-A chain from a rabbit embryonic aorta cDNA library. One of the cDNA clones was found to encode a novel PDGF-A chain, named PDGF-A3 in this report. PDGF-A3 arises from a single PDGF-A chain gene by alternative RNA splicing and differs from the sequences of previously reported endothelial- or the glioma-type transcripts by a 110 bp insertion. Expression of PDGF-A3 mRNA was selectively induced by Angiotensin II in the smooth muscle cell in vitro. Total PDGF-A mRNA is most enriched in embryonic aortas, but its expression is down-regulated with vascular development. PDGF-A mRNA is markedly increased in primary-cultured smooth muscle cells during the log-phase growth. Our results suggest that autocrine production of PDGF-A chains from the smooth muscle cell may play a role in early vascular development and in Angiotensin II-induced smooth muscle cell proliferation.

Cytogenetic studies were performed on two types of Japanese hagfish (Eptatretus okinoseanus) that eliminate about 45% (type A) and 55% (type B) of their DNA from presumptive somatic cells during the differentiation of somatic cells. The observations revealed inter- and intraindividual variations in the number of chromosomes in germ cells of both types of hagfishes. Although the modal number of chromosomes in the germ cells was 54 in both types, the percentage of cells with the modal number was rather low (38.6% [51/132] in five specimens of type A and 22.7% [25/110] in eight specimens of type B). In addition, one of seven type B specimens clearly had a modal number of 62 chromosomes. Another specimen of type B had a bimodal distribution of chromosome numbers, with peaks of 54 and 59 chromosomes. The observation of interindividual variations was supported by data on the amount of DNA in germ cells of type B specimens. However, these variations were rarely observed in somatic cells. These results suggest that supernumerary (B) chromosomes are maintained in germ cells and are eliminated together with some other chromosomes and/or chromatin from somatic cells.

Adult rabbit smooth muscles contain two types of myosin heavy chain (MHC) isoforms, SM1 and SM2 which are generated through alternative RNA splicing from a single gene (Nagai, R., Kuro-o, M., Babij, P. & Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737). We previously reported that the expression of SM1 and SM2 during vascular development is differentially regulated at the level of RNA splicing, whereby SM1 is constitutively expressed from early development but SM2 appear after birth (Kuro-o, M., Nagai, R., Tsuchimochi, H., Katoh, H., Yazaki, Y., Ohkubo, A. & Takaku, F. (1989) J. Biol. Chem. 264, 18272-18275). We also demonstrated that embryonic vascular smooth muscles contain a third type of MHC isoform, referred to as SMemb in this report, which comigrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with SM2. In the present study we have isolated and characterized a cDNA clone (FSMHC34) for SMemb. FSMHC34 encodes the light meromyosin region including the carboxyl terminus and showed 70% amino acid sequence identity with SM1 or SM2. SMemb is a nonmuscle-type MHC and identical with brain MHC, but clearly distinct from 196-kDa nonmuscle MHC in cultured smooth muscle cells. The expression of SMemb was predominant in embryonic and perinatal aortas, but down-regulated with vascular development. Interestingly SMemb was reexpressed in proliferating smooth muscle cells of arteriosclerotic neointimas. These results suggest that smooth muscle proliferation is coupled to the expression of SMemb and that dedifferentiation of smooth muscles toward the embryonic phenotype is involved in the mechanisms underlying atherosclerosis.

C- and R-banding analyses were performed on chromosomes of Salamandrella keyserlingii, in which 20 of 31 chromosome pairs could be identified. Banding patterns were compared between S. keyserlingii and specimens of Hynobius species of the family Hynobiidae. Between S. keyserlingii and six Hynobius species (2n = 56), there were four homoelogous and two partially homoeologous chromosome pairs. Between S. keyserlingii and H. retardatus (2n = 40), there were two homoeologous and two partially homoeologous chromosome pairs.

Two types of smooth muscle myosin heavy chain (MHC) isoforms, SM1 and SM2, were recently identified to have different carboxyl termini (Nagai, R., Kuro-o, M., Babij, P., and Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737). SM1 and SM2 are considered to be generated from a single gene through alternative RNA splicing. In this study we investigated expression of vascular MHC isoforms during development in rabbits at the mRNA, protein, and histological levels. In adults, all smooth muscle cells reacted with both anti-SM1 and anti-SM2 antibodies on immunofluorescence, suggesting the coexpression of SM1 and SM2 in a single cell. In fetal and perinatal rabbits, however, only anti-SM1 antibody consistently reacted with smooth muscles. Reactivity with anti-SM2 antibody was negative in the fetal and neonatal blood vessels and gradually increased during 30 days after birth. These developmental changes in SM1 and SM2 expression at the histological level coincided with mRNA expression of each MHC isoform as determined by S1 nuclease mapping, indicating that expression of SM1 and SM2 is controlled at the level of RNA splicing. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of myosin from fetal and perinatal aortas revealed the presence of large amount of SM2. Interestingly, fetal SM2 did not cross-react with our anti-SM2 antibody on immunoblotting. We conclude that expression of SM1 and SM2 are differentially regulated during development and that a third type of MHC isoform may exist in embryonic and perinatal vascular smooth muscles.

We previously reported the characterization of a rabbit uterus cDNA clone (SMHC29) which encoded part of the light meromyosin of smooth muscle myosin heavy chain (Nagai, R., Larson, D.M., and Periasamy, M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 1047-1051). We have now characterized a second cDNA clone (SMHC40) which also encodes part of the light meromyosin but differs from SMHC29 in the following respects. Nucleotide sequence analysis demonstrates that the two myosin heavy chain mRNAs are identical over 1424 nucleotides but differ in part of the 3'-carboxyl coding region and a portion of the 3'-nontranslated sequence. Specifically, SMHC40 cDNA encodes a unique stretch of 43 amino acids at the carboxyl terminus, whereas SMHC29 cDNA contains a shorter carboxyl terminus of 9 unique amino acids which is the result of a 39-nucleotide insertion. Recent peptide mapping of smooth muscle myosin heavy chain identified two isotypes with differences in the light meromyosin fragment that were designated as SM1 (204 kDa) and SM2 (200 kDa) type myosin (Eddinger, T. J., and Murphy, R.A. (1988) Biochemistry 27, 3807-3811). In this study we present direct evidence that SMHC40 and SMHC29 mRNA encode the two smooth muscle myosin heavy chain isoforms, SM1 and SM2, respectively, by immunoblot analysis using antibodies against specific carboxyl terminus sequences deduced from SMHC40 and SMHC29 cDNA clones.

To investigate the response of myosin isozyme transition in specialized myocardium to cardiac overload, we examined immunohistochemically the distribution of myosin isozymes in sinus node cells of overloaded canine atria, using the monoclonal antibodies CMA19 and HMC14, which are specific for atrial myosin heavy chain (alpha-HC) and ventricular myosin heavy chain (beta-HC), respectively. Overloading in canine right atria was induced by artificial tricuspid valve regurgitation and pulmonary stenosis. Right atrial mean pressure rose to 15-20 mm Hg (n = 4) 2 months after surgery. In the working myocardium, cardiac overload caused redistribution of myosin isozymes, alpha-HC to beta-HC. Compared with the normal right atria, fewer myocytes were labeled with CMA19, but more were labeled with HMC14. However, the reactivity of sinus node cells with CMA19 and HMC14 was not changed between normal and overloaded right atria, indicating no redistribution of myosin heavy chain isozymes, alpha-HC to beta-HC. These results suggest that isozymes in myosin heavy chains in the specialized myocardium are protected from overload effects by their firm cytoskeletal framework or other mechanisms.

To investigate the existence of heterogeneity of beta-type myosin isozymes (HC beta) in human hearts, immunohistochemical studies using monoclonal antibodies (MoAbs) raised against human ventricular myosin heavy chains were performed. Two types of MoAbs recognized some muscle fibers in the atrium, whereas both reacted with all ventricular muscle fibers. Since atrial muscle fibers reactive with each MoAb were found to be clearly different, the existence of two immunologically distinct HC beta (beta 1, and beta 2) was suggested in the atrium. By using affinity chromatography, two molecular variants of HC beta were isolated from the bovine atrium, and differences in the primary structure of beta 1 and beta 2 were confirmed by analysis of peptides produced by chymotryptic digestion. In pressure-overloaded human atria, myofibers containing beta 1 and/or beta 2 increased in accordance with decrement of myofibers containing alpha-type myosin isozyme (P less than 0.01). But they differed in expression during the developmental stage, since beta 2 did not exist in the early embryonic bovine heart, but beta 1 did. Thus, there are two distinct HC beta whose expression is regulated by at least two factors: pressure overload and developmental stage.

Cardiac muscles contain at least two isozymes--referred to as alpha(HC alpha) and beta(HC beta)--of the myosin heavy chain. The proportional ratio of these isozymes varies depending upon the developmental stage and the physiological and/or the hormonal milieu of the cell. Using monoclonal antibodies (MoAb) specific for human cardiac HC alpha and HC beta, we have examined the expression of these isozymes in fetal through adult cardiac tissues and investigated whether isozymic redistribution occurs in pressure overloaded human ventricles. We found that although HC alpha was expressed in the atrium from the early embryonic stage, in embryonic ventricular myofibers, only HC beta was expressed without expression of HC alpha, but some myofibers replace HC beta by HC alpha after birth, and these HC alpha containing ventricular myofibers were found to be decreased by pressure overload, which suggested that isozymic redistribution from HC alpha to HC beta also occurred in the ventricles, as well as the atrium. In addition, we also found two subtypes of HC beta (beta 1, beta 2) in the human heart. In the ventricle, both beta 1 and beta 2 was present in all myofibers; in contrast, some myofibers contained beta 1 or beta 2 or both with or without expression of HC alpha in the atrium. beta 1 and beta 2 were distinctive in their expression during the developmental stage, since beta 1 was present in the embryonic heart from the early developmental stage, whereas beta 2 was not present in the early embryonic heart, but began to be expressed in the late embryonic stage.(ABSTRACT TRUNCATED AT 250 WORDS)

To determine the presence and distribution of cardiac myosin isozymes in the human conduction system, we performed an immunohistochemical study using monoclonal antibodies CMA19 and HMC14, which are specific for myosin heavy chains of human atrial type (alpha-type) and ventricular type (beta-type), respectively. Serial frozen sections of human hearts were obtained from autopsy samples and examined by indirect immunofluorescence. Alpha-type was found in all myofibers of sinus node and atrio-ventricular node, and in 55.2 +/- 10.2% (mean +/- SD, n = 5) of the myofibers of ventricular conduction tissue, which consists of the bundle of His, bundle branches, and the Purkinje network. In contrast, beta-type was found in all myofibers of the atrio-ventricular node and ventricular conduction tissue, whereas almost all myofibers of the sinus node were unlabeled by HMC14. Although the number of ventricular myofibers labeled by CMA19 was small, the labeled myofibers were more numerous in the subepicardial region than in the subendocardial region. These findings show that the gene coding for alpha-type is expressed predominantly in specialized myocardium compared with the adjacent ordinary working myocardium.

The R-banding technique of Dutrillaux et al. (1973) was modified in order to analyze the chromosomes of salamanders in the genus Hynobius. Embryonic cells of Hynobius nigrescens Stejneger from Nakakubiki County, Niigata Prefecture, and Hakui County, Ishikawa Prefecture, were cultured in medium containing 5-bromodeoxyuridine (BrdU). Banded metaphases were obtained by the FPG (fluorescent-plus-Giemsa) technique (Perry and Wolff, 1974), with slight modifications. With this modified R-banding technique, multiple, clear DNA replication bands were obtained on the chromosomes, and 18 of 28 chromosome pairs could be identified easily by their replication patterns in embryos collected from Nakakubiki County. A distinct heteromorphism in banding pattern was detected on the long arm of chromosome 9 in these embryos, but the frequency of this variant was too low for chromosome 9 to be a sex chromosome. Chromosomes 1-14, except for 6, 12 (for which the data were not satisfactory), and 9 (the variant type from Nakakubiki County), had the same replication patterns in embryos obtained from Nakakubiki and Hakui Counties.

We have prepared monoclonal antibodies specific for either atrial or ventricular myosin and defined the isomyosin composition of myocardium in normal and overloaded human hearts. In the atrial myocardium, normal isozymic pattern was V1 dominant which converted to being V3 dominant in an overloaded condition. In contrast, normal isomyosin pattern of the ventricular myocardium was exclusively V3 dominant, and only a small change in the proportion of isomyosin was observed in an overloaded condition. From this, we conclude that isozymic changes in cardiac myosin could occur in the human heart to meet increased work induced by cardiac overload. However, the physiological importance of these isomyosin redistributions in human myocardium seems to be much greater in the atrium than in the ventricle, since larger amounts of V1 isomyosin which could be transformed to V3 isomyosin were present in the atrial myocardium.

An immunohistochemical study using monoclonal antibodies specific for the heavy chains of either human atrial (HC alpha) or ventricular (HC beta) myosin was performed to clarify the distribution of each isozyme in normal as well as pressure-overloaded human hearts. In normal human ventricles, all muscle fibers were stained by a monoclonal antibody (HMC14) specific for HC beta, whereas a small number of fibers reacted with a monoclonal antibody (CMA19) specific for HC alpha. In contrast, in normal human atria, almost all muscle fibers were stained by CMA19, and a relatively larger number of muscle fibers also reacted with HMC14. Furthermore, in pressure-overloaded atria, muscle fibers reactive with HMC14 were strikingly increased while those reactive with CMA19 showed a corresponding decrease. The extent of this isozymic redistribution was in good correlation with atrial pressure. These results not only confirmed the existence of isoforms of myosin heavy chain in human hearts, but also demonstrated that redistribution of iso-myosins could occur as an adaptation to pressure overload.