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Biomedical subjects

I Komuro

Publications and source records attributed to I Komuro.

At least 145 records · Page 8Linked to original sources
PubMed1994

Involvement of the renin-angiotensin system in the development of left ventricular hypertrophy and dysfunction.

OBJECTIVES: To elucidate the regulation of cardiac gene expression by mechanical stress and to analyse molecular mechanisms associated with the involvement of angiotensin II (Ang II) in the development of cardiac hypertrophy and dysfunction. METHODS: Neonatal rat cardiocytes were cultured in deformable silicone dishes, and mechanical stress was imposed on the cardiocytes by stretching them. In in vivo studies, spontaneously hypertensive rats (SHR) were treated with a non-peptide, specific Ang II type 1 receptor antagonist, TCV 116. RESULTS: Expression of the c-fos gene was rapidly induced, and fetal type genes such as skeletal alpha actin and beta myosin heavy chain genes were re-expressed by stretching. The mechanical stress decreased the expression of Ca(2+)-ATPase in the sarcoplasmic reticulum. With regard to signals for the development of cardiac hypertrophy, mechanical stress evoked c-fos expression via the activation of protein kinase C. The phosphorylation cascade (sequential activation of protein kinase C, Raf-1 kinase, mitogen-activated protein kinase kinase, mitogen-activated protein kinase and S6 kinase), which may be involved in protein synthesis and gene expression, was activated by mechanical stress in cardiocytes. Stretch-induced cardiac cellular hypertrophy was partially inhibited by TCV 116. TCV 116 treatment of SHR reduced left ventricular weight, left ventricular wall thickness, myocyte transverse diameter, V3 myosin heavy chain levels and the interstitial collagen volume fraction. CONCLUSIONS: These results indicate that AngII may, in part, mediate the stretch-induced hypertrophic growth of cardiomyocytes via the type 1 Ang II receptor.

Actins↗
PubMed1993

Csx: a murine homeobox-containing gene specifically expressed in the developing heart.

The molecular control of the differentiation process depends in part on lineage-restricted transcription factors that regulate expression of tissue-specific genes. Although significant progress has been made in molecular understanding of skeletal muscle differentiation, no information is available concerning the genes involved in development of the heart, the first organ to form in vertebrate embryos. Many vertebrate homeobox-containing genes have been shown to be expressed in broad regions of the mouse embryo, but no expression of a homeobox gene has been found in the most anterior region of the early embryo, the heart primordium. We report here on the cloning of a murine homeobox cDNA, Csx (cardiac-specific homeobox). The Csx homeodomain sequence is divergent from those of the Hox class genes but is related to that of Drosophila msh-2 (NK-4), which plays a key role in Drosophila heart formation. Csx is conserved in evolution and Csx homologs exist in all vertebrates examined. Transcripts of Csx are detected from the presomite stage (7.5 days postcoitum), when mesoderm differentiates into promyocardium. Csx expression is restricted in the myocardial cells from 8.5 days postcoitum through adult. Csx is not expressed in skeletal or smooth muscle or any other tissues examined. Expression of Csx precedes that of cardiac-specific genes in embryonic stem cells differentiating into beating myocardial cells in vitro. Although physiological function of Csx is yet to be determined, the temporal and spacial pattern of Csx expression raises a possibility that Csx may play a critical role in the differentiation of cardiac cells.

Aging↗
PubMed1993

Mechanical loading activates mitogen-activated protein kinase and S6 peptide kinase in cultured rat cardiac myocytes.

The molecular mechanisms by which overloaded cardiac myocytes increase the cell size (hypertrophy) remain unknown. We have previously shown that mechanical loading increased the protein synthesis and the expression of proto-oncogene c-fos mRNA (Komuro, I., Kaida, T., Shibazaki, Y., Kurabayashi, M., Katoh, Y. Hoh, E., Takaku, F., and Yazaki, Y. (1990) J. Biol. Chem. 265, 3595-3598; Komuro, I., Katoh, Y., Kaida, T., Shibazaki, Y., Kurabayashi, M., Hoh, E., Takaku, F., and Yazaki, Y. (1991) J. Biol. Chem. 266, 1265-1268). It has been known that both mitogen-activated protein (MAP) kinase and S6 kinase can be activated by many kinds of growth factors. To clarify whether MAP kinase(s) and S6 kinase(s) are associated with the intracellular signaling of cardiac hypertrophy induced by mechanical loading, we cultured neonatal rat cardiac myocytes in deformable dishes and imposed an in vitro mechanical loading by stretching the adherent myocytes. In this study, we demonstrated that 1) myocyte stretching maximally activated a kinase activity toward myelin basic protein (MBP) at 10 min after stretching, and the kinase activity returned to the control level at 30 min after stretching; 2) kinase assays in MBP-containing gel, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that stretch-induced MBP kinase activity mainly migrated at 42 kDa in the immunoprecipitated fraction of anti-MAP kinase antibody, suggesting that the stretching mainly increased the 42-kDa MAP kinase activity in cardiac myocytes; 3) phosphorylation of MAP kinase was induced after stretching cardiac myocytes; 4) when protein kinase C was depleted by preincubating myocytes with 100 nM 12-O-tetradecanoyl-phorbol-13-acetate for 24 h or 2 nM staurosporine for 30 min, stretch-induced MBP kinase activity was decreased by approximately 60-70% as compared with the kinase activity in myocytes without protein kinase C depletion; 5) although the receptor tyrosine kinases were depleted by preincubating myocytes with 50 microM tyrphostin or 20 microM genistein for 30 min, there was no change in the stretch-induced MBP kinase activity; 6) stretch-induced MBP kinase activity was partially dependent on transsarcolemmal influx of Ca2+; 7) myocyte stretching also increased S6 peptide (RRLSSLRA) kinase activity in the anti-S6 kinase II antibody immunoprecipitates. From these results, we conclude that myocyte stretching increases the activities of MAP kinase and S6 peptide kinase, which may play an important role in the induction of the specific genes and the increase in the protein synthesis.

Alkaloids↗
PubMed1993

Role of protein kinase system in the signal transduction of stretch-mediated protooncogene expression and hypertrophy of cardiac myocytes.

To examine the molecular mechanisms by which mechanical stimuli induce protooncogene expression and hypertrophy of cardiac myocytes directly, we cultured rat neonatal cardiac myocytes in deformable dishes and imposed mechanical load on adherent cultured cardiac myocytes by stretching the dishes. Myocyte stretching increased total cell RNA content and mRNA levels of c-fos and skeletal alpha-actin followed by the amino acid incorporation into cardiac proteins. CAT assay analysis indicated that the sequences containing serum response element were required for the efficient transcription of c-fos gene by stretching. This accumulation of c-fos mRNA by myocyte stretching was inhibited markedly by down-regulation of protein kinase C. Moreover, myocyte stretching increased inositol phosphate levels. These findings suggests that mechanical stimuli might directly induce protooncogene expression possibly via protein kinase C activation. Furthermore, we observed the activation of MAP kinase by myocytes stretching. This result suggests that MAP kinase activation induced by mechanical stimuli might increase the efficiency of protein synthesis on ribosomes induced by mechanical stimuli.

Actins↗
PubMed1993

Gtx: a novel murine homeobox-containing gene, expressed specifically in glial cells of the brain and germ cells of testis, has a transcriptional repressor activity in vitro for a serum-inducible promoter.

Although it is likely that a highly complex network of transcription factors acts in concert during mammalian brain development, relatively few such genes have been characterized to date. We describe here a novel murine homeobox gene, denoted Gtx, which in adult animals is specifically expressed within glial cells of the central nervous system, including the forebrain, and in germ cells of the testis. Gtx resides on chromosome 7 and does not cosegregate with any previously mapped homeobox gene. The amino acid sequence of the predicted protein encoded by Gtx is highly divergent from that of any other known homeobox genes. The Gtx homeodomain contains unique residues at positions predicted to contact DNA bases. It did not bind to known target sites for other homeobox genes in vitro but bound with high affinity to the MEF-2 motif, a binding site for the serum response factor-related proteins. GTX efficiently competed with RSRF to bind the MEF-2 element in vitro. Co-transfection of Gtx prevented the serum-induced activation of the MEF-2-containing reporter genes. Although the true biological role of Gtx is not known, these results suggest that Gtx is a novel cell-type specific homeobox gene that has the potential to act as a transcriptional repressor for a subset of serum-inducible genes.

Amino Acid Sequence↗
PubMed1992

Molecular cloning and characterization of the human cardiac Na+/Ca2+ exchanger cDNA.

The Na+/Ca2+ exchanger plays important roles in Ca2+ handling in many excitable cells. In particular, the Na+/Ca2+ exchanger is expressed at high levels in the cardiac sarcolemma and is the dominant mechanism of Ca2+ extrusion from the cells. In addition, the exchanger has been suggested to play key roles in digitalis action and in postischemic reperfusion injury of cardiac myocytes. We report here the isolation and characterization of the cDNA encoding the human cardiac Na+/Ca2+ exchanger. Twelve overlapping clones corresponding to 5.6 kilobases of the exchanger cDNA sequence were isolated from 5 x 10(5) phage plaques screened. The sequence predicted a 973-amino acid polypeptide with a putative leader peptide, 11 potential membrane-spanning regions, and one large putative cytoplasmic loop between the fifth and sixth transmembrane helices. When RNA was synthesized in vitro from the cloned cDNA and injected into Xenopus oocytes, it induced expression of Na+/Ca2+ exchange activity at high levels, confirming that this clone encodes the functional Na+/Ca2+ exchanger. Southern blot analysis indicated that the cardiac exchanger gene exists as a single copy in the human genome, although existence of other related genes cannot be ruled out. Northern blot and S1 mapping analyses revealed that the cardiac type exchanger mRNA is expressed most abundantly in the heart and next in the brain. The cardiac-type exchanger mRNA was also expressed in the retina and in skeletal and smooth muscles at very low levels. The levels of mRNA encoding the exchanger were significantly lower in fetal hearts than in adult hearts but were unchanged in the myocardium from patients with end-stage heart failure.

Amino Acid Sequence↗
PubMed1992

Role of protein kinase system in the signal transduction of stretch-mediated myocyte growth.

To examine the molecular mechanisms by which mechanical stimuli induce protooncogene expression, we cultured rat neonatal cardiocytes in deformable dishes and imposed an in vitro mechanical load by stretching the adherent cells. Myocyte stretching increased total cell RNA content and mRNA levels of c-fos and skeletal alpha-actin followed by activation of protein synthesis. CAT assay indicated that sequences containing a serum response element were required for efficient transcription of c-fos gene by stretching. This accumulation of c-fos mRNA was suppressed by protein kinase C inhibitors at the transcriptional level and was inhibited markedly by down-regulation of protein kinase C. Moreover, myocyte stretching increased inositol phosphate levels. These findings suggest that mechanical stimuli might directly induce protooncogene expression, possibly, via protein kinase C activation. Furthermore, we observed the activation of mitogen activated protein (MAP) kinase by myocyte stretching. This result suggest that MAP kinase activation might increase the efficiency of protein synthesis in ribosomes induced by mechanical stimuli.

Animals↗
PubMed1992

Molecular mechanism of hypertrophied failing heart--abnormalities of the diastolic properties and contractility.

The clinical syndrome of heart failure occurs as a consequence of the limitation of compensatory mechanisms, such as cardiac hypertrophy. To clarify transcriptional changes in specific genes in failing hearts, we examined the expression of cardiac Ca(2+)+Mg(2+)-dependent ATPase in the sarcoplasmic reticulum and transforming growth factor beta genes in the ventricles of rat hypertrophied heart, and the expression of guanine nucleotide-binding protein and "fetal" contractile protein genes in the ventricles of cardiomyopathic Syrian hamsters of Bio14.6. Northern blot analysis of total cellular RNA revealed that the mRNA levels of Ca(2+)+Mg(2+)-dependent ATPase were decreased by pressure overload and became 32% of sham in 1 month, and were correlated with corresponding protein levels. Transforming growth factor beta mRNA, a potent activator of collagen synthesis, was increased by pressure overload. The expression levels of the Gs alpha mRNA, which stimulated the adenylate cyclase, in Bio14.6 ventricles were lower than the levels in ventricles of the F1B hamster strain, and decreased as the stage of cardiomyopathy progressed. Moreover, re-expression of fetal mRNA was observed in the ventricle of cardiomyopathic Syrian hamsters of the Bio14.6 strain. These results indicate that reprogramming of cardiac gene expression both of myofibrillar and nonmyofibrillar components might occur in the failing heart.

Animals↗
PubMed1991

Mechanical loading stimulates cell hypertrophy and specific gene expression in cultured rat cardiac myocytes. Possible role of protein kinase C activation.

To examine the molecular mechanisms by which mechanical stimuli induce cardiac hypertrophy and specific gene expression, we cultured rat neonatal cardiocytes in deformable dishes and imposed an in vitro mechanical load by stretching the adherent cells. Myocyte stretching increased total cell RNA content and mRNA levels of c-fos and skeletal alpha-actin. Nuclear run-off transcription assay revealed that this increase in c-fos mRNA level by stretching at least partially reflects changes in the transcriptional status. The transfected chloramphenicol acetyltransferase gene linked to upstream sequences of the fos gene indicated that sequences containing a serum response element were required for efficient transcription by stretching and that sequences containing a cAMP/calcium response element might not be involved in the c-fos response to myocyte stretching. The accumulation of c-fos mRNA by stretching was suppressed by protein kinase C inhibitors at the transcriptional level and inhibited markedly by down-regulation of protein kinase C. Moreover, myocyte stretching increased inositol phosphate levels, and activation of protein kinase C by phorbol esters stimulated the expression of c-fos and skeletal alpha-actin genes. These findings suggest that mechanical stimuli (myocyte stretching) might directly induce cardiac hypertrophy and specific gene expression possibly via protein kinase C activation.

Actins↗
PubMed1991

Mechanisms of cardiac hypertrophy and injury--possible role of protein kinase C activation.

To examine the molecular mechanisms by which mechanical stimuli induced cardiac hypertrophy and injury, we cultured rat neonatal cardiocytes in deformable dishes and imposed an in vitro mechanical load by stretching the adherent cells. Myocyte stretching increased total cell RNA content and mRNA levels of c-fos. Marked accumulation of c-fos mRNA followed increases in intracellular Na+ and protein kinase C activation. The accumulation of c-fos mRNA by cardiocyte stretching was suppressed by protein kinase C inhibitors but not by stretch channel blockers. Moreover, myocyte stretching increased inositol phosphate levels, and activation of protein kinase C by phorbolesters stumulated the expression of c-fos. We also examined TGF beta expression in the heart. TGF beta is known to be stimulated by protein kinase C activation, and the mRNA level of TGF beta was increased in in vivo heart by pressure overload. Furthermore, collagen synthesis was stimulated by TGF beta in cultured fibroblasts from hearts. These findings suggest that hemodynamic overload may stimulate cardiac hypertrophy and induce cardiac injury (fibrosis) through protein kinase C activation.

Animals↗
PubMed1990

Functional identification of the transcriptional regulatory elements within the promoter region of the human ventricular myosin alkali light chain gene.

We have identified and functionally characterized DNA sequences that regulate the expression of the human ventricular/slow twitch isoform of myosin alkali light chain (VLC1) gene. By using primer extension and S1 nuclease mapping techniques, we have shown that the VLC1 gene is transcribed from the identical site in the ventricular and slow twitch skeletal muscles. Comparison of the VLC1 sequences from +1 to -1296 in the genes for human and mouse showed that the 5'-proximal flanking region, up to about 220 nucleotides, was highly conserved (83% homology). To determine the location of sites that may be important for the function of the VLC1 promoter, a series of transient expression vectors containing progressive deletions of the VLC1 gene 5'-flanking sequence fused to the bacterial chloramphenicol acetyltransferase (CAT) gene was introduced into myogenic and nonmyogenic cells. Deletion mutagenesis of sequences between -357 and +40 revealed the presence of positive and negative activity in all the cells tested. We demonstrated that the minimal promoter sequence required to generate muscle cell-specific expression is the region between -94 to -64 upstream from the cap site and a sequence element located between -107 and -94 was found to have a positive effect in both myogenic cells and nonmyogenic cells. These two proximal regions located between -107 and -64 appear to act together to determine the cell type-specific high level expression of the VLC1 gene in muscle cells. Competition gel retardation assays revealed that the CArG sequence located between -96 and -87 interacts specifically with nuclear extracts from myogenic and nonmyogenic cells and compete for binding with the CArG sequence present in the human cardiac alpha-actin gene and with the serum response element of the c-fos gene. These results strongly suggested that similar, if not identical, the CArG box binding proteins interact with the functionally different promoter element in the VLC1, cardiac alpha-actin, and c-fos genes.

Animals↗
PubMed1990

Stretching cardiac myocytes stimulates protooncogene expression.

Recently cellular protooncogenes have been found to be induced as an early response to pressure overload in cardiac hypertrophy. To examine whether mechanical stimuli directly induce specific gene expression in the heart, we cultured rat neonatal cardiocytes in elastic silicone dishes and stretched these adherent cells. Myocyte stretching stimulated expression of the protooncogene, c-fos, in a stretch length-dependent manner, followed by an increase in amino acid incorporation into proteins. c-fos mRNA levels were enhanced within 15 min by cardiocyte stretching, peaked at 30 min, and declined to undetectable levels by 240 min. In the presence of cycloheximide, a greater increase in c-fos mRNA was seen by stretching. The transfected chloramphenicol acetyltransferase gene linked to upstream sequences of the fos gene including its promoter was also activated by stretching cardiac myocytes. These results suggest that mechanical loading directly regulates gene transcription without the participation of humoral factors in cardiocytes.

Animals↗
PubMed1990

Cytoplasmic calcium ion elevating factor(s) in spontaneously hypertensive rat serum.

We have attempted to characterize the as yet unspecified circulating factor(s) in spontaneously hypertensive rat (SHR) serum that elevate(s) the cytoplasmic calcium ion concentration in vascular smooth muscle cells (VSMC). We measured the cytoplasmic calcium ion concentration in cultured VSMC before and after incubation with serum from SHR or Wistar-Kyoto rats (WKY). Incubation with serum from overtly hypertensive 8-week-old SHR, but not with serum from 8-week-old WKY, significantly elevated the cytoplasmic calcium ion concentration in VSMC from 8-week-old WKY and 4-week-old SHR in a dose- and an incubation period-dependent manner, although no significant elevation occurred in VSMC from 4-week-old WKY. Neither the heat-immobilized nor the trypsinized 8-week-old SHR serum elevated the cytoplasmic calcium ion concentration. Ultrafiltration procedures indicated that the molecular weight of the factor should be 10,000-30,000 daltons. Incubation with serum from 4-week-old SHR or WKY did not elevate the cytoplasmic calcium ion concentration. These results strongly suggest that: (1) a cytoplasmic calcium ion concentration elevating factor exists in serum from 8-week-old SHR but not from 4-week-old SHR, or 4- or 8-week-old WKY; (2) this factor should be of a protein nature, with a molecular weight of 10,000-30,000 daltons; (3) responsiveness to the factor appears in VSMC at a younger age in SHR than in WKY; (4) the factor elevates the cytoplasmic calcium ion concentration to the level determined a priori not by the strain but by the age of the rats from which the VSMC originate; and (5) the a priori determined level increases with the age of the rats.

Animals↗
PubMed1990

Molecular cloning of gene sequences from rat heart rapidly responsive to pressure overload.

Although pressure overload induces cardiac hypertrophy and reexpression of contractile protein isogenes, the molecular mechanism of both is not well understood. We demonstrated the early change in the translational activity of specific cardiac messenger RNA by two-dimensional gel electrophoresis of in vitro translational products. A total of over 400 translational products were detected by fluorography, and the relative predominance of eight species was increased by pressure overload whereas that of two translational products was decreased. We cloned four pressure-overload-responsive complementary DNA clones, pPO-1, -4, -5, and -6, by differential dot blot hybridization. Two clones were increased from the early period after the imposition of pressure overload; the other two were decreased. The expression pattern of each complementary DNA clone in the pressure-overloaded hearts was similar to that in fetal hearts. Pressure overload also induced the additional messenger RNA, which hybridized with pPO-4. This mRNA was also recognized in fetal, neonatal, and adult spontaneously hypertensive rat hearts. The induction of this transcript by pressure overload was not suppressed but, rather, stimulated by cycloheximide. These results suggest that there are some genes that rapidly respond to pressure overload and that may play some role in the development of cardiac hypertrophy.

Acute Disease↗
PubMed1990

Messenger RNA levels of guanine nucleotide-binding proteins are reduced in the ventricle of cardiomyopathic hamsters.

The expression of guanine nucleotide-binding protein (G-protein) genes (Gs alpha, Go alpha, Gi alpha 1, Gi alpha 2, and Gi alpha 3) was examined in the ventricle of cardiomyopathic Syrian hamsters of the Bio14.6 strain (10-35 weeks old). Northern blot analysis of total cellular RNA revealed that all G-protein genes except Gi alpha 1 were expressed in the ventricle of Syrian hamsters. Gs alpha and Gi alpha 2 genes were abundantly expressed. The expression levels of the Gs alpha and Gi alpha 2 messenger RNAs in Bio14.6 ventricles were lower than the levels in ventricles of the F1B hamster strain; the abundance of Go alpha and Gi alpha 3 messenger RNAs did not change markedly. Moreover, the messenger RNA levels of Gs alpha and Gi alpha 2 decreased as the stage of cardiomyopathy progressed. Since G-proteins are linked to adenylate cyclase, these alterations of G-protein messenger RNA levels may be related to reduced contractility of cardiomyopathic heart.

Animals↗
PubMed1990

The myosin gene switching in human cardiac hypertrophy.

Cardiac hypertrophy is associated with qualitative as well as quantitative changes in myocardial cells. To analyze the molecular basis of isozymic transitions of cardiac myosins in response to pressure overload, we have constructed and characterized two types of myosin heavy chain (MHC) cDNA clones, specifying alpha- and beta-MHCs, and two types of myosin alkali light chain cDNA clones, complementary to atrial type (ALC1) and ventricular type (VLC1) mRNAs from a human fetal heart cDNA library. Using the S1 nuclease mapping procedure, we showed that the MCH isozymic transitions from alpha- to beta-MHC in the pressure overloaded atria are produced by changes in the relative level of alpha- and beta-MHC gene expression. In addition, we observed that the expression of VLC1 gene is also induced in the atria subjected to severe pressure overload. Thus, it appears that the increased expression of VLC1 gene, together with the isogene switch from alpha- to beta-MHC gene, may participate in the adaptation of myocardium to new functional requirement. Then, to get a better understanding of the genetic mechanisms involved in the regulation of isogene expression, we have isolated and sequenced genomic clone for VLC1 isoform. Sequence analysis has identified multiple potential cis regulatory elements within a 686-bp upstream region. This region includes 28-bp alternating purine/pyrimidine sequences and two segment exhibiting homology to consensus sequence proposed for viral and cellular enhancer elements. In particular, a comparison of the VLC1 upstream gene sequence with those available for several muscle-specific genes revealed that CC(A + T-rich)6GG elements and CATTCCT sequence are conserved. These results suggested that CArG box (-96 to -87) has an important role in the positive regulation of the VLC1 gene and this element may be involved in the co-regulation of VLC1 and cardiac alpha-actin genes.

Adaptation, Physiological↗