Search PubMed⌕ Search

Biomedical subjects

I Komuro

Publications and source records attributed to I Komuro.

At least 127 records · Page 7Linked to original sources
PubMed1996

Mechanical stretch activates the stress-activated protein kinases in cardiac myocytes.

We have recently shown that mechanical stress activates a phosphorylation cascade of protein kinases including Raf-1 and the extracellular signal-regulated kinases (ERKs) in cultured cardiac myocytes partially through the enhanced secretion of angiotensin II. Osmotic stress in budding yeast has been shown to activate similar signaling molecules including Hog-1, a distant relative of the ERK family. In the present study, we examined whether mechanical stretch of cardiac myocytes activates the stress-activated protein kinases (SAPKs)/c-Jun NH2-terminal kinase, the mammalian homologs of yeast Hog-1 that regulate gene expression through activation of the transcription factor, AP-1. When cardiac myocytes of neonatal rats cultured on a deformable silicone dish were stretched, activity of SAPKs was increased from 10 min, peaked at 30 min, and gradually decreased thereafter. The increase in activity of SAPKs was proportional to the stretch. Unlike ERKs, the activation of SAPKs by stretching cardiac myocytes was not dependent on the secreted angiotensin II. The chelation of extracellular Ca2+ or down-regulation of protein kinase C did not attenuate activation of SAPKs by stretch. Transfection experiments using an AP-1 binding site-containing reporter gene revealed that stretch increases AP-1 activity in cardiac myocytes. In conclusion, like osmotic stress in yeast, mechanical stretch activates SAPKs in cardiac myocytes without the participation of angiotensin II. These results suggest that the activation of SAPKs may regulate gene expression during mechanical stress-induced cardiac hypertrophy.

Angiotensin II↗
PubMed1996

Mechanical stretch induces enhanced expression of angiotensin II receptor subtypes in neonatal rat cardiac myocytes.

Mechanical stress plays a pivotal role in the development of cardiac hypertrophy during hemodynamic overload, and angiotensin (Ang) II secreted from stretched myocytes plays an important role in mechanical stretch-induced hypertrophy. In the present study, we examined stretch-induced expression of Ang II receptors in an in vitro stretch model using 1-day-old rat myocytes. Both Ang II type 1 receptor (AT1-R) and type 2 receptor (AT2-R) mRNA levels were upregulated by myocyte stretching with similar time courses: significant increases were evident 6 hours after stretching, maximal levels (2.8- and 3.3-fold, respectively) were observed at 12 hours, and these were sustained for up to 18 hours. Ang II receptor expression in fibroblast-rich cultures was not affected by stretching. Conditioned medium in which myocytes were stretched for 12 hours significantly downregulated AT1-R and AT2-R mRNA levels in recipient myocytes, and this effect was almost completely blocked by AT1-R antagonists but not AT2-R antagonists. Stretch-induced expression of AT1-R and AT2-R mRNAs was further increased by 27% and 31%, respectively, after pretreatment with AT1-R antagonists, suggesting that Ang II secreted from stretched myocytes downregulates both AT1-R and AT2-R. Western blot and binding assays showed that the number of AT1-Rs and AT2-Rs increased by 2.4- and 2.6-fold, respectively, without affecting receptor affinities. Inositol phosphate response to 0.5 mumol/L Ang II was enhanced 2.1-fold in stretched myocytes. Nuclear runoff assays and treatment with actinomycin D revealed that stretch-induced upregulation of AT1-R was mainly due to increased transcription, whereas that of AT2-R resulted from a stabilizing effect on AT2-R mRNA metabolism. Stretch-induced changes in levels of Ang II receptors were inhibited by genistein but not by H-7, staurosporin, and protein kinase C depletion or by BAPTA-AM. Exposure to cycloheximide did not affect stretch-induced changes. These findings indicate that nonsecretory pathways activated by myocyte stretching upregulate the expression of Ang II receptor subtypes transcriptionally and posttranscriptionally through mechanisms involving stretch-activated tyrosine kinases independently of de novo protein synthesis and that the AT1-R-mediated action of Ang II is functionally enhanced in stretched cardiac myocytes.

Angiotensin II↗
PubMed1996

Molecular cloning and characterization of human cardiac homeobox gene CSX1.

Accumulating evidence has suggested that homeo-domain-containing proteins play critical roles in regulating the tissue-specific gene expression essential for tissue differentiation and in determining the temporal and spatial patterns of development. In order to elucidate the mechanisms of human heart development, we have isolated a human homologue of the murine cardiac homeobox gene Csx (also called Nkx-2.5) and denoted it as CSX1. The amino acid sequence of the CSX1 homeodomain is 100% and 67% identical to that of murine Csx/Nkx-2.5 and Drosophila tinman, respectively. CSX1 has at least three isoforms generated by an alternative splicing mechanism. One of these isoforms (CSX1a) encodes a protein of approximately 35 kD that possesses the homeodomain, whereas the other two (CSX1b and CSX1c) encode a truncated protein of approximately 12 kD that is identical to the CSX1a protein at the amino-terminal 112 amino acids but lacks the homeodomain. Northern blot analysis showed that CSX1 transcripts are abundantly expressed in both fetal and adult hearts, but no signal was detected in other human tissues examined. Amplification of each isoform by reverse transcriptase-polymerase chain reaction revealed that all of the three isoforms are expressed in fetal and adult hearts and that the homeobox-containing isoform CSX1a is most abundant. The homeodomain-containing protein encoded by CSX1a binds to Csx/Nkx-2.5 binding sequences and transactivates the sequence-containing luciferase reporter gene. Unexpectedly, the homeodomain-lacking protein encoded by CSX1b also transactivates the reporter gene, although CSX1b does not bind to the Csx/Nkx-2.5 binding sequences. The highly conserved homeodomain sequence in evolution and the restricted expression in the heart suggest that CSX1 plays an important role in the development and differentiation of the human heart and that there may be two different mechanisms in transcriptional regulation by the CSX1 protein, homeodomain-dependent and -independent mechanisms.

Base Sequence↗
PubMed1996

Rapid detection cytomegalovirus pneumonia in recipients of bone marrow transplant: evaluation and comparison of five survey methods for bronchoalveolar lavage fluid.

To detect cytomegalovirus-associated interstitial pneumonia (CMV-IP) in recipients of BMT in its earliest stage, five CMV methods were assessed for their usefulness using bronchoalveolar lavage fluid as the test specimen. Of the 43 cases enrolled in the study, PCR was positive in 12 cases, shell vial in eight, culture in eight and cytology in three. There were no positive cases in in situ hybridization. Based on this result, the 43 cases were classified into four groups: Group 1, three cases: positive in PCR, shell vial and cytology; Group 2, five cases: positive in PCR and shell vial; Group 3, four cases: positive only in PCR; and Group 4, 31 cases: negative in all CMV tests. Cases in Group 1 were judged as having the highest risk of overt CMV-IP. They were successfully treated with a combination of ganciclovir and immunoglobulin. Group 2 was diagnosed as having active CMV infection and ganciclovir monotherapy was effective for these patients. Groups 3 and 4 were not given anti-CMV therapy, but they were free from CMV-related manifestations throughout the study. The sensitivity and specificity of each survey method for the detection of Groups 1 and 2 were 1.0 and 0.89 in PCR, 1.0 and 1.0 in shell vial, 0.88 and 1.0 in culture, and 0.38 and 1.0 in cytology. Similarly, the positive and negative predictive values were 0.67 and 1.0 in PCR, 1.0 and 1.0 in shell vial, 1.0 and 0.97 in culture, and 1.0 and 0.88 in cytology. Thus, CMV survey on bronchoalveolar fluid was thought to be useful in detecting post BMT CMV-IP in its earliest stage.

Adolescent↗
PubMed1995

Stretching mesangial cells stimulates tyrosine phosphorylation of focal adhesion kinase pp125FAK.

Mechanical loading of cultured mesangial cells has been shown to induce both increased production of matrix proteins and cell proliferation. This in vitro phenomenon has been implicated in the progression of glomerular sclerosis observed in a variety of glomerular diseases. However, it is not yet known how cells sense mechanical stress and transfer this sense into biochemical and biological events. In this study, we show that mesangial stretching rapidly stimulates tyrosine phosphorylation of non-receptor tyrosine kinase pp125FAK. Since pp125FAK is a major constituent of focal adhesions, our findings suggest that the focal adhesion may be a site where mechanical forces are translated into biochemical events, and that pp125FAK may play an important role in this signaling cascade.

Animals↗
PubMed1995

Assignment of cardiac homeobox gene CSX to human chromosome 5q34.

Homeobox-containing genes play critical roles in regulating tissue-specific gene expression essential for tissue differentiation, as well as determining the temporal and spatial patterns of development. Recently, a human cardiac homeobox-containing gene, CSX, has been isolated. CSX is abundantly expressed in the human heart from fetal stages, suggesting that CSX plays an important role in human heart formation. In the present study, we have determined the chromosomal localization of CSX by fluorescence in situ hybridization techniques and systemic screening of a yeast artificial chromosome library using polymerase chain reaction. By these methods, CSX was mapped to 5q34 of human chromosome 5 near the boundary of 5q34 and 5q35. In this region, another homeobox-containing gene MSX2, which is expressed in various tissues including the conduction system of the developing heart, has been assigned. Localization of CSX and MSX2 to the same region of the human chromosome suggests that these genes may be coordinately regulated during human heart formation.

Animals↗
PubMed1995

Molecular mechanism of cardiac cellular hypertrophy by mechanical stress.

Mechanical stress is a major cause of cardiac hypertrophy. Although the mechanisms by which mechanical load induces cardiac cellular hypertrophy have long been a subject of great interest for cardiologists, the lack of a good in vitro system has hampered the understanding of the biochemical mechanisms. For these past several years, however, an in vitro cardiocyte culture system has made it possible to examine the biochemical basis for the signal transduction of mechanical stress. Passive stretch of cardiomyocytes cultured on silicone membranes activates protein kinase cascades of phosphorylation and induces an increase in protein synthesis and the expression of both immediate early genes such as c-fos, c-myc, c-jun, Egr-1, and late response genes such as beta-myosin heavy chain and skeletal alpha-actin. Although an important question regarding how mechanical stimulus is converted into biochemical signals remains unknown, the cultured cardiomyocyte is a good model to examine the signal transduction pathways of mechanical stress.

Actins↗
PubMed1995

Protein kinase cascade activated by mechanical stress in cardiocytes: possible involvement of angiotensin II.

Mechanical stress induces cardiac hypertrophy and expression of specific genes in the cardiac myocytes. External stimuli are generally transduced into the nucleus through the activation of a protein kinase cascade. We have previously shown that stretching cardiomyocytes stimulates the activity of protein kinase C (PKC), mitogen-activated protein (MAP) kinase and S6 protein kinase. In the present study, we examined two other kinases, Raf-1 kinase and MAP kinase kinase, which are supposed to lie between PKC and MAP kinase in the protein kinase cascade. Stretching cardiocytes by using the in vitro system induced hyperphosphorylation of Raf-1 kinase and activation of MAP kinase kinase. The protein kinases activated by mechanical stress are similar to those activated by growth factors. We examined the possible involvement of angiotensin II (Ang II) in the protein synthesis and gene expression induced by mechanical stress. CV11974, an Ang II-receptor antagonist, partially suppressed the increases in amino acid incorporation, c-fos gene expression and MAP kinase activity induced by stretching. These results suggest that a variety of protein kinases are activated by mechanical stress and that locally produced Ang II may in part play important roles in converting mechanical stimuli into biochemical signals.

Angiotensin II↗
PubMed1995

Angiotensin II partly mediates mechanical stress-induced cardiac hypertrophy.

We have previously shown that mechanical stress induces activation of protein kinases and increases in specific gene expression and protein synthesis in cardiac myocytes, all of which are similar to those evoked by humoral factors such as growth factors and hormones. Many lines of evidence have suggested that angiotensin II (Ang II) plays a vital role in cardiac hypertrophy, and it has been reported that secretion of Ang II from cultured cardiac myocytes was induced by mechanical stretch. To examine the role of Ang II in mechanical stress-induced cardiac hypertrophy, we stretched neonatal rat cardiac myocytes in the absence or presence of the Ang II receptor antagonists saralasin (an antagonist of both type 1 and type 2 receptors), CV-11974 (a type 1 receptor-specific antagonist), and PD123319 (a type 2 receptor-specific antagonist). Stretching cardiac myocytes by 20% using deformable silicone dishes rapidly increased the activities of mitogen-activated protein (MAP) kinase kinase activators and MAP kinases. Both saralasin and CV-11974 partially inhibited the stretch-induced increases in the activities of both kinases, whereas PD123319 showed no inhibitory effects. Stretching cardiac myocytes increased amino acid incorporation, which was also inhibited by approximately 70% with the pretreatment by saralasin or CV-11974. When the culture medium conditioned by stretching cardiocytes was transferred to nonstretched cardiac myocytes, the increase in MAP kinase activity was observed, and this increase was completely suppressed by saralasin or CV-11974. These results suggest that Ang II plays an important role in mechanical stress-induced cardiac hypertrophy and that there are also other (possibly nonsecretory) factors to induce hypertrophic responses.

Angiotensin II↗
PubMed1995

Mechanical stress activates protein kinase cascade of phosphorylation in neonatal rat cardiac myocytes.

We have previously shown that stretching cardiac myocytes evokes activation of protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and 90-kD ribosomal S6 kinase (p90rsk). To clarify the signal transduction pathways from external mechanical stress to nuclear gene expression in stretch-induced cardiac hypertrophy, we have elucidated protein kinase cascade of phosphorylation by examining the time course of activation of MAP kinase kinase kinases (MAPKKKs), MAP kinase kinase (MAPKK), MAPKs, and p90rsk in neonatal rat cardiac myocytes. Mechanical stretch transiently increased the activity of MAPKKKs. An increase in MAPKKKs activity was first detected at 1 min and maximal activation was observed at 2 min after stretch. The activity of MAPKK was increased by stretch from 1-2 min, with a peak at 5 min after stretch. In addition, MAPKs and p90rsk were maximally activated at 8 min and at 10 approximately 30 min after stretch, respectively. Raf-1 kinase (Raf-1) and (MAPK/extracellular signal-regulated kinase) kinase kinase (MEKK), both of which have MAPKKK activity, were also activated by stretching cardiac myocytes for 2 min. The angiotensin II receptor antagonist partially suppressed activation of Raf-1 and MAPKs by stretch. The stretch-induced hypertrophic responses such as activation of Raf-1 and MAPKs and an increase in amino acid uptake was partially dependent on PKC, while a PKC inhibitor completely abolished MAPK activation by angiotensin II. These results suggest that mechanical stress activates the protein kinase cascade of phosphorylation in cardiac myocytes in the order of Raf-1 and MEKK, MAPKK, MAPKs and p90rsk, and that angiotensin II, which may be secreted from stretched myocytes, may be partly involved in stretch-induced hypertrophic responses by activating PKC.

Amino Acid Sequence↗
PubMed1995

Interaction of cardiac myocytes and non-myocytes in mechanical stress-induced hypertrophy.

It is known that mechanical stress directly changes the conformation of the functional proteins, or directly activates enzymes such as phospholipase in the plasma membrane. The integrin-cytoskeleton complex may be an alternative candidate structure for a mechanoreceptor and a transducer. The cytoskeleton has been also shown to play an important role in secretion. Mechanical stress may stimulate the secretion of some cytokines or angiotensin II, which may generate multiple intracellular signals as a secondary event. External stimuli are generally transduced into the nucleus through the activation of protein kinase cascade. Stretching of cardiac myocytes stimulates the activity of PKC, Raf-1 kinase, MAP kinase kinase. MAP kinase and S6 kinase. In cardiac myocytes, mechanical stress directly induces gene expression as well as protein synthesis. Immediate early genes are first induced, and then fetal-type genes are reinduced. Both in hypertrophied hearts and in the experimental model of cardiac hypertrophy induced by pressure overload. Ca(2+)-ATPase content of cardiac myocytes is depressed. Reduced function of sarcoplasmic reticulum causes insufficient decrease of intracellular calcium in diastole and induces slowing of ventricular relaxation. In the interstitium of pressure overloaded hearts, the accumulation of collagen fiber is increased. The abnormal deposit leads to increased chamber stiffness and diastolic dysfunction. Furthermore, TGF-beta and tissue renin-angiotensin system are up-regulated in pressure overloaded hearts, both of which accelerate the interstitial fibrosis.

Animals↗
PubMed1994

Angiotensin II receptor antagonist TCV-116 induces regression of hypertensive left ventricular hypertrophy in vivo and inhibits the intracellular signaling pathway of stretch-mediated cardiomyocyte hypertrophy in vitro.

BACKGROUND: Previous studies have demonstrated that angiotensin II (Ang II) acts as a growth-promoting factor directly on cardiac myocytes and that angiotensin-converting enzyme inhibitor induces regression of hypertrophied hearts both in experimental animals and in humans. These results suggest that the renin-angiotensin system (RAS) is involved in the formation of left ventricular hypertrophy (LVH). To elucidate the role of RAS in the progression of cardiac hypertrophy, we evaluated the effect of an Ang II receptor antagonist on LVH in spontaneously hypertensive rats (SHRs) and investigated the molecular mechanisms by which antagonizing Ang II receptors reduces cell hypertrophy of myocytes using the in vitro model of mechanical stretch. METHODS AND RESULTS: In the in vivo study, we treated SHRs with the nonpeptide Ang II receptor antagonist TCV-116 (0.1, 1, or 10 mg/kg per day) or hydralazine (10 mg/kg per day). Blood pressure was measured by the tail-cuff method, and wall thickness of left ventricle was serially monitored using M-mode echocardiography. Rats were killed at the age of 13, 17, 21, or 25 weeks, and left ventricular (LV) weight, transverse diameter of cardiomyocytes, relative amount of V3 myosin heavy chain (MHC), and degree of interstitial collagen accumulation were examined. Untreated SHRs progressively developed severe hypertension, but treatment with TCV-116 or hydralazine inhibited the increase in blood pressure. Treatment with TCV-116 reduced LV weight, LV wall thickness, transverse diameter of myocytes, relative amount of V3 MHC, and interstitial fibrosis, whereas treatment with hydralazine slightly prevented an increase in LV wall thickness but did not exert significant reduction in other parameters. In the in vitro study, neonatal rat cardiomyocytes were cultured on deformable silicone dishes and mechanically stretched with or without pretreatment of CV-11974 (an active metabolite of TCV-116), and [3H]phenylalanine incorporation, activity of mitogen-activated protein (MAP) kinase, and c-fos mRNA expression were analyzed. Pretreatment of cultured cardiomyocytes with 10(-7) mol/L CV-11974 inhibited an increase in [3H]phenylalanine incorporation, MAP kinase activity, and c-fos gene expression induced by stretch of cardiomyocytes. CONCLUSIONS: The Ang II receptor antagonist TCV-116 induced regression of cardiac hypertrophy and had cardioprotective effects on hypertrophied myocardium in vivo, and antagonizing Ang II receptors inhibited intracellular signaling of stretch-mediated cardiomyocyte hypertrophy in vitro. These results suggest a crucial role of the cardiac RAS in the development of LVH produced by pressure overload.

Angiotensin II↗
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 c-fos 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 Ang II may, in part, mediate the stretch-induced hypertrophic growth of cardiomyocytes via the type 1 Ang II receptor.

Animals↗