Masahiko Kurabayashi

Expression of cellular oncogenes in the myocardium during the developmental stage and pressure-overloaded hypertrophy of the rat heart.

Proto-oncogenes have been revealed to participate in normal cell proliferation as well as in cell transformation. Since cardiac myocytes are terminally differentiated, they cannot divide except in the fetal period. To determine the role of cellular oncogenes in the growth of the heart, the expression pattern of eight cellular oncogenes during the developmental stage and pressure-overloaded hypertrophy of the rat hearts were examined in vivo. Northern blot analysis was performed with eight oncogene probes (myc, fos, Ha-ras, src, erbA, erbB, sis, myb). Pressure overload increased the levels of cellular (c-) fos, c-myc, and c-Ha-ras. An increase of c-fos and c-myc was detected at 30 minutes and 2 hours, respectively; the levels peaked at 8 hours, and they returned to baseline by 48 hours after aortic constriction. However, the level of c-Ha-ras showed a gradual increase. During the course of development, the expression of c-myc was detectable only in the embryonic stage, whereas the expression of c-fos was not detected in the fetal period, was increased after birth, and peaked in 200-day-old adults. The expression of c-Ha-ras was almost the same throughout the development. Cellular oncogenes were expressed in the heart in response to pressure overload and in a stage-specific manner. These results suggest that cellular oncogenes may participate in the normal developmental process and hypertrophy of hearts and that the cellular hypertrophy induced by pressure overload may share a similar mechanistic pathway with cell proliferation.

BTEB2, a Krüppel-Like Transcription Factor, Regulates Expression of the SMemb/Nonmuscle Myosin Heavy Chain B (SMemb/NMHC-B) Gene

We have recently characterized the promoter region of the rabbit embryonic smooth muscle myosin heavy chain (SMemb/NMHC-B) gene and identified the 15-bp sequence, designated SE1, located at -105 from the transcriptional start site as an important regulatory element for its transcriptional activity in a smooth muscle cell (SMC) line. In this study, we attempted to isolate cDNA clones encoding for the transcription factors that control the expression of the SMemb gene through binding to this cis-regulatory element. We screened a lambdagt11 cDNA library prepared from C2/2 cells, a rabbit-derived SMC line, by using a radiolabeled concatenated oligonucleotide containing SE1 as a probe. Sequence analysis revealed that one of the cDNA clones corresponds to the rabbit homologue of basic transcriptional element binding protein-2 (BTEB2), which has previously been identified as one of the Krüppel-like transcription factor. Gel mobility shift assays and antibody supershift analyses with nuclear extracts from C2/2 cells indicate that BTEB2 is a major component of nuclear factor:SE1 complexes. Furthermore, a glutathione S-transferase-BTEB2 fusion protein binds to the SE1 in a sequence-specific manner. In support of the functionality of BTEB2 binding, basal promoter activity and BTEB2-induced transcriptional activation were markedly attenuated by the disruption of the SE1. In adult rabbit tissues, BTEB2 mRNA was most highly expressed in intestine, urinary bladder, and uterus. BTEB2 mRNA levels were downregulated in rabbit aorta during normal development. Moreover, immunohistochemical analysis indicated a marked induction of BTEB2 protein in the neointimal SMC after balloon injury in rat aorta. These results suggest that BTEB2 mediates the transcriptional regulation of the SMemb/NMHC-B gene and possibly plays a role in regulating gene expression during phenotypic modulation of vascular SMC.

Molecular cloning and characterization of a Ca2+ + Mg2+-dependent adenosine triphosphatase from rat cardiac sarcoplasmic reticulum. Regulation of its expression by pressure overload and developmental stage.

To investigate the regulation of expression of cardiac Ca2+ + Mg2+-dependent ATPase (Ca2+-ATPase) in sarcoplasmic reticulum (SR), we isolated cDNA (pHA6) encoding a Ca2+-ATPase of rat cardiac SR. The clone consisted of 2,311 mRNA-derived nucleotides, which covered half the coding region and the entire 3-untranslated regions. The nucleotides and deduced amino acid sequences of pHA6 showed striking homology, 89 and 98%, respectively, to those of rabbit Ca2+-ATPase of the slow-twitch form. Northern blot analyses revealed that the mRNA levels of Ca2+-ATPase were decreased by pressure overload and became 32% of sham in 1 mo. During the developmental stage the mRNA levels were very low in the fetal period and steeply increased around birth. These changes in mRNA levels were correlated with the corresponding protein levels. These results suggest that the expression of cardiac Ca2+-ATPase in SR is regulated by pressure overload and the developmental stage, at least in part, at the pretranslational level.

Novel Biochemical Diagnostic Method for Aortic Dissection Results of a Prospective Study Using an Immunoassay of Smooth Muscle Myosin Heavy Chain

BACKGROUNDnAortic dissection is one of the most common aortic catastrophes. Although newer diagnostic methods as exemplified by image diagnostic techniques have greatly improved the diagnosis of aortic dissection, the diagnosis is still frequently missed today because the signs and symptoms of the disease are at times obscure. A reliable biochemical diagnostic method for aortic dissection would be beneficial.nnnMETHODS AND RESULTSnA novel biochemical diagnostic method for diagnosis of aortic dissection was developed that uses an immunoassay of monoclonal antibodies to smooth muscle myosin heavy chain. A prospective study was conducted to ascertain the usefulness of the method in the diagnosis of aortic dissection. Twenty-seven patients with aortic dissection admitted within the first 24 hours after onset were enrolled. Serial assay of serum smooth muscle myosin heavy chain showed significant elevations within the first 24 hours after onset of aortic dissection, with levels exceeding 10 ng/mL, with subsequent rapid reductions. The sensitivity of the assay within the first 12 hours was 90% with a specificity of 97%. Analysis of 65 patients with acute myocardial infarction showed that the method could accurately differentiate myocardial infarction from aortic dissection.nnnCONCLUSIONSnThe immunoassay of serum smooth muscle myosin heavy chain is a rapid and reliable biochemical method in the diagnosis of aortic dissection. The potential use of the method in clinical medicine is promising.

Regulation of Platelet-derived Growth Factor-A Chain by Krüppel-like Factor 5 NEW PATHWAY OF COOPERATIVE ACTIVATION WITH NUCLEAR FACTOR-κB

The transcription factor Krüppel-like factor 5 (KLF5) and its genetically downstream target gene platelet-derived growth factor-A (PDGF-A) chain are key factors in regulation of cardiovascular remodeling in response to stress. We show that KLF5 mediates a novel distinct delayed persistent induction of PDGF-A chain in response to the model agonist, phorbol ester, through a cis-element previously shown to mediate phorbol ester induction on to PDGF-A chain through the early growth response factor (Egr-1). Interestingly, the nuclear factor-κB (NF-κB) p50 subunit further cooperatively activates PDGF-A chain through protein-protein interaction with KLF5 but not Egr-1. RNA interference analysis confirmed that KLF5 and p50 are important for induction of PDGF-A chain. Collectively, we identify a novel regulatory pathway in which PDGF-A chain gene expression, under the control of KLF5, is cooperatively activated by the NF-κB p50 subunit and a pathophysiological stimulus.

Diagnostic Implications of Elevated Levels of Smooth-Muscle Myosin Heavy-Chain Protein in Acute Aortic Dissection: The Smooth Muscle Myosin Heavy Chain Study

Aortic dissection is an uncommon acute disease associated with high mortality and morbidity (1, 2). Initial management has a critical effect on survival. While diagnosis and treatment of the disease have greatly improved in recent years as a result of newer diagnostic (3) and therapeutic (4, 5) techniques, it is still difficult to recognize at clinical presentation (6). To aid in the initial diagnostic screening, an assay of circulating smooth-muscle myosin heavy-chain protein, a protein specific to smooth muscle that is released from damaged aortic medial smooth muscle at the onset of aortic dissection, was developed (7). Biochemical diagnosis of acute aortic dissection is attractive because it is rapid, noninvasive, and relatively easy to perform. A rapid 30-minute assay was developed for clinical use after initial studies that used an experimental assay showed promising results (8-10). Our study addressed the sensitivity and specificity of the rapid assay in acute aortic dissection. Methods The enzyme immunoassay of smooth-muscle myosin heavy-chain protein was developed with antibodies and reactions optimized for sensitive detection and minimal performance time (30 minutes). Cross-reactivity against aortic and uterine myosin was 100%; in contrast, cardiac and skeletal muscle showed cross-reactivity less than 0.05%. The measuring range of the assay was approximately 1.6 to 100 g/L. Within-run and between-run reproducibilities as measures of analytical precision were 6.2% 1.3% and 3.2% 1.4 %, respectively (coefficient of variance). Recovery as a measure of analytical accuracy (defined as the observed vs. the expected value when purified human smooth-muscle myosin heavy-chain protein was added to patient serum) was 93.9% 10.0%. All assays were performed by the diagnostics division of Yamasa Corp. (Tokyo, Japan). The technical specifications and details of the assay are available from the authors. We included patients with acute aortic dissection who presented to participating centers between August 1996 and March 1999. Eight major cardiovascular centers were selected for participation in the Smooth Muscle Myosin Heavy Chain Study because they had a large volume of early admissions for acute aortic dissection. Included patients had aortic dissection within 24 hours of onset of symptoms; the diagnosis was confirmed by imaging. Traumatic aortic dissections were excluded. Each center approved the study protocols, and patient consent was obtained. Single-specimen blood sampling was done at initial presentation. Protocols for documenting clinical characteristics, including age, sex, time of onset, time of admission, diagnosis, lesion site (according to DeBakey classification), and time of blood sampling, have been described elsewhere (9). We included 131 healthy volunteers presenting for an annual health examination as normal controls; we also included 48 patients with acute myocardial infarction to determine the specificity of the assay in distinguishing aortic dissection from other diseases that present with chest pain. We analyzed receiver-operating characteristic (ROC) curves to show sensitivity and specificity. Results are presented as the mean SD. Statistical analysis was done by using commercially available software (StatView 4.0, Abacus Systems, Berkeley, California). MannWhitney U tests were used for two-group comparisons. A P value less than 0.05 was considered statistically significant. Analysis for differences across time or between healthy volunteers and patients with aortic dissection are conservative because they do not adjust for differences among centers. Results We enrolled 95 consecutive patients with aortic dissection (58 men, 37 women; mean age, 64.7 13.1 years) within the first 24 hours after onset of symptoms. This sample of predominantly male patients, ranging from middle-aged to elderly, is typical for aortic dissection. In all cases, diagnosis was confirmed by imaging. Thirty-three patients had DeBakey type I lesions, 12 had DeBakey type II lesions, and 50 had DeBakey type III lesions. Patients presented 5.9 5.9 hours after symptom onset. Of importance, 35% of patients presented within 3 hours after onset and 65% presented within 6 hours after onset. Sensitivity and Specificity We compared serum levels of smooth-muscle myosin heavy-chain protein in patients with acute aortic dissection and 131 healthy volunteers (35 men, 13 women; mean age, 65.9 11.9 years). Values were significantly higher in patients with acute aortic dissection (22.4 40.4 g/L vs. 0.9 0.4 g/L, respectively; P <0.001). The highest levels (51.0 52.3 g/L) were seen in the 33 patients who presented within 3 hours after onset. Levels decreased significantly to 11.5 28.5 g/L in 29 patients in the next 3 hours (P <0.001) and decreased further to 3.3 5.2 g/L in 33 patients thereafter. During the initial 3 hours after symptom onset, sensitivity was 90.9% (95% CI, 85% to 96.8%) at a cutoff level of 2.5 g/L (the upper limit of the normal population) (Figure 1, top). Sensitivity decreased to 72.4% (CI, 65.3% to 79.5%) in the following 3 hours and decreased to 30.3% (CI, 23.9% to 36.7%) thereafter. The assay had a specificity of 98% and a diagnostic accuracy of 96% at the cutoff level of 2.5 g/L in patients with aortic dissection compared with healthy volunteers (Figure 1, bottom). Figure 1. Sensitivity and specificity of the smooth-muscle myosin heavy-chain assay. Top. Bottom. solid line dotted line To determine the specificity of the assay in distinguishing aortic dissection from diseases that present with similar symptoms, such as chest pain, we examined levels of smooth-muscle myosin heavy-chain protein in 48 patients with acute myocardial infarction who presented within 3 hours after onset (35 men, 13 women; mean age, 65.9 11.9 years). In these patients, the serum level of smooth-muscle myosin heavy-chain protein was 2.1 1.6 g/L (P <0.001 compared with acute aortic dissection). The assay had a specificity of 83% at the cutoff level of 2.5 g/L in patients with acute myocardial infarction (Figure 1, bottom). On the basis of these data, 2.5 g/L was set as the clinical decision limit because analysis of the ROC curve showed favorable sensitivity and specificity compared with normal volunteers and patients with acute myocardial infarction. Levels of smooth-muscle myosin heavy-chain protein that exceeded 10 g/L showed 100% specificity for aortic dissection. Analysis according to Type of Aortic Dissection In the first 3 hours after onset, all patients who had proximal lesions that were classified as DeBakey type I or II also had levels of smooth-muscle myosin heavy-chain protein that exceeded the clinical decision limit of 2.5 g/L. Conversely, patients who presented with definitive aortic dissection within the first 3 hours after onset and had levels of smooth-muscle myosin heavy-chain protein less than 2.5 g/L had distal lesions that were classified as DeBakey type III. Levels of smooth-muscle myosin heavy-chain protein were probably lower in patients with distal lesions because the abdominal aorta has less smooth muscle than the thoracic aorta. Analysis of samples taken within 3 hours of symptom onset confirmed that levels of smooth-muscle myosin heavy-chain protein were significantly higher in proximal lesions than in distal lesions (71.4 59.5 g/L vs. 31.8 36.7 g/L, respectively; P =0.03). The assay had superior sensitivity for proximal lesions 3 to 6 hours after onset and thereafter, which was confirmed by analysis of the ROC curve (Figure 2). Thirty-three patients, 16 of whom had proximal lesions, were tested within 3 hours after symptom onset. Figure 2. Levels of smooth-muscle myosin heavy-chain protein according to type of aortic dissection. Top. solid line dotted line Bottom. Discussion We found that the rapid assay of smooth-muscle myosin heavy-chain protein had a high sensitivity (90.9%) and acceptable specificity (98% compared with healthy controls, 83% compared with patients who had acute myocardial infarction) in patients with aortic dissection who presented within the first 3 hours after symptom onset. The assay performed best in patients with proximal lesions and was less sensitive in patients who presented at a later point in the disease and had decreased levels of smooth-muscle myosin heavy-chain protein. The sensitivity and specificity of this assay in the first 3 hours after onset are similar if not superior to those of transthoracic echocardiography (sensitivity, 59% to 85%; specificity, 63% to 96%) (11), conventional computed tomography (sensitivity, 83% to 94%; specificity, 87% to 100%) (3, 12), or aortography (sensitivity, 88%; specificity, 94%) (12). However, the assays sensitivity and specificity were lower than those of transesophageal echocardiography (sensitivity, 98% to 99%; specificity, 77% to 97%) (3, 12), helical computed tomography (both almost 100%) (13), or magnetic resonance imaging (both 98%) (14, 15). Because this assay is the first available biochemical diagnostic tool for aortic dissection, it is important to note that comparison with these established diagnostic methods (all of which are imaging procedures) provides only an estimate of its performance. Another important point is that biochemical testing can be done at a fraction of the cost of computed tomography or magnetic resonance imaging (approximately 10%) and is similar in cost to measuring cardiac enzymes (for example, myoglobin or troponin) (16, 17). The cost of a relatively inexpensive blood test is likely to outweigh the small risk for overlooking or failing to exclude the diagnosis of aortic dissection. In addition, manual or automated measurements can be performed easily in a manner similar to that of other conventional enzyme immunoassays. Important issues surround the practicality of this assay in clinical settings. This biochemical test would be most useful at the initial decision-making stage (triaging) in the emergency departm

Molecular cloning and characterization of human cardiac alpha- and beta-form myosin heavy chain complementary DNA clones. Regulation of expression during development and pressure overload in human atrium.

We have constructed and characterized two types of myosin heavy chain (MHC) cDNA clones (pHMHC2, pHMHC5) from a fetal human heart cDNA library. Comparison of the nucleotide and deduced amino acid sequences between pHMHC2 and pHMHC5 shows 95.1 and 96.2% homology, respectively. The carboxyl-terminal peptide and 3-untranslated (3-UT) regions are highly divergent and specific for these cDNA clones. By using the synthetic oligonucleotide probes that are complementary to the unique 3-UT regions of these cDNA clones, we demonstrate that pHMHC2 is exclusively transcribed in the atrium, whereas the mRNA for pHMHC5 is predominantly expressed in the ventricle. This result indicates that pHMHC2 and pHMHC5 code for alpha- and beta-form MHCs, respectively. Furthermore, we show that beta-form MHC mRNA is expressed in adult atrium at a low level but scarcely expressed in fetal atrium. Finally, we demonstrate that MHC isozymic transition in pressure-overloaded atrium is, at least in part, regulated at a pretranslational level.

Homeobox Protein Hex Induces SMemb/Nonmuscle Myosin Heavy Chain-B Gene Expression Through the cAMP-Responsive Element

Abstract— Recent studies have shown that the homeobox gene Hex plays an important role in inducing differentiation of vascular endothelial cells. In this study, we examined the expression of Hex in vascular smooth muscle cells (VSMCs) in vitro and in vivo. Immunohistochemistry showed a marked induction of Hex protein in neointimal VSMCs after balloon injury in rat aorta. Western and reverse transcriptase–polymerase chain reaction analyses demonstrated that Hex was abundantly expressed in cultured VSMCs, whereas it was undetectable in other cell types or in normal aorta. The expression pattern of Hex was similar to that of SMemb/NMHC-B, a nonmuscle isoform of myosin heavy chain that we have previously reported to be a molecular marker of dedifferentiated VSMCs. We next examined the role of Hex in SMemb gene transcription. Promoter analysis demonstrated that the sequence identical to consensus cAMP-responsive element (CRE) located at −481 of the SMemb promoter was critical for Hex responsiveness. Mutant Hex expression vector, which lacks the homeodomain, failed to stimulate SMemb gene transcription, suggesting the requirement of the homeodomain for its transactivation. Elecrophoretic mobility shift assay showed that Hex binds to a consensus binding sequence for homeobox proteins, but not to CRE. Cotransfection of protein kinase A expression vector increased the ability of Hex to stimulate SMemb promoter activity in a CRE-dependent manner. Overexpression of CRE binding protein (CREB), but not Mut-CREB which contains mutation at Ser133, strongly activated Hex-induced SMemb promoter activity. These results suggest that Hex mediates transcriptional induction of the SMemb/NMHC-B gene via its homeodomain, and Hex can function as a transcriptional modulator of CRE-dependent transcription in VSMCs.

Preferential Differentiation of P19 Mouse Embryonal Carcinoma Cells Into Smooth Muscle Cells Use of Retinoic Acid and Antisense Against the Central Nervous System–Specific POU Transcription Factor Brn-2

Investigation of the molecular mechanisms that control smooth muscle cell (SMC) development and differentiation is a prerequisite in understanding the regulatory mechanisms of physiological and pathological SMC-associated vascular processes. The pluripotent murine embryonal carcinoma P19 cell, whose developmental potential resembles that of early embryonic cells, can develop into cell types derived from the neuroectoderm, mesoderm, and endoderm. In the present study, we have shown a unique strategy to enhance SMC differentiation in P19 cells. Under chemical induction of high concentrations of retinoic acid (1 micromol/L), P19 cells showed optimum differentiation into SMCs. Because the P19 cells thus induced also showed differentiation into neuronal cells, a strategy to block neuronal lineage differentiation was developed using a stable transformant antisense RNA construct against Brn-2, a neuronal lineage-specific POU-domain transcription factor; thus, by specifically inhibiting neuronal differentiation, enhanced SMC differentiation by P19 cells was attained. SMC expression was confirmed by immunohistochemical staining, RNA analysis (RNase protection assay), and protein analysis (Western blot) using SMC-specific markers (eg, SM1 and calponin) and alpha-smooth muscle actin. Our results show that the pathway of SMC differentiation may provide an in vitro system useful in the investigation of SMC regulatory mechanisms (eg, transcriptional regulation) and in the further understanding of SMC development and differentiation.

Structure and Characterization of the 5′-Flanking Region of the Mouse Smooth Muscle Myosin Heavy Chain (SM1/2) Gene

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.