永井 良三

UNLABELLED: We compared myocardial viability evaluated by 18F-deoxyglucose (FDG) SPECT in 14 old myocardial infarction patients with that evaluated by 201TI SPECT and 123I-beta-methyl-iodophenyl pentadecanoic acid SPECT as imaging of fatty acid metabolism. FDG-SPECT was performed after oral administration of glucose. From each SPECT image which was divided into 7 segments, the degree of accumulation of each radioisotope (RI) was visually classified into four grades of defect score (ranging from 0 as normal to 3 as severe defect). The % uptake in the same area was also quantitatively calculated. RESULTS: The degree of accumulation of myocardial Rl relative to regional wall motion. FDG is the most wide for a range of accumulation of Rl of infarct area. Also, at FDG, in the area of wall motion had done a disorder, the degree of accumulation was higher than other two methods. In the infarcted area, the degree of accumulation on FDG-SPECT in the area of decreased wall motion was greater than that on the other two procedures. These results suggest that FDG-SPECT is useful for evaluating myocardial viability.

To evaluate the regional wall motion and the myocardial fatty acid metabolism at hibernating myocardium after revascularization (PTCA or CABG), we performed dual SPECT with 201Tl and 123I-beta-methyliodophenyl-pentadecanoic acid (BMIPP), and left ventriculography (LVG) in 34 patients with coronary artery disease before and 3 to 4 months after revascularization. In the SPECT, regional tracer uptake was estimated qualitatively (visual) and quantitatively (% uptake). Regional wall motion was estimated qualitatively (visual) and quantitatively (shortening fraction). At the 78 hibernating areas, the improvement of regional wall motion was more significantly (p < 0.05) correlated with that of regional tracer uptake of 123I-BMIPP (r = 0.63) than 201Tl (r = 0.39), and also correlated with the improvement of the difference between 201Tl and 123I-BMIPP regional uptake (r = 0.36). These results suggest that the improvement of wall motion at hibernating myocardium is more significantly correlated with the improvement of 123I-BMIPP than 201Tl uptake after revascularization.

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.

Romano-Ward syndrome, one of familial long QT syndromes, is an inherited disorder that causes sudden death from cardiac arrhythmias, specifically torsade de pointes and ventricular fibrillation. By linkage analyses, three LQT loci were previously mapped: LQT1 on chromosome 11p15.5, LQT2 on 7q35-36, LQT3 on 3p21-24. It was recently brought to light that LQT2 and LQT3 were caused by mutations of the gene encoding cardiac ion channels. Mutations in HERG on chromosome 7q35-36, encoding potassium channels (Ikr), cause LQT2, and block of Ikr is a known mechanism for drug-induced prolongation of cardiac action potentials, which provides a mechanistic link between LQT2 and certain forms of acquired LQT. Mutations in SCN5A on chromosome 3p21, encoding the human heart voltage-gated sodium-channel alpha-subunit, cause LQT3. Mutant channels show a sustained inward sodium current during membrane depolarization, which explains prolongation of cardiac action potentials.

Romano-Ward syndrome (RWS) is an autosomal dominant disorder characterized by prolongation of the electrocardiographic QT interval, with clinical manifestations that include recurrent syncope and sudden death from ventricular arrhythmias. In order to find a long QT locus in Japanese patients and to identify DNA markers useful for presymptomatic diagnosis, linkage analyses were undertaken in 13 Japanese families with RWS patients by means of two DNA markers located on 11p15.5. One of these marker loci, HRAS, was previously reported to be tightly linked to the LQT locus in another ethnic group. Our analyses of homogeneity suggest evidence for genetic heterogeneity of RWS with the Japanese population.

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.

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.

A 34-year-old woman with tuberous sclerosis presented with an increase of an abdominal mass and intermittent left flank pain on May 20, 1991. Computed tomography showed multiple bilateral renal masses with fatty density areas and a fatty density thrombus in the inferior vena cava, which extended through the right renal vein of the right kidney on ultrasonography. The inferior vena caval thrombus was also demonstrated by magnetic resonance imaging. Since marked deterioration of the right renal function was found on renography, right radical nephrectomy with thrombectomy was performed on July 2. Microscopically all tumors were identical with angiomyolipoma. She was discharged on Jury 20 and had been followed with good renal function at the outpatient clinic for more than 2 years. Follow up CT revealed no interval changes in the left renal masses.

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.

Smooth muscle myosin heavy chain (SMHC) isoforms, SM1 and SM2, are the products of alternative splicing from a single gene (P. Babij and M. Periasamy. J. Mol. Biol. 210: 673-679, 1989). We have previously shown that this splicing occurs at the 3'-end of the mRNA, resulting in proteins that differ at the carboxyterminal (R. Nagai, M. Kuro-o, P. Babij, and M. Periasamy. J. Biol. Chem. 264: 9734-9737, 1989). In the present study we demonstrate that additional SMHC isoform diversity occurs in the globular head region by isolating and characterizing two distinct rat SMHC cDNA (SMHC-11 = SM1B and SMHC-5 = SM1A). Sequence comparison of the two clones reveals that they are completely identical in their coding regions, except at the region encoding the 25/50 kDa junction of the myosin head, where the SM1B isoform contains an additional seven amino acids. This divergent region is located adjacent to the Mg(2+)-ATPase site, and differences in this region may be of functional importance. Ribonuclease protection analysis demonstrates that the corresponding SM1B and SM1A mRNA messages are coexpressed in all smooth muscle tissues; however, the proportion of the two mRNA present differs significantly between tissues. The SM1A-type mRNA predominates in most smooth muscle tissues, with the exception of intestine and urinary bladder, which contain greater proportions of the SM1B message. The differential distribution of these two isoforms may provide important clues toward understanding differences in smooth muscle contractile properties.

Cardiac functions are regulated by both contractile proteins and calcium regulatory proteins. Alterations of these are considered involved in impaired contractile and diastolic functions in hypertrophied hearts. In this study, we analyzed molecular changes during the development of cardiac hypertrophy. Cardiac hypertrophy was induced by constricting the pulmonary artery in rabbits or the aorta in rats. In rabbit right ventricular hypertrophy, protein synthesis was increased to 1.8 times the control 2-4 days after pulmonary constriction. This increase in protein synthesis could be classified as an increase in both capacity and efficiency of synthesis. beta-cardiac myosin heavy chain (beta-MHC) isoform was predominantly expressed and alpha-MHC was suppressed in pressure overload hypertrophy. The switch from alpha- to beta-MHC occurred at the mRNA level. Ca(2+)-ATPase of sarcoplasmic reticulum (SR) is important because it regulates intracellular Ca2+ levels during relaxation. In pressure-overload hypertrophy, the SR Ca(2+)-ATPase was markedly decreased in both the enzyme activities and mRNA levels, while in thyrotoxic hearts both were increased. Interstitial cells also undergo phenotypic modulation which was demonstrated by the induction of nonmuscle-type MHC in pressure-overload hypertrophy. The signal transduction system in cardiac hypertrophy was examined by stretching cardiac myocytes grown on deformable membranes. In our analysis, stretching myocytes stimulated protein kinase C, MAP-II kinase and S6 kinase, all of which may lead to the induction of fetal-type cardiac genes and accelerated protein synthesis. These analyses of subcellular adaptation in cardiac hypertrophy provide important insights into understanding molecular mechanisms of cardiac functions.

We analyzed the molecular basis of the pathogenesis of vascular disease.<BR>Firstly, we cloned three types of smooth muscle myosin heavy chain genes, SM1, SM2 and SMemb from rabbit cDNA library. Their expression during vascular development is differentially regulated at RNA levels. SM1 is constitutively expressed and SM2 appears after birth. Contrarily, SMemb is predominantly expressed in embryonic and perinatal stage. 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.<BR>Secondly, we have also cloned macrophage scavenger receptor genes and determined their primary structures. The macrophage scavenger receptors showed unusual ligandbinding properties indicating higher binding affinity to modified LDL, such as oxidized and acetylated LDL rather than to LDL. These scavenger receptors were expressed in foam cells of the atherosclerotic neointima. An understanding of the structure and function of this family of receptors will provide us new insights into the mechanism of the development of atherosclerosis.<BR>Finally, we investigated cell-mediated autoimmunity in Takayasu's disease. We found that the dominant populations of the infiltrating cells in the arterial tissues obtained from patients with Takayasu's disease were large granular lymphocytes which contained a lot of perform. Our results suggested that cell-mediated cytotoxicity plays an important role in the development of arterial tissue damage involved in Takayasu's disease.

Immunoassays for cardiac myosin light chains (LC) are important biochemical tests for diagnosis of acute myocardial infarction (AMI). LC appears in the serum 4-12 hours after AMI. The most unique characteristic of the time-course of LC is that the elevation of LC in the serum lasts for 1-2 weeks, which allows the retrospective diagnosis of AMI when cardiac enzymes in the serum are decreased to the normal level. This long time-course is due to the continuous liberation of LC from the infarcted myocardium, which could be confirmed by the cardiac scintigram using anti-myosin heavy chain antibody. The serum LC level is also useful in predicting the extent of the cardiac wall damage since the peak LC level reflected well the ventricular ejection fraction or the presence of mechanical complications, such as the formation of ventricular aneurysm. Immunoassays for cardiac LC have recently been improved by the introduction of ELISA using two monoclonal anti-light chain antibodies. ELISA can be finished within 3 hours, which will help to make cardiac LC measurement one of the most important biochemical tests for diagnosis of acute myocardial infarction.

心筋における収縮の最小単位である筋原線維を構成する収縮蛋白のミオシンと特異的に結合するモノクローナル抗体を用いた急性心筋梗塞の新しい観点に立った診断法が開発され,その臨床的有用性が高く評価されている.特に梗塞の大きさの正確な推定ばかりでなく,細胞構築の崩壊過程を中心とした梗塞部心筋の病態生理に関する情報が得られ,臨床で広く用いられるものと期待される.

We developed four types of immunoassays for cardiac myosin light chains (LC), which are two radioimmunoassays (RIA) for canine and human LC, and an immunoradiometric assay (IRMA) and an enzyme-linked immunosorbent assay (ELISA) for human LC. The first two assays make use of polyclonal antibodies and the last two use monoclonal antibodies. By using these immunoassays, we studied the release of cardiac LC into the serum following acute myocardial infarction (AMI). In experimental AMI in dogs, cardiac LC appeared in the serum within 4-12 hours, reached the maximum at 2-5 days and returned to normal at 7-10 days. This long time-course was suggested due to the continuous liberation of LC from the infarcted myocardium on the basis of a quick disappearance rate of LC from the circulation. The peak LC values were found to correlate well with the histological infarct size. Similar results were also obtained regarding the time-course of circulating LC in clinical patients with AMI. Thus LC measurement seems useful for diagnosis of AMI as well as for estimating the extent of myocardial damage. We also developed an IRMA and an ELISA for human LC by using anti-human LC monoclonal antibodies for a more rapid LC assay and for a consistent supply of antibodies. These assays showed sufficiently high sensitivities to measure 1-100 ng/ml of serum LC. Especially, serum LC can be assayed within 2.5 hours by our ELISA. Such progress in immunoassays for cardiac LC has made the measurement of LC an important laboratory test for the diagnosis of AMI.