永井 良三

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.

It is now established that cardiac myosin from hyperthyroid rabbit hearts (TXM) exhibits high Ca2+ ATPase activity. The high Ca2+ ATPase activity of TXM was completely retained in cardiac myosin subfragment-1 (S-1) (1.33 +/- 0.04 mumol Pi/mg per min; euthyroid, 0.51 +/- 0.04). Cardiac S-1 from hyperthyroid and euthyroid rabbits (TXS-1 and NS-1) had the same pattern in SDS-polyacrylamide gel electrophoresis. The possible influence of heavy and light chains of TXM on increasing the ATPase activity was examined by reconstitution in the S-1 preparation. Crosswise reconstitution was performed using cardiac S-1 heavy chain (90,000 daltons) and light chain 1 (LC1) (27,000 daltons) from hyperthyroid and euthyroid hearts. Reconstitution was verified by using radiolabeled LC1. More than 95% of S-1 was recovered with full ATPase activity. When TXS-1 was reconstituted with LC1 from euthyroid hearts, the reconstituted molecule retained high ATPase activity. On the other hand, NS-1 reconstituted with LC1 from hyperthyroid hearts failed to increase the ATPase activity. The ATPase activity of S-1 was determined by the source of the heavy chain. These results suggest that the high Ca2+ ATPase activity of cardiac myosin and S-1 from hyperthyroid animals arises from the molecular alteration of the heavy chain induced by thyroxine administration.

We studied serial changes in various myocardial enzymes and cardiac myosin light chain II (LCII) in plasma following coronary ligation in 14 conscious closed-chest dogs. Cytoplasmic enzymes [creatine phosphokinase (CPK) and supernatant glutamic oxaloacetic transaminase (sGOT)] reached maximum at 12-24 hr and returned to normal at 72-96 hr. The mitochondrial isozyme of GOT (mGOT) began to rise at 6-9 hr, peaked at 12-30 hr (4.8-42.2 IU/liter), and stayed higher at 96 hr than before infarction. Glutamate dehydrogenase (GLDH), another mitochondrial enzyme, began to elevate at 6-16 hr and reached maximum at 24-60 hr (6.2-20.5 U/liter); GLDH also showed higher levels at 96 hr than before infarction. N-Acetyl-beta-glucosaminidase (NAG), a lysosomal enzyme, showed a biphasic pattern in every case. The first peak appeared at 3-12 hr, and the second one at 36-72 hr. Myosin LCII began to rise at 3-9 hr, peaked at 30-120 hr (34-136 ng/ml), and remained elevated for 7 to 10 days. Determination of these myocardial enzymes or LCII in plasma is useful for the diagnosis of acute myocardial infarction.

The relationship between myocardial infarct size and serum cardiac light chain (LC) levels was studied in experimental and clinical myocardial infarction. In dogs with left anterior descending coronary artery occlusion, regression analysis showed good correlation between infarct size and LC II release, but CPK-MB release failed to correlate with infarct size because of a decreasing value of cumulative CPK with larger sized infarctions. In patients with acute myocardial infarction, Peak LC I levels correlated well with CPK release, since the phenomenon of the decreased CPK release in larger sized infarction was not so distinctive in human cases. Thus, LC determination may better quantitate the extent of myocardial damage as well as provide a specific and sensitive method for diagnosis of acute myocardial infarction.

心筋の構造蛋白であるミオシンのサブユニットの一つ,軽鎖Iをヒト心筋より抽出精製し,ラジオイムノアッセイによる軽鎖Iの血中での測定法を開発し,さらに急性心筋硬塞発症後の血中軽鎖I値の変動を検討した.抗軽鎖I抗体は,モルモットを精製した軽鎖Iにて免疫することにより作製した.軽鎖IはクロラミンT法により¹²⁵Iで標識し, B/F分離は二抗体法を用いた.アッセイの感度は0.2ng,骨格筋軽鎖との交叉反応は10.2%,成人健常者30名の血中軽鎖I値は3.7±0.9ng/ml(平均±標準偏差)であつた. 24例の急性心筋硬塞症において軽鎖Iは発作後4～16時間以内に上昇をはじめ, 30～144時間後に最高値となり(35.0±14.1ng/ml), 7～16日間高値を続け,各種の心筋酵素やミオグロビンとは全く異なる特異な経時的変動を示した.また前壁硬塞例の中で,異常Q波がV₁-V₃に出現した3例と, IおよびV₁-V₅に出現した5例を比較すると,後者において血中軽鎖Iの最高値, 24時間値, 7日値は高値を示した.血中軽鎖Iの測定は心筋硬塞の急性期のみでなく,発作後時間の経過した症例においても診断に有用であり,またこれにより硬塞の大きさを推定できる可能姓があると考えられる.

ATPase activity and synthesis of atrial and ventricular myosin were compared in the dog and rabbit heart. Atrial myosin showed enzymatic properties characterized by high Ca2+- and Mg2+-activated ATPase activities, low activation energy, lower rate of inactivation at alkaline pH, and no activation by N-ethylmalemide compared with the same properties in ventricular myosin. These findings suggest a difference in the myosin molecule at or near the active site involfing the SH-1 thiols. Also, actin activation of ATPase activities was studied to determine the physiological significance of this observation, since, in living muscle cell, MgATP is hydrolyzed by myosin under the activating effect of actin. Actin extracted from skeletal muscle was used in this experiment. Vmax for the actin-activated Mg2+-ATPase of atrial myosin was about twice that of ventricular myosin, whereas no significant difference was observed in the apparent dissociation constant for actin. This result suggests that the change in the enzymatic properties of myosin is reflected in the contractility of atrial and ventricular muscles. However, [3H]leucine incorporation into the myosin was approximately the same, suggesting that the difference in work load between atrium and ventricle is not associated with alteration of synthesis of cardiac myosin.

A sensitive radioimmunoassay for cardiac myosin light chain II (LCII) was developed, and changes of serum LCII levels were studied in experimental myocardial infarction in dogs. This radioimmunoassay employed an antiserum which was prepared in rabbits against canine LCII. In our assay, 0.2-5.0 ng of LCII were effectively measurable. In normal dogs, LCII concentration in serum was less than 20 ng/ml. The serum LCII level began to rise within 6 hr after ligation of the left anterior descending coronary artery, reaching maximum level at 3-5 days (40-320 ng/ml). In eight out of ten cases with coronary occlusion, LCII could be detected as long as 7 days after operation. In one sham-operated dog, LCII was detected at 2 and 3 days, but its concentrations were less than 30 ng/ml. When LCII was injected intravenously, it dissipated from the blood stream within 48 hr. The time course curves of serum LCII level had two characteristics that had not been observed in serum enzyme studies: 1) LCII level rose rapidly and stayed up during a long period after coronary occlusion, and 2) changes of serum LCII levels were biphasic in six out of ten dogs with coronary occlusion. These results, and our previous studies of synthesis rate of light chains, suggest that when a coronary artery is occluded LCII may first be released from a pool of uncombined LCII in myocardial cells, and then continuously liberated from cardiac myosin molecules. This radioimmunoassay can be expected to be useful when applied to clinical use.