藤田 英雄

Familial hypertrophic cardiomyopathy (FHC) is caused by missence mutations in β-myosin heavy chain or other various sarcomeric proteins. To elucidate the functional impact of FHC mutations in myosin heavy chain, we generated Dictyostelium discoideum myosin II mutants equivalent to human FHC mutations by site-directed mutagenesis, and characterized their molecular- basis motor function. The current mutants, i.e. R397Q, F506C, G575R, A699R, K703Q and K703W are equivalent to R403Q, F513C, G584R, G716R, R719Q and R719W FHC mutants respectively. We measured the molecular-basis force and the sliding velocity generated by these myosin mutants. The measurement revealed that the A699R, K703Q and K703W myosins exhibited the lowest level of force with their preserved actin-activated MgATPase activity. F506C mutant showed the least impairment of the motile and enzymatic activities. The motor function of R397Q and G575R myosins were classified as intermediate. These results suggest that ELC binding domain might be important for force production.

Recent studies have revealed that familial hypertrophic cardiomyopathy (FHC) is caused by missence mutations in myosin heavy chain or other sarcomeric proteins. To investigate the functional impact of FHC mutations in myosin heavy chain, mutants of Dictyostelium discoideum myosin II equivalent to human FHC mutations were generated by site-directed mutagenesis, and their motor function was characterized at the molecular level. These mutants, i.e., R397Q, F506C, G575R, A699R, K703Q, and K703W are respectively equivalent to R403Q, F513C, G584R, G716R, R719Q, and R719W FHC mutants. We measured the force generated by these myosin mutants as well as the sliding velocity and the actin-activated ATPase activity. These measurements showed that the A699R, K703Q, and K703W myosins exhibited unexpectedly weak affinity with actin and the lowest level of force, though their ATPase activity remained rather high. F506C mutant which has been reported to have benign prognosis exhibited the least impairment of the motile and enzymatic activities. The motor functions of R397Q and G575R myosins were classified as intermediate. These results suggest that the force level of mutant myosin molecule may be one of the key factors for pathogenesis which affect the prognosis of human FHC.

Distinct crossbridge kinetics among cardiac myosin isoforms have been proposed as the basis of differences in energetic characteristics. However, direct evidence for this hypothesis is lacking because of experimental difficulty. As a preliminary approach to this problem, we applied an in-vitro force measurement technique to directly observe force impulse generated by a single cardiac myosin molecule. The force measurement system was constructed with an inverted microscope coupled with a laser optical trap. With the feedback of the position signal to the driving circuit for a galvanomirror that steers the laser beam, trap stiffness was increased thus, isometric force measurement was made possible. We measured the force generated by the cardiac myosin V3 isoform purified from hypothyroid rat ventricular muscle. With very low myosin density and low adenosine triphosphate (ATP) concentration of the assay buffer, we successfully observed a single force impulse similar in shape to that of skeletal muscle myosin. With this approach, we will be able to gain a clear view of the molecular basis of cardiac mechanoenergetics.

MCI-154 (6-[4-(4′-pyridylamino)phenyl]-4,5-dihydro-3(2H)pyridazinone hydrochloride trihydrate) is a potent novel cardiotonic agent whose positive inotropism is shown to be mainly based on an increase in Ca2+ sensitivity of the contractile apparatus. To elucidate the exact mechanism through which this drug acts, we investigated the movement of the reconstituted thin filament on a myosin layer in vitro. Cardiac thin filaments were reconstituted from actin and tropomyosin-troponin complex purified from rat cardiac acetone powder separately. Double staining of the filament showed that tropomyosin-troponin complex was integrated along actin filament homogeneously. Thin filaments thus prepared were fluorescently labeled and made to slide on rat cardiac myosin fixed on a glass coverslip while varying the [Ca2+] of the medium (control, pH 7.2 at 25°C). When [Ca2+] was low, the filaments showed only brownian motion. However, above a certain level of [Ca2+] (the threshold [Ca2+]), the filaments started to slide, and the velocity increased, reaching the maximum velocity within a very narrow range of [Ca2+]. The regulation was completely abolished by using simple actin filaments without tropomyosin-troponin complex, demonstrating that the regulatory proteins are responsible for this Ca2+ regulation of the movement of the reconstituted thin filament. Under the control condition, addition of MCI-154 shifted the threshold [Ca2+] to a lower level (sensitization) in a concentration-related manner. And 10-4 mol/L of MCI-154 reversed the desensitization effect induced by either acidosis (pH 6.8), low temperature (15°C), or the addition of inorganic phosphate (10 mmol/L). However, the maximum sliding velocity was not affected by the drug under any condition. In conclusion, MCI-154 directly sensitized the contractile apparatus under not only physiological but also pathophysiological conditions. This in vitro motility assay technique using reconstituted thin filaments is a useful tool for studying the mechanism of action of Ca2+ sensitizers.

We attempted to introduce calcium regulation into in vitro motility assay. Cardiac thin filament was reconstituted from actin and tropomyosin-troponin complex purified from rat myocardium separately. Double staining of the filaments showed tropomyosin-troponin complex was integrated along actin filaments homogeneously. The reconstituted thin filaments were made to slide on cardiac myosin fixed on a glass coverslip in the presence of MgATP while varying free Ca2+ concentration of the medium ([Ca2+]). Filaments showed only Brownian motion when [Ca2+] was below 10-6.4 M. However, filaments slid at a constant velocity when [Ca2+] exceeded 10-6.4 M, showing that the sliding was regulated in an on-off manner. The threshold [Ca2+] increased to 10-5.0 M under acidic conditions, indicating a decrease in Ca2+ sensitivity of the contractile proteins. Simple actin filaments slid at a constant velocity independently of [Ca2+], demonstrating that the regulatory proteins were responsible for this on-off manner regulation. This new assay technique may be a powerful tool to directly evaluate the Ca2+ sensitivity of the contractile apparatus and to investigate how cardiac contraction is regulated by Ca2+. © 1995 Springer-Verlag.

1) TECは先端のカッターを回転させて粥腫を破砕し,破砕片を吸引回収する新しいカテーテルである。TECを用いた冠動脈形成術において,末梢塞栓を認めず,血栓除去効果は十分期待できる。2) TEC通過成功病変は21病変中8病変,TECのみで残存狭窄度50%以下となった病変は12病変であり,TECのみでは,粥腫除去効果は不十分である。3)バックアップの弱い9Fのガイドカテーテルとハイトルクフロッピーガイドワイヤーを使用した症例に,TEC通過不成功例があり,病変部のTEC通過にはガイドカテーテルの強い補助と専用ガイドワイヤーの使用が必要と考えられた。4)極端な屈曲を有する病変を除外することによりTECは比較的安全に施行し得た。5) TECとバルーンを使用した冠動脈形成術の3ヵ月後の再狭窄率は29%であった。翌日造影時の狭窄度は,3ヵ月後再狭窄のpredictorであった