MODULATION OF MYOFILAMENT CA2+ SENSITIVITY AS A POSITIVE INOTROPIC INTERVENTION

一个持续的挑战是开发出 通过增强肌丝增加心肌收缩力 对Ca 2+的反应性，而不是通过增加细胞的程度， Ca 2+负荷。二嗪酮衍生物EMD 53998（由E. 德国达姆施塔特默克公司），增加峰值力和换档速度 皮肤心肌纤维中的pCa-力关系;以及细胞中的pCa-力关系。 匀浆后，其表现出磷酸二酯酶（PDE）抑制活性。 然而，肌丝致敏的效力相对于 PDE抑制作用大于任何已知物质。 的影响 EMD 53998及其（+）EMD 57033和（-）EMD 57439， 对映异构体，测试收缩性能和Cai 单个、完整、豚鼠和犬心肌细胞的瞬态。 将细胞加载荧光染料indo-1，并浸泡在 Hepes缓冲液在25摄氏度。我们的目的是确定 光学对映体可分离PDE介导的效应 通过增加肌丝获得的抑制 对Ca 2+的反应。所有这三种物质都表现出明显的 增加抽搐幅度：最大的影响， 大约是其两种对映体的效果的总和。 蔡 瞬态，测量为410/490 nm indo-1荧光比 瞬态，增加了外消旋体及其（-）对映体，但 而不是（+）-对映体。在未受刺激的细胞中， （+）-对映异构体显着减少，这是伴随着 通过indo-1荧光的降低;（-）-对映体没有影响 任何一个参数 质量相似的效果， 用完整的狗心肌细胞做实验。 的分子机制 进一步研究了（+）-对映体在狗心脏中的作用 肌原纤维 EMD 57033刺激心肌细胞肌原纤维中的ATP酶活性， 其中肌钙蛋白原肌球蛋白已被提取，但不影响 Ca 2+与分离的肌钙蛋白C结合。此外，在运动性测定中， 含有分离的肌动蛋白和肌球蛋白，但缺乏Ca 2+和调节性蛋白质。 蛋白质，该物质增加肌动蛋白运动的速度沿着 肌球蛋白因此，在完整细胞中，EMD 53998的（+）-对映体具有 直接的肌丝效应不是简单地由于“Ca 2 +- 致敏作用”（即，对钙离子与肌钙蛋白C结合的下游效应 和细丝激活的调节蛋白调节）;而 对肌动蛋白-肌球蛋白相互作用的影响，即可能存在交叉桥 涉案

A persistent challenge has been the development of substances that increase myocardial contractility via an enhancement of myofilament responsiveness to Ca2+ rather than by increasing the extent of cellular Ca2+ loading. The diazinone derivative, EMD 53998 (designed by E. Merck, Darmstadt, Germany), increases the peak force and shift leftward the pCa-force relationship in skinned myocardial fibers; and in cell homogenates it exhibits phosphodiesterase (PDE) inhibitory activity. However, the potency of myofilament sensitization relative to that of PDE inhibition is greater than for any known substance. The effects of EMD 53998, and of its (+), EMD 57033 and (-), EMD 57439, enantiomers, were tested on the contractile properties and Cai transients of single, intact, guinea pig and dog cardiac myocytes. Cells were loaded with the fluorescent dye, indo-1, and bathed in a Hepes buffer at 25 degrees C. Our aim was to ascertain whether the optical enantiomers could separate the effect mediated through PDE inhibition from that obtained via an increased myofilament responsiveness to Ca2+. All three substances exerted a pronounced increase in twitch amplitude: the maximal effect of the racemate was approximately the sum of the effects of its two enantiomers. The Cai transient, measured as the 410/490 nm indo-1 fluorescence ratio transient, was increased by the racemate and its (-) enantiomer, but not by the (+)-enantiomer. In unstimulated cells resting length was significantly reduced by the (+)-enantiomer and this was accompanied by a decrease in indo-1 fluorescence; the (-)-enantiomer had no effect on either parameter. Qualitatively similar effects were obtained in experiments with intact dog cardiac cells. The molecular mechanism of the effect of the (+)-enantiomer was further studied in dog cardiac myofibrils. EMD 57033 stimulates the ATPase activity in myofibrils in which troponin-tropomyosin have been extracted, but does not affect Ca2+ binding to isolated troponin C. Furthermore, in a motility assay containing isolated actin and myosin, but devoid of Ca2+ and regulatory proteins, the substance increases the velocity of actin motion along myosin. Thus, in intact cells the (+)-enantiomer of EMD 53998 has direct myofilament effects which are not simply due to "Ca2+- sensitization" (i.e. effects downstream to Ca2+-binding to troponin C and regulatory protein modulation of thin filament activation); rather an effect on acto-myosin interaction, i.e. the cross-bridge is likely involved.