BASIS FOR ANESTHETIC-INDUCED FATALITIES

该项目的总体目标是确定以下生理基础 增强的兴奋性和对麻醉药固有的异常反应 恶性高热易感(MHS)人和猪的肌肉。1) 首先，我们将检验这样一种假设，即从新生儿培养的肌管 MHS肌肉表现出MHS肌肉的异常特征。 钙指示剂Fura的荧光成像将用于 确定MHS肌管肌浆钙是否异常 在接触氟烷后升高(这会引发MH发作)。这个 MHS和正常肌管的强度-时间关系将是 利用全细胞膜片钳技术确定 MHS肌管收缩阈值低于正常。2) MHS骨骼肌的兴奋-收缩(EC)偶联似乎 在横小管(TT)水平改变。我们将测试 MHS肌管在TT水平上是否存在功能缺陷 记录与EC耦合相关的电压相关TT信号，即， 慢激活、长时间钙电流(I-S)与膜内 电荷运动。全细胞膜片钳技术将用于 I-S及其电压依赖性电荷运动的刻画 钙通道的激活和失活与敏感性 拮抗剂来确定这些参数中的任何一个是否在 MHS肌管。丹曲烯和氮杂环烯，EC偶联抑制剂 预防MH发作，将通过研究确定它们是否会改变I-S 和/或电荷运动。3)最后，我们将检验假设 已知的MHS肌肉肌浆网(SR)异常改变 在TT级别上发挥作用。肌肉MH基因的杂合子，这是 SR钙通道的编码显示出一些相同的EC偶联 异常如纯合的MHS肌肉。仔猪肌管的培养 将使用MH基因的杂合子来确定是否 SR钙通道异常亚群的存在导致 更改了I-S和TTS中的电荷运动。SR对TT功能的影响 将通过记录全细胞i-S和电荷运动来进一步探索 在作用于肌管的三种药物中的一种情况下 SR钙通道：与SR钙通道结合的Ryanodine和 使它们处于低电导状态，氟烷或咖啡因 它们作用于肌质网钙通道，促进钙释放。总而言之， 这些研究将解决是否除了有良好记录的SR之外 钙通道缺陷，I-S和/或电荷也有异常 MHS肌肉运动和肌浆网钙通道异常是否可以 在技术转让层面上影响事件。关于EC联轴器缺陷的说明 对于理解MHS肌肉的基础将是重要的 启动网络管理，并有助于澄清尚未定义的部分 骨骼肌的EC偶联过程。

The overall goal of this project is to define the physiological basis for enhanced excitability and abnormal responses to anesthetics inherent in muscles of malignant hyperthermia susceptible (MHS) humans and pigs. 1) First we will test the hypothesis that myotubes cultured from neonatal MHS muscles exhibit abnormalities characteristic of MHS muscles. Fluorescence imaging of the calcium indicator fura will be used to determine whether myoplasmic calcium of MHS myotubes is abnormally elevated after exposure to halothane (which triggers MH episodes). The strength-duration relationship of MHS and normal myotubes will be determined using whole cell patch-clamp techniques to establish that the contractile threshold of MHS myotubes is lower than normal. 2) Excitation-contraction (EC) coupling in MHS skeletal muscles appears to be altered at the level of the transverse-tubules (TT). We will test whether MHS myotubes are functionally defective at the TT level by recording voltage-dependent TT signals associated with EC coupling, i.e., the slow activating, long lasting calcium current (I-s) and intramembrane charge-movement. Whole cell patch-clamp techniques will be used to characterize I-s and charge-movement as to voltage-dependence of activation and inactivation and sensitivity to calcium channel antagonists to determine whether any of these parameters are altered in MHS myotubes. Dantrolene and azumolene, EC coupling inhibitors which prevent MH episodes, will be studied to determine whether they alter I-s and/or charge-movement. 3) Finally, we will test the hypothesis that the known sarcoplasmic reticulum (SR) abnormality of MHS muscles alters function at the TT level. Muscles heterozygous for the MH gene, which codes for the SR calcium channel exhibit some of the same EC-coupling abnormalities as homozygous MHS muscles. Myotubes cultured from piglets heterozygous for the MH gene will be used to determine whether the presence of a subpopulation of abnormal SR calcium channels results in altered I-s and charge movement in the TTs. SR influence on TT function will be further explored by recording whole cell I-s and charge-movement of normal myotubes in the presence of one of three drugs which act at the SR calcium channel: Ryanodine which binds to SR calcium channels and holds them in a low conductance state, and halothane or caffeine both of which act at the SR calcium channel to enhance calcium release. In sum, these studies will address whether, in addition to the well documented SR calcium channel defect, there is also an abnormality in I-s and/or charge movement in MHS muscles and whether abnormal SR calcium channels can influence events at the TT level. Elucidation of the EC coupling defect in MHS muscles will be of importance for understanding the basis for initiation of NM and contribute to clarifying an as yet undefined portion of the EC coupling process of skeletal muscles.