Enhancement of Sarcolemmal Calcium Influx in a Novel Mouse Model of Malignant Hyperthermia
Enhancement of Sarcolemmal Calcium Influx in a Novel Mouse Model of Malignant Hyperthermia
复制标题
新型恶性高热小鼠模型中肌膜钙流入的增强
DOI：
10.1016/j.bpj.2018.11.2806
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发表时间：
2019
影响因子：
3.4
通讯作者：
Kaura V
中科院分区：
文献类型：
--
作者：
Kaura V
1型兰尼碱受体（RyR 1）是骨骼肌肌浆网上的钙离子释放通道，其基因突变可引起包括恶性高热（MH）在内的多种肌肉疾病。由于发病机制的主要潜在机制是由RyR 1通道的功能获得引起的过度活性Ca 2+释放，因此RyR 1的抑制有望成为这些疾病的有希望的治疗方法。我们最近开发了一种使用内质网（ER）中Ca 2+测量的RyR 1抑制剂的高效高通量筛选（HTS）平台，并成功地从充分表征的药物库中鉴定了三种新化合物（Murayama et al.，Mol Pharmacol，94：722-730，2018）。然而，只有oxolinic酸被发现是特定的RyR 1和其他两种化合物抑制RyR 1和RyR 2，心脏亚型。为了探索另一种RyR 1特异性抑制剂，我们从大量化合物的文库中进行了HTS。使用FlexStation 3荧光计，用R-CEPIA 1 er（遗传编码的ER Ca 2+指示剂）监测表达携带MH突变（R2163 C）的RyR 1的HEK 293细胞的ER Ca 2+。抑制RyR 1的化合物将通过防止Ca 2+经由突变体RyR 1泄漏而增加ER Ca 2+。我们成功地鉴定了几种RyR 1特异性化合物，它们在结构上不同于oxolinic acid或dantrolene，一种已知的RyR 1抑制剂。我们目前正在研究这些化合物如何抑制RyR 1通道。这些化合物可能是治疗RyR 1相关疾病的良好候选物。2581-Pos测定人体骨骼肌中的产热，从测量基础Ca 2 D运动Christopher J. Barclay，布拉德利S.劳尼柯尼斯昆士兰州大学，布里斯班，澳大利亚。哺乳动物的骨骼肌产生热量，用于维持体温。产热主要归因于肌浆网（SR）Ca 2+泵的ATP裂解活性。肌肉大部分时间处于休息状态，因此在这种状态下从肌肉收集热量提供了有效的恒定产热，这可能取决于RyR泄漏。最近，使用一种新的共聚焦成像技术在静息人体肌纤维中检测到通过兰尼碱受体（RyR）的SR Ca 2+泄漏，该技术利用了被困在皮肤纤维的密封管状（t-）系统中的Ca 2+敏感染料（Cully et al 2018，PNAS）。通过测量进入密封t系统的Ca 2+摄取来检测RyR Ca 2+泄漏是一组复杂事件的净结果。为了预测肌肉产生的热量，设计了一个模型，将系统分为t系统、SR、细胞质和交界空间的7个离散空间。这些空间之间的Ca 2+运动与RyR，PMCA，NCX和SERCA，EGTA缓冲Ca 2+在细胞质和交界处的空间;和SR Ca 2+缓冲钙螯合蛋白。利用Maple软件求解了描述系统的相关微分方程组。模型优化结果表明，影响t-系统Ca ~（2+）吸收的因素有PMCA浓度、PMCA Ca ~（2+）亲和力、pH和t-系统渗漏率。该模型确定SR泄漏率在0.01和0.02 s/cm-1之间，与实验数据一致（Cully等人，2018）。然后使用该模型来量化SR Ca 2+流出，以估计与SR和细胞质之间的Ca 2+循环相关的产热为0.4 W kg/m2，约为静止（或静息）人骨骼肌可能产热的一半。
Genetic mutations in type 1 ryanodine receptor (RyR1), a Ca2þ-release channel on the sarcoplasmic reticulum of skeletal muscle, cause various muscle diseases including malignant hyperthermia (MH). Because the main underlying mechanism of the pathogenesis is overactive Ca2þ release by gain-of-function of the RyR1 channel, inhibition of RyR1 is expected to be a promising treatment for these diseases. We have recently developed an efficient high-throughput screening (HTS) platform for RyR1 inhibitors using Ca2þ measurements in the endoplasmic reticulum (ER) and successfully identified three novel compounds from a library of well-characterized drugs (Murayama et al., Mol Pharmacol, 94: 722-730, 2018). However, only oxolinic acid was found to be specific to RyR1 and other two compounds inhibited both RyR1 and RyR2, a cardiac isoform. To explore another RyR1-specific inhibitor, we performed HTS from a library of larger numbers of compounds. ER Ca2þ of HEK293 cells expressing RyR1 carrying an MH mutation (R2163C) was monitored with R-CEPIA1er, a genetically encoded ER Ca2þ indicator, using FlexStation3 fluorometer. Compounds which inhibit RyR1 would increase ER Ca2þ by preventing Ca2þ leak via the mutant RyR1. We successfully identified several RyR1-specific compounds that are structurally different from oxolinic acid or dantrolene, a known RyR1 inhibitor. We are currently investigating how these compounds inhibit the RyR1 channel. These compounds may be good candidates for treatment of RyR1-related diseases.2581-Pos Determination of Heat Production in Human Skeletal Muscle from Measurements of Basal Ca2D Movements Christopher J. Barclay, Bradley S. Launikonis. Sch Biomed Sci, The University of Queensland, Brisbane, Australia. Skeletal muscle in mammals generates heat that is used to maintain body temperature. Heat generation is largely attributable to the ATP splitting activity of the sarcoplasmic reticulum (SR) Ca2þ pump. The muscle spends most of its time at rest, so harvesting heat from the muscle in this state provides an effectively constant generation of heat, likely dependent on RyR leak. Recently the SR Ca2þ leak through the ryanodine receptor (RyR) was detected in resting human muscle fibres using a novel confocal imaging technique utilizing a Ca2þsensitive dye trapped in the sealed tubular (t-) system of skinned fibres (Cully et al 2018, PNAS). Detection of RyR Ca2þ leak via measurements of Ca2þ uptake into the sealed t-system is the net outcome of a complex set of events. To allow the prediction of heat generated by the muscle a model was devised that divided the system into 7 discrete spaces across the t-system, SR, cytoplasm and junctional space. Ca2þ movements between these spaces were linked by the RyR, PMCA, NCX and SERCA, with EGTA buffering Ca2þ in the cytoplasm and junctional space; and SR Ca2þ buffered by calsequestrin. A system of inter-related differential equations to describe the system were solved using Maple software. Model optimization showed the factors that influence t-system Ca2þ uptake were PMCA density, PMCA Ca2þ affinity, pH and t-system leak rate. The model determined a SR leak rate between 0.01 and 0.02 s À1, consistent with experimental data (Cully et al 2018). The model was then used to quantify SR Ca2þ efflux to estimate heat production associated with Ca2þ cycling between SR and cytoplasm of $0.4 W kg À1, which is about half of the likely heat production of quiescent (or resting) human skeletal muscle.