Collective Ryanodine Receptor Operation at Release Sites

Ryanodine受体（RyRs）介导肌浆网（SR）的Ca诱导的Ca释放（CICR）。的 SR具有离散的Ca释放位点，每个位点具有RyR簇。在间歇期，单个RyR很少打开， 但当这样做时，它释放的Ca可能点燃局部的RyR间CICR（火花）。异常频繁或 大的火花可以引起传播的Ca波、室性心动过速和心源性猝死。 RyR在释放位点相互依赖地操作。集群中任何RyR的开放都是不变和动态的 （空间和时间）改变局部[Ca]（胞质和SR内）。这些[Ca]变化可能会激活邻近的 RyRs。在释放位点的聚集的RyR几何结构和CICR的固有正反馈构成了一个不断的， 有诱发钙波的风险因此，故障安全释放现场操作需要保持本地CICR处于检查状态。为 几十年来，各种单一RyR水平的机制（失活，适应，管腔钙调节）被提出 来解释释放位点的矛盾的CICR稳定性。最终发现这些单RyR过程 不够。我们的初步研究表明，原因是所需的CICR阴性对照来自 RyR的集体操作，而不是每个RyR彼此独立地起作用。集体RyR操作. 发布站点本质上仍然是一个黑盒子，即使我们知道很多关于它的组件，从单一的RyR 结构-功能到火花。 以前的模拟重要地揭示了“基本”Ca释放的细节。但是，这些应用了一些强大的 简化。例如，单RyR门控是复杂的，这只是在过去没有捕获。共同 使用池室模型（现在仍然是），其中所有RyR总是“看到”相同的[Ca]，但这实际上是 消除了驱动集体RyR的现象（动态纳米级时空[Ca]梯度） 释放现场的操作。因此，我们的基本原则是，当我们有所需的零件时，我们必须精心 在没有过去不切实际的假设的情况下组装它们。为此，我们开发了一种创新的混合动力汽车， 实验/计算方法。我们的初步研究已经揭示了以前未知的集体-RyR CICR控制机制，如恶性减员（有助于终止CICR），RyR招聘偏见（有效 缩小RyRCICR事件之间的距离）和长期封闭的耐药性（限制CICR在区域内的局部传播）。 站点）。这里检验的假设是：舒张期SR Ca的正常故障安全稳定性和致心律失常不稳定性 释放位点由集体RyR控制机制控制，该机制可以通过RyR治疗性地操纵， 靶向药物。并且，具体目标是1）识别促进细胞增殖的集体RyR控制机制。 舒张期稳定性正常人心室SR Ca释放位点和2）定义人心室SR Ca释放位点的集体RyR控制 促血管舒张释放位点和测试RyR靶向药物作为潜在的治疗集体控制 稳定器这项研究将为理解集体RyR操作提供一个强有力的机制基础， SR Ca释放位点，因此有望改变我们对致瘤性SR Ca释放位点的认识。

Ryanodine receptors (RyRs) mediate Ca-induced Ca release (CICR) from the sarcoplasmic reticulum (SR). The SR has discrete Ca release sites, each with a cluster of RyRs. During diastole, single -RyRs open infrequently, but when one does, the Ca it releases may ignite localized inter-RyR CICR (a spark). Abnormally frequent or large sparks can evoke propagating Ca waves, ventricular tachycardia and sudden cardiac death. RyRs operate inter-dependently at release sites. Opening of any RyR in a cluster invariably and dynamically (spatially & temporally) alters local [Ca]’s (cytosolic & intra-SR). These [Ca] changes may activate neighboring RyRs. The clustered RyR-geometry at release sites and the inherent positive feedback of CICR pose an ever- present risk of evoking a Ca wave. So, fail-safe release site operation requires keeping local CICR in check. For decades, various single RyR-level mechanisms (inactivation, adaptation, luminal Ca regulation) were proposed to explain the paradoxical CICR stability of release sites. These single-RyR processes were ultimately found to be insufficient. Our pilot studies indicate the reason is the required CICR negative control arises from the collective operation of RyRs, not each RyR acting independently of one another. Collective-RyR operation at release sites is still essentially a black box even though we know a lot about its components, from single-RyR structure-function to sparks. Previous simulations importantly revealed details of “elementary” Ca release. But, these applied some strong simplifications. For example, single-RyR gating is complex and this was just not captured in the past. Common pool compartment models were used (and still are), where all RyRs always “see” the same [Ca], but this literally eliminates the very phenomenon (dynamic nano-scale spatiotemporal [Ca] gradients) that drives collective-RyR operation at release sites. Thus, our foundational principle is, as we have the parts needed, we must meticulously assemble them without the unrealistic assumptions of the past. To this end, we developed an innovative hybrid experimental/computational approach. Our pilot studies have already revealed previously unknown collective-RyR CICR control mechanisms like pernicious attrition (which helps terminates CICR), RyR recruitment bias (that works to shrink inter-RyRCICR events) and long-closed resistance(whichlimits the local spread of CICR within arelease site). The hypothesis tested here is: The normal fail-safe stability and arrhythmogenic instability of diastolic SR Ca release sites are governed by collective-RyR control mechanisms that can be therapeutically manipulated by RyR- targeted drugs. And, the specific aims are 1) Identify collective-RyR control mechanisms that promote the diastolic stability normal human ventricular SR Ca release sites and 2) Define collective-RyR control at human arrhythmogenic diastolic release sites and test RyR-targeted drugs as potential therapeutic collective control stabilizers. This study will provide a strong mechanistic foundation for understanding collective -RyR operation at SR Ca release sites and so promises to transform our knowledge of arrhythmogenic SR Ca release sites.