原发性心室肌细胞肉质网 Ca2+ 释放的定量分析

Md Nure Alam Afsar, Mahmuda Akter, Christopher Y. Ko, Vasco Sequeira, Yusuf Olgar, Christopher N. Johnson
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引用次数: 0

摘要

在心脏中,离子通道产生电流,通过改变细胞内钙浓度(即[Ca2+])发出肌肉收缩信号。心脏的雷诺丁受体 2 型(RyR2)是主要的离子通道,负责通过从肌浆网(SR)释放 Ca2+ 来增加细胞内的[Ca2+]。及时释放 Ca2+ 是心脏功能正常所必需的,而功能障碍则会引起或导致心律失常等危及生命的疾病。对火花和波浪形式的 SR-Ca2+ 释放进行定量分析,可为了解 RyR2 开放情况以及影响或调节通道功能的因素提供宝贵的信息。在此,我们提供了一系列方案,概述了以下流程:(1)获得高质量的分离心肌细胞;(2)制备样本用于实验研究影响 RyR2 功能的因素;以及(3)数据采集和分析。值得注意的是,我们的方案利用了最近开发的肌球蛋白 ATP 酶抑制剂 Mavacamten 的效力。这使得我们有机会在[Ca2+]范围内描述小分子或重组蛋白/酶对 RyR2-Ca2+ 释放事件的影响。© 2024 Wiley Periodicals LLC.Basic Protocol 1: Cardiomyocyte isolation from mouseBasic Protocol 2: Preparation of cardiomyocytes for Ca2+ imagingBasic Protocol 3: Confocal microscopy and quantitative Ca2+ analysis using SparkMaster 2.
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Quantification of Sarcoplasmic Reticulum Ca2+ Release in Primary Ventricular Cardiomyocytes

In the heart, ion channels generate electrical currents that signal muscle contraction through changes in intracellular calcium concentration, i.e., [Ca2+]. The cardiac ryanodine receptor type 2 (RyR2) is the predominant ion channel responsible for increasing intracellular [Ca2+] by releasing Ca2+ from the sarcoplasmic reticulum (SR). Timely Ca2+ release is necessary for appropriate cardiac function, and dysfunction can cause or contribute to life-threatening diseases such as arrhythmia. Quantification of SR-Ca2+ release in the form of sparks and waves can provide valuable insight into RyR2 opening, and factors that influence or regulate channel function. Here, we provide a series of protocols that outline processes for (1) obtaining high-quality isolated cardiomyocytes, (2) preparing samples for experimentally investigating factors that influence RyR2 function, and (3) data acquisition and analysis. Notably, our protocols leverage the potency of the recently developed myosin ATPase inhibitor, Mavacamten. This affords the opportunity to characterize the effects of small molecules or reconstituted proteins/enzymes on RyR2-Ca2+ release events across a range of [Ca2+]. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Cardiomyocyte isolation from mouse

Basic Protocol 2: Preparation of cardiomyocytes for Ca2+ imaging

Basic Protocol 3: Confocal microscopy and quantitative Ca2+ analysis using SparkMaster 2

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