Heart Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States of America.
Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, Illinois, United States of America.
PLoS One. 2014 Jan 7;9(1):e83715. doi: 10.1371/journal.pone.0083715. eCollection 2014.
The functional role of inositol 1,4,5-trisphosphate (InsP3) signaling in cardiomyocytes is not entirely understood but it was linked to an increased propensity for triggered activity. The aim of this study was to determine how InsP3 receptors can translate Ca(2+) release into a depolarization of the plasma membrane and consequently arrhythmic activity. We used embryonic stem cell-derived cardiomyocytes (ESdCs) as a model system since their spontaneous electrical activity depends on InsP3-mediated Ca(2+) release. [InsP3]i was monitored with the FRET-based InsP3-biosensor FIRE-1 (Fluorescent InsP3 Responsive Element) and heterogeneity in sub-cellular [InsP3]i was achieved by targeted expression of FIRE-1 in the nucleus (FIRE-1nuc) or expression of InsP3 5-phosphatase (m43) localized to the plasma membrane. Spontaneous activity of ESdCs was monitored simultaneously as cytosolic Ca(2+) transients (Fluo-4/AM) and action potentials (current clamp). During diastole, the diastolic depolarization was paralleled by an increase of [Ca(2+)]i and spontaneous activity was modulated by [InsP3]i. A 3.7% and 1.7% increase of FIRE-1 FRET ratio and 3.0 and 1.5 fold increase in beating frequency was recorded upon stimulation with endothelin-1 (ET-1, 100 nmol/L) or phenylephrine (PE, 10 µmol/L), respectively. Buffering of InsP3 by FIRE-1nuc had no effect on the basal frequency while attenuation of InsP3 signaling throughout the cell (FIRE-1), or at the plasma membrane (m43) resulted in a 53.7% and 54.0% decrease in beating frequency. In m43 expressing cells the response to ET-1 was completely suppressed. Ca(2+) released from InsP3Rs is more effective than Ca(2+) released from RyRs to enhance INCX. The results support the hypothesis that in ESdCs InsP3Rs form a functional signaling domain with NCX that translates Ca(2+) release efficiently into a depolarization of the membrane potential.
肌醇 1,4,5-三磷酸（InsP3）信号在心肌细胞中的功能作用尚不完全清楚，但它与触发活动的倾向增加有关。本研究旨在确定 InsP3 受体如何将 Ca2+释放转化为质膜去极化，进而导致心律失常活性。我们使用胚胎干细胞衍生的心肌细胞（ESdC）作为模型系统，因为它们的自发电活动依赖于 InsP3 介导的 Ca2+释放。[InsP3]i 用基于 FRET 的 InsP3 生物传感器 FIRE-1（荧光 InsP3 反应元件）进行监测，并通过靶向核内表达 FIRE-1（FIRE-1nuc）或在质膜表达 InsP3 5-磷酸酶（m43）来实现亚细胞内[InsP3]i 的异质性。ESdC 的自发活动同时作为胞质 Ca2+瞬变（Fluo-4/AM）和动作电位（电流钳）进行监测。在舒张期，舒张去极化伴随着[Ca2+]i 的增加，自发活动受到[InsP3]i 的调节。用内皮素-1（ET-1，100nmol/L）或苯肾上腺素（PE，10μmol/L）刺激时，FIRE-1 FRET 比分别记录到 3.7%和 1.7%的增加和搏动频率增加 3.0 倍和 1.5 倍。FIRE-1nuc 缓冲 InsP3 对基础频率没有影响，而整个细胞（FIRE-1）或质膜（m43）中 InsP3 信号的衰减导致搏动频率降低 53.7%和 54.0%。在表达 m43 的细胞中，ET-1 的反应完全被抑制。从 InsP3Rs 释放的 Ca2+比从 RyRs 释放的 Ca2+更有效地增强 INCX。这些结果支持以下假设：在 ESdC 中，InsP3Rs 与 NCX 形成功能性信号域，将 Ca2+释放有效地转化为膜电位的去极化。
