Bovo Elisa, Nikolaienko Roman, Bhayani Siddharth, Kahn Daniel, Cao Quan, Martin Jody L, Kuo Ivana Y, Robia Seth L, Zima Aleksey V
Department of Cell and Molecular Physiology, Loyola University Chicago , Chicago, Illinois.
Department of Physiology and Biophysics, University of Illinois at Chicago , Chicago, Illinois.
Am J Physiol Heart Circ Physiol. 2019 Jun 1;316(6):H1323-H1331. doi: 10.1152/ajpheart.00031.2019. Epub 2019 Mar 22.
The type 2a sarco-/endoplasmic reticulum Ca-ATPase (SERCA2a) plays a key role in Ca regulation in the heart. However, available techniques to study SERCA function are either cell destructive or lack sensitivity. The goal of this study was to develop an approach to selectively measure SERCA2a function in the cellular environment. The genetically encoded Ca sensor R-CEPIA1er was used to measure the concentration of Ca in the lumen of the endoplasmic reticulum (ER) ([Ca]) in HEK293 cells expressing human SERCA2a. Coexpression of the ER Ca release channel ryanodine receptor (RyR2) created a Ca release/reuptake system that mimicked aspects of cardiac myocyte Ca handling. SERCA2a function was quantified from the rate of [Ca] refilling after ER Ca depletion; then, ER Ca leak was measured after SERCA inhibition. ER Ca uptake and leak were analyzed as a function of [Ca] to determine maximum ER Ca uptake rate and maximum ER Ca load. The sensitivity of this assay was validated by analyzing effects of SERCA inhibitors, [ATP]/[ADP], oxidative stress, phospholamban, and a loss-of-function SERCA2a mutation. In addition, the feasibility of using R-CEPIA1er to study SERCA2a in a native system was evaluated by using in vivo gene delivery to express R-CEPIA1er in mouse hearts. After ventricular myocyte isolation, the same methodology used in HEK293 cells was applied to study endogenous SERCA2a. In conclusion, this new approach can be used as a sensitive screening tool to study the effect of different drugs, posttranslational modifications, and mutations on SERCA function. The aim of this study was to develop a sensitive approach to selectively measure sarco-/endoplasmic reticulum Ca-ATPase (SERCA) function in the cellular environment. The newly developed Ca sensor R-CEPIA1er was used to successfully analyze Ca uptake mediated by recombinant and native cardiac SERCA. These results demonstrate that this new approach can be used as a powerful tool to study new mechanisms of Ca pump regulation.
2a型肌浆网/内质网钙ATP酶（SERCA2a）在心脏钙调节中起关键作用。然而，现有的研究SERCA功能的技术要么具有细胞破坏性，要么缺乏敏感性。本研究的目的是开发一种在细胞环境中选择性测量SERCA2a功能的方法。利用基因编码的钙传感器R-CEPIA1er来测量表达人SERCA2a的HEK293细胞内质网（ER）腔中的钙浓度（[Ca]）。内质网钙释放通道兰尼碱受体（RyR2）的共表达创建了一个钙释放/再摄取系统，该系统模拟了心肌细胞钙处理的某些方面。通过内质网钙耗竭后[Ca]再填充的速率来量化SERCA2a的功能；然后，在SERCA抑制后测量内质网钙泄漏。分析内质网钙摄取和泄漏与[Ca]的关系，以确定最大内质网钙摄取速率和最大内质网钙负荷。通过分析SERCA抑制剂、[ATP]/[ADP]、氧化应激、受磷蛋白和功能丧失性SERCA2a突变的影响，验证了该检测方法的敏感性。此外，通过体内基因递送在小鼠心脏中表达R-CEPIA1er，评估了在天然系统中使用R-CEPIA1er研究SERCA2a的可行性。分离心室肌细胞后，将HEK293细胞中使用的相同方法应用于研究内源性SERCA2a。总之，这种新方法可作为一种灵敏的筛选工具，用于研究不同药物、翻译后修饰和突变对SERCA功能的影响。本研究的目的是开发一种灵敏的方法，在细胞环境中选择性测量肌浆网/内质网钙ATP酶（SERCA）的功能。新开发的钙传感器R-CEPIA1er被用于成功分析重组和天然心脏SERCA介导的钙摄取。这些结果表明，这种新方法可作为研究钙泵调节新机制的有力工具。
