Novel fluorescence resonance energy transfer-based reporter reveals differential calcineurin activation in neonatal and adult cardiomyocytes
Novel fluorescence resonance energy transfer-based reporter reveals differential calcineurin activation in neonatal and adult cardiomyocytes
复制标题
DOI：
10.1113/jp270510
复制
发表时间：
2015-09-01
影响因子：
5.5
通讯作者：
Yue, David T.
中科院分区：
文献类型：
--
作者：
Bazzazi, Hojjat;Sang, Lingjie;Yue, David T.
磷酸酶钙调神经磷酸酶是许多钙信号传导途径的核心成分，将钙信号从质膜传递到细胞核。它在免疫、心脏和神经系统等多种系统中具有关键功能。鉴于钙调磷酸酶在正常和病理条件下的广泛重要性，阐明钙调磷酸酶活性时空动态的新工具将是非常宝贵的。在这里，我们开发了两种独立的基于基因编码荧光共振能量转移（FRET）的神经钙蛋白激活传感器，DuoCaN 和 UniCaN。两种传感器均表现出大动态范围和快速响应动力学，主要区别在于 FRET 对之间的连接结构。两种传感器都在 HEK293 细胞中进行了校准，它们的响应与 NFAT 易位到细胞核的相关性良好，从而验证了传感器读数的生物相关性。随后，这些传感器在新生大鼠心室肌细胞和急性分离的成年豚鼠心室肌细胞中表达。两种传感器都在肌细胞中表现出强烈的反应，并揭示了新生儿与成人肌细胞起搏速率变化期间钙调神经磷酸酶激活的动力学差异。最后，数学模型与定量 FRET 测量相结合，为响应肌细胞 Ca 瞬变的钙调神经磷酸酶激活的动力学和整合提供了新的见解。总之，DuoCaN 和 UniCaN 是了解钙调神经磷酸酶在正常和病理信号传导中的作用的有价值的新工具。
The phosphatase calcineurin is a central component of many calcium signalling pathways, relaying calcium signals from the plasma membrane to the nucleus. It has critical functions in a multitude of systems, including immune, cardiac and neuronal. Given the widespread importance of calcineurin in both normal and pathological conditions, new tools that elucidate the spatiotemporal dynamics of calcineurin activity would be invaluable. Here we develop two separate genetically encoded fluorescence resonance energy transfer (FRET)-based sensors of calcineurin activation, DuoCaN and UniCaN. Both sensors showcase a large dynamic range and rapid response kinetics, differing primarily in the linker structure between the FRET pairs. Both sensors were calibrated in HEK293cells and their responses correlated well with NFAT translocation to the nucleus, validating the biological relevance of the sensor readout. The sensors were subsequently expressed in neonatal rat ventricular myocytes and acutely isolated adult guinea pig ventricular myocytes. Both sensors demonstrated robust responses in myocytes and revealed kinetic differences in calcineurin activation during changes in pacing rate for neonatal versus adult myocytes. Finally, mathematical modelling combined with quantitative FRET measurements provided novel insights into the kinetics and integration of calcineurin activation in response to myocyte Ca transients. In all, DuoCaN and UniCaN stand as valuable new tools for understanding the role of calcineurin in normal and pathological signalling.