Institute of Cardiovascular Sciences, University of Manchester, Manchester Academic Health Sciences Centre, Manchester M13 9PT, UK; Department of Biochemistry, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt.
J Mol Cell Cardiol. 2013 Oct;63:57-68. doi: 10.1016/j.yjmcc.2013.07.007. Epub 2013 Jul 21.
Isoform 4 of the plasma membrane calcium/calmodulin dependent ATPase (PMCA4) has recently emerged as an important regulator of several key pathophysiological processes in the heart, such as contractility and hypertrophy. However, direct monitoring of PMCA4 activity and assessment of calcium dynamics in its vicinity in cardiomyocytes are difficult due to the lack of molecular tools. In this study, we developed novel calcium fluorescent indicators by fusing the GCaMP2 calcium sensor to the N-terminus of PMCA4 to generate the PMCA4-GCaMP2 fusion molecule. We also identified a novel specific inhibitor of PMCA4, which might be useful for studying the role of this molecule in cardiomyocytes and other cell types. Using an adenoviral system we successfully expressed PMCA4-GCaMP2 in both neonatal and adult rat cardiomyocytes. This fusion molecule was correctly targeted to the plasma membrane and co-localised with caveolin-3. It could monitor signal oscillations in electrically stimulated cardiomyocytes. The PMCA4-GCaMP2 generated a higher signal amplitude and faster signal decay rate compared to a mutant inactive PMCA4(mut)GCaMP2 fusion protein, in electrically stimulated neonatal and adult rat cardiomyocytes. A small molecule library screen enabled us to identify a novel selective inhibitor for PMCA4, which we found to reduce signal amplitude of PMCA4-GCaMP2 and prolong the time of signal decay (Tau) to a level comparable with the signal generated by PMCA4(mut)GCaMP2. In addition, PMCA4-GCaMP2 but not the mutant form produced an enhanced signal in response to β-adrenergic stimulation. Together, the PMCA4-GCaMP2 and PMCA4(mut)GCaMP2 demonstrate calcium dynamics in the vicinity of the pump under active or inactive conditions, respectively. In summary, the PMCA4-GCaMP2 together with the novel specific inhibitor provides new means with which to monitor calcium dynamics in the vicinity of a calcium transporter in cardiomyocytes and may become a useful tool to further study the biological functions of PMCA4. In addition, similar approaches could be useful for studying the activity of other calcium transporters during excitation-contraction coupling in the heart.
最近，质膜钙/钙调蛋白依赖性 ATP 酶（PMCA4）同工型 4 已成为心脏中几个关键病理生理过程的重要调节剂，例如收缩性和肥大。然而，由于缺乏分子工具，直接监测心肌细胞中 PMCA4 的活性和评估其附近的钙动力学仍然具有挑战性。在这项研究中，我们通过将 GCaMP2 钙传感器融合到 PMCA4 的 N 端，生成 PMCA4-GCaMP2 融合分子，从而开发了新型钙荧光指示剂。我们还鉴定了一种 PMCA4 的新型特异性抑制剂，这可能对研究该分子在心肌细胞和其他细胞类型中的作用有用。我们使用腺病毒系统成功地在新生和成年大鼠心肌细胞中表达了 PMCA4-GCaMP2。这种融合分子被正确靶向到质膜，并与 caveolin-3 共定位。它可以监测电刺激心肌细胞中的信号振荡。与突变失活的 PMCA4(mut)GCaMP2 融合蛋白相比，PMCA4-GCaMP2 在电刺激的新生和成年大鼠心肌细胞中产生更高的信号幅度和更快的信号衰减率。小分子文库筛选使我们能够鉴定出一种 PMCA4 的新型选择性抑制剂，我们发现该抑制剂可降低 PMCA4-GCaMP2 的信号幅度并延长信号衰减时间（Tau）至与 PMCA4(mut)GCaMP2 产生的信号相当的水平。此外，PMCA4-GCaMP2 而不是突变形式在β-肾上腺素能刺激下产生增强的信号。总之，PMCA4-GCaMP2 和 PMCA4(mut)GCaMP2 分别在泵的活性或失活状态下展示了泵附近的钙动力学。总之，PMCA4-GCaMP2 与新型特异性抑制剂一起提供了监测心肌细胞中钙转运体附近钙动力学的新方法，可能成为进一步研究 PMCA4 生物学功能的有用工具。此外，类似的方法可能有助于研究心脏兴奋-收缩耦联过程中其他钙转运体的活性。
