Reference : Development and characterization of a novel fluorescent indicator protein PMCA4-GCaMP...
Scientific journals : Article
Human health sciences : Cardiovascular & respiratory systems
Development and characterization of a novel fluorescent indicator protein PMCA4-GCaMP2 in cardiomyocytes.
Mohamed, Tamer M. A. [> >]
Abou-Leisa, Riham [> >]
Baudoin, Florence [> >]
Stafford, Nicholas [> >]
Neyses, Ludwig mailto [University of Luxembourg > Research Office]
Cartwright, Elizabeth J. [> >]
Oceandy, Delvac [> >]
Journal of molecular and cellular cardiology
Yes (verified by ORBilu)
[en] ATA ; Cardiomyocytes ; GCaMP2 ; GECI ; GFP-calmodulin-based GECI ; Local calcium ; NCX ; NRCM ; PMCA ; PMCA4 ; PMCA4 inhibitor ; SERCA ; aurintricarboxylic acid ; genetically encoded calcium indicator ; nNOS ; neonatal rat cardiomyocytes ; neuronal nitric oxide synthase ; plasma membrane calcium/calmodulin dependent ATPase ; sarcoplasmic reticulum ATPase ; sodium/calcium exchanger
[en] 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 beta-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.
Institute of Cardiovascular Sciences, University of Manchester, Manchester Academic Health Sciences Centre, Manchester M13 9PT, UK
(c) 2013.

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