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Updated: May 9, 2026
In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter
Published on: February 22, 2022
Development and characterization of a novel fluorescent indicator protein PMCA4-GCaMP2 in cardiomyocytes
Tamer M A Mohamed1, Riham Abou-Leisa, Florence Baudoin
1Institute 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.
Abstract:
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.
Insights
Researchers developed a novel tool, PMCA4-GCaMP2, to monitor calcium activity near a key heart protein, plasma membrane calcium/calmodulin dependent ATPase 4 (PMCA4). A new inhibitor was also found, aiding heart calcium dynamics research.
Area of Science:
- Cardiovascular Biology
- Molecular Cardiology
- Biophysics
Background:
- Plasma membrane calcium/calmodulin dependent ATPase 4 (PMCA4) is crucial for heart function, regulating contractility and hypertrophy.
- Directly measuring PMCA4 activity and local calcium dynamics in cardiomyocytes is challenging due to a lack of specific molecular tools.
Purpose of the Study:
- To develop novel molecular tools for monitoring PMCA4 activity and calcium dynamics in cardiomyocytes.
- To identify a selective inhibitor for PMCA4 to aid in studying its physiological roles.
Main Methods:
- Constructed a PMCA4-GCaMP2 fusion protein by linking GCaMP2 calcium sensor to PMCA4.
- Expressed PMCA4-GCaMP2 in neonatal and adult rat cardiomyocytes using an adenoviral system.
- Screened a small molecule library to identify a novel PMCA4 inhibitor.
Main Results:
- PMCA4-GCaMP2 localized to the plasma membrane and successfully monitored calcium oscillations in stimulated cardiomyocytes.
- The fusion protein exhibited higher signal amplitude and faster decay than an inactive mutant (PMCA4(mut)GCaMP2).
- A novel PMCA4 inhibitor reduced PMCA4-GCaMP2 signal amplitude and prolonged decay, mimicking the inactive mutant.
Conclusions:
- PMCA4-GCaMP2 and its inhibitor offer a new method to study calcium transport and dynamics near PMCA4 in cardiomyocytes.
- These tools advance the understanding of PMCA4's role in cardiac pathophysiology and excitation-contraction coupling.
- The approach may be adaptable for studying other calcium transporters involved in heart function.
