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Updated: Aug 23, 2026
Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes
Published on: September 18, 2017
Contractile arrest reveals calcium-dependent stimulation of SERCA2a mRNA expression in cultured ventricular
Ronald Vlasblom1, Alice Muller, René J P Musters
1Laboratory for Physiology, Institute for Cardiovascular Research (ICaR-VU), VU University Medical Center, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands.
Objective:
Downregulation of sarco-endoplasmic reticulum calcium ATPase 2a (SERCA2a) expression is a critical marker of pathological myocardial hypertrophy. The effects of calcium-dependent signaling and of contractile activity on the regulation of myocardial SERCA2a expression remain unclear. The present study dissociates effects of calcium-dependent signaling through calcineurin (CN) and calmodulin dependent protein kinase-II (CAMK-II), from effects of contractile activity in spontaneously contracting rat neonatal ventricular cardiomyocytes (NVCM) using 2,3-butanedione monoxime (BDM), which arrests contractions but maintains calcium fluxes.
Methods:
SERCA2a mRNA expression was analysed using Northern hybridisation in spontaneously contracting NVCM (control) and in NVCM treated with either BDM, L-type Ca2+-channel blocker (verapamil), CN-blocker (cyclosporin A; CsA), CAMK-II blocker (KN-93), or combinations thereof. Transient transfection of the CN-dependent transcription factor nuclear factor of activated T-lymphocytes (NFATc), coupled to GFP, was used to detect NFAT nuclear translocation. The effects of CN/CAMK-II-dependent signaling were further dissected into effects of the transcription factors NFATc4 and myocyte enhancer factor 2c (MEF2c) on the activity of various SERCA2a promoter fragments using transient transfection assays.
Results:
Treatment with BDM induced a 2.5-fold rise in SERCA2a mRNA, which was abolished by addition of verapamil and was reduced by addition of CsA (-40%) and KN-93 (-20%). NFAT nuclear translocation was similar in control and BDM-treated NVCM. SERCA2a promoter activity was stimulated by NFATc4 and MEF2c, but only when both factors were co-transfected.
Conclusion:
Following contractile arrest with BDM, upregulation of SERCA2a mRNA expression by CN/CAMK-II signaling becomes evident. This upregulation is likely the result of synergistic stimulation of SERCA2a promoter activity by NFATc4 and MEF2c. Contractile activity opposes this upregulation through distinct and independent pathways.
Insights
Contractile activity in cardiomyocytes opposes sarco-endoplasmic reticulum calcium ATPase 2a (SERCA2a) mRNA upregulation. Calcium signaling pathways involving calcineurin (CN) and calmodulin-dependent protein kinase-II (CAMK-II) promote SERCA2a expression.
Area of Science:
- Cardiovascular Biology
- Molecular Cardiology
- Cellular Physiology
Background:
- Sarco-endoplasmic reticulum calcium ATPase 2a (SERCA2a) expression is crucial for cardiac function and its downregulation is linked to pathological myocardial hypertrophy.
- The precise mechanisms by which calcium-dependent signaling and contractile activity regulate myocardial SERCA2a expression are not fully understood.
Purpose of the Study:
- To investigate the distinct roles of calcium-dependent signaling pathways (calcineurin [CN] and calmodulin-dependent protein kinase-II [CAMK-II]) and contractile activity in regulating myocardial SERCA2a expression.
- To dissociate the effects of calcium fluxes from mechanical contraction in cardiomyocytes.
Main Methods:
- Utilized rat neonatal ventricular cardiomyocytes (NVCM) with and without contractile activity (induced by 2,3-butanedione monoxime [BDM]).
- Analyzed SERCA2a mRNA expression via Northern hybridization following treatment with pharmacological inhibitors of L-type Ca2+-channels (verapamil), CN (cyclosporin A; CsA), and CAMK-II (KN-93).
- Assessed nuclear translocation of the CN-dependent transcription factor NFATc and evaluated SERCA2a promoter activity using transfection assays with NFATc4 and myocyte enhancer factor 2c (MEF2c).
Main Results:
- Contractile arrest with BDM significantly increased SERCA2a mRNA levels (2.5-fold), an effect diminished by CsA and KN-93, and abolished by verapamil.
- NFAT nuclear translocation remained unchanged between control and BDM-treated NVCM.
- Co-transfection of NFATc4 and MEF2c synergistically stimulated SERCA2a promoter activity.
Conclusions:
- Calcium-dependent signaling, mediated by CN and CAMK-II, upregulates SERCA2a mRNA expression, likely through synergistic action of NFATc4 and MEF2c on the SERCA2a promoter.
- Active contractile activity in cardiomyocytes exerts an independent inhibitory effect on SERCA2a mRNA expression.
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