Contractile arrest reveals calcium-dependent stimulation of SERCA2a mRNA expression in cultured ventricular cardiomyocytes.
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.


CONCLUSIONS
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.
Laboratory for Physiology, Institute for Cardiovascular Research (ICaR-VU), VU University Medical Center, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands.
Ronald
R
Vlasblom
Alice
A
Muller
René J P
RJ
Musters
Marian J
MJ
Zuidwijk
Cornelis
C
Van Hardeveld
Walter J
WJ
Paulus
Warner S
WS
Simonides
D000818:Animals; D015152:Blotting, Northern / Q000379:methods; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000235:genetics*; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D003931:Diacetyl / Q000031:analogs & derivatives* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation; D006352:Heart Ventricles; D064326:MEF2 Transcription Factors; D032383:Myocytes, Cardiac / Q000378:metabolism*; D018006:Myogenic Regulatory Factors / Q000235:genetics; D050778:NFATC Transcription Factors; D009419:Nerve Tissue Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection / Q000379:methods; D014700:Verapamil / Q000494:pharmacology
D002121:Calcium Channel Blockers; D004791:Enzyme Inhibitors; D064326:MEF2 Transcription Factors; D018006:Myogenic Regulatory Factors; D050778:NFATC Transcription Factors; D009419:Nerve Tissue Proteins; C477593:Nfatc4 protein, rat; D012333:RNA, Messenger; D014157:Transcription Factors; C004717:diacetylmonoxime; D014700:Verapamil; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D003931:Diacetyl; D002118:Calcium