Isoenzyme-selective regulation of SERCA2 gene expression by protein kinase C in neonatal rat ventricular myocytes.
Patients with cardiac hypertrophy and heart failure display abnormally slowed myocardial relaxation, which is associated with downregulation of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) gene expression. We previously showed that SERCA2 downregulation can be simulated in cultured neonatal rat ventricular myocytes (NRVM) by treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA). However, NRVM express three different PMA-sensitive PKC isoenzymes (PKCalpha, PKCepsilon, and PKCdelta), which may be differentially regulated and have specific functions in the cardiomyocyte. Therefore, in this study we used adenoviral vectors encoding wild-type (wt) and kinase-defective, dominant negative (dn) mutant forms of PKCalpha, PKCepsilon, and PKCdelta to analyze their individual effects in regulating SERCA2 gene expression in NRVM. Overexpression of wtPKCepsilon and wtPKCdelta, but not wtPKCalpha, was sufficient to downregulate SERCA2 mRNA levels, as assessed by Northern blotting and quantitative, real-time RT-PCR (69 +/- 7 and 61 +/- 9% of control levels for wtPKCepsilon and wtPKCdelta, respectively; P < 0.05 for each adenovirus; n = 8 experiments). Conversely, overexpression of all three dnPKCs appeared to significantly increase SERCA2 mRNA levels (dnPKCdelta > dnPKCepsilon > dnPKCalpha). dnPKCdelta overexpression produced the largest increase (2.8 +/- 1.0-fold; n = 11 experiments). However, PMA treatment was still sufficient to downregulate SERCA2 mRNA levels despite overexpression of each dominant negative mutant. These data indicate that the novel PKC isoenzymes PKCepsilon and PKCdelta selectively regulate SERCA2 gene expression in cardiomyocytes but that neither PKC alone is necessary for this effect if the other novel PKC can be activated.
Porter
Michael J
MJ
The Cardiovascular Institute and Department of Medicine, Loyola University Chicago Stritch School of Medicine, Maywood, IL 60153, USA.
Heidkamp
Maria C
MC
Scully
Brian T
BT
Patel
Nehu
N
Martin
Jody L
JL
Samarel
Allen M
AM
D000818:Animals; D000831:Animals, Newborn; D000252:Calcium-Transporting ATPases / Q000235:genetics* / Q000378:metabolism; D002273:Carcinogens / Q000494:pharmacology; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D019332:Endothelin-1 / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D006352:Heart Ventricles / Q000166:cytology / Q000201:enzymology; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000201:enzymology*; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D015398:Signal Transduction / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
C506948:Atp2a2 protein, rat; D002273:Carcinogens; D019332:Endothelin-1; D007527:Isoenzymes; D012333:RNA, Messenger; C497260:Prkcd protein, rat; C497257:Prkce protein, rat; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D013755:Tetradecanoylphorbol Acetate