Isoenzyme-selective regulation of SERCA2 gene expression by protein kinase C in neonatal rat ventricular myocytes

Michael J. Porter,1 Maria C. Heidkamp,1 Brian T. Scully,1 Nehu Patel,1 Jody L. Martin,1,2 and Allen M. Samarel1,2

The Cardiovascular Institute and Departments of 1Medicine and 2Physiology, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois 60153

Submitted 2 October 2002 ; accepted in final form 25 February 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (PKC{alpha}, PKC{epsilon}, and PKC{delta}), 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 PKC{alpha}, PKC{epsilon}, and PKC{delta} to analyze their individual effects in regulating SERCA2 gene expression in NRVM. Overexpression of wtPKC{epsilon} and wtPKC{delta}, but not wtPKC{alpha}, 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 wtPKC{epsilon} and wtPKC{delta}, respectively; P < 0.05 for each adenovirus; n = 8 experiments). Conversely, overexpression of all three dnPKCs appeared to significantly increase SERCA2 mRNA levels (dnPKC{delta} > dnPKC{epsilon} > dnPKC{alpha}). dnPKC{delta} 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 PKC{epsilon} and PKC{delta} 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.

heart; signal transduction; hypertrophy; transcription; mRNA stability; sarco(endo)plasmic reticulum Ca2+-ATPase


HEART FAILURE (HF) is the final common syndrome of most primary cardiovascular diseases, including coronary atherosclerosis, hypertension, cardiomyopathy, and valvular and congenital heart malformations (17). Despite the varied primary causes of HF, mechanical overload, increased wall stress, and neurohormonal activation are common features that precede the HF state. In response to these mechanical and neurohormonal stimuli, the process of ventricular remodeling is initiated, which is associated with numerous changes in myocardial gene expression and protein turnover. These genotypic alterations have been shown to have profound effects on myocardial performance. In particular, several studies have documented a significant reduction in sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA2) gene expression in ventricular tissue of patients undergoing heart transplantation for ischemic cardiomyopathy and other forms of HF (for review, see Ref. 3). SERCA2 downregulation clearly has functional consequences in the HF state, as even small alterations in the number of SR Ca2+ pumps affect SR Ca2+ loading, myocardial relaxation, and cardiac contractility (25).

Cultured neonatal rat ventricular myocytes (NRVM) have proven to be useful tools in understanding the cellular mechanisms regulating SERCA2 gene expression in response to neurohormonal and mechanical stimuli. For instance, previous studies have shown that SERCA2 gene expression is regulated by peptide growth factors (33), thyroid hormones (24), angiotensin II (28), endothelin-1 (ET) (23), and norepinephrine (9). Studies from our laboratory have shown that mechanical loading of NRVM reduced SERCA2 mRNA and protein levels compared with unloaded cells (4, 8). Importantly, reduced SERCA2 gene expression was reflected functionally by a significant prolongation of the intracellular Ca2+ concentration ([Ca2+]i) transient (4) and a significant reduction in SR pump activity (8) in cellular homogenates of mechanically loaded cells.

Several groups have also begun to evaluate the intracellular signaling pathways that regulate SERCA2 gene expression. At present, the most compelling evidence suggests that the Ras-Raf-MEK-ERK cascade is both necessary and sufficient to downregulate SERCA2 and that one or more isoenzymes of protein kinase C (PKC) may also be involved (27). Indeed, PKC activation was necessary for Ras-GTP loading in response to the hypertrophic agonists phenylephrine and ET (13), and phorbol 12-myristate 13-acetate (PMA), a direct activator of the conventional and novel PKCs, induced Ras activation via stimulation of guanine nucleotide exchange (31). PMA also markedly downregulated SERCA2 mRNA levels in NRVM (21, 23, 36), which could be prevented by the nonselective, PKC inhibitors staurosporine and chelerythrine (36). However, NRVM express three different phorbol ester-sensitive PKC isoenzymes (PKC{alpha}, PKC{epsilon}, and PKC{delta}) (38), which may be differentially regulated and have specific functions in the cardiomyocyte (26). This specificity is likely due to their differential activation by hypertrophic stimuli (5, 16, 35, 43) and their differential localization within the cell (19). Nevertheless, the role of each PKC isoenzyme in the regulation of SERCA2 gene expression remains unknown. Therefore, in this study we have used adenoviral vectors encoding wild-type, constitutively active, and kinase-defective forms of PKC{alpha}, PKC{epsilon}, and PKC{delta} to analyze their individual effects in regulating SERCA2 gene expression in NRVM.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents. PC-1 tissue culture medium was obtained from BioWhittaker (Walkersville, MD). Dulbecco's modified Eagle's medium (DMEM) was obtained from GIBCO BRL (Grand Island, NY). Medium 199, Ca2+-free, Mg2+-free (modified) Hanks' balanced salt solution (HBSS), acid-soluble calf skin collagen, and antibiotic/antimycotic solution were obtained from Sigma Chemical (St. Louis, MO). [32P]dCTP was purchased from Amersham Biosciences (Arlington Heights, IL). Monoclonal antibodies to PKC{alpha}, PKC{epsilon}, and PKC{delta} were obtained from Signal Transduction Laboratories (Lexington, KY). Horseradish peroxidase-conjugated goat anti-mouse IgG was from Bio-Rad (Hercules, CA). Real-time RT-PCR reagents were obtained from Amersham Biosciences, and Invitrogen (Carlsbad, CA). cDNA primers and probes were obtained from Integrated DNA Technologies (Coralville, IA). All other reagents were of the highest grade commercially available and were obtained from Sigma or Baxter S/P (Mc-Gaw Park, IL).

Cell culture. Animals used in these experiments were handled in accordance with the "Guiding Principles in the Care and Use of Animals," approved by the Council of the American Physiological Society. Ventricular myocytes were isolated from the hearts of 2-day-old Sprague-Dawley rats by collagenase digestion, as previously described (40). Myocytes were preplated for 1 h in serum-free PC-1 medium to reduce nonmyocyte contamination. The nonadherent NRVM were then plated at a density of 1,600 cells/mm2 onto collagen-coated 35- or 60-mm dishes and left undisturbed in a 5% CO2 incubator for 14–18 h. Unattached cells were removed by aspiration and washed twice in HBSS, and the attached cells were maintained in a solution of DMEM/medium 199 (4:1) containing antibiotic/antimycotic solution. Under these highdensity culture conditions, NRVM displayed synchronous [Ca2+]i transients and beating activity (~100–150 beats/min) within 24 h of plating. Cardiomyocytes were infected (60 min, 25°C with gentle agitation) with replication-defective adenoviruses (Adv) diluted in DMEM/medium 199. This medium was then replaced with virus-free DMEM/medium 199, and the cells were cultured for an additional 48–72 h.

Adenoviral constructs. Replication-defective adenoviruses encoding wild-type (wt) bovine PKC{alpha}, rat PKC{epsilon}, and rat PKC{delta} were constructed by first subcloning their respective cDNAs (kindly provided by Drs. Peter Parker and Peter Sugden, Imperial College of Science Technology and Medicine, Cambridge, UK) into pAC-CMV-pLpA-SR plasmid. The subcloned constructs were cotransfected along with pJM17 plasmid that contained adenoviral DNA into HEK-293 cells. After homologous recombination, the adenoviruses were plaque-purified, amplified by sequential infection of HEK-293 cells, and purified by double CsCl ultracentrifugation. Replication-defective adenoviruses encoding constitutively active (ca) PKC{epsilon} and PKC{delta} were generated as previously described (26, 42). Replication-defective adenoviruses encoding dominant negative (dn) mouse PKC{alpha} (10), rat PKC{delta} (10), and rabbit PKC{epsilon} (34) were obtained from Drs. Trevor Biden (Garvan Institute of Medical Research, St. Vincents Hospital, Darlinghurst, Sydney, Australia) and Peipei Ping (Dept. of Physiology, Univ. of California, Los Angeles, CA) and were amplified and purified. Finally, replication-defective adenoviruses encoding cytoplasmic (cyto) or nuclear-encoded (ne) {beta}-galactosidase ({beta}gal) were used to control for nonspecific effects of adenoviral infection (20). The multiplicity of viral infection (MOI) of each adenovirus was determined by dilution assay in HEK-293 cells.

Western blotting. NRVM were homogenized in lysis buffer (41). Equal amounts of extracted proteins (50 µg) were separated on 10% SDS-polyacrylamide gels with 5% stacking gels. Proteins were electrophoretically transferred to nitrocellulose membranes, and the Western blots were probed with antibodies specific for PKC{alpha}, PKC{epsilon}, and PKC{delta}. Primary antibody binding was detected with horseradish peroxidase-conjugated goat anti-mouse secondary antibody and visualized by enhanced chemiluminescence (ECL: Amersham).

mRNA analysis. Total cellular RNA was isolated by the method of Chomczynski and Sacchi (14) or by using the RNeasy mini kit (Qiagen, Valencia, CA). RNA was quantified by absorbance at 260 nm, and its integrity was determined by examining the 28S and 18S rRNA bands in ethidium bromide-stained agarose gels. Both extraction methods produced similar yields of undegraded, purified RNA. SERCA2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNAs were then analyzed by either Northern blotting (8) or real-time RT-PCR. For Northern blots, equal amounts of total RNA (10 µg) were separated by denaturing agarose gel electrophoresis. Blots were then sequentially hybridized with a 2.3-kb cDNA probe specific for rat SERCA2, kindly provided by Dr. Wolfgang Dillmann (University of California, San Diego), and a 1.2-kb cDNA probe specific for human GAPDH, clone pHcGAP (46) obtained from the American Type Culture Collection (Rockville, MD). In some Northern blotting experiments, the amounts of SERCA2 mRNA were quantified by scintillation spectroscopy (Instant Imager, Hewlett-Packard) and expressed relative to the amounts of GAPDH mRNA in each sample. To ensure equal loading of the gels, and also to verify that the various interventions had no effect on the "bystander transcript" GAPDH mRNA used in the real-time RT-PCR assay, some Northern blots were also probed with a 32P-labeled oligonucleotide probe specific for 18S rRNA (8).

For the real-time RT-PCR, cDNA was reverse-transcribed from the extracted RNA from a reaction mixture consisting of 5x First Strand buffer (5 µl), 0.1 M DTT (2 µl), pd(N)6 random hexamers (2 µl), 10 µM dNTPs (2 µl), RNase inhibitor (1 µl), and reverse transcriptase (1 µl). A volume equivalent to 1 µg of RNA was used per sample, and RNase-free diethyl pyrocarbonate (DEPC)-treated water was added to bring the final volume of the reaction mixture to 20 µl. The sample was heated to 42°C for 30 min, followed by 95°C for 5 min. The resultant cDNA was then stored at –80°C. All real-time PCR was performed with a Bio-Rad iCycler iQ Multi-Color real-time PCR detection system. Sample 96-well plates were loaded with 50 µl of reaction mixture per well. The mixture consisted of 1 µl of sample DNA, 21 µl of DEPC water, 25 µl of Platinum Quantitative PCR SuperMix-UDG, and 3 µl of a primer/dual-labeled probe combination specific for each gene of interest. TaqMan rodent GAPDH control reagents were obtained from Applied Biosystems (Foster City, CA). For the rat SERCA2 cDNA, the following primers were used: 5'-TCT GTC ATT CGG GAG TGG GG-3' and 5'-GCC CAC ACA GCC AAC GAA AG-3'. The rat SERCA2 cDNA fluorescent probe consisted of the following sequence: 5'-TGG CCA CTC ATG ACA ACC CG-3'.

Probes were labeled at the 5'-end with 6-carboxyfluorescein (6-FAM) and at the 3'-end with BHQ-1. Rat SERCA2 primer concentrations were 10 µM, and the probe concentration was 1 µM. Rodent GAPDH primer concentrations were 10 µM, and the probe concentration was 5 µM. PCR amplification was performed by cycling between 95 (15 s) and 60°C (60 s) for 45 cycles, using the 6-FAM fluorophore for quantification. All samples were run in triplicate, and the results were averaged. After the PCR, mRNA levels were expressed in threshold cycles (Ct) by the iCycler. The average Ct was then converted into an "input amount" by using a standard curve derived from serial dilutions of SERCA2 or GAPDH cDNA. For each plate in the experiment, the input amount for the gene of interest was standardized first to rat GAPDH mRNA and then to the appropriate adenovirus control (Adv-ne{beta}gal for the wtPKC overexpression studies, Adv-cyto{beta}gal for the dnPKC overexpression studies) derived from the same experiment.

Data analysis. Results are expressed as means ± SE. Normality was assessed using the Kolmogorov-Smirnov test, and homogeneity of variance was assessed using Levene's test. Data from multiple groups were compared by 1-way blocked analysis of variance (ANOVA) or 1-way blocked ANOVA on ranks followed by Dunnett's test, where appropriate. Data from two groups were compared by paired t-test or Wilcoxon signed rank test where appropriate. Differences among means were considered significant at P < 0.05. Data were analyzed using the SigmaStat statistical software package (ver. 1.0; Jandel Scientific, San Rafael, CA).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of ET, PMA, and Adv-wtPKCs on endogenous PKC levels. Initial experiments were conducted to ascertain the effects of ET and PMA on endogenous PKC isoenzyme levels over time. As shown in Fig. 1A, continuous exposure of NRVM to ET (100 nM, 48 h) had no effect on PKC{alpha} levels but partially reduced PKC{epsilon}, and to a lesser extent, PKC{delta}. In marked contrast, continuous exposure of cells to PMA (200 nM, 48 h) virtually eliminated PKC{alpha}, PKC{delta}, and PKC{epsilon} from the cells. Thus, although both agents acutely activated one or more PKC isoenzymes, only PMA completely downregulated them over time.



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Fig. 1. Effects of endothelin-1 (ET), phorbol 12-myristate 13-acetate (PMA), and wild-type protein kinase C (wtPKC) overexpression on endogenous PKC protein levels. A: neonatal rat ventricular myocytes (NRVM) were maintained in control medium or continuously treated with ET (100 nM, 48 h) or PMA (200 nM, 48 h). Cells were harvested, and equal amounts of extracted cellular protein (50 µg) were separated by SDS-polyacrylamide gel electrophoresis and Western blotting (WB). Blots were then probed with antibodies specific for PKC{alpha}, PKC{epsilon}, and PKC{delta}. The position of molecular mass markers is indicated to the right of each blot. B: NRVM were maintained in serumfree growth medium and then infected with adenovirus encoding nuclear-encoded {beta}-galactosidase (Adv-ne{beta}gal), Adv-wtPKC{alpha}, Adv-wtPKC{epsilon}, or Adv-wtPKC{delta} (25 MOI, 48 h). Cells were harvested and processed for Western blotting as described for A. MOI, multiplicity of infection.

 

We next examined the effects of Adv-mediated overexpression of each wtPKC on the expression levels of all of the phorbol ester-sensitive PKC isoenzymes expressed in NRVM. As shown in Fig. 1B, Adv-wtPKC{alpha} (25 MOI, 48 h) increased expression levels of PKC{alpha} 10- to 20-fold but had no significant effect on the expression of endogenous PKC{epsilon} or PKC{delta}. Similarly, Adv-wtPKC{epsilon} (25 MOI, 48 h) markedly increased PKC{epsilon} levels but also substantially increased PKC{alpha} in NRVM. Endogenous PKC{delta} levels, however, were unaffected by wtPKC{epsilon} overexpression. In marked contrast, overexpression of wtPKC{delta} (25 MOI, 48 h) substantially decreased endogenous PKC{alpha} and PKC{epsilon} levels compared with cells infected with Adv-ne{beta}gal. PKC{alpha}, PKC{epsilon}, and PKC{delta} levels in Adv-ne{beta}gal-infected NRVM (25 MOI, 48 h) were all similar to those observed in uninfected control NRVM (data not shown), indicating that adenovirus infection alone was not responsible for these changes.

Overexpression of the novel PKC isoenzymes downregulates SERCA2 mRNA levels. We then examined the effects of ET, PMA, and the individual Adv-wtPKCs on SERCA2 mRNA levels. As previously described (36), activation of all three phorbol ester-sensitive PKC isoenzymes with PMA (200 nM, 48 h) substantially reduced SERCA2 mRNA levels as assessed by Northern blotting (Fig. 2A). ET (100 nM, 48 h), a potent activator of PKC{epsilon}, and to a lesser extent, PKC{delta} (16), also caused SERCA2 downregulation, although its effect was somewhat less than that observed with PMA (Fig. 2B). Neither agent substantially affected the expression of GAPDH mRNA, because the expression of this transcript did not systematically change relative to the amount of total RNA or 18S rRNA.



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Fig. 2. Effects of ET, PMA, and wtPKC overexpression on sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) mRNA levels. NRVM were maintained under control conditions or treated with PMA (200 nM, 48 h) (A) or ET (100 nM, 48 h) (B). Total RNA (10 µg/lane) was size-fractionated on 1% agarose gels, and the resulting Northern blots were sequentially probed with 32P-labeled cDNA or oligonucleotide probes specific for SERCA2 and GAPDH mRNAs and 18S rRNA. C: NRVM were infected (25 MOI, 48 h) with Adv-ne{beta}gal, Adv-wtPKC{alpha}, Adv-wtPKC{epsilon}, or Adv-wtPKC{delta}. Northern blots were prepared as described for A and B.

 

We next evaluated the effects of the Adv-wtPKCs on SERCA2 mRNA levels. First, we found that the control adenovirus Adv-ne{beta}gal (25 MOI, 48 h) had no significant effect on SERCA2 mRNA levels compared with uninfected NRVM, indicating that adenovirus infection alone at this MOI did not produce nonspecific effects on SERCA2 gene expression (data not shown). Adv-wtPKC{alpha} (25 MOI, 48 h) was also without effect and in some experiments actually appeared to increase SERCA2 mRNA levels compared with Adv-ne{beta}gal (Fig. 2C). In contrast, Adv-wtPKC{delta} (25 MOI, 48 h), and to a lesser extent, Adv-wtPKC{epsilon} (25 MOI, 48 h), reduced steady-state levels of SERCA2 mRNA. None of the adenoviruses substantially affected the expression of GAPDH mRNA, because the expression of this transcript did not systematically change relative to the amount of total RNA or 18S rRNA.

To obtain a more sensitive and precise assessment of these relatively modest changes in SERCA2 mRNA, the effects of each adenovirus on SERCA2 mRNA levels (relative to the invariant GAPDH mRNA) were then quantitatively analyzed by real-time RT-PCR, and the results are depicted in Fig. 3. Adv-wtPKC{alpha} had no significant effect on SERCA2 mRNA levels compared with NRVM infected with Adv-ne{beta}gal. However, wtPKC{epsilon} and wtPKC{delta} overexpression reduced SERCA2 mRNA levels to 69 ± 7 and 61 ± 9% of control levels, respectively (P < 0.05 for each adenovirus; n = 9 experiments). The overall reduction in SERCA2 mRNA levels by overexpression of each novel PKC isoenzyme (30–40%) was somewhat less than the level of downregulation observed with PMA (55–60% reduction) (36) but was similar to that observed with ET (23) (Fig. 2B). Thus overexpression of either novel wtPKC was sufficient to downregulate SERCA2 gene expression in NRVM.



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Fig. 3. wtPKC{epsilon} and wtPKC{delta} overexpression downregulate SERCA2 mRNA. NRVM were infected with Adv-ne{beta}gal and Adv-wtPKCs as described in Fig. 2. Total RNA was then reverse-transcribed into cDNA, and real-time RT-PCR was performed by using 1 µg of cDNA from each sample. Oligonucleotide primers and dual-labeled fluorogenic probes specific for rat SERCA2 or rodent GAPDH were used. Relative quantities of amplified SERCA2 cDNA from each sample were standardized to GAPDH and then normalized to levels in Adv-ne{beta}gal-infected cells. Data are means ± SE; n = 8–9 experiments. *P < 0.05 vs. Adv-ne{beta}gal.

 

As a further check on the effects of PKC{epsilon} and PKC{delta} overexpression on SERCA2 mRNA levels, NRVM were also infected (10 MOI, 48 h) with replication-defective adenoviruses expressing constitutively active mutant forms of these isoenzymes. [PKC{delta} and PKC{epsilon} were rendered constitutively active by an amino acid deletion or a point mutation within their respective pseudosubstrate domains (49).] As shown in Fig. 4, overexpression of either novel caPKC isoenzyme was also sufficient to downregulate SERCA2 mRNA levels compared with uninfected or Adv-ne{beta}gal-infected cells.



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Fig. 4. Constitutively active (ca) PKC{epsilon} and caPKC{delta} overexpression downregulate SERCA2 mRNA. A: NRVM were maintained in control medium (uninfected) or infected (10 MOI, 48 h) with Adv-ne{beta}gal, Adv-caPKC{epsilon}, or Adv-caPKC{delta}. Total RNA (10 µg/lane) was size-fractionated on 1% agarose gels, and the resulting Northern blots were sequentially probed with 32P-labeled cDNA probes specific for SERCA2 and GAPDH mRNAs. B: quantitative analysis of 4 Northern blotting experiments, in which the SERCA2/GAPDH mRNA ratio was determined by scintillation spectroscopy and normalized to the ratio obtained in uninfected cells. Data are means ± SE. *P < 0.05 vs. uninfected cells. +P < 0.05 vs. Adv-ne{beta}gal.

 

Effects of Adv-dnPKC{alpha}, Adv-dnPKC{epsilon}, and Adv-dnPKC{delta} on PKC levels. Initial experiments were also conducted to ascertain the effects of adenovirus-mediated overexpression of each dnPKC on the expression levels of all of the phorbol ester-sensitive PKC isoenzymes. As shown in Fig. 5, Adv-dnPKC{alpha} (100 MOI, 72 h) increased immunoreactive PKC{alpha} but also reduced expression levels of endogenous PKC{epsilon}, and to a much lesser extent, PKC{delta}. Similarly, overexpression of dnPKC{epsilon} (100 MOI, 72 h) reduced endogenous PKC{alpha} expression but did not substantially affect endogenous PKC{delta}. Finally, overexpression of dnPKC{delta} (10 MOI, 72 h) increased the amount of immunoreactive PKC{delta} in NRVM but did not appear to substantially affect either endogenous PKC{alpha} or PKC{epsilon} levels. [Reduced MOI for dnPKC{delta} was used in this and subsequent experiments because higher concentrations caused cell detachment within 48–72 h, presumably as a consequence of apoptosis (22, 26).] As was noted above for Adv-ne{beta}gal, Adv-cyto{beta}gal (10–100 MOI, 72 h) also had no apparent effect on endogenous PKC{alpha}, PKC{epsilon},orPKC{delta} expression compared with uninfected control NRVM (data not shown).



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Fig. 5. Effects of dominant negative (dn) Adv-PKCs on PKC protein levels. NRVM were maintained in serum-free growth medium and then infected with Adv-cyto{beta}gal (10–100 MOI, 72 h), Adv-dnPKC{alpha} (100 MOI, 72 h), Adv-dnPKC{epsilon} (100 MOI, 72 h), or Adv-dnPKC{delta} (10 MOI, 72 h). Cells were harvested, and equal amounts of extracted cellular protein (50 µg) were separated by SDS-polyacrylamide gel electrophoresis and Western blotting. Blots were then probed with antibodies specific for PKC{alpha}, PKC{epsilon}, and PKC{delta}. The position of molecular mass markers is indicated to the right of each blot.

 

Effects of Adv-dnPKC{alpha}, Adv-dnPKC{epsilon}, and Adv-dnPKC{delta} on SERCA2 mRNA levels. As shown in Fig. 6, analysis of SERCA2 mRNA levels by Northern blotting indicated that overexpression of dnPKC{alpha} (100 MOI, 72 h), dnPKC{epsilon} (100 MOI, 72 h), or dnPKC{delta} (10 MOI, 72 h) appeared to increase SERCA2 mRNA levels compared with NRVM infected with Adv-cyto{beta}gal (100 MOI, 72 h). However, PMA (200 nM) added to the culture medium 24 h after adenovirus infection was still capable of downregulating SERCA2 mRNA in NRVM infected with each adenovirus. SERCA2 mRNA levels were then quantitatively analyzed by real-time RT-PCR, and the results are depicted in Fig. 7. All three dnPKCs significantly increased SERCA2 mRNA levels over time (dnPKC{delta} > dnPKC{epsilon} > dnPKC{alpha}). Adv-dnPKC{delta} produced the largest increase in SERCA2 gene expression (2.8 ± 1.0-fold; n = 11 experiments). As shown by Northern blotting, however, PMA treatment was still sufficient to significantly downregulate SERCA2 mRNA levels despite overexpression of each dominant negative mutant.



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Fig. 6. Effects of Adv-dnPKCs on SERCA2 gene expression. Representative Northern blot shows SERCA2 mRNA levels in cultured NRVM infected with Adv-cyto{beta}gal (100 MOI, 72 h), Adv-dnPKC{alpha} (100 MOI, 72 h), Adv-dnPKC{epsilon} (100 MOI, 72 h), or Adv-dnPKC{delta} (10 MOI, 72 h) with (+) and without (–) treatment with PMA (200 nM, 48 h). After cells were harvested, 10 µg of RNA from each sample were separated on a 1% denaturing agarose gel, and the resulting Northern blot was sequentially hybridized with 32P-labeled cDNA or oligonucleotide probes specific for SERCA2 and GAPDH mRNAs and 18S rRNA.

 


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Fig. 7. dnPKC overexpression upregulates SERCA2 mRNA levels. NRVM were infected with Adv-cyto{beta}gal (100 MOI, 72 h), Adv-dnPKC{alpha} (100 MOI, 72 h), Adv-dnPKC{epsilon} (100 MOI, 72 h), or Adv-dnPKC{delta} (10 MOI, 72 h) and treated with or without PMA (200 nM, 48 h). Total RNA was isolated and then reverse-transcribed into cDNA. Real-time RT-PCR was performed by using 1 µg of cDNA from each sample. Oligonucleotide primers and dual-labeled fluorogenic probes specific for rat SERCA2 or rodent GAPDH were used. Relative quantities of amplified SERCA2 cDNA from each sample were standardized to GAPDH and then normalized to levels in Adv-cyto{beta}gal-infected cells. Data are means ± SE; n = 10–13 experiments. *P < 0.05 vs. Adv-cyto{beta}gal for the effect of each Adv-dnPKC on basal SERCA2 mRNA levels. +P < 0.05 vs. respective untreated control cultures for the effect of PMA.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is now abundantly clear that the hypertrophic responses to neurohormonal and mechanical stimulation of NRVM in vitro and to hemodynamic overload of the intact ventricular myocardium in vivo share common signaling mechanisms (for review, see Ref. 30). A central role for PKC activation in these signaling pathways has been confirmed in transgenic experiments in which individual PKC isoenzymes or their activators have been overexpressed, resulting in selective features of cardiomyocyte hypertrophy, and in some instances, HF (6, 11, 44, 48). However, few studies have addressed how individual PKC isoenzymes may mediate specific alterations in gene expression that accompany cardiac hypertrophy and its transition to HF. In this report, we provide the first evidence indicating that specific PKC isoenzymes selectively regulate SERCA2 gene expression in cardiomyocytes.

Previous studies have demonstrated that NRVM stimulated with hypertrophic agonists (9, 23, 28, 32), PMA (21, 23, 36), or cyclic mechanical loading (8) undergo hypertrophy, and this growth response was accompanied by reduced SERCA2 gene expression. Conversely, we previously showed that mechanical unloading produced cardiomyocyte atrophy, which was accompanied by significant upregulation of SERCA2 mRNA and protein levels (4). One question raised by these studies was whether SERCA2 gene expression could be regulated independently of the hypertrophic response. However, we and others recently showed that adenovirally mediated overexpression of either PKC{epsilon} (7, 26, 42) or PKC{delta} (7, 26) was not sufficient to induce cardiomyocyte hypertrophy but, as demonstrated in the present report, was sufficient to downregulate SERCA2 mRNA levels. Conversely, overexpression of PKC{alpha} was sufficient to induce many features of NRVM hypertrophy in the absence of other stimuli (7) but was not sufficient to downregulate SERCA2 mRNA levels. These data therefore provide new evidence for isoenzyme-selective regulation of SERCA2 gene expression by PKC and also indicate that this regulation can occur independently of generalized effects on NRVM growth or atrophy. Nevertheless, a limitation of all of these adenoviral studies (including our own) is the lack of a documented dose-response relationship between levels of PKC overexpression, their impact on isoenzyme activation and translocation, and their effects on cardiomyocyte gene expression and growth. This is of some concern, because overexpressing PKC isoenzymes at different levels could conceivably trigger dichotomous phenotypes.

Our data demonstrating that both ET and PMA significantly reduced SERCA2 mRNA levels imply that these ET- and PMA-dependent effects were mediated by the activation of endogenous PKC{epsilon} and/or PKC{delta}. However, chronic exposure to phorbol esters is well known to induce activation, followed by downregulation of PKCs by intracellular proteolysis. Indeed, we demonstrate in Fig. 1A that chronic exposure to PMA substantially reduced endogenous PKC{alpha}, PKC{delta}, and PKC{epsilon} during 48 h of continuous exposure to the drug. However, in our previous paper (36), we compared SERCA2 mRNA levels in NRVM stimulated with PMA for only 30 min vs. cells that were treated continuously for up to 48 h and showed that continuous exposure to PMA was not necessary to reduce SERCA2 mRNA. In fact, the stimulus for SERCA2 mRNA downregulation was generated within 30 min of PMA exposure, but the reduction in SERCA2 mRNA took 12–24 h to become manifested. These results are consistent with a PKC-dependent signaling pathway in which only transient activation of PKC{delta} and/or PKC{epsilon} is required to initiate a cascade of events that ultimately leads to reduced SERCA2 mRNA. In other words, there must be a temporal element in the PKC stimulatory pathway that persists even after the PKC isoenzymes are downregulated during chronic PMA exposure. In further support of this conclusion is the observation that although ET acutely activated PKC{epsilon} and PKC{delta}, chronic ET treatment only partially reduced PKC{delta} and PKC{epsilon} protein levels over 48 h. It is worthwhile to mention, however, that agonist-induced reductions in individual PKC isoenzyme levels are best detected when protein samples are partitioned between soluble and particulate fractions, as shown by Sabri et al. (39).

Similarly, the specificity of Adv-wtPKC overexpression may have also been affected by the various compensatory changes in endogenous PKC isoenzyme expression that occurred over time. As demonstrated in Fig. 1B, adenovirally mediated wtPKC{delta} overexpression markedly reduced the expression levels of endogenous PKC{alpha} and PKC{epsilon}. Furthermore, we have previously shown that overexpression of caPKC{epsilon} increased the expression of endogenous PKC{delta}, whereas overexpression of caPKC{delta} markedly reduced PKC{epsilon} levels (26). In addition, PKC{delta} and PKC{epsilon} activities are also regulated by transphosphorylation within their respective hydrophobic and activation loop domains, providing another potential mechanism of cross talk between the various PKC isoenzymes (37). Therefore, it is conceivable that the loss of signals generated by endogenous PKC{alpha} and PKC{epsilon} might have contributed in some way to the observed reduction in SERCA2 mRNA. However, it should be pointed out that overexpression of dnPKC{alpha} and dnPKC{epsilon} increased, rather than decreased, SERCA2 mRNA levels, suggesting that the effect of wtPKC{delta} overexpression was due to a direct effect of PKC{delta} on the signaling pathways that regulate SERCA2 gene expression. In addition, overexpression of dnPKC{epsilon} substantially reduced endogenous PKC{alpha} but also increased (rather than decreased) SERCA2 mRNA, suggesting that the effects of the PKC{epsilon} adenoviruses were also direct.

As demonstrated in Figs. 6 and 7, SERCA2 gene expression under "basal" conditions appeared to be regulated by one or more PKC isoenzymes, because selective inhibition of each PKC increased SERCA2 mRNA levels in spontaneously contracting NRVM. dnPKC{delta} overexpression had by far the greatest quantitative effect in upregulating SERCA2 mRNA. The present results confirm our previous data, which showed that staurosporine and chelerythrine increased SERCA2 mRNA levels in spontaneously contracting NRVM (36). However, our previous results need to be interpreted with caution, especially in light of new information regarding the specificity and selectivity of these PKC inhibitors in cultured cells. For instance, staurosporine, even at the low concentration (10 nM) used in our previous study, inhibits many other signaling kinases (15), and chelerythrine may in fact have no inhibitory activity at all against PKC isoenzymes (18). Both agents activated the stress-activated protein kinases in NRVM (26) and induced apoptosis (26, 50). Indeed, these facts, along with the potential for selective inhibition of specific PKC isoenzymes, led to our use of the dnPKC adenoviruses in this system. However, it should be pointed out that dnPKC{epsilon} and dnPKC{alpha} overexpression also reduced expression levels of endogenous PKC{alpha} and PKC{epsilon}, respectively. These somewhat surprising results may explain why dnPKC{alpha} significantly increased basal SERCA2 mRNA levels, i.e., via an indirect effect on endogenous PKC{epsilon}. Future studies, including the use of isoenzymeselective, cell-permeant peptide inhibitors of PKC translocation, may help to clarify these issues (11).

In addition to the dnPKC mutants, we previously showed that inhibition of contractile activity (by blockade of Ca2+ influx through voltage-gated, L-type Ca2+ channels) also upregulated SERCA2 mRNA levels (4). We also found that a substantial proportion of endogenous PKC{delta} was found in the membrane fraction of quiescent NRVM (43), suggesting that this PKC isoenzyme is substantially activated even under basal conditions. Electrical stimulation of contraction caused the rapid translocation of additional PKC{delta}, along with PKC{epsilon}, from the cytoplasm to a Triton X-100-soluble membrane fraction (43). These data suggest that the regulation of SERCA2 gene expression by [Ca2+]i transients and contractile activity may also be dependent on activation of one or both of the novel PKC isoenzymes.

Although we provide evidence that PKC{epsilon} and PKC{delta} are both sufficient to downregulate SERCA2 mRNA, our data indicate that neither PKC alone is necessary for this effect if the other novel PKC can be activated. A similar redundancy in function has been demonstrated recently by Chen et al. (12) in transgenic mice overexpressing activator peptides of PKC{delta} and PKC{epsilon} in ventricular myocytes. Both transgenic lines demonstrated similar degrees of cardiomyocyte hypertrophy and upregulation of {beta}-myosin heavy chain and atrial natriuretic factor gene expression. However, the two lines differed dramatically with respect to their susceptibility to ischemic injury, indicating that PKC{delta} and PKC{epsilon} share common as well as distinct signaling functions in the heart.

In this regard, it is interesting to speculate on what signaling pathways may be operative downstream of PKC{epsilon} and PKC{delta} and how they may affect SERCA2 gene expression. First, there is evidence to indicate that SERCA2 is regulated at both the transcriptional (1, 2, 24, 45, 47) and posttranscriptional (29, 36) levels, so it is conceivable that the novel PKCs differentially influence SERCA2 mRNA levels by affecting the rate of transcription vs. posttranscriptional processing and stability of the SERCA2 mRNA. Second, there is also some evidence to suggest that PKC{delta} and PKC{epsilon} may differentially influence SERCA2 gene expression by activation of different MAPK cascades. Ho et al. (27) provide convincing evidence that SERCA2 expression in NRVM is regulated by the Ras-Raf-MEK-ERK cascade. The same group has recently indicated that the MKK6-p38MAPK pathway also reduces the activity of the rat SERCA2 promoter (1), leading to reduced SERCA2 mRNA and protein levels and slowed relaxation of the [Ca2+]i transient. Our laboratory has recently shown that adenovirally mediated overexpression of PKC{epsilon} predominantly activated ERKs, whereas PKC{delta} overexpression predominantly activated JNKs and p38MAPK under culture conditions identical to those used in the present experiments (26). These data are consistent with dual regulation of SERCA2 gene expression by different MAPK cascades at either the transcriptional or posttranscriptional level (36). Future experiments are necessary to identify the specific targets of novel PKC phosphorylation and to determine exactly how the SERCA2 gene is regulated during hypertrophy and HF.


    ACKNOWLEDGMENTS
 
We thank Alan G. Ferguson and Erika Szotek for excellent technical assistance.

These studies were supported by National Heart, Lung, and Blood Institute Grants R01-HL-34328 and R01-HL-63711 and by a gift to the Cardiovascular Institute from the Ralph and Marian Falk Trust for Medical Research. M. C. Heidkamp was the recipient of an National Institutes of Health National Research Service Award (F3-HL-68476), and Dr. Martin was the recipient of a James Beck/Patrick Scanlon, M.D. Scientist Development Award during the time these studies were performed.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. M. Samarel, The Cardiovascular Institute, Loyola Univ. Medical Center, Bldg. 110, Rm. 5222, 2160 South First Ave., Maywood, IL 60153 (E-mail: asamare{at}lumc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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