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Cardiovascular Research 2003 59(1):46-56; doi:10.1016/S0008-6363(03)00329-8
© 2003 by European Society of Cardiology
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Copyright © 2003, European Society of Cardiology

The MKK6–p38 MAPK pathway prolongs the cardiac contractile calcium transient, downregulates SERCA2, and activates NF-AT

Catherine Andrewsa, Peter D. Hob, Wolfgang H. Dillmannc, Christopher C. Glembotskib and Patrick M. McDonoughc,*

aGenetics Cell Biology/Development, University of Minnesota, 1445 Gortner Ave, St. Paul, MN 55108, USA
bDepartment of Biology, the SDSU Heart Institute, and the Molecular Biology Institute, San Diego State University, San Diego, CA 92182, USA
cDepartment of Medicine, University of California, San Diego, Mail code 0618, 9500 Gilman Drive, La Jolla, CA 92093-0618, USA

pmcdonough{at}ucsd.edu

* Corresponding author. Tel.: +1-858-534-9938.

Received 14 May 2002; accepted 13 February 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Our goal was to determine if the MKK6–p38 MAPK pathway regulates cardiac intracellular calcium ([Ca2+]i). We also tested if MKK6 might influence expression of SERCA2, a calcium regulatory molecule involved in relaxation, and the activity of nuclear factor of activated T-cells (NF-AT), a calcium-regulated transcription factor that participates in pathological responses to pressure-overload. Methods: Neonatal rat ventricular myocytes were transfected with MKK6(Glu), an activator of p38 MAPK. Green fluorescent protein (GFP) was used as transfection marker and [Ca2+]i was evaluated via indo-1. SERCA2 expression was assayed via Northern and Western techniques. The activity of the rat SERCA2 gene promoter and NF-AT-dependent gene expression were monitored with reporter genes. Myocyte contractility was regulated by electrical pacing. Results: MKK6(Glu) prolonged decay of the contractile calcium transients, downregulated SERCA2 expression, and reduced the activity of the rat SERCA2 gene promoter. Diastolic [Ca2+]i in myocytes pacing at 1–2 Hz was dramatically increased by MKK6(Glu). NF-AT-dependent gene expression was activated by MKK6(Glu) and by pacing of contractions in a synergistic manner. Overexpression of SERCA2 mitigated the effects of MKK6(Glu) on [Ca2+]i and NF-AT. Conclusions: The MKK6(Glu)–p38 MAPK pathway prolongs the decay phase of the cardiac contractile calcium by downregulating SERCA2, increasing diastolic [Ca2+]i which activates NF-AT. The ability of SERCA2 over-expression to reduce NF-AT activity represents a potential novel therapeutic effect of SERCA2 that should be further considered in the development of cardiac gene therapy strategies.

KEYWORDS Calcium (cellular); Signal transduction; Ca-pump; Gene expression; Gene therapy


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Pressure-overload initiates hypertrophic cardiac growth which, while initially compensatory, may lead to reduced contractility and heart failure. There are three broadly consistent observations associated with cardiac hypertrophy and failure. First, the intracellular calcium ([Ca2+]i) transients that drive contractions are prolonged [1], which is often associated with diminished expression of SERCA2, the enzyme responsible for re-sequestration of calcium into the sarcoplasmic reticulum (SR) during relaxation [2]. Second, mitogen-activated protein kinases (MAPKs) of the ERK, JNK, and p38 pathways are activated as part of the hypertrophic response [3]. p38 MAPK is of particular interest since this pathway can be growth promoting [4], pro-apoptotic [5], or anti-apoptotic [6] suggesting that disregulation of p38 play a role in cardiac pathologies. Third, activation of the calcium-sensitive transcription factor nuclear factor of activated T-cells (NF-AT) results in hypertrophy and heart failure [7]. It is unclear if p38 MAPK activation, SERCA2 downregulation, and NF-AT activation are independent or inter-related phenomena.

Our goal was to determine if the p38 pathway regulates [Ca2+]i in cardiac myocytes, and, if so, to investigate the mechanism(s) and consequences of altered [Ca2+]i regulation. Neonatal rat ventricular myocytes were utilized since co-transfection of GFP can be used as a transfection marker in these cells allowing evaluation of physiological function [8,9]. Our results suggest that the MKK6–p38 MAPK pathway prolongs the decay phase of the cardiac contractile transient by down-regulating SERCA2 and that these alterations in [Ca2+]i lead to activation of NF-AT. These effects were reversed by increasing SERCA2 expression, which is consistent with the ability of SERCA2 to prevent pressure-overload induced heart failure in vivo [10,11].


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
This study was approved by the University Animal Subjects Committee of San Diego State University and the Animal Subjects Program and the University of California, San Diego. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.1 Cell culture
Neonatal rat ventricular myocytes were dissociated and transfected via electroporation [8]. For [Ca2+]i measurements, 9x106 cells were used per transfection, and each sample was split onto two 35-mm fibronectin-coated glass coverslips. For electrical pacing of contractions, each transfection was plated into three 24-mm wells. Myocytes used for Western analysis were plated at a density of 0.3x106 cells per 35-mm well. Myocytes plated for Northern analysis were plated at a density of 4x106 cells per 100-mm dish. Cells were plated in medium containing a 1:1 mixture of DMEM–F12, supplemented with 10% FCS and antibiotics. After 18–20 h in culture, the medium was replaced with DMEM–F12 without serum additionally supplemented with 1 nM L-3,3-,5-triiodothyronine (T3), 5 µg/ml apo transferrin, 1 µg/ml insulin, and 0.1 nM selenium (maintenance medium) for 48–72 h prior to analysis. In experiments for analysis of SERCA2 promoter activity, the maintenance medium was supplemented with 10 nM T3.

2.2 Measurement of indo-1 fluorescence and calibration of [Ca2+]i
Cells plated on coverslips were loaded with indo-1, via a 25-min incubation at 37°C with 3 µM indo-1/AM. The coverslips were mounted on the stage of a Nikon Diaphot microscope interfaced to a Photon Technologies photometry system [12]. Data were collected at 20 Hz. For calibration, myocytes were incubated with 132 mM KCl, 10 mM HEPES, and 1 mM MgSO4 prepared with either no added calcium and 1 mM EGTA (for Rmin), or with 10 mM CaCl2 (for Rmax). The solution additionally contained 6 µM rotenone, 6 µM FCCP, and 20 µM ionomycin to de-energize the cells and equilibrate Ca2+ across the cellular membranes. Indo-1 ratios were calibrated by: [Ca2+]i=Kd(Sf2/Sb2)(R–Rmin)/(Rmax–R). The Kd value for indo-1 was assumed to be 250 nM [13]. Sf2/Sb2 is the ratio of indo-1 fluorescence at 485 nm in the absence vs. the presence of Ca2+ and was determined in vivo to be 4.39. Rmax and Rmin were 2.20 and 0.57, respectively.

2.3 Electrical pacing of contractions
Electrical pacing of contractions was accomplished as previously published [12,14]. Myocytes were cultured in the central two rows of multi-well culture dishes. Electrical impulses (60–80 V, 5 ms, 3 Hz) were delivered via a Grass Stimulator and a polarity reverser (which reversed the polarity of the pulses every 20 s). Silver–silver chloride electrodes were mounted in cell-free well edges of the culture dish and straps of agarose gel made up in culture medium were used to complete the circuits. In pacing experiments, myocytes were maintained in the absence of T3 as T3 elicited an unacceptably high rate of spontaneous contraction.

2.4 Plasmids and adenoviruses
pcDNA3 MKK6(Glu) (codes for activated MKK6, the p38-specific MAPKK was from R. Davis, University of Massachusetts, Worcester MA). 3.2SERCA2-luc was generated by digestion of the rat SERCA2 construct 3.2pBL-CAT3 with Hind3, which was then blunted and further digested with Xho. The released fragment was inserted into pGL2-basic which had been previously digested with MluI, blunted, and digested with Xho1. 0.6SERCA2-tk-luc was prepared by cloning a Hind III/Nae fragment of the rat SERCA2 promoter (corresponding to positions –562 to –163 upstream from the transcription start site) into the pT81 luc vector digested with Hind III and Sma I. The final construct contains –560 to –163 of the rat SERCA2 promoter, the Sma1 to Bgl II portion of the multiple cloning sequence of the pXP parent plasmid, the –81 to +52 portion of the thymidine kinase promoter, and the firefly luciferase reporter gene. pNF-AT-luc (contains a 3xconcatamer of the NF-AT binding site from human IL-2 promoter fused upstream from luciferase) was obtained from J. D. Molkentin. pcDNA3.1-asSERCA2, which encodes the entire rat SERCA2 cDNA in an anti-sense configuration, was constructed by digesting pCCI with Kpn and BamHI; the Kpn-BamHI fragment was ligated into pcDNA3.1(–) (Invitrogen). pACCMV.pLpA-SERCA2, which encodes SERCA2 in the sense orientation has been described previously [8]. ADV-MKK6(Glu) and ADV-GFP have also been described [15] and were generated via use of the pAdEasy-pAdTrack system [16].

2.5 Northern and Western blotting
SERCA2 mRNA levels were assayed via previously published procedures [17]. Ten micrograms of RNA was assayed per sample. SERCA2 mRNA levels were normalized by reprobing the blots for 28S ribosomal subunit. For analysis of SERCA2 protein, the myocytes were washed in ice cold PBS and lysed in 100 µl of a buffer consisting of 20 mM Tris (pH 7.4), 20 mM NaCl2, 100 µM EDTA, 1% Triton X-100, 12 mM deoxycholate, and 0.04% β-mercapto-ethanol. Samples corresponding to 20 µg protein were diluted with Laemmli buffer, subjected to electrophoresis on Novex Pre-Cast Gels (4–20% Tris/glycine), and transferred to nitrocellulose membrane according to the manufacturers’ instructions. Blotting was with a 1:1000 dilution of a rabbit polyclonal anti-SERCA2 antibody [17]; an HRP-conjugated donkey IG anti-rabbit conjugated antibody (Amersham) served as the secondary antibody and HRP activity was detected with the NEN Renaissance Chemiluminescence Reagent Plus kit. Western blots were reprobed with a 1:5000 dilution of a monoclonal antibody directed against {alpha}-sarcomeric actin (Sigma), which was detected with a 1:1000 dilution of an HRP-conjugated sheep anti-mouse Ig (Amersham).

2.6 Immunocytofluorescence
Myocytes on glass coverslips were fixed at 4°C for 15 min in a 1:1 mix of acetone and methanol. Blocking was with 10% (v/v) goat serum in Tris-buffered saline plus 0.1% (w/v) BSA. Myocytes were incubated with a 1:500 dilution of the SERCA2 polyclonal antibody [17] for 1 h, washed several times, and incubated with a 1:500 dilution of a TRITC-conjugated goat-anti-rabbit secondary antibody (Sigma). Photomicroscopy was performed with a Nikon Optiphot-2 microscope, a Nikon Fluor 100x 1.3 NA oil-immersion objective, and a SPOT camera (Diagnostic Instruments). Images were analyzed for fluorescence intensity using NIH Image.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 MKK6(Glu) prolongs the contractile calcium transient
The MKK6–p38 MAPK pathway participates in cardiac hypertrophic and failure conditions that feature altered [Ca2+]i regulation [4,5,18]. To test if the MKK6–p38 MAPK pathway modulates [Ca2+]i, myocytes were transfected with an expression plasmid for MKK6(Glu) along with a plasmid encoding GFP. GFP-positive myocytes were identified and monitored for [Ca2+]i while electrically stimulated at 0.3 Hz. This frequency was used in this initial characterization as it sufficiently resolves the duration and kinetics of the complete [Ca2+]i cycle. Control myocytes (transfected with pCMV6) and MKK6(Glu)-transfected myocytes paced at 0.3 Hz, displayed similar basal (Rbasal) and systolic (Rsys) indo-1 ratios (Fig. 1 and Table 1). However, the half-time of decay (tdecay) was 40% longer with MKK6(Glu).


Figure 1
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  		Fig. 1 Effect of MKK6(Glu) on [Ca2+]i. Myocytes were transfected with constructs at 15 µg/cuvette, maintained for 48 h in serum-free medium, and assayed for [Ca2+]i while electrically stimulated to contract at 0.3 Hz. (A) Data obtained for myocytes transfected with either pCMV6, MKK6(Glu), or MKK6(Glu) plus SERCA2. (B) Averaged indo-1 traces for control and MKK6(Glu)-transfected cells converted to [Ca2+]i and displayed on a linear scale (n = 80 cells per condition).

 

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  		Table 1 Effects of MKK6(Glu) on [Ca2+]i

 
Application of calibration constants obtained in vivo to the indo-1 data yielded estimates of 165 and 163 nM, for basal [Ca2+]i for pCMV6- or MKK6(Glu)-transfected myocytes, respectively, whereas systolic [Ca2+]i averaged 691 and 656 nM (Table 1). To better illustrate the effect of MKK6(Glu) on [Ca2+]i, indo-1 traces obtained from all of the cells studied were averaged and calibrated to [Ca2+]i (Fig. 1B). For the majority of the contractile cycle, [Ca2+]i was elevated in cells expressing MKK6(Glu). For the time period 0.5–1.0 s, post-stimulation, [Ca2+]i averaged 21% higher in the MKK6(Glu) cells. Eventually, the [Ca2+]i decayed back to the control value. Thus, the overall ability to regulate [Ca2+]i was not compromised, rather, there was a specific decline in the rate of Ca2+ removal from the cytoplasm.

3.2 MKK6(Glu) markedly increases diastolic calcium in myocytes contracting at 1–2 Hz
To test the effects of MKK6(Glu) at higher, more physiologically relevant frequencies, myocytes were paced to contract at 1 or 2 Hz. The myocytes were initially pre-paced at 0.3 Hz for 20–30 s. Next, pacing was paused for a few seconds, to allow the indo-1 ratio to decline to a basal value (Rbasal); the myocytes were then given a single ‘reference stimulation’. After the indo-1 ratio decayed again to the basal value, a train of pulses was sent at 1 or 2 Hz to the myocytes. Control-transfected myocytes paced at 1 Hz quickly reached a ‘plateau Rdia’, which was somewhat greater than the basal indo-1 ratio (Fig. 2A). Plateau Rdia was further increased at 2 Hz. Myocytes transfected with MKK6-Glu had dramatically elevated plateau Rdia values compared to controls. To quantify this, the plateau Rdia values were expressed as a percent of the magnitude of the first transient in the stimulus train (Rbasal was considered to be 0% and the peak of the initial transient, Rsys, was 100%; Fig. 2B). At 1 Hz, control plateau Rdia averaged 17±3% (mean±S.E., n = 15 cells) of the transient magnitude, whereas for MKK6(Glu)-transfected myocytes the plateau Rdia averaged 30±4% (mean±S.E., n = 10 cells, P<0.05). Notably, at 2 Hz, the plateau Rdia averaged 36±5% (n = 5) vs. 66±12% (n = 6), respectively (P<0.05 for MKK6-Glu vs. controls). Additionally, for MKK6(Glu) four out of the five cells paced at 2 Hz exhibited a positive staircase whereby the Rsys increased during the first four to six contractions by about 10%. At 2 Hz, there was a trend for plateau Rdia to increase with the tdecay of the reference transient (Fig. 2C). Furthermore, the data points for the control and MKK6(Glu) cells tended to cluster about the same regression line (Fig. 2C), suggesting that the transient decay may be a major determinant of diastolic [Ca2+]i. Calibration of these data yielded plateau diastolic [Ca2+]i values of 228 and 285 nM for control vs. MKK6(Glu) myocytes at 1 Hz and 311 nM vs. 471 nM, respectively, at 2 Hz. Thus, at 2 Hz, MKK6(Glu) elicited a 51% increase in diastolic [Ca2+]i.


Figure 2
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  		Fig. 2 Effect of MKK6(Glu) on myocytes paced at 1 or 2 Hz. (A) Indo-1 traces from control or MKK6(Glu)-transfected myocytes. The left y-axis depicts the indo-1 405/485 ratio; the right y-axis indicates [Ca2+]i. Dashed lines corresponding to Rbasal, Rsys, and Plateau Rdia are illustrated for myocytes paced at 1 Hz. (B) MKK(Glu) and contraction rate vs. Plateau Rdia. Each bar represents the mean±S.E. for n = 5–16 cells. (C) Plateau Rdia vs. tdecay for cells paced at 2 Hz. Plateau Rdia values were plotted against the tdecay of the corresponding reference contraction and fitted to a linear regression line. Control myocytes, filled circles; MKK6(Glu)-myocytes, open circles.

 
3.3 Expression of MKK6(Glu) reduces SERCA2 expression
Prolongation of transient decay often results from down regulation of SERCA2. To test if MKK6(Glu) modifies SERCA2 expression, myocytes were infected with adenoviruses that encode either GFP alone (ADV-GFP), or MKK6(Glu) plus GFP (ADV-MKK6(Glu) [15]. The viral titer of 45 MOI was used, which was sufficient to infect nearly 100% of the cultured myocytes. For myocytes infected with ADV-MKK6(Glu), SERCA2 mRNA expression (normalized to the 28 S ribosomal subunit) was diminished by approximately 40% vs. ADV-GFP after 1 day (Fig. 3A, day 1); SERCA2 mRNA was similarly downregulated by MKK6(Glu) on day 3. For three separate cell preparations, ADV-MKK6(Glu)-infected myocytes exhibited SERCA2 mRNA levels averaging 38±20% of the value for uninfected myocytes or 42±15% of the expression level for ADV-GFP infected cells.


Figure 3
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  		Fig. 3 MKK6(Glu) vs. SERCA2 expression. Myocytes were infected with ADV-GFP, or ADV-MKK6(Glu) and harvested 1 or 3 days after infection. (A) Northern data. (B) Western data. Protein samples were analyzed in duplicate after 3 days of infection.

 
As for mRNA, SERCA2 protein was diminished in ADV(Glu)-infected myocytes cells harvested on day 3 (Fig. 3B). In contrast, ADV-MKK6(Glu) increased expression of sarcomeric actin, which is consistent with the hypertrophic effect of MKK6(Glu) [5]. For three separate cell preparations, ADV-MKK6(Glu)-infected cells exhibited SERCA2/actin ratios that averaged 38±4% of the ratio for uninfected myocytes, and 44±8% of the ratio obtained with ADV-GFP-infected cells. Therefore, expression of SERCA2 relative to the expression of the contractile apparatus, is markedly reduced by MKK6-(Glu), which is consistent with its effect on [Ca2+]i. SERCA2 protein was not significantly downregulated on day 1, and only slightly affected on day 2 (data not shown), suggesting that the SERCA2 protein is relatively stable compared to the mRNA.

3.4 Caffeine-induced calcium release from the SR is similar in control and MKK6(Glu)-transfected myocytes
To test if MKK6(Glu) affects the calcium content of the SR, myocytes were paced at 0.3 Hz for 20 s to insure SR loading; pacing was then stopped and a reference contractile calcium transient was elicited. After decay back to baseline, caffeine was rapidly added (20 mM final concentration, mixing time less than 0.3 s). Caffeine-induced calcium release was virtually identical in control vs. MKK6(Glu)-transfected myocytes (Fig. 4), with the Rcaf averaging 1.67±0.07 (mean±S.E., n = 4) vs. 1.67±0.05 (n = 5) for control vs. MKK6(Glu) cells, respectively. This suggests that the degree of SR calcium loading is similar in ADV-GFP- vs. ADV-MKK6(Glu)-infected myocytes, which is consistent with absence of an effect of MKK6(Glu) on systolic [Ca2+]i.


Figure 4
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  		Fig. 4 Caffeine-induced calcium release in control and MKK6(Glu) expressing myocytes. Myocytes were transfected as in Fig. 1. Each cell prepaced at 0.3 Hz for 20 s, then given a reference stimulation (shown) followed by a rapid addition of caffeine (20 mM, final).

 
3.5 MKK6(Glu) downregulates activity of the SERCA2 gene promoter
SERCA2 transcription is downregulated during hypertrophic growth [19–21] to test if MKK6(GLU) regulates the SERCA2 gene promoter, myocytes were transfected with a reporter gene that features 3.2 kb of the rat SERCA2 5' flanking sequence (3.2SERCA2-luc). This portion of the SERCA2 gene confers hormonal-inducibility to reporter genes similar to endogenous SERCA2 [21,22]. Additional experiments were also performed with a construct that features a proximal segment of the SERCA2 promoter (–559 to –163 bp upstream from the transcription start site) ligated upstream from the minimal thymidine kinase promoter (0.6SERCA2-tk-luc), and with a construct containing the thymidine kinase promoter without SERCA2-derived sequences (tk-luc). To control for differences in transfection efficiency, results were normalized to β-galactosidase, expressed from a CMV-promoter β-galactosidase plasmid. MKK6-Glu reduced luciferase expression from 3.2SERCA2-luc by 40% (Fig. 5). MKK6(Glu) similarly reduced luciferase derived from 0.6SERCA2-tk-luc but had no effect on luciferase expression from the minimal tk-promoter-luciferase construct. These data suggest that MKK6(Glu) reduces the activity of the SERCA2 promoter, and this effect is mediated by the region –562 to –163 upstream from the transcription start site. Thus, it is likely that MKK6(Glu) reduces transcription of SERCA2.


Figure 5
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  		Fig. 5 MKK6(Glu) vs. SERCA2 promoter activity. Myocytes were cotransfected with reporter constructs (3.2SERCA2-luc, 559SERCA2-tk-luc or tk-luc) and with either control plasmid (pCMV6) or MKK6(Glu), as indicated. Plasmids input was 15 µg/cuvette except for CMV: β-galactosidase (12 µg/cuvette). Samples were harvested after 48 h in serum-free medium. Each bar represents the mean±S.E. of three samples. *P<0.05 vs. control, Student's t-test.

 
3.6 Over-expressing SERCA2 can cause reverse prolongation of the calcium transient by MKK6(Glu)
To test if co-expression of SERCA2 can compensate for the downregulating effects of MKK6(Glu), myocytes were cotransfected with a CMV-driven expression plasmid for SERCA2. Cotransfection with SERCA2 significantly reduced tdecay in the presence of MKK6(Glu), while having minimal effects on Rbasal and Rsys (Fig. 1 and Table 1). We have previously demonstrated that the SERCA2 expression plasmid, by itself, has no effect on Rbasal and Rsys [8] in this cell type. Thus, increasing SERCA2 expression via gene transfer techniques can compensate for the SERCA2 downregulating effects of MKK6(Glu), leading to an improvement in decay kinetics.

3.7 Activation of NF-AT-dependent gene expression by MKK6-Glu and contractile activity
The diastolic and systolic [Ca2+]i concentrations determined above are within the [Ca2+]i sensitivity range of the NF-AT system [23]. To test if MKK6[Glu]-mediated elevations in [Ca2+]i can activate NF-AT, myocytes were transfected with an NF-AT-sensitive reporter gene, and electrically paced at 3 Hz. In unpaced myocytes, which spontaneously contracted at less than 1 Hz, MKK6(Glu) upregulated NF-AT-dependent luciferase expression twofold on day 1 and day 2, and this increased to threefold by day 3 (Fig. 6A). For paced myocytes, virtually all cells were contracting in synchrony with the stimulator within 24 h. Pacing, by itself, increased luciferase expression, reaching a maximum of about 2.5-fold on day 3. The combination of MKK6(Glu) plus pacing produced a mean eightfold increase in luciferase by day 3 (the range was 2.5–14-fold for six cell preparations). Luciferase expression in response to MKK6(Glu) plus pacing was sensitive to cyclosporine (Fig. 4B). Cyclosporine also tended to reduce luciferase expression in unstimulated cells suggesting a degree of basal NF-AT activity. Thus, both MKK6(Glu) and pacing activate NF-AT, and these stimuli have strong synergistic effects.


Figure 6
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  		Fig. 6 Activation of NF-AT-dependent gene expression by MKK6(Glu) and pacing of contractions. (A) Time-course of NF-AT-dependent gene expression. Myocytes were transfected with pNF-AT-luc (15 µg/cuvette) plus either empty control plasmid (C), or MKK6(Glu) (M). Certain samples were also subjected to pacing at 3 Hz (P). Each bar represents the luciferase expression value, relative to control, for 2 (for day 1 and day 2) or five independent cell preparations (day 3). (B) Effect of cyclosporine (500 ng/ml, CSA). (C) Effects of 1 µM Bay K 8644. For B and C, each bar represents the mean±S.E. (n = 3). *P<0.05 vs. control, by Student's Newman–Keuls test; P<0.05 vs. MKK6+pacing (M+P).

 
Certain myocytes were also exposed to Bay K 8644, which elicits spontaneous calcium transients of irregular frequency in cardiac myocytes by increasing the ‘spark’ activity of the L-channel-ryanodine receptor complex [12,24]. While Bay K 8644 had little or no direct effect on NF-AT-dependent gene expression, it markedly enhanced the ability of MKK6(Glu) to activate NF-AT (Fig. 6C). Thus, pharmacological activation of the L-type calcium channels increases the ability of MKK6(Glu) to activate NF-AT in a manner similar to pacing of contractions.

3.8 Modulation of NF-AT-dependent gene expression by SERCA2
It was also tested if increasing SERCA2 expression would affect NF-AT activity. Over-expression of SERCA2 had little or no effect on NF-AT-dependent gene expression in unpaced cells (Fig. 7A). In contrast, for myocytes transfected with MKK6(Glu) and electrically paced, SERCA2 reduced NF-AT activity in a dose-dependent manner to a maximum of 30%. An input of 15 µg/cuvette of SERCA2 expression plasmid, which substantially corrects the calcium transient kinetics in the presence of MKK6(Glu) (Fig. 1 and Table 1), had a near-maximal effect to reduce NF-AT-dependent gene expression (Fig. 7A). Cotransfection with asSERCA2, which should reduce endogenous SERCA2, had the opposite effect, increasing NF-AT-dependent gene expression in the presence of MKK6(Glu) plus pacing by about 30%.


Figure 7
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  		Fig. 7 Regulation of NF-AT-dependent gene expression by SERCA2. Myocytes were transfected with NF-AT-luc, MKK6(Glu) (M) and with plasmids encoding SERCA2 in the (A) sense-(SERCA2); or (B) anti-sense (asSERCA2) orientation. Pacing (P) was at 3 Hz. Samples were harvested after 3 days in serum-free medium. Doses of the SERCA2 expression plasmids were 1, 15, and 80 µg/transfection. Each bar represents the mean±S.E. for n = 3 independent samples. *P<0.05 vs. control; P<0.05 vs. control and M+P in the absence of SERCA2 plasmid. (C) Photomicrographs of myocytes stained for SERCA2. Myocytes were transfected with 80 µg/cuvette of control plasmid (pCMV6), SERCA2, or asSERCA2. (D) Analysis of SERCA2 fluorescence in the transfected cells. Each bar represents the average data obtained from six to eight myocytes, normalized to the mean fluorescence of the control-transfected cells.

 
To confirm the effects of the transfection maneuvers on SERCA2, transfected myocytes were visualized for SERCA2 expression. Control-transfected myocytes displayed perinuclear SERCA2 staining and staining throughout the cytoplasm, with some evidence of a striated, myofibrillar pattern (Fig. 7C). Myocytes transfected with the SERCA2 expression plasmid were markedly brighter (by approx. 80%), and the cytoplasmic regions were labeled more extensively. Myocytes transfected with asSERCA2 exhibited just 60% of the control fluorescence (Fig. 7D).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
There are several implications to the obtained results (Fig. 8). Activation of p38 MAPK occurring in vivo as a result of stress, or in the present experiments by virtue of expression of MKK6(Glu), may lead to SERCA2 downregulation and prolongation of decay of the contractile calcium transient. This prolongation may result in an elevation of diastolic [Ca2+]i thereby increasing the activity of NF-AT, resulting in maladaptive phenotypic changes in cardiac function and structure. Gene transfer techniques that increase SERCA2 expression may act to inhibit this pathway by preventing the alterations in [Ca2+]i associated with SERCA2 downregulation.


Figure 8
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  		Fig. 8 Potential relationships between the MKK6–p38 MAPK pathway, SERCA2, and NF-AT. Increased cardiac p38 MAPK activity may downregulate SERCA2, leading to increased diastolic [Ca2+]i and activation of NF-AT. Increased SERCA2 expression, achieved via gene-transfer techniques, may reduce activation of NF-AT.

 
Reduction of SERCA2 activity via expression of an inhibitory phospholamban mutant leads to hypertrophy and failure [25], indicating that normal SERCA2 function must be maintained to insure cardiac health. Furthermore, increased expression of SERCA2 via transgenic or gene therapy approaches is protective against pressure-overload [10,11]. However, it has not previously been identified how alterations in SERCA2 activity could have deleterious or beneficial effects beyond the beat-to-beat regulation of cardiac function. Our results suggest that diminished SERCA2 expression may link activation of the MKK6–p38 MAPK pathway to NF-AT potentially resulting in pathologically relevant cardiac gene expression. Consistent with our results, expression of MKK6(Glu) leads to diminished SERCA2 expression in transgenic mice [26].

Ha-RasV12 or BXB-Raf, which activate ERK-MAPK, also prolong the decay phase of the cardiac contractile calcium transient [8]. However, in contrast to MKK6(Glu), Ras and Raf additionally reduce the magnitude of the calcium transient, most likely as a result of reducing the magnitude of the L-type calcium current [9]. Preliminary data suggests that MKK6(Glu) may increase the activity of the rat {alpha}1C gene promoter, whereas Ras and Raf have the opposite effect (Ho et al., unpublished). Relatedly, the magnitude of the contractile calcium transient and the SR-calcium load were normal in MKK6(Glu)-expressing myocytes, suggesting that MKK6(Glu) may upregulate compensatory mechanisms for maintaining SR calcium content. Transgenic inhibition of SERCA2 activity also does not alter the magnitude of the contractile calcium transient and is associated with increased L-channel function [25]. Furthermore, PMA, a strong activator of protein kinase C that reduces SERCA2, also does so without reducing transient magnitude [8,27].

The data of this study are consistent with the observation that pacing-induced upregulation of adenylosuccinate synthetase-1 is blocked by cyclosporine [28]. The data do not preclude the possibility that MKK6(Glu) activates NF-AT-dependent gene expression by additional, non-SERCA2-related, mechanisms, as it was not possible to completely prevent activation of NF-AT by SERCA2 overexpression. It is also possible that NF-AT-dependent gene expression in response to pacing is due, in part, to activation of p38 MAPK [29]; however, activation of p38 MAPK by pacing is transient and relatively small (about twofold) compared to that which is achieved with MKK6(Glu) (>10-fold [4]).

The situation in vivo is likely to be quite complex. For example, transgenic mice that express constitutively active calcineurin, the upstream activator of NF-AT, feature diminished SERCA2 expression [7] and p38 MAPK activity [30] suggesting that there are feed-back and feed-forward mechanisms that may be critical to the balance of compensatory vs. pathological responses to cardiac stress. Further research to elucidate these relationships will likely improve our understanding of the etiology of cardiovascular disease and suggest additional avenues for the development of gene therapeutic strategies.

Time for primary review 29 days.


   Acknowledgements
 
This work was supported by National Institutes of Health Grants HL-54030 (to P.M.M.), NL/HL-25073 (to C.C.G.), HL-63975 (to C.C.G.), HL-49434-01 (to W.H.D.), and an American Heart Association Western Affiliate Grant-in-Aid (0150725) to P.M.M. P.D.H. was the recipient of an American Heart Association California affiliate, predoctoral fellowship. We acknowledge Dr Bern Gloss for the gift of the 0.6SERCA2-tk-luc plasmid.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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