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Am J Physiol Cell Physiol 292: C2269-C2275, 2007. First published February 7, 2007; doi:10.1152/ajpcell.00441.2006
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MUSCLE CELL BIOLOGY AND CELL MOTILITY

Phenylephrine hypertrophy, Ca2+-ATPase (SERCA2), and Ca2+ signaling in neonatal rat cardiac myocytes

A. M. Prasad,1 H. Ma,2 C. Sumbilla,2 D. I. Lee,2 M. G. Klein,2 and G. Inesi1

1California Pacific Medical Center Research Institute, San Francisco, California; and 2Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland

Submitted 16 August 2006 ; accepted in final form 30 January 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We endeavored to use a basic and well-controlled experimental system to characterize the extent and time sequence of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) involvement in the development of cardiac hypertrophy, including transcription, protein expression, Ca2+ transport, and cytoplasmic Ca2+ signaling. To this end, hypertrophy of neonatal rat cardiac myocytes in culture was obtained after adrenergic activation with phenylephrine (PE). Micrographic assessment of myocyte size, rise of [14C]phenylalanine incorporation and total protein expression, and increased transcription of atrial natriuretic factor demonstrated unambiguously the occurrence of hypertrophy. An early and prominent feature of hypertrophy was a reduction of the SERCA2 transcript, as determined by RT-PCR with reference to a stable marker such as glyceraldehyde-3-phosphate dehydrogenase. Reduction of Ca2+-ATPase protein levels and Ca2+ transport activity to ~50% of control values followed with some delay, evidently as a consequence of a primary effect on transcription. Cytosolic Ca2+ signaling kinetics, measured with a Ca2+-sensitive dye after electrical stimuli, were significantly altered in hypertrophic myocytes. However, the effect of PE hypertrophy on cytosolic Ca2+ signaling kinetics was less prominent than observed in myocytes subjected to drastic SERCA2 downregulation with small interfering RNA or inhibition with thapsigargin (10 nM). We conclude that SERCA2 undergoes significant downregulation after hypertrophic stimuli, possibly due to lack of SERCA gene involvement by the hypertrophy transcriptional program. The consequence of SERCA2 downregulation on Ca2+ signaling is partially compensated by alternate Ca2+ transport mechanisms. These alterations may contribute to a gradual onset of functional failure in long-term hypertrophy.

calcium adenosinetriphosphatase; calcium transport

HYPERTROPHY OCCURS AS A RESPONSE of cardiac muscle cells to various stimuli, producing activation of specific gene profiles and protein expression and yielding significant enlargement of myocardial cells and overall size of the heart (4, 9). Pathological hypertrophy of the heart is a precursor to functional decompensation and failure (20). Clinical and animal studies indicate that inadequate function of the sarco(endo)plasmic reticulum Ca2+-transport ATPase (SERCA)2 and altered signaling are pathogenetic features of hypertrophy and failure (1, 13, 14, 19, 23, 25, 31, 38) and can be relieved by SERCA overexpression (5, 6, 10, 15, 21, 24). On the other hand, apparent inconsistencies are found in the literature, because in some studies the levels of Ca2+ transport protein appeared to be identical in nonfailing and failing human left ventricular myocardium (26), while in other studies the levels of both Ca2+-transport ATPase and related mRNA were shown to be reduced in cardiomyopathic hearts (8). In yet another case, SERCA2 protein levels were unchanged, while Ca2+ uptake and ATPase activity, as well as SERCA2 mRNA, were found to be reduced in failing human hearts (32). In a Dahl-S rat experimental model, the SERCA mRNA level was found to be reduced as the heart was undergoing transition from compensated hypertrophy to decompensated cardiac dilatation (28). We then considered that these apparent discrepancies may be related to heterogeneities in the type and stage of cardiac pathology, as well as to a sequential (i.e., nonsimultaneous) reduction of transcription, protein levels, and signaling alterations in the progression of hypertrophy. It may be relevant, in this regard, that long-term in vivo studies have shown that the onset of heart failure during pressure overload is faster in mice with chronically decreased SERCA2 Ca2+ pump activity (31).

In this connection, primary cultures of neonatal rat cardiac myocytes are a simpler, useful model (33) because they respond to appropriate stimuli through specific gene expression profiles and hypertrophy (31). Our laboratory (3, 16, 22, 27, 35) has been involved in overexpression of exogenous SERCA gene in cardiac myocytes, testing cell-specific promoters and characterizing functional consequences of overexpression. In this report we describe a series of experiments using this well-defined model to characterize the development of phenylephrine (PE)-induced hypertrophy. We planned to obtain a comprehensive evaluation of the consequences of hypertrophy with regard to changes of endogenous SERCA2 transcription, interference with Ca2+-ATPase expression and transport activity, and alteration of cytosolic Ca2+ signaling. We also performed comparative studies with myocytes subjected to SERCA gene silencing or SERCA inhibition with thapsigargin (TG) to establish quantitatively the contribution of Ca2+-ATPase to cytosolic Ca2+ signaling in neonatal myocytes.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Primary cell cultures. Neonatal cardiac myocytes were obtained from ten 1-day-old rats for each preparation. Harvesting of cardiac tissue was performed with protocols approved by the California Pacific Medical Center Research Institute and University of Maryland animal care and use committees. The minced ventricular muscle was incubated in 10 ml of medium (36) containing collagenase (0.357 mg/ml) and pancreatin (0.286 mg/ml) for 30 min. The first supernatant was then discarded, and the remaining muscle fragments were subjected to five consecutive incubations. Each time, the supernatant was transfered to a tube containing 1 ml of horse serum and centrifuged and the pellet resuspended in 2 ml of horse serum and maintained at 37°C under 5% CO2. The combined cell suspension was centrifuged again, and the pellets were resuspended in 20 ml of a 4:1 mixture of DMEM and medium 199 containing 5% FBS and 10% horse serum ("plating medium"). The dissociated myocytes were preplated in an uncoated P150 dish for 1 h at 37°C under 5% CO2, thereby eliminating nonmyocyte cells by adhesion to the plate. The unattached myocytes were then removed and plated (250 cells/mm2) on gelatin-coated dishes or laminin-coated glass surfaces and cultured under 5% CO2 in plating medium containing 0.1 mM bromodeoxyuridine.

Twenty four hours after plating, the attached myocytes were washed with phosphate-buffered saline (PBS). A 4:1 mixture of DMEM and medium 199 containing 0.1 mM bromodeoxyuridine and 5% FBS ("serum medium") was then added. Alternatively, a 4:1 mixture of DMEM and medium 199 containing 0.1 mM bromodeoxyuridine, 10 µg/ml insulin-transferrin-selenium (Mediatech), 0.1% BSA, 0.1 mM vitamin C, and 2 µg/ml vitamin B12 (but no FBS) was used ("serum-free medium"). The myocytes were then maintained at 37°C under 5% CO2.

Small interfering RNA construct and adenoviral vectors. DNA templates for the synthesis of silencing RNA were cloned into a pSilencer plasmid under the control of the U6 RNA Polymerase III promoter (–315 to +1) (Ambion). The selection of the coding sequence for targeting rat Ca2+-ATPase mRNA was done with the small interfering RNA (siRNA) Target Finder and Design Tool from Ambion. The potential siRNA target sequence was subjected to BLAST search (NCBI database) against expressed sequence tag EST libraries of rat to ensure that no other gene(s) was targeted. The target sequence for rat SERCA2a mRNA was 5'-AAGACTTACTAGTTAGAATTT-3'. It started at position 173 and had a GC content of 23.8%. To obtain transcription of a complementary sequence to the target, we designed the following sequence, where the segment in bold indicates the loop: sense template 5'-GACTTACTAGTTAGAATTTGGCTAAGAGCAAATTCTAACTAGTAAGTCTTTTT-3'; antisense template 3'-CCGGCTGAATGATCAATCTTAAACCGATTCTCGTTTAAGATTGATCATTCAGAAAAATTAA-5'.

The plasmid and the oligonucleotides were digested at the ApaI and EcoRI sites and then ligated together. The position of the DNA oligonucleotide was such that it was immediately preceded by the U6 promoter. The ligated DNA was transformed into competent DH5{alpha} cells, and the cells were selected for ampicillin resistance.

Adenoviral vectors were constructed with a pAd-lox plasmid containing an SV40 polyadenylation signal (12). The U6 promoter and siRNA construct were subcloned into the pAd-lox plasmid. Both the silencing construct and the control construct with just the promoter were cotransfected separately with purified {psi}5 adenovirus genome into CRE8 cells.

Protein synthesis. Protein synthesis was measured by L-[14C]phenylalanine incorporation as described by Simpson et al. (34). L-[14C]phenylalanine radioactive tracer [0.1 µCi per P35 culture plate (2.0 ml medium)] was added 15 h after plating. After a 72-h interval, the cells were washed with PBS, photographed by phase-contrast microscopy, and then denatured with 1.0 ml cold 10% TCA. After 1-h incubation at 4°C, the cells were rinsed twice with 1.0 ml of cold 10% TCA, 1.0 ml of 1% sodium dodecyl sulfate was added, and the cells were allowed to dissolve for 1 h at room temperature. Radioactivity was finally measured by scintillation counting. The results are expressed as radioactivity per cell, based on the cell counts by phase-contrast microscopy.

Real-time quantitative RT-PCR. Total RNA was isolated with the RNeasy mini Kit (Qiagen catalog no. 74104) with on-column DNase digestion with the RNase-free DNase set (Qiagen catalog no. 79254) according to the manufacturer's instructions. Primers and probes were designed with Beacon Designer 4.0 software (BD) and are shown in Table 1.


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  		Table 1. Real-time RT-PCR primers and probes

 
RT-PCR was performed by the SYBR Green method with an Applied Biosystems 7500 Fast Real-Time PCR System. The procedure was as follows: 1.0 µg of total RNA was used to synthesize cDNA by reverse transcription with the iScript cDNA Synthesis Kit (Bio-Rad) in a 20-µl volume. PCR amplification was performed in a total volume of 20 µl, containing 0.5 ng of the cDNA derived from reverse transcription, 25 pmol of each primer, and 10 µl of iQ SYBR Green Supermix. Each reaction was incubated for 2 min at 50°C and 10 min at 95°C and then subjected to 40 cycles, each involving denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min. In this way, the threshold cycle (CT) for fluorescence development was obtained. All samples were run in triplicate.

For comparative evaluation of transcription levels, standard plots of CT vs. cDNA derived from the total RNA of nontreated myocytes were first obtained for each gene of interest. The CT values obtained in subsequent experiments were then quantitated with reference to the standard curve obtained for each gene. The ratios of SERCA2 and atrial natriuretic factor (ANF) transcript levels in experimental (i.e., PE treated) and control samples were then compared with the ratios of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript levels in corresponding samples. GAPDH is widely considered to be a stable reference in this methodology.

Functional assays. After rinsing with PBS, the cultured cells were harvested by scraping in a resuspension medium (10 ml per 100-mm dish) containing 50 mM MOPS, pH 7.0, 10 mM NaF, 1 mM EDTA, 0.3 M sucrose, and protease inhibitors [0.4 mM Pefabloc SC (Roche), 0.5 mM dithiothreitol, 10 µg/ml aprotinin, 2 µg/ml leupeptin, 1 µg/ml pepstatin A]. Suspensions were centrifuged for 5 min at 2,000 g, and the cell pellets were frozen and stored at –70°C.

ATP-dependent (45Ca) Ca2+ transport was assayed with homogenates of cultured cells. The reaction conditions were as described by Sumbilla et al. (35). Transport by residual mitochondrial particle was inhibited with 1 µM ruthenium red and 5 mM NaN3 in the reaction medium. Control assays in the presence of 1 µM TG were performed to ensure that no additional activity remained after specific inhibition of SERCA.

Western blotting and immunofluorescence. Total protein was measured with a bicinchoninic acid assay kit (Pierce) after sonication of the harvested cells. Various protein components were separated in 7.5% polyacrylamide gels (18), transferred onto nitrocellulose paper, and stained with primary and secondary antibodies. Reactive bands were visualized by the Supersignal ECL Western blotting detection kit (Pierce), and densitometry was obtained in a NucleoVision workstation (Nucleotech) with Gel Expert software. Primary monoclonal antibodies for Western blots and immunostaining of whole cells were MA3-919 (1:250; Affinity Bioreagents) for rat SERCA2a, PA3-16782 (Affinity Bioreagents) for GAPDH, and MF-20 (Developmental Studies Hybridoma Bank, University of Iowa) for myosin.

Cytosolic Ca2+ transients. Cytosolic Ca2+ transients were measured in cells grown on special culture dishes (MatTeK, Ashland, MA) with laminin-coated glass coverslips . Cells were loaded for 5 min in fluo-4 and then washed with dye-free Ringer solution. Dishes containing loaded cells were placed in a special chamber mounted on an Olympus 1X70 inverted microscope and connected to a circulating bath with Ringer solution held at 30 ± 2°C. Measurements were performed with the Ion Wizard high-speed fluorescence imaging system with a MYO100 Myocam (Ion Optix, Milton, MA). Fluorescence emission from single cells was measured with 488-nm excitation for fluo-4, with a field stimulation of 9 V and 2-ms duration delivered most commonly at 1-Hz frequency. Time dependent/0 time fluorescence intensity ({Delta}F/Fo) signals were processed and analyzed with the customized software Ion Wizard 5.0 provided by Ion Optix. Data are shown as means ± SD, where n > 15. The threshold for statistical significance was set as P = 0.05 after a Student's two-tailed t-test.

In some cases, Ca2+ transients were observed under voltage clamp, with a Heka EPC-10 amplifier and Patchmaster software. Pipettes had resistance of 1.5–3 M{Omega}. Values for series resistances were 3–8 M{Omega} and were compensated up to 60%. The bathing solution contained (mM) 135 NaCl, 5 KCl, 20 HEPES, 2 CaCl2, 10 glucose, and 0.8 MgSO4, pH 7.4. Na+-free solutions contained an equimolar concentration of N-methyl-D-glucamine. The internal solution contained (mM) 100 Cs glutamate, 30 CsCl, 20 HEPES, 20 tetraethylammonium-Cl, 4 Mg-ATP, 3 K-phosphocreatine, 0.33 MgCl2, 0.1 EGTA, and 0.05 fluo-4, pH 7.2. The holding potential was –80 mV.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Cell growth and hypertrophy. Within 3 days after seeding, primary cultures prepared as described above contained at least 95% of cells identified as cardiac myocytes by immunostaining of specific endogenous proteins such as myosin or SERCA2 (3). As originally reported by Simpson (33), we obtained hypertrophic enlargement of myocytes cultured in serum-free medium after addition of 20 µM PE (Fig. 1). Quantitative assessment of overall protein expression, determined by [14C]phenylalanine incorporation, revealed a 69 ± 16% enhancement 3 days after exposure to PE. Upregulation of ANF is a prominent and specific consequence of hypertrophic stimuli in cardiac myocytes (7, 11, 17, 37). In our experiments, we determined by time-resolved RT-PCR that ANF transcription (compared with the standard stable transcript for GAPDH) was increased 2.97 ± 1.13- and 11.69 ± 1.95-fold after 3- or 7-day exposure to PE, respectively (Table 2). It is noteworthy that immunostaining of SERCA2 revealed no change in the percentage of cells identified as myocytes after 3- or 7-day exposure to PE, suggesting no isoform shift in our experiments. It is also apparent in Fig. 1 that nearly all cells in culture show hypertrophic enlargement, as expected of cardiac myocytes.


Figure 1
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  		Fig. 1. Hypertrophy of neonatal rat cardiac myocytes after exposure to phenylephrine (PE). A: control myocytes. B: myocytes exposed to 20 µM PE for 3 days. Myocytes were observed by phase-contrast microscopy with x10 magnification 4 days after plating.

 

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  		Table 2. Real-time RT-PCR

 
Downregulation of SERCA2. Time-resolved RT-PCR revealed that GAPDH transcription levels were not significantly changed by PE (Table 2). On the other hand, SERCA2 transcription was reduced to 0.62 ± 0.24 and 0.44 ± 0.13 after 3- or 7-day exposure to PE relative to control myocytes that were not exposed to PE (Table 2). In contrast, ANF transcription was markedly increased by PE (Table 2).

It is of interest that reduction of SERCA2 protein levels (as revealed by Western blots) became evident with significant time lag with respect to the effect of transcription. In fact, the SERCA protein levels were slightly reduced after 3 days of exposure to PE but became clearly reduced 7 days after exposure to PE (Table 3). A parallel pattern of reduction was demonstrated by measurements of ATP-dependent Ca2+ transport activity in homogenates of myocytes exposed to PE (Table 3). It is thus apparent that reduction of ATPase protein level and Ca2+ transport activity occurs with significant time lag relative to the transcriptional effect. As mentioned above, both in the RT-PCR measurements and the Western blots, the SERCA2 variations were always determined with reference to GAPDH transcript or protein, which is considered a methodological standard of constant transcription and expression.


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  		Table 3. SERCA2 protein levels and Ca2+ transport activity

 
Preliminary to evaluating the effect of PE-induced downregulation of SERCA on cytoplasmic Ca2+ signaling (see below), we optimized two alternative methods that interfere strongly with SERCA2 activity to establish to what extent Ca2+ signaling in neonatal myocytes is in fact dependent on SERCA2. In one case we used adenovirus vector delivery of cDNA templates for endogenous production of siRNA and interference with SERCA2 transcription. This intervention produced strong reduction of SERCA2 protein levels and Ca2+ transport activity within 3 days (Table 3), to an extent greater than observed in PE-treated myocytes. The low level of remaining Ca2+ transport activity is evidently due to residual SERCA protein that was present before gene silencing.

In the other case, we produced total inhibition of Ca2+ transport by exposing the myocytes to 10 nM TG. In preliminary experiments we established that the presence of 10 nM TG in the culture medium inhibited Ca2+-ATPase activity without reducing the ATPase protein level or myocyte survival. General toxic effects were produced by higher TG concentrations (Fig. 2). The comparative effects of SERCA downregulation or inhibition are shown in Fig. 3. It is thus apparent that PE hypertrophy is accompanied by partial reduction of SERCA protein level and Ca2+ transport activity, siRNA intervention produces a stronger reduction of SERCA protein level and Ca2+ transport activity, and TG produces total inhibition of Ca2+ transport activity without reducing the SERCA protein level. In all cases, the reference protein GAPDH remains at the same level.


Figure 2
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  		Fig. 2. Thapsigargin (TG) concentration dependence of Ca2+ transport inhibition and cell toxicity. Neonatal cardiac myocytes were cultured as described in METHODS and exposed to various concentrations of TG for 3 days. Cell counts were then obtained by phase-contrast microscopy, and myocytes were homogenized for determination of ATP-dependent Ca2+ transport. It is clear that exposure to 10 nM TG causes nearly total inhibition of Ca2+ transport and minimal cell toxicity. SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase.

 

Figure 3
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  		Fig. 3. Protein levels and ATP-dependent Ca2+ transport in homogenates of myocytes exposed to 20 µM PE, SERCA2 silencing (siSERCA2), or 10 nM TG. Control myocytes (lane 1, inset) are compared with myocytes exposed to PE for 3 (lane 2) or 7 (lane 3) days, siSERCA2 for 3 days (lane 4), or 10 nM TG (lane 5) for 3 days. Protein levels were measured by Western blotting and compared with the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein. ATP-dependent Ca2+ transport was measured as described in METHODS. siRNA, small interfering RNA.

 
SERCA activity and Ca2+ signaling transients. We performed a series of experiments to evaluate the effect of decreasing levels of Ca2+-ATPase transport activity on the kinetics of cytosolic Ca2+ transients elicited by field stimuli (1-Hz pulsing). In these experiments we used myocytes exposed for 7 days to PE, since at this time a reduction of SERCA protein levels and Ca2+ transport activity was more clearly evident. It is shown in Fig. 4 that the Ca2+ transients are moderately impaired in myocytes treated with PE, exhibiting a different shape of the curve that suggests partial compensation by alternative mechanisms for cytoplasmatic Ca2+ removal. The impairment is much more pronounced after SERCA silencing, and even more in myocytes treated with 10 nM TG. Average values for the Ca2+ signals yield a 0.188 ± 0.051-s half-width for control myocytes, 0.201 ± 0.023 for myocytes exposed to PE, 0.327 ± 0.034 after SERCA gene silencing, and 0.392 ± 0.058 after SERCA inactivation with TG. It is clear that the cytosolic Ca2+ transient kinetics are altered when Ca2+ transport activity by SERCA is reduced (compare Figs. 2 and 4), demonstrating that SERCA2 in fact plays an important role in neonatal myocytes. On the other hand, residual Ca2+ signaling is still observed even after complete SERCA inactivation, evidently due to alternative mechanisms for Ca2+ fluxes through the plasma membrane.


Figure 4
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  		Fig. 4. Alteration of Ca2+ signaling kinetics in myocytes exposed to PE, siSERCA2, or TG. Myocytes were exposed to 20 µM PE for 7 days, siSERCA2 for 3 days, or 10 nM TG for 3 days. Cytosolic Ca2+ transients were measured in single cells with fluo-4. Cells were subjected to field stimulation (1-Hz pulsing). Each trace represents the average of transients obtained from 30–70 cells over 5 different preparations. The fluorescence signal was normalized to the maximal change per each transient.

 
Inadequacy of the sarcoplasmic reticulum activity in the myocytes exposed to PE was further demonstrated by shortening the pulsing interval. It is shown in Fig. 5 that when a premature stimulus was delivered at a 200-ms interval to myocytes preconditioned with 1-Hz pulsing, the peak height (related to the amount of Ca2+ released) exhibited a greater reduction in PE-treated than control myocytes. This demonstrates that reuptake of Ca2+ into the sarcoplasmic reticulum is time limiting in hypertrophic myocytes.


Figure 5
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  		Fig. 5. Impaired recovery of Ca2+ signaling in a twin-pulse protocol. The myocytes were exposed to 20 µM PE for 7 days. Normal (A) and PE-treated (B) myocytes were preconditioned by pacing at 1.0 Hz, and then a premature stimulus was delivered after a 200-ms (rather than 1 s) interval. Filled symbols correspond to transients obtained during 1-Hz pacing, and open symbols correspond to twin pacing. Each trace represents the average of transients obtained from 20 cells over 2 different preparations. The fluorescence signal was obtained and normalized as for Fig. 4.

 
Finally, we obtained measurements in the absence of Na+, to evaluate how myocytes would handle the cytosolic Ca2+ transients in the absence of Na+/Ca2+ exchange. We found (Fig. 6) that after a series of voltage-clamp depolarizations the cytosolic Ca2+ concentration always returned to its basal level in control myocytes. In contrast, the basal cytosolic Ca2+ concentration underwent a progressive and definite rise in myocytes exposed to TG. A much milder rise was observed in myocytes exposed to PE. It is thus apparent that Na+/Ca2+ exchange sustains a complementary role in Ca2+ signaling and may provide some compensation when SERCA activity is reduced.


Figure 6
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  		Fig. 6. Ca2+ transients in Na+-free bathing solution. The myocytes were exposed to 20 µM PE for 7 days or 10 nM TG for 3 days. Control myocytes were examined after 3 or 7 days in culture, with no significant difference in the functional behavior. Myocytes were subjected to a series of voltage clamp-depolarizations to –10 mV at 2-s intervals (see METHODS). A: control myocytes. B: myocytes exposed to 20 µM PE for 7 days. C: myocytes exposed to 10 nM TG for 3 days. Each record is normalized to peak of its first transient.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We used a well-defined experimental system (i.e., neonatal rat myocytes) to characterize changes undergone by SERCA2 transcription, SERCA2 expression, Ca2+ transport activity, and cytoplasmic Ca2+ signaling as a consequence of hypertrophy induced by PE. In our experiments, the occurrence of hypertrophy was demonstrated by a clear enlargement of all myocytes in wide microscopic fields, increased [14C]phenylalanine incorporation for total protein synthesis, and strong upregulation of ANF transcription.

The earliest clear effect detected after exposure to PE was a reduction of SERCA2 transcription with reference to GAPDH transcription, which is widely used as a baseline standard in this type of measurement. It is noteworthy that the GAPDH transcript level was found to be unchanged after PE stimuli. On the other hand, the ANF transcript level was greatly increased. It should be pointed out that the reduced transcription noted in our experiments relates to total SERCA, including SERCA2a and SERCA2b, since our RT-PCR primers anneal with both isoforms.

Reduction of SERCA2 protein level (again with reference to GAPDH) followed with some time lag and was clearly established after 7 days of exposure to PE. Reduction of transport activity was also observed in parallel with the change in SERCA protein levels. The time sequence of these effects suggests that reduced transcription is primarily responsible for the lower SERCA levels, rather than a possible enhancement of SERCA protein degradation.

Regarding the effect of SERCA2 level reduction on cytosolic Ca2+ signaling after hypertrophy, we felt that it was first necessary to evaluate unambiguously the role of SERCA2 in normal neonatal myocytes with direct and specific procedures for interference with SERCA. To this end, we used SERCA2 transcription silencing as well as SERCA inhibition with TG. The use of these two alternative procedures under appropriate conditions ensured the specificity of the observed effects regarding SERCA, as opposed to general toxic effects. We found that the cytosolic Ca2+ transient kinetics are profoundly altered when Ca2+ transport activity by SERCA is reduced by either siRNA silencing or TG inhibition (compare Figs. 2 and 4). This demonstrates that SERCA2 plays an important role in neonatal myocytes. On the other hand, residual Ca2+ signaling is still observed even after complete SERCA inactivation, evidently due to alternative mechanisms for Ca2+ fluxes through the plasma membrane. In fact, in myocytes treated with TG to inhibit SERCA, repetitive stimuli under conditions interfering with Na+/Ca2+ exchange (i.e., absence of Na+) produce a progressive and prominent rise of resting cytosolic Ca2+ (Fig. 6).

Characterization of Ca2+ signaling in myocytes undergoing hypertrophy was obtained after 7-day exposure to PE, since at this time the SERCA protein levels and Ca2+ transport activity were more definitely reduced. At this time, a reproducible change was observed, consisting of an apparent reduction of Ca2+ release and reuptake kinetics (Fig. 5). This effect appears rather limited, considering that SERCA protein and transport activity were reduced by nearly 50%. This suggests that partial reduction of SERCA level and/or transport activity can be compensated to some extent by alternate transport mechanisms such as Na+/Ca2+ exchange. Compensation, however, becomes less adequate when the reduction of SERCA level and/or transport activity is >50%.

The complexity and sequential development of SERCA transcription, SERCA protein expression, and cytosolic Ca2+ signaling alterations explain apparently contradictory observations made under different conditions and sampling times. The sequence of alterations observed in our experiments indicates that reduction of the SERCA2 transcript is the first event following hypertrophic stimuli, possibly due to lack of SERCA gene involvement by the hypertrophy transcriptional program. Projection of the effects observed in cultured myocytes within a week after exposure to PE to the longer time frame of hypertrophy progression in vivo predicts a significant contribution of inadequate SERCA function to the development of functional failure (2, 29). This effect would likely be greater in adult cardiac muscle, where the role of SERCA in Ca2+ signaling is more prominent.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-69830.


   ACKNOWLEDGMENTS
 
Participation of Dr. Malini Seth in design of siRNA reagents previous to this work is gratefully acknowledged.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Inesi, California Pacific Medical Center Research Institute, 475 Brannan St., San Francisco, CA 94107 (e-mail: ginesi{at}cpmcri.com)

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.


   REFERENCES
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 DISCUSSION
 GRANTS
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