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Am J Physiol Heart Circ Physiol 289: H2484-H2490, 2005. First published July 29, 2005; doi:10.1152/ajpheart.00590.2005
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Protein kinase C and preconditioning: role of the sarcoplasmic reticulum

Ken Yamamura,1 Charles Steenbergen,2 and Elizabeth Murphy1

1Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park; and 2Department of Pathology, Duke University Medical Center, Durham, North Carolina

Submitted 3 June 2005 ; accepted in final form 23 July 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activation of protein kinase C (PKC) is cardioprotective, but the mechanism(s) by which PKC mediates protection is not fully understood. Inasmuch as PKC has been well documented to modulate sarcoplasmic reticulum (SR) Ca2+ and because altered SR Ca2+ handling during ischemia is involved in cardioprotection, we examined the role of PKC-mediated alterations of SR Ca2+ in cardioprotection. Using isolated adult rat ventricular myocytes, we found that addition of 1,2-dioctanoyl-sn-glycerol (DOG), to activate PKC under conditions that reduced myocyte death associated with simulated ischemia and reperfusion, also reduced SR Ca2+. Cell death was 57.9 ± 2.9% and 47.3 ± 1.8% in untreated and DOG-treated myocytes, respectively (P < 0.05). Using fura 2 fluorescence to monitor Ca2+ transients and caffeine-releasable SR Ca2+, we examined the effect of DOG on SR Ca2+. Caffeine-releasable SR Ca2+ was significantly reduced (by ~65%) after 10 min of DOG treatment compared with untreated myocytes (P < 0.05). From our examination of the mechanism by which PKC alters SR Ca2+, we present the novel finding that DOG treatment reduced the phosphorylation of phospholamban (PLB) at Ser16. This effect is mediated by PKC-{epsilon}, because a PKC-{epsilon}-selective inhibitory peptide blocked the DOG-mediated decrease in phosphorylation of PLB and abolished the DOG-induced reduction in caffeine-releasable SR Ca2+. Using immunoprecipitation, we further demonstrated that DOG increased the association between protein phosphatase 1 and PLB. These data suggest that activated PKC-{epsilon} reduces SR Ca2+ content through PLB dephosphorylation and that reduced SR Ca2+ may be important in cardioprotection.

calcium; protein kinase C-{epsilon}; 1,2-dioctanoyl-sn-glycerol; protein phosphatase 1

ISCHEMIC PRECONDITIONING (PC) refers to the phenomenon whereby intermittent brief episodes of ischemia and reperfusion reduce myocardial infarct size and contractile dysfunction induced by a subsequent sustained episode of ischemia. The signaling pathways involved in PC have been investigated intensively, and there is considerable support for the role of protein kinase C (PKC) in mediating PC (9, 24, 33, 38). However, the precise targets of PKC that mediate protection are poorly understood. It has been suggested that PKC mediates PC by modulating the mitochondrial ATP-sensitive K+ channel (37). PKC has also been reported to bind to or modulate the phosphorylation of several mitochondrial proteins (2, 3). Additional targets of PKC are likely involved in mediating cardioprotection. PKC has been shown to alter Ca2+ transients, contractility, and sarcoplasmic reticulum (SR) Ca2+ handling (6, 18, 29, 31, 35, 36); although there is some controversy, most studies suggest that activation of PKC decreases contractility, attenuates Ca2+ transients, and decreases SR Ca2+ content (6, 18, 29, 35, 36). Inasmuch as reduced SR Ca2+ loading before ischemia would reduce Ca2+ cycling and attenuate the rise in cytosolic Ca2+ concentration ([Ca2+]i) during ischemia and early reperfusion, such an alteration in SR Ca2+ could contribute to the cardioprotection observed with activation of PKC. Several studies have reported that altered SR Ca2+ handling is involved in the protection afforded by PC (8, 30, 39, 41).

SR Ca2+ handling plays a significant role in the regulation of cardiac contractility. Ca2+ uptake into the SR is mediated by the sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2), which is regulated by phospholamban (PLB). PLB inhibits SERCA2, leading to a reduced rate of Ca2+ uptake into the SR and a slower rate of relaxation. Phosphorylation of PLB causes dissociation of PLB from SERCA2, allowing faster rates of SR Ca2+ uptake and relaxation and enhanced contractility through increased SR Ca2+ loading. SR Ca2+ release is regulated by the SR Ca2+ release channel, which binds ryanodine and is therefore referred to as the ryanodine receptor. The mechanisms by which PKC alters SR Ca2+ are not well understood.

The aim of the present study was to examine the role of PKC modulation of SR Ca2+ in cardioprotection. Using adult rat cardiomyocytes, we found that addition of the PKC activator 1,2-dioctanoyl-sn-glycerol (DOG), under conditions that were cardioprotective, resulted in a decrease in SR Ca2+. We further examined the mechanism by which PKC might alter SR Ca2+ and found that activation of PKC resulted in a decrease in the phosphorylation of PLB and an increase in the association between PLB and protein phosphatase (PP) 1.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of cardiomyocytes. Ventricular myocytes were isolated from male Sprague-Dawley rats (8–10 wk, 250–300 g) by collagenase perfusion as described previously (16). Cardiomyocytes were resuspended in a Krebs-Henseleit buffer containing (in mmol/l) 1.25 CaCl2, 120 NaCl, 4.7 KCl, 25 NaHCO3, 1.2 KH2PO4, 1.2 MgSO4, 11 glucose, 20 creatine, 60 taurine, and 30 HEPES, with 0.1% bovine serum albumin (Sigma Aldrich). Animal studies were approved by the National Institute of Environmental Health Sciences Institutional Animal Care and Use Committee and conform with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996).

Simulated ischemia model. To examine whether DOG is protective in isolated adult rat cardiomyocytes, we subjected myocytes to simulated ischemia by pelleting the myocytes as described previously (1). Freshly isolated myocytes were divided into two experimental groups and treated with or without 3 µM DOG for 10 min, washed twice, and then pelleted and covered with mineral oil and incubated at 37°C for up to 4 h. Cell viability was assessed with trypan blue as a function of time as described elsewhere (1). Microscopic examination at x40 magnification was used to determine morphology (rod-shaped myocytes) and trypan blue staining.

Ca2+ measurements in isolated rat myocytes. Freshly isolated myocytes were placed on laminin-coated glass coverslips and allowed to attach for 30 min before they were loaded with fura 2-AM (Molecular Probes). Myocytes on coverslips were placed on the stage of a Nikon microscope connected to a Photon Technology International spectrofluorometer and superfused with modified Hanks' balanced salt solution. All fura-2 experiments were conducted at room temperature. Myocytes were treated with or without 3 µM DOG for 10 min and field stimulated at 0.5 Hz. For the chelerythrine protocol, myocytes were treated with 2 µM chelerythrine for 1 min and then with 3 µM DOG + 2 µM chelerythrine for 9 min. Studies were also done by using {epsilon}V1-2 peptide (amino acids 14–21 of PKC-{epsilon}), a selective PKC-{epsilon} inhibitor, which was synthesized at the Stanford Protein and Nucleic Acid Facility (Stanford, CA). The myocytes were treated with 1 µM {epsilon}V1-2 peptide or 1 µM control peptide for 20 min, and DOG was added for 10 min. Field stimulation was stopped just before caffeine (20 mmol/l) addition, and caffeine-releasable Ca2+ was used as a measure of SR Ca2+ content. [Ca2+]i was calculated from the following equation: [Ca2+]i = Kd x {beta} x (R – Rmin)/(Rmax – R), where R is the fluorescence ratio recorded at the two excitation wavelengths (340 and 380 nm), Kd represents the dissociation constant (224 nM), Rmin and Rmax are the fluorescence ratios under Ca2+-free and Ca2+-saturating conditions, and {beta} = F380,0 Ca2+/F380,saturating Ca2+, where F380 is fluorescence at 380 nm.

Western blot analysis. Myocytes were mixed with ice-cold lysis buffer containing (in mmol/l) 75 NaCl, 20 HEPES (pH 7.4), 2.5 MgCl2, 0.1 EDTA, 0.5 DTT, 0.1 Na3VO4, 1 NaF, and 20 glycerophosphate, with 0.1% Triton X-100 and protease inhibitors, homogenized, and snap frozen and stored in liquid nitrogen until Western blots were run. Proteins were separated by 14% SDS-PAGE and blotted onto nitrocellulose membranes (Invitrogen). Membranes were probed with the following primary antibodies: PLB (catalog no. MA3-922, Affinity Bioreagents; diluted 1:2,000) and Ser16-phosphorylated PLB (phospho-Ser16 PLB; RDI-PPHOSLAMBabR, Research Diagnostics; diluted 1:700). We found two immunoreactive bands at ~25 kDa, the molecular mass for the phospho-PLB pentamer. However, we confirmed in two ways that the upper band was nonspecific: 1) If we reprobed with total-PLB antibody after stripping was completed, the upper band was not recognized by total (nonphosphorylated) PLB antibody. 2) If we boiled the sample to convert the PLB pentamer to a monomer, the lower 25-kDa band was converted to the monomer band (5 kDa) detected with the phosphorylated PLB antibody, but the nonspecific upper 25-kDa band remained. For clarity, we show only the lower 25-kDa band, which was shown to be specific for PLB. The optical density of immunoreactive bands was quantified by using NIH Image.

Immunoprecipitation analysis. Lysates from myocytes were incubated with 2 µg of PP-1 antibody (Cell Signaling Technology) in a buffer containing (in mmol/l) 150 NaCl, 20 HEPES (pH 7.4), and 1 EDTA, with 0.1% Nonidet P-40, for 2 h at 4°C. Protein G-agarose (50 µl) was added, and the solutions were incubated at 4°C overnight. Pellets were rinsed four times, resuspended with sample buffer, boiled, and centrifuged, and the supernatants were subjected to immunoblotting (PLB mouse monoclonal, Affinity Bioreagents).

Statistical analysis. Values are means ± SE. For comparison between two groups, a Student's t-test was used. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Simulated ischemia study. Previous studies in in vivo and perfused heart models showed that the PKC activators result in cardioprotection that is comparable to the protective effect of ischemic PC (9, 38). Studies have also demonstrated that the protection afforded by a PKC activator can be blocked by a PKC peptide inhibitor ({epsilon}V1-2) (23). In this study, we demonstrate reduced cell death in DOG-treated myocytes after simulated ischemia. Before simulated ischemia, there was no difference in cell viability between untreated control and DOG (3 µM)-treated myocytes: 24.8 ± 3.4% and 24.0 ± 2.1%, respectively (Fig. 1). With simulated ischemia, cell death was increased in untreated control myocytes compared with myocytes treated with DOG. After 4 h of simulated ischemia, 70.7 ± 2.0% of control myocytes and 53.3 ± 1.7% of DOG-treated myocytes stained with trypan blue. The oxygenated control group retained a predominantly rod-shaped morphology and excluded trypan blue for the 4-h duration of the experimental protocol (Fig. 1, dashed line). These results indicate that 3 µM DOG can protect isolated myocytes.



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  		Fig. 1. Effect of 1,2-dioctanoyl-sn-glycerol (DOG) on simulated ischemia. Myocytes were treated for 10 min with or without 3 µM DOG and then subjected to 4 h of simulated ischemia. Cell viability was measured with trypan blue staining. Values are means ± SE; n = 4–7 rats. *Significantly different from control (P < 0.05).

 
Effect of DOG on caffeine-releasable SR Ca2+. We next investigated whether SR Ca2+ content was affected by activation of PKC with 3 µM DOG. We used caffeine-induced release of SR Ca2+, measured with fura 2 in isolated rat myocytes, as a measure of SR Ca2+ content. After rapid addition of caffeine (20 mmol/l), the amplitude of the caffeine-induced Ca2+ release was measured as an estimate of SR Ca2+ content. Figure 2 shows caffeine-releasable SR Ca2+ in untreated myocytes, in DOG-treated myocytes, and in myocytes treated with DOG + the PKC inhibitor chelerythrine. DOG treatment decreased caffeine-releasable SR Ca2+ (259 ± 45 and 448 ± 57 nmol/l in DOG-treated and control myocytes, respectively, P < 0.05), and chelerythrine blocked this DOG-induced reduction in SR Ca2+ content (259 ± 45 and 462 ± 72 nmol/l in DOG- and DOG + chelerythrine-treated myocytes, respectively, P < 0.05).



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  		Fig. 2. Sarcoplasmic reticulum (SR) Ca2+ estimated from caffeine-releasable Ca2+ in rat myocytes. DOG treatment decreased SR Ca2+, and the PKC inhibitor chelerythrine (CH) abolished the DOG effect. Fluorescence was converted to Ca2+ by calibration as described in MATERIALS AND METHODS. Dissociation constant was taken as 224 nM. Values are means ± SE; n = 4–6 rats. *Significantly different from control (P < 0.05). #Significantly different from DOG (P < 0.05).

 
[Ca2+]i transients. To address the mechanism responsible for the DOG-induced decrease in SR Ca2+ content, we investigated the effect of DOG on [Ca2+]i transient kinetics in myocytes stimulated at 0.5 Hz. Because the amplitude of the [Ca2+]i transient in DOG-treated myocytes was reduced (Fig. 3A), we examined the rate of Ca2+ uptake and release in stimulated DOG-treated myocytes. In DOG-treated myocytes, the half-decay time of the Ca2+ transient was prolonged compared with untreated myocytes (Fig. 3B): 172 ± 25 and 213 ± 25 ms at 0 and 10 min, respectively (P < 0.05). There was no difference in time to peak Ca2+ (Fig. 3C). Because the SR removes 92% of total activator Ca2+ in rat ventricular myocytes, the half-decay time can be used as an index of SR Ca2+ uptake (26). Therefore, the prolongation of the decay in the Ca2+ transient suggests that SR Ca2+ uptake is decreased by PKC activation.



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  		Fig. 3. Intracellular Ca2+ concentration transients. Myocytes loaded with fura 2 were stimulated at 0.5 Hz. A: typical single transient after 10 min without or with 3 µM DOG. B and C: half-decay time and time to peak shortening for the Ca2+ transient at 0 min (before treatment) and at 10 min with or without 3 µM DOG treatment. Values are means ± SE; n = 6 rats. *Significantly different from DOG at 0 min (P < 0.05).

 
Dephosphorylation at Ser16 on PLB. Ca2+ uptake is regulated by SERCA2 and PLB. Dephosphorylated PLB is an inhibitor of SERCA2, and phosphorylation of PLB relieves its inhibitory effects on SERCA2 (10, 21). We therefore examined whether DOG alters phosphorylation at Ser16 of PLB. Myocytes treated with DOG for 10 min showed a marked dephosphorylation of PLB compared with control myocytes (P < 0.05; Fig. 4). No difference in total PLB protein levels was detected between control and DOG-treated myocytes. Figure 5 shows a time course of PLB dephosphorylation at Ser16 after DOG treatment.



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  		Fig. 4. Top: representative Western blots. An antibody specific for Ser16-phosphorylated phospholamban (Phos-PLB) and an antibody that recognizes total PLB were used. DOG treatment for 10 min reduced phosphorylation of PLB at Ser16. Bottom: graphic representation of Western blot data. Values are means ± SE; n = 6–7 rats. *Significantly different from control (P < 0.05).

 


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  		Fig. 5. Time course of PLB dephosphorylation after DOG treatment. Top: Western blot for Ser16-phosphorylated PLB and total PLB. Middle and bottom: graphic representation of Western blot data. Values are means ± SE; n = 3–5 rats. *Significantly different from control (P < 0.05).

 
PP-1 and PLB. The DOG-dependent dephosphorylation of PLB is consistent with the DOG-induced reduction in SR Ca2+ content. Dephosphorylation of PLB is reported to be primarily mediated by PP-1 (25). To investigate whether PP-1 might be involved in PKC-dependent dephosphorylation of PLB, myocytes were treated with and without DOG for 10 min, extracted in lysis buffer, and immunoprecipitated to determine whether DOG increased the association between PP-1 and PLB. DOG treatment resulted in a significant increase in the association of PP-1 and PLB as determined by immunoprecipitation with PP-1 followed by PLB immunoblotting (Fig. 6). We also tested whether PKC-{epsilon} coimmunoprecipitated with PP-1, but we found no association in the presence or absence of DOG (data not shown).



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  		Fig. 6. Immunoprecipitation (IP) in isolated myocytes. Isolated myocytes were treated with or without 3 µM DOG for 10 min. Top: immunoprecipitated protein phosphatase (PP)-1 was immunoblotted with antibody for total PLB or PP-1. IP control #1, no lysate + IP antibody; IP control #2, lysate + protein G/no IP antibody. Bottom: mean densitometry data in arbitrary units (AU). Values are means ± SE; n = 6 rats. *Significantly different from control (P < 0.01).

 
PKC-{epsilon} regulates phosphorylation of PLB. Inasmuch as PKC-{epsilon} has been reported to mediate cardioprotection, we were interested in examining the role of PKC-{epsilon} in the dephosphorylation of PLB. We tested whether a PKC-{epsilon} isozyme-selective inhibitory peptide, {epsilon}V1-2 (20), would block the DOG-mediated decrease in PLB dephosphorylation. This eight-amino acid peptide, derived from the V1 region of PKC-{epsilon}, inhibits activation-induced translocation and function of PKC-{epsilon} in cardiomyocytes (20). Myocytes were treated for 20 min with 1 µM {epsilon}V1-2 peptide and then incubated with or without DOG for 10 min. The carrier peptide alone (1 µM) also was used as a negative control. Addition of DOG in the presence of an inactive control peptide resulted in a significant decrease in phosphorylation of PLB (Fig. 7), similar to that observed in Fig. 4. Addition of DOG in the presence of 1 µM {epsilon}V1-2 resulted in no change in phosphorylation of PLB. Interestingly, addition of {epsilon}V1-2 in the absence of DOG caused a slight but significant increase in phosphorylation of PLB/total PLB, suggesting a degree of basal activation of PKC-{epsilon} in normal cardiomyocytes.



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  		Fig. 7. PKC-{epsilon} modulates phosphorylation of PLB at Ser16. Top: Western blot of myocytes treated with PKC-{epsilon} isozyme-selective antagonist peptide {epsilon}V1-2 (PKC-{epsilon} inhibitor), PKC-{epsilon} inhibitor + DOG, control peptide, or control peptide + DOG. Myocytes were treated for 20 min with 1 µM PKC-{epsilon} inhibitor or control peptide; DOG was added for the last 10 min. Treatment with PKC-{epsilon} inhibitor blocked effects of DOG on phosphorylation of PLB at Ser16. Bottom: graphic representation of Western blot results for Ser16-phosphorylated PLB and total PLB. Values are means ± SE; n = 3 rats. *Significantly different from PKC-{epsilon} inhibitor + DOG (P < 0.05). #Significantly different from PKC-{epsilon} inhibitor (P < 0.01).

 
Effect of PKC-{epsilon} on caffeine-releasable SR Ca2+ and SR function. We further tested whether the PKC-{epsilon}-selective inhibitor peptide would affect the DOG-mediated decrease in caffeine-releasable SR Ca2+ described in Fig. 2. The PKC-{epsilon} inhibitor blocked the DOG-induced reduction in SR Ca2+ content: 188 ± 31 and 324 ± 44 nmol/l with DOG and PKC-{epsilon} inhibitor + DOG, respectively (P < 0.01; Fig. 8A).



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  		Fig. 8. PKC-{epsilon} inhibition blocks DOG-induced decrease in SR Ca2+. A: SR Ca2+ estimated from caffeine-releasable Ca2+ in DOG-treated myocytes with and without selective PKC-{epsilon} inhibitor. B and C: half-decay time and time to peak shortening for Ca2+ transient at 0 min (before treatment with PKC-{epsilon} inhibitor or DOG) and in DOG-treated myocytes with and without 1 µM PKC-{epsilon} inhibitor. Values are means ± SE; n = 3–4 rats. *Significantly different from control peptide + DOG (P < 0.01).

 
We also examined whether the PKC-{epsilon} inhibitor peptide would block the DOG-induced prolongation of the decay of the Ca2+ transients (Fig. 3). Addition of the PKC-{epsilon} inhibitor blocked the DOG-induced prolongation of the half-decay time of the Ca2+ transient (Fig. 8B).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
PKC and cardioprotection. A role for PKC in cardioprotection has been well established (9, 13, 17, 19, 24, 27, 38, 40). PC has been shown to result in the translocation of PKC, specifically PKC-{epsilon}, to membrane fractions, and inhibitors of PKC have been shown to block the protection afforded by PC (38). Furthermore, mice with moderate cardiac-specific overexpression of PKC-{epsilon} have been shown to be protected against ischemia-reperfusion injury compared with wild-type littermates (11). Similarly, hearts of mice overexpressing a peptide that enhances translocation and function of PKC-{epsilon} or pigs treated in vivo with this peptide by a single intracoronary infusion before or early during the ischemic period exhibit a >60% cardioprotection (13, 19). Despite the well-established role for PKC-{epsilon} in cardioprotection, the target mechanisms by which PKC mediates protection are just beginning to be elucidated. PKC has been shown to phosphorylate troponin I (5), and PKC has been suggested to activate the mitochondrial ATP-sensitive K+ channel (37). PKC-{epsilon} has also been reported to interact with components of the mitochondrial permeability transition pore (2) and to mediate phorbol ester-induced phosphorylation of connexin-43 (12). PKC-{epsilon} also forms a complex with Lck (32) or MAPK, resulting in phosphorylation of Bad (3).

PKC and SR Ca2+ content. PKC activation has also been reported to modulate cytosolic Ca2+, contractility, and SR Ca2+ (6, 18, 29, 31, 35, 36). Given the importance of cytosolic and SR Ca2+ in ischemia-reperfusion injury, we investigated whether SR Ca2+-handling proteins might be additional targets of PKC. Consistent with the majority of data (6, 18, 29, 35, 36), we found that addition of DOG results in a decrease in SR Ca2+ content (~65% decrease), which was abolished by addition of the PKC inhibitor chelerythrine or the selective PKC-{epsilon} inhibitor peptide. A decrease in SR Ca2+ would be expected to be cardioprotective, inasmuch as it would decrease SR Ca2+ cycling, reduce a potential trigger for arrhythmias, and potentially attenuate the rise in cytosolic Ca2+ during ischemia and reperfusion (8, 39). Previous data suggested that modulation of SR Ca2+ is important in cardioprotection (42). In addition, studies have suggested that SR Ca2+ cycling is an important determinant of survival of ischemic or metabolically inhibited myocytes (14, 22). Cave and Garlick (7) showed that addition of ryanodine and cyclopiazonic acid does not block the protection afforded by PC and concluded that a "functional" SR is not required for PC. However, these data do not rule out the hypothesis that reduced SR Ca2+ content is protective, inasmuch as ryanodine and cyclopiazonic acid will reduce SR Ca2+.

PKC and PLB. Activation of PKC has been reported to reduce SR Ca2+; however, the mechanism by which PKC alters SR Ca2+ content is not established. Although PKC can phosphorylate PLB in vitro, activation of PKC does not result in an increase of 32P incorporation into PLB in vivo under conditions where adrenergic receptors were inhibited (15). We found that DOG decreased phosphorylation of PLB at Ser16 by >80% (Figs. 4 and 5). The effect of DOG was rapid, because 1 min of treatment was sufficient to reduce phosphorylation of PLB. We further demonstrated that the effect of DOG was mediated by PKC-{epsilon}, because an isoform-specific inhibitor of PKC-{epsilon} blocked the DOG-dependent decrease in phosphorylation of PLB. In addition, DOG enhanced the interaction of PP-1 with PLB, providing additional information regarding the mechanism by which PKC reduces phosphorylation of PLB. PLB is primarily dephosphorylated by the action of PP-1 and PP-2a (25), and PP-1 is regulated by PP inhibitor-1, which is subject to protein kinase A regulation through phosphorylation at Thr35 (28). Interestingly PKC-{alpha} has recently been shown to influence binding of PP inhibitor-1 to PP-1 (4).

In conclusion, the present data have shown that activation of PKC under conditions that lead to cardioprotection reduces SR Ca2+ loading by a mechanism involving PKC-{epsilon}-mediated dephosphorylation of PLB. Activation of DOG is also shown to enhance binding of PP-1 to PLB, thereby resulting in decreased phosphorylation of PLB, which in turn leads to a decrease in SR Ca2+ content. A decrease in SR Ca2+ at the start of ischemia could lead to less SR Ca2+ cycling and could attenuate the rise in cytosolic Ca2+ during ischemia and reperfusion, which would be cardioprotective (34). A decrease in SR Ca2+ cycling would reduce the consumption of ATP; interestingly, a reduction in ATP consumption during ischemia has been shown in hearts with cardiac-specific overexpression of PKC-{epsilon} (11). SR Ca2+ is also important in the development of arrhythmias, and activation of PKC-{epsilon} has been reported to have antiarrhythmic efficacy (19). These findings lend support to the concept that modulation of SR function might be a new important cardioprotective strategy.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Daria Mochly-Rosen for the gift of PKC-{epsilon} inhibitory peptides and John Petranka for suggestions and help with technical procedures.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. Murphy, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, 111 T. W. Alexander Dr., Bldg. 101, MD F2-07, Research Triangle Park, NC 27709 (e-mail: murphy1{at}niehs.nih.gov)

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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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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