Role of G Proteins and Modulation of p38 MAPK Activation in the Protection by Nitric Oxide against Ischemia–Reoxygenation Injury

Biochemical and Biophysical Research Communications 286, 995–1002 (2001) doi:10.1006/bbrc.2001.5477, available online at http://www.idealibrary.com on

Role of G Proteins and Modulation of p38 MAPK Activation in the Protection by Nitric Oxide against Ischemia–Reoxygenation Injury Roby D. Rakhit, 1 Alamgir N. M. Kabir, James W. Mockridge, Adrian Saurin, and Michael S. Marber Department of Cardiology, Kings College London, St. Thomas’ Hospital, Lambeth Palace Road, London SE1 7AH, United Kingdom

Received July 27, 2001

Protein kinase C (PKC)-mediated regulation of the mitogen-activated protein kinases (MAPK) may play a role in the protection afforded by ischemic preconditioning (PC). Nitric oxide (NO) can influence MAPK activation via interaction with PKC or farnesylation of low-molecular-weight (LMWT) G proteins. However, we have recently reported the mechanism of NO-induced cardioprotection to be a PKC-independent process. Therefore, we investigated the role of LMWT G proteins and MAPK signaling in NO-induced cardioprotection against simulated ischemia–reoxygenation (SI-R) injury. Neonatal rat cardiomyocytes treated for 90 min with the NO donor S-nitroso-N-acetyl-L,L-penicillamine (SNAP) 1 mM were protected against 6 h of SI (hypoxic conditions at 37°C with 20 mM lactate, 16 mM KCl at pH 6.2) and 24 h reoxygenation under normal culture conditions. NOinduced protection was blocked by the G protein inhibitor ␣-hydroxyfarnesylphosphonic acid (␣HFP) 10 ␮M. We studied the time course of p42/44 and p38 MAPK dual-phosphorylation hourly during SI using phosphospecific antibodies. p38 was phosphorylated during SI and the peak phosphorylation was significantly delayed by SNAP pretreatment. The p38 inhibitor SB203580 1 ␮M, given during SI, protected against injury. Thus the delay in peak p38 activation may contribute to, rather than be the effect of, NO-induced cardioprotection. We have shown that p38␤ does not contribute to the total p38 signal in our extracts. Thus there is no detectable ␤ isoform. We conclude that the main isoform present in these cells and thought to be responsible for the observed phenomenon, is the ␣ isoform. © 2001 Academic Press Key Words: G proteins; mitogen-activated protein kinase; nitric oxide; preconditioning; p38.

1 To whom correspondence should be addressed at Waller Department of Cardiology, St. Marys Hospital, Praed Street, London W2 1NY, United Kingdom. Fax: ⫹44-(0)-20-7886-1763. E-mail: [email protected].

We have recently reported the cardioprotective effects of nitric oxide (NO) in a model of simulated ischemia–reoxygenation (SI-R) and early preconditioning (PC) in neonatal rat ventricular cardiomyocytes. The mechanism of this protection appears to be cGMP-dependent but independent of protein kinase C (PKC) or ATP-sensitive potassium channels (1). Recent evidence has shown that the downstream signaling mechanisms of IPC may involve the PKCmediated regulation of the mitogen activated protein kinases (MAPKs). In particular, the modulation of p38 MAPK activity both during PC ischemia and lethal ischemia may be pivotal in cardioprotection. However, the exact role of p38 in PC is controversial and remains unclear. NO can influence downstream MAPK activation at two levels. First, via the nitrosylation of critical thiol residues, it may activate specific PKC isoforms (2, 3). However, NO may also signal independent of PKC via the farnesylation of low-molecular-weight (LMWT) G proteins (4). The G proteins p21 ras, Rac 1 and Cdc42 are important molecular switches critical in regulating the activation of ERK, JNK and p38 signaling. G proteins must undergo a series of posttranslational modifications before they can localize at the cytoplasmic face of the plasma membrane in order to be active. An important posttranslational modification is the addition of a farnesyl group to the carboxyl-terminal end of the newly synthesized G protein. This step is catalyzed by the enzyme farnesyl transferase, which can be blocked by ␣-hydroxyfarnesylphosphonic acid (␣HFP) (5). Lander et al. have implicated p21 ras as a common signaling target for oxidative agents such as H 2O 2, hemin and nitric oxide in a rat pheochromocytoma cell line (6). This is supported by Aikawa et al. who have shown that H 2O 2 activates ERKs via Ras in cultured neonatal cardiac myocytes (7). Evidence also suggests that NO can differentially activate MAPKs in a Jurkat human

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T-cell leukemia line via a LMWT G-protein-dependent mechanism (4). We hypothesized that NO may influence MAPK kinase signaling independent of PKC by the direct interaction with LMWT G proteins and that modulation of MAPK phosphorylation during ischemia might be involved in the mechanism of protection. The aim of this study was to explore the downstream signaling mechanisms involved and, in particular, to interrogate MAPK signaling in the mechanism of NO-induced cardioprotection against SI-R injury. MATERIALS AND METHODS Materials

mM CaCl 2 䡠 2H 2O, 1.2 mM MgCl 2, 0.5 mM sodium EDTA 䡠 2H 2O, 20 mM sodium lactate and 16 mM KCl, pH 6.2). Near anoxic conditions were achieved using the BBLGasPak Pouch System (Becton– Dickinson, Cockeysville, U.S.A.). This system provides a compact microenvironment contained in an impermeable bag which, when properly activated and sealed, provides anaerobic conditions with an O 2 concentration of less than 1% within 2 h of incubation at 37°C. First, the liquid activating reagent (containing 5 g of the following: iron powder, calcium carbonate, citric acid and inert extender) is placed in the reagent channel of the GasPak Pouch. Then the 6- or 12-well plate is heat-sealed within the bag. The catalytic reaction which ensues consumes O 2 and produces CO 2. An anaerobic strip on the surface of the bag turns from blue to white once anoxic conditions are achieved. This method of producing hypoxia, after 6 h incubation at 37°C in 95% air, 5% CO 2, produced approximately 50% cell death in control cells.

Evaluation of Cell Viability

Dulbecco’s modified Eagle medium, medium 199 (M199), bovine serum albumin, horse serum, fetal calf serum (FCS), pancreatin, and penicillin/streptomycin were from Life Technologies, Inc. (Paisley). Collagenase was from Worthington. Hepes was from Sigma (Poole, UK). S-Nitroso-N-acetyl-L,L-penicillamine (SNAP) and SB203580 was obtained from Calbiochem. ␣HFP was obtained from Biomol Research Laboratories. Mouse monoclonal ERK 21 primary antibodies (Santa Cruz Biotechnology Inc); rabbit anti-mouse IgG secondary antibody (DAKO). Rabbit polyclonal phospho-specific JNK, p42/44 and p38 primary antibodies (New England Biolabs); swine antirabbit IgG secondary antibody (DAKO).

Isolation and Culture of Rat Ventricular Cardiomyocytes Neonatal rat ventricular cardiomyocytes were prepared from 1- to 2-day-old Sprague–Dawley rats as described previously (8). Briefly, cells from neonatal rat ventricles were dispersed in a series of incubations at 37°C in Hepes-buffered salt solution containing 0.6mg/ml pancreatin and 0.5 mg/ml collagenase. The dispersed cells were preplated for at least 30 min to minimize fibroblast contamination, and the unattached cells were re-plated on 6-well gelatin-coated plates at a density of 1–1.5 million cells/well. Fibroblast contamination was less than 5%. The cardiac myocytes were cultured at 37°C, in room air with 5% CO 2 in 4:1, Dulbecco’s modified Eagle medium: M199, supplemented with 10% horse serum, 5% FCS and 100 units/ml penicillin/streptomycin for the first 24 h. Thereafter, cells were maintained in an identical medium with a reduced serum concentration of 1% FCS. Under these conditions in excess of 80% of cells beat spontaneously for the duration of the experiment. Experiments were performed after 2 days in culture.

Recombinant Adenovirus Vector Growth and Transfection Recombinant adenoviruses encoding wild-type p38␣ or wild-type p38␤ driven by a cytomegalovirus promoter were generated as described previously (9, 10). Recombinant adenoviruses were tested for transgenic expression in cardiac myocytes by reverse transcriptase– polymerase chain reaction, Western blot, or kinase assays. The concentrated recombinant adenoviruses were prepared and titered as described (10). Cells maintained in serum-free medium were infected with adenoviruses at a multiplicity of infection of 10 plaque forming unit/cells for 1 h at 37°C in room air containing 5% carbon dioxide. Cells were then cultured in maintenance medium containing 1% FCS for an additional 48 –72 h before analysis.

Simulated Ischemia Model Cells were washed with PBS prior to addition of 1 ml ischemia buffer (118 mM NaCl, 24 mM NaHCO 3, 1 mM NaH 2PO 4 䡠 H 2O, 2.5

Cell viability was quantified using MTT (3-4,5-dimethylthiazol-2yl-2,5-diphenyltetrazolium bromide) bioreduction assay (11). Briefly, cell culture plates were washed with PBS, exposed to 5 mg/ml of MTT solution and placed in an incubator at 37°C for 20 –30 min. The dark blue crystals which formed were dissolved in stop solution containing 0.1 M HCl, 10% Triton ⫻ 100 and isopropanol and the absorbance read at 570 nm wavelength.

Measurement of Lactate Dehydrogenase (LDH) Activity Following the end of simulated ischemia, buffer was gently aspirated and saved for LDH determination. A spectrophotometric LDH enzyme assay was performed with a Sigma assay kit (TOX-7) (12).

Western Blotting Analysis for MAPK Phosphorylation Cells were harvested at specific time points indicated in experimental protocols. Cells were washed three times with PBS and harvested in 1 ml of sample buffer (250 mM Tris–HCl, pH 6.8, 4% SDS, 10% glycerol, and 2% ␤-mercaptoethanol) and then boiled for an additional 5 min. The cell extracts were then centrifuged for 5 min to remove insoluble material. The samples were then loaded on a 7.5% polyacrylamide gel and after one-dimensional separation were transferred to nitrocellulose membranes (Hybond C, Amersham, UK). Uniform protein loading was confirmed by Coomassie staining of identically loaded gels and blotting for ERK2 protein. The blots were probed with rabbit polyclonal phospho-specific JNK, p42/44 and p38 primary antibodies and a peroxidase-conjugated swine antirabbit IgG secondary antibody prior to detection with enhanced chemiluminescence (ECL, Little Chalfont, UK). These antibodies recognize the phosphorylation of key threonine and tyrosine residues within the activation loop of the MAPKs. PMA-treated cells were used as a positive control for phospho p42/44 and sorbitol treatment was used as a positive control for phospho p38.

Experimental Protocols (Fig. 1) Study 1: Effect of ␣HFP on SNAP-induced cardioprotection. We have previously characterized a dose–response relationship for SNAP-triggered protection in this model with a 1 mM dose providing robust and optimum protection (13). Therefore, cells were treated for 90 min with SNAP 1 mM prior to 30 min of reoxygenation in normal culture conditions and 6 h of SI using the BBLGasPak Pouch System. The effect of ␣HFP 10 ␮M, given during SNAP treatment, was studied on SNAP-induced protection. LDH was measured at the end of long ischemia and MTT bioreduction assay was measured after 24 h reoxygenation in normal culture conditions as already described.

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FIG. 1. Experimental protocols. The following experiments were performed on neonatal rat ventricular myocytes in primary culture. Study 1: S-nitroso-N-acetyl-L,L-penicillamine (SNAP)-induced protection. Cells are pretreated for 90 min with 1 mM SNAP followed by 30 min reperfusion (R) before 6 h simulated ischemia (SI). Effect of ␣-HFP. Study 2: effect of 90 min treatment with SNAP 1 mM on the phosphorylation status of JNK, p42/44, and p38 mitogen-activated protein kinases (MAPK). Study 3: effect of SNAP 1 mM pretreatment on the time course of phosphorylation of p38 and p42/44 MAPK during 6 h SI. Study 4: effect of the p38 MAPK inhibitor SB203580 (1 and 10 ␮M) given during 6 h SI.

Study 2: Effect of SNAP on MAPK phosphorylation. The effect of 90-min treatment with SNAP 1 mM was studied on the phosphorylation status of JNK, p42/44, and p38 MAPK using Western blotting with polyclonal phospho-specific antibodies as described under Materials and Methods. Cells were harvested at 15-min intervals during SNAP treatment. Study 3: Effect of SNAP on the time course of MAPK phosphorylation during 6 h simulated ischemia. We studied the effect of 90 min treatment with SNAP 1 mM, followed by 30 min reperfusion, on the phosphorylation of p42/44 and p38 MAPKs during 6 h SI. The phosphorylation status of JNK was not studied as the JNKs are known not to be activated during ischemia (14). Cells were cultured on 60 mm single plates and either untreated (controls) or treated with SNAP plus 30 min reperfusion. Control and SNAP-treated plates were placed in pairs into a BBLGasPak Pouch and cells were har-

vested for Western blotting analysis during SI at 1, 2, 3, 4, and 5 h intervals after bag closure. Study 4: The effect of SB 203580 during 6 h simulated ischemia. We studied the effect of the p38 inhibitor SB203580 at 1 and 10 ␮M doses given during 6 h of SI in order to corroborate recent data suggesting that p38 phosphorylation during lethal SI is deleterious. SB203580 1 ␮M was used because at this dose it is most specific for p38 as it does not appear to block cyclooxygenase or thromboxane synthase (15). Study 5: Assessment of the p38 isoforms present in cultured neonatal cardiocytes. The p38 isoforms, which are responsible for ischemic protection, remain controversial 16. We have used pan p38 and p38␤-specific antibodies to examine this question. In addition, to act as controls, we have probed neonatal cardiocytes that were trans-

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fected with the ␣, or ␤ isoforms of p38 and compared these to myocytes harvested during simulated ischemia at zero, 3 and 4 h.

Statistical Analysis Data are expressed as mean ⫾ SEM. The “n” numbers refer to the number of wells from which data was obtained from at least 4 separate experimental preparations. For the ␣HFP study MTT/LDH data comparisons between groups were analyzed using a Kruskal– Wallis nonparametric one-way ANOVA combined with Dunn’s multiple comparisons test. For the SB203580 study comparisons between groups were analyzed using a parametric one-way ANOVA combined with the Tukey–Kramer multiple comparisons test (Graphpad Instat 1998). A P value of ⬍0.05 was considered to be statistically significant.

RESULTS

␣HFP Inhibits SNAP-Induced Cardioprotection ␣HFP 10 ␮M given during 90 min SNAP treatment abrogated the cardioprotective effect of SNAP producing a reduction in MTT from 164.0 ⫾ 23.3 to 104.5 ⫾ 8.3% and an increase in LDH release from 68.9 ⫾ 4.7 to

FIG. 3. Effect of 90 treatment with SNAP 1 mM on phosphorylation of p42/44, p38, and JNK MAPKs. ERK2 protein blot was used to assess protein loading. Positive controls (⫹ve C) are shown in the first lane except for JNK for which a ⫹ve C was not available.

103.0 ⫾ 10.1% (Fig. 2). 90 min treatment with ␣HFP 10 ␮M alone had no detrimental effect on cell viability (data not shown). SNAP Treatment Alone Does Not Phosphorylate JNK and p38 MAPK PMA-treated cells were used as a positive control for phospho p42/44 and sorbitol-treated cells were used as a positive control for phospho p38. The results of these experiments are shown in Fig. 3. P42/44: high basal levels of phospho p42/44 have been noted in untreated cells (data not shown) so that interpretation of the effects of any intervention are difficult. SNAP treatment did not appear to have any effect on p42/44 phosphorylation compared to untreated cells. p38 and JNK: 90 min treatment of SNAP did not cause any detectable phosphorylation of p38 or JNK. SNAP Pretreatment Delays Peak p38 MAPK Phosphorylation during Ischemia Compared to Controls

FIG. 2. Effect of ␣HFP on SNAP-induced cardioprotection against simulated ischemia *P ⬍ 0.05, **P ⬍ 0.01.

The results of these experiments are summarized in Fig. 4. Blot for ERK2 protein showed even protein loading in control and SNAP-treated cells (Fig. 4A). p42/44: in untreated cells the basal phosphorylation seen was rapidly suppressed after 1 h of ischemia. After this time p44 phosphorylation was no longer detected with only a small amount of p42 phosphorylation remaining. Following SNAP treatment and 30 min reperfusion a similar response was seen (Fig. 4B). p38: phosphorylation was not detectable in untreated control cells but during ischemia phosphorylation of p38 occurred with a maximum peak at 3 h after which it became rapidly dephosphorylated. In SNAP pretreated cells there appeared to be a striking delay in p38 phosphorylation with a maximum peak at 4 h

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this implies that SB 203580 fails to protect early after ischemia. The reason for this is unclear. Similar discordant effects have been demonstrated with SB 203580 using 3 different methods of determining cell viability (29) and reflects the nature of the model/ pattern of protection. 6 h treatment with SB203580 1 and 10 ␮M alone had no detrimental effect on cell viability (data not shown). p38␣ Not ␤ Is Expressed during Simulated Ischemia Our experiments have shown that the p38 in our extracts shows none of the ␤ isoform. We have used neonatal cardiac myocytes, which have been transfected with p38␤ to act as a control. We hold that the main isoform in the cells we used thought to be responsible for the observed phenomenon are the ␣ isoform (see Fig. 8). DISCUSSION Implication of results

FIG. 4. Effect of SNAP on the time course of MAPK phosphorylation during severe hypoxia. C, untreated control; SI, simulated ischemia.

compared to controls (Fig. 4C). A similar pattern of delayed peak p38 phosphorylation has been shown in response to preconditioning of rat neonatal ventricular myocytes (30). A further experiment was performed in a different cell preparation comparing 3 and 4 h samples taken during ischemia in control and SNAP treated cells. A similar delay in p38 phosphorylation was seen in SNAP-treated cells (see Fig. 5).

The data presented lend support for the interaction of NO with MAPK signaling in the context of I/R. The finding that pharmacological early PC with SNAP is abrogated by ␣HFP suggests that the signaling responsible for affording protection is dependent on the farnesylation of low molecular G proteins. Since we have previously reported that the mechanism of NO-induced cardioprotection is PKC-independent, this represents a novel pathway by which NO might modulate MAPK signaling distal to PKC (Fig. 7). Study 2 investigated whether SNAP 1 mM when given for 90 min (the time course known to induce pharmacological PC) itself phosphorylated MAPKs and in particular p38 MAPK because it has been shown that PC ischemia can induce p38 phosphorylation in several studies (17–19). Also, a recent study has dem-

SB 203580 Protects during 6 h Simulated Ischemia The p38 inhibitor SB203580 1 and 10 ␮M given during 6 h of SI showed differential effects on the cell viability assays used (see Fig. 6). Using MTT bioreduction SB203580 1 ␮M was significantly cardioprotective increasing cell viability from 100% in control cells to 142.3 ⫾ 11.9%. SB203580 10 ␮M produced a nonsignificant increase in cell viability from 100% to 121.6 ⫾ 10.5%. However neither 1 or 10 ␮M doses had any cardioprotective effect on reducing LDH release compared to controls. As the LDH assay was performed immediately after the cessation of prolonged ischemia

FIG. 5. Effect of SNAP on the time course of p38 phosphorylation during severe hypoxia. Samples were harvested at 3 and 4 h during long simulated ischemia.

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FIG. 7. Schematic showing the interaction between NO and MAPK signaling. NO could either act via the activation of PKC or via the direct activation of LMWT G proteins.

to rather than be the effect of early pharmacological PC by NO. Role of p38 during Ischemia/Preconditioning Controversy exists over the role of p38 MAPK in the context of ischemia and adaptation to ischemia during PC. p38-MAPK and its downstream target MAPKAPK2 have been shown to be activated during ischemia and this activation is sustained or increased during reperfusion (14). Subsequently Maulik et al. showed in isolated perfused rat hearts that, in response to PC ischemic stress, p38 was phosphorylated which in turn activated MAPKAPK2 (19). This was

FIG. 6. Effect of SB203580 1 and 10 ␮M given during 6 h of simulated ischemia. ***P ⬍ 0.001.

onstrated p38 phosphorylation in human neutrophils after 20 min exposure to SNAP (20). However, 90 min SNAP exposure did not affect the high basal levels of p42/44 phosphorylation seen in untreated cardiomyocytes and did not induce JNK phosphorylation. Neither did it induce phosphorylation of p38. Study 3 went on to characterize the phosphorylation of p38 and p42/44 MAPKs during long SI. SNAP did not alter the rapid dephosphorylation of p42/44 seen in response to ischemia. However SNAP appeared to delay the time course of peak p38 phosphorylation during ischemia. Study 4 supports a deleterious role for p38 phosphorylation during long ischemia. Therefore, the delay in peak p38 phosphorylation during long SI may represent a protective phenomenon. However, what cannot be concluded from these studies is whether the delay in peak p38 phosphorylation is the cause or effect of NOinduced cardioprotection. Therefore, it is proposed that the delay in peak p38 phosphorylation may contribute

FIG. 8. Investigation of p38 isoforms present in extracts. The extracts do not contain any p38 ␤. Cells with p38 ␤-transfected acted as controls. We hold that the main isoform in cells responsible for this observation is the ␣ isoform.

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thought to represent a possible adaptive phenomenon because MAPKAPK2 can phosphorylate the cytoprotective heat shock proteins HSP25/27, which are thought to modulate cytoskeletal integrity (21). However, more recent studies, in agreement with our findings, have suggested a deleterious role for p38 activation during ischemia. A protective role for the p38 inhibitor SB203580 has so far been demonstrated in neonatal rat cardiac myocytes (22), the Langendorffperfused rabbit heart (18) and the in vivo pig heart (23) via a possible anti-apoptotic mechanism. At least 4 members of the p38 MAPK family have been identified and its dichotomous effects may be explained by the relative expression of a particular isoform. The isoforms ␣ and ␤ are both expressed in the heart and despite sharing approximately 74% sequence identity, they have been suggested to have opposing functions in cardiac myocytes. Using co-expression of individual wild-type or dominant negative p38 MAPK isoforms Wang et al. (24) showed that the ␣ isoform was proapoptotic whereas the ␤ isoform mediated a hypertrophic response. Therefore p38␤ may protect against whereas p38␣ may promote apoptotic cell death. In our experiments we have shown that during SI the p38␣ isoform is activated representing a novel mechanism contributing to cellular injury.

are necessary explore to this complex relationship more fully. CONCLUSION This study provides new data implicating a role for protein farnesylation of LMWT G proteins or other proteins requiring posttranslational farnesylation in the signaling mechanism of NO-induced cardioprotection. The modulation of MAPK activation through the delayed peak p38 phosphorylation seen during ischemia in response to pharmacological preconditioning by SNAP may represent a novel mechanism resulting in delayed cell death and further add to our understanding of the emerging cytoprotective properties of NO. ACKNOWLEDGMENTS This study was funded by the British Heart Foundation and Wellcome Trust. Dr. Yibin Wang, Department of Physiology, University of Maryland School of Medicine, provided the adenoviral vectors encoding p38-MAPK isotypes.

REFERENCES

NO and Activation of MAPKs The ability of NO to interact with thiol and metalloprotein groups in a diverse manner and, as already discussed, the ability to farnesylate key low molecular weight G proteins have implicated a possible interaction of NO with MAPK signaling. However, there is a paucity of data directly implicating NO as a modulator of MAPK activation in the myocardium. A recent study has demonstrated the ability of sodium nitroprusside to activate p42/44, p38 MAPK and MAPKAPK2 in adult rat cardiac myocytes via both cGMP-dependent and cGMP-independent mechanisms (25). Other data have focused on the cytokine regulation of iNOS. Evidence in cardiac myocytes suggests that the IL-1␤ and IFN␥ regulation of iNOS may involve the activation of p42/44 and p38 MAPKs (1, 14). The indirect modulation of MAPKs by NO is evidenced in a study by Wang et al. who showed that NO suppressed the angiotensin I-induced activation of ERK in cardiac fibroblasts (26). Several studies in non-cardiac cell types have shown that NO can directly activate MAPKs in mitogenic cell lines in the study of NO-induced apoptosis (27, 28). These studies either use high-output NO donors or NO generated by lipopolysaccharide-induced iNOS induction in order to study the cytotoxic effects of pathologically high levels of NO. It is possible that differential effects on MAPK activation may occur in response to physiological concentrations of NO in the context of cytostasis and cytoprotection. Clearly, further studies 1001

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