p38 Mitogen-activated Protein Kinase Pathway Protects Adult Rat Ventricular Myocytes against β-Adrenergic Receptor-stimulated Apoptosis EVIDENCE FOR Gi-DEPENDENT ACTIVATION

Abstract We have shown that stimulation of β-adrenergic receptors (β-AR) by norepinephrine (NE) increases apoptosis in adult rat ventricular myocytes (ARVMs) via a cAMP-dependent mechanism that is antagonized by activation of Giprotein. The family of mitogen-activated protein kinases (MAPKs) is involved in the regulation of cardiac myocyte growth and apoptosis. Here we show that β-AR stimulation activates p38 kinase, c-jun N-terminal kinases (JNKs), and extracellular signal-regulated kinase (ERK1/2) in ARVMs. Inhibition of p38 kinase with SB-202190 (10 μm) potentiated β-AR-stimulated apoptosis as measured by flow cytometry and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining. SB-202190 at this concentration specifically blocked β-AR-stimulated activation of p38 kinase and its downstream substrate MAPK-activated protein kinase-2 (MAPKAPK2). Pertussis toxin, an inhibitor of Gi/Go proteins, blocked the activation of p38 kinase and potentiated β-AR-stimulated apoptosis. Activation of Gi protein with the muscarinic receptor agonist carbachol protected against β-AR-stimulated apoptosis. Carbachol also activated p38 kinase, and the protective effect of carbachol was abolished by SB-202190. PD-98059 (10 μm), an inhibitor of ERK1/2 pathway, blocked β-AR-stimulated activation of ERK1/2 but had no effect on apoptosis. These data suggest that 1) β-AR stimulation activates p38 kinase, JNKs, and ERK1/2; 2) activation of p38 kinase plays a protective role in β-AR-stimulated apoptosis in cardiac myocytes; and 3) the protective effects of Gi are mediated via the activation of p38 kinase.


We have shown that stimulation of ␤-adrenergic receptors (␤-AR) by norepinephrine (NE) increases apoptosis in adult rat ventricular myocytes (ARVMs) via a cAMP-dependent mechanism that is antagonized by activation of G i protein. The family of mitogen-activated protein kinases (MAPKs) is involved in the regulation of cardiac myocyte growth and apoptosis.
Here we show that ␤-AR stimulation activates p38 kinase, c-jun N-terminal kinases (JNKs), and extracellular signal-regulated kinase (ERK1/2) in ARVMs. Inhibition of p38 kinase with SB-202190 (10 M) potentiated ␤-ARstimulated apoptosis as measured by flow cytometry and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining. SB-202190 at this concentration specifically blocked ␤-AR-stimulated activation of p38 kinase and its downstream substrate MAPK-activated protein kinase-2 (MAPKAPK2). Pertussis toxin, an inhibitor of G i /G o proteins, blocked the activation of p38 kinase and potentiated ␤-AR-stimulated apoptosis. Activation of G i protein with the muscarinic receptor agonist carbachol protected against ␤-ARstimulated apoptosis. Carbachol also activated p38 kinase, and the protective effect of carbachol was abolished by SB-202190. PD-98059 (10 M), an inhibitor of ERK1/2 pathway, blocked ␤-AR-stimulated activation of ERK1/2 but had no effect on apoptosis. These data suggest that 1) ␤-AR stimulation activates p38 kinase, JNKs, and ERK1/2; 2) activation of p38 kinase plays a protective role in ␤-AR-stimulated apoptosis in cardiac myocytes; and 3) the protective effects of G i are mediated via the activation of p38 kinase.
Apoptosis occurs in the myocardium of patients with endstage heart failure and myocardial infarction and in animal models of myocardial hypertrophy and failure (1)(2)(3)(4). We demonstrated that stimulation of ␤-adrenergic receptors (␤-AR) 1 induces apoptosis in adult rat ventricular myocytes (ARVMs) in vitro (5). This effect was mimicked by the adenylyl cyclase stimulator forskolin and blocked by inhibition of protein kinase A, suggesting a role for cAMP in ␤-AR-stimulated apoptosis in ARVMs. Likewise, Iwai-Kanai et al. (6) showed that ␤-AR stimulation increases apoptosis in a cAMP-protein kinase Adependent manner in neonatal rat cardiac myocytes. We further demonstrated that activation of G i inhibits ␤-AR-stimulated apoptosis in ARVMs (7).
Activation of ERK1/2 has been implicated in the regulation of cellular growth and survival (9, 10) and protects cells against cellular stress (18). In PC12 cells, activation of p38 and JNKs with concurrent inhibition of ERK1/2 was found to induce apoptosis (19). In cardiac cells, p38 kinase and JNKs appear to be involved in mediating growth and apoptosis (20 -22). The molecular mechanisms involved in ␤-AR-stimulated apoptosis in adult cardiac myocytes are largely unknown. This study was undertaken to define the role of MAPKs in ␤-AR-stimulated apoptosis and to test the hypothesis that the antiapoptotic effects of G i are mediated via the activation of MAPKs.

Myocyte Isolation and Culture
Calcium-tolerant ARVMs were isolated from the hearts of adult male Harlan Sprague-Dawley rats (200 -220 g) as described by Singh et al.
* Supported by Grants HL-057947 (to K. S.), HL-42539 and HL-61639 (to W. S. C.); a grant-in-aid from the American Heart Association (AHA), New England Affiliate (to K. S.); a Merit grant from the Department of Veterans Affairs (to K. S.); and a fellowship from the AHA, Massachusetts Affiliate (to C. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. (23). Briefly, hearts were perfused retrogradely with nominally Ca 2ϩfree Krebs-Henseleit bicarbonate buffer and were minced and dissociated in the same buffer containing 0.02 mg/ml trypsin and 0.02 mg/ml deoxyribonuclease. The cell mixture was filtered and sedimented through 60 g/ml bovine serum albumin (Sigma) to separate ventricular myocytes from nonmyocyte cells. The cell pellet was resuspended in ACCT medium consisting of Dulbecco's modified Eagle's medium with 2 mg/ml bovine serum albumin, 2 mM L-carnitine, 5 mM creatine, 5 mM taurine, 100 IU/ml penicillin, and 100 g/ml streptomycin.
The ARVMs were then plated in ACCT medium at a density of 30 -50 cells/mm 2 on 100-mm culture dishes (Fisher Scientific) or glass coverslips (Fisher Scientific) precoated with laminin (1 g/cm 2 , Becton-Dickinson). After 1 h, the dishes were washed with ACCT to remove the nonadherent cells. Experiments were performed following 16 h of culture.

Cell Treatments
The cells were pretreated with prazosin (PZ; 0.1 M, Sigma), followed by treatment with l-norepinephrine (NE; 10 M, Sigma) for 24 h to measure apoptosis or 15 min to assess MAPK activities. In some experiments propranolol (
In-gel MAPKs Assay-To confirm the activation of MAPKs, in-gel kinase assays were performed as described previously (25). The immune complexes, as prepared above, were washed three times with lysis buffer and separated on 10% SDS-PAGE polymerized with 0.5 mg/ml MBP. The gel was rinsed twice with 20% isopropanol in 100 mM Tris HCl, pH 8.0 (20 min each), followed by two washes (30 min each) in buffer B (100 mM Tris-HCl, pH 8.0, and 5 mM ␤-mercaptoethanol). The gel was denatured in buffer B containing 6 M guanidine HCl for 1 h followed by renaturation in buffer B containing 0.04% Tween 40 at 4°C for 16 h, with four to five changes with this buffer over the time period. The gel was then incubated in a kinase buffer C (20 mM HEPES, pH 7.2, 10 mM MgCl 2 , and 3 mM ␤-mercaptoethanol) for 30 min, followed by another incubation in the kinase buffer C containing 50 Ci of [␥-32 P]ATP (NEN Life Science Products) and 50 M ATP at room temperature for 1 h. The gel was then washed several times with 1% sodium pyrophosphate in 5% trichloroacetic acid. Radiolabeled MBP was detected by autoradiography and quantified using laser densitometer (Bio-Rad).
Western Blot Analysis-Activation of p38 kinase was also studied using phospho-specific antibodies (New England BioLabs). Total cell lysates (70 g) were resolved by 10% SDS-PAGE, and proteins were transferred to polyvinylidene difluoride membranes. The membranes were probed with phospho-specific p38 kinase antibodies and analyzed as described previously (25).

Measurement of Apoptosis
Flow Cytometry-Flow cytometry (fluorescence-activated cell sorter analysis) was performed on a FACS Star Plus using Lysis II software (Becton-Dickinson) as described previously (5,7). Briefly, the cells were trypsinized, fixed/porated in 70% ethanol at 4°C for 30 min, and resuspended in 1 ml of phosphate-buffered saline solution containing 0.1% Triton X-100, 50 g/ml RNase A (Life Technologies, Inc.), and 50 g/ml propidium iodide (Sigma). Apoptotic cells stained with propidium iodide exhibit reduced DNA content with a peak in the hypodiploid region. The percentage of apoptotic cells was determined as fraction of cells with hypodiploid DNA content.
TUNEL Staining-Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) was performed on cells plated on glass coverslips using a Roche Molecular Biochemicals in situ death detection kit according to the manufacturer's instructions. The percentage of TUNEL-positive myocytes (relative to total myocytes) was determined by counting 400 -500 cells in 20 randomly chosen fields per coverslip on each of three coverslips for each experiment.

Statistical Analysis
All data are expressed as mean Ϯ S.E. Comparisons between control and treatments were performed using Student's unpaired t tests. Statistical significance of multiple treatments was determined by analysis of variance and a post hoc Tukey's test. Probability (p) values of less than 0.05 were considered to be significant.

Activation of MAPKs via ␤-AR Stimulation-
To elucidate the effect of ␤-AR stimulation on MAPK signaling, the activities of ERK1/2, p38 kinase, and JNKs were measured in ARVMs treated with NE in the presence of the ␣ 1 -AR antagonist prazosin (NE/PZ). Initial characterization of the time course using the immune complex kinase assay indicated that activation of p38 kinase and JNKs was maximal at 15 min (data not shown), whereas activation of ERK1/2 was not evident until 60 min, and therefore subsequent experiments were performed at 15 (JNKs and p38) or 60 (ERK1/2) min after ␤-AR stimulation. ␤-AR stimulation increased the activities of p38 kinase and JNKs by 2.3 Ϯ 0.3-and 3.5 Ϯ 0.2-fold, respectively (Fig. 1A), and these effects were fully blocked by the ␤-AR antagonist propranolol (data not shown). ERK1/2 activity was increased 2.6 Ϯ 0.4-fold at 60 min (Fig. 1A). MAPK activation was then confirmed by in-gel kinase assay, using MBP as substrate (21,26), which showed that ␤-AR stimulation increased p38 kinase and JNKs activities by approximately 2-and 3-fold, respectively (Fig. 1B).
␤-AR stimulation also increased the number of TUNELpositive cells by 1.8 Ϯ 0.2-fold (Fig. 3B). After pretreatment with SB, ␤-AR stimulation increased the number of TUNELpositive cells by 3.4 Ϯ 0.5-fold, which was greater than the effect of ␤-AR stimulation alone (p Ͻ 0.05). Treatment with SB p38 Kinase Inhibits ␤-AR-stimulated Apoptosis alone had no effect on the number of apoptotic cells measured by flow cytometry or TUNEL staining.
Pharmacological Specificity of SB-202190 -The pyridinyl imidazole compound SB has been shown to inhibit JNKs activity at concentrations Ն 10 M (28,29). To elucidate the pharmacology of SB-202190 in ARVMs, we tested the effect of SB (10 M; a concentration that increases ␤-AR-stimulated apoptosis) on ␤-AR-stimulated p38 kinase and JNKs activation.
Due to the reversible nature of this compound, it was added during the immune complex kinase reaction. SB completely inhibited ␤-AR-stimulated p38 kinase activity (Fig. 5A) but caused only minimal inhibition of JNKs activation (Fig. 5B).
Role of G i Proteins in Mediating ␤AR Stimulation of p38 Kinase-To determine whether G i is involved in the ␤-AR stimulation of p38 kinase, ARVMs were pretreated with pertussis toxin (PTX; 1 g/ml, 3 h) to inactivate G i (6,24). PTX alone had no effect on p38 kinase activity (Fig. 6A)  To further assess the role of Gi, cells were pretreated with carbachol (CARB; 30 M, 30 min) to activate G i . CARB alone increased p38 kinase activity, and concurrent ␤-AR stimulation caused no further increase in p38 kinase activity (CARB, 2.6 Ϯ

FIG. 1. Activation of MAPKs by stimulation of ␤-adrenergic receptors.
ARVMs cultured for 24 h in defined media were pretreated with ␣ 1 -adrenergic receptor antagonist prazosin (PZ, 0.1 M) for 30 min followed by treatment with norepinephrine (NE, 10 M) for 15 or 60 min. The cell lysates were analyzed by immune complex kinase assay using GST-ATF2, GST-c-Jun, or MBP as substrate for p38 kinase, JNKs, and ERK1/2, respectively (A) and in-gel kinase assay using myelin basic protein as substrate for p38 kinase and JNKs (B). In A, the intensity of each band on the autoradiogram was quantified by densitometric scanning. Activity of MAPKs is shown as a fold increase in average of three independent experiments compared with unstimulated controls (CTL). *p Ͻ 0.05 versus CTL.  Inhibition of p38 Kinase Abolishes the Antiapoptotic Effect of Carbachol-As reported previously (7), pretreatment with CARB (30 M, 30 min) abolished ␤-AR-stimulated apoptosis (Fig. 7). Pretreatment with SB (10 M) completely blocked the protective effect of carbachol (as measured by flow cytometry). The combination of CARB and SB had no effect on apoptosis.
Involvement of ␤-AR Subtypes in Activation of p38 Kinase-We have shown previously (7) that stimulation of ␤ 1 -AR increases apoptosis, whereas stimulation of ␤ 2 -AR inhibits apoptosis. To study the involvement of ␤ 1 -or ␤ 2 -AR in the activation of p38 kinase, ARVM were treated with different concentrations of xamoterol (1 and 10 M) and clenbuterol (1 and 10 M). Xamoterol is a partial agonist for ␤ 1 -AR (30), whereas clenbuterol is a selective agonist for ␤ 2 -AR (31). The activation of p38 was evaluated by Western blot using phosphospecific p38 kinase antibodies and by immune complex kinase assays. Using phospho-specific p38 kinase antibodies, we found that xamoterol increased phosphorylation of p38 kinase by 2.0and 1.5-fold at 1 and 10 M concentrations, respectively (Fig. 8). This activation of p38 was inhibited 40 -90% by ␤ 1 -AR antagonist CGP-20712A. Clenbuterol increased p38 kinase activity by 1.3-and 2.2-fold at 1 and 10 M concentrations, respectively. Likewise, the immune complex kinase assay using MBP as substrate showed 4.0 Ϯ 1.7-and 6.7 Ϯ 2.3-fold increase in p38 kinase activity following a 15-min exposure to xamoterol (1 M) or clenbuterol (1 M), respectively. These data suggest that both ␤ 1 -and ␤ 2 -AR subtypes can couple to the activation of p38 kinase.

DISCUSSION
Stimulation of ␤-AR induces apoptosis in cardiac myocytes. This phenomenon has been shown to occur both in vitro in adult and neonatal rat cardiac myocytes (5,6) and in vivo in rat myocardium following infusion of isoproterenol (32). Recently, we found that stimulation of G i protein opposes ␤-AR-stimulated apoptosis (7). The present study demonstrates that ␤-AR stimulation activates three members of the MAPKs family, ERK1/2, JNKs, and p38 kinase and that the activation of p38 kinase plays a protective role in ␤-AR-stimulated apoptosis. Furthermore, the data suggest that the protective effects of G i stimulation are mediated via the activation of p38 kinase.
We found that activation of p38 kinase protects against ␤-AR-stimulated apoptosis. The p38 kinase pathway has been shown to either stimulate or inhibit apoptosis in various cell types (19,21,22,33). Activation of p38 kinase was shown to protect Jurkat cells against Fas ligation and UV irradiation induced apoptosis (35). Likewise, overexpression of MAPK kinase 6, a selective activator of p38 kinase, protected neonatal cardiac myocytes from either anisomysin-or constitutively active MAPK kinase kinase 1-induced apoptosis (22). In contrast, in neonatal cardiac myocytes, pharmacological inhibition of p38 kinase protected against ischemia-induced apoptosis (35), and overexpression of p38␣ exerted a proapoptotic action (21). Six isoforms for p38 kinase have been identified, of which heart muscle expresses predominantly the ␣ and ␤ isoforms (36). SB is a potent inhibitor of both the ␣ and ␤ 2 isoforms of p38 kinase (29). Thus, the differential effects of p38 inhibition on apoptosis in various cell types may reflect heterogeneity in the expression and/or activation of various p38 kinase isoforms.
PD-98059 at a concentration of 10 M fully inhibited ␤-ARstimulated activation of ERK1/2 but had no effect on apoptosis. Thus, it seems unlikely that ERK1/2 played a major role in opposing the apoptotic action of ␤-AR stimulation (18). These findings are in contrast to data in neonatal rat cardiac myocytes, where ERK1/2 has been shown to play an antiapoptotic role. For example, in neonatal myocytes, the ability of cardiotropin-1 to protect against serum deprivation-stimulated apoptosis was blocked by inhibition of ERK1/2 (18). Similarly, concurrent ␣ 1 -AR stimulation was found to oppose ␤-AR-stimulated apoptosis in neonatal rat cardiac myocytes, and this protective effect was blocked by inhibition of ERK1/2 (6). Both cardiotropin-1 and ␣ 1 -AR agonists activate ERK1/2 within 10 -15 min of stimulation, and the activity returns to basal levels in 30 min (18,37), whereas ␤-AR-stimulated activation of ERK1/2 was not detected until 60 min in ARVM. Thus, the p38 Kinase Inhibits ␤-AR-stimulated Apoptosis different roles of ERK1/2 in modulating apoptosis in ARVM versus neonatal rat myocytes may reflect differences in the kinetics of activation and/or coupling.
␤-AR stimulation caused the activation of JNKs in ARVM.
SB had only a minimal effect on the ␤-AR-stimulated activation of JNKs. Therefore, it is unlikely that JNKs contributed to the proapoptotic effect of SB that we observed. However, because there are no specific pharmacological inhibitors for JNKs, we p38 Kinase Inhibits ␤-AR-stimulated Apoptosis can not exclude a possible role for JNKs in ␤-AR-stimulated apoptosis.
We provide evidence for the involvement of G i proteins in the ␤-AR-stimulated activation of p38 kinase in ARVMs. PTX, which inhibits G i and potentiates ␤-AR-mediated apoptosis (7), abolished the ␤-AR-stimulated activation of p38 kinase. Likewise, treatment with carbachol, which stimulates G i in ARVMs via the activation of M2 muscarinic receptors and inhibits ␤-AR-mediated apoptosis, activated p38 kinase. These findings are consistent with the demonstration that activation of p38 kinase by endothelin-1 or epinephrine is PTX-sensitive in smooth muscle cells and osteoblasts (38,39). Likewise, p38 kinase and JNKs, but not ERK1/2, were activated in transgenic mice that overexpress a constitutively active mutant of G␣ i-2 (17). The precise link between ␤-AR stimulation, G i , and p38 activation requires further investigation.
Xamoterol and clenbuterol each stimulated p38 kinase activity, suggesting that both ␤ 1 -and ␤ 2 -AR subtypes can couple p38 kinase. There is evidence that ␤ 2 -AR (40) and ␤ 1 -AR (41,42) can couple to G i , and thus might participate in the activation of p38 kinase with ␤-AR stimulation.
The ability of ␤ 1 -AR to activate p38 kinase suggests that this subtype can activate both apoptotic and antiapoptotic pathways. ␤ 1 -AR may stimulate apoptosis via a PKA-calcium-dependent mechanism (5), and oppose apoptosis via the activation of p38 kinase. Simultaneous activation of apoptotic and antiapoptotic pathways in cardiac myocytes has been observed with daunomycin (43). Finally, it is possible that the apparent ability of both ␤ 1 -and ␤ 2 -AR subtypes to activate p38 kinase may reflect differential activation of p38 kinase isoforms, which may exert opposing effects on apoptosis in cardiac myocytes (21).
The mechanism by which activation of p38 kinase protects against ␤-AR-stimulated apoptosis is not yet clear. MAP-KAPK2, a downstream target of p38 kinase that is activated by ␤-AR stimulation in ARVMs, is one candidate to exert an antiapoptotic action. Support for this thesis comes from the observation that MAPKAPK2 can phosphorylate several other proteins, including heat shock proteins that have been shown to protect cells from apoptosis (44 -46). On the other hand, activation of p38 kinase may regulate the activities of Akt and/or caspases. Activation of Akt (47) and inhibition of caspases (34,48) are shown to have antiapoptotic effects. Further studies aimed at determining the molecular mechanisms by which p38 kinase opposes ␤-AR-stimulated apoptosis in cardiac myocytes may have important implications for the regulation of myocyte survival.