b 2 -Adrenergic Receptor-induced p38 MAPK Activation Is Mediated by Protein Kinase A Rather than by G i or G bg in Adult Mouse Cardiomyocytes*

Increasing evidence shows that stimulation of b -adre-nergic receptor (AR) activates mitogen-activated protein kinases (MAPKs), in addition to the classical G s - adenylyl cyclase-cAMP-dependent protein kinase (PKA) signaling cascade. In the present study, we demonstrate a novel b 2 -AR-mediated cross-talk between PKA and p38 MAPK in adult mouse cardiac myocytes expressing b 2 AR, with a null background of b 1 b 2 -AR double knockout. b 2 -AR stimulation by isoproterenol increased p38 MAPK activity in a time- and dose-dependent manner. Inhibiting G i with pertussis toxin or scavenging G bg with b ARK-ct overexpression could not prevent b 2 -AR-in-duced p38 MAPK activation. In contrast, a specific peptide inhibitor of PKA, PKI (5 m M ), completely abolished the stimulatory effect of b 2 -AR, suggesting that b 2 -AR- induced p38 MAPK activation is mediated via a PKA-de-pendent mechanism, rather than by G i or G bg . This con- clusion was further supported by the ability of forskolin (10 m M ), an adenylyl cyclase activator, to elevate p38 gene FBS-free gene-carrying full All experiments were performed after 24 of adenoviral In a subset of experiments, myocytes treated pertussis toxin ribosylate o The efficacy of PTX routinely examined the abolition of M 2 -muscarinic recep-tor-mediated inhibitory effect on b -AR contractile response p38 Statistical Evaluations— Comparisons between control and treatments were performed using Student’s unpaired t test or analysis of variance when appropriate. A value of p , 0.05 was considered to be statistically significant.

extracellular stimuli, such as growth factors, cytokines, mechanical stress, UV light, osmotic stress, and heat shock, can activate MAPK signaling cascades (5). Increasing evidence has shown that G protein-coupled receptors (GPCRs), e.g. ␤-adrenergic receptor (AR), also regulate MAPKs, particularly ERK1/2 MAPK. One major pathway of GPCR-mediated activation of MAPKs is dependent on "transactivation" of a panel of receptor tyrosine kinases (such as epidermal growth factor and insulin-like growth factor) (6 -8). Specifically, stimulation of GPCRs leads to the release of free G␤␥ dimers, which, in turn, activate these receptor tyrosine kinases by unidentified mechanisms, resulting in activation of ERK1/2 MAPKs (9,10).
A large body of evidence has demonstrated that activation of p38 MAPK, also called a stress-activated protein kinase, is associated with the onset of cardiac hypertrophy and cell death in response to in vivo pressure overload or ischemic/reperfusion injury (11)(12)(13). In G␣ q transgenic mice, the transition of hypertrophy to apoptosis is coincident with activation of p38 MAPK (14). This paradigm has been further manifested by p38 MAPK-induced apoptosis in cultured neonatal rat cardiac myocytes overexpressing p38 MAPK (15). These previous studies suggest an involvement of p38 MAPK in cardiac apoptosis. However, more recent studies have proposed that in cultured adult rat myocytes, p38 MAPK is activated by ␤-AR stimulation via a G i -dependent mechanism, protecting myocytes against ␤-AR/G s -mediated apoptosis (16). Thus, in contrast to the wealth of knowledge as to GPCR-mediated ERK1/2 activation, the role of GPCRs in regulating p38 MAPK activity and its physiological relevance remains controversial.
This study seeks to determine whether cardiac ␤ 2 -AR stimulation regulates p38 MAPK signaling and, if so, to explore underlying mechanisms and examine the possible interaction between the concurrent PKA and p38 MAPK signaling pathways. To selectively stimulate cardiac ␤ 2 -AR, we expressed the human ␤ 2 -AR in ventricular myocytes isolated from adult ␤ 1 ␤ 2 -AR double knockout (DKO) mice using adenoviral gene transfer (17). Here we demonstrate that "pure" ␤ 2 -AR stimulation activates p38 MAPK via a PKA-dependent mechanism, but independent of G i and G␤␥, and that the activated p38 MAPK provides a novel negative feedback to the PKA-mediated contractile response in adult mouse ventricular myocytes.

EXPERIMENTAL PROCEDURES
Isolation and Culture and Adenoviral Infection of Adult Mouse Cardiomyocytes-The investigation conforms to National Institutes of Health guiding principles in the care and use of animals. Single mouse cardiac myocytes were isolated from the hearts of 2-3-month-old ␤ 1 / ␤ 2 -AR DKO mice with an enzymatic technique, then cultured and infected with ␤ 2 -AR-adenoviral vector, adeno-␤ 2 -AR at a multiplicity of * 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.
ʈ To whom correspondence should be addressed: Laboratory of Cardiovascular Science, Gerontology Research Center, NIA, NIH, 5600 Nathan Shock Dr., Baltimore, MD 21224. Tel.: 410-558-8662; Fax: 410-558-8150; E-mail: xiaor@grc.nia.nih.gov. 1 The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; GPCR, G proteincoupled receptor; AR, adrenergic receptor; DKO, double knockout; SB, SB203580; PKA, cAMP-dependent protein kinase; m.o.i., multiplicity of infection; PKI, a specific peptide inhibitor of PKA; MEM, minimal essential medium; FBS, fetal bovine serum; PTX, pertussis toxin; ISO, isoproterenol; FSK, forskolin. infection (m.o.i.) of 100, as described previously (17). In a subset of experiments, myocytes were co-infected by adeno-␤ 2 -AR and adeno-␤ARK-ct (adenovirus vector carrying a ␤ARK carboxyl-terminal fragment) or infected by adeno-␤-Gal (adenovirus vector with reporter gene lacZ) as negative control, all at m.o.i. of 100. Before culture, myocytes were washed three times with minimal essential medium (MEM) containing 1.2 mM Ca 2ϩ , 2.5% fetal bovine serum (FBS) and 1% penicillinstreptomycin and then plated at 0.5 ϳ 1 ϫ 10 4 /cm 2 with the same medium in the culture dishes precoated with 10 g/ml mouse laminin. Following 1 h of culture (to achieve attachment), the culture medium was aspirated along with unattached cells. Adenovirus-mediated gene transfer was implemented by adding a minimal volume of the FBS-free MEM containing an appropriate titer of gene-carrying adenovirus. The full volume of FBS-free MEM was supplied after culture for another 1-2 h. All experiments were performed after 24 h of adenoviral infection. In a subset of experiments, myocytes were treated with pertussis toxin (PTX, 0.5 g/ml for 24 h) to ribosylate G i /G o proteins. The efficacy of PTX was routinely examined by the abolition of M 2 -muscarinic receptor-mediated inhibitory effect on ␤-AR contractile response (19).
p38 MAPK Phosphorylation-After culture for 24 h, cells were incubated with isoproterenol (ISO) or forskolin at 37°C for an indicated period of time (1 ϳ 60 min), then lysed with ice-cold lysis buffer (20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ␤-glycerol phosphate, 1 mM Na 3 VO 4 , 1 g/ml leupeptin, 2 g/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride). The lysates were centrifuged at 14,000 rpm for 10 min at 4°C. Total protein contents were measured by Bradford assay (Bio-Rad). Phosphorylation of p38 MAPK was measured by Western blotting as described previously (18). Briefly, 50 g of protein were denatured in 2% SDS, 10 mM dithiothreitol, 60 mM Tris, and 0.1% bromphenol blue, pH 6.8. The samples were separated by 12% Tris-glycine/SDS-polyacrylamide gel electrophoresis (Novex) and transferred to nitrocellulose membranes. After blocking with 5% fat-free dry milk in Western buffer (1ϫ Tris-buffered saline, 0.1% Tween 20) for 1 h at room temperature, membranes were incubated with 1:1000 phospho-p38 MAPK antibody overnight in Western Buffer with 5% bovine serum albumin at 4°C. After being washed twice, membranes were incubated with 1:2000 anti-IgG secondary antibody conjugated to horseradish peroxidase (New England Biolabs) for 1 h. Antigen-antibody complexes were visualized by LumiGLO Chemiluminescent Substrate (new United Kingdom Biolabs), and the relative level of phosphorylated p38 MAPK was quantified by densitometry (Molecular Dynamics, Sunnyvale, CA). The same membrane was striped in strip buffer (62.5 mM Tris, 100 mM ␤-mercaptoethanol, 2% SDS, pH 6.7) at 50°C for 30 min, then re-probed with a second primary antibody to determine the total p38 protein abundance using a similar procedure.
p38 MAPK Activity Assay-p38 MAPK activity was detected by using MAPK assay kits (New England Biolabs) following the instruction manual. Briefly, 200 g of total protein were immunoprecipitated with immobilized phospho-p38 MAPK (Thr 180 /Tyr 182 ) monoclonal antibody. After the immune complexes were washed three times with the lysis buffer and three times with the kinase buffer, the reaction was performed in kinase buffer containing 200 M ATP and 2 g of ATF-2 fusion protein as a substrate of p38 MAPK. The reaction was stopped by adding 4ϫ Laemmli sample buffer at the time indicated. The boiled samples were subjected to SDS-polyacrylamide gel electrophoresis, and the proteins were transferred to nitrocellulose membranes. After the membrane was blocked, anti-phospho-ATF2 antibody was used to probe the activity of p38 MAPK. In a subset of experiments, a p38 MAPK inhibitor, SB203580 (SB), at variable concentrations was added into the reaction mixture to determine its inhibitory effect on p38 MAPK activity.
Cell Contraction Measurements-Cells were placed on the stage of an inverted microscope (Zeiss, model IM-35), perfused with HEPES-buffered solution (1.0 mM CaCl 2 , 137 mM NaCl, 5 mM KCl, 15 mM dextrose, 1.3 mM MgSO 4 , 1.2 mM NaH 2 PO 4 , 20 mM HEPES, pH 7.4) at a flow rate of 1.8 ml/min and electrically stimulated at 0.5 Hz at 23°C. Cell length was monitored by an optical edge-tracking method using a photodiode array (model 1024 SAQ, Reticon) with a 3-ms time resolution. Cell contraction was measured by the percent shortening of cell length following electrical stimulation.
Materials and Antibodies-MEM, PTX, ISO, forskolin, and propranolol were purchased from Sigma. FBS and penicillin-streptomycin were purchased from Life Technologies, Inc. Laminin was purchased from Upstate Biotechnology. PKI (a cell membrane-permeable protein kinase A inhibitor 14 -22 amide) and SB203580 were purchased from Calbiochem. Rabbit polyclonal antibody against phospho-p38 was purchased from New England Biolabs. Mouse monoclonal antibody against SAPK/ p38 and anti-mouse IgG were purchased from Upstate Biotechnology. Adeno-␤ 2 -AR and adeno-␤ARK-ct were kindly provided by Robert J. Lefkowitz and Walter J. Koch at Duke University Medical Center, Durham, NC.
Statistical Evaluations-All data are presented as mean Ϯ S.E. Comparisons between control and treatments were performed using Student's unpaired t test or analysis of variance when appropriate. A value of p Ͻ 0.05 was considered to be statistically significant.

RESULTS
␤ 2 -AR Stimulation by ISO Activates p38 MAPK in a Timeand Dose-dependent Manner-To define the effect of ␤ 2 -AR stimulation on p38 MAPK, both the phosphorylation status and the activity of p38 MAPK in response to a ␤-AR agonist ISO were examined in adult ␤1␤2 DKO mouse cardiac myocytes expressing ␤ 2 -AR (450 Ϯ 48 fmol/mg protein, n ϭ 4). Stimulation of ␤ 2 -AR by ISO (1 M) increased p38 MAPK phosphorylation (an index of the kinase activation) in a time-dependent manner, as shown by the typical Western blot (Fig. 1A, top band) and the average data (Fig. 1B). Phosphorylation of p38 MAPK was transiently elevated following ␤ 2 -AR stimulation; it occurred significantly as early as 5 min, reached the peak of 1.7-fold augmentation at 15 min, and then gradually declined to the basal level at 60 min (Fig. 1, A and B). Concomitantly, phosphorylation of its substrate protein, ATF-2, was also enhanced by 1.8-fold in response to ␤ 2 -AR stimulation, with a similar temporal profile to that of p38 MAPK phosphorylation (Fig. 1, A and B). Pretreatment of cells with a ␤-AR antagonist, propranolol (10 M), 15 min prior to ISO, completely abolished ␤ 2 -AR-stimulated p38 MAPK activation (Fig.  1C). Furthermore, a p38 MAPK inhibitor, SB203580, antagonized a ␤ 2 -AR-induced increase in p38 MAPK activity in a dose-dependent fashion (Fig. 1D). Fig. 2 shows the average dose-response curve of p38 MAPK phosphorylation to ␤ 2 -AR stimulation; ISO dose-dependently increased p38 MAPK phosphorylation with an EC 50 of 10 nM and a maximal response at concentrations Ն 1 M. These results indicate that ISO-induced ␤ 2 -AR stimulation activates p38 MAPK in a time-and dose-dependent fashion.
␤ 2 -AR-induced p38 MAPK Activation Does Not Depend on G i or G␤␥ Signaling-Since ␤ 2 -AR couples to both G s and G i proteins (19 -21), we first asked whether G i signaling is essentially involved in the ␤ 2 -AR-induced p38 MAPK activation. To test this hypothesis, we treated cells with PTX to disrupt G i /G o signaling. While PTX treatment completely abolished the inhibitory effect of M 2 -muscarinic stimulation on ␤-AR-mediated positive contractile response in all of the cells examined (data not shown), it could not prevent the effect of ␤ 2 -AR stimulation to activate p38 MAPK (Fig. 3, A and B). Next, we determined whether the ␤ 2 -AR stimulatory effect is dependent on G␤␥, as often is the case for GPCR-mediated ERK1/2 MAPK activation (9, 10). As shown in Fig. 3, A and B, inhibiting G␤␥ subunits by overexpressing G␤␥ scavenger, ␤ARK-ct, could not abolish ␤ 2 -AR-induced p38 MAPK activation. In contrast, overexpression of ␤ARK-ct fully prevented ␤ 2 -AR-mediated anti-apoptotic effect (data not shown), indicating it is effective in blocking G␤␥. Thus, the effect of ␤ 2 -AR stimulation on p38 MAPK is independent of either G i or G␤␥ signaling.
We next examined the possible role of the classic G s -adenylyl cyclase-PKA signaling pathway in ␤ 2 -AR-induced p38 MAPK activation. Cells were treated with PKI (5 M), a specific peptide inhibitor of PKA, 15 min prior to ISO application. Fig. 4 shows that PKI fully prevented the effect of ␤ 2 -AR stimulation (1 M ISO for 15 min) on p38 MAPK. If PKA is sufficient to activate p38 MAPK, receptor-independent reagents induced PKA activation should also increase this MAPK activity. Indeed, direct stimulation of adenylyl cyclase by forskolin (FSK, 10 M), similar to ␤ 2 -AR stimulation by ISO, markedly increased p38 MAPK phos-phorylation. Moreover, PKI completely abolished the response of p38 MAPK to forskolin (Fig. 4, A and B). As a negative control, we further demonstrated that in cells transfected with adeno-␤-Gal, ISO had no effect on p38 MAPK activation, whereas FSK did increase the p38 MAPK activity in a PKI-sensitive manner (Fig.  4C). These results indicate that ␤ 2 -AR stimulation activates p38 MAPK via a PKA-dependent pathway.

p38 MAPK Activation Inhibits ␤ 2 -AR-mediated Contractile
Response-A predominant functional role of acute ␤-AR stimulation in cardiac myocytes is to increase contractility. To investigate the possible physiological relevance of ␤ 2 -AR-stimulated p38 MAPK activation, we measured cell contractile response to ␤ 2 -AR stimulation by ISO at a submaximal concentration (10 Ϫ9 M) in the presence or absence of the p38 MAPK inhibitor, SB203580. Since our data showed that SB203580, the p38 MAPK inhibitor, at 10 M effectively antagonized ␤ 2 -

FIG. 2. Dose response of p38 MAPK Phosphorylation to ␤ 2 -AR stimulation by ISO. Adeno-␤ 2 -AR-infected myocytes were incubated with ISO at variable concentrations for 15 min (see "Experimental
Procedures"). A shows a typical Western bolt with an anti-phospho-p38 antibody to determine p38 phosphorylation (top band) or with an anti-p38 MAPK antibody to show the total p38 MAPK (bottom band). Note that while the total p38 amount is highly comparable at different ISO concentrations, the phosphorylated p38 is increased by ISO in a dosedependent manner. B shows the average dose response of p38 phosphorylation to ISO (with a log EC 50 of Ϫ8 Ϯ 0.24) (n ϭ 3 independent experiments; *, p Ͻ 0.05 versus control value).

FIG. 3. Inhibition of G i signaling with PTX treatment or inhibition of G␤␥ with ␤ARK-ct does not alter ␤ 2 -AR-mediated p38 MAPK activation.
Adeno-␤ 2 -AR-infected myocytes were treated with PTX or co-transfected with adeno-␤ARK-ct (at m.o.i. 100, see "Experimental Procedures"). A shows that ␤ 2 -AR stimulation by ISO (1 M for 15 min) increases p38 phosphorylation to a similar extent in the presence and absence of PTX or overexpression of ␤ARK-ct; the total p38 protein levels are highly comparable among groups (bottom band). B summarizes the average data (n ϭ 3ϳ6 independent experiments; *, p Ͻ 0.01 versus the groups in the absence of ISO). AR-mediated p38 MAPK activation (Fig. 1D), we chose this concentration of SB203580 to determine the possible cross-talk between p38 MAPK and PKA in regulating cardiac contractility. SB203580 at 10 M markedly enhanced ␤ 2 -AR-induced contractile response, although SB alone at the concentration employed had no detectable effect on cell base-line contractility (Fig. 5, A and B). On average, the positive inotropic effect of ␤ 2 -AR stimulation was enhanced by ϳ60% (Fig. 5B). In fact, SB203580 even at a lower concentration (5 M) also significantly enhanced ISO-induced positive inotropic effect. Thus, inhibition of p38 MAPK potentiates ␤ 2 -AR-stimulated contractile response in adult mouse cardiac myocytes, suggesting that p38 MAPK provides a negative feedback to ␤ 2 -AR-stimulated, PKA-mediated contractile response. DISCUSSION There are three major findings in this study. First, we have demonstrated that in adult mouse cardiac myocytes, ␤ 2 -AR stimulation by ISO induces a time-and dose-dependent increase in p38 MAPK activation (Fig. 1). Second and most importantly, we have shown that the stimulatory effect of ␤ 2 -AR on p38 MAPK is mediated by a PKA-dependent pathway, rather than by G i or G␤␥ signaling (Fig. 2). Finally, the present results elucidate a novel role of p38 MAPK in regulating cardiac contractility, in addition to its chronic functional role in regulating cell growth and cell death (11)(12)(13)(14)(15)(16). Thus, the present study not only shows that ␤ 2 -AR sequentially activates the G s -adenylyl cyclase-PKA and p38 MAPK signaling pathways, but also documents an intriguing forward and retrograde crosstalk between those signaling pathways.
␤ 2 -AR Activates p38 MAPK by a PKA-, Rather than G i -or G␤␥-, dependent Mechanism-While previous studies have demonstrated that G␤␥ and G␣ q/11 increase p38 MAPK activity (22,23), more recent studies showed that in rat cardiac myocytes both ␤ 1 -AR and ␤ 2 -AR subtypes activate p38 MAPK in a PTX-sensitive manner (16). We determined the possible role of G i in ␤ 2 -AR-mediated p38 MAPK activation in adult mouse ventricular myocytes. Surprisingly, inhibition of G i with PTX cannot prevent the ␤ 2 -AR-induced p38 MAPK activation, although PTX treatment fully abolishes M 2 -mediated anti-adrenergic effect under the same experimental conditions. In sharp contrast, inhibition of PKA by a specific peptide inhibitor, PKI, completely abrogates the stimulatory effect of ␤ 2 -AR on p38 MAPK activation, strongly suggesting that PKA plays an essential role in ␤ 2 -AR-induced p38 MAPK activation. This conclusion is reinforced by the fact that direct stimulation of adenylyl cyclase by forskolin also markedly increases p38 activity in a PKI-sensitive manner, resembling ␤ 2 -AR stimulation.  Thus, ␤ 2 -AR-stimulated p38 MAPK activation is largely attributable to the G s -adenylyl cyclase-PKA, rather than a G i signaling pathway. The reason caused the different outcomes of the present study from that of the previous work in adult rat cardiac myocytes (16) is presently unclear. However, it might be related to the different species used. It has been shown that there are six p38 MAPK isoforms (24). Both ␣ and ␤ p38 MAPK isoforms co-exist in cardiac myocytes and elicit divergent functional roles (25,26). Whether rat and mouse cardiac myocytes have different predominant isoform(s) of p38 MAPK awaits further studies.
In principal, a dynamic interplay of protein phosphatases and protein kinases may be applicable to the cross-talk between PKA and p38 MAPK. Various protein kinases may regulate p38 MAPK activity by modulating the phosphorylation status of this MAPK. It has been well accepted that that MKK (MAPK kinase)3/6-mediated phosphorylation of this kinase at Thr-Glu-Tyr motif is essential for its activation. Recent evidence suggests that phosphorylation of intermediate signaling molecules, e.g. glia maturation factor, by PKA causes a robust increase in p38 MAPK activity (27). In addition, in HEK 293 cells, activation of PKA mediates phosphorylation of ␤ 2 -AR, resulting in a switch of the receptor coupling preference from G s to G i and release of G␤␥, then the resultant free G␤␥ subunits subsequently activate MAPKs (28). Nevertheless, under our experimental conditions, inhibiting free G␤␥ with ␤ARK-ct cannot prevent ␤ 2 -AR-stimulated p38 MAPK activation. Thus, G␤␥ subunits are not involved in ␤ 2 -AR-mediated, PKA-dependent p38 MAPK activation in cardiac myocytes.
PKA may enhance the activity of MAPKs by inhibiting Ser/ Thr protein phosphatases, particularly, phosphatase 1 (PP1). PKA inhibits PP1 either by direct inhibition via phosphorylation of its regulatory subunit or by indirect inhibition via phosphorylation and subsequent activation of endogenous phosphatase inhibitor 1 (29). More recently, it has been reported that PKA phosphorylates protein tyrosin phosphatases and inhibits their interaction with MAPKs, ERK1/2, and p38 MAPK (30), thereby resulting in enhanced activation of MAPKs, including p38 MAPK (31). Thus, the interaction between PKA and MAPKs signaling pathways is complicated and remains highly controversial. The exact mechanism underlying ␤ 2 -AR-stimulated, PKA-dependent p38 MAPK activation in adult mouse cardiac myocytes merits further investigations.
Physiological and Pathophysiological Relevance of ␤ 2 -ARinduced p38 MAPK Activation-Previous studies on p38 MAPK have been focused on multitude aspects of cell growth and cell death. Activation of p38 MAPK has been shown to enhance apoptosis in neonatal cardiac myocytes (26,32), but to protect against ␤-AR-mediated apoptosis in adult rat cardiac myocytes (16). A similar controversial situation has been reported in other cell types. The diversity in p38 MAPK functions may be related to the heterogeneity of this kinase or different experimental settings, e.g. different cell types or distinct developmental or differentiation status of the studied cells.
It has been well accepted that ␤ 2 -AR increases cardiac contractility through G s -adenylyl cyclase-PKA signaling cascade (18 -21, 33-35). Here we demonstrate, for the first time, that this PKA pathway subsequently induces a ␤ 2 -AR-stimulated p38 MAPK activation. Interestingly, the activated p38 MAPK provides a negative feedback to its upstream, PKA, mediated contractile response, because inhibition of p38 MAPK by SB significantly enhances ␤ 2 -AR-mediated contractile response. This observation is supported by a strikingly reduced ␤-AR contractile response in transgenic mice overexpressing p38 MAPK. 2 The opposing effects of PKA and p38 MAPK on cardiac contractility may have important physiological and pathophysiological relevance. Since increased p38 MAPK activity is associated with cardiomyopathy induced by overexpression of G␣ q (14), ischemic/reperfusion injury or in vivo pressure overload (11)(12)(13), the inhibitory effect of p38 MAPK on ␤-AR/PKAmediated contractile support may contribute, at least in part, to the defect of cardiac contractility under those pathological circumstances.
In summary, the present study provides the first documentation that in adult mouse cardiac myocytes, ␤ 2 -AR stimulation increases p38 MAPK activation via a PKA-dependent mechanism, rather than by G i or G␤␥ signaling. Furthermore, the present results reveal an important cross-talk between ␤ 2 -ARstimulated PKA and the concurrent p38 MAPK signaling cascades in regulating cardiac contractility, i.e. cardiac ␤ 2 -AR-PKA activated p38 MAPK provides a negative feedback to the PKA-mediated contractile response. These findings shed new light on understanding the close association of p38 MAPK activation with the onset of cardiac dysfunction and markedly diminished ␤-AR contractile support in response to pressure overload and ischemia or reperfusion injury (11)(12)(13).