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(Circulation Research. 2004;95:798.)
© 2004 American Heart Association, Inc.

Cellular Biology

Sustained ß₁-Adrenergic Stimulation Modulates Cardiac Contractility by Ca²⁺/Calmodulin Kinase Signaling Pathway

Wang Wang, Weizhong Zhu, Shiqiang Wang, Dongmei Yang, Michael T. Crow, Rui-Ping Xiao, Heping Cheng

From the Laboratory of Cardiovascular Sciences (W.W., W.Z., S.W., D.Y., R.-P.X., H.C), National Institute on Aging, National Institutes of Health; Department of Medicine (M.T.C.), Johns Hopkins University, School of Medicine, Baltimore, Md; and The Institute of Molecular Medicine (R.-P.X., H.C.), Peking University, Beijing, China.

Correspondence to Heping Cheng, PhD, Laboratory of Cardiovascular Sciences, NIA, NIH, Baltimore, MD 21224. E-mail chengp{at}grc.nia.nih.gov

	Abstract

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A tenet of ß₁-adrenergic receptor (ß₁AR) signaling is that stimulation of the receptor activates the adenylate cyclase-cAMP-protein kinase A (PKA) pathway, resulting in positive inotropic and relaxant effects in the heart. However, recent studies have suggested the involvement of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) in ß₁AR-stimulated cardiac apoptosis. In this study, we determined roles of CaMKII and PKA in sustained versus short-term ß₁AR modulation of excitation-contraction (E-C) coupling in cardiac myocytes. Short-term (10-minute) and sustained (24-hour) ß₁AR stimulation with norepinephrine similarly enhanced cell contraction and Ca²⁺ transients, in contrast to anticipated receptor desensitization. More importantly, the sustained responses were largely PKA-independent, and were sensitive to specific CaMKII inhibitors or adenoviral expression of a dominant-negative CaMKII mutant. Biochemical assays revealed that a progressive and persistent CaMKII activation was associated with a rapid desensitization of the cAMP/PKA signaling. Concomitantly, phosphorylation of phospholamban, an SR Ca²⁺ cycling regulatory protein, was shifted from its PKA site (¹⁶Ser) to CaMKII site (¹⁷Thr). Thus, ß₁AR stimulation activates dual signaling pathways mediated by cAMP/PKA and CaMKII, the former undergoing desensitization and the latter exhibiting sensitization. This finding may bear important etiological and therapeutical ramifications in understanding ß₁AR signaling in chronic heart failure.

Key Words: ß₁-adrenergic receptor • Ca²⁺/calmodulin-dependent protein kinase II • cAMP-dependent protein kinase • cardiac contractility • phospholamban

	Introduction

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As a prototypical member of G protein–coupled receptor (GPCR) superfamily, ß-adrenergic receptor (ßAR) plays a central role in sympathetic regulation of cardiac function.^1,2 Stimulation of ßAR by catecholamines induces robust chronotropic, inotropic, and relaxant effects via the Gs-adenylate cyclase-cAMP-protein kinase A (PKA) pathway.^3,4 This signaling pathway is also thought to be responsible for other functions of ßAR, such as regulation of metabolism, gene expression, cell growth, and apoptosis.² However, sustained ßAR activation under pathological conditions such as hypertension and congestive heart failure will result in downregulation and desensitization of ßAR attributable to the negative feedback of this pathway.^5–7

Recent studies have revealed unanticipated complexity of ßAR signal transduction. For ß₂AR subtype stimulation in the heart, a parallel activation of G_i protein counterbalances G_s-mediated contractile response. Whereas ß₁AR stimulated contractile response is thought to be mediated exclusively by the cAMP/PKA signaling pathway,^8,9 ßAR regulation of cell growth and remodeling involves mitogen-activated protein kinase (MAPK) cascades and phosphoinositol 3 kinase (PI3K) pathway.^10,11 Moreover, we have recently shown that sustained (24-hour) ß₁AR stimulation progressively activates Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), which is obligatory to cardiac apoptosis.¹² Therefore, in addition to the classic cAMP/PKA pathway, chronic ßAR stimulation under certain physiological and pathophysiological circumstances may evoke pathways other than cAMP/PKA.^13,14 These lines of evidence raise the question whether ß₁AR modulates cardiac contractility using different sets of signaling mechanisms in short-term versus prolonged receptor stimulation.

CaMKII is a widely expressed protein kinase that modulates various functions ranging from learning and memory of the nervous system, muscle contraction, cell secretion to gene expression.¹⁵ In the heart, CaMKII is the predominant isoform and plays a pivotal role in regulating cardiac performance and remodeling such as myocyte hypertrophy,¹⁶ apoptosis,¹² and heart failure.¹⁷ Furthermore, CaMKII modulates an array of key proteins involved in cardiac excitation-contraction (E-C) coupling and Ca²⁺ handling, such as the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) and its regulator, phospholamban (PLB), ryanodine receptor (RyR) Ca²⁺ release channels, and sarcolemmal L-type Ca²⁺ channels (LCC).^18–22 However, the involvement of CaMKII in ß₁AR modulation of myocardial contractility remains obscure.

The present study aimed at appraising roles of CaMKII and PKA in ß₁AR modulation of cardiac E-C coupling, with an emphasis on the signaling mechanism for the sustained ß₁AR stimulation. We found that both short-term and sustained ß₁AR stimulation are efficacious in mediating positive inotropic and relaxant effects in cardiac myocytes. Unlike the short-term ß₁AR stimulation, the sustained responses are mediated mainly by the CaMKII rather than the cAMP/PKA pathway. Furthermore, molecular integration of these two signaling pathways is mediated by dual site phosphorylation of key proteins involved in cardiac E-C coupling and its physiological regulation.

	Materials and Methods

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Isolation and Culture of Cardiac Myocytes
Cardiac myocytes were isolated from male Sprague-Dawley rat hearts (Charles River Laboratories, Wilmington, Mass) using standard enzymatic technique as described previously.²³ Freshly isolated myocytes were plated at a density of 0.5 to 1x10⁴/cm² in dishes precoated with 20 µg/mL laminin (Upstate Biotechnology). The culture medium (M199, SIGMA) containing (in mmol/L) creatine 5, L-carnitine 2, taurine 5, insulin-transferrin-selenium-X 0.1%, HEPES 25, and penicillin plus streptomycin 1%, were adjusted to pH 7.4 with NaOH at 37°C. For sustained ß₁AR stimulation, norepinephrine (100 nmol/L with vitamin C) was added along with ₁AR antagonist, prazosin (1 µmol/L), for 24 hours. All the antagonists were added at least 10 minutes before norepinephrine.

Dominant-Negative CaMKIIC Adenovirus Construction and Myocyte Infection
Dominant-negative CaMKIIC (DN-CaMKII) was generated by replacing the residue lysine43 with alanine (K43A) using the transformer site directed mutagenesis kit (Clontech). Adenoviral expression of ß-gal or the HA-tagged DN-CaMKII was performed at the multiplicity of infections (MOI) of 100. Twenty four hours after adenoviral infection, norepinephrine (100 nmol/L) was added.

Measurements of Cell Shortening and Ca²⁺ Transients
Measurements of cell contraction and Ca²⁺ transients were performed 24 hours after norepinephrine exposure. Normal cultured myocytes (24 hours) were used for short-term ß₁AR stimulation. Myocytes were field-stimulated at 0.5 Hz in perfusion solution containing (in mmol/L) NaCl 137, KCl 4.9, CaCl₂ 1, MgSO₄ 1.2, NaH₂PO₄ 1.2, glucose 15, and HEPES 20 (pH 7.4). Prazosin was added 10 minutes before short-term norepinephrine treatment. Protocols pertaining to specific experiments were given in the respective result figure

In indicator-unloaded myocytes, cell length was monitored by an optical edge tracking method at a 3-ms time resolution.²³ In myocytes loaded with the Ca²⁺ indicator fluo-4/AM (Molecular Probes, 20 µmol/L for 30 minutes), Ca²⁺ transients and cell shortening were measured with a confocal laser scanning microscope (LSM510, Carl Zeiss). Digital image analysis used customer-designed programs coded in Interactive Data Language (IDL).

Receptor Radioligand Binding Assay
Cardiac myocytes were homogenized and crude membranes were prepared by centrifuging at 35 000g for 20 minutes at 4°C twice. ß₁AR radioligand binding studies were performed in membranes (25 to 100 µg/tube) using the nonselective ßAR antagonist ligand ¹²⁵I-cyanopindolol (¹²⁵I-CYP, 1 to 300 pmol/L) as described previously.²⁴ Nonspecific binding was determined in the presence of 10 µmol/L propranolol. The maximal numbers of binding sites (B_max) and equilibrium dissociation constants (Kd) for ¹²⁵I-CYP were determined by Scatchard analysis.

Immunostaining
Fixed cells were incubated with anti-HA antibody (1:500, Covance Research Products Inc) at 4°C overnight, followed by Cy5-conjugated secondary antibody (1:1000, Jackson ImmunoResearch laboratories). Negative controls were obtained by incubating cells with only the secondary antibody.

CaMKII Activity and cAMP Accumulation
Cell lysate (500 µg protein) was first immunoprecipitated with anti-CaMKII antibody (1:100, Santa Cruz Biotechnology) in a Ca²⁺-free medium. The precipitated proteins were evaluated with CaMKII assay kits (Upstate Biotechnology Inc) using a specific peptide substrate (KKALRRQETVDAL) as previously described.¹² cAMP level was measured with the cAMP (³H) assay system (Amersham Biosciences) as described previously.²⁴

Western Blotting Analysis of PLB Phosphorylation
PLB phosphorylation was detected with the phosphorylation site-specific antibodies recognizing P-¹⁶Ser PLB or P-¹⁷Thr PLB (1:10000, PhosphoProtein Research). Total PLB was detected with monoclonal antibody against PLB (Upstate Biotechnology).

Statistics
Data were reported as mean±SEM. Student t test and ANOVA with repeated measurement were applied, when appropriate, to determine statistical significance of the differences. P<0.05 was considered statistically significant.

	Results

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Sustained ß₁AR Stimulation Induced Positive Inotropic and Relaxant Effects
To investigate possible modulatory effects of sustained ß₁AR stimulation on cardiac E-C coupling, enzymatically isolated rat ventricular myocytes were cultured in the presence of ß₁AR stimulation (norepinephrine 100 nmol/L plus the ₁AR blocker, prazosin 1 µmol/L) for 24 hours. Cell shortening measured in the continued presence of ß₁AR agonist was used as the end-point readout. Figure 1 shows typical examples and the average data of contractile response to short-term and sustained ß₁AR stimulation. Sustained ß₁AR stimulation (24 hours) elicited a 3.3-fold increase of the contraction amplitude (Figure 1C), which was accompanied by a 47% decrease of the 90% relaxation time (Figure 1D). Notably, the effects of sustained ß₁AR stimulation on cell contraction and relaxation were fully reversible on washout of the agonist, as was the case with short-term ß₁AR stimulation (Figure 1). That sustained contractile responses are fully and rapidly reversible excludes the possibility that they were due simply to a long-term "memory" formed during prolonged receptor stimulation.²⁵

View larger version (35K): [in this window] [in a new window]   Figure 1. Sustained ß1AR stimulation increased contraction amplitude and hastened relaxation in adult rat cardiomyocytes. A and B, Typical chart recordings of cell shortening (0.5 Hz pacing) under short-term (10 minutes, A) and sustained (24 hour, B) exposure of cells to norepinephrine (NE, 100 nmol/L plus prazosin 1 µmol/L). Experimental protocols are shown on the top of the recordings. Downward deflection indicates cell shortening. Individual traces at marked time points are shown below, on an expanded time scale. C and D, Average responses to short-term and sustained NE-induced contractile effects. n=120 to 150 cells from 10 hearts for each data point. P<0.001 vs control, P<0.001 vs NE+prazosin. T90 indicates time-from-peak to 90% relaxation.

As is the case with short-term ß₁AR stimulation, sustained contractile response of ß₁AR was sensitive to a nonselective ßAR blocker, propranolol (10 µmol/L), or a specific ß₁AR blocker, CGP 20712A (0.5 µmol/L). Pretreatment with propranolol or CGP 20712A prevents the increase of contractility by subsequent 24-hour norepinephrine stimulation (Table 1); ICI 118,551 (0.5 µmol/L), a ß₂AR specific antagonist, did not affect the 24-hour norepinephrine-induced response (data not shown). Radioligand binding assay of receptor density in crude membrane fraction revealed a trend of decrease (though statistically insignificant) in ß₁AR density after 24-hour norepinephrine stimulation (Table 2), consistent with previous in vitro reports.²⁶ When compared with the response to short-term ß₁AR stimulation (10 minutes), we found that sustained ß₁AR stimulation was equally efficacious in mediating the positive inotropic and relaxant effects (Figure 1). This observation is somewhat unexpected because it has been well established that cAMP/PKA signaling desensitizes within hours during prolonged ß₁AR stimulation.^5,27

View this table: [in this window] [in a new window]   Table 1. Effects of ßAR Blockers on Sustained ß1AR Contractile Responses

View this table: [in this window] [in a new window]   Table 2. ßAR Density and Affinity in Response to NE Exposure

Sustained ß₁AR Contractile Response Is Mediated by CaMKII Signaling Pathway
Whereas short-term and sustained ß₁AR stimulations elicited indistinguishable responses in terms of cell contraction, there is no a priori reason that the same PKA-dependent mechanism is responsible for the contractile responses in both cases. To appraise role of PKA in mediating the sustained ß₁AR responses, we used two inhibitors of the cAMP/PKA signaling: PKI, a membrane permeable peptide inhibitor of PKA, and Rp-cpt-cAMPS, an inhibitory cAMP analogue. We found that PKI treatment (10 µmol/L for 30 minutes) largely blocked the effect of short-term ß₁AR stimulation (Figure 2A and 2B) as expected. By contrast, PKI was unable to reverse the increase in contraction amplitude in cells exposed to ß₁AR stimulation for 24 hours (Figure 2A and 2B). Likewise, the cAMP antagonist Rp-cpt-cAMPS (100 µmol/L for 30 minutes) significantly inhibited the effects of short-term, but not sustained, ß₁AR stimulation (Figure 2B). These results indicate that sustained contractile response of ß₁AR stimulation is largely PKA-independent.

View larger version (33K): [in this window] [in a new window]   Figure 2. Effects of PKA and CaMKII inhibition on short-term and sustained ß1AR contractile responses. A, Typical examples of the effect of a membrane permeable peptide inhibitor of PKA, PKI (10 µmol/L), on contraction amplitude of cells subjected to short-term or sustained ß1AR stimulation by norepinephrine (NE 100 nmol/L plus prazosin 1 µmol/L). B, Average effects of PKI and Rp-cpt-cAMPS (100 µmol/L). n=20 to 35 cells from 3 to 4 hearts for each data point. P<0.001 vs baseline, NE+PKI and NE+Rp-cAMPS groups; P<0.001 vs baseline. C, Typical examples of the effect of the CaMKII inhibitor, KN93 (10 µmol/L). D, Average effects of KN93 and AIP (10 µmol/L). n=20 to 35 cells from 3 to 4 hearts for each data point. P<0.001 vs baseline; P<0.001 vs baseline, NE+KN93, and NE+AIP groups.

Recently, it has been shown that sustained ß₁AR-stimulated cardiomyocyte apoptosis requires activation of CaMKII signaling independently of PKA.¹² Previous studies have also established an important role for CaMKII modulation of phosphorylation and function of key proteins involved in cardiac E-C coupling, including LCC,^21,22 SERCA,¹⁸ and its regulator PLB,¹⁹ and RyR.^20,28 Next, we examined the involvement of CaMKII signaling in the contractile response to sustained ß₁AR stimulation. As shown in Figure 2C and 2D, the inotropic effect of sustained ß₁AR stimulation was reversed by KN93 (10 µmol/L for 30 minutes), a synthetic CaMKII inhibitor, whereas the effect of short-term ß₁AR stimulation was unaffected by KN93. KN92 (10 µmol/L), an inactive analogue of KN93, exerted no significant effects on either short-term or sustained ß₁AR stimulation (data not shown). Similar to KN93, a cell-permeable peptide inhibitor of CaMKII, myristoylated autocamtide-2 related inhibitory peptide (AIP, 10 µmol/L for 30 minutes), completely abolished the positive inotropic effect of sustained ß₁AR stimulation, without affecting those of short-term ß₁AR stimulation (Figure 2D). Thus, inhibition of cAMP/PKA and CaMKII preferentially blocked contractile modulation by short-term and sustained ß₁AR stimulation, respectively, indicating that the initial responses depend mainly on PKA, whereas the sustained responses require CaMKII signaling.

CaMKII-Dependent Increase of Ca²⁺ Transients in Response to Sustained ß₁AR Stimulation
To further investigate cellular mechanisms underlying the contractile responses in sustained versus short-term ß₁AR stimulation, we measured Ca²⁺ transients and the corresponding cell contraction in Ca²⁺ indicator-loaded myocytes, using confocal microscopy. Figure 3A shows typical micrographs of cellular Ca²⁺ transients and shortenings from cells that underwent short-term and sustained ß₁AR stimulation in the absence or presence of PKA or CaMKII inhibitor. Both short-term and sustained ß₁AR stimulation increased Ca²⁺ transient amplitude (F/F₀ from 2.7±0.1 to 5.5±0.2 or 6.0±0.2 for cells with short-term or sustained ß₁AR stimulation, respectively, n=37 to 55 cells) and contractile amplitude (from 2.5±0.3% to 12.2±0.6% or 11.1±0.6% for cells with short-term or sustained ß₁AR stimulation, respectively, n=33 to 55; Figure 3). The short-term ß₁AR stimulation-induced Ca²⁺ and contractile responses were blocked by Rp-cpt-cAMPS but not by AIP (Figure 3). In contrast, the sustained ß₁AR stimulation–induced increases in Ca²⁺ transients and contraction were resistant to Rp-cpt-cAMPS, but were reversed by AIP (Figure 3). These data indicate that both cAMP/PKA and CaMKII signaling pathways of ß₁AR stimulation augment cell contraction by enhancing intracellular Ca²⁺ transients.

View larger version (43K): [in this window] [in a new window]   Figure 3. CaMKII-dependent increase of Ca2+ transients in response to sustained ß1AR stimulation. A, Linescan confocal images of Ca2+ transients in cells subjected to short-term (NE, 10 minutes) or sustained (NE, 24 hours) ß1AR stimulation with or without AIP (10 µmol/L) or Rp-cpt-cAMPS (100 µmol/L). B and C, Average results. Ca2+ transient amplitude was indexed by F/F0. n=30 to 40 cells from 3 to 4 hearts for each data point; *P<0.05 vs basal, P<0.001 vs basal.

Effects of Dominant-Negative CaMKII on Short-Term and Sustained ß₁AR Stimulation
To confirm that the sustained ß₁AR contractile effect is CaMKII-dependent, we resorted to molecular and genetic manipulation of the CaMKII signaling system. An adenovirus carrying the HA-tagged CaMKII gene with a dominant-negative mutation (DN-CaMKII, mutation K43A) that abrogates the kinase activity was constructed and infected cardiac myocytes in culture. Twenty-four hours after adenoviral infection at MOI of 100, nearly 100% cells showed intense immunostaining of DN-CaMKII as visualized by an anti-HA antibody (Figure 4A). Western Blotting analysis revealed that CaMKII-specific phosphorylation of PLB at ¹⁷Thr was also markedly reduced in the DN-CaMKII expressing myocytes (Figure 4B). Figure 4C and 4D show that sustained ß₁AR stimulation in DN-CaMKII-expressing cells failed to elicit significant increases of Ca²⁺ transient amplitude or contraction amplitude. However, no significant difference between ß-gal and DN-CaMKII expression groups was found in contractile and Ca²⁺ responses to short-term ß₁AR stimulation (Figure 4C and 4D). These data corroborate that sustained ß₁AR stimulation enhances Ca²⁺ transients and cell contraction through a CaMKII pathway, whereas the initial ß₁AR response is largely CaMKII-independent.

View larger version (32K): [in this window] [in a new window]   Figure 4. Effects of DN-CaMKII expression on short-term and sustained ß1AR responses. A, Confocal imaging of HA immunofluorescence in rat cardiac myocytes expressing either ß-gal or HA-tagged DN-CaMKII. B, Expression of DN-CaMKII reduced PLB phosphorylation at 17Thr (P-17Thr). C and D, Ca2+ and contractile responses to short-term and sustained ß1AR stimulation in cells expressing ß-gal or DN-CaMKII. n=33 to 43 cells from 4 to 5 hearts for each data point; *P<0.05, P<0.001 between groups.

Switch of cAMP and CaMKII Signaling During Sustained ß₁AR Stimulation
To better characterize the aforementioned signaling shift during ß₁AR stimulation, we directly measured cellular cAMP accumulation and CaMKII activation over a wide period of time (from 10 minutes up to 24 hours). ß₁AR stimulation elicited a rapid increase of cellular cAMP production that reached its peak in 10 minutes. In the continued presence of the ß₁AR agonist, however, cAMP production was reduced by 66% at 3 hours after stimulation (Figure 5A), and returned to basal level at the steady state (12 hours). The gradual decay of cAMP is consistent with the notion that cAMP/PKA signaling undergoes substantial desensitization during prolonged receptor stimulation.^5–7

View larger version (37K): [in this window] [in a new window]   Figure 5. cAMP formation, CaMKII activity, and site-specific PLB phosphorylation during prolonged ß1AR stimulation. A, Time course of cAMP response to norepinephrine (NE, 100 nmol/L plus prazosin 1 µmol/L). n=5. B, Time course of CaMKII activation in response to norepinephrine (NE, 100 nmol/L plus prazosin 1 µmol/L); n=4. C and D, Western blotting of PLB phosphorylation at the PKA-dependent site (P-16Ser, C) and the CaMKII-dependent site (P-17Thr, D). n=4, *P<0.05 vs control, P<0.001 vs control.

Parallel measurement of CaMKII activity revealed a distinctly different temporal pattern for CaMKII response during ß₁AR stimulation. CaMKII activity rose exponentially (time constant =15 minutes) without an initial overshoot (Figure 5B). The plateau was reached after 1 hour stimulation and was stable for at least 24 hours. Overall, the gradual sensitization of CaMKII signaling roughly mirrored the desensitization of cAMP/PKA signaling, indicating a shift from a cAMP/PKA-dominant signaling to a CaMKII-dominant signaling. The slow and nondecremental activation of CaMKII provides the basis for the sustained contractile and Ca²⁺ responses to ß₁AR stimulation.

Phospholamban (PLB) as a Molecular Integrator of ß₁AR-Stimulated PKA and CaMKII Signals
The SR protein PLB, in its unphosphorylated form, serves as a constitutive inhibitor of the SR Ca²⁺ ATPase. PKA and CaMKII can independently phosphorylate PLB at ¹⁶Ser and ¹⁷Thr, respectively, and either site phosphorylation is sufficient to reverse its inhibition on SERCA activity and subsequently elicit positive inotropic and relaxant responses.²⁹ Thus, PLB with its dual-site phosphorylation might operate as a molecular integrator of both short-term and sustained ß₁AR signaling.

To explore this possibility, we examined the site-specific phosphorylation of PLB in response to sustained and short-term ß₁AR stimulation. The phosphorylation at PKA-dependent site (P-¹⁶Ser) was increased by 7.2±0.9-fold (n=4, P<0.001 versus control) at 10 minutes after exposure to norepinephrine, but was then diminished toward the basal level during sustained ß₁AR stimulation (Figure 5C). Conversely, phosphorylation at the CaMKII-dependent site (P-¹⁷Thr) was significantly increased in response to sustained, but not short-term, ß₁AR stimulation (Figure 5D). These data support the idea that dual site phosphorylation by PKA and CaMKII in effector proteins (eg, PLB) serves to integrate the dual signaling pathways of ß₁AR stimulation.

Effect of cAMP/PKA on CaMKII Mediated ß₁AR Contractile Response
Because ß₁AR-stimualted cAMP/PKA signaling precedes the CaMKII signaling, it might be argued that activation of CaMKII pathway in sustained ß₁AR stimulation is still dependent on the initial PKA activation. However, preinhibition of cAMP/PKA with Rp-cpt-cAMPS (100 µmol/L) did not influence the sustained ß₁AR-stimulated contractile and relaxant responses or the blockade effect of AIP (Figure 6A). Conversely, direct activation of cAMP/PKA pathway by forskolin (1 µmol/L), an adenylate cyclase activator, or cpt-cAMP (100 µmol/L), an active cAMP analogue, elicited no CaMKII-dependent component in the sustained inotropic response (Figure 6B and 6C). Thus, cAMP/PKA signaling appears to be neither sufficient nor necessary for ß₁AR activation of CaMKII.

View larger version (37K): [in this window] [in a new window]   Figure 6. Role of cAMP/PKA in ß1AR activation of CaMKII signaling. A, Preinhibition of cAMP/PKA did not alter the CaMKII-dependent contractile response at 24 hours ß1AR stimulation. n=20 to 35 cells from 3 rats. P<0.001 between groups. B and C, Contractile responses to forskolin (1 µmol/L, 1 or 24 hours) or cpt-cAMP (100 µmol/L, 1 or 24 hours) were insensitive to CaMKII blockers. n=15 to 20 cells from 3 rats. P<0.001 vs other groups.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Time-Dependent Shift of ß₁AR Dual Signaling Pathways
We have systematically examined the signaling mechanisms underlying cardiac contractile modulation by short-term and sustained ß₁AR stimulation (24 hours), using myocyte culture combined with genetic manipulation, confocal imaging, and biochemical measurements. In contrast to the anticipated receptor desensitization,^5–7,27 we demonstrated that short-term and sustained ß₁AR stimulation similarly enhance Ca²⁺ transients and contraction and accelerate relaxation (Figures 1 and 3). Despite phenomenological similarities, inhibition of CaMKII by specific inhibitors or adenoviral expression of DN-CaMKII exerts profound inhibitory effects on the sustained, but not short-term, ß₁AR responses, whereas inhibition of the cAMP/PKA pathway preferentially blocks the responses to short-term ß₁AR stimulation (Figures 2 to to 4). By tracking cAMP production and CaMKII activation over an extended time course (Figure 5), we have uncovered that, CaMKII activity rose to a plateau that does not show any noticeable decay, whereas the cAMP/PKA signaling subsides in the continued presence of ß₁AR agonist. These results indicate that ß₁AR signaling undergoes a time-dependent switch from the PKA-dominant pathway to the CaMKII-dominant pathway after receptor stimulation.

Because inhibition of the cAMP/PKA pathway did not alter the responses to sustained receptor stimulation and receptor-independent cAMP/PKA signal failed to elicit CaMKII-dependent response (Figure 6), activation of the CaMKII pathway may not be consequential to the transient cAMP/PKA activation. In other words, different pathways initiated from the same GPCR may manifest desensitization or sensitization independently.^6,12,30 As compared with ß₁AR-stimulated CaMKII activation in ß₁ß₂AR double knockout mouse cardiac myocytes overexpressing ß₁AR (3-fold, 60 minutes), the elevation of CaMKII activity by native ß₁AR in heart cells is modest (35% over baseline) yet exhibits faster kinetics ( 15 minutes) (Figure 5B). These quantitative discrepancies might reflect differences in the receptor density or the coupling efficiency of ß₁AR to downstream signaling pathways in these two systems.

Molecular Integration of ß₁AR-Stimulated PKA and CaMKII Signals
That short-term and sustained ß₁AR contractile and Ca²⁺ responses are virtually indistinguishable indicates a seamless integration of ß₁AR-stimulated PKA and CaMKII signals at the molecular and cellular levels. Indeed, we detected that a shift of PLB phosphorylation from the PKA-dependent site (¹⁶Ser) in short-term ß₁AR stimulation to the CaMKII-dependent site (¹⁷Thr), which correlates well with the time-dependent changes in cAMP production and CaMKII activation in sustained ß₁AR stimulation. Because either site phosphorylation of PLB is sufficient to release its inhibition on the SERCA,^28,31,32 PLB serves as a key molecular integrator of the dual signaling pathways of ß₁AR stimulation. Disinhibition of SERCA activity by PLB phosphorylation will enhance SR Ca²⁺ recycling, accelerate relaxation of Ca²⁺ transients and cell contraction, and subsequently increase the SR Ca²⁺ load,¹² contributing to both the positive inotropic and relaxant effects of ß₁AR stimulation. It is noteworthy that the relaxant effect of sustained norepinephrine exposure was not significantly influenced by CaMKII inhibitors (Figure 2C), suggesting the possibility for a differential regulation of peak contraction and relaxation by sustained ß₁AR stimulation.

In addition to PLB, previous studies have also shown that LCC Ca²⁺ currents are regulated by both PKA and CaMKII.^21,33 Our preliminary data suggested the enhancement of LCC Ca²⁺ currents during initial (10 to 30 minutes) and sustained (3 to 6 hours) ß₁AR stimulation is mediated by PKA- and CaMKII-dominant mechanisms, respectively, suggesting LCC serves as another molecular integrator of the PKA and CaMKII signals of ß₁AR stimulation. In addition, it has been documented that either PKA or CaMKII can phosphorylate RyRs,^20,34 although functional consequence of such phosphorylation remains controversial.^34–36

Functional Significance of ß₁AR Signaling Switch
The finding of time-dependent switch between two signaling pathways of ß₁AR stimulation bears important implications in understanding physiological modulation of cardiac function as well as the etiology and pathophysiology of cardiac diseases associated with chronically exaggerated ßAR signaling. First, the present result reassures that short-term ß₁AR stimulation, as in fight-or-flight response or during exercise, is largely mediated by the cAMP/PKA pathway, rapid activation of which enables a beat-to-beat regulation of cardiac performance. However, the cAMP/PKA signaling desensitizes nearly completely (Figure 5A) and is unlikely to play any major role for more enduring responses. In contrast, the CaMKII signaling does not undergo any appreciable desensitization over the period of observation (up to 24 hours). If this can be extrapolated to chronic ß₁AR stimulation in vivo, a corollary is that long-term ß₁AR effects, beneficial or toxic, must be due primarily to CaMKII-dependent signal transduction. This concept resonates with several lines of evidence found in previous studies. First, ßAR-stimulated cardiac cell hypertrophy is largely PKA-independent but requires CaMKII activation.^37,38 Second, CaMKII, but not PKA activation is obligatory to ß₁AR-mediated cardiac apoptosis.¹² Third, whole-animal and clinic data hint on that cardiac toxic effects of chronically enhanced ßAR stimulation are likely PKA-independent.³⁹ Emerging evidence also suggests that increased CaMKII activity in human heart failure might play a compensatory role for the decreased cardiac contractility in failing hearts.^40,41

Possible Mechanisms Underlying CaMKII Activation
The exact mechanisms underlying CaMKII activation and the switch of signaling pathways remain unknown. Preliminary observations showed that the ß₁AR CaMKII-dependent response is insensitive to G_i/G_o inhibition by pertussis toxin (1.5 µg/mL, added 3 hours before norepinephrine). Results in Figure 6 further suggest that direct and sustained activation of the cAMP/PKA signaling failed to activate CaMKII, and that blockage of cAMP/PKA does not prevent CaMKII from activation after ß₁AR stimulation; thus, the ß₁AR-cAMP signaling is neither necessary nor sufficient for the activation of ß₁AR-induced CaMKII signaling. Apart from the cAMP/PKA pathway, some evidences support a direct coupling between LCC and GPCRs including ß₂AR.^42,43 Overexpression of G_s has also been shown to activate LCC currents via PKA-independent mechanisms.⁴⁴ If LCC could be activated in a PKA-independent manner, LCC Ca²⁺ entry might be responsible for the gradual CaMKII activation during ß₁AR stimulation. In this scenario, the cAMP-dependent activation of LCC may differ from ß₁AR-dependent, but cAMP-independent, LCC activation because sustained cAMP-dependent activation of LCC by forskolin or active cAMP analogue does not express the CaMKII signaling even after 24 hours. Recent studies have also hinted on a G protein–independent GPCR signaling. For instance, the carboxyl terminus of ß₁AR can directly interact with PDZ-motif containing proteins such as PSD-95 and Ras exchanger regulatory factor.^45,46 By analogy, ß₁AR might directly activate element(s) of the CaMKII pathway through G protein–independent mechanisms. Future investigations are warranted to explore these possibilities.

In summary, ß₁AR modulation of cardiac E-C coupling invokes dual signaling pathways mediated by cAMP/PKA and CaMKII, respectively. The cAMP/PKA signaling is biphasic with a prominent early peak, whereas the CaMKII signaling is slow but persistent. During the signaling switch, the inotropic and relaxant responses are maintained at the steady state because of the convergence of PKA and CaMKII signals onto common effector proteins involved in E-C coupling and intracellular Ca²⁺ regulation. As a result, the effects of sustained ß₁AR stimulation (eg, inotropy, cell growth, and cell death) are due primarily to CaMKII, rather than PKA signaling. These findings provide mechanistic insights into ßAR modulation of cardiac function and GPCR signaling, and suggest new concepts for mechanistic understanding and therapeutic treatment of cardiac conditions such as hypertension and chronic heart failure that are associated with sustained elevation of endogenous catecholamines.

	Acknowledgments

 
This work was supported by NIH intramural research programs (R.-P.X., H.C.). We thank Dr Edward G. Lakatta for critical comments and Bruce Ziman for cell isolation.

	Footnotes

 
This manuscript was sent to Hans Michael Piper, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Original received April 26, 2004; revision received September 2, 2004; accepted September 7, 2004.

	References

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