Published online before print September 18, 2008, doi: 10.1161/CIRCRESAHA.108.176024
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2008 American Heart Association, Inc.
Cellular Biology |
Nuclear 
1-Adrenergic Receptors Signal Activated ERK Localization to Caveolae in Adult Cardiac Myocytes
From the Cardiovascular Research Center, Sanford Research/University of South Dakota, Sioux Falls.
Correspondence to Timothy D. O'Connell, Cardiovascular Research Center, Sanford Research/USD, 1100 E 21st St, Suite 700, Sioux Falls, SD 57105. E-mail oconnelt{at}sanfordhealth.org
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We previously identified an
1-AR-ERK (1A-adrenergic receptor–extracellular signal-regulated kinase) survival signaling pathway in adult cardiac myocytes. Here, we investigated localization of 1-AR subtypes (1A and 1B) and how their localization influences 1-AR signaling in cardiac myocytes. Using binding assays on myocyte subcellular fractions or a fluorescent 1-AR antagonist, we localized endogenous 1-ARs to the nucleus in wild-type adult cardiac myocytes. To clarify 1 subtype localization, we reconstituted 1 signaling in cultured 1A- and 1B-AR double knockout cardiac myocytes using 1-AR–green fluorescent protein (GFP) fusion proteins. Similar to endogenous 1-ARs and 1A- and 1B-GFP colocalized with LAP2 at the nuclear membrane. 1-AR nuclear localization was confirmed in vivo using 1-AR-GFP transgenic mice. The 1-signaling partners Gq and phospholipase Cβ1 also colocalized with 1-ARs only at the nuclear membrane. Furthermore, we observed rapid catecholamine uptake mediated by norepinephrine-uptake-2 and found that 1-mediated activation of ERK was not inhibited by a membrane impermeant 1-blocker, suggesting 1 signaling is initiated at the nucleus. Contrary to prior studies, we did not observe 1-AR localization to caveolae, but we found that 1-AR signaling initiated at the nucleus led to activated ERK localized to caveolae. In summary, our results show that nuclear 1-ARs transduce signals to caveolae at the plasma membrane in cardiac myocytes.
Key Words: 1-adrenergic receptors • cardiac myocytes • ERK
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cardiovascular disease is the leading killer in the United States, accounting for 1.4 million deaths a year. Five million Americans experience heart failure, leading to 970 000 hospitalizations annually, a number that has tripled in the last 25 years.1 In heart failure, increased activation of the sympathetic nervous system is correlated with pathophysiologic remodeling of the heart,2 which has led to the therapeutic use of β-adrenergic receptor (AR) antagonists in heart failure. However, the general conclusion that inhibition of catecholamine activation of ARs is beneficial in heart failure is disputed by clinical trials with
1-AR antagonists. The Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT) demonstrated that the 1-antagonist doxazosin increased the risk of heart failure by 80% and stroke by 26% leading to termination of the trial.3,4 Similar detrimental effects were seen in the Vasodilator-Heart Failure Trials (V-HeFT), in which prazosin was compared with other vasodilators for the prevention of death in heart failure.5
All 3 1-AR subtypes (A, B, and D) are expressed in the heart6–9; however, cardiac myocytes only express the 1A and 1B.9 Using 1A- and 1B-AR double knockout mice (1ABKO), we demonstrated previously that 1-ARs are required for postnatal hypertrophy and adaptation to pathological stress.9,10 In 1ABKO mice, we found that aortic constriction induced dilated cardiomyopathy that led to heart failure and death.9 More recently, we identified an 1A-AR-ERK (1A-AR–extracellular signal-regulated kinase) survival signaling pathway in adult cardiac myocytes,11 and knockout of this 1A-ERK pathway could explain the maladaptive response to aortic constriction in 1ABKO mice. In summary, our data demonstrate that 1-ARs are protective in the heart, which agrees with clinical trials where 1-antagonists increased the incidence of heart failure.
Here, we examined the mechanisms of 1-AR survival signaling and expanded on our previous demonstration of 1-AR localization to the nucleus in adult cardiac myocytes.11 To circumvent the lack of 1-AR subtype–specific antibodies and ligands, we developed a reconstitution system by expressing 1-AR-GFP (1-AR–green fluorescent protein) fusion proteins in cultured 1ABKO cardiac myocytes to examine 1-AR localization and survival signaling. With no endogenous 1-ARs, 1ABKO cardiac myocytes provide the ideal model for localization and signaling experiments. Earlier work in the mouse heart suggests that 1-ARs localize to the plasma membrane, possibly in caveolae and that 1-AR signaling can be modified by caveolin-3.12–15 These findings fit the classic "outside-in" model of G protein–coupled receptor (GPCR) localization and signaling because the immediate signaling partners (Gq and phospholipase [PL]Cβ1) are also localized to caveolae.16,17 In contrast, our previous data using 1-AR-GFP fluorescent fusion proteins suggested a nuclear localization for 1-ARs in adult cardiac myocytes.11 Our identification of a GPCR (1-AR) localized to the nucleus is not without precedence. Recently, functional endothelin-A, endothelin-B and β-ARs were identified on the nucleus in adult cardiac myocytes.18,19
Using our reconstitution system, we set out to clarify 1-AR localization and study the mechanisms behind 1-AR-ERK survival signaling in adult cardiac myocytes. In this report, we found that endogenous 1-ARs localized to the nucleus in wild-type (WT) adult mouse cardiac myocytes, which was confirmed in vivo using a cardiac-specific 1-AR transgenic mouse. Confocal microscopy and cellular fractionation demonstrated that the 1-AR subtypes, Gq, and PLCβ1 localized to the nuclear membrane in adult cardiac myocytes. Furthermore, we found no evidence that either 1-AR subtype localized to the plasma membrane or caveolae. Finally, activation of functional 1-ARs on the nucleus led to accumulation of activated (phosphorylated) ERK in caveolae at the plasma membrane. Our results present a provocative new model by demonstrating that 1-AR signaling initiated at the nucleus results in activated ERK localization in caveolae at the plasma membrane.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The procedures for generating adenoviral constructs,11 culture of adult mouse cardiac myocytes,10,11 localization of
1-ARs by confocal microscopy,11 isolation18,19 and enrichment20 of nuclei, and measurement of 1-AR expression9 have been described elsewhere. Methods describing the generation of 1-AR-GFP transgenic mice, catecholamine uptake assay, treatment of cardiac myocytes with CGP-12177A, and detection of the extraneuronal monoamine transporter (EMT/OCT3) are available in the expanded Materials and Methods section of the online data supplement at http://circres.ahajournals.org. The use of animals in this study conformed to the Public Health Service Guide for Care and Use of Laboratory Animals and was approved by The University of South Dakota Institutional Animal Care and Use Committee. |
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Endogenous
1-Adrenergic Receptors Localize to the Nucleus in WT Adult Mouse Cardiac MyocytesHere, we set out to determine
1-AR subtype localization in adult cardiac myocytes. To examine 1-AR subcellular distribution, we isolated membrane, cytosolic, and nuclear fractions from freshly isolated WT adult mouse cardiac myocytes and measured 1-AR binding with 3H-prazosin. Membrane, cytosolic, and nuclear fractions were validated by Western blots for caveolin-3, GAPDH, and LAP2, respectively (Figure 1a). Binding assays with 3H-prazosin indicated that 80% of total 1-AR binding was detected in the nuclear fraction. Binding detected in the membrane fraction was likely a result of 1-ARs localized to endoplasmic reticulum, because SERCA2 was detected in the membrane fraction (Figure 1a). To verify this finding, we examined endogenous 1-AR localization in cultured WT adult cardiac myocytes by labeling 1-ARs with a fluorescent, subtype-nonselective 1-AR antagonist, BODIPY-prazosin, which overcomes the lack of subtype-specific 1-AR antibodies. BODIPY-prazosin labeling revealed that endogenous 1-ARs were concentrated around the nucleus but not on the plasma membrane in WT cardiac myocytes (Figure 1b). To verify that 1-ARs were expressed on the nucleus in cardiac myocytes, isolated nuclei from WT adult cardiac myocytes were also labeled with BODIPY-prazosin, and a strong signal was detected on the nuclear membrane (Figure 1b, insets). In summary, these data suggest that endogenous 1-ARs localize primarily to the nucleus in WT adult cardiac myocytes.
|
The 1A and 1B Subtypes Colocalize With the Nuclear Membrane Protein LAP2 in Adult Mouse Cardiac Myocytes
To test whether the 1A and 1B subtypes localize to the nucleus, we assessed colocalization of both 1 subtypes with the inner nuclear matrix protein LAP2. Again, to overcome the lack of subtype-specific antibodies, we expressed 1-fluorescent fusion proteins (1-GFP) in cultured 1ABKO adult cardiac myocytes, which lack endogenous 1-ARs.11 In this reconstitution system, the 1-GFP fusion proteins are expressed at only 2.5- to 3-fold over endogenous 1-AR levels.11 Confocal microscopy revealed an intense GFP signal for both the 1A and 1B and, as expected, an intense fluorescent signal (red) for LAP2 on the nuclear membrane (Figure 2a). Using Imaris image analysis software, we demonstrated that both 1 subtypes colocalize with the nuclear matrix protein LAP2 (Figure 2a, yellow).
|
To confirm the confocal microscopy results, cellular fractions were isolated from cultured 1ABKO adult cardiac myocytes expressing the 1-GFP fusion proteins. Membrane, cytosolic, and nuclear fractions were validated by Western blots for caveolin-3, GAPDH, and LAP2, respectively (Figure 2b). As expected, both the 1A-GFP and 1B-GFP were detected predominantly in the isolated nuclear fraction (Figure 2b). In summary, the results obtained in our reconstitution system by confocal microscopy and subcellular fractionation demonstrated that the 1A and 1B subtypes were expressed in the nuclear membrane and confirmed the nuclear localization of endogenous 1-ARs in WT adult cardiac myocytes.
Prazosin Induces 1-Adrenergic Receptors to Move off the Nuclear Membrane in Adult Mouse Cardiac Myocytes
In cultured WT adult cardiac myocytes, BODIPY-prazosin labeling also identified a small population of nonnuclear 1-ARs (Figure 1b). We hypothesized that this was attributable to long-term incubation with BODIPY-prazosin, which induces β-arrestin–dependent receptor internalization (movement off the membrane).21 To test this, we expressed the 1-GFP fusion proteins in cultured 1ABKO cardiac myocytes and examined the 1-subtype localization throughout a 16-hour treatment with unlabeled prazosin. Indeed, prazosin induced translocation of both the 1A-GFP and 1B-GFP off the nucleus, whereas vehicle treatment had no effect (Figure 3). This effect was seen at 2 hours and was consistent throughout treatment for the 1A-GFP (Figure 3, time course). In summary, the nonnuclear 1-AR population observed with BODIPY-prazosin staining is likely attributable to 1-AR translocation off the nuclear membrane, possibly by receptor desensitization, rather than a subpopulation of nonnuclear 1-ARs.
|
1-Adrenergic Receptors Localize to the Nucleus in Adult Mouse Cardiac Myocytes In Vivo
To determine whether the nuclear 1-AR localization we observed in cultured adult cardiac myocytes was similar in vivo, we generated cardiac-specific 1A-GFP transgenic mice and used an antibody against GFP to identify 1A-subtype localization in whole heart sections. Heart sections from the 1A-GFP transgenic mice stained for GFP, identified the 1A subtype at the nucleus (Figure 4), which parallels results from our culture model. Saturation binding analysis indicated that the 1A-GFP was overexpressed 7-fold in the heart (Figure Ia in the online data supplement). Functionally, 1A-GFP overexpression increased 1-mediated phosphorylation of ERK, as determined by Western blot (supplemental Figure Ib). However, we did not detect a hypercontractile phenotype, as previously reported in 1A-Tg mice with 170-fold overexpression, nor did we detect a hypertrophic phenotype, as observed with overexpression of the 1B subtype (data not shown).12,22,23
|
The 1A and 1B Subtypes Colocalize With Gq and PLCβ1 in the Nuclear Membrane in Adult Mouse Cardiac Myocytes
If functional 1-ARs are expressed on the nuclear membrane in adult cardiac myocytes, then the 1-signaling partners Gq and PLCβ1 should colocalize to the nucleus as well. To test this, we used immunocytochemistry to determine whether Gq and PLCβ1 were also expressed on the nuclear membrane. In cultured 1ABKO cardiac myocytes expressing 1A-GFP or 1B-GFP, we found that both Gq and PLCβ1 localized to the nuclear membrane, as well as the plasma membrane (Figure 5a and 5b, respectively). However, image analysis revealed that both Gq and PLCβ1 colocalized with the 1 subtypes only at the nuclear membrane.
|
To confirm the immunocytochemical analyses performed above, cellular fractionation and Western blot analyses were performed on freshly isolated WT adult mouse cardiac myocytes (Figure 5c). Western blots for caveolin-3, GAPDH, and LAP2 validated the purity of the membrane, cytosolic, and nuclear fractions (Figure 5c). Blotting the WT cell fractions for Gq and PLCβ1 detected both signaling molecules in the nuclear fraction (Figure 5c). In summary, Gq and PLCβ1 colocalized with both 1-AR subtypes only in the nuclear membrane (defined by LAP2 staining), which supports the hypothesis that 1-ARs could signal at the nucleus in adult cardiac myocytes.
1-Adrenergic Receptors Do Not Localize to Caveolae in Adult Mouse Cardiac Myocytes
The colocalization of the 1-AR subtypes, Gq, and PLCβ1 to the nuclear membrane suggests that 1-ARs signal from the nucleus not the plasma membrane. However, previous research suggested that 1-ARs localize to caveolae in rats17 and adult mice.12 Furthermore, others demonstrated that increasing caveolin-3 expression inhibits 1-ERK signaling in cardiac myocytes, whereas knocking out caveolin-3 leads to hyperactivation of ERK signaling.14,15 Here, we immunolabeled caveolin-3 to determine whether the 1-AR subtypes localized to caveolae. In cultured 1ABKO cardiac myocytes expressing the 1-GFP fusion proteins, caveolin-3 localized to the plasma membrane and t-tubules, whereas both 1-AR subtypes remained on the nucleus (Figure 6). The failure to observe caveolin-3 colocalization with either 1-AR subtype provides evidence against a direct interaction of 1-ARs with caveolin-3 in adult cardiac myocytes. These results also suggest that the modulation of 1-AR signaling by caveolin-3 likely occurs with signaling molecules downstream of the receptor itself.
|
Catecholamine Uptake and Activation of Nuclear 1-Adrenergic Receptors in Adult Mouse Cardiac Myocytes
To activate nuclear 1-ARs, norepinephrine or other catecholamines must enter the cardiac myocyte, a process known as norepinephrine-uptake-2,24 which is facilitated by extraneuronal monoamine transporter (EMT/OCT3).25 To measure catecholamine uptake in WT cardiac myocytes, we used a fluorescent catecholamine analog that fluoresces only when transported inside a cell. We found that catecholamine uptake occurred almost immediately, peaked at 30 minutes and was antagonized by addition of norepinephrine 15 minutes before catecholamine uptake measurement, indicating specificity (Figure 7a).
|
To demonstrate the functionality of nuclear 1-ARs, we examined 1-AR mediated activation of ERK in cultured WT adult cardiac myocytes. Specifically, we compared the ability of CGP-12177A, a membrane impermeable 1-antagonist26–29 and prazosin, which freely crosses the plasma membrane, to block phenylephrine (PE) (1-agonist) induced phosphorylation of ERK in cultured WT adult cardiac myocytes. Prazosin blocked PE-mediated activation of ERK, whereas CGP-12177A did not (Figure 7b), indicating that 1-AR mediated activation of ERK requires intracellular agonist and receptor.
In cultured WT adult cardiac myocytes, we detected EMT/OCT3 on both the plasma and nuclear membranes by immunocytochemistry (Figure 7c). Furthermore, we demonstrated that inhibiting EMT/OCT3 with corticosterone, an EMT/OCT3 antagonist, prevented 1-mediated activation of ERK (Figure 7c). Because our results indicate that 1-ARs, Gq, and PLCβ1 localize to the nucleus, the results with CGP-12177A and corticosterone inhibition of EMT/OCT3 are best explained by 1 signaling at the nucleus.
Phosphorylated ERK Localizes to Caveolae at the Plasma Membrane in Adult Mouse Cardiac Myocytes
To investigate the mechanism of 1-mediated activation of ERK, we examined phosphorylated ERK localization following PE treatment. In cultured 1ABKO cardiac myocytes expressing 1A-GFP or 1B-GFP, PE led to phospho-ERK localization at the plasma membrane (Figure 8a). The 1A-GFP was more efficacious than 1B-GFP in activating ERK (Figure 8b), a difference we reported previously.11,30 Furthermore, in cardiac myocytes expressing the 1A-GFP, PE induced phospho-ERK plasma membrane localization within 5 minutes, which persisted for up to 3 hours (Figure 8c). The localization of phospho-ERK to the plasma membrane was surprising, because the receptor and its signaling molecules were localized to the nuclear membrane. However, short-term PE treatment, which activated ERK at the plasma membrane, did not translocate 1-ARs from the nucleus, suggesting 1-AR signaling was initiated at the nucleus, with a postreceptor signal translocated to the plasma membrane.
|
To determine whether phosphorylated ERK localized to caveolae at the plasma membrane, we colocalized phospho-ERK and caveolin-3 following 1-AR stimulation in cultured 1ABKO cardiac myocytes expressing the 1A-GFP (Figure 8d). Following PE treatment, phospho-ERK and caveolin-3 colocalized at the plasma membrane. To verify the caveolar localization of phospho-ERK, we disrupted the assembly of caveolae using filipin.31 Increasing concentrations of filipin reduced the colocalization of phospho-ERK and caveolin-3, suggesting an interaction between phospho-ERK and caveolin-3 (Figure 8d). A putative caveolin-binding domain is present in ERK and previous research has shown an interaction between ERK and caveolins,32–34 but we could not confirm a direct interaction between ERK and caveolin-3 by coimmunoprecipitation. However, our results demonstrating a loss of phospho-ERK and caveolin-3 colocalization following filipin treatment indicates that ERK likely localizes to caveolae, possibly via some intermediate binding partner. Furthermore, although 1-ARs do not localize to caveolae (Figure 6), 1-AR signaling could still be modified indirectly by caveolin-3 at the plasma membrane.14,15 In summary, our data suggest a novel model for 1-AR function in cardiac myocytes, where activation of nuclear 1-ARs leads to activated ERK localized to caveolae at the plasma membrane.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Here, we examined the localization of both the
1A and 1B subtypes and the mechanisms regulating 1A-AR-ERK survival signaling in adult cardiac myocytes. Our data indicate that both the 1A and 1B subtypes localize to and signal at the nuclear membrane. Furthermore, our data suggest that 1-signal transduction from the nucleus to the plasma membrane results in activated ERK localization to caveolae in adult cardiac myocytes. The following lines of evidence support this novel paradigm for 1-AR signaling in cardiac myocytes.
Using binding assays on subcellular fractions and a fluorescent 1 antagonist, we localized endogenous 1-ARs to the nucleus in WT adult cardiac myocytes, which was confirmed using 1-GFP fusion proteins in cultured 1ABKO cardiac myocytes and in vivo with 1-AR-GFP transgenic mice (Figures 1 through 4
). Furthermore, we colocalized the 1-signaling partners Gq and PLCβ1 with both 1-AR subtypes in the nuclear membrane (Figure 5), suggesting the possibility of 1 signaling at the nucleus. We failed to detect functional 1-ARs at the plasma membrane using confocal microscopy or functional studies examining 1-AR–mediated activation of ERK (Figures 6 and 7b). However, activation of nuclear 1-ARs led to accumulation of activated (phosphorylated) ERK at the plasma membrane in adult cardiac myocytes, whereas the receptor remained at the nucleus (Figure 8a). Finally, we demonstrated that phosphorylated ERK localizes to caveolae at the plasma membrane, which might provide a mechanism for caveolin-3 regulation of 1-AR signaling (Figure 8d).
Previously, others hypothesized that 1-ARs are expressed in caveolae at the plasma membrane in cardiac myocytes.16,17,35 These reports relied on isolation of caveolar membrane fractions, followed by immunoblots to detect 1-signaling partners and functional examination of inositol phosphate generation. However, some of these reports acknowledged the failure to identify 1-ARs in caveolae, possibly because of basic (pH 11) conditions used to isolate the caveolar membrane fractions.16,35 Alternatively, our data indicated that 1-ARs are expressed on the nuclear membrane in cardiac myocytes. The use of BODIPY-prazosin and 1-GFP fusion proteins in our reconstitution system avoided the technical challenges of isolating caveolar membrane fractions. Prior research also demonstrated that overexpression of caveolin-3 suppressed 1-AR signaling,15 and caveolin-3 knockout increased activation of ERK.14 In contrast to others,12,17 we demonstrated that these effects were probably not attributable to a direct interaction of 1-ARs and caveolin-3. However, our finding that activated ERK localized to caveolae following 1-AR stimulation provides a mechanism where caveolin-3 might regulate 1-AR signaling. Earlier studies in cardiac-specific 1A-AR transgenic mice suggested that 1-ARs are expressed on the plasma membrane in cardiac myocytes in vivo based on immunohistochemical detection of the 1A subtype in ventricular tissue sections.12 However, we observed 1A localization to the nucleus in our 1A-GFP Tg mouse. This discrepancy might be attributable to the 170-fold level of overexpression examined in prior reports.12 In summary, our results, both in vivo and in vitro, indicate that 1-ARs localize to the nuclear membrane in adult cardiac myocytes.
Nuclear localization of GPCRs in adult cardiac myocytes is not unique to 1-ARs. Functional endothelin-A and -B receptors and β-ARs, as well as their cognate signaling proteins, are also localized to the nuclear membrane of adult cardiac myocytes.18,19 Both of these reports directly demonstrated the functionality of their respective receptors on isolated nuclei, detecting a calcium transient in response to endothelin treatment18 and cAMP accumulation in response to isoproterenol treatment.19 Here, we localized 1-ARs, Gq, and PLCβ1 to the nuclear membrane, and our experiments examining 1-mediated ERK activation indicate that 1-AR signaling was initiated at the nucleus in adult cardiac myocytes.
If 1-ARs localize to and activate signaling at the nucleus, then 1-agonists must enter the myocyte to initiate signaling. Norepinephrine entry into cardiac myocytes is facilitated by norepinephrine-uptake-2,24 which is mediated by extraneuronal monoamine transporter (EMT/OCT3).25 A previous report demonstrated that 3H-norepinephrine is taken up by neonatal rat cardiac myocytes and accumulates in the nucleus.36 We confirmed catecholamines are taken up in adult cardiac myocytes and detectable inside the cell within 5 minutes (Figure 7a). In support of the results that demonstrate agonists like norepinephrine enter the myocyte to activate 1-ARs, we found that CGP-12177A, a membrane impermeable 1-AR antagonist, was unable to block PE-induced activation of ERK in cardiac myocytes, whereas prazosin did (Figure 7b). Finally, we also identified EMT/OCT3 on both the plasma and nuclear membranes and confirmed its role in catecholamine uptake in adult cardiac myocytes (Figure 7c).
In total, our data suggest a new model for 1-AR survival signaling. We propose that ligand enters the cardiac myocyte, binds to 1-ARs, and activates 1 signaling at the nucleus, which is transduced from the nucleus to the plasma membrane, causing activated ERK localization to caveolae and protection from cell death. This novel 1-signaling model does not fit the classic outside-in GPCR signaling model or the "caveolae signaling hypothesis" as described previously.33 However, some details of this new model remain to be determined. First, the mechanism regulating 1-signal transduction from the nucleus to the plasma membrane is unknown but could involve protein kinase C, which is activated by 1-ARs, translocates to caveolae when activated,37 and could lead to ERK phosphorylation. Second, potential downstream targets of 1-ERK signaling in cardiac myocytes are unidentified. Previous work in neonatal cardiac myocytes suggested that targets could include the survival/transcription factor GATA4 and the Bcl-2 family member Bad.38,39 However, we recently demonstrated that 1 signaling does not activate GATA4 and that GATA4 was not required for 1-mediated survival signaling in adult cardiac myocytes.40
In summary, we found that the 1A and 1B subtypes localized to and activated signaling at the nuclear membrane in adult cardiac myocytes. Activation of these nuclear 1-ARs led to accumulation of activated ERK at the plasma membrane, where caveolin-3 might regulate downstream 1-AR signaling. These results present a provocative new model for 1-AR signaling, where nuclear 1-ARs signal to the plasma membrane. This unique 1-AR-ERK survival-signaling pathway challenges the classic model of 1-AR localization and signaling in adult cardiac myocytes.
|
Acknowledgments |
|---|
We acknowledge Paul C. Simpson for critical discussion of the manuscript.
Sources of Funding
This work was supported by the Pharmaceutical Research and Manufacturers of America Foundation (to C.D.W.), American Heart Association Grant 0435338Z (to T.D.O.), the South Dakota State Legislature 2010 Grant (to T.D.O.), and the NIH grant P20 RR-017662 (to T.D.O.).
Disclosures
None.
|
Footnotes |
|---|
Original received November 2, 2007; resubmission received March 20, 2008; revised resubmission received August 28, 2008; accepted September 4, 2008.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. American Heart Association. Heart Disease and Stroke Statistics - 2005 Update. Dallas, Tex: American Heart Association; 2005.
2. Cohn JN, Levine TB, Olivari MT, Garberg V, Lura D, Francis GS, Simon AB, Rector T. Plasma norepinephrine as a guide to prognosis in patients with chronic congestive heart failure. N Engl J Med. 1984; 311: 819–823.[Abstract]
3. ALLHAT. Diuretic versus alpha-blocker as first-step antihypertensive therapy: final results from the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT). Hypertension. 2003; 42: 239–246.
4. ALLHAT. Major cardiovascular events in hypertensive patients randomized to doxazosin vs chlorthalidone: the antihypertensive and lipid-lowering treatment to prevent heart attack trial (ALLHAT). ALLHAT Collaborative Research Group. JAMA. 2000; 283: 1967–1975.
5. Loeb HS, Johnson G, Henrick A, Smith R, Wilson J, Cremo R, Cohn JN. Effect of enalapril, hydralazine plus isosorbide dinitrate, and prazosin on hospitalization in patients with chronic congestive heart failure. The V-HeFT VA Cooperative Studies Group. Circulation. 1993; 87 (suppl VI): VI-78–VI-87.[Medline] [Order article via Infotrieve]
6. Turnbull L, McCloskey DT, O'Connell TD, Simpson PC, Baker AJ. Alpha 1-adrenergic receptor responses in alpha 1AB-AR knockout mouse hearts suggest the presence of alpha 1D-AR. Am J Physiol Heart Circ Physiol. 2003; 284: H1104–H1109.
7. Yang M, Reese J, Cotecchia S, Michel MC. Murine alpha1-adrenoceptor subtypes. I. Radioligand binding studies. J Pharmacol Exp Ther. 1998; 286: 841–847.
8. Alonso-Llamazares A, Zamanillo D, Casanova E, Ovalle S, Calvo P, Chinchetru MA. Molecular cloning of alpha 1d-adrenergic receptor and tissue distribution of three alpha 1-adrenergic receptor subtypes in mouse. J Neurochem. 1995; 65: 2387–2392.[Medline] [Order article via Infotrieve]
9. O'Connell TD, Ishizaka S, Nakamura A, Swigart PM, Rodrigo MC, Simpson GL, Cotecchia S, Rokosh DG, Grossman W, Foster E, Simpson PC. The alpha(1A/C)- and alpha(1B)-adrenergic receptors are required for physiological cardiac hypertrophy in the double-knockout mouse. J Clin Invest. 2003; 111: 1783–1791.[CrossRef][Medline] [Order article via Infotrieve]
10. O'Connell TD, Swigart PM, Rodrigo MC, Ishizaka S, Joho S, Turnbull L, Tecott LH, Baker AJ, Foster E, Grossman W, Simpson PC. alpha-1-Adrenergic receptors prevent a maladaptive cardiac response to pressure overload. J Clin Invest. 2006; 116: 1005–1015.[CrossRef][Medline] [Order article via Infotrieve]
11. Huang Y, Wright CD, Merkwan CL, Baye NL, Liang Q, Simpson PC, O'Connell TD. An alpha1A-adrenergic-extracellular signal-regulated kinase survival signaling pathway in cardiac myocytes. Circulation. 2007; 115: 763–772.
12. Lin F, Owens WA, Chen S, Stevens ME, Kesteven S, Arthur JF, Woodcock EA, Feneley MP, Graham RM. Targeted 1A-adrenergic receptor overexpression induces enhanced cardiac contractility but not hypertrophy. Circ Res. 2001; 89: 343–350.
13. Petrashevskaya NN, Bodi I, Koch SE, Akhter SA, Schwartz A. Effects of alpha1-adrenergic stimulation on normal and hypertrophied mouse hearts. Relation to caveolin-3 expression. Cardiovasc Res. 2004; 63: 561–572.
14. Woodman SE, Park DS, Cohen AW, Cheung MW, Chandra M, Shirani J, Tang B, Jelicks LA, Kitsis RN, Christ GJ, Factor SM, Tanowitz HB, Lisanti MP. Caveolin-3 knock-out mice develop a progressive cardiomyopathy and show hyperactivation of the p42/44 MAPK cascade. J Biol Chem. 2002; 277: 38988–38997.
15. Koga A, Oka N, Kikuchi T, Miyazaki H, Kato S, Imaizumi T. Adenovirus-mediated overexpression of caveolin-3 inhibits rat cardiomyocyte hypertrophy. Hypertension. 2003; 42: 213–219.
16. Morris JB, Huynh H, Vasilevski O, Woodcock EA. alpha(1)-Adrenergic receptor signaling is localized to caveolae in neonatal rat cardiomyocytes. J Mol Cell Cardiol. 2006; 41: 17–25.[CrossRef][Medline] [Order article via Infotrieve]
17. Fujita T, Toya Y, Iwatsubo K, Onda T, Kimura K, Umemura S, Ishikawa Y. Accumulation of molecules involved in alpha1-adrenergic signal within caveolae: caveolin expression and the development of cardiac hypertrophy. Cardiovasc Res. 2001; 51: 709–716.
18. Boivin B, Chevalier D, Villeneuve LR, Rousseau E, Allen BG. Functional endothelin receptors are present on nuclei in cardiac ventricular myocytes. J Biol Chem. 2003; 278: 29153–29163.
19. Boivin B, Lavoie C, Vaniotis G, Baragli A, Villeneuve LR, Ethier N, Trieu P, Allen BG, Hebert TE. Functional beta-adrenergic receptor signalling on nuclear membranes in adult rat and mouse ventricular cardiomyocytes. Cardiovasc Res. 2006; 71: 69–78.
20. Boheler KR, Chassagne C, Martin X, Wisnewsky C, Schwartz K. Cardiac expressions of - and β-myosin heavy chains and sarcomeric -actins are regulated through transcriptional mechanisms. J Biol Chem. 1992; 367: 12979–12985.
21. Pediani JD, Colston JF, Caldwell D, Milligan G, Daly CJ, McGrath JC. Beta-arrestin-dependent spontaneous alpha1a-adrenoceptor endocytosis causes intracellular transportation of alpha-blockers via recycling compartments. Mol Pharmacol. 2005; 67: 992–1004.
22. Milano CA, Dolber PC, Rockman HA, Bond RA, Venable ME, Allen LF, Lefkowitz RJ. Myocardial expression of a constitutively active 1B-adrenergic receptor in transgenic mice induces cardiac hypertrophy. Proc Natl Acad Sci U S A. 1994; 91: 10109–10113.
23. Zuscik MJ, Chalothorn D, Hellard D, Deighan C, McGee A, Daly CJ, Waugh DJ, Ross SA, Gaivin RJ, Morehead AJ, Thomas JD, Plow EF, McGrath JC, Piascik MT, Perez DM. Hypotension, autonomic failure, and cardiac hypertrophy in transgenic mice overexpressing the 1B-adrenergic receptor. J Biol Chem. 2001; 276: 13738–13743.
24. Obst OO, Rose H, Kammermeier H. Characterization of catecholamine uptake2 in isolated cardiac myocytes. Mol Cell Biochem. 1996; 163–164: 181–183.
25. Schomig E, Lazar A, Grundemann D. Extraneuronal monoamine transporter and organic cation transporters 1 and 2: a review of transport efficiency. Handb Exp Pharmacol. 2006: 151–180.
26. Brahmadevara N, Shaw AM, MacDonald A. Alpha1-adrenoceptor antagonist properties of CGP 12177A and other beta-adrenoceptor ligands: evidence against beta(3)- or atypical beta-adrenoceptors in rat aorta. Br J Pharmacol. 2004; 142: 781–787.[CrossRef][Medline] [Order article via Infotrieve]
27. Brahmadevara N, Shaw AM, MacDonald A. Evidence against beta 3-adrenoceptors or low affinity state of beta 1-adrenoceptors mediating relaxation in rat isolated aorta. Br J Pharmacol. 2003; 138: 99–106.[CrossRef][Medline] [Order article via Infotrieve]
28. Levin MC, Marullo S, Muntaner O, Andersson B, Magnusson Y. The myocardium-protective Gly-49 variant of the beta 1-adrenergic receptor exhibits constitutive activity and increased desensitization and down-regulation. J Biol Chem. 2002; 277: 30429–30435.
29. Staehelin M, Simons P, Jaeggi K, Wigger N. CGP-12177. A hydrophilic beta-adrenergic receptor radioligand reveals high affinity binding of agonists to intact cells. J Biol Chem. 1983; 258: 3496–3502.
30. Stanasila L, Perez JB, Vogel H, Cotecchia S. Oligomerization of the alpha 1a- and alpha 1b-adrenergic receptor subtypes. Potential implications in receptor internalization. J Biol Chem. 2003; 278: 40239–40251.
31. Xiang Y, Rybin VO, Steinberg SF, Kobilka B. Caveolar localization dictates physiologic signaling of beta 2-adrenoceptors in neonatal cardiac myocytes. J Biol Chem. 2002; 277: 34280–34286.
32. Das M, Das S, Das DK. Caveolin and MAP kinase interaction in angiotensin II preconditioning of the myocardium. J Cell Mol Med. 2007; 11: 788–797.[CrossRef][Medline] [Order article via Infotrieve]
33. Engelman JA, Chu C, Lin A, Jo H, Ikezu T, Okamoto T, Kohtz DS, Lisanti MP. Caveolin-mediated regulation of signaling along the p42/44 MAP kinase cascade in vivo. A role for the caveolin-scaffolding domain. FEBS Lett. 1998; 428: 205–211.[CrossRef][Medline] [Order article via Infotrieve]
34. Song KS, Li S, Okamoto T, Quilliam LA, Sargiacomo M, Lisanti MP. Co-purification and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains. Detergent-free purification of caveolae microdomains. J Biol Chem. 1996; 271: 9690–9697.
35. Lanzafame AA, Turnbull L, Amiramahdi F, Arthur JF, Huynh H, Woodcock EA. Inositol phospholipids localized to caveolae in rat heart are regulated by alpha1-adrenergic receptors and by ischemia-reperfusion. Am J Physiol Heart Circ Physiol. 2006; 290: H2059–H2065.
36. Buu NT, Hui R, Falardeau P. Norepinephrine in neonatal rat ventricular myocytes: association with the cell nucleus and binding to nuclear alpha 1- and beta-adrenergic receptors. J Mol Cell Cardiol. 1993; 25: 1037–1046.[CrossRef][Medline] [Order article via Infotrieve]
37. Rybin VO, Xu X, Steinberg SF. Activated protein kinase C isoforms target to cardiomyocyte caveolae: stimulation of local protein phosphorylation. Circ Res. 1999; 84: 980–988.
38. Liang Q, Wiese RJ, Bueno OF, Dai YS, Markham BE, Molkentin JD. The transcription factor GATA4 is activated by extracellular signal-regulated kinase 1- and 2-mediated phosphorylation of serine 105 in cardiomyocytes. Mol Cell Biol. 2001; 21: 7460–7469.
39. Valks DM, Cook SA, Pham FH, Morrison PR, Clerk A, Sugden PH. Phenylephrine promotes phosphorylation of Bad in cardiac myocytes through the extracellular signal-regulated kinases 1/2 and protein kinase A. J Mol Cell Cardiol. 2002; 34: 749–763.[CrossRef][Medline] [Order article via Infotrieve]
40. Huang Y, Wright CD, Kobayashi S, Healy CL, Elgethun M, Cypher AL, Liang Q, O'Connell TD. GATA4 is a survival factor in adult cardiac myocytes, but is not required for {alpha}1A-adrenergic receptor survival signaling. Am J Physiol Heart Circ Physiol. 2008; 295: H699–H707.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||