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Am J Physiol Heart Circ Physiol 288: H1193-H1202, 2005. First published November 4, 2004; doi:10.1152/ajpheart.00109.2004
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Inhibition of {beta}-adrenergic receptor trafficking in adult cardiocytes by MAP4 decoration of microtubules

Guangmao Cheng, Fei Qiao, Thomas N. Gallien, Dhandapani Kuppuswamy, and George Cooper, IV

Gazes Cardiac Research Institute, Cardiology Division, Medical University of South Carolina, and Department of Veterans Affairs Medical Center, Charleston, South Carolina

Submitted 3 February 2004 ; accepted in final form 27 October 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Decreased {beta}-adrenergic receptor ({beta}-AR) number occurs both in animal models of cardiac hypertrophy and failure and in patients. {beta}-AR recycling is an important mechanism for the {beta}-AR resensitization that maintains a normal complement of cell surface {beta}-ARs. We have shown that 1) in severe pressure overload cardiac hypertrophy, there is extensive microtubule-associated protein 4 (MAP4) decoration of a dense microtubule network; and 2) MAP4 microtubule decoration inhibits muscarinic acetylcholine receptor recycling in neuroblastoma cells. We asked here whether MAP4 microtubule decoration inhibits {beta}-AR recycling in adult cardiocytes. [3H]CGP-12177 was used as a {beta}-AR ligand, and feline cardiocytes were isolated and infected with adenovirus containing MAP4 (AdMAP4) or {beta}-galactosidase (Ad{beta}-gal) cDNA. MAP4 decorated the microtubules extensively only in AdMAP4 cardiocytes. {beta}-AR agonist exposure reduced cell surface {beta}-AR number comparably in AdMAP4 and Ad{beta}-gal cardiocytes; however, after agonist withdrawal, the cell surface {beta}-AR number recovered to 78.4 ± 2.9% of the pretreatment value in Ad{beta}-gal cardiocytes but only to 56.8 ± 1.4% in AdMAP4 cardiocytes (P < 0.01). This result was confirmed in cardiocytes isolated from transgenic mice having cardiac-restricted MAP4 overexpression. In functional terms of cAMP generation, {beta}-AR agonist responsiveness of AdMAP4 cells was 47% less than that of Ad{beta}-gal cells. We conclude that MAP4 microtubule decoration interferes with {beta}-AR recycling and that this may be one mechanism for {beta}-AR downregulation in heart failure.

myocardium; hypertrophy; tubulin; adenovirus

TEN YEARS AGO (32), we reported that the progressive contractile abnormalities characteristic of severe pressure overload cardiac hypertrophy are attributable to a remarkable extent to a densification of the microtubule component of the cardiocyte extramyofilament cytoskeleton. In subsequent work reviewed elsewhere (6), we and others observed that increased microtubule network density, which imposes a viscous load on active myofilaments, is seen in myocardium hypertrophying in response to pressure overloading of either ventricle in each of the five mammalian species examined to date, including humans. This microtubule network densification, which in our hands has tight specificity for severe pressure overload hypertrophy, which fails to maintain normal ventricular wall stress, appears to have a major role in the contractile dysfunction found in this distinct but common hemodynamic setting, because in each of our animal models microtubule depolymerization restored normal contractile function on the levels of the isolated cell, isolated tissue, and in vivo intact heart.

We have now found several potentially synergistic bases for this microtubule network densification (6): 1) there is transcriptional upregulation of the two genes encoding the {beta}1- and {beta}2-tubulin isoforms, whose expression is ordinarily minor in the heart of the adult, accounting for the persistent increase in {beta}-tubulin synthesis in pressure overload hypertrophy, and the hypertrophic regulation of these genes mimics their developmental cardiac regulation; 2) microtubule-associated protein 4 (MAP4), the major cardiac microtubule binding and stabilizing fibrous microtubule-associated protein, is both markedly upregulated in hypertrophy and decorates microtubules far more densely than in control hearts; and 3) microtubules in pressure hypertrophied cardiocytes are greatly stabilized compared with those in control cells. Of these three potential bases for the microtubule network densification characteristic of pressure overload cardiac hypertrophy, the second and third are the most important (31).

We then turn here from the direct mechanical effects of microtubule network densification on cardiocyte global constitutive properties to a consideration of the effects of this cytoskeletal alteration on unique microtubule-related functions. That is, given the central role of microtubules in localizing and regulating cellular constituents, it would seem quite unlikely that the marked changes in the microtubule network that we find in the hypertrophied cardiocyte would be without major specific effects on cellular homeostasis. Especially interesting here in the context of intracellular trafficking are the potential effects of the combination that we find in hypertrophy of microtubule network densification, MAP4 upregulation, and extensive MAP4 decoration of microtubules, because increased microtubule decoration with MAP4 inhibits the microtubule interactions of each of the major families of microtubule-associated motor proteins, causing inhibition of intracellular microtubule-based transport, and in cells whose microtubules are extensively decorated with MAP4, vesicle transport as well as steady-state vesicle position are quite abnormal (2, 16, 18).

In asking whether the alterations in microtubule network structure now defined have a role in known functional alterations of the pressure hypertrophied cardiocyte, we report here our studies of the potential role of altered microtubules in the {beta}-adrenergic receptor ({beta}-AR) downregulation found in the substantially pressure-overloaded myocardium where these cytoskeletal changes occur. Our hypothesis was that the extensive microtubule decoration by MAP4 that we see in hypertrophy would impede microtubule-based transport, especially that of G protein-coupled receptors via microtubule plus end-directed kinesin motor proteins. That is, given that {beta}-AR resensitization after agonist activation involves receptor internalization and endosomal transport, {beta}-AR downregulation in heart failure could occur, at least in part, through reduced receptor recycling after activation and {beta}-arrestin binding. We tested this hypothesis initially by studying muscarinic acetylcholine receptor internalization and recovery after agonist stimulation in neuroblastoma cells as a simplifying first step and found that receptor recovery after agonist exposure is microtubule dependent and that MAP4 decoration of microtubules inhibits receptor recovery (4). Because it is likely that this result reflects a general effect of MAP4 microtubule decoration rather than one specific to the muscarinic receptor in neuroblastoma cells, we extended this work here to the effects of MAP4 decoration of microtubules on {beta}-AR downregulation in cardiocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal models. All animal usage was under protocols approved by the institutional animal care committee in accordance with National Institutes of Health guidelines.

Construction of recombinant adenoviruses. The pTG3602 plasmid system for generating replication-defective recombinant adenoviruses via homologous recombination with a bacterial system (3, 24) was used to construct adenoviruses for MAP4 and {beta}-galactosidase ({beta}-gal) as a control for nonspecific effects of adenovirus infection. The MAP4 cDNA construct was generated by PCR using specific oligonucleotide primers and full-length human MAP4 cDNA (a gift from J. C. Bulinski, Columbia University) as a template (2). The primer for the NH2 terminus also contained a sequence for an epitope tag of amino acids 410–419 of human c-Myc, and KpnI restriction sites were included at the end of each primer. The sequence of the full-length MAP4 construct was confirmed by DNA sequencing. The KpnI fragment was subcloned into the pAD.CMV-Link.1 shuttle plasmid and sequenced to identify clones with correct orientation. The MAP4 shuttle vector construct was digested with NheI and ApaI and recombined with the ClaI-linearized viral DNA vector T63602 [GenBank] in Escherichia coli strain BJ5183. The recombinant adenovirus DNA containing the MAP4 (AdMAP4) expression cassette was linearized and transfected into the HEK-293 human epithelial kidney cell line using Lipofectamine reagent. An adenovirus expressing bacterial {beta}-gal (Ad{beta}-gal) was generated in the same way as described above. Each adenovirus was plaque purified, expanded, purified by CsCl gradient centrifugation, dialysed, and titered via the detection of visible plaque formation in HEK-293 monolayers.

Murine cardiocyte isolation and culture. Adult mouse cardiocytes were isolated as we have described previously (12). Briefly, mice (25–35 g) of random sex were anesthetized with ketamine (100 mg/kg ip) and xylazine (5 mg/kg ip), heparinized (200 IU ip), intubated, and placed on a respirator. The hearts were rapidly excised, cannulated via the aorta, and perfused in Langendorff mode at 80-cmH2O pressure at 37°C. Hearts were initially perfused for 5 min with 1.8 mM Ca2+-Tyrode solution containing (in mM) 137 NaCl, 5.4 KCl, 1.8 CaCl2, 0.5 MgCl2, 10 HEPES, and 10 glucose at pH 7.4 and then in Ca2+-free buffer for 5 min. This was followed by perfusion of the heart with Ca2+-free buffer containing 0.05% collagenase D (Boehringer-Mannheim; Indianapolis, IN) and 0.01% protease XIV (Sigma; St. Louis, MO). After the hearts were palpably flaccid at 6–8 min, the digestion solution was washed out with Ca2+-free Tyrode solution for 30 s. The hearts were removed from the cannula, and the left ventricle including septum was separated, minced, and gently agitated, allowing cardiocytes to be dispersed in the Ca2+-free Tyrode solution. After 15 min, the cardiocytes were resuspended at room temperature in oxygenated 0.1 mM Ca2+-Tyrode buffer at pH 7.4.

Feline cardiocyte isolation and culture. Adult feline cardiocytes were isolated as before (25) via retrograde coronary artery perfusion with a collagenase solution (Liberase Blendzyme 1, Roche Molecular Biochemicals; Indianapolis, IN) and maintained at 37°C on laminin-coated dishes (Becton-Dickinson; San Jose, CA) with 5% CO2 in serum-free Piper’s medium [medium 199 buffer supplemented with 5 mM creatine, 5 mM taurine, 2 mM carnitine, 100 µM ascorbic acid, 0.25 mM phenylalanine, 0.2% (wt/vol) fraction V BSA, 10 µM cytosine arabinofuranoside, 0.1 µM insulin, and 100 µg/ml penicillin with streptomycin].

Adenovirus-mediated gene transfer. Adult feline cardiocytes were infected with AdMAP4 in Piper’s medium (2 ml/35-mm plate) at the indicated multiplicity of infection (MOI) for 24 h. The cells were then rinsed in serum-free medium and incubated for a further 48 h to permit transgene expression. Parallel control cultures were infected at the same MOI with Ad{beta}-gal. Over a MOI range of 10–100 plaque-forming units (pfu)/cell of AdMAP4 or Ad{beta}-gal, there was after 48 h >90% cardiocyte infection as determined by immunofluorescence microscopy using either our polyclonal MAP4 antibody (4) or a {beta}-gal antibody; there was no observable cytotoxicity.

Transgenic mice from the FVB/N strain. For generating transgenic mice having {alpha}-myosin heavy chain promoter-driven cardiac-restricted overexpression of MAP4, which we have reported before (31), the cDNA encoding human MAP4, confirmed by DNA sequencing, was the same as that used for constructing the AdMAP4 virus. The MAP4 cDNA was fused at the 5' end to a sequence encoding the 9-amino acid immunodominant epitope of influenza hemagglutinin antigen. Two MAP4 mouse lines were made, both of which demonstrated homogeneous expression of the transgene in all ventricular cardiocytes via immunofluorescence microscopy of transmural frozen sections of the left ventricle. Third generation or later adult mice heterozygous for the transgene were used for these studies.

[3H]CGP-12177 binding assays. Cardiocyte cell surface {beta}-AR density was assayed in terms of the binding of 4-(3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole-2-on hydrochloride ([3H]CGP-12177; Perkin-Elmer; Boston, MA; specific activity 45 Ci/mmol), which is specific to cell surface {beta}-ARs (27). Following established methods (7, 15), the binding assays were performed at 4°C to avoid both receptor endocytosis and reappearance of the receptors on the cell surface after agonist-induced internalization. Nonspecific binding was determined by the addition of 10 µM propranolol, a {beta}-AR antagonist, to triplicate wells and represented <10% of total counts. This value was subtracted from the total binding to obtain specific plasmalemma-bound [3H]CGP-12177. Radioactivity of the solution was measured in a scintillation counter. The results were expressed as femtomoles per milligram of protein in each sample as determined by a bicinchoninic acid assay (Pierce Biotechnology; Rockford, IL). The binding data (Bmax and Kd) from saturation experiments were obtained via nonlinear regression analysis using Graphpad Prism software (GraphPad Software; San Diego, CA), and the curves were plotted.

Myocardial sarcolemmal {beta}-AR density was assayed for control and hypertrophied right ventricles in a membrane-enriched fraction (26). Feline right ventricular hypertrophy was induced by pulmonary artery banding as before (32). Four weeks later, the right ventricular free wall was dissected from sham-operated control and hypertrophied hearts and minced in ice-cold buffer [10 mM Tris·HCl (pH 7.4) and 250 mM sucrose]. The tissue was then homogenized (Polytron setting 8 for 30 s), filtered through gauze, and centrifuged at 40,000 g at 4°C for 30 min. The supernatant was discarded, and the pellet was homogenized (Polytron setting 8 for 20 s) in incubation buffer [120 mM Tris·HCl (pH 7.4) and 40 mM MgCl2] before centrifugation at 40,000 g at 4°C for 30 min. The final pellet was resuspended and gently homogenized in incubation buffer for the {beta}-AR binding assay, performed in triplicate with [3H]CGP-12177 in the presence (nonspecific) and absence (total) of 10 µM propranolol. Aliquots of these membrane-rich fractions (400 µg protein in each) were incubated with [3H]CGP-12177 for 2 h at room temperature in a final volume of 0.5 ml. The incubation was stopped by dilution with 5 ml of ice-cold washing buffer [50 mM Tris·HCl (pH 7.4) and 10 mM MgCl2], followed by rapid filtration through Whatman GF/C glass fiber filters. The filters were washed three times with 5 ml of ice-cold washing buffer, and the radioactivity retained on the filters was counted with a scintillation counter. The data were generated as described above for cardiocytes.

Determination of cAMP. Cellular cAMP was assessed as before (7) via a radioimmunoassay method according to the manufacturer’s instructions (Cyclic AMP [3H] Assay System, Amersham Biosciences; Piscataway, NJ). Briefly, the cells were harvested in 5 ml of ethanol after the treatment protocols described in Fig. 11 were completed. The cells were then homogenized on ice and kept at room temperature for 5 min before centrifugation. The supernatants were collected, and the precipitates were washed with 2.5 ml of ethanol-H2O (2:1) and centrifuged. The supernatants were pooled and evaporated to dryness at 55°C under a stream of nitrogen. The residues were dissolved in 0.5 ml of 0.05 M Tris·HCl (pH 7.5) and 4 mM EDTA before centrifugation to remove insoluble residues, and the supernatants were used in the cAMP assay system. These data were normalized to protein content as determined by a bicinchoninic acid assay (Pierce Biotechnology).



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  		Fig. 11. Adenovirus-mediated MAP4 overexpression inhibits {beta}-AR-stimulated cAMP synthesis. Feline left ventricular cardiocytes plated on laminin-coated 150-mm dishes (1.5 x 105 cells/dish) were infected with AdMAP4 or Ad{beta}-gal at a MOI of 10 pfu/cell for 24 h and allowed to express the protein for a further 48 h. The cells were then treated with 1 µM isoproterenol for 45 min, after which they were washed 3 times and allowed to recover for 1 h at 37°C in Piper’s medium. The cardiocytes were then reexposed to 1 µM isoproterenol and harvested at 0, 5, 10, or 20 min of drug exposure for measurement of cellular cAMP content. Results are given as means ± SE of 6 independent experiments performed in duplicate. Accumulation of cAMP at 5 and 10 min of {beta}-AR stimulation was significantly inhibited in the AdMAP4-infected left ventricular cardiocytes. *P < 0.05, significant difference from the corresponding Ad{beta}-gal value by Student’s t-test.

 
Immunofluorescence confocal microscopy. The cells were fixed and stained as described previously (4). Briefly, cardiocytes were extracted for 1 min in a microtuble stabilizing buffer (2 mM EGTA, 0.1 mM EDTA, 1 mM MgSO4, and 100 mM MES; pH 6.75) containing 1% Triton X-100, rinsed three times in the same buffer with no detergent, and fixed in 3.7% formaldehyde in this buffer for 30 min. After each coverslip was blocked with 10% donkey serum in 0.10 M glycine and 0.05 M PBS for 30 min at room temperature, the cells were incubated at 4°C overnight in a 1:50 dilution of primary antibody in PBS. After being washed three times with PBS, the cells were incubated at room temperature for 2 h in a 1:50 dilution of fluorescein-conjugated secondary antibody in 2% normal donkey serum in PBS. Optical sections (0.1 µm) were acquired by confocal laser microscopy (Olympus BX50WI).

Immunoblots. Cardiocytes or myocardial lysates were prepared for immunoblotting as before (4). For cardiocytes, 0.5 x 106 cells were harvested and homogenized in 0.5 ml of a lysis buffer [2% NP-40, 100 mM Tris·HCl (pH 7.40), 10 mM EGTA, 0.35 M NaCl, and 10 mM dithiothreitol] and 5 µl of a protease inhibitor cocktail (P 8340, Sigma). After incubation on ice for 20 min, the cell lysates were centrifuged at 16,000 g at 4°C for 10 min. The supernatants were boiled for 5 min and centrifuged at 16,000 g at 4°C for 30 min, and these supernatants were mixed with an equal volume of 2x SDS sample buffer and boiled for 5 min. An equal amount of total protein (10 µg) was loaded in each lane. The blots were incubated for 16 h with our MAP4 primary antibody (4). After blots were incubated with peroxidase-labeled secondary antibody, specific protein bands were detected by enhanced chemiluminescence (Perkin-Elmer). For myocardium, with the exception that NP-40 was not included in the lysis buffer, the procedure for immunoblots from extirpated hearts was identical.

Data analysis. Means ± SE are given for the numerical data. Any statistical comparisons are specified in individual figures.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Initial studies. Because this was a new area of cardiac research, it was important to know at the outset if the hypotheses that MAP4 microtubule decoration interferes with {beta}-AR recycling and that this may be one cause of {beta}-AR downregulation in heart failure were tenable. To learn this, we needed to answer three questions. First, do we see {beta}-AR downregulation in pressure overload cardiac hypertrophy in a setting where microtubule network densification exists? Using our standard feline model of right ventricular pressure overload hypertrophy, we found, as expected from a large body of work on {beta}-ARs in cardiac hypertrophy and failure, and as shown in Fig. 1, that {beta}-AR density was reduced to 48 ± 3% of that in control right ventricles in hypertrophied right ventricular myocardium. These cats (Fig. 1) with 1 mo of right ventricular pressure overload hypertrophy had a right ventricular systolic pressure of 62.0 ± 2.3 mmHg (control = 22.6 ± 1.9 mmHg, P < 0.001), a right ventricular end-diastolic pressure of 9.0 ± 2.6 mmHg (control = 1.8 ± 0.9 mmHg, P < 0.01), and a right ventricle-to-body weight ratio of 1.18 ± 0.04 g/kg (control = 0.55 ± 0.07 g/kg, P < 0.001). Our previous data (29) show that cats with this duration and marked degree of right ventricular hypertrophy have without exception major densification of the right ventricular cardiocyte microtubule network. Second, is {beta}-AR recovery from agonist-induced downregulation microtubule dependent? To answer this question, we depolymerized the microtubules of isolated feline cardiocytes with 1 µM colchicine for 1 h at 37°C (32) before and during a 20-min exposure to 1 µM isoproterenol and during a subsequent 20-min isoproterenol washout. As would be expected from the study of Limas and Limas (15) and our own study of neuroblastoma cells (4), we found in adult cardiocytes, as shown in Fig. 2, that {beta}-AR recovery from isoproterenol-induced downregulation is markedly inhibited by colchicine-induced microtubule depolymerization. Third, because MAP4 microtubule decoration both stabilizes microtubules (18, 25) and inhibits microtubule-based transport (2, 4, 16), does microtubule stabilization itself affect microtubule-dependent {beta}-AR recovery from agonist-induced downregulation? To answer this question, we stabilized the microtubules of isolated feline cardiocytes with 10 µM taxol for 3 h at 37°C (33) before and during a 20-min exposure to 1 µM isoproterenol and during a subsequent 20-min isoproterenol washout. Figure 2 shows that taxol, which we have found to increase microtubule network density and stability in cardiocytes without causing increased MAP4 microtubule decoration (25), does not alter {beta}-AR recovery from agonist-induced downregulation. Thus these initial studies show that {beta}-AR downregulation is present in markedly pressure overload hypertrophied myocardium having microtubule network densification, and cardiocyte {beta}-AR recovery from agonist-induced downregulation is strongly dependent on the presence but not on stabilization of the cardiocyte microtubule network.



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  		Fig. 1. {beta}-Adrenergic receptor ({beta}-AR) density in control and pressure overload hypertrophied feline right ventricles (RVs). RVs from 3 sham-operated control and 3 pulmonary artery-banded cats were prepared and assayed as described in MATERIALS AND METHODS. There is a marked reduction in {beta}-AR density in the membrane-enriched fraction from hypertrophied myocardium. *P < 0.001, significant difference from control by Student’s t-test.

 


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  		Fig. 2. {beta}-AR recovery after agonist-induced downregulation in isolated feline cardiocytes. {beta}-AR internalization after isoproterenol exposure is similar in the control, colchicine-treated, and taxol-treated groups of cardiocytes. {beta}-AR recovery to the cell surface after isoproterenol washout is markedly inhibited by colchicine-induced microtubule depolymerization but is unaffected by taxol-induced microtubule stabilization. Statistical comparisons were by one-way ANOVA followed by Scheffé’s S-procedure. *P < 0.001, significant difference from control recovery; {dagger}P = not significant for a difference from control recovery.

 
MAP4 overexpression and microtubule decoration in AdMAP4-infected isolated cardiocytes. We have shown that there is a striking increase in MAP4 in pressure overload hypertrophied myocardium (25). Furthermore, and in distinct contrast to what is seen in normal cardiocytes, there is extensive MAP4 decoration of the dense microtubule network in hypertrophied cardiocytes (25). To study the effects of MAP4 microtubule decoration on {beta}-AR transport as a more isolated variable, we began with adenovirus-mediated MAP4 overexpression in normal cardiocytes. The data shown in Fig. 3 validate this model system: MAP4 protein is expressed in AdMAP4-infected cells in excess of the MAP4 level in Ad{beta}-gal-infected control cells (immunoblot lane 1 vs. lane 2), and it decorates the microtubules extensively (Fig. 3, A vs. B).



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  		Fig. 3. microtubule-associated protein 4 (MAP4) overexpression and microtubule decoration in isolated adult feline cardiocytes. The cardiocytes were infected with AdMAP4 at a multiplicity of infection (MOI) of 10 plaque-forming units (pfu)/cell for 24 h and allowed to express the protein for a further 48 h. For the confocal micrographs, our MAP4 primary antibody (1:100) and a FITC-conjugated secondary antibody (1:200) were used to visualize MAP4 (green); an {alpha}-tubulin primary antibody (B-5–1-2) (1:100) and a Cy3-conjugated secondary antibody (1:200) were used to visualize microtubules (red). Comparison of an Ad{beta}-gal-infected control cell (A) with an AdMAP4-infected cell (B) shows overlapping areas of immunoreactivity representing MAP4 microtubule decoration (yellow) solely in the AdMAP4-infected cell. Bar = 20 µm. In the immunoblot (C), there is much greater MAP4 expression in AdMAP4-infected cells (lane 2) than there is in Ad{beta}-gal-infected cells (lane 1).

 
{beta}-AR recovery in isolated right ventricular feline cardiocytes. The binding of [3H]CGP-12177 to isolated feline cardiocytes at 4°C is shown in Fig. 4. The binding data were calculated using nonlinear regression analysis (see MATERIALS AND METHODS) and yielded one class of sites with a Kd value of 2.76 ± 0.15 nM and a Bmax value of 25.9 ± 0.5 fmol/mg protein; these values are similar to those published elsewhere (15, 26). This binding curve for isolated adult feline cardiocytes indicated that [3H]CGP-12177 binding occurs in a saturatable manner. It has been shown in isolated rat cardiocytes (15) that there is a single {beta}-AR class with no apparent change in affinity after isoproterenol treatment, indicating that any reduction in receptor binding activity would be due to reduced sarcolemmal receptor density. For the present study, we repeated this Scatchard analysis of [3H]CGP-12177 binding in triplicate for isolated feline adult ventricular cardiocytes before, immediately after, and after recovery from isoproterenol treatment under the same conditions as those used by Limas and Limas (15) (Fig. 4). As seen in that study of rat cardiocytes, we found here in our cat cardiocytes three parallel lines with identical slopes (data not shown), indicating that isoproterenol caused no change in sarcolemmal {beta}-AR affinity.



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  		Fig. 4. Affinity and saturability of specific [3H]CGP-12177 binding sites on feline cardiocytes. Freshly isolated cardiocytes were plated in Piper’s medium on laminin-coated multiwell dishes at a density of 1.5 x 105 cells/16-cm2 well. After 4 h, the medium was exchanged for one containing known concentrations of [3H]CGP-12177, where a given concentration of [3H]CGP-12177 with or without 10 µM propranolol was applied to each of 3 wells at 4°C for 16 h. Each well was then washed 3 times with 2 ml PBS, the buffer was replaced by 1 N HCl at 25°C for 30 min, the cells were scraped off the plates, and the solution was neutralized with 1 N NaOH in a scintillation vial before we assayed the protein concentration and performed scintillation counting. Specific [3H]CGP-12177 binding, where each point is the mean ± SE of 6 separate experiments performed in triplicate, was obtained by subtracting nonspecific binding for each concentration of [3H]CGP-12177 in the presence of propranolol from total binding in the absence of propranolol. The Scatchard plot of specific binding for these feline cardiocytes is shown in the inset.

 
Having characterized sarcolemmal {beta}-AR density in normal adult feline cardiocytes and found a concurrence with what would be expected from published data (15, 26), we were in a position to examine the effects of MAP4 microtubule decoration on {beta}-AR trafficking in these same cells after transfection with either Ad{beta}-gal as a virus infection control or AdMAP4 as an inducer of MAP4 microtubule decoration. Figure 5 shows that a 45-min exposure to 1 µM isoproterenol caused very similar reductions in the specific binding of [3H]CGP-12177 to cell surface {beta}-ARs in cells infected with either Ad{beta}-gal or AdMAP4. However, sarcolemmal {beta}-AR recovery after a 60-min isoproterenol washout was inhibited in the AdMAP4-infected cells (Ad{beta}-gal = 82.3 ± 4.4% of the preisoproterenol value and AdMAP4 = 63.7 ± 3.6% of the preisoproterenol value, P < 0.01).



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  		Fig. 5. MAP4 overexpression inhibits {beta}-AR recycling after agonist-induced downregulation in adult feline RV cardiocytes. To evaluate the role of MAP4 microtubule decoration in {beta}-AR recycling, adult feline cardiocytes were infected with AdMAP4 or Ad{beta}-gal at a MOI of 10 pfu/cell for 24 h and allowed to express the protein for a further 48 h. The cells were then treated with 1 µM isoproterenol for 45 min, washed 3 times, and allowed to recover for 1 h at 37°C. [3H]CGP-12177 (6 nM) binding was determined in separate triplicate samples of both groups of cells before agonist exposure (control), after agonist exposure (isoproterenol), and after agonist washout (recovery). Results are given as means ± SE of 6 independent experiments. {beta}-AR recovery was significantly inhibited in the AdMAP4-infected RV cardiocytes. *P < 0.01, significant difference from corresponding Ad{beta}-gal recovery by Student’s t-test.

 
Isolated left ventricular cardiocytes from FVB/N wild-type and MAP4 transgenic mice. We extended this work from acutely transfected isolated cells to cardiocytes from the hearts of transgenic mice for several reasons. First, the effects of a transient increase in a protein in terminally differentiated isolated cells do not necessarily mimic the effects of the long-term presence of that protein. Second, the effects of that protein in the same cell might well be quite different when that protein has been synthesized and assembled in the in vivo as opposed to the in vitro context. Third, this gave us the opportunity to confirm our findings in cardiocytes from a different species and cardiac ventricle. However, the isolation of viable adult murine cardiocytes has been more difficult in our hands than comparable cell isolations from other species, such that it was necessary to ascertain the quality and viability of the murine cardiocytes prior to the pharmacological assays. In our present mouse cardiocyte isolation procedure, noncardiocyte cells and cellular debris are eliminated from the suspensions during three washes in medium 199 containing 0.1 µM Ca2+. As shown in Fig. 6, the percentage of rod-shaped cardiocytes was 80–90% for the isolates from both wild-type and MAP4 transgenic left ventricles. Furthermore, the cardiocytes were quiescent at 32° C in 1.2 mM Ca2+ and exhibited stable mechanical function during electrically stimulated contractions for several hours (12). During the pharmacological assays used here, the number of viable rod-shaped cardiocytes did not decrease significantly.



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  		Fig. 6. Phase-contrast micrographs of freshly isolated left ventricular cardiocytes in medium 199 from FVB/N wild-type (A and B) and MAP4 transgenic (C and D) mice. The MAP4 transgenic lines used in this study exhibited homogeneous expression of the transgene in 100% of the ventricular cardiocytes. A and C: x100 magnification; B and D: x400 magnification.

 
MAP4 overexpression and microtubule decoration in left ventricular cardiocytes isolated from MAP4 transgenic mice. In an approach analogous to that shown in Fig. 3 for AdMAP4-infected cardiocytes, we next validated the cardiac phenotype of the MAP4 transgenic mice in terms of MAP4 microtubule decoration. As shown in Fig. 7, the FVB/N immunoblot (lane 1) and cardiocyte (Fig. 7A) showed relatively little MAP4 protein and minimal MAP4 microtubule decoration. The MAP4 transgenic immunoblot (lane 2) and cardiocyte (Fig. 7B) showed a major increase in MAP4 protein and extensive MAP4 microtubule decoration.



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  		Fig. 7. Myocardial MAP4 overexpression and microtubule decoration in transgenic mice. For the confocal micrographs, left ventricular cardiocytes from adult wild-type FVB/N (A) and MAP4 transgenic (B) mice were enzymatically isolated and fixed. Our MAP4 primary antibody (1:100) and a FITC-conjugated secondary antibody (1:200) were used to visualize MAP4 (green); an {alpha}-tubulin primary antibody (B-5–1-2) (1:100) and a Cy3-conjugated secondary antibody (1:200) were used to visualize microtubules (red). Overlapping areas of immunoreactivity representing MAP4 microtubule decoration (yellow) are seen only in the cardiocyte from the MAP4 transgenic mouse. Bar = 20 µm. In the immunoblot (C), there is much greater MAP4 expression in MAP4 transgenic myocardium (lane 2) than there is in wild-type myocardium (lane 1).

 
{beta}-AR recovery in wild-type and MAP4 transgenic left ventricular cardiocytes. Figures 8 and 9 show the saturation binding of [3H]CGP-12177 to isolated murine cardiocytes at 4°C. The binding data, which are similar to other murine myocardial data (5, 11), were calculated using nonlinear regression analysis (see MATERIALS AND METHODS) and yielded one class of sites for both types of cells, with similar Kd (2.54 ± 0.21 nM for wild type and 2.72 ± 0.20 nM for MAP4 transgenic) and Bmax (39.9 ± 1.1 fmol/mg protein for wild type and 40.2 ± 1.0 fmol/mg protein for MAP4 transgenic) values for these two types of cardiocytes. As in feline cardiocytes, these binding curves for wild-type and transgenic murine cardiocytes indicated that [3H]CGP-12177 binding occurs in a saturatable manner. Thus baseline properties of the myocardial {beta}-ARs were not affected by prolonged MAP4 overexpression and microtubule decoration.



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  		Fig. 8. Affinity and saturability of specific [3H]CGP-12177 binding sites on FVB/N wild-type murine cardiocytes. Suspensions of freshly isolated left ventricular cardiocytes were incubated with known concentrations of [3H]CGP-12177, where a given concentration of [3H]CGP-12177 with or without 10 µM propranolol was applied to each of three 35-mm dishes at 4°C for 16 h. At the end of the incubation, the contents of each well were filtered under vacuum through Whatman GF/C 25-mm filters on a Millipore manifold (15). The filters were then washed 4 times with 5 ml ice-cold washing buffer [50 mM Tris·HCl (pH 7.4) and 10 mM MgCl2] and scintillation counted. Specific [3H]CGP-12177 binding, where each point is the mean ± SE of 5 separate experiments performed in duplicate, was obtained by subtracting nonspecific binding at a particular concentration of [3H]CGP-12177 in the presence of propranolol from total binding in the absence of propranolol. The Scatchard plot of specific binding for the FVB/N wild-type cells is shown in the inset.

 


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  		Fig. 9. Affinity and saturability of specific [3H]CGP-12177 binding sites on MAP4 transgenic murine cardiocytes. The experimental conditions were identical to those specified for FVB/N wild-type cardiocytes in Fig. 8. The Scatchard plot of specific binding for the MAP4 transgenic cells is shown in the inset.

 
Having established that sarcolemmal {beta}-AR properties are similar in wild-type and MAP4 transgenic cardiocytes, we were in a position to examine the effects of long-term MAP4 microtubule decoration on {beta}-AR trafficking in these same cells. Figure 10 shows that a 15-min exposure to 1 µM isoproterenol caused very similar reductions in the specific binding of [3H]CGP-12177 to cell surface {beta}-ARs in wild-type and MAP4 transgenic cardiocytes. However, sarcolemmal {beta}-AR recovery after a 30-min isoproterenol washout was strongly inhibited in the MAP4 transgenic cells (wild type = 70.2 ± 4.3% of the preisoproterenol value and MAP4 transgenic = 44.0 ± 2.1% of the preisoproterenol value, P < 0.01).



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  		Fig. 10. MAP4 overexpression inhibits {beta}-AR recycling after agonist-induced downregulation in MAP4 transgenic murine left ventricular cardiocytes. Freshly isolated adult wild-type FVB/N and MAP4 transgenic mouse cardiocytes were placed in medium 199 containing 0.1 mM CaCl2; the medium was equilibrated with 100% O2 for 30 min before use. The cells were then treated with 1 µM isoproterenol for 15 min, washed 3 times, and allowed to recover for 30 min at 37°C. [3H]CGP-12177 (6 nM) binding was determined in separate triplicate samples of both groups of cells before agonist exposure (control), after agonist exposure (isoproterenol), and after agonist washout (recovery). Results are given as means ± SE of 4 independent experiments. {beta}-AR recovery was significantly inhibited in left ventricular cardiocytes from the MAP4 transgenic mice. *P < 0.01, significant difference from corresponding wild-type recovery by Student’s t-test.

 
Effect of MAP4 overexpression on {beta}-agonist-induced cAMP accumulation. The physiological consequence of ligand binding to the {beta}-AR is stimulation of adenyl cyclase, causing an increase in cAMP. While the data given in Figs. 5 and 10 show that MAP4 microtubule decoration inhibits cardiac {beta}-AR recycling after agonist stimulation, they do not show whether this has any functional significance in terms of this physiologically relevant {beta}-AR activity. Because Figs. 5 and 10 also show that baseline {beta}-AR density and internalization upon agonist exposure are unaffected by MAP4 microtubule decoration, and because Fig. 2 shows that this internalization is not microtubule dependent, we did not anticipate that initial {beta}-AR agonist exposure in cardiocytes having extensive MAP4 microtubule decoration would affect cAMP accumulation. This was borne out experimentally: when Ad{beta}-gal-infected and AdMAP4-infected cardiocytes were treated with 1 µM isoproterenol, the peak increase in cAMP at 5 min of drug exposure was identical (107.3 ± 19.3% increase for Ad{beta}-gal and 109.9 ± 19.9% increase for AdMAP4), and there was no difference between these two groups of cells at any other time during this 45-min isoproterenol exposure. However, we would expect that defective receptor recycling would follow {beta}-AR agonist exposure in cells having extensive MAP4 microtubule decoration, with a consequent reduction in functional sarcolemmal {beta}-ARs. Figure 11 shows this to be the case. When Ad{beta}-gal-infected and AdMAP4-infected cardiocytes were treated with isoproterenol for 45 min, allowed to recover for 1 h without drug, and then restimulated with isoproterenol, baseline cAMP levels were the same in the two groups of cells, but agonist-induced cAMP accumulation was inhibited in the AdMAP4-infected cardiocytes.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study is our first attempt to determine whether the microtubule network densification, stabilization, and MAP4 decoration that we find in pressure overload cardiac hypertrophy have specific effects based on alterations of normal microtubule functions as opposed to nonspecific cytomechanical effects based on altered intracellular frictional dissipation during contraction (30). In the initial exploratory test of this idea reported here (4), we found that MAP4 microtubule decoration does in fact inhibit the normal microtubule-based trafficking of the activated G protein-coupled muscarinic receptor in neuroblastoma cells.

In now taking this question to the heart, we have focused on the G protein-coupled adrenergic receptor, because it is well established that {beta}-AR responsiveness is reduced due to {beta}-AR desensitization and downregulation both in patients with cardiac hypertrophy and failure and in animal models of these conditions (1, 8, 10, 14, 21, 28, 35). In parallel, increased concentrations of tubulin and MAP4, with MAP4 decoration of microtubules, are found in the heart as early as 2 days after pressure overloading and persist for at least 6 mo thereafter (25, 31). We initially examined the potential role of microtubule network densification and MAP4 decoration in {beta}-AR desensitization. There were several reasons for this choice. First, in terms of immediate {beta}-agonist-induced cAMP generation in leukocytes that is independent of microtubule-based {beta}-AR recycling, microtubule depolymerization causes cellular hyperresponsiveness to {beta}-agonists (23). Second, and in direct contrast, decreased responsiveness of this G protein-coupled receptor to {beta}-agonists has long been known to occur in cardiac hypertrophy, especially with the transition to failure, and this has been linked to {beta}-AR desensitization (5) in a model of left ventricular pressure overload hypertrophy wherein we find increased microtubules (12). Desensitization of the activated {beta}-AR is caused by receptor binding of and phosphorylation by {beta}-AR kinase ({beta}ARK), a member of the family of G protein-coupled receptor kinases, but only after cytosolic {beta}ARK is membrane targeted by binding to receptor-associated G protein {beta}{gamma}-subunits (13, 20). Finally, there is clear evidence that tubulin binds to G proteins (22, 34).

Because in preliminary studies we had found a shift of {beta}ARK to cardiac microtubules in a model of pressure overload cardiac hypertrophy in which {beta}-AR desensitization is known to occur (5), we tested the hypothesis that microtubule binding of {beta}ARK and/or microtubule binding of the {beta}ARK-G protein-{beta}-AR complex in cardiac hypertrophy desensitizes the {beta}-AR either by localizing {beta}ARK to the receptor or by altering the kinase-G protein-receptor interaction. However, in these studies, we found that while there was a shift of {beta}ARK to the microtubule fraction in cardiac hypertrophy, there was no correlation between the extent of the shift of {beta}ARK to cardiac microtubules and either the extent of cardiac hypertrophy and/or failure or the extent of {beta}-AR desensitization. Further, we found no evidence that a tubulin-{beta}ARK complex forms in vivo. However, while these preliminary studies precluded a role for altered cardiocyte microtubules in {beta}-AR desensitization, the present data do support a role for microtubule alterations in the {beta}-AR downregulation found in the substantially pressure-overloaded myocardium where these cytoskeletal changes occur.

In setting the stage for this study, Fig. 1 shows that in our feline model of cardiac hypertrophy wherein microtubule network densification and microtubule MAP4 decoration are prominent, there is a striking reduction of sarcolemmal {beta}-AR density. Figure 2 shows that, as would be expected from our data in neuroblastoma cells (4), internalization of the activated {beta}-AR is not microtubule dependent, but recovery of these {beta}-ARs to the sarcolemma is strongly microtubule dependent. Furthermore, microtubule stabilization by a means other than by MAP4 microtubule decoration does not affect either {beta}-AR internalization or {beta}-AR recovery.

After feline cardiocyte microtubule network densification and MAP4 decoration had been effected by adenovirus-mediated gene transfer rather than by hemodynamic overloading (Fig. 3) and after the {beta}-AR ligand binding parameters for normal adult feline cardiocytes had been established (Fig. 4), we were in a position to ask whether {beta}-AR trafficking in these cardiocytes is affected by MAP4 microtubule decoration itself. Figure 5 shows this to be the case: while as would be predicted from Fig. 2, {beta}-agonist-induced {beta}-AR internalization is unaffected by MAP4 microtubule decoration, sarcolemmal recovery of the {beta}-ARs after {beta}-agonist exposure is inhibited.

We then took this question to the in vivo setting of the hearts of transgenic mice for two reasons: first, the effects of a transient increase in MAP4 microtubule decoration in isolated cells do not necessarily mimic the effects of this cytoskeletal alteration in a long-term process such as cardiac hypertrophy; and second, the effects of this cytoskeletal alteration in the same cell might well be quite different in the in vivo as opposed to the in vitro context. After we were satisfied that we could isolate a high percentage of viable cardiocytes from control and MAP4 transgenic murine left ventricles (Fig. 6) and that the microtubules in cardiocytes from the nonmosaic MAP4 transgenic left ventricles were extensively decorated by MAP4 (Fig. 7), we could do studies analogous to those done on the AdMAP4-infected feline cardiocytes. Thus after the {beta}-AR ligand binding parameters for normal and transgenic murine left ventricular cardiocytes had been established (Figs. 8 and 9), we asked whether {beta}-AR trafficking in these cardiocytes is affected by MAP4 microtubule decoration itself. We found that just as in the AdMAP4-infected feline cardiocytes, {beta}-agonist-induced {beta}-AR internalization is unaffected by MAP4 microtubule decoration, but sarcolemmal recovery of the {beta}-ARs after {beta}-agonist exposure is strongly inhibited (Fig. 10).

To then relate these studies of experimental MAP4 microtubule decoration more directly to the pathological MAP4 microtubule decoration found in severe pressure overload hypertrophy (25), we compared sarcolemmal {beta}-AR recovery after isoproterenol exposure and withdrawal in cardiocytes isolated from the hypertrophied right ventricle and the normally loaded left ventricle of a cat with the same degree of right ventricular hypertrophy as that described for the cats in Fig. 1. While the result that we obtained does not establish a causal relationship to MAP4 microtubule decoration, recovery of the right ventricular cardiocyte {beta}-ARs following {beta}-agonist exposure was only 43% of that for the left ventricular cardiocytes from the same cat.

Finally, because the physiologically relevant end point for myocardial {beta}-AR stimulation is cAMP generation, and because this is reduced in failing myocardium despite persistently high adrenergic input (9), we wanted to establish whether the new MAP4 microtubule decoration-dependent mechanism for {beta}-AR downregulation identified here might have functional significance. The data in Fig. 11 suggest that this is the case. For cardiocytes unexposed to catecholamines during 72 h in culture, generation of cAMP in response to initial {beta}-agonist exposure is unaffected by MAP4 microtubule decoration, consistent with the other data given here showing that {beta}-AR signaling and internalization are not microtubule dependent. However, on reexposure of the same cardiocytes to a {beta}-agonist, where the response now depends on the extent of prior {beta}-AR trafficking, cAMP generation is inhibited by MAP4 microtubule decoration. Of practical interest, these experimental conditions are somewhat analogous to the persistent {beta}-agonist stimulation experienced by the failing heart.

In conclusion, this study identifies a new mechanism for the decreased {beta}-AR responsiveness to adrenergic input that is characteristic of cardiac hypertrophy and failure. However, it is certainly not operating exclusively to cause {beta}-AR downregulation in this setting, because there are a number of other well-defined alterations of the {beta}-AR signaling cascade in the failing heart (17, 19). Indeed, the data from this study reinforce this point. As seen in Fig. 1, sarcolemmal {beta}-AR density is reduced to 48 ± 3% of control in the severely pressure-overloaded feline myocardium that would be expected to exhibit multiple defects of the {beta}-AR signaling cascade. However, when the experiment shown in Fig. 1 was repeated in MAP4 transgenic mouse myocardium, where presumably the single defect adversely affecting the {beta}-AR signaling cascade is MAP4 microtubule decoration, sarcolemmal {beta}-AR density was also reduced, but in this case only to 71 ± 5% of control. Thus while certainly not the sole cause for the reduced {beta}-AR responsiveness characteristic of hypertrophied and failing myocardium, the microtubule network alterations in severe pressure overload cardiac hypertrophy represent a unique mechanism additive to those already established. Furthermore, given the generality of the effects of MAP4 decoration of microtubules on motor-dependent microtubule-based transport, we would anticipate that a number of other intracellular transport processes will be found to be quite abnormal in the hypertrophied and failing heart.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Program Project Grant HL-48788 and by Merit and Research Enhancement Award Program awards from the Research Service of the Department of Veterans Affairs.


   ACKNOWLEDGMENTS
 
We thank Mary Barnes for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Cooper, IV, Gazes Cardiac Research Institute, PO Box 250773, Medical Univ. of South Carolina, 114 Doughty St., Charleston, SC 29403 (E-mail: cooperge{at}musc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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