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Molecular Endocrinology, doi:10.1210/me.2003-0125
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Molecular Endocrinology 18 (7): 1840-1849
Copyright © 2004 by The Endocrine Society

Chronic Cardiac-Specific Thyrotoxicosis Increases Myocardial ß-Adrenergic Responsiveness

Suzy D. Carvalho-Bianco, Brian W. Kim, Julie X. Zhang, John W. Harney, Rogério S. Ribeiro, Balazs Gereben, Antonio C. Bianco, Ulrike Mende and P. Reed Larsen

Thyroid Section, Division of Endocrinology, Diabetes and Hypertension (S.D.C.-B., B.W.K., J.W.H., R.S.R., A.C.B., P.R.L.); Cardiology Division of the Brigham and Women’s Hospital (J.X.Z., U.M.); Harvard Medical School, Boston, Massachusetts 02115; and Department of Endocrine- and Behavioral Neurobiology (B.G.), Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest H-1083, Hungary

Address all correspondence and requests for reprints to: P. Reed Larsen, M.D., Division of Endocrinology, Diabetes and Hypertension, Brigham & Women’s Hospital, 77 Avenue Louis Pasteur, Room 550, Boston, Massachusetts 02115.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Whereas many cardiac symptoms of thyrotoxicosis resemble those of the hyperadrenergic state, circulating catecholamines are reduced or normal in this condition. To test the hypothesis that the thyrotoxic heart is hypersensitive to catechol-amines, we studied ß-adrenergic signaling in a transgenic (TG) mouse in which the human type 2 iodothyronine deiodinase (D2) gene is expressed in myocardium. Because D2 converts T4 to T3, the active form of thyroid hormone, the D2 TG mouse exhibits mild, chronic thyrotoxicosis that is limited to the myocardium. In the current study, we determined that cAMP accumulation in response to either norepinephrine or forskolin treatment was increased in isolated ventricular myocardiocytes and membrane-enriched fractions prepared from these D2 TG hearts as compared with wild type. This increase in adenylyl cyclase (AC) Vmax could not be explained by changes in AC isoform expression or changes in the long or short forms of stimulatory G-protein Gs{alpha}, which were approximately 10% decreased in D2 TG membranes. However, Western analysis and ADP-ribosylation studies suggest that the increase in AC Vmax is mediated by a decrease in the expression of inhibitory G proteins (Gi{alpha}-3 and/or Go{alpha}). These data suggest that cardiac thyrotoxicosis leads to increased ß-adrenergic responsiveness of cardiomyocytes via alterations in the regulatory G-protein elements of the AC membrane complex.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
WHEREAS THE MOST prominent cardiac manifestations of thyrotoxicosis are also seen in the hyperadrenergic state, circulating catecholamine levels in thyrotoxicosis are normal or low. Based on these observations, it has been hypothesized that thyrotoxicosis increases myocardial sensitivity or responsiveness to ß-adrenergic stimuli (1). Studies documenting the effectiveness of ß-adrenergic blockade in ameliorating the cardiac symptoms of thyrotoxicosis have lent credence to this hypothesis (2, 3). However, conflicting data exist as to whether patients with thyrotoxicosis have augmented increases in heart rate or systolic blood pressure in response to catecholamine administration, and attempts to prove that adrenergic sensitization occurs in thyrotoxic animals have also met with mixed results (4, 5, 6). In spite of numerous studies showing changes in the elements of the adenylyl cyclase (AC) complex in response to thyroid hormone, it remains unclear whether cardiac ß-adrenergic responsiveness is increased in thyrotoxicosis.

Several experimental factors have limited the interpretation of previous work in this field. The administration of pharmacological, rather than physiological, quantities of thyroid hormone has been an experimental paradigm. Such treatments often result in an acutely negative caloric balance, with associated muscle wasting, significant weight loss, and/or cardiac hypertrophy (7). These changes are not typical of human thyrotoxicosis, which typically develops over many months. It has also become clear that the choice of endpoint used to assess ß-adrenergic responsiveness must be considered, because certain features of the thyrotoxic cardiac phenotype, contractility being the most notable, have been shown to be augmented by thyrotoxicosis through nonadrenergic pathways (8). Finally, and most importantly, few studies have been able to distinguish between the direct effects of thyroid hormone on myocardial tissue and the indirect cardiovascular effects that result from systemic thyrotoxicosis. These indirect, compensatory effects occur as the heart responds to a decrease in systemic vascular resistance and to changes in neurohormonal input from the central nervous system (6, 9). Only the heterotopically transplanted rat heart model (the unloaded heart model) has directly addressed this obstacle (10). However, this method required surgical intervention, and the transplanted unloaded hearts exhibited a reduction in cell protein compared with the native euthyroid hearts (11). Considering that most of the published data are limited in regard to one or more of the above considerations, it is perhaps not surprising that the fundamental question of the nature of the changes in myocardial ß-adrenergic responsiveness in thyrotoxicosis remains controversial.

The murine model of cardiac thyrotoxicosis used for the current study is one model that overcomes all of these limitations. Unlike humans, rodents do not express the type 2 iodothyronine deiodinase (D2) in their myocardium (12, 13). D2 converts T4 to T3, and tissues that express D2 may therefore be more sensitive to changes in circulating T4. We took advantage of this interspecies difference to create a transgenic (TG) mouse model of cardiac-specific thyrotoxicosis, i.e. a mouse with cardiac thyrotoxicosis mediated by TG expression of D2, but with normal serum thyroid hormones and thus systemic euthyroidism (14). Briefly, D2 is overexpressed in the myocardium under the control of the {alpha}-myosin heavy chain (MHC) promoter, leading to a modest, but chronic, increase in myocardial thyroid status reflected by an increase in thyroid hormone-regulated genes such as HCN2 (hyperpolarization-activated cyclic nucleotide-gated channel) (14, 15). Whereas the cardiac phenotype of the D2 TG mouse is not apparent in vivo, examination of the isolated perfused D2 TG hearts reveals findings typically seen in thyrotoxicosis, such as tachycardia and a decrease in both creatine and phosphocreatine (16, 17). This suggests that the cardiac-specific thyrotoxicosis seen in this model can be compensated for by autonomic regulation and/or other factors.

In the current study, we used the D2 TG murine model of cardiac-specific thyrotoxicosis to investigate the direct effects of thyrotoxicosis on cardiac ß-adrenergic signaling. We compared the function of the AC complex in D2 TG and wild-type mice, studying cAMP accumulation in isolated cardiomyocytes and ventricular membranes. In addition to AC function, we examined the expression of the critical postreceptor signaling elements including AC isoforms and both the stimulatory (Gs{alpha}) and inhibitory (Gi{alpha} and Go{alpha}) heterotrimeric G proteins.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
cAMP Accumulation in Fresh Cardiomyocytes from D2 TG vs. Wild-Type Mice
Accumulation of cAMP in response to ß-adrenergic receptor (ß-AR) stimulation with norepinephrine (NE) was studied in isolated cardiomyocytes from D2 TG mice and their wild-type littermates. Basal (unstimulated) cAMP accumulation was not different between groups. Stimulation with NE led to an approximately 2.4-fold increase in cAMP accumulation in D2 TG cardiomyocytes relative to wild type (P < 0.05; Fig. 1AGo), whereas the EC50 for NE was not significantly altered (data not shown).



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  		Fig. 1. cAMP Accumulation in Response to NE or Forskolin in Freshly Isolated Ventricular Cardiomyocytes

Cardiomyocytes were diluted to 50,000 cells/ml and incubated with CaCl2, BDM, IBMX, adenosine deaminase, and increasing concentrations of NE (panel A) or forskolin (panel B) at 37 C for 30 min. Results are the mean ± SD of four to five measurements. *, P < 0.05 vs. wild-type cells at the same concentration of NE/forskolin. WT, Wild-type.

 
To define the mechanism by which ß-adrenergic responsiveness was increased in the D2 TG cardiomyocytes, we studied direct AC activation by forskolin. Accumulation of cAMP in response to forskolin was approximately 70% greater in the isolated D2 TG cardiomyocytes at 10–5 M forskolin (P < 0.05; Fig. 1BGo), suggesting that a postreceptor change might be responsible for the enhanced responsiveness to catecholamines.

Characterization of AC Kinetics in Ventricular Membranes of D2 TG vs. Wild-Type Mice
To further characterize possible differences in the ß-adrenergic signaling elements in D2 TG cardiomyocytes, forskolin-stimulated cAMP accumulation was studied in membrane-enriched fractions prepared from ventricular tissue. Such preparations allow manipulation of the cellular components that might alter AC activity such as ATP, GTP, Mg2+, and Ca2+, while also virtually eliminating effects on cAMP accumulation mediated via cross-talk by cytosolic proteins (18). This system also allows for the determination of apparent Michaelis-Menten constant (Km) and maximum velocity (Vmax) of the AC complex in response to ATP.

Under the assay conditions, cAMP production was linear with the amount of membrane protein used and time of incubation (Fig. 2Go, A and B). Accumulation of cAMP in response to increasing concentrations of forskolin was enhanced in the D2 TG membranes (Fig. 2CGo), consistent with the results from intact cardiomyocytes. At every forskolin concentration there was a significantly higher cAMP accumulation in the D2 TG membranes, and the maximal response (10–3 M forskolin) was approximately 75% higher in the D2 TG membranes. This difference was similar in two different lines of D2 TG mice and was observed in both males and females (data not shown).



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  		Fig. 2. cAMP Accumulation in Response to Forskolin in Ventricular Membranes

A, Aliquots of pooled ventricular membranes from wild-type (WT) and D2 TG mice were incubated at 30 C for 30 min. cAMP production was stimulated by 10–5 M forskolin, in a 10 mM Tris-HCl buffer (pH 8.0), containing 10 mM propranolol, 1 mM IBMX, 0.4 U/ml adenosine deaminase, 1 mM GTP, 5 mM MgCl2, and 0.5 mM ATP. B, Same assay conditions as in panel A except that 5 µg ventricular membrane protein were employed and the incubation time varied up to 90 min. C, Assays were performed with 5 µg of the membrane-enriched fractions incubated in Tris buffer, pH 8.0, at 30 C for 30 min with IBMX, adenosine deaminase, MgCl2, GTP, ATP, propranolol, BSA, protease inhibitors ± increasing concentrations of forskolin. For panels A–C, results are the mean ± SD of four to five measurements. *, P < 0.05 vs. wild-type membranes at the same concentration of forskolin.

 
The greater cAMP accumulation in membranes from D2 TG ventricles was also present when the membranes were stimulated with 0.1 mM forskolin + increasing concentrations of ATP (Fig. 3AGo). cAMP accumulation was approximately 1.8-fold higher in the D2 TG membranes (P < 0.05) at 1 mM ATP, the con-centration resulting in a maximal response. The Lineweaver-Burk replot of the data resulted in a Vmax that was approximately 70% greater in the D2 TG membranes (P < 0.05; Fig. 3BGo), whereas the Km values were not significantly different in the two groups. Again, the effect was not gender specific (data not shown).



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  		Fig. 3. cAMP Accumulation in Response to ATP in Ventricular Membranes

A, Same as in Fig. 2Go except that incubations were performed in the presence of 10–4 M forskolin ± increasing concentrations of ATP. B, Lineweaver-Burk replot of the data in panel A. Results are the mean ± SD of four to five measurements. *, P < 0.05 vs. wild-type samples at the same ATP concentrations.

 
Characterization of Ca2+ Sensitivity of AC Activity in Ventricular Membranes of D2 TG vs. Wild-Type Mice
The existence of nine AC isoforms sharing similar topology but differing in regulatory properties, tissue distribution, and even in relative abundance indicates that this system has the potential for extreme plasticity (19, 20). These isoforms are differentially regulated by Gs, Gi, Ca2+, protein kinase A (PKA), protein kinase C, and other cellular components. Five isoforms of AC (IV–VII and IX) have been found in mouse heart (21), and isoforms V and VI have been reported to be predominant (22).

AC isoforms V and VI are directly inhibited by micromolar concentrations of Ca2+, whereas other isoforms are significantly less sensitive (or not sensitive) to Ca2+ (23). We took advantage of this fact to try to further discriminate as to which AC isoforms were affected by D2 transgene expression. If the increased AC Vmax in D2 TG ventricular membranes involved AC V and/or VI, these membranes should exhibit heightened sensitivity to Ca2+ inhibition. To test this hy-pothesis, we compared the Ca2+ sensitivity of forskolin-stimulated cAMP accumulation in D2 TG and wild-type membranes. For the 0 free Ca2+ (baseline) samples, EGTA was added to the forskolin-stimulated cAMP accumulation assay mixture to chelate any residual Ca2+ in the membrane preparation. Addition of EGTA led to an increase in the absolute value of cAMP accumulation in response to 10–5 M forskolin in both D2 TG and wild-type membranes (compare 0 free Ca2+ samples in Fig. 4Go vs. 10–5 M forskolin samples in Fig. 2Go). Whereas cAMP accumulation in the wild-type membranes was increased approximately 3.5-fold by the addition of EGTA, the increase was about 5.9-fold in the D2 TG membranes, suggesting increased calcium sensitivity in the D2 TG membranes. As expected, addition of defined free Ca2+ concentrations to the EGTA-containing buffer decreased forskolin-stimulated cAMP accumulation in both D2 TG and wild-type membranes (Fig. 4Go). At 200 µM free Ca2+, cAMP accumulation fell to approximately 40% in the wild-type group, whereas it fell to about 25% in the D2 TG group (P < 0.001). These data indicate that forskolin-stimulated cAMP accumulation in D2 TG membranes was significantly more sensitive to calcium inhibition than that in wild-type membranes. This is consistent with the hypothesis that the contribution of Ca2+-sensitive AC isoforms (V and VI) to cAMP generation is increased in the D2 TG membranes.



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  		Fig. 4. Forskolin-Stimulated cAMP Accumulation in Response to Ca2+ in Ventricular Membranes

Same as in Fig. 3Go except that incubations were performed with 1 mM EGTA and in the presence of 10–5 M forskolin ± increasing concentrations of CaCl2. The total amount of CaCl2 to yield the exact desired free Ca2+ was calculated by the Chelator software developed by Theo J. M. Schoenmakers (University of Nijmegen, The Netherlands) and available at Biotechniques Software Library (http://www.biotechniques.com/). Results are the mean ± SD of four to five measurements. *, P < 0.05 vs. 0 free calcium. WT, Wild-type.

 
Expression of AC Isoforms and G Proteins in Purified Membranes
The simplest explanation for the increased AC Vmax and increased Ca2+ sensitivity would be an increase in AC V/VI expression in the D2 TG ventricular membranes. This was tested by real-time PCR, which was used to quantitate AC IV, V, VI, and VII isoform mRNAs in the D2 TG and wild-type myocardial tissue. ß-Actin and cyclophilin A were used as housekeeping genes. Regardless of which housekeeping gene was used, there was no difference in mRNA for AC isoforms (Table 1Go). Although changes less than 0.5-fold in magnitude may be difficult to demonstrate using this technique, the data suggest that the increase in AC activity is mediated via posttranscriptional changes or through modulation of proteins that affect AC activity. To investigate these possibilities, Western analysis was performed using specific antiserum against AC V/VI isoforms, the two most abundant AC isoforms expressed in the myocardium. There was no statistically significant difference between the two groups (Fig. 5AGo).


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  		Table 1. Myocardial Levels of AC mRNA

 


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  		Fig. 5. Quantification of Ventricular AC-V/VI and Gs{alpha}

Purified ventricular membranes were processed for Western analysis with antibodies against ACV/VI (panel A) or Gs{alpha} (panel B). The signal intensity of the indicated bands (left gray arrow) was quantified by direct chemiluminescent imaging. The results are shown below each gel and the values are the mean ± SD. Both experiments were repeated three times with similar results. *, P < 0.05 vs. wild type.

 
Next we sought to investigate whether the amounts of the AC-modulating G proteins Gs{alpha} and Gi{alpha}/Go{alpha} were altered in the D2 TG hearts. Surprisingly, Western analysis revealed that there were small but significant decreases in both the long (~10%; P < 0.05) and short (~10%; P < 0.05) forms of Gs{alpha} protein in D2 TG membranes (Fig. 5BGo). The Gi subfamily has at least three distinct gene products, Gi{alpha}-1, Gi{alpha}-2, and Gi{alpha}-3 (24), which were assessed by Western analysis. There was a decrease of approximately 30% in Gi{alpha}-3/Go{alpha} in the D2 TG membranes (P < 0.05; Fig. 6AGo), whereas Gi{alpha}-1 and Gi{alpha}-2 isoforms were not altered (Fig. 6Go). Further evidence for reduced expression of the Gi{alpha}/Go{alpha} protein in the D2 TG membranes was obtained by evaluating the levels of heterotrimeric Gi{alpha}/Go{alpha} in the myocardium by pertussis toxin (PTX)-catalyzed ADP ribosylation in purified membranes (Fig. 6Go) (25, 26). The 40-kDa [32P]nicotinamide adenine dinucleotide (NAD)+ PTX-ribosylated substrate was decreased by approximately 50% in myocardial membranes from D2 TG mice, suggesting a reduced availability of Gi{alpha}/Go{alpha} (Fig. 6Go). Note that Gß levels are not changed in these membranes, excluding the possibility that the reduced labeling represents a decrease in heterotrimer.



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  		Fig. 6. Quantification of Ventricular Gi{alpha}-1,2 and Gi{alpha}-3/Go{alpha}

Purified ventricular membranes were processed for Western analysis with antibodies against Gi{alpha}-1,2, Gi{alpha}-3/Go{alpha}, or Gß proteins (panel A). The signal intensity of the indicated bands (left gray arrow) was quantified by direct chemiluminescent imaging. The results are shown below each gel, and the values are the mean ± SD. The experiment was repeated three times with similar results. *, P < 0.02 vs. wild-type samples. B, SDS-PAGE of purified D2 TG or wild-type ventricular membrane proteins after ADP ribosylation using PTX and [32P]NAD+. As indicated, two different amounts of ventricular membrane protein were used. The Gi{alpha}/Go{alpha} band is indicated by a left gray arrow. After exposure to the film, the gel region corresponding to the band of interest was excised and counted directly in a ß-counter. The results are shown below the gel, and the values are the mean ± SD. The experiment was repeated four times with similar results. *, P < 0.05 vs. wild-type membranes.

 
To discriminate between the Gi{alpha} and Go{alpha} components of the adrenergic signaling pathway, real-time PCR was used to evaluate Go{alpha} mRNA levels, with the finding that there was no significant difference between the wild-type and D2 TG groups (ratio of D2 TG/wild type, 0.96 ± 0.12; similar results using cyclophilin A and ß-actin housekeeping genes). Specific primers for Gi{alpha}-3 could not be designed based on existing sequence information using Beacon Designer software.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Our results indicate that D2 TG ventricular cardiomyocytes exhibit an increase in ß-adrenergic responsiveness, defined as an increased AC Vmax, which is likely mediated by changes in the expression of regulatory G proteins. The novelty of these results is heightened by the fact that the chronically thyrotoxic D2 TG cardiomyocytes were harvested from systemically euthyroid mice, such that the increase in AC Vmax must stem directly from the effects of intracellularly generated T3. The current study therefore represents the first assessment of the direct effects of chronic, tissue-specific thyrotoxicosis on the ß-adrenergic signaling pathway in the heart.

The increase in forskolin-stimulated cAMP accumulation was similar in magnitude to that observed with NE, suggesting that the major changes in D2 TG ß-adrenergic signaling occur at a postreceptor level (Fig. 1Go). This is not surprising considering that increases in ß-AR number have not consistently been found in experimental thyrotoxicosis (27, 28, 29), and in one study the acute increase in ß-AR number did not persist in longer-term thyrotoxicosis (30). Furthermore, changes in ß-AR number do not necessarily correlate with catecholamine-stimulated cAMP accumulation (31, 32, 33). A postreceptor mechanism is also indirectly supported by the observations that mice overexpressing AC isoforms V (34) or VI (35) in their hearts have a proportional increase in catecholamine-stimulated myocardial cAMP accumulation in an otherwise normal myocardium. On the other hand, cardiac hypertrophy or heart failure has been observed with overexpression of ß-AR or Gs{alpha} isoforms (36, 37, 38, 39). These deleterious changes, which do not occur in the D2 TG hearts (14), may result because increases in ß-AR cause constitutive activation of the signaling pathway, whereas postreceptor changes may not abrogate the ligand dependence of the system.

An increase in the amount of AC would have been a plausible mechanism for increased cardiac ß-adrenergic responsiveness. Such a mechanism has been suggested to occur in brain (40) and brown fat (41, 42). Whereas it is possible that thyrotoxicosis might alter the expression of various AC isoforms selectively, there were no significant differences in AC IV-VII mRNA (Table 1Go) or in AC V and VI protein levels (Fig. 5AGo) in D2 TG vs. wild-type animals. It is notable, however, that forskolin-stimulated cAMP accumulation in the D2 TG ventricular membranes was significantly more sensitive to inhibition by Ca2+ (Fig. 4Go), indicating an even more pronounced functional predominance of isoforms AC V and VI in the D2 TG group. These data indicate that regulation of AC, rather than AC itself, is the critical target in cardiac-specific chronic thyrotoxicosis.

Gi is the major inhibitory pathway with respect to AC function (43, 44), and in cardiomyocytes, Gi{alpha}-2 is believed to be the isoform primarily linked to the muscarinic receptors (45, 46). Gi{alpha}-2 protein is not increased in the D2 TG membranes (Fig. 6AGo), suggesting that the cardiac muscarinic pathway is not a major target of thyroid hormone.

The finding of a decrease in the amount of the inhibitory Gi{alpha}-3/Go{alpha} proteins in the D2 TG ventricular membranes, together with the finding of no change in Go{alpha} mRNA, suggests that a decrease in Gi{alpha}-3 is the mechanism by which adrenergic responsiveness is increased in the D2 TG hearts. Such a decrease in Gi{alpha}-3 could explain the increased sensitivity of the AC complex to both NE and forskolin. The fact that the predominant isoforms of AC in the heart, AC V and VI, are also the most sensitive to inhibition by the Gi{alpha} pathway (23) lends further support to this hypothesis.

The proposed effect of thyroid hormone (D2 expression) on the Gi{alpha} pathway can explain why differences in cAMP generation between D2 TG and wild-type cardiomyocytes are only seen with higher amounts of adrenergic stimulators (NE or forskolin) (Fig. 1Go). Whereas cardiac ß-1 adrenergic receptors are predominantly coupled to G{alpha}s and AC VI (47), ß-2 adrenergic receptors can be coupled to either G{alpha}s or to Gi{alpha}, depending on the degree of receptor phosphorylation by PKA (48). Under basal conditions, ß-2 adrenergic receptors are predominantly coupled to G{alpha}s. However, as cAMP-stimulated PKA activity increases, phosphorylation of the ß-2 adrenergic receptors shifts their coupling predominance to the inhibitory Gi{alpha} pathways, thus providing feedback inhibition. According to our model, the major effect of D2-mediated cardiac thyrotoxicosis is to decrease the efficacy of this feedback mechanism by decreasing the amount of Gi{alpha}. However, the data do not allow us to evaluate the possibility that the effects of thyroid hormone may also affect feedback inhibition via Gi{alpha} at the level of constitutively active Gi{alpha}-coupled receptors. In either case, the difference between D2 TG and wild-type cardiomyocytes is unmasked only when the cAMP generation system is maximally activated.

Several previous studies using pharmacological doses of thyroid hormones and acute treatment protocols have examined the relationship between regulatory G protein levels in the heart and thyroid status. Rats treated with L-T4 in their drinking water for 18 d had no difference in Gs{alpha}, Gi{alpha}-2 and Gi{alpha}-3 protein, or mRNA compared with untreated rats, as well as no increase in forskolin-stimulated AC activity (49). In a separate study of rats made thyrotoxic with injection of T3 for 5 d, there was no correlation between thyroid status and Gs{alpha} (the major target of cholera toxin-catalyzed [32P]ADP ribosylation) (50). The only previous study in which Gi{alpha} was found to be reduced by thyrotoxicosis involved immature rats treated with T3 during 2–21 d postpartum. However, in these animals there was a decrease in the amounts of Gi{alpha}-2 and Gi{alpha}-3 and an increase in the long form of Gs{alpha} (29).

Given that none of these studies showed a decrease in Gs{alpha}, it is intriguing that the D2 TG mice had an approximately 10% decrease in the long and short forms of Gs{alpha} (Fig. 5BGo). One could speculate that this decrease represents a compensatory mechanism. It is also possible that the intensity or duration of thyrotoxicosis, or the maturity of the animal at the time of onset of thyrotoxicosis, is critical with regard to G protein expression. In any case, the decrease in Gs{alpha} clearly does not prevent the thyroid hormone-induced increase in AC Vmax.

Because D2 is normally expressed in the human heart, the current data have implications for both normal human cardiac physiology and the cardiac manifestations of systemic thyrotoxicosis. It is conceivable that when adrenergic input to the heart increases, such as during stress, the expression of the cAMP-responsive D2 gene increases, therefore increasing the local conversion of T4 to T3. This increase in cardiac thyroid status would, in turn, amplify the ß-adrenergic responsiveness of the heart, operating in a positive feed back loop such as the one recently described in brown adipocytes (51).

In conclusion, the current data indicate that chronic thyrotoxicosis directly increases the ß-adrenergic responsiveness of cardiomyocytes via alterations in the regulatory elements of the AC complex, specifically by decreasing the Gi{alpha} pathway-mediated feedback inhibition of the AC complex.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animals
All aspects of animal care and experimentation were approved by the Institutional Animal Care and Use Committee of the Beth Israel Deaconess Medical Center and the Brigham and Women’s Hospital. Animals were maintained on a 12-h light/12-h dark schedule (light on at 0600 h) and fed laboratory chow and water ad libitum if not otherwise indicated. The expression of the human D2 gene transgene was directed to the heart by placing it under the control of the {alpha}-MHC promoter (14). The genotyping was determined by PCR using a 21-bp sequence complementary to a region of the {alpha}-MHC promoter next to the insertion point as the upstream primer and a 19-bp fragment complementary to a carboxyl terminus sequence of the D2 cDNA as the downstream primer. Tailing for genotyping is performed before the animals are 21 d old. Genotyping was confirmed by measuring D2 activity in the ventricles used in the experiments as has been previously described (52). Cardiomyocytes were isolated from both 2-month-old and 1-yr-old animals. Other experiments were performed with 2- to 3-month-old animals.

Isolation of Ventricular Cardiomyocytes and Stimulation of cAMP Accumulation (See Below)
Male or female mice were anesthetized by ethrane inhalation and heparinized (~5000 U ip). The chests were opened, hearts were quickly removed, and the aorta was cannulated for retrograde (Langendorff) perfusion of the coronary arteries with Tyrode’s buffer containing 126 mM NaCl, 4.4 mM KCl, 1 mM MgCl2, 4 mM NaHCO3, 30 mM 2,3-butane-dione monoxime (BDM), 10 mM HEPES, and 11 mM Glucose (pH 7.3). After the blood had been washed out, the hearts were digested with Tyrode’s buffer containing 0.4 mg/ml collagenase II (Worthington Biochemical Corp., Freehold, NJ; 266 U/mg) and 0.3 mg/ml hyaluronidase II (Sigma Chemical Co., St. Louis, MO) for 15 min. The atria were trimmed away, and the ventricles were minced and transferred to 10 ml Tyrode’s buffer containing 0.02 mg/ml trypsin IX, 0.02 mg/ml deoxyribonuclease I, and 0.3 mM CaCl2. After 10 min incubation in a 37 C shaking water bath, myocytes were released by gentle centrifugation. The resulting supernatant containing the cell suspension was transferred to another tube containing 10 ml of a 1:1 mixture of Tyrode’s solution and DMEM plus 0.9 mM CaCl2 and 2% FBS. If needed, the trypsin digestion was repeated until the majority of the ventricular myocytes were released. The cell suspensions were pooled and centrifuged at 50 x g for 3 min. The pellet was then carefully resuspended with 10 ml of Tyrode’s solution/ DMEM and cells were allowed to settle by gravity for 10–15 min. The final pellet of freshly isolated cardiomyocytes was then resuspended in Tyrode’s buffer (0.45 mM CaCl2 and 15 mM BDM), counted, adjusted to a final density of 40,000–50,000 cells/ml, and incubated at 37 C for 30 min in a 5% CO2 controlled environment in the presence of the phosphodiesterase inhibitor IBMX (1 mM), adenosine deaminase (0.5 U/ml), and increasing concentrations of NE or forskolin. cAMP accumulation was stopped by the addition of 1.5% perchloric acid.

Isolation of Membrane-Enriched Fractions and Stimulation of cAMP Accumulation
Membrane-enriched fractions from WT and TG mice ventricles were isolated as described previously (31, 42). Mice hearts were excised, and the ventricle was separated from the atria and immediately frozen in liquid nitrogen. Four to five frozen heart ventricles were pooled in a cold pestle and homogenized in 5 mM Tris-HCl (pH 8.0) buffer containing 0.25 M sucrose, 1 mM EDTA, and protease inhibitors (10 µg/ml aprotinin, 10 µg/ml leupeptin, and 50 µg/ml PMSF). Homogenates were centrifuged at 15,000 x g for 15 min. The pellets were washed in the same buffer, and final pellets were resuspended in assay buffer (10 mM Tris-HCl, pH 8.0, containing 0.25 M sucrose, 1 mM EGTA, and protease inhibitors as above). Membrane fractions were frozen at –70 C in small aliquots. For each experiment, an aliquot was defrosted and diluted to the appropriate protein concentration in assay buffer. One milliliter of this membrane solution was incubated at 30 C for 30–60 min with 1 mM IBMX, 0.4 U/ml adenosine deaminase, 5 mM MgCl2, 0.1% BSA, 0.1 µM GTP, and 10 µM propranolol. cAMP accumulation was stimulated by increasing concentrations of forskolin or ATP. When studying the dose response to forskolin, 1 mM ATP was also included in the incubation solution. When studying AC kinetic properties, concentrations of ATP varied from 0–2 mM, and 0.1 mM forskolin was present in the incubation solution. In the experiments with Ca2+, membranes were diluted in assay buffer without EGTA, and an exact final concentration of 1 mM EGTA was added to the assay tube. Forskolin (10–5 M) was present in the incubation solution. The total CaCl2 added to obtain each exact desired concentration of free Ca2+ was calculated by a computer program (The Chelator). cAMP accumulation was stopped by the addition of 3% perchloric acid.

Quantification of cAMP Content in Cells and Membranes
cAMP in all samples was quantified by a solid-phase RIA Kit (NEK-033) from New England Nuclear (Boston, MA). Defrosted samples were briefly centrifuged, pellets were discarded, and the pH in supernatants was adjusted to 5–6 with 15% KHCO3. Solutions (10–50 µl) were incubated overnight at 4 C with [125I]cAMP and anti-cAMP antibody. The cAMP-antibody complexes were precipitated and counted. cAMP concentration was estimated from the standard curves. Results are expressed as cAMP content/h·105 cells or cAMP content/ h·mg of membrane protein.

Real-Time PCR of AC Isoform mRNA
For total RNA extraction, animals were euthanized via CO2, and ventricular tissue was immediately harvested and frozen in liquid nitrogen. Frozen hearts were pulverized in liquid nitrogen, and the resulting frozen fragments were homogenized in Trizol Reagent (Invitrogen, Carlsbad CA) using a Brinkmann homogenizer (Brinkmann Instruments, Inc., Westbury, NY). RNA was extracted from homogenized tissue via the Trizol protocol supplied by the manufacturer. RNA pellets were treated with deoxyribonuclease I (Invitrogen) according to the instructions of the manufacturer, and the samples were repurified using Trizol. RNA concentration was estimated by measuring the absorption at 260 nm (A260), with purity being estimated via A260/A280 ratio between 1.8–2.1 and degradation being assessed via Tris-acetate-EDTA agarose electrophoresis. cDNA synthesis was performed using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) according to the instructions of the manufacturer. RNA (5 µg) was reverse transcribed using oligo dT primers to generate real-time PCR template cDNA. cDNA content was quantified and checked for purity and condition via spectrophotometry and gel electrophoresis. Real-time PCR was performed using the iCycler iQ real-time PCR detection system (Bio-Rad Laboratories, Inc., Hercules CA) and iQ SYBR green supermix (Bio-Rad) according to the instructions of the manufacturer at the 30 {lambda} scale. Data analysis was performed using the iCycler system software, with fold change of a given target gene from TG samples to wild-type samples being expressed relative to a housekeeping gene (ß-actin and cyclophilin A were used in this study). Melt curve analysis was performed for each primer set. Specific primer sets for murine AC IV (sense, 5'-CTGGACACTGGTGATGCTAAG; antisense, 5'-AGGCTGCGTAGTATTTGAAGG), V (sense, 5'-GCCTGCTCCGTGTTCCTG; antisense, 5'-CCGTTGTTGCTGAAGTCTATGG), VI (sense, 5'-CCTCCTGGTTCCCAAAGTG; antisense, 5'-GGTGGCTCCGCATTCTTG), VII (sense, 5'-TGCTGCTCCAAGTCTGATG; antisense, 5'-CACAGGCGAAGTCGTAGC), G-protein {alpha}-subunit o (sense, 5'-GGACAGACTGACCACCATC; antisense, 5'-AGAGGAAGGATTGCCAACC0). ß-actin (sense, 5'-TTTGTTTTGGCGCTTTTGACTC; antisense, 5'-TGGGAGGGTGAGGGACTTC), and cyclophilin A (sense, 5'-CGGCAGGTCCATCTACGG; antisense, 5'-CCATCCAGCCATTCAGTCTTG) were designed using published nucleotide sequences and Beacon Designer version 2.0 software (Premier Biosoft, Palo Alto, CA). To generate standard curves, serial 5-fold dilutions of a cDNA stock (cDNA was prepared from TG ventricular tissue as described) were prepared for use as templates. Amplification was performed with housekeeping or target gene-specific primers. Standard curves were determined for housekeeping and target genes on every experimental plate, with each sample being run in triplicate (standard curve points) or two triplicates (unknown quantity points). Results were expressed as mean of fold change ± SD.

Quantitation of AC, Gs{alpha}, Gi{alpha}, and Gß Proteins by Western Blotting
Western blot analysis was used to investigate changes in the total amount of isoforms V/VI of AC, the long and short isoforms of the Gs{alpha} protein, and isoforms 2 and 3 of the Gi{alpha} proteins. This was done using ventricular membranes isolated from TG and WT mouse ventricles that were further purified through a sucrose cushion, as described for the ADP-ribosylation assay. Equal amounts of protein (10–30 µg/lane) were size fractionated by SDS-PAGE, followed by transfer to polyvinylidene fluoride membranes (PVDF, Immobilon-P by Millipore Corp., Bedford, MA). Immunoblots were performed using rabbit antibodies as described below.

AC V/VI: a 1:200 Dilution of C-17 from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
This is an affinity purified polyclonal antibody raised against a peptide mapping at the C terminus of mouse AC V/VI that partially cross reacts with AC I (not present in the myocardium) but not other AC isoforms).

Gi{alpha}-1 and Gi{alpha}-2: a 1:1000 Dilution of 371723 from Calbiochem (La Jolla, CA).
This is a Protein A affinity-purified polyclonal antibody raised against a synthetic peptide mapping at the C terminus of Gi{alpha}-1 and Gi{alpha}-2, the specificity of which was confirmed with lysates from bacterially produced recombinant G proteins.

Gi{alpha}-3 and Go{alpha}: a 1:1000 Dilution of Calbiochem 371726, an Antiserum that Was Raised against a Synthetic Peptide Mapping at the C Terminus of Gi{alpha}-3, but Which Also Recognizes Go{alpha} because Both Share the Terminal Four Residues.
The specificity of this antiserum was confirmed using bacterial lysates containing recombinant G proteins.

G{alpha}s: a 1:500 Dilution of C-18 from Santa Cruz.
This is an affinity purified polyclonal antibody raised against a peptide mapping at the C terminus of rat G{alpha}s. It also reacts with mouse G{alpha}s, but not other G{alpha} subunits.

Gß: a 1:500 Dilution of SC25413 from Santa Cruz.
This is a rabbit polyclonal antibody raised against a recombinant protein corresponding to amino acids 1–300 mapping at the amino terminus of Gß. Membranes were incubated for 1 h at room temperature, washed, and incubated for 1 h with horseradish peroxidase-labeled antirabbit IgG (Roche Molecular Biochemicals, Mannheim, Germany). The antibodies were then detected by chemiluminescence (ECL+Plus, Amersham Biosciences, Piscataway NJ) and visualized by exposure to photographic film. The membranes were also scanned by direct chemiluminescent imaging using the high-performance gel and blot imager Typhoon 9410 (Amersham Biosciences), and the intensity of the bands of interest was quantified using ImageQuant 5.2 software (Molecular Dynamics, Inc., Sunnyvale, CA/Amersham). In preliminary ex-periments, we ascertained that the signal obtained was in the linear range. Blots were stripped and reprobed for consistency.

Functional State of Gi Proteins by ADP Ribosylation
The functional state of Gi/Go protein was assayed based on the susceptibility of membrane proteins to ADP ribosylation by PTX with the presence of [32P]NAD+. The ribosylated Gi/Go was quantified through SDS-PAGE and reflects the total amount of the Gi/Go capable of activation. Ventricular membranes used for this assay were obtained as described above, and then purified through a sucrose cushion as follows: 500 µl of the membrane-enriched fraction were loaded over a 2-ml layer of 0.67 M sucrose buffer (10 mM Tris-HCl, pH 7.4; 0.67 M sucrose; and 1 mM EDTA) and then centrifuged at 20,000 x g for 20 min at 4 C. The membrane interface was collected and centrifuged again at 100,000 x g for 1 h at 4 C. The pellets were resuspended in 10 mM Tris-HCl, pH 8.0, containing 0.25 M sucrose, 1 mM EGTA, and protease inhibitors. Before the assay, the membranes were incubated with 0.6% lubrol for 30 min at 4 C to solubilize the G proteins. Immediately before ribosylation, the ADP-ribosyltransferase activity in the S1 subunit of the PTX was activated by incubation in 25 mM Tris-HCl buffer, pH 8.0, containing 20 mM dithiothreitol, 0.12% sodium dodecyl sulfate, and 15 µg BSA for 20 min at 30 C. Then, the activated toxin was transferred to a 25 mM Tris, pH 8.0, buffer containing 15–25 µg membrane protein, 1 mM EDTA, 10 mM thymidine, 1 mM GTP, 0.5 mM ATP, and 5–10 µM [32P]NAD+ (~4–5 x 106 cpm/tube). After 30 min at 30 C, the reaction was stopped by the addition of 150 mM NaCl, followed by 1 ml of ice-cold acetone. After 10 min on ice, samples were centrifuged at 2,500 rpm for 30 min at 4 C and supernatants were discarded. Pellets were washed with 1 ml of ice-cold 20% TCA and centrifuged at 2500 rpm for 30 min at 4 C. Pellets were extracted with ice-cold ethyl ether and centrifuged again. Supernatants were aspirated, and remaining ether was evaporated at room temperature (RT). The pellets were then dissolved in loading buffer with 1% sodium dodecyl sulfate and applied to a 12% SDS-PAGE. The dried gels were exposed to photographic film, and later the gel region corresponding to the band of interest was excised and counted directly in a ß-counter. Results are expressed as arbitrary units.

Statistical Analysis
Experiments containing multiple groups were analyzed by ANOVA followed by the Bonferroni test for multiple comparisons. Student’s t test was used when experiments contained only two groups. The 5% threshold was used to reject the null hypothesis.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grants DK44128 and HL52320 and Grant 9930032N from the American Heart Association. B.W.K. was a recipient of a grant from the Endocrine Fellows Foundation and DK07529 training grant.

Abbreviations: AC, Adenylyl cyclase; ß-AR, ß-adrenergic receptor; BDM, butane-dione monoxime; D2, type 2 iodothyronine deiodinase; IBMX, 3-isobutyl-1-methylxanthine; MHC, myosin heavy chain; NAD, nicotinamide adenine dinucleotide; NE, norepinephrine; PKA, protein kinase A; PTX, pertussis toxin; TG, transgenic.

Received for publication April 8, 2003. Accepted for publication April 16, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Dillmann WH 2002 Cellular action of thyroid hormone on the heart. Thyroid 12:447–452[CrossRef][Medline]
  2. Steinbock M, Grossman W 1971 Tachycardia in thyrotoxicosis. Ann Intern Med 75:973
  3. Wiener L, Stout BD, Cox JW 1969 Influence of ß sympathetic blockade (propranolol) on the hemodynamics of hyperthyroidism. Am J Med 46:227–233[CrossRef][Medline]
  4. Rutherford JD, Vatner SF, Braunwald E 1979 Adrenergic control of myocardial contractility in conscious hyperthyroid dogs. Am J Physiol 237:H590–H596
  5. Hoit BD, Khoury SF, Shao Y, Gabel M, Liggett SB, Walsh RA 1997 Effects of thyroid hormone on cardiac ß-adrenergic responsiveness in conscious baboons. Circulation 96:592–598[Abstract/Free Full Text]
  6. Klein I, Levey GS 2000 The cardiovascular system in thyrotoxicosis. In: Braverman LE, Utiger RD, eds. Werner and Ingbar’s the thyroid: a fundamental and clinical text, 8th ed. Baltimore: Lippincott, Williams & Wilkins; 586–604
  7. Hohl CM, Wetzel S, Fertel RH, Wimsatt DK, Brierley GP, Altschuld RA 1989 Hyperthyroid adult rat cardiomyocytes. I. Nucleotide content, ß- and {alpha}-adrenoreceptors, and cAMP production. Am J Physiol 257:C948–C956
  8. Ojamaa K, Kenessey A, Klein I 2000 Thyroid hormone regulation of phospholamban phosphorylation in the rat heart. Endocrinology 141:2139–2144[Abstract/Free Full Text]
  9. Silva JE 2000 Catecholamines and the sympathoadrenal system in hypothyroidism. In: Braverman LE, Utiger RD, eds. Werner and Ingbar’s the thyroid: a fundamental and clinical text. Baltimore: Lippincott Williams & Wilkins; 820–823
  10. Ojamaa K, Samarel AM, Kupfer JM, Hong C, Klein I 1992 Thyroid hormone effects on cardiac gene expression independent of cardiac growth and protein synthesis. Am J Physiol 263:E534–E540
  11. Klein I, Hong C 1986 Effects of thyroid hormone on cardiac size and myosin content of the heterotopically transplanted rat heart. J Clin Invest 77:1694–1698
  12. Croteau W, Davey JC, Galton VA, St Germain DL 1996 Cloning of the mammalian type II iodothyronine deiodinase. A selenoprotein differentially expressed and regulated in human and rat brain and other tissues. J Clin Invest 98:405–417[Medline]
  13. Salvatore D, Bartha T, Harney JW, Larsen PR 1996 Molecular biological and biochemical characterization of the human type 2 selenodeiodinase. Endocrinology 137:3308–3315[Abstract]
  14. Pachucki J, Hopkins J, Peeters R, Tu H, Carvalho SD, Kaulbach H, Abel ED, Wondisford FE, Ingwall JS, Larsen PR 2001 Type 2 iodothyronine deiodinase transgene expression in the mouse heart causes cardiac-specific thyrotoxicosis. Endocrinology 142:13–20[Abstract/Free Full Text]
  15. Pachucki J, Burmeister LA, Larsen PR 1999 Thyroid hormone regulates hyperpolarization-activated cyclic nucleotide-gated channel (HCN2) mRNA in the rat heart. Circ Res 85:498–503[Abstract/Free Full Text]
  16. Lortet S, Heckmann M, Ray A, Rossi A, Aussedat J, Grably S, Zimmer HG 1995 Energy metabolism response to calcium activation in isolated rat hearts during development and regression of T3-induced hypertrophy. Mol Cell Biochem 151:99–106[CrossRef][Medline]
  17. Bak MI, Ingwall JS 1998 Regulation of cardiac AMP-specific 5'-nucleotidase during ischemia mediates ATP resynthesis on reflow. Am J Physiol 274:C992–C1001
  18. Kessler PD, Cates AE, Van Dop C, Feldman AM 1989 Decreased bioactivity of the guanine nucleotide-binding protein that stimulates adenylate cyclase in hearts from cardiomyopathic Syrian hamsters. J Clin Invest 84:244–252
  19. Iyengar R 1993 Molecular and functional diversity of mammalian Gs-stimulated adenylyl cyclases. FASEB J 7:768–775[Abstract]
  20. Taussig R, Tang WJ, Hepler JR, Gilman AG 1994 Distinct patterns of bidirectional regulation of mammalian adenylyl cyclases. J Biol Chem 269:6093–6100[Abstract/Free Full Text]
  21. Defer N, Best-Belpomme M, Hanoune J 2000 Tissue specificity and physiological relevance of various isoforms of adenylyl cyclase. Am J Physiol 279:F400–F416
  22. Feldman AM 2002 Adenylyl cyclase: a new target for heart failure therapeutics. Circulation 105:1876–1878[Free Full Text]
  23. Patel TB, Du Z, Pierre S, Cartin L, Scholich K 2001 Molecular biological approaches to unravel adenylyl cyclase signaling and function. Gene 269:13–25[CrossRef][Medline]
  24. Hadcock JR, Malbon CC 1993 Agonist regulation of gene expression of adrenergic receptors and G proteins. J Neurochem 60:1–9[CrossRef][Medline]
  25. Ribeiro-Neto F, Mattera R, Grenet D, Sekura RD, Birnbaumer L, Field JB 1987 Adenosine diphosphate ribosylation of G proteins by pertussis and cholera toxin in isolated membranes. Different requirements for and effects of guanine nucleotides and Mg2+. Mol Endocrinol 1:472–481[Abstract]
  26. Kopf GS, Woolkalis MJ 1991 ADP-ribosylation of G proteins with pertussis toxin. Methods Enzymol 195:257–266[Medline]
  27. Williams LT, Lefkowitz RJ, Watanabe AM, Hathaway DR, Besch Jr HR 1977 Thyroid hormone regulation of ß-adrenergic receptor number. J Biol Chem 252:2787–2789[Abstract/Free Full Text]
  28. Stiles GL, Lefkowitz RJ 1981 Thyroid hormone modulation of agonist-ß-adrenergic receptor interactions in the rat heart. Life Sci 28:2529–2536[CrossRef][Medline]
  29. Novotny J, Bourova L, Malkova O, Svoboda P, Kolar F 1999 G proteins, ß-adrenoreceptors and ß-adrenergic responsiveness in immature and adult rat ventricular myocardium: influence of neonatal hypo- and hyperthyroidism. J Mol Cell Cardiol 31:761–772[CrossRef][Medline]
  30. Zwaveling J, Batink HD, Taguchi K, de Jong S, Michel MC, Pfaffendorf M, van Zwieten A 1998 Thyroid status affects the rat cardiac ß-adrenoceptor system transiently and time-dependently. J Auton Pharmacol 18:1–11[CrossRef][Medline]
  31. Zolk O, Kilter H, Flesch M, Mansier P, Swynghedauw B, Schnabel P, Bohm M 1998 Functional coupling of overexpressed ß1-adrenoceptors in the myocardium of transgenic mice. Biochem Biophys Res Commun 248:801–805[CrossRef][Medline]
  32. Heubach JF, Trebess I, Wettwer E, Himmel HM, Michel MC, Kaumann AJ, Koch WJ, Harding SE, Ravens U 1999 L-type calcium current and contractility in ventricular myocytes from mice overexpressing the cardiac ß2-adrenoceptor. Cardiovasc Res 42:173–182[Abstract/Free Full Text]
  33. Liggett SB, Tepe NM, Lorenz JN, Canning AM, Jantz TD, Mitarai S, Yatani A, Dorn II GW 2000 Early and delayed consequences of ß(2)-adrenergic receptor overexpression in mouse hearts: critical role for expression level. Circulation 101:1707–1714[Abstract/Free Full Text]
  34. Tepe NM, Lorenz JN, Yatani A, Dash R, Kranias EG, Dorn II GW, Liggett SB 1999 Altering the receptor-effector ratio by transgenic overexpression of type V adenylyl cyclase: enhanced basal catalytic activity and function without increased cardiomyocyte ß-adrenergic signalling. Biochemistry 38:16706–16713[CrossRef][Medline]
  35. Gao M, Ping P, Post S, Insel PA, Tang R, Hammond HK 1998 Increased expression of adenylylcyclase type VI proportionately increases ß-adrenergic receptor-stimulated production of cAMP in neonatal rat cardiac myocytes. Proc Natl Acad Sci USA 95:1038–1043[Abstract/Free Full Text]
  36. Iwase M, Bishop SP, Uechi M, Vatner DE, Shannon RP, Kudej RK, Wight DC, Wagner TE, Ishikawa Y, Homcy CJ, Vatner SF 1996 Adverse effects of chronic endogenous sympathetic drive induced by cardiac GS {alpha} overexpression. Circ Res 78:517–524[Abstract/Free Full Text]
  37. Iwase M, Uechi M, Vatner DE, Asai K, Shannon RP, Kudej RK, Wagner TE, Wight DC, Patrick TA, Ishikawa Y, Homcy CJ, Vatner SF 1997 Cardiomyopathy induced by cardiac Gs {alpha} overexpression. Am J Physiol 272:H585–H589
  38. Engelhardt S, Hein L, Wiesmann F, Lohse MJ 1999 Progressive hypertrophy and heart failure in ß1-adrenergic receptor transgenic mice. Proc Natl Acad Sci USA 96:7059–7064[Abstract/Free Full Text]
  39. Gao MH, Lai NC, Roth DM, Zhou J, Zhu J, Anzai T, Dalton N, Hammond HK 1999 Adenylylcyclase increases responsiveness to catecholamine stimulation in transgenic mice. Circulation 99:1618–1622[Abstract/Free Full Text]
  40. Wagner JP, Seidler FJ, Lappi SE, McCook EC, Slotkin TA 1994 Role of thyroid status in the ontogeny of adrenergic cell signaling in rat brain: ß receptors, adenylate cyclase, ornithine decarboxylase and c-fos protooncogene expression. J Pharmacol Exp Ther 271:472–483[Abstract/Free Full Text]
  41. Granneman JG 1995 Expression of adenylyl cyclase subtypes in brown adipose tissue: neural regulation of type III. Endocrinology 136:2007–2012[Abstract]
  42. Carvalho SD, Bianco AC, Silva JE 1996 Effects of hypothyroidism on brown adipose tissue adenylyl cyclase activity. Endocrinology 137:5519–5529[Abstract]
  43. Katada T, Bokoch GM, Northup JK, Ui M, Gilman AG 1984 The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. Properties and function of the purified protein. J Biol Chem 259:3568–3577[Abstract/Free Full Text]
  44. Cerione RA, Staniszewski C, Gierschik P, Codina J, Somers RL, Birnbaumer L, Spiegel AM, Caron MG, Lefkowitz RJ 1986 Mechanism of guanine nucleotide regulatory protein-mediated inhibition of adenylate cyclase. Studies with isolated subunits of transducin in a reconstituted system. J Biol Chem 261:9514–9520[Abstract/Free Full Text]
  45. Rudolph U, Spicher K, Birnbaumer L 1996 Adenylyl cyclase inhibition and altered G protein subunit expression and ADP-ribosylation patterns in tissues and cells from Gi2 {alpha}–/– mice. Proc Natl Acad Sci USA 93:3209–3214[Abstract/Free Full Text]
  46. Nagata K, Ye C, Jain M, Milstone DS, Liao R, Mortensen RM 2000 G{alpha}(i2) but not G{alpha}(i3) is required for muscarinic inhibition of contractility and calcium currents in adult cardiomyocytes. Circ Res 87:903–909[Abstract/Free Full Text]
  47. Ostrom RS, Violin JD, Coleman S, Insel PA 2000 Selective enhancement of ß-adrenergic receptor signaling by overexpression of adenylyl cyclase type 6: colocalization of receptor and adenylyl cyclase in caveolae of cardiac myocytes. Mol Pharmacol 57:1075–1079[Abstract/Free Full Text]
  48. Zamah AM, Delahunty M, Luttrell LM, Lefkowitz RJ 2002 Protein kinase A-mediated phosphorylation of the ß2-adrenergic receptor regulates its coupling to Gs and Gi. Demonstration in a reconstituted system. J Biol Chem 277:31249–31256[Abstract/Free Full Text]
  49. Levine MA, Feldman AM, Robishaw JD, Ladenson PW, Ahn TG, Moroney JF, Smallwood PM 1990 Influence of thyroid hormone status on expression of genes encoding G protein subunits in the rat heart. J Biol Chem 265:3553–3560[Abstract/Free Full Text]
  50. Krawietz W, Werdan K, Erdmann E 1982 Effect of thyroid status on ß-adrenergic receptor, adenylate cyclase activity and guanine, nucleotide regulatory unit in rat cardiac and erythrocyte membranes. Biochem Pharmacol 31:2463–2469[CrossRef][Medline]
  51. de Jesus LA, Carvalho SD, Ribeiro MO, Schneider M, Kim SW, Harney JW, Larsen PR, Bianco AC 2001 The type 2 iodothyronine deiodinase is essential for adaptive thermogenesis in brown adipose tissue. J Clin Invest 108:1379–1385[CrossRef][Medline]
  52. Steinsapir J, Harney J, Larsen PR 1998 Type 2 iodothyronine deiodinase in rat pituitary tumor cells is inactivated in proteasomes. J Clin Invest 102:1895–1899[Medline]



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