Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation Research
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation Research. 1995;76:852-860

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Loh, E.
Right arrow Articles by Galper, J. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Loh, E.
Right arrow Articles by Galper, J. B.
(Circulation Research. 1995;76:852-860.)
© 1995 American Heart Association, Inc.

Articles

Decreased Adenylate Cyclase Activity and Expression of Gs{alpha} in Human Myocardium After Orthotopic Cardiac Transplantation

Evan Loh, Joey V. Barnett, Arthur M. Feldman, Gregory S. Couper, Dorothy E. Vatner, Wilson S. Colucci, Jonas B. Galper

From the Department of Medicine (Cardiovascular Division), Hospital of the University of Pennsylvania, Philadelphia (E.L.); Department of Medicine (Cardiovascular Division) and Cardiac Surgery, Brigham and Women's Hospital and Harvard Medical School, Boston, Mass (G.S.C., W.S.C., J.B.G.); Cardiovascular Division, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tenn (J.V.B.); Division of Cardiology, University of Pittsburgh (Pa) (A.M.F.); and Children's Service, Massachusetts General Hospital, Boston (D.E.V.).

Correspondence to Evan Loh, MD, Cardiovascular Division, Hospital of the University of Pennsylvania, 3400 Spruce St, Philadelphia PA 19104.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract We studied several aspects of guanine nucleotide–stimulated adenylate cyclase function in patients after orthotopic cardiac transplantation. In 28 patients, adenylate cyclase activity was measured in endomyocardial biopsy samples obtained just before and at monthly intervals after cardiac transplantation. In biopsies obtained >=6 months after transplantation, basal adenylate cyclase activity was decreased by 67% (n=12; P<.05), GTP{gamma}S-stimulated adenylate cyclase activity was decreased by 78% (n=12; P<.05), Mn+2+forskolin–stimulated adenylate cyclase activity was decreased by 80% (n=8; P<.05), and Mn+2-stimulated adenylate cyclase activity (a measure of activity of the catalytic subunit of adenylate cyclase) was decreased by 83% (n=8, P<.05). Western blot analysis demonstrated that 6 months after cardiac transplantation, the level of Gs{alpha} protein was decreased by 61±12% (n=8; P<.001). There was no change in the level of Gi{alpha} as assessed by pertussis toxin–catalyzed ADP-ribosylation (n=4; P=NS). With the use of the quantitative polymerase chain reaction, a 50±10% (n=6; P<.001) reduction in the steady-state level of Gs{alpha} mRNA was observed. There was no change in the level of mRNA for Gi-3{alpha}. Thus, after orthotopic cardiac transplantation in humans, guanine nucleotide–stimulated adenylate cyclase activity is decreased in parallel with decreased levels of Gs{alpha} protein and mRNA.


Key Words: cardiac transplantation • adenylate cyclase • guanine nucleotide regulatory proteins


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The autonomic nervous system regulates the contractile state of the heart in part through ß-adrenergic and muscarinic cholinergic receptors that stimulate and inhibit adenylate cyclase activity, respectively.1 2 ß-Adrenergic receptors are coupled to the catalytic subunit of adenylate cyclase via a stimulatory guanine nucleotide regulatory protein (G-protein, Gs{alpha}), and muscarinic cholinergic receptors are coupled to the catalytic unit of adenylate cyclase via an inhibitory G-protein, Gi{alpha}. These G-proteins are heterotrimers consisting of {alpha}, ß, and {gamma} subunits, all of which exist in multiple isoforms.3

There is evidence that changes in G-protein levels can influence the responses of the heart to autonomic stimulation. For example, the growth of cultured chick atrial cells in medium supplemented by lipoprotein-depleted serum resulted in an increase in parasympathetic responsiveness and a decrease in sympathetic responsiveness associated with increased levels of muscarinic receptors and Gi{alpha} and decreases in ß-adrenergic receptor number and Gs{alpha}.4 5 Alterations in the expression and/or activity of cardiac G-proteins may also occur in human pathological states. In myocardium from patients with dilated cardiomyopathy, increased Gi{alpha} activity is associated with a decreased adenylate cyclase response to guanine nucleotides that is normalized by treatment of the membranes with pertussis toxin to inactivate Gi{alpha}.6 The demonstration that Gi-3{alpha} mRNA is increased by 44% in failing myocardium further suggests that increased Gi{alpha} activity in failing myocardium is due, at least in part, to increased transcription.7 Thus, levels of G-proteins that can influence the balance between sympathetic and parasympathetic responsiveness of the heart may be regulated by extrinsic factors that act at the level of gene expression.

Several studies have suggested that orthotopic transplantation or denervation of the heart may be associated with abnormalities in the coupling of ß-adrenergic receptors to the stimulation of adenylate cyclase.8 9 We previously found that the ability of the guanine nucleotide guanylyl-imidodiphosphate (GppNHp) to stimulate adenylate cyclase activity was blunted in membranes from transplanted human hearts.10 The purpose of the current study was to elucidate the molecular mechanism(s) responsible for altered G-protein function in the heart after cardiac transplantation and thereby test the hypothesis that transplantation of the heart in humans results in alterations in the expression and/or function of G-proteins. Adenylate cyclase activity, the levels of Gs{alpha} (by Western blotting) and Gi{alpha} (by pertussis toxin–catalyzed ADP-ribosylation) expression, and the steady-state levels of Gs{alpha} and Gi{alpha} mRNA (by quantitative polymerase chain reaction [PCR]) were determined in endomyocardial biopsy samples from 28 patients who underwent successful cardiac transplantation. A unique aspect of this study is that endomyocardial biopsies were taken from the donor heart both before and after transplantation (at monthly intervals), thus allowing each heart to serve as its own control.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Materials
[14C]AMP, [32P]ATP, and [32P]NAD were from Du Pont–New England Nuclear. 125I-Protein A was from Amersham Searle. Lima bean trypsin inhibitor, soybean trypsin inhibitor, ATP, and GTP were from Sigma. Pertussis toxin was from List Biochemicals. Antibody to the {alpha}-subunit of Gs{alpha} generated against the amino acid sequence of peptide A-572 was the generous gift of Drs S.M. Mumby and A.G. Gilman, Department of Pharmacology, Southwestern Graduate School, University of Texas Health Science Center, Dallas.

Patient Selection
After informed consent was obtained, 28 patients undergoing orthotopic cardiac transplantation at the Brigham and Women's Hospital between February 1990 and February 1991 were enrolled in this study. The mean age of the donor hearts was 27±2 years (range, 16 to 48 years). Eighty percent of the hearts were from male donors, and 20% of the hearts were from female donors. Patients were included in this study only if a pretransplantation biopsy of the donor heart was obtained. To control for the potential confounding effects of rejection, each posttransplantation biopsy was reviewed histologically, and any sample with evidence of myocyte rejection,11 including perivascular inflammation without myocyte necrosis, was excluded from the analysis.

Tissue Acquisition
Immediately before implantation of the cardiac allograft, a single right ventricular endomyocardial biopsy was obtained from the donor heart. Biopsies were then immediately frozen in liquid N2. After transplantation, in addition to the standard three to five biopsies to monitor for myocyte rejection, a single extra serial right ventricular endomyocardial biopsy was obtained from each patient via a standard right internal jugular approach or femoral vein approach. Biopsies were frozen immediately in liquid N2. In all of the data presented, the pretransplantation biopsies were used as each subject's own control, thus allowing for paired comparisons.

Adenylate Cyclase Assay
Biopsy samples were Dounce homogenized, frozen, and thawed twice in a buffer consisting of 50 mmol/L Tris (pH 7.4), 10 mmol/L MgCl2, 75 mmol/L NaCl, 1.6 mmol/L EDTA, 3.2 mmol/L dithiothreitol, 40 µmol/L EGTA, 50 µg/mL lima bean protease inhibitor, 50 µg/mL soybean protease inhibitor, and 50 µg/mL leupeptin. Aliquots were taken for protein determination by the method of Lowry et al.12 Adenylate cyclase activity was determined as described by Salomon,13 as modified by Neer.14 The reaction mixture contained 1 to 2 µCi of [32P]ATP, 50 mmol/L Tris (pH 7.6), 11 mmol/L MgCl2, 75 mmol/L NaCl, 1 mmol/L EDTA, 150 mmol/L sucrose, and 1 mmol/L cAMP. GTP{gamma}S, Mn+2, and forskolin were added to a final concentration of 100 µmol/L, 1 mmol/L, and 70 µmol/L, respectively. After incubation at 37°C for 5 minutes, the reaction was stopped by boiling for 2 minutes and the addition of 1 mL of solution containing unlabeled 100 µmol/L cAMP, 200 µmol/L ATP, and [14C]cAMP (1000 cpm/1 mL). cAMP was isolated by the method of Salomon.13 Each point was assayed in triplicate. Fractions containing cAMP were eluted and counted in a Beckman liquid scintillation counter. Recovery of [32P]cAMP, as determined by the ratio of 32P to 14C, ranged from 50% to 60%. These assay conditions were shown to be linear with respect to both time and protein concentration.

Pertussis Toxin–Catalyzed ADP-Ribosylation
Homogenates of endomyocardial biopsy specimens were prepared in 50 mmol/L Tris-HCl (pH 7.6), 10 mmol/L MgCl2, 0.2 mol/L sucrose, 1.0 mmol/L dithiothreitol, and 1 mmol/L EDTA by dounce homogenization and frozen and thawed twice. Protease inhibitors (soybean protease inhibitor, lima bean protease inhibitor, and leupeptin) were present at 50 µg/mL. The ability of pertussis toxin to catalyze the incorporation of 32P-ribose from [32P]NAD+ into proteins was determined as described previously except that homogenates were made 1% in cholate before incubation with pertussis toxin.15 Protein was determined by the method of Lowry et al12 and ranged from 20 to 40 µg per assay. Autoradiographs were quantitated by scanning densitometry and the intensity of each peak normalized to the intensity of the peak obtained from biopsies of the heart just before transplantation. These assay conditions were shown to be linear with respect to protein concentration.

Immunoblotting
Immunoblotting was carried out by a modification of the method of Towbin et al.16 Endomyocardial biopsy homogenates were solubilized by boiling in 2% sodium dodecyl sulfate (SDS) in Laemmli sample buffer. Samples containing 60 µg total protein were subjected to gel electrophoresis on 9% acrylamide gels.17 The gels were equilibrated in transfer buffer (39 mmol/L glycine, 48 mmol/L Tris-OH, 0.0375% [wt/vol] SDS, 20% methanol) and electrophoretically transferred to nitrocellulose at 150 V for 90 minutes. The dried fibers were incubated with 3% bovine serum albumin in 10 mmol/L Tris (pH 7.6) and 150 mmol/L NaCl for 60 minutes at room temperature to decrease nonspecific binding and subsequently incubated overnight at 4°C with specific antiserum to Gs{alpha}18 diluted 1:250 with the same buffer. The nitrocellulose filters were washed two times with 10 mmol/L Tris, 150 mmol/L NaCl, and 0.05% Nonidet P-40 for 10 minutes, and again with 10 mmol/L Tris and 150 mmol/L NaCl for 10 minutes.

125I-protein A (100 000 to 150 000 cpm/mL) in 10 mmol/L Tris, 150 mmol/L NaCl containing 3% bovine serum albumin was incubated with the blots for 1 hour at room temperature, followed by a repeat cycle of washes as described above. The dried filters were then exposed to Kodak XAR film with enhancing screens for 2 to 5 days at -20°C. Autoradiographs were quantitated by scanning densitometry and the intensity of each peak normalized to the intensity of the peak obtained from biopsies of the heart just before transplantation. These assay conditions were shown to be linear with respect to protein concentration.

Quantitative PCR Protocol
Steady-state levels of mRNA in human endomyocardial biopsy samples were assessed by means of PCR, as previously described.7 19 20 21 In brief, total RNA was isolated with the use of a modification of the acid guanidinium thiocyanate/phenol/chloroform extraction (RNAzol B, Cinna/Biotex, Inc).22 Frozen ventricular myocardium (3 to 5 mg) was homogenized with a polytron (Brickman Instruments Co, Inc) and 1.5 mL of RNAzol B, and the concentration of the resulting RNA was assessed spectrophotometrically (DU65, Beckman Instruments, Inc). First-strand cDNA was then synthesized by reverse transcription of 1 µg of total RNA with the use of oligo-dT primers according to manufacturer's instructions (Boehringer Mannheim Pharmaceuticals). Oligonucleotide primers complementary to selected regions of the mammalian gene encoding Gs{alpha}, Gi-3{alpha}, Gi-2{alpha}, ß-myosin heavy chain, phospholambam, and sarcoplasmic reticulum Ca2+-ATPase were synthesized on a DNA synthesizer (Applied Biosystems, Inc) by the Johns Hopkins University School of Medicine Protein-Peptide Facility, as previously described.7 19 20 21 The cDNA was amplified in a TempCycler (Coy Corp) with 2.5 U Thermus aquaticus DNA polymerase (Perkin-Elmer/Cetus Corp) in the presence of 13 pmol of each primer, and 100 µL of 10 mmol/L Tris-HCl containing 50 mmol/L KCl, 1.5 mmol/L MgCl2 0.001% (wt/vol) gelatin, and 200 µmol/L of each dNTPs. The 3' primer of each primer pair was end-labeled with [{gamma}-32P]ATP by the use of T4 polynucleotide kinase (Pharmacia LKB Biotechnology). Therefore, each synthesized DNA strand was radiolabeled and could be recognized by autoradiography after size separation on an agarose gel containing 3% (wt/vol) NuSieveGTG (FMC Bioproducts), 0.5% (wt/vol) Seakem LE, Trisacetate/EDTA, and ethidium bromide. The identity of each PCR product was confirmed by sequence analysis by the deoxy chain determination method (Sequenase, 2.0, United States Biochemical Corp) after asymmetrical amplification (1:50 dilution of 5' primer). To quantify the amount of cDNA in each sample, a known amount of control RNA was included in the reverse transcription reaction. This control RNA was prepared by in vitro transcription of a synthetic template that had sequences complementary to the two primers used to amplify the cDNA of interest. In addition, the template contained sequences for the bacteriophage T7 promoter on the 5' end for transcription into RNA and a polyadenine tract at the 3' end to facilitate reverse transcription by oligo-dT. The synthetic DNA template also contained internal base-pair sequences in order that the amplification product of the control cDNA synthesized from the template was 72 bp. Because the amplification products of the control cDNA were substantially different in size, they could be separated electrophoretically. Amplification products from both the control DNA and the cDNA of interest were visualized with indirect UV irradiation and cut out from the gel, and radioactivity in each band was determined by Cerenkov counting.

Amplification curves were constructed by removing 10 µL from each reaction mixture in successive cycles (16-30) of amplification and plotting radioactivity in the excised bands against amplification cycles, as shown previously.19 Alternatively, standard curves were obtained by including varying amounts of control RNA and total RNA in each PCR reaction and plotting radioactivity against molecules of control RNA. Amplification curves were performed to ensure that all measurements were obtained during the exponential phase of PCR amplification. Since the efficiency of amplification for each primer was variable, the amount of control RNA in the reverse transcription reaction was adjusted for each primer pair in order that the ratios of control RNA and 1 µg of total cardiac RNA would result in colinearity of the sample RNA and control RNA products in both the amplification and standard curves. Colinearity was observed over several orders of magnitude, and nonspecific amplification was not observed for any of the primer pairs presented in this report. Therefore, the amount of mRNA in the sample from ventricular myocardium could be assessed in relation to the amount of product from the control RNA. The differences between groups were assessed by means of the Student's t test.

Statistical Analysis
Data were analyzed by paired t test analysis or the Wilcoxon signed rank order test, as appropriate.23 Data from individual patients (Figs 1ADown, 2ADown, and 3ADown) represent the mean±SEM of triplicate determinations. Differences were considered significant if the null hypothesis could be rejected at the .05 probability level.



View larger version (22K):
[in this window]
[in a new window]
  		Figure 1. Bar graphs showing effect of transplantation on basal and GTP{gamma}S-stimulated adenylate cyclase activity. Homogenates from endomyocardial biopsies obtained at the indicated time intervals were prepared and adenylate cyclase activity under control conditions or in the presence of 10-4 mol/L GTP{gamma}S determined as described in "Materials and Methods." Data are plotted as picomoles cAMP generated per milligram protein per minute at the indicated intervals and represent the mean±SEM of three replicate determinations from each biopsy sample. A, Time course of a representative patient. B, Mean±SEM of 12 patients similar to the patient in A. Data were pooled into three groups: pretransplantation (Pre-Tx), 1 to 5 months posttransplantation, and 6 to 12 months posttransplantation. indicates total adenylate cyclase activity in the presence of GTP{gamma}S (10-4 mol/L); {square}, basal adenylate cyclase activity; {blacksquare}, stimulated activity (total adenylate cyclase activity [in the presence of GTP{gamma}S] minus basal activity). Data for the Wilcoxon signed rank order analysis were obtained for the 1- to 5-month posttransplantation interval by using the value for each patient that was closest to 6 months; for the 6- to 12-month interval, the value for each patient that was closest to 12 months was used. *P<.05 vs Pre-Tx.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 2. Bar graphs showing effect of cardiac transplantation on Mn+2+forskolin–stimulated adenylate cyclase activity. Homogenates from endomyocardial biopsies obtained at the indicated time intervals were prepared and adenylate cyclase activity determined as described in "Materials and Methods" in the presence of Mn+2 (10-3 mol/L) plus forskolin (7x10-5 mol/L). Data are plotted as in Fig 1Up. A, Time course of the effect of Mn+2+forskolin in the representative patient described in Fig 1AUp is presented as the mean±SEM of three replicate determinations from each biopsy sample. B, Mean±SEM of 8 patients similar to the patient in A is shown; data were pooled as in Fig 1AUp. {square} indicates total adenylate cyclase activity in the presence of Mn+2+forskolin; {blacksquare}, stimulated activity (total adenylate cyclase activity [in the presence of Mn+2+forskolin] minus basal activity). Data for the Wilcoxon signed rank order analysis were obtained as described in Fig 1Up. *P<.05 vs pretransplantation (Pre-Tx).



View larger version (16K):
[in this window]
[in a new window]
  		Figure 3. Bar graphs showing effect of cardiac transplantation on Mn+2-stimulated adenylate cyclase activity. Homogenates of endomyocardial biopsies obtained at the indicated time intervals were prepared and adenylate cyclase activity in the presence of 10-3 mol/L Mn+2 determined as described in "Materials and Methods." Data are plotted as in Fig 2Up. A, Time course of the effect of cardiac transplantation on Mn+2 stimulation of adenylate cyclase activity in the representative patient described in Figs 1AUp and 2AUp, presented as the mean±SEM of three replicate determinations for each biopsy sample. B, Mean±SEM of adenylate cyclase activity in the presence of Mn+2 of the same 8 patients described in Fig 2BUp. Data were pooled as described previously. {square} indicates total adenylate cyclase activity in the presence of Mn+2; {blacksquare}, stimulated activity (total adenylate cyclase activity [in the presence of Mn+2] minus basal activity). Data for the Wilcoxon signed rank order analysis were obtained as described in Fig 1Up. *P<.05 vs pretransplantation (Pre-Tx).


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
To determine the effect of cardiac transplantation on adenylate cyclase activity, we compared the ability of GTP{gamma}S, Mn+2+forskolin, and Mn+2 to stimulate adenylate cyclase activity in homogenates of right ventricular endomyocardial biopsies taken from the transplanted heart just before implantation of the cardiac allograft and in serial biopsies obtained at monthly intervals after transplantation.

Effects of Transplantation on Basal Adenylate Cyclase Activity
Initially we studied the effect of transplantation on basal adenylate cyclase activity. Data from a representative patient (Fig 1AUp) demonstrated that basal adenylate cyclase activity decreased during the 8 months after cardiac transplantation to 5±2% of its pretransplantation levels. This decrease in basal activity over time was representative of 12 patients studied in triplicate. Data summarized in Fig 1BUp represent results from 12 patients grouped into three separate time intervals: pretransplantation, early posttransplantation (1 to 5 months), and late posttransplantation (6 to 12 months). Analysis of these data with the Wilcoxon signed rank order test demonstrated that basal adenylate cyclase activity decreased by 55% in the early posttransplantation period (P<.05 compared with pretransplantation levels) and by 67% in the late posttransplantation period (P<.05 compared with pretransplantation levels).

Effect of Transplantation on GTP{gamma}S-Stimulated Adenylate Cyclase Activity
The effect of cardiac transplantation on the ability of GTP{gamma}S to stimulate adenylate cyclase activity is demonstrated in data from the representative patient summarized in Fig 1AUp. As a result of the finding that basal adenylate cyclase activity decreased after cardiac transplantation, total adenylate cyclase activity in the presence of GTP{gamma}S and GTP{gamma}S-stimulated adenylate cyclase activity above basal levels were compared. Both decreased during the 8 months after transplantation to 3±3% and 2±3% of the pretransplantation levels, respectively. This decrease in GTP{gamma}S-stimulated activity was typical of 12 patients studied in triplicate. Analysis of the data for 12 patients summarized in Fig 1BUp by means of the Wilcoxon signed rank order test demonstrated that in the early (1 to 5 months) posttransplantation period, total adenylate cyclase activity in the presence of GTP{gamma}S and GTP{gamma}S-stimulated adenylate cyclase activity above basal levels decreased by 59% and 67%, respectively, compared with matched pretransplantation biopsies (P<.05; Fig 1BUp). In the late (6 to 12 months) posttransplantation period, total adenylate cyclase activity in the presence of GTP{gamma}S and GTP{gamma}S-stimulated adenylate cyclase activity above basal levels decreased by 71% and 78%, respectively, compared with matched pretransplantation biopsies (P<.05; Fig 1BUp).

Effect of Cardiac Transplantation on Mn+2+Forskolin–Stimulated Adenylate Cyclase Activity
To further evaluate changes in adenylate cyclase activity, endomyocardial biopsies were assayed in the presence of Mn+2 (1 mmol/L) plus forskolin (70 µmol/L). In a representative experiment from a single patient, both total adenylate cyclase activity in the presence of Mn+2+forskolin and Mn+2+forskolin–stimulated adenylate cyclase activity over basal levels decreased during the 8 months after transplantation to 3±3% and 3±2%, respectively, compared with values seen in pretransplant homogenates (Fig 2AUp). These data are typical of findings in 8 other patients studied in triplicate. The Mn+2+forskolin–stimulated adenylate cyclase activity in 8 patients is summarized in Fig 2BUp. Wilcoxon signed rank order analysis demonstrated that in the early posttransplantation period, total adenylate cyclase activity in the presence of Mn+2+forskolin and Mn+2+forskolin–stimulated adenylate cyclase activity above basal levels were decreased by 41% and 40%, respectively, compared with matched pretransplantation controls (P<.05; Fig 2BUp) and were decreased in the late posttransplantation period by 79% and 80%, respectively, compared with matched pretransplantation controls (P<.05; Fig 2BUp). Because the magnitude of forskolin stimulation of adenylate cyclase activity is dependent on Gs{alpha},24 these data do not distinguish between a decrease in adenylate cyclase activity due to a change in levels or function of G-proteins after cardiac transplantation or a change in the catalytic subunit of adenylate cyclase after cardiac transplantation.

Effect of Cardiac Transplantation on Mn+2-Stimulated Adenylate Cyclase Activity
Since Mn+2 acts directly on the catalytic subunit of adenylate cyclase,14 we studied the effect of cardiac transplantation on Mn+2-stimulated adenylate cyclase activity. In the representative patient shown in Fig 3AUp, total adenylate cyclase activity in the presence of Mn+2 and Mn+2-stimulated adenylate cyclase activity above basal levels decreased during the 8 months after cardiac transplantation to 3±3% and 3±2% of pretransplantation values, respectively. This change was typical of 8 other patients. In 8 subjects studied early posttransplantation, total adenylate cyclase activity in the presence of Mn+2 and Mn+2-stimulated adenylate cyclase activity over basal levels analyzed by Wilcoxon signed rank order analysis decreased by 34% and 15%, respectively, compared with matched pretransplantation controls (P<.05; Fig 3BUp). In the late posttransplantation period, total Mn+2-stimulated and Mn+2-stimulated adenylate cyclase activity above basal levels decreased by 77% and 83%, respectively, compared with matched pretransplantation controls (P<.05; Fig 3BUp). These data suggest a possible abnormality in the function of the catalytic subunit of adenylate cyclase in human ventricular myocardium after cardiac transplantation.

Decrease in the Levels of Gs{alpha} After Cardiac Transplantation
One possible mechanism for the decrease in GTP{gamma}S-stimulated adenylate cyclase activity in transplanted heart is that denervation and transplantation may alter the levels of G-protein {alpha}-subunits. To test this hypothesis, the relative levels of Gs{alpha} were determined in biopsies taken at the time of transplantation and at various times after transplantation by Western blot analysis with the use of a specific antibody directed against the {alpha}-subunit of Gs{alpha}.18 This antibody consistently identified a single band that migrated with an apparent molecular weight of 52 kD (Fig 4ADown). In the experiment shown in Fig 4ADown, relative levels of Gs{alpha} decreased markedly compared with control between 4 and 6 months after transplantation. The mean values of the relative levels of Gs{alpha} as measured by the 52-kD band in 10 patients similar to that shown in Fig 4ADown are summarized in Fig 4BDown. Relative levels were unchanged from those of controls until 3 months posttransplantation but decreased to 36±12% of the pretransplantation level by 6 months (paired t test; P<.001).



View larger version (26K):
[in this window]
[in a new window]
  		Figure 4. Effect of cardiac transplantation on relative levels of Gs{alpha}. Equal quantities of tissue from endomyocardial biopsies obtained at the indicated intervals were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, electrotransferred onto nitrocellulose, and incubated with specific antibody to Gs{alpha} followed by exposure to 125I-protein A, as described in "Materials and Methods." Blots were dried, autoradiographed, scanned by an LKB densitometer, and analyzed as described in "Materials and Methods." A, Results of a typical experiment. B, Graph showing mean of 10 experiments similar to that in A. The intensity of the 52-kD bands in 10 patients similar to that shown in A was determined at the indicated intervals and data normalized to levels of the 52-kD band in the pretransplantation (Pre-Tx) biopsy. Data are plotted as the mean±SEM percentage of the Pre-Tx density. Biopsy data were pooled for values at 1 month, 2 to 3 months, 4 to 5 months, and 6 to 7 months after cardiac transplantation. *P<.001 vs Pre-Tx.

Lack of Change in the Level of Pertussis Toxin Substrate After Transplantation
To determine whether cardiac transplantation is associated with changes in relative levels of Gi{alpha}, pertussis toxin–catalyzed ADP-ribosylation in the presence of [32P]NAD+ and 1% cholate (to unmask any membrane-associated pertussis toxin substrates) was performed. Pertussis toxin–catalyzed ADP-ribosylation demonstrated a single band that migrated at an apparent molecular weight of 40 kD on SDS–polyacrylamide gel electrophoresis (Fig 5Down). There was no significant change in the relative level of pertussis toxin ADP-ribosylated substrate in biopsies obtained 1 to 6 months after cardiac transplantation compared with matched control biopsy samples (posttransplantation levels were 95±10% of pretransplantation levels; n=4; P=NS).



View larger version (59K):
[in this window]
[in a new window]
  		Figure 5. Effect of cardiac transplantation on levels of pertussis toxin (PT) substrate. Human heart biopsies were homogenized and equal amounts of protein ADP-ribosylated in the presence of [32P]NAD+, PT, and 1% cholate, as described in "Materials and Methods." Samples were subjected to polyacrylamide gel electrophoresis followed by autoradiography. This experiment is typical of four similar experiments. The upper band represents creatine phosphokinase, which is added to the reaction mixture as part of an ATP-regenerating system and is often ribosylated nonspecifically in these studies. Pre indicates pretransplantation; Post-Tx, posttransplantation.

Change in the Steady-State Level of Gs{alpha} mRNA After Transplantation
To determine whether the decrease in Gs{alpha} protein levels after cardiac transplantation was reflected in a change in the steady-state level of mRNA encoding Gs{alpha}, six matched pairs of endomyocardial biopsy samples obtained before transplantation and late after transplantation (12±1 months posttransplantation) were analyzed by quantitative PCR (TableDown, Fig 6Down). Absolute levels of Gs{alpha} mRNA were decreased by 50±10% compared with pretransplantation control samples (P<.01; n=6). When normalized to ß-myosin heavy chain mRNA levels, there was also a significant decrease in the steady-state levels of Gs{alpha} mRNA (P<.01; n=6). Preliminary data suggest that there was no change in the level of Gi-3{alpha} mRNA levels after cardiac transplantation. This was consistent with the observed lack of change in Gi{alpha} protein level determined by pertussis toxin–catalyzed ADP-ribosylation (data not shown). Finally, no change in the expression of mRNA encoding the cardiac contractile proteins, phospholambam, and sarcoplasmic reticulum Ca2+-ATPase was demonstrated, supporting the specificity of the change in Gs{alpha} mRNA expression.


View this table:
[in this window]
[in a new window]
  		Table 1. Changes in mRNA Expression After Orthotopic Heart Transplantation1



View larger version (10K):
[in this window]
[in a new window]
  		Figure 6. Plot of the quantitative assessment of the {alpha}-subunit of the stimulatory guanine nucleotide–binding protein (Gs{alpha}), demonstrating mRNA measurements and representative samples of RNA from the right ventricular myocardium of a patient before and after orthotopic heart transplantation. The closed symbols indicate the radioactivity in the amplification product from the RNA standard ({blacksquare}, pretransplant control mRNA; {bullet}, posttransplant control mRNA). The open symbols indicate the radioactivity in the Gs{alpha} amplification product from total RNA ({square}, pretransplant Gs{alpha} mRNA; {circ}, posttransplant Gs{alpha} mRNA).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The major findings of this study are that >=6 months after successful orthotopic heart transplantation in humans, (1) basal and guanine nucleotide–stimulated adenylate cyclase activity, (2) the expression of Gs{alpha} but not Gi{alpha} protein, and (3) the steady-state level of Gs{alpha} but not Gi{alpha} mRNA are markedly reduced in the myocardium. We previously found, using unmatched samples obtained from patients 1 to 21 months after transplantation, that guanine nucleotide–stimulated adenylate cyclase activity was blunted, although there was no apparent alteration in basal activity.10 The goal of the current study was to elucidate the mechanism for these effects by directly examining the protein levels of Gs{alpha} and Gi{alpha} and their respective mRNAs.

An important aspect of the present study is that biopsies were taken from the donor heart just before transplantation and at frequent intervals after transplantation. This permitted each posttransplantation measurement to be normalized to its own pretransplantation "control" value. Since basal and stimulated adenylate cyclase activities showed substantial patient-to-patient variability, this "paired-control" approach proved to be important in allowing us to detect changes between the pretransplantation and posttransplantation periods.

The major finding of this study is that expression of the 52-kD form of Gs{alpha} can be regulated in human myocardium. There is prior evidence that Gs{alpha} levels in the heart may be regulated in animal models of pathological states. In canine pressure-overload hypertrophy, Chen et al25 found 30% decreases in both Gs{alpha} protein and mRNA associated with decreased basal, GTP, and GppNHp-stimulated adenylate cyclase activity. Conversely, in myopathic Syrian hamsters, Gs{alpha} functional activity determined by the cyc- complementation assay was decreased, although Gs{alpha} levels determined by Western blot analysis were unchanged.26 Our finding that the steady-state level of Gs{alpha} mRNA is reduced after cardiac transplantation in humans suggests that control of Gs{alpha} protein expression in this situation occurs, at least in part, at the level of gene expression. It is unlikely that the changes in Gs{alpha} mRNA are attributable to the use of quantitative PCR for measuring mRNA levels in endomyocardial biopsy samples since this technique has been shown to be sensitive and reproducible.21 Furthermore, recent studies have shown a high correlation between mRNA levels determined by quantitative PCR, Northern blot analysis, and RNase protection techniques.27 To our knowledge, this is the first demonstration that cardiac Gs{alpha} can be regulated at the mRNA level in humans.

The decrease in adenylate cyclase response to GTP{gamma}S appears to precede the decrease in Gs{alpha} protein levels. It would be of interest to determine whether the early decrease in GTP{gamma}S-stimulated adenylate cyclase activity and late decrease in Gs{alpha} levels correlate temporally with decreases in the functional activity of Gs{alpha}. Unfortunately, attempts to assess Gs activity by performing reconstitution studies with the adenylate cyclase of S49 lymphoma cell cyc- mutants were not successful because of the limited amount of protein available from the biopsies.

A second major finding of this study is that the activity of the catalytic unit of adenylate cyclase is decreased after cardiac transplantation in humans. Although the Mn+2+forskolin response depends on the presence of both Gs{alpha} and the catalytic subunit of adenylate cyclase, Mn+2 exerts a more direct effect on the catalytic subunit. Thus, the early decrease in adenylate cyclase activity, which precedes the decrease in Gs{alpha} protein levels, could be due to changes in the activity of the catalytic subunit. Recent studies have suggested that the activity of the catalytic subunit may be regulated or altered by changes in the isoform of the subunit expressed. It has been demonstrated that the catalytic subunit of adenylate cyclase exists in at least six isoforms.28 29 30 Also, different isoforms of adenylate cyclase are expressed at different stages of development in Dictyostelium.31 Furthermore, since various isoforms of the catalytic subunit of adenylate cyclase demonstrate different rates of cAMP synthesis and differential regulation of the ß-subunit of Gs{alpha}, the expression of alternative isoforms of the catalytic subunit of adenylate cyclase after cardiac transplantation could potentially also account for differences in basal and stimulated activity. Little data on the regulated expression of specific isoforms of the catalytic subunit of adenylate cyclase activity have appeared. However, most recently downregulation of mRNA levels of two distinct forms of adenylylcyclase, types V and VI, has been recently reported in failing mammalian hearts.32

Only a few studies have appeared that support the conclusion that alterations in the catalytic subunit of adenylate cyclase may be associated with pathological states of the heart. In hearts with hypertrophy due to aortic banding, Chen et al25 found that forskolin-stimulated adenylate cyclase activity decreased only after progression to overt heart failure, whereas Gs{alpha} mRNA levels were decreased in the hypertrophy stage that preceded failure. Recently Bristow et al33 demonstrated a decrease in Mn+2-stimulated adenylate cyclase activity in left ventricular myocardium from patients with idiopathic dilated cardiomyopathy and right ventricular myocardium from patients with primary pulmonary hypertension.

Several mechanisms could account for the observed decrease in Gs{alpha} expression in the transplanted hearts. Studies have suggested that innervation of the heart or neuronal input to the heart may regulate G-protein levels and function as well as calcium channel number.34 35 Ogawa et al35 demonstrated that coculture of neonatal rat ventricular myocytes with sympathetic ganglia resulted in an increase in the number of calcium channels in the ventricular cells. In idiopathic dilated cardiomyopathy, a condition in which sympathetic stimulation of the heart is increased, levels of pertussis toxin substrates increased 30% to 40%,36 with an associated decrease in both basal and GTP{gamma}S-stimulated adenylate cyclase activity. Hence, denervation of the transplanted heart could account for the decreased level of the 52-kD form of Gs{alpha}. However, the role of denervation or alterations in neurohormonal stimuli in mediating these changes should be tempered by recent data suggesting that after intracoronary tyramine infusion, increases in coronary sinus levels of norepinephrine are observed from the transplanted human heart, implying that limited sympathetic reinnervation may occur late after cardiac transplantation in some patients.37 38 Furthermore, any hypothesis regarding the role of innervation in regulating G-protein levels and adenylate cyclase activity must also take into account the possibility that corticosteroids or immunosuppressive agents, which are included in the regimens of all these patients, may affect the expression of Gs{alpha} or the catalytic subunit of adenylate cyclase.

The physiological consequence of the decreased adenylate cyclase activity described here after denervation and transplantation of the heart is unclear. After cardiac transplantation in humans, although cardiac transplant recipients may be able to perform most activities without limitation, the maximal exercise oxygen consumption that they are able to achieve is chronically impaired.39 Although no data exist in humans with regard to contractile reserve in these patients, their chronotropic response to exercise is impaired, with reductions in both the rate of increase and maximum achieved heart rates.40 41 This attenuated heart rate response has been attributed to the loss of direct sympathetic innervation of the sinoatrial node, a view supported by the observation that the heart rate response to exogenous ß-adrenergic agonists is normal or even increased.42 43 The basis for the discrepancy between reduced adenylate cyclase responses reported here and the apparently preserved heart rate response of the transplanted heart is unclear.

Orthotopic cardiac transplantation is an important therapeutic modality for the treatment of end-stage congestive heart failure in humans. Our data demonstrate that profound changes in adenylate cyclase activity and the levels of Gs{alpha} protein and mRNA occur in human ventricular myocardium after successful cardiac transplantation. The relation of these changes to alterations in autonomic balance and the physiological responses of the transplanted heart remain to be established.


*    Acknowledgments
 
This study was supported by National Institutes of Health (NIH) grants HL-36014 and HL-42539; HL-39719 (Dr Feldman); and HL-37404, HL-38070, and HL-45332 (Dr Vatner). Dr Loh is the recipient of a Physician Scientist Award (K11-HL-02514) from the NIH. Dr Feldman is an Established Investigator of the American Heart Association and was supported by a grant from the W.W. Smith Charitable Trust. Dr Colucci is an Established Investigator of the American Heart Association. We would like to thank Drs Thomas W. Smith and Eva J. Neer for their insightful comments, the staff of the Cardiac Catheterization Laboratory for their help and patience, Paula McColgan, and Georgina Roman for expert typing.

Received September 19, 1994; accepted February 13, 1995.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 
1. Feldman AM. Experimental issues in assessment of G-protein function in cardiac diseases. Circulation. 1991;84:1852-1861. [Free Full Text]

2. Gilman AG. G proteins and dual control of adenylate cyclase. Cell. 1984;36:577-579. [Medline] [Order article via Infotrieve]

3. Neer RJ, Clapham DE. Role of G-protein subunits in transmembrane signalling. Nature. 1988;333:129-134. [Medline] [Order article via Infotrieve]

4. Haigh LS, Leatherman GF, O'Hara DS, Smith TW, Galper JB. Effect of low density lipoproteins and mevinolin on cholesterol content and muscarinic responsiveness in cultured chick atrial cells: regulation of levels of muscarinic receptors and guanine nucleotide regulatory proteins. J Biol Chem. 1988;263:15608-15618. [Abstract/Free Full Text]

5. Barnett JV, Haigh LS, Marsh JD, Galper JB. Effects of low density chick arterial cells: regulation of ß-adrenergic receptors on {alpha}s. J Biol Chem. 1989;264:10779-10786. [Abstract/Free Full Text]

6. Feldman AM, Cates AE, Veazey WB, Hershberger RE, Bristow MR, Baughman KL, Baumgartner WA, Van Dop C. Increase of the 40,000-mol wt pertussis toxin substrate (G protein) in the failing human heart. J Clin Invest. 1988;82:189-197.

7. Feldman AM, Ray PE, Bristow MR. Expression of {alpha} subunits of G-proteins in failing human heart: a reappraisal of utilizing quantitative polymerase chain reaction. J Mol Cell Cardiol. 1991;23:1355-1358. [Medline] [Order article via Infotrieve]

8. Lurie KG, Bristow MR, Reitz B. Increased ß-adrenergic receptor density in an experimental model of cardiac transplantation. J Thorac Cardiovasc Surg. 1983;86:195-201. [Abstract]

9. Vatner DE, Lavallee M, Amano J, Finizola A, Homcy CJ, Vatner SF. Mechanisms of supersensitivity to sympathomimetic amines in the chronically denervated heart of the conscious dog. Circ Res. 1985;57:55-64. [Abstract/Free Full Text]

10. Denniss AR, Marsh JD, Quigg RJ, Gordon JB, Colucci WS. ß-Adrenergic receptor number and adenylate cyclase function in denervated transplanted and cardiomyopathic human hearts. Circulation. 1989;79:1028-1034. [Abstract/Free Full Text]

11. Billingham ME. Endomyocardial biopsy diagnosis of acute rejection in cardiac allografts. Prog Cardiovasc Dis. 1990;33:1-8.

12. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275. [Free Full Text]

13. Salomon Y. Adenylate cyclase assay. Adv Cyclic Nucl Res. 1979;10:35-55. [Medline] [Order article via Infotrieve]

14. Neer EJ. Interaction of soluble brain adenylate cyclase with manganese. J Biol Chem. 1978;253:1498-1502. [Abstract/Free Full Text]

15. Barnett JV, Shamah SV, Lassegue B, Griendling KK, Galper JB. Muscarinic cholinergic stimulation of inositol phosphate production in cultured embryonic chick arterial cells: evidence of a role of two guanine nucleotide-binding proteins. Biochem J. 1990;271:437-442. [Medline] [Order article via Infotrieve]

16. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. Proc Natl Acad Sci U S A. 1979;76:4350-4354. [Abstract/Free Full Text]

17. Laemmli UK. Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature. 1980;227:680-685.

18. Mumby SM, Kahn RA, Manning DR, Gilman A. Antisera of designed specificity for subunits of guanine nucleotide-binding regulatory proteins. Proc Natl Acad Sci U S A. 1986;83:265-269. [Abstract/Free Full Text]

19. Feldman AM, Ray PE, Silan CM, Mercer JA, Minobe W, Bristow MR. Selective gene expression in failing human heart: quantification of steady-state levels of messenger RNA in endomyocardial biopsies using the polymerase chain reaction. Circulation. 1991;83:1866-1872. [Abstract/Free Full Text]

20. Ladenson PW, Sherman SI, Baughman KL, Ray PE, Feldman AM. Reversible alterations in myocardial gene expression in a young man with dilated cardiomyopathy and hypothyroidism. Proc Natl Acad Sci U S A. 1992;89:5251-5255. [Abstract/Free Full Text]

21. Feldman AM, Weinberg EO, Ray PE, Lorell BH. Selective changes in cardiac gene expression during compensated hypertrophy and the transition to cardiac decompensation in rats with chronic aortic banding. Circ Res. 1993;73:184-192. [Abstract]

22. Chomczynski P, Sacchi N. Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159. [Medline] [Order article via Infotrieve]

23. Colton T, ed. Statistics in Medicine. Boston, Mass: Little, Brown & Co; 1974:219-221.

24. Seamon KB, Daly JW. Forskolin: its biological and chemical properties. In: Greengard P, Robison GA, eds. Advances in Cyclic Nucleotide and Protein Phosphorylation Research. New York, NY: Raven Press Publishers; 1986:1-150.

25. Chen L, Vatner DE, Vatner SF, Huttinger L, Homcy C. Decreased Gs mRNA levels accompany the fall in Gs and adenylate cyclase activities in compensated left ventricular hypertrophy. J Clin Invest. 1991;87:293-298.

26. Kessler PD, Cates AE, Van Dop C, Feldman AM. Decreased bioactivity of the guanine nucleotide-binding protein that stimulates adenylate cyclase in hearts from cardiomyopathic Syrian hamsters. J Clin Invest. 1989;84:244-252.

27. Bristow MR, Minobe WA, Port JD, Ray PE, Feldman AM. Decreased ß1-adrenergic receptor levels in the failing human heart. J Clin Invest. 1994;92:2737-2745.

28. Tang WJ, Krupinski J, Gilman AG. Expression and characterization of calmodulin-activated (type I) adenylyl cyclase. J Biol Chem. 1991;266:8595-8603. [Abstract/Free Full Text]

29. Tang WJ, Gilman AG. Type-specific regulation of adenylate cyclase by G-protein ß{gamma} subunits. Science. 1991;254:1500-1503. [Abstract/Free Full Text]

30. Bakalyar HA, Reed RR. Identification of a specialized adenylyl cyclase that may mediate odorant detection. Science. 1990;250:1403-1406. [Abstract/Free Full Text]

31. Pitt GS, Milona N, Borleis K, Lin KC, Reed RR, Devreotes PN. Structurally distinct and stage-specific adenylyl cyclase genes play different roles in Dictyostelium development. Cell. 1992;69:305-315. [Medline] [Order article via Infotrieve]

32. Ishikawa Y, Sorota S, Kiuchi K, Shannon RP, Komamura K, Katsushika S, Vatner DE, Vatner SF, Homcy CJ. Down regulation of adenylylcyclase types V and VI mRNA levels in pacing-induced heart failure in dogs. J Clin Invest. 1994;93:2224-2229.

33. Bristow MR, Minobe W, Rasmussen R, Laurabbee P, Skirl L, Klein JW, Anderson FL, Murray J, Mestroni L, Karwande SW, Fowler M, Ginsburg R. ß-Adrenergic neuroeffector abnormalities in the failing human heart are produced by local rather than systemic mechanisms. J Clin Invest. 1989;89:803-815.

34. Barnett JV, Taniuchi M, Yang M, Galper JB. Co-culture of embryonic chick heart cells and ciliary ganglia produces parasympathetic responsiveness in embryonic chick heart cells. Biochem J. 1993;292(pt 2):395-399.

35. Ogawa S, Barnett JV, Sen L, Galper JB, Smith TW, Marsh JD. Sympathetic innervation in vitro increase L-type calcium channel expression in rat cardiac myocytes. J Clin Invest. 1992;89:1085-1093.

36. Böhm M, Gierschik P, Jakobs K, Pieske B, Schabel P, Ungerer M, Erdmann E. Increase of Gi{alpha} in human hearts with dilated but not ischemic cardiomyopathy. Circulation. 1990;82:1249-1265. [Abstract/Free Full Text]

37. Stark RP, McGinn AL, Wilson RF. Chest pain in cardiac recipients: evidence of reinnervation after cardiac transplantation. N Engl J Med. 1991;3324:1791-1794.

38. Wilson RF, Christensen BV, Olivari MT, Simon A, White CW, Laxson DD. Evidence for structured sympathetic reinnervation after orthotopic cardiac transplantation in humans. Circulation. 1991;83:1210-1220. [Abstract/Free Full Text]

39. Stevenson LW, Sietsema K, Tillisch JH, Lem V, Walden J, Kobashigawa JA, Moriguchi J. Exercise capacity for survivors of cardiac transplantation or sustained medical therapy for stable heart failure. Circulation. 1990;81:78-85. [Abstract/Free Full Text]

40. Savin WM, Haskell SC, Schroeder JS, Stinson EB. Cardiorespiratory responses of cardiac transplant patients to graded symptom-limited exercise. Circulation. 1980;62:55-60. [Abstract/Free Full Text]

41. Pope SE, Stinson EB, Daughters GT, Schroeder JS, Ingels NB, Alderman EL. Exercise response of the denervated heart in long-term cardiac transplant recipients. Am J Cardiol. 1980;96:213-218.

42. Quigg RJ, Rocco MB, Gauthier DF, Creager MA, Hartley LH, Colucci WS. Mechanism of the attenuated peak heart rate response to exercise after orthotopic cardiac transplantation. J Am Coll Cardiol. 1989;14:338-344. [Abstract]

43. Yusuf S, Theodoropoulos S, Mathias CJ, Dhalla N, Wittes J, Mitchell A, Yacoub M. Increased sensitivity of the denervated transplanted human heart to isoprenaline both before and after ß-adrenergic blockade. Circulation. 1987;75:696-704.[Abstract/Free Full Text]




This article has been cited by other articles:


Home page
 J. Immunol.Home page
G. Colombo, S. Gatti, F. Turcatti, A. Sordi, L. R. Fassati, F. Bonino, J. M. Lipton, and A. Catania
Gene Expression Profiling Reveals Multiple Protective Influences of the Peptide {alpha}-Melanocyte-Stimulating Hormone in Experimental Heart Transplantation
J. Immunol., September 1, 2005; 175(5): 3391 - 3401.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Loh, E.
Right arrow Articles by Galper, J. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Loh, E.
Right arrow Articles by Galper, J. B.

Circulation Research Home | Subscriptions | Archives | Feedback | Authors | Help | AHA Journals Home | Search
Copyright © 1995 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited.