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(Circulation. 1995;92:286-289.)
© 1995 American Heart Association, Inc.

Articles

Elevation of Circulating and Ventricular Adrenomedullin in Human Congestive Heart Failure

Michihisa Jougasaki, MD, PhD; Chi-Ming Wei, MD, PhD; Linda J. McKinley, MA; John C. Burnett, Jr, MD

From the Cardiorenal Research Laboratory, Division of Cardiovascular Diseases, Mayo Clinic and Foundation, Rochester, Minn.

	Abstract

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Background Adrenomedullin (ADM) is a newly discovered vasodilating and natriuretic peptide that may play an important role in cardiorenal regulation. Although ADM was originally isolated from human pheochromocytoma, ADM-like immunoreactivity has also been widely detected in various tissues, including the cardiovascular system.

Methods and Results In view of reports that ADM circulates in the body and that ADM gene and ADM-like immunoreactivity are present in the heart, the present study was designed to determine the plasma concentration of ADM in healthy subjects and in patients with congestive heart failure (CHF) and to investigate the immunohistochemical presence and localization of ADM in normal and failing human hearts. Plasma ADM concentration was 13.2±2.3 pg/mL in healthy subjects (n=11) and increased to 47.3±6.7 pg/mL in patients with CHF (n=11, P<.05 versus normal). Human cardiac tissues were obtained from five patients with end-stage CHF undergoing cardiac transplantation. Five normal donor hearts that were used for cardiac transplantation served as sources for normal atrial tissues. Normal ventricular myocardium was also obtained by endomyocardial biopsy from the right ventricles of these donor hearts immediately before cardiac transplantation. Positive immunostaining was detected within the myocardia in both atria and ventricles of healthy and severely failing human transplanted hearts and was more intense in the atria than in the ventricles. Although there were no significant differences in the intensity of immunoreactivity between normal and failing atria, ADM immunoreactivity was significantly more intense in the ventricular myocytes from failing hearts compared with normal hearts.

Conclusions The present study demonstrates that plasma concentration of ADM is increased in patients with CHF and that ADM is present in the human heart. ADM immunoreactivity is markedly increased in the failing human ventricle, suggesting that ventricular ADM expression may be influenced by the circumstances associated with CHF. This supports a potential role for this newly identified vasoactive and natriuretic peptide, ADM, in the neurohumoral activation that characterizes human CHF.

Key Words: adrenomedullin • radioimmunoassay • heart failure

	Introduction

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Adrenomedullin (ADM) is a newly discovered potent endogenous vasodilating peptide originally isolated from extracts of human pheochromocytoma.¹ More recently, ADM production and secretion have been reported in endothelial cells.² In addition, studies have established that ADM is also a potent natriuretic and diuretic peptide.³ Since ADM has been reported to be present in the heart,⁴ this newly characterized peptide could function like atrial natriuretic peptide (ANP) in the control of cardiorenal homeostasis.⁵

Chronic congestive heart failure (CHF) is a pathophysiological state in which activated vasodilating and natriuretic factors are opposed by coactivation of local and circulating vasoconstrictive and sodium-retaining factors. Indeed, investigations have supported a role for neurohumoral activation as important to the progression of ventricular dysfunction.⁶ Recently, ADM has been reported to be present in normal human plasma,⁷ and its plasma concentration has been reported to be increased in hypertension.⁸ To date, the plasma concentration of ADM in patients with CHF remains undefined. Also, although ADM immunoreactivity is detectable in mammalian hearts,^{4 9 10} its presence and localization in the human heart in the presence and absence of CHF also remain undefined. Our hypothesis is that like ANP, ADM, which is natriuretic and vasorelaxing, is increased in the plasma of human subjects with CHF and may have an important role in pathophysiology of CHF. Therefore, the first objective of the present study was to determine the concentration of circulating ADM in humans with CHF. The second objective was to investigate by immunohistochemistry the localization of ADM in the hearts of humans with and without CHF.

	Methods

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Study Subjects for Circulating ADM Concentration
Eleven patients (6 men and 5 women, 66±4 years old [mean±SEM]) with severe CHF and 11 age-matched subjects without cardiac disease (4 men and 7 women, 64±3 years old) were studied. All patients with severe CHF had a complete physical and laboratory evaluation and were classified by New York Heart Association (NYHA) functional class criteria according to their cardiac symptoms after physical examination and laboratory evaluation. Among the 11 patients with CHF studied, 4 were classified as NYHA class III and 7 as NYHA class IV. The ejection fraction determined by echocardiography was 17±2%. The causes of the ventricular dysfunction in these patients with CHF included idiopathic dilated cardiomyopathy and ischemic cardiomyopathy. All patients with CHF were on treatment, which included digitalis, diuretics, and/or vasodilators. Eleven age-matched subjects without cardiac disease were free of ventricular dysfunction. Patients with renal failure were excluded from the present study. Venipuncture for measurement of plasma concentration of ADM was performed with the patient in the supine position.

Quantification of Plasma ADM Concentration
Blood samples for ADM assay were collected in chilled tubes containing EDTA and immediately placed on ice. After centrifugation at 2500 rpm at 4°C for 15 minutes, the plasma was decanted and stored at -20°C until analyzed. Plasma (1 mL) was extracted on C-18 Bond Elute cartridges and eluted with 95% methanol containing 1% TFA. Concentrated eluates were then assayed with a specific and sensitive radioimmunoassay for ADM (Phoenix Pharmaceuticals). Samples and standards were incubated with 100:1 antibody raised against human ADM(1-52) at 4°C for 24 hours. ¹²⁵I-labeled ADM (100 µL) was added and incubated another 24 hours at 4°C. Free and bound fractions were then separated by addition of a second antibody and centrifuged. Radioactivity of the pellet was measured with a gamma counter. Minimal detectable concentration for this assay is 0.5 pg per tube, and the half-maximal inhibition dose of radioiodinated ligand binding by ADM was 10 pg per tube. Recovery was 72±2%, and intra-assay and interassay variations were 10% and 12%, respectively.

Healthy and CHF Subjects for Immunohistochemistry
Human cardiac tissue was obtained from five patients (four men and one woman, 47±2 years old) with end-stage CHF (NYHA class IV) undergoing cardiac transplantation at the Mayo Clinic. Cardiac index and ejection fraction were decreased (1.9±0.2 L · min^-1 · m^-2 and 12±1.2%, respectively), and pulmonary capillary wedge pressure was increased (28.2±3.7 mm Hg). The causes of CHF included idiopathic dilated cardiomyopathy and ischemic cardiomyopathy. Cardiac tissues included both atria and ventricles from these patients. Atrial sections were taken from the appendages and free walls of both atria, and full-thickness sections of human ventricular myocardium were obtained from the middle third of the free wall of both left and right ventricles from failing hearts. Five normal donor hearts that were used for cardiac transplantation at the Mayo Clinic served as the source for normal atrial tissue sections. Normal ventricular myocardium was also obtained by endomyocardial biopsy from the right ventricles of these donor hearts immediately before cardiac transplantation and was processed in an identical manner.

Immunohistochemical Staining
Immunohistochemical studies were performed by the indirect immunoperoxidase method as described previously.^{3 4 11 12} The tissues were immediately fixed with 10% buffered formalin. These tissues were embedded in paraffin, and sections 6 µm thick were cut and mounted on glass slides treated with silica. The slides were incubated at 60°C and deparaffinized with graded concentrations of xylene and ethanol. To block the activity of endogenous peroxidase, the slides were incubated with 0.6% hydrogen peroxide in methanol for 20 minutes at room temperature. After being washed, they were incubated with 5% goat serum (Dako) for 10 minutes at room temperature to reduce nonspecific background staining and were then incubated with rabbit polyclonal anti-ADM antiserum (Peninsula) at a dilution of 1:800 in humidified chambers for 24 hours at room temperature. The antiserum to ADM showed no cross-reactivities with ANP, brain natriuretic peptide (BNP), or C-type natriuretic peptide. All the treated slides were incubated for 30 minutes with second antibody–horseradish peroxidase conjugate (Tago) at a dilution of 1:100. The final reaction was achieved by incubating the sections with freshly prepared reagent containing 3-amino-9-ethylcarbazole (Sigma) dissolved in dimethylformamide and sodium acetate. The sections were counterstained with hematoxylin, mounted, and reviewed with an Olympus microscope. Two trained independent observers reviewed these sections without any knowledge as to the respective groups from which the tissues originated. Especially in the ventricular tissues, endomyocardial regions of the transplanted hearts were compared with the normal ventricular tissues, because the biopsy specimens from the donor heart were endomyocardial tissues. The presence of ADM immunoreactivity was assessed by microscopic examination and evaluated to quantify the degree of staining of ADM (0, no staining of ADM; 0.5, minimal; 1.0, mild density; 1.5, moderate density; and 2.0, maximal density) and percentage of area of positive staining in the entire section examined.

To examine the immunohistochemical specificity of the reaction between antiserum and tissue, absorption tests were performed. The anti-ADM antiserum was preincubated with 10^-6 mol/L of human ADM(1-52) (Phoenix Pharmaceuticals) overnight. After centrifugation at 4500 rpm for 10 minutes, the supernatant was used instead of primary antiserum. The specificity was further confirmed by substitution of nonimmune rabbit serum (Dako) or PBS for primary antiserum.

Statistical Analysis
Values are expressed as mean±SEM. Statistical comparisons between each group were performed by ANOVA for repeated measures followed by Fisher's least-significant-difference test of repeated measures when appropriate, and statistical comparisons between groups were made by factorial ANOVA followed by Fisher's least-significant-difference test of repeated measures. Statistical significance was accepted for P<.05.

	Results

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Circulating ADM Concentration
Plasma ADM concentration was 13.2±2.3 pg/mL in healthy age-matched subjects (n=11). Plasma ADM concentration increased to 47.3±6.7 pg/mL in patients with severe CHF (n=11, P<.05 versus healthy group) (see Fig 1).

View larger version (9K): [in this window] [in a new window]   Figure 1. Graph showing plasma concentrations of adrenomedullin (ADM) in age-matched healthy subjects and in patients with congestive heart failure (CHF). *P<.05 vs between groups.

Immunohistochemistry
Immunohistochemistry for ADM demonstrated unequivocal evidence of ADM positivity within the atrial and ventricular myocytes from both healthy and severely failing hearts. ADM immunoreactivity was observed within the cytoplasm of myocytes and was distributed widely in the peripheral cytoplasm. There were some indications that ADM immunoreactivity was located in the perinuclear region. ADM immunohistochemical scores and percentage distribution (percent positive staining area) are reported in the Table. ADM immunoreactivity was more intense in the atria than in the ventricles from both healthy hearts and severely failing hearts. Although ADM immunoreactivity in the atria was at similar intensities in both healthy hearts and severely failing hearts, it was significantly more intense in the ventricular myocytes from severely failing hearts compared with normal hearts. There was no evidence of immunoperoxidase activity in the endocardium, epicardium, pericardium, or connective tissues. Fig 2 illustrates representative immunohistochemical staining for ADM in the atrial and ventricular myocardia from a healthy and a severely failing human heart. The sections treated with preabsorbed antiserum, nonimmune rabbit serum, or PBS instead of primary antibody as a negative control failed to show immunoperoxidase activity within the myocardia.

View this table: [in this window] [in a new window]   Table 1. ADM Staining Score and Area in Normal (Donor) and CHF (Recipient) Heart and Aorta

View larger version (0K): [in this window] [in a new window]   Figure 2. Immunohistochemical staining for adrenomedullin in atrium and ventricle in human normal and transplanted heart. NRS indicates nonimmune rabbit serum; CHF, congestive heart failure. Original magnification x400.

	Discussion

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ADM is a newly discovered amino acid peptide that has potent vasodilating¹ and natriuretic effects.³ Previous reports have demonstrated that circulating ADM is increased in patients with essential hypertension.⁸ The present study documents for the first time that the plasma concentrations of circulating ADM are increased in patients with CHF compared with those in healthy subjects. We also report for the first time the presence and distribution of ADM immunoreactivity in human atrial and ventricular myocardium and its elevation in failing human ventricular myocardium.

The mechanism of circulating ADM elevation in patients with CHF is unknown. At present, the synthesis, secretion, and metabolism of ADM are not fully understood. There may be an increase in the production and secretion of ADM and/or a decrease in degradation of ADM in patients with CHF. Although the major source of circulating ADM is unclear, previous investigators measured plasma concentration of ADM from various sites in humans without ventricular dysfunction and found that there was no step-up of ADM in the coronary sinus, suggesting that the heart is not the main source of circulating ADM.¹³ This may be in contrast to ANP, which is produced and secreted by the heart and whose concentration in plasma is increased in patients with CHF secondary to enhanced myocardial production.¹⁴ Similarly, Yasue et al¹⁵ reported that the secretion of BNP from the ventricle is increased in proportion to the severity of the ventricular dysfunction. Although there is no evidence that the major source of circulating ADM is the heart, the observation in the present study of increased ventricular ADM immunoreactivity suggests that the failing ventricular myocardium may be characterized by increased production and secretion of ADM, like ANP and BNP. Further studies are necessary to address this issue.

Plasma ADM concentrations were significantly higher in patients with severe CHF than in the healthy subjects without ventricular dysfunction. Since we chose an age-matched group for control subjects, the elevation of plasma ADM in patients with CHF is not considered to be an age effect but rather is secondary to the neurohumoral activation that characterizes human CHF.

Recently, ADM was reported to be present within the myocardium in both atria and ventricles of normal dogs.⁴ To date, no reports have described the immunohistochemical presence and distribution of ADM in the human heart, although ADM mRNA and ADM immunoreactivity by radioimmunoassay have been reported in normal human hearts.^{9 16} The present study demonstrates for the first time by immunohistochemistry that ADM is present in the human heart. Although the immunohistochemical localization of ADM within the heart is consistent with local cardiovascular production, another explanation is that the immunoreactivity may represent binding of endogenous ADM of extracardiovascular origin to the cardiovascular sites of action.

In the present study, we show that ADM immunoreactivity is significantly more intense in the ventricular myocytes from severely failing hearts than in those from normal hearts. As discussed above, the production of ADM, especially in the ventricles, may be increased in patients with CHF. The fact that ADM is increased in the ventricles suggests that ventricular ADM expression may be influenced by the circumstances associated with CHF. It is also known that ventricular production of the natriuretic peptides ANP and BNP secondary to ventricular dilatation and myocyte hypertrophy is markedly augmented in patients with CHF.^{12 15} Like the natriuretic peptides, ventricular expression of ADM may be enhanced in patients with CHF, and ventricular ADM may contribute, at least in part, to the rise in the plasma concentrations of ADM in patients with CHF. The demonstration that ADM mediates its biological actions via generation of cAMP¹⁷ may relate to CHF. It is tempting to speculate that the activation of ADM in CHF could be a compensatory response to enhance myocardial contractility via cAMP. To date, no direct action of ADM on myocardial contractility has been reported. In addition, like the natriuretic peptide in evolving CHF, ADM may also play a compensatory role in the kidney to maintain sodium balance during the early stage of ventricular dysfunction in the maintenance of optimal intravascular volume and cardiac filling pressures despite ventricular dysfunction.¹⁸ Furthermore, on the basis of its known cardiorenal actions, one could also speculate on a role for exogenous ADM as a therapeutic agent to improve left ventricular function by reducing both preload and afterload in patients with CHF.

In the present study, normal ventricular myocardium was obtained by endomyocardial biopsy from the right ventricles of donor hearts immediately before cardiac transplantation. In contrast, whole ventricular tissues of the transplanted hearts were used for the immunohistochemistry. Since the biopsy specimens were endomyocardial tissues, we compared these biopsy specimens with the endomyocardial region of the transplanted ventricles. However, the difference in immunoreactivity may be related to the different methods used to obtain tissue rather than a true pathophysiological difference. The difference in obtaining ventricular tissues between normal and failing heart is therefore a limitation of the present study.

In summary, the present study demonstrates that circulating ADM is increased in patients with CHF, that ADM is present in the human heart, and that its immunoreactivity is markedly increased in the failing human ventricle, suggesting that ventricular ADM expression may be influenced by the circumstances associated with CHF. This supports a potential role for this newly identified vasoactive and natriuretic peptide in the neurohumoral activation that characterizes human CHF.

	Acknowledgments

 
This work was supported by National Heart, Lung, and Blood Institute grants HL-36634 and HL-03174-01, the American Heart Association, Minnesota Affiliate (MN-94-GB-20), and the Mayo Foundation. We acknowledge the technical assistance of Denise M. Heublein and Sharon M. Sandberg and the collaboration of Dr Christopher G.A. MacGregor.

	Footnotes

 
Reprint requests to Michihisa Jougasaki, MD, PhD, Cardiorenal Research Laboratory, Mayo Clinic and Foundation, 915 Guggenheim, 200 First St, SW, Rochester, MN 55905. E-mail jburnett@mayo.edu.

Received April 4, 1995; revision received May 11, 1995; accepted June 4, 1995.

	References

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				C-M Yu, B M Y Cheung, R Leung, Q Wang, W-H Lai, and C-P Lau Increase in plasma adrenomedullin in patients with heart failure characterised by diastolic dysfunction Heart, August 1, 2001; 86(2): 155 - 160. [Abstract] [Full Text] [PDF]

				D. J Autelitano, R. Ridings, and F. Tang Adrenomedullin is a regulated modulator of neonatal cardiomyocyte hypertrophy in vitro Cardiovasc Res, August 1, 2001; 51(2): 255 - 264. [Abstract] [Full Text] [PDF]

				A. M. Richards, R. Doughty, M. G. Nicholls, S. MacMahon, N. Sharpe, J. Murphy, E. A. Espiner, C. Frampton, T. G. Yandle, and for the Australia-New Zealand Heart Failure Group Plasma N-terminal pro-brain natriuretic peptide and adrenomedullin: Prognostic utility and prediction of benefit from carvedilol in chronic ischemic left ventricular dysfunction J. Am. Coll. Cardiol., June 1, 2001; 37(7): 1781 - 1787. [Abstract] [Full Text] [PDF]

				M. Jougasaki and J. C Burnett Jr. Continuing insights into the heart as an endocrine organ: adrenomedullin and cardiac fibroblasts Cardiovasc Res, March 1, 2001; 49(4): 695 - 696. [Full Text] [PDF]

				Y. Tomoda, K. Kikumoto, Y. Isumi, T. Katafuchi, A. Tanaka, K. Kangawa, K. Dohi, and N. Minamino Cardiac fibroblasts are major production and target cells of adrenomedullin in the heart in vitro Cardiovasc Res, March 1, 2001; 49(4): 721 - 730. [Abstract] [Full Text] [PDF]

				H. Romppanen, J. Puhakka, G. Foldes, I. Szokodi, O. Vuolteenaho, H. Tokola, M. Toth, and H. Ruskoaho Endothelin-1-Independent and Angiotensin II-Independent Induction of Adrenomedullin Gene Expression Hypertension, January 1, 2001; 37(1): 84 - 90. [Abstract] [Full Text] [PDF]

				K. Terata, H. Miura, Y. Liu, F. Loberiza, and D. D. Gutterman Human coronary arteriolar dilation to adrenomedullin: role of nitric oxide and K+ channels Am J Physiol Heart Circ Physiol, December 1, 2000; 279(6): H2620 - H2626. [Abstract] [Full Text] [PDF]

				R. W. Troughton, L. K. Lewis, T. G. Yandle, A. M. Richards, and M. G. Nicholls Hemodynamic, Hormone, and Urinary Effects of Adrenomedullin Infusion in Essential Hypertension Hypertension, October 1, 2000; 36(4): 588 - 593. [Abstract] [Full Text] [PDF]

				J. G. Lainchbury, D. M. Meyer, M. Jougasaki, J. C. Burnett Jr., and M. M. Redfield Effects of adrenomedullin on load and myocardial performance in normal and heart-failure dogs Am J Physiol Heart Circ Physiol, September 1, 2000; 279(3): H1000 - H1006. [Abstract] [Full Text] [PDF]

F. Piquard, A. Charloux, B. Mettauer, E. Epailly, E. Lonsdorfer, S. Popescu, J. Lonsdorfer, and B. Geny Exercise-Induced Increase in Circulating Adrenomedullin Is Related to Mean Blood Pressure in Heart Transplant Recipients J. Clin. Endocrinol. Metab., August 1, 2000; 85(8): 2828 - 2831. [Abstract]