Molecular mechanism of contractile dysfunction in cardiac allograft rejection

JOURNAL

OF SURGICAL

52, 472-475 (19%)

RESEARCH

Molecular Mechanism of Contractile Dysfunction in Cardiac Allograft Rejection ABIGAIL K. HANNA, Departments Presented

M.D., MICHAEL LOUIE, B.A., JEAN MILLER, B.A., ALEC HIRSCH, B.A., BRUCE T. LIANG, M.D., AND VERDI J. DISESA, M.D.

of Medicine and Surgery, School

at the Annual

Meeting

of Medicine,

of the Association

University

for Academic

Day4 Day5 Day6

Isoproterenol

Isograft

Allograft

Isograft

535?34 500+25 752 + 56

363?65 325? 37 333 * 70

138& ss* 147+

Allograft 18 4 16

91? so* 92*

n 9 12 14

7 4 7

(% increase in CAMP in response to forskolin or isoproterenol + standard error. All results P < 0.03 except Day 4 forskolin and Day 5 isoproterenol.) No significant difference was noted between isografts and allografts stimulated with carbachol and R-PIA. These data suggest that a primary alteration in adenylyl cyclase activity may be a component of the molecular basis of reversible contractile dysfunction in cardiac allograft rejection. 0 1992 Academic Press, Inc.

INTRODUCTION Acute allograft rejection is a common occurrence in patients who undergo cardiac transplantation. Rejection may be complicated by deterioration in cardiac contractile performance that frequently requires inotropic support during treatment with intensified immunosuppression. Reversal of acute rejection results in improvement OOZZ-4804/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

Surgery, Colorado

Philadelphia,

Springs, Colorado,

Pennsylvania November

19104 20-23, 1991

in myocardial performance, often within hours. The mechanism of this form of reversible heart failure is unknown. Previous work in our laboratory [l] demonstrated impaired contractile response to P-adrenergic stimulation with isoproterenol in cardiac allografts that histologically showed early rejection. These studies suggested that rejection might cause alterations in a ,&adrenergic receptor-mediated pathway important in regulation of cardiac contractile performance. Isoproterenol stimulates the contractile apparatus of the cardiac myocyte via a P-receptor on the surface of the cell membrane that is linked to the adenylyl cyclase pathway (Fig. 1). Guanine nucleotide binding proteins (G-proteins), ubiquitous transmembrane molecular units, link the receptor to adenylyl cyclase. G-proteins are of two types: stimulatory, G, and inhibitory, Gi. Activated G-protein either stimulates or inhibits adenylyl cyclase, an enzyme that resides on the cytosolic surface of the cell membrane and catalyzes the conversion of ATP to cyclic AMP. Cyclic AMP is a cytosolic second messenger that is coupled to the contractile apparatus via a protein kinase, leading ultimately to an increase in the intracellular calcium and enhanced myocyte contractility. Other investigators have noted alterations in this pathway associated with heart transplantation. Denniss and coinvestigators [2] observed that G-protein-stimulated adenylyl cyclase activity was markedly reduced in transplanted hearts. Gulick and colleagues [3] studied cardiac myocytes cultured in the presence of supernatants of immune-activated cells. The supernatants contained both the cytokines interleukin-1 (IL-l) and tumor necrosis factor (TNF) and inhibited P-adrenergicmediated increases in cultured myocyte contractility and intracellular accumulation of CAMP. These changes occurred in the absence of alteration of P-receptor density or ligand binding affinity. Pagani [4] studied the effect of intravenous IL-l and TNF on the canine heart in vivo. Within the first 4 hr after infusion of these cytokines, there was a marked decrease in cardiac output and stroke work. By 72 hr after the infusion, the dogs re-

Alterations in the fi-adrenergic receptor adenylyl cyclase pathway are well known in heart failure. To determine if an alteration in this pathway occurs during the reversible phase of cardiac allograft rejection, we used a rat heterotopic heart transplant model. Lewis rats received either isografts or Lewis Brown Norway allografts. Cardiac grafts and native hearts were explanted 4, 5, or 6 days later. Receptor-mediated modulation of adenylyl cyclase activity was investigated using isoproterenol, forskolin, and the muscarinic and adenosine receptor agonists carbachol and R-N6-(C2phenyl-isopropyl)-adenosine (R-PIA), respectively. Allografts demonstrated evidence of histological rejection and a significantly impaired response to forskolin and isoproterenol on all days: Forskolin

of Pennsylvania,

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lsoproterenol

GTP-

GDP 1

FIG. 1. The adenylyl cyclase pathway. A receptor on the cell membrane is activated by binding isoproterenol, carbachol, or R-PIA. This activates the G-protein complex to catalyze GTP to GDP which, in turn, stimulates the adenylyl cyclase enzyme to convert ATP to cyclic AMP. Forskolin bypasses the membrane receptor-G-protein complex.

gained normal cardiac function. Similar observations were made by Hosenpud and colleagues [5] who examined the inhibitory effects of IL-l on cardiac contraction in an isolated heart model. These and our own observations suggested that cardiac allograft rejection might cause alterations in the adenylyl cyclase pathway that are mediated by cytokines and that can lead to reversible contractile dysfunction. The present study was undertaken to test this hypothesis directly. METHODS

We directly examined the adenylyl cyclase pathway in the rat heterotopic heart transplant model. Using a modification of the technique of Ono and Lindsey [6] the donor heart was anastomosed to the infrarenal abdominal aorta and infrahepatic inferior vena cava of the recipient. Grafts were performed in Lewis rats using Lewis X Brown Norway F, (allograft) or Lewis (isograft) donors. In this model, allograft rejection is histologically detectable on Day 4 and leads to complete necrosis of the organ by Days 6-8. This strain combination was selected for this and our previous work since the immunologic rejection response is well worked out and occurs predictably over a short period of time. However, the interval between transplantation and rejection is not so short that analysis of the stages of rejection is impossible. All animals received humane care in accordance with National Institutes of Health guidelines and were maintained in a virus-free environment. Animals were anesthetized with methoxyfluorane inhalation anesthetic via nose cone. After removal of the

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anterior chest wall, the donor organ was rapidly excised and immersed in cold saline for myocardial preservation. The abdominal great vessels of the recipient were exposed by a midline laparotomy. Anastomoses were performed using microsurgical technique and 18X magnification on the dissecting microscope. The grafted hearts and the native hearts of the recipients were explanted on post-transplant Day 4, 5, or 6. The animals were anesthetized and the grafted and native hearts arrested with chilled normal saline and rapidly removed. Cross-sectional samples of the ventricles were immediately placed in formalin and subsequently stained with hematoxylin and eosin for histologic examination. Stained specimens were examined by light microscopy for the presence of lymphocyte infiltrates, myocyte necrosis, and hemorrhage. Mild rejection was defined as lymphocyte infiltration alone. Moderate rejection required both the presence of lymphocytes and evidence of myocyte necrosis. This was usually manifested by disruption of myofibers and hyper eosinophilia. Severe rejection included these features and in addition showed hemorrhage and perivascular infiltrate. A sample of the ventricular tissue was suspended in ice-cold buffer solution for biochemical analysis; the remainder was frozen in liquid nitrogen for future studies. Biochemical studies were done after homogenization of cardiac tissue in buffered EDTA solution [7]. The homogenates were filtered, yielding a membrane preparation on which adenylyl cyclase activity was assayed. Protein quantification was determined by established methods. ATP prepared by phosphorylation of adenine and therefore low in contaminating GTP was used as a substrate. Membrane homogenates were placed in a buffered solution containing unlabeled cyclic AMP, radiolabeled (tritiated, 3H) ATP, and radiolabeled (‘“C) cyclic AMP. GTP at a concentration of 100 pM was added to all preparations. In order to stimulate selectively various components of the P-receptor G-protein adenylyl cyclase pathway, various other reagents were added to different aliquots of the reaction mixture. Isoproterenol, a direct stimulant of the P-receptor, was added at a concentration of 100 yM. Forskolin, a direct stimulant of adenylyl cyclase activity that bypasses the P-receptor and G-protein complex, was added at a concentration of 1.0 pM. Carbachol, a muscarinic receptor stimulant, and R-PIA, an adenosine receptor stimulant, both of which increase inhibitory G-protein activity, were added at concentrations of 1.0 mM and 1.0 pM, respectively. The concentrations of reagents were based on our experience using these assays in rat cardiac tissue. Adjustments to specific concentrations were made on the basis of results of initial pilot experiments. After appropriate periods of incubation, the reaction was loaded on a separation column and the cyclic AMP peak collected. Recovery of tritiated cyclic AMP was determined by measurement of the ratio of tritium to carbon-14 in an aliquot of the supernatant in a Beckman liquid scintillation counter.

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JOURNAL

TABLE Percentage

Increase

RESEARCH:

1

in CAMP in Response and Isoproterenol

Forskolin

Day 4 Day 5 Day 6

OF SURGICAL

to Forskolin

Isoproterenol

isograft

allograft

isograft

allograft

I2

535 f 34 500 + 25 752 5 56

363 f 65 325 237 333 + 70

138 f 18 88k 4 147 + 16

91+9 80+12 92+14

7 4 7

Note. All results are P < 0.03 except Day 4 forskolin

and Day 5

VOL.

52, NO. 5, MAY

1992

tion, there was a trend toward impaired response to isoproterenol in the native heart of an allograft recipient on Day 6, at which time cyclic AMP production in the native heart of the allograft recipient was 579 L 22 compared to 718 & 12 in the isograft recipient (P = 0.037). No consistent difference was noted between isografts and allografts stimulated with carbachol and R-PIA (inhibitory G-protein stimulators).

DISCUSSION

isoproterenol.

Both the native and grafted hearts from pairs of allograft and isograft recipients were batch assayed using the prepared reagents. Since the adenylate cyclase pathway assays must be performed on fresh tissue, only four hearts, that is, isograft transplant, isograft native, allograft transplant, and allograft native hearts could be analyzed for each assay run. Thus, baseline cyclic AMP synthesis after GTP stimulation varied with each run. Statistical analysis using ANOVA was performed on both absolute data and data expressed as a percentage of change in cyclic AMP production over that with baseline stimulation with GTP. A logarithmic model using least squares means was used for analysis of absolute data. The results of statistical analysis were identical for both absolute and percentage data, but for clarity of presentation, only the percentage changes are reported. Since several comparisons were made for each ANOVA, a P value of ~0.03 was considered significant.

RESULTS Histologic

Studies

Day 4 allografts had evidence of a moderate lymphocytic infiltrate with sheets of lymphocytes dissecting between myocytes. There was perivascular cuffing and evidence of myocyte disruption. Day 5 allografts had more pronounced infiltration of lymphocytes. On Day 6, severe lymphocytic infiltrate was accompanied by hemorrhagic necrosis. Day 6 allografts had minimal pulsation on palpation through the abdominal wall. Isograft controls had normal myocardial architecture on Days 4, 5, and 6.

Adenylyl

Cyclase Pathway

Functional

Assay

Results are summarized in Table 1. Allografts demonstrated significantly impaired response to isoproterenol (P-receptor) and forskolin (direct adenylyl cyclase) stimulation when compared with isograft controls. In addi-

This study demonstrated defects in @-agonist stimulated and primary adenylyl cyclase synthetic function in cardiac allografts suffering moderate rejection. This observation is consistent with our previous work showing a blunted response to P-adrenergic stimulation in allografts at a similar phase of rejection. The data are also consistent with studies by other investigators that suggest that immune mediators produce alterations in the b-receptor-G-protein-adenylyl cyclase pathway [2-51. Since this pathway is important in the regulation of myocardial contraction [8-lo], it is likely that these alterations are important components of the molecular basis of contractile dysfunction that can be observed in allograft rejection. One of the more interesting features of this contractile dysfunction is its rapid reversibility. The fact that contractile dysfunction can be reversed suggests that it must not be primarily due to massive myocyte necrosis. We have performed preliminary experiments also in the rat heterotopic transplant model, which suggest that the defect in adenylyl cyclase function that we have identified is in fact reversible when rejection is reversed. This preliminary observation lends further credence to our hypothesis that this pathway is important in the physiology of heart transplant rejection. The reasons for the alterations in adenylyl cyclase function during acute rejection are incompletely understood. As noted, data from other laboratories suggest that immune mediators including tumor necrosis factor and interleukin-1 may have important effects on this pathway. It is not clear how either of these cytokines produce these changes, nor what the specific defect in adenylyl cyclase function is. In our experiments, when end-stage rejection occurred in the allograft (Day 6), the native heart of the recipient appeared to develop a similar defect in adenylyl cyclase function. This may be further evidence that a soluble factor or factors such as immune cytokines are mediating these changes. One possible hypothesis is that immune mediators such as tumor necrosis factor, interleukin 1 or other cytokines are released from cells infiltrating the graft. On Day 6, when the immunologic response is maximal, there may be sufficient release of mediators to affect function in the native heart. Observations made by Pagani [4] and Hosen-

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FAILURE

pud [5] showing that soluble cytokines can affect cardiac function are consistent with this hypothesis. These data may have therapeutic implications for patients with heart transplants. Inotropic drugs are often required for temporary support of patients suffering transplant rejection which is associated with contractile failure. The most commonly used inotropic drugs produce their effects by action on myocardial P-adrenergic receptors. Our data suggest that the efficacy of this approach may be limited since the process of rejection alters the response to P-agonists and adenylyl cyclase, the ultimate target of P-receptor stimulation. Our studies raise the possibility that phosphodiesterase inhibitors, drugs that decrease the catabolism of cyclic AMP, may be more effective in the setting of heart transplant failure due to rejection. Our data are certainly consistent with numerous clinical observations that the combination of a catecholamine P-agonist and a phosphodiesterase inhibitor such as amrinone act synergistically in augmenting cardiac output both in heart transplantation and other instances of acute heart failure. Direct investigations of the effects of phosphodiesterase inhibitors in this model may be fruitful. These experiments have identified a defect in the /3receptor-G-protein-adenylyl cyclase pathway caused by heart transplant rejection. Preliminary data also suggest that the defect may be reversible and therefore importantly related to reversible cardiac allograft dysfunction. These observations may have significant implications regarding the physiology and treatment of heart transplant rejection.

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REFERENCES 1.

2.

DiSesa, V., Masetti, P., Disco, M., Schoen, F., Marsh, J., and Cohn, L. The mechanism of heart failure caused by cardiac allograft rejection. J. Thorac. Cardiouasc. Surg. lOl(3): 446, 1991. Denniss, A., Marsh, J., Quigg, R., Gordon, J., and Colucci, W. P-adrenergic receptor number and adenylyl cyclase function in denvervated transplanted and cardiomyopathic human hearts. Circulation 79(5): 11,028, 1989.

3.

Chung, M., Gulick, T., Rotondo, R., Schreiner, G., and Lange, L. Mechanism of cytokine inhibition of &adrenergic agonist stimulation of cyclic AMP in rat cardiac myocytes. Circ. Res. 79(3): 753, 1990.

4.

Pagani, F., Baker, L., Knox, M., Cheng, H., Fink, M., and Visner, M. Tumor necrosis alpha causes diastolic creep and reversible left ventricular dysfunction in conscious dogs. Surg. Forum 41: 40, 1990. Hosenpud, J., Campbell, S., Mendelson, D. Interleukin-1 induced myocardial depression in an isolated beating heart preparation. J. Heart Transplant. S(6): 460, 1989. Ono, K., and Lindsey, E. Improved technique of heart transplantation in rats. J. Thorac. Cardiovasc. Surg. 5’7: 225, 1969.

5.

6. 7.

8. 9.

10.

Liang, B., and Galper, J. Reconstitution of muscarinic cholinergic inhibition of adenylyl cyclase activity in homogenates of embryonic chick hearts by membranes of adult chick hearts. J. Biol. Chem. 262(6): 2494, 1987. Robishaw, J., and Foster, K. Role of G-proteins in the regulation of the cardiovascular system. Annu. Rev. Physiol. 51: 229, 1989. Horn, E., and Bilezikian, J. Mechanisms of abnormal transmembrane signaling of the P-adrenergic receptor in congestive heart failure. Circulation (Supplement,J 82(2): 26, 1990. Bristow, M., Hershberger, R., Port, J., Gilbert, E., Sandoval, A., Rasmussen, R., Cates, A., and Feldman, A. @-adrenergic pathways in nonfailing and failing ventricular myocardium. Circulation (Supplement) 82(2): 12, 1990.