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(Circulation. 1995;92:2183-2189.)
© 1995 American Heart Association, Inc.

Articles

Cardiac ß-Adrenergic Neuroeffector Systems in Acute Myocardial Dysfunction Related to Brain Injury

Evidence for Catecholamine-Mediated Myocardial Damage

Michel White, MD; Robert J. Wiechmann, MD; Robert L. Roden, MSc; Mary Beth Hagan, RN; Mary M. Wollmering, MD; J. David Port, PhD; Elizabeth Hammond, MD; William T. Abraham, MD; Eugene E. Wolfel, MD; JoAnn Lindenfeld, MD; David Fullerton, MD; Michael R. Bristow, MD, PhD

From the Cardiology and Cardiothoracic Surgery Divisions, University of Colorado Health Sciences Center, Denver, and the University of Utah School of Medicine, Salt Lake City.

Correspondence to Michael R. Bristow, MD, Division of Cardiology, University of Colorado Health Sciences Center, B-139, 4200 E 9th Ave, Denver, CO 80262.

	Abstract

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Background Ten percent to 20% of potential cardiac donors with brain injury and no previous cardiac history have myocardial dysfunction. We assessed components of the ß-receptor–G-protein–adenylyl cyclase complex as well as the contractile response in 10 explanted acutely failing human hearts (donor heart dysfunction [DHD]) and compared the results with 13 age-matched nonfailing (NF) organ donor controls.

Methods and Results As measured by echocardiography, all DHD hearts exhibited a decreased shortening fraction (16±2%, mean±SEM). Although total and subpopulation ß-receptor densities measured by [¹²⁵I]iodocyanopindolol (ICYP) were similar in the DHD and NF groups, DHD hearts exhibited a 30% decrease in maximum isoproterenol-stimulated adenylyl cyclase activity and a 50% decrease in the maximal response to zinterol. DHD hearts also exhibited decreases in adenylyl cyclase maximal stimulation by forskolin (211±25 [DHD] versus 295±23 [NF] pmol cAMP · min^-1 · mg^-1, P<.05) and 5'-guanylylimidodiphosphate (12.5±1.8 [DHD] versus 19.6±3.2 [NF] pmol cAMP · min^-1 · mg^-1, P<.05), but there was no significant decrease in adenylyl cyclase stimulation by Mn²⁺, a direct activator of adenylyl cyclase. Right ventricular trabeculae removed from DHD hearts exhibited a profound decrease in the contractile response to isoproterenol (8.7±1 [DHD] versus 22±2 [NF] mN, P<.001) as well as reduced calcium responses (7.2±1.6 [DHD] versus 14±3 [NF] mN, P=.03). Morphological examination of two hearts revealed some ultrastructural evidence suggestive of catecholamine-mediated injury, but there was no difference in tissue creatine kinase activity between the two groups.

Conclusions Compared with NF hearts, DHD hearts exhibit marked uncoupling of ß₁- and ß₂-adrenergic receptors from adenylyl cyclase and contractile response stimulation as well as decreased intrinsic systolic function. Thus, acute myocardial dysfunction accompanying brain injury is characterized by marked alterations in ß-adrenergic signal transduction as well as changes in the contractile apparatus, and this profile is markedly different from what occurs in the chronically failing human heart.

Key Words: heart failure • catecholamines • receptors • adrenergic • beta • proteins

	Introduction

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Cardiac transplantation is now a widely accepted treatment for patients with end-stage heart failure.¹ The major limitation of transplantation is no longer clinical outcome but rather an increasingly limited pool of organ donors.² Approximately 10% to 20% of subjects with severe closed-head injury who have no evidence of cardiac contusion or chest trauma have myocardial dysfunction severe enough to exclude the heart from organ donation.³ This scenario defines a type of acute heart failure related to severe brain injury, a clinical syndrome we call DHD.⁴

One of the consequences of traumatic head injury is the initiation of a cascade of deleterious events that appear to relate to activation of the sympathetic nervous system.^{5 6} In animal models of increased intracranial pressure, cerebral injury results in marked perturbations of hemodynamics⁷ and histological evidence of myocardial damage.^{8 9} In humans, the increase in circulating catecholamine levels reflects the severity of the neurological insult and is a physiological marker of patient outcome.⁶ Previous studies have also demonstrated alterations in ß-adrenergic signal transduction in model systems exposed to high levels of catecholamines.^{10 11 12 13 14} In cultured heart cells, short-term (1 to 4 hours) exposure to catecholamines generally causes uncoupling followed by downregulation of ß-adrenergic receptors^{10 11} as well as calcium overload in isolated cardiac myocytes.¹² Similarly, in in vivo models, chronic elevation of norepinephrine also causes uncoupling of myocardial ß-adrenergic receptors from mechanical response^{13 14 15} and adenylyl cyclase stimulation.¹⁴

Changes in the ß-receptor–G-protein–adenylyl cyclase complex in explanted human hearts exhibiting depressed LV systolic function from DHD or in other settings of acute heart failure have not been previously investigated. On the basis of results of the present investigation, we postulate that cardiac organ donors exhibiting acute LV systolic dysfunction related to brain injury (DHD) are subjected to massive sympathetic discharge that results in acute-type desensitization phenomena of the ß-adrenergic receptor pathways. As previously reported in model systems, these acute desensitization changes consist primarily of receptor uncoupling and changes in G-protein function as opposed to the more chronic desensitization changes that are related to altered gene expression.¹⁶ The acute desensitization changes in ß-adrenergic signal transduction are probably an adaptive cellular response that partially reduces harmful sympathetic stimulation. However, these desensitization phenomena will impair the ability of the heart to respond to the stress of brain injury, which includes the need for increased cardiac output to compensate for peripheral vascular collapse.

	Methods

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Explanted Human Hearts
Human hearts were obtained from the Stanford, Utah, and Colorado Cardiac Transplant Programs via the Northern California, Intermountain, and Colorado Organ Procurement Agencies. Ten DHD hearts were harvested from potential organ donors excluded from heart donation because of depressed LV systolic function defined as a shortening fraction 25%. In all DHD cases, the echocardiogram obtained as a part of the organ donation process revealed diffuse hypokinesis without segmental wall motion abnormalities, suggesting myocardial infarction or cardiac contusion. Thirteen age-matched NF hearts obtained from local organ donors were excluded from heart donation because of age, body size, or ABO blood type incompatibility. None of the NF hearts harvested from organ donors had a known cardiac history, and in all cases, the echocardiogram revealed normal LV systolic function (shortening fraction >25%). Written consent for organ donation for research purposes was obtained from a family member for all procured DHD and NF hearts.

The explanted hearts were rapidly removed and immediately immersed in ice-cold oxygenated physiological salt solution in <30 minutes. Portions of the LVs and RVs of two hearts were removed and cut into 1-mm³ cubes, fixed overnight in Karnovsky's fixative, and processed routinely into plastic. Sections of the blocks were cut at 1 µm, stained with toluidine blue, and examined histologically and ultrastructurally in a transmission electron microscope (JEOL 100SX).

Biochemical Studies
Aliquots (5 to 6 g) of LV and RV free walls were dissected free of epicardial fat and endocardial tissue and placed in 10 mmol/L Tris/1 mmol/L EGTA buffer pH 8.0. The tissue was finely minced with scissors and homogenized. A crude membrane fraction was made by extracting the contractile proteins in 0.5 mol/L KCl and repeatedly washing a 50 000g pellet.

Total ß-adrenergic receptor density was radiolabeled with ICYP as previously described.¹⁷ Briefly, seven increasing concentrations of ICYP between 3.124 and 150 pmol/L in the presence or absence of 1 µmol/L (-)-propranolol were used to construct the specific binding curve. Maximum binding and K_d were determined by nonlinear least-squares fit of the specific binding curve.¹⁷ The percentages of ß₁-receptor and ß₂-receptor were determined by computer modeling of ICYP-betaxolol or ICYP-CGP 20712A¹⁷ competition curves using either 50 or 100 pmol/L ICYP to maintain the radioligand concentration at 3xK_d to 5xK_d.

A preparation suitable for measuring hormone-stimulated adenylyl cyclase activity was prepared as previously described.¹⁷ For the adenylyl cyclase assays, myocardial membranes (0.05 to 0.125 mg/tube) were suspended in 100 mmol/L Tris buffer, pH 7.3, at 30°C and exposed to the agonist drug of choice.^{17 18} The standard reaction mixture included 0.1 mmol/L MgATP with 0.5 mmol/L MgCl₂ in excess, 10^-5 mol/L GTP, 10 mmol/L phosphocreatinine, 1 mmol/L cAMP, and 1.75 U creatine kinase per incubation tube. A second assay condition consisted of the above mixture minus GTP plus 10^-6 mol/L (-)-propranolol [Gpp(NH)p condition]. A third assay was used to assess Mn²⁺ stimulation and consisted of the Gpp(NH)p condition minus MgCl₂ (Mn²⁺ condition). Recovery was determined by trace labeling with [³H]cAMP, and the reaction was started by trace labeling with [³²P-]ATP. Newly formed [³²P]cAMP was recovered by the technique of Salomon et al.¹⁹

Tissue norepinephrine, dopamine, and epinephrine levels were measured radioenzymatically with kits obtained from Amersham (Cat-a-Kit). The protein concentration for calculation of ß-receptor density was determined by the Peterson modification²⁰ of the method of Lowry et al,²¹ and the protein concentration in adenylyl cyclase assays was measured by the method of Lowry et al.²¹ Soluble creatine kinase activity was measured in the supernatant of the 1085g centrifugation used to process a particulate fraction of adenylyl cyclase assays by a spectrophotometric technique.¹⁷

Contractile Response of Isolated RV Trabeculae
The contractile response of isolated human cardiac preparations was assessed as previously described.^{17 18} Trabeculae of uniform size (1 to 2x6 to 8 mm) were carefully dissected and mounted in an eight-chamber muscle bath. After equilibration, tension was applied and adjusted to each strip of muscle to achieve maximal contraction (about 1 to 1.5 g). The trabeculae were then field-stimulated by a 5-ms pulse at 10% above threshold. After a 2-hour equilibrium period, full dose-response curves to isoproterenol were performed with 0.3 or 0.5 log unit dose increments between 10 nmol/L and 10 µmol/L. After completion of the isoproterenol dose-response curve and washout of isoproterenol, the maximal response to calcium was measured by administration of calcium chloride at a final concentration of 2.5, 5, and then 10 mmol/L. Tension was recorded as the stimulated tension minus baseline tension, and the maximum response was taken as the greatest amount of net tension produced at any point in the dose-response curve. The maximum tension and the concentration of isoproterenol that produced 50% of the maximum developed tension (EC₅₀) were computed by nonlinear regression analysis.

Statistical Methods
The continuous variables were compared by unpaired two-tailed Student's t test, and the discrete variables were computed by ² test. The saturation curves and the ICYP-CGP 20712A competition curves were analyzed by a nonlinear least-squares curve-fitting procedure.¹⁷ The differences between the dose-response curves to isoproterenol were analyzed by ANOVA with repeated measures. All probabilities (P values) of P<.05 were considered statistically significant, whereas values of .05<P<.10 were considered to be statistically marginal. Values are expressed as mean±SEM unless specified otherwise.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Study Population
The characteristics of the DHD population (n=10) and NF donors (n=13) are presented in Table 1. In the DHD group, the causes of brain death were motor vehicle accident 6, gunshot wound 2, anoxic encephalopathy 1, and miscellaneous head trauma 1. In the NF group, the causes of death were motor vehicle accident 6, intracranial hemorrhage 3, anoxic encephalopathy 2, and gunshot wound 2. The average calculated shortening fraction measured by echocardiography was 15.8±6.4% (range, 10% to 25%). The subjects were intubated between 10 and 48 hours (median, 21 hours). Nine DHD subjects received dopamine, and one DHD donor received norepinephrine alone.

View this table: [in this window] [in a new window]   Table 1. Characteristics of the Study Population

The control group consisted of 13 NF donors 24±12 years old (range, 1 to 39 years) intubated for 24 to 144 hours (median, 48 hours). In all cases, the shortening fraction calculated by echocardiography was >25%. The systolic and diastolic blood pressures and heart rate computed hourly and averaged for each subject as well as the range of observations for each subject plus between-group comparisons are also presented in Table 1. Mean blood pressures and heart rate were similar for the two groups, but DHD donors exhibited larger fluctuations in systolic and diastolic blood pressures expressed by a significantly larger range of observations (both P<.05). ECGs revealed evidence of brain injury (repolarization changes, including T-wave conversion) in both groups.

ß-Adrenergic Receptors in DHD and NF Organ Donors
Total ß-adrenergic receptor density and ß₁- and ß₂-subpopulation characteristics are illustrated in Fig 1. The DHD group had marginally higher total ß-receptor and subpopulation densities compared with the age-matched NF organ donors. Maximum ß-AR B_max was 105±8.3 fmol/mg (DHD) versus 97±7.6 fmol/mg (NF) for LVs and 113±9 fmol/mg (DHD) versus 107.6±8.9 fmol/mg (NF) for RVs (P=NS). The ß₁-subpopulation receptor density was 80.1±6.7 fmol/mg (DHD) versus 74.7±5.8 fmol/mg (NF) for LVs and 92.6±9 fmol/mg (DHD) versus 86.3±7.9 fmol/mg (NF) for RVs (both P=NS). Similarly, the ß₂-receptor population was 24.9±2.9 fmol/mg (DHD) versus 21.7±2.6 fmol/mg (NF) for LVs and 20±4 fmol/mg (DHD) versus 18.8±3.2 fmol/mg (NF) for RV myocardial membrane preparations (both P=NS). No differences were observed in antagonist affinity (ICYP K_d) between the two groups in either ventricle (14.8±3.3 pmol/L [DHD] versus 14±2.9 pmol/L [NF] for LVs and 13.9±4.2 pmol/L [DHD] versus 9.6±2.6 pmol/L [NF] for RVs, P=NS).

View larger version (26K): [in this window] [in a new window]   Figure 1. Bar graph showing total and subpopulation ß-receptor density from LV and RV for DHD and for NF organ donors.

Stimulation of Adenylyl Cyclase
The basal activity and net maximum adenylyl cyclase stimulation in response to different agonists are presented in Table 2. DHD hearts exhibited a trend toward lower basal adenylyl cyclase activity in RVs, and this reduction reached borderline significance (P=.09) when LVs and RVs were pooled for analysis. DHD hearts exhibited a 30% decrease in maximum adenylyl cyclase activity in response to isoproterenol in LVs and RVs (both P<.05). Similarly, there were 54% and 45% decreases, respectively, in maximum adenylyl cyclase stimulation in response to the ß₂-agonist zinterol for LVs and RVs. In DHD LVs, the isoproterenol EC₅₀ measured on each individual dose-response curve was shifted rightward, by 6.8-fold (1.14±0.6x10^-6 mol/L [NF] versus 7.7±4.7x10^-6 mol/L [DHD], P=.13) and by 4.3-fold in the RVs (1.58±6.1x10^-7 mol/L [NF] versus 3.7±1.3x10^-6 mol/L [DHD], P=NS).

View this table: [in this window] [in a new window]   Table 2. Adenylyl Cyclase Data as Net Maximum Stimulation (Agonist Stimulation Minus Basal Activity, pmol cAMP · min-1 · mg-1) or Stimulation Ratio Under Standard, Gpp(NH)p, and Mn2+ Conditions1

Histamine-mediated adenylyl cyclase activity was assessed in eight NF and nine DHD hearts. Maximal activity was decreased by 30% in LVs (P=.15) and by 46% and 37% in RVs and pooled LVs and RVs, respectively (both P<.05). Similarly, there were 26%, 20%, and 22% reductions in adenylyl cyclase stimulation in response to forskolin for LVs, RVs (P=.08), and pooled LVs and RVs, respectively (P<.05). Under standard assay conditions, adenylyl cyclase stimulation in response to NaF was not different in DHD versus NF ventricles.

Adenylyl cyclase stimulation in response to various agonists examined under Gpp(NH)p and Mn²⁺ assay conditions are also presented in Table 2. Under the Gpp(NH)p condition, DHD LVs and RVs exhibited significantly lower maximum adenylyl cyclase stimulation in response to Gpp(NH)p, by 37% and 58%, respectively (both P<.05). NaF-mediated adenylyl cyclase stimulation tended to be decreased, by 15% and 25% in DHD LVs and RVs, respectively; however, neither value reached statistical significance. In the DHD group, the ratio of Gpp(NH)p to NaF was decreased by 19%, 45%, and 28% in LVs (P=.15), RVs (P<.05), and pooled LVs and RVs (P=.07), respectively. Adenylyl cyclase stimulation under Mn²⁺ assay conditions was performed in seven NF and nine DHD hearts. DHD hearts exhibited significantly lower basal adenylyl cyclase activity in both ventricles. However, when the catalytic unit of adenylyl cyclase was pharmacologically probed by MnCl₂, NF and DHD donors exhibited similar degrees of adenylyl cyclase stimulation in both ventricles.

Myocardial Catecholamines and Soluble Creatine Kinase Activity
NF and DHD hearts exhibited similar levels of myocardial norepinephrine, dopamine, and epinephrine. Mean norepinephrine levels were 996±157 (NF) versus 850±112 (DHD) ng/g wet wt, and intramyocardial epinephrine concentration 96±31 (NF) versus 97±27 (NF) ng/g wet wt, for both LV and RV myocardium combined. Both groups had elevated levels of myocardial dopamine (516±99 [NF] versus 581±109 [DHD] ng/g wet wt for both LVs and RVs combined). There were no differences in myocardial content of catecholamines between LVs and RVs. Finally, the DHD and NF groups had similar amounts of viable myocardium as assessed by myocardial levels of creatine kinase (1097±66 [NF] versus 1015±94 [DHD] IU/g wet wt).

Contractile Responses of Isolated RV Trabeculae
The contractile responses of isolated RV trabeculae harvested from NF and DHD groups are illustrated in Figs 2 and 3. Ten DHD and 10 NF hearts were studied, with respective totals of 32 and 31 individual trabeculae. DHD trabeculae exhibited a marked decrease in maximum developed tension in response to isoproterenol (8.7±1 versus 22±2 mN, P<.001) as well as a significantly lower maximum contractile response to calcium (7.2±1.6 versus 14±3 mN, P=.03) (Fig 2). The profound decrease in the contractile response to isoproterenol and the somewhat more modest decrease in the tension response to calcium resulted in a 33% lower isoproterenol-to-calcium ratio in the DHD group (1.8±0.17 [DHD] versus 2.7±0.63 [NF], P=.06). Full dose-response information from 19 trabeculae harvested from eight NF hearts and 24 trabeculae obtained from nine DHD hearts was available and was analyzed. The isoproterenol EC₅₀ computed for each individual trabecula was 150-fold higher in DHD than in NF (isoproterenol EC₅₀, 4.4±1x10^-8 mol/L [NF] versus 7.5±3.8x10^-6 mol/L [DHD], P=.034). The contractile response to isoproterenol was also analyzed by constructing a curve averaging the data at each concentration of isoproterenol (Fig 3). These two dose-response curves were markedly different when analyzed as a whole by ANOVA computed on the data obtained at 10 nmol/L, 1 µmol/L, and 10 mmol/L (F ratio=31.9 for NF versus DHD, P<.001), and the DHD hearts yielded a 35-fold rightward shift of isoproterenol EC₅₀ (6.9x10^-7 mol/L [DHD] versus 2.06x10^-8 mol/L [NF]).

View larger version (16K): [in this window] [in a new window]   Figure 2. Bar graph showing maximum net tension responses (stimulated minus basal) of isolated RV trabeculae to isoproterenol (ISO) and calcium. ISO/calcium indicates isoproterenol-to-calcium ratio. *P<.05, **P<.001, P=.06.

View larger version (14K): [in this window] [in a new window]   Figure 3. Graph showing net contractile response of RV trabeculae to increasing concentrations of isoproterenol. Computed isoproterenol EC50 is 3.9±1.4x10-8 mol/L for NF organ donors () and 4.9±3.6x10-6 mol/L for DHD ().

Histological and Ultrastructural Findings in Myocardium
Sections 1 µm thick of myocardium from two hearts were examined histologically. These sections showed myocytes without myofilament loss. The cells had focally severe contraction band formation involving some of most sections (Fig 4). Ultrastructural examination of the same sections revealed disorganized aggregates of actin filaments in areas corresponding to the contraction bands. Some myocytes had dilated sarcoplasmic reticulum. Myocytes exhibited no other abnormal features; the microcirculation was unremarkable.

View larger version (130K): [in this window] [in a new window]   Figure 4. A toluidine blue–stained 1-µm-thick plastic-embedded section of myocardium from one of the study patients. Marked contraction band formation is noted. Magnification x400.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The type of acute heart failure in brain-injured organ donors that we call DHD is a unique model for investigating acute heart failure and acute desensitization changes in the myocardial ß-adrenergic–G-protein–adenylyl cyclase complex. This acutely failing human heart model allowed us to observe in vitro changes in the ß-adrenergic neuroeffector systems in hearts exposed to a short and intense sympathetic discharge,^{5 8 9} in contrast to the lower-level elevation in adrenergic activity that characterizes chronic heart failure. Moreover, DHD is an important problem in cardiac transplantation, since it reduces an already inadequate donor pool.²

In this model of acute heart failure, we observed no DHD-related changes in total, ß₁-, or ß₂-adrenergic receptor densities or affinity for the radiolabeled antagonist ICYP compared with age-matched NF organ donors. However, we observed profound abnormalities in adenylyl cyclase stimulation in response to ß-receptor and G-protein pharmacological probes. The acutely failing hearts also exhibited marked abnormalities in the contractile response of isolated RV trabeculae to isoproterenol and to a lesser extent to calcium. DHD also exhibited ultrastructural findings typical of catecholamine-mediated myocardial damage, but the amount of functioning myocardium assessed by creatine kinase measurement was similar in DHD and NF controls. Tissue norepinephrine levels, a marker of adrenergic neuron density, were not reduced in DHD ventricles.

In contrast to cultured heart cells, in which downregulation preceded by uncoupling generally occurs with ß-agonist exposure,^{10 11} the behavior of myocardial ß-adrenergic receptors in the intact heart exposed to elevated systemic levels of catecholamines appears to be variable and probably species-dependent.^{14 22 23} In humans, 72 hours of dopamine infusion in NF organ donors does not decrease total myocardial ß-receptor density compared with donors not exposed to dopamine.²⁴ Similarly, 48 hours of a dobutamine infusion does not change ß-receptor density in patients with heart failure.²⁵ Accordingly, our observations on a lack of ß-receptor downregulation after short-term exposure to ultrahigh levels of sympathetic activity and short durations of myocardial dysfunction are in general agreement with previous studies performed in humans. In ß₁-receptor downregulation as described in chronic heart failure^{17 18} or more recently in aging,²⁶ the magnitude and duration of increased adrenergic activity or other factors needed to produce ß-adrenergic receptor downregulation are unknown, but from the above observations would appear to be longer than several weeks. Finally, since both DHD and NF groups were exposed to elevated levels of circulating and no doubt locally released catecholamines, it is possible that both the DHD and control groups had undergone some degree of desensitization, including receptor downregulation, before harvest of the explanted hearts.

The major finding in these acutely failing, diffusely hypocontractile hearts was the degree of uncoupling from functional response of both ß₁- and ß₂-adrenergic receptors. In fact, DHD ventricles exhibited the same magnitude of decrease in isoproterenol-mediated stimulation of adenylyl cyclase but a 50% greater decrease in zinterol-mediated adenylyl cyclase stimulation compared with end-stage chronic heart failure^{17 18} despite no decrease in both ß₁- and ß₂-adrenergic receptors. DHD ventricles also exhibited a 6- to 22-fold rightward shift of the isoproterenol–adenylyl cyclase dose-response curve, which does not occur in chronic heart failure.^{17 18} Also, RV trabeculae harvested from DHD hearts yielded a decrease in maximum contractile response to isoproterenol that is at least as great as that observed in chronic heart failure plus a 35- to 150-fold increase in the isoproterenol EC₅₀, indicating markedly reduced ß₁-receptor agonist binding affinity. Thus, it appears that, unlike chronic heart failure due to idiopathic dilated cardiomyopathy,^{17 18} DHD ventricles exhibit uncoupling of ß₁-adrenergic receptors, a more profound uncoupling of ß₂-receptors from adenylyl cyclase, and a significant decrease in ß₁- and ß₂-receptor agonist binding affinity as deduced from the position of isoproterenol–adenylyl cyclase (primarily ß₂) and muscle contraction (primarily ß₁) dose-response curves. The mechanism(s) responsible for this altered agonist binding affinity in DHD has not been directly investigated. However, phosphorylation of receptors or other changes in signal transduction occurring at the receptor level and mediated by ultrahigh levels of endogenous catecholamine agonists is a likely explanation.

The prominent uncoupling of ß-adrenergic receptors in DHD hearts is most likely caused by altered G-protein activity. In fact, DHD hearts exhibited a significant decrease in the adenylyl cyclase response to Gpp(NH)p and forskolin. The magnitude of impairment in adenylyl cyclase activity in response to these two pharmacological probes is in agreement with the finding described in the chronic norepinephrine infusion model¹⁴ and is quite similar to the changes previously reported in end-stage chronic heart failure.^{17 18} In contrast to Gpp(NH)p and forskolin, we observed no statistically significant decrease in the response to NaF in DHD hearts compared with NF controls, and no differences were observed between DHD and NF hearts in the adenylyl cyclase response to Mn²⁺. Mn²⁺ is a relatively selective probe for the adenylyl cyclase catalytic unit, since activation is not affected by the presence of G proteins.²⁷ Accordingly, in DHD ventricles, our results support an alteration in G-protein function, perhaps an increase in the functional activity of G_i,^{18 28} without a change in the catalytic unit of adenylyl cyclase.

The contractile response findings are consistent with desensitization to ß-agonist stimulation plus myocardial damage produced by catecholamine toxicity. Although no previous human data exist, several experimental studies have shown a depression of myocardial mechanical response by endogenous or exogenous elevation of systemic or regional adrenergic activity.^{14 15 16 29} Similarly, massive sympathetic discharge induced by brain damage in anesthetized rabbits results in typical catecholamine-induced myofibrillar damage and causes a significant decrease in LV performance assessed 2 hours after the initial insult.³⁰ The decrease in maximum contractile response to calcium is also in agreement with a previous investigation showing that 14 days of continuous norepinephrine infusion results in an 18% decrease in maximal inotropic response to calcium in isolated rabbit LV papillary muscles.²⁹ Thus, unlike in chronic human heart failure,^{17 18} DHD ventricles are characterized by a decrease in the contractile response to both ß-agonists and calcium. This suggests that the contractile apparatus is relatively more damaged via catecholamine-mediated myocardial injury in DHD than in chronic heart failure, based on the normal maximal systolic tension response to calcium in the latter.^{17 18}

Both DHD and NF organ donor ventricles exhibited similar myocardial levels of catecholamines and similar dopamine/norepinephrine ratios. Both groups exhibited elevated myocardial dopamine and epinephrine levels, reflecting prior exposure to intravenous dopamine²⁴ and high levels of circulating epinephrine.^{6 7} Therefore, a few days of exposure to high systemic and cardiac sympathetic activity does not cause myocardial depletion of catecholamines such as occurs in chronic heart failure.^{17 18 31} This indicates that intramyocardial depletion of norepinephrine, like ß₁-receptor downregulation, is a more chronic process in human cardiac tissue.

Our findings are also in agreement with the recent work of Shivalkar et al,⁹ since we also report features typical of catecholamine toxicity in histological analysis of two DHD hearts. Thus, our data complement that study⁹ by demonstrating that profound ß-adrenergic receptor desensitization may contribute to DHD and to the poor functional recovery of these hearts when transplanted. However, an important observation of our study is that despite these changes, creatine kinase levels in DHD ventricles did not differ from NF controls, indicating that abnormal contractile function may be reversible.³²

Our results may have important clinical implications. The average age of the DHD hearts was 22 years; younger hearts exhibit an increased myocardial sensitivity to ß-agonist stimulation in vivo as well as higher ß-adrenergic receptor density than older hearts,²⁵ and in that regard they may be more vulnerable to a sudden increase in sympathetic activity. ß-Blockade has been beneficial in reducing less severe cardiac damage in the setting of brain injury³³ or in other conditions characterized by a sudden increase in sympathetic activity, such as acute myocardial infarction.^{34 35} Early myocardial protection by ß-blockade, especially in younger donors, could help expand the pool of donors for transplantation. Finally, since high doses of dopamine cause norepinephrine release³⁶ and therefore may increase cardiac adrenergic drive, dopamine may not be the ideal agent for stabilization of blood pressure in organ donors.

As is the case for any clinical investigation, some potential limitations may have influenced our results. The criteria for DHD were based on echocardiographically determined shortening fraction, which is a load-dependent measurement of systolic function routinely obtained during the evaluation of cardiac organ donors.³ However, in all cases, the echocardiogram was recorded after stabilization of the potential donors and several hours before the time of explant. As for the DHD hearts, half of our NF donors were exposed to dopamine for several hours. This could not be avoided because of the nature of the study, but it is unlikely to have significantly influenced our observations.²⁴ Finally, the hypothesis that the DHD hearts were exposed to higher adrenergic drive remains speculative, since circulating catecholamines were not measured. However, circulating norepinephrine and epinephrine levels have been markedly elevated in previous studies that measured them in the setting of severe brain injury.^{6 33 37}

In summary, acutely failing human hearts do not exhibit ß-receptor downregulation but rather a marked uncoupling of both ß₁- and ß₂-adrenergic receptors from adenylyl cyclase and contractile response. The degree of uncoupling is quantitatively higher than the changes previously described in end-stage chronic heart failure. DHD hearts also exhibit histological features of catecholamine toxicity. Although calcium responses indicate that the contractile apparatus is damaged, the normal amount of viable myocardium as assessed by creatine kinase activity indicates that these changes may be reversible. In DHD, detrimental changes in cardiac ß-adrenergic signal transduction as well as the histological features of catecholamine-mediated myocardial injury and damage to the contractile apparatus might be preventable by pharmacological interventions, which could increase the cardiac transplantation donor pool.

	Selected Abbreviations and Acronyms

 

DHD = donor heart dysfunction Gpp(NH)p = 5'-guanylylimidodiphosphate ICYP = [125I]iodocyanopindolol LV = left ventricular, left ventricle NF = nonfailing RV = right ventricle

	Acknowledgments

 
This study was supported by NIH grant HL-13108 and by a Research Fellowship from the Heart and Stroke Foundation of Canada awarded to Michel White. The authors wish to thank Frank Stewart and Jill Jones for their assistance in the preparation of the manuscript.

Received February 19, 1995; revision received May 2, 1995; accepted May 6, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 

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