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J Thorac Cardiovasc Surg 1999;117:365-374
© 1999 Mosby, Inc.
CARDIOTHORACIC TRANSPLANTATION |
From the Department of Surgery,a University of Louisville, Louisville, Ky; the Department of Cardiothoracic Surgery,b Allegheny University of the Health Sciences, MCP, Hahnemann School of Medicine, Philadelphia, Pa; the Departments of Surgery,c Physiology,f and Internal Medicine,e Medical College of Virginia/Virginia Commonwealth University, Richmond, Va; and the Department of Biological Sciences,d Mary Washington University, Fredericksburg, Va.
Supported in part by grants from the United States Public Health Service (grant GM3529 [E.R.J.]), from the National Institutes of Health (grant HL26302 [A.S.W.]), and from the American Heart Association (grant AHA94010440 [A.S.W.]).
Read at the Seventy-eighth Annual Meeting of The American Association for Thoracic Surgery, Boston, Mass, May 3-6, 1998.
Received for publication April 6, 1998. Revisions requested May 27, 1998. Revisions received Sept 21, 1998. Accepted for publication Sept 21, 1998. Address for reprints: Thomas Yeh, Jr, MD, PhD, Division of Cardiovascular Surgery, University of Louisville, 201 Abraham Flexner Way, No 1200, Louisville, KY 40202.
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Abstract |
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-actins, egr-1, and heat shock protein 70 were significantly increased. Sham-operated animals did not exhibit these changes. |
Introduction |
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1 The myocardial dysfunction accompanying brain death can be so severe that an estimated 20% of donors are eliminated from consideration to avoid early graft failure. 2 Once accepted for transplantation, organs with occult injury have the potential to result in the death of the recipient. The typical delay in onset of this contractile dysfunction suggested to us that the altered expression of select myocardial genes might play a role. If so, then defining the molecular changes induced by brain death would be an important first step in the rescue or protection of these hearts.
A newly emerging concept is that hypertrophy, a compensatory response of the heart to injury underlies cardiac pathologic conditions 3 and may play a role in altered function. Hypertrophy results not only in a quantitative increase in myocyte size but also in qualitatively different patterns of gene expression and is known to alter performance without changing morphology. 4 Because catecholamines induce hypertrophy in cell culture 5,6 and are elevated in brain death, 2,7,8 perhaps brain death initiates a hypertrophic response in vivo, which adversely affects cardiac function.
We tested the hypothesis that acute brain death from increased ICP alters the expression of left ventricular (LV) myocardial genes important in contractility. The physiologic, electrocardiographic, and histologic parameters were assessed at timed intervals with a balloon expansion model of increased ICP in rabbits. Because sympathetic stimulation of cardiomyocytes affects several subcellular compartments and to more fully characterize the response of the myocardium, expression levels of mRNAs encoding cardiac myofilaments, adrenergic receptors, sarcoplasmic reticulum proteins, and heat shock protein 70 (hsp70) were measured. Additionally, immediate early genes known to be regulated by adrenergic receptor activation were assayed as early markers of transcriptional change.
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Methods |
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Mechanical ventilation through a tracheostomy was normalized with serial arterial blood gas measurements. Pulse and blood pressure were continually monitored through a carotid arterial line and recorded at baseline and periodically until death. Invasive intrathoracic monitoring was avoided by design to minimize catecholamine release as a result of surgical stress. Electrocardiographic recordings (limb leads) were taken at baseline and periodically after brain injury. Except for histologic features, all data were generated from the same animals.
Animals were randomly assigned to 1 of 3 groups: untreated naive rabbits (n = 4), experimental increased-ICP rabbits (n = 9), and sham-operated rabbits (n = 9). Untreated, naive rabbits were anesthetized and rapidly killed to establish baseline mRNA expression levels (time = 0). Experimental (increased-ICP) animals had a 5-mm burr hole placed in the right parietal calvarium, and an 8F balloon-tipped catheter was inserted between the dura and calvarium. At time point 0, the balloon was rapidly inflated with 3 mL saline solution to produce increased ICP. Brain death occurred within minutes of balloon inflation, as assessed by episodic electroencephalograms. Sham-operated rabbits also underwent burr hole placement, but no intracranial catheter was inserted to avoid any increased ICP. Animals in both experimental and control groups were killed at 1, 2, and 4 hours (n = 3 per time point for a total; n = 9 per experimental group). Hearts were rapidly excised, rinsed with cold heparinized saline solution, and the right and left ventricles were dissected. Myocardial tissue was snap frozen in liquid nitrogen and stored at –70°C for mRNA analysis.
mRNA analysis
Total RNA was isolated with the guanidinium isothiocyanate/CsCl method. 9 LV tissue (0.5 g) was pulverized under liquid nitrogen, homogenized in 4 mol/L guanidinium isothiocyanate/CsCl (9 mL) using a tissue homogenizer (maximum speed, 1 minute; PT10-3; Polytron, Brinkman Instruments, Westbury, NY), and adjusted to 2 mol/L cesium chloride (1 g/2.5 mL). The homogenate was layered onto a 5.7 mol/L cesium chloride cushion and centrifuged at 210,000g for 16 to 20 hours at 20°C. The RNA pellet was resuspended in water and stored at –70°C.
The integrity of mRNA and appropriate hybridization and wash stringencies were confirmed by Northern blot. Slot blot hybridization was used for quantitative analyses. 10 Four micrograms of total RNA from each specimen were immobilized on nitrocellulose membrane. Membranes were prehybridized at 42°C overnight in hybridization buffer (4 x SSC, 50 mmol/L NaH2PO4, 0.2% sodium dodecylsulfate, 5 x Denhardt's solution, 200 mg/mL transfer RNA, 50% formamide) and then sequentially hybridized with a series of specific complementary [32P]-labeled probes. Between probings, membranes were stripped at 80°C for 2 minutes in 1% glycerol and exposed to film overnight to verify the absence of signal.
Oligonucleotide probes were radiolabelled with [32P]-adenosine triphosphate (ATP) and the T4 polynucleotide kinase reaction. Complementary DNAs were radiolabelled with [32P]-deoxycytidine triphosphate and a random primed Klenow reaction. Specific gene probes and hybridization conditions tested are summarized in Table I. Total poly(A)+ RNA was estimated by hybridization with a [32P]-poly d(T) probe (Clontech Labs Inc, Palo Alto, Calif).
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Quantitation of systemic catecholamines
Plasma epinephrine and norepinephrine were measured before and at 0, 1, 5, 10, 15, 30, 45, and 60 minutes after induction of increased ICP and every 60 minutes thereafter. Levels were determined by electrochemical detection with an electrochemical detector (Coulochem II; ESA, Inc, Bedford, Mass).* Catecholamines were extracted from plasma with alumina oxide and separated by high-pressure liquid chromatography with an isocratic mobile phase comprised of 85% NaH2PO4 and 15% acetonitrile adjusted to pH 3.0 with H3PO4 . Run time was approximately 28 minutes. The apparatus was standardized daily at the 50, 200, and 700 pg/mL concentration level with a mean coefficient of variation (n = 5) of 6.4% and 4.4% for epinephrine and norepinephrine, respectively. The limit of detection was 10 pg/mL.
Histologic evaluation
To evaluate histologic injury, a separate group of hearts (n = 4) were harvested. One hour after increased ICP (or sham operation) and systemic heparinization (1000 units, intravenously), excised hearts were immersed in saline solution (4°C) and sequentially perfused: (1) with saline solution until residual blood was removed, (2) for 2 minutes with modified Karnovsky's fixative (1% glutaraldehyde, 1.5% paraformaldehyde in 0.1 mol/L sodium-cacodylate buffer, pH 7.2), and (3) for 3 minutes with 3% glutaraldehyde and 1.5% paraformaldehyde in the same buffer. Two-millimeter transverse sections were immersed in fixative (3% glutaraldehyde and 1.5% paraformaldehyde) for 1 hour at 21°C, then overnight at 4°C. Each specimen was dehydrated and embedded in paraffin blocks. Three nonadjacent 7-µm sections (taken from separate blocks) were stained with hematoxylin and eosin.
These sections were evaluated by a cardiac pathologist blinded to experimental group. A glass template was used to identify 8 regions per section. Each region was semiquantitatively evaluated for 4 characteristics: the presence of contraction bands, myocytolysis, pyknotic nuclei, and loss of myofibrillar striations. Respectively, scores of 0, 0.5, 1.0, 1.5, and 2.0 indicated that 0%, 1% to 25%, 26% to 50%, 51% to 75%, and 76% to 100% of cells within that section manifested the indicated characteristic.
Statistical analyses
Statistical analyses were performed with SAS (Statistical Analysis Systems, Cary, NC). Hemodynamic and catecholamine data (both of which were repeated measures over time) were analyzed between groups by rank-ordered 2-way analysis of variance (ANOVA) and over time by rank-ordered 1-way ANOVA. Results were rank ordered because variances within groups were dissimilar (PROC MIXED; SAS). Histologic results were analyzed by ANOVA comparing 2 groups and modeling for the variation of multiple samplings from each animal (PROC GLM; SAS). mRNA results were analyzed with 2-way ANOVA, treating each time point as an independent group because different animals were killed to generate each time point (PROC GLM; SAS). A trend toward statistical significance was accepted at P 
.10.
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11(bradycardia and hypertension, associated with increased ICP) occurred in all increased-ICP animals. Bradycardia was inconsistently observed in the increased-ICP group, lasting only a few beats. Tachycardia was invariably present (Fig l, A). At 1 minute, the heart rate of increased-ICP animals increased from a mean of 240 beats/min to 295 beats/min. By 5 minutes, heart rate fell to 247 beats/min and gradually declined to 190 beats/min over 4 hours, a level significantly lower than baseline.
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). Mean arterial blood pressure increased significantly from a baseline value of 73 mm Hg (systolic/diastolic, 93/64 mm Hg) to a peak of 96 mm Hg (systolic/diastolic, 118/87 mm Hg) by 1 minute. By 5 minutes, mean arterial pressure dropped to 52 mm Hg (systolic/diastolic, 68/34 mm Hg) and by 4 hours to 26 mm Hg (systolic/diastolic, 42/20 mm Hg), significantly below baseline levels. Sham-operated animals showed no significant change in heart rate over 4 hours and manifested significantly higher mean arterial pressure when compared with the increased-ICP group. Electrocardiographic ST segments were also evaluated. Abnormal ST segments were observed in all rabbits in the increased-ICP group, with no change in sham-operated animals over time. Electrocardiograms showed sustained ST segment depression, ST segment elevation, or T-wave inversion, which persisted variably over the course of the experiment.
Systemic catecholamine levels
The pattern of response of systemic catecholamine levels was uniform in all increased-ICP animals, with a massive spike in systemic levels of both epinephrine and norepinephrine 1 minute after balloon inflation. Systemic epinephrine levels (Fig 2, A) started at a baseline level of 146 pg/mL, were significantly elevated 5-fold (722 pg/mL) at 1 minute, dropped below baseline (33 pg/mL) by 5 minutes, and were significantly decreased to undetectable levels (<10 pg/mL) by 2 hours. Systemic norepinephrine (Fig 2, B) behaved similarly, starting at a baseline level of 387 pg/mL, increasing 7- to 8-fold (P < .05) by 1 minute (3042 pg/mL), dropping below baseline at 5 minutes (286 pg/mL), and decreasing significantly to 25% of baseline levels by 1 hour (81 pg/mL). The decrease in catecholamine levels preceded the decline in heart rate but was concurrent with the decline in mean arterial pressure. No significant changes in systemic levels of epinephrine or norepinephrine were found in sham-operated animals.
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-actin were found 4 hours after increased ICP (Fig. 4, A and B) compared with sham-operated animals. In contrast, no differences in ß –myosin heavy chain (MHC) mRNA expression levels were found between increased-ICP and sham-operated hearts (Fig. 4, D). Levels of messages encoding -MHC significantly increased in both increased-ICP and sham-operated hearts by 4 hours (Fig. 4, D); however, no significant differences were detected between groups.
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1-adrenergic receptor or ß1-adrenergic receptor mRNA levels (Fig. 4, E and F).
Levels of mRNAs encoding sarcoplasmic reticulum proteins. Blots were sequentially hybridized to CaATPase, phospholamban, and ryanodine receptor cDNA probes (Fig. 4, G, H, and I, respectively). When mRNAs in increased-ICP hearts were compared with sham-operated hearts, a trend towards a significant increase (P = .076) in CaATPase mRNA level was found 4 hours after increased ICP (Fig. 4, G). The relative levels of mRNAs encoding the calcium-release channel (ryanodine receptor) and phospholamban were not significantly different between groups (Fig. 4, H and I).
Levels of mRNAs encoding immediate early genes and cellular stress response genes. The expression levels of immediate early genes, c-fos, c-jun, and egr-1, were assayed (Fig. 4, J, K, and L, respectively). Increased ICP specifically increased levels of mRNA encoding the transcription factor egr-1 relative to sham-operated controls at 1 and 4 hours. A trend toward higher expression levels of c-fos mRNA was present in increased-ICP hearts compared with sham-operated controls. No significant differences in c-jun mRNA levels were found between increased-ICP and sham-operated controls. A significant increase in hsp70 mRNA was found 4 hours after increased-ICP hearts relative to sham-operated controls (Fig. 4, M).
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Discussion |
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-actins, and hsp70 were also noted (Table III). Increased ICP elicits a molecular response similar to 1-adrenergic receptor stimulation.
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7 Whereas increased mechanical load and -adrenergic receptor stimulation lead to hypertrophy, ischemia reperfusion injury does not.
The phenotype of hypertrophy, whether initiated by increased hemodynamic load or adrenergic receptor activation, is characterized by selective changes in gene expression (Table III). 12 mRNAs for c-fos and c-myc 13,14 increase concurrently with increased expression of hsp70. 15 Transient increases in -actin mRNAs and lasting increases in ß-MHC mRNAs are also found. 12,16 Hypertrophy as the result of -adrenergic receptor activation can be further distinguished from that induced by mechanical load by the induction of egr-1. 13,14 In our study, the increase in egr-1 implicates catecholamines in the observed changes in gene expression; however, the prolonged peak in egr-1 mRNA levels differs from that associated with -adrenergic receptor activation alone. The significance of this finding is currently under investigation.
ß-Adrenergic receptors can be regulated by (1) receptor sequestration, (2) decreased levels of mRNAs encoding the receptor, and (3) receptor uncoupling from its second messenger. In previous studies of brain death from increased ICP in rabbits, Bittner and colleagues 8 documented uncoupling of the b-adrenergic receptor from its adenylate cyclase second messenger pathway (with no change in the number of cell surface receptors). In our study, mRNA levels of ß-adrenergic receptor did not change in response to increased ICP. Thus the prolonged egr-1 activation observed by us and the uncoupling of ß-adrenergic receptor demonstrated by others suggests an imbalance in - and ß-adrenergic receptor stimulation. Whether this contributes to the myocardial dysfunction observed in hearts after brain death is not known.
The role of ischemia reperfusion in this phenomenon cannot be discerned histologically because ischemia-reperfusion injury and catecholamines both have been reported to cause contraction band necrosis. At the molecular level, ischemia-reperfusion injury has been reported to increase the expression of c-fos, hsp70, 17,18 and sarcoplasmic reticulum proteins (CaATPase and phopholamban). 19,20 In this study, the increase in hsp70 mRNA levels is modest and late. This, in conjunction with the rapid dissipation of elevated catecholamines and the pressor response, argues that the contribution of ischemia reperfusion is small.
The phenotype of congestive heart failure, a scenario also associated with elevated catecholamines, bears note because it differs from that observed in brain death. Unlike brain death, congestive heart failure is associated with chronic elevation of systemic catecholamines 21and depressed levels of mRNAs encoding proteins of the sarcoplasmic reticulum, -actin and ß-MHC. 22 Increased ICP results in the transient elevation of systemic catecholamines followed by rapid dissipation to subnormal levels, and no significant changes in mRNAs encoding sarcoplasmic reticulum proteins are found (Table III). We suspect that these differences reflect the levels and temporal course of circulating catecholamines.
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Conclusion |
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-adrenergic receptor activation. 14 Although the functional significance of these molecular changes is unknown, defining these changes may provide important clues to ameliorate the functional deterioration of myocardial function after brain death. |
Appendix: Discussion |
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Dr Yeh. We are currently in the process of analyzing brain death in the presence of adrenergic blockade. Although it will obviously be impossible to use adrenergic blockade in clinical donors before head injury, I believe these data may have broader implications in the realm of surgical stress. Controlling catecholamine release during general anesthesia, operation, weaning from cardiopulmonary bypass, and emergence from anesthesia may prove to be very important.
Dr Verdi J. DiSesa (Chicago, Ill). If I understood you correctly, you are measuring mRNA transcription, not gene expression, in terms of protein levels. Is that correct?
Dr Yeh. That is correct.
Dr DiSesa. Because you are measuring mRNA and not protein, as you know, protein levels are a balance between synthesis and degradation. Maybe what you are really seeing is a compensatory mechanism for some change in the subsequent protein metabolism of these brain-injured animals.
Dr Yeh. We have not planned on pursuing that at the moment. We are currently investigating the effects of adrenergic blockade, but I think it will be important to confirm that changes in mRNA levels are ultimately reflected in altered protein levels.
Mr Magdi Yacoub (London, England). In choosing the mechanism, the neurohumoral factors that are implicated in this, you concentrated on sympathetic -agonists, but there could be other neural or, indeed, humoral peptidergic or other. Have you looked at any of these?
Dr Yeh. I think you are absolutely right to question this. Although we have not investigated other neurohumoral factors, brain injury profoundly alters the hormonal milieu of the body. I believe that one of the prime candidates may be thyroid hormone (which we plan on investigating).
Mr Yacoub. My second question is again about the target genes. You have looked at a variety of contractile proteins, for example, in phospholamban and calcium-handling proteins, but what about other more fundamental things like G proteins? Virginia Owens in our group has been looking at G protein expression and found significant changes associated with brain death. Have you looked at that?
Dr Yeh. No, we did not examine G proteins.
Dr Michael Grosso (Moorestown, NJ). After the first 30 minutes, it appeared that the blood pressure was significantly depressed and remained so for the remaining period of the experiment. How much do you think hypoperfusion of the myocardium contributed to your findings? Also, if you had taken a little bit different approach and corrected that hypotension, as we do in the clinical setting of donor treatment, would that have altered your findings?
Dr Yeh. That is an excellent question. The in vivo model mandated by our hypothesis makes it difficult to discern a mechanism. I think there are 3 possible explanations. Ischemia-reperfusion injury is one of those, although the limited induction of heat shock protein argues against this. Altered myocardial loading from the observed alterations in blood pressure is another. Finally, catecholamines may be exerting a direct trophic effect on myocardial gene expression.
We intentionally did not correct hypotension with catecholaminergic agents because we wanted to study the unmodified phenomenon of brain death. Clinically, exogenous inotropic stimulation has stabilized many donors, but would not it be interesting if part of what we were doing was to supply an exogenous trophic influence while the heart and maintain an "optimal" pattern of gene expression?
Dr Juan C. Chachques (Paris, France). Did you study LV function in your model? I do not see any parameters of LV function related with the LV adrenergic expression.
Dr Yeh. We intentionally avoided invasive intrathoracic monitoring in this model simply to minimize the catecholamine release accompanying thoracotomy; therefore we have no sophisticated functional analyses of these hearts. Nevertheless, this type of analysis will ultimately be central in defining exactly what is a "favorable" pattern of gene expression.
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Acknowledgments |
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812 and p251-1 ßMHC, to Dr L. Jones (University of Indiana) for pGEM3z-PLB7, to Dr L. Kedes (University of Southern California) for LK295 (skeletal -actin), LK300 (cardiac -actin), to Dr T. Curran (Roche Institute) for pSp65-cfos, to Dr R. J. Lefkowitz (Duke University) for pTZ18R 1-adrenergic receptor and pSP65 ß1-adrenergic recptor, and to Dr V. Sukhatme for pUC13.191. Egr-1, pH-2.3, and hsp70 were purchased from American Type Culture Collection. |
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1-adrenergic receptor-stimulated hypertrophy of cultured rat heart myocytes. J Clin Invest 1990;85:1206-14. 1-mediated response. J Biol Chem 1990;265:13809-17. 1-adrenergic receptor signaling in cardiac cells. EMBO J 1993;13:5131-9. 1-adrenoceptors. Cardiovasc Res 1996;32:374-83. This article has been cited by other articles:
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  A. H. Harken Brain death leads to abnormal contractile properties of the human donor right ventricle J. Thorac. Cardiovasc. Surg., July 1, 2006; 132(1): 10 - 11. [Full Text] [PDF]
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 N. M. Banki, A. Kopelnik, M. W. Dae, J. Miss, P. Tung, M. T. Lawton, B. J. Drew, E. Foster, W. Smith, W. W. Parmley, et al. Acute Neurocardiogenic Injury After Subarachnoid Hemorrhage Circulation, November 22, 2005; 112(21): 3314 - 3319. [Abstract] [Full Text] [PDF]
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 S. C. Stoica, D. K. Satchithananda, C. Atkinson, S. Charman, M. Goddard, and S. R. Large Heat shock protein, inducible nitric oxide synthase and apoptotic markers in the acute phase of human cardiac transplantation Eur. J. Cardiothorac. Surg., December 1, 2003; 24(6): 932 - 939. [Abstract] [Full Text] [PDF]
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T. Yeh Jr, A. S. Wechsler, L. Graham, K. E. Loesser, D. A. Sica, L. Wolfe, and E. R. Jakoi Central sympathetic blockade ameliorates brain death-induced cardiotoxicity and associated changes in myocardial gene expression J. Thorac. Cardiovasc. Surg., December 1, 2002; 124(6): 1087 - 1098. [Abstract] [Full Text]
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S. C. Stoica, D. K. Satchithananda, J. Dunning, and S. R. Large Two-decade analysis of cardiac storage for transplantation Eur. J. Cardiothorac. Surg., October 1, 2001; 20(4): 792 - 798. [Abstract] [Full Text] [PDF]
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F. Farhat, D. Loisance, J.-P. Garnier, and M. Kirsch Norepinephrine release after acute brain death abolishes the cardioprotective effects of ischemic preconditioning in rabbit Eur. J. Cardiothorac. Surg., March 1, 2001; 19(3): 313 - 320. [Abstract] [Full Text] [PDF]
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 D. LYN, X. LIU, N. A. BENNETT, and N. L. EMMETT Gene expression profile in mouse myocardium after ischemia Physiol Genomics, April 27, 2000; 2(3): 93 - 100. [Abstract] [Full Text] [PDF]
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denotes a trend toward significance (P < .1). Sham-operated controls are indicated by the solid line. Increased-ICP animals are indicated by the dashed line.
ICP contrasted to