Ann Thorac Surg 1995;59:1182-1186
© 1995 The Society of Thoracic Surgeons
Novel Technique to Bioassay Endocardium-Derived Nitric Oxide From the Beating Heart
Berent Discigil, MD,
Paul J. Pearson, MD, PhD,
Yeow L. Chua, MD,
Paulo R. B. Evora, MD, PhD,
John F. Seccombe, MD,
Hartzell V. Schaff, MD
Cardiac Surgical Research and Section of Cardiovascular Surgery, Mayo Clinic and Mayo Foundation, Rochester, Minnesota
Accepted for publication February 1, 1995.
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Abstract
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Nitric oxide is a potent vasodilator and antiplatelet substance released by the vascular endothelium. In the current study, isolated rabbit hearts were perfused retrograde in the aortic root with a balanced salt solution using a Langendorff technique. To perfuse the right cardiac chambers, an inflow cannula was placed in the superior vena cava and an outflow cannula in the right ventricular apex via the pulmonary artery. To detect endocardial vasodilator production, right heart perfusate was used to bathe a ``bioassay'' segment of canine coronary artery denuded of endothelium. Perfusate from unstimulated hearts did not alter smooth muscle tone in the bioassay tissue. Calcium inophore, a potent stimulus for endothelial nitric oxide production, produced relaxation of the bioassay smooth muscle when added to the cardiac perfusate but not when applied directly to the bioassay segment. Cardiac effluent vasodilator activity was abolished by removal of the endocardium or addition of nitric oxide synthesis inhibitors, but not by prostanoid inhibitors. These experiments describe a practical method to bioassay endocardial nitric oxide production in the beating heart.
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Introduction
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The nitric oxide radical has been identified as the mediator of endothelium-dependent vasodilation [1] first described by Furchgott and Zawadzki in 1980 [2]. Nitric oxide now is considered to be a ubiquitous and important modulator of vascular tone [3]. However, the nitric oxide radical also has the important properties of inhibiting platelet adhesion to the endothelium [4] and preventing platelet aggregation [5]. These antithrombogenic properties of endothelium-derived nitric oxide would be particularly important in the cardiac chambers, where myocardial dysfunction can lead to conditions potentiating thrombus formation [6].
Although endocardial endothelium has a different morphology than vascular endothelium [7], recent studies have shown that cultured porcine endocardial cells can release an unstable humoral agent, which has relaxing effects on the endothelium-denuded pig coronary artery; this substance is indistinguishable from endothelium-derived nitric oxide [8]. However, cultured and native endothelial cells do not always exhibit comparable physiology [9, 10]. The present experiments were undertaken to develop a bioassay system to detect the production of endocardium-derived nitric oxide from the isolated, perfused, beating rabbit heart. This technique may be a useful tool to study the effect of drug–receptor interactions, physiochemical stimuli, or other pathologic conditions on the production of nitric oxide by the endocardium.
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Material and Methods
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Isolated Heart Preparation
Adult female New Zealand white rabbits (weighing 3 to 4 kg) were anesthetized by intramuscular injection of ketamine hydrochloride (88 mg/kg; Aveco Co, Fort Dodge, IA), acepromazine maleate (3 mg/kg; Fort Dodge Laboratories, Fort Dodge, IA), and xylazine (12 mg/kg; Mobay Co, Shawnee, KS) and anticoagulated with heparin (1,000 units, intravenous injection; Upjohn, Kalamazoo, MI). When an adequate level of anesthesia was achieved, cardiectomy was performed through a median sternotomy. Beating hearts were immediately placed in cool, Krebs-Ringer bicarbonate solution of the following millimolar composition: NaCl, 118.3; KCl, 4.7; MgSO4, 1.2; KH2PO4, 1.22; CaCl2, 2.5; NaHCO3, 25.0; and glucose, 11.1. Hearts then were suspended from a Langendorff perfusion column [11], and retrograde perfusion was established at 37°C with an overflow system to maintain a constant coronary perfusion pressure of 50 mm Hg [12]. The delay between harvest of the heart and initiation of perfusion was less than 1 minute. The nonrecirculated physiologic salt solution was bubbled with 95% oxygen and 5% carbon dioxide gas mixture to achieve an oxygen tension between 400 and 450 mm Hg, and the solution was infused through a 0.2-µm cardioplegia filter to remove all particulate debris (CPS-O2; PALL Biomedical, Glen Cove, NY) (Fig 1
).
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Fig 1. . Apparatus for bioassay of vasodilators from the isolated perfused heart. The cardiac chambers are perfused through a separate constant-flow perfusion loop, and vasoactivity of effluent from the cardiac chambers is bioassayed on a ring of epicardial coronary artery smooth muscle. (Ao = aorta; KRBG = Krebs-Ringer bicarbonate solution; PA = pulmonary artery; SVC = superior vena cava.)
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After retrograde perfusion was established, the sinus node was crushed and pacing electrodes attached. The heart was paced 50% above threshold at a rate of 210 beats/min [13].
Right Cardiac Chambers Perfusion Circuit
To create a perfusion circuit to bioassay vasoactive factors released by the right heart endocardium, the inferior vena cava was ligated and the tricuspid valve leaflets were excised. A perfusion cannula was secured into the superior vena cava for perfusion inflow, and another cannula was placed at the base of the right ventricle via the pulmonary artery for perfusion outflow. From the epicardial surface of the heart, the coronary sinus was opened at the interatrial groove just before entering the right atrium, and the effluent was diverted outside the heart. From the endocardial surface, the coronary sinus ostium was sutured closed. Perfusion (physiologic salt solution, 95% O2/5% CO2, pH = 7.4) through the right cardiac chambers was commenced at a constant flow of 8 mL/min using a roller pump (Minipuls 2; Gilson Medical Electronics, France). Before entering the heart, the solution was passed through a heat exchanger to maintain perfusate at 37°C. The perfusate flowed out of the heart via the pulmonary artery cannula, and the vasoactivity of the effluent was bioassayed on a segment of canine coronary artery smooth muscle in a manner similar to that described by Cocks and associates [9] (see Fig 1
).
Bioassay Detector Tissue
Heartworm-free mongrel dogs (25 to 30 kg) of either sex were anesthetized with intravenous pentobarbital sodium (30 mg/kg bolus injection; Fort Dodge Laboratories) and exsanguinated. The heart was excised and the left circumflex artery dissected free. A coronary artery ring 5 mm in length was prepared for bioassay experiments; endothelium of the rings was removed by rubbing the intimal surface with a pair of watchmakers' forceps. The ring was suspended directly in a perfusion of physiologic salt solution (8 mL/min) by means of two steel stirrups passed through its lumen; one stirrup was connected to an isometric force transducer (Grass FT03; Grass Instrument Company, Quincy, MA). The coronary artery bioassay ring was placed at its optimal length tension by progressively stretching the ring until contraction to potassium ions (20 mol/L) was maximal [14]. The absence of endothelium on the bioassay ring was confirmed by the lack of relaxation to calcium ionophore A23187 (10-6 mol/L) administered directly to the bioassay ring during contraction to prostaglandin F2
(2 x 10-6 mol/L). Calcium ionophore causes endothelium-dependent vasodilation of the canine coronary artery with an intact endothelium [14].
Bioassay Technique
The bioassay system was arranged so that the coronary artery smooth muscle could be superfused with physiologic salt solution (direct superfusion) or with physiologic salt solution that first had passed through the right cardiac chambers of the isolated rabbit heart (endocardial superfusion) (see Fig 1). The time required for effluent exiting the heart to reach the bioassay ring was approximately 0.5 second. To bioassay the production of vasodilator by the endocardium, the bioassay ring was contracted by adding prostaglandin F2 (2 x 10-6 mol/L) to the perfusate fluid. The vasoconstrictor action of prostaglandin F2 on the canine coronary artery smooth muscle is concentration-dependent. To prevent dilution of the perfusate containing prostaglandin F2 as it passed through the right heart by the small but significant venous return from the thebesian veins, prostaglandin F2 (2 x 10-6 mol/L) also was added to the aortic perfusion fluid during superfusion experiments. Prostaglandin F2 in the Langendorff perfusate caused no perceptible impairment in cardiac function. Indomethacin (10-5 mol/L) also was added to the physiologic salt solution at the beginning of the experiment to block the synthesis of prostanoids.
In some experiments, the endocardium of the beating heart was removed by infusing 2 mL of a 1% solution of Triton X-100 (diluted in physiologic salt solution) into the superior vena cava cannula [8]. Triton X-100 at this concentration caused no perceptible impairment in cardiac function. The effectiveness of endocardial disruption was confirmed with scanning electron microscopy of the endocardium at the conclusion of the experiment. Samples of the right ventricle were prepared for study of the endocardium by fixation in Trump's EM fixative solution; material was rinsed in 0.1 mol/L phosphate buffer, dehydrated in series of alcohols, and then coated with gold/palladium (Fig 2).
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Fig 2. . Scanning electron micrographs of the endocardial surface of rabbit right ventricle fixed in Trump's EM fixative and coated with gold/palladium. (A) Untreated ventricle (original magnification, x500) showing an undulating continuous sheet of endothelial cells. (B) Ventricle treated with Triton-X 100 (original magnification, x1,700). The plasmalemma of endothelial cells is perforated with numerous small pores.
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At the end of each experiment, the ability of the bioassay vascular smooth muscle to relax was tested by infusing sodium nitroprusside (10-4 mol/L) directly onto the bioassay ring during contraction with prostaglandin F2.
All animals received humane care in compliance with the ``Principles of Laboratory Animal Care'' formulated by the National Society for Medical Research and the ``Guide for the Care and Use of Laboratory Animals'' prepared by the National Academy of Sciences (NIH publication 85-23, revised 1985).
Drugs
The following drugs were used: calcium ionophore A23187 free acid; prostaglandin F2, trissalt; indomethacin; NG-nitro-L-arginine; sodium nitroprusside; and Triton X-100 (t-Octylphenoxypoly-ethoxyethanol) (all from the Sigma Chemical Company, St. Louis, MO). The concentrations of drugs are expressed as final molar concentration in the physiologic salt solution.
Data Analysis
The results are expressed as means ± standard error of the mean. In all experiments, n refers to the number of animals from which hearts or bioassay rings were taken. Relaxations are expressed as percent change in tension from the contraction of the bioassay ring to prostaglandin F2. Statistical evaluation of the data was performed by Student's t test for either paired or unpaired observations. Values were considered to be significantly different when p was less than 0.05.
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Results
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Physiologic salt solution with prostaglandin F2 (2 x 10-6 mol/L) infused through the stainless steel cannula (direct superfusion) induced a stable contraction in the canine coronary artery bioassay ring of 5.14 ± 0.78 g (mean ± standard error of the mean; n = 6). A bolus injection of the calcium ionophore A23187 (1 mL of a 10-6 mol/L solution) into the perfusate of the direct line caused no change in tension in the deendothelialized canine coronary artery bioassay ring. During contraction with prostaglandin F2, the superfusion of the bioassay ring was switched from the direct line to the cannula from the right cardiac chambers (endocardial superfusion) (Fig 3). There was no change in tension when the contracted bioassay ring was exposed to the endocardial perfusate. However, when the calcium ionophore A23187 was infused as a bolus injection into the right cardiac chambers, the bioassay ring exhibited a relaxation of 60.2% ± 12.2% of the initial prostaglandin F2 contraction (mean ± standard error of the mean; n = 6; p < 0.05) in the presence of indomethacin.
Addition of NG-nitro-L-arginine (10-4 mol/L) to the physiologic salt solution caused no change in contraction of the bioassay ring to prostaglandin F2. NG-nitro-L-arginine (infused for 45 minutes through the cardiac chambers) did not alter the response of the coronary artery ring to endocardial superfusion (ie, no relaxation). However, NG-nitro-L-arginine completely blocked the relaxation induced by infusion of A23187 into the right cardiac chambers: 2.6% ± 1.4% of initial prostaglandin F2 contraction (n = 6; p < 0.05) (Fig 4). NG-nitro-L-arginine did not alter the ability of the bioassay ring to relax to sodium nitroprusside given as a bolus injection (1 mL of a 10-4 mol/L solution) during direct superfusion.
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Fig 4. . Bioassay of calcium ionophore-induced release of endocardium-derived nitric oxide. Effluent from cardiac chambers perfused in the presence of calcium ionophore A23187 (10-6 mol/L) was used to bathe the canine coronary artery bioassay ring, which had been contracted with prostaglandin F2 (2 x 10-6 mol/L). Values are presented as means ± standard error of the mean and represent percent relaxation of the bioassay ring. Hearts were pretreated with either indomethacin or indomethacin plus NG-nitro-L-arginine. Asterisk denotes significant difference between each relaxation (p < 0.05).
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Removal of the endocardium with Triton X-100 did not alter the response of the coronary artery bioassay ring to endocardial superfusion. However, endocardial removal completely blocked the relaxation induced by infusion of A23187 into the right cardiac chambers: 76.5% ± 23.5% versus 4.4% ± 0.2% of initial prostaglandin F2 contraction before and after Triton X-100 treatment, respectively.
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Comment
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The endocardium modulates performance of underlying myocardium [8, 15–17] and can synthesize prostaglandins [18]. Smith and associates [8] have demonstrated that removal of the endocardium produces an unusual and characteristic negative inotropic effect on ferret papillary muscles; in their experimental preparations, treatment with Triton X-100 destroyed the endocardial monolayer and caused a reduction in muscle twitch duration and peak isometric tension with no change in Vmax. Further, these investigators found that substance P induced release of an endothelium-derived relaxing factor–like compound from endocardium of ferret papillary muscles and from cultured porcine endocardial cells. Effluent from cultured endocardial cells reversed the negative inotropic effect of removal of the endocardium [8].
The present study demonstrates production of endocardial-derived nitric oxide from the cardiac chambers of the beating heart. Because it prevents platelet adhesion to endothelium [4] and inhibits platelet aggregation [5], in addition to promoting platelet disaggregation, nitric oxide could serve a vital antithrombotic function in the cardiac chambers.
There are three important aspects of the experiment that support the conclusion that nitric oxide is released from the endocardium. First, increases in intracellular calcium concentrations play an important role in release of nitric oxide from endothelial cells [19], and in our study, release of vasodilator from the right cardiac chambers occurred in direct response to exposure to the calcium ionophore A23187. Second, release of vasodilator from the right cardiac chambers was inhibited by NG-nitro-L-arginine, a competitive inhibitor of nitric oxide synthesis from L-arginine [20]. The finding that indomethacin did not prevent vasodilator synthesis indicates that the substance is not a prostanoid; this is additional evidence that the vasodilator is nitric oxide.
The vasodilator was produced by the intima, as evidenced by a complete absence of vasodilator production after endocardial disruption.
In the present model, we could not demonstrate a basal release of endothelium-derived nitric oxide (ie, release in the absence of stimulation by an agonist) from the cardiac chambers, as is typically present in other blood vessels [21]. However, the high partial pressure of oxygen of the perfusate used in this experiment could have generated oxygen-derived free radicals, which inactivate nitric oxide [22].
Nitric oxide production by vascular endothelial cells is a universally important mechanism of vascular tone and platelet aggregation in mammals. Indeed, endothelium-dependent vasodilation is even present in the vasculature of lower vertebrates [23]. We now provide evidence of nitric oxide production by vascular tissue in which the nitric oxide radical would not necessarily function as a vasodilator, but as an antithrombotic agent.
In conclusion, we describe a technique to bioassay the production of nitric oxide from the endocardium of the beating heart. Such a model will be useful to define the effect of drugs, physiochemical stimuli, and cardiac function on the production of endocardial-derived nitric oxide. As thrombus formation in the cardiac chambers is a major clinical problem, such a model also could help elucidate mechanisms responsible for intracardiac thrombogenesis.
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Acknowledgments
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This work was supported in part by grant-in-aid research award MHA-108 from the Minnesota Heart Association.
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Footnotes
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Address reprint requests to Dr Schaff, Section of Cardiovascular Surgery, Mayo Clinic and Mayo Foundation, 200 First St SW, Rochester, MN 55905.
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References
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Abstract
Introduction
