Effects of l-Arginine and Nω-Nitro-l-Arginine Methyl Ester on Cardiac Perfusion and Function After 1-Day Cold Preservation of Isolated Hearts
Background Coronary flow responses to endothelium-dependent (acetylcholine [ACh] or 5-hydroxytryptamine [5-HT]) and endothelium-independent (adenosine [ADE] or nitroprusside [NP]) vasodilators may be altered before and after 1-day hypothermia during the perfusion of arginine vasopressin (AVP), d-arginine (D-ARG), l-arginine (L-ARG), or nitro-l-arginine methyl ester (L-NAME).
Methods and Results Four groups of guinea pig hearts (37.5°C [warm]) were perfused for 6 hours with AVP, L-ARG, L-NAME, or nothing (control). Five heart groups (cold) were perfused with AVP, D-ARG, L-ARG, L-NAME, or nothing (control), but after 2 hours they were perfused at low flow for 22 hours at 3.7°C and again for 3 hours at 37.5°C. ADE, butanedione monoxime, and NP were given for cardioprotection before, during, and after hypothermia. In warm groups, L-ARG did not alter basal flow or ADE, ACh, 5-HT, or NP responses, whereas L-NAME and AVP reduced basal flow and the ADE response, abolished ACh and 5-HT responses, and increased the NP response. In cold groups after hypothermia, L-ARG did not alter basal flow, but L-NAME, AVP, D-ARG, and control reduced flow. In the postcold L-ARG group, ACh increased peak flow, but NP did not increase flow in other cold groups. Effluent L-ARG and L-CIT in the cold control group fell from 64±9 and 9±1 μg/L at 1 hour to 36±5 and 5±1 μg/L at 25 hours, respectively. Left ventricular pressure and cardiac efficiency improved more in the postcold L-ARG group than in the postcold D-ARG, AVP, and L-NAME groups.
Conclusions Endogenous effluent levels of L-ARG and L-CIT decrease after 24 hours in isolated hearts, whereas perfusion of L-ARG improves cardiac performance, basal coronary flow, and vasodilator responses. In contrast, L-NAME, L-ARG, and AVP limit flow and performance but maintain a partial vasodilatory response to NP. Sustained release of NO may account for improved performance after L-ARG after hypothermia.
Most experimental models of cardiac preservation have focused on return of mechanical function as an indicator of the quality of protection after cold storage1 or cold perfusion.2 Suboptimal global myocardial perfusion, however, is also an important factor limiting contractile function on normothermic reperfusion; the supply of nutrients and O2 may not meet the metabolic demand.3 In addition, restoration of mechanical function may be dependent not only on maintenance of normal perfusion but also on the adequacy of coronary flow reserve. Vasodilation can be mediated through specific agents that produce effects directly on vascular smooth muscle cells or indirectly through vascular endothelial cells.4 5 Prolonged cold storage has been reported to abolish endothelium-dependent relaxing responses in isolated porcine coronary arteries.6 Basal coronary flow is reduced and the coronary flow response to vasodilators is severely blunted or absent on reperfusion after 1-day hypothermic perfusion without added myocardial protection.3
The common experimental approach to long-term cardiac preservation is to flush the coronary bed with a high-K+ or intracellular-type storage solution and to store hearts hypothermically.7 8 9 An alternative approach to protect ex vivo hearts is to perfuse them with a cold, but normal, extracellular ionic solution containing metabolic inhibitors and vasodilators. We have reported in isolated guinea pig hearts that the cardiac depressant drug and vasodilator BDM, when infused before, during, and initially after 1 day of hypothermic perfusion with a modified Krebs-Ringer solution, restores coronary flow and cardiac function much better than a cold high-K+– or a cold low-Ca2+–containing solution.3 The improvement in myocardial perfusion, vasodilator responsiveness, and contractile function after BDM treatment is enhanced even more when ADE10 or nitrobenzylthioinosine11 is given during rewarming with NP just before and initially after normothermic reperfusion.
In a previous study,10 our best protocol for cardiac and vascular protection was to infuse BDM before, during, and initially after hypothermia and to infuse ADE and NP only initially during warm reperfusion. In the present study, each hypothermia group was perfused continuously with an extracellular solution containing not only BDM and NP but also ADE to attempt additional myocardial protection. Because this drug combination resulted in improved responses to a variety of vasodilators after hypothermia, the role of endothelium-relaxing factor, or NO, in maintenance of vascular responsiveness after 1-day hypothermic preservation could be more clearly defined.
In the presence of these cardioprotective agents, we examined mechanical and metabolic function and the coronary flow responses to various vasodilators after prolonged hypothermic perfusion in the presence of a substrate and a substrate inhibitor of NOS. Coronary responses to drugs that have endothelium-dependent and -independent actions on vascular smooth muscle tone were tested: ADE, ACh, 5-HT, and NP. These responses were tested in nine groups that were continuously infused in the absence (control) or presence of L-ARG, the biologically active NOS substrate; D-ARG, the inactive enantiomer of L-ARG; L-NAME, an analogue of L-ARG and a blocker of NOS; or AVP, primarily an endothelium-independent V1 receptor agonist that served as a vasoconstrictor control for L-NAME. L-NAME is effectively a vasoconstrictor in this model because of NOS-inhibited basal vasodilation. In addition, coronary effluent concentrations of L-ARG and L-CIT, the coproduct with NO of NOS, were measured at 1 and 25 hours in the cold control group.
Preparation and Measurements
Approval from the institutional animal studies committee was obtained before initiation of this study. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication No. 85-23, revised 1985). Albino English short-haired guinea pigs (400 to 600 g) were injected intraperitoneally with 10 mg ketamine and 1000 U heparin, and they were decapitated when unresponsive to noxious stimulation. Isolation and preparation of hearts as used for this study have been detailed in recent reports.3 10 11 12 13 The inferior and superior venae cavae were cut after thoracotomy, and the aorta was cannulated distal to the aortic valve. Each heart was immediately perfused and then excised. All hearts were perfused at a constant aortic root perfusion pressure of 55 mm Hg during normothermic perfusion. The perfusate, a modified Krebs-Ringer solution, was filtered (5-μm pore size) in-line (Astrodisc, Gelman Scientific) and had the following control composition (in mmol/L): Na+ 137, K+ 5, Mg2+ 1.2, Ca2+ 2.5, Cl− 134, HCO3− 15.5, H2PO4− 1.2, pyruvate 2, glucose 11.5, mannitol 16, glutamate 0.05, and EDTA 0.05 plus insulin 5 U/L. LV pressure was measured isovolumetrically with a transducer connected to a thin, saline-filled latex balloon that was inserted into the LV through the mitral valve from a cut in the left atrium. Balloon volume was adjusted to maintain a diastolic LV pressure of 0 mm Hg during the initial control period. Two pairs of bipolar electrodes (Teflon-coated silver, diameter of 125 μm) were placed in each heart to monitor intracardiac electrograms from which spontaneous sinoatrial rate and atrioventricular conduction time were measured as noted previously.14 Coronary sinus effluent was collected by placing a cannula into the right ventricle through the pulmonic valve after ligation of the venae cavae. Coronary inflow (aortic) was measured at a constant temperature with a transit time, in-line ultrasonic flowmeter (Research Flowmeter T106, Transonic Systems Inc).
Coronary inflow and outflow (coronary sinus) O2 tensions were measured continuously on-line (Instech 203B) and verified simultaneously off-line with an intermittently self-calibrating analyzer system (Instrumentation Labs 813) as described previously.3 10 11 12 13 O2 consumption and % O2E were measured in all studies to assess the direct vasodilatory response of drugs apart from the response caused by metabolic factors (eg, a decrease in coronary flow and O2 delivery secondary to decreased contractility and O2 consumption). Use of this measurement is based on the assumption that local metabolites are produced in proportion to myocardial O2 consumption and that local metabolites are major factors that regulate coronary flow.15 % O2E, O2 consumption, and cardiac efficiency were calculated as reported previously.10 The rate×pressure product per minute O2 consumption is a relative index of cardiac efficiency.
Perfusate and bath temperature were maintained at 37.5±0.1°C before and after hypothermia with a thermostatically controlled water circulator. During the 22-hour hypothermic perfusion period, perfusate and bath temperature were maintained at 3.7±0.1°C. A switch to hypoperfusion at 3.7°C was accomplished by the use of a separate refrigerated jacket and perfusion circuit (VWR 1160) placed in parallel with the warm perfusion circuit. Normothermic perfusion for 4 hours at 37.5±0.1°C after cold perfusion was reinstated by switching back to the warm circuit. Warm and cold perfusion circulation circuits were temperature equilibrated in advance. Time to reach half of the temperature decrease from 37.5°C to 3.7°C was 5 minutes. On lowering of the temperature, at 15°C, cardiac perfusion was switched from constant pressure to a low constant flow (1.7 mL·g−1·min−1), which is approximately one fourth of the baseline normothermic flow during constant pressure perfusion. Perfusion pressure, which was monitored throughout hypothermia at constant flow, averaged 23±2 mm Hg. Calculated coronary vascular resistance was ≈25% higher during hypothermia than during normothermia. On raising temperature at 25°C after hypothermia, cardiac perfusion was returned to the constant pressure (55 mm Hg) mode. Time to reach half of the temperature rise from 3.7°C to 37.5°C was 3 minutes. Warm and cold perfusate solutions were equilibrated with a gas mixture of 96% O2/4% CO2. For hearts in all groups during the initial normothermic period, mean coronary arterial (inflow) pH averaged 7.44±0.02 (±SEM), Pco2 was 27±1 mm Hg, and Po2 was 567±12 mm Hg; samples, collected at 3.7°C during the hypothermic period and measured at 37°C, had values of 7.15±0.02, 47±2 mm Hg, and 787±16 mm Hg, respectively. There were no significant differences in these values among the cold groups at each of the two temperatures.
Electrograms, heart rate, atrioventricular conduction time, outflow O2 tension, coronary flow, LV pressure, and perfusion pressure were recorded on FM tape for later detailed analysis. All measured variables were displayed on a fast writing (3 kHz), thermal-array eight-channel recorder (Astro-Med MT9500). Calculated variables were computed with a software program (Microsoft Excel). Hearts were weighed immediately after each experiment (28 hours for posthypothermia groups and 6 hours for the time/treatment warm control groups), and dehydrated weights were determined to calculate dry heart weight expressed as a percentage of wet heart weight.
Measurement of L-ARG and L-CIT
The substrate for NOS, L-ARG, and the coproduct with NO from NOS, L-CIT, were measured in 10 coronary effluent samples of the cold control group with the use of HPLC. The HPLC system consisted of a Laboratory Data Control (LDC) Constametric III G pump, a Gilson Automatic Sampler model 231, and an electrochemical detector (model BAS LC-4B, Bioanalytical Systems, Inc). The column (Beckman Ultra-sphere ODS 5 μm, 4.6 mm×25 cm) was perfused at a mobile phase flow rate of 1.5 mL/min. The detector potential was set at +0.7 V. The mobile phase consisted of 800 mL of 0.1 mol/L sodium acetate, adjusted to pH 5.7, plus 260 mL acetonitrile. L-ARG and L-CIT were detected electrochemically as the OPA derivatives. The OPA reagent consisted of 25 mL of 0.1 mol/L borate buffer, pH 9.5, 50 μL 2-methyl-2-propanethiol, 2.5 mL methanol, and 135 mg OPA. All chemicals were HPLC grade. The chromatographic data were collected on a Hewlett Packard 3393A integrator and stored on a Hewlett Packard 9122 disk drive. Coronary effluent samples (2 mL) were prepared and analyzed as follows: To each 0.5-mL sample, we added 25 μL methyl-l-arginine (2 μg/mL) as an internal standard. Three milliliters of ethanol was added to each sample, mixed, and centrifuged. The supernatant was transferred to a clean tube and evaporated to dryness under a stream of air at 40°C. The dried residue was redissolved in 2.0 mL of mobile phase, and 400 μL was mixed with 40 μL of the OPA reagent for exactly 2.00 minutes before injection of 100 μL into the HPLC. L-ARG and L-CIT concentrations were calculated from standard curves of the respective peak height-versus-concentration ratio. The standard curve data were derived using perfusate that had not passed through the isolated heart. The standard curves for L-ARG and L-CIT were linear over the concentration range studied. The limit of detectability for L-ARG and L-CIT was 1 ng/mL perfusate. The absolute retention times for L-CIT and L-ARG were 10.3 and 15.0 minutes, respectively.
Fig 1⇓ is a schema of the protocol. Once isolated, each heart was assigned to one of four normothermia (warm) groups or to one of five hypothermia (cold) groups (12 to 16 hearts per group; total of 120 hearts). In addition to the two control groups (warm and cold), two groups (warm and cold) were infused continuously with 100 μmol/L L-ARG, two groups with 0.1 IU/L (367 IU/mg) AVP, and two groups with 100 μmol/L L-NAME (Sigma-Aldrich Chemical) beginning at hour 2. D-ARG was infused only to a cold group.
After control and vasodilator-response readings were taken, all nine groups were infused with 10 mmol/l BDM (Sigma-Aldrich Chemical) and 10 μmol/L ADE for 1 hour in warm groups or in cold groups for 0.5 hour during induction of hypothermia, during 22 hours of hypothermia, and for 0.5 hour after return to normothermia. NP (Nipride, Abbott Labs) was also infused during this last 0.5-hour period in both warm and cold groups. After discontinuation of BDM, ADE, and NP, all groups were perfused normothermically at constant perfusion pressure for an additional 2.5 hours. Thus, warm and cold groups were treated identically except for the 22-hour period of low-flow hypothermic perfusion with BDM, ADE, and NP in the cold groups.
Peak coronary flow responsiveness was tested in all hearts temporarily arrested with ADE. A bolus of ADE (0.2 mL of a 200 μmol/L solution) was injected directly into the aortic (coronary perfusion) cannula for assessment of this response. Endothelium-dependent responses were tested with 1 μmol/L 5-HT (warm L-NAME and cold AVP and D-ARG groups only) or 1 μmol/L ACh (other six groups), and endothelium-independent responses were tested with 100 μmol/L NP (all nine groups). Each drug, except ADE, was given in 3-minute infusions between hour 1 and 1.5 (D1) before initiation of continuous infusion of L-ARG, D-ARG, L-NAME, or AVP and before induction of cardiac hibernation with BDM, ADE, and hypothermia. These responses were tested again (D2) between hour 4.5 and 5 (warm groups) or hour 26.5 and 27 (cold groups) during continued infusion of L-ARG, D-ARG, L-NAME, or AVP but after discontinuation of BDM, ADE, and NP. Maximal (m), steady-state (s), and ventricularly paced (p) (240 min−1) flow responses to ACh are displayed. The initial AChm response is a peak flow response that occurs before the slowed atrial rate results in a relative vasoconstriction through autoregulatory mechanisms; AChs is the response that occurs during the slowed atrial rate (150 min−1); and AChp is the response that occurs during pacing at 240 min−1 that nearly corresponded to the resting heart rate (230±3 min−1). In the three groups given 5-HT, heart rate was not affected, so for these three groups, there are no AChm or AChs values. EPI (0.5 μmol/L) was infused to test inotropic and chronotropic responsiveness. As noted above, BDM, ADE, and NP were infused in each cold group because of their protective effects during the perihypothermic period. Higher concentrations of NP and ADE than those reported in this study did not increase coronary flow. We have found that isolated guinea pig hearts perfused normothermically for 24 hours, with or without drug protection, exhibit only very slow atrial beating; in addition, hearts stored (no flow) hypothermically at 3.5°C for 22 hours have no detectable function when reperfused (D.F. Stowe, MD, PhD, unpublished observations).
All variables were measured during the last minute of (1) an initial control (C1) period at hour 0.5, (2) during the initial ADE bolus and the 3-minute infusions of epinephrine, ACh, or 5-HT and NP between hour 1 and 1.5 (D1), (3) before (hour 2) and every 0.5 hour during continuous infusions of L-ARG, D-ARG, L-NAME, or AVP (warm and/or cold groups), (4) during initial (hour 3) and final infusions of BDM and ADE (at hour 3.5 or 25.5), (5) during infusions of BDM and ADE and NP at hour 3.5 or 25.5, (6) every 0.5-hour period, and (7) in all groups, during the final bolus ADE and repeat infusions of ACh, or 5-HT, NP, and EPI (D2) at hour 4.5 to 5 or hour 26.5 to 27. Because rewarming provoked ventricular dysrhythmias in a few hearts at ≈25°C, each heart in the cold groups received prophylactically one bolus injection of 0.1 mL of 10 mg lidocaine HCl during rewarming at 25°C to reduce the occurrence of such dysrhythmias.
Dry heart weight expressed as a percentage of wet heart weight for each group was for warm control, 13.4±0.5%; warm AVP, 13.0±0.4%; warm L-NAME, 12.6±0.3%; cold control, 13.6±0.5%; cold L-ARG, 13.6±0.4%; cold D-ARG, 12.2±0.2%; cold AVP, 13.2±0.2%; and cold L-NAME, 15.5±0.8%. The cold L-NAME group exhibited a significant water loss compared with the other groups (P<.05).
Only original summarized data are shown. Results of long-term hypothermia studies in which there was no treatment or treatment with BDM alone during hypothermia or with ADE or NP on reperfusion as well as results of drug-free time control studies have been published previously.3 10 11
All data are expressed as mean±SEM. Mean values were considered significant at P<.05. For data expressed over time (Figs 2 to 5), data of the six groups displayed were compared for variability at each time interval with the use of two-way ANOVA (CLR ANOVA, Clear Lake Research). Data from each of the five cold groups are compared with those of the L-ARG warm group. In addition, L-ARG, D-ARG, AVP, and L-NAME cold groups are compared with the cold control group. For clarity of presentation, time graph data for LV pressure, cardiac efficiency, coronary flow, and % O2E of the warm control, AVP, and L-NAME groups are not displayed in Figs 2 to 5.
For coronary flow (Figs 6⇓ and 7⇓) and % O2E (Figs 8⇓ and 9⇓), vasodilator responses to ADE, ACh, or 5-HT and NP were tested with the use of one-way ANOVA with repeated measures. The data for all nine groups are displayed in these graphs. The following comparisons were made: Figs 6⇓ and 8⇓, initial vasodilator responses to ADE, ACh, or 5-HT and NP versus C1 (initial controls); Figs 7⇓ and 9⇓, basal (C2) responses after 4 hours (warm groups) or 26 hours (cold groups) versus initial controls (C1) within each group; final responses to ADE, ACh, or 5-HT and NP versus C2; and responses to vasodilators at 4 or 26 hours compared with warm and cold control groups. Fisher’s least significant difference test was used to compare mean values. Selected comparisons between posthypothermia and prehypothermia data are noted in the text. Software programs were run on compatible computers (Macintosh, Apple Computer Inc).
Cardiac Rhythm, Mechanical Function, and Efficiency Over Time
Heart rate (231±3 min−1) and atrioventricular conduction time (57±1 ms) were statistically similar for all cold groups before and after hypothermia, and these values were similar to those of the four warm time control groups (details not displayed). Continuous administration of either L-ARG, D-ARG, L-NAME, or AVP had no effect on atrial rate or atrioventricular time. There were no ventricular dysrhythmias in any warm group or in any postcold group once normothermic reperfusion conditions were attained. Atrioventricular dissociation, with slowed ventricular response rate (<100 min−1), occurred in most hearts in the presence of ADE infused with BDM for 1 hour in the warm groups and before, during, and initially after hypothermic perfusion in the cold groups; otherwise, hearts were in sinus rhythm.
LV developed (systolic minus diastolic) pressure (Fig 2⇓) was similar in all groups initially (C1) and increased similarly during initial EPI infusion by ≈16%. In the warm groups perfused over 5 hours, LV pressure gradually decreased by 14%, and the repeat response to EPI was blunted (P<.05). Of the four warm groups, the L-ARG group served as the time and temperature control (Fig 2⇓). L-ARG and D-ARG alone had no significant effect on LV pressure (data not displayed). Treatment with BDM and ADE between 2.5 and 3.5 hours (warm groups) and 2.5 and 25.5 hours (cold groups) nearly abolished LV developed pressure in all groups. During the posthypothermia period between 26 and 28 hours (ie, after discontinuation of BDM, ADE, and NP), LV developed pressure in the postcold control and L-ARG–treated groups returned to 90±3% of that in the warm L-ARG–treated group, but LV pressure returned to only 71±3% of that in the postcold D-ARG–, AVP-, and L-NAME–treated groups. Diastolic LV pressure, set initially to 0 mm Hg in each group by adjusting balloon volume during diastole, increased (5±1 mm Hg) during treatment with BDM and ADE but remained at 0 mm Hg after discontinuation of treatment in all groups (data not shown).
Relative cardiac efficiency (Fig 3⇓), an index of O2 consumed per unit of developed LV pressure per beat, was initially similar (C1) and tended to increase with EPI in all groups. Initiation of continuous treatment with L-ARG, D-ARG, AVP, or L-NAME had no significant effect on cardiac efficiency (data not shown). During treatment with BDM and ADE, cardiac efficiency could not be accurately measured because heart rate was erratic and LV pressure was nearly abolished (Fig 3⇓). In the postcold control and L-ARG–treated groups, efficiency returned approximately to the warm L-ARG level, whereas it was significantly reduced in the postcold D-ARG– (−40±5%), AVP- (−44±7%), and L-NAME– (−48±8%) treated groups.
Coronary Flow and Oxygen Extraction Over Time
Coronary flow (Fig 4⇓) doubled in response to bolus injection of ADE and increased slightly during initial BDM and ADE treatment at hour 2.5. The second response to ADE was marked by a decreased flow response in all groups but especially in the postcold groups. Initiation of L-ARG or D-ARG had no effect on basal coronary flow, whereas flow decreased with initiation of AVP (−26±3% and −25±4%) and L-NAME (−24±3% and −25±3%) in warm and cold groups, respectively (data not shown). Basal flow during normothermic reperfusion at the 28th hour had returned to a level intermediate in the postcold L-ARG group between that of the warm L-ARG group and the other postcold groups.
% O2E (Fig 5⇓) increased moderately during EPI infusion in all groups and decreased markedly in each group on infusion of BDM and ADE between hour 2.5 and 3.5 (warm groups) and hour 2.5 and 25.5 (cold groups). On normothermic reperfusion, % O2E was lower in warm and postcold L-ARG groups and higher in the L-NAME group than in the postcold control group.
Coronary Flow and Oxygen Extraction Responses to Vasodilators
Figs 6 to 9 detail responses to brief infusions of ADE, ACh, or 5-HT and NP on absolute and percent changes in coronary flow and % O2E before (Figs 6⇓ and 8⇓) and after (Figs 7⇓ and 9⇓) either a 1-hour (warm groups) or a 23-hour (cold groups) infusion of BDM and ADE (and NP) during continued treatment with L-ARG, D-ARG, AVP, or L-NAME. Coronary flow (Fig 6⇓) was initially (C1) similar in all groups and increased, on average, 111±4% with bolus injection of ADE, 67±4% with AChm, 25±3% with AChs and AChp/5-HT, and 35±3% with NP. The flow increase with 5-HT (used in warm L-NAME and cold D-ARG and AVP groups) was similar to that of AChp. Infusion of NP increased the atrial rate to 260±4 min−1. Accompanying these increases in coronary flow were decreases in % O2E that were similar among all groups (Fig 8⇓). Combined for all groups, % O2E decreased 48±5% with AChs, 35±4% with AChp/5-HT, and 39±4% with NP from the initial controls (C1). % O2E was not in a steady state during bolus injection of ADE or during the initial maximal flow response to AChm and so was not recorded.
Figs 7⇑ and 9⇓ display the percent change in coronary flow and percent change in % O2E from initial values (C1) during continuous infusions of L-ARG, D-ARG, AVP, or L-NAME but after termination of treatment (C2) with BDM, ADE, and NP for 1 hour (warm groups) or 23 hours (cold groups). Fig 7⇑ shows that in the warm groups, L-ARG had no significant effect, whereas AVP and L-NAME similarly decreased basal flow (C2). In addition, the flow response to ADE was blunted in the presence of AVP or L-NAME, the response to ACh was blunted by AVP, and the responses to ACh or 5-HT (warm L-NAME group) were abolished by L-NAME. The flow response to NP was reduced only by AVP and was unchanged by L-ARG or L-NAME; however, the absolute change in response to NP (change from C1) was greater (P<.01) in the presence than in the absence of L-NAME.
Fig 7⇑ shows further that in the postcold groups, basal flow was significantly reduced in the control group but was not additionally lowered in the D-ARG, AVP, or L-NAME groups. However, flow was increased significantly in the L-ARG group (C2) after hypothermia compared with the cold control group and to a level comparable to those of the warm control and L-ARG groups. The flow responses to ADE bolus were smaller in each postcold group, and flow was increased only by AChm in the L-ARG group and by NP in the postcold control, AVP, and L-NAME groups. AChp/5-HT and NP flow responses were smaller in each postcold group compared with those in the warm groups (data not shown). However, flow during AChm, AChs, and NP was higher during L-ARG treatment than in control, and flow during AChm, AChs, and AChp/5-HT was higher during L-ARG than during L-NAME treatment (data not given). NP did not additionally increase flow in the postcold L-ARG group, and the magnitude of the flow increase by NP (from C2) in the postcold control, D-ARG, AVP, and L-NAME groups was similar.
Fig 9⇑ shows that % O2E increased as flow decreased in the warm AVP or L-NAME groups (C2). % O2E fell as flow was increased by AChp from a reduced basal state in the warm AVP group, but % O2E remained elevated as flow remained reduced with 5-HT in the warm L-NAME group. NP decreased % O2E similarly in all warm groups. In the postcold groups, % O2E was elevated in the control, D-ARG, AVP, and L-NAME groups but was decreased in the L-ARG group (C2). % O2E fell additionally only in the postcold L-ARG group with AChs and remained unchanged with AChp/5-HT. Just as basal % O2E was reduced more in the postcold L-ARG group than in the control group, responses to AChp/5-HT and NP were greater in the postcold L-ARG group although unchanged from C2 levels. The effect of NP to cause a relative increase in flow in postcold D-ARG, AVP, and L-NAME groups was accompanied by decreases in % O2E.
Fig 10⇓ provides a summary of the time-dependent decrease in coronary effluent concentrations of L-ARG and L-CIT in the cold control group. The levels of L-ARG and L-CIT were decreased by −36±9% and −43±9%, respectively, after 24 hours of cardiac perfusion with Krebs’ solution in the cold control group.
The primary aim of the present study was to investigate the role of NO in the maintenance of cardiac perfusion and mechanical function during 1-day hypothermic preservation of isolated guinea pig hearts. A secondary aim was to examine the myocardial and vascular protective effects of a normal ionic perfusate containing ADE, BDM, and NP. We found that LV pressure development, basal and initial (AChm) endothelium-induced coronary flow, cardiac work efficiency, and % O2E were better restored in the presence of L-ARG, a substrate for NO, than in the presence or absence of D-ARG, the inactive enantiomer of L-ARG, or L-NAME, an inhibitor of NOS. We also found that the postcold control, AVP, D-ARG, and L-NAME groups exhibited similar effects on basal coronary flow and % O2E, with equivalently reduced responses to ADE and NP and no response to ACh or 5-HT. These results suggest that less NO is produced or is less effective and that exogenous L-ARG partially restores NO levels. Although L-ARG improved perfusion and cardiac performance, it did not improve the test response to NP. This suggests that the beneficial effects of L-ARG are related to enhanced basal function of the NO guanylyl cyclase/cGMP pathway. Moreover, we observed that endogenous levels of both L-ARG and L-CIT are reduced after 1 day of continuous cold perfusion. This indicates that NO production is likely reduced because of reduced availability of the NO substrate L-ARG. That groups treated with AVP, a direct vasoconstrictor, and L-NAME, an indirect, endothelium-dependent “vasoconstrictor,” had similar vasoconstrictor tone and elicited a similar response to NP as the untreated control group might have suggested that blockade of NOS does not worsen perfusion after long-term hypothermic preservation if it were not for the observation that treatment with L-ARG improved basal flow and myocardial function but not the response to the exogenous NO donor NP.
Another focus of this study was to test the combination of BDM and ADE given initially before, during, and initially after long-term hypothermia, with NP initially during rewarming. This myocardial protective solution gave the best return of basal coronary flow, LV pressure, and cardiac efficiency of any previous study from this laboratory. The earlier studies demonstrated that return of cardiac function was better when only BDM was added to a normal ionic solution compared with a low-Ca2+ or high-K+ solution3 and that there was additional functional improvement when ADE or nitrobenzylthioinosine and NP were added during the rewarming period after hypothermic perfusion with BDM alone.10 11 The results of the present study indicate that continuous infusion of ADE, along with BDM, during hypothermia protects better than administration of BDM alone during hypothermia. Moreover, the present study shows that the improved recovery after protection with BDM plus ADE and NP, though independent of the presence of AVP or L-NAME, was additionally enhanced when L-ARG was administered.
Role of Controls
The four warm groups served as controls for time, temperature, and drugs. (1) To control for effects of L-ARG, AVP, or L-NAME on vasodilator responses after hypothermia, these drugs were infused continuously and similarly in warm and cold groups before, during, and after hypothermia. L-ARG had no basal effects on any variable or response to vasodilators compared with previous drug-free time control studies.3 11 The warm control and warm L-ARG groups exhibited no significant differences in any variable measured. The warm L-ARG group was chosen as the control for the time studies (Figs 2 through 5⇑⇑⇑⇑), and the warm control group was chosen as the control for the vasodilator response studies (Figs 6 through 9⇑⇑⇑⇑). AVP was given at a concentration to mimic reduced flow by L-NAME and was used to compare changes in basal flow and % O2E induced by the direct-acting vasodilator NP in the presence of L-NAME. AVP has a major effect on cardiac smooth muscle V1 receptors to cause vasoconstriction16 but also has a small effect on endothelial V2 receptors to promote vasodilatation,17 an effect that appears to be masked by vasoconstriction in this study. Compared with L-NAME, AVP decreased flow and % O2E responses to ACh and NP initially (C1). However, during administration of AVP (C2), the absolute increase in flow and the decrease in % O2E with ACh and NP were of the same magnitude as before AVP. These effects of AVP were completely reversible (data not shown). L-NAME completely blocked the responses to 5-HT but, like the AVP response, effectively maintained the response to NP on the basis of the absolute change in responses obtained with L-NAME (C2).
(2) To control for time-dependent changes in variables related to hypothermic preservation with cardioprotective agents, the four warm groups were treated in a manner identical to the five cold groups except that there was no 22-hour period of low-flow hypothermic perfusion. BDM and ADE were given for 1 hour and NP was given for the second 0.5 hour in the warm groups to mimic the 0.5-hour prehypothermic and posthypothermic treatment periods in the cold groups. BDM, ADE, and NP treatment, once terminated, had no lasting adverse effect on any variable measured compared with treatment-free warm controls.3 11 BDM has completely reversible effects.3 10 11 12 13 18
(3) To control for possible variant effects of different endothelium-dependent vasodilators, 5-HT was given instead of ACh in one warm (L-NAME) and two cold groups (D-ARG and AVP). Because ACh has direct negative chronotropic effects and 5-HT does not, heart rate effects could be better controlled with 5-HT. L-NAME similarly blocked the flow increase to 5-HT as it did to ACh, as also shown by others.19
Comparison of Treatment Effects on Coronary Flow and Oxygen Extraction
Although interstitial NO was not measured directly, this study indicates indirectly that NO is produced by isolated hearts after ≤5 hours of normothermic perfusion because there was a persistent response to ACh or 5-HT. After 25 to 27 hours, a period that included 22 hours of low-flow hypothermic perfusion, effluent levels of L-ARG and L-CIT fell and there was no response to ACh or 5-HT, whereas the vasodilatory response to NP was attenuated. Basal flow was reduced and % O2E was increased similarly after hypothermia in the absence or presence of D-ARG or L-NAME, and there were no differences among the control, D-ARG, and L-NAME groups in their response to vasodilators. This suggests that L-NAME had no additional effect to block NO synthesis after the hypothermic period because no response to ACh remained in the cold control group as well.
The additional finding that a similar reduction in basal flow and an increase in % O2E occurred in the cold AVP group as well as in the cold control, D-ARG, and L-NAME groups suggests that AVP was also incapable of producing an added vasoconstrictor effect after hypothermia. However, because the responses to NP were similar after hypothermia in these four groups, this indicates that an exogenous NO donor, such as NP, remains capable of producing vasodilation after hypothermia. It appears that either the time period of low-flow hypothermic perfusion or both duration and hypothermia are responsible for the attenuated responses to vasodilators because these were the only parameters that differed between the warm and cold groups. Nevertheless, hypothermia is protective because hearts perfused normothermically for 27 hours have no mechanical function, are maximally vasoconstricted, and exhibit no response to ADE, EPI, ACh, 5-HT, or NP (D.F. Stowe, MD, PhD, unpublished results).
NO is normally constitutively expressed and accounts for a portion of basal coronary flow.20 21 22 This was shown indirectly in the present study by the reduced flow after initial administration of L-NAME in both the warm and cold groups and by luminal release of L-CIT into the coronary effluent. In the isolated heart, L-CIT is most likely derived only from L-ARG via catalysis by NOS. Although L-ARG can be converted to glutamic acid to enter the tricarboxylic acid cycle via α-ketoglutarate, this pathway likely produces minimal product in aerobic hearts with adequate carbon (dextrose) substrate. Excess L-ARG did not alter initial basal flow or enhance responses to ACh or 5-HT before hypothermia. L-ARG normally has no effect on vascular tone because NOS has a low Km value for L-ARG, so the enzyme is normally saturated. However, after hypothermia, basal flow and the peak flow response to ACh were improved by L-ARG. Thus, L-CIT and NO production may be diminished after 1 day of cold crystalloid perfusion in isolated hearts, and the addition of L-ARG to cold preservation solutions may be beneficial in maintenance of basal perfusion. The administration of intracoronary L-ARG during reperfusion has been shown to reduce infarct size in cat and dog models after myocardial ischemia and to better preserve endothelial function in isolated coronary rings.23 24 Similarly, warm reperfusion after a long period of cold perfusion may contribute to endothelial and vascular reperfusion injury. Diminished response to endothelium-dependent vasodilation has been demonstrated for reperfusion after coronary occlusion25 and after cold storage.6 Our study suggests that the NO-generating system can be made at least partially functional if the substrate L-ARG is furnished.
The postcold L-ARG group did not show an improved response to NP compared with the other cold groups. It is possible that stimulation of guanylyl cyclase by endogenous NO saturates this mechanism so that exogenously administered NO in the form of NP has no additional effect. Dysfunctional NOS, altered metabolism of NO, dysfunctional guanylyl cyclase, or any other factor regulating vascular tone could be involved because the response to NP in all postcold groups after hypothermia was attenuated. The vasoconstriction induced indirectly by L-NAME afforded a better response to ADE and to NP than did the comparable vasoconstriction induced directly by AVP on the V1 receptor–activated phospholipase C phosphoinositide mechanism.26 Guanylyl cyclase might become more sensitive to nitrovasodilators if it is less stimulated by endogenous NO when NOS is blocked by L-NAME. Indeed, it has been reported that vasorelaxation in response to nitroglycerin in rat aortic segments is greater in the absence than in the presence of endothelium.27 It was suggested that relaxation is due to competition for activation of guanylyl cyclase between endogenously released NO by intact endothelium and nitroglycerin or its NO-like product.27 Another possibility is that this effect might be due to upregulation of guanylate cyclase for NO released by NP or to blockade by L-NAME of endogenous NO feedback inhibition of NOS.
Comparison of Treatment Effects on LV Pressure and Cardiac Efficiency
Myocardial function was better preserved after hypothermia in the presence of L-ARG, which also improved basal flow and % O2E, than in the presence of D-ARG, L-NAME, or AVP. Although controversial, nitrosyl compounds28 and AVP16 have little or no known direct effects on myocardial fibers, as also demonstrated in the present study. D-ARG, like L-ARG, had no direct effect on any heart rate or LV pressure. If basal myocardial perfusion is reduced sufficiently after hypothermia by any cause, myocardial contractile function would be expected to decrease when % O2E approaches a maximum because O2 and nutrient supply decrease relative to demand.29 The reduced cardiac efficiency observed after hypothermia was due to a greater decrease in MVo2 than in the heart rate×LV pressure product. The increase in % O2E after hypothermia reflects a relative increase in coronary vascular resistance that could be a result of vasoconstriction, edema, or global or regional obstruction.
Protective Effects of BDM, ADE, and NP During Hypothermia
One method of protecting the myocardium during hypothermic preservation is to infuse a reversible intracellular metabolic inhibitor without changing extracellular ionic composition, as in a “cardioplegic” solution. BDM was selected as a cardioprotective agent because concentrations of ≤10 mmol/L have minimal chronotropic or dromotropic effects but marked negative inotropic and vasodilatory effects.10 11 12 13 Improved contractile function after cold preservation with BDM may result indirectly not only from functional and metabolic depression3 but also directly from its effect on intracellular calcium handling. BDM has little effect on the slow Ca2+ inward current but acts more so on “downstream” factors involved in excitation/contraction coupling. Although its specific site or sites of action have not been fully elucidated,12 30 31 evidence indicates that the major effect of BDM in cardiac muscle is to reversibly decrease myofibrillar Ca2+ sensitivity, with a lesser effect to alter the uptake or release of Ca2+ from the sarcoplasmic reticulum.12 However, BDM does not decrease responsiveness of troponin C to Ca2+.31 Because BDM also causes vasodilation,3 10 11 12 13 it may protect the myocardium by enhancing O2 supply as well as by reducing O2 demand. The vascular effect of BDM is independent of NO, prostacyclin, and cGMP pathways.18
In the present study, ADE was infused with BDM before and during hypothermia as well as during rewarming to promote maximal vasodilatation and cardioprotection and to determine whether this would enhance contractile function after hypothermia. Indeed, hearts hypothermically perfused with BDM and ADE exhibited better mechanical and metabolic function on normothermic reperfusion than previous postcold groups perfused only with BDM given before, during, and after hypothermia or with the addition of ADE and NP given only during the initial warm reperfusion period.10 The negative chronotropic effect of ADE appears to be mediated through A1 receptors coupled to K+ channels through a pertussis toxin–sensitive G protein that results in membrane hyperpolarization. A negative inotropic effect is also mediated through A1 receptors and coupled to adenylyl cyclase activity.32 ADE has been shown to improve cardiac contractile function during reperfusion after ischemia33 34 and during continuous cold perfusion.35 Some effects of ADE can be mimicked by substituting nitrobenzylthioinosine, a nucleoside transport inhibitor, for ADE during the initial normothermic reperfusion period.11 However, the most important beneficial effect of ADE during preservation in this model, with hearts already metabolically depressed by hypothermia and BDM, may be its vasodilatory effect. ADE relaxes vascular smooth muscle in an endothelium-independent fashion through Gs-coupled A2 receptor stimulation of adenylyl cyclase to form cAMP; this effect ultimately leads to a reduced Ca2+ effect on contractile proteins.32 The beneficial effect of ADE may be at least partially endothelium dependent because the flow response to ADE was found to be reduced in the presence of L-NAME.36 This is also demonstrated in our study. NP may maintain vasodilation in a manner different from that of ADE and thus could add to the vasodilatory effect of NP. In vitro, NP, unlike ADE, has no negative inotropic effect and only a small positive chronotropic effect. Other investigators have reported that endogenous vasodilation by endothelium-derived NO is attenuated during early reperfusion after ischemia37 38 and myocardial ischemia reperfusion injury may be decreased by the administration of NO donors.39
The isolated heart preparation was used in this study as a tool to understand mechanisms of vascular dysfunction and to test new methods of preserving hearts for long periods of time. Our results show that it is possible to partially restore endothelial function due to decreased NO production and its effect by supplying L-ARG. Supplying exogenous NO donors continuously may be as effective or better in improving perfusion and function after hypothermia; this was not tested. Because these hearts are crystalloid perfused, a possible limitation of this model is less coronary and mechanical reserve compared with hearts in vivo. It could not be distinguished if reduced coronary flow and increased oxygen extraction after hypothermia were due to global or regional hypoperfusion or whether the hypoperfusion was a result of regional edema or emboli. Also, blood-borne factors (eg, platelets, neutrophils, heme, hormones) may play a significant role in reperfusion dysfunction after long-term hypothermia. It will be important to examine such approaches for long-term cardiac preservation by using in vivo animal models to validate the potential success of transplanting the human donor heart harvested many hours or days earlier.
Selected Abbreviations and Acronyms
|% O2E||=||percent oxygen extraction|
|HPLC||=||high-performance liquid chromatography|
|L-NAME||=||nitro-l-arginine methyl ester|
|LV||=||left ventricular, left ventricle|
|NOS||=||nitric oxide synthase|
This work was supported in part by grants from the VA Merit Review Program (8204-04P), American Heart Association, Wisconsin Affiliate (88-GA-06), and National Institutes of Health (HL-34708) and by Anesthesiology Research Training Grant GM-08377. We are grateful to James S. Heisner for his help in conducting animal protocols, to Kimberly Stommel for HPLC analysis, to Jolene Andryk and Susan Lawrence for laboratory assistance, and to Edith Sulzer for literature retrieval and other secretarial assistance.
Portions of this work have appeared in abstract form (FASEB J. 1993;7:A718, Anesth Analg. 1993;76:S419, and FASEB J. 1996;10:A156).
- Received August 22, 1996.
- Revision received November 7, 1996.
- Accepted November 14, 1996.
- Copyright © 1997 by American Heart Association
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