Delayed Preconditioning-Mimetic Actions of Nitroglycerin in Patients Undergoing Exercise Tolerance Tests
Background— Nitroglycerin (NTG) induces delayed preconditioning (PC)-mimetic effects in animal models and in humans during coronary angioplasty. We tested the hypothesis that NTG mitigates ischemia and enhances functional capacity during an exercise tolerance test (ETT) in patients with coronary artery disease.
Methods and Results— Twenty-eight patients with stable angina and ischemia documented by a stress test were randomized in a double-masked, crossover design to receive a titrated intravenous infusion of NTG or normal saline over 4 hours. At 24 to 28 hours after study medication infusion, each patient underwent 2 ETTs separated by a 1-week washout period. Compared with control patients, pretreatment with NTG was associated with a dose-dependent increase in exercise duration averaging 40 seconds (412±19 versus 372±24 seconds, P=0.014) and an improvement in ECG manifestations of ischemia, as shown by a decrease in maximal ST-segment depression (1.84±0.14 versus 1.63±0.13 mm, P=0.011), sum of ST-segment depressions in 12 leads (7.64±1.01 versus 6.61±0.83 mm, P=0.027), and time to resolution of ST-segment depression (229±30 versus 207±28 s, P=0.018). These benefits occurred despite an increase in myocardial workload after NTG, as indicated by a higher peak rate-pressure product (24 492±1054 versus 22 536±1019 mm Hg/min, P=0.015).
Conclusions— NTG produces a late PC-mimetic effect that mitigates the ECG manifestations of ischemia during exercise and improves exercise capacity. To our knowledge, this is the first study to demonstrate that NTG can alleviate exercise-induced ischemia 24 hours after its administration, long after the hemodynamic effects have subsided. The finding that nitrate-induced late PC ameliorates a common manifestation of coronary artery disease has potentially significant implications for the management of this disorder and for the design of clinical trials.
Received October 19, 2004; revision received January 31, 2005; accepted February 9, 2005.
The late phase of preconditioning (PC) is a genetic reprogramming of the heart whereby exposure to a mild stress upregulates a battery of cardioprotective genes, resulting in enhanced tolerance to ischemia 24 to 72 hours later.1 Experimental studies have demonstrated that increased nitric oxide (NO) availability is both necessary to trigger the late phase of ischemia-induced PC and sufficient to reproduce this adaptation in the absence of ischemia.2,3 That is, inhibition of NO synthase prevents ischemia from eliciting a late PC state,4 whereas administration of exogenous NO (ie, NO donors, such as nitroglycerin [NTG]) mimics the phenotype of ischemia-induced late PC.3,5–7 NTG has been demonstrated to exert late PC-mimetic effects in humans as well. Leesar et al8 randomized 66 patients to receive a 4-hour intravenous infusion of NTG or placebo on the day before percutaneous transluminal coronary angioplasty (PTCA) and found a decrease in ST-segment elevation, regional wall-motion abnormalities, and chest pain score during balloon inflation in patients pretreated with NTG. Although that study8 demonstrated the ability of NO donors to elicit late PC in humans, the primary end point was electrocardiographic (shift in the ST segment) rather than clinical. The concept that ST-segment elevation accurately reflects the severity of myocardial ischemia has been questioned.9 Furthermore, although alleviation of ischemia during PTCA is conceptually important as proof of the PC-mimetic properties of nitrates, this end point has limited practical implications because most patients undergoing PTCA do not require protective interventions.
Consequently, the present study was undertaken to determine whether NTG exerts late PC-mimetic actions that affect an important clinical outcome rather than simply an ECG end point. To this end, we tested whether the same form of therapy that induces delayed protection against the ischemia associated with PTCA8 also induces delayed protection against the ischemia associated with physical exercise in patients with chronic stable angina. We reasoned that if nitrates elicit a late PC cardiac phenotype resulting in genuine antiischemic actions, then this effect would manifest itself not only in reduced ST-segment depression but also in improved functional capacity and exercise tolerance.
The study was performed according to the 1983 revision of the 1975 Declaration of Helsinki, adhered to local guidelines for good clinical practice, and was approved by the local institutional review board committee. All patients provided written, informed consent. Health Insurance Portability and Accountability Act authorization forms were obtained from all patients who enrolled after April 2003.
Thirty subjects with chronic stable angina were enrolled at a single center (Veteran’s Administration Hospital, Louisville, Ky). Candidates included patients with objective evidence of coronary artery disease (CAD), as evidenced by a positive exercise tolerance test (ETT) and/or a positive exercise/rest Tc-99 sestamibi perfusion single positron emission computed tomography test. Patients with unstable coronary syndromes, advanced heart failure (New York Heart Association class III or IV), ECG evidence of preexcitation, atrial fibrillation, pacemaker dependency, left bundle-branch block, or resting ST-segment depression ≥1 mm and those receiving digoxin therapy were excluded from the study. The first ETT study was performed at least 2 weeks after the qualifying stress test. Two patients did not complete the protocol because they developed atrial fibrillation and refractory angina, respectively; thus, the final analysis was performed in 28 patients. In all patients, long-acting nitrates were stopped at least 1 week before the first ETT and for the remainder of the study. β-Blockers and nondihydropyridine calcium channel inhibitors were discontinued 48 hours before each ETT. All other medications were maintained constant throughout the study. After the end of the study, patients were managed at the discretion of the treating physician.
Patients were randomized in a crossover, double-masked design to receive a 4-hour infusion of intravenous NTG or normal saline (placebo) on the day before the ETT. The ETT was performed 24 to 28 hours after the end of the infusion. The infusion of NTG was started at 10 μg/min and titrated up to 80 μg/min while avoiding hypotension (systolic blood pressure <100 mm Hg or mean arterial pressure <70 mm Hg), dizziness, and disabling headache. Each patient underwent 2 symptom-limited ETTs that were separated by a 1-week washout period. For 3 days before each ETT, patients were discouraged from engaging in activities that might induce angina or require the symptomatic relief of short-acting nitrates. Twenty-five patients underwent the standard Bruce protocol on a Quinton Q stress exercise treadmill (model MP200-115, Quinton). Three patients, who had very poor functional capacity and were exercised with the modified Bruce protocol during their qualifying ETT, underwent a modified Bruce protocol during the study. Twelve-lead ECG tracings were obtained every 30 seconds. Blood pressure was recorded at baseline, every 3 minutes during exercise, and every 2 minutes during recovery.
Data Collection and Study End Points
All outcomes were adjudicated and the final analysis was performed by 3 independent investigators (M.C., H.J., M.A.) who were blinded during both ETT performance and data analysis. Exercise-induced ischemia was defined as the new development of horizontal or downsloping ST-segment depression (measured 0.08 second after the J-point) of ≥1 mm versus the baseline tracing. In all patients, ST-segment depression was measured by 2 independent, blinded observers (M.C., H.J.); measurements were taken manually in increments of 0.5 mm in 3 consecutive beats, and the 3 values were averaged. When a discrepancy in measurements was present, a third observer (M.A.) blindly reanalyzed the disputed ECG tracing, and a consensus was reached. Intraobserver variability for the 2 observers who analyzed all ECG tracings (M.C., H.J.) was 4% and 5%, respectively, with an interobserver variability of 5%. Twenty-three patients had a normal baseline ECG. Five patients had minimal ST-segment depression at rest, compatible with our enrollment criteria, ie, <1 mm; in these subjects, the end point was additional ST-segment depression (of at least 1 mm) below the resting value.
The lead with the greatest ST-segment deviation was used for subsequent analysis of maximal ST-segment depression and time to onset of 1-mm ST-segment depression. The sum of ST-segment depressions in 12 leads at peak heart rate (HR) was calculated in 19 patients who had a normal baseline ECG and in whom all 12 leads were readily interpretable (free of artifacts, wandering baseline, and ectopy) at peak exercise. As was the case for ST-segment depression, hemodynamics and exercise duration were also analyzed by 2 independent, blinded observers (M.C., H.J.); there was no discrepancy between the 2 observers with regard to these variables. The primary end points were total exercise duration and maximal ST-segment depression. Secondary end points included time to normalization of ST-segment depression, time to onset of 1-mm ST-segment depression, peak rate-pressure product (RPP), sum of ST-segment depressions in all 12 leads at peak exercise, maximal ST/HR index (the difference between ST-segment depression at peak exercise and ST-segment depression at baseline divided by the difference between HR at peak exercise and HR at baseline10), and frequency of ectopy during and after exercise.
Data are presented as mean±SEM. A 2-tailed, paired, Student t test was used to compare continuous variables. Categorical data were compared by means of the χ2 test. A value of P<0.05 was considered statistically significant.
Of the 30 patients enrolled in the study, 2 did not complete the protocol and were excluded from the final analysis. The demographic, angiographic, and clinical characteristics of the remaining 28 patients are shown in Table 1. Twenty-four patients underwent coronary angiography after the conclusion of the study; 4 patients had adequate control of angina with medical therapy and declined invasive procedures.
The 4-hour infusion of NTG was not associated with any complications. The peak NTG infusion rate achieved was 34.1 μg/min (or 16.64 μg · min−1 · m−2), and the average rate was 32.9 μg/min, corresponding to a mean cumulative dose of 7.9 mg per patient. During NTG infusion, HR increased from a baseline of 69±5 bpm to a maximum of 78±6 bpm, which was significantly (P=0.02) higher than in control patients (67±4 bpm). In addition, systolic blood pressure decreased from a baseline of 129±7 mm Hg to a nadir of 101±6 mm Hg, which was significantly (P=0.035) lower than in control patients (126±12 mm Hg). However, no significant differences in HR or blood pressure were present between the 2 groups at the start of exercise (76±3 versus 75±2 bpm and 139±4 versus 142±4 mm Hg, respectively) or at peak exercise (136±3 versus 140±4 bpm and 166±4 versus 175±4 mm Hg) (Table 2).
ECG Signs of Ischemia
The maximal ST-segment depression achieved during exercise was significantly less in patients pretreated with NTG than in patients pretreated with normal saline (1.63±0.13 versus 1.84±0.14 mm, respectively, P=0.011) (Figure 1A). Of the 28 patients, 14 (50%) exhibited less ST-segment depression after pretreatment with NTG, whereas the remainder showed similar (11 patients) or increased (3 patients) ST-segment depression. When the maximal exercise-induced ST-segment shift was normalized to the maximal exercise-induced increase in HR (ST/HR index), a significant difference was still found in favor of the NTG-pretreated group (2.66±0.27 versus 3.21±0.26×10−2 mm/min, P=0.018) (Figure 1B). NTG pretreatment was also associated with a significantly shorter time to resolution of ST-segment depression (207±28 versus 229±30 seconds, P=0.018) (Figure 2 and Table 2). However, the time to 1-mm ST-segment depression did not differ between the 2 groups (190±15 seconds after NTG versus 178±18 seconds after normal saline, P=0.33).
The sum of ST-segment depressions in all 12 leads at peak HR was measured in 19 patients whose baseline 12-lead ECGs were completely normal and who had no ectopy or artifacts in any of the 12 ECG leads at peak exercise. In this subset, the sum of ST-segment depressions was significantly lower after NTG pretreatment (6.61±0.83 mm) than after placebo (7.64±1.01 mm, P=0.027) (Figure 1C). Using the sum of ST-segment depressions increased the sensitivity of the analysis for detecting improvement in ECG manifestations of ischemia, from 50% to 68% (13/19) of patients.
The order of treatment allocation (NTG first versus normal saline first) did not affect the ECG manifestations of ischemia. The 15 patients who received NTG before the first ETT exhibited a trend toward less maximal ST-segment depression (1.85±0.20 mm after NTG versus 2.04±0.17 mm after normal saline, P=0.064); a similar trend was observed in the 13 patients who received NTG before the second ETT (1.5±0.21 versus 1.77±0.23 mm, P=0.11). Although these differences did not achieve statistical significance (probably owing to the small sample sizes), they were directionally similar and had comparable magnitude in the 2 subsets of patients.
In the 25 patients who underwent ETTs with the standard Bruce exercise protocol, pretreatment with NTG was associated with a significant increase in exercise duration compared with placebo treatment (412±19 versus 372±24 seconds, P=0.014) (Figure 3). The remaining 3 patients were exercised with the modified Bruce protocol and showed no significant differences in exercise duration (800±176 seconds after NTG versus 802±143 seconds after normal saline). Overall, 72% (18/25) of the patients who underwent the Bruce protocol demonstrated an improvement in functional capacity after pretreatment with NTG, with the duration of exercise increasing by an average of 40 seconds. All 7 patients who received the highest dose of NTG showed improvement in exercise duration, with an average increase of 73 seconds (P=0.035), as opposed to 5 of 9 patients at the lowest dose (0 to 10 μg · min−1 · m−2) and 6 of 9 patients at the intermediate dose (10 to 20 μg · min−1 · m−2) (Figure 4). In addition, a significantly higher RPP was achieved after NTG pretreatment (24 492±1054 versus 22 536±1019 mm Hg/min, respectively, P=0.015), indicating an increase in myocardial workload associated with increased exercise duration (Table 2).
The functional effects of NTG pretreatment were not related to whether NTG was given before the first or the second ETT. In the 13 patients who received NTG before the first ETT, exercise duration was increased by 40 seconds (P=0.09) versus 39 seconds (P=0.07) in the 12 patients who received NTG before the second ETT (the 3 patients who underwent a modified Bruce protocol were not included in this analysis).
Angina and Arrhythmias
There was no difference in the occurrence of angina between NTG- and normal saline–pretreated patients (25/28 and 26/28 patients, respectively, P=0.65). Similarly, no difference in the frequency of ectopic beats during or after exercise was noted between the 2 ETTs. NTG pretreatment was associated with a short run of ventricular bigeminy in 1 patient and a brief episode of atrial fibrillation during recovery in 1 patient with a known prior history of paroxysmal atrial fibrillation.
Although pretreatment with NTG has been shown to produce a delayed cardioprotective effect that improves tolerance to ischemia during PTCA,8 it is unknown whether this form of pharmacological PC can alleviate ischemia induced by physical exercise, arguably the most common form of ischemia in patients with CAD.
Using a crossover, double-masked, placebo-controlled design, we randomized 28 patients with stable angina and objective evidence of myocardial ischemia to pretreatment with intravenous NTG or placebo 24 to 28 hours before an ETT. We found that patients pretreated with NTG were able to exercise for a longer duration (+40 seconds [+11.7%]) and achieved a higher RPP (+1956 mm Hg/min [+9.3%]) with less ischemia (−0.21 mm [−12.8%] in maximal ST-segment depression). Thus, not only did NTG pretreatment augment exercise tolerance compared with placebo treatment, but it also decreased the ECG manifestations of ischemia at a given level of workload.
Previous studies have documented the ability of nitrates to alleviate exercise-induced ischemia when the drugs were given during exercise.11–13 To our knowledge, this is the first study to demonstrate that NTG can alleviate exercise-induced ischemia 24 hours after its administration, long after the hemodynamic effects of this NO donor have subsided. The mechanism whereby nitrates induce late PC has been investigated in experimental models.2,14 These studies have shown that NO donors activate a signaling pathway that includes protein kinase C, Src tyrosine kinases, and Janus kinases, leading to the recruitment of latent transcription factors (nuclear factor-κB, STAT1, and STAT3), which in turn cause upregulation of cardioprotective genes, such as inducible NO synthase, cyclooxygenase-2, and heme oxygenase-1, that render the heart resistant to ischemia/reperfusion injury.15 NO donors appear to induce late PC by a mechanism that involves NO-dependent generation of oxidant species such as peroxynitrite.15
Because of the considerable variability in response to exercise among patients with CAD, we elected to use a crossover design that enabled each patient to serve as his own control, thereby enhancing the power of the study. The interval between the 2 ETTs (1 week) was selected because it facilitated patient recruitment, minimized dropouts, and allowed any PC effect elicited by the first ETT to dissipate before the second ETT. Time-course studies in animal models have demonstrated that late PC starts to manifest itself 12 hours after the stimulus, becomes fully expressed at 24 hours, and persists for ≈72 hours, disappearing within 4 to 6 days.16,17 Hence, a carryover of the NTG effect to the second ETT in patients who received this drug before the first ETT seems highly unlikely. In support of this conclusion, the improvement in maximal ST-segment depression, exercise duration, and other manifestations of ischemia was similar in patients who received NTG before the first ETT vis-à-vis those who received NTG before the second ETT (see Results).
The possibility that exposure to the first ETT enhanced exercise performance on the second ETT, regardless of treatment, by inducing either a “training/familiarity effect” or a delayed-PC effect (caused by the ischemia associated with the first ETT) is highly unlikely because the interval separating the 2 ETTs was relatively long (1 week), and all patients had undergone at least 1 ETT before entering the study. Such a possibility is further ruled out by the fact that (1) exercise duration on the placebo tests was similar, irrespective of whether normal saline was given before the first or the second ETT (370±23 versus 374±32 seconds, respectively, P=0.92), and (2) exercise duration was slightly less on the second ETT (382±19 seconds) than on the first (402±25 seconds), which is the opposite of what would be expected if a delayed-PC effect or training/familiarity effect had developed after the first ETT. Furthermore, the fact that exercise duration on all placebo tests combined, regardless of whether they were the first or second ETT (372±24 seconds), was similar to that on the qualifying ETT (359±25 seconds) demonstrates that the qualifying ETT did not exert a training effect. Because oral nitrates were discontinued 1 week before the first ETT and for the rest of the study and because all medications and clinical conditions were maintained constant during the study, it is unlikely that nitrate tolerance or a rebound effect from nitrate withdrawal biased the results.
The 4-hour intravenous infusion protocol for NTG was chosen because this treatment has previously been shown to induce late PC in patients undergoing PTCA.8 Although intravenous infusion of NTG would not be a practical prophylactic therapy in the majority of patients with stable exertional angina, our results provide proof of principle for the concept that nitrates can elicit a protective adaptation that results in improved exercise tolerance at a distance of 24 hours from their administration.
Previous Studies of Late PC in Humans
The first study to document the existence of a late-PC phenomenon in humans was performed in patients undergoing PTCA.8 Recently, and despite previous claims to the contrary,18 exercise-induced ischemia has also been shown to trigger a late-PC effect in humans.19 However, unlike the results obtained with NTG-induced late PC in the present study, in that study exercise-induced late PC did not increase exercise duration, a parameter that is considered one of the most potent predictors of risk during stress testing.20
In a recent study by Crisafulli et al,21 10 patients with stable angina underwent serial bicycle exercise tests to assess exercise-induced early and late ischemic PC as well as the late PC-mimetic effects of a 10-mg cumulative dose of transdermal NTG pretreatment. The authors found that 48 hours after transdermal administration of NTG (given over 8 hours), the appearance of 1-mm ST-segment depression was delayed and the duration of exercise was prolonged. That study, however, was not blinded and lacked a proper control group. Furthermore, although NTG pretreatment was reported to improve exercise duration by a degree similar to that observed in our study (+10% to 15%), no differences were noted in the maximal ST-segment depression compared with baseline.21 The present investigation is the first to demonstrate a late PC-mimetic effect of nitrates against exercise-induced ischemia in a randomized, blinded, controlled design.
Potential Study Limitations
The improvement in exercise duration afforded by NTG pretreatment in this study may appear to be relatively modest (+40 seconds). However, this effect is conceptually important because it demonstrates the ability of nitrates to improve exercise tolerance many hours after their hemodynamic actions have disappeared. Furthermore, this improvement in exercise duration compares favorably with that observed previously with other antiischemic therapies. For example, ranolazine was found to increase exercise duration by 24 seconds, although (unlike in the present study) this effect was observed on the background of medical therapy with calcium channel blockers and β-blockers.22 This benefit was observed in a large cohort of 823 patients with stable angina who underwent repeated ETTs with the modified Bruce protocol, however (as opposed to the standard Bruce protocol used in this study); in that study, the increased duration of exercise was not associated with increased myocardial workload, as estimated by the RPP.22 In a study of 351 patients with stable angina randomized to 10-week therapy with amlodipine, atenolol, or their combination, exercise duration on repeated bicycle ergometer tests increased by 30, 18, and 24 seconds, respectively.23 In a crossover study of 100 patients with stable angina undergoing repeated ETTs (Bruce protocol), total exercise time increased by 38, 13, and 55 seconds, respectively, in patients receiving amlodipine, atenolol, or their combination.24 In another study of 170 patients with stable angina, diltiazem CD was associated with a 16-second increase in total exercise duration on repeated ETTs when added to existing antianginal therapy.25
Our study was performed in a selected cohort of patients at the Veteran’s Administration Hospital. Nevertheless, the demographic characteristics of the patients (Table 1) suggest that this group was representative of the general population with CAD. Because of the nature of the population, almost all subjects were men. It is therefore unknown whether our findings are applicable to women as well. Our study group included only 28 patients. Nevertheless, the crossover design enabled each patient to serve as his own control, thereby enhancing the power of the study. Changes in oxygen supply due to stenosis dilation and/or collateral recruitment may result in changes in myocardial perfusion redistribution that are independent of PC; however, these mechanisms should have occurred equally in both ETTs in this randomized, crossover trial. We were also unable to control for potential confounding effects of silent ischemia; however, again, the randomized and crossover design of the study should have resulted in an even distribution of silent ischemia between the 2 treatments. Finally, it could be speculated that the late-PC effect was caused by the hypotension associated with NTG. This conjecture, however, is unlikely because (1) there is no experimental evidence that hypotension per se induces late PC, (2) a late-PC effect can be recruited by nonhypotensive doses of NO donors,25 and (3) we found no correlation between the decrease in blood pressure induced by NTG and the effect of NTG on exercise duration or ST-segment depression (data not shown).
The present study has conceptual and practical implications with respect to late PC. From a conceptual standpoint, our results indicate that nitrates can induce a clinically significant late-PC effect in humans. Our previous study of NTG-induced late PC8 used ST-segment shifts as the primary end point; however, whether ST-segment shifts are a faithful index of myocardial ischemia has been questioned.9 The present finding that NTG pretreatment enhances 2 non-ECG end points, ie, exercise duration and maximal RPP achieved, indicates that nitrate-induced late PC is a genuine form of protection rather than an ECG curiosity. From a practical standpoint, the finding that nitrates can produce a delayed increase in exercise capacity among patients with stable angina has potentially broad implications for the management of CAD and also for the design of clinical trials.
In summary, the data reported herein reveal a novel action of nitrates that was heretofore unrecognized, namely, their ability to alleviate exercise-induced ischemia even after their hemodynamic actions have dissipated. This late PC-mimetic effect results in tangible clinical improvement, not simply in ECG changes, implying that late PC can be harnessed to combat the clinical manifestations of CAD. It has been traditionally thought that the protective effects of nitrates against exercise-induced ischemia dissipate quickly after the hemodynamic actions of these drugs cease. Our study demonstrates that this long-held belief is incorrect and that nitrates continue to exert protective actions long after they have been cleared from the body.
This study was supported in part by National Institutes of Health grants HL-55757, HL-68088, HL-70897, and HL-76794.
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