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Ann Thorac Surg 2002;74:2147-2155
© 2002 The Society of Thoracic Surgeons
a Department of Thoracic and Cardiovascular Surgery, Heinrich-Heine-University, Duesseldorf, Germany
b Research Group Experimental Surgery, Heinrich-Heine-University, Duesseldorf, Germany
Accepted for publication June 7, 2002.
* Address reprint requests to Dr Sunderdiek, Department of Thoracic and Cardiovascular Surgery, Heinrich-Heine-University, Moorenstr 5, 40225 Duesseldorf, Germany
e-mail: usunderdiek{at}aol.com
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Abstract |
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Methods. In isolated, blood-perfused rabbit hearts, the effects of IP (3 minutes of no flow ischemia and 8 minutes of reperfusion) during 30 minutes of coronary hypoperfusion and 60 minutes of reperfusion were investigated. In two groups (n = 8 each) with and without (control group) preconditioning, ventricular function was assessed by load-insensitive measures: slope of the end-systolic pressure–volume relation (Emax), slope of the stroke work/end-diastolic volume relation (Mw), and end-diastolic pressure–volume relation. External efficiency was calculated, and contractile efficiency was assessed using the reciprocal of the myocardial oxygen consumption–pressure–volume area relationship. To investigate the possible role of adenosine, the adenosine A1 receptor antagonist DPCPX (2.5 µmol/L) was administered before preconditioning in a third group (n = 7).
Results. The effects of hypoperfusion on systolic function, diastolic function (dP/dtmin, end-diastolic pressure–volume relation), external efficiency, and contractile efficiency were similar in both the IP and control groups. Lactate efflux was significantly reduced after preconditioning (p = 0.02). During reperfusion, recovery of systolic function and coronary flow were significantly improved in the IP group compared with controls: aortic flow, 85% versus 63% (p = 0.01); dP/dtmax, 91% versus 67% (p = 0.001); pressure–volume area, 97% versus 68% (p = 0.01); Emax, 74% versus 62% (p = 0.03); and Mw, 94% versus 84% (p = 0.04). Release of creatine kinase was reduced in the IP group, 9.6 ± 1.3 U · 5 min-1 · 100 g-1 wet weight, versus controls, 12.7 ± 2.7 U · 5 min-1 · 100 g-1 wet weight (p = 0.04). During reperfusion, contractile efficiency (p = 0.03) and external efficiency (p = 0.02) recovered better in preconditioned than in untreated hearts. Recovery was less pronounced in the DPCPX group compared with the IP group (p, not significant).
Conclusions. The results, derived from load-insensitive measures, confirm that IP provides protection after episodes of severe hypoperfusion by attenuating systolic dysfunction without improving diastolic dysfunction and reduces the severity of anaerobic metabolism as well as ischemic injury. Contractile efficiency and external efficiency both indicate improved energetics after IP (oxygen utilization by the contractile apparatus). The protective effect, at least in part, is mediated by adenosine A1 receptors.
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Introduction |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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The cardioprotective effect, namely, reduction in infarct size, seems to be mediated by adenosine [7]. Because it is unknown whether protection against postischemic ventricular dysfunction is also mediated by adenosine A1 receptors, we also investigated the effects of a selective adenosine A1 receptor antagonist, DPCPX.
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Material and methods |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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The rabbits were anesthetized with intravenous administration of sodium pentobarbital (30 mg/kg) and mechanically ventilated after a tracheotomy. After sternotomy, the hearts were rapidly excised, immediately connected to a modified Langendorff apparatus, and perfused with a modified crystalloid, erythrocyte-enriched Krebs-Henseleit solution containing the following (in millimoles per liter): NaCl, 119; NaHCO3, 25; KCl, 4.7; CaCl2, 1.8; MgCl2, 1.2; EDTA (ethylenediaminetetraacetic acid), 0.5; and glucose, 11. The buffer was equilibrated with 20% oxygen, 5% carbon dioxide, and 75% nitrogen at 37°C, giving a pH of 7.4. Albumin (4 g/100 mL) and bovine erythrocytes were added to obtain a hemoglobin concentration of 10 g/100 mL. Concentration of Ca2+ was held constant at 2.5 mmol/L.
A saline solution–filled latex balloon was inserted into the left ventricular (LV) cavity through the left atrium. The balloon was connected to a "systemic" circuit. Aortic flow was assessed with an ultrasonic flow probe (T 206; Transonic Systems Inc, Ithaca, NY). Aortic pressure (afterload) was measured with a pressure transducer (P23 II; Statham). The circuit permitted changes in afterload and preload without alterations in coronary perfusion pressure. A 3F micro-tip manometer (SPR-249; Millar Instruments Inc) inserted into the balloon was used to measure LV pressure. Sonomicrometry (System 6; Triton Technology, Inc) using two ultrasonic crystals glued to either side of the balloon measured LV dimensions. Different balloon sizes were selected depending on the heart size.
Total coronary venous flow was drained and measured, and the difference in arteriovenous oxygen content was continuously measured using absorption spectrophotometry (Avox Systems, Inc). Coronary perfusion pressure was held constant at 80 mm Hg throughout the study except for the period of coronary hypoperfusion.
Determination of myocardial edema
At the end of the study, two myocardial tissue samples weighing around 0.5 g were taken from the left ventricle of each heart. Each sample was weighed and dried for 24 hours at 80°C to determine the wet to dry weight ratio. Percent myocardial water was defined as follows:
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Experimental protocols
Control conditions (perfusion pressure of 80 mm Hg) were recorded after a 20-minute period for stabilization of LV function. Then the hearts were randomly assigned to one of three groups. Control hearts (n = 8) underwent 30 minutes of normothermic hypoperfusion at a perfusion pressure of 30 mm Hg followed by 60 minutes of reperfusion at a perfusion pressure of 80 mm Hg (Fig 1).
In the second group (IP group) (n = 8), hearts were preconditioned by a single 3-minute period of global no-flow ischemia, followed by 8 minutes of reflow prior to the sustained 30 minutes of hypoperfusion and 60 minutes of reperfusion. In the third group (n = 7), the adenosine A1 antagonist DPCPX (2.5 µmol/L) was administered 5 minutes before the 3-minute period of no-flow ischemia and 8 minutes of reflow, which again was followed by global hypoperfusion and reperfusion (see Fig 1).
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Data acquisition
The following variables were registered continuously: aortic flow, coronary flow, LV pressure, and inner diameter. Heart rate and first derivative of LV pressure (dP/dt) were derived from the pressure signal. At steady-state conditions after preload alterations, the variables were simultaneously stored digitally for later analysis at a sampling rate of 300 Hz.
Calculations and statistical analysis
Computer-assisted analysis of hemodynamic data was performed. The end-systolic pressure–volume relation, the pressure–volume area (PVA), and the ventricular stiffness (monoexponential fitting of the end-diastolic pressure–volume relation [EDPVR]) were calculated with the help of a custom-made computer program (EASYDAT) and, if appropriate, using equations suggested by Mirsky [8]. The EDPVR is approximated by the equation
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The slope of the end-systolic pressure–volume relation (end-systolic elastance, or Emax) was calculated as suggested by Suga [9]:
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Stroke work (SW) (mm Hg · mL) was defined as the area bound by the pressure–volume loop and was calculated from the ventricular LV peak pressure and stroke volume. The stroke work–end-diastolic volume relation, also termed preload-recruitable stroke work relation, was fit to the equation
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Coronary flow was normalized to 100 g wet weight. Coronary resistance was calculated from coronary artery pressure and normalized blood flow. Myocardial oxygen consumption (MVO2) was calculated according to the Fick principle from normalized coronary flow and the difference in arteriovenous oxygen content (AVO2 difference) (mL O2/100 mL blood):
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As an index of external efficiency, the ratio of stroke work and MVO2 per beat was determined at the same LV inner diameters, ie, the same preload. Contractile efficiency was determined as the inverse slope of the MVO2–pressure–volume area relationship, and the MVO2 for the unloaded contraction was assessed as the intercept of the MVO2–pressure–volume area relationship with the MVO2 axis.
Data are expressed as the mean ± the standard deviation. Statistical analysis was performed with a statistical software package (Systat). Repeated-measures analysis of variance was used to test differences in hemodynamic variables within any given group. If significant overall effects were encountered, further analysis was performed using the Bonferroni correction. Differences were considered to be significant at a p value of less than 0.05.
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Results |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Myocardial oxygen consumption, metabolic function, and cardiac efficiency
Total MVO2, was significantly reduced during hypoperfusion and recovered to 89% in the control group, 96% in the IP group, and 99% in the DPCPX group (Table 2).
During hypoperfusion, MVO2 for the unloaded contraction was unchanged in the control group and in the DPCPX group, but it was reduced (-16%) in the preconditioned hearts. After 60 minutes of reperfusion, MVO2 for the unloaded contraction was increased in the control group (13%) and in the DPCPX group (10%) but remained nearly unchanged in the IP group (p = NS).
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Contractile efficiency showed no change for the control group during hypoperfusion compared with baseline, but it was decreased by 14% during reperfusion (see Table 2). In the IP group, contractile efficiency was slightly increased during hypoperfusion and even higher during reperfusion (p = 0.03 versus controls). In the DPCPX group, contractile efficiency remained essentially unaffected. External efficiency was severely decreased during hypoperfusion and recovered significantly in the IP group compared with the control group (p = 0.02). The recovery was significantly different between groups by percentage values.
Myocardial edema and creatine kinase release
After 60 minutes of reperfusion, edema had formed in the untreated hearts. The wet to dry weight ratio was 82% ± 1% compared with 78% ± 1% in the IP group (p = 0.02 versus controls) and 79% ± 2% in the DPCPX group (p = 0.01 versus controls).
There was no notable CK or lactate dehydrogenase release (>5 U · 5 min-1 · 100 g-1 wet weight) before ischemia in any of the three groups. After 30 minutes of hypoperfusion, levels were somewhat lower in the preconditioned hearts (p = NS), but after 60 minutes of reperfusion, release of these markers was significantly lower in the IP group compared with the untreated group (p = 0.04) (see Table 3).
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Comment |
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The major finding in this study was that postischemic recovery of LV systolic performance and cardiac contractility assessed using dP/dtmax and load-insensitive measures such as Emax (systolic elastance) and Mw (slope of the stroke work–end-diastolic volume relation) were improved after IP together with the energetic state. In turn, the beneficial effect of IP on functional recovery was less pronounced after treatment with the A1 receptor antagonist DPCPX, a finding suggesting that the IP–induced cardiac protection is mediated by way of A1 receptors.
Ischemic preconditioning
Although many details of IP are well described [10], it is not clear whether the enhanced recovery of contractile function is a consequence of smaller ischemic injury, reduced stunning, or both. In the globally ischemic isolated rabbit heart [5, 11] and in in vivo porcine hearts [12], no beneficial effect of IP on postischemic ventricular dysfunction independent of reduction in infarct size was demonstrated. In contrast to those results, we found enhanced recovery of systolic function after IP, as assessed under controlled loading conditions.
The most favored current hypothesis for the protective effects of IP describes endogenous factors, such as adenosine, norepinephrine, endothelin, and bradykinin, initiating intracellular pathways by activation of phospholipases C and D leading to the activation of protein kinase C isotypes [13]. Protein kinase activation and phosphorylation of a target protein seem an important step in rendering the heart resistant to sustained ischemic stress [14]. The most likely candidates for effector proteins are ion channels, ie, the KATP channel and Na+/H+ exchanger, and myofibrillar proteins. It is speculated that Protein Kinase C mediates phosphorylation of troponin I and partially prevents its proteolytic breakdown. At least in the perfused rat heart model [15], IP limited the reversible breakdown of myofilaments and contributed to the mechanism by which it curtails stunning.
Ventricular function and energetics during coronary hypoperfusion
Coronary hypoperfusion results in a state of moderate ischemia causing reversible contractile dysfunction. Such ischemia-induced down-regulation of contractile function is characterized by reduced myocardial energy demand, and therefore, the metabolic integrity (metabolic adaptation: recovery of creatine phosphate content, attenuation of net lactate production or lactate consumption, recovery from acidosis to normal) of the ischemic myocardium can be prolonged [16]. In general, the myocardium regains contractile function, but the ability of the ischemic myocardium to resist an irreversible ischemic injury critically depends on a number of factors, including duration and severity of ischemia.
In one study [17] the effects of IP on ventricular factors during low-flow ischemia have been investigated. In the present study, coronary perfusion pressure was reduced to 37.5% of control. A more drastic reduction in coronary perfusion pressure would have resulted in severe myocardial dysfunction and irreversible injury [16]. Within this protocol, we did observe a beneficial effect of IP in terms of lactate production. Thus, we provide additional evidence that IP exerts part of its cardioprotective effects already during the index ischemia and not only during reperfusion.
Systolic and diastolic ventricular function
In many studies, ventricular function was assessed only by LV developed pressure or end-diastolic pressure [1, 6, 11, 18]. Because these variables are preload and afterload dependent, their use is of limited value. In the present study, preload-insensitive measures such as the systolic elastance, Emax, and the preload-recruitable stroke work relation, Mw, were used. The slopes of both relations were shifted to the right, and the slopes were decreased during hypoperfusion. During reperfusion, the relations shifted leftward and the slopes were increased, meaning functional recovery. This recovery was more pronounced after preconditioning, a finding indicating that IP did attenuate postischemic dysfunction when assessed by load-insensitive measures. Some previous studies [3, 19] that are in accord with our results speculated that analysis of the effects of IP might be confounded by the fact that repetitive preconditioning-results in stunning before the index ischemia and therefore may limit or mask a beneficial effect of preconditioning on postischemic myocardial function. In fact, in this study LV peak pressure was significantly decreased after the preconditioning measure, but total recovery (compared with baseline) after the index ischemia was improved compared with the untreated hearts.
Diastolic function, as assessed using different measures, was not markedly improved by IP. At first sight, this finding could mean that IP does not affect diastolic properties. On the other hand, recovery of diastolic properties might have a delayed time course compared with systolic properties, and thus it might have been overlooked because of our relatively short reperfusion period. Finally, more than one cycle of IP either might have caused more severe deterioration during diastole or might have initiated better recovery during reperfusion. Whether or not more preconditioning cycles would have provided better protection of systolic and diastolic properties was beyond the scope of this study.
Coronary vasculature
It has been suggested that IP might limit the vascular consequences of ischemia-reperfusion injury. Potent cardiovascular effects of adenosine on ischemia-reperfusion injury are derived from reversal of endothelin-induced vasoconstriction [20], inhibition of neutrophil functions, reduction in platelet aggregation, and replenishment of high energy stores in endothelial and myocardial cells [21]. However, in buffer-perfused models [1, 6, 11] where the coronary reserve is almost exhausted, it is unlikely that IP can exert physiologically important protective effects on vascular function because of the high rate of coronary flow and the lack of plasma proteins [21]. In our model we used an erythrocyte-containing perfusate enriched with albumin, which secures an almost physiological coronary flow. At a constant coronary perfusion pressure, coronary flow was better preserved after IP in our model, which indicates an attenuation of vascular stunning.
In a comparable study [17] involving a canine model of regional left anterior descending coronary artery perfusion, IP resulted in significantly increased adenosine production during coronary hypoperfusion leading to increased coronary flow together with improved metabolic and mechanical function.
Ventricular energetics
Few studies have examined the energetics of IP. Preconditioning reduced the energy demand during ischemia, and adenosine triphosphate utilization is lower possibly because of lower metabolic requirements for contractile activity [22]. Our results demonstrated reduced anaerobic glycolysis during ischemia in the preconditioned hearts. As glycogenolysis is also slower, both effects cause reduced intracellular acidosis during ischemia and markedly reduced accumulation of purine nucleosides.
The PVA is a measure of total mechanical work [9]. Analysis of the MVO2–PVA relationship allows partitioning MVO2 for the unloaded contraction, which was only slightly reduced in the IP hearts compared with the untreated hearts. This demonstrates no altered oxygen demand for basal energy utilization, Ca2+ handling, or both during hypoperfusion and reperfusion. The PVA–related energy demand, described as contractile efficiency, was slightly improved during hypoperfusion and significantly preserved during reperfusion in the IP hearts compared with the untreated hearts. Similarly, the ratio between stroke work and oxygen consumption per beat, ie, external efficiency, was almost preserved in the IP group compared with controls, whereas the deterioration was significant in the untreated group. These notable findings together indicate ameliorated oxygen consumption by the contractile apparatus or better preserved energy transfer and function of the contractile proteins. These results might be explained by an attenuated myofilament degradation [15] and other reduced ischemic injuries, expressed in lower CK and lactate dehydrogenase release during ischemia and reperfusion after preconditioning.
Role of adenosine a1 receptors
Our results showed that the A1 receptor antagonist DPCPX blunted the protective effect of IP on ventricular and energetic function in part, thus demonstrating that the protection afforded by preconditioning is adenosine dependent. These data are supported by previous studies showing that adenosine A1 receptor activation [23] contributes to the protective effect of IP. However, as DPCPX did not entirely blunt the protective effect of preconditioning, it is concluded that preconditioning might also be mediated by way of triggers other than adenosine.
Critique of methods
Some limitations of the present study must be recognized. These results represent only the first 60 minutes of reperfusion and do not reflect the potential effects during reperfusion beyond this time. After 60 minutes of reperfusion, CK levels in the preconditioned hearts were significantly lower than in the unprotected hearts, and accordingly, we did not find any marked necrosis. Because 180 minutes of reperfusion is suggested to produce necrotic areas control and preconditioned hearts in a separate set of experiments were subjected to 180 minutes of reperfusion and showed no differences compared with the hearts reperfused for 60 minutes. As all hearts in this study were subjected to the same reperfusion time, a systematic error can theoretically be ruled out.
For application of IP, it is desirable to maximize the extent of protection attained. Increasing the number of cycles of transient ischemia and reperfusion used to induce IP may achieve this goal [24] and might also affect diastolic recovery. However, in a separate small series in this study, we found that every cycle of global ischemia compromised ventricular function, and within the reperfusion or so-called stabilization period of 5 minutes between every ischemic cycle, ventricular function improved to 90% to 95% of the control function data. With two or more ischemic cycles, ventricular dysfunction is more pronounced, and the following hypoperfusion period results in more severe myocardial dysfunction. Therefore, we decided to apply only one IP cycle.
We used only one concentration of the adenosine A1 receptor antagonist (2.5 µmol/L) that was shown to be effective in blocking the effects of preconditioning in rabbit myocardium in concentrations ranging from 0.2 µmol/L [25] to 2.5 and 5 µmol/L [1, 11] to 10 µmol/L [26]. Using an average concentration, we provided evidence that most of the postischemic protection was blunted. Thus, we cannot determine whether or not a higher concentration would have completely prevented the preconditioning effect.
Clinical application
The use of IP for protection during ischemia as well as for rapid recovery of impaired myocardial function after ischemia has gained popularity. In fact, some studies support the evidence that human myocardium can be preconditioned during a cardiac operation [27]. However, during surgical procedures on the heart, global ischemia is most often induced by cardioplegic arrest. The results from human trials with cardioplegia and additional IP are not encouraging [6], and other factors such as cardiopulmonary bypass might act as a preconditioning stimulus [28]. However, with the simple intermittent aortic cross-clamping technique, IP might play a beneficial role, as use of this cardioprotective method might very well limit myocardial necrosis during coronary artery operations [29]. However, the discussion about whether or not IP could play an important role in cardiac surgery will continue.
Conclusions
Although the influence of IP on postischemic ventricular dysfunction has been investigated before, the current study explores the phenomenon in a different fashion. We used preload- and afterload-insensitive measures such as Emax and Mw to describe the beneficial effects on systolic function. Our measures did not exhibit any positive effect of preconditioning on diastolic function. We provided some insight into cardiac energetics by showing that IP reduces the severity of anaerobic metabolism and improves contractile efficiency during brief episodes of coronary hypoperfusion and reperfusion in the blood-perfused rabbit heart, thus indicating better preserved energy conversion to contraction. Finally, the important role of adenosine A1 receptors in mediating IP is emphasized.
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Acknowledgments |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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References |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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