Effect of preconditioning temperature on cardioprotection during global ischemia-reperfusion in the rat heart

Abstract

BACKGROUND

Myocardial ischemic preconditioning (PC) is a protective intervention that aims to reduce the deleterious effects of ischemia-reperfusion injury.

OBJECTIVES

: To assess the comparative efficacy of ischemic PC induced at hypothermic, normothermic and hyperthermic temperatures on the postischemic recovery of left ventricular contractile and coronary vascular functions in the aortic perfused rat heart model.

METHODS

An isolated aorta-perfused (Langendorff) rat heart model was used. Hearts were studied in eight different groups (n=5 each). Unprotected ischemia for 60 min served as control. For the remaining seven groups, ischemia was preceded by PC at 10°C, 20°C, 30°C, 34°C, 37°C, 40°C and 42°C achieved by two episodes of 5 min ischemia at the designated PC temperature and 10 min of reperfusion, respectively. The postischemic recovery in contractile (maximum developed pressure and left ventricular end-diastolic pressure) and coronary vascular functions (coronary flow and coronary vascular resistance) was assessed at the end of 30 min reperfusion.

RESULTS

Hyperthermic PC provided optimal preservation and resulted in a 25% increase in the myocardial and 15% increase in the coronary vascular tolerance to ischemia. Normothermic PC resulted in a 21% increase in myocardial and 14% increase in coronary vascular tolerance to ischemia. Hypothermic PC was comparatively less effective and resulted in 11% increase in myocardial and 15% increase in the coronary vascular tolerance to ischemia. The temperature-dependence of PC may be summarized as: PC-42°C > PC-40°C > PC-37°C > PC-34°C > PC-30°C > PC-20°C > PC-10°C.

CONCLUSIONS

The results of the present study indicate that the extent of reversibility of the ischemic damage depends on the PC temperature and that optimal preservation occurred at the ideal PC temperature between 40°C and 42°C.

Ischemia-reperfusion injury is known to be deleterious to the myocardium (1,2). However, during open heart surgical procedures, hearts must undergo prolonged periods of induced ischemic arrest followed by reperfusion. To minimize the extent of the ischemic damage, several cardioprotective interventions have been used during open heart surgical procedures to reduce such an injury. These interventions include hypothermia, and hypothermic or normothermic cardioplegia.

More recently, the experimental role of ischemic preconditioning (PC) has been identified. PC originated from the hypothesis proposed by Swain et al (3), who showed that a few repetitive ischemic episodes did not cause a cumulative depletion of high-energy phosphate compounds. PC was also reported to limit infarct size (4,5), reduce ischemia and reperfusion arrhythmias (6), and to improve contractile and coronary vascular functions (7).

Many studies have tested the possible additive cardioprotection when ischemic PC was combined with hypothermia and cardioplegia. When ischemic PC was added to hypothermic global ischemia, functional reperfusion recovery was either unaffected (8) or improved (9). Furthermore, the addition of cardioplegia had no beneficial effect compared with hypothermic cardioplegia alone (8). A slight improvement in functional recovery reflected by a decrease in end-diastolic pressure was observed when ischemic PC preceded a prolonged period of normothermic cardioplegic arrest (10). However, other investigators reported no effect of such a combination on functional recovery (11). On the other hand, when ischemic PC was combined with an impaired cardioplegic delivery, an increase in the functional recovery was reported (12). Moreover, preconditioning the heart before longer hypothermic cardioplegic periods (ranging from 2.5 h to 12 h) was shown to improve left ventricular contractile functions (13). Coronary vascular functions were reported to either improve (13) or remained unaffected by the same protocol (14).

A recent study by Lu et al (15) studied the additive beneficial effects of ischemic PC in patients undergoing cold crystalloid cardioplegic arrest. They observed a significant reduction of both ST-segment elevation and creatine kinase isoenzyme release. Moreover, they showed that contractility was enhanced with improved postoperative functional recovery. On the other hand, Cremer et al (16) reported adverse effects of combining ischemic PC with cold cardioplegic arrest in patients undergoing open heart surgery. Perrault et al (17) observed no additional beneficial effect on the metabolic indices of the heart when ischemic PC was combined with warm cardioplegic arrest. They observed an increase in the release of creatine kinase isoenzyme and in the total production of lactate in the preconditioned patients compared with those given only warm cardioplegia (17).

PC for all these studies of global ischemia was achieved at normothermic temperature. There is only one study (18) of regional ischemia in which PC temperature was manipulated from normothermic to hypothermic with significant reduction in the infarct size.

The main objective of the present study was to evaluate the comparative efficacy of different PC temperatures, ranging from hypothermic to hyperthermic temperatures, on cardioprotection during prolonged periods of global ischemia in a surgically relevant rat heart model of retrograde aortic perfusion.

METHODS

Experimental model

Hearts were obtained from male Wistar rats weighing between 400 g and 500 g. They were lightly anesthetized with a mixture of ether and O₂, and heparinized intravenously (1000 U/kg body weight) through the femoral vein. The abdominal cavities were opened and then the hearts were rapidly removed and immersed in cold (4°C) Krebs-Henseleit solution. Hearts were mounted on the perfusion assembly through the aortic cannula and immediately perfused retrogradely with oxygenated (95% O₂ and 5% CO₂) Krebs-Henseleit solution (117.86 mM NaCl, 5.59 mM KCl, 2.40 mM CaCl₂•2H₂O, 20.00 mM NaHCO₃, 1.19 mM KH₂PO₄, 1.20 mM MgCl₂•6H₂O, 12.11 mM glucose, 300 mOsm/L, pH 7.35). The preparation was stable for a period of several hours during which various experimental protocols were carried out. A jacketed perfusion reservoir was used and the temperature of the perfusion medium was controlled by circulating temperature-controlled water (RMS Lauda, Germany and Techne Circulator, USA).

The perfusion pressure (PP) for baseline control conditions was kept constant at 50 mmHg. PP was measured immediately downstream from the flow probe in a branch of the aortic cannula using a Statham pressure transducer (P23XL, Hugo Sachs Elektronik, Germany). Constant PP (and also a rapid preset change in this value) was ensured electronically by a PP control module of the perfusion assembly (Module PPCM type 671, Hugo Sachs Electronik, Germany). This system permits accurate adjustment of PP between 5 mmHg and 150 mmHg to an accuracy of ±1 mmHg. The level of perfusate in the jacketed perfusion oxygenator was kept at 10 cm H₂O pressure above the level of the heart. The PP control module senses this pressure and adjusts the control PP of 50 mmHg inclusive of the hydrostatic pressure of the perfusate in the oxygenator. The level of perfusate in the oxygenator was properly controlled and adjusted to 10 cm H₂O pressure by the liquid level control module (Module LLC type 661, Hugo Sachs Electronik, Germany).

Experimental protocols

All hearts were perfused for 20 min before monitoring and recording the baseline left ventricular and coronary vascular hemodynamic data. Following this, they were divided into eight groups (n=5 each). They were all subjected to global ischemia at 34°C for 60 min followed by reperfusion for 30 min at the same temperature during which the postischemic recovery in contractile and coronary vascular functions was assessed. In seven of these groups, ischemia was preceded by PC at different temperatures ranging from 10°C to 42°C. PC was induced as two episodes of 5 min global ischemia at the desired PC temperature, which varied from hypothermic (10°C, 20°C, 30°C and 34°C) to normothermic (37°C) to hyperthermic (40°C and 42°C) temperatures. Each ischemic PC episode was followed by reperfusion for 10 min (Figure 1). To obtain a perfect temperature control during the PC periods, the heart was immersed in the temperature-controlled jacketed bath filled with isotonic saline of the desired temperature. This helped to maintain the desired level of myocardial temperature within a few seconds of immersion during the ischemic PC periods. Myocardial temperature was continuously monitored by a temperature-sensitive thermister needle probe (Thermalert TH-5, Physitemp, USA) inserted into the intraventricular septum.

Figure 1.

Schematic representation of experimental protocols. I Global ischemia; PC Preconditioning; R Reperfusion; T Preconditioning temperature (10°C, 20°C, 30°C, 34°C, 37°C, 40°C or 42°C)

Assessment of left ventricular global contractility

Left ventricular global contractility was assessed by continuously monitoring the left ventricular maximum developed pressure (Pmax), and the left ventricular end-diastolic pressure (LVEDP). An alcohol-filled latex balloon was inserted into the left ventricular cavity and secured there. The balloon was attached to a pressure transducer which converted the mechanical pressure energy into electrical signals that were amplified by a DC-Bridge amplifier (DC-BA) of the pressure module (DC-BA type 660, Hugo-Sachs Electronik, Germany) interfaced to a personal computer for on-line monitoring of left ventricular pressure and its derivatives. Under basal control conditions, the LVEDP was kept constant at 5 mmHg by suitably adjusting the volume of the balloon with 50% alcohol. Left ventricular Pmax was derived from the on-line acquisition of left ventricular systolic pressure by a Max-Min module (MMM type 668, Hugo-Sachs Electronik, Germany). This module converts the output from the DC-BA to Pmax by subtracting LVEDP from the left ventricular systolic pressure.

Assessment of coronary hemodynamics

Coronary flow was measured continuously by the electromagnetic flow probe attached to the inflow of the aortic cannula. The probe was attached to a flow meter which was interfaced to a personal computer. The coronary flow in mL/min was continuously monitored as well as computed. The coronary vascular resistance was computed every 10 s along with the hemodynamic data by an online data acquisition program (Isoheart software V 1.524-S, Hugo-Sachs Electronik, Germany).

Statistical analysis

All data in the present study were expressed as mean ± SEM for n values. Mean values were calculated using Excel for windows (Version 7.0, Microsoft Corporation, USA). The statistical significance of differences between different group means were assessed by one-way ANOVA. Post hoc analysis of differences between two group means was performed using unpaired t test (two-tailed). The level of difference between a group mean and that of its own control was assessed using the paired Student’s t test (two-tailed). Differences were considered significant when P<0.05.

RESULTS

Effect of PC temperature on left ventricular global contractile functions

There were no significant differences in any of the contractility parameters during the baseline control conditions between any group (Table 1). Postischemic reperfusion recovery in global contractility (Pmax) was significantly reduced in all the experimental groups (Table 1). Postischemic recovery in Pmax was 45% after unprotected ischemia-reperfusion (I); 54%, 53%, 56% and 64% after hypothermic PC temperatures of 10°C, 20°C, 30°C and 34°C, respectively; 66% after normothermic PC at 37°C; and 69% and 70% after hyperthermic PC temperatures of 40°C and 42°C, respectively. However, compared with unprotected ischemia, recovery in Pmax was significantly better after hypothermic, normothermic and hyperthermic PC protocols. There were no significant differences between the protective efficacy of hypothermic and normothermic PC. Hyperthermic PC produced a significantly (P<0.05) better postischemic recovery in Pmax compared with that of hypothermic PC when the post-ischemic recovery in Pmax reached to an optimum value of 70% after PC at 42°C (Table 1). There were no significant differences between the protective efficacy of normothermic and hyperthermic PC. There was an approximately sevenfold increase in LVEDP after unprotected ischemia when it increased from a mean control value of 4.8±0.3 mmHg to 33.8±1.7 mmHg. Hypothermic PC decreased this excessive rise and the mean postischemic values of LVEDP recorded were 23.5±2.7 mmHg, 27.5±3.5 mmHg and 19.3±3.5 mmHg after 10°C, 20°C and 30°C PC, respectively (Table 1). These values were significantly (P<0.01) lower when compared with those of unprotected ischemia. Similarly, normothermic PC (37°C) was equally effective in reducing the excessive rise of postischemic LVEDP. There were no significant differences between the preservative efficacy of hypothermic and normothermic PC (Table 1). However, hyperthermic PC further significantly (P<0.01 or P<0.001) improved the recovery in the postischemic LVEDP. The lowest mean postischemic value recorded was 13.1±3.7 mmHg after PC at 40°C.

TABLE 1.

Effect of preconditioning (PC) temperature on the postischemic recovery in global contractility

	Pmax			LVEDP
	Control	Reperfusion		Control	Reperfusion
	mmHg	mmHg	%	mmHg	mmHg	%
I (n=5)	92±4	41±4***	45±4***	4.8±0.3	33.8±1.7***	707±46***
PC-10°C (n=5)	107±6	57±4***†	54±3***	4.9±0.1	23.5±2.7**†	496±63**†
PC-20°C (n=5)	109±6	57±8**	53±8**	5.4±0.2	27.5±3.5**	512±69**†
PC-30°C (n=5)	107±4	59±5**†	56±5***	4.7±0.1	19.3±3.5*††	417±80*†
PC-34°C (n=5)	108±6	68±6*††	64±7**†	4.9±0.2	16.3±3.3*††	337±68*††
PC-37°C (n=5)	111±6	74±8***††	66±4**††‡	4.9±0.2	17.6±3.4*††	364±78*††
PC-40°C (n=5)	118±6	81±5**†††‡‡§¶	69±4**††‡	5.0±0.3	13.1±3.7†††§	260±70*†††‡§
PC-42°C (n=5)	106±5	74±6*††‡	70±6**††‡	4.8±0.2	17.3±2.9*††	336±77*††

The data were computed at 30 min reperfusion and expressed as mean ± SEM.

^*P<0.05,

^**P<0.01,

^***P<0.001 compared with respective controls;

^†P<0.05,

^††P<0.01,

^†††P<0.001 compared with ischemia at 34°C (I);

^‡P<0.05,

^‡‡P<0.01 compared with PC-10°C;

^§P<0.05 compared with PC-20°C;

^¶P<0.05 compared with PC-30°C. LVEDP Left ventricular end-diastolic pressure; % Per cent of respective control; Pmax Left ventricular maximum developed pressure

The pattern of postischemic recovery in Pmax and LVEDP for all the protocols of the present study is shown in Table 1. There was a linear but inverse relationship in the postischemic recovery of Pmax (r²=0.81) and LVEDP (r²=0.80) with graded increase in the PC temperature from 10°C to 42°C. With the increase in the PC temperature left ventricular Pmax significantly increased and LVEDP significantly decreased in a linear fashion. The major protective effect of PC on the left ventricular systolic and diastolic functions was evident at higher PC temperatures.

Effect of PC temperature on coronary flow and coronary vascular resistance

Reperfusion recovery in coronary flow was significantly reduced in all the experimental groups when compared with respective baseline control values (Table 2). Major reduction occurred after unprotected ischemia-reperfusion. Compared with baseline controls, postischemic recovery in coronary flow was 44% after unprotected ischemia; 49%, 47% and 59% after hypothermic PC at 10°C, 20°C and 30°C, respectively; 58% after normothermic (37°C) PC; and 61% and 59% after hyperthermic PC at 40°C and 42°C, respectively. Compared with hypothermic PC, hyperthermic PC offered a better postischemic recovery in coronary flow.

TABLE 2.

Effect of preconditioning (PC) temperature on the postischemic recovery in coronary hemodynamics

	CF			CVR
	Control	Reperfusion		Control	Reperfusion
	mL·min–1	mL·min–1	%	mmHg·mL–1min–2	mmHg·mL–1min–2	%
I (n=5)	10.2±0.6	4.5±0.5***	44±3***	5.2±0.3	12.1±1.3**	232±15***
PC-10°C (n=5)	11.1±0.5	5.6±0.6***	49±4***	4.6±0.2	10.0±1.3**	214±21**
PC-20°C (n=5)	12.2±0.3	5.7±0.6***	47±5***	4.2±0.1	8.0±0.6**†	192±13**
PC-30°C (n=5)	10.9±0.2	6.4±0.5**†	59±6**†	4.7±0.1	8.3±0.5**†	177±15**††
PC-34°C (n=5)	11.3±0.7	6.3±1.0***	55±6**	4.6±0.3	9.3±1.5*	195±23*
PC-37°C (n=5)	13.3±0.5	7.7±0.5***††‡§	58±2***††	3.9±0.2	7.1±0.5***††	179±6***†
PC-40°C (n=5)	12.1±0.4	7.4±0.8**†	61±6**†	4.4±0.2	7.7±1.3*†	175±22*†
PC-42°C (n=5)	12.9±0.4	7.7±0.5***††‡§	59±2***††§	4.0±0.1	7.0±0.5**††	173±7***††

The data were computed at 30 min reperfusion and expressed as mean ± SEM.

^*P<0.05,

^**P<0.01,

^***P<0.001 compared with respective controls;

^†P<0.05,

^††P<0.01 compared with ischemia at 34°C (I);

^‡P<0.05 compared with PC-10°C;

^§P<0.05 compared with PC-20°C. CF Coronary flow; CVR Coronary vascular resistance; % Per cent of respective control

Coronary flow changes were accompanied by a significant increase in the coronary vascular resistance. Coronary vascular resistance increased more than twofold after unprotected ischemia. With hypothermic PC, coronary vascular resistance showed a gradual decline from 232% after unprotected ischemia to 177% of control after PC at 30°C. Normothermic (37°C) and hyperthermic PC did not further improve the postischemic recovery in coronary vascular resistance, which remained around 179% of the control at the PC temperatures of 40°C and 42°C (Table 2).

There was a linear relationship in the postischemic recovery of coronary flow (r²=0.72) and coronary vascular resistance (r²=0.75) with graded increase in the PC temperature from 10°C to 42°C (Table 2). Coronary flow increased and coronary vascular resistance decreased with the graded increase in the PC temperature and the optimum effect is evident at PC temperatures of 40°C to 42°C (Table 2).

DISCUSSION

The results of the present study indicate that the PC temperature plays an important role in determining the postischemic recovery of myocardial and coronary vascular functions. Unprotected ischemia-reperfusion produced serious derangements in the left ventricular systolic and diastolic functions terminating in the left ventricular contracture development and irreversibility of the ischemic damage (Table 1). Coronary vascular resistance was considerably increased, leading to the development of the low-reflow phenomenon (Table 2), indicative of coronary vascular damage. PC (between 10°C and 42°C) before the onset of prolonged ischemia reduced the extent of left ventricular contractile and vascular dysfunction, but failed to completely normalize these functions. This reduced postischemic recovery is a measure of myocardial stunning and necrosis. The extent of reversibility of the ischemic damage depended on the PC temperature. In general, hyperthermic PC provided better preservation than normothermic PC, and normothermic PC provided better preservation than the hypothermic PC (Tables 1 and 2). These ‘dose-response’ effects may be summarized as: hyperthermic PC preservation > normothermic PC preservation > hypothermic PC preservation.

PC at 37°C, when combined with sustained ischemia-reperfusion, resulted in a 21% increase in myocardial and 14% increase in coronary vascular tolerance to ischemia. These results are in agreement with earlier reported studies (8,9). There is still a great controversy about the exact mechanism(s) responsible for the beneficial protective effects of normothermic ischemic PC. The proposed mechanisms of protection include reduced ATP utilization and improvement of metabolic status (4,19); maintenance of ionic homeostasis and prevention of Ca²⁺ loading (19); and release of various neuroendocrine and paracrine substances such as adenosine (20), acetylcholine (21), bradykinin (22), endothelin (23), angiotensin II (24) and opioids (25). The effect of these substances may be mediated by the activation of protein kinase C, which in turn will open ATP-sensitive potassium channels and thereby reduce the cellular Ca²⁺ overload (26).

PC improves the coronary vascular functions as indicated by the improved postischemic recovery of coronary flow and coronary vascular resistance. This hemodynamic effect may be secondary to the improvement in myocardial contractility. The available evidence indicates that both endothelium-dependent coronary flow and vasodilatory reserve is improved by ischemic PC (27). The vasodilatory response to acetylcholine was better preserved by ischemic PC under both acute and chronic reperfusion conditions; indicating better preservation of vascular endothelium by PC. The improvement in the postischemic coronary flow may also be secondary to the improvement of the functional recovery in the isolated perfused rat hearts after ischemic PC (28).

Hypothermic PC provided a measure of protection to the myocardium and coronary vasculature, and resulted in 11% increase in the myocardial and 15% increase in the coronary vascular tolerance to ischemia. However, when compared with normothermic PC, this protection occurred to a lesser extent. Using the experimental conditions of the present study, it is not possible to explain this limited beneficial effect of hypothermic PC. However, recent reports in the literature (29,30) indicate that cooling followed by rewarming during short periods induces cold-shock proteins in the mammalian cells as well as improving antioxidant reserve, which may provide additive protection to the compromised tissues. Transient cold shock has been reported (29) to induce heat-shock proteins, which may have a cellular protective role during stressful situations. Cold-induced expression of heat-shock proteins has been reported to be mediated through the activation of heat-shock factor 1. Hypothermia preceding ischemia has also been shown to preserve myocardial functions and ATP stores, signalling mitochondrial biogenesis and heat-shock protein induction (29). Dote et al (18) recently reported that hypothermia during PC increases the threshold of ischemic PC and it required several cycles of hypothermic ischemic PC before the beneficial effects were evident. In the present study, two cycles of hypothermic PC of 5 min each were enough to show the beneficial effects. The observed differences may be explained by the type of experimental models used in these studies. Dote et al (18) used the perfused rabbit heart model of regional ischemia and evaluated the effect of hypothermic PC on the infarct size reduction, whereas the present study described the effects of ischemic PC during global ischemia-reperfusion.

Graded increases in PC temperatures from hypothermic to hyperthermic ranges increased the myocardial and coronary vascular tolerance to ischemia-reperfusion, and this effect had a linear relationship (Tables 1 and 2). A major improvement in the postischemic left ventricular systolic and diastolic functions occurred after hyperthermic PC. Coronary vascular resistance was considerably reduced, aiding a significant improvement in the postischemic coronary flow, indicating a better preservation of coronary vasculature by hyperthermic PC.

Hyperthermic (40°C to 42°C) PC temperatures produced the optimal preservation, resulting in a 25% increase in the myocardial and 15% increase in the coronary vascular tolerance to ischemia. The cellular mechanism of heat-shock-mediated cardioprotection is still not clear. Recent studies, however, indicate the expression of heat stress-related antioxidative genes and proteins in the heart following exposure to hyperthermia. The ischemic tolerance of the isolated perfused hearts, which were harvested after several hours of whole body hyperthermia, was found to be significantly improved, and this improvement was linked to the expression of various heat-shock proteins (31). Synthesis of stress-induced heat-shock proteins has also been demonstrated in isolated perfused hearts subjected to nonpreconditioned ischemia at different temperatures (32). The degree of induction of heat-shock protein HSP71 was dependent on the ischemic temperature, namely, 42°C > 37°C > 34°C > 31°C > 4°C, correlating very well with the functional recovery results of the present study where preservation after ischemic PC was graded as PC-42°C > PC-40°C > PC-37°C > PC-34°C > PC-30°C > PC-20°C > PC-10°C (Tables 1 and 2). A direct correlation between the amount of heat-shock protein and the degree of myocardial protection following whole body hyperthermia has also been demonstrated by Hutter et al (33). Isolated perfused hearts subjected to 15 min of hyperthermic perfusion at 42°C and followed by no-flow normothermic ischemia showed a significantly enhanced functional recovery during the reperfusion period and this recovery was correlated with increased synthesis of heat-shock proteins HSP72. This is the only report in the literature which is close – although not identical – to the PC protocols of the present study. In addition, several other stress-induced antioxidant genes and stimulation of antioxidant enzyme activities may reflect the heart’s endogenous response to cope with the ischemic stress (34). Although the induction of heat-shock proteins and other protective proteins after ischemic PC at higher temperatures, as used for the present study, is yet to be characterized, indirect reported evidence indicate a good correlation between the ischemic temperature and the degree of induction of various heat-shock and other stress-induced protective proteins, which may explain the PC temperature-dependent enhanced left ventricular postischemic functional recovery observed in the present study. The mechanism of stress protein protection against myocardial ischemic-reperfusion injury is still not clear and is hypothetical.

Ischemic PC, particularly at hyperthermic temperatures, provided a significant preservation to the coronary vasculature by enhancing the postischemic recovery of coronary flow and reducing coronary vascular resistance. Although the mechanism of this protection is not clear from the results of the present study, recent reports indicate the induction of heat-shock proteins in the coronary vascular endothelial cells by ischemic PC (35). PC also activates the ATP-sensitive potassium channels in the vascular smooth muscle causing hyperpolarization and coronary vasodilation (36), and attenuates postischemic leukocyte adhesion and migration possibly by the release of adenosine (37).