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Am J Physiol Heart Circ Physiol 282: H1961-H1969, 2002. First published February 14, 2002; doi:10.1152/ajpheart.01032.2001
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Vol. 282, Issue 6, H1961-H1969, June 2002

Cardiac preconditioning with 4-h, 17°C ischemia reduces [Ca2+]i load and damage in part via KATP channel opening

Qun Chen1,5, Amadou K. S. Camara1, Jianzhong An1, Matthias L. Riess1,2, Enis Novalija1,2, and David F. Stowe1,2,3,4

Anesthesiology Research Laboratories, Departments of 1 Anesthesiology and 2 Physiology, and 3 Cardiovascular Research Center, The Medical College of Wisconsin, Milwaukee, 53226; 4 Research Service, Veterans Affairs Medical Center, Milwaukee, Wisconsin 53295; and 5 Xuzhou Medical College, Xuzhou, People's Republic of China 221002


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Brief ischemia before normothermic ischemia protects hearts against reperfusion injury (ischemic preconditioning, IPC), but it is unclear whether it protects against long-term moderate hypothermic ischemia. We explored in isolated guinea pig hearts 1) the influence of two 2-min periods of normothermic ischemia before 4 h, 17°C hypothermic ischemia on cardiac cytosolic [Ca2+], mechanical and metabolic function, and infarct size, and 2) the potential role of KATP channels in eliciting cardioprotection. We found that IPC before 4 h moderate hypothermia improved myocardial perfusion, contractility, and relaxation during normothermic reperfusion. Protection was associated with markedly reduced diastolic [Ca2+] loading throughout both hypothermic storage and reperfusion. Global infarct size was markedly reduced from 36 ± 2 (SE)% to 15 ± 1% with IPC. Bracketing ischemic pulses with 200 µM 5-hydroxydecanoic acid or 10 µM glibenclamide increased infarct size to 28 ± 3% and 26 ± 4%, respectively. These results suggest that brief ischemia before long-term hypothermic storage adds to the cardioprotective effects of hypothermia and that this is associated with decreased cytosolic [Ca2+] loading and enhanced ATP-sensitive K channel opening.

cold storage; 5-hydroxydecanoic acid; glibenclamide; isolated hearts; guinea pig


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

BRIEF ISCHEMIA before a much longer period of normothermic ischemia is protective against myocardial injury and is called ischemic preconditioning (IPC) (15). This has been the focus of many studies (2, 5, 18, 22, 40). Cytosolic [Ca2+] overload on reperfusion is associated with cardiac dysfunction and myocardial damage during ischemia and reperfusion (23, 35). Hearts preconditioned by brief ischemia exhibit decreased cytosolic [Ca2+] loading during ischemia-reperfusion (2, 41). Many studies suggest that mitochondrial ATP-sensitive potassium (KATP) channels play a key protective role in normothermic ischemia-reperfusion injury (5, 6, 14, 18, 30).

Moderate hypothermia protects against cardiac ischemia by prolonging the time to stunning or permanent damage (4, 26, 29). Hypothermia has the disadvantage of causing cytosolic [Ca2+] overloading during storage, which leads to reduced diastolic ventricular compliance and contractility on rewarming and reperfusion (10, 24, 32). It remains unclear whether brief ischemia also protects hearts against reperfusion injury after long-term moderately cold storage (4, 10, 28, 33, 34). The effect of IPC after moderately cold storage on cytosolic [Ca2+] loading has not been reported. Moreover, the role of sarcolemmal and mitochondrial KATP channels in eliciting cardioprotection of IPC after cold storage is not known.

Moderate hypothermia (17°C) is widely used to protect hearts during open-heart surgery with circulatory arrest, and we have reported that two 2-min periods of global IPC before 30 min of ischemia at 37°C partially protected isolated guinea pig hearts against ischemia-reperfusion injury (2, 18, 19). Thus in the present study we used the same IPC protocol to test these hypotheses: 1) brief ischemia before 17°C storage for 4 h affords additive cardioprotection via a preconditioning mechanism; 2) protection elicited by IPC is related to decreased cytosolic [Ca2+] loading during and after cold storage; and 3) protection by IPC results from sarcolemmal or mitochondrial KATP channel opening.


    METHODS
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INTRODUCTION
METHODS
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Isolated heart preparation and measurements. The investigation conformed to the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health (NIH No. 85-23, Revised 1996). Prior approval was obtained from the Medical College of Wisconsin Animal Studies Committee. Our preparation and measurements have been described in detail (1, 2, 18, 24, 26, 35, 36). Forty guinea pig hearts of either sex were prepared by the Langendorff method and perfused at 55 mmHg with Krebs-Ringer solution. The perfusate had the following control composition (in mmol/l): 137 Na+, 4.5 K+, 2.4 Mg2+, 2.5 Ca2+, 134 Cl-, 15.5HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, 1.2H2PO<UP><SUB>4</SUB><SUP>−</SUP></UP>, 11.5 glucose, 2 pyruvate, 16 mannitol, 0.05 EDTA, 0.1 probenecid, including 5 U/l insulin were administered. Isovolumetric left ventricular pressure (LVP) and LV first derivative of pressure (LV dP/dt) were measured with diastolic LVP set to 0 mmHg initially. Coronary inflow was measured ultrasonometrically, and coronary effluent Na+, K+, Ca2+, PO2, PCO2, and pH were measured off-line. Coronary outflow (coronary sinus) O2 tension was also measured continuously on-line with an O2 clark-type electrode. Myocardial O2 consumption (MVO2) was calculated as (coronary flow/g) × (arterial PO2 - venous PO2) × 24 µl O2/ml at 760 mmHg; and cardiac work efficiency was calculated as LVDP × HR/MVO2, where HR is heart rate.

Measurement of cytosolic and noncytosolic free Ca2+ in intact hearts. We have published these methods and calibrations in detail (1, 2, 24, 26, 35, 36). In brief, intracellular Ca2+ was measured by spectrofluorometry at the LV free wall using a trifurcated fiber optic cable to direct the excitation and emission wavelengths. Hearts were loaded with the Ca2+-sensitive dye indo 1-AM. Indo-1 fluorescence (F) intensity was corrected for background F and the changes in F due to hypothermia, ischemia, and reperfusion in six additional hearts, wherein the same cooling, storage, and warming protocols were used as for Ca2+ determination except that only the vehicle was perfused. Calibration curves were derived using modifications of the standard equation for fluorescent indicators used by Brandes et al. (3). In brief, total intracellular ([Ca2+]tot) was calculated from the total F385 to total F456 ratio (Rtot), minimum ratio (Rmin), and maximum ratio (Rmax), and dissociation constant (Kd) according to the equation: [Ca2+]tot = S456 × Kd [(Rtot - Rmin)/(Rmax - Rtot)], where S is a calibration constant.

Noncytosolic F (mostly mitochondrial) was measured at the end of each experiment (425 min) after perfusing hearts with 100 µmol/l MnCl2 for 10 min to quench F derived from the cytosolic compartment; this does not alter the LVP transients. Details of this subtraction method have been published previously (24, 26, 35). In the present study quenching of cytosolic Ca2+ transients gave a baseline noncytosolic [Ca2+] of 194 ± 5 nM, and cytosolic [Ca2+] accounted for about 65% of total [Ca2+] averaged for all groups. [Ca2+] data displayed are cytosolic [Ca2+] only. Baseline total cell systolic [Ca2+] for each group was 228 ± 6 nM for control (CON), 226 ± 4 nM for IPC, 242 ± 5 nM for IPC + 5-hydroxydecanoate (5-HD), and 226 ± 5 nM for IPC + glibenclamide (GLB); baseline total cell diastolic [Ca2+] values were 160 ± 4 nM for CON, 152 ± 3 nM for IPC, 166 ± 3 nM for IPC + 5-HD, and 152 ± 4 nM for IPC + GLB. These values were not different among groups. The Kd is inversely proportional to temperature. Free indo-1 reduces the fluorescence ratio F385/F456 in a nearly linear fashion by 0.30, 0.23, and 0.16 per 10°C fall, so that Kd increased 28% at 27°C (305 nmol/l), 44% at 17°C (354 nmol/l), and 67% at 7°C (385 nmol/l) (24, 26). The linear relationship (y = mx + b) for temperature and Kd was Kd = -4.6°C + 423.8 (r2 = 0.99). Loss of membrane integrity in infarcted cells on reperfusion could result in the leakage of indo-1 and lower signal intensities; however, because this is a ratiometric determination of [Ca2+], both F signals degrade similarly so that the F ratio is relatively unchanged and remains no less than half the postloaded signal strength and fivefold greater than the unloaded signal strength (24, 26, 35).

Cumulative effluent creatine kinase (CK) concentration, a marker of cell demise and membrane integrity, was measured (Creatine Kinase Flex reagent cartridge, Dade Behring Dimension; Newark, DE; sensitivity >10 U/l) by collecting the effluent continuously on reperfusion and spot sampling the collected effluent at 5 and 15 min. CK release was expressed as (U/1,000 ml) · ml collected per minute wet heart weight (U · g-1 · min-1). The 2,3,5-triphenyltetrazolium chloride staining technique was used to determine infarct size. The infarcted and noninfarcted tissues of left and right ventricles were carefully dissected and weighed so that percent infarct size was expressed as a percentage of total heart weight (1, 5).

Protocol. Hearts were randomly divided into four groups of 10 hearts each: nontreated hypothermic ischemia controls (CON), IPC, IPC + GLB, and IPC + 5-HD. Initial background (before indo-1 loading) measurements were obtained after 30 min of stabilization. Each heart was then loaded with indo-1 dye for 30 min followed by a 20-min washout of residual dye. Ca2+ transient recordings were obtained every 1 to 5 min during normothermia, cooling, and rewarming, and once per hour during hypothermic storage. Systolic LVP (mmHg) before indo-1 loading was not different among groups: CON, 90 ± 3; IPC, 90 ± 5; IPC + 5-HD, 92 ± 4; and IPC + GLB, 91 ± 4. Thus systolic LVP decreased by 25-30% after indo-1 loading and washout (Table 1, baseline), but there were no differences among groups. Perfusate and bath were maintained at 37°C before and after hypothermic ischemia in each group by a heated water circulator and at 17°C during ischemia by a parallel, refrigerated water circulator. All hearts in each group were subjected to 4 h of global ischemia at 17°C, and it took 15 min (100-115 min) for hearts to cool down from 37°C to 17°C before ischemia, and 10 min (355-365 min) to warm up from 17°C to 37°C after ischemia. The IPC protocol was two 2-min periods of global ischemia separated by 5 or 6 min of reperfusion at 37°C, and IPC was initiated beginning at 85 min and ending before the onset of cooling (Fig. 1, A and B, and Fig. 2A). 5-HD (200 µmol/l) and GLB (10 µmol/l) were perfused from 5 min before IPC until the onset of global hypothermic ischemia (80-115 min, Fig. 2A). In preliminary experiments (n = 4), GLB or 5-HD given before hypothermic storage did not alter any measured functional, metabolic, or structural variable compared with the CON hypothermia group (data not shown). Bradykinin (BK, 10 nmol/l) and sodium nitroprusside (SNP, 100 µmol/l) were perfused for 3 min after 30-45 min of reperfusion to assess maximally stimulated endothelial [Ca2+] and endothelial and nonendothelial-dependent vasodilation. BK increased diastolic [Ca2+] by about 16% for all groups; nitroprusside had no effect on diastolic [Ca2+] (see Fig. 1). MnCl2 was perfused for quenching noncytosolic [Ca2+] (see METHODS). LVP and LV dP/dt, coronary flow, and coronary sinus PO2 were measured continuously before and after hypothermic ischemia. Cytosolic [Ca2+] and LVP were recorded continuously at all temperatures to 17°C and back to 37°C.

                              
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  		Table 1.   Cardiac effects of 4-h 17°C cold ischemia in CON, IPC, IPC + 5-HD, and IPC + GLB groups on diastolic [Ca2+], heart rate, and LVP variables



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  		Fig. 1.   Time course of diastolic [Ca2+] (A) and systolic-diastolic left ventricular pressure (LVP) (B) before, during, and after 4 h of hypothermic global ischemia (ISC) at 17°C in control (CON) and ischemic preconditioning (IPC). Note that diastolic [Ca2+] increased in each group during cooling from 37°C to 17°C, but that diastolic [Ca2+] increased more after CON during hypothermic ISC reperfusion compared with IPC. Diastolic [Ca2+] increased in each group after perfusion with bradykinin (BK) but not sodium nitroprusside (SNP). Systolic-diastolic LVP decreased during transient global ISC in the IPC group and was zero in each group during hypothermic ISC. Systolic-diastolic LVP was depressed on rewarming and reperfusion in two groups, but increased more in IPC compared with CON. RP, reperfusion.



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  		Fig. 2.   Time course of diastolic LVP (A) before, during, and after 4 h of cold global ISC at 17°C. Diastolic LVP decreased during cold ISC and increased during initial reperfusion in each group compared with baseline value at perfusion 80 min. Diastolic LVP was markedly elevated throughout reperfusion in the CON group, whereas it gradually returned to the baseline value after reperfusion 10 min in the IPC, IPC + 5-hydroxtdecanoate (5-HD), and IPC + glibenclamide (GLB) groups. B: effluent creatine kinase (CK) release at reperfusion 15 min and infarct size determined by percent whole heart weight at 70 min of reperfusion. Treatment with IPC significantly decreased infarct size and CK release compared with CON, IPC + 5-HD, and IPC + GLB groups. There were no differences in CK release and infarct size among CON, IPC + 5-HD, and IPC + GLB groups.

All data were expressed as means ± SE. One-way analysis of variance for repeated measures (Super Anova 1.11 software for Macintosh from Abacus Concepts; Berkeley, CA) was used to assess within-group differences over time. Among groups data were compared at discrete time points before cooling at 37°C (baseline, at 80 min), after 4 h of hypothermic ischemia (at 355 min), and during rewarming and reperfusion (at 357, 365, 385, and 415 min). Two-way analysis of variance was used to assess among-group differences at these time points. If F values for the analysis of variance were significant, Tukey's multiple-comparison post hoc tests were used to differentiate within or among group differences. Differences among means were considered significant when P < 0.05.


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ABSTRACT
INTRODUCTION
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DISCUSSION
REFERENCES

Figure 1, A and B, and Fig. 2A display changes in diastolic [Ca2+], developed LVP, and diastolic pressure before, during, and after cold ischemia and reperfusion in CON and IPC groups. Diastolic [Ca2+] increased incrementally during hypothermic ischemia, rose abruptly and markedly during initial rewarming and reperfusion, and remained elevated throughout reperfusion in the CON group. The two 2-min periods of IPC before the index cold ischemia significantly decreased the diastolic [Ca2+] overloading both during hypothermic ischemia and during rewarming and reperfusion. BK increased diastolic [Ca2+]; SNP had no effect. MnCl2 decreased total [Ca2+] (see METHODS). After 10 min of reperfusion, diastolic [Ca2+] returned to baseline in the IPC, but not in CON, IPC + 5-HD, and IPC + GLB groups (Fig. 1A and Table 1). Developed LVP (systolic - diastolic LVP) was lower than the baseline value in each group during cold ischemia and reperfusion, but it was higher in the IPC group than in CON, IPC + 5-HD, and IPC + GLB groups at 10 min of reperfusion and remained higher in the IPC group than in the CON group at 60 min of reperfusion (Fig. 1B and Table 1). Diastolic LVP increased similarly in each group on initial reperfusion compared with the baseline values. Diastolic LVP remained markedly elevated in the CON group throughout reperfusion, whereas it gradually returned to nearly the baseline value in the treated groups after 10 min of reperfusion without significant differences among groups (Fig. 2A).

Table 1 summarizes changes in indexes of cytosolic [Ca2+], HR, and LVP in CON, IPC, IPC + 5-HD, and IPC + GLB groups before and at the end of cold ischemia, and at 2, 10, and 60 min of reperfusion. Systolic LVP was depressed during early rewarming and reperfusion and increased during later reperfusion with no significant differences among the groups. Systolic [Ca2+], which is the same as diastolic [Ca2+] during cold ischemia, was increased markedly by the end of ischemia compared with preischemic diastolic [Ca2+] values, and rose abruptly and markedly on initial reperfusion at 2 min (24°C); at this time, the rise in systolic [Ca2+] was less in IPC and IPC + GLB groups than in CON and IPC + 5-HD groups. After 10 and 60 min of reperfusion, systolic [Ca2+] had returned to baseline values in each group. HR was zero during cold ischemia and lower than baseline values in each group at 2 and 10 min of reperfusion, whereas it returned to baseline value beginning after 10 min of reperfusion without significant differences among groups. Rate-pressure product (RPP, HR × developed LVP) was zero during cold ischemia and much lower than the baseline value in each group at 2 min of reperfusion, whereas it was higher in the IPC group than in CON, IPC + 5-HD, and IPC + GLB groups at 10 min of reperfusion. RPP remained lower than baseline values in each group at 60 min of reperfusion, but there were no significant differences among groups.

Table 2 displays changes in maximal (LV dP/dtmax), minimal (LV dP/dtmin), MVO2, cardiac efficiency, and coronary flow in CON, IPC, IPC + 5-HD, or IPC + GLB groups before and during hypothermic ischemia and reperfusion. LV dP/dtmax and LV dP/dtmin was nil during ischemia and returned to 38% and 28% of its preischemic value by 10 min of reperfusion in the IPC group; LV dP/dtmax was higher in the IPC than in the CON (25%) group but not different from the IPC + 5-HD (34%) or IPC + GLB (30%) groups. LV dP/dtmin was higher in the IPC group by 10 min of reperfusion than that in the CON (16%), IPC + 5-HD (19%) or IPC + GLB (18%) groups. There were no differences in LV dP/dtmax among groups, but LV dP/dtmin was still higher in the IPC group than in the CON group at 60 min of reperfusion. MVO2 was higher after IPC than CON at 2, 10, and 60 min of reperfusion. Cardiac efficiency was nil at 2 min of reperfusion and remained depressed at 10 min of reperfusion, but it was higher in the IPC group than in other groups. At 60 min of reperfusion cardiac efficiency was restored similarly in all groups. Coronary flow was higher in the IPC group than in CON group at 30 and 60 min of reperfusion. On reperfusion at 30 min, coronary flow responses to BK and SNP were increased significantly in each group, but were increased more after IPC compared with the CON group.

                              
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  		Table 2.   Cardiac effects of 4-h cold ischemia at 17°C in CON, IPC, IPC + 5-HD, and IPC + GLB groups on dLVP/dtmax, dLVP/dtmin, coronary flow, MVO2, and cardiac efficiency

Heart weight was not different among groups: CON, 1.51 ± 0.01 g; IPC, 1.51 ± 0.02 g; IPC + 5-HD, 1.50 ± 0.01 g; and IPC + GLB, 1.52 ± 0.06 g. Infarct size was much smaller in the IPC group than in the CON, IPC + 5-HD, and IPC + GLB groups, whereas there were no differences in infarct size among CON, IPC + 5HD, and IPC + GLB groups (Fig. 2B). At 5 min of reperfusion, effluent CK release (U · g-1 · min-1) was not different among groups: CON, 2.28 ± 0.31; IPC, 2.67 ± 0.53; IPC + 5-HD, 1.79 ± 0.67; and IPC + GLB, 2.16 ± 0.24. At 15 min of reperfusion, it was much lower in the IPC group than in CON, IPC + 5-HD, and IPC + GLB groups (Fig. 2B).


    DISCUSSION
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ABSTRACT
INTRODUCTION
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DISCUSSION
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This study demonstrates for the first time in intact hearts that two brief periods of global IPC applied before sustained 4 h of cold global ischemia at 17°C improved mechanical and metabolic function, coronary flow, and cardiac efficiency. In addition, IPC significantly reduced cytosolic [Ca2+] loading on reperfusion, decreased infarct size, and reduced CK release. Furthermore, the reduction of infarct size by IPC was blocked by 5-HD and by GLB; functional and metabolic improvements by IPC were attenuated, but not completely inhibited by 5-HD and by GLB. This work demonstrates that IPC adds to the cardioprotection of moderate hypothermia during prolonged ischemia and that IPC-induced protection is associated with decreased [Ca2+] loading and caused, in part, by KATP channel opening.

IPC and hypothermic ischemia-reperfusion injury. We have shown in the same model that IPC was demonstrated by significantly increased contractility and improved relaxation after 30 min of 37°C global ischemia, that this was associated with improved association of cytosolic [Ca2+] and function, and that KATP channel blockade reversed the protective effects of IPC (2, 18). Ogino et al. (20) showed that IPC (5 min of global ischemia and 10 min of reperfusion at 37°C) before 6 h of cold global ischemia at 6°C primarily improved diastolic relaxation. We observed similarly that IPC before hypothermic ischemia improved cardiac diastolic relaxation with little effect on systolic function. These studies show the powerful cardioprotective effect of hypothermia alone. The extent of added protection by IPC before hypothermic ischemia, however, is relatively small compared with that observed in normothermic hearts with shorter-term ischemia. The beneficial effect of IPC plus hypothermia was manifested by improved diastolic relaxation.

Several different temperatures, animal species, ischemia periods, and IPC protocols have been used to examine additive cardioprotective effects of IPC and hypothermia (4, 10, 28, 33, 34). In severe hypothermia studies, it was reported that IPC (5 min of global ischemia plus 5 min of reperfusion at 37°C) before 115, 135, and 160 min of global ischemia at 20°C added to the hypothermia-induced functional protection in isolated rat hearts (4). In contrast, IPC (two cycles, 3 min of global ischemia plus 5 min of reperfusion at 37°C) improved postischemic cardiac function in isolated rat hearts after 25 min of global ischemia at 37°C, but not in hearts at 3.5, 4, and 5 h of cold ischemia at 6°C (33). In studies on mild hypothermia, IPC (one cycle, 2 min of global ischemia plus 5 min of reperfusion at 37°C) improved cardiac function after 75 min of global ischemia at 23°C, but not after 5 h of cold ischemia at 6°C in isolated rat hearts (28). IPC (two cycles, 5 min of global ischemia and 10 min of reperfusion at 34°C) improved postischemic cardiac function in isolated rat hearts after 60 min of global ischemia at 34°C but not after 60 min of ischemia at 10°C (9). IPC in vivo rats also showed additive cardioprotection with mild hypothermia (8, 34).

Taken together, these studies indicate that IPC adds to the protection of hypothermia but that differences in IPC protocol, duration of ischemia, and temperature lead to variable results. Because hypothermia itself is so cardioprotective, IPC can be shown to occur only after long-term ischemia when the protective effects of hypothermia alone wane. In this study, we selected 4-h, 17°C ischemia because the functional return of control hearts was <50% of the baseline values, indicating that the cardioprotection of hypothermia diminishes over time. Because high K+ solutions add to the protection of hypothermia (26), we perfused hearts only with normal ionic Krebs-Ringer solution. We reported that two 2-min periods of global ischemia before 30 min of ischemia provided significant cardioprotection in isolated guinea pig hearts (2, 18). Thus we used the same IPC protocol before cold ischemia and were able to show not only improved cardiac function and perfusion, but also reduced diastolic [Ca2+] overloading and a smaller infarct size.

IPC and cytosolic [Ca2+] on hypothermic ischemia reperfusion. Postischemic reperfusion injury likely results in large part from cytosolic [Ca2+] overloading. We have detailed the time course of change in contractility and relaxation with cytosolic [Ca2+] during ischemia and reperfusion at 37°C in intact guinea pig hearts (2, 35). We found that high systolic [Ca2+] was associated with reduced contractile force and impaired relaxation on reperfusion and that IPC (two 2-min periods of ischemia) simultaneously reduced systolic and diastolic [Ca2+] overloading and improved systolic and diastolic function. In the present study we observed that the higher cytosolic [Ca2+] after hypothermic ischemia was also associated with depressed cardiac function. In contrast to normothermic ischemia, systolic [Ca2+] loading occurred only during the initial reperfusion and then returned to preischemic values during later reperfusion. However, diastolic [Ca2+] was markedly increased and remained elevated throughout the reperfusion. IPC before hypothermic ischemia significantly reduced diastolic [Ca2+] loading and improved diastolic function. We have also reported that cardioplegia decreases diastolic [Ca2+] loading and improves relaxation after 4 h of hypothermic storage at 3°C in isolated guinea pig hearts (26) and that hearts perfused at 17°C (no ischemia) had markedly elevated diastolic [Ca2+] and impaired relaxation function (24). Taking these results together, we conclude that diastolic [Ca2+] overloading plays a key role in the development of impaired myocardial relaxation during hypothermic ischemia and normothermic reperfusion and that IPC improves cardiac relaxation by reducing diastolic [Ca2+] loading.

IPC, hypothermic ischemia reperfusion injury, and KATP channel opening. IPC confers protection to the myocardium through a signal-transduction pathway that encompasses both a "trigger" phase, which occurs before sustained ischemia, and a "mediator" phase, which occurs during prolonged ischemia (6, 21). For example, it was reported that the mitochondrial KATP channel inhibitor 5-HD, bracketing either 5 min of IPC ischemia or 5 min of 10 µM diazoxide (a mitochondrial KATP channel opener) perfusion, blocked the anti-infarction effect of IPC or diazoxide infusion in isolated rabbit hearts. However, 5-HD starting just 5 min before index ischemia did not affect the cardioprotection of IPC. Thus it was suggested that mitochondrial KATP channels mainly serve as the trigger effect (21). However, it was also found that 5-HD attenuated cardioprotection in in vivo rats when administered either 5 min before the IPC stimulus, during the reperfusion phase of the IPC stimulus, or 5 min before reperfusion during prolonged ischemia; it was concluded that activation of mitochondrial KATP channels is an important downstream regulator of myocardial protection with effects lasting into the reperfusion period following prolonged ischemia (6). Another study suggested that mitochondrial KATP channel opening both triggered and mediated cardioprotection in isolated rabbit hearts (38). Thus it has been suggested that opening KATP channels in the mitochondrial inner membrane is an essential step leading to protection against infarction by ischemic preconditioning during normothermic ischemia (13).

In our study of long-term cold ischemia in intact guinea pig hearts, the IPC-induced effect to reduce infarct size was blocked when bracketed by either mitochondrial KATP channel inhibition or combined sarcolemmal KATP and mitochondrial KATP inhibition. Because mitochondrial KATP channels may open during a trigger phase and during a mediator phase (38), we perfused 5-HD or GLB from 5 min before IPC until the onset of global ischemia to block any KATP channel opening during both brief ischemia (IPC) and the long-term (index) cold ischemia. However, although lower concentrations of 5-HD are claimed to be specific for mitochondrial KATP channels in rat (5) and rabbit (21) hearts in vitro at 200 µM, in guinea pig hearts, 5-HD may have an additional effect on the sarcolemmal KATP channel (16, 17). Thus our results suggest that, like IPC before normothermic ischemia, the protection against infarction by IPC before moderate hypothermic ischemia is modulated by mitochondrial KATP and/or sarcolemmal KATP channels.

Others have also reported that the IPC-induced smaller infarct size after short-term normothermic ischemia is not necessarily associated with better functional return (27, 31, 39). IPC reduced infarct size in isolated mouse hearts after 20 min of global ischemia and reperfusion but did not improve cardiac functional return (39). The adenosine-enhanced effect of IPC to reduce infarct size was blocked by either 5-HD or GLB, but the improvement in functional indexes was only blocked by HMR-1883 (a selective sarcolemmal KATP channel inhibitor) and not by 5-HD or GLB (31). Also, KATP channels were reported to play an essential role in late IPC against infarction, but not in late IPC against stunning (27). Our findings are consistent with these reports in that the improved functional effect induced by IPC before hypothermia was also partially blocked by 5-HD and GLB. Thus it appears that the improved cardiac function provided by IPC before hypothermia is mediated in part by opening sarcolemmal KATP and/or mitochondrial KATP channel after long-term cold ischemia and reperfusion.

IPC, cytosolic [Ca2+], and KATP channel opening on hypothermic ischemia reperfusion. We found that inhibiting KATP channel opening with 5-HD or GLB during IPC blocked the effect of IPC to reduce diastolic [Ca2+] overloading during cold ischemia and initial rewarming and reperfusion. In 37°C, 30-min ischemia studies, we reported that mitochondrial [Ca2+] ([Ca2+]m) and diastolic [Ca2+] were elevated during warm ischemia and initial reperfusion (35), that IPC reduced diastolic [Ca2+] loading (2), and that protection was abolished by GLB (18). Wang et al. (37) also showed that IPC before 37°C, 25-min ischemia in rat hearts reduced cytosolic and [Ca2+]m loading during ischemia, and that the decrease in cytosolic and [Ca2+]m loading was blocked by 5-HD. Our present results extend these studies by showing that IPC decreases diastolic [Ca2+] loading in association with KATP channel opening during 4-h, 17°C ischemia and reperfusion.

The connection between reduced [Ca2+] overload and KATP channel opening is not clear, but it is likely that slowed or reversed Na+/Ca2+ exchange secondary to Na+/H+ exchange is responsible in part for [Ca2+] loading after normothermic (1) and hypothermic (25) ischemia. Sarcoplasmic reticular dysfunction also likely plays a role. It is well known that L-type Ca2+ channel opening triggers a rapid release of Ca2+ from the sarcoplasmic reticulum to initiate mechanical contraction. Subsequent activity of Ca2+-ATPase in the sarcoplasmic reticulum membrane transports a large fraction of the released Ca2+ back into the sarcoplasmic reticulum lumen, resulting in a rapid decrease in [Ca2+] and muscle relaxation. The resting diastolic [Ca2+] is restored via sarcoplasmic reticulum Ca2+ uptake by Ca2+ pump in the sarcoplasmic reticulum, together with Ca2+ extrusion by the Na+/Ca2+ exchange and Ca2+ pump in the sarcolemmal membrane (11). Indeed, it was reported that hypothermia alone caused a significant rise of cytosolic [Ca2+] because of dysfunctional sarcoplasmic reticulum handling Ca2+ in rats (12). It also was found that Na+-K+-ATPase activity of sarcolemmal vesicles was not depressed after 4 h of ischemia at 4°C or after 40 min of reperfusion at 37°C in rabbit hearts (7); however, sarcoplasmic reticulum Ca2+-ATPase activity was markedly lowered after hypothermic ischemia and reperfusion. It was suggested that [Ca2+] loading during hypothermic ischemia and reperfusion resulted primarily from dysfunctional Ca2+ handling ability in the sarcoplasmic reticulum (7). A Ca2+ paradox may also lead to diastolic [Ca2+] loading and diastolic dysfunction (11). IPC before Ca2+ paradox improved diastolic function and reduced diastolic [Ca2+] loading via improved sarcoplasmic reticulum Ca2+-handling ability during the Ca2+ paradox (11). By inference, our results suggest that IPC decreases diastolic [Ca2+] loading by improving sarcoplasmic reticulum Ca2+ handling ability and or reducing Na+/H+ exchange secondary to opening KATP channel during hypothermic ischemia and reperfusion.

In summary, these results confirm that IPC before moderate hypothermic ischemia exerts additive cardioprotection with moderate hypothermia and that this is associated with decreased cytosolic [Ca2+] overloading and is due, in part, to opening of KATP channels. This protective effect of IPC may be clinically important because moderate hypothermia (17°C) is widely used to protect hearts during open-heart surgery (7).


    ACKNOWLEDGEMENTS

The authors thank Dr. Gopu Varadarajan, Dr. Ming Tao Jiang, Jim Heisner, Samhita Shahane Rhodes, and Anita Tredeau for valuable contributions to this study.


    FOOTNOTES

The research was supported in part by National Institutes of Health Grants R01-HL-58691 and RO1-5T32 GM-08377. Portions of this work have appeared in abstract form (FASEB J 16: A463, 2001. Biophys J 80: 608a, 2001.)

Address for reprint requests and other correspondence: D. F. Stowe, M4280, 8701 Watertown Plank Rd., Medical College of Wisconsin, Milwaukee Regional Medical Center, Milwaukee, WI 53226 (E-mail: dfstowe{at}mcw.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published February 14, 2002;10.1152/ajpheart.01032.2001

Received 27 November 2001; accepted in final form 11 February 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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