(Circulation. 1997;95:2559.)
© 1997 American Heart Association, Inc.
Articles |
Changes in Ischemic Tolerance and Effects of Ischemic Preconditioning in Middle-aged Rat Hearts
the Department of Geriatric Medicine, Keio University School of Medicine, Tokyo, Japan.
Correspondence to Masato Tani, MD, PhD, FACC, FJCC, Department of Geriatric Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-Ku, Tokyo 160, Japan.
Abstract
Background Although both clinical and animal studies have shown that ischemic tolerance is reduced in the senescent myocardium, it has not been clarified when myocardium becomes more vulnerable to ischemia. Preconditioning protects the hearts of young adult animals of various species, but its effects are not identical in human studies. We investigated whether ischemic tolerance and the effect of preconditioning decreased in isolated hearts of middle-aged rats.
Methods and Results The hearts of young adult rats (12 weeks old: group Y, n=44) and middle-aged rats (50 weeks old: group M, n=44) were subjected to global ischemia for 15, 20, or 25 minutes followed by reperfusion. Hearts were also subjected to preconditioning and then to 20 (group Y, n=22) or 15 (group M, n=22) minutes of ischemia followed by reperfusion. Left ventricular developed pressure (LVDP) was decreased by 40% to 60%, and the level of ATP was decreased by 60% to 70% in group M compared with group Y. Preconditioning increased LVDP (% LVDP, 40.5% to 72.4%) and levels of high-energy phosphates (ATP, 11.8 to 14.1; creatine phosphate, 17.0 to 23.1 µmol/g dry wt) and reduced left ventricular end-diastolic pressure (LVEDP, 32.8 to 10.3 mm Hg), creatine kinase release (257 to 132 U/g dry wt), and ryanodine-sensitive sarcoplasmic reticulum Ca2+ release after ischemia in group Y. Preconditioning exerted opposite effects in group M (% LVDP, 45.9% to 15.8%; LVEDP, 21.0 to 28.5 mm Hg; ATP, 14.1 to 8.5 µmol/g dry wt; and CK release, 176 to 332 U/g dry wt). Preconditioning was associated with increases in the incidence of reperfusion-induced ventricular fibrillation (0% to 62.5%) and the rate of sarcoplasmic reticulum Ca2+ release in group M.
Conclusions These results indicate that hearts became more vulnerable to ischemia with age and that the beneficial effects of preconditioning were reversed in middle-aged rat hearts.
Key Words: aging • reperfusion • sarcoplasmic reticulum • creatine kinase • ischemia
Several studies1 2 3 4 have reported that the rates of morbidity and mortality are increased in elderly patients after acute myocardial infarction. Animal studies5 6 7 8 9 have shown age-related decreases in the recovery of cardiac function and heart rate and increases in the release of CK and lactate dehydrogenase after ischemia-reperfusion. We previously found that hearts from senescent Fischer 344 rats (100 weeks old) were more susceptible to ischemia than hearts from young adult rats (24 weeks old) (M.T., MD, PhD, et al, unpublished data, 1996). These studies indicated an age-related decrease in myocardial ischemic tolerance.
Temporary ischemia and reperfusion are unavoidable during cardiac surgery. Despite an increased incidence of noncardiac complications and previous episodes of myocardial damage in the elderly, the outcomes of elderly patients have been found to be satisfactory after cardiac surgery compared with those of middle-aged patients.10 11 12 13 14 However, for further improvement in myocardial preservation or protection during any kind of ischemic insult, it is important to determine when the susceptibility of the myocardium to ischemia-reperfusion injury increases and to identify methods that protect the less tolerant myocardium.
Murry et al15 reported that several brief cycles of ischemia-reperfusion in dogs increased the tolerance of the myocardium to subsequent prolonged ischemia. Although this phenomenon, which is called "preconditioning," has been confirmed in several species, including rats, rabbits, and pigs,16-18 its mechanism has not been clarified. We recently found that the beneficial effects of PC were associated with inhibition of the inappropriate opening of the SR Ca2+ release channel during ischemia in young adult rats (12 to 20 weeks old).19 20
Although PC is being considered for clinical use, its effects in humans are controversial.21 22 Experimental data on the effects of PC have usually been obtained using young adult animals,15 16 17 18 whereas clinical studies involve subjects of various ages.
We investigated the relative tolerance to ischemia of hearts isolated from young (12 weeks old) and middle-aged (50 weeks old) Fischer 344 rats. We also investigated the effects of PC on the recovery of cardiac function and metabolites, the release of CK in the coronary effluent, the incidence of reperfusion-induced ventricular tachyarrhythmias, and the rate of ryanodine-sensitive SR Ca2+ release in the hearts of young and middle-aged rats.
Methods
Hearts were removed from young adult (group Y: 12 weeks old; body mass, 200 to 220 g; n=66) and middle-aged (group M: 50 weeks old; body mass, 330 to 380 g; n=66) male Fischer 344 rats (Charles River, Japan). Age-related pathological changes in the kidney and increased incidence in proteinuria have been reported in these rats, although the incidence of neoplastic lesions does not increase with age in this rat strain, and the survival curve shows almost no decline until rats reach 70 to 80 weeks of age.5 23 24 25 26 We used 50-week-old rats as the middle-aged model because proteinuria occurs in 65% of 50-week-old Fischer 344 rats but does not occur in 12- and 24-week-old rats. The initial washout perfusion was performed at 37°C by the Langendorff technique with a solution gassed with O2/CO2 (95%/5%) and containing 118 mmol/L NaCl, 25 mmol/L NaHCO, 4.7 mmol/L KCl, 1.2 mmol/L MgSO4, 1.2 mmol/L KH2PO4, 1.75 mmol/L CaCl2, 0.5 mmol/L EDTA, 11 mmol/L glucose, and 5 mmol/L pyruvate.
Perfusion Protocols
Effect of Age on Ischemic Tolerance
Thirty-two hearts from each age group were subjected to 20 minutes of recirculating perfusion followed by 15, 20, or 25 minutes of sustained global ischemia and 30 minutes of reperfusion. Sustained global ischemia was induced for 
25 minutes because a preliminary study showed that the no-reflow phenomenon was minimal when ischemia lasted for <25 minutes. Hearts were frozen during normoxic perfusion or after each period of ischemia-reperfusion (eight rats in each group) with Wollenberger clamps cooled in liquid nitrogen and were stored in liquid nitrogen for assays of metabolites.
Effect of Age on PC
PC was induced in 16 hearts from each age group by use of three 5-minute periods of global ischemia separated by 5 minutes of perfusion before induction of 20 minutes (group Y) or 15 minutes (group M) of sustained global ischemia and 30 minutes of reperfusion. The periods of sustained global ischemia selected yielded similar results for the recovery of LV function, the release of CK, and the incidence of reperfusion-induced arrhythmias in both age groups. Eight hearts from each group were frozen and stored in liquid nitrogen, as described above, after PC followed by sustained global ischemia or after 30 minutes of reperfusion for assays of metabolites.
Analysis of LV Function
A plastic catheter with a latex balloon tip was inserted into the left ventricle through the left atrium to record LV pressure. Hearts were paced at 5 Hz during preischemic control perfusion with an electrical stimulator (Nihon Koden Inc) via wires attached to the aorta and the right atrium. The LVEDP was adjusted to 9 mm Hg by filling the balloon with fluid. LVSP, LVDP (LVDP=LVSP-LVEDP), and LV peak positive dP/dt (+dP/dt) during isovolumic contraction were used as indices of LV systolic function; LVEDP and the peak negative dP/dt (-dP/dt) were used as indices of LV diastolic function. LV function was measured before the induction of sustained ischemia. Pacing was turned off during ischemic PC and sustained global ischemia to avoid inducing excessive ventricular tachyarrhythmias during reperfusion27 and was turned on after 25 minutes of reperfusion for determinations of the postischemic recovery of LV function.
Analysis of VT and VF
An epicardial ECG was recorded throughout the experimental period from three platinum electrodes attached directly to the left atrium, the right ventricle, and the apex of the left ventricle. Surface ECGs were analyzed to determine the incidence of VT or VF. VF was diagnosed if individual QRS deflections could no longer be distinguished from one another and the heart rate could not be determined on the ECG. VT was defined as 
6 consecutive premature ventricular complexes.
Analysis of Cardiac Metabolites and CK Release
Levels of ATP, creatine phosphate, and lactate in neutralized perchloric acid extracts obtained from frozen hearts were determined by standard enzymatic procedures.28 The results are expressed in micromoles per gram of dry weight of tissue. The coronary effluent obtained during 30 minutes of reperfusion from eight hearts in each group was stored, and CK activity was determined by ADP-dependent dephosphorylation of creatine phosphate. The results are expressed in units per gram of dry weight of tissue.
Analysis of Rates of SR 45Ca2+ Uptake and Release
Preparation of Heart Homogenates
A 20-minute period of ischemia and 30 minutes of reperfusion with or without PC were induced in 18 hearts from group Y, and periods of 15 minutes of ischemia and 30 minutes of reperfusion with or without PC were induced in 18 hearts from group M. Crude SR was prepared before induction of sustained global ischemia (before PC; n=6) and after sustained global ischemia with (n=6) or without PC (n=6).16 Hearts were placed in an ice-cold 0.9% NaCl solution and blotted, and the large vessels, fatty tissue, and atria were removed. The remaining tissue was weighed and then homogenized in 9 mL of an ice-cold solution containing 1 mol/L KCl, 10 mmol/L NaN3, and 10 mmol/L imidazole (pH 7.0) with a Polytron homogenizer (PT 10 probe; Brinkmann) at setting 5 for 15 seconds. The homogenate was filtered through a layer of cheesecloth to yield a crude SR preparation. The protein concentration of heart homogenates was determined by the method of Lowry et al.29
Measurement of SR 45Ca2+ Uptake and Release Rates
The rate of SR 45Ca2+ uptake was measured at 37°C in 4 mL of a solution containing the heart homogenate (350 to 450 µg of protein), 100 mmol/L KCl, 6 mmol/L MgCl2, 15 mmol/L potassium oxalate, 10 mmol/L NaN3, and 30 mmol/L Tris-HCl (pH 7.0). The reaction was initiated by adding ATP and CaCl2 with 0.4 µCi of 45Ca2+. The final concentrations of ATP and CaCl2 were 5 and 0.2 mmol/L, respectively. After 0, 0.5, 1, and 1.5 minutes, 0.2-mL portions of the reaction mixture were filtered through 0.45-µm filters (Millipore). The filters were washed with 15 mL of a solution containing 100 mmol/L KCl, 1 mmol/L EGTA, and 10 mmol/L histidine (pH 6.4), and the filter-associated radioactivity was determined with a liquid scintillation spectrometer (LS9801, Beckman Instruments Japan). The rate of 45Ca2+ uptake was calculated from the linear regression of uptake determined at the four time points. The rate of 45Ca2+ uptake was also determined in the presence of 625 µmol/L ryanodine.27 The difference between the rates of SR 45Ca2+ uptake with and without ryanodine was defined as the rate of ryanodine-sensitive SR Ca2+ release.19 20
Statistical Analysis
Data are presented as mean±SE. Between-group differences and differences among time points within groups were analyzed by two-way ANOVA followed by Bonferroni test. Differences in the incidence of VT or VF during reperfusion were analyzed by 
2 test followed by Fisher's exact test. A value of P<.05 was considered statistically significant.
Results
Effect of Age on Ischemic Tolerance
Recovery of LV Function
There were no significant differences in the preischemic indices of LV function between groups (Tables 1
and 2). Hearts stopped beating within 3 minutes of the onset of ischemia, and the intraventricular pressure increased 10 to 20 minutes after the onset of ischemia. The increases in intraventricular pressure after 15, 20, and 25 minutes of ischemia were greater in group M than in group Y (data not shown). LVSP tended to be lower after ischemia followed by reperfusion in group M than in group Y; the difference was significant only after 15 and 20 minutes of ischemia. LVDP was lower after ischemia followed by reperfusion in group M due to a marked increase in LVEDP, although the difference in LVEDP between groups Y and M after 25 minutes of ischemia was not significant.
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LV peak positive dP/dt and peak negative dP/dt were lower after ischemia followed by reperfusion in group M than in group Y.
Reperfusion-Induced VT and VF
The incidence of reperfusion-induced VT during reperfusion after 20 minutes of ischemia was higher in group M than in group Y, but there was no difference between groups after 15 or 25 minutes of ischemia (Tables 1
and 2).
Myocardial Energy Metabolites and CK Release
There were no significant differences in the concentrations of myocardial energy metabolites after control normoxic perfusion (before ischemia) between groups Y and M (Table 3). Concentrations of ATP and creatine phosphate after ischemia-reperfusion were higher for all ischemic periods in group Y than in group M. There was no significant difference in the lactate level before or after ischemia-reperfusion between groups. CK release was significantly higher for each period of ischemia in group M than in group Y.
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Effect of Age on PC
Recovery of LV Function
PC did not significantly affect the recovery of LVSP after 20 minutes of ischemia followed by reperfusion in group Y, but recovery of LVSP after 15 minutes of ischemia and reperfusion in group M was inhibited by PC (Tables 1, 2, and 4). PC inhibited the increase in LVEDP after ischemia followed by reperfusion in group Y but enhanced the increase in LVEDP in group M. PC enhanced recovery of LVDP, LV peak positive dP/dt, and LV peak negative dP/dt in group Y but inhibited recovery of these indices in group M.
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Reperfusion-Induced VT and VF
PC did not alter the incidences of reperfusion-induced VT and VF in group Y but tended to increase their incidences in group M (Tables 1, 2, and 4).
Myocardial Concentrations of Energy Metabolite and CK Release
Levels of ATP, creatine phosphate, and lactate after 15 and 20 minutes of ischemia tended to be lower in both groups in hearts with PC than in hearts without PC (Table 5), although only the change in the lactate level after ischemia in group M was significant. PC increased levels of HEP after 20 minutes of ischemia and 30 minutes of reperfusion in group Y but decreased these levels after 15 minutes of ischemia and reperfusion in group M.
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PC reduced CK release in the coronary effluent in group Y but increased CK release in group M.
Rate of SR 45Ca2+ Uptake in the Absence and Presence of Ryanodine and Ryanodine-Sensitive SR 45Ca2+ Release
The rates of SR 45Ca2+ uptake with or without ryanodine and ryanodine-sensitive SR 45Ca2+ release before induction of ischemia were somewhat higher in group Y than in group M, but only the rate of ryanodine-sensitive SR 45Ca2+ release was significantly higher (Figure). PC inhibited the increases in the rates of SR 45Ca2+ uptake with ryanodine and ryanodine-sensitive SR 45Ca2+ release after ischemia in group Y but enhanced the increases in these rates after ischemia in group M.
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P<.05 vs 12-week-old rat hearts, 
P<.05 vs hearts without PC in the same age group.Discussion
The present study is the first to show that the myocardium becomes less tolerant of ischemia-reperfusion before animals reach old age. The beneficial effects of PC in young adult hearts were lost or reversed in middle-aged rat hearts.
Effect of Age on Ischemic Tolerance
Studies have shown that the recovery of LV function5 6 7 8 9 10 and heart rate6 are decreased and the release of CK, lactate dehydrogenase, and protein2 is increased after ischemia-reperfusion in hearts from aged animals (72- to 100-week-old rats, 28- to 38-month-old rabbits, and 7.1±0.45-year-old sheep) compared with hearts from young adult animals. The myocardial concentrations of HEP have been found to be lower during normoxic perfusion in aged animals than in young adult animals.30 Thus, the level of ATP is decreased, at least during the early period of ischemia. This decrease may be responsible for the decrease in ischemic tolerance in the senescent myocardium, because the depletion of ATP during ischemia accelerates rigor-bond formation.31 In the present study, the increase in intraventricular pressure after ischemia was greater in 50-week-old than in 12-week-old rat hearts. There were no significant differences in the preischemic myocardial levels of HEP between 12- and 50-week-old rats, although the ATP level was slightly lower in the 50-week-old rats. Ataka et al8 reported that intracellular Ca2+ showed a greater increase in hearts from aged rabbits (28 to 38 months old) than in hearts from young animals (18 to 25 weeks old). Narayanan32 and Frolkis et al6 ,33 reported that the rate of SR Ca2+ accumulation decreased with age at 12 months in the rat myocardium. In the present study, the preischemic rate of ryanodine-sensitive SR Ca2+ release was decreased in 50-week-old rat hearts. This finding would appear to rule out an elevation of intracellular Ca2+, although we did not determine the rates after identical ischemic periods in both age groups. It has been 34 35 that H+ accumulated during ischemia is exchanged for extracellular Na+ via the Na+/H+ exchanger, resulting in an increase in intracellular Na+ and in activation of the reverse mode of Na+/Ca2+ exchange. The kinetic properties of these exchangers may change with age. Increased intracellular Ca2+ may not be a primary cause of ischemic contracture but may accelerate it by activating cross-bridge cycling31 and can be responsible for increased myocardial damage.36 The release of CK after 15 minutes of ischemia was relatively low in group M compared with CK release after 20 minutes of ischemia in group Y, although the recovery of LV function was similar, suggesting that myocardial stunning may have been a more important contributor to depressed functional recovery in group M.
Myocardial levels of HEP during reperfusion were decreased in 50-week-old rat hearts in the present study, which may have been responsible for the impaired recovery of contractile function. Utilization of hypoxanthine, a degradation product of adenine nucleotide, by the salvage pathway increases in the aged myocardium30 during normoxic perfusion, although no further increase is observed during reperfusion. Therefore, depletion of substrates for the salvage pathway or suppression of production of ATP via a de novo pathway may occur in 50-week-old rat hearts because poorer recovery of contractile function should result in decreased energy consumption. Snoeckx et al7 reported that decreased levels of creatine phosphate in the aged hypertrophied myocardium in spontaneously hypertensive rats may be due to hypoperfusion of the subendocardium, but we found no significant increase in the lactate level in 50-week-old rat hearts in the present study.
Rao et al37 showed that the myocardial activity of superoxide dismutase, an antioxidant enzyme, decreased with age in Fischer 344 rats; there were no changes in catalase and glutathione peroxidase activity. A decrease in the ability to scavenge oxygen-free radicals with age may lead to decreased ischemic tolerance. However, we previously found that extracellular administration of superoxide dismutase with catalase or substrates for the glutathione redox pathway had little effect on the recovery of cardiac function38 in hearts from young adult animals.
Effect of Age on PC
PC had beneficial effects on the recovery of LV function in 12-week-old Fischer 344 rats, which is consistent with our previous data obtained in Sprague-Dawley rats.19 PC also improved recovery of HEP and reduced CK release in the coronary effluent. PC had the opposite effects in 50-week-old rat hearts; it also increased the incidence of reperfusion-induced VT/VF. We also found that vulnerability of myocardium to ischemia increased sometime between 24 and 50 weeks of age in Fischer 344 rats (M.T., MD, PhD, et al, unpublished data, 1996). Recently, Abete et al39 reported that the effect of PC was lost in senescent (24-month-old) Wistar rats whereas a beneficial effect was observed in 6-month-old rats. Their data support our observation that PC was not effective in myocardium that was less tolerant of ischemia. The difference in the effects of PC in 12- and 50-week-old rat hearts was not related to decreases in HEP at the end of sustained ischemia in preconditioned hearts because similar nonsignificant changes in these levels were observed in both age groups. Studies40 41 have shown that repeated episodes of brief ischemia do not cause cumulative myocardial damage in young adult rats or in dogs of unknown age. However, it is possible that the three episodes of 5-minute ischemia used for PC could have caused cumulative effects in the vulnerable myocardium because LV function showed a slight but progressive depression with increasing episodes of brief ischemia in 50-week-old rat hearts, although there was no significant increase in CK release during PC in either age group (data not shown). The PC effect might have been preserved in 50-week-old rat hearts if the duration or the number of ischemic episodes had been reduced. However, we previously showed that three episodes of 5-minute ischemia were needed to induce the PC effect in young Sprague-Dawley rats.19
Adenosine receptors are involved in the PC effect in dogs, rabbits, and pigs but probably not in rats because a specific antagonist of adenosine receptors failed to abolish the cardioprotective effects of PC in rats.42 Nitta et al43 showed that production of heat shock protein 70 is reduced in middle-aged rats. Reduced production of stress-induced proteins in middle-aged rats may have been due to the loss of the beneficial effect of PC, although stress-induced proteins were not measured in the present study. However, Thornton et al44 demonstrated that inhibition of protein synthesis did not cancel out the protective effect of PC. Murry et al45 suggested that free radicals produced during brief episodes of ischemia may precondition the myocardium. However, according to this theory, the effect of PC should have been enhanced, not reversed, in middle-aged rats because the activities of some free-radical scavenging enzymes have been found to decrease with age.37
We19 previously showed that the beneficial effects of PC were associated with inhibition of both the increase in SR Ca2+ uptake in the presence of ryanodine and the inappropriate opening of the SR Ca2+ channel during ischemia and reperfusion. Increased SR Ca2+ uptake may offer protection by lowering the cytosolic free Ca2+ concentration in ischemic, reperfused myocardium. However, SR Ca2+ uptake is an active process, and these measurements of uptake were performed in the presence of sufficient ATP with an in vitro assay system, which is quite different from conditions of myocardium subjected to ischemia. Furthermore, the net activation of SR Ca2+ uptake in the ischemic myocardium may accelerate depletion of ATP. Zucchi et al46 recently found that PC had similar effects on Ca2+-induced Ca2+ release from the SR, but they did not investigate the correlation between SR Ca2+ release and recovery of contractile function. These previous studies used crude SR preparations because the yield or recovery of SR is affected by the purification procedure, including high-speed centrifugation, especially in the ischemic myocardium.47 48 Differences in Ca2+ loading conditions of the SR between young and middle-aged rats may affect measurements of Ca2+ release. However, we did not observe any difference between the calcium content of purified SR from young and middle-aged rat hearts in a separate study (M.T., MD, PhD, et al, unpublished data, 1995), although we did not apply this observation to the interpretation of data in the present study. The results of the present study using heart homogenates from 12-week-old rats are consistent with these previous reports.19 46 PC inhibited functional and metabolic recovery and increased CK release during reperfusion in association with an increase in the rate of ryanodine-sensitive SR Ca2+ release in middle-aged rat hearts in the present study. These findings suggest that there may be a cause-effect relationship between the effect of PC and the opening of the SR Ca2+ release channel during ischemia. We used a relatively high Ca2+ concentration for the assay of SR Ca2+ uptake, which prohibited analysis of the role of phospholamban.
We did not measure cytosolic Ca2+ in the present study, but we speculate that the increase in inappropriate Ca2+ release from middle-aged rat hearts may have an adverse effect by enhancing the excessive increase in cytosolic Ca2+ during ischemia-reperfusion.
Clinical Implications
Both clinical and animal studies have indicated that the senescent myocardium is less tolerant of acute ischemia than the myocardium in younger adult subjects.1 2 3 4 5 6 7 8 9 10 However, the outcomes of cardiac operations, such as coronary artery bypass grafting and open heart surgery for valvular heart diseases, are satisfactory despite more comorbid conditions in elderly patients,10 11 12 13 14 although ischemic insult is unavoidable during these procedures. Therefore, we hypothesized that the myocardium becomes vulnerable to ischemia-reperfusion in middle age. The present results strongly support this hypothesis.
PC has been found to protect the myocardium from injury during ischemia-reperfusion in a variety of animal species. However, its effect in humans is not identical.22 23 Clinical studies have been performed with patients of various ages, whereas laboratory studies have usually been conducted using young adult animals of the same age. Thus, the response of the myocardium to PC may vary with age. The present results suggested that the procedure commonly used to induce PC in experimental models with young animals may not be acceptable in middle-aged animals with decreased ischemic tolerance. Additional studies are needed to clarify the appropriate regimen of PC for less tolerant myocardium in large animals of various ages before clinical trials are undertaken, even though clinical data suggest that PC is operative in most patients.
Selected Abbreviations and Acronyms
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Acknowledgments
This study was supported in part by grants from the Keio Health Consulting Center, Tokyo, Japan, and from the Ministry of Education, Science and Culture, Japan.
Footnotes
Presented in part at the 17th International Society for Heart Research, European Section, Bologna, Italy, June 18-20, 1996; the Satellite Symposium of the XIV World Congress of the International Society for Heart Research, Urayasu, Japan, November 29-30, 1996; and the 69th Scientific Sessions of the American Heart Association, New Orleans, La, November 11-13, 1996, and previously published in abstract form (J Mol Cell Cardiol. 1996;28:A72 and Circulation. 1996;94[suppl I]:I-363).
Received September 4, 1996; revision received December 10, 1996; accepted January 2, 1997.
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