Reduced Efficacy of Volatile Anesthetic Preconditioning with Advanced Age in Isolated Rat Myocardium

The study protocol was approved by the Animal Care Committee of the University of California, Davis (Davis, California), and all experiments were conducted in accordance with guidelines of animal care from the National Institutes of Health.

Rat hearts were obtained from male Fischer 344 rats (National Institute on Aging, National Institutes of Health, Bethesda, Maryland) of the following age groups: 2–4, 10–12, and 20–24 months. Anesthesia was first obtained with an intraperitoneal injection of sodium thiopental (65 mg/kg) along with 1,000 U heparin. Sodium thiopental was chosen for initial anesthesia because this drug has been shown not to influence preconditioning. 17When no response to tail clamping was obtained, the heart was excised via  thoracotomy and placed in an iced solution of Krebs-Henselet buffer (127 mm NaCl, 4.7 mm KCl, 1.25 mm MgCl², 2.5 mm CaCl², 25 mm NaHCO³, and 10 mm glucose). The aorta was quickly cannulated and aerobically perfused with Krebs-Henselet buffer on a nonrecirculating Langendorff apparatus at a perfusion pressure of 140 ± 10 cm H²O. The perfusate was continuously oxygenated with a 95% O²–5% CO²mixture, and its temperature was kept constant at 37 ± 0.5°C with a water-jacketed column. The time from excision to cannulation was less than 60 s in all hearts. After cannulation, pacing wires were placed in the right atrium.

Twelve rats from each age group were randomly selected. Six hearts in each age group served as controls and underwent a 30-min equilibration period, followed by 15 min of perfusion, 25 min of global ischemia, and finally, 60 min of reperfusion. The other six hearts in each age group underwent a 30-min equilibration period, 10 min of sevoflurane exposure, and a 5-min washout period before the 25 min of global ischemia and 60 min of reperfusion. The sevoflurane was delivered at 2.5% to the gas mixture via  a standard Sevotec5 variable bypass vaporizer (Datex-Ohmeda, Milwaukee, WI) at a concentration of 0.4 ± 0.02 mm measured in the liquid phase by gas chromatography (Varian, Walnut Creek, CA). The concentration is approximately 0.9 minimum alveolar concentration (MAC) for young rats and 1.1 MAC for middle-aged rats. Global ischemia was induced by stopping all flow of the Krebs-Henselet buffer perfusate to the heart. Atrial pacing at 5 Hz was used during all phases of the experiment, except during the period of global ischemia. Any episodes of ventricular fibrillation were mechanically converted when they occurred.

²³Na, ¹⁹F, and ³¹P nuclear magnetic resonance were used to measure intracellular Na (Naⁱ), intracellular Ca ([Ca]ⁱ), and intracellular pH (pHⁱ) and high-energy phosphates, respectively. 18,19All three nuclei were measured in separate hearts. To measure Naⁱ, 7.5 mm dysprosium triethylenetetraminehexaacetic acid (DyTTHA) was substituted iso-osmotically for NaCl in the perfusate, and Ca was added to reach a perfusate concentration of 2.5 mm as measured by Ca electrode. To measure [Ca]ⁱ, hearts were perfused with a perfusate containing the acetoxymethyl ester of 5F-1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (FBAPTA) at 2.5 μm. FBAPTA was then washed out of the extracellular space with control solution for 15 min before measurement of [Ca]ⁱ. ²³Na, ¹⁹F and ³¹P experiments were conducted using a Bruker AMX400 spectrometer (Bruker, Rheinstetten, Germany). ²³Na, ¹⁹F, and ³¹P spectra were generated from the summed free induction decays of 1,000, 1,500, and 148 excitation pulses (90, 45, and 60°) using 2,000-, 2,000-, and 4,000-word data files and ±4,000-, ±5,000-, and ±4,000-Hz sweep widths, respectively. For all nuclei, data files were collected over 5-min intervals. Because the nuclear magnetic resonance signal intensity reflects the time average for the interval over which data are collected, data are represented in time as corresponding to the midpoint of the 5-min acquisition interval.

Intracellular Na (in mEq/kg dry weight) was calculated from the calibrated area under the unshifted peak of the ²³Na spectra after subtracting out the extracellular peak. 18,19At the end of the experiment, hearts were weighed wet and dried to a constant weight (at least 48 h) at 65°C to determine the dry weight. [Ca]ⁱ(in nmol/l cell water) was calculated as the product of the ratio of the areas of the Ca-bound and Ca-free peaks in the FBAPTA spectrum and the 500 nm Ca–FBAPTA dissociation constant. 18,19pHⁱwas determined from the chemical shift of the inorganic phosphate resonance (with reference to control phosphocreatine) calibrated at 37°C. High-energy phosphates are reported as percent of control peak height. 18,19 

To measure left ventricular pressures, a latex balloon was filled with water and connected to a pressure transducer (Medex, Dublin, CA) using PE40 tubing. The balloon was inserted into the left ventricle via  the left atrial appendage through the mitral valve. The balloon volume was adjusted during the equilibration period to yield a left ventricular end-diastolic pressure (LVEDP) of 5–10 cm H²O. Pressures were recorded using Powerlab 4/20 hardware with amplifiers (ADInstruments, Colorado Springs, CO) and Chart for Windows v4.0.4 software (ADInstruments). Hemodynamic measurements included LVEDP, left ventricular end-systolic pressure (LVESP), and left ventricular developed pressure (LVDP). LVDP was defined as LVESP minus LVEDP.

The run-off from the coronary sinus was collected for the first 10 min of reperfusion. This was placed in aliquots and stored at −80°C until analysis could take place. After thawing, the amount of creatine kinase (CK) was determined using a CK-10 kit (Sigma Diagnostics, St. Louis, MO) and a Shimadzu UV-VIS recording photospectrometer (Shimadzu, Columbia, MD). Units are expressed as U/g dry weight. 19 

At the end of reperfusion, the hearts were quickly taken down from the Langendorff apparatus and sliced into 2-mm sections. The sections were immersed in 2% 2,3,5-triphenyltetrazolium chloride staining solution and placed in a 37°C incubator for 20 min. Noninfarcted myocardium stains a bright red that is caused by reduction of 2,3,5-triphenyltetrazolium chloride by dehydrogenases present in viable tissue. 20,21After 20 min, the sections were rinsed off with water and placed on transparent Petri dishes. They were then scanned into a computer using Adobe Photoshop 5.0LE software (Adobe, San Jose, CA). Standard computer planimetric analysis, using NIH image 1.62 (National Institutes of Health, Bethesda, MD), was used to determine the infarct area. Because the entire heart was at risk from global ischemia, the infarct size was expressed by dividing the necrotic area by the total slice area to obtain the percent necrosis.

Analysis of variance for repeated measures was used to test for differences between treatments. When differences between treatments were found, the unpaired t  test was used to determine the times at which differences between treatments occurred. The t  test was used only across treatments for a particular time. Under these conditions, the t  test and multiple-comparison tests provide identical results for two treatments. Data are reported as mean ± SEM. For all comparisons, differences were considered significant when P  was less than 0.05.

Average hemodynamic variables during the preischemic period are presented in table 1. There was no significant difference in preischemic hemodynamic variables between the APC hearts and their respective controls for any of the three age groups. Hemodynamic measurements after 60 min of reperfusion are presented in table 1. The percentage of LVDP recovery was calculated by dividing the measurement after 60 min of reperfusion by the average LVDP during the preischemic period. There was a significant difference between control and APC hearts in the young adult (16 ± 2 vs.  38 ± 7%) and middle-aged (10 ± 1 vs.  18 ± 1%; P < 0.05) groups but not the aged hearts (9 ± 1 vs.  12 ± 2%; P < 0.05). Figure 1displays the percent LVDP recovery of each age group at 10-min intervals for the entire 60 min of reperfusion. The other observation to reach statistical significance was LVESP in the middle-aged groups (table 1).

Creatine kinase and infarct size measurements are summarized in table 1. Significant differences between control and APC hearts were found in the young adult and middle-aged groups only. Likewise, the difference in infarct size was only significant in the young adult and middle-aged groups. No differences between control and APC hearts were found in the aged group for CK measurements and infarct size.

Figure 2demonstrates a significant difference in ATP between control and APC hearts in the young adult group but not the middle-aged or aged group. Similarly, phosphocreatine, a substrate of ATP synthesis, recovered better in young adult hearts but not in middle-aged or aged hearts in APC treated groups after reperfusion (fig. 3). Inorganic phosphate, an ATP breakdown product, was significantly lower at the end of ischemia and reperfusion when compared with control groups and in young adult hearts of the APC groups but not in middle-aged adult or aged hearts (fig. 4).

Figure 5shows that ischemia causes increases in [Ca]ⁱduring ischemia and reperfusion in all three age groups. APC attenuates the increases in [Ca]ⁱin young adult and middle-aged hearts, but not in aged hearts. [Ca]ⁱlevels were significantly different between control and APC hearts in the young adult group at the end of ischemia (1,505 ± 242 vs.  502 ± 79 nm; P < 0.05) and reperfusion (1,339 ± 145 vs.  385 ± 43 nm; P < 0.05). Middle-aged hearts also showed less [Ca]ⁱaccumulation at the end of reperfusion when exposed to sevoflurane (1,786 ± 133 vs.  1,039 ± 87 nm for control and APC, respectively; P < 0.05). No differences were found between control and APC hearts in the aged group.

Intracellular pH was measured during ischemia with and without APC as one variable required to assess the effect of APC on pH regulation during and after ischemia. Figure 6shows that at the end of 25 min of sustained ischemia, the pHⁱdecreased in both control and APC hearts in all the three age groups, but the pHⁱdecreased less at the end of ischemia in young adult and middle-aged hearts than in aged hearts. The recovery of pHⁱafter prolonged ischemia and 60 min of reperfusion was not different in any of the three age groups between control and APC hearts.

Figure 7shows that APC blunts the increases in Naⁱin young adult and middle-aged hearts during ischemia. We also demonstrated that APC does not prevent the increase in Naⁱin aged myocardium. Significant differences between control and APC hearts were found in the young adult and middle-aged groups only at the end of ischemia. There was no significant difference between control and APC in aged hearts during ischemia and reperfusion.

The percentage of the world’s population that is elderly is increasing. As a result, the number of elderly patients undergoing surgical procedures is increasing. 22This has important implications for intraoperative cardioprotection because there are metabolic and structural differences in the senescent myocardium. 23IPC is one protection strategy that has been shown to have decreased effectiveness in the aged heart. 3,4APC shares similarities with IPC, but the effects of age on this phenomenon have yet to be studied.

We investigated the effects of APC with sevoflurane using isolated perfused rat hearts from three different age groups: young adult, 3–4 months old; middle-aged, 10–12 months old (12 months for a rat is approximately equal to a 50 yr for a human); and aged, 20–24 months old (24 months for a rat is approximately equal to 75 yr for a human). Benefits of APC have been previously demonstrated in this model using young rats. Specifically, Li et al.  16and Mathur et al.  24showed a greater recovery of systolic function, as measured by LVDP, and diastolic relaxation, as measured by LVEDP, in APC hearts after global ischemia. In addition, Li et al.  16examined myocardial tissue under light microscopy and found a 50% reduction in the infarction area with APC hearts.

Our hemodynamic measurements in the young adult and middle-aged rats were consistent with these two studies. Systolic function, as measured by LVDP, recovered to a greater extent in the APC hearts after 60 min of reperfusion. Diastolic relaxation, as measured by LVEDP, tended to be improved in the young adult and middle-aged APC hearts, but it did not reach statistical significance in aged hearts. One difference between our study and those of Li et al.  16and Mathur et al.  24is that our protocol included a 5-min washout period before global ischemia. Although this may have decreased the overall benefit of sevoflurane administration, it created a true preconditioning model. Cope et al.  25first demonstrated decreased infarct size in rabbit hearts exposed to volatile anesthetics using a washout period. This correlates with the “early phase” of preconditioning originally shown by Murry et al.  1 

In addition to hemodynamic recovery, we also compared tissue damage among groups. CK release was used as an indirect measurement of cellular injury. APC hearts in the young adult and middle-aged groups had significantly less CK releases than controls, presumably because of less myocardial damage. We confirmed this by using the accepted standard for measuring tissue damage, infarct size analysis. APC hearts in the young adult and middle-aged groups had almost 50% and 30% reduced infarct sizes, respectively, compared with controls.

None of the benefits of APC seen in the young adult and middle-aged groups could be demonstrated in the aged group. There was no difference between APC and control hearts in any hemodynamic measurement, CK release, or infarct size. Schulman et al.  4exposed rats from three different age groups (3, 12, and 18–20 months) to an IPC protocol before prolonged regional ischemia and failed to demonstrate any benefit in the oldest rats. Fenton et al.  3also failed to show IPC benefit in rats aged 22 months. If the mechanisms of IPC and APC are similar, their effects on the senescent myocardium would likely be comparable.

Although the exact mechanism of APC is unknown, its protective benefits may be related to decreased [Ca]ⁱ. Increased [Ca]ⁱhas been identified as a causal factor in reperfusion injury. 18,19,26The ischemic chain of events leading to cell damage or necrosis is presumed to begin with increased anaerobic metabolism, leading to intracellular acidosis and decreased pHⁱ. This activates an Na–H exchanger (NHE1) to maintain pH balance and causes increased Naⁱ. 27,28[Ca]ⁱthen increases as a result of impaired or reversed Na–Ca exchange. 18,19,26A cascade of events is then initiated by a prolonged increase in [Ca]ⁱ, leading to increased ATP consumption, inefficient ATP synthesis, cytoskeletal damage, and cell death. 29–35In two articles, Varadarajan et al. , 30,32showed that previous exposure to sevoflurane in intact guinea pig hearts resulted in decreased cytosolic and mitochondrial calcium loading and improved indices of left ventricular function.

The results of this study are consistent with the above hypothesis. We demonstrated that anaerobic metabolism causes an increase in Hⁱand a decrease in pHⁱ. This causes Na influx via  NHE1. The increased Naⁱthen actives the Na–Ca exchanger and causes [Ca]ⁱaccumulation. The whole cascade consumes high-energy phosphates, which was demonstrated here by decreasing phosphocreatine and ATP, at the same time increasing inorganic phosphate production at the end of ischemia. 18,19,28,29The increased Na influx from the extracellular space and the accumulation of Naⁱcan independently inhibit the mitochondria state 3 respiration, cause irreversible damage to mitochondria during ischemia, and further decrease ATP synthesis. 36Our results show that APC blunts the decrease in pHⁱ, as well as the increase in Naⁱin young adult hearts. High-energy phosphates are better preserved in younger hearts using APC, with significantly less [Ca]ⁱaccumulation during ischemia and reperfusion.

Together with our functional and histologic data, our results support the hypothesis that increased [Ca]ⁱplays a major role in myocardial cell damage. APC, like IPC, failed to protect aged myocardium from ischemia–reperfusion injury. This further supports the hypothesis that one of the cardioprotective mechanisms of APC is to maintain intracellular calcium homeostasis. Our results are also consistent with the report by Ataka et al.  33that cytoplasmic calcium accumulates to a greater extent in aged rabbit hearts. This may be because of a decreased ability of the cardiac sarcoplasmic reticulum to uptake excess calcium. 34,35Na–Ca exchanger activity may also be altered with aging. 37This reduced ability of the aged cells to handle calcium may contribute to the failure of APC in the senescent rat heart. Findings of Tricarico et al.  38indicate that K^ATPchannels from aged rats show decreases in open state probability and in the number of functional channels. K^ATPchannels from aged rats also exhibit greater sensitivity to inhibitors, an effect that is redox sensitive. Therefore, it is possible that changes in K^ATPchannel activation are responsible for the observed results in senescent myocardium. 38 

One of the limitations of our study was the difference in baseline pressures between the age groups. Compared with the young adult and middle-aged groups, the aged hearts had a significantly higher LVESP. The failure of APC could have been due to this relative hypertension. However, in two studies of IPC in hypertensive rats, IPC was still effective in one 39and had an increased effect in the other. 40Therefore, it seems unlikely that the failure of APC in the aged rats was due solely to their increased baseline pressures.

Our study was also limited by applying the same dose of sevoflurane to all age groups even though the MAC value decreases with age. Although the most effective dose to elicit APC protective mechanisms is still debatable, it is possible that this dose may vary with age. Further studies would be needed to determine whether higher, or possibly lower, doses could activate APC pathways in aged hearts. Also, we only used sevoflurane in this study, and we do not know whether all the volatile anesthetics are equal regarding APC.

Finally, we were unable to measure the mitochondrial calcium concentrations with the nuclear magnetic resonance spectroscopy we used. Because APC may act at the mitochondrial level, this could be an important measurement. However, numerous studies have reported that increased [Ca]ⁱhas a positive correlation with increased mitochondrial calcium.

In conclusion, we have demonstrated improved myocardial functional recovery; less cellular damage; decreased infarct size; minimized Hⁱ, Naⁱand [Ca]ⁱincreases; and better-preserved high-energy phosphates with APC in young adult and middle-aged isolated rat hearts. However, these beneficial effects of APC could not be reproduced in aged rat hearts. This is consistent with the observed decrease in efficacy of IPC in the senescent myocardium.