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CARDIOVASCULAR ANESTHESIA

Increasing Heart Size and Age Attenuate Anesthetic Preconditioning in Guinea Pig Isolated Hearts

Departments of Anesthesiology and Physiology, Cardiovascular Research Center, Medical College of Wisconsin, and the VA Medical Center Research Service, Milwaukee, Wisconsin

Address correspondence and reprint requests to Matthias L. Riess, MD, PhD, Medical College of Wisconsin, M4280, 8701 Watertown Plank Road, Milwaukee, WI 53226. Address e-mail to mriess{at}mcw.edu.

	Abstract

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Anesthetic preconditioning (APC) reduces myocardial ischemia/reperfusion injury. Recent investigations have reported that older hearts are not susceptible to APC. We investigated if increasing heart size with age determines the susceptibility to APC in young guinea pigs. Langendorff-prepared guinea pig hearts of different weights (1.1–2.2 g) and ages (2–7 wks) were exposed to 1.3 mM sevoflurane for 15 min followed by 30 min washout (APC; n = 20) before 30 min global ischemia and 120 min reperfusion. Control hearts (n = 20) were not subject to APC. Left ventricular pressure was measured isovolumetrically and infarct size was determined by triphenyltetrazolium staining. Functional data were not different between groups at the beginning of the experiments nor did they correlate with heart weight or age. At 120 min reperfusion, left ventricular pressure, coronary flow, and tissue viability showed significant negative correlations with increasing heart weight and age in APC but not in control hearts; i.e., APC improved function and attenuated infarct size better in smaller/younger hearts than in larger/older hearts. Thus, increasing age and heart size attenuate the susceptibility for APC even in younger guinea pigs. This may have important implications for further basic science research and the possible clinical applicability of APC in humans.

	Introduction

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Murry et al. in 1986 (1) first described the phenomenon of ischemic preconditioning (IPC) as a means of cardioprotection. Four short duration circumflex artery occlusions before sustained occlusion for 40 min and reperfusion were able to prepare, i.e., "precondition," the heart and reduce ischemia/reperfusion (IR) injury. In a fashion similar to IPC, anesthetic preconditioning (APC), i.e., temporary exposure to a volatile anesthetic, can also trigger an acute cardioprotective memory effect that lasts beyond its elimination (2–4).

Most basic science research in this area uses models of younger animals or tissue. Protection against IR injury, however, would be most beneficial for patients of advanced age because most cardiac complications in humans occur later in life. Older hearts have generally less tolerance to stress than do younger hearts (5). Therefore, preconditioning in the older myocardium is an important issue that may differ considerably from preconditioning in the younger myocardium, so that cardioprotection may need to be tailored to the aged population (6). A recent study by Sniecinski and Liu (7) indeed showed reduced efficacy of APC with advanced age; they isolated rat hearts from 3 different age groups up to 24-months-old and found that cardioprotection by APC was significantly reduced with increasing age.

It is unclear if a reduction in cardioprotection is purely related to aging, i.e., occurs only later in life, or if it happens much earlier, i.e., correlates with normal cardiac development before and during adolescence. The aim of the present study was to determine the possible effect of increasing heart weight and age during adolescence on the success of APC. We measured several cardiac variables before and after IR injury in Langendorff-prepared hearts of different weights harvested from guinea pigs aged 2 to 7 weeks (i.e., before they reached full sexual maturity) to evaluate the possible relationship of heart weight and age on APC.

	Methods

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The investigation conformed to the Guide for the Care and Use of Laboratory Animals and was approved by the Medical College of Wisconsin Animal Use and Care Committee. We used 40 albino English short-haired guinea pigs at ages between 2 and 7 wk (body weight 250 to 400 g), i.e., before the onset of sexual maturity (personal communication with the breeder, Kuiper Rabbit Ranch, Gary, IN). When unresponsive to noxious stimulation after intraperitoneal injection of ketamine (100 mg/kg) and heparin (3000 U/kg) the animals were decapitated and the hearts were excised, cannulated, and perfused retrograde through the aorta at a constant pressure of 55 mm Hg and at 37°C as previously described (8,9). Left ventricular pressure (LVP) was measured isovolumetrically with a saline-filled latex balloon inserted into the left ventricle. Attention was paid to inserting the latex balloon unfilled to prevent inadvertent preconditioning by stretching (10). At the beginning of each experiment, the balloon volume was adjusted to achieve a diastolic LVP of 0 mm Hg, so that any subsequent increase in diastolic LVP reflected an increase in diastolic contracture.

Characteristic data derived from LVP measurements were developed (systolic–diastolic) LVP, and the first derivative of LVP, dLVP/dt_max, as an index of contractility. Spontaneous heart rate was monitored with bipolar electrodes placed in the right atrial and ventricular walls. Coronary flow (CF) was measured at constant temperature and perfusion pressure by an ultrasonic flowmeter (T106X; Transonic, Ithaca, NY) placed directly into the aortic inflow line.

Sevoflurane (sevo) (Abbott Laboratories, North Chicago, IL) was bubbled into the perfusate using an agent-specific vaporizer (Vapor 2000; Dräger Medizintechnik GmbH, Lübeck, Germany) placed in the O₂-CO₂ gas mixture line. Samples of coronary perfusate were collected to measure sevo concentrations by gas chromatography. After stabilization, each experiment lasted 195 min. Hearts were assigned randomly to 1 of 2 groups: untreated ischemic control hearts (CON, n = 20) were not exposed to sevo. APC hearts (n = 20) were exposed to 1.3 ± 0.1 mM sevo (8.9 ± 0.7 Vol% at 37°C) for 15 min, followed by a 30-min washout period before ischemia. Although this sevo concentration is too high to use for maintenance of general anesthesia, it may be achieved temporarily during mask induction (11). This concentration was chosen to maximize the concentration-dependent cardioprotection by APC in this model (8,9).

Each heart then underwent 30 min of global no-flow ischemia by clamping the aortic inflow; this was followed by 120 min of reperfusion. At the end of each experiment, each heart was removed and ventricular infarct size was determined by staining with 0.1% 2,3,5-triphenyltetrazolium chloride followed by cumulative planimetry as described previously (8).

All analog signals were digitized (PowerLab/16 SP; ADInstruments, Castle Hills, Australia) and recorded at 200 Hz (Chart & Scope v3.6.3; ADInstruments) on a Power Macintosh Computer G4 (Apple, Cupertino, CA) for later analysis using MATLAB (MathWorks, Natick, MA) and Microsoft Excel (Microsoft Corporation, Redmond, WA) software. Group data were expressed as mean ± sem and were compared by unpaired Student's t-test. Statistical significance was accepted when P < 0.05 (two-tailed). Regression-correlation analyses were used to determine the relationships between heart weight or age and other measured variables in preconditioned and non-preconditioned hearts before and during sevo exposure and after 120 min reperfusion.

	Results

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The measured heart weights (CON: 1.63 ± 0.06 g; APC: 1.66 ± 0.05 g) and the animals' ages (CON: 4.4 ± 0.3 wks; APC: 4.6 ± 0.3 wks) were not different between the two groups, and heart weight correlated significantly (r² = 0.92) with age in this model. Table 1 shows that there were no significant differences between the two groups for any measured cardiac variable before treatment (baseline). None of the measured variables correlated with heart weight or age before treatment (baseline; data not shown). Sevo exposure (APC) temporarily depressed developed LVP, contractility (Table 1), and heart rate (data not shown), and increased CF (Table 1). Neither sevo concentrations nor any of the sevo-induced functional changes correlated with heart weight or age, and all changes were completely reversible with washout of sevo before ischemia (data not shown).

At 120 min reperfusion, averaged APC hearts exhibited a higher developed LVP, improved CF, and attenuated ventricular infarct size compared with averaged nontreated CON hearts (Table 1). However, developed LVP, contractility, and CF showed significant negative correlations with increasing heart weight and age in APC hearts, but not in CON hearts, at 120 min reperfusion (Table 2 and Fig. 1 A to C). Ventricular infarct size at 120 min reperfusion correlated positively with heart weight and age in APC hearts but negatively in CON hearts (Table 2 and Fig. 1 D).

View larger version (28K): [in this window] [in a new window]   Figure 1. Effect of heart weight (wt) on developed (systolic–diastolic; A) left ventricular pressure (LVP), dLVP/dtmax (B) as an index of contractility, coronary flow (C) and ventricular infarct size (D) in APC and CON hearts (n = 20 each) at 120 min reperfusion.

	Discussion

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Our study in guinea pig isolated hearts demonstrates that the ability of the myocardium to be protected against IR injury by APC decreases with increasing heart size and age during adolescence. Although some investigators have found that age does not influence myocardial tolerance to ischemia or the protective effect of IPC (12), other studies have questioned the ability of aged myocardium to be susceptible to preconditioning at all (13,14). Schulman et al. (15), for example, reported that aged rat hearts could not be preconditioned by ischemic or pharmacological means and that middle-aged rat hearts had only a blunted response to preconditioning as compared with young adult hearts. They suggested defects in the signaling cascade of preconditioning in aged hearts.

The study by Sniecinski and Liu (7) points in a similar direction for APC. They examined the effect of APC in isolated perfused rat hearts from three different age groups, young adults, middle-aged, and aged. None of the benefits of APC in the younger hearts, e.g., better preserved mechanical function and reduced infarction on reperfusion, could be demonstrated in the aged group. This was supported by measurements of intracellular Na⁺ and pH that showed better protection during ischemia in younger and middle-aged hearts than in the aged group. On the other hand, the observed decreases in phosphocreatine, adenosine triphosphate, and inorganic phosphate during ischemia and on reperfusion were not different between APC and control experiments in aged and middle-aged hearts as opposed to young hearts. These differential results suggest that some of the cardioprotective effects by APC may be lost even earlier in life.

Although many investigators choose the rat heart as a model to study cardiac preconditioning, the guinea pig heart is believed to be closer in design to the human heart with regard to action potential, Ca²⁺ flux, and other characteristics (16–19). Our results indicate that the cardioprotective effect of APC in guinea pig hearts decreases as a function of heart weight and age before and during adolescence. This was demonstrated by less improved vascular function as well as mechanical function and less attenuated infarct size on reperfusion with increasing heart weight and age. Although improved coronary function is usually associated with mechanical cardioprotection and attenuated infarct size after APC in this model, we have previously shown that good CF alone is necessary, but not sufficient, to ensure good recovery of contractile function and reduction in infarct size on reperfusion (20).

Neither heart weight nor age had an effect on any measured cardiac variable before or during preconditioning. For example, CF (per g heart weight) were identical in lighter or younger hearts when compared with heavier or older hearts, respectively; therefore, sevo delivery (CF times sevo concentration) was identical in the different groups and independent of weight or age. Also, the decreases in developed LVP and contractility during sevo exposure were independent of weight or age which excludes a less efficient delivery of the anesthetic to heavier and older hearts. This is important for the interpretation of the data on reperfusion because it excludes the possibility that older and heavier hearts were preconditioned differently from younger and lighter hearts. Furthermore, a possible size- or age-related effect, per se, on the degree of IR injury as suggested, e.g., by decreasing infarct sizes in control hearts with age in both our study and the one by Sniecinski and Liu (7), would have to apply to both the APC and CON hearts and can therefore not be responsible for the observed differences between the APC and the CON groups.

We can only speculate the mechanisms that underlie our observations, especially because the exact triggering mechanism of APC is not yet fully elucidated (21). However, because APC seems to share a variety of common pathways with other cardioprotective mechanisms (22), it is possible that this effect is also a result of alterations in the triggering factors, e.g., the formation of oxygen free radicals, and in the expression and activation of proteins critical for cardioprotection, such as heat shock proteins, nitric oxide synthase, and mitogen activated protein kinases (6,23). The findings from this study may have important implications not only for future basic research with preconditioning models but also for the possible application of APC in the clinical situation (24). Clearly, the role of cardiac development and aging in APC and the mechanisms involved need to be thoroughly investigated, particularly if susceptibility to APC should indeed be reduced much earlier in life than previously thought.

The authors thank Srinivasan G. Varadarajan, MD, Assistant Professor, James S. Heisner, B.S., Research Technologist, Mohammed Aldakkak, MD, Postdoctoral Research Fellow, and André Heinen, MD, Postdoctoral Research Fellow, all from the Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin, for their valuable assistance.

	Footnotes

 
Supported, in part, by grants No. HL 58691, AG 022171 (to Dr. Stowe), and HL 073246–01 (to Dr. Camara) from the National Institutes of Health (Bethesda, MD) and by grants No. 0355608Z (to Dr. Stowe) and 0425661Z (to Dr. Rhodes) from the American Heart Association (Dallas, TX).

Presented, in part, at the 26th Annual Meeting of the Society of Cardiovascular Anesthesiologists, Honolulu, Hawaii, April 24–28, 2004.

Accepted for publication July 27, 2005.

	References

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