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Am J Physiol Heart Circ Physiol 282: H1978-H1987, 2002. First published February 7, 2002; doi:10.1152/ajpheart.00929.2001
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Vol. 282, Issue 6, H1978-H1987, June 2002

Cardioprotective effect of ischemic preconditioning is preserved in food-restricted senescent rats

Pasquale Abete1, Gianluca Testa1, Nicola Ferrara3,4, Domenico De Santis1, Pina Capaccio1, Luisa Viati1, Claudio Calabrese5, Francesco Cacciatore5, Giancarlo Longobardi4, Mario Condorelli2, Claudio Napoli2,6, and Franco Rengo1,4

1 Cattedra di Geriatria and 2 Cattedra di Medicina Interna, Dipartimento di Medicina Clinica, Scienze Cardiovascolari ed Immunologiche, Università degli Studi di Napoli "Federico II," Naples; 3 Cattedra di Geriatria, Dipartimento delle Malattie del Metabolismo dell'Invecchiamento, Seconda Università di Napoli, Naples; 4 Centro Medico di Telese Terme, Fondazione Salvatore Maugeri, Istituto di Ricovero e Cura a Carattere Scientifico, Benevento; 5 Unità Operativa Riabilitazione, ASL4 Basso Molise, Fondazione Salvatore Maugeri, Larino/Termoli, Italy; and 6 Department of Medicine, University of California, San Diego, California


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ischemic preconditioning (PC) has been proposed as an endogenous form of protection against-ischemia reperfusion injury. We have shown that PC does not prevent postischemic dysfunction in the aging heart. This phenomenon could be due to the reduction of cardiac norepinephrine release, and it has also been previously demonstrated that age-related decrease of norepinephrine release from cardiac adrenergic nerves may be restored by caloric restriction. We investigated the effects on mechanical parameters of PC against 20 min of global ischemia followed by 40 min of reperfusion in isolated hearts from adult (6 mo) and "ad libitum"-fed and food-restricted senescent (24 mo) rats. Norepinephrine release in coronary effluent was determined by high-performance liquid chromatography. Final recovery of percent developed pressure was significantly improved after PC in adult hearts versus unconditioned controls (85.2 ± 19% vs. 51.5 ± 10%, P < 0.01). The effect of PC on developed pressure recovery was absent in ad libitum-fed rats, but it was restored in food-restricted senescent hearts (66.6 ± 13% vs. 38.3 ± 11%, P < 0.05). Accordingly, norepinephrine release significantly increased after PC in both adult and in food-restricted senescent hearts, and depletion of myocardial norepinephrine stores by reserpine abolished the PC effect in both adult and in food-restricted senescent hearts. We conclude that PC reduces postischemic dysfunction in the hearts from adult and food-restricted but not in ad libitum-fed senescent rats. Despite the possibility of multiple age-related mechanisms, the protection afforded by PC was correlated with increased norepinephrine release, and it was blocked by reserpine in both adult and food-restricted senescent hearts. Thus caloric restriction may restore PC in the aging heart probably via increased norepinephrine release.

aging; caloric restriction; rat heart


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MORTALITY AND MORBIDITY for coronary heart disease increase with advancing age with an age-related poor prognosis for acute myocardial infarction (11). Several hypotheses have been postulated to explain this phenomenon, including the presence of frequent comorbidity and the low number of elderly patients with myocardial infarction treated with thrombolysis (10, 33, 51). Moreover, several experimental studies have demonstrated an age-related reduced tolerance to myocardial ischemia-reperfusion injury (7, 19) or stunning (2). One possible hypothesis is that endogenous protective mechanisms may decrease with age. Indeed, it has been demonstrated that preconditioning, the endogenous mechanism by which brief episodes of ischemia and reperfusion protect the heart against a more prolonged episode of ischemia (28, 37, 40), is reduced with aging (5, 15, 32, 45, 50). Similarly, clinical equivalents of preconditioning, such as preinfarction angina (3, 22) and the "warm-up phenomenon" (31, 41), appear to be less effective to protect the heart against myocardial ischemia in elderly patients.

In the murine model, the release of norepinephrine by intramyocardial adrenergic nerves seems to represent one of the mediators involved in preconditioning (8, 52). Interestingly, it has been proposed that age-related reduction of preconditioning may be due to norepinephrine release reduction in response to preconditioning stimulus in hearts from senescent animals (5). Accordingly, it is well established that norepinephrine release from rat cardiac nerves progressively decreases with age (13, 36).

It is widely well known that both exercise and caloric restriction profoundly affect physiological and pathophysiological modifications induced by aging and markedly increase life span in mice and rats (9, 29, 34, 54). However, the biological mechanism by which exercise and caloric restriction may exert their antiaging action is not yet understood. We have recently demonstrated that exercise training is able to restore preconditioning in the heart from senescent rats possibly through an increase of norepinephrine release in response to preconditioning stimulus (1). Moreover, we have also shown that cardioprotective effect of preinfarction angina is preserved in elderly patients with a high level of physical activity (4).

Caloric restriction attenuate several age-related modifications linked to adrenergic system (16, 18). In particular, it has been demonstrated that cardiac synaptosomal P2 fraction from food-restricted rats possessed a higher norepinephrine content (26), and the age-related reduction of norepinephrine release from cardiac nerve ending increases in hearts from food-restricted senescent animals (46). To our knowledge, no data are available on the effect of caloric restriction in preconditioning in aging heart. One hypothesis should be that caloric restriction may restore the protective effect afforded by preconditioning in the aging heart. This mechanism could be linked to the restoring of norepinephrine release in senescent animals in response to preconditioning stimulus.

Thus the goal of the present study was to evaluate the early effect of preconditioning on ischemia-reperfusion injury in isolated hearts from adult and both ad libitum-fed and food-restricted senescent rats. We also performed additional experiments in rats with depleted norepinephrine stores induced by reserpine to verify the possible role of norepinephrine release in the preconditioning phenomenon.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental procedure. Langendorff-perfused isolated hearts from male Wistar rats aged 6 (adult) and 24 (senescent) mo were studied. Animal care was performed according to the "Position of the American Heart Association on Research Animal Use." The animals were anesthetized with diethylether, and heparin sodium (200 IU) was injected intravenously. Hearts then were rapidly excised and attached via the aorta to a modified Langendorff perfusion apparatus. The basal perfusion medium (37°C, pH 7.4, perfusion pressure 66 mmHg) contained (in mM) 117 NaCl, 4.6 KCl, 0.8 NaH2PO4, 25 NaHCO3, 1 MgCl2, 2 CaCl2, and 5 glucose. The perfusion liquid was oxygenated by a mixture of 5% CO2-95% O2. Left ventricular pressure was measured using an intraventricular balloon attached to a 1-mm-diameter cannula and connected to a Statham P23 pressure amplifier and to a low-level preamplifier and direct differentiator (OTE Biomedica model 2080 pressure meter) to obtain the rise and fall in pressure (±dP/dt). Isovolumic loading conditions were established by setting left ventricular end-diastolic pressure at ~5 mmHg. Heart electrogram was obtained by an atraumatic epicardial electrode (0.8 mm diameter, silver wire) attached to the free wall of the right ventricle (to avoid affecting left ventricular function). The electrical signal was obtained from a bioelectric amplifier (OTE Biomedica model 2077 electrocardiogram amplifier). Pacing wires were fixed to the pulmonary outflow tract, and the hearts were paced at 6 Hz (pacer off during ischemia and drug infusion). Pacing was resumed 3 min after the start of reperfusion, and the hearts were defibrillated when necessary. Coronary flow rate was measured in graduated cylinders at intervals of 5 min before, during, and after infusion and during reperfusion. Coronary flow rate was related to wet ventricular weight (ml · min-1 · g-1) in both age groups. During the 20-min ischemic period, perfusion pressure was 0 mmHg, and the hearts were maintained at 37°C in a thermostated chamber. After a 20-min ischemic period, creatine kinase release in the coronary effluent was similar to that observed during the baseline period in all groups studied (2). Developed pressure (DP, mmHg), end-diastolic pressure (EDP, mmHg), +dP/dt, and coronary flow rate (CFR, ml · /min-1 · g-1) were monitored and recorded at intervals of 5 min on a Harvard oscillograph at a paper speed of 0.1 mm/s. A small number of hearts were perfused for 80 min to demonstrate only the preparation stability and were not counted in the present study.

Caloric restriction in senescent rats. Adult rats were fed ad libitum, whereas senescent rats were fed either ad libitum or restricted to 60% of the food intake of ad libitum-fed rats from the time of weaning. The senescent rats were shipped at 12 mo of age just 1 yr before use. In our institution, rats were housed in a barrier facility, in standard filter-topped cages, one rat per cage. Room temperature was set at 21 ± 1°C, the light-dark cycle was 12 h, and humidity was controlled at 40-65%. The animal were fed a pasteurized rodent diet and autoclaved water adjusted to pH 3.

Experimental design. After 20 min in which electrical and mechanical parameters were stabilized, the hearts were divided into three protocol groups of 21, comprising seven each from ad libitum-fed adult (n = 7) and senescent (n = 7) and food-restricted senescent (n = 7) rats.

Control hearts (n = 21) were from ad libitum-fed adult (n = 7) and senescent (n = 7) and food-restricted senescent (n = 7) rats in which ischemic perfusion was performed for 20 min and reperfused for 40 min (standard ischemia-reperfusion insult group, Control). Preconditioned hearts (n = 21) were from ad libitum-fed adult (n = 7) and senescent (n = 7) and food-restricted senescent (n = 7) rats treated with preconditioning transient ischemic stimulus for 2 min followed by 10 min of reperfusion (window) and then a standard ischemia-reperfusion insult (preconditioning group). Reserpinized preconditioned hearts (n = 21) were from ad libitum-fed adult (n = 7) and senescent (n = 7) and food-restricted senescent (n = 7) rats treated with preconditioning transient ischemic stimulus for 2 min followed by 10 min of reperfusion (window) and then a standard ischemia-reperfusion insult from reserpinized rat (0.15 mg/kg intraperitoneally 24 h beforehand) (reserpinized preconditioning group, Res-PC).

This preconditioning protocol (1 episode for 2 min) was used on the basis of previously published data (5), indicating that one or multiple cycles of preconditioning stimulus do not statistically differ to induce myocardial protection in senescent hearts.

Analysis of arrhythmias. The incidence of ventricular fibrillation (stated as a signal in which individual QRS complexes could not be distinguished from one another and for which a rate could not be determined) during reperfusion was determined before pacing (started at 3 min of reperfusion) in all groups. Late ventricular fibrillation was defined as ventricular fibrillation that occurred after initial conversion to paced rhythm.

Norepinephrine assay. Norepinephrine levels were determined by collecting coronary effluent after 2 min of transient global ischemia accumulated over the preconditioning window and corrected to the wet ventricular weight in both age groups (pmol · ml-1 · g-1). The effluent was collected in chilled tubes containing 3% perchloric acid (final concentration) and frozen at -80°C. The assay was performed by high-performance liquid chromatography (Beckman; Fullerton, CA). Briefly, 50 µl of each sample (Beckman model 210 injector) were separated on an ultrasphere ODS (3-mm particles) reverse-phase column (Beckman, Altex Division; San Ramon, CA), detected by dual electrode (ox-redox, +0.25; -0.25 V) colormetry (ESA 5100A Coulochem System; Bedford, MA), and finally matched to a standard control.

Statistical analysis. Results are expressed as means ± SD. A one-way ANOVA was performed to separately test the main effects of age and caloric restriction in adult and in ad libitum-fed and food-restricted senescent rats. A one-way ANOVA was also done to compare the functional parameters (DP, EDP, +dP/dt, CFR) and ventricular fibrillation at specific time points and in the different protocol groups in adult and in ad libitum-fed and food-restricted senescent rats. The same analysis was performed to compare norepinephrine release at baseline and after ischemia in adult and in ad libitum-fed and food-restricted senescent rats in the presence and in the absence of reserpine. If the F ratios were significant, post hoc Scheffé's test was applied. Comparison within groups was performed by using paired samples t-test. Values <0.05 (P < 0.05) were considered as significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of age and caloric restriction on body weight and left ventricular weight. Age effect was significant between adult and ad libitum-fed senescent rats and between adult and food-restricted senescent rats in terms of body weight, left ventricular wet weight, and the ratio of left ventricular weight to body weight (P < 0.01 and P < 0.05, respectively) (Table 1). A statistical significant effect of caloric restriction on body weight and left ventricular weight-to-body weight ratio was also present between ad libitum-fed and food-restricted senescent rats (P < 0.05) (Table 1).

                              
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  		Table 1.   Body and left ventricular weights in adult and ad libitum-fed and food-restricted senescent rats

Effect of ischemia and reperfusion in hearts from adult and ad libitum-fed and food-restricted senescent rats. DP and +dP/dt rapidly decreased, whereas EDP increased after 20 min of normothermic global ischemia in hearts from all groups. However, in senescent rats, EDP increase was more pronounced in both ad libitum and food-restricted senescent than in adult rats (36.1 ± 16.1 mmHg in adult hearts vs. 50.4 ± 9.5 mmHg in hearts from ad libitum-fed and 50.0 ± 9.9 mmHg from food-restricted senescent rats, P < 0.05) (Table 2). DP in hearts from adult rats recovered by approx 50%, from ad libitum-fed senescent rats by approx 30%, and from food-restricted senescent rats by approx 35% (see Figs. 1 and 2). CFR did not significantly differ among the three groups.

                              
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  		Table 2.   Hemodynamic parameters of ischemia (20 min) and reperfusion (40 min) in hearts from adult and ad libitum-fed and food-restricted senescent rats (standard ischemia-reperfusion insult group)



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  		Fig. 1.   Changes in developed pressure (DP, mmHg) during baseline (B), ischemia, and reperfusion in adult and in ad libitum-fed or food-restricted senescent hearts subjected to ischemia for 20 min and reperfused for 40 min (standard ischemia-reperfusion insult, IR, control; A) treated with preconditioning transient ischemic stimulus for 2 min followed by 10 min of reperfusion (PC; B) and then a standard ischemia-reperfusion insult after pretreatment with reserpine (reserpinized preconditioning group, Res-PC; C). * P < 0.05 vs. ad libitum-fed and food-restricted senescent hearts; dagger  P < 0.01 vs. adult hearts; Dagger  P < 0.05 vs. food-restricted senescent hearts.



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  		Fig. 2.   Bar graphs showing percent recovery of DP (%) from baseline to 40 min of reperfusion in adult and in ad libitum-fed or food-restricted senescent hearts subjected to ischemia for 20 min and reperfused for 40 min (standard ischemia-reperfusion insult, IR, control), treated with preconditioning transient ischemic stimulus for 2 min followed by 10 min of reperfusion (PC), and then a standard ischemia-reperfusion insult after pretreatment with reserpine (reserpinized preconditioning group, Res-PC). * P < 0.05 vs. ad libitum-fed and food-restricted senescent hearts; dagger  P < 0.01 vs. Control and Res-IP; Dagger  P < 0.05 vs. Control and Res-IP.

Effect of preconditioning transient ischemia on ischemia-reperfusion injury in hearts from adult and ad libitum-fed and food-restricted senescent rats. In the adult group, preconditioning determined an increase of both DP (81 ± 18 vs. 51 ± 10 mmHg, P < 0.01) and contractility (+dP/dt: 2,157 ± 201 vs. 1,290 ± 146 mmHg/s, P < 0.01) with respect to ischemia-reperfusion control (Tables 2 and 3). In a similar way, in the adult group EDP significantly recovered with respect to the control group, returning close to baseline values (11.5 ± 7.9 vs. 36.1 ± 16.1 mmHg, P < 0.01) (Tables 2 and 3). In preconditioned hearts from ad libitum-fed senescent rats, neither DP nor EDP improved with respect to controls (Tables 2 and 3). However, in preconditioned hearts from food-restricted senescent rats, both DP and +dP/dt showed a significant improvement with respect to controls (66 ± 12 vs. 38 ± 11 mmHg and 1,654 ± 165 vs. 970 ± 170 mmHg/s, respectively, P < 0.05) (Tables 2 and 3). Similarly, in preconditioned hearts from food-restricted senescent rats, EDP was also significantly lower with respect to controls (20.0 ± 11.2 vs. 50.0 ± 9.9 mmHg, P < 0.05) (Tables 2 and 3). Figure 2 shows that preconditioning in hearts from adult but not from ad libitum-fed senescent rats improved the percentage of final recovery of DP (85.2 ± 10.1% vs. 38.3 ± 10.2%, P < 0.01). In contrast, in hearts from food-restricted senescent hearts, percentage final recovery of DP showed a recovery similar to that of adult ones (66.6 ± 12.0%). These data clearly indicate that caloric restriction may restore the protective effect of preconditioning in senescent hearts against ischemia-reperfusion injury.

                              
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  		Table 3.   Hemodynamic parameters of transient ischemia preconditioning stimulus (2 min) on ischemia (20 min) and reperfusion (40 min) in hearts from adult and ad libitum-fed and food-restricted senescent rats (preconditioning group).

Preconditioning stimuli on ischemia-reperfusion injury in hearts from reserpinized adult and ad libitum-fed and food-restricted senescent rats: the possible role of the adrenergic pathway. The role of alpha 1-adrenergic stimulation by endogenous norepinephrine was preliminary evaluated by studying the effect of prazosin (1 µmol/l) perfused for 10 min before and during 10 min of reperfusion following the ischemic preconditioning stimulus in isolated hearts from adult rats (n = 5, data not shown). The specific alpha 1-adrenergic blockade abolished the protective effect of preconditioning on postischemic dysfunction with the respective preconditioned controls (87.7 ± 9% vs. 41.6 ± 12%, P < 0.01). Here, reserpine pretreatment experiments (0.15 mg/kg ip) were performed to study the effect of partially depleted norepinephrine stores on preconditioning in the rat. Reserpine eliminated the protective effect of preconditioning in hearts from both adult and food-restricted senescent rats. EDP final recovery was 11.5 ± 7.9 mmHg in the absence and 38.1 ± 11 mmHg in the presence of reserpine in preconditioned adult hearts, (P < 0.01) (Tables 3 and 4). Similarly, in preconditioned hearts from food-restricted senescent rats, the EDP final recovery was 20.0 ± 11.2 mmHg in the absence and 51.5 ± 9.5 mmHg in the presence of reserpine (P < 0.05) (Tables 3 and 4). Figure 2 shows that in hearts from reserpinized rats, the protective effects of preconditioning on the percentage of final recovery of DP was abolished in hearts from both adult and food-restricted senescent rats. Because the protective effect of preconditioning was abolished by reserpine in hearts from adult and food-restricted senescent rats, norepinephrine release after preconditioning stimulus seems to play a pivotal role on restoring preconditioning mechanism in food-restricted senescent hearts.

                              
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  		Table 4.   Hemodynamic parameters of transient ischemia preconditioning stimulus (2 min) on ischemia (20 min) and reperfusion (40 min) after pretreatment with reserpine in adult and ad libitum-fed and food-restricted senescent rats (reserpinized preconditioning group, Res-PC)

Effects of preconditioning on reperfusion-induced ventricular fibrillation. The incidence of reperfusion-induced ventricular fibrillation was higher in both ad libitum-fed and food-restricted senescent than adult hearts in all groups (P < 0.05), and preconditioning was not able to reduce them in all groups studied (Fig. 3A). Moreover, late ventricular fibrillation was significantly reduced by preconditioning in adult (2/7 vs. 5/7, P < 0.01) and in food-restricted senescent (3/7 vs. 7/7, P < 0.01) but not in ad libitum-fed senescent hearts (7/7 vs. 7/7, P = not significant).


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  		Fig. 3.   Incidence of reperfusion-induced ventricular fibrillation (VF) (A) and late VF (B) in adult and in ad libitum-fed or food-restricted senescent hearts subjected to ischemia for 20 min and reperfused for 40 min (standard ischemia-reperfusion insult, IR, control), treated with preconditioning transient ischemic stimulus for 2 min followed by 10 min of reperfusion (PC), and then a standard ischemia-reperfusion insult after pretreatment with reserpine (reserpinized preconditioning group, Res-PC). * P < 0.05 vs. ad libitum-fed and food-restricted senescent hearts; dagger  P < 0.01 vs. Control and Res-IP; Dagger  P < 0.01 vs. ad libitum-fed senescent hearts.

Norepinephrine release after preconditioning stimulus. Norepinephrine background obtained during the 10 min before preconditioning stimulus from coronary effluent was similar in hearts from adult and from ad libitum-fed and food-restricted senescent rats (ANOVA = P < 0.617, not significant) (Fig. 4). After 2 min of global ischemia (transient ischemic stimulus), norepinephrine concentrations in coronary effluent accumulated over the 10-min reperfusion preconditioning window significantly increased in hearts from adult (from 0.34 ± 0.09 to 4.1 ± 1.3 pmol · ml-1 · g-1; P < 0.01) and from food-restricted senescent (from 0.43 ± 0.09 to 3.05 ± 1.1 pmol · ml-1 · g-1; P < 0.001) rats but not in those from ad libitum-fed senescent rats (from 0.35 ± 0.07 to 1.1 ± 0.7 pmol · ml-1 · g-1; P < 0.231, not significant) (n = 4 for each group) (Fig. 4). Reserpine pretreatment significantly reduced norepinephrine release in response to preconditioning in all groups (from 0.41 ± 0.06 to 0.61 ± 0.08 pmol · ml-1 · g-1 in adult hearts, from 0.38 ± 0.07 to 0.51 ± 0.06 pmol · ml-1 · g-1 in ad libitum-fed senescent hearts, and from 0.31 ± 0.06 to 0.78 ± 0.1 pmol · ml-1 · g-1 in food-restricted senescent hearts) (n = 4 for each group) (Fig. 4). These data confirm that norepinephrine is primarily implicated in the preconditioning phenomenon and that restoration of preconditioning in food-restricted senescent hearts might be due to the restoration of norepinephrine release in response to preconditioning stimulus.


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  		Fig. 4.   Norepinephrine (NE) release from coronary effluent at baseline and over the 10-min reperfusion preconditioning window after 2 min of ischemic preconditioning stimulus in hearts from adult and from ad libitum-fed or food-restricted rats. * P < 0.01 vs. baseline; dagger  P < 0.001 vs. baseline.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Preconditioning improved both mechanical and electrical parameters in adult but not in hearts from ad libitum-fed senescent animals. Here, our results demonstrate for the first time that preconditioning is preserved in food-restricted senescent animals. In addition, caloric restriction seems to restore preconditioning in hearts from senescent animals through an involvement of the adrenergic pathway in response to preconditioning.

Ischemic myocardial tolerance and aging. Mortality and morbidity for coronary heart disease significantly increase with age (11, 33, 51). Before the use of thrombolysis, comorbidity has been considered the more reasonable cause of the age-related increase of mortality for coronary artery disease. However, this hypothesis is not afforded by some adverse baseline and additional in-hospital characteristics in elderly patients with acute myocardial infarction (51). In addition, the under use of thrombolytic therapy in the absence of marked contraindications might affect the prognosis of elderly patients (10). However, it has been demonstrated that elderly patients with first myocardial infarction receiving thrombolytic therapy show a higher mortality than in younger patients (33, 39). This clinical evidence has been validated by animal studies clearly showing an age-related decrease in myocardial ischemic tolerance (2, 7, 19). In the present study, the more severe arrhythmias observed in senescent hearts can itself generate significant tissue oxidative stress and may contribute the more severe impairment observed in the aging heart (12).

Preconditioning and aging. One possibility is that the most powerful endogenous mechanism against myocardial ischemia, i.e., preconditioning (28, 37, 40), may be reduced with aging. This intriguing phenomenon is mediated by numerous mediators, including adenosine receptors, norepinephrine, bradykinin, sarcolemmal and mitochondrial ATP-sensitive K+ channels, opioid receptors, and several others (28, 37, 40). These mediators alone or in combination may stimulate protein kinase C and other components of the kinase cascade as intracellular mediators of preconditioning (28).

We first demonstrated that the protective effect of preconditioning on electrical and mechanical function following ischemia-reperfusion injury was reduced in senescent hearts (5). A reduction of norepinephrine release in response to ischemic stimulus was involved for the age-related decrease of preconditioning (5). Successively, it has also been demonstrated a reduction of preconditioning in middle-aged rat hearts related to an impairment of ryanodine-sensitive sarcoplasmic reticulum receptor (50). In addition, preconditioning with two 5-min ischemia and 5-min reperfusion cycles significantly reduced necrosis development and enhanced reperfusion contractile function in young hearts but not in aged hearts (15). Moreover, it has also been demonstrated that the aging heart could not be preconditioned neither by ischemic stimulus nor pharmacological means such as adenosine A1 agonist, protein kinase C analog, and the mitochondrial ATP-sensitive K+ channel opener diazoxide (45). Accordingly, Tani et al. (49) attributed the age-related reduction of preconditioning to a failure of protein kinase C isoforms translocation in response to preconditioning stimulus.

Caloric restriction and aging heart. Exercise training, as well as caloric restriction, has been widely described as antiaging intervention (29, 34, 54). In particular, caloric restriction increases the life span of rodents (9), retards the severity of some age-related diseases (53), and attenuates the physiological decline of several organs, including the heart (27, 34, 54). Specifically, caloric restriction enhances arterial baroreflexes (17) and isoproterenol sensitivity (20) and prevents the age-related impairments in diastolic function (48).

Aging, caloric restriction, norepinephrine, and preconditioning . In the rat experimental model, one of the possible mechanisms of preconditioning is the release of norepinephrine in response to a preconditioning stimulus by alpha 1-adrenoreceptor stimulation (8, 52). The abolition of a preconditioning mechanism by prazosin and reserpine strongly suggest that the endogenous release of catecholamines mediates the effect of preconditioning. The age-related decline of tissue catecholamines due to several mechanisms, including a related diminished ability for catecholamine synthesis (13, 36), could explain the reduction of preconditioning in the aging heart. It has been demonstrated that antiaging intervention such as exercise training is able to restore norepinephrine release from cardiac adrenergic terminations in response to stress stimulus (35). Accordingly, it has been recently shown that exercise training reestablishes preconditioning in trained senescent rats through an increase of norepinephrine in response to a preconditioning stimulus (1). Caloric restriction affects sympathetic system in several ways. First, restriction of caloric intake is accompanied by a reduction in plasma norepinephrine (17, 52). Second, caloric restriction attenuates several age-related modifications linked to the adrenergic system, including beta -adrenergic receptors in the liver (24), lung (44), and heart (16) and alpha 1-adrenergic receptors in the aorta (18). In addition, Kim et al. (26) demonstrated that the norepinephrine content of hearts from food-restricted rats was higher than controls as well as the cardiac synaptosomal P2 fraction from food-restricted rats possessed a higher norepinephrine content than the P2 fraction of ad libitum-fed rat controls. Subsequently, Snyder et al. (46) demonstrated that aging reduces the capacity of cardiac adrenergic nerve terminals to release norepinephrine, and this age-related reduction is significantly blunted by dietary restriction. Accordingly, in our experimental conditions, cardiac norepinephrine release in response to preconditioning stimulus is reduced in senescent than in adult animals. However, cardiac norepinephrine release in response to preconditioning stimulus is restored by dietary restriction in senescent animals, and this might be, at least in part, one of the mechanism by which dietary restriction restores preconditioning in the aging heart. The absence of preconditioning in adult and food-restricted senescent animals with depleted norepinephrine stores by reserpine confirms the above hypothesis.

Other possible mechanisms. Obviously, other possible pathophysiological mechanisms may explain the reestablishment of preconditioning in the aging heart by dietary restriction. A very complex cascade of events simultaneously act in the preconditioned heart (40), and a detailed analysis of such mechanisms are beyond the scope of the present paper. Recently, it has been demonstrated that the age-related reduction of preconditioning might be attributed to a decrease in the release of calcitonin gene-related peptide in response to preconditioning stimulus (32). In this regard, it is also known that food restriction interferes with calcitonin metabolism and could modulate the expression of the calcitonin gene in the aging heart (23). Finally, the pivotal role of oxygen radicals in the aging process (6) and ischemia-reperfusion injury (21) is widely accepted. Dietary restriction also reduces the extent of oxidative damage in the heart supporting antioxidant defenses (26).

Clinical implications. It is well known that excess body weight increases the risk of death from any cause and from cardiovascular disease among adult and old patients (30, 42, 47). The relationship between overweight and coronary heart disease might be explained by higher serum cholesterol levels and the prevalence of diabetes and hypertension among overweight subjects (42). However, these conditions do not totally explain this relationship, especially in elderly patients with coronary heart disease. For example, although aging promotes low-density lipoprotein oxidation (38), cholesterol levels decrease progressively with advancing age (14). Age-related higher mortality for coronary heart disease may in part be due to the preconditioning reduction as consequence of the decrease in norepinephrine release in response to preconditioning stimuli. A reduction of body mass index through a caloric restriction in elderly patients may restore the preconditioning in the aging heart by restoring norepinephrine release in response to preconditioning stimuli.

In conclusion, in senescent hearts, preconditioning fails to afford protection against ischemia-reperfusion injury. However, in food-restricted senescent animals, caloric restriction seems to be able to restore preconditioning. This could be due to an increase in norepinephrine release in response to preconditioning stimulus. The absence of the protection in adult and food-restricted senescent hearts after norepinephrine depletion by reserpine administration seems to support this working hypothesis.


    ACKNOWLEDGEMENTS

This study was supported by Consiglio Nazionale delle Ricerche-Progetto Finalizzato Invecchiamento


    FOOTNOTES

Address for reprint requests and other correspondence: P. Abete, Dipartimento di Medicina Clinica e Scienze Cardiovascolari ed Immunologiche, Cattedra di Geriatria, Università degli Studi di Napoli "Federico II" Via S. Pansini, 5 80131 Napoli, Italy (E-mail: p.abete{at}cds.unina.it).

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 7, 2002;10.1152/ajpheart.00929.2001

Received 25 October 2001; accepted in final form 31 January 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 282(6):H1978-H1987
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