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Ann Thorac Surg 2000;70:621-626
© 2000 The Society of Thoracic Surgeons

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Original articles: cardiovascular

Age dependence of heat stress mediated cardioprotection

^a Department of Cardiothoracic Surgery, National Heart and Lung Institute, Imperial College, Harefield Hospital, Harefield, Middlesex, United Kingdom

Address reprint requests to Prof Sir Yacoub, Department of Cardiothoracic Surgery, National Heart and Lung Institute, Harefield Hospital, Harefield, Middlesex, UB9 6JH United Kingdom
e-mail: caroline.gray{at}harefield.nthames.nhs.uk

	Abstract

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Background. To study the influence of age on heat stress cardioprotection, functional recovery, nucleotide concentrations, and heat stress protein 70 (Hsp70) levels were compared in the heat stressed (HS) and control (C) hearts at different ages, in a protocol mimicking donor heart preservation for transplantation.

Methods. Control and heat stressed (24 hours before experiment) rat hearts were divided into three age groups: (I) 1 month, (Y) 4 months, and (M) 16 months (n = 6). Left ventricle balloon catheter was used to determine systolic and end-diastolic pressure/volume relations before and after 4 hours of cardioplegic arrest at 4°C. Another identical set of isolated hearts underwent 5 minutes of normoxic perfusion to obtain preischemic Hsp70 content and metabolite concentrations.

Results. The postischemic recovery was highest in group HS-Y as compared to C-Y, HS-I, C-I, HS-M, and C-M. There were no differences in preischemic adenine nucleotides or creatine metabolite concentrations between the three age groups. In contrast, the nicotinamide adenine dinucleotide (oxidized form) (NAD⁺) and nicotinamide adenine dinucleotide phosphate (oxidized form) (NADP⁺) concentrations were significantly raised in group HS-Y. Hsp70 content was increased in all HS groups with no difference between the age groups.

Conclusions. Improved postischemic functional recovery after cardioplegic arrest was observed in the young adult HS hearts. This was associated with highest NAD⁺ and NADP⁺ concentrations and did not correlate with increased Hsp70 content.

	Introduction

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The aging process of the myocardium is associated with biochemical and physiological alterations [1]. The deterioration of calcium handling [2], nucleotide metabolism [3], enzyme activity [4], mechanical and endothelial function [5, 6] with age is well established. The effect of age on the heat stress response and its ability to protect the myocardium following ischemia and reperfusion have not been adequately studied [7, 8].

Heat stress proteins (HSP) are induced by a variety of stimuli. They contribute to maintaining the metabolic and structural integrity of the cell, as a protective response to external stresses. They are also known to protect the myocardium from ischemia/reperfusion injury [9]. We have previously shown that the beneficial effects associated with their expression include improved endothelial and mechanical recovery of the ischemic heart [10]. However the biochemical mechanisms, underlying this protective effect is not clearly defined. Alterations in the cell metabolism [11] and chaperone function [9] of cells overexpressing heat shock proteins, as well as inhibition of apoptotic pathways [12], are thought to play an important role.

Little is known about specific age-related changes in nucleotide metabolism and their relationship with cardiac function following cold cardioplegic arrest of the heat stressed heart. We aim to evaluate the association between the concentration of adenine and pyridine nucleotides on the postischemic recovery of the heat stressed rat heart in three defined age populations, using a protocol mimicking donor preservation for heart transplantation. The age groups studied include 1, 4, and 16 months, which corresponds to the infant, young adult, and mature adult hearts in humans.

	Material and methods

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Animals
In all studies, animals received humane care in compliance with the "Principles of Laboratory Animals’ Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication No. 80-23, revised 1985). Male Sprague-Dawley rats were used in all experiments to avoid a potential lack of homogeneity related to the hormonal cycle. Two sets of rats were studied, control and heat stressed, in which animals were divided into three age groups: (I) 1 month, less than or equal to 100 g; (Y) 4 months, less than or equal to 450 g, and (M) 16 months, less than or equal to 800 g (n = 6 in each group).

Induction of heat stress
Rats were anesthetized with an intraperitoneal injection of sodium pentobarbitone (50 mg/kg), then placed on a temperature-controlled heating pad (IMS K-Temp control unit; Congleton, Cheshire, UK) set at 42°C until body temperature reached 42°C. Body temperature was monitored with a rectal temperature probe and maintained between 42°C and 42.5°C for 20 minutes as previously described [13]. On recovery, the animals were rehydrated with normal saline (intraperitoneal, 10 ml/kg) and left to recover for experiments 24 hours later. Control animals did not undergo this procedure.

Experimental protocol
Rats were anesthetized with diethyl ether, the femoral vein was exposed and sodium heparin (200 IU) was injected. The hearts were rapidly excised and placed in ice-cold Krebs-Henseleit buffer. The aorta was immediately attached to the Langendorff apparatus and perfused with filtered Krebs-Henseleit buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 24 mM NaHCO₃, 11 mM glucose, 1.2 mM CaCl₂, pH 7.4) at a constant pressure of 100 cm of H₂O and continuously gassed with 95% O₂/5% CO₂ at 37°C, as described previously [14]. Control and heat stressed hearts were used to study metabolic and functional changes before and following 4 hours of cardioplegic arrest as at 4°C. All hearts were initially perfused for a total of 20 minutes with assessment of cardiac function in the last 5 minutes (Fig 1).

View larger version (13K): [in this window] [in a new window]   Fig 1. Experimental protocol.

After taking readings, the hearts were arrested by infusion of 10 ml of St1 (St. Thomas’ Hospital No. 1 cardioplegic fluid). St1, supplied as a concentrate (David Bull Labs, Victoria, Australia), was diluted (1:50) in Ringer’s solution (Travenol Laboratories, Norfolk, UK) and filtered before use. After infusion of cardioplegic solution, hearts were maintained under hypothermic conditions (4°C) for 4 hours as shown in Figure 1. Hearts were then reperfused for 20 minutes, at the end of which postischemic function was evaluated. Another series of control and heat stressed hearts in all three age groups (n = 6 in each group) were freeze-clamped after 5 minutes of normoxic perfusion to obtain preischemic baseline metabolic concentrations and heat shock protein 70 (Hsp70) content.

High-performance liquid chromatography
At the end of perfusion, hearts were freeze-clamped using aluminium clamps precooled in liquid nitrogen and freeze-dried overnight. About 40 mg of left ventricle was extracted at 4°C with 0.6 M perchloric acid (25 µl/mg dry tissue ratio), centrifuged (13,000 rpm for 3 minutes at 4°C) extracts were immediately analyzed using high-performance liquid chromatography with application of the reverse phase method, using a Hewlett-Packard series 1100 chromatography (Hewlett-Packard, Reading, UK). The analytical column (150 x 4.6 mm ID) was packed with 3 µm octa-decyl silica (ODS) Hypersil (Shandon, Cheshire, UK). Chromatographic conditions were as follows: Buffer A consisted of 150 mM KH₂PO₄ containing 150 mM KCl (pH 6.0); buffer B consisted of a 15% (v/V) solution of acetonitrile in buffer A. The composition of the mobile phase changed from 0% to 100% of buffer B according to the gradient curve, as previously described [17]. Sample peaks were integrated and quantified using the HP-Chemstation chromatography data system (Hewlett-Packard).

Assessment of heat-shock protein expression
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis assessed the induction of Hsp70 and Western immunoblotting as previously described [10]. Whole heart homogenates were solubilized in 1% w/v sodium dodecyl sulfate and assayed for total protein using the Bradford assay, denatured by heating at 100°C in Laemmli buffer, and separated on 10% sodium dodecyl sulfate gels until the bromophenol blue tracking dye reached the end of the gel. Gels were equilibrated for 30 minutes in transfer buffer, and transfer of the proteins was performed for 1 hour at 500 mA. Western blots were blocked using 3% w/v non-fat dried milk (Marvel; Premier Beverages, Stafford, UK) in phosphate buffered saline containing 0.05% w/v Tween-20 for 1 hour to block nonspecific binding sites. The blots were then probed with mouse antibodies specific to inducible Hsp70 (Bioquote Ltd, York, UK) diluted to a final concentration of 1:1,000 for 1 hour. Blots were washed three times and incubated with secondary horseradish-peroxidase-conjugated rabbit anti-mouse antibody for 1 hour. The result was visualized using an enhanced chemiluminescence detection system (Amersham, Bucks, UK). Hyperfilm myoperoxidase was exposed to blots treated with enhanced chemiluminescence for 30 seconds and developed in an automatic film processor. Following enhanced chemiluminescence exposure, antibodies were removed from blots by incubation in a solution of 2% w/v sodium dodecyl sulfate, 6.25% v/v 1 M Tris-HcL (hydrochloride), pH 6.8, and 0.7% v/v 2-mercaptoethanol. Proteins were then visualized by staining with 0.01% amido black in a solution of methanol, water, and acetic acid (ratio of 45:45:10 volume/volume). Amido black-stained blots and enhanced chemiluminescence films were scanned using a Molecular Dynamics 300A laser densitometer and Hsp70 levels determined as a proportion of a known amount of Hsp70 protein standard using the Quantity One software package (PDI, Huntington, NY).

Statistics
Values are presented as means ± standard error of the mean. Statistical comparison between different age groups was performed using a two-way analysis of variance followed by Student-Newmann-Keuls test. A value of p less than 0.05 was considered a significant difference.

	Results

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Western blotting
All age groups showed the capacity to overexpress Hsp70 when subjected to heat stress. There was no significant difference in Hsp70 expression between the age groups. All C hearts across the three age groups demonstrated similar Hsp70 contents as shown in Figure 2. The HS hearts showed an increased level of expression between 2 to 3 times higher than that of the C hearts of each group.

View larger version (17K): [in this window] [in a new window]   Fig 2. Total content of Hsp70 in heat-stressed and control hearts subjected to cardioplegic arrest, 4 hours of ischemia, at 4°C and reperfusion in three age groups. Values represent the mean (± SEM).

View larger version (17K): [in this window] [in a new window]   Fig 3. Percentage change in diastolic stiffness coefficient for heat-stressed (HS) and control (C) 1-, 4-, and 16-month-old hearts. (Values represent the mean (± SEM.) *p < 0.05 versus control.

View larger version (16K): [in this window] [in a new window]   Fig 4. Percentage recovery of left ventricular developed pressure in heat-stressed and control 1-month-old hearts subjected to cardioplegic arrest, 4 hours of ischemia at 4°C, and reperfusion. Values represent the mean (± SEM). (LV = left ventricle; LVDP = left ventricular developed pressure.)

View larger version (17K): [in this window] [in a new window]   Fig 5. Percentage recovery of left ventricular developed pressure in heat-stressed and control 4-month-old hearts subjected to cardioplegic arrest, 4 hours of ischemia at 4°C, and reperfusion. Values represent the mean (± SEM). *p < 0.05 versus control. (LV = left ventricle; LVDP = left ventricular developed pressure.)

View larger version (18K): [in this window] [in a new window]   Fig 6. Percentage recovery of left ventricular developed pressure in heat-stressed and control 16-month-old hearts subjected to cardioplegic arrest, 4 hours of ischemia at 4°C, and reperfusion. Values represent the mean (± SEM). (LV = left ventricle; LVDP = left ventricular developed pressure.)

View larger version (18K): [in this window] [in a new window]   Fig 7. Percentage recovery of maximal developed pressure in heat-stressed and control hearts subjected to cardioplegic arrest, 4 hours of ischemia at 4°C, and reperfusion in different age groups. Values represent the mean (± SEM). *p < 0.05 versus control. (LVDP = left ventricular developed pressure.)

	Comment

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Heat stress proteins are a small family of gene products, which are expressed as an intracellular response to stress. The accumulation of heat stress proteins in cardiac cells was found to enhance myocardial resistance to ischemia/reperfusion injury. However, the exact mechanism of the cytoprotective effects of these proteins have yet to be fully elucidated [9].

We and others have shown the role of catalase [13] and other antioxidant factors [18] in the heat stress response. This study suggests that heat stress may also improve the antioxidant capacity of the myocardium via increase of the NAD⁺/NADP⁺ pool. We have previously reported that the concentration of NADP⁺ is increased by heat stress, however this present study suggests that this effect may also be age-dependent [11]. We have previously observed altered NAD⁺ and NADP⁺ content in hearts of different ages [3]. These differences could be the result of changes in the NAD⁺/NADP⁺ synthesis or breakdown enzyme activities.

NAD⁺ and NADP⁺ act as essential cofactors of redox reactions and serve as major electron acceptors in the oxidation of fuel molecules for the generation of energy. NAD⁺ is necessary for mitochrondrial oxidative phosphorylation and glycolysis [19]. Both nicotinamide adenine dinucleotide (reduced form) (NADH) and nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) are also involved in glutathione metabolism, a major component of the cellular antioxidant mechanisms. The activation of antioxidant enzymes by heat stress/whole-body hyperthermia has been shown to be important in the acquisition of cardioprotection [18].

We have observed no significant decline or increase in the level of Hsp70 after heat stress with age. Several investigators have documented attenuation in the heat stress response of aged tissue. Fargnoli and colleagues reported a decrease in expression of Hsp70 messenger ribonucleic acid (mRNA) and protein levels following heat shock of confluent cultures of lung and skin fibroblasts [20]. Similar findings were reported by Locke and Tanguay in the aged myocardium of rats [8]. The findings of the present study differ from the above-mentioned investigations. This apparent discrepancy may be explained by the differences in the experimental age groups. In those studies, animals of 22 months and older were used to represent the aged or senescent heart [21, 22]. This investigation used three specifically defined age groups of 1, 4, and 16 months, which represented the infant, young adult, and mature adult heart. Elderly hearts were not included in this study, as donor hearts for cardiac transplantation are never aged 60 years or over.

Deterioration in function observed following cardioplegic arrest was relatively small in the present study, confirming substantial protection of the myocardium during cardiac arrest under hypothermic conditions. Together with our earlier experience of this model, these findings suggest that small changes in function observed here cannot be attributed to necrosis, but more likely represent stunning of the myocardium. Although the pathogenesis of myocardial stunning has not been clearly defined, the generation of free radicals during reperfusion is thought to play the most significant role [23]. Heat stress mediated cardioprotection is mainly associated with damage incurred by necrosis, hence the lack of improved cardiac function in the hearts of groups I and M despite increased levels of Hsp70 [24]. The enhanced postischemic function observed in the young adult HS hearts of group Y may be attributed to the increased NAD⁺ and NADP⁺ content and, therefore, improved capacity against free-radical mediated contractile dysfunction.

We have previously shown the increased resistance of the infant heart to cold cardioplegic arrest compared to the young and mature adult heart [3]. The difference in our present findings could result from a balloon catheter being used instead of working mode for assessment of mechanical function, and the conditions of ischemia and reperfusion could be less severe. Variation in the size of the hearts makes it difficult to compare cardiac function at different age groups using the balloon technique.

In conclusion, heat stress pretreatment enhances the recovery of the young adult heart, but not of the infant or mature adult hearts following cold cardioplegic arrest, when the heart is well protected during ischemia and functional deterioration is small. This was associated with increased NAD⁺ and NADP⁺ levels and did not correlate with increased Hsp70 expression. Because both NAD⁺ and NADP⁺ could be involved in the antioxidant mechanisms in the myocardium, increased antioxidant capacity and prevention of stunning could be responsible for the observed age-dependent differences.

	References

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