INTRODUCTION

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COLD-INDUCED STRESS is a phenomenon associated with an increase in inducible heat shock protein expression in various tissues (31, 32). Particularly in brown adipose tissue, cold-induced stress or hypothermia also induces mitochondrial biogenesis (20, 29, 30). At the transcriptional level this is characterized by coordinated increases in expression of nuclear- and mitochondrial-encoded genes regulating mitochondrial membrane proteins (28). Although this signaling has been well characterized in brown adipose fat, it remains relatively unexplored in other mammalian tissues. This is surprising because stress responses in the heart secondary to heat shock or ischemia have been a major focus of investigation with respect to enhancement of tissue resistance to subsequent ischemia (14, 21, 33, 35, 40). Furthermore, hypothermia either singly or accompanied by cardioplegia is regularly employed in myocardial protection during heart surgery. The operative mechanism is a putative reduction in myocardial high-energy phosphate utilization during ischemia (15). Hypothermic induction of a stress response, when applied before ischemia, has not been investigated in the heart.

In this study, we propose that even a mild, relatively brief exposure to hypothermia can improve resistance to a subsequent prolonged ischemic insult and that this response is associated with an alteration in signaling for mitochondrial biogenesis. As hypothermia can produce alterations in myocardial performance and energy utilization even during subsequent rewarming, cardioplegia was used during ischemia to negate this effect. Studies were performed in a perfused heart preparation, a model that has been used frequently to characterize other stress-related phenomena, including heat shock (33, 35, 40). Cardiac function and ATP preservation were measured to demonstrate that improved ischemic resistance occurred in this model. Additionally, Northern blot analyses of expression of an inducible heat shock protein (HSP70-1) gene (10, 16, 23), as well as genes regulating major constitutive mitochondrial membrane proteins [adenine nucleotide translocator isoform 1 (ANT₁) and -subunit F₁-adenosinetriphosphatase (-F₁-ATPase)] (11, 36, 44, 47) were performed to index signaling for mitochondrial biogenesis and cold adaptation.

	MATERIALS AND METHODS

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Preparation of Isolated Heart

The analog signals were continuously recorded on a pressurized ink-chart recorder (Gould, Cleveland, OH) and an on-line computer (Macintosh, Biopac Analog Signal Acquisition System). To characterize cardiac function, left ventricular developed pressure (LVDP) is defined as peak systolic pressure (PSP) minus end-diastolic pressure (EDP). The product of heart rate and LVDP [pressure-rate product (PRP), mmHg/min] was calculated to provide an estimate of myocardial work. Myocardial O₂ consumption (MO₂) was calculated as CF × [(Pa_O2  Pv_O2) × (c_O2/760)], where CF is coronary flow (ml · min¹ · g wet tissue¹), (Pa_O2  Pv_O2) is the difference in the partial pressure of O₂ (mmHg) between perfusate and coronary effluent, and c_O2 is the Bunsen solubility coefficient of O₂ in perfusate at 37°C (22.7 µl O₂ · atm¹ · ml perfusate¹) (37, 38). O₂ extraction was calculated as MO₂ divided by the O₂ content in the perfusate. Wet weight of the heart was determined at the conclusion of each experiment after trimming the great vessels and fat and blot drying the heart with nine-layer cotton gauze. Procedures followed were in accordance with institutional and National Institutes of Health guidelines.

Lactate, pH, and CO₂ Measurements

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ATP and Metabolites

RNA Isolation

Northern blot analysis. For Northern blot analysis, 15 µg of RNA were denatured and electrophoresed in a 1% formaldehyde agarose gel, transferred to a nitrocellulose transfer membrane (Micron Separations, Westboro, MA), and cross-linked to the membrane with short-wave UV cross linker. The prehybridizing and hybridizing solutions contained 50% formamide, 1× Denhardt's solution, 6× sodium chloride-sodium phosphate-EDTA, and 1% sodium dodecyl sulfate (SDS). cDNA probes were labeled with [³²P]dCTP by random primer extension (PRIME-IT II, Stratagene, La Jolla, CA) and added to the hybridizing solution to a specific activity. Hybridization was carried out at 42°C for 18 h. The blots were then washed several times with a final wash in 1× standard sodium citrate and 0.1% SDS at 65°C. The relative amount of mRNAs was evaluated using a PhosphorImager (model 400S, Molecular Dynamics, Sunnyvale, CA). The same size area at each band was taken to measure the intensity and the same size area at the closest upstream position of each band was taken as the background of the image, respectively. The blots were exposed on Kodak X-o-mat film (Eastman Kodak, Rochester, NY) at 70°C. RNA loading was normalized by comparison to that of 28S ribosomal RNA. Adenine nucleotide translocase isoform 1 (ANT₁) mRNA levels were detected using a 1.4-kilobase (kb) cDNA fragment cloned from the human skeletal muscle [American Type Culture Collection (ATCC), Rockville, MD] (11, 36, 44, 47). -F₁-ATPase mRNA levels were detected using a 1.8-kb cDNA fragment cloned from human HeLa cell line (ATCC) (11, 36, 46, 47). HSP70-1 mRNA levels were detected using a 1.7-kb cDNA fragment cloned from human hippocampus (ATCC) (10, 16, 23, 35). To compare different mRNA levels in the same myocardial sample, we analyzed aliquots of 15 µg total RNA from the myocardium by means of sequentially reprobing the membranes with 28S, ANT₁, -F₁-ATPase, and HSP70-1 cDNA probes.

Experimental Protocols

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Myocardial temperature. Ischemia starts at 0 min and ends at 120 min, then reperfusion follows. Before ischemia, the temperature is stable at 37°C in control hearts (C) but the temperature decreases to about 31°C progressively from baseline of 37°C in hypothermia-exposed hearts (H; P < 0.05, n = 10/group). Application of cardioplegia transiently decreases the temperature. There is no significant difference in temperature between groups during ischemia and reperfusion.

Myocardial temperatures were not measured routinely in each experiment to avoid potential problems associated with traumatic introduction of needle-mounted temperature probes. In parallel experiments, myocardial temperature was monitored with a Khuri regional tissue temperature monitor (Vascular Technology) to determine changes in the myocardial temperature profile with use of our standard experimental protocol in 10 hearts of the control and hypothermia-treated groups (Fig. 1).

Seventy-seven hearts were used for the experimental protocols. During several points in the protocol, some hearts were quickly frozen in liquid N₂ for measurement of metabolites related to energy utilization. The hearts fell into the following groups: control; after 20 min of hypothermia; after 120 min of ischemia in control and preischemic hypothermic groups; and after 15 min of reperfusion in both groups. These groups are indicated in Tables 1 and 2. Samples for RNA measurement were obtained after 20 min of either normothermic or hypothermic perfusion and at 45 min of reperfusion in control and preischemic hypothermic groups. Myocardial samples were also taken from five hearts in situ for a nonperfused control group.

Statistical Analysis

	RESULTS

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Functional Parameters

Effect of hypothermia on baseline. Heart weight and left ventricular balloon volume were similar in the control (7.98 ± 0.57 g and 1.5 ± 0.05 ml, respectively) and hypothermia groups (7.94 ± 0.61 g and 1.6 ± 0.06 ml, respectively). Under baseline conditions, there were no significant differences between the control and hypothermia groups in EDP, LVDP, LV ±dP/dt_max, heart rate, PRP, CF, MO₂, and O₂ extraction. Hemodynamic results are summarized in Table 1. Twenty minutes of hypothermia decreased LVDP, LV +dP/dt_max, LV dP/dt_max, heart rate, PRP, coronary flow, and MO₂, but the left ventricular EDP increased. No significant changes in these parameters occurred in the control group (Table 1) during 20 min of perfusion at 37°C.

Functional recovery during reperfusion. In Table 1, the data demonstrate that the preischemic hypothermia provided superior functional recovery compared with that observed in control hearts. The preischemic hypothermic hearts were characterized by higher LVDPs, higher LV dP/dt_max values, and lower EDP values.

Ischemic contracture. As noted in MATERIALS AND METHODS, a specific balloon volume was adjusted and then maintained throughout the protocol, allowing comparisons of LVP under constant end-diastolic volume. After CP solution was injected, the LVP was always near 0 mmHg. The beginning of ischemic contracture was defined by an initial rise of >2 mmHg in LVP. Ischemic contracture started significantly later in preischemic hypothermic hearts (97.5 ± 3.6 min) than in control hearts (67.3 ± 3.3 min).

Energy Metabolism

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Effects of preischemic hypothermia on accumulation of lactate, CO2, and myocardial pH. Differences in CO2 content (dCO2) or in lactate concentration (dLactate) between coronary effluent and inflow perfusates (n = 23 in C group, n = 18 in H group), as well as myocardial pH (n = 6/group), were determined as described in MATERIALS AND METHODS.

Table 2 summarizes data relevant to ATP and its principal metabolites. Myocardial ATP, ADP, AMP, adenosine, total nondiffusible nucleotide (TNN), and total diffusible nucleoside (TDN) concentrations were not affected by exposure to hypothermia. Although ATP was depleted substantially regardless of preischemic temperature at end ischemia, levels were significantly higher in the hearts exposed to hypothermia. AMP levels were similarly increased in both groups. Adenosine and TDN concentrations were elevated in both groups but to a lesser extent in the hypothermic group (P < 0.05). At 15 min of reperfusion, AMP returned to levels similar to baseline in both groups. Levels of ATP, ADP, and TNN were significantly higher and TDN was lower in hearts exposed to hypothermia.

-F₁-ATPase, ANT₁, and HSP70-1 mRNAs

View larger version (81K): [in this window] [in a new window]   Fig. 3.   A representative Northern blot. Each lane was loaded with 15 µg total RNA from ventricular myocardium and probed specifically for 28S, adenine nucleotide translocase isoform 1 (ANT1), -subunit of F1-adenosinetriphosphatase (-F1-ATPase), and inducible heat shock protein (HSP70-1). Samples were taken from hearts in situ (I), hearts at control baseline (Bc) and after 20 min of hypothermia (Bh), and hearts after 2 h of ischemia and 45 min of reperfusion in C and H groups (see text).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Steady-state mRNA levels. All transcript levels are relative to 28S band intensity and normalized to Bc lane. A: RNA levels for nuclear-encoded genes for ANT1 and -F1-ATPase. Hypothermic exposure yields no differences in these levels (Bc vs. Bh; n = 6/group). Ischemia and reperfusion result in decreased levels in C hearts (n = 9) but no significant change in H hearts (n = 5). B: mRNA levels for HSP70-1. Exposure to hypothermia does not alter these levels. Ischemia and reperfusion yield a substantial increase in these levels in C hearts and an even greater increase in H hearts.

Hypothermia did not alter the pattern of expression for any of these genes (Fig. 4) within 20 min of exposure. Ischemia followed by reperfusion did affect expression of the three genes. Postischemic steady-state levels of -F₁-ATPase and ANT₁ mRNA were markedly diminished in controls (Fig. 4A). Hypothermic exposure ameliorated this subsequent postischemic decrease in steady-state levels of mRNA for the two mitochondrial proteins (Fig. 4A). HSP70-1 mRNA steady-state levels were elevated as a result of ischemia and reperfusion (Fig. 4B). However, the increase in HSP70-1 mRNA was substantially higher after reperfusion in the group that had been exposed to prior hypothermia.

	DISCUSSION

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Cardioplegic arrest in this perfused heart model removes discrepancies in myocardial performance between the experimental groups during early ischemia. Thus differences in high-energy phosphate depletion should not be due to discrepancies in contractile energy cost during ischemia. Furthermore, repeated application of cold cardioplegia and rewarming at 34°C produced comparable temperature responses between the groups (Fig. 1), negating another possible source of discrepancy in ATP utilization. Hypothermia results in alterations in contractile state and rates of energy expenditure in the intact heart. A reduction in O₂ consumption during preischemic hypothermia in this study exemplifies a decrease in ATP synthesis and utilization. The reductions in LVDP and LV dP/dt_max indicate that there is a concomitant decrease in contractile state during mild hypothermia. To our knowledge, studies evaluating contractile function and the specific effects of mild hypothermia in the isolated perfused rabbit heart have not been previously performed and are thus not available for comparison. Although the reduction in O₂ consumption during hypothermia is probably related principally to the decrease in contractile performance, an energy-sparing effect may also contribute to the decrease in ATP depletion during subsequent ischemia. Monroe et al. (34) demonstrated that under conditions of constant peak systolic pressure, lowering temperature from 38 to 32°C resulted in a greater area under the pressure-volume curve with no significant change in O₂ consumption. This implies that at lower temperature more cardiac work can be achieved for the same energy cost and that ATP utilization is more efficient. Accordingly, ATP depletion and accumulation of its degradation products are greater in the control hearts during ischemia. These are indications that a smaller imbalance between ATP production and utilization occurs in the cold-stressed group. Net CO₂ and lactate accumulation during ischemia reflect, respectively, aerobic and anaerobic ATP production. These values support, although do not prove, the notion that the greater ATP imbalance in the control group is due to greater high-energy phosphate utilization and not to decreased ATP production. A reduction in ATP utilization initiated during hypothermia might continue or perhaps may influence high-energy phosphate utilization during the subsequent ischemic period.

Restoration of ATP levels after reperfusion is higher in the hypothermia-exposed hearts. Improved ATP repletion after reperfusion also occurs consistently after other forms of preischemic stress exposure. However, modes of stress are inconsistent with respect to ATP preservation during the ischemic episode. For example, ischemic preconditioning produces either no effect or a reduction in ATP preservation, depending on the model under study (22, 46). This implies that the mechanisms that control ATP preservation and restoration may vary according to the mode of stress applied. Decreased purine loss or increased salvage during reperfusion in hypothermic exposed hearts probably contributes to improved ATP restoration because ATP degradation was decreased during ischemia. Maintenance of mitochondrial membrane function may also enhance ATP restoration after ischemia (22, 46).

The mechanisms responsible for decreased ATP utilization and their contribution to preservation of contractile function in the hypothermic hearts after ischemia remain highly speculative. Disturbances in excitation-contraction coupling and contractile apparatus, generated by free radical formation and Ca²⁺ overload, prevail as proposed causes of myocardial damage during ischemia and reperfusion (5). Profound hypothermia at temperatures <15°C and of several hours duration exacerbates cellular Ca²⁺ overload and induces peroxide and free radical formation (4, 25, 43). However, the effects of mild or short periods of hypothermia on these processes have not been examined. Conceivably, brief, relatively mild decreases in temperature can induce rapid enzyme changes consistent with cold adaptation, which would improve Ca²⁺ and free radical handling during a subsequent ischemic episode. Circumstantial evidence supporting this notion is provided in studies of tissue from cold-adapted or hibernating species (8, 12). Cold-adapted tissue demonstrates high rates of cardiac sarcoplasmic reticulum Ca²⁺ uptake (27), which presumably accounts for low cytosolic Ca²⁺ levels noted in hibernating animals. Reduction in cytosolic Ca²⁺ would effectively reduce activation of a variety of ATPases as well as catalytic proteases (1, 3, 6). Similarly, exposure to low environmental temperatures induces increases in antioxidant enzyme levels in a variety of tissues. Thus cold-adapted tissues display mechanisms that combat deleterious effects of oxidative stress present during extreme cold and/or ischemia.

Changes in heat shock protein and mitochondrial membrane protein gene expression demonstrate that an adaptive process has occurred. The isolated perfused heart has frequently been used as a model for characterization of heat shock protein gene expression, although questions concerning the appropriateness of this model in such investigations have been raised. Knowlton et al. (21) demonstrated that even a single ventricular stretch or hampering of systolic shortening resulted in a rapid increase in HSP70 expression in erythrocyte-perfused rabbit heart. However, this finding could not be reproduced by Myrmel et al. (35), who found no change in HSP70 expression in rat hearts after isovolumic perfusion at 65 mmHg for 30 min. Delcayre et al. (9) did find that augmentation of coronary perfusion pressure in beating or KCl-arrested isolated hearts perfused for 2 h produced increases in protein synthesis as well as HSP68 mRNAs. The discrepancies between these studies may be related to level and duration of perfusion pressure. In the present protocol, perfusion was performed with constant aortic pressure and isovolume, thus minimizing alterations in systolic shortening and diastolic stretch. Accordingly, this procedure resulted in no detectable increase in expression of HSP70-1 over 50 min in either normothermic or hypothermic hearts before ischemia.

This is the first report of a hypothermia-induced alteration in heat shock protein gene expression after ischemia and reperfusion in hearts. A primary objective was to determine if an alteration in steady-state transcript levels could be induced by hypothermia-induced stress before ischemia. Cold stress is known to influence induction of heat shock proteins in various tissues (13, 19, 31). The response is highly variable and can extend from induction to suppression according to tissue and/or temperature (13, 19, 31). Furthermore, the heat shock protein response to cold occurs in some tissues only on recovery to normal temperature (26). Studies of cold-induced alterations in expression of the HSP70 family of proteins and their RNAs after ischemia have been reported in brain only (24, 42). These studies demonstrate the extreme temperature variability of the heat shock response to cold. Although deep hypothermia (15°C) represses ischemic induction of HSP72 mRNA relative to ischemia at 23°C in pig brain, even lower expression occurs at 29°C (42). The mechanism of these temperature-dependent responses to cold stress in brain has not been elucidated (42).

Mitochondrial biogenesis can be initiated by cold-induced stress in brown adipose tissues (20, 31, 32). In several studies this has been characterized by increases in steady-state mRNAs for the uncoupling protein, a specific component of the mitochondrial membrane in brown adipose tissue. Other studies (28) have demonstrated coordinated gene expression for the adenine nucleotide translocator and the -subunit for F₁-ATPase with the uncoupling protein. This is consistent with reports that imply that the -F₁-ATPase subunit in particular can be used as a reporter gene for mitochondrial biogenesis (2, 17, 18, 24, 26, 41). The cold-induced stress response in brown adipose tissue, including HSP70 and uncoupling protein induction, can be specifically blocked through -adrenoreceptor antagonism, implying that signaling is mediated by norepinephrine (30). These responses, including activation and regulation of mitochondrial membrane protein genes by cold-induced stress, have not been studied in other tissues. The coordinate expression of HSP70 with the uncoupling protein in brown adipose tissue compelled us to investigate whether alterations in steady-state levels of transcripts from genes regulating these important constitutive mitochondrial proteins could be induced by hypothermia followed by ischemia in the heart. A decrease in transcript levels for the -F₁-ATPase subunit gene has previously been documented by Heads et al. (14) after prolonged ischemic preconditioning, ischemia, and reperfusion protocol in rabbit myocardium (14). In this study a decrease in steady-state mRNA levels for both genes controlling these constitutive mitochondrial membrane proteins after reperfusion was detected only in the normothermic group. This finding implies that hypothermia directly or indirectly induced either an increase in transcription or a stabilization of these mRNAs.

In summary, these data suggest that exposure to a brief period of mild hypothermia improves resistance to injury during a subsequent period of prolonged ischemia with cardioplegic arrest. This response is associated with maintenance of steady-state mRNA levels for the adenine nucleotide translocator and the -F₁-ATPase subunit, as well as an elevation in expression of HSP70-1. These results imply that hypothermia induces an adaptive response, which is apparent in the postischemic period. These signals are associated with mitochondrial biogenesis in other tissues and are usually followed by an increase in mitochondrial protein synthesis consistent with cold adaptation. Because of the brief time course of events in this study, it is unlikely that increased protein synthesis occurred rapidly enough to effect the preservation of function and ATP associated with the elevated signal. However, reduction in injury may contribute to preservation of signaling for mitochondrial biogenesis. Mitochondrial dysfunction and damage have been documented in various models of myocardial O₂ deprivation and repletion, and recovery is related to content of proteins participating in oxidative phosphorylation (15). Factors regulating synthesis of these proteins are complex and involve coordination of both nuclear and mitochondrial genes (45). Intuitively, maintenance of signaling for mitochondrial biogenesis and protein synthesis seems necessary for recovery of respiratory function after injury. As relatively little is known concerning the role of mitochondrial biogenesis in myocardial recovery, the implications of the hypothermia-induced preservation in signaling have not been elucidated but remain an area for future investigations.