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Am J Physiol Heart Circ Physiol 280: H426-H433, 2001;
0363-6135/01 $5.00
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Vol. 280, Issue 1, H426-H433, January 2001

Activation of the heat shock response: relationship to energy metabolites. A 31P NMR study in rat hearts

J. Chang1, A. A. Knowlton2, F. Xu1, and J. S. Wasser1

1 Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station 77843; and 2 Baylor College of Medicine and Veterans Affairs Medical Center, Houston, TX 77030


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Heat shock factor (HSF), the transcription factor for the heat shock proteins, is activated by cardiac ischemia, but the mechanism of activation is unknown. Ischemia is accompanied by changes in the energy state and acid-base conditions. We hypothesized that decreased ATP and/or intracellular pH (pHi) might activate HSF. To test this hypothesis, we perfused rat hearts within an NMR spectrometer. NMR data showed that after 6.5, 13, and 20 min of ischemia, ATP dropped to 62.7, 23.1, and 6.9% of the control level, and pHi was 6.16, 5.94, and 5.79, respectively. Reperfusion after ischemia partially restored ATP levels, and this was associated with greater activation of HSF1. HSF1 was also activated after 6.5 min of ischemia. Activation of HSF1 was less after 13 min of ischemia and barely detectable after 20 min of ischemia. In conclusion, 1) a moderate decrease in intracellular ATP correlates with activation of HSF1 in the heart; and 2) a severe depletion in ATP correlates with an attenuation in HSF1 activation, and the restoration of ATP leads to greater activation of HSF1, suggesting that a critical ATP level is required for activation of HSF1.

heat shock factor 1; intracellular pH; ATP; free energy of ATP hydrolysis; ischemia; reperfusion; rat heart; cardiac energetics


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ORGANISMS RESPOND TO SUBLETHAL STRESSES such as ischemia, heat, and hypoxia with the synthesis of heat shock proteins (HSPs) (8, 14, 20, 21). HSPs are a protective response to stress. Mammalian hearts pretreated with heat show enhanced resistance to ischemia (12, 46). HSPs protect native proteins from denaturation, act as molecular chaperones, and refold denatured proteins (5, 39, 44). The overexpression of several different HSPs can protect the heart from stresses such as hypoxia and ischemia-reperfusion and even improve postischemic recovery of the high-energy phosphate pool and correction of metabolic acidosis (35).

Expression of heat shock genes is controlled by the heat shock transcription factor (HSF). At least four different HSFs have been described, but HSF1 appears to be the one predominantly activated by stresses that injure the cell. HSF binds to heat shock element (HSE) (29, 30, 37). Multiple HSEs are present in the promoter of the HSP genes. When cells are exposed to stress, HSF converts from its inactive monomeric form in the cytoplasm to a functional trimeric form that moves to the nucleus, is phosphorylated, and specifically binds to the HSE, which is the first step to initiate HSP synthesis (25, 27).

Mammalian myocardium is highly sensitive to ischemia or hypoxia. In the heart, ischemia and hypoxia are characterized by a rapid depletion of high-energy phosphate compounds with intracellular acidosis and a decrease in cardiac function. Although ischemia and/or hypoxia are known to increase HSP mRNA and protein, the biochemical factors that trigger activation of HSF in the heart are undefined (7, 13, 21, 26, 31). Changes in intracellular high-energy phosphate compounds and pH may be the potential triggers for the heat shock response. Several studies (4, 6, 28, 45) have shown a relationship between the changes in intracellular pH (pHi) and ATP and the heat shock response in cell lines and the rat kidney. No one has demonstrated an association between cellular energy state and/or intracellular acid-base condition and the initiation of stress response in the intact heart. The purpose in the present study was to determine the relationship between myocardial energetics and activation of the heat shock response in isolated rat hearts. We hypothesized that ATP acted as a regulator of the initiation of the heat shock response in the isolated perfused rat heart during ischemia and reperfusion. We found that a moderate decrease in intracellular ATP correlated with the activation of HSF1 and that a severe depletion in ATP was accompanied by attenuation in HSF1 activity.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Male Sprague-Dawley rats (mean body wt 228 ± 9 g, n = 43) were obtained from commercial suppliers (Harlan, Indianapolis, IN) and maintained on standard chow and water ad libitum. The experimental protocol was approved by the Animal Welfare Committee of Texas A&M University.

Langendorff perfusion preparation. Rats were heparinized (500 U ip) and anesthetized with pentobarbital sodium (110 mg/kg ip). The chest was opened, and the heart removed and placed in ice-cold Ringer solution [(in mM) 120.0 NaCl, 4.7 KCl, 18.0 NaHCO3, 11.0 glucose, 1.5 MgSO4, and 2.5 CaCl2, equilibrated with 95% O2-5% CO2 gas]. Retrograde perfusion (Langendorff) was quickly established with a perfusion pressure of 95~100 cmH2O. The pH and temperature of the perfusate were 7.4 and 37°C, respectively. We inserted a fluid-filled latex balloon into the left ventricle via the left atrium and connected it to a pressure transducer to monitor cardiac hemodynamic function. The initial left ventricle diastolic pressure was adjusted to 6 mmHg by adjusting balloon volume.

Hemodynamic data. We collected cardiac hemodynamic data at 30-s intervals throughout the experiments with the use of a pressure transducer connected to a computerized data-acquisition system [Micron Millenium notebook computer, LabView software and a PCMCIA analog-to-digital card (National Instrument, Austin, TX)]. With this system, data for positive and negative maximal rates of pressure development (±dP/dtmax), maximum and minimum ventricular developed pressure (Pmax and Pmin, respectively), and heart rate were generated.

NMR perfusion system. The heart was mounted vertically in a 20-mm NMR tube filled with Ringer solution equilibrated with 95% O2-5% CO2 gas. We inserted a Tygon tube into the NMR tube for gas equilibration of the solution surrounding the heart and perfused the hearts via a gravity-flow system from a water-jacked reservoir. The level of Ringer solution in the reservoir was maintained constant at 95 cmH2O and equilibrated with 95% O2-5% CO2 gas.

NMR spectroscopy. The perfused hearts were placed in the bore of a high-field NMR spectrometer (Bruker WB300, field strength 8.2 Tesla). Phosphorus NMR (31P NMR) spectra of the hearts were collected using a 20-mm broadband variable temperature probe. Probe temperature was maintained at 37 ± 0.3°C.

Phosphorus spectra were acquired at 121 MHz with the use of the following acquisition parameters: recycle delay, 1 s; pulse angle, 90° (60 µs); spectral width, 3,649.635 Hz; data table size, 4 k; number scans, 128; receiver gain, 800; and acquisition time, 0.561152 s. Total time required to generate one signal with an adequate signal-to-noise ratio was 3.25 min.

Data handling and analysis. We analyzed the spectral data using NUTS (version 5.097, 1995), an NMR data processing program (Acorn NMR, San Rafael, CA). We set line broadening to 15 Hz. The relative concentrations of metabolites were obtained by integration of peak areas after appropriate baseline correction.

The pHi was calculated from the chemical shift of inorganic phosphate (Pi) relative to phosphocreatine (PCr) with the use of the following equation for the rat heart from the study of Brindle et al. (9): pHi = 6.72 + log[(sigma  - 3.27)/(5.69 - sigma )], where sigma  is the chemical shift of Pi relative to PCr (in parts per million).

The free energy of ATP hydrolysis (Delta GATP) was calculated from the following series of equations (3, 19, 36), where R is the gas constant, and T is temperature (in K)
&Dgr;G<SUB>ATP</SUB><IT>=&Dgr;G</IT><SUP>o</SUP><SUB>obs[ATP]</SUB><IT>+RT×</IT>ln([ADP][P<SUB>i</SUB>]<IT>/</IT>[ATP]) (1)
Because we cannot directly obtain [ADP] from NMR spectra, the [ADP] was calculated from the creatine kinase reaction (see Eq. 2), which is assumed to be close to equilibrium throughout the experiment.
[ADP]<IT>=</IT>[ATP][Cr]<IT>/</IT>[PCr][H<SUP><IT>+</IT></SUP>]<IT>K</IT><SUB>ck</SUB> (2)
where Cr is creatine and Kck is the equilibrium constant for the creatine kinase reaction.

We calculated Delta GATP (in kJ/mol) (Eq. 3) by combining Eqs. 1 and 2
&Dgr;G<SUB>ATP</SUB><IT>=</IT>−<IT>30.5+8.31×310 </IT>ln([Cr][P<SUB>i</SUB>]<IT>/</IT>[PCr][H<IT>+</IT>]<IT>K</IT><SUB>ck</SUB>) (3)
where Kck = 1.66 × 109 M-1, Delta Gobs[ATP]0= -30.5 kJ/mol, R = 8.31 J/mol K, and T = 310 K.

The total tissue content of Cr (PCr + Cr, creatine pool 25.44 mM) was assumed to remain constant during experiments. Therefore, control rat heart cytosolic concentrations are Pi = 0.88 mM, PCr = 14.16 mM, and Cr =11.28 mM, respectively (3, 19, 41).

Gel mobility shift assay. Essentially, the method of Benjamin et al. was followed with modifications as previously described (7, 33, 42). Ventricles were rapidly dissected and freeze-clamped in liquid nitrogen. Tissue samples (0.2 g) were suspended in 1 ml of lysis buffer [20 mM HEPES (pH 7.9), 1.66 M KCl, 1.5 mM MgCl2, 0.4 mM sodium orthovanadate, 0.4 mM NaF, 0.5 mM phenylmethylsulfonyl fluoride, 1.0 mM dithiothreitol, 0.2 mM EDTA, 20% (vol/vol) glycerol, 2 µg/ml leupeptin, 10 µg/ml pepstatin, and 0.01 U/ml aprotinin], homogenized on ice, and then processed as previously described (42). Samples were aliquoted and stored at -80°C until analyzed. Two single-stranded complementary HSE fragment oligonucleotides containing the 5'-nGAAn-3' repeats were synthesized (5'-CTAGAAGCTTCTAGAAGCTTCTAG-3') and then annealed and end labeled with [gamma -32P]ATP (0.6 µCi/µl).

Samples of the extract (30 µg) were incubated with 32P-labeled HSE at room temperature for 30 min and loaded onto a 4% nondenaturing acrylamide gel. Cold competition and supershift studies were carried out as previously described (42). The gel was dried and exposed to an X-ray film at -70°C for 8-16 h or overnight. The films were scanned, and the density of each sample was measured (SigmaGel, Jandel). To compare the differences among the pooled densitometry from different X-ray films, the densitometry of each sample was normalized by the average densitometry of control samples on the same film.

Experimental protocol. The heart was perfused in the magnet bore for approximately a 20- to 30-min stabilization period. We then subjected the hearts to one of the following experimental protocols, as summarized in Fig. 1: control hearts were perfused for 32.5 min (n = 6 hearts); ischemic hearts were perfused for 13 min followed by 6.5, 13, or 20 min of ischemia (n = 6 hearts/group); and ischemia-reperfusion hearts were perfused 13 min and then underwent 13 min of ischemia/16 min of reperfusion (n = 6 hearts) or 20 min of ischemia/10 min of reperfusion (n = 5 hearts). To keep the total perfusion time of the two ischemia-reperfusion groups consistent, hearts were reperfused for different times. Total time required to generate each signal with an adequate signal-to-noise ratio was 3.25 min. Therefore, we designed experimental time points based on this signal requirement period, for example, the 6.5- and 13-min ischemia protocols are two and four times 3.25 min. For severe ischemia, we added a 20-min ischemia protocol. At the end of the experiment, the heart was rapidly removed from the NMR bore and snap frozen in liquid nitrogen. For comparison, two hearts were perfused for 13 min at 43°C as a heat shock treatment.


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  		Fig. 1.   This diagram summarizes the experimental time points. Stabilization perfusion, ischemia, and reperfusion were as indicated in diagram. The control group was perfused for 32.5 min. Hearts in the 3 ischemia groups were perfused for 13 min followed by 6.5, 13, and 20 min of global ischemia, respectively. For the 2 groups of ischemia-reperfusion, hearts were perfused 13 min followed by 13 min of ischemia/16 min of reperfusion and 20 min of ischemia/10 min of reperfusion, respectively. n, number of rat hearts used in each group.

Statistical analysis. Hemodynamic data in individual groups were analyzed by an ANOVA of repeated measures. Data for HSF activity comparisons were analyzed by Kruskal-Wallis one-way analysis of variance (ANOVA) on ranks followed by a Dunn's test. All statistical analyses were performed by SigmaStat software (version 2.03, SPSS, Chicago, IL). We considered P < 0.05 as statistically significant. Data were presented as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Myocardial ATP. ATP level remained stable throughout the perfusion in the control experimental group and during all control periods in the ischemia and ischemia-reperfusion experimental groups (Fig. 2). ATP levels fell to 62.7 and 23.1% of control values after 6.5 min and 13 min of ischemia, respectively (P < 0.05). After 20 min of ischemia, ATP fell further to 6.9% of the control level. ATP levels among 6.5-, 13-, and 20-min ischemia groups were also significantly different (P < 0.05). We observed the different degrees of recovery of ATP in two separate reperfusion protocols. At the end of reperfusion, the ATP level was 68.8% of control values in 13-min ischemia/16-min reperfusion hearts, which was significantly improved from end ischemia (P < 0.05). After 20 min of ischemia/10 min of reperfusion, we found that ATP was only 33.6% of control values, a partial recovery compared with end ischemia (P < 0.05). But the two ATP recovery levels were still significantly lower than control levels (P < 0.05).


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  		Fig. 2.   Myocardial ATP changes during ischemia and ischemia (isch)-reperfusion (reperf). For simplicity, data are shown at 3-min intervals instead of NMR 3.25-min acquisition time. *P < 0.05 compared with last control time point; **P < 0.05 compared with last ischemic time point.

Myocardial PCr. The PCr level remained stable during the control perfusions. PCr levels fell to 8.3% of control and 0% of control values after 6.5 and13 min of ischemia, respectively (P < 0.05, Fig. 3). We observed a recovery of PCr in two reperfusion experiments. After 13 min of ischemia/16 min of reperfusion, we found that PCr returned to 92.9% of control values [P = not significant (NS)]. However, PCr was only 20.6% of control values after 20 min of ischemia followed by 10 min of reperfusion, which was significantly different from both control and end ischemia (P < 0.05).


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  		Fig. 3.   Myocardial phosphocreatine (PCr) changes during ischemia and ischemia-reperfusion. *P < 0.05 compared with last control time point; **P < 0.05 compared with last ischemic time point.

Myocardial free energy of ATP hydrolysis. Delta GATP was calculated with the use of Eq. 3 as discussed in METHODS. Delta GATP levels were stable throughout control perfusions. The values ranged from -61.8 to -65.2 kJ/mol (Fig. 4). Delta GATP decreased to -54.3 kJ/mol (86.8% of control values) and -43.5 kJ/mol (69.8% of control values) after 6.5 (P < 0.05) and 13 min of ischemia (P < 0.05), respectively. By the end of 20 min of ischemia, Delta GATP declined to -50.5 kJ/mol (81% of control values, P < 0.05). Delta GATP returned to -62.89 kJ/mol (100.3% of control values, P = ns) after 13 min of ischemia/16 min of reperfusion. With 20 min of ischemia/10 min of reperfusion, Delta GATP recovered to -54.1 kJ/mol (86.7% of control values), which was significantly different from the end ischemia (P < 0.05) but still lower than control (P < 0.05).


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  		Fig. 4.   Myocardial free energy of ATP hydrolysis changes during ischemia and ischemia-reperfusion. *P < 0.05 compared with last control time point; **P < 0.05 compared with last ischemic time point.

Intracellular pH. pHi levels were unchanged throughout control perfusion. pHi fell from 7.0 to 6.16, 5.94, and 5.79 (P < 0.05 for all compared with control) at the end of 6.5, 13, and 20 min of ischemia, respectively (Fig. 5). pHi levels returned to 6.88 and 6.98 after 13 min of ischemia/16 min of reperfusion and 20 min of ischemia/10 min of reperfusion, respectively (P = NS compared with control for both values).


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  		Fig. 5.   Myocardial intracellular pH (pHi) changes during ischemia and ischemia-reperfusion. *P < 0.05 compared with last control time point; **P < 0.05 compared with last ischemic time point.

Heat shock transcription factor activation. With the gel mobility shift assay, we assessed HSF activation for each experimental protocol. As shown in a representative gel shift (Fig. 6), we observed that HSF was activated after 6.5 min of ischemia (B; left and right lanes) compared with control (A, left and right lanes). Less activation of HSF was observed after 13 min of ischemia (C, left and right lanes) compared with 6.5 min of ischemia, and a further attenuation of HSF activation was observed after 20 min of ischemia (D, left and right lanes). With reperfusion, 13 min of ischemia followed by 16 min of reperfusion led to the strongest HSF activity (E, left and right lanes). With 20 min of ischemia/10 min of reperfusion (F, left and right lanes), HSF activity was similar to that observed after 6.5 min of ischemia alone (B, left and right lanes) or perhaps slightly higher. Figure 7 summarizes the degree of HSF activation with different protocols. HSF activation was greater in the 6.5 min of ischemia and two ischemia-reperfusion groups compared with controls (P < 0.05). All of our control hearts showed some basal HSF activity. Several controls were done to show the specificity of the gel shifts. As shown in Fig. 8, the addition of an excess cold competing oligonucleotide (B; left and right lanes) abolished the shift seen with the same ischemia-reperfusion sample (A; left and right lanes). Anti-HSF1 and HSF2 antibodies were used to detect which HSF was activated by ischemia. As shown in Fig. 8, addition of anti-HSF1 (C; left and right lanes) resulted in a supershift, whereas anti-HSF2 had no effect (D; left and right lanes). Similar results were observed with heat-shocked rat hearts (F-I, left and right lanes).


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  		Fig. 6.   Gel mobility shift assay showing activation of heat shock factor (HSF) in ischemia, ischemia-reperfusion, and heat-shocked rat hearts. A (left and right lanes): control perfusion; B (left and right lanes): 6.5 min of ischemia; C (left and right lanes): 13 min of ischemia; D (left and right lanes): 20 min of ischemia; E (left and right lanes): 13 min of ischemia/16 min of reperfusion; F (left and right lanes): 20 min of ischemia/10 min of reperfusion; G: perfusion with 43°C perfusate for heat shock of 13 min. NS, nonspecific binding. Arrows indicate shift bands: HSF and NS bands, respectively.



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  		Fig. 7.   HSF activity in control, ischemia, and ischemia-reperfusion rat hearts. Data are expressed as a percentage of control average intensity. To compare HSF activities of hearts at different gel shift X-ray films, the intensities of all samples within each film were normalized (divided) by the average intensity of the control hearts at the same film. n, heart number. *P < 0.05 compared with control.



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  		Fig. 8.   Cold competition and supershift assay for ischemia-reperfusion and heat-shocked rat hearts. A-D (left and right lanes of all): ischemia-reperfusion samples (A: ischemia-reperfusion samples; B: addition of excess cold heat shock element oligonucleotide and positive HSF shift bands were competed; C: antibody to HSF1 showing positive supershift; D: antibody to HSF2 showing negative supershift). E-I (left and right lanes of all): heat-shocked samples (E: heat-shocked samples; F and H: antibody to HSF1 showing positive supershift; G and I: antibody to HSF2 showing negative supershift). Arrows indicate supershift HSF1, HSF shift, and NS bands, respectively.

Hemodynamics. As shown in Table 1, all measured hemodynamic variables were stable during the control perfusions. Typical hemodynamic changes were observed with ischemia and ischemia-reperfusion. With ischemia, heart rate, +dP/dt max, and Pmax decreased, whereas -dP/dt max and Pmin increased (P < 0.05 compared with control for all groups), consistent with loss of both systolic and diastolic function. By the end of 13 min of ischemia/16 min of reperfusion, heart rate, Pmax, -dP/dt max, and Pmin returned to control levels, but +dP/dt max showed only partial recovery (P < 0.05 vs control). We did not observe hemodynamic recovery after 20 min of ischemia/10 min of reperfusion. The lack of hemodynamic recovery correlated with severe ATP depletion. With reperfusion, all parameters showed partial recovery in 13 min of ischemia/16 min of reperfusion hearts, but no recovery was observed in the 20-min ischemia/10-min reperfusion group.

                              
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  		Table 1.   Hemodynamic variables


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

HSP70 and heat shock response. It is thought that HSP70 and ATP levels are involved in the regulation of the heat shock response (1, 4, 6, 24, 27, 40), but the exact relationship of metabolic state to HSF activation is not well defined. Under unstressed conditions, HSP70 binds to HSF and represses HSF. HSP70 also interacts with unfolded proteins, functioning as a molecular chaperone, and releases them with hydrolysis of ATP (16). It has been suggested that the release of HSP70 from its substrates might be inhibited with depletion of ATP (4-6). ATP depletion also amplifies the concentration of aggregated or denatured proteins; this then further decreases the free HSP70 pool, which leads to dissociation of the HSP70-HSF complex and initiates heat shock response (6). However, the release of HSF from HSP70 is also ATP dependent (1): a decrease in the ATP level should decrease the free HSP70 pool and inhibit the dissociation of the HSP70-HSF complex. Failure to release HSF from HSP70 would then result in no activation of HSF and no increase in HSPs. This ATP/HSF paradox would explain the present finding that a moderate reduction in ATP correlates with activation of HSF1 and a severe depletion in ATP correlates with attenuation in HSF1 activation.

Cardiac energy metabolites and activation of HSF. 31P NMR spectroscopy permits continuous monitoring of cardiac energy metabolites, including ATP, PCr, and pHi, and therefore facilitates exploration of the relationship between cardiac energy metabolites, pHi, and the initiation of heat shock response. We observed stepwise reductions of ATP in hearts subject to 6.5, 13, and 20 min of ischemia. Our results showed that a 37% reduction in the ATP level was correlated with activation of HSF1. This result supports the hypothesis that the rapid decrease in [ATP] during ischemia could inhibit the efficiency of HSP70 releasing from its substrate proteins and lead to heat shock response (5, 6). Meanwhile, more denatured or malfolded proteins would accumulate in the hearts during ischemia (32) and bind to HSP70 (32). All of these would promote HSP70 dissociation from HSF; however, the severe depletion of ATP paradoxically attenuated HSF1 activation. We found that hearts in which ATP levels were depleted by 77% (13 min of ischemia) had less HSF activation than in those with ATP levels depleted by 37% (6.5 min of ischemia). The activity of HSF was almost abolished with a 93% reduction in ATP (20 min of ischemia).

These results support the hypothesis that the process of HSF activation is ATP dependent (1, 6). Mildly reduced ATP levels activate HSF, but a greater reduction in ATP may block the dissociation of the HSP70-HSF complex and attenuate the initiation of heat shock response. Finally, when ATP is depleted to a critical level, there is insufficient ATP available to release HSP70 from HSF, and heat shock response is blocked. Activation of HSF was observed again when we reperfused hearts, which partially restored high-energy phosphate levels. We propose that intracellular ATP is a complex regulator of the heat shock response during ischemia and ischemia-reperfusion in rat hearts. Other adenosine phosphates, such as ADP and AMP, have proportionately large increases according to the equilibrium constant for adenylate kinase during ischemia, which may lead to activation of some important protein kinases, such as protein kinase A and protein kinase C, but their roles in the regulation of heat shock response are uncertain (or contradictory) (11, 22, 34).

Nishizawa et al. (33) reported that HSF activation was reached a peak at 6 min of global myocardial ischemia and was abolished after 20 min of ischemia; however, they did not measure high-energy phosphates or pH. Benjamin et al. (6) reported that a decrease in ATP to 30% of control induced the DNA-binding activity of HSF in rotenone-treated myogenic cells. In renal ischemia, a fall in cortical ATP to 35~50% of control values resulted in activation of HSF (45). A similar relationship between cell energy depletion and HSF activation was demonstrated in central nervous system astrocytes (17). In a cell-free extract, addition of enough ATP could reduce the binding activity of HSF to HSE, and removal of ATP could restore HSF-HSE bindings (38). Both our observations and those of others support that the ATP level regulates HSF activity. We suggest that this relation between HSF and ATP is complex: a moderate reduction in ATP initiates HSF1 activation, but HSF1 binding activity is attenuated with severe ATP depletion, and the restoration of ATP leads to the activation of HSF1 again in rat hearts in vitro.

Delta GATP is the available energy that can be derived from ATP hydrolysis under given conditions. Changes in the intracellular environment, such as pHi, PCr, and Pi, could cause changes of Delta GATP and influence cardiac function. Because Kammermeier et al. (19) found that the reduction of Delta GATP is closely associated with early cardiac hypoxic failure without changes in the ATP level, we wonder whether the change in Delta GATP without changes in the ATP level correlated with HSF activation. In our experiments, we observed reductions in both Delta GATP and ATP during ischemia. It could be that we either missed the differential period between Delta GATP and ATP or it did not occur under our conditions. Our control Delta GATP values of approximately -62 to -63 kJ/mol were in the range of other published papers (3, 41). The Delta GATP data in ischemic hearts may implicate a high Delta GATP requirement for the activation of the heat shock response. The 20 min of ischemia Delta GATP was higher than that of 13 min of ischemia in our protocol. One possible reason for this was that the PCr level was below the NMR detection limit during late ischemia for some of the hearts, and we replaced zero with the group mean to make the calculations. But this technical limitation should not have any impact on our conclusions because HSF activation occurred as early as 6 min of ischemia. Other biochemical assays or methods, such as high-performance liquid chromatography, may help to define the status of Delta GATP in the late ischemic period.

pHi and activation HSF. The role of pHi in the activation of HSF is not clear. Typical changes in pHi were observed in our ischemia and ischemia-reperfusion protocols. During ischemia, pHi decreased dramatically. By the end of reperfusion, pHi in both reperfusion groups had completely recovered to control levels. We did not test whether the change of pHi alone had any effects on HSF activation in our experimental design. In the present study, HSF activation was observed when pHi was at both control (at two end-reperfusion time points) and acidic levels. Therefore, we cannot identify whether pHi is a causal factor, but we also cannot exclude its additive effect. Severe acidosis may have a synergistic effect on HSF activation via an indirect pathway. Benjamin et al. (6) reported that a pHi of 6.7 alone without ATP changes failed to induce HSF-DNA binding activity in C2C12 cells. A similar absence of effect was observed in Drosophila melanogaster salivary glands when pHi dropped to 6.84 (15). However, a very steep reduction in pHi (6.0) induced HSF activation in HeLa cells, which the authors attributed to conformational changes in proteins caused by the severe acidosis (28). With 13 and 20 min of ischemia, similar changes to the pH changes occurred in the perfused hearts, and these hearts had less to no activation of HSF compared with 6.5 min of ischemia. This severely acidotic state correlated with very low ATP levels.

Reperfusion and HSF activation. It is not surprising that reperfusion, which generates oxygen-derived free radicals, caused the same or greater vigorous activation of HSF than ischemia alone, as shown in Figs. 7 and 8. Experiments by other investigators have demonstrated that oxidative stress can induce the heat shock response (10). Endothelial cells subjected to oxidative stress increased HSP70 mRNA and HSP70 protein (2, 18). Rat hearts perfused with oxidants had increased HSP70 mRNA (23). A similar high HSF activity after reperfusion was also observed in rat hearts (33) and rat livers (43). We found that the activity of HSF in 20-min ischemia/10-min reperfusion hearts where the ATP level is only ~33.6% of control values is similar to the activity observed in 6-min ischemia alone hearts (ATP is 62.7% of control level), indicating that oxidative stress may have additive or synergistic effect on HSF activation. Interestingly, further recovery in the ATP level to 68.8% of control values with reperfusion (after 13 min of ischemia/16 min of reperfusion) led to even higher HSF activation, which supports our conclusion again that a critical ATP level is important for activation of HSF. The strong activation of HSF after reperfusion may reflect both the restoration in the ATP level and oxidative stress factors.

Our results support the hypothesis that intracellular ATP levels act as a regulator of the heat shock response in the isolated perfused rat heart during ischemia and ischemia-reperfusion. A moderate reduction in ATP correlates with activation of HSF1, a severe depletion in ATP correlates with an attenuation in HSF1 activation, and the restoration of ATP leads to greater activation of HSF1 again.


    ACKNOWLEDGEMENTS

The authors thank Yanding Gao and Neal Stolowich for technical assistance with the NMR spectrometer.


    FOOTNOTES

This study was supported in part by National Heart, Lung, and Blood Institute Grant HL-58515 (to A. A. Knowlton).

Present address of J. Chang: Div. of Cardiology, Dept. of Internal Medicine, Univ. of Texas Houston Medical School, 6431 Fannin, Houston, TX 77030.

Address for reprint requests and other correspondence: A. A. Knowlton, Cardiology Research, Baylor College of Medicine and VA Medical Center, 151C, 2002 Holcombe, Houston, TX 77030 (E-mail: annek{at}bcm.tmc.edu).

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.

Received 6 March 2000; accepted in final form 1 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 280(1):H426-H433


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