Journal of Surgical Research 139, 176 –181 (2007)
doi:10.1016/j.jss.2006.07.041
Heat Stress Attenuates ATP-Depletion and pH-Decrease
During Cardioplegic Arrest
Sebastian Vogt, M.D., Ph.D.,*,1 Dirk Troitzsch, M.D.,* Hashim Abdul-Khaliq, M.D.,†
and Rainer Moosdorf, M.D., Ph.D.*
*Biomedical Research Center, Cardiovascular Research Lab and Heart Surgery, University Hospital, Philipps-University Marburg,
Marburg, Germany; and †German Heart Institute Berlin, Berlin, Germany
Submitted for publication June 21, 2006
Background. The capacity of heat stress induction to
improve myocardial tolerance against ischemia is well
known. We investigated cardiac energy metabolism
after hsp 72 ⴙ/73 ⴙ induction in isolated perfused neonatal rabbit hearts subjected to prolonged cold cardioplegic ischemia.
Methods. Hearts from neonatal rabbits were excised,
isolated perfused and arrested by 2-h cold cardioplegic ischemia. Rectal temperature of eight neonatal
rabbits was raised to 42.0 to 42.5°C for heat shock protein expression in a whole body water bath for 15 min
before the onset of arrest. Another set of eight rabbits
without hyperthermia pretreatment served as control.
Recovery of left ventricle function was assessed by aortic flow, cardiac output, and max dP/dt. Status of highenergy phosphates was measured by 31phosphorus nuclear magnetic resonance-spectroscopy.
Results. Immunoblot analysis revealed clear hsp
72ⴙ/73ⴙ induction after a brief period of systemic hyperthermia. Heat stress pretreatment resulted in a better recovery of left ventricular function (aortic flow and
cardiac output improvement P < 0.05, max dP/dt P <
0.01) than in controls at 60 min after reperfusion. During
ischemia and reperfusion, myocardial energy metabolism was better preserved in hearts after hsp induction
as a consequence of increased ␥-, ␣-, and -ATP as well as
phosphocreatine-values over controls. The ischemiainduced pH-decrease was attenuated.
Conclusion. These data contribute to the evidence of
heat stress mediated beneficial effects on functional
myocardial recovery and improved cardiac energy
metabolism after prolonged cold cardioplegic ischemia. More importantly, the attenuation of ischemic
1
To whom correspondence and reprint requests should be addressed at Klinik für Herzchirurgie, Universitätsklinikum, PhilippsUniversität Marburg, Baldinger Strae 1, D-35043 Marburg, Germany. E-mail: vogts@med.uni-marburg.de.
0022-4804/07 $32.00
© 2007 Elsevier Inc. All rights reserved.
pH reduction and better restoration suggest an involvement of mitochondrial membrane potential
alterations. © 2007 Elsevier Inc. All rights reserved.
Key Words: heat shock proteins; high energy phosphates.
INTRODUCTION
Induction of Heat Shock-Proteins (hsp) results in a
cytoprotective effect. Different forms of acute and
chronic stress induce production of abnormal and misfolded cellular proteins, which is neutralized by overexpression of these molecular chaperones, even in intact
organs (e.g., in the heart). Our group demonstrated an
improved functional recovery of the cardioplegic arrested
ischemic hearts, when hsp 72 and 73 were induced after
short term hyperthermia prior to ischemia [1]. These
mechanisms, which confer protection against myocardial ischemia-reperfusion injury, have beneficial influence on high-energy-phosphate (HEP) metabolism [2].
Improved mechanical recovery after heat shock was
associated with an decreased rate of HEP depletion
and increased recovery of ATP and phosphocreatine
levels during reperfusion. In an excellent paper Sammut et al. hypothesized an increased mitochondrial
respiratory enzyme activity caused by hyperthermic
stress. This study provides evidence for heat-stressmediated enhancement of mitochondrial energetic capacity [3]. Especially, the enzyme activities of respiratory chain complexes I, IV, and V were significantly
increased. Only little is known about the simultaneous
time course of the myocardial pH-value and HEP content monitored during heat shock protected ischemiareperfusion-injury. This circumstance is caused by
technical problems. Until now, the only method to estimate HEP content and pH on parallel is the 31phos-
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VOGT ET AL.: HEAT SHOCK PROTEINS AND HIGH ENERGY PHOSPHATES
phorus nuclear magnetic resonance ( 31P-NMR) analysis. However, there are limitations in the assay.
Jayakumar reports about difficulties to quantitate
changes in intracellular inorganic 31P-NMR signals because of very broad peaks in the spectra [2]. Additionally, the relationship between pH and the chemical
shift of inorganic phosphate at 4°C was not well established. In our experimental setting we reinvestigate
the HEP-alterations depending on hsp 72/73 induction
with special focus on the time course of pH-value during ischemia and reperfusion injury.
MATERIALS AND METHODS
Procedures for all animal were carried out in conformance with
the Guidelines for the Care and Use of Laboratory Animals published
by the National Institutes of Health. Hearts from neonatal New
Zealand White rabbits (in each group n ⫽ 8, body weight 0.16 – 0.2
kg, age 8 –10 days) were perfused using a Langendorff perfusion
apparatus. Heat shock treatment was identical to the setting described [1]. After being anesthetized a group of rabbits were gently
heat stress treated with a temperature-controlled water bath and
electric heating pad (heating rate of 0.5– 0.7°C/min, rectal temperature 42.0 – 42.5°C for 15 min, time of recovery 1 h). Hearts were
rapidly excised and a cannula was placed into the aorta for perfusion
in the non-recirculating Langendorff technique. The isolated working heart system with split circulation set up (Hugo Sachs Elektronik, March-Hugstetten, Germany) provides a physiological left
ventricular pressure-volume work for neonatal rabbits and function
mode was performed. Krebs-Henseleit buffer solution were used for
heart perfusion. Cardioplegic arrest was induced by commercially
available crystalloid cardioplegic solution (modified “Eppendorf cardioplegia”, Fresenius AG, Bad Hamburg, Germany).
The experimental protocol was performed in two groups of eight
neonatal rabbit hearts and identical with previous experiments [1].
Cold cardioplegic arrest (control group) was compared with cold
cardioplegic arrest after whole-body hyperthermia and heat shock
protein induction. Perfusion for 15 min in Langendorff technique and
equilibration in “working heart mode” for 20 min were required for
steady state conditions. Global hypothermic arrest was induced by
177
left atrial inflow/aortic outflow-clamping and retrograde single-dose
infusion of cold crystalloid Eppendorf cardioplegic solution (4°C,
volume 20 mL, aortic pressure between 30 and 50 mmHg, myocardial temperature 12–14°C, duration 2 h). Hearts were reperfused
for 15 min in Langendorff mode and converted back to the working
heart mode by restoring left atrial and aortic flows.
Heat-shock protein analysis of constitutive hsp73 ⫹ and inducable
hsp72 ⫹ was performed by myocardial biopsies from left heart tissue
before the onset of cold cardioplegic arrest and after 60-min reperfusion. Evaluation with Western blot analysis and densitometry by a
gel doc-reader (Software Quantity one; Bio-Rad Laboratories, Hercules, CA).
The status of myocardial HEP were measured by time-resolved
31
P-NMR spectroscopy according [4]. Whole perfusion system were
placed into a superconducting magnet (4.7 Tesla; Biospec, Bruker,
Germany). A rectangular surface coil adopted to the phophorussignal (81 Mhz) was positioned upon the hearts. Signals for ATPpeaks (␣, , and ␥), Phosphocreatine, and inorganic phosphorus were
registered in equal time intervals. Intracellular pH was calculated
using the calibrated chemical shift of the inorganic phosphate peak
in the spectra. Data are plotted as mean ⫾ SEM. Statistical analysis
within the groups were performed using paired Student’s t-test and
between the two groups using the unpaired Student’s with a MannWhitney rank sum test as a non-parametric second step. Differences
were considered statistically significant at P ⬍ 0.05 and marked in
the figures with asterisks.
RESULTS
Immunoblot analysis of the biopsies from the left
ventricles revealed high-level expression of heat shock
proteins (hsp 72 ⫹/73 ⫹, Fig. 1–3). We found this expression in a time dependent manner. For the investigations in this report a heat stress of 15 min at 42.0 to
42.5°C was chosen and an optimal time of normothermic recovery of 60 min followed. After recovery the
hearts were harvested and hemodynamic baseline data
were evaluated. Values were equal in each group (Table 1).
When cardioplegic arrest was induced, after 60 min
reperfusion the aortic flow and cardiac output were
FIG. 1. Densitometric analysis of hsp 72 ⫹/73 ⫹ expression detected by Western blot technique of control and the group after hyperthermic
stress (n ⫽ 8 in each group). Data were evaluated before ischemia and after reperfusion. When animals were exposed to hyperthermic stress
a clear expression of hsp 72 ⫹/73 ⫹ is seen.
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JOURNAL OF SURGICAL RESEARCH: VOL. 139, NO. 2, MAY 15, 2007
TABLE 1
Hemodynamic Data of Both Groups After Myocardial
Recovery at 60 min of Reperfusion
LV max. dp/dt (mmHg/s)
Baseline
60 min reperfusion
Aortic flow (mL/min)
Baseline
60 min reperfusion
Cardiac output (mL/min)
Baseline
60 min reperfusion
Heart rate (beats/min)
Baseline
60 min reperfusion
FIG. 2. Examples for immuno-dot-analysis of 72 kDa-hspexpression after 15 min heat shock exposure (see “Materials and
Methods”) at different time intervals for subsequent recovery: mon
Ab: anti-hsp 70; clones brm-22 and h 5147, Sigma-Aldrich; detection
by horseradish-peroxidase-staining and 4-chloro-1-naphtol (SigmaAldrich). With increased time of recovery, the hsp-expression is
found to be increased. After optimization protocol of post heat shock
hemodynamic data (data not shown) further investigations were
carried out with 60-min recovery time.
increased by 25.3% and 25.8%, respectively (P ⬍ 0.05).
Contractility (dP/dt max) was reduced in the control
group by 54.9% (P ⬍ 0.01). Heart rate between the
groups and after reperfusion showed no differences. In
31
P-NMR-analysis, initial data of HEP and pH were
equal. During cardioplegic arrest different reductions
in HEP was found. At final ischemia, higher levels of
phosphocreatine (Fig. 4) and ATP-derivates were detected after heat shock protein induction (P ⬍ 0.05,
Fig. 4). After reperfusion, increased ATP peak areas
and phosphocreatine values were measured, which returned to baseline. In this group, values were superior
to the controls (P ⬍ 0.05). These data underline the
FIG. 3. Example for comparative immuno-dot-analysis of hsp
72 ⫹-expression between heat shock compromised group and sham
group. Two animals were sacrificed initially and investigated for
hsp-expression before starting heat shock exposure (control *).
Control group*
HT-group*
P-value
970 ⫾ 100
550 ⫾ 70
970 ⫾ 200
900 ⫾ 150
NS
⬍0.01
33.3 ⫾ 2.1
23.2 ⫾ 3.4
34.5 ⫾ 2.2
30.1 ⫾ 3.2
NS
⬍0.05
35.8 ⫾ 3.0
23.8 ⫾ 2.7
37.1 ⫾ 2.6
33.2 ⫾ 2.4
NS
⬍0.05
210 ⫾ 8
205 ⫾ 9
208 ⫾ 10
219 ⫾ 13
NS
NS
* Values as mean ⫾ SD; control group (n ⫽ 8, without heat shock
pre-treatment) and HT-group (n ⫽ 8, with heat pre-treatment); LV
max. dp/dt, maximum rate of left ventricular pressure rise; NS, no
significance.
heat shock response mediated beneficial effect on preservation of HEP during cold cardioplegic ischemia and
restoration of myocardial energy metabolism during
reperfusion. Parallel to these findings, the pH-decrease
during ischemia is attenuated and pH-values returned to
baseline data in reperfusion (P ⬍ 0.05, Fig. 5).
DISCUSSION
Molecular chaperones such as the heat shock family
of stress proteins participate in different cellular processes, especially in cytoprotection. There is a stressinducible network, both at the transcriptional and
posttranscriptional levels. At the present time we discuss their clinical significance in physiological and
pathophysiological conditions such as ischemic preconditioning and aging [5]. Expression of hsps is an evolutionary relict and found in all species, but the hsp
genes vary in the pattern of their expression. It correlates with the resistance to stress [6]. Up-regulation of
protein expression establishes a defense system to
maintain cellular protein homeostasis and tolerance to
ischemia, hypoxia, and radical oxygen species responsible for cardiovascular disorders [7]. Currie and coworkers suggest a therapeutic value for the heat-shock
response by its association with enhanced post-ischemic
ventricular recovery and limitation of tissue necrosis during coronary occlusion and reperfusion [8, 9]. Although
they found an absence of changes in energy metabolism
[10] the increase of heat shock protein 70 mRNA in rat
heart exposed to oxidant stress suggested an involvement of mitochondrial respiratory enzyme function
[11]. Further investigations by Sammut and coworkers recover contribution of heat stress to the enhancement of cardiac mitochondrial complex activity
VOGT ET AL.: HEAT SHOCK PROTEINS AND HIGH ENERGY PHOSPHATES
179
FIG. 4. Results of the 31P-NMR spectroscopy of both groups during perfusion, cold cardioplegic ischemia and reperfusion: on the base an
example of ECG-triggered 31P-NMR-spectrum is documented. The relating time courses of each signal for HEP is plotted above. With ongoing
ischemia a reduction of the myocardial HEP contents was registered, the restoration of these compounds begins during reperfusion.
Differences between hsp 72 ⫹/73 ⫹ induction and control were found in the depth of HEP reduction in ischemia and restoration of them in
reperfusion. These data suggest beneficial effects on myocardial energy metabolism in or after ischemia.
[3, 12]. Our group contributes to the importance of
cardiac heat shock protection by confirmation of clear
correlation between heat shock protein induction and
improved myocardial performance after cardioplegic
arrest [1]. In this setting we addressed the enhanced
protection of myocardial energy metabolism by heat
stress pretreatment in newborn rabbit hearts. Concluding the results, we found higher myocardial HEP
levels (␥-, ␣-, and -ATP, phosphocreatine) at final
ischemia when hsp 72 and hsp 73 were induced (Fig. 1).
After reperfusion the ATP- and phosphocreatine-peak
areas are increased in the hsp 72 ⫹/73 ⫹ group over
control and reveal improved myocardial HEP-contents
by heat shock (Fig. 4). More importantly, the fall of
ischemic induced intracellular myocardial pH-values
was attenuated (Fig. 5).
These data correlate with previous experiments of
Jayakumar and co-workers [2] and confirm the beneficial changes of heat stress in HEP metabolism. In their
study it was difficult to define the intracellular inorganic 31P-NMR signals and additionally the chemical
shift of inorganic phosphate at low temperatures was
not established. We found an acute course of inorganic
phosphate peak sufficient for intracellular pH calculation. Moreover, Kost worked out pH standardization protocols for 31P-NMR-spectroscopy at low temperatures
[13]. Based on his measurements Bock et al. and Binzoni
et al. defined a pH change of ⫺0.015 to ⫺0.016 pH
units/°C [14, 15] that seems to be negligable for our investigation. Therefore, we point out an attanuation of
pH-decrease in cardioplegic induced myocardial arrest.
The importance of pH-decrease is very often discussed
in the context of severity of ischemic tissue damage.
Otherwise, the myocardial pH-value is relevant for the
proton motive force, which itself is determined by the
mitochondrial membrane potential. Three enzyme complexes of the mitochondrial respiratory chain (NADHubiquinone oxidoreductase ⫽ complex I, cytochrome c
reductase ⫽ complex III, and cytochrome c oxidase ⫽
complex VI) are electron transport-driven proton pumps
that transfer reducing equivalents from nutrition to
dioxygen accompanied by transfer of protons across the
inner mitochondrial membrane, and generate a proton
motive force. The ATP-Synthase (complex V) in mito-
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FIG. 5. Evaluation of myocardial pH-values during the experimental protocol. The data were calculated from the chemical shift
between the signals of inorganic phosphate to phosphocreatine. After
cold cardioplegic ischemia in both groups a pH reduction was found.
In contrast to control, after hsp 72 ⫹/73 ⫹-induction the pH reduction
was attenuated and in reperfusion the pH returned back to physiological values.
chondria uses this driving element to produce ATP
from ADP and inorganic phosphate. When an increased activity of cytochrome c oxidase is known in
case of hypoxia [16] on the one hand and the mitochondrial membrane potential drops sharply in parallel
with mechanical cardiac performance in ischemia [17],
then our findings can only be interpreted as an involvement of hsp 72 ⫹/73 ⫹ in its function as chaperones for
cytochrome c oxidase-translation. Cytochrome c oxidase is the terminal enzyme of the mitochondrial respiratory chain which transfers electrons from cytochrome c to molecular oxygen, coupled with the uptake
of protons from the matrix forming water and translocating protons across the mitochondrial inner membrane. It is considered terminating enzyme complex for
electron transfer and preservation of mitochondrial membrane potantial driving the activity of ATP-synthase and
ATP-production. Corresponding to this point Piel et al.
found increased ventricular cytochrome oxidase I
mRNA and protein content in case of hypoxia. The
Heme a,a3 content, as essential element of cytochrome
c oxidase, and turnover number was also increased
[16]. In our investigation the elevated myocardial level
of HEP when hsp were induced, underline the thesis.
Therefore, it seems to be evident that the beneficial
action of hsp expression is caused by induction of protein
translation and internalization [18] of the apoenzyme
into the mitochondrium resulting in higher enzyme activities of cytochrome c oxidase. If other mitochondrial
enzyme complexes are involved in this mechanism still
remains open. It is suggested that this effect also concerns the level of gene expression and contains a pivotal importance in delayed ischemic preconditioning
[19]. At least, we have to extent our view on other
further hsp species: Selective temporal induction of
different hsp (e.g., 70, 60, and 32) might cooperate and
participate in the regulative defense program of mitochondrial enzyme complex induction [20, 21]. Moreover, hsp 90 is involved in up-regulation of NOsynthase-activity and NO production, which itself
participate in post-ischemic myocardial oxygenation
and oxygen consumption by modulation of mitochondrial electron transport [22]. Occasionally, even these
mechanisms make oxygen radical generation in reperfusion of the ischemic heart more understandable [23].
Considering clinical relevance, the pH-dependence of the
mitochondrial membrane potential and HEP-production
is directly linked with the parameters of contractility.
Hence, the report of Hata et al. in 1994, about a rapid
correction of acidosis resulting in a stunned myocardium, could be explained by our results [24]. Therefore,
consequences for strategies in cardiac resuscitation
and intensive care might be expected.
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