Download Activation of Heat-Shock Factor by Stretch-Activated

Activation of Heat-Shock Factor by Stretch-Activated
Channels in Rat Hearts
Jiang Chang, MD, PhD; Jeremy S. Wasser, PhD; Richard N.M. Cornelussen, PhD; A.A. Knowlton, MD
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Background—Previously, we have observed that the isolated, erythrocyte-perfused rabbit heart has increased levels of
heat-shock protein (HSP) 72 after a mild mechanical stress. We hypothesized that stretch-activated ion channels (SACs)
mediated this increase.
Methods and Results—To test this hypothesis, we subjected isolated, perfused rat hearts to mechanical stretch. Gel
mobility shift assay showed that heat-shock factor (HSF) was activated in hearts with mechanical stretch, but not in
controls. Supershift experiments demonstrated that HSF1 was the transcription factor. Northern blots revealed the
concomitant increase in HSP72 mRNA in stretched rat hearts. In a separate set of experiments, gadolinium, an inhibitor
of SACs, was added to the perfusate. Gadolinium inhibited the activation of HSF and decreased HSP72 mRNA level.
Because gadolinium can inhibit both SACs and L-type calcium channels, we perfused a group of hearts with diltiazem,
a specific L-type calcium channel blocker, to eliminate the involvement of L-type calcium channels. Diltiazem failed
to inhibit the activation of HSF.
Conclusions—Stretch in the rat heart results in activation of HSF1 and an increase in HSP72 mRNA through SACs. This
represents a novel mechanism of HSF activation and may be an important cardiac signaling pathway for hemodynamic
stress. (Circulation. 2001;104:209-214.)
Key Words: stretch 䡲 ion channels 䡲 calcium 䡲 proteins
C
ells from all organisms respond to stress by synthesizing
heat-shock, or stress, proteins (HSPs). HSPs are a group
of highly conserved proteins classified by their molecular
weights. Many studies have demonstrated that HSPs play an
important role in the protection of stressed organisms.1– 4
The regulation of heat-shock response is mediated by ⱖ1
cytosolic proteins known as heat-shock factors (HSFs) that
interact with a specific regulatory element, the heat-shock
element, in the promoter regions of the HSP gene.5 HSF
activation is one of the earliest responses observed in many
different cells exposed to stresses, such as ischemia, hypoxia,
hyperthermia, and mechanical stretch.6 –9
Mechanical stretch and pressure overload have been shown
to induce HSP72 expression in a variety of different cells and
tissues.8,10 –12 Several studies reported that stretch resulted in
HSF1 activation and HSP72 expression in rat aorta,8,13,14 but
few data are available for the heart.9,15 Previously, we
observed that even a single myocardial stretch can cause
increased expression of HSP72 in the isolated erythrocyteperfused rabbit heart.15 The mechanism is unknown, but
several research groups have postulated that stretch-activated
ion channels (SACs) may act as mechanotransducers to
mediate stretch-induced gene expression.16,17 As yet, however, there is no direct evidence that stretch-induced HSP72
expression is mediated by SACs.
Therefore, the purpose of this study was 3-fold. First, we
used 2 different standard isolated heart perfusion models
(Langendorff and working) to demonstrate that stretch alone
was sufficient to induce the heat-shock response in rat hearts.
These models eliminated any influence of circulating neural
and/or humoral factors. Second, we tested the hypothesis that
the induction of HSP72 mRNA was via the activation of HSF,
specifically HSF1. Third, we used gadolinium, an SAC
blocker, to test the hypothesis that the stretch-induced heatshock response was mediated by SACs.
Methods
Langendorff Perfusion
Male Sprague-Dawley rats (228⫾9 g, n⫽43; Harlan Inc, Indianapolis, Ind) were heparinized and anesthetized with sodium pentobarbital. The heart was removed and placed in ice-cold Ringer’s
solution (mmol/L: NaCl 120.0, KCl 4.7, NaHCO3 18.0, glucose 11.0,
MgSO4 1.5, and CaCl2 2.5, equilibrated with 95% O2/5% CO2).
Retrograde perfusion (Langendorff) was quickly established with a
perfusion pressure of 95 to 100 cm H2O. The pH and temperature of
Received January 18, 2001; revision received March 14, 2001; accepted March 21, 2001.
From Texas A&M University, Department of Veterinary Physiology and Pharmacology, College Station (J.C., J.S.W.), and Baylor College of Medicine
and VA Medical Center, Houston (R.N.M.C., A.A.K.), Tex. Dr Chang is now at the Department of Cell Biology, Baylor College of Medicine, Houston,
Tex. Dr Cornelussen is now at the Cardiovascular Research Institute Maastricht, Department of Physiology, Maastricht University, Maastricht, the
Netherlands.
Guest Editor for this article was Derek M. Yellon, PhD, DSc, Hon MRCP, University College London Hospitals and Medical School, London, UK.
Correspondence to Dr A.A. Knowlton, Cardiology Research, Baylor College of Medicine, VA Medical Center 151C, 2002 Holcombe, Houston, TX
77030. E-mail [email protected]
© 2001 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
209
210
Circulation
July 10, 2001
the perfusate were 7.4 and 37°C, respectively. A PE90 tubing was
inserted into the left ventricle (LV) and through the apex for
thebesian drainage. We placed a fluid-filled latex balloon into the LV
via the left atrium and connected it to a transducer to monitor
pressures. Initial LV end-diastolic pressure (LVEDP) was adjusted to
10 mm Hg by adjustment of balloon volume. The heart was
submerged in a water-jacketed constant-temperature chamber that
contained Ringer’s solution equilibrated with 95% O2/5% CO2.
The protocol was approved by the Animal Welfare Committee of
Texas A&M University.
Working Heart Perfusion
The heart was perfused by the Langendorff method first without
balloon or ventricular drain. The left atrium was connected to a
second cannula and perfused at a preload pressure of 10 to 15
cm H2O. Afterload was set at 50 to 70 cm H2O by adjustment of the
height of the aortic outflow line above the heart. A compliance
chamber was added to the aortic outflow line. The heart was perfused
in the working mode (ejecting LV).
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Maximum Ventricular Developed Pressure
To monitor cardiac contraction, we collected cardiac maximum
ventricular developed pressure (Pmax) throughout the experiments
with a pressure transducer connected to a computerized dataacquisition system (Labview).
Gel Mobility Shift Assay
As described previously, a single-strand self-complementary oligonucleotide containing the 5⬘-nGAAn-3⬘ repeats was synthesized
(5⬘-CTAGAAGCTTCTAGAAGCTTCTAG-3⬘), annealed, and endlabeled with [␥-32P]ATP.18,19 Because HSF is normally present in the
cell in an inactive form, we were able to use whole-cell lysates for
our studies. Samples were processed as previously described. Cold
competition and supershift studies were done as previously
described.
Figure 2. A, GMSA in Langendorff-perfused hearts (B lanes) and
nonperfused rat hearts (A lanes). B, Effects of varying stretch
(LVEDP) on HSF activation in Langendorff-perfused hearts:
10 mm Hg LVEDP (lane B), 7 to 8 mm Hg LVEDP (lane C), and
5 mm Hg LVEDP (lane D). Heart in lane B showed strongest
HSF activity shift band, followed by lanes C and D, indicating
that different amount of stretch can influence HSF activity. Lane
A, nonperfused heart. NS indicates nonspecific binding; HSF,
activation with band shift.
RNA Isolation and Northern Blot Analysis
After each experiment, heart ventricles were rapidly dissected and
freeze-clamped in liquid nitrogen. Total RNA isolation was performed with a STAT-60 kit (Tel-test, Inc). Northern blotting was
performed as described previously.6,15
Experimental Protocols
All hearts were perfused for a 20-minute stabilization period followed by 1 of 7, 60-minute experimental protocols (Figure 1). As a
positive control, 2 working hearts were perfused at 43°C for 13
minutes.
Statistical Analysis
Data were analyzed by 1-way ANOVA, followed by the StudentNewman-Keuls test (SigmaStat software, SPSS Inc). A value of
P⬍0.05 was considered significant. Data are presented as
mean⫾SEM.
Results
HSF Activation Was Observed in Langendorff
Stretched Hearts but Not in Working Hearts
Figure 1. Experimental protocols for Langendorff and working
perfusions. All hearts were perfused for 20-minute stabilization
period. Hearts in Langendorff mode were perfused with gadolinium (SAC and L-type calcium channel blocker) or diltiazem (specific L-type calcium channel blocker) for 60 minutes. To impose
mechanical stretch in working perfusion mode, aortic cannula
with small diameter (⬍1 mm ID) was used to mimic aortic stenosis. Two hearts were perfused at 43°C for 13 minutes as positive controls. n⫽hearts/group.
Previously, we observed that a mechanical stretch induced the
expression of HSP7215 in the rabbit heart. In the present
study, we again demonstrated that mechanical stretch, which
included stretch from insertion of the apical drain and
placement of the ventricular balloon, initiated the heat-shock
response in the Langendorff perfusion. The gel mobility shift
assays (GMSAs) showed that this stretch activated HSF
(Figure 2A). In pilot experiments, the effects of different
amounts of stretch on HSF activity were examined. This was
done by varying the initial LVEDP in Langendorff perfusions. As shown in Figure 2B, an LVEDP of 10 mm Hg
correlated with the strongest HSF activation (lane B in Figure
2B), followed by an LVEDP of 7 to 8 mm Hg (lane C in
Figure 2B). Only minimal HSF activation occurred in the
Chang et al
Heat-Shock Factor and Stretch-Activated Channels
211
Figure 3. A, GMSA in Langendorff (A lanes),
working perfusion with aortic stenosis (B lanes),
and working perfusion (C lanes). B, Cold competition and supershift assay for stretched rat
hearts. A lanes, Langendorff perfusion; B lanes,
addition of excess cold heat-shock element
oligo; C lanes, antibody (1:5, vol/vol) to HSF1
showing positive supershift (SS); D lanes, antibody (1:5, vol/vol) to HSF2 showing no supershift bands. Abbreviations as in Figure 2.
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heart with an LVEDP of 5 mm Hg (lane D in Figure 2B).
Thus, the increase in HSF activation induced by stretch is
correlated with the amount of stretch imposed on the heart. In
contrast, working hearts, which are closer to a physiological
perfusion, showed little activation of HSF (C lanes in Figure
3A) compared with the Langendorff- perfused hearts (A lanes
in Figure 3A). To further test that mechanical stretch can
induce the activation of HSF, we perfused a separate set of
hearts in the working mode using an aortic cannula with a
small diameter (⬍1 mm in ID) to mimic aortic stenosis. LV
afterload was thereby increased, and the ventricular wall
stress was enhanced. This resulted in a degree of activation of
HSF similar to that observed in Langendorff hearts (B lanes,
Figure 3A).
Specific activation of HSF was confirmed by the cold
heat-shock element competition assay (B lanes, Figure 3B).
The addition of antibodies to HSF1 and HSF2 showed a
supershift only with anti-HSF1 (C lanes, Figure 3B), indicating that it is HSF1 that is activated by stretch. HSF2 was not
activated (no supershift bands in D lanes, Figure 3B).
␮mol/L), if anything, increased activation of HSF (lane G,
Figure 6). Figure 6 summarizes HSF binding activities in
hearts from all of the experimental protocols: no perfusion
(A), working perfusion (B), Langendorff perfusion (C), 10
␮mol/L gadolinium (D), 50 ␮mol/L gadolinium (E), 1
␮mol/L diltiazem (F), and 3 ␮mol/L diltiazem (G). As a
positive control, hearts with heat shock (43°C for 13 minutes)
were analyzed and showed similar degrees of activation of
HSF (H lanes, Figure 6).
Analysis in HSP72 mRNA Expression
After 60 minutes, we found that the HSP72 mRNA level in
Langendorff-perfused rat hearts increased compared with
nonperfused control rat hearts (Figure 7). Northern blotting
showed that gadolinium attenuated this increase at both doses
(Figure 7). No dose-dependent inhibition was observed. This
observation is consistent with the GMSA, which showed that
HSF activity was decreased by 50% by gadolinium.
Gadolinium but Not Diltiazem Inhibits Mechanical
Stretch–Induced Activation of HSF
We perfused hearts in the Langendorff mode with gadolinium, an SAC blocker. As shown in Figure 4 (C and D lanes)
and Figure 5 (B and C lanes), gadolinium 10 and 50 ␮mol/L
markedly decreased HSF activation. Densitometric measurement of the GMSA showed a 45% and a 50% decrease in
HSF binding activity, respectively, compared with Langendorff perfusion without gadolinium (Figure 5A, P⬍0.05).
Although the high dose of gadolinium tended to suppress
HSF activation more than the low dose, this was not statistically significant.
Because gadolinium can block both SACs and L-type
calcium channels, it was important to identify whether HSF
activation was mediated by L-type calcium channels. To test
this, hearts were perfused with diltiazem, a specific L-type
calcium channel blocker. Two different concentrations (1 and
3 ␮mol/L) of diltiazem were used, and both failed to inhibit
the activation of HSF (D lanes in Figure 5B, and F and G
lanes in Figure 6), indicating that gadolinium attenuates HSF
activity via SACs. The high concentration of diltiazem (3
Figure 4. Gadolinium effect on Langendorff-perfused (stretched)
rat hearts. A, Nonperfusion; B, Langendorff perfusion; C, Langendorff perfusion with gadolinium (10 ␮mol/L); and D, gadolinium
(50 ␮mol/L). Abbreviations as in previous figures.
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Circulation
July 10, 2001
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Figure 5. A, Comparison of HSF activation in (bar A) Langendorff (n⫽4) and (bar B) Langendorff with 10 ␮mol/L gadolinium
(n⫽2), (bar C) with 50 ␮mol/L gadolinium (n⫽3), and (bar D) with
1 ␮mol/L diltiazem (n⫽3) rat hearts. *P⬍0.05 vs control Langendorff. B, Representative gel shift for gadolinium and diltiazem
effects on Langendorff (stretched) rat hearts. Abbreviations as in
previous figures.
Maximum Ventricular Developed Pressure
Pmax from all groups was stable during perfusions (Figure 8).
There was no significant difference in Pmax between the start
and completion of perfusion for any of the groups. Furthermore, there was no significant difference in Pmax among the
gadolinium, diltiazem, and working-mode groups compared
with Langendorff-perfused control hearts.
Discussion
In the present study, we demonstrated that stretch initiated the
heat-shock response in rat hearts by activating HSF1, and this
was followed by an increase in HSP72 mRNA. The intensity
of HSF activation correlated with the amount of stretch,
suggesting that the magnitude of stretch influences the degree
of HSF1 activation. HSF1 activation was blocked by gadolinium, but not by diltiazem, which indicates that the stretchinduced heat-shock response was mediated by SACs. An
alternative perfusion system, the working heart, was used to
determine whether perfusion alone was sufficient to activate
HSF. Perfusion in the working mode, which is closer to
physiological perfusion and does not use an apical drain or
ventricular balloon, did not activate HSF; however, HSF
could be activated by increasing afterload (aortic stenosis),
which effectively added an acute stretch.
Mechanical stresses, such as hemodynamic overload or
mechanical stretch, alter the protein expression pattern in
cardiac and skeletal muscles. For instance, Peterson and
Lesch20 first reported that stretch could accelerate protein
synthesis and amino acid transport. A similar phenomenon
was observed in rat hearts subjected to high aortic pressures.21
Subsequent studies showed that the HSP72 gene and the
“immediate early” (IE) genes, such as c-fos, c-myc, c-jun, JE,
and Egr-1, were involved in the early response to mechanical
stresses, followed by reexpression of fetal contractile protein
genes, such as skeletal ␣-actin, ␤-myosin heavy chain
(MHC), and atrial natriuretic peptide (ANP) genes.9,22–25
How cells sense mechanical stimuli, transmit, and how this
translates into gene expression is not understood. Increased
sympathetic nervous activity and increased catecholamine
levels have been suggested as potential mediators for induction of cardiac hypertrophy and IE gene expression.26,27 Since
Vandenburgh and Kaufman28 reported that mechanical
stretch without confounding factors caused increased protein
synthesis in cultured skeletal muscle cells, however, many
studies have demonstrated that mechanical stimuli alone can
directly induce gene expression, including HSPs.9,10,12,15,23,24
In our study, we found that mechanical stretch and isovolumic contraction are sufficient to initiate the heat-shock
response without neural and humoral factors. These observations extend our previous observations in the erythrocyteperfused rabbit heart, in which other factors, such as cytokines, could have had an effect, and demonstrate the
activation of HSF via SACs in response to stretch.15
It has been postulated that SACs may function as a
mechanotransducer between mechanical load and alterations
in protein synthesis in cardiac hypertrophy.16,29 Mechanosensitive ion channels have been found in a broad variety of cell
types.30 Activation of stretch-dependent channels results in
cation influx, which is associated with gene expression and
protein synthesis.16,31 We found that gadolinium decreased
HSF activity by half and had a similar effect on HSP72
mRNA levels. Although other studies have shown a dose
response,31 the small sample size may account for the lack of
dose-dependent inhibition in the present study, or alternatively, a second activating pathway may be involved. Gadolinium 10 ␮mol/L may have completely inhibited the SACs,
even though it did not abolish HSF1 activation. A 5-fold
increase in concentration had no further effect; thus, a second
pathway may be involved in activation of HSF1 with stretch.
Because it is known that gadolinium also blocks L-type
calcium channels,31,32 we used diltiazem, a specific L-type
calcium channel blocker, to exclude its effects, and it did not
block the activation of HSF. Similar observations, ie, the
noninvolvement of L-type calcium channels in the stretch
response, were made by Laine et al33 and Komuro et al.23 We
did not observe a negative inotropic effect with gadolinium in
the concentrations used.
Gadolinium does not affect the stretch-induced expression
of IE genes in cultured neonatal cardiac myocytes.17 It has
been shown that multiple second messenger factors, such as
mitogen-activated protein kinases and protein kinase C, may
be involved in the signal transductions for IE gene expression
via the activation of serum response factor–P62TCF com-
Chang et al
Heat-Shock Factor and Stretch-Activated Channels
213
Figure 6. Comparison of HSF activity in nonperfused (A), working perfusion (B), Langendorff
perfusion (C), Langendorff with gadolinium (D,
10 ␮mol/L; E, 50 ␮mol/L), Langendorff with diltiazem (F, 1 ␮mol/L; G, 3 ␮mol/L), and working
perfusion (43°C for 13 minutes) (H). Abbreviations as in previous figures.
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plex.25 The activation of these pathways and induction of the
IE genes lead to cardiac hypertrophy.30 Induction of HSPs is
not per se associated with hypertrophy, nor does overexpression of the HSPs result in hypertrophy.34 Thus, the activation
of the heat-shock response by stretch may involve pathways
distinct from the hypertrophic response. A recent study
showed that mitogen-activated protein kinase specific inhibitors did not affect stretch-induced HSF1 activation in vascular smooth muscle cells, and our previous work suggested
that protein kinase C does not play an important role in
stretch-induced HSP72 expression.12,15
There are several additional differences between our work
and the studies of stretch-induced IE genes. Not only are we
studying a different set of genes, but we also used the intact,
perfused heart as a model. The use of isolated cardiac
myocytes, although an excellent model, may eliminate or
modulate extracellular signaling. Furthermore, we have focused on the adult heart, and responses in the neonatal heart,
in which the induction of IE genes has primarily been studied,
may be different. Thus, differences in induction of the
heat-shock response and the IE genes may reflect differences
in overall regulation and/or differences in models.
Others have observed that volume overload could precondition the heart, and both this stretch-induced preconditioning
and classic preconditioning were blocked by gadolinium.35
They suggested that volume overload, “stretch,” preconditioned the heart through the activation of SACs. Because brief
cardiac ischemia/reperfusion results in the upregulation of
HSP726 and short ischemia could lead to a transient cardiac
dilation (stretching), we suggest that the activation of SACs
may be part of the common pathway in the initiation of the
cellular stress response in the heart. Certainly, other important factors are present in ischemia, such as denatured
Figure 7. Northern blot of HSP72 mRNA present in
Langendorff-perfused rat hearts with and without gadolinium.
proteins, but stretch may be a factor, particularly in the
nonischemic region of the ventricle. Previously, we reported
that we did not detect a further increase in HSP72 mRNA
after ischemia in erythrocyte-perfused hearts compared with
control perfusions.6 We may have already maximally induced
the heat-shock response. With the current understanding of
cytokines, it may be that in this preparation, which used
erythrocytes from cows in the slaughterhouse, cytokines may
have had an additive effect; however, perfusion without
stretch, but with the erythrocytes, did not induce HSP72.36
More recently, knowing the effect of stretch, we took measures, such as reduced balloon volume/LVEDP (6 mm Hg), to
prevent stretch. This resulted in minimal activation of HSF in
control hearts, and we were able to correlate changes in ATP
during ischemia/reperfusion with the degree of activation of
HSF.19
In conclusion, we demonstrate that stretch is sufficient to
activate HSF1 and increase HSP72 mRNA expression and
that SACs are involved in the activation of HSF1 and
upregulation of HSP72 mRNA. This represents a novel
pathway for the activation of HSF1. Although these results
have implications for researchers using the isolated, perfused
rat heart preparation, the overall implications are far greater.
Stretch is a common phenomenon in both the normal and
diseased cardiovascular system. Increased stretch occurs with
hypertension, heart failure, and myocardial infarction, for
Figure 8. Pmax at start and end of experiments. There were no
significant differences in developed pressure for gadoliniumtreated, diltiazem-treated, and 2 working-mode hearts vs
Langendorff-perfused control hearts. No difference in Pmax was
found between start and end of experiment within each group.
214
Circulation
July 10, 2001
example, and SACs may be an important signaling pathway
for the induction of the stress response in these settings.
Furthermore, the heart has higher levels of HSP72 than many
other tissues (unpublished results), and this may be related to
the repetitive stretch that occurs as part of normal cardiac
function.
Acknowledgments
This study was supported by NHLBI grants HL-45257 (Dr Wasser)
and HL-58515 (Dr Knowlton). We thank Dr Heinrich Taegtmeyer
for advice on the working heart model and Feng Xu for technical
assistance.
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Circulation. 2001;104:209-214
doi: 10.1161/01.CIR.104.2.209
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