(Circulation. 2000;102:1703.)
© 2000 American Heart Association, Inc.
Basic Science Reports |
Confocal Microscopic Localization of Constitutive and Heat Shock–Induced Proteins HSP70 and HSP27 in the Rat Heart
From the Department of Anatomy and Neurobiology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada.
Correspondence to Dr R. William Currie, Department of Anatomy and Neurobiology, Dalhousie University, Halifax, NS B3H 4H7, Canada. E-mail wcurrie{at}is.dal.ca
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Abstract |
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Background—Heat-shock treatment of rats elevates expression of heat-shock proteins, which play a role in improving the contractile recovery and reducing infarct size in hearts after ischemic injury. However, the location of these proteins in the heart is unknown.
Methods and Results—Anesthetized rats were heat-shocked by elevation of body temperature to 42°C to 42.5°C for 15 minutes, followed by 24 hours of recovery. Control and heat-shocked hearts were extirpated and perfused briefly with saline followed by 2% paraformaldehyde in PBS. Confocal immunofluorescence microscopy of control hearts revealed that HSP27 was localized in cardiomyocytes in a pattern reminiscent of Z bands and was colocalized with neuronal markers in somata and axons. No obvious change in HSP27 content or distribution occurred after heat shock. Confocal microscopy revealed little or no HSP70 in control hearts. After heat shock, HSP70 was detected neither in cardiomyocytes nor in neuronal elements within the heart, but HSP70 was abundant in small blood vessels found between the ventricular cardiomyocytes.
Conclusions—Heat shock induces a cell type–specific expression of HSP70 in blood vessels but not myocytes or intrinsic cardiac neurons, suggesting that blood vessels play a primary role in myocardial protection.
Key Words: immunohistochemistry • heat shock proteins • nervous system, autonomic • endothelium • myocytes
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Introduction |
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Many heat-shock proteins are constitutively expressed and some are highly inducible by a variety of stressors.1 2 After heat-shock proteins are induced, cells in such organs as the heart and brain can show remarkable resistance to subsequent stress. For example, heat shock–induced expression of HSP70 is correlated with enhanced recovery of myocardial contractility after ischemic injury.3 4 In fact, transgenic overexpression of rat and human HSP70 plays a direct role in protection of the mouse myocardium from ischemic injury.5 6 Similarly, HSP27, a member of the small heat-shock protein family, confers thermoresistance to NIH/3T3 cells7 8 and increases resistance to oxidative stress and cytotoxicity induced by tumor necrosis factor-
.9 Recent work suggests that heat-shock proteins play a role in protecting the function of endothelial cells after ischemia-reperfusion injury. Transfection of an expression vector containing human HSP70 cDNA into endothelial cells resulted in significant protection from hypoxia-reoxygenation injury.10 In hearts, heat-shock treatment significantly improves myocardial and endothelial functional recovery after cardioplegic arrest.11 In addition, isolated endothelial cells have higher levels of HSP70 than do myocytes from heat-shocked hearts,12 suggesting that myocardial protection is mainly a result of expression of HSP70 in endothelial cells. Other heat-shock proteins may also be involved in endothelial cell–related myocardial protection. In cultured endothelial cells, HSP27 is associated with protecting microfilaments from disruption and aggregation in response to ischemia-like injury.13 However, the specific cell types that express HSP27 and HSP70 in the heart are as yet unknown. Thus, our first objective was to determine the distribution of HSP27 and HSP70 in the atrial and ventricular myocardium of normal rat hearts and in hearts 24 hours after heat-shock treatment.
In the central nervous system, HSP70 is not normally expressed14 but is highly inducible in neurons by such stressors as ischemia15 and seizures.14 HSP27 is constitutively expressed in many sensory and motor neurons of the brain stem and spinal cord of the adult rat.16 It is therefore likely that both of these proteins are either constitutively expressed or can be induced in the peripheral nervous system. However, the distribution of these proteins in intracardiac neuronal elements has not been determined. Moreover, neither the presence of HSP27 nor that of HSP70 has been reported in neurons with somata intrinsic to the heart. Thus, a second objective was to determine whether HSP27 and HSP70 were constitutively expressed or could be induced by heat shock in elements of the intracardiac nervous system.
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Methods |
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Experimental Animals
Male Sprague-Dawley rats (225 to 300 g; Charles River Inc, St Constant, Québec, Canada) were cared for in accordance with the Guide to the Care and Use of Experimental Animals of the Canadian Council on Animal Care. Rats were randomly assigned to a control group (n=21) or a heat-shock group (n=21). Rats in the heat-shock group were injected with 50 mg/kg sodium pentobarbital IP and placed on a temperature-controlled heating pad (50°C) until body temperature, monitored with a rectal thermometer, reached 42°C. Core body temperature was maintained between 42°C and 42.5°C for 15 minutes. Animals were then allowed to recover for 24 hours. Control animals were anesthetized with sodium pentobarbital but not heated.
Tissue Preparation and Fixation
At 24 hours after heat shock, animals were given an overdose of
sodium pentobarbital (100 mg/kg), and hearts were removed and perfused
briefly via the aorta with 100 mmol/L PBS. Once the blood was
cleared from the heart, the heart was either frozen (-70°C) for
later extraction of proteins or prepared for immunohistochemistry. For
immunohistochemistry, the atria were separated from the ventricles and
pinned flat. Atria and ventricles were fixed for 24 hours by immersion
in a solution of 2% paraformaldehyde in 100
mmol/L PBS. Ventricles were then immersed overnight in 30% sucrose in
100 mmol/L phosphate buffer, then sectioned at 40 µm on a
freezing microtome. Sections were stored in Millonig’s buffer until
processed for immunofluorescence analysis.
Whole-mounts of atria were processed similarly.
2D Gel Electrophoresis and Western Analysis
Ventricular samples from control (n=4) and
heat-shocked (n=4) rats were analyzed by 2D gel electrophoresis
followed by Western analysis, as previously
described.6 17 Briefly, 
1 mg of ventricular
protein was loaded onto each isoelectric focusing gel and separated
overnight. Then gels were equilibrated in SDS buffer, and the proteins
were separated by electrophoresis in the second dimension on either
7.5% (for HSP70) or 12% (for HSP27) SDS-polyacrylamide gels.
The proteins were transferred overnight onto Immunobilon PVDF membranes
(Millipore). Membranes were incubated in PBS containing 5% skim milk
powder and reacted overnight at 4°C with either the primary rabbit
polyclonal antibody specific for HSP27 (1:1000 dilution; StressGen) or
the primary monoclonal antibody specific for HSP70 (1:1000 dilution;
StressGen). After a washing in PBS, membranes were incubated for 1 hour
with peroxidase-conjugated goat antibody raised against rabbit or mouse
IgG in 100 mmol/L PBS. Membranes were reacted in PBS containing
4-chloro-1-naphthol (0.05%). Membranes were digitally photographed,
counterstained with amido black, and photographed again. Densitometric
analysis of immunoprecipitate on membranes was done with
Bio-Rad Molecular Analyst version 1.5 software.
Immunofluorescence
Atrial whole-mounts (n=34) were dissected to remove the
endocardium, allowing penetration of antibodies to the intrinsic
cardiac nervous system.18 Atria were dehydrated in a
graded series of ethanol solutions and cleared in xylene. The tissue
was rehydrated and incubated in a 4% solution of Triton-X 100 in PBS
for 48 hours to improve antibody penetration. Free-floating
whole-mounts were incubated in a blocking solution of 2.5% normal
donkey serum to reduce nonspecific staining.
Double-label immunohistochemistry was performed with an initial 48 hours of incubation of the primary antibody directed against either protein gene product 9.5 (PGP 9.5), a general neuronal marker; tyrosine hydroxylase (TH), an enzyme in the synthesis pathway for catecholamines used to identify sympathetic postganglionic projections; or choline acetyltransferase (ChAT), an enzyme in the synthesis pathway for acetylcholine, used to identify parasympathetic preganglionic projections and intracardiac neurons. Atria were incubated in the appropriate secondary anti-IgG antibody conjugated to a fluorescent tag as previously described.18 Double-labeling was performed by a second 48 hours of incubation of primary antibody directed against either HSP27 or HSP70. The atria were then incubated in the appropriate secondary antibody directed against the second primary antibody.
Images of whole-mount atria and ventricular sections were captured with a confocal microscope (Zeiss LSM 510), and a 3D view of the tissue was reconstructed from optical sections. Anatomic figures were compiled from confocal images with Adobe Photoshop 6.0 software.
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Results |
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Western analysis of proteins separated by 2D gel electrophoresis revealed a constitutive level of HSP27 in control rat hearts (Figure 1A
, 1A'). After heat-shock
treatment, the amount of HSP27 appeared to be increased (Figure 1B, 1B'). Two immunoreactive isoforms of HSP27 (Figure 1A, 1B) were apparent among other proteins visualized after the
membrane had been counterstained with amido black (Figure 1A',
1B'). The semiquantitative analysis of the immunoreactive spots
revealed a modest (nonsignificant) increase in HSP27 after heat-shock
treatment (Figure 1C).
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A relatively low constitutive level of HSP70 was revealed in control
rat hearts by Western analysis (Figure 2A, 2A'). After heat-shock treatment (24
hours), HSP70 was increased (Figure 2B, 2B'). Immunoreactive
HSP70 (Figure 2A, 2B) was apparent among other proteins
visualized after the membrane had been counterstained with amido black
(Figure 2A', 2B'). The semiquantitative analysis of the
immunoreactive spots revealed a significant increase in HSP70 after
heat-shock treatment (Figure 2C).
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Confocal microscopy of whole-mount atria revealed intense HSP27
immunoreactivity (HSP27-IR) within neuronal somata and axons (Figure 3). Double-labeling of whole-mount atria
from control (Figure 3A, 3A') and heat-shocked (Figure 3B, 3B') rats revealed positive PGP 9.5 immunoreactivity (PGP
9.5-IR; Figure 3A, 3B) and HSP27-IR (Figure 3A', 3B')
that was colocalized within neurons and axon bundles. Similarly, in
atria from control (Figure 3C, 3C') and heat-shocked (Figure 3D, 3D') rats, positive ChAT immunoreactivity (ChAT-IR; Figure 3C, 3D) and HSP27-IR (Figure 3C', 3D') overlapped in
neuronal somata and axons. In all instances of colocalization in
neuronal somata, HSP27 appeared to concentrate within the cytoplasm,
leaving the nucleus relatively unreactive. PGP 9.5, ChAT, and TH
immunohistochemistry has been shown to label all neuronal somata and
axons within the heart.18
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Immunofluorescence confocal microscopy of
whole-mount atria revealed no clear constitutive or inducible HSP70-IR
in either cardiomyocytes or neuronal elements of the
intrinsic cardiac nervous system (Figure 4). Atria were double-labeled for HSP70
and either PGP 9.5 or TH to show elements of the sympathetic cardiac
innervation. In control (Figure 4A, 4A', 4C, 4C') and
heat-shocked (Figure 4B, 4B', 4D, 4D') rat atria, PGP 9.5-IR
(Figure 4A, 4B) and TH immunoreactivity (TH-IR; Figure 4C, 4D) were detected in neural elements, but no HSP70-IR was
detected in neuronal elements or cardiomyocytes in either
control (Figure 4A', 4C') or heat-shocked (Figure 4B',
4D') atria. However, HSP70-IR was apparent in the walls of blood
vessels within the atria after heat shock (Figure 5). Double-labeling revealed that after
heat shock, PGP 9.5-IR (Figure 5A) was detected in axons
adjacent to blood vessels, whereas HSP70-IR was associated with blood
vessels (Figure 5B).
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Control and heat-shocked hearts were double-labeled with antibodies
against HSP27 and HSP70. In whole-mounts of control atria, HSP27-IR
(Figure 6A) was found in neuronal
elements of the heart and possibly in association with blood vessels,
whereas no clear HSP70-IR (Figure 6A) was detected. After heat
shock, the atrial distribution of HSP27-IR (Figure 6B) was
similar to its distribution in control tissue; that is, it was located
in neuronal elements and blood vessels. However, in contrast to the
control situation, after heat shock, HSP70-IR (Figure 6B) was
present in the walls of blood vessels coursing within the atria.
Double-labeling for HSP25 and HSP70 revealed minimal colocalization of
the 2 proteins, with the possible exception of some colocalization of
HSP27-IR and HSP70-IR in scattered microvessels.
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In ventricular sections, HSP27-IR was present within
cardiomyocytes, axons, and terminals (Figure 6C
).
Sections of heat-shocked ventricles were positive for HSP27-IR (Figure 6C). The HSP27-IR within axons and terminals was colocalized
with ChAT-IR (Figure 6C). HSP27-IR in cardiomyocytes
occurred in a banding pattern transverse to the long axis of these
cells reminiscent of the Z banding of the contractile proteins.
No HSP70-IR was observed within control hearts (Figure 6D, 6E),
but neuronal processes and nerve terminals with varicosities were
immunoreactive for PGP 9.5 (Figure 6D) and TH (Figure 6E). After heat shock, intense HSP70-IR was found in blood
vessels (Figure 6F, 6G) but was not colocalized with neuronal
elements containing PGP 9.5-IR (Figure 6F) or TH-IR (Figure 6G). HSP70-IR showed occasional branching patterns that
intermingled with and coursed parallel to the myocytes in sections cut
longitudinally in relation to myocyte orientation (Figure 6F).
In ventricular tissue cut transversely to the long axis of
myocyte orientation, it became more apparent that elements labeled
positively for HSP70 were situated between the myocytes (Figure 6G).
Confocal microscopy of ventricular sections revealed
HSP70-IR associated with apparent interstitial capillaries
(Figure 7). In sections of ventricle from
control rats, no positive HSP70-IR was observed in neural elements,
cardiomyocytes, or blood vessels (Figure 7A), but
after heat shock, intense HSP70-IR was observed in capillaries between
the myocytes (Figure 7B; see also Figure 6F). To
determine independently the microvascular pattern in the
ventricular myocardium, Evans blue dye
dissolved in a gelatin-saline solution was perfused into the
coronary arteries. In sections cut longitudinally to the long
axis of the myocytes, long striations of Evans blue dye–filled vessels
were observed with occasional branching (Figure 7C, 7D). In
sections transverse to the long axis of the myocytes, small-diameter,
dye-filled vessels were seen situated between myocytes (not shown). The
overall distribution pattern of HSP70-IR (Figures <6F, 7B) in
heat-shocked ventricle was similar to that of the Evans blue dye in
microvessels (Figure 7C, 7D).
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Finally, ventricles from control and heat-shocked rats were
double-labeled with antibodies against HSP27 and HSP70. In control
ventricular sections, confocal microscopy revealed HSP27-IR
(Figure 8A) in
cardiomyocytes, but no HSP70-IR (Figure 8A') was
detected. After heat shock, the distribution of HSP27-IR (Figure 8B) was similar to that in control hearts. HSP27-IR appeared to
be located in cardiomyocytes, neuronal elements, and
possibly blood vessels. After heat shock, HSP70-IR (Figure 8B')
appeared to be associated with blood vessels coursing between the
cardiomyocytes in the ventricles. Double-labeling for HSP25
and HSP70 revealed minimal colocalization of the 2 proteins.
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Discussion |
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Within 24 hours of recovery from hyperthermia, a modest increase in HSP27 and a robust increase in HSP70 were detected in the rat heart by Western analysis. HSP27 was localized primarily in cardiomyocytes and in neuronal somata and processes in both control and heat-shocked hearts. HSP70 was not detected in control hearts, but after heat shock, HSP70 was localized within blood vessels in the atria and ventricles.
HSP27 is a constitutive protein of human, rabbit, and rat hearts.19 20 21 In cultured cardiomyocytes, overexpression of HSP27 is associated with resistance to ischemic injury.22 23 Recently, overexpression of HSP27 in cultured cardiac cells has been shown to protect against apoptotic stimuli as well as thermal and hypoxic stress.24 Our finding that HSP27 was present in cardiomyocytes provides more precise anatomic localization of this protein, in the Z bands of the contractile apparatus.
The finding of HSP27 in neuronal somata and axons in the heart is novel. Constitutive HSP27 is seen in many, but not all, neurons of motor nuclei in the brain stem and spinal cord of the rat.16 Among these, HSP27 is expressed in preganglionic parasympathetic cholinergic neurons of the nucleus ambiguus that project to the heart in the vagus nerves.25 Because heat-shock proteins are synthesized in neuronal somata and transported antegradely to axonal terminals,26 27 we anticipated finding HSP27-IR colocalized with ChAT-IR in axons in the heart. The vagal preganglionic fibers innervate intrinsic cardiac neurons, and not cardiomyocytes directly. In the present study, the somata of many intrinsic cardiac neurons in the atria demonstrated ChAT-IR and HSP27-IR. In addition, HSP27 was also localized in processes and terminals in the atria and ventricles, but it was not possible to differentiate between preganglionic and intrinsic cardiac processes, because both contained ChAT and HSP27.
Intracardiac neuronal elements of adrenergic phenotype were identified by TH-IR, and their axons and terminals were particularly numerous in ventricular tissue. However, we could not determine whether intracardiac projections of sympathetic postganglionic neurons contained HSP27, because the primary antibodies against TH and HSP27 used in our study were developed in the same host (mouse).
The distribution of HSP27 in the intracardiac nervous system after heat shock appeared to be similar to that of control hearts. In particular, all neuronal somata that displayed PGP 9.5-IR or ChAT-IR also contained HSP27-IR. Constitutive expression of HSP27 in neural tissues in the heart may indicate that this protein functions to help resist physiological stress on neural control of cardiac function. Although the precise role of HSP27 in cardiac neurons is unclear, it may aid in axonal transport and synaptic function27 28 and could act to protect or stabilize neuronal function after ischemia, possibly helping to suppress arrhythmias.29 30
In contrast to the pattern of HSP27 labeling, HSP70-IR was not detected in the intracardiac nervous system either in control hearts or after heat shock. Thus, HSP70 appears not to have a role in neural control of the heart on an ongoing basis, or after heat shock.
Low levels of HSP27 also appeared to be present in microvessels of the heart. However, HSP27-IR is not usually detected in cerebral blood vessels.16 17 31 Conversely, constitutive HSP27 has been detected in vascular endothelial cells in culture.13 32 33 Therefore, blood vessels may express varying levels of HSP27, depending on local, organ-specific factors.
Heat shock–induced HSP70 in the heart was localized in the blood vessels (confirming the work of Amrani et al12 ), but not in cardiomyocytes or neurons. The basis for this expression pattern is unclear at present. Perhaps endothelial cells are exposed to the highest burden of reactive oxygen radicals during the heat-shock treatment. Alternatively, HSP70 mRNA may reach higher concentration followed by greater translation in the smaller endothelial cells than in the larger myocytes or neurons.
Finding HSP70 in blood vessels in the heart is interesting for several reasons. First, it suggests that cells have a cell type–specific response to stress, ie, endothelial cells in the heart appear to be more sensitive to heat shock than are cardiomyocytes or neurons. However, each of these cell types can express HSP70 after metabolic injury.34 35 36 Second, cells, in this case endothelial, appear to respond to stress according to their local environment. After hyperthermia, cells in blood vessels in the heart express HSP70 (present study; Amrani et al12 ), whereas in the brain HSP70 is localized not in blood vessels but mostly in glia and in some neurons.37 This suggests that the protective role of HSP70 may be through different cell types and cell-cell interactions in different organs. Third, caution should be used in extrapolating from studies of cells in culture (eg, cardiomyocytes) to intact organs that include vascular and neuronal elements. Although considerable information has been gleaned about the effects of overexpressing HSP70 in cardiomyocytes and the resulting cellular protection, in the intact heart after heat shock, myocardial protection is most likely due to high levels of HSP70 in blood vessels. Fourth, as discussed by Gray et al,38 HSP70 in vascular cells of heart has important implications for myocardial protection. If blood vessels are protected from free-radical injury during reperfusion, then the whole organ may be protected. Finally, our findings suggest that therapies designed to increase protective proteins, such as HSP70, should be directed at the blood vessels.
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Acknowledgments |
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This work was supported by grants to Drs Currie and Smith from the Heart and Stroke Foundation of New Brunswick. We thank Brenda Ross for technical assistance and Dr R.J. Wassersug for critical comments on the manuscript.
Received March 27, 2000; revision received May 2, 2000; accepted May 5, 2000.
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C. R. Hampton, A. Shimamoto, C. L. Rothnie, J. Griscavage-Ennis, A. Chong, D. J. Dix, E. D. Verrier, and T. H. Pohlman HSP70.1 and -70.3 are required for late-phase protection induced by ischemic preconditioning of mouse hearts Am J Physiol Heart Circ Physiol, July 11, 2003; 285(2): H866 - H874. [Abstract] [Full Text] [PDF]
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M. B. Harris, M. A. Blackstone, H. Ju, V. J. Venema, and R. C. Venema Heat-induced increases in endothelial NO synthase expression and activity and endothelial NO release Am J Physiol Heart Circ Physiol, June 5, 2003; 285(1): H333 - H340. [Abstract] [Full Text] [PDF]
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R. N.M. Cornelussen, F. A. van Nieuwenhoven, L. H.E.H. Snoeckx, A. A. Knowlton, J. P. Leger, F. M. Smith, and R. W. Currie Presence of Heat Shock Protein 72 in Cardiomyocytes After Heat Stress Response Circulation, November 27, 2001; 104 (22): e123 - e123. [Full Text] [PDF]
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T. Ooie, N. Takahashi, T. Saikawa, T. Nawata, M. Arikawa, K. Yamanaka, M. Hara, T. Shimada, and T. Sakata Single Oral Dose of Geranylgeranylacetone Induces Heat-Shock Protein 72 and Renders Protection Against Ischemia/Reperfusion Injury in Rat Heart Circulation, October 9, 2001; 104(15): 1837 - 1843. [Abstract] [Full Text] [PDF]
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