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J Appl Physiol 93: 484-488, 2002. First published March 29, 2002; doi:10.1152/japplphysiol.00009.2002
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Vol. 93, Issue 2, 484-488, August 2002

Heat-induced force suppression and HSP20 phosphorylation in swine carotid media

Matthew J. O'Connor and Christopher M. Rembold

Cardiovascular Division, Departments of Internal Medicine and Physiology, University of Virginia Health System, Charlottesville, Virginia 22908


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In vascular smooth muscle, cyclic nucleotide-dependent phosphorylation of heat shock protein 20 (HSP20) on serine-16 (Ser16) has been suggested to cause force suppression, i.e., reduced force with only minimal myosin regulatory light chain (MRLC) dephosphorylation. We hypothesized that heat pretreatment also suppresses force by increasing HSP20 phosphorylation. After heat pretreatment of swine carotid artery at 44.5°C for 4 h and reduction to 37°C for 1 h, Ser16-HSP20 phosphorylation was increased and histamine-induced increases in contractile force were suppressed. Subsequent addition of nitroglycerin induced additive force suppression. Heat and nitroglycerin induced a similar relation between Ser16-HSP20 phosphorylation and force. Heat pretreatment induced a small, but significant, increase in total HSP20 immunostaining. These results demonstrate that vascular smooth muscle responds to thermal stress by increasing Ser16-HSP20 phosphorylation in addition to a possible small increase in total HSP20 concentration. The resulting heat-induced reduction in force should be considered "force suppression" because histamine-induced increases in MRLC phosphorylation were not significantly altered by heat pretreatment. These processes may bring about a resistance to contractile agonists, which could have clinical significance in conditions such as hyperthermia and/or sepsis with vasodilatory shock.

guanosine 3',5'-cyclic monophosphate; heat shock proteins; nitric oxide; vascular smooth muscle


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

VASCULAR SMOOTH MUSCLE CONTRACTION primarily involves pathways that increase myosin regulatory light chain (MRLC) phosphorylation. Stimuli typically increase myoplasmic calcium concentration ([Ca2+]i) and activate myosin light chain kinase, with consequent phosphorylation of the myosin regulatory light chains (MRLC) on serine (Ser)-19 (Ser19) (8). Some stimuli also may reduce myosin light chain phosphatase activity and thereby increase Ser19-MRLC phosphorylation (21). These processes can be termed "activation." Ser19-MRLC phosphorylation increases myosin's actin-activated ATPase activity and is associated with contraction (reviewed in Ref. 11).

Relaxation is typically hypothesized to be the reversal of activation, i.e., "deactivation." Removal of contractile agonists or the addition of some relaxing agents can cause relaxation by either reducing [Ca2+]i-dependent myosin light chain kinase activity (6, 13) or by increasing myosin light chain phosphatase activity (5).

There is also a novel form of smooth muscle relaxation that does not involve deactivation mechanisms. Elevations in concentrations of cGMP (1, 10) or cAMP (18) can reduce smooth muscle tone, whereas MRLC phosphorylation levels remain elevated in the presence of excitatory stimuli. We term this process "force suppression" to separate it from mechanisms that reduce force by reducing MRLC phosphorylation.

Cyclic nucleotide-induced relaxation was found to be associated with phosphorylation of heat shock protein 20 (HSP20) on Ser16 (2, 3, 15). More recently, Ser16-HSP20 phosphorylation was shown to specifically and temporally correlate with force suppression rather than the deactivation form of relaxation (15, 18). A region of HSP20 (residues 110-121) has sequence homology with troponin I, and peptides from this region bound thin filaments, reduced actin activated myosin S1 ATPase activity, and relaxed skinned swine carotid artery (15). We hypothesized that binding of Ser16-phosphorylated HSP20 to the thin filament may "turn off" thin filaments so that phosphorylated myosin does not interact with the thin filament (i.e., a model similar to skeletal muscle troponin I). This would explain low force with elevated MRLC phosphorylation.

HSP20 is a member of the heat shock protein superfamily and is known to provide resistance to heat treatment in cells (24). HSP20 is primarily a cytosolic protein in swine carotid (19) and rat cardiac myocytes (23). Heat treatment (44.5°C) of cultured rat cardiac myocytes induced partial redistribution to the nucleus (23). These results suggested that heat treatment could be a tool to manipulate HSP20 in intact smooth muscle tissues. In this paper, we pretreated swine carotid arterial tissues with elevated temperature and assessed the effect on Ser16-HSP20 phosphorylation, MRLC phosphorylation, and contractile force. We tested the hypothesis that heat pretreatment suppresses force by increases in HSP20 phosphorylation.


    MATERIALS AND METHODS
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tissues. Swine common carotid arteries were obtained from a slaughterhouse and transported at 0°C in physiological salt solution. Physiological salt solution contained (in mM) 140 NaCl, 4.7 KCl, 5 MOPS, 1.2 Na2HPO4, 1.6 CaCl2, 1.2 MgSO4, and 5.6 D-glucose, pH adjusted to 7.4 at 37°C. Dissection of medial strips, mounting, and determination of the optimum length for stress development at 37°C were performed as described in RESULTS and in Ref. 16. The intimal surface was mechanically rubbed to remove the endothelium.

Heat pretreatment experimental protocol. Tissues were first equilibrated at 37°C (16). This involved a "warm-up" 109 mM K+ contraction ~30 min after mounting, repeated stretching to ~1 × 105 N/m2 (~10 g for a 10 mg tissue; typically tissues are stretched 6 times until a stable force is obtained), a release to ~0.2 × 105 N/m2 (~2 g), and a second 109 mM K+ contraction ~120 min after mounting. This protocol sets the muscle to the optimal length for force generation. The latter K+ contraction was used for force normalization. Tissues were then exposed to temperatures of 44.5, 41, or 37°C (control) for 2 or 4 h (solutions were replaced if evaporation was observed). Temperature was changed by switching the tissue bath jacket supply between two water circulators set at different temperatures. This procedure changed bath temperature to the desired temperature within 5 min. After the exposure to different temperatures, all tissues were returned to 37°C for 60 min. Tissues were then either 1) frozen, 2) contracted with 10 µM histamine for 10 min and then frozen, or 3) contracted with 10 µM histamine for 10 min, then relaxed by addition of 10 µM nitroglycerin for 20 min, and then frozen.

Antibodies. Rabbit anti-HSP20 antibody was made commercially via repeated injection of gel-purified recombinant HSP20 (sequence confirmed by mass spectroscopy). After confirmation of an antigenic response, serum was collected and frozen for future use. Specificity was verified as described previously (18).

Measurement of HSP20 and MRLC phosphorylation. Swine carotid arteries were first thermally and then pharmacologically treated, followed by freezing in an acetone-dry ice slurry (16). After air drying, the tissues were homogenized in a buffer containing 1% SDS, 10% glycerol, and 20 mM dithiothreitol (20 mg wet wt/ml buffer). Full-strength, half-strength, and quarter-strength dilutions of samples were then separated on one-dimensional isoelectric focusing gels [ampholytes were a 50:50 mixture of isoelectric point (pI) 4-6.5 and pI 5-8 for HSP20 and a 50:50 mixture of pI 4-6.5 and pI 4.5-5.4 for MRLC], blotted to nitrocellulose, immunostained with our rabbit polyclonal anti-HSP20 antibody (1:5,000) or rabbit polyclonal anti-MRLC antibody (1:4,000 in 1% bovine serum albumin and 0.01% sodium azide), and detected with enhanced chemiluminescence (17). The dilutions ensured that the enhanced chemiluminescence detection system was in the linear range (26). Immunoblots were scanned on a Hewlett-Packard flatbed scanner and quantitated with UNSCANIT software.

Phosphorylation was determined by change in the pI for each phosphorylation species. We find two HSP20 phosphorylation sites in the swine carotid; therefore, there are four immunoreactive species: unphosphorylated at pI 6.3, monophosphorylated on a protein kinase C (PKC) site at pI 6.0, monophosphorylated on Ser16 at pI 5.9, and diphosphorylated on Ser16 and the PKC site at pI 5.7 (see blot in Fig. 1 of Ref. 18; phosphorylation at Ser16 confirmed by mass spectroscopy sequencing of the pI 5.7 isoform). In our experience, HSP20 is >90% phosphorylated on the PKC site regardless of treatment with histamine, nitroglycerin, forskolin, and heat. Typically, the level of HSP20 that is unphosphorylated (pI 6.3) is <10% and the level of HSP20 monophosphorylated at Ser16 (pI 5.9) is <1% of total HSP20. Therefore, Ser16-HSP20 phosphorylation was reported as the percentage of diphosphorylated HSP20 (pI 5.7) in relation to the sum of PKC monophosphorylated HSP20 (pI 6.0) plus diphosphorylated HSP20 (pI 5.7). For MRLC, phosphorylation was determined as the percentage of phosphorylated smooth muscle MRLC in relation to total smooth muscle MRLC (i.e., nonmuscle MRLC were ignored). MRLC phosphorylation is reported as suprabasal MRLC phosphorylation.

Statistics. Comparisons between multiple groups were performed in Sigmastat by ANOVA testing with Student-Newman-Kuels pairwise post hoc testing. Paired t-testing was performed if there were two groups. Significance was defined as P < 0.05. Data are presented in the text and figures as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of heat pretreatment on contractile force. As detailed in MATERIALS AND METHODS, tissues were first equilibrated at 37°C, then exposed to higher temperature for a certain duration, and then returned to 37°C for 60 min before pharmacological treatment and freezing. Representative force tracings of the response to histamine stimulation followed by nitroglycerin-induced relaxation are shown in Fig. 1. Heat pretreatment at 44.5°C for 4 h slowed force development and reduced the steady-state contraction induced by 10 µM histamine compared with 37°C controls. Heat pretreatment at 44.5°C for only 2 h also slowed the rate of contraction but had less effect on the sustained contraction. Nitroglycerin induced a relaxation regardless of heat pretreatment.


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  		Fig. 1.   Heat pretreatment reduces contractile force. Representative force tracings of swine carotid artery tissues that had been pretreated at 37°C (control; top tracing), 44.5°C for 2 h (middle tracing), or 44.5°C for 4 h (bottom tracing). Tissues were all stimulated with 10 µM histamine for 10 min and then relaxed by addition of 10 µM nitroglycerin. Force was measured on a curvilinear recorder.

Quantitative measurements of the effects of heat pretreatment are shown in Fig. 2. Heat pretreatment for 4 h at 44.5°C significantly reduced 10 µM histamine-induced force generation compared with 37°C controls (Fig. 2B). Heat pretreatment for 2 h at 44.5°C did not significantly reduce 10 µM histamine-induced force measured 10 min after addition of histamine. Nitroglycerin induced a significant reduction in force regardless of prior heat pretreatment. However, the relative relaxation induced by 10 µM nitroglycerin did not depend on prior heat pretreatment (Fig. 2A). Equivalent relative relaxation indicates that the reduced force induced by heat pretreatment was additive with nitroglycerin.


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  		Fig. 2.   Duration-dependent 44.5°C heat pretreatment reduced contractile force without altering relative nitroglycerin-induced relaxation. Swine carotid artery tissues were either unheated (37°C control; left), heated at 44.5°C for 2 h (middle), or heated at 44.5°C for 4 h (right). Contractile force [as a percentage of a 109 mM extracellular K+ (K109) contraction performed before heat treatment] was measured 1) 10 min after stimulation with 10 µM histamine (Hist) and 2) 20 min after relaxation by the addition of 10 µM nitroglycerin (NTG) to the histamine-contracted tissues (Hist+NTG; B). A: relative relaxation induced by nitroglycerin as a percentage of the histamine contraction. Values are means ± SE; n = 4-13 tissues. * Significant difference (P < 0.05) between histamine stimulation and histamine and nitroglycerin treatment. # Significant difference (P < 0.05) between the indicated treatment and the 37°C histamine-treated tissues. (ANOVA P values were 0.30 for relative relaxation and P < 0.001 for force.)

Effect of heat pretreatment on Ser16-HSP20 and MRLC phosphorylation. Because a significant effect on sustained histamine-induced force was observed only with heat pretreatment for 4 h at 44.5°C, we evaluated Ser16-HSP20 and MRLC phosphorylation with this protocol. Pretreatment at 44.5°C for 4 h significantly increased Ser16-HSP20 phosphorylation in unstimulated tissues to 0.26 ± 0.06 mol Pi/mol HSP20 without significantly increasing suprabasilar MRLC phosphorylation or force (Fig. 3).


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  		Fig. 3.   Biochemical correlates of heat-induced reductions in force: heat pretreatment increased serine-16 (Ser16)-heat shock protein 20 (HSP20) phosphorylation (Phos) without altering myosin regulatory light chain (MRLC) phosphorylation. Swine carotid artery tissues were pretreated for 4 h at either 44.5°C or 37°C . After a return to 37°C for 1 h, tissues were frozen either without activation (left), 10 min after activation with 10 µM histamine (middle), or after activation with 10 µM histamine for 10 min followed by relaxed induced by addition of 10 µM nitroglycerin for 20 min (right). Frozen tissues were then processed for measurement of MRLC phosphorylation (B) and Ser16-HSP20 phosphorylation (A). Force (C) was normalized to that elicited with 109 mM extracellular K+ depolarization before heat pretreatment. Values are means ± SE; n = 4-13 tissues. Symbols without error bars represent errors smaller than the size of the symbol. unstim, Unstimulated. * Significant difference (P < 0.05) between 37 and 44.5°C pretreatment. # Significant difference (P < 0.05) between the indicated treatment and the 37°C unstimulated tissues. (ANOVA P values were <0.001 for HSP20 phosphorylation, 0.02 for MRLC phosphorylation, and <0.001 for force.)

Compared with unstimulated tissues, 10 µM histamine stimulation significantly increased both contractile force and suprabasilar MRLC phosphorylation without altering Ser16-HSP20 phosphorylation. Prior heat pretreatment at 44.5°C heat pretreatment reduced 10 µM histamine-induced contraction; however, histamine-induced increases in suprabasilar MRLC phosphorylation did not significantly differ from the histamine-induced 37°C control response (Fig. 3). These results suggest that the attenuation of histamine-induced force by 44.5°C heat pretreatment cannot be explained by reduced MRLC phosphorylation. These data suggest that heat pretreatment induced force suppression rather than deactivation as the mechanism for reduction in force.

In unheated tissues, addition of 10 µM nitroglycerin to histamine-stimulated tissues induced a relaxation associated with an increase in Ser16-HSP20 phosphorylation to 0.19 ± 0.04 mol Pi/mol HSP20, a value similar to that observed previously (18). In 44.5°C heat-pretreated tissues, nitroglycerin induced a relaxation that was associated with an additional increase in Ser16-HSP20 phosphorylation to 0.59 ± 0.03 mol Pi/mol HSP20.

Effect of other heat pretreatment on Ser16-HSP20 phosphorylation. We evaluated two other heat treatment protocols. 1) Tissues were pretreated at 44.5°C for 2 h, contracted with 10 µM histamine, and then relaxed by addition of 10 µM nitroglycerin. This protocol generated Ser16-HSP20 phosphorylation values of 0.37 ± 0.04 mol Pi/mol HSP20, which was significantly greater than that observed in unheated tissues. Force did not significantly differ from that observed without heat pretreatment (Fig. 2). 2) Tissues were pretreated at 41°C for 4 h and contracted with 10 µM histamine. This protocol generated Ser16-HSP20 phosphorylation values of 0.09 ± 0.03 mol Pi/mol HSP20 and force of 0.84 ± 0.17% of 109 mM K+ (both values not significantly different from unheated tissues).

Dependence of histamine-induced force on Ser16-HSP20 phosphorylation. The relation between mean Ser16-HSP20 phosphorylation and mean force from all the tissues that were stimulated with histamine (with or without nitroglycerin) is shown in Fig. 4. The heat pretreatment response (triangles and squares in Fig. 4) was similar to the nitroglycerin response (filled symbols in Fig. 4), suggesting a similar mechanism of action. The correlation between increases in Ser16-HSP20 phosphorylation and decreases in force suggests that Ser16-HSP20 phosphorylation could be mediating the reduction in force.


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  		Fig. 4.   Dependence of mean contractile force on mean Ser16-HSP20 phosphorylation in tissues maximally stimulated with histamine. Mean data demonstrate the relation between Ser16-HSP20 phosphorylation (plotted on a log scale) and contractile stress in tissues that were either stimulated with 10 µM histamine or stimulated with 10 µM histamine and relaxed with 10 µM nitroglycerin (H+NTG). Some tissues were untreated (data from Fig. 3), and some had been pretreated at 41°C for 4 h, 44.5°C for 2 h, or 44.5°C for 4 h (data from Fig. 3 and the text) as detailed in the legend of Fig. 3. All heat-treated tissues were returned to 37°C for 1 h before histamine stimulation. Overall there was a good inverse correlation between Ser16-HSP20 phosphorylation and contractile force with heat pretreatment and nitroglycerin-induced relaxation (R2 = 0.82). Values are means ± SE. Symbols without error bars represent errors smaller than the size of the symbol.

Effect of heat pretreatment on total HSP20 concentration. We evaluated the effect of heat pretreatment on total HSP20 immunostaining. Our experimental design involves dissection of multiple tissues from a given carotid artery. We loaded the homogenates from all the tissues from a given artery onto a common gel. This procedure allowed normalization of immunostaining after heat treatment with unheated tissues from the artery. Total HSP20 immunostaining was determined by summing the intensity from all phosphorylated and unphosphorylated species. Mean data showed that heat pretreatment at 44.5°C for 4 h increased HSP20 immunostaining by a small, but significant, 36 ± 14% compared with 37°C controls (paired t-test; n = 16).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

These data demonstrate that heat pretreatment of swine carotid artery increased Ser16-HSP20 phosphorylation and suppressed force, i.e., a reduction in force without a significant reduction in MRLC phosphorylation (Fig. 3). Heat pretreatment for a longer duration or higher level induced higher levels of Ser16-HSP20 phosphorylation and enhanced force suppression. Heat pretreatment increased Ser16-HSP20 phosphorylation more than nitroglycerin. The effects of heat pretreatment and nitroglycerin also appear to be additive, both on Ser16-HSP20 phosphorylation and force (Figs. 2 and 3). The relation between Ser16-HSP20 phosphorylation and force was similar with heat pretreatment and nitroglycerin (Fig. 4). These data suggest that Ser16-HSP20 phosphorylation is suppressing force regardless of the mechanism that increases its Ser16 phosphorylation.

Ser16-HSP20 phosphorylation has been shown to be one of several mechanisms responsible for cyclic nucleotide-induced relaxation. The molecular mechanism responsible for the HSP20-associated force suppression is not yet understood, but it may involve binding of phosphorylated HSP20 to thin filaments in a manner similar to troponin I (15). However, it is clear that mechanisms other than HSP20 can induce force suppression. Elevated extracellular Mg2+ concentration induced force suppression (4) without increases in Ser16-HSP20 phosphorylation (17). Furthermore, as noted in the introduction, there are other mechanisms whereby cyclic nucleotides reduce smooth muscle force via deactivation (i.e., reduced [Ca2+]i) rather than force suppression (reviewed in Ref. 14). It is possible that heat pretreatment may increase Ser16-HSP20 phosphorylation via increases in cyclic nucleotides or some other mechanisms; however, cyclic nucleotides were not tested in this study.

We found that heat pretreatment induced a small, but significant, increase in total HSP20 immunostaining. This suggests an increase in total HSP20 concentration that may involve increased production or reduced degradation of HSP20. Heat and other cellular stresses are known to induce synthesis of other heat shock proteins (25). The significance of increased total HSP20 immunostaining is unclear, but it could represent a cytoprotective response to heat or other cellular damage. For example, it is possible that HSP20 phosphorylation-dependent force suppression may prevent arterial damage caused by maximal contraction.

Our observation that heat pretreatment suppressed force may have clinical correlates. In systemic hypotension caused by vasodilatory shock, there is a general resistance to pressor agents (9). Shock is associated with high nitric oxide (NO) levels and fever: both cause HSP20 phosphorylation. It is possible that resistance to pressor agents in shock may be mediated by phosphorylated HSP20, although this will require further study. Similarly, profound hyperthermia, such as occurs in heat stroke and the neuroleptic malignant syndrome, can progress to systemic hypotension and cardiovascular collapse (20). Hyperthermia is known to increase endogenous production of NO (7). NO synthase has been shown to be activated by cytokines released during periods of cell stress (12, 22). It is possible that hyperthermia could induce Ser16-HSP20 phosphorylation via increases in NOS activity and increased NO concentration.

In summary, we found that heat pretreatment of swine carotid media is sufficient to increase Ser16-HSP20 phosphorylation and suppress force without addition of exogenous NO donors or forskolin. These effects were additive with the NO donor nitroglycerin.


    ACKNOWLEDGEMENTS

The authors thank Rongrong Fan, Roger Shih, and Marcia Ripley for technical support and Mike Kurilla for help with production of recombinant HSP20. Dr. Subah Packer graciously supplied the MRLC antibody. Smithfield (Smithfield, VA) donated the swine carotid arteries.


    FOOTNOTES

Grants from the Mid Atlantic American Heart Association and the Jeffress Trust supported this research.

Address for reprint requests and other correspondence: C. M. Rembold, Box 801395, Cardiovascular Div., Univ. of Virginia Health System, Charlottesville, VA 22908-1395 (E-mail: crembold{at}virginia.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.

March 29, 2002;10.1152/japplphysiol.00009.2002

Received 8 January 2002; accepted in final form 25 March 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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J APPL PHYSIOL 93(2):484-488
8750-7587/02 $5.00 Copyright © 2002 the American Physiological Society


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