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Am J Physiol Heart Circ Physiol 291: H638-H647, 2006. First published March 24, 2006; doi:10.1152/ajpheart.00144.2006
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Heat shock-induced cardioprotection activates cytoskeletal-based cell survival pathways

Hongguang Wei,3 Wendy Campbell,2 and Richard S. Vander Heide1,2,3

1Department of Pathology, John D. Dingell Veterans Affairs Medical Center, and Departments of 2Pathology and 3Medicine, Wayne State University Medical School, Detroit, Michigan

Submitted 8 February 2006 ; accepted in final form 8 March 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
To define better the subcellular mechanism of heat shock (HS)-induced cardioprotection, we examined the effect of HS, as well as selective expression of individual HS proteins (HSPs), on cell injury in neonatal rat ventricular myocytes (NRVM). HS was induced in NRVM by a rapid elevation of temperature to 42°C for 20 min followed by 20–24 h of recovery at 37°C. Other NRVM were infected with a replication-deficient adenovirus encoding HSP27 or HSP70. On the same day, all groups were subjected to metabolic inhibition (MI). Cell injury was assayed by measurement of the percentage of total lactate dehydrogenase released, the percentage of cells staining with trypan blue, or TdT-mediated dUTP nick-end labeling, whereas cell signaling was assayed by immunoblot analysis and coimmunoprecipitation. Before MI, the viability of all treated groups did not differ significantly from control NRVM. HS resulted in a significant increase in HSP70 and HSP27 expression. Infection with either virus caused a significant increase in selective HSP content compared with control NRVM. HS protected NRVM from injury. Selective expression of HSP27 or HSP70 alone was not protective in NRVM, but dual infection with both viral vectors (HSP27 + HSP70) was protective. HS and HSP27 + HSP70 expression caused increased paxillin localization in the membrane fraction, which persisted in response to MI, compared with control NRVM. HS increased the integrin-paxillin-focal adhesion kinase interaction, whereas targeted inhibition of focal adhesion kinase activity abolished the integrin-paxillin association and resulted in an increase in cell death. HS and HSP27 + HSP70 expression increased the association of members of the focal adhesion complex and protected NRVM against irreversible injury. Cytoskeletal-based signaling pathways at focal adhesion junctions may represent a unique pathway of cardioprotection.

heat shock protein; neonatal rat ventricular myocytes; metabolic inhibition

SEVERE MYOCARDIAL ISCHEMIA results in irreversible injury of myocytes, which is manifest classically as coagulation necrosis. Despite many years of active research, the exact series of subcellular events underlying the transition from reversible to irreversible injury remains elusive. It is well known that native heat shock (HS) protects against ischemic injury. Although numerous studies in a variety of model systems have shown that increased expression of selective individual HS proteins (HSPs), specifically, inducible HSP70 (HSP70i), protects ischemic myocardium (3, 12, 20–22), the precise subcellular mechanism whereby HSPs achieve cardioprotection is not clear. Unraveling the mechanism is complicated by the fact that native HS and/or nonselective activation of HSPs (i.e., chemical stimulators) may activate several members of the large HSP family and/or other cell-signaling proteins/pathways that may be cardioprotective through a different mechanism.

HSPs are known to function as "chaperone proteins," i.e., proteins that facilitate protein folding and translocation at the subcellular level (4). In addition, chaperone proteins play a role in the inhibition of apoptosis and necrosis, which could account for their cardioprotective effect. For example, HSP70, as well as other HSPs, inhibits caspase-dependent and caspase-independent apoptotic stimuli (7). HSP70 has been reported to block stress kinases, including JNK, whereas HSP70 and HSP90 have been shown to block the formation and activation of the Apaf-1 complex and, therefore, the subsequent activation of caspase-9 (7, 17). HSP90 is a chaperone for pyruvate dehydrogenase kinase (PDK1) and Akt, members of an important antiapoptotic pathway involving activation of phosphatidylinositol 1,4,5-trisphosphate (PIP3) kinase (6). Finally, HSP27 has been reported to inhibit cytochrome c-dependent activation of procaspase-9 (9).

Moreover, the HSPs are known to associate with cytoskeletal proteins. This interaction may be important for at least two reasons. 1) In simple cell systems, HSP27 has been shown to stabilize certain cytoskeletal structures, which in turn have been associated with increased resistance to stress (4, 11, 14). 2) Because irreversible ischemic injury in the myocardium is thought to involve critical lesions in the cytoskeletal support system (8, 15, 32, 38), proteins that could bind to and protect these critical proteins would be predicted to protect against lethal cell injury (8, 15, 32, 36, 38).

Focal adhesion kinase (FAK) has become recognized as a key mediator of cell survival signaling pathways. FAK is a nonreceptor protein tyrosine kinase that is localized to focal adhesions and costameres and is activated in response to cell adhesion, integrin clustering, and growth factor stimulation (29). FAK plays a critical homeostatic role, because it transduces extracellular matrix-derived survival signals (2). FAK binds to integrins (cell surface receptors) as well as several intracellular proteins that are important in signal transduction, including paxillin, tensin, p130CAS, talin, and vinculin (28, 33). Paxillin (cytoskeletal/adapter protein) and p130CAS (docking protein) act as docking/adapter proteins, which concentrate and facilitate cell-signaling proteins at the focal adhesion junction (28). The potential importance of this pathway in cell survival is highlighted by recent studies showing that disruption of the FAK-paxillin interaction localized at the focal adhesion has dire consequences for cell survival (34, 35).

Therefore, emerging evidence suggests that more than one potential target, perhaps a pathway of signaling molecules, could be responsible for the cardioprotective effects of HS and/or HSPs. However, it is difficult to define the role that signaling molecules might play in whole animal models of ischemic injury, because such models do not selectively increase or decrease expression exclusively in the myocytes. Selective overexpression of HSPs in a cultured ventricular myocyte system provides a reproducible model system whereby subcellular targets and/or binding proteins can be better examined.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
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All experiments conformed to the standards outlined in the National Institutes of Health "Guide for the Care and Use of Laboratory Animals" (DHEW Publ. No. NIH 85-23, revised 1985). The animal protocol for these studies was approved by the Animal Investigation Committee of Wayne State University (A04-12-05), which is accredited by Association for Assessment and Accreditation of Laboratory Animal Care International.

Isolation of neonatal myocytes. For each isolate, the ventricular portion of 9–12 hearts from 1- to 2-day-old rats was pooled and gently agitated overnight at 4°C with trypsin (0.1 g in 100 ml) in Hanks’ balanced salt solution. On the next day, the myocytes were digested further with serial incubations in collagenase (0.1 g in 100 ml of Hanks’ balanced salt solution). The final cell isolate was centrifuged for 3 min at 1,000 rpm at 4°C. The resulting supernatant was discarded, and the cells were resuspended in ice-cold DMEM, transferred to a 50-ml conical tube, and centrifuged again for 3 min at 1,000 rpm at 4°C. The resulting supernatant was discarded, and the cell yield was determined using a hemocytometer.

Cell culture. After isolation and purification, the myocytes were resuspended in DMEM [supplemented with 10% FBS and containing antibiotics (penicillin-streptomycin and gentamicin) to inhibit bacterial growth] and cultured on 100-mm plates for 30 min to reduce fibroblast contamination. After they were preplated, the cells were cultured in standard six-well plates or 35-mm dishes (Corning, Corning, NY). After 24 h of culture, the medium was changed to DMEM without FBS. Immunofluorescent staining for muscle-specific actin confirmed that >95% of the plated cells were myocytes (data not shown).

Construction of recombinant adenovirus. The 0.76-kb coding region of the human HSP27 cDNA [provided by Drs. L. Weber and E. Hickey, University of Nevada (Reno)] was used to make the construct for the HSP27 virus. The appropriate fragments were cloned between the enhancer/promoter of the cytomegalovirus immediate-early genes and the simian virus 40 polyadenylation signal of the pACCMV. The pLpA shuttle vector was provided by Dr. R. Gerard. Replication-deficient adenovirus was generated through homologous recombination of two plasmids (pJM17, a bacterial plasmid that contains the full-length adenoviral genome, and the shuttle vector) after cotransfection into E1-transformed human embryonic kidney (HEK)-293 cells to produce E1-deleted adenovirus encoding the appropriate transgene. The confluent HEK-293 cells were infected, harvested, and concentrated through CsCl ultracentrifugation to generate viral stocks. The viral stocks were desalted through a Sepharose CL4B column (Sigma Chemical) into Tris-buffered solution, plaque titered, divided into aliquots, and stored at –70°C with 10% glycerol.

Rat HSP70i was cloned into the multiple cloning site of the adenoviral shuttle plasmid pACCMVpLpASR. This plasmid contains the 5' end of the adenovirus serotype 5 genome (map units 0–17), where the E1 region has been substituted with the human cytomegalovirus enhancer/promoter followed by the multiple cloning sites from pAC19 and the polyadenylation region from simian vacuolating virus 40. The resulting plasmid was cotransfected with pJM17, a plasmid that contains the complete adenovirus 5 genome, into the HEK 293-cell using the calcium phosphate transfection method. Infectious viral particles containing the inserted HSP70 were generated by in vivo recombination in the HEK-293 cells and isolated as single plaques 7 days later. The viral stocks were then desalted through a Sepharose CL4B column into Tris-buffered solution, plaque titered, divided into aliquots, and stored at –70°C with 10% glycerol.

FAK-related nonkinase (FRNK) virus was a generous gift from Dr. Allen Samarel (Loyola University Medical School, Chicago, IL).

Experimental design/protocol. Myocytes were divided into three main groups: control, adenovirus infected, and heat shocked. Data from at least three separate cell isolations were averaged for all cell injury data [i.e., lactate dehydrogenase (LDH) release and trypan blue (TB) counts]. Western blot data were generated in parallel from the isolates that were used to derive the cell injury data.

Adenoviral infection. Myocytes were infected for 48 h with replication-deficient adenovirus containing the cDNA of HSP70i, HSP27, or FRNK (see above). Control myocytes were cultured without adenovirus infection or with an empty adenovirus (without cDNA). After 48 h of incubation, the myocytes were each split into two equal halves (i.e., 3 wells from each 6-well plate). One-half of each plate was used for Western blot analysis of protein expression and cell-signaling analysis and the other for assay of cardioprotection from simulated ischemic injury.

Induction of HS. In the other major experimental group, HS was induced by a rapid increase in the temperature of the culture plates to 42°C for 20 min followed by 20–24 h of recovery at 37°C. Control myocytes were cultured in parallel at 37°C but were not subjected to HS.

Metabolic inhibition. After infection or HS, all groups were subjected to simulated ischemia and/or reperfusion via a metabolic inhibition (MI) protocol. To induce MI, the culture medium was removed and replaced with fresh PBS containing 3.0 mM iodoacetic acid to inhibit glycolysis and 3.0 mM amobarbital to inhibit mitochondrial respiration. Pilot experiments were undertaken to study cell injury/cell protection. The results showed that reproducible significant amounts of irreversible oncosis developed after 150 min of MI (data not shown). In some experiments, NRVM were assayed for cell death at the end of MI; in others, the ischemic buffer was exchanged with fresh hypotonic oxygenated culture medium containing glucose without the chemical inhibitors for 30 min to simulate reperfusion. [Hypotonic buffer is used to expose irreversible injury (38).] The resulting proportion of live and dead cells was determined by measurement of LDH release with a commercially available kit (Sigma, St. Louis, MO) or assessment of the number of TB-positive cells (see Cell injury assay).

Western blot procedures. Myocytes were harvested for protein analysis by standard Western blot techniques. Briefly, the cells were washed twice with PBS, lysed with whole cell lysis buffer (50 mM Tris·HCl, pH 8.0, 150 mM NaCl, 0.02% sodium azide, 100 µg/ml PMSF, 1 µg/ml aprotinin, and 1% Triton X-100), and stored at –20°C. For analysis of membrane fractions, myocytes were separated into three fractions according to our previous methodology (40). Western blot analysis of the membrane fraction showed the absence of positive staining for myosin heavy chain compared with cytoskeletal and cytosolic fractions and Na+-K+-ATPase enrichment compared with the other two fractions, confirming the nature of our membrane fraction (data not shown). Samples were thawed, and each lane was loaded with an equal amount of protein (as determined by bicinchoninic acid protein assay) and subjected to SDS-protein electrophoresis. Equal protein loading was confirmed in all blots by amido black staining of the nitrocellulose membrane and in selected blots by anti-{alpha}-actin staining. After electrophoresis, the proteins were transferred to nitrocellulose membranes, which were blocked with 5% nonfat milk in Tris-buffered saline + Tween 20 and then incubated with one of the following primary antibodies: 1) monoclonal anti-mouse primary antibody to HSP70 (catalog no. SC-24, Santa Cruz Biotechnology), 2) polyclonal anti-goat HSP27 antibody (catalog no. SC-1048, Santa Cruz Biotechnology), 3) monoclonal anti-mouse paxillin antibody (catalog no. 610052, Transduction Laboratories), 4) rabbit anti-FAK antibody (catalog no. 06-543, Upstate Biotechnology), or 5) rabbit anti-FAKpY397 antibody (catalog no. 44-624G, Biosource). The membranes were incubated with goat anti-mouse (HSP70 and paxillin) or donkey anti-goat (HSP27) IgG (Santa Cruz Biotechnology) at a 1:1,000 dilution, and final protein expression was detected using a standard chemiluminescence system (Amersham, Arlington, IL).

Coimmunoprecipitation. Myocytes were cultured on standard six-well or 35-mm culture plates. After infection with virus, induction of HS, or induction of MI, cells from two 35-mm dishes were rinsed with PBS at room temperature, and immunoprecipitation lysis buffer [150 mM NaCl, 1% Triton X-100, 50 mM Tris·HCl, pH 7.5, and a cocktail of protease inhibitors (10.4 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBST), 8 µM aprotinin, 0.2 mM leupeptin, 0.4 mM bestatin, 0.15 mM pepstatin A, and 0.14 mM E-64) and phosphatase inhibitors (1 mM Na3VO4 and 10 nM okadaic acid)] was added to the cultured myocytes. The myocytes were scraped from the culture dish, passed through a 26.5-gauge needle, and placed in a microcentrifuge tube. The lysates were incubated on ice for 45 min and centrifuged at 10,000 g for 10 min at 4°C. Approximately 0.4 ml of the supernatant containing equal amounts of protein (confirmed by amido black staining of nitrocellulose membranes) was incubated with 4 µl of mouse polyclonal anti-beta1-integrin antibody (catalog no. 610467, BD Transduction) for 3 h at 4°C. Protein A/G-agarose (25 µl) was added, and the lysate was rocked gently overnight at 4°C. The cell pellet was collected by centrifugation at 1,000 g for 30 s at 4°C and washed four times with immunoprecipitation lysis buffer. After the final wash, the supernatant was discarded, and the pellet was resuspended in sample buffer and subjected to SDS-PAGE. The separated proteins were transferred to nitrocellulose membranes and probed for paxillin with the mouse monoclonal antibody (1:5,000 dilution) or anti-FAK antibody (Tyr397; 1:1,000 dilution) followed by secondary antibody conjugated to peroxidase (1:1,000 dilution; Roche Diagnostics). Membranes were probed with the same chemiluminescence system used for routine Western blots. For quantitative Western blot analysis, films were scanned and data are reported in arbitrary units and/or as percent elevation over the control cells.

Cell injury assay. In some experiments (to determine the amount of cell injury induced by the MI protocol followed by reperfusion), LDH release was measured as an indication of irreversible (lethal) cell injury. LDH is normally retained in the cytosol until the sarcolemmal membrane is ruptured; then it is free to diffuse into the surrounding medium. After completion of the MI protocol, each well of the culture dish was assayed for LDH. The attached cells in each well were extracted, and the resulting extracts were analyzed for LDH. Total LDH was considered the sum of the LDH released into the medium during the MI protocol plus the residual LDH in the attached cells. The percent LDH release was calculated by dividing the amount released into the medium by the total LDH (released + cell content) for each experimental group. This allowed the amount of LDH release to be normalized for the number of cells in each dish. Each well was assayed in duplicate, and the results were averaged for each group. In other experiments, TB or TdT-mediated dUTP nick-end label (TUNEL) staining was used as an indication of cell death. TB, a vital dye excluded by viable cells with intact cell membranes, is well documented as an indicator of lethal oncotic cell death. Briefly, the cells were gently removed from the culture surface by treatment with trypsin and neutralized with serum; then a small volume of 1% TB was added to cells from each dish. TUNEL staining was performed as indicated in the manufacturer’s instruction kit (ApopTag, Chemicon International). For TB staining, the cells were counted immediately after addition of the dye to ensure exclusion of nonspecific stained cells, which appear over time. A cell was considered TB positive when the entire cytoplasm was diffusely stained any shade of blue and TUNEL positive when unequivocal bright nuclear staining was identified. The total number of viable cells and positive (dead) cells was counted using an inverted immunofluorescent microscope (model TE300, Nikon), and the resulting data are presented as the percentage of TB- or TUNEL-positive cells. All cell counts were performed in a blinded fashion by two different observers to ensure objectivity.

Statistics. Because each myocyte isolate generates control and experimental groups, each isolate serves as its own control. Values are means ± SE. For Western blot data, films were scanned and data are reported in arbitrary units and/or percent increase over the control cells. Western blot data originate from one gel. Statistically significant differences between groups were tested using ANOVA followed by correction for multiple comparisons or the paired t-test (when only 2 groups were compared). In all analyses, P < 0.05 was considered statistically significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of HS and adenoviral infection on HSP expression in NRVM. HS caused a robust increase in HSP70 expression (Fig. 1A). HS also increased HSP27 expression, but to a lesser degree than HSP70 expression (Fig. 1A). HS induced a large increase in HSP70 expression in all three subcellular compartments (cytosolic, membrane, and cytoskeletal; Fig. 1B). Infection with HSP27 adenovirus increased HSP27 expression in all three compartments but did not significantly increase HSP70 expression. Infection with HSP27 + HSP70 resulted in coexpression of HSP27 and HSP70 in the same myocyte population (Fig. 1C).


Figure 1
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  		Fig. 1. Heat shock (HS) expression in neonatal rat ventricular myocytes (NRVM). A: expression of HS proteins 27 and 70 (HSP27 and HSP70) in lysates from control NRVM and NRVM subjected to HS. Lysate from control NRVM showed low baseline HSP27 and HSP70 expression. HS induced increases in HSP70 and HSP27 expression. Results are representative of 3 similar Western blots. B: HSP27 and HSP70 expression from NRVM subjected to HS or infected with HSP27 virus (V). HS increased expression of HSP27 and HSP70 in all cell compartments (cytosolic, membrane, and cytoskeletal), with HSP70 showing a more robust response. Infection with HSP27 adenovirus induced a selective increase in HSP27, but no appreciable increase in HSP70, expression in all compartments. Multiplicity of infection (MOI) was 20 for HSP27 virus. C, control. Results are representative of 3 similar Western blots. C: expression of HSP27 and HSP70 in lysates from NRVM infected with HSP27 and HSP70 vectors. When NRVM were infected with each vector alone, expression was selective for the appropriate HSP. However, in NRVM infected with both vectors, expression of HSP27 and HSP70 increased simultaneously in the same myocyte population. Western blot (membrane) was stained with HSP27 and HSP70 primary antibodies to show simultaneous HSP expression. Multiplicities of infection were 20 and 4 for HSP27 and HSP70, respectively. Results are representative of 3 similar Western blots.

 
Effect of HS on response to lethal cell injury. First, we sought to confirm that HS would protect isolated NRVM from simulated ischemia-reperfusion injury. Previous studies showed that incubation of myocytes with inhibitors of glycolysis (iodoacetic acid) and mitochondrial respiration (amobarbital) causes significant irreversible injury (36, 37, 39). Myocytes were subjected to HS (see MATERIALS AND METHODS) and allowed to recover for 20–24 h before the onset of ischemia-reperfusion. After 150 min of MI followed by simulated reperfusion, control myocytes released 34.8 ± 3.1% of total LDH. In contrast, myocytes subjected to HS before ischemia-reperfusion released significantly less LDH: 26.9 ± 2.4% (n = 10, P < 0.004; Fig. 2A).


Figure 2
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  		Fig. 2. Effect of native HS on cell injury. A: NRVM were subjected to HS and then to 150 min of metabolic inhibition (MI) followed by 30 min of reperfusion. NRVM subjected to HS before simulated ischemia-reperfusion (Isch/Rep) injury retained more lactate dehydrogenase (LDH), indicating less lethal injury. *Significantly different from control (P ≤ 0.004, by paired t-test). B: NRVM were subjected to HS and then to 10 or 60 min of MI followed by 30 min of reperfusion. Apoptosis was significant after 10 min of MI followed by reperfusion and increased when MI was extended to 60 min. NRVM subjected to HS before simulated ischemia-reperfusion injury showed fewer TdT-mediated dUTP nick-end label-positive cells, indicating less myocyte injury. *Significantly different from control.

 
To determine whether HS also protected against apoptotic cell death, using the TUNEL assay as an indicator of apoptotic death, we conducted additional experiments. Ten minutes of MI followed by reperfusion resulted in significant apoptotic cell death (20.0 ± 3.2%). As expected, as the duration of MI was increased to 60 min, the amount of apoptotic cell death increased: 46.4 ± 1.25 and 20.0 ± 3.2% at 10 and 60 min, respectively. When myocytes were exposed to HS before MI, the amount of apoptotic cell death was significantly inhibited at 10 and 60 min: 20.0 ± 3.2 and 8.7 ± 2.8% at 10 min for control and HS, respectively (P < 0.02), and 46.4 ± 1.3 and 17.6 ± 3.3% at 60 min for control and HS, respectively (P < 0.05).

Effect of adenoviral-mediated HSP expression on lethal cell injury. One of the primary aims of the study was to determine more precisely the subcellular mechanism of HSP-induced cardioprotection. Therefore, the ability to achieve cardioprotection with selective expression of single HSPs was an important prerequisite for further meaningful subcellular investigation. Infection of NRVM with HSP27 or HSP70 adenovirus resulted in a marked and selective increase in the appropriate HSP (Fig. 1C). However, after 150 min of MI followed by reperfusion, LDH release was not significantly different between control myocytes and myocytes selectively expressing HSP27 or HSP70 (data not shown). To confirm the lack of protection, we conducted additional experiments in which NRVM were subjected to 150 min of MI and cell injury was assayed by TB exclusion, rather than by LDH release. Similar to the LDH data, cells infected with HSP27 or HSP70 alone did not show significant protection (i.e., reduction in TB-positive cells) compared with control NRVM [P = not significant (NS) by ANOVA; Fig. 3A].


Figure 3
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  		Fig. 3. Effect of selective HSP overexpression on lethal cell injury. A: NRVM were infected with a single virus designed to increase expression of HSP27 or HSP70 and then to 150 min of MI. Positive staining for trypan blue (TB) indicates dead cells. Despite significantly higher levels of HSP expression, infection with HSP27 or HSP70 virus before MI did not significantly protect against lethal cell injury [P = not significant (NS), by ANOVA]. B: NRVM were infected with both viral vectors simultaneously to increase expression of HSP27 and HSP70 in the same population and then to 150 min of MI. In contrast to A, dual expression of HSP27 and HSP70 resulted in significant protection against the same 150-min lethal insult. *Significantly different from control (P ≤ 0.04, by paired t-test).

 
Effect of HSP27 + HSP70 expression on lethal cell injury. Because HS provided significant protection against lethal cell injury in NRVM and selective expression of HSP27 or HSP70 alone did not, we reasoned that HS-induced protection might be more complex than an increase in the expression of a single HSP (at least HSP27 and/or HSP70). Therefore, we infected NRVM with HSP27 + HSP70 adenoviral vectors to determine whether expression of more than one HSP simultaneously in the same myocyte would provide better protection than either HSP alone. Dual infection resulted in substantially increased expression of HSP27 and HSP70 (Fig. 1C). Furthermore, myocytes expressing both HSPs were protected compared with control cells when subjected to 150 min of MI (35.6 ± 2.4 vs. 28.0 ± 1.4% for control vs. dual expression, P ≤ 0.04; Fig. 3B).

Effect of HS on beta1-integrin/paxillin binding. Integrins are cell surface receptors known to be physically linked to certain members of the focal adhesion complex, including FAK and paxillin (10). Paxillin is physically linked to the cytoplasmic tail of the transmembrane-spanning integrin receptors. Therefore, activation of integrin receptors should increase the integrin-paxillin association. If focal adhesion-related molecules such as paxillin are important in cell survival signaling, they should be assembled/activated by external stress. To determine whether integrin was present and linked to other members of the focal adhesion complex, we compared the effect of HS on integrin-FAK and integrin-paxillin interactions. As shown in Fig. 4, HS increased the integrin-FAK interaction. Moreover, the Western blot in Fig. 4 shows that HS increased the amount of FAK phosphorylated at Tyr397, which occurs when FAK becomes activated. Figure 5 shows that HS also increased the integrin-paxillin interaction. The sum of these results is consistent with myocyte stress causing assembly of an activated signaling complex that localizes at the cell membrane-adhesion complex.


Figure 4
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  		Fig. 4. Effect of myocardial stress on integrin-associated focal adhesion kinase (FAK) activation in NRVM. Control NRVM and NRVM subjected to HS were exposed to 10 min of MI. Cell extracts from each indicated condition were immunoprecipitated (IP) with antibody to beta1-integrin and then probed for FAK phosphorylated at Tyr397 (indicative of activated FAK). Results are representative of 3 similar Western blots (WB). NRVM subjected to prior stress (HS) contained significantly more integrin-associated activated FAK after 10 min of MI than control NRVM.

 

Figure 5
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  		Fig. 5. Effect of myocardial stress on integrin/paxillin interaction in NRVM. Control NRVM and NRVM subjected to HS were exposed to 10 min of MI. Cell extracts from each condition were immunoprecipitated with antibody to beta1-integrin and then probed for paxillin. Results are representative of 3 similar Western blots. NRVM subjected to prior stress (HS) contained significantly more integrin-associated paxillin after 10 min of MI than control NRVM.

 
Effect of HS and viral infection on paxillin localization in response to MI. Paxillin is known to be important in cell adhesion and, recently, has been hypothesized to occupy a central location in cell-signaling pathways. On the basis of the results described above, we investigated further the biology of paxillin in response to stress. In response to HS, paxillin was more prevalent in the membrane fraction (Fig. 6A). Next, we compared the effect of 10 min of MI on membrane paxillin levels in control myocytes and myocytes subjected to prior HS. Compared with control myocytes, the HS-induced paxillin effect persisted after onset of the MI insult. The total cell content of paxillin did not change over 30 min of ischemia in the control and HS groups (Fig. 6B). The data suggest that HS-induced cardioprotection is not secondary to inhibition of protein degradation but likely results from stabilization of critical signaling molecules.


Figure 6
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  		Fig. 6. Effect of myocardial stress on paxillin content and localization in NRVM. A: control NRVM and NRVM subjected to HS were exposed to 10 min of MI. Results are representative of 3 similar Western blots. NRVM subjected to HS contained significantly more paxillin in the membrane fraction at earlier time points after the onset of MI than control NRVM. B: control NRVM and NRVM subjected to HS were exposed to 30 min of MI. Results are representative of 3 similar Western blots. In contrast to the membrane fraction (A), MI did not have a significant effect on total paxillin content in NRVM subjected to HS or control NRVM (P = NS by ANOVA). C: paxillin content in the membrane fraction of NRVM infected with viral vectors designed to increase expression of HSP27 alone, HSP70 alone, or HSP27 + HSP70. Compared with HSP27 or HSP70 alone, NRVM expressing both HSPs together (HSP27/HSP70) contained significantly more paxillin in the membrane fraction at earlier time points after the onset of MI than NRVM expressing either HSP alone (by t-test).

 
Infection with HSP27 or HSP70 alone did not affect paxillin expression in the membrane fraction at baseline or during a subsequent MI stress (Fig. 6C). However, infection with HSP27 + HSP70 resulted in a dramatic increase in membrane-associated paxillin (Fig. 6C). The paxillin changes (only in HS- and HSP27 + HSP70-infected myocytes) correlated with protection from lethal injury, suggesting that the paxillin change may be associated with a cardioprotective response. Because the membrane fraction (as prepared in our laboratory) contains the sarcolemmal membrane as well as other proteins attached to the sarcolemmal membrane and potentially important in intracellular signaling pathways, the data are consistent with paxillin being assembled into a membrane-based signaling complex.

Effect of FAK disruption on association with paxillin and ischemic cell death. FAK is known to interact/associate with integrin receptors and paxillin in the focal adhesion complex. Because the above-mentioned data suggest that a stress-induced shift in paxillin localization is associated with increased cell survival, it would be predicted that disruption of paxillin from the hypothesized signaling complex would prevent any potential cardioprotective effect of paxillin and may even worsen cell survival in response to a lethal insult. The data in Fig. 7A show that infection of NRVM with FRNK virus, which contains an FAK-related nonkinase and, therefore, functions as a competitive inhibitor of FAK (13), downregulates activated FAK compared with NRVM infected with empty virus. Treatment with FRNK virus disrupted the normal association between beta1-integrin and paxillin (P < 0.02; Fig. 7B). Infection with FRNK adenovirus had no detrimental effect on cell viability in the absence of MI compared with empty virus: 29.5 ± 3.5 and 26.3 ± 1.0% for empty virus and FRNK virus, respectively, at 48 h of infection (n = 3, P = NS; Fig. 7C). However, infection of NRVM with FRNK virus resulted in significantly more injury in response to 60 min of MI than infection of NRVM with empty virus: 64.3 ± 2.7 and 50.3 ± 2.4%, respectively (n = 4, P < 0.05; Fig. 7C).


Figure 7
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  		Fig. 7. Effect of FAK-related nonvirus kinase (FRNK) infection on FAK activation (A), integrin-paxillin interaction (B), and cell death (C) in NRVM. A: NRVM were infected with FRNK or empty virus (control). FRNK-infected NRVM contained substantially less activated FAK than NRVM infected with empty virus. Amount of total FAK in both groups was not different (not shown). B: NRVM were infected with FRNK or empty adenovirus (control), and cell extracts were immunoprecipitated with antibody to beta1-integrin and then probed for paxillin. Results are representative of 3 similar Western blots. Treatment with FRNK virus disrupted the normal association between beta1-integrin and paxillin. C: NRVM were infected with empty virus or FRNK virus and cultured under aerobic conditions (–MI, left) for 48 h, and cell death was assayed using trypan blue permeability. FRNK adenovirus did not adversely affect NRVM compared with empty virus. NRVM infected with FRNK or empty virus were exposed to 60 min of MI (right), and cell death was assayed by trypan blue permeability. NRVM infected with FRNK suffered more lethal injury than NRVM infected with empty virus, suggesting that sustained integrin-FAK-paxillin interaction is important in maintaining cell viability under conditions of potentially lethal stress (n = 4, P < 0.05).

 

   DISCUSSION
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 ABSTRACT
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The results of the present study show significantly less injury in myocytes exposed to HS before sustained MI than in myocytes exposed only to the sustained metabolic insult. This cardioprotection was from traditional oncotic cell death as well as apoptotic cell death (Fig. 2). At least one previous study in isolated myocytes showed that HS, rather than selective adenoviral-induced overexpression, induces cardioprotection (42). As expected, we measured a robust increase in HSP70 expression, as well as a smaller, but significant, increase in HSP27 expression (Fig. 1) (25). Our results and those of previous studies suggest that the cardioprotective effect of HS at least correlates with and may be directly due to increased expression of HSP27 and/or HSP70, but the subcellular mechanism is not clear.

Role of focal adhesion complex/paxillin in HS-induced protection. The most intriguing finding of the present study was the greater amount of FAK and paxillin associated with beta-integrin in myocytes protected by HS than in those that did not show protection (i.e., control, non-HS myocytes). Furthermore, the increase in FAK and paxillin associated with beta-integrin in protected cells persisted longer throughout a subsequent episode of sustained MI than in non-HS-protected myocytes (Figs. 4 and 5). These results suggest that HS causes a change in paxillin localization and/or FAK activation and that this change is at least related to the underlying pathway and/or mechanisms of cardioprotection or may even be an integral part of a signaling pathway leading to protection.

It is well established that the sarcolemmal membrane contains numerous receptors and potential signaling molecules and serves as a "gathering point" for docking proteins such as paxillin, FAK, talin, and vinculin. FAK, paxillin, and vinculin are commonly seen interacting with each other at focal adhesion complexes. Furthermore, FAK has been linked to other signaling molecules, which in turn results in activation of protein kinase B (Akt). Akt has well-known antiapoptotic actions as well as recently described cardioprotective effects (5, 30). Therefore, it is possible that HS (or potentially any forms of myocardial stress) causes assembly of a membrane-based signaling complex that activates cardioprotective pathways or sustains activation of cell survival pathways after episodes of stress (i.e., ischemia). This hypothesis would predict that other forms of protection would produce similar changes in FAK and paxillin and, furthermore, that disruption of paxillin and/or FAK would interrupt the potential cell survival pathway and, thereby, exacerbate ischemic injury.

Effect of selective HSP expression on lethal cell injury and paxillin localization. We sought to determine whether other cardioprotective interventions would have a similar effect on paxillin. On the basis of the increases in HSP27 and HSP70 mediated by HS-induced protection, we determined whether selective expression of HSP27 or HSP70 alone would be cardioprotective in our model system. However, adenoviral-mediated expression of HSP27 or HSP70 alone did not achieve cardioprotection in our model system (Fig. 3A). Others have shown that selective expression of HSP27 is protective in adult, but not neonatal, isolated myocytes (22), so it is not surprising that no protection was measured in HSP27-infected myocytes. The reason for lack of protection with HSP70 expression in NRVM is not clear. One possible explanation may lie in the nature of the model system. In this study, MI was used to simulate ischemia. Many of the previously published studies showing protection with overexpression of HSP70 in cultured neonatal myocytes utilized hypoxia and reoxygenation. The combination of inhibitors used in this study caused a rapid and profound depletion of ATP levels within 10 min of exposure (data not shown). Hypoxia-reoxygenation reduces energy levels over a much longer duration. Therefore, it is possible that rapid reduction of energy levels may somehow affect the ability of HSPs to protect. However, our model system is capable of utilizing MI to measure a significant protective effect as shown with HS (Fig. 2). Nevertheless, the expression of either virus alone did not result in a significant change in paxillin localization. These results generated further intrigue regarding the correlation between altered paxillin localization and cardioprotection.

Effect of dual HSP expression on lethal cell injury and paxillin localization. One potential explanation for the lack of protection in myocytes infected with HSP27 or HSP70 alone is that HS-induced cardioprotection is more complex than simply an increase in expression of individual HSPs. Because we had both viral reagents, we decided to test this hypothesis by infecting NRVM with HSP27 + HSP70 to achieve simultaneous expression of both HSPs in the same myocyte. Coinfection with both viral reagents resulted in a significant increase in HSP27 and HSP70 in the same population (Fig. 1C). Furthermore, dual infection resulted in protection against MI injury (Fig. 3B). Most interestingly, the anticipated shift in paxillin localization in response to infection and subsequent MI was observed in myocytes infected with HSP27 + HSP70. These results are consistent with all the other discussed results; i.e., when protection from lethal injury is present in this model system, it correlates with a change in paxillin localization in response to MI.

Effect of paxillin disruption on lethal cell injury and paxillin localization. The experiments utilizing selective expression of HSPs confirmed the observations from the HS experiments; i.e., protected myocytes exhibit a shift in paxillin localization to the membrane fraction that persists during exposure to MI. Furthermore, the virus experiments confirmed that, without protection, no alteration in paxillin localization is identified (i.e., experiments with HSP27 or HSP70 alone). However, it would also be predicted that disruption of paxillin and/or its ability to bind to and activate partner proteins would prevent activation of the proposed cell survival signals and, therefore, exacerbate lethal/ischemic injury. Therefore, we decided to take advantage of other members of the focal adhesion complex that bind to paxillin, specifically, FAK. FAK is a membrane-bound nonreceptor tyrosine kinase that is known to interact with paxillin. Inhibition of FAK binding to paxillin and/or inhibition of FAK activity should prevent activation of potential downstream cardioprotective and/or survival signaling partners. To inhibit FAK activation of paxillin, we used an adenovirus expressing FRNK; the noncatalytic COOH-terminal region of FAK acts as a competitive inhibitor of FAK protein (13). The results shown in Fig. 7 documented that infection with the FRNK virus prevented FAK activation as well as normal interaction between beta1-integrin and paxillin. If paxillin is prevented from binding/activating its normal binding partners, then we would predict that the response to lethal injury would be intensified. Indeed, the extent of cell death in response to MI was greater in FRNK- than in empty virus-infected myocytes (Fig. 7C).

Possible mechanism(s) of HS/HSP-induced cardioprotection. The data from this study strongly implicate paxillin as an important biological/molecular marker that may be causally related to the observed cardioprotection. However, whether the HS-induced effects on paxillin and the corresponding cardioprotection reported in this study are causally related is not known. One possible mechanism of protection (see above) would involve linking of FAK to other signaling molecules, which in turn would result in activation of protein kinase B (Akt). Akt has well-known antiapoptotic actions as well as recently described cardioprotective effects (5, 30). A recent study showed that paxillin could directly inhibit Src kinase-triggered cardiomyocyte apoptosis (24).

HSPs have also been reported to utilize their chaperone function to protect key members of known signaling pathways (27, 31). Specifically, HSP70 and HSP90 have been shown to protect members of the Src kinase family of nonreceptor tyrosine kinases, Raf, and other serine/threonine kinases (4). Coupled with recent data showing that protein kinase B (Akt) may play an important role in cell survival signaling (1, 16, 23, 26, 41), it is possible that HS (and, by extrapolation, generalized stress) may limit or reduce lethal injury through HSP-mediated protection of key signaling molecules involved in cell survival and/or antiapoptotic pathways. Such candidate proteins are many and include Akt, BAD, Bcl, JNK, endothelial nitric oxide synthase, PI3-kinase, glycogen synthase, paxillin, FAK, and gp130. However, HSP90 expression/localization was not measured in this study.

It may be that HSPs act directly to protect members of a cytoskeletal-based cell survival pathway. Recently, HSP72 was shown to interact with paxillin and facilitate the reassembly of focal adhesions in kidney epithelial cells during recovery from ATP depletion (19). HSP72 was recently shown to inhibit FAK degradation in ATP-depleted renal epithelium (18). Although our results suggest that protection and/or stabilization of signaling molecules is more important than inhibition of protein degradation in ventricular myocytes, we did not directly measure protein degradation.

In summary, exposure to HS induces a protective response that leads to less oncotic and apoptotic cell death in NRVM. Furthermore, HS is associated with a shift in paxillin localization to the membrane fraction and assembly of an integrin-paxillin-FAK signaling complex, both of which persist during a subsequent period of sustained MI. Interventions that did not result in cardioprotection (i.e., increased expression of HSP27 or HSP70 alone) did not show any alteration in paxillin localization. Infection with HSP27 + HSP70 restored the paxillin effect and cardioprotection. Infection with FRNK, a competitive inhibitor of FAK, resulted in inhibition of FAK activation and disruption of paxillin binding to beta1-integrin, which exacerbated lethal injury. We conclude that HS-induced cardioprotection may result from direct activation of prosurvival/antiapoptotic signaling pathways and/or utilization of the chaperone function of HSPs to protect critical members of intracellular prosurvival/antiapoptotic signaling pathways.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
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 GRANTS
REFERENCES
 
This research was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-59563-A2 (to R. S. Vander Heide).


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. S. Vander Heide, Dept. of Pathology, Wayne State Univ. Medical School, 540 East Canfield Ave., Detroit, MI 48201 (e-mail: rvanderh{at}med.wayne.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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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