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Am J Physiol Heart Circ Physiol 292: H2220-H2226, 2007. First published January 5, 2007; doi:10.1152/ajpheart.01080.2006
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Influence of PKC-{alpha} overexpression on HSP70 and cardioprotection

Sonya D. Coaxum, Tina M. Griffin, Jody L. Martin, and Ruben Mestril

Department of Physiology and Cardiovascular Institute, Loyola University Medical Center, Maywood, Illinois

Submitted 2 October 2006 ; accepted in final form 16 December 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Recent research has indicated that the protein kinase C (PKC) isoforms and the heat shock proteins (HSPs) are involved in cardioprotection. We have investigated the possible interaction between these two protein families. We have found that adenoviral-mediated expression of PKC-{alpha} in neonatal rat ventricular myocytes (NRVM) not only increases the expression of HSP70 but also protects against simulated ischemia-reperfusion. In addition, Western blots of PKC-{alpha}-infected NRVM indicated that other HSPs are not induced in the same manner as HSP70. In an effort to determine the mechanism of induction of HSP70 by PKC-{alpha}, we tested a chimeric construct that linked the luciferase reporter gene to the 5'-promoter region of HSP70 in myogenic H9c2 cells. When PKC-{alpha} was expressed, the 5'-promoter region of the HSP70 responded robustly, indicating that PKC-{alpha} induction of HSP70 expression is through transcription activation. Electrophoretic mobility shift assay determined that overexpression of PKC-{alpha}, PKC-{delta}, or PKC-{epsilon} did not induce activation of heat shock factor-1 (HSF-1). Therefore, induction of HSP70 by PKC-{alpha} is independent of heat shock factor-1 activation. We also measured cellular injury by assessing creatine kinase (CK) release from NRVM after simulated ischemia to determine cardioprotection. NRVM infected with the wild-type adenoviral construct AdwtPKC-{alpha} released 54% less CK than control NRVM. Experiments using small interfering RNA against HSP70 indicate that loss of PKC-{alpha}-induced HSP70 expression results in increased CK release or a loss of protection. Our results show that there is a close interaction between PKC-{alpha} and HSP70, independent of heat shock factor-1 activation, and that the protection conferred by PKC-{alpha} overexpression is mediated by the transcriptionally induced expression of HSP70.

heat shock proteins; neonatal rat ventricular myocytes; ischemia-reoxygenation; simulated ischemia

THE HEAT SHOCK PROTEINS (HSPs), also known as the stress proteins, have been well documented to be cardioprotective (21). In several studies in which a transgenic mouse approach was used, different HSPs, such as HSP70 (17, 20, 25, 26), HSP27 (11), {alpha}B-crystallin (27), and HSP32 (also known as heme oxygenase-1) (29, 30), have been shown to be cardioprotective against ischemia-reperfusion injury. However, the mechanism of HSP-mediated cardioprotection remains unclear.

Another protein family that has been found to be cardioprotective is the protein kinase C (PKC) family. The different PKC isoforms in cardiac tissue have distinct functions in the heart. Two of these isoforms, PKC-{delta} and PKC-{epsilon}, have clearly been shown to be cardioprotective (7, 8, 16, 31). In contrast, some recent studies have reported that PKC-{delta} activation increases damage during ischemia-reperfusion injury and may be associated with apoptosis (3, 10).

Interestingly, studies have suggested that PKC may mediate protection induced by heat stress (14, 15, 18). However, the mechanism of PKC involvement in HSP-mediated cardioprotection has not been elucidated. Nonetheless, there is strong evidence of an association between PKC and HSPs. For example, it has been reported that the overexpression of PKC-{epsilon} in transgenic mice upregulates HSP70 compared with nontransgenic mice (24). Furthermore, studies have shown that overexpression of HSP70 inhibits PKC activity and, subsequently, inhibits heat shock factor-1 (HSF-1) phosphorylation in human epidermoid A-431 cells (6). In addition, our previous study found that adenoviral-mediated expression of HSP70 and HSP90 in rat neonatal cardiomyocytes differentially modulated the expression of PKC isoforms (4).

Since it is clear that HSPs and PKC play a role separately in mediating cardioprotection during ischemia-reperfusion injury, it is important to evaluate the potential relation between these two protein families. In the present study, we attempted to investigate whether HSP expression is modulated by PKC levels in rat neonatal ventricular myocytes. This would permit us to determine whether the PKC signaling cascade is involved in modulating HSP-mediated cardioprotection. Therefore, we utilized adenoviral-mediated overexpression of PKC-{alpha} in rat neonatal ventricular myocytes and examined the subsequent expression levels of HSP70. Interestingly, we found that an increase in PKC-{alpha} expression results in an increase in HSP70 gene transcription but that this transcriptional activation is HSF-1 independent. We also found that overexpression of PKC-{alpha} protects cardiomyocytes against simulated ischemia and that this protection is directly mediated by PKC-{alpha}-induced expression of HSP70.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cultured cell conditions. Neonatal cardiomyocytes were prepared from 1- to 2-day-old Sprague-Dawley rat hearts by enzyme digestion, as previously described by us (20). Cells were plated onto six-well plates at 1 x 106 cells/well or 100-mm plates at 6 x 106 cells/plate. This study was conducted in accordance with animal care guidelines issued by the National Institutes of Health. All protocols were approved by the Animal Care and Use Committee of Loyola University Medical Center.

The culture and maintenance of the embryonic rat heart-derived cell line H9c2 (2-1) (American Type Culture Collection, Rockville, MD) has been previously described (20). Cells were plated into six-well plates at 2 x 106 cells/ml. Selected plates of transfected H9c2 cells were treated with PMA (catalog no. 524400, Calbiochem) at a final concentration of 200 nM for 18 h.

Adenoviral infection protocol. The cDNAs for PKC-{alpha}, PKC-{epsilon}, and PKC-{delta} were kindly provided by Drs. P. Parker and P. Sudgen (Imperial College of Technology and Medicine, Cambridge, UK). Viral constructs were created by us as previously described (22). Neonatal rat ventricular myocytes were infected with virus at a multiplicity of infection of 10:1 as previously described by us (9).

Construction of HSP70-firefly luciferase chimeric plasmid. The HSP70-firefly luciferase plasmids were generated by using the firefly luciferase reporter vector pGL3-promoter vector (catalog no. E1761, Promega). The 5'hsp70-firefly luciferase plasmid was generated by excision of the SV40 promoter from pGL3 and insertion in its place of the promoter region (–880 to +80) of the rat HSP70 gene (19) in the Bgl II and Hind III sites of pGL3 in the proper orientation. This construct placed the reporter firefly luciferase gene under the transcriptional control of the rat HSP70 promoter.

Transfection protocols. Transfection of H9c2 cells for dual-luciferase reporter assays was performed by using a high-efficiency calcium phosphate transfection protocol (2). Briefly, 16 µg of 5'hsp70-firefly luciferase plasmid DNA + 4 µg of pRL-TK vector (containing the HSV-TK-Renilla luciferase gene) plasmid DNA for a total of 20 µg of plasmid DNA were mixed with 0.5 ml of 0.25 M CaCl2. An additional 0.5 ml of 2x BES-buffered saline was added, and the mixture was incubated for 20 min at room temperature. Then 1 ml of calcium phosphate-DNA mixture was added drop by drop to the plate of H9c2 cells while the plate was gently swirled. After 24 h of incubation, cells were treated and processed as required for the dual-luciferase reporter assay.

Transfection of neonatal rat ventricular myocytes for small interfering RNA (siRNA) inhibition followed manufacturer's instructions for the transfecting reagent (Lipofectamine 2000, Invitrogen). Briefly, the reagent (7.2 µl/250 µl per well) and siRNA (1 nM/250 µl per well) were mixed with an equal volume of transfection mix. The total transfection mix was added drop by drop at 500 µl per well to six-well plates and returned to the incubator for 6 h. After incubation, the cells were processed as required for creatine kinase (CK) assay. siRNA oligonucleotides were obtained from Ambion (catalog no. 16704) and Santa Cruz Biotechnology (catalog no. SC-37007).

Western blot analysis. After appropriate treatment, cells were harvested as previously described by us (9). Protein concentrations were determined by Bio-Rad protein assay using a microplate reader. Each protein sample was loaded at 40 µg per lane on 8% SDS-polyacrylamide gels and transferred to nitrocellulose. Blots were reacted with primary antibodies to HSP70i (catalog no. SPA810, Stressgen), HSP90 (catalog no. SPA845, Stressgen), HSF-1 (catalog no. SPA950, Stressgen), and actin (catalog no. 69100, MP Biochemicals) and subsequently reacted with the corresponding secondary antibodies. All secondary antibodies were horseradish peroxidase conjugates. Blots were developed by enhanced chemiluminescence kit (Pierce) before exposure to X-ray film.

Electrophoretic mobility shift assay. Electrophoretic mobility shift assay (EMSA) was performed as described by us (5). Briefly, after treatment, 100-mm plates were rinsed, cells were scraped into cold PBS and spun at 14,500 rpm (Minispin Plus, Eppendorf), and the liquid was aspirated. Nuclear fractions were obtained from pellets according to the manufacturer's instructions for extraction (NE-PER, Pierce). The resulting fractions, after radiolabeled oligonucleotide binding, were electrophoresed in a 5% acrylamide gel at 100 V. Gels were then soaked in 5% glycerol for 20 min at room temperature, dried onto filter paper, and exposed to X-ray film.

Dual-luciferase reporter assay. Luciferase assay was performed as described by the manufacturer (Dual Luciferase Reporter Assay System, catalog no. E1910, Promega). Briefly, plasmid construct was generated by introduction of the 5'-promoter region from the rat HSP70 gene into the multiple cloning region of the pGL3 vector (firefly luciferase reporter vector) provided with the kit and as described above. The control luciferase vector (HSV-TK-Renilla luciferase reporter vector, catalog no. E2241, Promega), the parental firefly luciferase reporter vector (pGL3-promoter vector, catalog no. E1761, Promega), and the hsp70-firefly luciferase chimeric plasmid were grown in HB101 cells (Zymo Research), purified by column (Qiagen), and then transfected as described above. At 24 h after transfection, cells were infected with the wild-type adenoviral construct AdwtPKC-{alpha} or treated with PMA. On the following day, cells were rinsed gently in 1x PBS and then scraped into a microcentrifuge tube in lysis buffer. Cells were subjected to two freeze-thaw cycles and spun (Minispin Plus), and supernatants were transferred to a clean tube. Borosilicate tubes were prepared with luciferase substrate in buffer. Samples were assayed one at a time as follows: sample (20 µl) was added, and firefly luciferase activity was read on a luminometer (Lumat LB9501, Berthold); then Stop & Glo reagent provided by the manufacturer was added, and Renilla luciferase activity, which is used to control for transfection efficiency, was read. Therefore, data are presented as a ratio of firefly-to-Renilla luciferase activity in relative units.

Simulated ischemia protocol. After treatment, six-well culture plates were divided into sets. One set was changed to control buffer (in mM: 1.13 CaCl2, 5 KCl, 0.3 KH2PO4, 0.5 MgCl2, 0.4 MgSO4, 128 NaCl, 0.3 Na2HPO4, 4 NaHCO3, 10 glucose, and 10 HEPES, pH 7.2), and the other was changed to ischemic buffer (in mM: 1.13 CaCl2, 5 KCl, 0.3 KH2PO4, 0.5 MgCl2, 0.4 MgSO4, 128 NaCl, 4 NaHCO3, and 10 HEPES, pH 7.2). Control samples were left in the incubator, while ischemic samples were set in an anaerobic jar (GasPak System, Becton Dickinson). Hypoxia was maintained with a hydrogen-and-carbon dioxide-generating envelope for 16 h within the same incubator. A methylene blue indicator left with the plates verified oxygen depletion. This system is capable of depleting and maintaining contained oxygen to 0.2–0.6% within 60 min (28). Supernatants were collected and labeled, and cells were then scraped into 1 ml of PBS. Supernatants and cells were lysed by sonication, spun, and then used for detection of CK activity.

CK assay. Supernatants and cell extracts were sonicated for 10 s each. CK reagent (Diagnostic Chemicals) was reconstituted in 10 ml of buffer provided by the manufacturer. Two parts of each sample were mixed with one part of reconstituted reagent and read for 7 min at 340 nm by microplate reader (Molecular Devices). CK was determined as a fraction of CK release vs. total sample. Results are presented as percent total CK compared with control. All points were normalized for protein concentration as determined by bicinchoninic acid assay (Pierce).

Statistical analysis. Values are means ± SE. Statistical significance was assessed by analysis of variance followed by Bonferroni's t-test. Results were interpreted to be significantly different when P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Our previous studies showed that certain HSPs interact closely with particular PKC isoforms in cardiomyocytes (4). This has led us to further investigate the relation between two members of these protein families. We have therefore studied the relation between PKC-{alpha} and the inducible HSP70. We found that adenoviral-mediated overexpression of the wild-type PKC-{alpha}, which significantly increases PKC-dependent phosphorylation (data not shown), is able to induce expression of the inducible form of HSP70 in neonatal rat ventricular myocytes. As shown by Western blot analysis in Fig. 1, A and B, protein extracts from neonatal rat ventricular myocytes infected with the adenoviral construct containing the wild-type PKC-{alpha} induce a significant increase of the inducible HSP70. This result confirms our previous finding of cross talk between the HSPs and the PKC isoforms, although this effect of PKC-{alpha} was found not to affect the expression of other HSPs, HSP90 and HSP25. Results obtained from analysis of HSP90 expression during PKC-{alpha} overexpression in neonatal rat ventricular myocytes are shown in Fig. 1, C and D. Therefore, in contrast to the significant increase of HSP70 expression (Fig. 1, A and B), HSP90 (Fig. 1, C and D) and HSP25 (data not shown) expression is not increased by PKC-{alpha} overexpression. Proteomic analysis has indicated cross talk between PKC-{epsilon} and HSP70 (24). Additional studies showed that phorbol esters, such as PMA, are able to induce the expression of HSP70 and that this is potentially a posttranscriptional effect (13).


Figure 1
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  		Fig. 1. Western blot analysis of protein extracts from neonatal rat ventricular myocytes. A: cardiomyocytes were uninfected or infected with a control adenoviral construct (AdSR) or adenoviral construct containing the wild-type PKC-{alpha} isoform (AdwtPKC{alpha}). Blots were reacted with an antibody specific for the inducible heat shock protein 70 (HSP70) and beta-actin. B: quantification of scanned immunoblots. Values are means ± SE (n = 3). *P < 0.01 vs. control. C: cardiomyocytes were uninfected or infected with AdSR or AdwtPKC{alpha}. Blots were reacted with an antibody specific for the inducible HSP90 and beta-actin. D: quantification of scanned immunoblots from C. Values are means ± SE (n = 3).

 
To address the possible mechanism of induction of HSP70 expression by PKC-{alpha}, we proceeded to generate a chimeric construct that contains the 5'-promoter region of HSP70 that includes the necessary transcriptional signals for proper gene expression linked to the luciferase reporter gene. This construct permits us to determine whether PKC-{alpha} induces HSP70 expression by activating its transcription. This construct was then transfected into the H9c2 myogenic cells to test whether our chimeric construct responded to PKC-{alpha} overexpression. Figure 2 presents our results using this approach. Although the 5'-promoter region of HSP70 responds strongly to an increase in PKC-{alpha}, treatment with the phorbol ester PMA has a lesser effect on this construct. This result indicates that although the PKC isoforms induced by PMA treatment (including PKC-{alpha}) increase HSP70 protein levels modestly, the sole overexpression of PKC-{alpha} has a significantly stronger effect on inducing transcription activation of HSP70. This demonstrates that PKC-{alpha} overexpression directly induces HSP70 transcriptional activation.


Figure 2
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  		Fig. 2. Luciferase assays of H9c2 cell extracts transfected with the chimeric construct containing the 5'-promoter region (5'hsp70) or the parental firefly luciferase reporter vector (pGL3). Each was subsequently infected with AdwtPKC{alpha} or treated with PMA (200 nM for 18 h). Values are means ± SE (n = 7). *P < 0.01 vs. 5'hsp70 control. #P = not significant vs. pGL3 control.

 
Since heat shock gene expression is controlled by the HSF-1, we investigated whether increased expression of the wild-type PKC-{alpha} might result in an increase in HSF-1 expression. As shown in Fig. 3, A and B, this does not seem to be the case. Western blot analysis of adenoviral-infected neonatal rat cardiomyocyte protein extracts shows that an increase in expression of the wild-type PKC-{alpha} actually decreases the overall expression of HSF-1.


Figure 3
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  		Fig. 3. A: Western blot analysis of protein extracts from neonatal rat ventricular myocytes. Cardiomyocytes were uninfected or infected with AdSR or AdwtPKC{alpha}. Blots were reacted with an antibody specific to heat shock factor-1 (HSF-1) and actin. B: quantification of scanned immunoblots vs. control. *P < 0.01. C: electrophoretic mobility shift assay (EMSA) of nuclear extracts from neonatal rat ventricular myocytes. Cardiomyocytes were infected with adenoviral constructs containing the wild-type PKC isoforms ({alpha}, {delta}, and {epsilon}) or AdSR. Uninfected cardiomyocytes were heat shocked (HS) at 42°C for 30 min. Controls for DNA binding reaction are as follows: no extract, non-100 molar excess of noncompetitive octomer (Oct)-1 oligonucleotide, and 100 molar excess of nonradiolabled HSF oligonucleotide. Nuclear extracts were reacted with a 32P-labeled oligonucleotide containing the DNA heat shock element (HSE) for the rat HSP70 promoter. Arrow indicates position of the HSE-HSF-1 complex. D: control EMSA of C on nuclear extracts from neonatal rat ventricular myocytes. Controls for DNA binding reaction are as follows: no extract, 100 molar excess of noncompetitive HSF-1 oligonucleotide, or 100 molar excess of nonradiolabeled Oct oligonucleotide. Nuclear extracts were reacted with a 32P-labeled oligonucleotide containing Oct-1. Arrow indicates position of the Oct complex.

 
We then analyzed whether an increase in HSF-1 transcriptional activity during increased wild-type PKC-{alpha} expression was responsible instead. We performed EMSAs to analyze whether overexpression of different PKC isoforms altered HSF-1 transcriptional activity. For this purpose, we prepared nuclear extracts from neonatal rat cardiomyocytes that were infected with adenoviral constructs expressing the wild-type PKC-{alpha}, PKC-{delta}, and PKC-{epsilon}, as well as our control adenoviral construct AdSR, or cells heat shocked at 42°C for 30 min. Nuclear extracts were reacted with the 32P-labeled heat shock DNA element from the rat inducible HSP70 promoter (19). To demonstrate the specificity of the formation of the heat shock element (HSE)-HSF-1 shifted complex, we included three lanes, one where no nuclear extract was added and two where the nuclear extract from heat shocked cardiomyocytes was reacted with a 100x molar excess of the nonlabeled oligonucleotide for the octamer-1 (Oct-1) binding element or the HSE-I, before addition of the 32P-labeled HSE-I oligonucleotide. As shown in Fig. 3C, an HSE-HSF-1 complex is detected only in the nuclear extract from heat-shocked cardiomyocytes and not in any of the nuclear extracts from cardiomyocytes expressing the PKC isoforms, including PKC-{alpha}, which we already saw induces HSP70 expression. In addition, to assess that the nuclear extracts from the cardiomyocytes infected with the adenoviral constructs containing the PKC isoforms were not inactive, we used the same extracts in another EMSA using a 32P-labeled Oct-1 oligonucleotide (Fig. 3D). This result indicates that induction of HSP70 expression by PKC-{alpha} would seem to be independent of HSF-1 activation, since PKC-{alpha} overexpression results in a decrease in HSF-1 expression and in the absence of any HSF-1 activation that would produce HSF-1 binding to the HSE sites on the promoter of the HSP70 gene.

We were also aware that PKC-{alpha} had previously been shown to confer protection against simulated ischemia-reoxygenation (31). We confirmed this by measuring cell damage by the release of CK after simulated ischemia-reoxygenation. As shown in Fig. 4, neonatal rat ventricular myocytes infected with adenoviral vectors expressing the wild-type PKC-{alpha} and the inducible HSP70 show less cellular damage than cardiomyocytes infected with our control adenoviral vector (AdSR). These results suggest that the protective effect of increased PKC-{alpha} may involve its ability to induce HSP70 expression.


Figure 4
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  		Fig. 4. Creatine kinase (CK) released after simulated ischemia by neonatal rat ventricular myocytes that were infected with AdSR, AdwtPKC{alpha}, and inducible HSP70 (Adhsp70) genes. Results are from 3 independent experiments. *P < 0.01 vs. AdSR.

 
To investigate the potential contribution of HSP70 expression to the cardioprotective effect of an increase in PKC-{alpha} expression, we used an HSP70 siRNA approach to reduce PKC-{alpha}-induced HSP70 expression. For this purpose, we used a commercially available siRNA kit against HSP70 (Santa Cruz Biotechnology). A representative immunoblot blot is shown in Fig. 5A. Our results clearly show that we are able to reduce by >50% the expression of HSP70 induced by adenoviral-mediated PKC-{alpha} overexpression using siRNA (Fig. 5B). In addition, adenoviral-mediated overexpression of PKC-{alpha} in neonatal rat ventricular myocytes in the presence of a transfected control siRNA (AdwtPKC-{alpha} + siCo) shows a significant decrease of CK release after simulated ischemia-reoxygenation, an indication of significantly less cellular damage. However, as shown in Fig. 5C, in cardiomyocytes transfected with HSP70 siRNA (AdwtPKC-{alpha} + sihsp70), there is no decrease in CK release after simulated ischemia-reoxygenation compared with our control adenoviral construct (AdSR). This result shows that the induction of HSP70 by PKC-{alpha} is a requirement for protection against simulated ischemia in neonatal rat ventricular myocytes.


Figure 5
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  		Fig. 5. A: Western blot protein extracts from neonatal rat ventricular myocytes infected with AdSR or AdwtPKC{alpha}. Some cardiomyocytes were subsequently transfected with a control small interfering RNA (siRNA) or HSP70 siRNA. B: quantification of scanned immunoblots, presenting HSP70 expression normalized to actin expression. Values are means ± SE (n = 3). *P < 0.05 vs. siRNA transfected control. C: CK released after simulated ischemia by neonatal rat ventricular myocytes infected with AdSR and AdwtPKC{alpha}. One group infected with AdwtPKC{alpha} was transfected with control siRNA (AdwtPKC{alpha} + siCo) and another group with HSP70-siRNA (AdwtPKC{alpha} + sihsp70). Values are means ± SE (n = 3). *P < 0.01 vs. AdSR.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In a previous study, we investigated the complex and close interaction between HSPs and PKC isoforms in cardiac tissue (4). Both of these protein families have been implicated in mediating cardioprotection (1, 7, 8, 16, 31). Our interest was to investigate how PKC-{alpha}, a conventional PKC isoform in cardiac tissue, is able to protect against ischemia-reperfusion injury. Interestingly, we found that increased expression of wild-type PKC-{alpha} induced the expression of HSP70, but not HSP90 or HSP25 (Fig. 1). A previous study showed that PMA-induced PKC activation results in increased HSP70 and HSP90 expression (13) and that the induction of HSP70 and HSP90 was mainly due to mRNA stabilization in human peripheral blood monocytes. Surprisingly, we found that the PKC-{alpha} induction of HSP70 in a myogenic cell line (H9c2) is mainly by transcriptional activation, as shown by our luciferase assay experiments (Fig. 2). This would seem to be consistent with previous studies that postulated that PKC activation is responsible for HSF-1 activation resulting in HSP synthesis (12, 23). Nonetheless, when we examined the level of expression of HSF-1 during an adenoviral-mediated increase in PKC-{alpha} expression, we found a decrease in the total protein level for HSF-1 (Fig. 3, A and B). In addition, we found that adenoviral-mediated expression of PKC-{alpha} in rat neonatal cardiomyocytes did not activate HSF-1, as seen in our EMSA experiments, where there is no binding of HSF-1 to HSE on the promoter of HSP70 (Fig. 3C). This is an important finding, since this indicates that induction of HSP70 is not due to the simple overexpression of an exogenous protein that is recognized by the cell as a general stress resulting in HSF-1 activation. Therefore, the PKC-{alpha}-induced expression of HSP70 in rat neonatal ventricular myocytes is specific to this HSP and is independent of HSF-1 activation. One possible way by which PKC-{alpha} might induce HSP70 transcription is by activation of Sp1, a transcriptional factor known to have two binding sites on the HSP70 promoter; further studies are needed to elucidate this point.

We also found that adenoviral-mediated overexpression of PKC-{alpha} in rat neonatal ventricular myocytes protects against simulated ischemia to almost the same level as does the overexpression of HSP70 (Fig. 4). Since we know that increased expression of PKC-{alpha} induces HSP70 expression, we examined whether the protective effect seen during PKC-{alpha} overexpression maybe due to its ability to induce HSP70 expression. Although the siRNA against HSP70 did not totally eliminate the PKC-{alpha} induction of HSP70 expression in rat neonatal ventricular myocytes (Fig. 5, A and B), this reduction in PKC-{alpha}-induced HSP70 was sufficient to cause a loss in protection against simulated ischemia (Fig. 5C). This indicates that the protection after PKC-{alpha} overexpression is due to the level of induced HSP70.

In conclusion, our present results show that overexpression of wild-type PKC-{alpha} confers protection to neonatal rat ventricular myocytes during simulated ischemia-reoxygenation and that this protective effect is mediated by the ability of PKC-{alpha} to induce HSP70 expression. In addition, we have found that PKC-{alpha} increases HSP70 expression in a transcriptional manner and that this induction of HSP70 is independent of HSF-1 activation.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-61339 and HL-67971, a grant-in-aid from the American Heart Association National Center, and the John and Marian Falk Trust for Medical Research.


   ACKNOWLEDGMENTS
 
Present address of S. D. Coaxum: Dept. of Nephrology CSB 829, Medical University of South Carolina, 96 Jonathan Lucas St., Charleston, SC 29425 (e-mail: coaxum@musc.edu).


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Mestril, Cardiovascular Institute, Loyola Univ. Medical Center, 2160 S. First Ave., Bldg. 110, Rm. 5224, Maywood, IL 60153 (e-mail: rmestri{at}lumc.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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