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Cardiovascular Research 2000 46(1):139-150; doi:10.1016/S0008-6363(00)00007-9
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Copyright © 2000, European Society of Cardiology

Hypoxia differentially regulates stress proteins in cultured cardiomyocytes

Role of the p38 stress-activated kinase signaling cascade, and relation to cytoprotection

Rachid Kacimi, Jamila Chentoufi, Norman Honbo, Carlin S. Long and Joel S. Karliner*

Cardiology Section, VA Medical Center, 4150 Clement Street, San Francisco, CA 94121, USA

* Corresponding author. Tel.: +1-415-750-2112; fax: +1-415-750-6950 joel.karliner{at}med.va.gov

Received 6 August 1999; accepted 13 December 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Summary and conclusions
 References
 
Objective: Stress proteins (heat shock proteins, HSPs) are molecular chaperones that have been shown to enhance the survival of cells exposed to environmental stress. We sought to investigate the effects of hypoxia on the levels of HSP27 and heme oxygenase-1 (HO-1 or HSP32) in an established model of rat neonatal cardiac myocytes in culture. Methods: Myocytes were subjected to hypoxia (<0.5% O2 for 16 h). Studies of cell viability and nuclear morphology showed no evidence of cell death under these conditions. Results: Messenger RNA analysis demonstrated constitutive expression of HSP27 and low levels of HO-1. Hypoxia strongly induced HO-1 mRNA without affecting HSP27 mRNA. In parallel to mRNA levels, hypoxia increased HO-1 protein level without affecting HSP27. To further assess the signaling pathways implicated in HO-1 induction, we used inhibition experiments. The tyrosine kinase inhibitor tyrphostin and the mitogen-activated protein kinase inhibitor PD98059 did not prevent HO-1 induction, while the protein kinase C inhibitor chelerythrine partially blocked this response. The p38 stress-activated kinase inhibitor SB203580 was the most potent in suppressing hypoxia-induced HO-1. In vitro kinase assays, cell labeling and immunoprecipitation showed activation of signaling pathways downstream of p38 stress-activated kinase as revealed by an increase in phosphorylation of MAPKAPK-2/3 kinases and HSP27. Conclusions: These data show a differential pattern of hypoxia-induced HSP expression and implicate the stress kinase in HO-1 induction. Thus, selective regulation of HSP levels may play a role in the cardioprotective mechanisms that participate in the adaptive response to hypoxia-induced stress.

KEYWORDS HSPs: stress proteins or heat shock proteins; HO-1: Heme oxygenase-1 or a 32-kDa heat shock protein (HSP32); PMA: phorbol 12-myristate 13-acetate; PKC: protein kinase C; IL-1β: interleukin-1β; MTT: Tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; RT-PCR: reverse transcriptase polymerase chain reaction; ERK: extracellular signal-regulated protein kinase; MAP kinase: mitogen-activated protein kinase; p38/RK: a MAP kinase called stress-activated protein kinase (SAPK) or reactive kinase (RK); MAPKAPK-2/3: MAP kinase kinase-2/3; PI: propidium iodide; PBS/T: phosphate buffered saline–Tween 0.05; PD98059: MEK-1 inhibitor; SB203580: p38/RK stress kinase inhibitor; BCA: Bicinchoninic acid reagent


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 5 Summary and conclusions
 References
 
Stress proteins (heat shock proteins, HSPs) appear to be involved intimately in diverse biological processes. While they can be induced by environmental stress in cell models and in tissues, several HSPs are constituitively expressed in eukaryotic species ranging from insects such as Drosophila melanogaster to mammals [1–3]. Some HSPs appear to be involved in basic cellular functions such as protein folding, protein trafficking and membrane translocation and they may have a more general role as molecular chaperones [4–7]. However, the precise functional role for some HSPs in protein–protein interactions and/or the repair of stress-induced damage to proteins after exposure to environmental stress, particularly to reduced oxygen tension, remains to be established.

Induction and accumulation of HSPs has been correlated with the acquisition of tolerance to stress. It has been suggested that some HSPs might induce a transient protection against damage caused by heat or chemical stress, UV radiation, starvation, hypoxia or ischemia [3,8–11]. Thus, HSP induction represents a biologically important cellular response to external stress.

The induction of HSP70 has been implicated in myocardial protection and has been reported in response to a variety of cardiac insults such as ischemia–reperfusion and heart failure in vivo and to hypoxia and reoxygenation in cardiac myocytes in vitro [12–17]. Recently, strong evidence for a role of HSP70 in myocardial protection against ischemic injury was established in transgenic mice overexpressing human and rat inducible HSP70 [18,19]. The cellular and temporal pattern of HSP70 induction varies depending on the nature and severity of cardiac injury [17–21] and has also been found to match the known histopathology for various cardiac insults, thus establishing the usefulness of HSP70 induction as a marker of cellular stress in the cardiovascular system.

However, HSP70 protein induction as a marker of cellular stress and/or cytoprotection is likely a part of a broader, coordinated response to an hypoxic insult. We hypothesized that other stress proteins undergo modification that could be important in regulating the response to hypoxic injury in cardiac myocytes. To explore this hypothesis and to better understand the modulation and the potential function of HSPs in the heart, we have analyzed additional features of the myocardial cell response to hypoxia. Among these are steady state levels and regulation of other stress proteins such as the small heat shock proteins, namely HSP27 and HSP32 (heme oxygenase-1, HO-1). We also addressed the signaling pathways which could mediate induction and/or activation of small HSPs during oxidative stress in an established culture model of neonatal rat cardiac myocytes. We further investigated in parallel whether there is a link between HSP expression and cell survival and/or cytoprotection.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 5 Summary and conclusions
 References
 
2.1 Cell culture
Primary cardiac myocyte cultures were prepared by enzymatic dissociation of ventricular tissue from 1-day-old neonatal rats according to methods described previously [22–25]. The euthanasia protocol was approved by the Animal Studies Committee of the San Francisco VA Medical Center. Myocytes were seeded on to 100-mm plastic dishes at final density of 400 cells/mm2. Through culture day 3, cells were kept in MEM containing 5% bovine calf serum supplemented with 1.5 mM B12, 50 U penicillin, and 0.1 mM bromodeoxyuridine to prevent low-level nonmyocardial cell proliferation as previously described [22]. On day 3, cells were placed in serum-free defined medium containing 10 µg/ml insulin, 10 µg/ml transferrin and 0.1% BSA. Under these conditions, myocyte cultures showed <10% contamination with other myocardial cell types as confirmed by immunofluorescence microscopy and flow cytometry analysis using an MF-20-FITC mouse monoclonal antibody against striated muscle myosin [24]. Experiments were initiated on day 4, which was 24 h after the change to serum-free conditions.

2.2 Cell treatment and experimental hypoxia
After overnight incubation, fresh serum-free medium was added. Agonists, inhibitors, antioxidants or vehicle were then added, and cells were returned to the incubator or placed in the hypoxia chamber [24]. The exposure to hypoxia was essentially as described earlier in our laboratory [23–26]. Briefly, low oxygen tension was achieved in an airtight plexiglass humidified chamber (Anaereobic Environment, Sheldon, OR, USA), which was maintained at 37°C and continuously gassed with a mixture of 99% N2–1% CO2–0% O2. Cells were placed into the hypoxia chamber on culture day 4 and remained for 12–16 h. Maintenance of the desired O2 concentration was routinely monitored during incubation using an oxygen sensor (Controls Katharobic System, Sheldon, OR, USA). For concurrent normoxic conditions, cells were placed in a Forma Scientific incubator gassed with 99% air–1% CO2 at 37°C.

2.3 Electrophoresis and immunoblotting
After each treatment period, cardiac myocytes plated on 100-mm dishes were washed with cold PBS, and scraped into 500 µl lysis buffer consisting of 20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% NP-40, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonylfluoride (PMSF), 50 mM sodium fluoride (NaF), and 5 mg/ml aprotinin. Total protein was quantitated by BCA reagent. Ten micrograms of protein was loaded per lane, and were electrophoresed on 10–12% sodium dodecylsulfate polyacrylamide gels (SDS–PAGE) as previously described [26]. The transfer onto enhanced chemiluminescence (ECL)-nylon membranes (Amersham) was made in 48 mM Tris, 150 glycine, and 10% methanol using a Transblot apparatus (Bio-Rad, San Diego, CA, USA) at 100 V for 1 h at 4°C. The membranes were saturated in 10 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween 20, and 5% non-fat dry milk for 1 h at room temperature and then probed with specific polyclonal HSP antisera (1:1000), anti-p38 (1:500), phospho-specific p38 MAP kinase (1:500) in the same buffer for 1 h at room temperature with gentle agitation. Membranes were washed three times with 10 mM Tris, 150 mM NaCl, and 0.1% Tween 20. Bound antibodies were identified after incubation with peroxidase-conjugated anti-rabbit antibodies (1/2000 dilution in saturation buffer) for 1 h at room temperature. Membranes were then rewashed three times and the position of the individual proteins in separate lanes was detected by chemiluminescence ECL (Amersham) or Super Signal (Pierce, Rockford, IL, USA) using radiographic film (X-Omat AR-5, Eastman Kodak). To compare the degree of expression for the different HSPs and to confirm the equivalent protein loads in each lane, the immunoblots were sequentially reprobed with each of the HSP antibodies. The first set of antibodies was removed by stripping at 55°C (2% SDS, 75 mM Tris–HCl, pH 6.8, 100 mM 2-mercaptoethanol for 30 min); membranes were then washed and reprobed.

2.4 Immunofluorescence microscopy
After each treatment, cells were washed twice in PBS, and cardiac myocytes plated on glass coverslips and fixed in acetone–methanol (1:1) for 10 min at –20°C. Fixed cells were permeabilized with 0.5% (v/v) Triton-X100 in PBS, and incubated with HSP antibodies (1/1000 dilution in PBS–0.1% Tween 20, 3% BSA) for 1 h at room temperature or overnight at 4°C. After three washes with PBS–Tween for 5 min, the cells were reincubated with a secondary antibody, fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Vector, Burlingame, CA, USA) for 1 h at room temperature. Cells were rewashed in PBS on a rocking platform. Immunostained cells were embedded in Vectashield mounting medium (Vector) and immediately analyzed using fluorescence microscopy [Axiophot-2 coupled to a Kodak camera (63x objective, Carl Zeiss, Germany)]. Negative control experiments used preimmune serum as primary antibody.

2.5 In vitro kinase assay
Stress kinase p-38 and MAPKAPK-2 activities were measured using either phospho-specific antisera detected by Western blotting or by an immunocomplex kinase assay. Briefly, cells were lysed by scraping in ice-cold lysis buffer (1% v/v Triton X-100, 20 mM Tris, pH 7.5, 5 mM EDTA, 1 mM PMSF, 10 mM aprotinin, 10 mM leupeptin, 1 mM sodium orthovanadate and 50 mM NaF). Lysates were passed three times through a 25-gauge needle, kept on ice for 30 min and centrifuged for 15 min at 12 000 g at 4°C to pellet insoluble material. Aliquots of the supernatant were assayed for protein concentration by BCA reagent. Samples were normalized by total protein and volume adjusted to 1 ml with lysis buffer. To specifically determine the stress kinase activity, cell lysates were immunoprecipitated with 2 µg of antibody (either anti-p38 or anti-MAPKAPK-2 antisera) per 100 µg protein at 4°C for 1 h. Protein A or G beads (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) were added to each sample for 1 h. The beads were centrifuged, the supernatant was removed and beads washed four times with lysis buffer and two times with kinase buffer (50 mM Tris–HCl, pH 7.4, 30 mM ATP, 10 mM MgCl2, 1 mM DTT). p-38 or MAPKAP-2 immunocomplexes were incubated in 50 µl kinase buffer with 10 µCi {gamma}[32P]ATP for 30 min at 30°C. The substrates were ATF-2 for p38 kinase or recombinant HSP27 for the MAPKAP-2 kinase assay. Reactions were stopped by boiling in 2xsample buffer (150 mM Tris–HCl, pH 6.8, 25% glycerol, 2.5% SDS, 10% mercaptoethanol, 0.3% bromophenol blue) for 5 min to dissociate the proteins from the antibodies. Samples were subjected to SDS–PAGE; gels were then dried and exposed to autoradiographic X-Omat Kodak films.

2.6 Metabolic labeling and immunoprecipitation
Cardiac myocytes plated in six-well culture plates (Costar, Cambridge, MA, USA) were washed twice with phosphate-free medium, then incubated in serum-free medium and labeled with 25 µCi/ml of orthophospho-[32P] in phosphate-free medium for 4 h. Labeled myocytes were subjected to hypoxia for the times indicated. Cells were washed in ice-cold PBS and then lysed in 20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM orthovanadate, 50 mM NaF, and the complete inhibitor of protease cocktail (Boehringer, Indianapolis, IN, USA). Radiolabeled protein in cell lysates was counted and a standard amount of radioactivity was used in the immunoprecipitation assay described above. The lysate supernatants were precleared by incubation with normal rabbit serum and protein A–agarose and were immunoprecipitated with rabbit antisera to HSP27 overnight at 4°C with agitation. Immunocomplexes were recovered by binding to protein A–agarose. Beads were washed with lysis buffer and the immunoprecipitates were analyzed by 12% SDS–PAGE under reducing conditions as described above. HSP27 phosphorylated bands were analyzed by densitometric scanning.

2.7 Measures of cell viability
(a) Percentages of live and dead cells were measured through a two-color fluorescence assay (Molecular Probes, Eugene, OR, USA) as previously described in our laboratory [23,24,26].

(b) The MTT assay which measures metabolic activity of viable cells was performed as previously described [27].

(c) Gel electrophoresis was used to determine nucleosomal DNA fragmentation. Treated myocytes were washed two times with cold PBS, harvested and centrifuged for 30 s at 500 g, resuspended in lysis buffer (10 mM EDTA, 50 mM Tris–HCl, pH 8 containing lauryl sarcosine and 0.5 mg/ml proteinase K), and incubated for 1 h at 37°C. RNase A (0.5 mg/ml) was then added and incubation continued for a further 1 h at 37°C. The resulting lysate was electrophoresed on a 2% agarose gel for 2 h at 40 V and DNA visualized by poststaining with ethidium bromide and examination under UV illumination.

(d) Fluorescence microscopy was performed on viable cells after staining with Hoechst H33342 [GenBank] and/or propidium iodide in the dark for 30 min at 37°C. Dead cells either apoptotic (showing condensed and fragmented nuclei) or necrotic could be easy differentiated from normal cells.

2.8 Statistical analysis
Significant differences were determined by either Student's two tailed t-test for comparison of the means of two samples or analysis of variance (ANOVA) for the comparison of more than two sample means followed by post-hoc testing for multiple comparisons between two sample means. The significance level was set at P<0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 5 Summary and conclusions
 References
 
3.1 HSP steady-state protein levels
Western blot analysis showed that hypoxia (<0.5% O2 for 16 h) selectively increased HO-1 protein level without affecting HSP27 protein levels. As expected, hypoxia also stimulated HSP70i. The hypoxia-induced HO-1 and HSP70i protein level was sustained after 6 h of reoxygenation, and declined after 1 day of reoxygenation, although levels were still elevated vs. control. Furthermore, prolonged exposure to 36 h of hypoxia resulted in a reduced HO-1 protein level compared with 16 h of hypoxia although the level still exceeded control. We also found no induction of HSP70i and a decrease in constituitive HSP27 expression after 36 h of hypoxia (Fig. 1).


Figure 1
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  		Fig. 1 Time course effect of hypoxia on HSP27, HO-1, and HSP70i protein levels. Rat neonatal ventricular myocytes were subjected to normoxia, hypoxia (<0.5% O2) for 16–36 h or 16 h of hypoxia+6–20 h reoxygenation. Equal amounts of protein from each cell lysates were subjected to SDS–PAGE followed by immunoblotting using specific antibodies as described in Methods. Shown is a representative western blot analysis. Scanning densitometry of 12 separate experiments is shown below. CT: control; HX: hypoxia, HxR: hypoxia +reoxygenation; *=P<0.05 vs. control.

 
Indirect immunofluorescence showed immunolocalization of HSP27, HO-1, and HSP70 in quiescent cardiac myocytes (Fig. 2). We confirmed the Western blot results, finding a marked induction of HO-1 by hypoxia without any change in HSP27 immunofluorescence. Semi-quantitative RT-PCR analysis revealed that hypoxia sharply induced HO-1 mRNA expression in cardiac myocytes (data not shown). Conversely, we noted that unstimulated rat cardiac myocytes exhibit constituitive expression of HSP27 mRNA. Hypoxia (<0.5% O2 for 12–16 h) had no substantial effect on HSP27 mRNA expression (data not shown).


Figure 2
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  		Fig. 2 Immunofluorescence analysis of myocardial stress proteins and effect of hypoxia. Cardiac myocytes were subjected to normoxia or hypoxia (<0.5% O2) for 16 h and immunofluorescence analysis was performed as indicated in Methods. During normoxia there is a low level of HO-1. Hypoxia increased immunostaining of HO-1 in the cytoplasm and the perinucleus as indicated by the arrows. No effect of hypoxia was observed in the immunostaining distribution of constituitively expressed HSP27. The figure is representative of data obtained from three to five separate experiments. Negative controls in each experiment with either secondary antibody or serum showed no immunofluorescence (data not shown).

 
3.2 HSPs and stress-dependent signal transduction
3.2.1 Role of kinases in hypoxia induced HSP expression
We next asked which kinase signaling pathways mediate hypoxia-induced HSP levels. We studied the contribution of protein kinase C, and the individual kinases of the MAP (mitogen-activated protein) kinase family, including ERK (extracellular-signal regulated kinase) JNK/SAPK (c-Jun NH2-terminal kinase/stress-activated protein kinase) and p38 during hypoxia -induced activation of HSPs in myocytes. The concentration selected for each inhibitor was based on reported concentration–response studies by others [28,29]. In preliminary experiments, we found that these concentrations did not induce any significant cytotoxicity with or without hypoxia during the time indicated as determined by MTT uptake and cell viability (data not shown). Treatment with the PKC inhibitor chelerythrine, the MAP kinase inhibitor PD98059, or the p38/RK stress activated kinase inhibitor SB203580, substantially inhibited HSP70i induction by hypoxia. However, PD98059 did not prevent HO-1 induction, while chelerythrine partially blocked this response. SB203580 was the most potent in suppressing hypoxia-induced HO-1 (Fig. 3). We also found that tyrosine kinase inhibitor tyrphostin substantially blocked HSP70i and partially inhibited HO-1 (data not shown).


Figure 3
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  		Fig. 3 Inhibition of increased HO-1 and HSP70i protein expression stimulated by hypoxia in cardiac myocytes. Cardiac myocytes were treated with chelerythrine (1 µM), PD98059 (10 µM), SB203580 (10 µM) or vehicle under normoxia or hypoxia for 16 h. Cell lysates were analyzed for HO-1 and HSP70i protein expression using SDS–PAGE and immunoblotting as described above. Shown above is a representative immunoblot. The bar graphs below are from five separate inhibition experiments; *=P<.05 vs. control.

 
3.2.2 Effects of PMA and 1L-1β on HSP levels:
We also tested the effect of PMA, a PKC activator and the pro-inflammatory cytokine IL-1β, both of which can act as pro-oxidants relative to HSP expression [30–32]. We found that PMA induced HO-1 protein expression as did IL-1β but to a lesser amount). The combination of either IL-1β and PMA with hypoxia did not modify this increase in protein level compared to stimulation with either agent alone (Fig. 4). In contrast to H0-1 mRNA induction by both PMA and IL-1β, neither had any detectable effect on HSP27 protein probably due to the high level constituitive of HSP27.


Figure 4
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  		Fig. 4 Effect of PMA and IL-1β and their combination with hypoxia on HSP27 and HO-1 protein expression: cardiomyocytes were treated with either vehicle (PBS–BSA–DMSO), PMA (100 nM), IL-1β (10 ng/ml) or each of these agents in combination with hypoxia (<0.5% O2) for 16 h at 37°C. Whole cell extracts were subjected to immunoblotting analysis. A representative Western blot is shown above. The bar graph below shows scanning densitometry results of three separate experiments. PMA and IL-1β are less potent inducers of HO-1 compared to hypoxia. Reox: reoxygenation for 6 h; *=P<0. 05 vs. control.

 
3.3) Stimulation of HSP27 phosphorylation in response to hypoxia
MAP kinase-activated protein kinases 2/3 (MAPKAP-2/3, downstream kinases in the p38 stress protein signaling pathway), are major isoenzymes responsible for small HSP phosphorylation [33,34]. In vitro kinase assays showed that hypoxia enhanced p38 phosphorylation and MAPKAP-2/3 activity compared to control (Fig. 5A and B). As noted earlier, we found that hypoxia did not modify steady-state HSP27 protein levels. Accordingly, we next considered the possibility that hypoxia could modulate HSP27 phosphorylation. Using cell labeling with 32P-orthophosphoric acid and immunoprecipitation, we found an increase of HSP27 phosphorylation in hypoxic myocytes compared to control (Fig. 5C). These observations implicate a post-translational mechanism of HSP27 regulation during hypoxia, and make it likely that HSP27 phosphorylation could play a role in the stress response to hypoxia in cardiac myocytes.


Figure 5
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  		Fig. 5 Hypoxia-stimulated p38 kinase, MAPKAP kinase 2 activity and HSP27 phosphorylation in cardiac myocytes. Myocytes were exposed to normoxia or hypoxia for times ranging between 6 and 12 h and then to reoxygenation for 0.5–2 h. Upper set of figures: (A) Western blot using phospho-specific antisera against active p38 stress kinase. (B) Autoradiograph showing the MAPKAP Kinase 2 activity determined by phosphorylation of recombinant HSP27 substrate using an in vitro kinase assay as described in Methods. (C) Autoradiograph of HSP27 phosphorylation after immunoprecipitation in a cell labeling assay as described in Methods. Lower set of bar graphs: all data are scanning densitometry results of three to five separate experiments. Note that p-p38 exhibits a biphasic response to reoxygenation that is not reflected either in MAPKAP kinase 2 activity or HSP27 phosphorylation. CT: control; HX: hypoxia; HxR: hypoxia–reoxygenation, p-p38; phospho-active p38 kinase; p-HSP27: phosphorylated HSP27. *=P<.05 vs. control.

 
3.4 Cell viability and morphology
3.4.1 Biochemical analysis of cell death induced by hypoxia and/or reoxygenation
In order to analyze alterations in cell metabolism and cell death induced by hypoxia and reoxygenation, we used two widely employed methodologies based on MTT uptake and cellular morphology. Using the MTT assay as an index of metabolic activity of viable cells, we observed no evidence of a decline in the metabolic activity of surviving cells during exposure to hypoxia for 12–16 h. However, after 20 h of reoxygenation (with change to fresh medium to allow cells to recover from hypoxic stress) MTT uptake was decreased significantly by 35% compared to control (n=16, P<0.001). After more prolonged exposure to hypoxia for 36 h, a reduction in myocyte metabolic activity was evident with a decrease of MTT uptake of 59% (n=6, P<0.001) (Fig. 6A).


Figure 6
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  		Fig. 6 Effect of hypoxia on metabolic activity and cell viability. Cardiac myocytes were subjected to normoxia, hypoxia for 12–16 h followed by reoxygenation, or hypoxia for 36 h. (A) Metabolic activity of viable cells was measured by the MTT assay. Optical density values were expressed as mean±S.E.M. (B) Photograph using double staining with calcein and ethidium homodimer fluorescent dyes. Normal living cells show green fluorescence (calcein uptake). Dead cells (either apoptotic or necrotic) exhibit red staining of DNA stained (ethidium homodimer). (C) Bar graph showing cell viability score (live/dead kit) expressed as percentage. Values are mean±S.E.M.; *=P<0.05 vs. control.

 
3.4.2 Morphology of cell death by fluorescence microscopy
Live/dead assays showed that after 12–16 h of hypoxia (<0.5% O2), there was no enhancement of cell death versus control cardiac myocytes (Fig. 6B and C). However, prolonged hypoxia for 36 h induced 50% cell death, and beyond this time the percentage of dead cells increased further. Similar results were obtained by trypan blue exclusion (data not shown). These data are consistent with the MTT uptake findings.

3.4.3 Nuclear morphology and DNA degradation (necrosis or apoptotic index)
Fig. 7A shows the behavior of electrophoresed DNA isolated from cardiac myocytes during hypoxia. We were unable to detect any sign of DNA degradation or nucleosomal laddering after 16 h of exposure to hypoxia, and after 20 h of reoxygenation in our cardiac myocyte cultures. Moreover, nuclear staining showed preservation of nuclear integrity in hypoxic cardiomyocytes compared to control (Fig. 7B). Taken together the nuclear index and the viability assay make it likely that up to 16 h of hypoxic stress did not induce detectable apoptosis (programmed cell death) or necrosis. However, after cardiomyocytes were exposed to prolonged hypoxia beyond 30 h, gel electrophoresis revealed DNA degradation and laddering. In addition to laddering, we also found diffuse DNA smearing, indicating fragments of many sizes as is usually observed in cells dying through a non-apoptotic process (Fig. 7C). This observation was confirmed by dual staining of the nuclei which is consistent with ongoing apoptotic and some necrotic cell death (Fig. 7B).


Figure 7
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  		Fig. 7 Effect of hypoxia on DNA fragmentation and nuclear morphology in cardiac myocytes: neonatal cardiac myocytes were subjected to either hypoxia or normoxia for indicated time. Reoxygenation was for 36 h. Nucleosomal DNA degradation was examined by gel electrophoresis. (A) No DNA degradation up to 16 h hypoxia or during reoxygenation; Mr1: {lambda}DNA marker, Mr2: {varphi}X174, CT: control, HX: hypoxia, Reox: reoxygenation. (B) Morphological assessment of cell death by nuclear double stain analyzed under fluorescence microscopy shows live cells with intact nuclei (Blue, Hoescht). At 36 h of hypoxia, dead cells are detectable. Apoptosis is indicated by nuclear condensation, fragmented nuclei and/or spotted nuclear bodies (in red). Presumably necrotic cells stained by propidium iodide are shown in red with intact nuclei. Data are representative of four to six separate experiments. (C) Prolonged hypoxia for 24 and 36 h is characterized by apoptosis as shown by DNA degradation and laddering; Lanes: 1, control; 2 and 4, 24 h hypoxia; 3 and 5, 36 h hypoxia.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
5 Summary and conclusions
 References
 
4.1 HSP protein expression
A major aim of the present study was to explore HO-1 regulation during changes in redox balance elicited by hypoxia and to compare the HO-1 response to other stress proteins such as HSP70i and HSP27. Heme oxygenase (HO) is a rate-limiting enzyme in heme degradation that cleaves heme to form biliverdin, carbon monoxide, and iron [35]. Two isoforms of HO have been identified, the inducible (HO-1) and the constituitive form (HO-2) [35]. HO-1 is induced by variety of exogenous and environmental stimuli such as ultraviolet light, hydrogen peroxide, heat shock, heavy metals, cytokines and lipopolysaccharides [35–37]. The precise function of HO is not fully understood, but a growing body of evidence points to its role in cytoprotection [38,39]. Although HO-1 has been recognized as a protein responding to oxidative stress, the signaling pathways involved and the regulation of its gene expression are poorly understood.

Using Northern blot and RT-PCR analysis we found a low level of HO-1 and substantial constituitive HSP27 mRNA expression. Knöll et al. reported that in a porcine model, ischemia–reperfusion induced new HSP27 mRNA synthesis as measured by a nuclear run-on assay [40]. Our experiments revealed that hypoxia has no effect on steady-state HSP27 mRNA expression, so that these observations may not be incompatible, as we do not have any measurements of mRNA half-life. In the rat brain, an increase in both HSP27 mRNA and protein has been described after thrombotic injury [41]. These differences between heart and brain may reflect different regulation in different tissues. However, consistent with previous reports [42,43], hypoxia markedly induced HO-1 mRNA expression and HO-1 protein de novo. This induction was not modified by reoxygenation but decreased significantly with 36 h of exposure to hypoxia. Furthermore, immunolocalization showed that HSPs are mainly present in the cytoplasm and the perinucleus in cardiac myocytes. In accord with the mRNA and protein data, hypoxic stress led to a sharp increase in HO-1 immunostaining in the cytoplasm and the perinucleus.

4.2 Stress kinase signaling cascade is implicated in hypoxia induced HSP expression
The signaling pathway that leads to the activation of stress protein genes is not completely understood. One of the goals of this study was to determine which signaling cascades are responsible for HSP induction during hypoxic stress. We and others have already shown that multiple kinase cascades (i.e., protein kinase C, tyrosine kinase, mitogen-activated protein kinase (ERKs), and their homologues the stress-activated kinases (SAPKs; JNK/p38), are indeed activated in hypoxic cells including cardiomyocytes [24,25,28,44–47]. In the present study we found for the first time that in cardiac myocytes p38 stress kinase inhibition substantially reduced hypoxia-induced HO-1 levels with no effect on HSP27. Although our data suggest a likely role for stress kinase in HO-1 induction, they do not exclude other signaling pathways or signaling cross-talk. For example, HO-1 induction by hypoxia was also partially blocked by either PKC or tyrosine kinase inhibition. In parallel we suggest that HSP70i induction by hypoxia may also involve a number of pathways since it is sensitive to multiple kinase inhibitors.

It has been shown that phosphorylation on serine residues mediated by stress kinases modulates the activity of HSP27 [48]. Moreover, Clerk et al. recently reported that oxidative stress induced by H2O2 stimulates multiple mitogen-activated protein kinase subfamilies resulting in phosphorylation of HSP27 in neonatal ventricular myocytes [49]. Consistent with these prior observations, we found that phosphorylation of HSP27 is an important post-translational modification in cultured cardiac myocytes in response to hypoxia. We observed that hypoxia induced phosphorylation of HSP27 through a signaling pathway involving the p38 stress kinase and MAPKAP-2/3 stress activated kinases. This posttranslational regulation of HSP27 may play a role in cardioprotection from hypoxia-induced stress.

4.3 Cell survival and cytoprotective role of HSPs
Several reports have described alterations in cardiomyocyte viability in response to hypoxia [24–28,50,51]. However, many of these studies were done in cultured cardiomyocytes plated at high density and/or under an additive metabolic stress consisting of glucose deprivation or serum without any supplementation. These conditions induce injury but are different from hypoxic stress. Under our conditions cells are plated at a relatively low density (400 cells/mm2) and can be maintained for at least 2 weeks in culture without any deterioration. Cells were serum starved to eliminate the ‘bias’ of growth factor 24 h before any treatment, but also were kept in supplemented defined medium with glucose which enhances cell survival in culture [52].

Our data showing an increase of HO-1 protein level during hypoxia (16 h of <0.5% O2) without evidence of cell death suggests that HO-1 may be cytoprotective during hypoxia-induced stress. The differential regulation of HSPs during hypoxic stress in cardiac myocytes (induction of HO-1 and HSP70i and increased phosphorylation of HSP27) suggests that tight regulation of HSPs is correlated with cytoprotection and/or the response to stress as has been shown in other cell models [2,3,35,36]. Recent reports using retroviral gene transfer in vitro and transgenic techniques have confirmed that HO-1 overexpression is protective against oxidative stress induced by heme and H2O2 in vitro or ischemic brain injury in vivo [38,39].

In contrast to its short-term effects, long term hypoxia (<0.5% O2) over 24 h and its consequent metabolic stress induced cell injury. This was evidenced by an increase in cell death, either apoptosis or necrosis, in parallel to depletion of constituitive HSP27 and a decrease in inducible HO-1. Thus, it is likely that at this later stage of severe stress the defense mechanisms of cardiac myocytes are overwhelmed, suggesting that the cytoprotective effect of stress protein induction is limited by the intensity and the severity of the environmental stress.


   5 Summary and conclusions
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Summary and conclusions
References
 
Cardiac myocytes exposed to a physiologically relevant decrease in oxygen tension exhibited a differential pattern of HSP expression with both transcriptional and posttranslational regulation. Both HSP70i and HO-1 were induced by hypoxia while constituitive HSP27 mRNA and protein expression were not modified. We also showed for the first time a link between de novo HO-1 synthesis, HSP27 phosphorylation and involvement of the p38 stress-activated kinase pathways in response to hypoxia. Further elucidation of how HSPs are regulated and how they exert their cardioprotective effects, e.g., on intracellular organelles such as microfilaments [10], microtubules [53] and actin [54], or on inhibition of caspase activity and subsequent apoptosis [55], will lead to additional understanding of the adaptive response to hypoxia-induced stress.

Time for primary review 33 days.


   Acknowledgements
 
This work was supported by Program Project Grant HL-25847 from the National Heart, Lung, and Blood Institute and The Research Service, Department of Veterans Affairs


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Summary and conclusions
 References
 

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