AMP-Activated Protein Kinase Protects Cardiomyocytes against Hypoxic Injury through Attenuation of Endoplasmic Reticulum Stress

Reagents.

AICAR (Toronto Research Chemicals, Inc., Canada) was used as a pharmacological activator of AMPK. Tunicamycin (Tm), an inhibitor of the synthesis of all N-linked glycoproteins, and thapsigargin (Tg), an irreversible inhibitor of ER Ca²⁺-ATPase, were used as agents inducing ER stress (Sigma, St. Louis, MO). 8-(p-sulfophenyl)-theophylline (8-SPT) was used as a nonselective adenosine receptor antagonist (Sigma). Anti-BiP/GRP78 and anti-CHOP/GADD153 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-caspase 12 and anti-α-actinin (sarcomeric) antibodies were purchased from Sigma. Anti-phosphorylated AMPK (Thr172), anti-AMPK, anti-phosphorylated eukaryotic elongation factor 2 (eEF2) (Thr56), anti-phosphorylated 4E binding protein 1 (4E-BP1) (Thr37/46), anti-phosphorylated eukaryotic initiation factor 2α (eIF2α) (Ser51), and anti-cleaved poly(ADP-ribose) polymerase (PARP) (Asp214) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-phosphorylated acetyl-coenzyme A carboxylase (ACC) (Ser79) was purchased from Upstate Biotechnology (Charlottesville, VA). Anti-α-tubulin monoclonal antibody was purchased from Calbiochem (Germany). All other chemicals were reagents of molecular biology grade obtained from standard commercial sources.

Cell culture and in vitro hypoxic model.

Primary cultures of neonatal rat cardiomyocytes were prepared from ventricles of 1-day-old Wistar rats (Kiwa Jikken Dobutsu, Japan) as described previously (39). Care of all animals in the present study accorded with the Osaka University Animal Care guidelines. Briefly, hearts were treated with trypsin and collagenase for 30 min. Isolated cells were collected by centrifugation and resuspended in Medium 199 (M-199) containing 10% fetal calf serum (FCS). Cultures were enriched with myocytes by preplating for 90 min to deplete the population of nonmyocytes. Nonattached cells were plated onto plastic culture dishes at an appropriate cell density. The cells were cultured in M-199 containing 10% FCS at 37°C in 95% air-5% CO₂ for 30 h. The culture medium was then changed to fresh M-199 containing 1% FCS and cells were cultured for 16 h. Subsequently, the cells were treated with reagents or incubated in a hypoxic culture chamber. Medium was changed to fresh M-199 containing 1% FCS 1 h before exposure to reagents or hypoxia in order to obtain consistent ER stress response data (29).

Hypoxia was achieved by using an AnaeroPack System anaerobic jar (Mitsubishi Gas Chemical, Japan) equipped with an AnaeroPack (oxygen-absorbing and carbon dioxide-generating envelope) and a methylene blue indicator to monitor oxygen depletion. The AnaeroPack System is capable of depleting the concentration of oxygen down to <1% in 1 h and providing a 5% CO₂ atmosphere without changing medium pH. Cultured neonatal rat cardiomyocytes were subjected to hypoxic conditions in the anaerobic jar at 37°C for the time periods indicated, while controls were left in normoxic conditions at 37°C for the same time periods.

Generation of recombinant adenovirus and protocol for adenoviral infection.

AMPK is an αβγ heterotrimer, the activation of which requires phosphorylation of threonine-172 on its catalytic α subunit. Therefore, cDNA encoding α1, which contains a mutation that changes threonine-172 to alanine, was used to construct the recombinant adenovirus expressing a dominant-negative form of the α1 subunit of AMPK (Ad-dnAMPK) according to a protocol described elsewhere (1). The cDNA was kindly donated by T. Kadowaki (Tokyo University, Tokyo, Japan) (66, 68).

We used adenovirus-mediated gene transfer to express this mutant in cultured primary rat cardiomyocytes. At 22 h after plating, cardiomyocytes were infected with adenovirus diluted in M-199 containing 10% FCS at a multiplicity of infection (MOI) of 20 and incubated for 8 h. After removal of viral suspension, cardiomyocytes were incubated with M-199 containing 1% FCS for 16 h and stimulated with reagents or exposed to hypoxia. Adenovirus vector expressing β-galactosidase (Ad-β-gal) was used as a control. Efficiency of infection, determined by lacZ gene expression in cultured cardiomyocytes, was consistently >90% with this method.

siRNA and transfection.

Silencing of CHOP, caspase 12, and eEF2 kinase gene expression in primary neonatal rat cardiomyocytes was achieved by the small interfering RNA (siRNA) technique (12). The sequences of the siRNA duplexes were selected from the coding regions of the target mRNAs. Duplexes of 21-nucleotide siRNA with two 3′-overhanging TTs were designed and synthesized by Genedesign (Japan). The sense strand of siRNA used to silence the rat CHOP gene (CHOP siRNA) was CCUGUCCUCAGAUGAAAUUdTdT (where dT is deoxythymidine) (regions 172 to 192). The sense strand of siRNA used to silence the rat caspase 12 gene (caspase 12 siRNA) was GCACAUUCCUGGUCUUUAUdTdT (regions 743 to 763). The sense strand of siRNA used to silence the eEF2 kinase gene (eEF2 kinase siRNA) was GGCAGUCCAUGAUUUUAGUdTdT (regions 2116 to 2136). Nonspecific siRNA (ATCCGCGCGATAGTACGTAdTdT) was purchased from B-Bridge International (Japan) and used as a negative control (unrelated siRNA).

Transfection of cultured cardiomyocytes was carried out by electroporation using a Nucleofector device (Amaxa, Germany) according to proposed protocol (13). Briefly, after isolation of cardiomyocytes, 2 × 10⁶ cardiomyocytes were resuspended in 100 μl of Nucleofector solution (rat cardiomyocyte Nucleofector kit; Amaxa) together with 1.5 μg of a green fluorescent protein (GFP)-coding plasmid (pCMV-GFP) and 1.5 μg of siRNA duplexes for each target. The cells were then placed in an electroporation cuvette. Transfection of siRNA duplexes specific for each target transcript was carried out. Immediately after electroporation, 400 μl of prewarmed M-199 containing 10% FCS was added to the cuvette and the cells were transferred into culture plates containing prewarmed M-199 with 10% FCS. At the optimal time of gene silencing (48 h posttransfection), cells were exposed to reagents or hypoxia.

Western blot analysis.

Neonatal rat cardiomyocytes were cultured at a cell density of 1 × 10⁶ in 60-mm plastic culture dishes in duplicate. After culture with M-199 containing 10% FCS for 30 h, the medium was changed to M-199 containing 1% FCS and incubated for 16 h. Thereafter, cardiomyocytes were stimulated with reagents or incubated under hypoxic conditions. After stimulation, cells were immediately lysed in lysis buffer (62.5 mmol/liter Tris-HCl, pH 6.8; 2% [wt/vol] sodium dodecyl sulfate; 10% glycerol; 50 mmol/liter dithiothreitol; 0.1% bromophenol blue). The samples were separated on a 4% to 20% gradient polyacrylamide gel (Daiichi Kagaku, Japan), electrotransferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA), and processed for immunoblot analysis essentially as described previously (39). An enhanced chemiluminescence system was used for detection. Band intensity was analyzed using NIH image software (version 1.62).

Northern blot analysis.

Portions (10 μg) of total RNA were prepared by the acid guanidinium thiocyanate-phenol-chloroform method. Total cellular RNA was loaded in each lane and size fractionated by 1% formaldehyde-agarose gel electrophoresis. Probes for BiP and CHOP were kindly donated by T. Katayama (Osaka University, Suita, Japan). Probes for ATF6 and XBP1 were kindly donated by K. Mori (Kyoto University, Kyoto, Japan). Probe for GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was kindly donated by K. R. Chien (University of California, San Diego, CA). Probe for VEGF was generated as described previously (19). Band intensity was analyzed using NIH image software (version 1.62).

Immunostaining.

For immunostaining, cardiomyocytes were grown on glass coverslips coated with gelatin. Cells were incubated under hypoxic conditions. Cells were fixed with 2% (wt/vol) formaldehyde for 15 min at room temperature and permeabilized with 0.1% Triton X-100-phosphate-buffered saline (PBS). Following three PBS washes, the glass coverslips were incubated in 1% bovine serum albumin-PBS for 1 h to block nonspecific binding sites. Subsequently, the cells were stained overnight at 4°C in 0.05% Tween 20 and 1% bovine serum albumin-PBS with primary antibodies for each target, followed by incubation for 1 h at room temperature with fluorescence-conjugated goat anti-mouse and goat anti-rabbit secondary antibodies. Then, the cells were stained with 0.5 mg/ml of Hoechst 33258 (Nacalai Tesque, Japan) to visualize nuclei. Cardiomyocytes were viewed by fluorescence microscopy, and pictures were taken using a Zeiss Axioskop2 (Carl Zeiss, Germany) with equal exposure times. To count positive cells for each primary antibody, a total of 200 to 300 Hoechst-stained nuclei (magnification of ×200) were counted for each experimental group. We measured the extent of fluorescence by using MetaMorpho microscope image analysis software (version 6.3; Molecular Devices, Japan). Cells with densities greater than the mean + standard deviation (SD) of the control group were considered positive.

Measurement of cell viability.

Cells were plated at a density of 1 × 10⁵ cells per well on 96-well plates for the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Cell viability was quantitated with an MTS cell respiratory assay (Promega, Madison, WI) according to the manufacturer's instructions. Viability was expressed as the absorbance at 490 nm measured with a spectrophotometer (Immuno-Mini NJ-2300; Inter Med, Japan).

Apoptosis assays.

We used the cleaved PARP antibody for Western blot analyses and immunofluorescence analyses (52). PARP can be cleaved by many interleukin-converting enzyme-like caspases and is one of the main cleavage targets of caspase 3. Apoptosis was also confirmed by fluorescence microscope examination using Hoechst 33258 to visualize cell nuclei.

ATP measurement.

Cells were plated at a density of 5 × 10⁵ cells per well on 96-well plates. Intracellular ATP contents in cardiomyocytes were measured using an ATP assay kit (Calbiochem, Germany). After exposure to hypoxia or normoxia for the indicated time periods, culture medium was removed. Cells were treated with 100 μl of nuclear releasing reagent for 5 min at room temperature with gentle shaking. Then, the cell lysate was treated with 1 μl ATP monitoring enzyme. Luminescence was measured using FluorChem-IS8000 (Alpha Innotech Corporation, San Leandro, CA). Amounts of ATP were determined from a standard curve constructed with known concentrations of ATP. Decreases in ATP levels were determined by comparing these results with levels for the normoxic control.

Measurement of percent CPK.

Cells were plated at a density of 1 × 10⁶ cells per well onto 60-mm plastic culture dishes for the creatine phosphokinase (CPK) assay. Enzyme release determinations were made 30 h after exposure to hypoxia. Determination of CPK in cultured conditioned medium was performed with a commercially available kit (CPK2 kit; Wako, Japan) by monitoring absorbance changes at 560 nm. We then added 0.5 ml of 0.1% Triton X-100-PBS to the adherent cells on the 60-mm plastic culture dishes. After lysing pellets by incubating the cells for 5 min at 37°C and centrifuging for 2 min at 11,000 × g, we measured CPK activities from lysed cells as described above.

Leucine incorporation.

For measurement of incorporation of [³H]leucine, cells were plated at a density of 1 × 10⁵ cells per well on 96-well plates. To examine the effects of AICAR on protein synthesis under hypoxic conditions, the cells were preincubated with or without AICAR, and then [³H]leucine (1 μCi/ml) was added to the culture medium and the cells were exposed to hypoxia. The cells were washed three times with PBS and then treated with 5% trichloroacetic acid at 4°C for 10 min to precipitate proteins. The precipitates were dissolved in 0.1 N NaOH, and aliquots were tested with a scintillation counter.

Statistical analysis.

Values are presented as means ± SDs. Statistical analysis was performed with the unpaired Student t test. Findings of P values of <0.05 were considered significant.

Induction of the unfolded protein responses in cultured neonatal rat cardiomyocytes after exposure to hypoxia.

We first examined transcriptional and translational induction of an ER-specific chaperone, BiP, and a transcriptional factor, CHOP, both of which are markers of UPR (2, 71), in cultured neonatal rat cardiomyocytes under conditions of oxygen depletion. Cardiomyocytes were exposed to hypoxia in an anaerobic jar for various periods of time. Expression levels of BiP and CHOP mRNAs were induced by exposure to hypoxia (Fig. 1A). Expression levels of BiP and CHOP were also increased at the protein level during hypoxia (Fig. 1B). In contrast, neither BiP mRNA nor CHOP mRNA was induced under normoxic conditions (data not shown). Furthermore, we examined the expression levels of the upstream components of UPR (32, 69), ATF6 and XBP1, and a well-known hypoxia-inducible gene, the VEGF gene (33). ATF6 and XBP1 mRNA levels increased in a progressive manner during the time course (Fig. 1C). Both ATF6 and XBP1 mRNA expression levels followed a similar pattern to BiP, CHOP, and VEGF mRNA expression levels during the time periods. Because expression of ATF6, XBP1, BiP, and CHOP occurs specifically under conditions in which ER function is disturbed, these findings indicate that hypoxia disturbed ER function in cultured neonatal rat cardiomyocytes.

To confirm activation of UPR following exposure to hypoxia and to investigate the extent of UPR in each cell, we examined the expression levels of BiP and CHOP by use of immunohistochemical staining in cultured neonatal rat cardiomyocytes. Hypoxia increased the levels of expression of both BiP and CHOP proteins (Fig. 2). Higher magnification on microscopic examination revealed that BiP and CHOP proteins were immunostained in the paranuclear and nuclear regions of cardiomyocytes, respectively (Fig. 2A and B). BiP was constitutively expressed in the cytosol under control conditions but significantly increased in expression in the cytosol after exposure to hypoxia (Fig. 2A). On the other hand, CHOP was undetectable in most cells under normoxic conditions but was strongly induced in nuclei by hypoxia (Fig. 2B). Quantitative analysis revealed large numbers of BiP- or CHOP-positive cells among cardiomyocytes after exposure to hypoxia (Fig. 2C and D, respectively). We thus confirmed that UPR was induced by hypoxia in our model by demonstrating up-regulation of BiP and CHOP expression. Taken together, these findings suggest that ER stress is one of the pathological changes induced by hypoxia in cardiomyocytes.

Exposure to hypoxia induced ER stress-associated apoptotic signaling in cardiomyocytes.

Because caspase 12, which is confined to the cytoplasmic side of the ER (46), is expressed at high levels in rat heart (45) and is believed to play an important role in ER stress-mediated cell death (46), we examined the activation of caspase 12 after exposure to hypoxia. As shown in Fig. 3A, caspase 12 was cleaved during hypoxia, indicating activation of caspase 12 in cardiomyocytes following exposure to hypoxia.

To clarify the involvement of ER stress in apoptotic cell death, double-fluorescence immunohistochemical staining of CHOP and cleaved PARP, a marker of apoptosis, was performed. Strong induction of CHOP during hypoxia colocalized with staining of cleaved PARP and condensed nuclei revealed by Hoechst staining (Fig. 3B), indicating that CHOP was associated with apoptosis.

Knockdown of CHOP and caspase 12 using siRNA duplexes yielded cardioprotective effects following exposure to hypoxia.

To estimate transfection efficacy, 1.5 μg of a GFP-coding plasmid (pCMV-GFP) was cotransfected with 1.5 μg of unrelated siRNA duplexes or of siRNA duplexes cognate to GFP (GFP siRNA, purchased from Amaxa) by electroporation as described in Materials and Methods. After 48 h of incubation, cells were fixed and immunostained with anti-GFP antibody and with Hoechst 33258 for nuclear staining. We analyzed the findings for more than 300 Hoechst-stained cells and counted GFP-positive cells by fluorescence microscopy. A total of 16% of cardiomyocytes were transfected with pCMV-GFP. GFP siRNA duplexes efficiently suppressed transfected GFP gene expression, with an 83% reduction in the percentage of GFP-positive cells with GFP siRNA duplexes from that with unrelated siRNA duplexes (Fig. 4A and B). We also confirmed by Western blot analysis that GFP expression was barely detectable in GFP siRNA-transfected cardiomyocytes (Fig. 4C). It thus appeared that siRNA duplexes were almost completely transfected in cardiomyocytes transfected with pCMV-GFP and that GFP expression was a marker of transfection with siRNA duplexes.

To investigate whether siRNA duplexes for CHOP or caspase 12 knock down the target gene expression specifically, immunofluorescence experiments to determine target protein depletion were performed after exposure to 16 h of hypoxia. Figures 4D and E show that the CHOP siRNA and caspase 12 siRNA duplexes specifically reduced CHOP and caspase 12 expression levels, respectively, without modifying the expression levels of other proteins. No morphological changes were observed with phase-contrast micrographs of the CHOP siRNA- or caspase 12 siRNA-transfected cardiomyocytes under normoxic conditions (data not shown).

To confirm the effects of CHOP and caspase 12 knockout against hypoxic injury, after 48 h posttransfection with CHOP siRNA or caspase 12 siRNA duplexes, the cardiomyocytes were exposed to normoxia or hypoxia for 36 h. After fixing the cells, we counted GFP-positive cells undergoing apoptosis, which were labeled with the cleaved PARP antibody in nuclei by use of fluorescence microscopy. Figure 4F shows the percentages of cells undergoing apoptosis among GFP-positive cells. The expression of cleaved PARP was barely detectable in cardiomyocytes under normoxic conditions. Under conditions of hypoxia, the percentage was dramatically attenuated in CHOP siRNA- or caspase 12 siRNA-transfected cardiomyocytes compared to that in unrelated siRNA-transfected cardiomyocytes. These findings suggest that ER stress plays a role in inducing apoptosis during hypoxia.

AMPK protected cardiomyocytes against hypoxic injury through attenuation of ER stress.

We investigated the roles played by AMPK during hypoxia in cultured neonatal rat cardiomyocytes. As shown in Fig. 5A, the exposure of cardiomyocytes to hypoxia resulted in rapid and sustained phosphorylation of AMPK and ACC, a downstream target of AMPK (24). We next assessed the activation of AMPK in cardiomyocytes in the presence or absence of AICAR, a chemical activator of AMPK. Western blot analysis revealed that exposure of cardiomyocytes to AICAR resulted in rapid phosphorylation of AMPK in a time-dependent (Fig. 5B) and dose-dependent (data not shown) fashion. Pretreatment with AICAR 1 h before exposure to hypoxia accelerated the phosphorylation status of AMPK during hypoxia compared with that in the untreated group (Fig. 5C).

To clarify whether AMPK promotes survival of cardiomyocytes in conditions yielding hypoxic injury, we performed an MTS cell respiration assay to assess cell viability and measured percent CPK release in culture medium to detect cell injury after exposure to hypoxia for 30 h. Relative MTS activity of the cells in the AICAR-pretreated group was significantly higher than that in the untreated group (Fig. 6A). Similarly, percent CPK release in the culture medium in the AICAR-pretreated group was significantly less than that in the untreated group (Fig. 6B). To confirm the effects of activation of AMPK by AICAR, we generated and performed infection of Ad-dnAMPK in cardiomyocytes. Ad-dnAMPK (20 MOI) effectively blocked AICAR (0.5 mM)- and hypoxia-induced phosphorylation of ACC in cardiomyocytes (Fig. 6C). However, Ad-dnAMPK (20 MOI) did not completely block the activation of AMPK induced by more than 1 mM of AICAR (Fig. 6C). Figures 6D and E show that the protective effects of AICAR against hypoxia were abolished by overexpression of dnAMPK and that the inhibitory effects of Ad-dnAMPK were attenuated by a further dose of AICAR pretreatment, indicating that the cardioprotective effects of AICAR are mediated in an AMPK-dependent manner.

To examine whether AMPK protects cardiomyocytes against hypoxia-induced apoptosis through attenuation of ER stress, we assessed the effects of AICAR on the extent and time course of induction of ER stress-associated proapoptotic signaling, such as that through CHOP and cleavage of caspase 12 during hypoxia. We also performed Western blot analysis of cell extracts by using anti-cleaved PARP antibody to assess cellular apoptosis. Pretreatment with AICAR attenuated the induction of CHOP mRNA (Fig. 7A) and protein (Fig. 7B to D) as well as cleavage of caspase 12 (Fig. 7B) during hypoxia. To confirm that these effects of AICAR were mediated in an AMPK-dependent manner, we examined the effects of AICAR on ER stress-mediated apoptotic signaling in cardiomyocytes infected with Ad-dnAMPK. Induction of CHOP and cleavage of caspase 12 were diminished by pretreatment with AICAR in Ad-β-gal-infected cardiomyocytes (Fig. 7E and G). In contrast, the effects of AICAR were abolished in Ad-dnAMPK-infected cardiomyocytes (Fig. 7F and H), indicating that the protective effects of AICAR against ER stress-associated apoptotic signaling are mediated in an AMPK-dependent manner. Moreover, as shown in Fig. 7I, pretreatment with AICAR significantly decreased the expression of cleaved PARP during hypoxia, and these cardioprotective effects of AICAR were abolished in Ad-dnAMPK-infected cardiomyocytes. In addition, the inhibitory effects of dnAMPK were attenuated by a further dose of AICAR pretreatment. These findings indicate that pretreatment with AICAR diminishes apoptotic cell death and promotes survival.

Intracellular ATP content did not affect cardioprotection by AICAR during hypoxia.

Depletion of intracellular ATP content is a crucial feature of ischemic processes, and degree of depression of ATP level is one of the determinants of apoptosis (9). Because AMPK is known to preserve energy during hypoxia (25), we examined whether the cardioprotective effects of AICAR against hypoxia-induced injury are due to AMPK-dependent ATP conservation during hypoxia. To investigate whether AICAR pretreatment preserves intracellular ATP content, ATP levels were measured after exposure of cardiomyocytes to hypoxia for various time periods in the presence or absence of AICAR. Figure 8 shows the time course of hypoxia-induced ATP depletion in cardiomyocytes. Intracellular ATP contents did not differ significantly between the AICAR-untreated group and the AICAR-pretreated group at either 10 h or 20 h after exposure to hypoxia. These findings indicate that the mechanism of cardioprotection by AICAR is not dependent on ATP conservation.

AMPK decreased the rate of protein synthesis during hypoxia via inactivation of eEF2.

Recent reports have indicated that activation of AMPK leads to increased phosphorylation of eEF2, which inhibits elongation of translation in its phosphorylated state and thus leads to inhibition of protein synthesis in cardiomyocytes (28). We also confirmed that treatment of cardiomyocytes with 0.5 mM AICAR for 24 h caused inhibition of the protein synthesis rate by 21.5% under normoxic conditions, as assessed by examining incorporation of [³H]leucine. Reduced rates of protein synthesis under conditions of ER stress serve to reduce the load of substrates presented to the folding machinery in the ER lumen, with mitigation of the consequences of insults to cellular homeostasis (53). We therefore hypothesized that regulation of eEF2 by AMPK might play a role in the attenuation of ER stress-associated apoptosis under conditions of hypoxia.

First, we measured the activities of key molecules related to translation, such as eEF2, 4E-BP1, and eIF2α, in cardiomyocytes during hypoxia. As shown in Fig. 9A, eIF2α, but not 4E-BP1, was phosphorylated during hypoxia. eEF2 was uniquely phosphorylated during hypoxia in biphasic fashion. Phosphorylation of eEF2 during hypoxia was significantly attenuated by Ad-dnAMPK (Fig. 9B), but phosphorylation of eIF2α during hypoxia was not attenuated by Ad-dnAMPK (data not shown). Moreover, as shown in Fig. 9C and D, AICAR phosphorylated eEF2, but not eIF2α, in a dose- and time-dependent fashion in cardiomyocytes. To further examine the effects of AMPK on protein synthesis during hypoxia, cardiomyocytes were exposed to hypoxia for 8 h with or without AICAR pretreatment and incorporation of [³H]leucine was examined. The rate of protein synthesis during hypoxia was significantly and dose-dependently decreased by pretreatment with AICAR (Fig. 9E). This decrease in rate of protein synthesis was completely prevented by Ad-dnAMPK, and the inhibitory effects of dnAMPK were attenuated by a further dose of AICAR pretreatment (Fig. 9F).

Taken together, these findings suggest that AMPK might protect cardiomyocytes against ER stress-induced apoptosis during hypoxia via inhibition of protein synthesis through inactivation of eEF2.

Attenuation of eEF2 phosphorylation using eEF2 kinase siRNA duplexes abolished the cardioprotective effects of AICAR during hypoxia.

To confirm that eEF2 plays a role in cardioprotection by AICAR during hypoxia, experiments with siRNA duplexes to attenuate the phosphorylation of eEF2 during hypoxia and AICAR treatment in cardiomyocytes were performed. Because eEF2 kinase is the only kinase that phosphorylates eEF2 (5), we suspected that knockdown of eEF2 kinase using siRNA duplexes would attenuate the phosphorylation of eEF2 during hypoxia and AICAR treatment. As shown in Fig. 10A, eEF2 kinase expression was specifically knocked down in eEF2 kinase siRNA-transfected cardiomyocytes. The fluorescence intensity of the eEF2 protein was affected in neither eEF2 kinase siRNA- nor unrelated siRNA-transfected cardiomyocytes (Fig. 10B). Fluorescent phosphorylated eEF2 (p-eEF2) production increased after 4 h of hypoxia with AICAR treatment in the unrelated siRNA-transfected cardiomyocytes (Fig. 10C). In contrast, fluorescent p-eEF2 production was abolished even after exposure to 4 h of hypoxia with AICAR treatment in eEF2 kinase siRNA-transfected cardiomyocytes (Fig. 10C), indicating that knockdown of eEF2 kinase attenuated the phosphorylation of eEF2 during hypoxia with AICAR treatment. No morphological differences between eEF2 kinase siRNA- and unrelated siRNA-transfected cardiomyocytes were observed with phase-contrast micrographs under normoxic conditions (data not shown).

To investigate the roles played by eEF2 in mediating the cardioprotective effects of AICAR against hypoxia-induced ER stress and cell death, unrelated siRNA- or eEF2 kinase siRNA-transfected cardiomyocytes were incubated for 36 h under hypoxic conditions with or without pretreatment with AICAR. Cells under conditions of ER stress were assessed by immunofluorescence analyses with CHOP antibody using fluorescence microscopy. In addition, cells undergoing apoptosis were assessed by immunofluorescence analyses with cleaved PARP antibody using fluorescence microscopy. Pretreatment with AICAR dramatically decreased the percentage of CHOP-positive and cleaved PARP-positive cells in the unrelated siRNA-transfected cells (Fig. 10D and E). However, knockdown of eEF2 kinase abolished these cardioprotective effects of AICAR (Fig. 10D and E). These findings demonstrate that eEF2 inactivation plays a crucial role in mediating the cardioprotective effects of AICAR.

The cardioprotective effects of AICAR were not mediated by adenosine.

AICAR has been proposed to increase tissue adenosine levels by entering the de novo nucleotide biosynthetic pathway (44), and several reports have indicated that the protective effects of AICAR are mediated by an adenosine-dependent mechanism (11). Therefore, to determine whether the protective effects of AICAR are independent of adenosine, we used 8-SPT as a nonselective adenosine receptor antagonist. The cytoprotective effects of AICAR were not diminished by 8-SPT (100 μM) (Fig. 11A and B), indicating that these effects are independent of adenosine.

AICAR attenuated Tm- and Tg-induced apoptotic signaling in cardiomyocytes.

In cardiomyocytes, we found that BiP and CHOP were induced by ER stress inducers, including Tm and Tg, in a dose- and time-dependent fashion (data available on request) and that exposure to Tm and Tg induced the activation of caspase 12 (data available on request). Therefore, we examined whether AICAR attenuated Tm- and Tg-induced apoptotic signaling, as well as hypoxia-induced apoptotic signaling, in neonatal rat cardiomyocytes. As shown in Fig. 12A and B, induction of CHOP mRNA by Tm or Tg was significantly attenuated by pretreatment with AICAR 1 h before exposure to Tm or Tg, compared with results obtained with lack of pretreatment. These findings support the hypothesis that AMPK plays a cardioprotective role under conditions of ER stress.