Protein Kinase Cδ Activation Induces Apoptosis in Response to Cardiac Ischemia and Reperfusion Damage

Heart attacks caused by occlusion of coronary arteries are often treated by mechanical or enzymatic removal of the occlusion and reperfusion of the ischemic heart. It is now recognized that reperfusion per se contributes to myocardial damage, and there is a great interest in identifying the molecular basis of this damage. We recently showed that inhibiting protein kinase Cδ (PKCδ) protects the heart from ischemia and reperfusion-induced damage. Here, we demonstrate that PKCδ activity and mitochondrial translocation at the onset of reperfusion mediates apoptosis by facilitating the accumulation and dephosphorylation of the pro-apoptotic BAD (Bcl-2-associated death promoter), dephosphorylation of Akt, cytochrome c release, PARP (poly(ADP-ribose) polymerase) cleavage, and DNA laddering. Our data suggest that PKCδ activation has a critical proapoptotic role in cardiac responses following ischemia and reperfusion.

Reperfusion after coronary occlusion is one of the leading causes of cardiac injury and cell death (1). A variety of signaling pathways participate in myocardial responses to ischemia and reperfusion and the damage that occurs involves both necrotic and apoptotic cell death (2,3). Apoptosis, otherwise known as programmed cell death, is an energy-dependent process (4) that requires the participation of specific cascades of tightly regulated enzymes that function to remove injured, dying, or unnecessary cells (3). Apoptosis can be activated by a number of stimuli such as DNA damage (5) and oxidative stress (6 -8). Unlike apoptosis, necrosis is an energy-independent process that often occurs in response to uncontrolled cell rupturing, leading to damage of surrounding cells and a subsequent inflammatory response (9).
Current research of the molecular events that control the apoptotic process in response to ischemia and reperfusion implicated pathways involving death receptor-induced apoptosis by, for example, up-regulation of their ligands such as Fas (10 -12) and tumor necrosis factor-␣ (13). Other works described the effects of differential expression and activity of the Bcl-2-related proteins, such as Bak, BAD, Bax, Bid, Bcl-2, and Bcl-x L (14 -18). In addition, signaling kinases including p38 MAPK (19), JNK (6), Erk (20), Akt (21), and protein kinase C (PKC) 1 (reviewed in Ref. 3) have also been suggested to play a role in apoptosis. These enzymes and others cooperate to induce downstream events resulting in cytochrome c release, caspase activation, PARP cleavage, and DNA laddering (22)(23)(24). However, how these receptors, kinases, and other enzymes influence apoptosis caused by ischemia and reperfusion injury is still under investigation.
One family of proteins that has been shown to mediate ischemia and reperfusion damage is PKC. Since PKC was first identified in 1977 (25), PKC activity has been shown to affect numerous signal transduction processes including differentiation, tumor progression, proliferation, secretion, as well as apoptosis (3,26). A hallmark of PKC activation involves the movement, or translocation, of PKC from the cytosol to membranes in response to various stimuli (27). In isolated adult cardiac myocytes, ischemia induces the activation and translocation of two PKC isozymes, PKC⑀ and ␦ (28,29). However, using PKC isozyme-specific peptides that modulate individual isozyme translocation and activity (29), we showed that PKC⑀ and ␦ activities have opposing consequences in response to ischemia-induced cell damage (30,31). Activating PKC⑀ during ischemia leads to cardioprotection (29,32,33), whereas activating PKC␦ with a PKC␦-specific activator enhanced cell damage (29). Recent evidence indicates that PKC␦, but not PKC⑀, also participates in reperfusion injury. Delivery of the PKC␦selective inhibitor peptide, ␦V1-1, to hearts ex vivo or in vivo (30,31) during the onset of reperfusion was cardioprotective. In contrast, delivery of ⑀RACK peptide to activate PKC⑀ translocation during reperfusion had no cardioprotective effect (30). Our data suggest that activation of PKC␦ during reperfusion mediates cardiac damage. However, questions remain regarding the molecular basis of the damage that occurs to the heart in response to PKC␦ activity.
Many studies suggest a role for PKC␦ in apoptosis (reviewed in Ref. 34). In both LNCaP prostate cancer cells and CaCo-2 human colon-cancer cells, phorbol 12-myristate 13-acetate-induced translocation of overexpressed PKC␦ results in apoptosis (35,36). In addition, PKC␦-induced apoptosis occurs in response both to UV as well as etoposide-induced damage (37,38). PKC␦ translocates to the mitochondria, resulting in the release of cytochrome c, in response to various pro-apoptotic stimuli (39 -41). Therefore, the pro-apoptotic activity of PKC␦ may mediate mitochondrial dysfunction. In this study, we provide evidence that PKC␦ translocation to the mitochondria during reperfusion plays a key role in the regulation of the mitochondrial involvement in the signaling pathways leading to apoptosis and cell death.

Isolation and Rat Heart Perfusion
Male Wistar rats (250 -300 g) were heparinized (2000 units/kg intraperitoneally) and then anesthetized with sodium pentobarbital (100 mg/kg intraperitoneally). The hearts were rapidly excised and then perfused with an oxygenated (95% O 2 , 5% CO 2 ) Krebs-Henseleit buffer containing (in mmol/liter) NaCl 120, KCl 5.8, NaHCO 3 25, NaH 2 PO 4 1.2, MgSO 4 1.2, CaCl 2 1.0, and dextrose 10, pH 7.4, at 37°C in a Langendorff coronary perfusion system as previously described (30). The hearts were perfused with a constant coronary flow rate of 10 ml/min throughout the experiment. After a 10-min equilibration, perfusion was stopped for 30 min to induce global ischemia, during which time the hearts were immersed into a 37°C heated bath of Krebs-Henseleit buffer. After the ischemic period, the flow was restored; and reperfusion commenced for 15, 30, 60, or 120 min with or without 500 nM ␦V1-1 (29), conjugated to the cell permeable peptide, Tat (amino acids 47-57, Tat-␦V1-1 for short) (42), for the first 15 min of reperfusion. In studies reported in Fig. 5B, reperfusion was studied for up to 240 min. Following reperfusion, the hearts were removed from the apparatus and homogenized for mitochondria isolation, sliced into sections, or snap-frozen with liquid nitrogen, stored at Ϫ70°C, and subsequently used for protein analysis. All animal protocols were approved by the Institutional Animal Care and Use Committee of Stanford University and the Case Western Reserve University.

Isolation of Subsarcolemmal Mitochondria from Rat Heart
Immediately following each experimental protocol, the hearts were immersed and rinsed in ice-cold homogenization buffer A (210 mM mannitol, 70 mM sucrose, 1.0 mM EDTA, and 5.0 mM MOPS at pH 7.4). Hearts (0.9 -1.2 g) were minced and homogenized in 20 ml of homogenization buffer/g of tissue using a polytron homogenizer (low setting, 2 s, three times) as previously described (43,44). The homogenate was filtered through cheesecloth and centrifuged at 500 ϫ g for 5 min (5°C). The resulting supernatant was filtered through cheesecloth and centrifuged at 10,000 ϫ g for 10 min (5°C) to obtain the mitochondrial pellet and the cytosolic extract (supernatant). The mitochondrial pellet was rinsed and resuspended in buffer A at a final concentration of ϳ20 mg/ml. Protein determinations were carried out using the bicinchoninic acid method (Pierce) with bovine serum albumin as a standard. Mitochondria and cytosolic extracts were stored at Ϫ70°C.

Western Blot Analysis
Detection of PKC␦ and cytochrome c-PKC␦ levels were assessed using 30 g of mitochondrial protein/lane resolved by 4 -15% SDS-PAGE and transferred to nitrocellulose. Proteins from cytosolic extracts were resolved by 4 -20% SDS-PAGE and transferred to nitrocellulose. After transfer, the membranes were washed three times in phosphatebuffered saline with 0.05% Tween 20 (PBST). The membranes were then blocked for 30 min in PBST with 5.0% milk and probed with polyclonal anti-PKC␦ (Cell Signaling Technology), cytochrome c, adenine nucleotide translocase (obtained from Dr. Schmidt, Hormel Institute), and glyceraldehyde-3-phosphate dehydrogenase (Chemicon International) antibodies, followed by secondary anti-IgG rabbit antibody linked to alkaline phosphatase (Tropix). Protein bands were visualized using the CSPD system (Tropix). Densitometry was performed using the NIH ImageJ software program (available at rsb.info.nih.gov/ij/) to assess the relative mitochondrial and cytosolic content of PKC␦ and cytochrome c, respectively.
Tissue fractionation-Frozen heart tissues were thawed on ice in homogenization buffer B (20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 10 mM EGTA, 250 mM sucrose). The tissues were then minced and ground by pestle in the presence of molecular grinding resin (Geno Technology) to produce homogenates. The homogenates were centrifuged at 100,000 ϫ g for 45 min (4°C) to separate the cytosolic soluble fraction from membranous proteins. After the soluble fractions were isolated, the pellets were then rehomogenized in buffer B containing 1% Triton X-100, incubated on ice for 30 min, and subsequently centrifuged at 100,000 ϫ g for 45 min (4°C). The Triton-soluble particulate fraction was isolated, and the protein concentrations were determined by Bradford assay. For PKC␦ translocation, ϳ5 g of soluble and particulate fractions were resolved by 10% SDS-PAGE and transferred to nitrocellulose membrane. After transfer, the blots were blocked with a 5% milk solution in Tris-buffered saline (pH 7.5) containing 0.05% Tween 20 (TBST) and probed with polyclonal anti-PKC␦ (Cell Signaling Technology) and anti-PKC␣ (Santa Cruz Biotechnology) antibodies followed by secondary anti-IgG rabbit antibody linked to horseradish peroxidase (Amersham Biosciences). Protein bands were visualized using ECL and quantitated using NIH ImageJ software.

Detection of Apoptosis-related Proteins
To produce total cell lysates, frozen heart tissues were thawed on ice in homogenization buffer B containing 1% Triton X-100. The tissues were then minced and ground by pestle in the presence of molecular grinding resin (Geno Technology), and the homogenates were incubated on ice for 30 min and centrifuged at 100,000 ϫ g for 45 min (4°C) to pellet detergent-insoluble debris. Approximately 30 g of protein was resolved by 8 -12.5% SDS-PAGE and transferred to nitrocellulose membrane. After transfer, the nitrocellulose blots were blocked with a 5% milk solution in TBST. The blots were then probed with primary rabbit polyclonal antibodies against BAD, phospho-BAD (Ser 136 ), Bax, Bcl-x L proteins (Santa Cruz Biotechnology), Akt, phospho-Akt (Ser 473 ), caspase 3, and PARP proteins (Cell Signaling Biotechnology) and a mouse monoclonal antibody against Bcl-2 (Stressgen Biotechnologies). Primary antibodies were followed by corresponding secondary antibodies linked to horseradish peroxidase. The protein bands were visualized and quantitated as previously described.

Fluorescence Microscopy: PKC␦ and Mitochondria Co-localization
To assess PKC␦ and mitochondria co-localization, rat heart sections were immediately frozen in optimal cutting temperature compound (Tissue-Tek), and 5-micron-thick sections were then cut on a cryostat (Stanford Histology Research Core Laboratory) and mounted on glass slides. The sections were washed with PBS and fixed with 4% formaldehyde for 20 min. Following fixation, the heart sections were incubated with 150 nM MitoTracker Red CMXRos (Molecular Probes) for 15 min in PBS, washed with PBS, and incubated with blocking buffer C (1% normal donkey serum in PBS, 0.1% Triton X-100) for 1 h. The sections were then incubated with polyclonal anti-PKC␦ (Santa Cruz Biotechnology) in buffer C for 3 h at room temperature and subsequently with Alexa Fluor 488 goat anti-rabbit fluorescein isothiocyanate secondary (Molecular Probes) in buffer C for 1 h at room temperature. The slices were then washed with PBS, 0.1% Triton X-100 and dried for 15 min at room temperature, and the coverslips (Corning) were mounted using Vectashield (Vector Laboratories). Fluorescence images of mitochondria (MitoTracker) and PKC␦ (fluorescein isothiocyanate) were obtained using a laser scanning confocal microscope (Pascal, Zeiss) with a 63ϫ water objective (1.2 numerical aperture). The slides were imaged on the stage of an inverted microscope (Axiovert 100 M) using the 568-nm laser line for the red label and the 488-nm line for green. Emission was collected through a 585-nm-long pass filter for the red label, and a 505-550-nm band pass filter for the green. Image analysis was performed using Metamorph software (Universal Imaging).

Apoptosis Assays
TUNEL Staining-To observe the incidence of apoptosis in damaged tissue, we subjected isolated perfused rat hearts to 30-min of ischemia and then 60 min of reperfusion. Tat-␦V1-1 was perfused during the first 15 min of reperfusion. The rat hearts were immediately frozen in optimal cutting temperature compound (Tissue-Tek) after ischemia/ reperfusion, and 5-m-thick sections were then cut on a cryostat. The sections were fixed with 4% formaldehyde and blocked with 1% normal donkey serum. The sections were first incubated with mouse monoclonal anti-␣-actinin antibody (Sigma) overnight at 4°C and subsequently with goat anti-mouse conjugated with fluorescein isothiocyanate for distinction between cardiomyocytes and other cells in the myocardium. Thereafter, terminal deoxynucleotidyl TUNEL staining was performed for detection of apoptotic cells according to the manufacturer's instructions (Roche Applied Science) and counterstained with 4,6-diamidino-2-phenylindole (Sigma). Tissues samples from normoxic control hearts were used as negative control. The tissues were imaged using fluorescent microscopy. TUNEL-positive nuclei were counted in a total of 1,500 myocytes over several random fields and expressed as percentages of the total number of nuclei.
DNA Laddering-To observe the appearance of DNA laddering, ϳ100 mg of ex vivo rat heart tissue following ischemia and reperfusion was lysed and DNA extracted as described (45). 20 g of DNA was resolved on a 1.8% agose gel subsequently stained with SYBR Green I nucleic acid gel stain per the manufacturer's instructions (Molecular Probes) and visualized by UV transillumination using the ChemiDoc XRS system (Bio-Rad). The gel shown represents two independent experiments.

Statistical Analysis
The results represent the means Ϯ standard deviation of four to five independent experiments (unless otherwise noted). The values are from a two-tailed t test where p Յ 0.05 was considered significant.

␦V1-1 Inhibits PKC␦ Translocation during Reperfusion-We
reported that inhibiting PKC␦ activity during reperfusion after ischemia, using Tat-␦V1-1 (␦V1-1), improved myocardial cell survival both ex vivo as well as in vivo (30,31). To identify the molecular events affected by inhibiting PKC␦ activation during ischemia and reperfusion, we first confirmed previous observations (28,29) that PKC␦ translocates from the cell soluble to the cell particulate fraction in response to ischemia (Fig. 1B, I 30  versus N). However, we observed that significant increases in PKC␦ translocation occur during reperfusion (Fig. 1B, I 30 ver-sus I 30 /R 60 ). Delivering 500 nM of ␦V1-1 at the onset of reperfusion resulted in a significant decrease in PKC␦ translocation, whereas delivering the Tat carrier alone had no effect (data not shown). The ␦V1-1 effect was specific for PKC␦, because treatment with ␦V1-1 did not affect ␣PKC (Fig. 1C). Therefore, reperfusion-induced PKC␦ translocation in this rat Langendorff preparation is inhibited with ␦V1-1 administration during the initiation of reperfusion.
␦V1-1 Inhibits Reperfusion-induced PKC␦ Translocation to Mitochondria and Cytochrome c Release-In cultured myeloid leukemia, muscle, and liver cells, PKC␦ was shown to translocate to mitochondria upon insulin, H 2 O 2 , or 12-O-tetradecanoylphorbol-13-acetate treatment (40,41,46). We therefore determined whether our observed increased translocation of PKC␦ to the particulate fraction is due, at least in part, to its translocation to the mitochondria. As shown in Fig. 2A, ischemia followed by 120 min of reperfusion induced a 2-fold increase in the level of PKC␦ in the mitochondrial fraction over values observed for perfused normoxic controls. No significant alteration in the level of PKC␦ in the mitochondrial fraction was observed during ischemia alone. Although PKC␦ was observed in the mitochondrial fraction from perfused control hearts, the level was not dependent on the duration of the perfusion period (data not shown). Importantly, when hearts were treated during the first 15 min of reperfusion with ␦V1-1, no reperfusion-induced increase in mitochondrial PKC␦ levels was observed. In addition, the Tat carrier peptide alone did not inhibit PKC␦ translocation to the mitochondria (not shown). The purity of the mitochondrial fraction and equal protein loading were confirmed by probing both mitochondrial and cytosolic fractions for the mitochondrial protein adenine nucleotide translocase and the cytosolic marker glyceraldehyde-3phosphate dehydrogenase. Therefore, PKC␦ translocation to the mitochondria during reperfusion appears to be specific.
To further establish whether PKC␦ and mitochondria colocalized in tissue, we labeled fixed heart tissue sections from each condition with the mitochondrial-selective dye Mito-Tracker CMXRos (red) and a fluorescein isothiocyanate-conjugated secondary antibody against primary anti-PKC␦ (green) (Fig. 3). Using confocal laser fluorescence microscopy, we observed a dramatic increase in the distribution of co-localized PKC␦ and mitochondria (yellow) in hearts reperfused after global ischemia (Fig. 3C), whereas very little co-localization was evident in normoxic (Fig. 3A) and ischemic hearts (Fig.  3B). Importantly, and correlating with our fractionation stud- ies studies (Fig. 2), when we inhibited PKC␦ translocation at the beginning of reperfusion using ␦V1-1, the co-localization of PKC␦ with mitochondria was greatly inhibited (Fig. 3D).
Western blot analysis also revealed a significant increase in the cytosolic content of cytochrome c during reperfusion (Fig.  2B), concurrent with PKC␦ translocation to mitochondria ( Fig.  2A). This increase in cytosolic cytochrome c was blocked by the addition of ␦V1-1 at the onset of reperfusion (Fig. 2B), whereas the Tat carrier alone had no effect (not shown). Probably because of mechanic disruption of the mitochondria during isolation, a significant level of cytochrome c is present in cytosolic extracts prepared from perfused normoxic control hearts (Fig.  2B). Nevertheless, the increase during reperfusion appears specific, given that it is dependent on PKC␦ translocation, and the mitochondrial protein adenine nucleotide translocase does not appear in the cytosolic fraction under any of the experimental conditions (Fig. 2C).
␦PKC Inhibition during Reperfusion Decreases Caspase 3 and PARP Cleavage and DNA Fragmentation-Release of cytochrome c from the mitochondria into the cytosol induces caspase activation involved in apoptosis (reviewed in Ref. 47). We found a significant increase in caspase 3 cleavage and activation, as shown by a decrease in procaspase 3, in response to reperfusion (Fig. 4A). Furthermore, reperfusion led to the increase of another apoptotic marker, cleaved PARP, as shown by a decrease in full-length PARP in response to reperfusion (Fig. 4B). PARP becomes inactivated upon cleavage and can no longer participate in DNA repair or maintain genomic stability (48). Administration of ␦V1-1 during reperfusion inhibited caspase 3 activation and PARP inactivation (Fig. 4).
To confirm that ischemia and reperfusion induced apoptosis, we examined tissue sections of hearts for cleaved DNA using the TUNEL assay (49). Indeed, reperfusion following ischemia induced a significant increase in TUNEL-stained nuclei; treating hearts with ␦V1-1 caused a greater than 70% reduction in TUNEL staining (Fig. 5A). We confirmed that reperfusion induced apoptosis by measuring DNA fragmentation (45). We observed significant DNA laddering in hearts exposed to 4 h of reperfusion following 30 min of global ischemia (Fig 5B). Treating hearts with ␦V1-1 to inhibit reperfusion-induced PKC␦ translocation led to a marked inhibition of DNA ladder formation (Fig. 5B). Therefore, our findings support previous data suggesting that ischemia and reperfusion induces myocardial cell apoptosis (31,50,51) that is mediated by PKC␦ translocation to the mitochondria and the activation of the apoptotic effectors involving cytochrome c release, caspase 3 activation, PARP cleavage, and DNA fragmentation.  7. A model depicting the mechanism of PKC␦-induced apoptosis in response to cardiac reperfusion. Reperfusion after cardiac ischemia alters the ratio of pro-and anti-apoptotic proteins with increases in pro-apoptotic BAD levels and decreases in the levels of anti-apoptotic Bcl-2, Bcl-x L , and phosphorylated BAD and Akt. PKC␦ translocation to the mitochondria correlates with increased levels of BAD while decreasing BAD and Akt phosphorylation. This, in turn, leads to cytochrome c release, caspase 3 activation, PARP cleavage, and subsequent DNA fragmentation. Our data suggest that inhibition of PKC␦ translocation is sufficient to block reperfusion-induced cascades of events that lead to myocyte death by apoptosis.
PKC␦ Activation during Reperfusion Affects BAD Protein Levels, BAD Phosphorylation, and Akt Activity-To further investigate the role of PKC␦ in myocardial cell apoptosis, we examined the levels and activity of the Bcl-2-related proteins: a pro-apoptotic protein, BAD, as well as anti-apoptotic proteins Bcl-2, Bcl-x L , and Akt. Ischemia and reperfusion caused significant increases in the levels of BAD protein (Fig. 6A), as well as considerable reductions in Bad phosphorylation (Fig. 6B) and the levels of anti-apoptotic Bcl-2 and Bcl-x L (Fig. 6, D and E). These results suggest that ischemia and reperfusion induced changes in the ratios and activity of pro-and anti-apoptotic proteins. When we specifically inhibited PKC␦ activity by treating hearts with ␦V1-1 during reperfusion, only, the levels of BAD were reduced, whereas BAD phosphorylation increased to the levels present during basal perfused conditions, with no changes in the ischemia and reperfusion-induced decreases in Bcl-2 or Bcl-x L . Therefore our results suggest that increased PKC␦ translocation correlates with a concurrent increase in BAD protein and decrease in phosphorylated BAD, but changes in Bcl-2 and Bcl-x L are independent of PKC␦ activity.
The phosphatidylinositol 3-kinase-related kinase Akt plays a part in the survival pathways of many different cell types (52,53) by phosphorylating BAD, which enables its binding to cytosolic 14-3-3 and keeps it away from the mitochondria (54,55). In the heart, active phosphorylated Akt has been shown to protect cardiac cells from ischemia and reperfusion damage and apoptosis (21,56). We therefore determined whether ischemia and reperfusion caused changes in Akt activity and whether PKC␦ inhibition would have any Akt-specific effects. There was a significant decrease in active Akt, as shown by a decrease in phosphorylated Akt during reperfusion (Fig. 6C) (no changes in fold levels of Akt occurred). Importantly, we found that inhibiting PKC␦ translocation during reperfusion blocked the decrease in Akt phosphorylation (Fig. 6C), which may lead to inhibition of BAD phosphorylation. Therefore these data suggest that PKC␦ activity may mediate ischemia and reperfusion-induced apoptosis by specifically decreasing the activity and phosphorylation of pro-survival Akt and pro-apoptotic BAD. DISCUSSION Our results show that ischemia and reperfusion cause translocation of PKC␦ to mitochondria, which in turn affects the activity of downstream apoptotic factors through the release of cytochrome c. In addition, reperfusion induced significant increases in the levels of pro-apoptotic BAD and decreases in BAD and Akt phosphorylation, as well as anti-apoptotic Bcl-2 and Bcl-x L protein levels. Using ␦V1-1 to specifically inhibit PKC␦ translocation at the onset of reperfusion, we observed inhibition of cytochrome c release, caspase 3 activation, PARP cleavage, and apoptosis-related DNA fragmentation. In addition, inhibiting PKC␦ translocation at reperfusion attenuated the rise in the levels of pro-apoptotic BAD and led to increased BAD phosphorylation and the activity of the pro-survival kinase Akt. However, inhibiting PKC␦ had no effect on the decline in the levels of the anti-apoptotic proteins Bcl-2 and Bcl-x L . Therefore, our data demonstrates that PKC␦ increases ischemia and reperfusion-induced apoptosis by affecting the balance between the pro-apoptotic and the anti-apoptotic enzymes.
Cell survival and death relies on the balance of pro-and anti-apoptotic proteins (57). Increases in BAD protein levels with parallel decreases in anti-apoptotic Bcl-2 in isolated porcine lung endothelial cells was shown to be sufficient for apoptosis induction (58). Moreover, Schimmer et al. (59) observed that overexpression of BAD causes apoptosis in Cos cells, whereas in cardiac myocytes, H 2 O 2 induces an increase in BAD protein levels that lead to apoptosis as well (60). Increasing the amount of anti-apoptotic Bcl-2 through overexpression in cardiac myocytes has also been shown to inhibit apoptosis caused by ischemia and reperfusion both in vitro and in vivo (61,62). In our study, we show that ischemia and reperfusion induced an increase in pro-apoptotic BAD but decreases in anti-apoptotic Bcl-2 and Bcl-x L proteins; however, the changes in BAD levels only were dependent on PKC␦ activation. We also observed changes in the phosphorylation state of BAD, an event that correlates with BAD inactivation because of its phosphorylation by Akt and sequestration by 14-3-3 (54,55,63). Previous studies support our finding that changes in the ratios and phosphorylation states of pro-and anti-apoptotic proteins are sufficient in determining cell fate (59 -62). How PKC␦ activation regulates the phosphorylation state of BAD and Akt has not yet been determined. It is also not clear whether PKC␦ is the Akt and/or BAD kinase. As for regulation of BAD levels by PKC␦, it is possible that the inhibition of proteosome function that we previously reported (64) and other unknown signaling enzymes are contributors.
In summary, we show that reperfusion causes translocation of PKC␦ to mitochondria where it enhances the release of cytochrome c, leading to the propagation of further downstream apoptotic effects (Fig. 7). More importantly, the selective PKC␦ inhibitor, ␦V1-1, not only attenuates translocation of PKC␦ to the mitochondria but also prevented the cascade of events that lead to apoptosis; inhibiting PKC␦ lead to a decrease in BAD protein levels as well as an increase in BAD and Akt phosphorylation. Coupled with the ischemia and reperfusion-induced decreased Bcl-2 and Bcl-x L , the increase in Akt and BAD phosphorylation along with the decrease in BAD levels may shift cells toward a survival state. Lowering BAD levels and increasing phosphorylated BAD may block the release of cytochrome c, possibly preventing Bax homodimerization (65,66). Once cytochrome c release is blocked, caspase 3 activation that induces PARP cleavage and DNA fragmentation are also inhibited (Fig. 7). Therefore, blocking PKC␦ translocation appears to be an effective means of salvaging the reperfused myocardium after an ischemic insult.