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Cardiovascular Aging
-activating peptide mimics ischemic preconditioning in aged hearts through GSK-3
but not F1-ATPase inactivation1Intercollege Program in Physiology and 2Department of Kinesiology, Pennsylvania State University, University Park, Pennsylvania; and 3Department of Medicine, University of Wisconsin, Madison, Wisconsin
Submitted 31 March 2007 ; accepted in final form 2 August 2007
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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activation limits ischemia (I)-reperfusion (R) damage and mimics the protection associated with ischemic preconditioning. We sought to determine whether local delivery of PKC activator peptide 
-receptor for activated C-kinase (-RACK) is sufficient to produce a similarly protected phenotype in aged hearts. Langendorff-perfused hearts isolated from adult (5 mo; n = 9) and aged (24 mo; n = 9) male Fisher 344 rats were perfused with -RACK conjugated to Tat (500 nM) or Tat only (500 nM) for 10 min before global 31-min ischemia. Western blotting was used to measure mitochondrial targeting of PKC, PKC
, phospho (p)-GSK-3
(Ser9) and GSK-3 in hearts snap-frozen during I. Recovery of left ventricular developed pressure was significantly improved by -RACK (P < 0.01) and infarct size reduced in 24-mo rats vs. age-matched controls (60% vs. 34%; P < 0.01). Mitochondrial PKC levels were 30% greater during I with -RACK in aged vs. control rats (P < 0.01). Interestingly, mitochondrial GSK-3 levels were threefold greater in aged vs. adult rats during I, and -RACK prevented this increase (P < 0.01). Mitochondrial p-GSK-3 levels were also greater in aged rats after -RACK (P < 0.01), and subsequent inhibition of GSK-3 with SB-216763 (3 µM) before I/R elicited protection similar to that of -RACK (n = 3/group). Mitochondrial proteomic analysis further identified group differences in the F1-ATPase -subunit, and coimmunoprecipitation studies revealed a novel interaction with PKC. F1-ATPase-PKC association was affected by -RACK in adult but not aged rats. Our results provide evidence, for the first time, for PKC-mediated protection in aged rat heart after I/R and suggest a central role for mitochondrial GSK-3 but not F1-ATPase as a potential target of PKC to limit I/R damage with aging.
ischemia-reperfusion injury; proteomics; mitochondria; senescence
Multiple lines of evidence suggest that activation of PKC-dependent signaling pathways plays a pivotal role in cardioprotection and resistance to ischemic insult. Translocation of PKC to the mitochondria is thought to induce opening of mitochondrial ATP-sensitive K+ (KATP) channels, as well as inhibit the mitochondrial permeability transition pore (MPT) (5, 30, 37), thereby protecting against Ca2+-overload injury and apoptosis. In this regard, our recent observation (33) of reduced PKC levels in the aged heart at baseline and after ischemia may contribute to the mechanism of ischemic intolerance and reduced efficacy of IPC with advancing age. Additional evidence supporting the importance of PKC in cardioprotection is data from Inagaki and colleagues (25, 26), who demonstrated that acute activation of PKC conjugated to the cell-permeating carrier peptide Tat immediately before global ischemia is sufficient to reduce infarct size in young adult rats, suggesting that IPC can be mimicked through pharmacological manipulation of PKC (25). However, the efficacy of acute PKC activation has yet to be tested in the aged population. Given the diminished effectiveness of IPC in the aged heart, combined with our observation of age-related reductions in cardiac PKC levels, the extent to which specific PKC activation can confer cardioprotective benefit to the aged heart may be limited.
The major purpose of the present investigation was therefore to test the hypothesis that acute pharmacological activation of PKC before ischemia would be sufficient to harness cardioprotective signaling reserves in the aged rat heart. Because the mitochondria are critically involved in transducing the coordinated cellular response to ischemia (29, 38), we utilized a proteomic approach within the mitochondrial compartment to identify potential targets of PKC-mediated cardioprotection affected by senescence. For instance, glycogen synthase kinase-3 (GSK-3) has been identified as an important downstream effector of PKC-mediated protection in cardiac mitochondria through inhibition of the MPT (5, 30, 37), and recent evidence from our laboratory (23, 33) suggests that age-related increases in active GSK-3 during reperfusion are highly correlated with ischemia-reperfusion (I/R) injury. Here, we present evidence for the first time that acute augmentation of mitochondrial PKC levels during ischemia as a result of local delivery of a PKC-activating peptide is highly effective in reducing infarct size in the aged heart. Our data further suggest a novel interaction of the mitochondrial F1-ATPase -subunit with PKC, as well as adaptive responses in the activation of mitochondrial GSK-3 during ischemia.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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PKC activator.
A PKC activator [-receptor for activated C-kinase (-RACK)] derived from PKC [amino acids 85 to 92 (HDAPIGYD)] conjugated reversibly to the carrier peptide Tat [amino acids 43–58 (YGRKKKRRQRRR)] by disulfide bond through free cysteines at the NH2 terminus was provided by KAI Pharmaceuticals (see Refs. 7, 26). -RACK promotes binding of PKC to its specific RACK anchoring protein, thus enhancing translocation and/or activation (7, 26). Vehicle consisted of Tat only.
Isolated heart preparation. Animals were anesthetized with pentobarbital sodium (40 mg/kg body wt ip), and hearts were rapidly excised by midline thoracotomy and rinsed in cold (4°C) saline. After isolation and rinsing, hearts were secured to a Langendorff apparatus and perfused at 85 mmHg with a modified Krebs-Henseleit buffer containing (in mM) 1.75 CaCl2, 117.4 NaCl, 4.7 KCl, 1.2 MgSO4, 1.3 KH2PO4, 24.7 NaHCO3, 11.0 glucose, 0.5 pyruvate, and 0.5 EDTA as previously described in our laboratory (23, 33). During a 20-min equilibration period, pacing at 260 beats/min was established and balloon volume was adjusted to yield an end-diastolic pressure (EDP) of 5–6 mmHg. Functional data [LV developed pressure (LVDP; calculated as LV systolic pressure – LVEDP), LVEDP, and positive and negative change in pressure over time (+dP/dt and –dP/dt)] were assessed with the Ponemah Physiology platform (Gould Instrument Systems, Valley View, OH).
Protocol of isolated heart studies.
After equilibration, -RACK conjugated to Tat (500 nM) or Tat peptide vehicle alone (500 nM) was administered in the perfusate 10 min before global ischemia (see Fig. 1). Hearts were then subjected to 31-min global ischemia as described previously (23, 33). Pacing was reinitiated 1 min after restoration of flow, and hearts were reperfused for 30 min. Administration of the PKC activator before global ischemia is based on previous studies in which PKC-modulating peptides were shown to be cardioprotective with this exposure duration and concentration (7, 26), readily distributing throughout cardiac tissue. In a separate series of studies, adult and aged hearts (n = 3/group) were perfused with the GSK-3 inhibitor SB-216763 (3 µM; Tocris) 10 min before ischemia in an identical fashion to the protocol associated with -RACK administration. Creatine kinase release (Stan Bio) was assessed in coronary effluents to ensure that significant injury was induced by the experimental protocol. After reperfusion LVs were isolated, weighed, halved, and frozen in liquid N2. All LV sections were stored at –80°C until tissue preparation.
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2-h reperfusion, and the area of infarction was assessed exactly as described previously (33). Briefly, after reperfusion, LVs were isolated and frozen at –20°C for 30 min. LVs were sliced transversely and stained with 1% triphenyltetrazolium chloride (TTC) in phosphate buffer for 30 min at 37°C. Digital images of infarcted LV were analyzed with National Institutes of Health (NIH) ImageJ software. Tissue sample preparation. Cytosolic and mitochondrial samples were prepared exactly as described previously in our laboratory (22, 23, 33). Briefly, frozen LV samples were homogenized with a glass-glass grinder in 10 vols of lysis buffer and subjected to serial centrifugations of 10,000 and 100,000 g. The 10,000 g pellet (mitochondrial fraction) was resuspended and subjected to 21,000 g centrifugation for 10 min. The resultant supernatant was defined as the mitochondrial fraction. The 100,000 g supernatant was defined as the cytosolic fraction. All protein concentrations were determined by the Bradford method (11). Citrate synthase activity was also assessed in mitochondrial homogenates at baseline and during ischemia with a modification of the Srere method (45) and used as a marker of mitochondrial yield and viability.
Western blotting.
Western blotting was performed according to well-established procedures in our laboratory (31, 32). Briefly, equal amounts of cytosolic (cytochrome c) or mitochondrial [PKC, phospho (p)-GSK-3 (Ser9), GSK-3] protein were electrophoresed on SDS-polyacrylamide gels (PAGE) and transferred to polyvinylidene difluoride membranes. Membranes were blocked and incubated with rabbit polyclonal antibodies against PKC (1:1,200) or PKC (1:1,500) for 3 h at room temperature or p-GSK-3(Ser9), GSK-3, and cytochrome c (1:1,000) overnight at 4°C. Immunoreactive bands were detected with horseradish peroxidase-linked anti-rabbit IgG (1:15,000) and enhanced chemiluminescence (ECL; Amersham). Densitometry was performed with Scion Image (NIH).
To control for total GSK-3 protein levels, membranes were initially incubated with p-GSK-3 antibodies and then stripped by 30-min incubation (70°C) in buffer containing 62.5 mM Tris·HCl pH 6.80, 7% 2-mercaptoethanol, and 2% SDS. Membranes were then probed for total levels of GSK-3 as described above and expressed as a ratio. To control for minor differences in protein loading, membranes were stained with Sypro Ruby (PKC, PKC) or Ponceau S (p-GSK-3, GSK-3) and densitometry values were adjusted as described previously (43). All group Western blot data are presented relative to preischemic values in adult animals.
Two-dimensional gel electrophoresis and mass spectrometry.
Mitochondrial homogenates were isolated from LV of untreated adult and aged rats as described above and subjected to two-dimensional electrophoresis (2-DE). Proteins (200 µg/sample) were separated in the first dimension by isoelectric point on immobilized pH gradient (IPG) strips (Bio-Rad) with a linear pH gradient from 3 to 10 (9). SDS-PAGE was performed on IPG strips with 12% separating gels, which were later stained with Sypro Ruby and imaged (Fluor-S, Bio-Rad). Protein images were analyzed with PD Quest software (Bio-Rad version 7.2), and matched sets (n = 3 hearts/group) were generated.
Mass spectrometry analysis. Protein spots were excised and subjected to trypsin (Promega) digestion according to standard procedures. Peptide sequencing of selected tryptic peptides was carried out in conjunction with the Penn State Mass Spec Core Facility, using the Applied Biosystems 4700 Proteomics Analyzer (MALDI TOF-TOF) and GPS Explorer 3.5. Identification of peptides from the mass spectroscopy (MS/MS) spectra was done by the Mascot algorithm.
Assessment of PKC-F1-ATPase -subunit associations.
Mitochondrial samples (400 µg) from adult and aged hearts treated with -RACK or vehicle were diluted to 4 ml with RIPA buffer (10 mM Tris·HCl, pH 7.2, 150 mM NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% SDS) and incubated with 5 µl of rabbit polyclonal antibodies against PKC for 2 h (4°C). After 30-min incubation with protein A Sepharose, the beads were washed three times according to previously published procedures from our laboratory (22). Samples were electrophoresed on 7.5% polyacrylamide gels, and resulting membranes were probed with F1-ATPase -subunit (1:3,000).
Statistical analyses.
All data are presented as means ± SE and were analyzed with the Statistical Analysis System (SAS). Baseline data and infarct size were compared with a two-way ANOVA. A two-way ANOVA with repeated measures on one factor (age x reperfusion time) was used to analyze functional recovery, and a two-way ANOVA (age x group) was used to analyze Western blotting data. The Tukey-Kramer method was employed for all post hoc comparisons. An 
-level of P 
0.05 was defined as statistically significant.
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RESULTS |
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-RACK. LV and body weights were significantly greater in aged animals compared with adults (P < 0.01). As we reported previously (23, 33), aging also resulted in a small but significant decrease in –dP/dt (P < 0.05). No significant group differences for any baseline variables of interest were observed between adult and aged animals treated with -RACK vs. Tat only.
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activation improves postischemic recovery and reduces infarct size in aged rat heart.
Recovery of LVDP after ischemia is presented in Fig. 2. A significant reduction in recovery of LVDP was observed in aged vs. adult (P < 0.05). In contrast, hearts perfused with -RACK before ischemia showed improved recovery compared with hearts perfused with Tat only (P < 0.01). The efficacy of -RACK was particularly apparent during the early period of reperfusion such that at minute 5 recovery of LVDP was 50% greater in aged and adult hearts (P < 0.01). Parallel reductions in EDP were also observed with -RACK (P < 0.01; Fig. 3).
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activation with -RACK before ischemia was highly effective in reducing infarct size in the aged group from 60% in age-matched controls to 34% (P < 0.01). A similar pattern was observed for integrated creatine kinase release (not shown) and cytosolic cytochrome c levels (Fig. 5). Collectively, these data are consistent with a protective role for acute PKC activation in the aged male rat heart.
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activation alters mitochondrial PKC levels during ischemia.
Figure 6A shows that mitochondrial PKC levels are reduced during ischemia relative to baseline in aged but not adult male rats (40%; P < 0.01), suggesting that the aged male heart is unable to maintain mitochondrial PKC levels to the same extent as adult rats during ischemia. However, -RACK administration before ischemia was effective in preserving mitochondrial PKC levels (Fig. 6A). Specifically, augmentation of cardiac mitochondrial PKC levels during ischemia was observed in both aged and adult rats after -RACK vs. age-matched controls perfused with Tat only (Fig. 6A; P < 0.01). While a significant age-related increase was observed in mitochondrial PKC levels at baseline and during ischemia (Fig. 6B; P < 0.01), PKC levels were unaffected by -RACK in aged rats. Finally, mitochondrial citrate synthase activity was assessed as an index of mitochondrial viability, and observed values were similar in adult and aged hearts at baseline (118.8 ± 7.8 vs. 117.2 ± 10.6 µmol·g wet wt–1·min–1). Citrate synthase activity during ischemia was not significantly different from preischemic values (adult 122.8 ± 7.5 vs. aged 104.03 ± 7.5 µmol·g wet wt–1·min–1) and was unaffected by -RACK treatment (adult 126.42 ± 8.9 vs. aged 109.4 ± 13.15 µmol·g wet wt–1·min–1).
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-mediated cardioprotection.
One downstream mechanism by which PKC may confer cardioprotection includes phosphorylation and inactivation of mitochondrial GSK-3 (29). Our previous studies (23, 33) indicated age-related increases in both basal p-GSK-3 and GSK-3, which were confirmed here (Fig. 7). During ischemia, we observed significantly greater mitochondrial p-GSK-3 levels in aged vs. adult in Tat only-treated hearts (Fig. 7A; P < 0.05). Unexpectedly, we also observed an age-dependent increase in active mitochondrial GSK-3 of approximately threefold (Fig. 7B; P < 0.01), suggesting greater GSK-3 activation with aging. Importantly, mitochondrial p-GSK-3 levels were significantly increased in aged and adult rats after -RACK, thereby increasing the p-GSK-3-to-GSK-3 ratio. Prior exposure to -RACK also prevented translocation of GSK-3 to the mitochondria in aged rats, presumably limiting mitochondrial GSK-3 activation (Fig. 7B; P < 0.01). Interestingly, perfusion of hearts with the GSK-3-specific inhibitor SB-216763 before ischemia resulted in improved recovery of LVDP in aged hearts similar to that observed for -RACK (Fig. 8). Together, our results support an important role for mitochondrial GSK-3 as a potential target to limit I/R damage with aging following acute PKC activation.
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-subunit, a key enzyme involved in ATP synthesis and energy metabolism. The molecular size and isoelectric point (pI) of this spot were consistent with the F1-ATPase -subunit and based on at least 20 mass peak identifications.
Coimmunoprecipitation studies further revealed a significant interaction of the F1-ATPase -subunit with PKC in mitochondrial homogenates (Fig. 9A), suggesting that alterations in ischemia-dependent phosphorylation (via PKC) of the F1-ATPase -subunit may contribute to age-dependent alterations in ischemic tolerance. Interestingly, aging was associated with a significant reduction in F1-ATPase interaction with PKC during ischemia (P < 0.05). Furthermore, while a significant effect of -RACK was also observed on reduction of F1-ATPase -subunit interactions with PKC in adult hearts (Fig. 9B), -RACK was without effect in aged hearts.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-activating peptides to trigger cardioprotection was uncertain. Key findings of the present investigation include the observation that acute activation of PKC augmented mitochondrial PKC during ischemia, abrogated mitochondrial GSK-3 levels, and reduced infarct size in the aged male rat heart. Proteomic analysis revealed a possible mechanistic role for the mitochondrial F1-ATPase -subunit in the genesis of age-related reductions in ischemic intolerance and a novel F1-ATPase-PKC association. Together, our results suggest for the first time that acute manipulation of mitochondrial PKC is sufficient to improve ischemic tolerance in the aged male rat heart, the cellular basis of which is likely to involve reductions in necrotic and apoptotic cell death.
A major purpose of the present investigation was to determine the efficacy of acute PKC activation to limit I/R damage in the aged male rat heart. Recent studies have identified PKC as a key modulator of cardioprotection and a novel therapeutic target for infarct size reduction and improved ischemic tolerance in adult animals (15, 25, 26), thereby mimicking the effects of IPC. Here, we extended these results to the aged male heart and observed reversal of age-related reductions in recovery of LVDP in hearts treated with -RACK compared with controls. Most dramatic differences were observed early during the reperfusion period, when initiation of apoptotic cell death signaling is likely to occur. Infarct size was also significantly reduced by 50%, and attenuation of additional markers of cell death including creatine kinase and cytochrome c release was observed in aged hearts treated with -RACK. Collectively, these data provide important evidence in support of the potential clinical application of acute PKC activation in the treatment of acute coronary syndrome in the aged male population.
While the mechanism(s) by which PKC limits infarct size is incompletely understood, particularly with regard to its role in IPC (39), localization of PKC to the mitochondria and phosphorylation of downstream targets is a critical step in engaging cell survival signaling (18, 41). However, aging studies to date have focused on signaling during the reperfusion period, and it may be that events during ischemia could provide important clues to understanding the cellular basis of ischemic intolerance with aging. Previous findings from our laboratory (22, 23, 31–33) suggest that mitochondrial PKC levels are indeed reduced in the aged rat heart during reperfusion following ischemia. Here we demonstrate that augmentation of mitochondrial PKC levels after -RACK can mimic the protective effects of IPC in the aged myocardium. However, given findings from several recent studies utilizing PKC modulatory peptides (13, 19, 26, 27), it is unclear whether the PKC-activating effect mediated by -RACK reflects altered translocation of PKC to the mitochondria or activation of constitutively expressed PKC and hence greater association with its RACK. A limitation of the present study is that PKC levels were examined at only one time point during ischemia and the effects of -RACK treatment on the temporal activation of PKC during ischemia remain unknown. Alternatively, differences in PKC proteolytic activity or sensitivity to lipid activators may be altered by -RACK to enhance the activating effect of PKC under conditions of I/R. Future studies targeting the mechanism by which -RACK alters PKC activation, particularly in the aged heart, are therefore indicated. Nevertheless, it is important to note that effects of -RACK on reversing age-related reductions in ischemic tolerance were independent of changes in PKC levels in aged hearts. This observation is important since PKC activation on reperfusion is known to increase apoptotic cell death and I/R injury (26, 33).
A proposed convergence point for many cardioprotective signals, including PKC, is inactivation of mitochondrial GSK-3 and associated apoptotic signaling. Specifically, inhibition of the MPT by PKC is thought to involve phosphorylation and inhibition of mitochondrial GSK-3 (29, 48). Disruption of this model by aging is supported by our recent observations (23, 33) of I/R-dependent changes in mitochondrial p-GSK-3. Here we also observed threefold greater levels of active mitochondrial GSK-3 only in aged hearts during ischemia. Interestingly, inhibition of GSK-3 resulted in a degree of cardioprotection similar to that observed for acute PKC activation in aged hearts. In adult hearts, GSK-3 inhibition was less efficacious than -RACK during the very early minutes of reperfusion, which may suggest a more robust effect of PKC activation on GSK-3 inhibition in aged vs. adult hearts. Our observations of age-related reductions in mitochondrial PKC (and higher active GSK-3) during ischemia may predispose to elevated reactive oxygen species and lowering of the MPT threshold, thereby increasing apoptotic cell death known to occur in aged hearts (34, 40). The age-dependent increase in active mitochondrial GSK-3 demonstrated here may also represent translocation of GSK-3 away from the nucleus, the impact of which may impinge upon transcription of cardioprotective genes. However, we can only speculate as to the mechanism of PKC-GSK-3 association since our studies did not directly address this issue.
Because signaling pathways associated with cardioprotection ultimately converge at the mitochondria (29, 38), we focused on the mitochondrial cellular compartment to identify additional mechanistic targets of PKC-mediated cardioprotection in the aged heart with a proteomic approach. The mitochondria play a critical role in the maintenance of cell survival, due to both maintenance of ATP production and reductions in apoptotic cell death (for review see Refs. 17, 20). Proteomic studies have revealed novel protein interactions involved in cardioprotection including PKC-ERK (16, 46, 48), and evidence has emerged regarding the role of signaling modules as a critical concept in the regulation of biological phenotypes (6, 46–48).
A key protein identified in our proteomic analysis is the F1-ATPase -subunit, which has recently been identified as part of a novel macromolecular supercomplex (3) that may provide for the structural identity of the mitochondrial KATP channel. In the absence of O2, F1-ATPase can operate in reverse to maintain mitochondrial membrane potential at the expense of ATP. Thus age-related reductions in the F1-ATPase -subunit may represent a compensatory adaptation to prevent excessive ATP hydrolysis during ischemia. Alternatively, age-related reductions may hinder ATP synthesis on reperfusion and exacerbate ischemic intolerance (35, 36). Although some studies have shown no role for F1-ATPase in IPC protection (10, 21), others have found that IPC leads to F1-ATPase inhibition in early ischemia (2, 24). Recently, Akt-dependent phosphorylation of F1-ATPase has also been demonstrated and localized to the -subunit, which also binds 14-3-3 proteins in plants and ATPase inhibitor protein IF-1 (8, 14, 42). Our observation that the F1-ATPase -subunit interacts with PKC suggests that alterations in ischemia-dependent phosphorylation (via PKC) of the F1-ATPase -subunit may contribute to alterations in ischemic tolerance. In this regard, Van Eyk and colleagues (4) recently demonstrated significant reductions in F1-ATPase -subunit phosphorylation following pharmacological induction of IPC with the KATP channel agonist diazoxide, suggesting that the reduction is cardioprotective. If this is so, our observation of age-related reductions in F1-ATPase -subunit-PKC association may represent an adaptive response by the aged myocardium as a cellular strategy to maintain ischemic tolerance. That -RACK decreased F1-ATPase -subunit-PKC association in adult but not aged hearts further suggests that additional targets of -RACK, i.e., GSK-3, underlie the protective effect of acute PKC following I/R in aged males. Clearly, future studies are indicated to validate the role of F1-ATPase -subunit phosphorylation by PKC in cardioprotection.
In summary, we present here for the first time evidence that acute augmentation of mitochondrial PKC levels during ischemia through local peptide delivery is protective to the aged heart. Our data further suggest a novel interaction of the mitochondrial F1-ATPase -subunit with PKC, as well as adaptive responses in mitochondrial GSK-3 during ischemia. Collectively, our findings extend the protective reach of PKC therapeutics to a model of senescence and suggest that PKC may represent an important target in the treatment of ischemic cardiovascular disease in the aged population.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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effect of drug (P < 0.01).
effect of ischemia (P < 0.05).
