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Cardiovascular Research 2006 70(2):325-334; doi:10.1016/j.cardiores.2006.02.009
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Copyright © 2006, European Society of Cardiology

Acute PKC{delta} inhibition limits ischaemia–reperfusion injury in the aged rat heart: Role of GSK-3β

John C. Kostyaka, J. Craig Huntera and Donna H. Korzicka,b,*

aIntercollege Program in Physiology, The Pennsylvania State University, University Park, PA 16802, United States
bThe Department of Kinesiology, The Pennsylvania State University, University Park, PA 16802, United States

* Corresponding author. 106 Noll Laboratory, The Pennsylvania State University, University Park, PA 16802, USA. Tel.: +1 814 865 5679; fax: +1 814 865 4602. Email address: dhk102{at}psu.edu

Received 12 December 2005; revised 3 February 2006; accepted 7 February 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective Age is a leading risk factor for the development of ischaemic heart disease and failure. However, the efficacy of cardioprotective strategies designed to rescue the aged myocardium remains controversial. We have previously demonstrated increased levels of basal cardiac protein kinase C{delta} (PKC{delta}) with ageing, a well-known mediator of apoptotic cell death following ischaemia and reperfusion (I/R) in adult hearts. Our objective was to determine the contribution of PKC{delta} signaling mechanisms to reperfusion injury in the aged heart using local delivery of a novel PKC{delta} inhibitory peptide (KID1-1).

Methods Contractile responses were assessed in hearts isolated from adult (4 months, n=38) and aged (24 months, n=45) male Fisher 344 rats treated with either KID1-1 (500 nM) or Tat vehicle peptide (500 nM) upon reperfusion for 10 min following 31-min global ischaemia.

Results Recovery of left ventricular (LV) developed pressure was significantly improved by KID1-1 and associated with smaller infarct size in 24 months vs. age-matched controls (p<0.005). We also observed significant reductions in DNA laddering and cytochrome c and caspase 3 levels in aged hearts treated with KID1-1. Interestingly, KID1-1 attenuated mitochondrial and nuclear PKC{delta} levels during reperfusion in aged vs. age-matched controls (p<0.01). Further, increases in mitochondrial phosphorylated glycogen synthase kinase-3β (pGSK-3β) levels were hastened in aged and adult hearts following KID1-1 (p<0.05), increasing the pGSK-3β/GSK-3β ratio.

Conclusions These results provide novel evidence for cardioprotection through acute PKC{delta} inhibition in aged rat heart following I/R. Our results also suggest, for the first time, a key role for mitochondrial GSK-3β as a cellular basis for the protection associated with PKC{delta} inhibition with ageing.

KEYWORDS Senescence; Myocardial infarction; GSK-3β; Protein kinase B (Akt473); Apoptosis


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The deleterious effects of ageing on the cardiovascular system are well-documented and epidemiological evidence indicate that over 80% of the US population 75 years and older are afflicted with some form of cardiovascular disease [1]. A hallmark characteristic of the aged heart includes reductions in ischaemic tolerance, which have been repeatedly demonstrated in both animal [2–4] and human [5–7] models of ischaemia–reperfusion (I/R) injury. In contrast to the adult heart, however, the efficacy of therapeutic manipulations employed to improve ischaemic stress tolerance reserves in the aged myocardium has proven controversial (for review, see [8]). For example, while ischaemic preconditioning (IPC) has yielded robust cardioprotection across all species tested to date, the aged heart is apparently refractory to IPC-mediated benefit [8]. Efforts to limit I/R damage in the aged heart have also been hampered by the paucity of information on age-related adaptive responses in protective signaling pathways known to limit apoptotic cell death.

One approach currently being investigated to limit I/R injury in the adult myocardium involves use of peptide modulators designed to limit translocation and subsequent activation of the delta isoform of protein kinase C (PKC{delta}) [9–11]. Acute inhibition of PKC{delta} translocation to mitochondrial targets at the onset of reperfusion following ischaemia in vivo or in vitro dramatically improves functional recovery, reduces infarct size and limits post-ischaemic apoptotic cell death [12]. In the aged heart, we have previously demonstrated that both cytosolic and mitochondrial PKC{delta} levels are increased under basal conditions [13], which may exacerbate age-related increases in post-ischaemic apoptotic cell death. However, it is currently unknown whether further increases in PKC{delta} translocation occur in the aged heart upon reperfusion and, more importantly, whether or not acute PKC{delta} inhibition would be effective in limiting I/R damage with advancing age.

A major purpose of the current study was to determine whether alterations in the subcellular distribution of PKC{delta} impact infarct size and/or multiple markers of apoptotic cell death following I/R in the aged rat heart. For these studies, a novel PKC{delta} translocation peptide inhibitor (KID1-1) reversibly conjugated to the HIV carrier protein Tat was employed [9] and administered immediately upon reperfusion following global ischaemia. Because PKC{delta} inhibition is also likely to alter the fate of downstream signaling proteins critical to cell survival, it was also the purpose of this investigation to examine the cellular mechanism by which PKC{delta} inhibition confers benefit to the aged heart against reperfusion injury. Here we provide evidence for the first time that inhibition of PKC{delta} translocation to both mitochondrial and nuclear cellular compartments is associated with cardioprotection in aged hearts, and coincident with adaptive responses in glycogen synthase kinase-3β (GSK-3β) signaling.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Experimental animals
Adult (4 months, n=38) and aged (24 months, n=45) male Fisher 344 rats were used in this study according to the schema presented in Fig. 1. All rats were housed with a 12-h light/dark cycle and food and water provided ad libitum. Animal handing was in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and approval from The Pennsylvania State University Institutional Animal Care and Use Committee (IACUC).


Figure 1
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  		Fig. 1 Protocol of the isolated heart studies. Following equilibration, hearts were subjected to 31 min of ischaemia followed by 30, 60, 110 or 240 min of reperfusion for Western blotting (n=4/adult group, n=5/per aged group per time point), infarct size assessment (n=6/group) and DNA laddering studies (n=3/adult group, n=4/aged group), respectively. KID1-1 or vehicle (Tat-only) was delivered at the onset of reperfusion for 10 min. For baseline biochemistry studies, hearts were snap frozen during the equilibration period at min 30 (adult n=4, aged n=5).

 
2.2 PKC{delta} inhibitor
A PKC{delta} inhibitor (KID1-1, amino acids 8–17 [SFNSYELGSL]) conjugated reversibly to the carrier peptide Tat (amino acids 43–58 [YGRKKKRRQRRR]) by disulfide bond as described in [9,11] was provided by KAI Pharmaceuticals. KID1-1 inhibits PKC{delta} binding to its specific anchoring protein, RACK (receptors for activated C kinase), thus interfering with translocation and activation. Vehicle consisted of Tat-only.

2.3 Isolated heart preparation
Animals were anesthetized with sodium pentobarbital (35 mg/kg, i.p.). Hearts were perfused in Langendorff mode under constant pressure (85 mm Hg) with a modified Krebs–Henseleit (K–H) 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, gassed continuously with 95% O2–5% CO2 and heated to 37 °C as previously described in our laboratory [14]. Following equilibration, a water-filled latex balloon attached to a force transducer (Becton Dickinson) was inserted into the left ventricle (LV) and balloon volume adjusted to obtain a minimum pressure of 5–6 mm Hg, while hearts were paced at 260 bpm. Functional data (LV developed pressure, LVDP; calculated as [LV systolic – LV end diastolic pressure (EDP)], LVEDP, dP/dt and –  dP/dt) were assessed using the Ponemah Physiology platform (Gould Instrument Systems, Valley View, OH).

2.4 Protocol of the isolated heart studies
Following a 30-min equilibration period, adult and aged hearts were subjected to 31 min of global no-flow ischaemia (without pacing) followed by either KID1-1 (500 nM) or Tat vehicle peptide (500 nM) for 10 min upon reperfusion (see Fig. 1). Intracellularly acting PKC-modulating peptides have been previously shown to readily distribute throughout cardiac tissue following this exposure duration [15]. Likewise, the dose used in the current study was previously found to confer maximum protective benefit in adult rat myocardium [12], preventing the cascade of events leading to apoptosis. Finally, hearts were reperfused for 30 or 60 min to assess the temporal nature of PKC translocation and activation of downstream signaling events with advancing age. The rationale for utilizing variable reperfusion periods was based on recent studies demonstrating distinct PKC{delta} translocation events (i.e. early vs. late) to the mitochondrial cellular fraction in conjunction with propagation of downstream apoptotic effectors [11].

2.5 Infarct size assessment
For infarct size assessment, post-ischaemic hearts were reperfused for a total period of 110 min (n=6 hearts/treatment group) and LV slices (n=5–6/heart) stained using 1% triphenyltetrazolium chloride (TTC) as previously described by Downey [16]. Average infarct area for each slice (~5–6 slices/heart) was determined and multiplied by the corresponding slice weight to get infarct volume. Infarct volumes for each slice were then added to achieve total infarct size.

2.6 Tissue sample preparation and subcellular fractionation
Tissue samples were prepared without deviation from established methods in our laboratory [13]. Briefly, frozen LV sections were minced and homogenized by glass–glass grinder. Nuclear, mitochondrial and cytosolic separations were performed as described in [13] using differential ultracentrifugation. For total homogenates, isolated LVs were homogenized by polytron in the presence of 1% Triton-X and subjected to 100,000 x g ultracentrifugation. The supernatant was defined as the total fraction. Protein concentrations were determined by the method of Bradford [17] and stored at – 80 °C.

2.7 Western blotting
Western blotting was performed according to previously published procedures from our laboratory [18]. Briefly, equal amounts of protein per lane were electrophoresed on SDS-polyacrylamide gels. Following blocking, samples were probed with antibodies against pGSK-3βSer9 (1:1000, 30 µg), total GSK-3β (1:1000, 30 µg), pAktser473 (1:1000, 45 µg) or total Akt (1:1000, 45 µg). pAkt ser473 and pGSK-3β data were normalized to total Akt and GSK-3β levels, respectively. Additionally, Western blotting was performed to assess cytosolic cytochrome c (Pharmigen, 1:1000, 9 µg) and cleaved caspase 3 (1:750, 26 µg). Finally, PKC{delta} (Santa Cruz, 1:1200, 7.5 µg) levels were assessed in cytosolic, mitochondrial and nuclear cellular compartments. Proteins were visualized using enhanced chemiluminescence (Amersham) and densitometry performed using Scion Image. All antibodies were purchased from Cell Signaling unless otherwise specified.

2.8 DNA laddering
In a separate set of experiments, DNA laddering was assessed in Langendorff-perfused hearts (n=3–4/group) as described by He and colleagues [19] with minor modifications. Post-ischaemic hearts were reperfused for 240 min to allow for the progression of apoptosis [11].

2.9 Statistical analysis
All variables of interest were reported as mean±standard errors. Baseline contractile function and infarct size were analyzed by two-way ANOVA (age x drug), while post-ischaemic contractile function and Western blotting data were analyzed by three-way ANOVA (age x drug x reperfusion time) with repeated measures using the Statistical Analysis System (SAS) and Mixed procedure, which accounts for unbalanced designs and multiple main effects. For analysis of functional responses, each heart served as its own control and n=8 representative hearts/experimental group without missing data points were analyzed. The Duncan multiple range method was used for all post hoc comparisons on significant interactive effects. An {alpha} level of p<0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Baseline morphological and hemodynamic parameters
Baseline characteristics for adult and aged rats are shown in Table 1. There was a significant effect of age on increasing body weight (BW) (p<0.05), LV weight (p<0.05) and LV weight/BW ratios (p<0.05). No significant differences were observed in LVDP or + dP/dt. As expected, ageing significantly reduced –  dP/dt (p<0.001).


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  		Table 1 Baseline morphologic and hemodynamic parameters

 
3.2 Acute PKC{delta} inhibition improves post-ischaemic recovery and reduces infarct size in aged rat heart
Representative contractile responses in adult and aged hearts are presented in Fig. 2 (n=8/group). Recovery of LVDP following global ischaemia was significantly reduced in aged vs. adult hearts (closed circles; p<0.001). Acute PKC{delta} inhibition via KID1-1 significantly improved early recovery of LVDP in adult hearts (open circles) and throughout the entire reperfusion period in aged hearts (open triangles) compared to age-matched controls (age x drug x reperfusion time interaction; p<0.03). Group differences in recovery of LVDP were most apparent during the first 10 min of reperfusion and a similar pattern of response was observed for + dP/dt (not shown).


Figure 2
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  		Fig. 2 Acute PKC{delta} inhibition upon reperfusion with KID1-1 improves recovery of LVDP following 31 min of global ischaemia in the adult and aged male rat heart and reverses age-related reductions. Adult hearts administered KID1-1 (n=8) displayed significantly greater recovery of LVDP compared to age-matched controls (n=8) during early recovery. Significantly greater recovery of LVDP was also observed in aged hearts (n=8) relative to age-matched controls following KID1-1 (n=8). A significant effect of age was also observed on decreasing recovery of LVDP in hearts isolated from aged vs. adult hearts in the absence of KID1-1. *Effect of age vs. adult (p<0.001), {dagger}effect of drug in aged (p<0.001), {ddagger}effect of drug in adult (p<0.001). Abbreviations: LVDP, left ventricular developed pressure. Values represent mean±S.E.

 
As expected, EDP was significantly higher in both adult and aged hearts during reperfusion when compared to pre-ischaemic control values (Fig. 3; p<0.001). Further, aged hearts had a significantly higher EDP compared to adult hearts during the early reperfusion period (Fig. 2; age x drug x reperfusion time interaction; p<0.01). Acute PKC{delta} inhibition with KID1-1 attenuated the early post-ischaemic rise in EDP in adult hearts, and in aged hearts following 5, 10 and 30 min of reperfusion, respectively (Fig. 3).


Figure 3
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  		Fig. 3 PKC{delta} inhibition via KID1-1 reduces end diastolic pressure (EDP) in aged hearts following 31 min of global ischaemia. *Effect of age (p<0.05), {dagger}effect of drug (p<0.05). Abbreviations: R, reperfusion. Values represent means±S.E.

 
In a subset of experiments, hearts (n=6/group) were subjected to TTC staining following 110 min of reperfusion to assess size of infarction. As seen in Fig. 4, infarct size was significantly greater in aged vs. adult hearts (47%±0.05 vs. 35%±0.03; p<0.05) and paralleled recovery of LV function. Importantly, KID1-1 administration at the onset of reperfusion reduced infarct size in both adult (23%±0.03; p<0.005) and aged (27%±0.04; p<0.005) hearts when compared to age-matched controls. Interestingly, KID1-1 reperfusion abolished the age-dependent increase in infarct size.


Figure 4
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  		Fig. 4 PKC{delta} inhibition reduces infarct size in adult and aged rat hearts. Advancing age increases while KID1-1 attenuates infarct size following ischaemia–reperfusion in male rat heart (n=6/group). Hearts were stained with triphenyltetrazolium (TTC) following 31 min of ischaemia and 110 min of reperfusion. *Effect of age (p<0.05), {dagger}effect of drug (p<0.05). Values are means±S.E.

 
3.3 Acute PKC{delta} inhibition alters the sub-cellular distribution of PKC{delta} in aged male F344 rat hearts subjected to ischaemia at reperfusion
At baseline, mitochondrial PKC{delta} levels were ~40% greater in aged when compared to adult hearts (Fig. 5A; p<0.01). Age-related increases in mitochondrial PKC{delta} levels observed at R30 (p<0.05). A late increase in mitochondrial PKC{delta} levels was observed in adult hearts at R60 (~40% from baseline; p<0.05). Importantly, KID1-1 administration in aged hearts attenuated the early increase in PKC{delta} observed at R30, while reducing mitochondrial PKC{delta} levels at R60 in adult hearts (p<0.01).


Figure 5
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  		Fig. 5 Representative immunoblots for mitochondrial (panel A), cytosolic (panel B) and nuclear (panel C) PKC{delta} levels prior to and following 31 min of global ischaemia (n=4–5/group for a given time point). Ageing increased mitochondrial and cytosolic PKC{delta} levels, while reducing nuclear PKC{delta}. Administration of KID1-1 attenuated aged-related increases and decreases in mitochondrial and nuclear PKC{delta} levels during reperfusion, respectively. *Effect of age (p<0.05), {dagger}effect of drug (p<0.05), {ddagger}effect of reperfusion. Abbreviations: CTL, control; R, reperfusion. Values are means±S.E.

 
Cytosolic PKC{delta} levels are presented in Fig. 5B. At baseline, aged hearts exhibited a two-fold increase in cytosolic PKC{delta} (p<0.01) when compared to adult which persisted throughout reperfusion (p<0.05). Reperfusion-induced increases in cytosolic PKC{delta} in adult hearts were observed at both R30 and R60 (p<0.05), and PKC{delta} levels did not differ in adult hearts perfused with KID1-1 at either R30 or R60. Interestingly, PKC{delta} inhibition with KID1-1 increased cytosolic PKC{delta} levels in aged hearts (p<0.05) at R30, which corresponded to drug-induced reductions in mitochondrial PKC{delta} levels as noted above. In the nuclear homogenate, significant age-related reductions (~20%) were observed in PKC{delta} levels at baseline (Fig. 5C; p<0.01). Upon reperfusion, a significant increase in PKC{delta} was observed in aged but not adult hearts, which persisted at R30 and R60 (p<0.05). Acute PKC{delta} inhibition with KID1-1 blocked the reperfusion-induced increase in nuclear PKC{delta} levels in aged hearts (p<0.05) throughout reperfusion. Effects of KID1-1 on PKC{delta} levels in adult hearts were not observed (Fig. 5C).

3.4 Acute PKC{delta} inhibition attenuates apoptotic signaling in aged rat hearts subjected to ischaemia–reperfusion
As can be seen in Fig. 6A, DNA fragmentation was enhanced in aged compared to adult hearts, while KID1-1 perfusion attenuated age-related increases in DNA laddering. Reductions in DNA laddering by KID1-1 were also associated with reversal of ischaemia-induced increases in cytosolic cytochrome c levels at R30, as well as cleaved caspase 3 levels at R60, from both adult and aged hearts (p<0.05; Fig. 6B and C).


Figure 6
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  		Fig. 6 Local delivery of KID1-1 at the onset of reperfusion following 31 min of global ischaemia reduces DNA fragmentation (panel A), cytosolic cytochrome c (panel B) and cytosolic cleaved caspase 3 levels (panel C) in aged rat hearts (n=4–5/group for a given time point). Abbreviations: CTL, control; R, reperfusion. Values are means±S.E.

 
3.5 Acute PKC{delta} inhibition alters pAkt473 and pGSK-3β levels in the aged heart
Fig. 7 displays immunoblotting results for pAkt473, Akt and pAkt473/Akt ratios performed on total LV homogenates. No differences were noted between adult and aged rat hearts in pAkt473 or Akt at baseline. However, reperfusion resulted in reduced pAkt473 levels in adult hearts to ~80% of pre-ischaemic control values (p<0.03), with greater reductions observed in aged hearts (age x drug x reperfusion interaction; p<0.001). PKC{delta} inhibition with KID1-1 upon reperfusion maintained pAkt473/Akt ratios to baseline levels at R30 in aged hearts.


Figure 7
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  		Fig. 7 Acute PKC{delta} inhibition with KID1-1 abolishes the reperfusion-induced decrease in pAkt473 in aged myocardium. Representative immunoblots for pAkt473 and total Akt (panel A) and the pAkt473/AKT ratio (panel B) in adult and aged hearts (n=4–5/group). *Effect of age (p<0.05), {dagger}effect of drug (p<0.05), {ddagger}effect of reperfusion (p<0.05). Abbreviations: CTL, control; R, reperfusion. Values presented are means±S.E.

 
Fig. 8 presents mitochondrial pGSK-3β(Ser9), GSK-3β levels and pGSK-3β/GSK-3β ratios for all experimental groups. While ageing was associated with increased levels of both pGSK-3β(Ser9) and GSK-3β prior to ischaemia, when compared to adult controls (Fig. 8A; p<0.05), age-related differences in pGSK-3β/GSK-3β ratios were not observed (Fig. 8B). Upon reperfusion, further increases in mitochondrial GSK-3β occurred in aged but not adult hearts at R30 (p<0.05) and were unaffected by KID1-1. Reperfusion-induced increases in pGSK-3β(Ser9) levels were observed in adult and aged hearts at R60; KID1-1 treatment hastened this response such that mitochondrial pGSK-3β(Ser9) levels were significantly increased above baseline in both adult and aged hearts at R30. Consequently, age-related reductions in the pGSK-3β/GSK-3β ratio observed at R30 were reversed by KID1-1 (p<0.05).


Figure 8
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  		Fig. 8 Acute PKC{delta} inhibition with KID1-1 reverses reperfusion-induced reductions in mitochondrial pGSK-3β(ser9) in the aged myocardium. Representative immunoblots for pGSK-3β(ser9) and total GSK-3β (panel A) and the pGSK-3β(ser9)/GSK-3β ratio (panel B) in adult and aged hearts (n=4–5/group). *Effect of age (p<0.05), {dagger}effect of drug (p<0.05), {ddagger}effect of reperfusion (p<0.05). Abbreviations: CTL, control; R, reperfusion. Values presented are means±S.E.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
It has long been established that ageing reduces myocardial tolerance to ischaemia in both animals [4,20] and humans [5,6,21]. Recently, PKC{delta} has emerged as a potential therapeutic target in the prevention of apoptosis following myocardial ischaemia in the adult heart, but efficacy in aged hearts was uncertain. We therefore used an ex vivo approach to explore the effects of a novel PKC{delta} inhibitory peptide, KID1-1, on post-ischaemic LV function and apoptotic cell death in the isolated aged rat heart. Key findings include the observations that KID1-1 reduced infarct size, attenuated reperfusion-related cytochrome c and caspase 3 levels, and diminished the extent of DNA laddering in the aged rat myocardium. Significant effects on downstream effectors including mitochondrial GSK-3β were also apparent. Collectively, our results suggest, for the first time, a central role for PKC{delta} as a key mediator of cell death in the aged heart following ischaemia and reperfusion (I/R), the cellular basis of which involves both mitochondrial and nuclear PKC{delta} localization.

4.1 PKC{delta} inhibition reduces I/R injury in aged myocardium
Results from the current study extend the protective benefit of acute PKC{delta} inhibition during early reperfusion to the aged heart, a finding previously observed in adult rat myocardium [12]. Recovery of LVDP in aged hearts treated with KID1-1 was statistically indistinguishable from adult hearts treated with vehicle-only and was accompanied by a ~20% decrease in infarct size. Our results are intriguing since previous experimental manipulations (namely IPC) designed to protect the aged heart from I/R injury have been largely ineffective and remain controversial [8]. One logical conclusion associated with the current study is that, while aged hearts lack the ischaemic stress tolerance reserves necessary to harness innate cardioprotection, the aged heart is still capable of mounting a protective response through inhibition of cellular effector(s) activated by PKC{delta}.

4.2 PKC{delta} inhibition alters the cellular localization of PKC{delta} following I/R
Translocation of PKC from the cytosol to different cellular compartments is a hallmark of PKC activation [22]. Translocation of PKC{delta} to mitochondria in response to I/R has also been associated with enhanced apoptotic cell death in a number of different models [23–25], initiated in part by mitochondrial ROS production [10]. Given known age-related increases in oxidant stress [26], we logically hypothesized that enhanced translocation of PKC{delta} from the cytosol to the mitochondria may have exaggerated I/R damage in aged when compared to adult myocardium. Indeed, we observed age-dependent increases in mitochondrial and cytosolic PKC{delta} levels, which persisted throughout reperfusion. In contrast, reperfusion with KID1-1 was associated with a small but significant decrease in mitochondrial PKC{delta} levels at R30, while cytosolic PKC{delta} was increased during this same time period. One interpretation of these findings is that PKC{delta} translocation was inhibited by KID1-1 at this time. Churchill and Szweda [10] recently demonstrated increases in mitochondrial PKC{delta} within 5 min of reperfusion in adult hearts, while Murriel et al. observed increases as late as 120 min into reperfusion which were effectively blocked by KID1-1 [11]. Our results are consistent with these previous findings and further suggest that age-related increases in mitochondrial PKC{delta} during reperfusion may also underlie, at least in part, known reperfusion-induced mitochondrial dysfunction with advancing age.

Apoptosis has also been associated with translocation of PKC{delta} from the cytosol to the nucleus and subsequent PKC{delta} proteolytic cleavage by caspase 3 [27]. Studies in several different cell types also suggest that PKC{delta} translocation to the nucleus is necessary to engage the apoptotic programme [28–30]. In the present study, we provide the first evidence that nuclear PKC{delta} levels are increased during reperfusion relative to baseline in aged but not adult myocardium. Importantly, KID1-1 prevented reperfusion-induced nuclear PKC{delta} translocation, highlighting a potential protective mechanism for acute PKC{delta} inhibition in the aged heart. That reperfusion did not induce similar increases in nuclear PKC{delta} levels in adult hearts suggests that PKC{delta} translocation to the nucleus is not an integral step in the progression of apoptosis in adult myocardium. Alternatively, it may be that the ischaemic stress in adult hearts was not sufficient to elicit reperfusion-dependent alterations in nuclear PKC{delta} localization. Nevertheless, because acute PKC{delta} inhibition early in reperfusion significantly reduced DNA fragmentation and presumably the extent of apoptosis in aged hearts, we chose to next explore the effects of PKC{delta} inhibition on key markers of apoptotic signaling in the aged myocardium.

4.3 Effects of PKC{delta} inhibition on downstream apoptotic effectors
Initial events underlying mitochondrial-dependent apoptosis include release of cytochrome c from the outer leaflet of the inner mitochondrial membrane to the cytosol [31], and subsequent activation of effector caspases including caspase 3 [32]. Effectors of caspase 3 also include PKC{delta} [33,34], positioning PKC{delta} as an integral death signal. Here, we observed increases in both cytochrome c and cleaved caspase 3 levels during reperfusion that were similarly attenuated by KID1-1. Reductions in caspase 3 levels with acute PKC{delta} inhibition have been demonstrated previously in adult rats [11] and results from the current study further suggest a requisite role for PKC{delta} in the progression of apoptotic cell death with ageing. Reperfusion-induced decreases in the pro-survival signal pAkt473 were also significantly greater in aged vs. adult hearts. KID1-1 abolished these group differences, restoring the pAkt473/Akt ratio to pre-ischaemic levels. Interestingly, KID1-1 reduced infarct size in adult hearts without discernable effects on Akt phosphorylation. It may be that, under our experimental conditions, alternative protective pathways independent of Akt were likely sufficient to limit reperfusion injury in these hearts as recently observed in mice with constitutively active phosphatidylinositol 3 kinase [36].

An important downstream target in the apoptotic signaling cascade includes GSK-3β, whereby phosphorylation of GSK-3β at Ser9 by Akt, PKC or additional kinases results in inactivation [35,36]. While GSK-3β has been previously implicated as a key mediator of apoptotic signaling in a variety of tissues [35,37–39], Juhaszova et al. [37] recently demonstrated that GSK-3β may also play a pivotal role in myocardial apoptotic signaling as a point of integration localized to the mitochondrial permeability transition. Here, PKC{delta} inhibition increased pGSK-3β/GSK-3β in adult hearts ~2.5-fold over baseline, while completely blocking reperfusion-induced decreases observed in aged hearts. To the best of our knowledge, our data are the first to demonstrate an age-related reduction in pGSK-3β/GSK-3β signaling in the aged heart and reversal by acute PKC{delta} inhibition. That reperfusion-induced increases in pGSK-3β/GSK-3β occurred in distinction from changes in pAkt473, particularly in adult hearts, raises the possibility of direct mechanistic linkages between PKC{delta} and GSK-3β. While direct PKC{delta}-GSK-3β interactions have been observed in multiple cell lines [40], future studies investigating the interactive effects of PKC{delta} inhibition on GSK-3β phosphorylation during I/R are warranted.

4.4 Conclusions
In the present study, we have revealed potential mechanisms for the cardioprotection associated with acute PKC{delta} inhibition in the aged rat myocardium. Administration of KID1-1 improved recovery of LVDP, decreased infarct size and attenuated the activation of three markers of apoptosis in the aged myocardium following I/R. Coordinate with reductions in cell death, we also provide novel evidence demonstrating increases in pAkt473 as well as pGSK-3β(Ser9) in the aged myocardium through PKC{delta} inhibition. Of interest is the apparent influence of PKC{delta} on GSK-3β signaling with advancing age. Future explorations of this potential interaction could further our understanding of apoptotic signaling in the aged myocardium under conditions of I/R injury and acute coronary syndrome, and importantly, lead to the development of protective therapeutic interventions to preserve ischaemic tolerance in the aged human heart.


   Acknowledgements
 
The authors wish to thank Jennifer Novotny and Amy Simpson for their invaluable assistance with the graphic presentation of results. This project was supported by the following: KAI Pharmaceuticals (DHK), NIH KO1 AG00875 (DHK), NIH T32 GM008619 (JCH) and an American College of Sports Medicine Foundation Grant (JCK).


   Notes
 
Time for primary review 22 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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