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Am J Physiol Regul Integr Comp Physiol 292: R800-R809, 2007. First published September 28, 2006; doi:10.1152/ajpregu.00374.2006
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Sex Differences in Renal and Cardiovascular Function: Physiology and Pathophysiology

Estrogen deficiency decreases ischemic tolerance in the aged rat heart: roles of PKC{delta}, PKC{epsilon}, Akt, and GSK3beta

J. C. Hunter,1 J. C. Kostyak,1 J. L. Novotny,1 A. M. Simpson,1 and Donna H. Korzick1,2

1Intercollege Program in Physiology, and 2The Department of Kinesiology, The Pennsylvania State University, University Park, Pennsylvania

Submitted 31 May 2006 ; accepted in final form 27 September 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The mechanisms underlying the age-dependent reversal of female cardioprotection are poorly understood and complicated by findings that estrogen replacement is ineffective at reducing cardiovascular mortality in postmenopausal women. Although several protective signals have been identified in young animals, including PKC and Akt, how these signals are affected by age, estrogen deficiency, and ischemia-reperfusion (I/R) remains unknown. To determine the independent and combined effects of age and estrogen deficiency on I/R injury and downstream PKC-Akt signaling, adult and aged female F344 rats (n = 12/age) with ovaries intact or ovariectomy (Ovx) were subjected to I/R using Langendorff perfusion (31-min global-ischemia). Changes in cytosolic (s), nuclear (n), mitochondrial (m) PKC ({delta}, {epsilon}) levels, and changes in total Akt and mGSK-3beta phosphorylation after I/R were assessed by Western blot analysis. Senescence increased infarct size 50% in ovary-intact females (P < 0.05), whereas no differences in LV functional recovery or estradiol levels were observed. Ovx reduced functional recovery to a greater extent in aged compared with adult rats (P < 0.05). In aged (vs. adult), levels of m- and nPKC(-{delta}, -{epsilon}) were markedly decreased, whereas mGSK3beta levels were increased (P < 0.05). Ovx led to greater levels of sPKC(-{delta}, -{epsilon}) independent of age (P < 0.05). I/R reduced p-Akt(Ser473) levels by 57% and increased mGSK-3beta accumulation 1.77-fold (P < 0.05) in aged, ovary-intact females. These data suggest, for the first time, that estrogen alone cannot protect the aged female myocardium from I/R damage and that age- and estrogen-dependent alterations in PKC, Akt, and GSK-3beta signaling may contribute to loss of ischemic tolerance.

senescence; ischemia-reperfusion; female

THE SENESCENT MYOCARDIUM HARBORS several alterations, which negatively affect its reserve capacity to combat mechanical or ischemic stresses, even in the absence of overt cardiovascular pathology (28). That women surpass men in cardiovascular disease prevalence at approximately the same age as menopause in adults suggests that decreased plasma estradiol contributes to the age-dependent decrease in ischemic tolerance in females (1). Although animal models using surgical menopause clearly support cardioprotection from estrogen replacement therapy (ERT) (8, 15, 17), recent clinical trials have failed to demonstrate any protection from ERT in postmenopausal women (2, 7, 41). In fact, the incidence of cardiovascular disease actually increased in women receiving estrogen or estrogen plus progesterone in the Women's Health Initiative compared with those receiving placebo (2, 41). Taken together, these findings have forced a reevaluation of estrogen-dependent cardioprotection and have highlighted the paucity of research investigating the potentially adverse interactive effects of estrogen deficiency and advanced age on cardioprotective signaling in females.

Although the majority of research into cardioprotective signaling has been conducted on male animals, results of these studies have suggested several candidate signaling pathways, which may be negatively affected by senescence in females (23, 45). Such pathways include protein kinase C (PKC) and Akt, which may both converge on GSK3beta to promote cell survival (23). Two members of the PKC family, PKC{delta} and PKC{epsilon}, have repeatedly been shown to mediate opposing effects following ischemia-reperfusion (I/R) injury (5, 20, 34). In contrast to PKC{epsilon}, which mediates protection by inhibiting the mitochondrial permeability transition, PKC{delta} promotes cell death by enhancing cytochrome c release and inactivating Akt (5, 6, 30, 34). In fact, recent studies have shown that activation of PKC{epsilon} prior to ischemia combined with inhibition of PKC{delta} upon reperfusion demonstrates additive protection (20). Of interest, reductions in PKC activation with age have been demonstrated in response to both ischemic preconditioning and phenylephrine (25, 46) in males. Similarly, Akt activation has been shown to be decreased in hearts of aged females, which may account for ischemic intolerance observed in senescent females (11). Given that GSK3beta is downstream of both PKC{epsilon} and Akt, it is also a likely target for age-dependent modifications, which may negatively affect ischemic tolerance. Because PKC and Akt can be activated by estrogen (3, 37), these data collectively suggest that alterations in PKC, Akt, and GSK3beta activation may underlie the age-dependent reduction in female cardioprotection.

We have previously shown basal age-dependent alterations in PKC signaling distribution in females; however, whether these proteins are affected by I/R remains unknown (19). Although previous studies have demonstrated estrogen-dependent cardioprotection, an important limitation of these studies is that surgically induced menopause in adult animals was used to mimic the long-term effects of aging on the hormonal milieu. As such, the combined effects of senescence and estrogen deficiency on PKC- and Akt-mediated protection from I/R remains uncertain. Accordingly, the purpose of the present study was to assess the independent and combined effects of age and estrogen deficiency on recovery from I/R and on activation of PKC{epsilon}, PKC{delta}, Akt, and GSK3beta following I/R.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal care. Adult (5 mo) and aged (23 mo) female (n = 12/age) Fischer-344 rats were obtained from Harlan Sprague Dawley (Indianapolis, IN). Rats were exposed to a 12:12-h light-dark cycle and received food and water ad libitum. All animal handling and utilization were in accordance with the Penn State University Animal Care and Utilization Committee.

Surgical ovariectomy. Adult (4 mo) and aged (22 mo) female F344 rats (n = 4/group) underwent surgical ovariectomy (Ovx), according to standard procedures. Briefly, each ovary and part of the oviduct was removed, and animals were allowed to recover for 5 wk before experimental intervention. Successful surgery was confirmed by assessing circulating levels of 17beta-estradiol.

I/R protocol. 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, according to well-established procedures in our laboratory (25, 27). During a 15-min equilibration period, pacing at 260 beats/min was established, and balloon volume was adjusted to 5–6 mmHg. After equilibration, hearts in the I/R group were subjected to 31-min global, normothermic ischemia, as we described previously (27). Pacing was reinitiated 1 min after restoring flow, and hearts were reperfused for 30 min. In a subset of hearts (n = 6 adult, n = 8 aged), a greater than fourfold increase in creatine kinase release (Stan Bio) was observed in coronary effluent, assuring that significant injury was induced by our experimental protocol (not shown). Following reperfusion left ventricles (LV) were isolated, weighed, halved and frozen in liquid nitrogen. LV from control hearts were harvested similarly immediately after the equilibration period. All LV sections were stored at –80°C until tissue preparation.

Triphenyltetrazolium chloride staining. To assess infarct size, a subset of hearts (n = 4 adult and aged female) underwent 47 min normothermic ischemia followed by 2 h reperfusion and the area of infarction was assessed exactly as described (27). Briefly, after reperfusion, LV were isolated and frozen at –20°C for 30 min. LV 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 using National Institutes of Health (NIH) ImageJ software.

Tissue sample preparation. Cytosolic, nuclear, and mitochondrial samples were prepared exactly as described previously (19, 27). Briefly, frozen LV samples were homogenized by glass-glass grinder in 10 vol of buffer A containing (in mM) 250 sucrose; 10 Tris·HCl, pH 7.4, 1 EDTA, pH 7–8, 1 orthovanadate; 1 NaF; 0.3 PMSF; 5 µg/ml each of leupeptin and aprotinin; and 0.5 µg/ml pepstatin A and subjected to serial centrifugations of 1,000 g, 10,000 g, and 100,000 g. The 1,000 g pellet (nuclear fraction) and the 10,000 g pellet (mitochondrial fraction) were washed in buffer A and recentrifuged. The final pellets were resuspended in buffer B containing (in mM): 150 NaCl; 20 Tris·HCl, pH 7.4, 10 EDTA, pH 7–8, 1 orthovanadate; 1 NaF; 0.3 PMSF and 0.5 µg/ml pepstatin A, 5 µg/ml each of leupeptin and aprotinin, and 1% NP-40 and subjected to a 21,000 g centrifugation for 10 min. The resultant supernatants were defined as the nuclear and mitochondrial fractions. The 100,000 g supernatant was defined as the cytosolic fraction.

Total homogenates were prepared as described previously (19, 27). Briefly, LV tissue was homogenized on ice in cold lysis buffer containing (in mM) 20 Tris, pH 7.5, 2 each of EDTA and EGTA, pH 7.5–8.0, 5 NaF, 0.3 PMSF, as well as 5 µg/ml each of leupeptin and aprotinin, 0.5 µg/ml pepstatin A, 1 µM vanadate, 0.03% 2-mercaptoethanol, and 1% Triton X-100. After 30 min agitation on ice, samples were subjected to a 100,000 g centrifugation at 4°C, and the supernatant was taken as the total fraction. All protein concentrations were determined by the method of Bradford (9).

Western blot analysis. Western blot analysis was performed according to well-established procedures in our laboratory (25, 26). Briefly, equal amounts of cytosolic (PKC and cytochrome c), nuclear (PKC), mitochondrial (PKC, GSK3beta), or total homogenate (Akt) were electrophoresed on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Membranes were blocked and incubated with rabbit polyclonal antibodies against PKC{delta} (1:1,200) or PKC{epsilon} (1:1,500) for 3 h at room temperature or against p-Akt(Ser473), Akt, p-GSK3beta(Ser9), GSK3beta, cytochrome c (1:1,000) overnight at 4°C. Immunoreactive bands were detected using HRP-linked anti-rabbit IgG (1:15,000) and enhanced chemiluminescence (Amersham). Densitometry was performed using Scion Image (NIH).

To control for total GSK and Akt protein levels, membranes were initially incubated with phospho-specific antibodies [p-Akt (Ser473)], or p-GSK3beta (Ser9), and then stripped by 30 min incubation (70°C) in buffer containing 62.5 mM Tris·HCl pH 6.80, 7% 2-mercaptoethanol, 2% SDS. Membranes were subsequently probed for total levels of Akt and GSK3beta, as described above.

To control for minor differences in protein loading, all membranes were stained with Sypro Ruby (PKC{delta}, PKC{epsilon}) or Ponceau S (p-Akt, p-GSK3beta, Akt, GSK3beta), and densitometry values were adjusted, as described previously (39). All samples were normalized to adult female control.

Statistical analyses. All data are presented as means ± SE and analyzed using the Statistical Analysis System (SAS). Baseline data 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 blot analysis data. The Tukey-Kramer method was employed for all post hoc comparisons. An alpha level of P ≤ 0.05 was defined as statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Rat heart characteristics. Table 1 shows morphological and baseline cardiac functional data for adult and aged female rats with gonads intact or following ovariectomy. LV and body weights were significantly greater in aged animals compared with adults. As reported previously, Ovx led to a further increase in body weight in aged females (P < 0.05), and a small but significant increase in left ventricular developed pressure (LVDP) and +dP/dt in both adult and aged animals (P < 0.05) (38).


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  		Table 1. Morphological and baseline functional characteristics

 
Recovery after I/R. Recovery of LVDP (A and B), +dP/dt (C and D), and –dP/dt (E and F) are displayed in Fig. 1. Although a modest reduction in LVDP recovery (23%) was observed in adult Ovx females, the effects of estrogen deficiency were more pronounced in aged animals, resulting in a significant reduction in LVDP recovery throughout reperfusion (P < 0.05) (Fig. 1B). Reductions in +dP/dt and –dP/dt were also observed early in reperfusion (63% and 69%, respectively; P < 0.05) in Ovx (compared with intact) aged females (Fig. 1, D and F). The only age-dependent reduction in postischemic function in females with ovaries intact was noted with respect to –dP/dt (P < 0.05) (Fig. 1F). A similar pattern for age-related reductions in coronary flow during reperfusion following Ovx were also observed (group x reperfusion time interaction, P < 0.001; Fig. 2).


Figure 1
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  		Fig. 1. Functional recovery after ischemia/reperfusion (I/R). Hearts were subjected to 31-min normothermic global ischemia, and recovery of function was followed throughout 30 min of reperfusion. To quantify recovery, the following variables were measured: percent recovery of left ventricular developed pressure (LVDP; A and B), +dP/dt (C and D), and –dP/dt (E and F). For clarity, adult recovery (A, C, E) is presented separately from aged recovery (B, D, F). All results are presented as means ± SE. *P < 0.05 aged different from adults with ovaries intact. {dagger}P < 0.05 effect of ovariectomized (Ovx) within aged vs. adult.

 

Figure 2
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  		Fig. 2. Relative coronary flows after I/R. Hearts were subjected to 31-min normothermic global ischemia, and coronary effluent was collected throughout 30 min of reperfusion. Values were normalized to left ventricular (LV) weight. All data are presented as means ± SE. *P < 0.05 different from adult, {dagger}P < 0.05 effect of Ovx within aged.

 
Figure 3 shows that Ovx had no effect on recovery of end diastolic pressure (EDP) during reperfusion in adult females (A), whereas EDP remained elevated two- to threefold in aged Ovx (vs. ovary-intact) females throughout reperfusion (P < 0.05; B). Of interest, increasing the time of ischemia to 47 min or increasing the perfusate-free Ca2+ concentration to 2 mM reduced functional recovery (50% and 30%, respectively) similarly in both adult and aged gonad-intact females (data not shown). Despite the lack of age-dependent functional decrements in females with ovaries, infarct size was significantly increased by 50% (P < 0.05) in aged (vs. adult) females (Fig. 4). It is important to note that increased area of infarction in aged females was observed despite comparable plasma estradiol levels (Table l, P = NS). Importantly, Ovx in aged animals further increased infarct size by ~35% as compared with ~25% increase in adult animals, which corresponded to subsequent decrements in LV functional recovery (Fig. 1B). A similar pattern was observed for cytosolic cytochrome c levels (Fig. 5). Finally, the area of infarction in adult females subjected to 47 min ischemia was comparable with the infarction observed in adult males after 31 min ischemia (data not shown), consistent with the concept of female-dependent cardioprotection.


Figure 3
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  		Fig. 3. Recovery of end-diastolic pressure (EDP) after I/R. Hearts were subjected to 31-min normothermic global ischemia, and recovery of EDP was followed throughout 30 min of reperfusion. All data are presented as means ± SE. {dagger}P < 0.05 effect of Ovx within age.

 

Figure 4
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  		Fig. 4. Area of infarction after ischemia and reperfusion. Hearts were subjected to 47-min ischemia followed by 2-h reperfusion and stained with triphenyltetrazolium chloride (TTC). Data are presented as group means ± SE. *P < 0.05 different from adult. {dagger}P < 0.05 effect of Ovx within aged.

 

Figure 5
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  		Fig. 5. Cytosolic cytochrome c release after I/R. Hearts were subjected to 31-min global ischemia, and cytochrome c was assessed by Western blot analysis after reperfusion. Equal amounts of cytosolic protein (20 µg protein/lane) were loaded per lane and electrophoresed on 18% polyacrylamide gels and immunoblotted with antibodies against cytochrome c. All data are presented as means ± SE. *P < 0.05 different from adult . {dagger}P < 0.05 effect of Ovx within age.

 
PKC subcellular distribution. Figure 6A shows that aged females had significantly higher levels of cytosolic PKC{delta} compared with adult females in control rats (1.4-fold), and in post-I/R ovariectomized rats (1.65-fold) (P < 0.05). Conversely, cytosolic PKC{epsilon} was reduced in post-I/R aged (vs. adult) rats with and without ovaries (30%, P < 0.001) (Fig. 6B). Furthermore, Ovx significantly increased post-I/R cytosolic PKC{epsilon} in both adult and aged female rats (1.5-fold, P < 0.001).


Figure 6
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  		Fig. 6. Effects of age, Ovx, and I/R on cytosolic PKC{delta} and PKC{epsilon} levels. Hearts were subjected to 31-min ischemia/30-min reperfusion or served as perfused controls. Equal amounts of cytosolic protein were loaded per lane and electrophoresed on 7.5% polyacrylamide gels and immunoblotted with antibodies against PKC{delta} (A) or PKC{epsilon} (B). All data are normalized to adult female control and expressed as means ± SE. R30 denotes 30 min of reperfusion; Ovx R30 denotes 30 min of reperfusion in ovariectomized females. Statistical differences were detected by a two-way ANOVA on age (adult, aged) and group (Ctrl, R30, Ovx R30). *P < 0.05 vs. adult female within group; {ddagger}P < 0.05 Ovx R30 vs. R30 within age. §P < 0.05 vs. control within age.

 
Figure 7A demonstrates that nuclear PKC{delta} levels were unchanged by I/R and Ovx in aged female rat hearts. Levels of nuclear PKC{delta} were higher in adult (vs. aged) intact (1.4-fold, P < 0.005), and Ovx (1.25-fold, P = 0.076) animals after I/R. In contrast, senescence was associated with lower levels of nuclear PKC{epsilon} at baseline (40%) and after I/R in both intact and Ovx animals (55%; P < 0.05) (Fig. 7B). Additionally, in adult and aged females, levels of nuclear PKC{epsilon} were significantly lower in post-I/R Ovx compared with control rats (P < 0.05).


Figure 7
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  		Fig. 7. Effects of age, Ovx, and I/R on nuclear PKC{delta} and PKC{epsilon} levels. Hearts were subjected to 31-min of ischemia/30-min of reperfusion or served as perfused controls. Equal amounts of nuclear protein were loaded per lane and electrophoresed on 7.5% polyacrylamide gels and immunoblotted with antibodies against PKC{delta} (A) or PKC{epsilon} (B). All data are normalized to adult female control and expressed as means ± SE. R30 denotes 30 min of reperfusion; Ovx R30 denotes 30 min of reperfusion in ovariectomized females. Statistical differences were detected by a two-way ANOVA on age (adult, aged) and group (Ctrl, R30, Ovx R30). *P < 0.05 vs. adult female within group. {dagger}P < 0.05 R30 vs. Ctrl within age. §P < 0.05 Ovx R30 vs. Ctrl within age.

 
I/R led to a 26% decrease in mitochondrial PKC{delta} levels in intact adult female rats (P < 0.05) but had no effect in aged rats (Fig. 8A). Furthermore, the combination of Ovx and I/R led to a significant decrease in both adult and aged mitochondrial PKC{delta} (P < 0.05). Figure 8B demonstrates that aged (intact) females had 45% less mitochondrial PKC{epsilon} at baseline compared with adult (intact) females (P < 0.005). This difference was negated after I/R by the 40% reduction in mitochondrial PKC{epsilon} in adult (but not aged) intact females. Similar differences (from control) in mitochondrial PKC{epsilon} levels were noted in post-I/R Ovx females.


Figure 8
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  		Fig. 8. Effects of age, Ovx, and I/R on mitochondrial PKC{delta} and PKC{epsilon} levels. Hearts were subjected to 31-min ischemia/30-min reperfusion or served as perfused controls. Equal amounts of mitochondrial protein were loaded per lane and electrophoresed on 7.5% polyacrylamide gels and immunoblotted with antibodies against PKC{delta} (A) or PKC{epsilon} (B). All data are normalized to adult female control and expressed as means ± SE. R30 denotes 30 min of reperfusion; Ovx R30 denotes 30 min of reperfusion in ovariectomized females. Statistical differences were detected by a two-way ANOVA on age (adult, aged) and group (Ctrl, R30, Ovx R30). *P < 0.05 vs. adult female within group. {dagger}P < 0.05 R30 vs. Ctrl within age. {ddagger}P < 0.05 Ovx R30 vs. R30 within age. §P < 0.05 Ovx R30 vs. Ctrl within age.

 
Akt activation. In agreement with previous reports, the present study demonstrated higher levels of p-Akt (Ser473) in the total homogenate (1.7-fold) in females compared with males, independent of age (data not shown) (11). Age-dependent increased levels of p-Akt (Ser473) were noted at baseline in total (1.56-fold) and nuclear homogenates (not shown); however, this pattern was reversed after I/R in gonad-intact females (P < 0.05) (Fig. 9A). Additional age-dependent differences were noted in the response to Ovx in which only adult animals demonstrated a reduction (38%) in post-I/R p-Akt levels after Ovx (compared with post-I/R intact females) (P < 0.05). Although total levels of Akt were lower in aged vs. adult rats at all time points studied (P < 0.05, Fig. 9B), correcting for total levels of Akt (Fig. 9C) did not change the pattern of Akt activation observed in Fig. 9A.


Figure 9
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  		Fig. 9. Effects of age, Ovx, and I/R on Akt activation. Hearts were subjected to 31-min ischemia/30-min reperfusion or served as perfused controls. Equal amounts of total homogenate protein were loaded per lane and electrophoresed on 10% polyacrylamide gels and immunoblotted with antibodies against p-Akt (Ser473) (A), Akt (B), and p-Akt (Ser473) was expressed as a ratio to Akt (C). All data are normalized to adult female control and expressed as means ± SE. R30 denotes 30 min of reperfusion; Ovx R30 represents 30 min of reperfusion in ovariectomized females. Statistical differences were detected by a two-way ANOVA on age (adult, aged) and group (Ctrl, R30, Ovx R30). *P < 0.05 vs. adult female within group. {dagger}P < 0.05 R30 vs. Ctrl within age. {ddagger}P < 0.05 Ovx R30 vs. R30 within age. §P < 0.05 Ovx R30 vs. Ctrl within age.

 
GSK3beta phosphorylation. Similar to ovariectomy-dependent changes in post-I/R levels of p-Akt, phosphorylation of post-I/R mitochondrial GSK3beta was significantly reduced by Ovx only in adult animals (Fig. 10A). It is important to note that total levels of mitochondrial GSK3beta were 1.6- to 2.7-fold greater in senescent (vs. adult) females in all groups (Fig. 10B) and that in intact females, I/R significantly increased total levels of mitochondrial GSK3beta only in the aged (1.76-fold, P < 0.001). Unexpectedly, total levels of post-I/R mitochondrial GSK3beta were significantly reduced by Ovx in both adult and aged myocardium (58%, P < 0.05). Taken together, Fig. 10C shows that the relative inactivation of GSK3beta is greatly reduced (52–67%) in aged vs. adult rats independent of ovary status (P < 0.005). Of interest and consistent with changes in p-Akt levels, we observed significantly greater p-GSK3beta in females vs. males (n = 4; P < 0.05; data not shown).


Figure 10
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  		Fig. 10. Effects of age, Ovx, and I/R on GSK3beta phosphorylation. Hearts were subjected to 31-min ischemia/30-min reperfusion or served as perfused controls. Equal amounts of mitochondrial protein were loaded per lane and electrophoresed on 10% polyacrylamide gels and immunoblotted with antibodies against p-GSK3beta (Ser9) (A), GSK3beta (B), and p-GSK3beta (Ser9) was expressed as a ratio to GSK3beta (C). All data are normalized to adult female control and expressed as means ± SE. R30 denotes 30 min of reperfusion; Ovx R30 denotes 30 min of reperfusion in ovariectomized females. Statistical differences were detected by a two-way ANOVA on age (adult, aged) and group (Ctrl, R30, Ovx R30). *P < 0.05 vs. adult female within group. {dagger}P < 0.05 R30 vs. Ctrl within age. {ddagger}P < 0.05 Ovx R30 vs. R30 within age. §P < 0.05 Ovx R30 vs. Ctrl within age.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Postmenopausal women are especially vulnerable to ischemic insult and ~80,000 more cardiovascular deaths occur yearly in aged women than in aged men. The age-dependent reversal of female cardioprotection may be independent of sex hormone levels, as aged women in placebo-controlled trials have not demonstrated the well-documented cardioprotective advantage of estrogen-replacement observed in animals (2, 8, 15, 18, 41). This discrepancy may be due to the use of surgical menopause in adult animal models, which may obscure important age-estrogen deficiency interactions. Accordingly, we sought to determine the individual and combined effects of age and estrogen deficiency on functional recovery from I/R and associated signaling in female rats. Key findings are as follows: 1) age was associated with greater infarct size independent of changes in plasma estradiol levels and in spite of similar functional recovery between adult and aged groups, 2) Ovx resulted in a more pronounced reduction in postischemic functional recovery in aged vs. adult rats, and 3) profound reductions in mitochondrial and nuclear PKC{epsilon} were observed in aged rats and were associated with increased levels of active mitochondrial GSK3beta independent of estrogen deficiency. These results suggest, for the first time, that estrogen alone is insufficient to protect the aged female myocardium from I/R damage.

The majority of studies investigating age-dependent decrements in ischemic tolerance have been conducted in male animals and have demonstrated reduced ischemic tolerance with age (29, 46). In contrast to studies by Willems et al. (49), who studied aging in female mice, we found that recovery of systolic function following I/R was not different between adult and aged female F344 rats with ovaries intact after either 31 or 47 min of ischemia. A notable difference between the present study and those of Willems and colleagues (49) is the concentration of free Ca2+ (1.25 mM vs. 1.9 mM, respectively), which has been shown to blunt postischemic recovery (14). Although our follow-up studies using 2 mM free Ca2+ reduced functional recovery by ~30% (not shown), this reduction was comparable in adult and aged, gonad-intact females. Therefore, the discrepancy between our findings and those of Willems et al. (49) is more likely due to species differences or differences in age-related changes in plasma estradiol levels (not reported in the latter study).

The preservation of post-I/R systolic function in spite of greater infarct size in gonad-intact aged animals is intriguing for several reasons. First, although stunning may account for the preservation of postischemic function observed in adult and aged hearts, this interpretation is confounded by the more than four-fold increase in postischemic creatine kinase release (not shown) and the presence of necrotic (TTC) staining. Of interest, this apparent disconnect between function and damage is reminiscent of heart failure in women in which systolic function can be preserved despite the presence of pathological hypertrophy (12, 44). However, further studies are required to conclude whether this disconnect truly occurs following I/R in females. Second, the age-dependent increase in area of infarction in the present study was noted in the absence of significant changes in plasma estradiol with age. Although it is important to note that we did not control for day of estrus, since aged female rats were likely in a state of constant estrus (33), our results suggest that preserved levels of estrogen are not sufficient to prevent age-dependent increases in post-I/R area of infarction.

To determine the combined effects of senescence and estrogen deficiency, adult and aged females were ovariectomized 5 wk before I/R. The Ovx-induced increase in body weight in aged females resulted in an LV-to-body weight ratio comparable to aged male F344 rats (reported previously in Ref. 27). That this increase in body weight was not observed in adults may suggest that estradiol may be more important in maintaining LV/body wt in aged vs. adult rats. The relative decrease in LV mass (to body weight) in the aged due to Ovx may have contributed to the greater reductions in post-I/R recovery by limiting the reserve capacity of the heart. Furthermore, Ovx resulted in greater ischemic intolerance in aged (vs. adult) rats, suggesting that aged females may be more dependent on the cardioprotective effects of endogenous estrogens than adults. Although previous studies have shown I/R-dependent functional decrements with Ovx in adult females (16, 24), to our knowledge, we are the first to address recovery from I/R in senescent, Ovx females. Taken together, these findings might have clinical implications, as it is most often the aged who experience both estrogen deficiency and ischemic cardiac episodes.

Previously, we demonstrated in male rats that age-associated reductions in ischemic tolerance were ameliorated with PKC{delta} inhibition in a GSK3beta-dependent manner (27). These results, together with the opposing cardioprotective effects of PKC{delta} and PKC{epsilon} (20), led us to investigate whether perturbations in PKC{epsilon}, PKC{delta}, Akt, or GSK3beta signaling may underlie age- and estrogen-dependent differences in I/R recovery.

Here, the observation that significant I/R-induced PKC translocation occurred in intact adult (but not intact aged) rats is intriguing and consistent with previous studies demonstrating age-dependent blunting of PKC translocation in response to phenylephrine or ischemic preconditioning (25, 45). These data suggest that reduced translocation of PKCs may underlie the decreased ischemic tolerance (i.e., increased size of infarction) observed with age in female rat hearts. This situation may be further exacerbated by the present findings that I/R in combination with Ovx tends to shift PKC partitioning from the nuclear and mitochondrial fractions to the cytosolic fraction. The cytosolic fraction has classically been thought of as an inactive pool of PKC. However, we and others have demonstrated active PKC in the cytosol using phospho-specific antibodies that detect Ser/Thr phosphorylation at the activation loop and hydrophobic domains of PKC{delta} and PKC{epsilon} (26, 36, 42). Furthermore, studies have demonstrated that PKC{delta} may be active in the cytosol due to tyrosine phosphorylation (43). Although PKC phosphorylation was not measured in the present study, it is conceivable that the increased cytosolic partitioning of PKC due to Ovx leads to activation of novel signals downstream of phosphorylated cytosolic PKC{delta} and PKC{epsilon}, which may exacerbate myocardial damage.

Further compromising the ability of the aged heart to respond to an ischemic stress is the observation in the current study that following I/R, aged females have lower levels of PKC ({delta} and {epsilon}) at the nucleus compared with adult females. Because both PKC{delta} and PKC{epsilon} promote cardiac hypertrophy (10, 13), these findings may represent age-dependent reductions in the ability of the aged heart to induce compensatory ventricular remodeling (47). These results are consistent with several studies that have documented a reduced cardiac hypertrophic response in aged rats following pressure or volume overload (4, 21).

Like PKC{delta} and PKC{epsilon}, Akt promotes hypertrophy and is involved in cardioprotection (23, 31). Furthermore, Akt has been shown to be activated by estrogen (11). Akt can mediate protection at the mitochondria by phosphorylating and inactivating GSK3beta and Bad, both of which promote apoptosis when active (23, 34). Our present findings of age-dependent increases in total p-Akt contrast the findings of Camper-Kirby and colleagues (11), who noted a significant age-dependent decrease in nuclear p-Akt levels. This discrepancy is likely due to differences in species and/or plasma estradiol levels (no age-dependent reductions in estradiol levels were noted in the present study). Our observation that I/R reduced levels of p-Akt (Ser473) in aged, but not adult, gonad-intact females supports a protective role for Akt and suggests that the decrease in active Akt with senescence may contribute to the increased area of infarction observed in aged (vs. adult) rat hearts. Because chronic Akt activation also mediates hypertrophy (31), the age-dependent increase in basal p-Akt levels may represent an underlying mechanism of the relative hypertrophy observed with aging.

One target of Akt that has been proposed as a convergence point for cardioprotective signaling is GSK3beta (23). This model is supported by the present observation of I/R- and ovariectomy-dependent changes in pGSK3beta, which mirror changes in pAkt in adult female rats. In contrast, changes in p-Akt levels are disconnected from changes in p-GSK3beta levels in the aged, which might suggest that Akt-dependent GSK3beta phosphorylation is dysfunctional in the aged. Alternatively, GSK3beta phosphorylation may be occurring independently of Akt, as proposed by Juhaszova and colleagues (22). That mitochondrial levels of total GSK3beta were elevated in aged (vs. adult) intact females at baseline and were significantly increased by I/R only in the aged may contribute to the increased area of infarction observed in the aged, gonad intact females. Of interest, recent studies suggest that GSK3beta can enhance ER{alpha}-mediated transcription (32). The age-dependent increase in mitochondrial GSK3beta in the present study may represent a translocation away from the nucleus. If this is so, several cardioprotective proteins that are modulated by estrogen [such as nitric oxide synthase (35), ATP-dependent K+ channels (40), and heat shock proteins (48)] may show diminished expression, thus contributing to the decrease in ischemic tolerance in the aged.

In summary, our results demonstrate that senescence, independent of alterations in plasma estradiol levels, leads to reduced ischemic tolerance in the female rat myocardium and that ischemic intolerance is exacerbated by estrogen deficiency to a greater extent in the aged. Our results further suggest that alterations in post-I/R distribution and activation of PKC{delta}, PKC{epsilon}, Akt, and GSK3beta may underlie age- and estrogen deficiency-dependent reductions in both acute recovery and long-term remodeling of the female rat heart.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This project was supported by Pennsylvania Life Sciences Tobacco Settlement Funding (to D. H. Korzick), American Heart Association Grant 0555421U (to D. H. Korzick), National Institutes of Health Grant T32 GM008619 (to J. C. Hunter), and an National Institute on Aging Animal Allocation Award.

Present address for J. C. Hunter: Department of Physiology and Pharmacology, University of Western Ontario, London, ON.


   ACKNOWLEDGMENTS
 
The authors wish to thank Ann Albert for her expert assistance with Akt signaling studies.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. H. Korzick, 106 Noll Laboratory, The Pennsylvania State Univ., Univ. Park, PA 16802 (E-mail: dhk102{at}psu.edu)

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.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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