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.

The effects of estrogen deficiency on the loss of cardioprotection with advancing age are complex and poorly understood. A major focus of the current study was to uncover a cardioprotective role for rapid, nongenomic estrogen receptor (ER) signaling in the aged female myocardium. We hypothesized that selective ERalpha activation in aged females would reduce infarct size in part, through reversal of age-associated reductions in mitochondrial protein kinase Cepsilon (PKCepsilon). Hearts isolated from adult (6 month old) and aged (23-24 months old) female F344 rats with ovaries removed (n = 20 per group) were subjected to ischemia/reperfusion (47 min global ischemia). Rats were injected sc with the ERalpha agonist propylpyrazole triol (PPT) or vehicle 45 min before heart isolation (5 microg/kg). Infarct size was greatest in aged vs. adult ovariectomized rats, significantly reduced by PPT, and the protection reversed by prior administration of the ER inhibitor ICI 182,780 (3 mg/kg). Increased ERalpha particulate targeting occurred after PPT in conjunction with reversal of age-related reductions in nuclear PKCepsilon, mitochondrial PKCepsilon and pAkt (P < 0.05). PPT also increased mRNA levels for the PKCepsilon anchoring protein, receptor for activated C kinase2 (RACK2; P < 0.05). Our data suggest, for the first time, that selective ERalpha activation reduces ischemic injury in the aged, estrogen-deficient heart through a mechanism involving nongenomic redistribution of ERalpha and PKCepsilon activation. A novel feed-forward transcriptional mechanism to potentially enhance PKCepsilon-RACK2 interactions was also observed. Collectively, our findings may provide key insight into developing targeted therapeutic interventions in postmenopausal women to reduce ischemia/reperfusion injury, including selective ERalpha mimetics.

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

The present study sought to determine whether the protein catabolic response in skeletal muscle produced by chronic alcohol feeding was exaggerated in aged rats. Adult (3 mo) and aged (18 mo) female F344 rats were fed a nutritionally complete liquid diet containing alcohol (36% of total calories) or an isocaloric isonitrogenous control diet for 20 wk. Muscle (gastrocnemius) protein synthesis, as well as mTOR and proteasome activity did not differ between control-fed adult and aged rats, despite the increased TNF-α and IL-6 mRNA and decreased IGF-I mRNA in muscle of aged rats. Compared with alcohol-fed adult rats, aged rats demonstrated an exaggerated alcohol-induced reduction in lean body mass and protein synthesis (both sarcoplasmic and myofibrillar) in gastrocnemius. Alcohol-fed aged rats had enhanced dephosphorylation of 4E-BP1, as well as enhanced binding of raptor with both mTOR and Deptor, and a decreased binding of raptor with 4E-BP1. Alcohol feeding of both adult and aged rats reduced RagA binding to raptor. The LKB1-AMPK-REDD1 pathway was upregulated in gastrocnemius from alcohol-fed aged rats. These exaggerated alcohol-induced effects in aged rats were associated with a greater decrease in muscle but not circulating IGF-I, but no further increase in inflammatory mediators. In contrast, alcohol did not exaggerate the age-induced increase in atrogin-1 and MuRF1 mRNA or the increased proteasome activity. Our results demonstrate that, compared with adult rats, the gastrocnemius from aged rats is more sensitive to the catabolic effects of alcohol on protein synthesis, but not protein degradation, and this exaggerated response may be AMPK-dependent.