No abstract

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.

Studies on the role of mitochondrial fission/fusion (MFF) proteins in the heart have been initiated recently due to their biological significance in cell metabolism. We hypothesized that the expression of MFF proteins is affected by post-infarction remodeling and in vitro cardiomyocyte hypertrophy, and serves as a target for the Na(+)/H(+) exchanger 1 (NHE-1) inhibition. Post-infarction remodeling was induced in Sprague-Dawley rats by coronary artery ligation (CAL) while in vitro hypertrophy was induced in cardiomyocytes by phenylephrine (PE). Mitochondrial fission (Fis1, DRP1) and fusion (Mfn2, OPA1) proteins were analyzed in heart homogenates and cell lysates by Western blotting. Our results showed that 12 and 18 weeks after CAL, Fis1 increased by 80% (P < 0.01) and 31% (P < 0.05), and Mfn2 was reduced by 17% (P < 0.05) and 22% (P < 0.05), respectively. OPA1 was not changed at 12 weeks, although its expression decreased by 18% (P < 0.05) with 18 weeks of ligation. MFF proteins were also affected by PE-induced hypertrophy that was dependent on mitochondrial permeability transition pore opening and oxidative stress. The NHE-1-specific inhibitor EMD-87580 (EMD) attenuated changes in the expression of MFF proteins in both the models of hypertrophy. The effect of EMD was likely mediated, at least in part, through its direct action on mitochondria since Percoll-purified mitochondria and mitoplasts have been shown to contain NHE-1. Our study provides the first evidence linking cardiac hypertrophy with MFF proteins expression that was affected by NHE-1 inhibition, thus suggesting that MFF proteins might be a target for pharmacotherapy with anti-hypertrophic drugs.

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.