Evidence of Altered Mitochondrial Protein Expression After Chronic Ethanol Consumption in the Aged Estrogen-Deficient Female Rat Heart.

BACKGROUND: Estrogen loss has been implicated to increase the risk of alcoholic cardiomyopathy in postmenopausal women. The purpose of this study was to identify novel mitochondrial protein targets for the treatment of alcoholic cardiomyopathy in aged women using a state-of-the-art proteomic approach. We hypothesized that chronic ethanol (EtOH) ingestion exacerbates maladaptive mitochondrial protein expression in the aged female heart. METHODS: Adult (3 months) and aged (18 months) F344 ovary-intact or ovariectomized (OVX) rats were randomly assigned an EtOH or control Lieber-DeCarli "all-liquid" diet for 20 weeks. Proteomic analyses were conducted in mitochondria isolated from left ventricles using isobaric tags for relative and absolute quantification (iTRAQ) 8plex labeling and mass spectrometry (n = 3 to 5/group). RESULTS: After EtOH, significant differences (false discovery rate <5%) were observed in electron transport chain components (NADH dehydrogenase [ubiquinone] flavoprotein 2) as well as proteins involved in lipid metabolism (2,4 dienoyl-CoA reductase) and cellular defense (catalase), suggesting a possible link to congestive heart failure. Directional changes in protein levels were confirmed by Western blotting. Additionally, EtOH significantly reduced state 3 mitochondrial respiration in all groups, yet only reduced respiratory control index in the aged OVX rat heart (p < 0.05). CONCLUSIONS: Collectively, the data reveal that EtOH-induced changes in the mitochondrial proteome exacerbate cardiac dysfunction in aged and estrogen-deficient hearts, but not in adult. In conclusion, iTRAQ is a powerful tool for investigating new mitochondrial targets of alcoholic cardiomyopathy.

Acute adiponectin delivery is cardioprotective in the aged female rat heart.

AIM: The aged, post-menopausal female heart is characterized by reduced ischemic tolerance, and few therapies currently exist to limit ischemic damage. Adiponectin (APN), a cytokine produced in adipose tissue, limits infarct size and improves functional recovery after ischemia/reperfusion injury in adult hearts. The aim of the present study was to extend these previous studies and determine the cardioprotective efficacy of APN treatment in aged female rats. METHODS: Hearts were isolated from adult (age 6-7 months; n = 10), aged (age 23 months; n = 14) and aged ovariectomized (n = 10) female rats, and subjected to ischemia/reperfusion injury. On ischemia, hearts were infused with 9 µg of APN or vehicle. Adiponectin receptor 1, adiponectin receptor 2 and adenosine monophosphate-dependent kinase (AMPK) were assessed by western blotting, tumor necrosis factor-α and nicotinamide adenine dinucleotide phosphate oxidase levels by real time polymerase chain reaction. Non-reducing western blotting for APN multimers in visceral adipose was also carried out. RESULTS: APN infusion successfully improved post-ischemic left ventricular developed pressure (∼10-15%) and attenuated the rise in end diastolic pressure in all groups (P < 0.05). With ischemia/reperfusion injury, phospho-AMPK increased in all groups with additive effects of APN on increasing phospho-AMPK abundance in aged ovary-intact female rats only (P < 0.001). Age-associated increases in pre-ischemic tumor necrosis factor-α mRNA were unaffected by APN, whereas nicotinamide adenine dinucleotide phosphate oxidase 2 mRNA levels were attenuated by APN in adult and aged ovariectomized female rats. An age-associated decrease in cardiac adiponectin receptor 2 was observed in conjunction with elevated high molecular weight APN in adipose. CONCLUSIONS: The present data suggest that APN might be a relevant therapy for protecting the aging female heart, albeit through divergent mechanisms that are likely influenced by age-associated estrogen availability.

Estrogen receptor beta does not influence ischemic tolerance in the aged female rat heart.

INTRODUCTION: Ischemic heart disease remains the leading cause of morbidity and mortality in aged women, with a 2- to 3-fold increase in incidence following menopause. Clinical trials have failed to demonstrate cardioprotective benefit from chronic estrogen (E(2)) replacement therapy, yet protective effects of E(2) have been demonstrated in adult animal models and are mediated by the estrogen receptor (ER) subtypes ERα and ERß. AIMS: The aim of this study was to determine the effects of acute ERß activation on ischemia/reperfusion (I/R) injury in adult, aged, and aged E(2)-deficient female rats. METHODS: Hearts were isolated from adult (6 months; n = 9), aged (24 months; n = 13), and aged ovariectomized (OVX; n = 14) female Fischer 344 rats and subjected to 47 min of global I and 60 min of R. Rats were acutely treated with the ERß-agonist diarylpropionitrile (DPN; 5 µg/kg) or vehicle 45 min prior to I/R; ERß mRNA and protein levels were also assessed. RESULTS: Acute treatment with DPN had no effect on functional recovery following I/R injury in adult, aged, or aged OVX female rats. Additionally, we were unable to detect ERß mRNA or protein in the adult or aged female rat myocardium. CONCLUSIONS: Here, for the first time, our data suggest that acute ERß activation does not impact ischemic tolerance in the adult or aged female Fischer 344 rat myocardium and this likely due to a lack of detectable ERß.

Comparison of the agar block and Lieber-DeCarli diets to study chronic alcohol consumption in an aging model of Fischer 344 female rats.

INTRODUCTION: Post-menopausal women have a greater risk of developing alcoholic complications compared to age-matched men. Unfortunately, animal models of chronic ethanol consumption with estrogen deficiency are lacking. Here, we characterize the ability of the agar block and Lieber-DeCarli models of chronic ethanol consumption to produce elevated blood alcohol content (BAC) and liver pathology in the F344 postmenopausal animal model of aging. METHODS: Adult (3 mo) and aged (18 mo) F344 ovary-intact or ovariectomized rats were administered ethanol for 14-20 weeks as follows: diet 1, standard chow access, 10% ethanol in drinking water, and 40% ethanol in agar blocks; diet 2, diet 1 plus low phytoestrogen chow (known to affect ethanol metabolism) for the final 4 weeks; diet 3, Lieber-DeCarli all liquid diet with 36% kcal ethanol. Control animals were matched isocalorically with dextrin. RESULTS: For the agar block diet, average BAC was 13±4 mg/dL across groups. BAC was unaffected by reducing dietary phytoestrogen content (12±4 mg/dL), which is known to interfere with ethanol metabolism. Liver pathology was unaffected by the agar block diet. In contrast, the Lieber-DeCarli diet resulted in BAC of 45±5 mg/dL in conjunction with more severe hepatopathology.223 DISCUSSION: We conclude that the Lieber-DeCarli diet produces greater BAC and hepatopathology to study the effects of chronic ethanol administration in the F344 postmenopausal rodent model of aging when compared to an ethanol agar block diet.

Quantitative proteomic analysis reveals novel mitochondrial targets of estrogen deficiency in the aged female rat heart.

The incidence of myocardial infarction rises sharply at menopause, implicating a potential role for estrogen (E(2)) loss in age-related increases in ischemic injury. We aimed to identify quantitative changes to the cardiac mitochondrial proteome of aging females, based on the hypothesis that E(2) deficiency exacerbates age-dependent disruptions in mitochondrial proteins. Mitochondria isolated from left ventricles of adult (6 mo) and aged (24 mo) F344 ovary-intact or ovariectomized (OVX) rats were labeled with 8plex isobaric tags for relative and absolute quantification (iTRAQ; n = 5-6/group). Groups studied were adult, adult OVX, aged, and aged OVX. In vivo coronary artery ligation and in vitro mitochondrial respiration studies were also performed in a subset of rats. We identified 965 proteins across groups and significant directional changes in 67 proteins of aged and/or aged OVX; 32 proteins were unique to aged OVX. Notably, only six proteins were similarly altered in adult OVX (voltage-dependent ion channel 1, adenine nucleotide translocator 1, cytochrome c oxidase subunits VIIc and VIc, catalase, and myosin binding protein C). Proteins affected by aging were primarily related to cellular metabolism, oxidative stress, and cell death. The largest change occurred in monoamine oxidase-A (MAO-A), a source of oxidative stress. While acute MAO-A inhibition induced mild uncoupling in aged mitochondria, reductions in infarct size were not observed. Age-dependent alterations in mitochondrial signaling indicate a highly selective myocardial response to E(2) deficiency. The combined proteomic and functional approaches described here offer possibility of new protein targets for experimentation and therapeutic intervention in the aged female population.

Age-dependent reductions in mitochondrial respiration are exacerbated by calcium in the female rat heart.

BACKGROUND: Cardiovascular disease mortality increases rapidly after menopause by poorly defined mechanisms. OBJECTIVE: Because mitochondrial function and Ca(2+) sensitivity are important regulators of cell death after myocardial ischemia, we sought to determine whether aging and/or estrogen deficiency (ovariectomy) increased mitochondrial Ca(2+) sensitivity. METHODS: Mitochondrial respiration was measured in ventricular mitochondria isolated from adult (6 months; n = 26) and aged (24 months; n = 25), intact or ovariectomized female rats using the substrates α-ketoglutarate/malate (complex I); succinate/rotenone (complex II); ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine/antimycin (complex IV). State 2 and 3 respiration was initiated by sequential addition of mitochondria and adenosine diphosphate. Ca(2+) sensitivity was assessed by Ca(2+)-induced swelling of de-energized mitochondria and reduction in state 3 respiration. Propylpyrazole triol (PPT) was administered intraperitoneally 45 minutes before euthanasia to assess mitochondrial protective effects through estrogen receptor (ER) α activation. RESULTS: Aging decreased the respiratory control index (RCI; state 3/state 2) for complexes I and II by 12% and 8%, respectively, independent of ovary status (P < 0.05). Of interest, Ca(2+) induced a greater decrease (18%-30%; P < 0.05) in complex I state 3 respiration in aged and ovariectomized animals, and mitochondrial swelling occurred twice as quickly in aged (vs adult) female rats (P < 0.05). Pretreatment with PPT increased RCI by 8% and 7% at complexes I and II, respectively (P < 0.05) but surprisingly increased Ca(2+) sensitivity. CONCLUSIONS: Age-dependent decreases in RCI and sensitization to Ca(2+) may explain in part the age-associated reductions in female ischemic tolerance; however, protection afforded by ER agonism involves more complex mechanisms.

Ouabain increases iNOS-dependent nitric oxide generation which contributes to the hypertrophic effect of the glycoside: possible role of peroxynitrite formation.

In addition to inotropic effects, cardiac glycosides exert deleterious effects on the heart which limit their use for cardiac therapeutics. In this study, we determined the possible contribution of ouabain-induced iNOS stimulation to the resultant hypertrophic as well as cytotoxic effects of the glycoside on cultured adult rat ventricular myocytes. Myocytes were treated with ouabain (50 µM) for up to 24 h. Ouabain significantly increased gene and protein levels of inducible nitric oxide synthase (iNOS) which was associated with significantly increased release of NO from myocytes as well as increased total release of reactive oxygen species (ROS), superoxide anion (O(2) (-)), and increased peroxynitrite formation as assessed by protein tyrosine nitration. Administration of ouabain was also associated with increased levels of myocyte toxicity as determined by myocyte morphology, trypan blue staining and lactate dehydrogenase (LDH) efflux. The nonspecific NOS inhibitor Nω-nitro-L: -arginine methyl ester and the more selective iNOS inhibitor 1400W both abrogated the increase in LDH release but had no significant effect on either morphology or trypan blue staining. Ouabain also significantly increased both myocyte surface area and expression of atrial natriuretic peptide indicating a hypertrophic response with both parameters being completely prevented by NOS inhibition. The effects of iNOS inhibitors were associated with diminished ouabain tyrosine nitration as well as abrogation of ouabain-induced p38 and ERK phosphorylation. Our study shows that ouabain is a potent inducer of NO formation, iNOS upregulation, and increased production of ROS. Inhibition of ouabain-dependent peroxynitrite formation may contribute to the antihypertrophic effect of iNOS inhibition possibly by preventing downstream MAPK activation.

Targeting the mitochondrial permeability transition: cardiac ischemia-reperfusion versus carcinogenesis.

Cardiovascular diseases and cancer continue to be major causes of death worldwide, and despite intensive research only modest progress has been reached in reducing the morbidity and mortality of these awful diseases. Mitochondria are broadly accepted as the key organelles that play a crucial role in cell life and death. They provide cells with ATP produced via oxidative phosphorylation under physiological conditions, and initiate cell death through both apoptosis and necrosis in response to severe stress. Oxidative stress accompanied by calcium overload and ATP depletion induces the mitochondrial permeability transition (mPT) with formation of pathological, non-specific mPT pores (mPTP) in the mitochondrial inner membrane. Opening of the mPTP with a high conductance results in matrix swelling ultimately inducing rupture of the mitochondrial outer membrane and releasing pro-apoptotic proteins into the cytoplasm. The ATP level is the determining factor in deciding whether cells die through apoptosis or necrosis. Cardiac cells undergoing ischemia followed by reperfusion (IR) possess exactly the same conditions mentioned above to induce mPTP opening. Due to its critical role in cell death, inhibition of mPTP opening has been accepted as a major therapeutic approach to protect the heart against IR. In contrast to cardiac IR, cancer cells exhibit less sensitivity to pore opening which can be in part explained by increased expression of mPTP compounds/modulators and metabolic remodeling. Since the main goal of chemotherapy is to provoke apoptosis, mPT induction may represent an attractive approach for the development of new cancer therapeutics to induce mitochondria-mediated cell death and prevent cell differentiation in carcinogenesis. This review focuses on the role of the mPTP in cardiac IR and cancer, and pharmacological agents to prevent or initiate mPT-mediated cell death, respectively in these diseases.

mTOR mediates RhoA-dependent leptin-induced cardiomyocyte hypertrophy.

Obesity is associated with increased leptin production which may contribute to cardiac hypertrophy. However, the mechanism of leptin-induced cardiac hypertrophy remains incompletely understood. The Rho family (RhoA, Rac1, and Cdc42) and mammalian target of rapamycin (mTOR) have recently emerged as important regulators of cell growth. We therefore explored the roles and interrelationships of phosphatidylinositol 3-kinase (PI3K), mTOR, and the Rho family in the regulation of actin polymerization and leptin-induced hypertrophy in cultured neonatal rat ventricular myocytes. Five minutes treatment with leptin (3.1 nM) resulted in activation of RhoA and Rac1 (by 330 and 160%, respectively, P < 0.05) which was significantly attenuated by AG-490 (50 µM) and LY294002 (10 µM), specific inhibitors of JAK2 and PI3K, respectively. However, Cdc42 activity was unaffected by leptin. The hypertrophic effect of leptin was associated with an increase in phosphorylation of p70(S6K), the major target of mTOR, by 110% (P < 0.05). The specific mTOR inhibitor rapamycin (10 nM) attenuated leptin-induced RhoA and Rac1 activation. Furthermore, the leptin-induced decrease in the G/F-actin ratio, a measure of actin polymerization, was blunted by rapamycin. Leptin produced activation of the transcriptional factor GATA4 which was attenuated by the RhoA inhibitor C3, the p38 MAPK inhibitor SB203580 (10 µM) as well as rapamycin. Our results demonstrate a critical role for PI3K/mTOR/p70(S6K) in leptin-induced RhoA activation resulting in cardiomyocyte hypertrophy associated with GATA4 stimulation.

Expression of mitochondrial fusion-fission proteins during post-infarction remodeling: the effect of NHE-1 inhibition.

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.

A novel chimeric natriuretic peptide reduces cardiomyocyte hypertrophy through the NHE-1-calcineurin pathway.

AIMS: Natriuretic peptides (NPs) inhibit cardiomyocyte hypertrophy through a cyclic GMP (cGMP)-dependent process, although these effects are associated with substantial vasodilatation. In this study, we used CU-NP, a non-vasodilatating novel NP synthesized from the ring structure of human C-type NP (CNP) and both C- and N-termini of urodilatin, and investigated whether it can directly modulate cardiomyocyte hypertrophy. METHODS AND RESULTS: Experiments were carried out in cultured neonatal rat ventricular myocytes exposed to phenylephrine, angiotensin II, or endothelin-1 in the absence or presence of CU-NP. CU-NP produced a concentration- and time-dependent increase in intracellular cGMP levels. The hypertrophic responses to all agonists were abrogated by 10 nM CU-NP. CU-NP treatment also prevented increased activity, gene and protein expression of sodium-hydrogen exchanger-1 (NHE-1) as well as elevations in intracellular Na(+) concentrations caused by hypertrophic agents. In addition, these effects were associated with a more than two-fold increase in activity of the Ca(2+)-dependent protein phosphatase calcineurin that peaked 6 h after addition of hypertrophic stimuli. Early (1-3 h) calcineurin activation was unaffected by CU-NP, although activation at 6 and 24 h was prevented by CU-NP as was the resultant translocation of the transcriptional factor NFAT into nuclei. CONCLUSION: Our study demonstrates a direct anti-hypertrophic effect of the chimeric peptide CU-NP via NHE-1 inhibition, thereby preventing calcineurin activation and NFAT nuclear import. Thus, CU-NP represents a novel fusion peptide of CNP and urodilatin that has the potential to be developed into a therapeutic agent to treat cardiac hypertrophy and heart failure.

Nitric oxide inhibits endothelin-1-induced neonatal cardiomyocyte hypertrophy via a RhoA-ROCK-dependent pathway.

Although nitric oxide (NO) has received extensive attention as an anti-hypertrophic agent the mechanisms underlying its regulation of endothelin-1 (ET-1) have not been fully elucidated. Since RhoA has been identified as an important mediator of cardiac hypertrophy and is inhibited by NO in vascular tissue, we sought to determine whether the anti-ET-1 effects of NO in cardiomyocytes were mediated via inhibition of the RhoA-ROCK cascade in the context of cardiac hypertrophy. Neonatal rat ventricular myocytes were cultured in the presence of ET-1 (10 nM) with or without pre-treatment with the NO donor S-nitroso-n-acetylpenicillamine (SNAP; 100 microM), 8-Br-cGMP (cGMP; 100 microM), the RhoA inhibitor C3 exoenzyme (C3; 30 ng/ml), or the ROCK inhibitor Y-27632 (10 microM). ET-1-induced cardiomyocyte hypertrophy was prevented by pre-treatment with SNAP, cGMP, C3, or Y-27632. The hypertrophic response to ET-1 was associated with significantly increased gene and protein expression of both NOS2 and NOS1 although NOS3 was unaffected. ET-1 treatment for 15 min increased membrane-bound RhoA 2.6-fold (p<0.05), which was prevented by both SNAP and cGMP (p<0.05). These effects were associated with a complete abrogation of ET-1-induced phosphorylation of the downstream target of RhoA, cofilin-2, that was mimicked by direct inhibition of RhoA and ROCK. In addition, confocal microscopy and Western blotting revealed that 24 h ET-1 treatment reduced the G- to F-actin ratio 67% (p<0.05) which was prevented by SNAP, cGMP, C3 and Y (p<0.05). Taken together, these results suggest that the anti-hypertrophic effects of NO are due, in part, to cGMP-dependent inhibition of the RhoA-ROCK-cofilin signalling pathway. These findings may be important in understanding the mechanisms of anti-ET-1 and anti-hypertrophic effects of NO as well as in the development of novel RhoA-targeted therapeutic interventions for treating cardiac hypertrophy.

Local delivery of PKCepsilon-activating peptide mimics ischemic preconditioning in aged hearts through GSK-3beta but not F1-ATPase inactivation.

In adult heart, selective PKCepsilon activation limits ischemia (I)-reperfusion (R) damage and mimics the protection associated with ischemic preconditioning. We sought to determine whether local delivery of PKCepsilon activator peptide psiepsilon-receptor for activated C-kinase (psiepsilon-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 psiepsilon-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 PKCepsilon, PKCdelta, phospho (p)-GSK-3beta (Ser(9)) and GSK-3beta in hearts snap-frozen during I. Recovery of left ventricular developed pressure was significantly improved by psiepsilon-RACK (P < 0.01) and infarct size reduced in 24-mo rats vs. age-matched controls (60% vs. 34%; P < 0.01). Mitochondrial PKCepsilon levels were 30% greater during I with psiepsilon-RACK in aged vs. control rats (P < 0.01). Interestingly, mitochondrial GSK-3beta levels were threefold greater in aged vs. adult rats during I, and psiepsilon-RACK prevented this increase (P < 0.01). Mitochondrial p-GSK-3beta levels were also greater in aged rats after psiepsilon-RACK (P < 0.01), and subsequent inhibition of GSK-3beta with SB-216763 (3 muM) before I/R elicited protection similar to that of psiepsilon-RACK (n = 3/group). Mitochondrial proteomic analysis further identified group differences in the F(1)-ATPase beta-subunit, and coimmunoprecipitation studies revealed a novel interaction with PKCepsilon. F(1)-ATPase-PKCepsilon association was affected by psiepsilon-RACK in adult but not aged rats. Our results provide evidence, for the first time, for PKCepsilon-mediated protection in aged rat heart after I/R and suggest a central role for mitochondrial GSK-3beta but not F(1)-ATPase as a potential target of PKCepsilon to limit I/R damage with aging.

Acute PKCdelta inhibition limits ischaemia-reperfusion injury in the aged rat heart: role of GSK-3beta.

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 Cdelta (PKCdelta) 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 PKCdelta signaling mechanisms to reperfusion injury in the aged heart using local delivery of a novel PKCdelta 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 PKCdelta levels during reperfusion in aged vs. age-matched controls (p<0.01). Further, increases in mitochondrial phosphorylated glycogen synthase kinase-3beta (pGSK-3beta) levels were hastened in aged and adult hearts following KID1-1 (p<0.05), increasing the pGSK-3beta/GSK-3beta ratio. CONCLUSIONS: 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.

Age- and sex-dependent alterations in protein kinase C (PKC) and extracellular regulated kinase 1/2 (ERK1/2) in rat myocardium.

Cardiovascular morbidity and mortality increase significantly with advancing age, with proportionally higher rates occurring in aged women when compared to aged men. The signaling alterations responsible for age-related reductions in ischemic stress reserves, particularly in aged women, are poorly understood. Accordingly, we sought to determine whether alterations in the cellular location and formation of specific protein kinase C (PKC)-extracellular regulated 1/2 (ERK1/2) signaling modules (SMS) might provide insight into known age- and sex-related differences in cardiovascular disease outcomes. Cytosolic (Cyto), mitochondrial (Mito) and nuclear (Nuc) fractions were isolated from left ventricles of male (M) and female (F) adult (6 mo), castrated or aged (23 mo) F344 rats by centrifugation. Western blotting was used to assess PKC (alpha, delta, epsilon), p-ERK1/2 and p-Bad(Ser112) levels, and immunoprecipitation to assess PKC-ERK1/2 SMS. Cyto-PKCalpha levels increased with age (p<0.0001), whereas increases in cyto-PKCalpha-ERK1/2 SMS were only observed in aged F (60%; p<0.01). Mito-PKCdelta and Mito-PKCdelta-ERK1/2 SMS increased in M and F with age (p<0.0001); however increases in Cyto-PKCdelta were only observed in aged M (80% p<0.0001). It is important to note that Nuc- and Mito-PKCdelta-ERK1/2 SMS were 3.5- and 4.8-fold greater in males versus females, respectively (p<0001). Increases in Mito-PKCepsilon-ERK1/2 SMS (216%) were also specific to aged M (p<0.0001), however, Mito-p-Bad(Ser112) levels were decreased with age in both M and F. Differences in sex hormone status could not fully account for observed age-related differences in PKC. Collectively, our results provide novel evidence for age and sex-related differences in the magnitude and distribution of cardiac PKC-ERK1/2 SMS consistent with previously described pathological and protective phenotypes, respectively.

Protein kinase C distribution and translocation in rat myocardium: Methodological considerations.

INTRODUCTION: Protein kinase C (PKC) is an important modifier of several cardiovascular phenomena, including cardioprotection, apoptosis, and hypertrophy. Although pharmacological activation of PKC is often assessed by translocation, the effects of isolation procedures on left ventricular (LV) PKC distribution have not been systematically examined. Accordingly, we sought to determine whether homogenization methods (Polytron, glass-glass tissue grinder), detergent selection and concentration, or centrifugation protocols affect PKC (alpha, epsilon) distribution or phorbol-12-myristate-13-acetate (PMA)-induced translocation. METHODS: Hearts of male F344 or Wistar rats were Langendorff perfused with either 100 nM PMA or vehicle, and LV cytosolic and particulate PKC (alpha, epsilon) distributions were assessed by differential centrifugation and Western blotting. RESULTS: Following 100000 xg centrifugation of the homogenate, resuspension of the pellet (P(1)) in 0.1% sodium dodecyl sulfate (SDS) increased electrophoretic mobility of PKC (alpha, epsilon) such that PKCepsilon comigrated with a nonspecific band. Resuspension of P(1) in Triton X-100 (TX) did not affect mobility but decreased P(1) PKC (alpha, epsilon) levels in a TX-concentration-dependent manner; however, this decrease was found to be due to differential protein solubilization. Decreased levels of PKC (alpha, epsilon) were also noted in soluble and P(2) (supernatant of 100000 xg centrifugation of P(1)) fractions due to increased Polytron burst and total homogenization times. Interestingly, the P(2) fraction also revealed Polytron-dependent decreases (47% vs. glass-glass tissue grinder; p<0.05) in PKCepsilon following an initial 1000 xg centrifugation and an increased PMA-dependent translocation of PKC (alpha, epsilon; 2.4-fold and 1.6-fold, respectively, vs. P(1); p<0.05). DISCUSSION: Taken together, these results suggest that PKC isolation procedures should be carefully considered when designing or comparing LV PKC studies due to the potential effects isolation may have on PKC distribution and translocation.