Inhibition of programmed necrosis limits infarct size through altered mitochondrial and immune responses in the aged female rat heart.

Both advancing age and estrogen loss exacerbate acute myocardial infarction in the female heart. However, the mechanistic underpinnings of age-related differences in cell death after ischemia-reperfusion (I/R) injury in female subjects and reductions in cardioprotective reserve capacity remain largely unexplored. The aim of the present study was to determine the efficacy of programmed necrosis inhibition on infarct size reduction and preservation of left ventricular (LV) function after I/R injury with female aging. Fischer 344 rats were ovariectomized (OVX) at 15 mo and studied at 24 mo (MO OVX) versus adult rats with intact ovaries (6 mo). After in vivo coronary artery ligation (55-min ischemia and 2- or 6-h reperfusion), necrostatin-1 (Nec-1; 3.5 or 5.7 mg/kg) delivered upon reperfusion significantly reduced infarct size by 37% and improved LV function in the MO OVX group ( P < 0.01). Although age-associated elevations in cyclophilin D and mitochondrial acetylation ( P < 0.001) were unaffected by Nec-1, profound reductions in IL-1, IL-6, and TNF-α ( P < 0.05) as well as cardiac immune cell infiltration were observed in MO OVX but not adult rats. We conclude that chronic inflammation and postmenopausal estrogen deficiency conspire to exacerbate acute infarction through a mechanism involving exaggerated mitochondria-mediated programmed necrosis through receptor-interacting protein 1 signaling. Modulatory effects of programmed necrosis inhibition on proinflammatory cytokine production after I/R reveal a potentially important mechanistic target to restore and preserve cardiac function in the OVX aged female heart. NEW & NOTEWORTHY Myocardial infarct size reduction by inhibition of programmed necrosis in aged female subjects suggests a dominant cell death pathway. Alterations in mitochondrial protein levels and acetylation underscore a mitochondria-dependent mechanism, whereas the profound cytokine reduction in aged subjects alone points to a divergent role for immune modulation of programmed necrosis and viable therapeutic target.

Age and ischemia differentially impact mitochondrial ultrastructure and function in a novel model of age-associated estrogen deficiency in the female rat heart.

Altered mitochondrial respiration, morphology, and quality control collectively contribute to mitochondrial dysfunction in the aged heart. Because myocardial infarction remains the leading cause of death in aged women, the present study utilized a novel rodent model to recapitulate human menopause to interrogate the combination of age and estrogen deficiency on mitochondrial ultrastructure and function with cardiac ischemia/reperfusion (I/R) injury. Female F344 rats were ovariectomized (OVX) at 15 months and studied at 24 months (MO OVX; n = 40) vs adult ovary intact (6 months; n = 41). Temporal declines in estrogen concomitant with increased visceral adipose tissue were observed in MO OVX vs adult. Following in vivo coronary artery ligation or sham surgery, state 3 mitochondrial respiration was selectively reduced by age in subsarcolemmal mitochondria (SSM) and by I/R in interfibrillar mitochondria (IFM); left ventricular maximum dP/dt was reduced in MO OVX (p < 0.05). Elevated cyclophilin D and exacerbated I/R-induced mitochondrial acetylation in MO OVX suggest permeability transition pore involvement and reduced protection vs adult (p < 0.05). Mitochondrial morphology by TEM revealed an altered time course of autophagy coordinate with attenuated Drp1 and LC3BII protein levels with age-associated estrogen loss (p < 0.05). Here, reductions in both SSM and IFM function may play an additive role in enhanced susceptibility to regional I/R injury in aged estrogen-deficient female hearts. Moreover, novel insight into altered cardiac mitochondrial quality control garnered here begins to unravel the potentially important regulatory role of mitochondrial dynamics on sustaining respiratory function in the aged female heart.

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.

Chronic alcohol consumption disrupts myocardial protein balance and function in aged, but not adult, female F344 rats.

The purpose of this study was to assess whether the deleterious effect of chronic alcohol consumption differs in adult and aged female rats. To address this aim, adult (4 mo) and aged (18 mo) F344 rats were fed a nutritionally complete liquid diet containing alcohol (36% total calories) or an isocaloric isonitrogenous control diet for 20 wk. Cardiac structure and function, assessed by echocardiography, as well as myocardial protein synthesis and proteolysis did not differ in either alcohol- versus control-fed adult rats or in adult versus aged control-fed rats. In contrast, cardiac function was impaired in alcohol-fed aged rats compared with age-matched control rats. Additionally, alcohol feeding decreased cardiac protein synthesis that was associated with decreased phosphorylation of 4E-BP1 and S6K1. This reduction in mammalian target of rapamycin (mTOR) kinase activity was associated with reduced eIF3f and binding of both Raptor and eIF4G to eIF3. Proteasome activity was increased in alcohol-fed aged rats with a coordinate elevation in the E3 ligases atrogin-1 and muscle RING-finger protein-1 (MuRF1). These changes were associated with increased regulated in development and DNA damage response 1 (REDD1) and phosphorylation of AMP-activated protein kinase (AMPK) but no increase in AKT or forkhead transcription factor (FOXO)3 phosphorylation. Finally, markers of autophagy (e.g., LC3B, Atg7, Atg12) and TNF-α were increased to a greater extent in alcohol-fed aged rats. These data demonstrate that aged female rats exhibit an enhanced sensitivity to alcohol compared with adult animals. Our data are consistent with a model whereby alcohol increases proteolysis via FOXO-independent increase in atrogin-1, which degrades eIF3f and therefore impairs formation of a functional preinitiation complex and protein synthesis.

Age-related differences in cardiac ischemia-reperfusion injury: effects of estrogen deficiency.

Despite conflicting evidence for the efficacy of hormone replacement therapy in cardioprotection of postmenopausal women, numerous studies have demonstrated reductions in ischemia/reperfusion (I/R) injury following chronic or acute exogenous estradiol (E2) administration in adult male and female, gonad-intact and gonadectomized animals. It has become clear that ovariectomized adult animals may not accurately represent the combined effects of age and E2 deficiency on reductions in ischemic tolerance seen in the postmenopausal female. E2 is known to regulate the transcription of several cardioprotective genes. Acute, non-genomic E2 signaling can also activate many of the same signaling pathways recruited in cardioprotection. Alterations in cardioprotective gene expression or cardioprotective signal transduction are therefore likely to result within the context of aging and E2 deficiency and may help explain the reduced ischemic tolerance and loss of cardioprotection in the senescent female heart. Quantification of the mitochondrial proteome as it adapts to advancing age and E2 deficiency may also represent a key experimental approach to uncover proteins associated with disruptions in cardiac signaling contributing to age-associated declines in ischemic tolerance. These alterations have important ramifications for understanding the increased morbidity and mortality due to ischemic cardiovascular disease seen in postmenopausal females. Functional perturbations that occur in mitochondrial respiration and Ca(2+) sensitivity with age-associated E2 deficiency may also allow for the identification of alternative therapeutic targets for reducing I/R injury and treatment of the leading cause of death in postmenopausal women.

Divergent effects of aging and sex on vasoconstriction to endothelin in coronary arterioles.

OBJECTIVE: The risk for cardiovascular disease increases with advancing age; however, the chronological development of heart disease differs in males and females. The purpose of this study was to determine whether age-induced alterations in responses of coronary arterioles to the endogenous vasoconstrictor, endothelin, are sex-specific. METHODS: Coronary arterioles were isolated from young and old male and female rats to assess vasoconstrictor responses to endothelin (ET), and ETa and ETb receptor inhibitors were used to assess receptor-specific signaling. RESULTS: In intact arterioles from males, ET-induced vasoconstriction was reduced with age, whereas age increased vasoconstrictor responses to ET in intact arterioles from female rats. In intact arterioles from both sexes, blockade of either ETa or ETb eliminated age-related differences in responses to ET; however, denudation of arterioles from both sexes revealed age-related differences in ETa-mediated vasoconstriction. In arterioles from male rats, ETa receptor protein decreased, whereas ETb receptor protein increased with age. In coronary arterioles from females, neither ETa nor ETb receptor protein changed with age, suggesting age-related changes in ET signaling occur downstream of ET receptors. CONCLUSIONS: Thus, aging-induced alterations in responsiveness of the coronary resistance vasculature to endothelin are sex-specific, possibly contributing to sexual dimorphism in the risk of cardiovascular disease with advancing age.

Aging accentuates alcohol-induced decrease in protein synthesis in gastrocnemius.

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.

From syncitium to regulated pump: a cardiac muscle cellular update.

The primary purpose of this article is to present a basic overview of some key teaching concepts that should be considered for inclusion in an six- to eight-lecture introductory block on the regulation of cardiac performance for graduate students. Within the context of cardiac excitation-contraction coupling, this review incorporates information on Ca(2+) microdomains and local control theory, with particular emphasis on the role of Ca(2+) sparks as a key regulatory component of ventricular myocyte contraction dynamics. Recent information pertaining to local Ca(2+) cycling in sinoatrial nodal cells (SANCs) as a mechanism underlying cardiac automaticity is also presented as part of the recently described coupled-clock pacemaker system. The details of this regulation are emerging; however, the notion that the sequestration and release of Ca(2+) from internal stores in SANCs (similar to that observed in ventricular myocytes) regulates the rhythmic excitation of the heart (i.e., membrane ion channels) is an important advancement in this area. The regulatory role of cardiac adrenergic receptors on cardiac rate and function is also included, and fundamental concepts related to intracellular signaling are discussed. An important point of emphasis is that whole organ cardiac dynamics can be traced back to cellular events regulating intracellular Ca(2+) homeostasis and, as such, provides an important conceptual framework from which students can begin to think about whole organ physiology in health and disease. Greater synchrony of Ca(2+)-regulatory mechanisms between ventricular and pacemaker cells should enhance student comprehension of complex regulatory phenomenon in cardiac muscle.

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.

Exaggerated coronary vasoreactivity to endothelin-1 in aged rats: role of protein kinase C.

OBJECTIVE: The interaction between advanced age and increased susceptibility to ischemic insult is well documented. Age-related increases in coronary vascular resistance, in part due to impaired dilator responses, have been reported. Our aim was to determine the role of endothelin-1 (ET-1) on enhanced constrictor responses in aged coronary arteries (CAs) and whether protein kinase C (PKC) signaling mechanisms impact ET-1 responses. METHODS: Vasoreactivity was assessed in CAs isolated from aged (24 months; n=16) and adult (4 months; n=21) male F344 rats following ET-1 (10(-10)-10(-8)) with and without specific ETA/ETB receptor antagonists (BQ-123, 1 microM; BQ-788, 30 nM) or the PKC inhibitor bisindolylmaleimide (Bis; 10(-6) M). Constrictor responses to KCl (80 mM) were also measured and voltage-gated Ca2+ channel (VGCC) determined in isolated coronary smooth muscle cells. Dilator responses to acetylcholine (ACH) and sodium nitroprusside (SNP) were assessed. RESULTS: Passive diameter was greater (357+/-19 vs. 309+/-9; p<0.02) while spontaneous tone was similar in 24 months vs. 4 months. ET-1 resulted in greater constriction in 24 months vs. 4 months (79% vs. 67%; p<0.01). Group differences persisted following selective ETB inhibition with BQ-788 (p<0.02), while BQ-123 abolished contractile responses to ET-1. Importantly, inhibition of ET-1 constriction by Bis occurred in 24 months but not 4 months (p<0.01). Constrictor responses to KCl and peak VGCC current density were similar in 24 months vs. 4 months (48% vs. 50%). No age-related differences were observed in ACH- or SNP-mediated dilation. Western blotting revealed increases in Ca2+-sensitive PKCalpha, -betaI, and -betaII levels with age, while eNOS and ETA receptor protein levels were unchanged. CONCLUSION: Aberrant ETA constrictor responses and directional changes in PKC are likely to contribute to coronary vascular pathology with advanced age.

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.

Exercise and hypercholesterolemia produce disparate shifts in coronary PKC expression.

INTRODUCTION: Sedentary lifestyle and high-fat, high-cholesterol diets are each associated with elevated risk for coronary heart disease (CHD); however, the mechanisms by which they increase risk are unclear. Specific PKC isoforms have been implicated in the development of CHD, regulation of coronary vasoreactivity, as well as exercise-induced cardioprotection. Thus, diet and physical inactivity may increase CHD risk by altering coronary protein kinase C (PKC) isoform profiles. PURPOSE: To determine whether coronary PKC isoform profiles are altered in a model of early CHD and whether exercise can prevent these changes. METHODS: Male and female Yucatan miniature swine were either fed a normal (NF) or high-fat (HF) diet (8 vs 46% kilocalories from fat) and remained sedentary (Sed) or were treadmill-trained (Ex) at 75% of; VO2max (6 mph, 60 min) for 16 wk. Groups were as follows: NFSed (N=8/N=7), NFEx (N=8/N=7), HFSed (N=8/N=7), and HFEx (N=8/N=7). Western blotting was performed on right coronary conduit artery (CCA) segments (>1 mm I.D.) to measure total protein levels of PKC-alpha, -betaI, -betaII, -delta, -epsilon, and -zeta. RESULTS: HF diet increased total cholesterol by more than sixfold with no increase in triglycerides. Hypercholesterolemia increased PKC-betaII and -epsilon protein levels in CCA of both male and female pig; Ex had no effect on this response. Ex-induced increases in PKC-betaI, PKC-delta, and PKC-zeta were observed in HF male pigs. Female pigs had higher baseline amounts of PKC-alpha (25%), PKC-betaI (33%), PKC-betaII (39%), and PKC-epsilon (29%), whereas male pigs had higher amounts of PKC-delta (308%). Further analyses revealed a direct relationship between androgens and PKC-delta levels. CONCLUSION: Hypercholesterolemia and exercise exert disparate effects on coronary PKC expression. Observed sex differences in PKC protein profiles may also contribute to altered cardiovascular risk patterns in males versus females.

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.

Chronic exercise improves myocardial inotropic reserve capacity through alpha1-adrenergic and protein kinase C-dependent effects in Senescent rats.

We have previously demonstrated that alpha(1)-adrenergic (AR)-mediated contraction is diminished in the senescent rat heart, in part due to alterations in protein kinase C (PKC) signaling. Since chronic exercise training (EX) can exert independent effects on increasing alpha(1)-AR contraction in the adult rat heart, we sought to determine whether age-related defects in alpha(1)-AR contraction could be reversed by chronic EX. We further hypothesized that improved alpha(1)-AR contraction by EX may be PKC dependent. Adult (4 months; Y) and aged (24 months; O) male F344 rats were treadmill-trained (n = 12-13/group; TR) at approximately 70% of VO(2max) for 12 weeks or remained sedentary (YSED, YTR, OSED, OTR). Training status was verified by plantaris citrate synthase activity and left ventricular (LV) contractile responses (dP/dt) to alpha(1)-AR stimulation were assessed in Langendorff-perfused hearts using the alpha(1)-AR agonist phenylephrine (PE; 10(-5) M) with and without the PKC inhibitor chelerythrine (CE; 10(-6) M). alpha(1)-AR stimulation elicited greater increases in LV dP/dt in hearts isolated from OTR (4525.4 +/- 224.1 mmHg/s) versus OSED (3658.9 +/- 291.0 mmHg/s), while CE abolished PE-induced effects (OTR, 4069.2 +/- 341.2) versus (OSED, 3608.9 +/- 321.2) (p < .01). Upon western blotting, phosphospecific antibodies directed at PKCepsilon (pSer(729)) revealed greater levels in LV isolated from YTR versus YSED, and EX ameliorated aged-related reductions in OSED (p < .001). Basal PKCepsilon mRNA levels were also greater in YTR and OTR versus YSED (p < .01). PE-induced increases in phosphor-PKCdelta (pThr(507)) levels observed in OSED were attenuated in OTR (p < .03). Chronic EX was also associated with significant reductions in PKCalpha (pSer(657)) levels following PE in OTR (p < .002). The results indicate that age-related reductions in alpha(1)-AR contraction can be partially reversed by EX in the rat heart. These results further suggest that alterations in PKC levels underlie, at least in part, EX-induced improvements in alpha(1)-AR contraction.

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.

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.

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.

Increased estrogen receptor ß in adipose tissue is associated with increased intracellular and reduced circulating adiponectin protein levels in aged female rats.

BACKGROUND: Obesity and associated metabolic and cardiovascular disease risk are correlated with reduced circulating adiponectin (APN) levels. Metabolic and cardiovascular disease risk is also increased after menopause and may be linked to disturbances in estrogen receptor (ER) signaling in adipose. OBJECTIVE: We hypothesized that age-associated estrogen (E(2))-deficiency alters the ERα/ß ratio in adipose tissue and increases risk for metabolic disease via APN-ac activated mechanisms. METHODS: Visceral adipose was isolated from adult (6 months) and aged (24 months) female Fisher 344 rats (n = 5-6/group) with ovaries intact or removed by surgical ovariectomy (OVX) and subjected to western blotting. RESULTS: Notably, weight was greatest in aged OVX rats (P < 0.01) and associated with a 2-fold increase in ERß protein versus adult intact rats (P < 0.001). ER levels were increased in aged OVX versus adult OVX rats. Intra-adipocyte APN was also increased in aged OVX rats versus all groups (P < 0.01), whereas circulating APN levels decreased in aged OVX versus adult OVX rats (P < 0.05). Endoplasmic reticulum protein of 44 kDa (Erp44) levels remained the same (P = 0.09). Adiponectin receptor-1 (AdipoR1) and peroxisome proliferator-activated receptor-α (PPAR-α) were also unchanged. AdipoR2, PPAR-γ, and phosphorylated adenosine monophosphate-dependant kinase (pAMPK) to total AMPK ratio all decreased with age (P < 0.05). CONCLUSIONS: Collectively, these data suggested that age-associated increases in ERß paired with decreased PPAR-γ levels might predispose E(2)-deficient postmenopausal women for increased adiposity and associated metabolic and cardiovascular disease risk. Reduced circulating APN and AdipoR2 levels might contribute to age and E(2)-deficiency linked disease progression.