Delivery may affect arterial elasticity in women.

BACKGROUND: Estrogen is considered to be cardioprotective, but estrogen replacement therapy for postmenopausal women has not shown results for either primary or secondary cardiovascular event prevention. During normal pregnancy, women have significantly higher levels of estrogen and it may be endogenous estrogen that helps prevent atherosclerosis. METHODS AND RESULTS: The present cross-sectional study examined the association between pregnancy followed by delivery and clinical atherosclerosis using the brachial-ankle pulse wave velocity (PWV). A total of 2,560 women undergoing annual health screening at the Institute of Hyogo Prefecture Health Promotion Association in Japan were recruited. Pregnancy history (the age of menarche/menopause and the number of gravida/para), conventional coronary risk factors, and brachial-ankle PWV were recorded. Multivariate linear regression by stepwise selection analysis demonstrated that women who had 1 or more deliveries had a significantly lower PWV, independent of age and other conventional coronary risk factors. CONCLUSIONS: Pregnancy followed by delivery may decrease arterial stiffness and prevent the progress of atherosclerosis in women. The contribution of such a pregnancy followed by delivery-related decrease in arterial stiffness to the reduction of cardiovascular disease in women should be further evaluated.

Darbepoetin alfa exerts a cardioprotective effect in autoimmune cardiomyopathy via reduction of ER stress and activation of the PI3K/Akt and STAT3 pathways.

Dilated human cardiomyopathy is associated with suppression of the prosurvival phosphatidylinositol-3-kinase (PI3K)/Akt and STAT3 pathways. The present study was carried out to determine if restoration of the PI3K/Akt and STAT3 activity by darbepoetin alfa improved cardiac function or reduced cardiomyocyte apoptosis in rabbit autoimmune cardiomyopathy induced by a peptide corresponding to the second extracellular loop of the ss(1)-adrenergic receptor (ss(1)-EC(II)). We found that ss(1)-EC(II) immunization produced progressive LV dilation, systolic dysfunction and myocyte apoptosis as measured by TUNEL, single-stranded DNA antibody, and active caspase-3. These changes were associated with activation of p38 mitogen-activated protein kinase (MAPK), endoplasmic reticulum stress markers (GRP78 and CHOP), and increased cleavage of procaspase-12, as well as decreased phosphorylation of Akt and STAT3, and decreased Bcl2/Bax ratio. As expected, darbepoetin alfa treatment increased phosphorylation of Akt and STAT3. It also increased the myocardial expression of erythropoietin receptor which was reduced in the failing myocardium, and improved cardiac function in the ss(1)-EC(II)-immunized animals. The latter was associated with reductions of myocyte apoptosis and cleaved caspase-3, as well as reversal of increased phosphorylation of p38-MAPK, increased ER stress, and decline in Bcl2/Bax ratio. The anti-apoptotic effects of darbepoetin alfa via Akt and STAT activation were also demonstrated in cultured cardiomyocytes treated with the anti-ss(1)-EC(II) antibody. These effects of darbepoetin alfa in vitro were prevented by LY294002 and STAT3 peptide inhibitor. Thus, we conclude that darbepoetin alfa improves cardiac function and prevents progression of dilated cardiomyopathy probably by activating the PI3K/Akt and STAT3 pathways and reducing ER stress.

Adoptive passive transfer of rabbit beta1-adrenoceptor peptide immune cardiomyopathy into the Rag2-/- mouse: participation of the ER stress.

Auto-antibodies against the beta(1)-adrenoceptors are present in 30-40% of patients with dilated cardiomyopathy. Recently, a synthetic peptide corresponding to a sequence of the second extracellular loop of the human beta(1)-adrenoceptor (beta(1)-EC(II)) has been shown to produce endoplasmic reticulum (ER) stress, myocyte apoptosis and cardiomyopathy in immunized rabbits. To study the direct cardiac effects of anti-beta(1)-EC(II) antibody in intact animals and if they are mediated via beta(1)-adrenoceptor stimulation, we administered IgG purified from beta(1)-EC(II)-immunized rabbits to recombination activating gene 2 knock-out (Rag2(-/-)) mice every 2 weeks with and without metoprolol treatment. Serial echocardiography and cardiac catheterization showed that beta(1)-EC(II) IgG reduced cardiac systolic function after 3 months. This was associated with increase in heart weight, myocyte apoptosis, activation of caspase-3, -9 and -12, and increased ER stress as evidenced by upregulation of GRP78 and CHOP and cleavage of ATF6. The Rag2(-/-) mice also exhibited increased phosphorylation of CaMKII and p38 MAPK. Metoprolol administration, which attenuated the phosphorylation of CaMKII and p38 MAPK, reduced the ER stress, caspase activation and cell death. Finally, we employed the small-interfering RNA technology to reduce caspase-12 in cultured rat cardiomyocytes. This reduced not only the increase of cleaved caspase-12 but also of the number of myocyte apoptosis produced by beta(1)-EC(II) IgG. Thus, we conclude that ER stress plays an important role in cell death and cardiac dysfunction in beta(1)-EC(II) IgG cardiomyopathy, and the effects of beta(1)-EC(II) IgG are mediated via the beta(1)-adrenergic receptor.

Cardiomyocyte apoptosis in autoimmune cardiomyopathy: mediated via endoplasmic reticulum stress and exaggerated by norepinephrine.

Evidence suggests that the autoimmune cardiomyopathy produced by a peptide corresponding to the sequence of the second extracellular loop of the beta(1)-adrenergic receptor (beta(1)-EC(II)) is mediated via a biologically active anti-beta(1)-EC(II) antibody, but the mechanism linking the antibody to myocyte apoptosis and cardiac dysfunction has not been well elucidated. Since the beta(1)-EC(II) autoantibody is a partial beta(1)-agonist, we speculate that the cardiomyopathy is produced by the beta(1)-receptor-mediated stimulation of the CaMKII-p38 MAPK-ATF6 signaling pathway and endoplasmic reticulum (ER) stress, and that excess norepinephrine (NE) exaggerates the cardiomyopathy. Rabbits were randomized to receive beta(1)-EC(II) immunization, sham immunization, NE pellet, or beta(1)-EC(II) immunization plus NE pellet for 6 mo. Heart function was measured by echocardiography and catheterization. Myocyte apoptosis was determined by terminal deoxytransferase-mediated dUTP nick-end labeling and caspase-3 activity, whereas CaMKII, MAPK family (JNK, p38, ERK), and ER stress signals (ATF6, GRP78, CHOP, caspase-12) were measured by Western blot, immunohistochemistry, and kinase activity assay. beta(1)-EC(II) immunization produced progressive LV dilation, systolic dysfunction, and myocyte apoptosis. These changes were associated with activation of GRP78 and CHOP and increased cleavage of caspase-12, as well as increased CaMKII activity, increased phosphorylation of p38 MAPK, and nucleus translocation of cleaved ATF6. NE pellet produced additive effects. In addition, KN-93 and SB 203580 abolished the induction of ER stress and cell apoptosis produced by the beta(1)-EC(II) antibody in cultured neonatal cardiomyocytes. Thus ER stress occurs in autoimmune cardiomyopathy induced by beta(1)-EC(II) peptide, and this is enhanced by increased NE and caused by activation of the beta(1)-adrenergic receptor-coupled CaMKII, p38 MAPK, and ATF6 pathway.

Endothelial lipase gene polymorphism is associated with acute myocardial infarction, independently of high-density lipoprotein-cholesterol levels.

BACKGROUND: Endothelial lipase (EL) is a major determinant of high-density lipoprotein-cholesterol (HDL-C) metabolism and promotes monocytes recruitment. The local expression of EL could influence atherogenesis directly, in addition to its systemic role in HDL metabolism. The EL gene has a common 584C/T polymorphism, but it is unclear whether this polymorphism is associated with HDL-C levels or acute myocardial infarction (AMI). METHODS AND RESULTS: A case-control study of 107 AMI patients and 107 control subjects was conducted. T allele frequency was lower in the AMI group than in controls (0.18 vs 0.26, p<0.05). No significant association was found between the 584C/T polymorphism and HDL-C levels. Multivariate regression analyses showed that the association of the T allele with AMI was statistically significant and independent of other risk factors when age, sex, hypertension, hypercholesterolemia, and diabetes mellitus were included in the analyses (odds ratio (OR), 0.52; 95% confidence interval (95% CI) 0.28-0.98; p=0.04). However, when smoking status was included, the association of the T allele with AMI did not remain statistically significant (OR, 0.61; 95% CI 0.32-1.18; p=0.14). CONCLUSIONS: The 584C/T polymorphism of the EL gene was associated with AMI independently of HDL-C levels and thus may be involved in the pathogenesis of AMI.

Cardiac sympathetic neuroprotective effect of desipramine in tachycardia-induced cardiomyopathy.

Cardiac sympathetic transmitter stores are reduced in the failing heart. In this study, we proposed to investigate whether the reduction of cardiac sympathetic neurotransmitters was associated with increased interstitial norepinephrine (NE) and reactive oxygen species in congestive heart failure (CHF), using a microdialysis technique and salicylate to detect .OH generation. Rabbits with and without rapid ventricular pacing (340 beats/min) were randomized to receive desipramine (10 mg/day) or placebo for 8 wk. Rapid pacing produced left ventricular dilation and systolic dysfunction. The failing myocardium also showed reduced tissue contents of NE and tyrosine hydroxylase protein and activity. In contrast, myocardial interstitial NE was increased in CHF (0.89 +/- 0.11 ng/ml) compared with the sham-operated animals (0.26 +/- 0.03 ng/ml). In addition, cardiac oxidative stress was increased in CHF animals as measured by myocardial interstitial .OH radical, tissue oxidized glutathione, and oxidized mitochondrial DNA. Desipramine treatment produced significant NE uptake inhibition as evidence by an exaggerated pressor response and a greater increase of myocardial interstitial NE in response to intravenous NE infusion but no significant effects on cardiac function or hemodynamics in sham-operated or CHF animals. However, desipramine treatment attenuated the reductions of tissue NE and tyrosine hydroxylase protein and activity in CHF. Desipramine also prevented the reduction of tyrosine hydroxylase produced by NE in PC12 cells. Thus the reduction of cardiac sympathetic neurotransmitters is related to the increased interstitial NE and tissue oxidative stress in CHF. Also, normal neuronal uptake of NE is required for NE or its oxidized metabolites to exert their neurotoxic effects.

Norepinephrine-induced oxidative stress causes PC-12 cell apoptosis by both endoplasmic reticulum stress and mitochondrial intrinsic pathway: inhibition of phosphatidylinositol 3-kinase survival pathway.

Norepinephrine (NE) induces endoplasmic reticulum (ER) unfolded protein response and reduces maturation and translocation of NE transporter to cell membrane via enhanced formation of reactive oxygen species in PC-12 cells. In the present study, we investigated whether ER stress is also implicated in the proapoptotic effect of NE. We found that the apoptotic effect of NE was associated with increased processing of ER-resident pro-caspase-12, cleavage of caspase-9 and -3, and mitochondrial release of cytochrome c. ER stress was evidenced by upregulation of ER chaperone GRP78 and transcription factor CHOP and the translocation of XBP-1 from the ER to the nucleus by NE. NE also reduced phospho-Akt (Ser473), indicating suppression of the phosphatidylinositol 3-kinase (PI3-kinase)/Akt survival pathway. Similar results were produced by thapsigargin. NGF, which promotes the PI3-kinase/Akt activity, reduced the effects of NE and thapsigargin on apoptosis and activation of caspase-12 and -3. However, the effects of NE, but not of thapsigargin, were abolished by pretreatment with SOD and catalase. In contrast, the PI3-kinase inhibitors LY-294002 and wortmannin abolished the protective effects of both SOD/catalase and NGF on NE-induced apoptosis. The functional importance of caspase-12 activation was supported by the use of Z-ATAD-FMK, which reduced the NE-induced processing of caspase-12 and cell apoptosis, but the caspase-12, -9, and -3 inhibitors had no effects on the increase in cytosolic cytochrome c produced by NE. In contrast, the release of mitochondrial cytochrome c was abolished by SOD/catalase and NGF. These results indicate that NE induced cell apoptosis by both ER stress and a mitochondrial death pathway and that the effects of NE were mediated via oxidative stress and inhibition of the PI3-kinase/Akt survival pathway.

Norepinephrine induces endoplasmic reticulum stress and downregulation of norepinephrine transporter density in PC12 cells via oxidative stress.

Cardiac norepinephrine (NE) uptake is reduced in cardiomyopathy. This change is associated with a decrease of NE transporter (NET) receptor and can be reproduced in PC12 cells by extracellular NE. To study whether this effect of NE is mediated via impaired glycosylation and trafficking of NET in the endoplasmic reticulum (ER), we measured the distribution of glycosylated 80-kDa NET and unglycosylated 46-kDa NET in the membrane and cytosolic fractions of PC12 cells. We found that NE decreased glycosylated NET in both membrane and cytosolic fractions and increased cytosolic unglycosylated NET protein. Similar results were produced by tunicamycin and thapsigargin, two agents that induce ER stress by inhibiting N-glycosylation of membrane proteins and disrupting calcium homeostasis, respectively. Also, like the ER stressors, NE not only increased phosphorylation of both the alpha-subunit of eukaryotic initiation factor-2 and its upstream RNA-dependent protein kinase-like ER kinase over 12 h of treatment but also increased ER chaperone molecule glucose-regulated protein 78 and the nuclear transcription factor C/EBP homologous protein. Antioxidants superoxide dismutase and catalase prevented the downregulation of NET proteins and induction of ER stress signals produced by NE but not by tunicamycin or thapsigargin. The results indicate that the downregulation of membrane NET by NE is mediated by decreased N-glycosylation of NET proteins secondary to induction of ER stress pathways by NE-derived oxidative metabolites. Interventions involving the ER stress pathways may provide novel therapeutic strategies for the treatment of sympathetic dysfunction in heart failure.

Extracellular norepinephrine reduces neuronal uptake of norepinephrine by oxidative stress in PC12 cells.

Cardiac norepinephrine (NE) uptake activity is reduced in congestive heart failure. Our studies in intact animals suggest that this effect on the cardiac sympathetic nerve endings is caused by oxidative stress and/or NE toxic metabolites derived from NE. In this study, we investigated the direct effects of NE on neuronal NE uptake activity and NE transporter (NET), using undifferentiated PC12 cells. Cells were incubated with NE (1-500 microM) either alone or in combination of Cu(2+) sulfate (1 microM), which promotes free radical formation by Fenton reaction for 24 h. NE uptake activity was measured using [(3)H]NE. Cell viability was determined with the use of Trypan blue exclusion and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay, and cellular oxidative stress by dichlorodihydrofluorescein fluorescence and the GSH/GSSG ratio. Cell viability was reduced by NE >100 microM. At lower doses, NE produced oxidative stress and a dose-dependent reduction of NE uptake activity without affecting cell viability significantly. Cu(2+), which has no direct effect on NE uptake activity, potentiated oxidative stress and reduction of NE uptake activity produced by NE. This decrease of NE uptake activity was associated with reductions of NE uptake binding sites and NET protein expression by using the radioligand assay and Western blot analysis, but no changes in NET gene expression. In addition, the free-radical scavenger mannitol, and antioxidant enzymes superoxide dismutase and catalase, reduced oxidative stress and attenuated the reductions of NE uptake activity and NET protein produced by NE/Cu. Thus our results support a functional role of oxidative stress in mediating the neuronal NE uptake reducing effect of NE and that this effect of NE on NET is a posttranscriptional event.

Arg389Gly polymorphism of the human beta1-adrenergic receptor in patients with nonfatal acute myocardial infarction.

BACKGROUND: We sought to investigate the relation between the Arg389Gly polymorphism in the human beta1-adrenergic receptor (ADRB1) gene and acute myocardial infarction (AMI). It was previously reported that augmented sympathetic activity might play an important role as a trigger of AMI by enhanced hemodynamic or mechanical forces through ADRB1 activation. Recently, a common polymorphism has been identified at amino acid position 389 (Arg or Gly) of the human ADRB1, within a region important for receptor-Gs protein coupling and subsequent agonist-stimulated adenylyl cyclase activation. METHODS: To investigate the relation between the Arg389Gly polymorphism in the ADRB1 gene and AMI, we genotyped 354 patients with AMI and 354 age- and sex-matched control subjects by use of polymerase chain reaction amplification and the restriction fragment length polymorphism analysis. RESULTS: The prevalence of the Arg389 homozygote (CC) genotype was significantly more frequent in patients with AMI than in control subjects (68.1% vs 47.2%, P <.0001). In logistic regression models, the odds ratio (OR) of Arg389 homozygote (CC) versus Arg389Gly heterozygote (CG) + Gly389 homozygote (GG) genotypes between control subjects and patients with AMI was 2.86 (95% CI 1.92-4.26, P =.0001). The association of the Arg389Gly polymorphism of ADRB1 with AMI was statistically significant and independent of other risk factors. CONCLUSION: Our findings suggest that the genotype of Arg389Gly polymorphism in the human ADRB1 gene is associated with AMI.

Suppressive effect of the Gly389 allele of the beta1-adrenergic receptor gene on the occurrence of ventricular tachycardia in dilated cardiomyopathy.

Beta-1-adrenergic receptor (beta1-AR) blockers reduce both the incidence of sudden death and the ventricular volume in heart failure. In vitro, the Gly389 variant of beta1-AR mediates less adenylyl cyclase activities than the Arg389 variant, so Arg389Gly polymorphism was investigated with regard to the genesis, progression, or arrhythmogenesis of dilated cardiomyopathy (DCM). Allele and genotype frequencies of the Arg389Gly polymorphism were determined in 163 DCM patients and 157 age- and sex-matched controls. There were no differences in genotype and allele frequencies between patients and controls. Echocardiograms, left ventriculograms and 24h-Holter electrocardiograms were evaluated in the DCM patients and none of the clinical indices, other than ventricular tachycardia (VT), differed among the 3 genotypes. The Gly389 allele was more frequent in the VT(-) group than in the VT(+) group (0.46 vs 0.24, p=0.001). In univariate analysis, the odds ratio for VT in patients carrying 1 or 2 copies of the Gly389 allele was 0.29 ([95% confidence interval, 0.13-0.64], p=0.002), when compared with the Arg389 homozygotes. The Gly389 variant supressed the occurrence of VT in DCM, suggesting that this allele confers a decreased risk of sudden death.