Clinical trial and epidemiological data support that the cardiovascular effects of estrogen are complex, including a mixture of both potentially beneficial and harmful effects. In animal models, estrogen protects females from vascular injury and inhibits atherosclerosis. These effects are mediated by estrogen receptors (ERs), which when bound to estrogen can bind to DNA to directly regulate transcription. ERs can also activate several cellular kinases by inducing a “rapid” non-nuclear signaling cascade. However, the biologic significance of this rapid signaling pathway has been unclear.
Methods and Results
Here, we develop a novel transgenic mouse in which rapid signaling is blocked by over-expression of a peptide that prevents ERs from interacting with the scaffold protein, striatin (the Disrupting Peptide Mouse, DPM). Microarray analysis of ex vivo-treated mouse aortas demonstrates that rapid ER signaling plays an important role in estrogen-mediated gene regulatory responses. Disruption of ER-striatin interactions also eliminates the ability of estrogen to stimulate cultured endothelial cell migration and to inhibit cultured vascular smooth muscle cell growth. The importance of these findings is underscored by in vivo experiments demonstrating loss of estrogen-mediated protection against vascular injury in the DPM mouse following carotid artery wire injury.
Taken together, these results support that rapid, non-nuclear ER signaling contributes to the transcriptional regulatory functions of ER, and is essential for many of the vasoprotective effects of estrogen. These findings also identify the rapid ER signaling pathway as a potential target for the development of novel therapeutic agents.
cardiovascular diseases; hormones; molecular biology; signal transduction
Although cigarette smoking is a risk factor for heart failure (HF), smokers with HF have lower mortality rates during/following hospitalization compared to nonsmokers. We examined vascular endothelial function in chronic smokers and nonsmokers with HF as it relates to this smoker’s paradox.
Brachial artery flow mediated dilation (FMD), a measure of conduit vessel endothelial function, was measured in 33 smoking and nonsmoking patients with HF versus controls. In addition, soluble endoglin (sEng), a circulating mediator of endothelial function, was measured in a separate group of 36 smoking and non-smoking patients with HF versus controls.
FMD was significantly lower in smokers without HF compared the nonsmokers without HF (p<0.05). FMD was significantly higher in smokers with HF vs. non-smokers with HF (p<0.05) and did not differ from values seen in nonsmokers without HF (p>0.05). There were no differences in sEng between smokers and nonsmokers without HF (p>0.05). sEng was lower in smokers with HF vs. non-smokers with HF (p < 0.05) and did not differ from values seen in nonsmokers without HF (p>0.05).
Smokers with HF had higher brachial FMD and lower sEng than nonsmokers with HF and values were comparable to nonsmokers without HF. These findings offer novel insight into the smoker’s paradox and suggest that improved short-term outcome in patients hospitalized with HF may in part be mediated by preservation of vascular endothelial function in this setting.
Estrogen receptors are best known as ligand-activated transcription factors that regulate vascular cell gene expression. For many years now, a rapid signaling pathway mediated by cell membrane–associated estrogen receptors also has been recognized, but the physiological relevance of this pathway has remained unclear. In this issue of the JCI, Chambliss et al. provide new data to indicate that activation of non-nuclear estrogen receptor signaling regulates processes central to cardiovascular health and disease. These investigators show that an estrogen-dendrimer conjugate (EDC), which activates estrogen receptors but remains non-nuclear, stimulates vascular EC migration in vitro and protects against vascular injury in vivo. They show further that the vascular benefits of EDC in vivo occur selectively in the vasculature, without stimulating the uterus or enhancing growth of breast cancer xenografts. Taken together, these findings indicate that activation of non-nuclear estrogen receptor signaling regulates vascular events of physiological relevance and suggest that translation of these findings into clinically relevant therapeutic interventions is a logical next goal.
Cyclic GMP (cGMP) signaling attenuates cardiac remodeling, but it is unclear which cGMP effectors mediate these effects and thus might serve as novel therapeutic targets. Therefore, we tested whether the cGMP downstream effector, cGMP-dependent protein kinase G Iα (PKGIα), attenuates pressure overload–induced remodeling in vivo.
Methods and Results
The effect of transaortic constriction (TAC)–induced left ventricular (LV) pressure overload was examined in mice with selective mutations in the PKGIα leucine zipper interaction domain. Compared with wild-type littermate controls, in response to TAC, these Leucine Zipper Mutant (LZM) mice developed significant LV systolic and diastolic dysfunction by 48 hours (n=6 WT sham, 6 WT TAC, 5 LZM sham, 9 LZM TAC). In response to 7-day TAC, the LZM mice developed increased pathologic hypertrophy compared with controls (n=5 WT sham, 4 LZM sham, 8 WT TAC, 11 LZM TAC). In WT mice, but not in LZM mice, phosphodiesterase 5 (PDE5) inhibition with sildenafil (Sil) significantly inhibited TAC-induced cardiac hypertrophy and LV systolic dysfunction in WT mice, but this was abolished in the LZM mice (n=3 WT sham, 4 LZM sham, 3 WT TAC vehicle, 6 LZM TAC vehicle, 4 WT TAC Sil, 6 LZM TAC Sil). And in response to prolonged, 21-day TAC (n=8 WT sham, 7 LZM sham, 21 WT TAC, 15 LZM TAC), the LZM mice developed markedly accelerated mortality and congestive heart failure. TAC induced activation of JNK, which inhibits cardiac remodeling in vivo, in WT, but not in LZM, hearts, identifying a novel signaling pathway activated by PKGIα in the heart in response to LV pressure overload.
These findings reveal direct roles for PKGIα in attenuating pressure overload–induced remodeling in vivo and as a required effector for the cardioprotective effects of sildenafil.
heart failure; nitric oxide; protein kinase G; remodeling heart failure; signal transduction
Background. Cardiovascular disease is the leading cause of premature mortality in autosomal dominant polycystic kidney disease (ADPKD). We examined peripheral augmentation index (AIx) as a measure of systemic vascular function and circulating markers of vascular inflammation in patients with ADPKD.
Methods. Fifty-two ADPKD patients with hypertension and estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m2, 50 ADPKD patients with hypertension and eGFR ≥60 mL/min/1.73 m2, 42 normotensive ADPKD patients with eGFR ≥60 mL/min/1.73 m2 and 51 normotensive healthy controls were enrolled in this study. AIx was measured from peripheral artery tone recordings using finger plethysmography. Serum levels of soluble intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule-1, P-selectin, E-selectin, soluble Fas (sFas) and Fas ligand (FasL) were measured as markers of vascular inflammation.
Results. AIx was higher in all three patient groups with ADPKD compared to healthy controls (P < 0.05). AIx was similar between the normotensive ADPKD patients with eGFR ≥60 mL/min/1.73 m2 and hypertensive ADPKD patients with eGFR <60 mL/min/1.73 m2 (P > 0.05). ICAM, P-selectin, E-selectin and sFas were higher and FasL lower in all ADPKD groups compared to controls (P < 0.05). ICAM, P-selectin and E-selectin were similar between the normotensive ADPKD patients with eGFR ≥60 mL/min/1.73 m2 and hypertensive ADPKD patients with eGFR < 60 mL/min/1.73 m2 (P > 0.05). According to multiple regression analysis, predictors of AIx in ADPKD included age, height, heart rate and mean arterial pressure (P < 0.05). Vascular inflammatory markers were not predictors of AIx in ADPKD.
Conclusions. Systemic vascular dysfunction, manifesting as an increase in AIx and vascular inflammation is evident in young normotensive ADPKD patients with preserved renal function. Vascular inflammation is not associated with elevated AIx in ADPKD.
hypertension; inflammation; kidney disease; vascular function
Progressive left ventricular (LV) dysfunction induces expression of the cytokine transforming growth factor-beta (TGFb1). Endoglin (CD105) is a TGFb1 co-receptor that is released into the circulation as soluble endoglin (sEng). The objective of this study was to assess serum levels of sEng in heart failure and to identify the predictive value of sEng for detecting elevated left ventricular end-diastolic pressures (LVEDP). We measured sEng levels in 82 consecutive patients with suspected LV dysfunction referred for determination of left heart filling pressures by cardiac catheterization. Among these subjects sEng levels correlated with LVEDP (R=0.689; p<0.0001) irrespective of LV ejection fraction (LVEF). Using a receiving operative characteristic (ROC) curve, sEng levels predicted an LVEDP≥16mmHg with an area-under-the-curve (AUC) of 0.85, exceeding measured AUCs for both atrial- and brain-natriuretic peptide, currently used biomarkers for heart failure diagnosis (ANP:0.68; BNP:0.65, p<0.01 vs sEng respectively). In 10 subjects receiving medical therapy for heart failure guided by invasive hemodynamic monitoring, decreased pulmonary capillary wedge pressure was associated with a reduced sEng level (R=0.75, p=0.008). Finally, compared to 25 healthy controls, sEng levels were elevated in subjects with suspected LV dysfunction (3589±588 vs 4257±966 pg/mL, respectively, p<0.005) and correlated directly with New York Heart Association class (NYHA; R=0.501, p<0.001). In conclusion, circulating levels of sEng are elevated in patients with increased LVEDP and NYHA class, irrespective of LVEF. Soluble endoglin levels also decrease in association with reduced cardiac filling pressure after diuresis. These findings identify circulating sEng as a sensitive measure of elevated left heart filling pressure.
heart failure; cardiac filling pressure; soluble receptors
Almost one-third of patients with hypertrophic cardiomyopathy (HC) have an abnormal blood pressure response (ABPR) to exercise and this is associated with a greater risk of sudden cardiac death (SCD). In this study, we examined the association between steady (mean arterial pressure) and pulsatile (pulse pressure) blood pressure components as they relate to ABPR in patients with HC (n = 70). All patients completed a standard Bruce protocol during symptom limited stress testing with concurrent hemodynamic measurements. PP was significantly higher in HC patients with ABPR (n = 19) compared to HC patients with a normal BP response to exercise (p<0.05). According to binary logistic regression, resting PP was a significant predictor of ABPR in patients with HC (p<0.05). MAP was not significantly different between groups, nor was it a predictor of ABPR in HC. Those within the highest tertile of resting PP were 4.8 times more likely to have an ABPR compared to those within the lowest PP tertile (95% CI = 1.24 – 18.2, p<0.05). In conclusion, elevations in resting PP may identify HC individuals at higher risk for having an ABPR during exercise.
Although typically derived from the contour of arterial pressure waveform, augmentation index (AIx) may also be derived from the digital pulse volume waveform using finger plethysmography (peripheral arterial tonometry, PAT). Little is known regarding the physiologic correlates of AIx derived from PAT. In this study, we investigated the relation of PAT-AIx with measures of ventricular-vascular coupling.
Pulse volume waves were measured via PAT and used to derive AIx. Using 2-dimensional echocardiography, effective arterial elastance index (EaI) was estimated as end systolic pressure / stroke volume index. Left ventricular (LV) end-systolic elastance index (ELVI) was calculated as end systolic pressure / end systolic volume index. Ventricular-vascular coupling ratio was defined as EaI/ELVI.
Given the bi-directional nature of ventricular-vascular uncoupling as measured by echocardiography, patients were separated into 3 groups: low EaI/ELVI (<0.6, n = 21), optimal EaI/ELVI (mean 0.6–1.2, n = 16) and high EaI/ELVI (>1.2, n = 10). Adjusting for potential confounders (age, mean arterial pressure, height, and heart rate) patients with optimal EaI/ELVI had lower AIx (1±4%, p<0.05) compared to those with low EaI/ELVI (13±4%) and high EaI/ELVI (19±5%).
Abnormal ventricular-vascular coupling, arising from either increased effective arterial elastance or increased ventricular elastance, is associated with increased AIx as measured by PAT. Additional research is needed to examine other vascular correlates of PAT-AIx.
augmentation index; effective arterial elastance; Left ventricular end-systolic elastance
Background. Augmentation index (AIx) is traditionally obtained from pressure waveforms via arterial applanation tonometry. We sought to evaluate the association between
AIx obtained from peripheral arterial tonometry (PAT) with cardiovascular risk factors (CRF) and coronary artery disease (CAD).
Methods. 186 patients were enrolled in the study. The presence or absence of CRFs and CAD was assessed in each subject. AIx was calculated by an automated algorithm averaging pulse wave amplitude data obtained via PAT. Central blood pressures were assessed in a subset of patients undergoing clinically indicated cardiac catheterization. Results. An association was observed between AIx and age, heart rate, systolic blood pressure, mean arterial pressure, pulse pressure, body weight and body mass index. AIx was significantly lower in patients with <3
CRFs compared to those with >5 CRFs ( P = .02). CAD+ patients had significantly higher AIx compared to CAD− patients ( P = .008). Area under the ROC curve was 0.604 (P < .01). In patients undergoing cardiac catheterization, after adjusting for age, height and heart rate, AIx was a significant predictor of aortic systolic and pulse pressures (P < .05) Conclusion. AIx derived from PAT correlates with cardiac risk factors and CAD. It may be a useful measure of assessing overall risk for coronary artery disease.
Coronary artery disease (CAD) risk is not fully revealed by traditional risk factors. Identification of a simple, non-invasive tool that allows for detection of high risk CAD patients and can be applied in large population/clinical settings would prove valuable.
In this study, finger peripheral arterial tonometry (PAT) was used to measure pulse wave amplitude (PWA) during reactive hyperemia (RH) and taken as a measure of microvascular endothelial function in 42 men with stable CAD and well-controlled LDL-cholesterol levels. Plasma levels of high sensitivity C-reactive protein (hs-CRP) and lipoprotein-associated phospholipase A2 (Lp-PLA2) were measured and used to re-classify men into high-risk (elevated hs-CRP and Lp-PLA2), moderate risk (either elevated hs-CRP or Lp-PLA2) or low risk (low hs-CRP and Lp-PLA2) groups.
PWA-RH was significantly lower in the high risk group (1.3±0.04) compared to the moderate risk (1.6±0.07, p<0.05) and low risk (2.0±0.1, p<0.05) groups. According to binary logistic regression, PWA-RH was a significant predictor of high-risk status amongst men with CAD (p<0.05).
Measurement of peripheral microvascular endothelial function with PAT may be able to distinguish high-risk from moderate and low-risk men with stable CAD and well-controlled LDL-cholesterol levels and thus aid in residual risk stratification in this at risk cohort.
endothelial function; inflammation; cardiovascular
Patients with hypertrophic cardiomyopathy (HC) have coronary microvascular dysfunction which is an independent predictor of adverse left ventricular remodeling, systolic dysfunction, and mortality in these patients. Whether these defects in vasomotor function are localized to the coronary arteries or whether systemic vasomotor dysfunction is present in HC patients has not yet been adequatley examined. We tested the hypothesis that patients with HC (n = 46) have altered peripheral vascular endothelial function. Subjects without coronary artery disease (CAD− n = 46) and subjects with CAD (CAD+, n = 46), served as negative and positive controls, respectively. Conduit artery endothelium-dependent vasomotion was assessed with ultrasound by measuring flow-mediated dilation (FMD) of the brachial artery. FMD was lower in HC patients compared to CAD− (p<0.05) but was similar to CAD+ patients (p=NS). In conclusion, vasomotor dysfunction in HC is not restricted to the coronary vasculature. Patients with HC have impaired peripheral conduit vessel endothelial function and the magnitude of impairment is similar to that seen in older patients with advanced CAD.
cardiomyopathy; flow mediated dilation; vascular function
The ratio of pulse wave amplitude (PWA) during reactive hyperemia compared to baseline as measured by peripheral arterial tonometry is as a non-invasive measure of microvascular endothelial function referred to as the pulse wave amplitude reactive hyperemia index (PWA-RHI). Whether upstream conduit vessel structure may affect downstream resistance vessel PWA has not been clearly examined. We tested the hypothesis that digital PWA is influenced by brachial artery diameter (BAD) and that this association would influence comparison of PWA-RHI between genders.
Measures of vascular structure and microvascular function were carried out in 115 patients varying in cardiovascular risk profiles (average age 57 yrs, male n = 79, CAD n = 43). PWA was assessed using modified finger photoplethysmography (peripheral arterial tonometry, PAT) at baseline and following 5 minutes of brachial artery occlusion. Brachial artery diameter (BAD) was assessed using high-resolution ultrasonography.
There was a negative association between BAD and PWA-RHI and (r = −0.34, p<0.05). Women had greater PWA-RHI and smaller BAD compared with men (p<0.05). When co-varying for BAD, there were no longer gender differences in PWA-RHI. Moreover, when a subgroup of men and women without CAD (n = 40) matched for BAD were examined, there were no gender differences in PWA-RHI.
PWA-RHI obtained from PAT is associated with BAD. Studies examining gender differences in microvascular endothelial function with PAT may need to correct for BAD as a potential confounder.
endothelial function; peripheral arterial tonometry; vascular structure; flow mediated dilation
Low circulating levels of high density lipoprotein cholesterol (HDL-C) are associated with increased risk for cardiovascular events. HDL-C has a variety of poorly understood atheroprotective effects, including altering lipid metabolism and reducing inflammation. Increased arterial stiffness is an important predictor of subsequent cardiovascular risk. Therefore, in the current study, we sought to determine whether HDL-C levels are associated with carotid arterial stiffness. In addition we examined potential correlates of this association such as inflammatory factors, cardio-respiratory fitness and body fat percentage.
Carotid artery β stiffness was measured by ultrasound in 47 (23 yrs old) healthy pre-hypertensive men. Low HDL-C was defined as <1.0 mmol/L. Body fat was measured by air displacement plethysmograpy. Cardio-respiratory fitness was measured using a maximal exercise test with metabolic gas analysis and inflammatory markers consisted of C-reactive protein (CRP), white blood cell (WBC) count, and absolute neutrophil count.
Men with low HDL-C had significantly higher carotid artery stiffness, CRP, WBC count, neutrophil count, body fat, fasting glucose and lower cardio-respiratory fitness (p<0.05). Co-varying for cardio-respiratory fitness, % body fat, and glucose had no effect on group differences in carotid artery stiffness. Co-varying for inflammatory markers resulted in groups having similar carotid artery stiffness.
Pre-hypertensive men with low HDL-C have higher carotid artery stiffness when compared with those with higher HDL-C. The detrimental effects of low HDL-C on large artery stiffness in pre-hypertensive men may be mediated by inflammation and not by cardio-respiratory fitness or body fat levels.
arterial stiffness; C-reactive protein; maximal oxygen consumption; body fat
Smoking is an established cardiovascular risk factor that impairs endothelial function and reduces exercise capacity. Peripheral vascular endothelial function correlates with exercise capacity, but whether this association prevails in smokers is unknown. The purpose of this investigation was to examine the association between endothelial function and exercise capacity in chronic smokers and non-smoking controls.
Brachial artery flow mediated dilation (FMD, endothelium-dependent) following 5-minutes of upper arm occlusion was compared in 26 smokers (age 58±1 yrs; 15 female; BMI = 28±1) and 39 non-smokers (age 58±1 yrs; 24 female; BMI = 28±1) using ultrasound. Exercise treadmill time (ETT) was recorded from a standard Bruce protocol during symptom limited stress testing.
There was a significant positive association between FMD and ETT in smokers (r = 0.60, p<0.05) and non-smokers (r = 0.28, p<0.05). FMD was significantly lower in smokers vs. non-smokers (8.9 ± 0.9 vs. 12.6 ± 0.7%, p<0.05). ETT was significantly lower in smokers (425 ± 35 sec) versus non-smokers (522 ± 25 sec, p<0.05). After adjusting for FMD, there were no longer group differences in ETT. When patients were matched according to FMD, there were no differences in ETT between smokers and non-smokers.
Peripheral endothelial dysfunction is a correlate of low exercise capacity in smokers and non-smokers alike. Future research is needed to examine if improving endothelial function will lead to concomitant increases in exercise capacity in chronic smokers.
flow mediated vasodilation; exercise testing; smoking; vascular function
Estrogen has both rapid and longer-term direct effects on cardiovascular tissues mediated by the two estrogen receptors, ERα and ERβ. Previous work identified that estrogen regulates the expression of inducible nitric oxide synthase (iNOS) in vascular smooth muscle cells (VSMC). ERβ knockout mice have vascular dysfunction due to dysregulation of iNOS expression and these mice are hypertensive (Zhu et al, Science 2002;295:505-508). Here we report studies to examine the differential regulation of iNOS gene expression by ERα and ERβ. Immunoblotting and RT-PCR studies revealed that different VSMC lines expressed different levels of ERα and ERβ protein and mRNA. VSMC from different vascular beds were studied, including aortic VSMC expressing ERα and radial VSMC expressing ERβ. E2 inhibited NO production and iNOS protein expression in aortic VSMC. Human iNOS promoter reporter studies revealed suppression of iNOS reporter activity by E2 in aortic VSMC, and stimulation of iNOS reporter activity by E2 in radial arterial VSMC. In heterologous expression studies of COS-7 cells lacking endogenous ER, E2 treatment of COS-7 cells did not alter iNOS reporter activity in the presence of ERα, while reporter activity increased 2.3 fold in the presence of ERβ. Similar experiments in COS-7 cells using the selective estrogen receptor modulator raloxifene showed that raloxifene caused a reduction in iNOS reporter activity with ERα co-expression, and an increase with ERβ co-expression. Rat VSMC expressing ERβ but not ERα also showed increased iNOS reporter activity with E2 treatment, an effect lost when ERα was introduced into the cells. Taken together, these data support that hiNOS transcription is positively regulated by ERβ and negatively regulated by ERα in VSMC, supporting differential actions of these two estrogen receptors on a physiologically relevant gene in vascular smooth muscle cells.
vascular smooth muscle cell; hormone; estrogen receptor α; estrogen receptor β; nitric oxide synthase
Diabetic Autonomic Neuropathy (DAN), a major complication of diabetes mellitus, is characterized in part by impaired cardiac parasympathetic responsiveness. Parasympathetic stimulation of the heart involves activation of an acetylcholine-gated K+ current, IKAch, via a (GIRK1)2/(GIRK4)2 K+ channel. Sterol regulatory element binding protein-1 (SREBP-1) is a lipid-sensitive transcription factor. We describe a unique SREBP-1-dependent mechanism for insulin regulation of cardiac parasympathetic response in a mouse model for DAN. Compared to WT mice, Ins2Akita type I diabetic mice demonstrated a decrease in the negative chronotropic response to carbamylcholine characterized by a 2.4 fold decrease in duration of bradycardia; a 52±8% decrease in atrial expression of GIRK1 (P<0.01) and a 31.3±2.1% decrease in SREBP-1 (P<0.05). Myocytes from atria of Akita mice exhibited a markedly decreased carbamylcholine stimulation of IKAch with a peak value of −181±31 pA/pF compared to −451±62 pA/pF (P<0.01) for cells from WT mice. Insulin treatment of Akita mice reversed the impairment in parasympathetic response, increased the expression of GIRK1, SREBP-1 and IKAch activity in atrial myocytes from these mice to levels in WT mice. Insulin treatment of cultured atrial myocytes stimulated GIRK1 expression 2.68±0.12 fold (P<0.01) while overexpression of DN-SREBP-1 reversed this insulin effect. Finally, adenoviral expression of SREBP-1 in Akita atrial myocytes reversed the impaired IKAch to levels in cells from WT. These results support a unique molecular mechanism for insulin regulation of GIRK1 expression and parasympathetic response via SREBP-1 which might play a role in the pathogenesis of DAN in response to insulin deficiency in the diabetic heart.
Diabetic autonomic neuropathy; SREBP; insulin deficiency; GIRK channel
Many patients taking statins often complain of muscle pain and weakness. The extent to which muscle pain reflects muscle injury is unknown.
We obtained biopsy samples from the vastus lateralis muscle of 83 patients. Of the 44 patients with clinically diagnosed statin-associated myopathy, 29 were currently taking a statin, and 15 had discontinued statin therapy before the biopsy (minimal duration of discontinuation 3 weeks). We also included 19 patients who were taking statins and had no myopathy, and 20 patients who had never taken statins and had no myopathy. We classified the muscles as injured if 2% or more of the muscle fibres in a biopsy sample showed damage. Using reverse transcriptase polymerase chain reaction, we evaluated the expression levels of candidate genes potentially related to myocyte injury.
Muscle injury was observed in 25 (of 44) patients with myopathy and in 1 patient without myopathy. Only 1 patient with structural injury had a circulating level of creatine phosphokinase that was elevated more than 1950 U/L (10× the upper limit of normal). Expression of ryanodine receptor 3 was significantly upregulated in patients with biopsy evidence of structural damage (1.7, standard error of the mean 0.3).
Persistent myopathy in patients taking statins reflects structural muscle damage. A lack of elevated levels of circulating creatine phosphokinase does not rule out structural muscle injury. Upregulation of the expression of ryanodine receptor 3 is suggestive of an intracellular calcium leak.
Parasympathetic stimulation of the heart, which provides protection from arrhythmias and sudden death, involves activation of the G protein–coupled inward rectifying K+ channel GIRK1/4 and results in an acetylcholine-sensitive K+ current, IKACh. We describe a unique relationship between lipid homeostasis, the lipid-sensitive transcription factor SREBP-1, regulation of the cardiac parasympathetic response, and the development of ventricular arrhythmia. In embryonic chick atrial myocytes, lipid lowering by culture in lipoprotein-depleted serum increased SREBP-1 levels, GIRK1 expression, and IKACh activation. Regulation of the GIRK1 promoter by SREBP-1 and lipid lowering was dependent on interaction with 2 tandem sterol response elements and an upstream E-box motif. Expression of dominant negative SREBP-1 (DN–SREBP-1) reversed the effect of lipid lowering on IKACh and GIRK1. In SREBP-1 knockout mice, both the response of the heart to parasympathetic stimulation and the expression of GIRK1 were reduced compared with WT. IKACh, attenuated in atrial myocytes from SREBP-1 knockout mice, was stimulated by SREBP-1 expression. Following myocardial infarction, SREBP-1 knockout mice were twice as likely as WT mice to develop ventricular tachycardia in response to programmed ventricular stimulation. These results demonstrate a relationship between lipid metabolism and parasympathetic response that may play a role in arrhythmogenesis.
Black tea has been shown to improve endothelial function in patients with coronary artery disease and recent data indicate the polyphenol fraction of black tea enhances endothelial nitric oxide synthase (eNOS) activity through p38 MAP kinase (p38 MAPK) activation. Since the mechanisms for this phenomenon are not yet clear, we sought to elucidate the signaling events in response to black tea polyphenols. Bovine aortic endothelial cells (BAECs) exposed to black tea polyphenols demonstrated eNOS activation that was inhibited by the estrogen receptor (ER) antagonist ICI 182,780 and siRNA-mediated silencing of ER expression. Consistent with this observation, black tea polyphenols induced time-dependent phosphorylation of ERα on Ser-118 that was inhibited by ICI 182,780. Phosphorylation of ERα on Ser-118 was due to p38 MAP kinase (p38 MAPK) as it was inhibited by SB203580 and over-expression of dominant-negative p38 MAPKα. Conversely, constitutively-active MKK6 induced p38 MAPK activation that recapitulated the effects of polyphenols by inducing ERα phosphorylation and downstream activation of Akt, and eNOS. The key role of ERα Ser-118 phosphorylation was confirmed in eNOS-transfected COS-7 cells as polyphenol-induced eNOS activation required co-transfection with ERα subject to phosphorylation at Ser-118. This residue appeared critical for functional association of ERα with p38 MAPK as ERα with Ser-118 mutated to alanine could not form a complex with p38 MAPK. These findings suggest p38 MAP kinase-mediated eNOS activation requires ERα and these data uncover a new mechanism of ERα activation that has broad implications for NO bioactivity and endothelial cell phenotype .
Right ventricular (RV) failure is a major cause of mortality in acute or chronic lung disease and left heart failure. The objective of this study was to demonstrate a percutaneous approach to study biventricular hemodynamics in murine models of primary and secondary RV pressure overload (RVPO) and further explore biventricular expression of two key proteins that regulate cardiac remodeling: calcineurin and transforming growth factor beta 1 (TGFβ1).
Adult, male mice underwent constriction of the pulmonary artery or thoracic aorta as models of primary and secondary RVPO, respectively. Conductance catheterization was performed followed by tissue analysis for changes in myocyte hypertrophy and fibrosis.
Both primary and secondary RVPO decreased biventricular stroke work however RV instantaneous peak pressure (dP/dtmax) and end-systolic elastance (Ees) were preserved in both groups compared to controls. In contrast, left ventricular (LV) dP/dtmax and LV-Ees were unchanged by primary, but reduced in the secondary RVPO group. The ratio of RV:LV ventriculo-arterial coupling was increased in primary and reduced in secondary RVPO. Primary and secondary RVPO increased RV mass, while LV mass decreased in primary and increased in the secondary RVPO groups. RV fibrosis and hypertrophy were increased in both groups, while LV fibrosis and hypertrophy were increased in secondary RVPO only. RV calcineurin expression was increased in both groups, while LV expression increased in secondary RVPO only. Biventricular TGFβ1 expression was increased in both groups.
These data identify distinct effects of primary and secondary RVPO on biventricular structure, function, and expression of key remodeling pathways.
Clinical studies of hormone replacement therapy to prevent cardiovascular diseases have heightened interest in the cardiovascular effects of progestins. However, the role of the progesterone receptor (PR) in vascular biology has not been studied in vivo. We studied ovariectomized female PR knockout (PRKO) mice and their wild-type (WT) littermates using the mouse carotid artery injury model. Placebo-treated PRKO mice showed significantly greater vascular medial hypertrophy and vascular smooth muscle cell (VSMC) proliferation in response to vascular injury than did WT mice. Progesterone had no significant effect in the PRKO mice, but worsened the response to injury in WT mice. VSMCs cultured from PRKO mouse aortae were markedly hyperproliferative, and their growth was not affected by progesterone. In contrast to the in vivo findings, progesterone inhibited proliferation of WT-derived VSMCs. Furthermore, reintroduction of PR into PRKO-derived VSMCs using adenoviral methods restored progesterone-mediated inhibition of proliferation to these cells. This effect was reversed by the PR antagonist, RU 486. Thus, the effects of PR and progesterone differ markedly between cultured VSMCs and intact blood vessels. These data demonstrate a direct role for the PR in regulating the response to vascular injury and VSMC proliferation.
Estrogen is an important vasoprotective molecule that causes the rapid dilation of blood vessels by activating endothelial nitric oxide synthase (eNOS) through an unknown mechanism. In studies of intact ovine endothelial cells, 17β-estradiol (E2) caused acute (five-minute) activation of eNOS that was unaffected by actinomycin D but was fully inhibited by concomitant acute treatment with specific estrogen receptor (ER) antagonists. Overexpression of the known transcription factor ERα led to marked enhancement of the acute response to E2, and this was blocked by ER antagonists, was specific to E2, and required the ERα hormone-binding domain. In addition, the acute response of eNOS to E2 was reconstituted in COS-7 cells cotransfected with wild-type ERα and eNOS, but not by transfection with eNOS alone. Furthermore, the inhibition of tyrosine kinases or mitogen-activated protein (MAP) kinase kinase prevented the activation of eNOS by E2, and E2 caused rapid ER-dependent activation of MAP kinase. These findings demonstrate that the short-term effects of estrogen central to cardiovascular physiology are mediated by ERα functioning in a novel, nongenomic manner to activate eNOS via MAP kinase–dependent mechanisms.