[Update of the ESC guidelines 2018 on cardiovascular diseases during pregnancy : Most important facts]. / Update 2018 der ESC-Leitlinie zu kardiovaskulären Erkrankungen in der Schwangerschaft : Die wichtigsten Fakten.

Heart diseases are the most common cause of maternal death during pregnancy in Western countries. The current ESC guidelines 2018 for the management of cardiovascular diseases during pregnancy is a guide for any physician facing the challenge of caring for pregnant women with cardiovascular diseases. Among the new concepts compared to 2011, are recommendations to classify maternal risk due to the modified World Health Organization (mWHO) classification, introduction of the pregnancy heart team, guidance on assisted reproductive therapy, specific recommendations on anticoagulation for low-dose and high-dose requirements of vitamin K antagonists and the potential use of bromocriptine in peripartum cardiomyopathy. The Food and Drug Administration (FDA) categories A-D and X should no longer be used. Therefore, the table of drugs was completed with detailed information from animal and human studies on maternal and fetal risks. The new findings on specific heart diseases are presented in detail in the respective chapters.

[Gender and cardiovascular diseases : Why we need gender medicine]. / Geschlecht und Herz-Kreislauf-Erkrankungen : Warum wir Gendermedizin brauchen.

Gender medicine is concerned with the question of why diseases are expressed differently in the genders. It takes differences between men and women into account, which are often neglected by traditional medicine. Sex differences can also be found in cardiovascular diseases; therefore, risk factors for cardiovascular diseases have a different significance depending on the sex. Diabetic diseases tend to promote the occurrence of coronary heart disease (CHD) more strongly in women than in men. Myocardial infarctions affect women 10 years later than men and young women are often treated too late, possibly because myocardial infarction is consider to be a "male disease". The number of cases of coronary syndrome is significantly increasing, particularly in young women. Some of the diseases which predominantly occur in women are takotsubo cardiomyopathy, microcirculation disorders and spontaneous coronary artery dissection. Pharmacological treatment of CHD is principally the same in men and women but attention must be paid to differences in the pharmacokinetics of important drugs. Coronary dilatation has comparable effects in both men and women but more complications occur in women. Cardiac failure with impaired left ventricular systolic function affects more men than women in the Western world but the opposite is true for cardiac failure with preserved left ventricular ejection fraction. Hypertrophic and dilatative cardiomyopathies are more frequent in men. Many of the drugs used to treat cardiac failure have different actions in men and women. Too little attention is paid to the pharmacokinetics and pharmacodynamics in women when testing active agents; however, awareness of the differences that need to be considered is growing.

[Sex and gender differences in pharmacotherapy]. / Geschlechterunterschiede in der Pharmakotherapie.

Many drugs have act differently in women and men. Biological differences between women and men lead to sex differences in pharmacokinetics, i.e., in drug absorption, distribution in tissues, metabolism by liver enzymes, and excretion via the kidney and intestine. In addition there are sex differences in pharmacodynamics, leading to a different efficacy of drugs in women and men. The biological differences between women and men may be caused by sex-specific gene expression, by sex-specific epigenetic modifications, and finally by the effect of sex hormones. In addition, gender plays a role in drug efficacy as a sociocultural dimension that may lead to differences between women and men. Frequently drugs are only tested on animals of one sex and thereby optimized for one sex. This is based on the notion that sex differences are not important for clinical drug effects. Furthermore, to date, sex and gender differences have been underestimated in clinical studies, and phase III studies were not prospectively designed to assess sex differences in drug effects. In addition, women and men use drugs differently with respect to compliance, adherence, and self-medication with over-the-counter drugs. Further, it is known that male and female physicians treat women and men as patients differently. In conclusion, drug therapy is not yet optimized for both genders. However, there is increasing awareness that differences between women and men should be respected in order to provide optimal drugs in optimal doses for both genders.

Impact of sex on clinical outcome in early Multiple Sclerosis.

BACKGROUND: Previous evidence suggests sex differences in the clinical course of relapsing remitting multiple sclerosis (RRMS), but comprehensive early-stage prospective studies are lacking. We aim to quantify the impact of sex on clinical outcomes in early-stage RRMS. METHODS: Utilizing prospective cohort data, we assessed the impact of biological sex on time-to-relapse, disability progression (Expanded Disability Status Scale [EDSS]), extremity function (Nine-Hole Peg Test, Timed-25-food walk test), cognition (Paced Auditory Serial Addition Test, Symbol Digit Modalities Test), quality-of-life (Hamburg Quality of Life Questionnaire in Multiple Sclerosis, Short-Form-36), fatigue (Fatigue Severity Scale, Fatigue Scale for Motor and Cognitive functions), and depression (Beck Depression Inventory-II) in clinically isolated syndrome (CIS) or RRMS patients. Inclusion was within 12 months of symptom onset. Linear, negative binomial, mixed, and Cox models estimated male vs. female effects at the four-year follow-up including baseline-to-follow-up course. RESULTS: We included 149 patients (65.1 % female). Eighty-five completed four-year follow-up. No sex differences in time-to-relapse emerged (HR = 0.91;95 %CI = 0.53-1.58). Males had no increased risk of EDSS worsening (OR = 0.75;95 %CI = 0.21-2.35) compared to females. Similarly, minor/no sex differences emerged in other outcomes. CONCLUSIONS: Four years after first manifestation, neither disease activity (disability progression and relapse rate) nor patient-reported outcomes showed sex-related disparities in this early-MS-cohort. GOV IDENTIFIER: NCT01371071.

eNOS phosphorylation and translocation are altered in male but not female mice by increased activation of the Gαq protein.

Little is known about sex-dependent physiological and pathophysiological differences in cardiac endothelial nitric oxide synthase (eNOS) expression and activation. Therefore, we investigated cardiac morphology and eNOS protein expression, including its translocation-dependent activation and phosphorylation, in cardiac tissue of male and female wild-type mice and transgenic heart-failure mice having a cardiac-specific, 5-fold overexpression of the Galphaq protein. In addition, we measured calcineurin protein expression. Heart-to-body weight ratio was increased in Galphaq mice. Female wild-type mice showed higher eNOS protein expression and activation (translocation and phosphorylation) than did wild-type males. In cardiac tissue of Galphaq mice, these sex-dependent differences remained or were enhanced. Protein expression of the catalytic subunit calcineurin A, which has been shown to dephosphorylate eNOS, was higher in wild-type males than in wild-type females. These differences were increased in the Galphaq mice model. We conclude that sex differences exist in cardiac eNOS protein expression and phosphorylation. Increased activation of the Galphaq protein appears to alter eNOS protein expression and phosphorylation only in males.

[Implications of gender-specific aspects in the therapy of cardiovascular diseases]. / Therapeutische Implika- tionen gender-spezifischer Aspekte von Herzkrankheiten.

In cardiovascular diseases e.g. heart failure and coronary artery disease gender differences are evident in etiology, pathophysiology, clinical presentation, prognosis and response to treatment. Diabetes and hypertension are the major risk factors in women. Mechanisms leading to apparent diabetes or its clinical pre-stage are different in women and men and according to this result in different therapeutic implications. Diastolic heart failure is more frequent in women and effects and side effects of important groups of active pharmaceutical substances are, at least to some extent, different. Atrial fibrillation and ventricular arrhythmia differ in frequency of occurrence; drug induced tachycardia with QT interval prolongation is particularly frequent in women. Underlying pathomechanisms responsible for gender differences in pharmacotherapy are on the one hand differences in pharmacokinetic mechanisms. Particularly drugs which are metabolised via cytochrome P 450 CYP 3A pathway show different kinetics in women and men. In addition, important differences are evident in pharmocodynamics caused by effects of sex steroid hormones or products of X-chromosomal genes. The evidence of estrogen and testosterone receptors in cardiomyocytes and the vascular system, interaction of sex steroid hormones with cellular pathways and the role of X-chromosomal genes are the focus of basic research. Interactions of sex steroid hormone receptors with other nuclear receptors e.g. PPARs ("peroxisome proliferator-activated receptors") are another important underlying mechanism. The knowledge of different pharmacokinetic mechanisms has to be taken into consideration in pharmacotherapy of cardiovascular diseases, for example by adjustment of drug dosages in women, necessary in different groups of pharmaceutical substances or in the long run, gender differences in effects and side effects of drugs. In drug development both aspects have to be considered. There is more than one good reason to intensify basic and clinical research and research on health care on gender differences in cardiovascular diseases.

Aldosterone synthase (CYP11B2) -344 C/T polymorphism is associated with left ventricular structure in human arterial hypertension.

OBJECTIVES: This study examined the association between the -344 C/T polymorphism of the human aldosterone synthase promoter and left ventricular structure in arterial hypertension. BACKGROUND: Because of conflicting results from different studies, the mechanism of such an association, if any, has not been determined. METHODS: We examined the aldosterone synthase promoter genotype in 120 young (age: 26 +/- 3 years) male, white subjects with normal or mildly elevated blood pressure. Left ventricular structural parameters and urinary sodium excretion over 24 h before and after additional oral sodium load (6 g/day over 1 week) were determined. RESULTS: Hypertensive subjects with the CC genotype had a greater left ventricular end-diastolic diameter but smaller relative wall thickness than those with the TT genotype (54 +/- 2 vs. 50 +/- 4 mm, and 0.37 +/- 0.07 vs. 0.44 +/- 0.06 mm, respectively; p < 0.05). Hypertensive subjects with the TT genotype (n = 15) had a greater increase in urinary sodium excretion after oral sodium load than those with the CC genotype (n = 11) (135 +/- 95 vs. 24 +/- 133 mmol/liter/day; p < 0.05). Serum aldosterone levels were found to be decreased after oral sodium load in hypertensive subjects with the TT and CT genotypes only (-37 +/- 45 and -38 +/- 51 pg/ml, respectively; all p < 0.01) but not in those with the CC genotype (-12 +/- 30 pg/ml, n.s.). Such differences were not found in normotensive subjects. CONCLUSIONS: Hypertensive subjects with the -344 CC genotype of the aldosterone synthase promoter are characterized by a pattern of early eccentric left ventricular hypertrophy. Differences in renal sodium handling across the genotypes might contribute to this finding.

Effect of the angiotensin II type 2-receptor gene (+1675 G/A) on left ventricular structure in humans.

OBJECTIVES: Our study goal was to analyze whether gene variants of angiotensin II type 2-receptor (AT2-R) modulate the effects of angiotensin II on the left ventricle (LV). BACKGROUND: Experimental data suggest that angiotensin II modifies ventricular growth responses via angiotensin II type 1-receptors (AT1-R) and AT2-R. METHODS: In 120 white, young male subjects with normal or mildly elevated blood pressure, we assessed plasma angiotensin II and aldosterone concentrations (RIA), 24-h urinary sodium excretion, 24-h ambulatory blood pressure and LV structure (two-dimensional guided M-mode echocardiography). The intronic +1675 G/A polymorphism of the X-chromosomal located AT2-R gene was investigated by single-strand conformational polymorphism analysis and DNA-sequencing. RESULTS: Hypertensive subjects with the A-allele had a greater LV posterior (11.0 +/- 1.3 vs. 9.9 +/- 1.3 mm, p < 0.001), septal (11.8 +/- 1.4 vs. 10.1 +/- 1.2 mm, p < 0.001) and relative wall thickness (0.44 +/- 0.06 vs. 0.39 +/- 0.06, p < 0.01) as well as LV mass index (138 +/- 23 vs. 120 +/- 13 g/m2, p < 0.001) than those with the G-allele. Confounding factors (i.e., body mass index and surface area, plasma angiotensin II, sodium excretion, systolic and diastolic ambulatory blood pressure) were similar between the two genotypes. In normotensive subjects, relative wall thickness (0.36 +/- 0.05 vs. 0.35 +/- 0.05) and LV mass index (115 +/- 21 vs. 112 +/- 17 g/m2) were nearly identical across the two genotypes, with similar confounding variables. CONCLUSIONS: Our data indicate that the X-chromosomal located +1675 G/A-polymorphism of the AT2-R gene is associated with LV structure in young male humans with early structural changes of the heart due to arterial hypertension.

Activation of the cardiac renin-angiotensin system and increased myocardial collagen expression in human aortic valve disease.

OBJECTIVES: We sought to determine whether the cardiac renin-angiotensin system (RAS) is activated in human aortic valve disease depending on left ventricular function, and we analyzed the concomitant regulation of the extracellular matrix components. BACKGROUND: In animal models with pressure or volume load, activation of the cardiac RAS increases fibrosis. In human aortic valve disease, the ventricular collagen protein content is increased, but only scarce data on the activation state of the cardiac RAS and its effects on collagen and fibronectin messenger ribonucleic acid (mRNA) are available. METHODS: In left ventricular biopsies from patients with aortic valve stenosis (AS) and aortic valve regurgitation and from control subjects, we quantitated mRNAs for angiotensin-converting enzyme (ACE), chymase, transforming growth factor-beta1 (TGF-beta1), collagen I, collagen III and fibronectin by reverse-transcription polymerase chain reaction. Proteins were localized by immunohistochemistry; ACE activity was determined by high performance liquid chromatography; and TGF-beta protein by quantitative enzyme immunoassay. RESULTS: Protein, ACE and TGF-beta1 mRNA were significantly increased in patients with AS and AR (1.5- to 2.1-fold) and correlated with each other. The increase occurred also in patients with normal systolic function. Collagen I and III and fibronectin mRNAs were both upregulated about twofold in patients with AS and AR. In AS, collagen and fibronectin mRNA expression levels were positively correlated with left ventricular end-diastolic pressure and inversely with left ventricular ejection fraction (LVEF). CONCLUSIONS: In human hearts, pressure and volume overload increases cardiac ACE and TGF-beta1 in the early stages. This activation of the cardiac RAS may contribute to the observed increase in collagen I and III and fibronectin mRNA expression. The increase in extracellular matrix already exists in patients with a normal LVEF, and it increases with functional impairment.

Spectrum of clinical phenotypes and gene variants in cardiac myosin-binding protein C mutation carriers with hypertrophic cardiomyopathy.

OBJECTIVES: We studied the clinical and genetic features of hypertrophic cardiomyopathy (HCM) caused by mutations in the myosin-binding protein C gene (MYBPC3) in 110 consecutive, unrelated patients and family members of European descent. BACKGROUND: Mutations in the MYBPC3 gene represent the cause of HCM in approximately 15% of familial cases. MYBPC3 mutations were reported to include mainly nonsense versus missense mutations and to be characterized by a delayed onset and benign clinical course of the disease in Japanese and French families. We investigated the features that characterize MYBPC3 variants in a large, unrelated cohort of consecutive patients. METHODS: The MYBPC3 gene was screened by single-strand conformational polymorphism analysis and sequencing. The clinical phenotypes were analyzed using rest and 24-h electrocardiography, electrophysiology, two-dimensional and Doppler echocardiography and angiography. RESULTS: We identified 13 mutations in the MYBPC3 gene: one nonsense, four missense and three splicing mutations and five small deletions and insertions. Of these, 11 were novel, and two were probably founder mutations. Patients with MYBPC3 mutations presented a broad range of phenotypes. In general, the 16 carriers of protein truncations had a tendency toward earlier disease manifestations (33 +/- 13 vs. 48 +/- 9 years; p = 0.06) and more frequently needed invasive procedures (septal ablation or cardioverter-defibrillator implantation) compared with the 9 carriers of missense mutations or in-frame deletions (12/16 vs. 1/9 patients; p < 0.01). CONCLUSIONS: Multiple mutations, which include missense, nonsense and splicing mutations, as well as small deletions and insertions, occur in the MYBPC3 gene. Protein truncation mutations seem to cause a more severe disease phenotype than missense mutations or in-frame deletions.

Molecular biology of angiotensin receptors and their role in human cardiovascular disease.

The actions of angiotensin II in the cardiovascular system are transmitted by two known and possibly some unknown angiotensin receptor types. AT1 and AT2 both correspond to G-protein-coupled receptors with seven hydrophobic transmembrane domains, several N-glycosylation sites and a potential G-protein binding site. Cloning of coding regions and promoter sequences contributed to the understanding of receptor protein function and regulation. Angiotensin receptors with atypical binding properties for the known AT1- and AT2-specific ligands are expressed on human cardiac fibroblasts and in the human ulcrus. In several animal models, receptors with high affinity for angiotensin (1-7) have been described. AT1 stimulation is mediated by the generation of phospholipid-derived second messengers, activation of protein kinase C, the MAPkinase pathway and of immediate early genes. Recently, phosphorylation and dephosphorylation of tyrosine kinases have been associated with AT1- and AT2-mediated signal transduction. ATR are regulated by phosphorylation, internalization, modification of transcription rate and mRNA stability. Regulation is highly cell and organ specific and includes upregulation of ATR in some pathophysiological situations where the renin angiotensin system is activated. Whereas the function of AT1 in the cardiovascular system is relatively well established, there is little information regarding the role of AT2. Recent hypotheses suggest an antagonism between AT1 and AT2 at the signal transduction and the functional level. Transgenic animal models, particularly with targeted disruption of the AT1 and AT2 genes, suggest the contribution of both genes to blood pressure regulation. Genetic polymorphisms have been described in the AT1 and AT2 gene or neighbored regions and are used to analyze the association between gene defects and cardiovascular diseases. AT1 antagonists are now being introduced into the treatment of hypertension and potentially heart failure, and more interesting pharmacological developments are expected from the ongoing basic studies.

Analysis and functional characterization of alternatively spliced angiotensin II type 1 and 2 receptor transcripts in the human heart.

Expression levels of angiotensin II type 1 and type 2 receptors (AT1, AT2) vary at different cardiac localizations and are regulated in cardiac diseases. Differential splicing of the 5' untranslated exons of the primary AT1 mRNA transcripts may modulate translational efficiency and thus receptor expression. We therefore searched for AT1 and AT2 mRNA splice patterns specific to chamber localization or to cardiac performance and analyzed their effect on protein expression in transfection experiments. The exon composition of the AT1 and AT2 mRNA transcripts in normal and diseased human hearts were analyzed using a reverse transcription polymerase chain reaction followed by HPLC quantitation of the amplificates. We compared atrial (n=18) and ventricular (n=28) samples and endomyocardial biopsies (n=10) from patients with normal and severely impaired cardiac function and one donor heart, which was not used for transplantation. AT1 transcripts with the exon composition 1/2/5 and 1/5 represented about 93-98% of all AT1 mRNAs; transcript 1/2/3/5 represented 8% in the atria and 2% in ventricles. Since exon 2 reduces translational efficiency in vitro, the ratios of transcripts with and without exon 2, (1/2/5+1/2/3/5) to (1/5), were compared. These were 1.24+/-0.07 in normal atria, 0.96+/-0.09 in atria from failing hearts (P<0.05), 0.68 in the left ventricle of the donor heart, and 0.58+/-0.03 in failing left ventricles. Endomyocardial biopsy specimens showed significant differences between controls and heart failure (controls 0.63+/-0.04 vs. heart failure 0.52+/-0.02, P<0.05). Of the two identified AT2 transcripts, mRNA 1/2/3 was the most abundant in the human heart (92%). Luciferase reporter gene assays were performed to test the effect of the various 5' untranslated regions (5' UTRs) on protein expression. Among the constructs which contained the AT1 promoter/AT1 5' UTRs the plasmid Ex 1/2/5 exhibited 27% lower luciferase activity than Ex 1/5 (n=24, P<0.001), and Ex 1/2/3/5 expressed only 35.9% of Ex 1/5 activity (P<0.001). Among the reporter gene plasmids with the AT2 promoter/AT2 5' UTRs the construct Ex 1/2/3 expressed a 31% lower luciferase activity than Ex 1/3 (n=20, P<0.001). In conclusion, alternative splicing may represent a mechanism of ATR regulation in vivo. In the human heart, AT1 splice patterns differ distinctly between atria and ventricles and to a lesser degree between controls and failing hearts. This may lead to differences in AT1 mRNA translation into protein in the various cardiac areas and under different pathophysiological conditions.

Decreased expression of ventricular angiotensin receptor type 1 mRNA after human heart transplantation.

Myocardial angiotensin receptors of type 1 (AT1) are downregulated at the protein and mRNA levels in human heart failure. No data are available for the transplanted human heart, which frequently exhibits functional alterations. The aim of the present study was the quantitation of ventricular AT1 mRNA content in endomyocardial biopsies from patients after heart transplantation. For the determination of AT1 mRNA we used a novel quantitative reverse transcription polymerase chain reaction with low variance (6%) based on an internal AT1 cRNA standard, liquid-phase hybridization of polymerase chain reaction products in microtiter plates, and quantitation by enzyme-linked immunosorbent assay. Right ventricular biopsies from 16 patients after heart transplantation (left ventricular ejection fraction 67 +/- 7%) were compared with 12 patients with normal cardiac function (left ventricular ejection fraction 62 +/- 5%). A 46% lower AT1 mRNA content was found in biopsies from the 16 patients after heart transplantation than in the 12 controls (heart transplantation, 200 +/- 25 AT1 mRNA copies/ng RNA; controls, 368 +/- 50; P < 0.01). When AT1 mRNA content was related to the stably expressed GAPDH mRNA, a 49% decrease was detected (AT1/GAPDH: patients, 2.4 +/- 0.25; controls, 4.7 +/- 0.6; P < 0.006). No association between the extent of AT1 downregulation and clinical or hemodynamic variables was detected. In the human heart ventricular AT1 is downregulated after orthotopic heart transplantation. The decrease in AT1 mRNA is not associated with altered systolic function. This may partially reflect a loss of autonomic nerves and thus altered nervous control of the heart.

Adenovirus-mediated overexpression and stimulation of the human angiotensin II type 2 receptor in porcine cardiac fibroblasts does not modulate proliferation, collagen I mRNA expression and ERK1/ERK2 activity, but inhibits protein tyrosine phosphatases.

The contribution of the angiotensin (Ang) II type 2 receptor (AT2R) to cardiac hypertrophy is still controversial. Here we examined the effect of overexpressing the human AT2R in cultured porcine cardiac fibroblasts (pFib) on proliferation, procollagen I mRNA expression, and - as putatively underlying signal-transduction pathways - on mitogen-activated protein kinase ERK1/ERK2 and phosphotyrosine phosphatase activities. As quantitated by 125I-(Sar1,Ile8)-Ang II binding, transduction of cardiac fibroblasts with the adenoviral AT2R expression vector led to a six- to tenfold higher AT2 than endogenous Ang II type 1 receptor (AT1R) expression. The overexpressed AT2R had the same apparent molecular mass as the endogenous AT2R in rat PC12W cells. Proliferation was not significantly lower in AT2R expressing pFib than in antisense-transduced controls (TA2) upon stimulation with Ang II (AT2R 110.5+/-4.8% vs. TA2 110.2+/-5.5%), Ang II plus the AT1R blocker Irbesartan (97.1+/-1.4% vs. 108.0+/-5.0; P=0.052) and the partial AT2R antagonist CGP42112 at the agonistic concentration of 50 nM (92.1+/-2.7% vs. 99.8+/-3.1%; P=0.053). Procollagen Ialpha2 (COL1A2) mRNA levels were quantitated by (a) northern blot analysis and (b) reverse transcriptase polymerase chain reaction. COL1A2/GAPDH (a) and COL1A2/beta-actin (b) ratios revealed no differences between AT2R-transduced fibroblasts and antisense controls when stimulated with Ang II (1 microM, 24 h) plus Irbesartan and 10 ng/ml transforming growth factor beta1. Ang II stimulation of the endogenous AT1R increased extracellular signal regulated kinase 1/2 activities. This response was reduced by Irbesartan, but PD123319 had no effect. Time course and magnitude of Ang II stimulated ERK1/ERK2 activation was identical in AT2R-transduced and control cells. Also, neither simultaneous nor Ang II pre-stimulation, suggested to induce gene expression of the MAP kinase phosphatase 1, modulated phorbol myristate acetate-stimulated ERK1/ERK2 activation in AT2R-transduced pFib, in AT2R-transduced human umbilical vein endothelial cells, and in PC12W cells. By the use of a tyrosine phosphatase assay we observed an inhibition of phosphotyrosine phosphatase activity by 30.8% (P=0.009, n=5) after 5 min Ang II stimulation of AT2R-expressing pFib. Immunoprecipitation-tyrosine phosphatase assays revealed that inhibition of phosphotyrosine phosphatase 1B, which regulates insulin signaling, contributed to this effect. In conclusion, stimulation of the overexpressed human AT2R in porcine cardiac fibroblasts inhibited tyrosine phosphatase activity but had no significant effect on fibroblast functions related to cardiac fibrosis. It is conceivable that possible antifibrotic AT2R effects are species specific and/or require the interaction between fibroblasts and cardiomyocytes, probably via paracrine factors, or mechanical load.

Expression of ACE mRNA in the human atrial myocardium is not dependent on left ventricular function, ACE inhibitor therapy, or the ACE I/D genotype.

The activity of the cardiac renin-angiotensin system is altered in human heart failure, but the regulatory mechanisms are unknown. We analyzed whether angiotensin-converting enzyme (ACE) mRNA expression in heart failure is altered in the atrial myocardium, and whether a correlation exists between atrial ACE mRNA expression and the parameters of left ventricular function. We also investigated whether the use of ACE inhibitors or the ACE I/D genotype modulates the atrial ACE mRNA content. For this purpose patients who were to undergo routine cardiac surgery were selected in a prospective manner according to left ventricular function and ACE inhibitor therapy. Samples of atrial myocardium were taken, and ACE mRNA expression was determined by internally standardized reverse transcription polymerase chain reaction. Atrial ACE mRNA expression did not differ in patients with left ventricular ejection fraction higher than 55% (2423+/-199 copies/ng RNA) and those with a value less than 45% (2661+/-143 copies/ng RNA, n.s.). ACE mRNA expression also did not differ in patients using ACE inhibitors (2585+/-175 copies/ng RNA) and those not using ACE inhibitors (2476+/-185 copies/ng RNA). Furthermore, atrial ACE mRNA expression was not affected by the ACE genotype (DD 2573+/-203, ID 2472+/-215, II 2563+/-249 copies/ng RNA). We conclude that the regulation of atrial ACE mRNA expression occurs predominantly by local mechanical or para- or autocrine factors.

Reduced atrial angiotensin receptor type 1 mRNA content in end-stage human heart failure: assessment by a novel quantitative PCR-ELISA technique.

The number of atrial angiotensin II binding sites is reduced in end-stage human heart failure. The goals of our study were the development of a quantitative polymerase chain reaction for angiotensin II receptor type 1 mRNA to determine the angiotensin receptor type 1 (AT1) mRNA content in the atria of patients with end-stage heart failure. We established a quantitative PCR based on coamplification of AT1 wild-type and an internal standard in the same PCR, followed by liquid-phase hybridization of PCR products in microtiter plates and quantitation by ELISA. Glyceraldehyde phosphate dehydrogenase mRNA in the same samples was used to relate the AT1 mRNA content to a stably expressed reference gene. Atrial samples from 11 patients with end-stage heart failure obtained at cardiac transplantation were compared with atrial samples from 11 patients with normal cardiac function undergoing routine cardiac surgery. A PCR/ELISA system with a variance of about 6% after reverse transcription and a linear measuring range was established. In the samples from 11 patients with end-stage heart failure a 58% decrease in AT1 mRNA content was found in comparison with 11 controls (heart failure: 185,680 +/- 196,912 AT1 mRNA copies/microgram RNA, controls: 440,555 +/- 268,456, P < 0.02). When AT1 mRNA content was related to glyceraldehyde phosphate dehydrogenase mRNA, a 65% decrease was detected (AT1/glyceraldehyde phosphate dehydrogenase: heart failure: 4.84 +/- 5.18; controls: 13.74 +/- 7.77; P < 0.005). Standardization of PCR resulting in a low coefficient of variance, high reproducibility, and large sample capacity is possible using optimal internal standardization and the liquid-phase hybridization/ELISA system for detection. The optimized PCR procedure indicated downregulation of atrial AT1 in end-stage human heart failure, suggesting a reduced capacity of the atria to respond to angiotensin II stimulation in end-stage heart failure.