PP085. Angiotensin II type 1 receptor antibodies increase angiotensin II sensitivity in pregnant rats.

INTRODUCTION: Pregnant women who subsequently develop preeclampsia are highly sensitive to infused angiotensin (Ang) II; the sensitivity persists postpartum. Activating autoantibodies against the Ang II type 1 (AT1) receptor are present in preeclampsia. In vitro and in vivo data suggest that they could be involved in the disease process. OBJECTIVES: The aim of the study was to show if AT1-AB generated by immunisation alters Ang II sensitivity in pregnant rats. METHODS: We generated and purified activating antibodies against the AT1 receptor (AT1-AB) by immunizing rabbits against the AFHYESQ epitope of the second extracellular loop, which is the binding epitope of endogenous activating autoantibodies against AT1 from patients with preeclampsia. We then purified AT1-AB using affinity chromatography with the AFHYESQ peptide. RESULTS: We were able to detect AT1-AB both by ELISA and a functional bioassay. We then passively transferred AT1-AB into pregnant rats, alone or combined with Ang II. AT1-AB activated protein kinase C-alpha and extracellular-related kinase 1/2. Passive transfer of AT1-AB alone or Ang II (435ng/kg per minute) infused alone did not induce a preeclampsia-like syndrome in pregnant rats. However, the combination (AT1-AB plus Ang II) induced hypertension, proteinuria, intrauterine growth retardation, and arteriolosclerosis in the uteroplacental unit. We next performed gene-array profiling of the uteroplacental unit and found that hypoxia-inducible factor 1alpha was upregulated by Ang II plus AT1-AB, which we then confirmed by Western blotting in villous explants. Furthermore, endothelin 1 was upregulated in endothelial cells by Ang II plus AT1-AB. We show that AT1-AB induces Ang II sensitivity. CONCLUSION: Our mechanistic study supports the existence of an "autoimmune-activating receptor" that could contribute to Ang II sensitivity and possibly to preeclampsia.

Relation between circulating angiotensin II type 1 receptor agonistic autoantibodies and soluble fms-like tyrosine kinase 1 in the pathogenesis of preeclampsia.

CONTEXT: Placental and circulatory soluble fms-like tyrosine kinase 1 (sFlt1) has proven to be elevated in pregnant women with preeclampsia, a disease characterized by hypertension, proteinuria, and endothelial dysfunction. Recent studies also demonstrated an autoantibody against the angiotensin II type 1 (AT1) receptor (AT1-AA) in that disease. OBJECTIVE: Both factors are discussed as key players in the etiology of preeclampsia. However, it has not yet been clarified whether these two circulating factors correlate and whether synergy determines the severity of pathology. DESIGN: AT1-AA was retrospectively determined by a bioassay and sFlt1 by an ELISA. PATIENTS: Serum from second-trimester pregnancies with normal or abnormal uterine perfusion and in women at term with or without pregnancy pathology was analyzed. RESULTS: Most of the preeclamptic patients were characterized by high sFlt1 levels and the presence of AT1-AA, although the agonistic effects of the antibody did not correlate with the sFlt1 concentrations (P = 0.85). Although AT1-AA was also detected in second-trimester pregnancies evidencing abnormal uterine perfusion without later pathology, sFlt1 was not significantly elevated in these pregnancies, compared with those with normal uterine perfusion. However, whereas women with abnormal perfusion and later pregnancy pathology did not differ in AT1-AA, compared with those with normal outcome, sFlt1 was significantly increased. Again, the two factors did not correlate (P = 0.15). CONCLUSIONS: We conclude that AT1-AA bioactivity and sFlt1 concentrations do not correlate, are not mutually dependent, and are thus probably involved in distinct pathogenetic mechanisms. Both factors in combination may not be causative for the early impaired trophoblast invasion and pathological uterine perfusion.

Norepinephrine-induced interleukin-6 increase in rat hearts: differential signal transduction in myocytes and non-myocytes.

Continuous i.v. infusion of norepinephrine in rats has been shown to induce early interleukin (IL)-6 mRNA expression in the left ventricle (LV) which was followed by hypertrophy and fibrosis. In this study, two approaches were used. In the first, NE (0.1 mg/kg per hour) was infused i.v. in rats for several time periods, and freshly obtained ventricular myocardium was dissociated into myocyte (MC) and non-myocyte (NMC) fractions. Second, isolated adult MCs and fibroblasts were treated with NE (10 microM). NE infusion (4 h, in vivo) caused an 11-fold increase in IL-6 mRNA in both cell populations. In vitro treatment of isolated adult MCs for 2 h and of fibroblasts for 1 h with NE induced a 3.5- and 23-fold maximum increase, respectively, in IL-6 mRNA. After in vivo NE treatment, the expression of the mRNA of the transcriptional factor of IL-6, C/EBP-beta, was elevated earlier (after 45 min of NE infusion) than IL-6 mRNA (after 4 h) and was seen in MCs and NMCs. The mRNAs of both receptors of IL-6, the soluble IL6R and gp130, were increased subsequently to IL-6 mRNA. Gp130 was elevated after 24 h and, like IL6R, predominantly in NMCs. In contrast, the IL6R protein and the downstream regulator STAT3 were increased only in MCs after 24 h of NE infusion. The mRNA of C/EBP-delta, which is regulated by STAT3, was elevated only in myocytes.

Expression of alpha1-adrenergic receptor subtypes in heart cell culture.

Three alpha1-AR subtypes have been cloned so far and are designated as alpha1a, alpha1b,, and alpha1d. Organ-specific distribution pattern and subtype-specific effects are known but not fully understood. To address a cell-type specific expression pattern in the heart we investigated expression pattern of alpha1-AR subtypes on RNA- and protein-level in heart tissue, cultured cardiomyocytes and non-myocytes of the rat. Each alpha1AR-subtype mRNA was present in neonatal and adult rat heart culture but the relative distribution pattern was significantly different. While the alpha1a-AR subtype is preferentially expressed in adult cardiomyocytes, the alpha1b-AR subtype was preferentially expressed in the non-myocyte cell fraction. The RT-PCR results were confirmed by Western-blotting (alpha1b) and immunocytochemical studies. Incubation with an alpha1-agonist (phenylephrine) for 72 h led to a significant reduction of the alpha1b-AR in neonatal heart cell culture on both mRNA and protein level. In contrast, incubation with an alpha1-antagonist (prazosin) induced a 1.6 fold upregulation of the alpha1a-AR mRNA without significant effects on radioligand binding and functional assay. The results indicate a distribution pattern of the alpha1-AR subtype which is specific for cell type and ontogeny of the rat heart and may be regulated by adrenergic agents.

The upregulation of angiotensin II receptor AT(1) in human preeclamptic placenta.

The human placenta has been considered to possess a locally generated renin-angiotensin system (RAS), which may play a physiological role in the regulation of uteroplacental blood circulation. The changes in the expression of such a placental RAS during pregnancy could be important for the physiological and pathophysiological aspects of some clinical disorders, such as pregnancy-induced hypertension, preeclampsia. In the present study, the alterations of expression and localization of placental angiotensin II receptor subtypes, namely AT(1) in patients with preeclampsia (elective caesarean delivery) were investigated and compared with controls (vaginal delivery and elective caesarian delivery) using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry respectively. Results from RT-PCR analysis revealed an upregulated expression of placental mRNA for AT(1) receptor subtype in patients with preeclampsia when compared with those in controls. In addition, there was also a significant activation of placental expression of angiotensinogen mRNA in patients with preeclampsia. Results from Western blot showed that the expression of AT(1) receptor was also upregulated. Immunohistochemical results further demonstrated that increased immunoreactivity for placental AT(1) receptor was predominantly localized to the thin layers of syncytiotrophoblasts and, to a less extent, the capillaries of the term placental villi. These data indicate that upregulation of placental RAS components, notably AT(1) receptor in the syncytiotrophoblasts, could play a pathophysiological role in patients with preeclampsia.

Acute hemodynamic effects during immunoadsorption in patients with dilated cardiomyopathy positive for beta 1-adrenoceptor autoantibodies.

In two recent studies immunoadsorption improved left ventricular function in patients with dilated cardiomyopathy (DCM) who were positive for cardiostimulatory autoantibodies against the human beta 1-adrenoceptor (beta 1-AAB). In this study we invasively measured acute hemodynamics during immunoadsorption. Three patients with DCM, who were positive for beta 1-AAB (NYHA-class III, left ventricular ejection fraction < 0.3), were treated with 1 immunoadsorption session daily on 5 consecutive days. Immunoadsorption induced a strong decrease of cardiac index, whereas systemic vascular resistance increased. Baseline levels were reached again several hours after therapy ended. We conclude that during immunoadsorption substantial hemodynamic changes occur in patients positive for beta 1-AAB.

Autoantibodies against the beta1 adrenoceptor from patients with dilated cardiomyopathy prolong action potential duration and enhance contractility in isolated cardiomyocytes.

Autoantibodies against the beta1-adrenoceptor (beta1-AAB) from patients with dilated cardiomyopathy (DCM) increase the beating frequency of cultured neonatal rat cardiomyocytes. This effect is accompanied by only a small increase in cAMP production. Here we have investigated whether beta1-AAB affect electrophysiological properties and cell shortening of isolated cardiomyocytes by interacting with the beta1-adrenoceptor. Beta1-AAB were obtained during immunoadsorption of patients with DCM and were used for experiments in isolated myocytes cultured from neonatal rat hearts, or freshly isolated from adult rat ventricles or from human right atria. The unselective beta -adrenoceptor agonist (-)-isoprenaline was studied for comparison. Immunoglobulin G (IgG) antibodies increased the spontaneous beating frequency of neonatal rat cardiomyocytes to a lesser degree than (-)-isoprenaline, but both effects were maximum and stable after 2 min. In rat ventricular and human atrial myocytes, IgG increased action potential duration (APD) in a concentration-dependent manner with larger effects on late than on early repolarization phases. Similar effects were obtained with purified beta1-AAB, whereas flow through of the chromatography column was ineffective. (-)-isoprenaline prolonged APD to the same extent during plateau and late phase of repolarization. beta1-AAB increased L-Type Ca2+ current in correspondence with the prolongation of APD. The effects of beta1-AAB and (-)-isoprenaline on APD were strongly attenuated after preincubation of the myocytes with the selective beta1-adrenoceptor antagonist (-)-bisoprolol. In addition, beta1-AAB increased cell shortening in ventricular myocytes from adult rat hearts. Beta1-AAB enhancing the beating frequency of cultured cardiomyocytes, increase L-Type Ca2+ current, APD and contractility in freshly isolated cardiomyocytes mediated via beta1-adrenoceptors. These effects may contribute to beta1-adrenoceptor-mediated cardiotoxicity in heart failure.

Decreased oxidative stress in patients with idiopathic dilated cardiomyopathy one year after immunoglobulin adsorption.

OBJECTIVES: In a substudy to a recently reported investigation that demonstrated the benefit of immunoglobulin adsorption (immunoadsorption) for patients with idiopathic dilated cardiomyopathy (IDC), we tested whether this benefit is associated with a reduction of oxidative stress. BACKGROUND: The progression of cardiomyopathy is believed to be related to the increase of oxidative stress. Therefore, reduction of oxidative stress could be one of the effects of immunoadsorption for improvement of cardiac performance and clinical status. METHODS: Plasma markers for oxidative stress-thiobarbituric acid-reactive substances (TBARS), lipid peroxides (LPO), anti-oxidized low-density lipoprotein-autoantibodies (anti-oxLDL-AB), thiol groups and vitamin E-were compared in 31 patients, of whom 16 underwent immunoadsorption and 15 received conventional treatment (controls). All patients received a daily supplement of vitamins, minerals and trace elements. RESULTS: After one year, TBARS (p = 0.026), LPO (p = 0.026) and anti-oxLD

HELLP syndrome in the 18th week of gestation in association with elevated angiotensin AT(1)-receptor autoantibodies.

We present a 24-year-old woman with a twin pregnancy who was with a typical HELLP syndrome at the 18th week of pregnancy. One fetus was dead, while the other was severely growth retarded. Our patient had agonistic autoantibodies directed at the angiotensin AT(1)-receptor. Termination of the pregnancy proved necessary. This report is the first to our knowledge associating HELLP syndrome with angiotensin AT(1)-receptor antibodies. Since the antibodies may have a pathogenic significance, their removal could permit the prolongation of pregnancy in preeclamptic and HELLP syndrome patients.

A monoclonal antibody against the immunodominant epitope of the ribosomal P2beta protein of Trypanosoma cruzi interacts with the human beta 1-adrenergic receptor.

Monoclonal antibodies were raised against a recombinant ribosomal P2beta protein of Trypanosoma cruzi. One of these reacted with the C terminus of this protein (peptide R13, EEEDDDMGFGLFD) and epitope mapping confirmed that this epitope was the same as the one defined by the serum of immunized mice, and similar to the previously described chronic Chagas' heart disease (cChHD) anti-P epitope. Western blotting showed that the monoclonal antibody recognized the parasite ribosomal P proteins, as well as the human ribosomal P proteins. Electron microscopy showed that it stained different structures in parasite and human cells. Interestingly, surface plasmon resonance measurements indicated that the affinity for the parasite ribosomal P protein epitope (R13) was five times higher than for its human counterpart (peptide H13, EESDDDMGFGLFD). Since the human epitope contained an acidic region (EESDD) similar to the AESDE peptide recognized by cChHD patients in the second extra-cellular loop of the human beta1-adrenergic receptor, the biological activity of the antibody was assessed on neonatal rat cardiomyocytes in culture. The monoclonal antibody had an agonist-like effect. These results, together with the fact that the monoclonal reacted in Western blots with the different isoforms of the heart beta1-adrenergic receptor, confirm the possible pathogenic role of antibodies against the parasite ribosomal P protein based on their cross-reaction with the human beta1-adrenergic receptor.

Immunohistological changes in dilated cardiomyopathy induced by immunoadsorption therapy and subsequent immunoglobulin substitution.

BACKGROUND: Immunoadsorption (IA) and subsequent immunoglobulin (Ig) G substitution represent an additional therapeutic approach in dilated cardiomyopathy (DCM). It remains to be elucidated whether this treatment modulates myocardial inflammation, which is possibly a causal factor of ventricular dysfunction. METHODS AND RESULTS: From 25 DCM patients (EF <30%), 12 patients were randomized for IA therapy and subsequent IgG substitution at 1-month intervals until month 3. Before (<7 days) and after IA therapy, right ventricular biopsies were obtained from all patients. Biopsies were also obtained at intervals of 3 months from 13 patients without IA/IgG treatment (controls). IA/IgG treatment induced improvement in left ventricular ejection fraction from 21.3+/-1.7% (+/-SEM) to 27.0+/-1.3% (P<0.01 versus baseline/controls) and reduction of the beta-receptor autoantibody serum levels (P<0.01 versus baseline/controls). The number of CD3 cells decreased from 5.7+/-0.8 to 2.9+/-0.5 cells/mm(2) (P<0.01 versus baseline/controls). This decline was paralleled by a decrease in CD4 (P<0.01 versus baseline/controls) and CD8 (P<0.05 versus baseline/controls) lymphocytes. The number of leukocyte common antigen-positive cells (leukocytes) was reduced from 20.0+/-3.2 to 9.9+/-2.8 cells/mm(2) (P<0.01 versus baseline/P<0.05 versus controls). HLA class II expression decreased from 2.1+/-0.7% to 1.1+/-0.4% (P<0.05 versus controls/baseline). The number of immunopositive cells and the expression of HLA class II in controls remained stable. In both groups, the degree of fibrosis remained unchanged. CONCLUSIONS: IA and subsequent IgG substitution mitigate myocardial inflammation in DCM.

Inducible nitric oxide synthase in the myocard.

Recognition of significance of nitric oxide synthases (NOS) in cardiovascular regulations has led to intensive research and development of therapies focused on NOS as potential therapeutic targets. However, the NOS isoform profile of cardiac tissue and subcellular localization of NOS isoforms remain a matter of debate. The aim of this study was to investigate the localization of an inducible NOS isoform (NOS2) in cardiomyocytes. Employing a novel immunocytochemical technique of a catalyzed reporter deposition system with tyramide and electron microscopical immunocytochemistry complemented with Western blotting and RT-PCR, we detected NOS2 both in rat neonatal and adult cultured cardiomyocytes and in the normal myocard of adult rats as well as in the human myocard of patients with dilative cardiomyopathy. NOS2 was targeted predominantly to a particulate component of the cardiomyocyte--along contractile fibers, in the plasma membrane including T-tubules, as well as in the nuclear envelope, mitochondria and Golgi complex. Our results point to an involvement of NOS2 in maintaining cardiac homeostasis and contradict to the notion that NOS2 is expressed in cardiac tissue only in response to various physiological and pathogenic factors. NOS2 targeting to mitochondria and contractile fibers suggests a relationship of NO with contractile function and energy production in the cardiac muscle.

Phosphorylation of phospholamban at threonine-17 in the absence and presence of beta-adrenergic stimulation in neonatal rat cardiomyocytes.

The site-specific phospholamban phosphorylation was studied with respect to the interplay of cAMP- and Ca(2+)signaling in neonatal rat cardiomyocytes. To elucidate the signal pathway(s) for the activation of Ca(2+)/calmodulin-dependent protein kinase (CaMKII) we studied Thr17 phosphorylation of phospholamban in dependence of Ca(2+)channel activation by S(-)-Bay K8644 and in dependence of the depletion of the sarcoplasmic reticulum Ca(2+)stores by ryanodine or thapsigargin in the absence or presence of beta -adrenergic stimulation. The isoproterenol (0.1 microM)-induced Thr17 phosphorylation was potentiated 2.5-fold in presence of 1 microM S(-)-Bay K8644. Interestingly, S(-)-Bay K8644 alone was also able to induce Thr17 phosphorylation in a dose- and time-dependent fashion. Ryanodine (1.0 microM) reduced both the isoproterenol (0.1 microM) and S(-)-Bay K8644-(1 microM) mediated Thr17 phosphorylation by about 90%. Thapsigargin (1 microM) diminished the S(-)-Bay K8644 and isoproterenol-associated Thr17 phosphorylation by 53.5+/-6.3% and 92. 5+/-11.1%, respectively. Ser16 phosphorylation was not affected under these conditions. KN-93 reduced the Thr17 phosphorylation by S(-)-Bay K8644 and isoproterenol to levels of 1.1+/-0.3% and 8.6+/-2. 1%, respectively. However, the effect of KN-93 was attenuated (47. 8+/-3.6%) in isoproterenol prestimulated cells. Protein phosphatase inhibition by okadaic acid increased exclusively the Ser16 phosphorylation. In summary, our results reflect a cross-talk between beta -adrenoceptor stimulation and intracellular Ca(2+)at the level of CaMKII-mediated phospholamban phosphorylation in neonatal rat cardiomyocytes. We report conditions which exclusively produce Thr17 or Ser16 phosphorylation. We postulate that Ca(2+)transport systems of the sarcoplasmic reticulum are critical determinants for the activation of CaMKII that catalyzes phosphorylation of phospholamban.

Modulation of beta1-adrenoceptor activity by domain-specific antibodies and heart failure-associated autoantibodies.

OBJECTIVES: Our study attempted to gain further understanding of the allosteric effects of human autoantibodies on beta1-adrenergic receptor (beta1-AR) function. BACKGROUND: Recently, we reported on the existence of activating anti-beta1-AR antibodies in patients with dilated cardiomyopathy (DCM 26% prevalence) or ischemic cardiomyopathy (ICM, 10% prevalence); however, their functional effects have not yet been thoroughly characterized. METHODS: In this study we detected functionally active receptor-antibodies in 8 out of 30 DCM patients. Their immunological and functional properties were analyzed using both synthetic receptor-peptides and intact recombinant human beta1-AR, and were compared with those of heterologous antibodies to selected beta1-AR domains generated in rabbits and mice. RESULTS: Rabbit, mouse, and human anti-beta1-AR against the second extracellular domain preferentially bound to a native receptor conformation and impaired radioligand binding to the receptor. However, their functional effects differed considerably: Rabbit and mouse antibodies decreased both basal and agonist-stimulated cAMP production, whereas the patient antibodies (n = 8) increased basal, and six of them also increased agonist-stimulated receptor activity (i.e., acted as receptor-sensitizing agents). Two out of eight human anti-beta1-AR increased basal but decreased agonist-stimulated receptor activity (i.e., acted as partial agonists). CONCLUSIONS: Antibodies against the same small beta1-AR domain can have very divergent allosteric effects, ranging from inhibitory to agonist-promoting activities. Activating autoantibodies were associated with severe cardiac dysfunction and thus might be involved in the development and/or course of human cardiomyopathy.

Midterm follow-up of patients who underwent removal of a left ventricular assist device after cardiac recovery from end-stage dilated cardiomyopathy.

OBJECTIVE: Cardiac recovery in end-stage idiopathic dilated cardiomyopathy recently occurred after temporary support with a left ventricular assist device. We report the case histories of patients who underwent removal of the device more than 4 years ago. METHODS: Since June 1994, 23 patients with end-stage idiopathic dilated cardiomyopathy who were supported by a left ventricular assist device or biventricular assist device for 1 to 26 months (mean, 6 months) underwent removal of the device after complete or extensive cardiac recovery, as revealed by echocardiography. RESULTS: Seven patients (group A) had recurrent cardiac failure after 4 to 24 months. Transplantation was performed in 6 patients, and one died while on the waiting list. Three patients died of noncardiac causes within a period of 4 months and 3 days after removal of the assist device. Stable cardiac recovery occurred in 13 patients (group B) for 3 to 49 months (mean, 23 months). At the time of implantation, there were no significant differences between the groups with regard to age, hemodynamics, left ventricular ejection fraction, left ventricular internal diameter in diastole, and autoantibody levels. The increase of ejection fraction and the decrease of left ventricular internal diameter in diastole after 2 months were highly significant. The patients in group A had longer histories of heart failure and first cardiac symptoms and duration of assist when compared with group B. Group B demonstrated a quicker cardiac recovery on the assist device, and thus support was shorter. Also, the degree of recovery at assist device explantation was more complete in group B. The age at the time of device placement was the only influencing factor for duration on the assist device. The probability of recurrence of heart failure was influenced by the duration of heart failure. CONCLUSIONS: In selected patients with idiopathic dilated cardiomyopathy, lasting recovery can be achieved after unloading with a left ventricular assist device. Lasting cardiac recovery seems to be related to functional normalization and a more rapid recovery during the unloading period.

Modulation of cardiocyte functional activity by antibodies against trypanosoma cruzi ribosomal P2 protein C terminus.

Antibodies against the Trypanosoma cruzi ribosomal P2beta protein (TcP2beta) have been associated with the chronic cardiac pathology of Chagas' disease in humans. Using synthetic peptides spanning the entire TcP2beta molecule, we investigated their epitope recognition by antibodies from mice chronically infected with T. cruzi and from mice immunized with two recombinant TcP2betas. We found clear differences in epitope recognition between antibodies from T. cruzi-infected mice and mice immunized with two different recombinant TcP2betas associated with different schedules of immunization. Major epitopes recognized by antibodies from mice immunized with recombinant glutathione S-transferase (GST) or histidine (Hist) fusion TcP2beta (GST-TcP2beta or Hist-TcP2beta) are located in the central and hinge regions of the molecule. Nevertheless, mice immunized with Hist-TcP2beta were also able to elicit antibodies against the TcP2beta C terminus, a region which is highly conserved in both T. cruzi and mammal ribosomal P proteins. Strikingly, antibodies from infected animals recognized only the TcP2beta C terminus. By using these antisera with distinct profiles of epitope recognition, it could be shown that only C terminus-specific antibodies were able to increase the beating frequency of cardiomyocytes from neonatal rats in vitro by selective stimulation of the beta1-adrenergic receptor. Thus, antibodies against the TcP2beta C terminus elicited in the absence of infection are able to modulate a functional activity of host cells through a molecular mimicry mechanism.

Autoantibodies against the angiotensin receptor (AT1) in patients with hypertension.

Sera from patients with malignant essential hypertension (n = 14), malignant secondary hypertension mainly attributable to renovascular diseases (n = 12) and renovascular diseases without malignant hypertension (n = 11) and from normotensive healthy blood donors (n = 35) were studied for the presence of autoantibodies against G-protein-coupled cardiovascular receptors. Autoantibodies against the angiotensin II receptor (AT1) were detected in 14, 33, 18 and 14% of patients with malignant essential hypertension, malignant secondary hypertension, renovascular diseases and control patients, respectively. Sensitivity of the enzyme immunoassay was assessed as 5 microg/ml IgG. Patients did not show antibodies against bradykinin (B2) or angiotensin II subtype 2 (AT2) receptors. Autoantibodies affinity-purified from positive patients localized AT receptors in Chinese hamster ovary transfected cells, and displayed a positive chronotropic effect on cultured neonatal rat cardiomyocytes. These results demonstrate the existence of autoantibodies against a functional extracellular domain of human AT1 receptors in patients with malignant hypertension, and suggest that these autoantibodies might be involved in the pathogenesis of malignant hypertension.