Alicja Bukowska

Angiotensin II Receptor Blockade Reduces Tachycardia-Induced Atrial Adhesion Molecule Expression

Background— Increased levels of inflammatory markers are predictors of thromboembolic events during atrial fibrillation (AF). Increased endocardial expression of adhesion molecules (ie, vascular cell adhesion molecule [VCAM] and intercellular adhesion molecule [ICAM]) could be an important link between initiation of inflammatory and prothrombogenic mechanisms responsible for thrombus development at the atrial endocardium (endocardial remodeling). Methods and Results— Tissue microarrays were used to screen right atrial tissue specimens obtained from 320 consecutive patients for differences in atrial expression of the prothrombogenic proteins VCAM-1, ICAM-1, thrombomodulin, plasminogen activator inhibitor-1, and von Willebrand factor. An in vitro organotypic human atrial tissue model and a pig model of rapid atrial pacing were used to determine the therapeutic impact of angiotensin II receptor blockade. Immunohistochemical analyses showed that all prothrombogenic proteins are expressed by endocardial cells. Using multivariable analysis, only the intensity of VCAM-1 expression was increased in patients with AF (P=0.03). Increased atrial VCAM-1 expression was confirmed by Western blotting in patients with persistent and paroxysmal AF (persistent AF 207±42% versus sinus rhythm 100±16%, P=0.028; paroxysmal AF 193±42%, P=0.024 versus sinus rhythm). In vitro pacing of ex vivo human atrial tissue slices confirmed that rapid activation causes VCAM-1 upregulation (mRNA and protein levels). Pacing-induced VCAM-1 expression was abolished by olmesartan. To confirm this finding in vivo, VCAM-1 expression was determined in 14 pigs after rapid atrial pacing (600 bpm). Atrial tachycardia caused an upregulation of VCAM-1 expression, which was prevented by irbesartan, consistent with the observed increase in plasma levels of angiotensin II. Alterations in the in vivo VCAM-1 expression were more pronounced in the left atrium (>5-fold compared with sham) than in the right atrium (3.5-fold compared with sham). Conclusions— AF and rapid atrial pacing both increase endocardial VCAM-1 expression, which can be attenuated by angiotensin II receptor blockade. This provides evidence that angiotensin II plays a pathophysiological role in prothrombotic endocardial remodeling.

Acute atrial tachyarrhythmia induces angiotensin II type 1 receptor-mediated oxidative stress and microvascular flow abnormalities in the ventricles.

Aims Patients with paroxysmal atrial fibrillation (AF) often present with typical angina pectoris and mildly elevated levels of cardiac troponin (non ST-segment elevation myocardial infarction) during an arrhythmic event. However, in a large proportion of these patients, significant coronary artery disease is excluded by coronary angiography. Here we explored the potential underlying mechanism of these events. Methods and results A total of 14 pigs were studied using a closed chest, rapid atrial pacing (RAP) model. In five pigs RAP was performed for 7 h (600 b.p.m.; n = 5), in five animals RAP was performed in the presence of angiotensin-II type-1-receptor (AT1-receptor) inhibitor irbesartan (RAP+Irb), and four pigs were instrumented without intervention (Sham). One-factor analysis of variance was performed to assess differences between and within the three groups. Simultaneous measurements of fractional flow reserve (FFR) and coronary flow reserve (CFR) before, during, and after RAP demonstrated unchanged FFR (P = 0.327), but decreased CFR during RAP (RAP: 67.7 ± 7.2%, sham: 97.2 ± 2.8%, RAP+Irb: 93.2 ± 3.3; P = 0.0013) indicating abnormal left ventricular (LV) microcirculation. Alterations in microcirculatory blood flow were accompanied by elevated ventricular expression of NADPH oxidase subunit Nox2 (P = 0.039), lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1, P = 0.004), and F2-isoprostane levels (P = 0.008) suggesting RAP-related oxidative stress. Plasma concentrations of cardiac troponin-I (cTn-I) increased in RAP (RAP: 613.3 ± 125.8 pmol/L vs. sham: 82.5 ± 12.5 pmol/L; P = 0.013), whereas protein levels of eNOS and LV function remained unchanged. RAP+Irb prevented the increase of Nox2, LOX-1, and F2-isoprostanes, and abolished the impairment of microvascular blood flow. Conclusion Rapid atrial pacing induces AT1-receptor-mediated oxidative stress in LV myocardium that is accompanied by impaired microvascular blood flow and cTn-I release. These findings provide a plausible mechanism for the frequently observed cTn-I elevation accompanied with typical angina pectoris symptoms in patients with paroxysmal AF and normal (non-stenotic) coronary arteries.

Immunohistochemical evidence for impaired neuregulin-1 signaling in the prefrontal cortex in schizophrenia and in unipolar depression.

Abstract:  In the central nervous system (CNS), neuregulin‐1 (NRG‐1) proteins function in neuronal migration, differentiation, and survival of oligodendrocytes. The NRG‐1 gene codes for at least 15 different isoforms, which may be classified on the basis of their molecular structure. At least two different haplotypes of the NRG‐1 gene may be associated with schizophrenia. An abnormal expression pattern of NRG‐1 mRNA was found in the prefrontal cortex of schizophrenic patients in comparison to controls. We here show that the NRG‐1α isoform is significantly reduced in white matter of the prefrontal cortex in schizophrenia but not in affective disorder. In the prefrontal gray matter, the density of NRG‐1α expressing neurons was reduced in individuals with schizophrenia and in unipolar patients. We studied brains of 22 schizophrenics, 12 patients with affective disorders (7 unipolar and 5 bipolar), and 22 matched controls. NRG‐1α immunoreactive material was detected with a polyclonal antiserum against the synthetic peptide from α‐type EGF‐like domain of human NRG. The demonstrated decreased number of NRG‐1 immunoreactive neurons in the brains of schizophrenics and patients with unipolar depression points to an important role of this NRG‐1α splice variant in neuropsychiatric disorders. Reduced NRG‐1α protein concentrations were found in brains of schizophrenics after Western blot analysis. The diminished expression of NRG‐1α strongly supports an early neurodevelopmental component to schizophrenia.

Cigarette smoking induces atrial fibrosis in humans via nicotine

Background: Cigarette smoking (CS) promotes endothelial dysfunction and atherosclerosis in the vascular bed. The impact of smoking on atrial myocardium is not defined in humans. Objective: To determine the effect of CS on the development of interstitial fibrosis in atrial myocardium. Design: Case–control study. Patients: 95 patients (46 smokers and 49 non-smokers) undergoing coronary artery bypass grafting (CABG). Main outcome measures: Amount of atrial fibrosis, collagen I, III and IV expression pattern, and quantitative reverse transcriptase-PCR. Occurrence of postoperative atrial fibrillation (AF). Results: In the study population, patient age correlated significantly with the amount of atrial fibrosis (r = 0.18; p<0.05). Nicotine misuse (pack years) was identified as the only factor related to atrial fibrosis in smokers (r = 0.311; p<0.05). The amount of fibrosis was higher in patients with postoperative AF (22.9% (6.2%) vs. 27.0% (8.2%); p<0.05). To show a causal relationship between CS and atrial fibrosis, atrial tissue slices from non-smokers (n = 8) were cultured in the presence of nicotine base (185 and 740 nmol/l). Nicotine base induced mRNA expression of collagen III (up to 10-fold) in a concentration-dependent manner resembling the immunohistological collagen expression pattern observed in CS. Conclusion: CS contributes to the development of atrial fibrosis via nicotine. Atrial fibrosis by itself has been shown to provide an arrhythmogenic substrate, which may increase the likelihood of the occurrence of atrial arrhythmias, including postoperative AF.

Localization of neuregulin-1α (heregulin-α) and one of its receptors, ErbB-4 tyrosine kinase, in developing and adult human brain

Abstract Using immunohistochemistry, Western blot analysis, and RT-polymerase chain reaction, we studied the distribution of neuregulin-1 splice variant α (NRG-1α) and one of its putative receptors, ErbB-4 tyrosine kinase, in human brain. In the pre- and perinatal human brain immunoreactivity was confined to numerous neurons, with the highest cell density found in cortical gray matter, hypothalamus and cerebellum. In the adult brain, single cortical gray and white matter neurons showed NRG-1α immunoreactivity. Occasionally, immunoreactive oligodendrocytes were observed. NRG-1α-expressing neurons were also found in the hypothalamus, hippocampus, basal ganglia and brain stem. Application of two antibodies recognizing α and β isoforms revealed a different distribution pattern in that many cortical and hippocampal pyramidal neurons were labeled. ErbB-4 immunoreactivity was expressed in both neurons and oligodendrocytes. Our data show that NRG-1α expression is lower in the adult human brain than in the developing brain, and, therefore, support a role for NRG-1α in brain development.

Effect of Telmisartan on Nitric Oxide–Asymmetrical Dimethylarginine System: Role of Angiotensin II Type 1 Receptor and Peroxisome Proliferator Activated Receptor γ Signaling During Endothelial Aging

Telmisartan, in addition to blocking angiotensin (Ang) II type 1 receptor (AT1R), activates peroxisome proliferator activated receptor γ (PPARγ) signaling that interferes with nitric oxide (NO) system. Because aging of endothelial cells (ECs) is hallmarked by a reduction in NO synthesis, we hypothesized that telmisartan increases NO formation by regulated asymmetrical dimethylarginine (ADMA)-dimethylarginine dimethylaminohydrolase (DDAH)-system through blocking AT1R and activating PPARγ signaling. To test this hypothesis, ECs were cultured with telmisartan, eprosartan, Ang II, and GW9662 (PPARγ antagonist) until the twelfth passage. During the process of aging, PPARγ protein expression decreased significantly, whereas the expression of AT1R increased. Telmisartan reversed these effects and dose-dependently decreased reactive oxygen species and 8-iso-prostaglandin (PG) F2α formation. This effect was associated with an upregulated activity and protein expression of DDAH, accompanied by a decrease in ADMA concentration, an increase in NO metabolites, and delayed senescence. Blockade of PPARγ signaling by GW9662 or PPARγ small-interference RNA prevented the effect of telmisartan on ADMA-DDAH-NO system. Coincubation with Ang II did not affect the effect of telmisartan-delayed senescence, whereas Ang II itself accelerated endothelial aging. Moreover, AT1R blocker eprosartan that did not influence PPARγ protein expression had no effect on ADMA system and senescence. We have demonstrated that telmisartan mainly by activating PPARγ signaling can alter the catabolism and release of ADMA as an important cardiovascular risk factor. We therefore propose that telmisartan translationally and posttranslationally upregulated DDAH expression via activation of PPARγ signaling, causing ADMA to diminish and increase NO synthesis sufficient to delay senescence.

Mitochondrial Dysfunction and Redox Signaling in Atrial Tachyarrhythmia

Accumulating evidence links calcium-overload and oxidative stress to atrial remodeling during atrial fibrillation (AF). Furthermore, atrial remodeling appears to increase atrial thrombogeneity, characterized by increased expression of adhesion molecules. The aim of this study was to assess mitochondrial dysfunction and oxidative stress–activated signal transduction (nuclear factor-κB [NF-κB], lectin-like oxidized low-density lipoprotein receptor [LOX-1], intercellular adhesion molecule-1 [ICAM-1], and hemeoxgenase-1 [HO-1]) in atrial tissue during AF. Ex vivo atrial tissue from patients with and without AF and, additionally, rapid pacing of human atrial tissue slices were used to study mitochondrial structure by electron microscopy and mitochondrial respiration. Furthermore, quantitative reverse transcription polymerase chain reaction (RT-PCR), immunoblot analyses, gel-shift assays, and enzyme-linked immunosorbent assay (ELISA) were applied to measure nuclear amounts of NF-κB target gene expression. Using ex vivo atrial tissue samples from patients with AF we demonstrated oxidative stress and impaired mitochondrial structure and respiration, which was accompanied by nuclear accumulation of NF-κB and elevated expression levels of the adhesion molecule ICAM-1 and the oxidative stress-induced markers HO-1 and LOX-1. All these changes were reproduced by rapid pacing for 24 hours of human atrial tissue slices. Furthermore, the blockade of calcium inward current with verapamil effectively prevented both the mitochondrial changes and the activation of NF-κB signaling and target gene expression. The latter appeared also diminished by the antioxidants apocynin and resveratrol (an inhibitor of NF-κB), or the angiotensin II receptor type 1 antagonist, olmesartan. This study demonstrates that calcium inward current via L-type calcium channels contributes to oxidative stress and increased expression of oxidative stress markers and adhesion molecules during cardiac tachyarrhythmia.

The impact of rapid atrial pacing on ADMA and endothelial NOS

BACKGROUND The endothelial nitric oxide synthase (eNOS) inhibitor asymmetric dimethylarginine (ADMA) is a well-established risk factor for oxidative stress, vascular dysfunction, and congestive heart failure. The aim of the present study was to determine the impact of rapid atrial pacing (RAP) on ADMA levels and eNOS expression. METHODS AND RESULTS ADMA levels were studied in 60 age- and gender-matched patients. Thirty five patients had persistent atrial fibrillation (AF)≥ 4months. In AF-patients, parameters were studied before and 24h after electrical cardioversion. Moreover, ADMA, eNOS expression, and calcium-handling proteins were studied in pigs subjected to RAP as well as in endothelial cell (EC) cultures. ADMA level was significantly higher in AF compared to sinus rhythm patients (p=0.024). ADMA was highest in AF-patients, who also showed elevated troponin T (TnT) levels. Moreover, ADMA showed a significant linear correlation to TnT (r=0.47; p<0.01). After electrical cardioversion ADMA returned to normal within 24h. In pigs, RAP for 7h increased ADMA levels (p=0.018) and TnI (p<0.05), and reduced mRNA expression of ventricular and aortic eNOS (-80%; p<0.05) compared to sham-control. However, ADMA per se did not affect eNOS mRNA level in EC cultures. CONCLUSION The current study shows that acute and persistent episodes of atrial tachyarrhythmia are associated with elevated ADMA levels accompanied by increased ischemic myocardial markers. Moreover, RAP increases ADMA and down-regulates eNOS expression in an ADMA-independent manner. We conclude that the combination of these two separate and potentially synergistic mechanisms may contribute to long-term vascular injury during atrial tachyarrhythmia.

Atrial fibrillation and rapid acute pacing regulate adipocyte/adipositas-related gene expression in the atria

PURPOSE Atrial fibrillation (AF) has been associated with increased volumes of epicardial fat and atrial adipocyte accumulation. Underlying mechanisms are not well understood. This study aims to identify rapid atrial pacing (RAP)/AF-dependent changes in atrial adipocyte/adipositas-related gene expression (AARE). METHODS Right atrial (RA) and adjacent epicardial adipose tissue (EAT) samples were obtained from 26 patients; 13 with AF, 13 in sinus rhythm (SR). Left atrial (LA) samples were obtained from 9 pigs (5 RAP, 4 sham-operated controls). AARE was analyzed using microarrays and RT-qPCR. The impact of diabetes/obesity on gene expression was additionally determined in RA samples (RAP ex vivo and controls) from 3 vs. 6 months old ZDF rats. RESULTS RAP in vivo of pigs resulted in substantial changes of AARE, with 66 genes being up- and 53 down-regulated on the mRNA level. Differential expression during adipocyte differentiation was confirmed using 3T3-L1 cells. In patients with AF (compared to SR), a comparable change in RA mRNA levels concerned a fraction of genes only (RETN, IGF1, HK2, PYGM, LOX, and NR4A3). RA and EAT were affected by AF to a different extent. In patients, concomitant disease contributes to AARE changes. CONCLUSIONS RAP, and to lesser extent AF, provoke significant changes in atrial AARE. In chronic AF, activation of this gene panel is very likely mediated by AF itself, AF risk factors and concomitant diseases. This may facilitate the development of an AF substrate by increasing atrial ectopic fat and fat infiltration of the atrial myocardium.

Coagulation factor Xa induces an inflammatory signalling by activation of protease-activated receptors in human atrial tissue

Activated factor X (FXa) is an important player in the coagulation cascade responsible for thrombin generation, which is activated during atrial fibrillation. Increasing evidence suggests that FXa influences cell signalling in various cell types by activating protease-activated receptors (PARs). It is so far not known if molecular effects of FXa affect atrial signal transduction. To study the effects of FXa, human atrial tissue slices were cultivated with FXa up to 24h. Additionally, rapid pacing was applied at 4Hz to resemble atrial fibrillation. The inhibitory impact of FXa antagonist (Rivaroxaban), protease-activated receptor 1 antagonist (SCH79797), and protease-activated receptor 2 antagonist (GB83) were analysed under experimental conditions. The exposure of atrial tissue to FXa resulted in the 1.7 fold upregulation of PAR2-mRNA, activation of MAP kinases (ERK1/2) and NF-κB signalling. Furthermore FXa increased the expression of adhesion molecule ICAM-1 (1.82 ± 0.20), chemokine IL-8 (1.94 ± 0.20), as well as prothrombotic molecule PAI-1 (1.52 ± 0.17). The combination of rapid pacing and FXa caused significant upregulation of PAR1 (2.82 ± 0.22), PAR2 (2.66 ± 0.40), ICAM-1 (2.13 ± 0.25), IL-8 (2.22 ± 0.24), LOX-1 (2.59 ± 0.35), and PAI-1 (2.65 ± 0.52) at the mRNA level. Rivaroxaban and GB83 prevented upregulation of PARs, ICAM-1, LOX-1, IL-8, and activation of MAP kinases. The elevation in the expression of PAI-1 was hindered in the presence of SCH79797, or Rivaroxaban. The present study indicates that FXa mediates inflammatory signalling in atrial tissue. Importantly, FXa and tachyarrhythmia act synergistically to increase expression of protease-activated receptors and inflammatory mediators. Rivaroxaban prevented effectively FXa-induced molecular effects in human atrial tissue particularly during rapid pacing.