Identification of Mineralocorticoid Receptors, Aldosterone, and Its Processing Enzyme CYP11B2 on Parasympathetic and Sympathetic Neurons in Rat Intracardiac Ganglia

Recent interest has focused on the mineralocorticoid receptor (MR) and its impact on the myocardium and the performance of the heart. However, there is a lack of evidence about MR expression and its endogenous ligand aldosterone synthesis with specific regard to the intrinsic cardiac nervous system. Therefore, we looked for evidence of MR and aldosterone in sympathetic and parasympathetic neurons of intracardiac ganglia. Tissue samples from rat heart atria were subjected to conventional reverse-transcriptase polymerase chain reaction (PCR), Western blot, and double immunofluorescence confocal analysis of MR, corticosterone-inactivating enzyme 11β-hydroxysteroid-dehydrogenase-2 (11β-HSD2), aldosterone, and its processing enzyme CYP11B2 together with the neuronal markers vesicular acetylcholine transporter (VAChT) and tyrosine hydroxylase (TH). Our results demonstrated MR, 11β-HSD2, and CYP11B2 specific mRNA and protein bands in rat heart atria. Double immunofluorescence labeling revealed coexpression of MR immunoreactivity with VAChT in large diameter parasympathetic principal neurons. In addition, MR immunoreactivity was identified in TH-immunoreactive small intensely fluorescent (SIF) cells and in nearby VAChT- and TH-immunoreactive nerve terminals. Interestingly, the aldosterone and its synthesizing enzyme CYP11B2 and 11β-HSD2 colocalized in MR– immunoreactive neurons of intracardiac ganglia. Overall, this study provides first evidence for the existence of not only local expression of MR, but also of 11β-HSD2 and aldosterone with its processing enzyme CYP11B2 in the neurons of the cardiac autonomic nervous system, suggesting a possible modulatory role of the mineralocorticoid system on the endogenous neuronal activity on heart performance

Histopathological Changes in the Kidney following Congestive Heart Failure by Volume Overload in Rats

Background. This study investigated histopathological changes and apoptotic factors that may be involved in the renal damage caused by congestive heart failure in a rat model of infrarenal aortocaval fistula (ACF). Methods. Heart failure was induced using a modified approach of ACF in male Wistar rats. Sham-operated controls and ACF rats were characterized by their morphometric and hemodynamic parameters and investigated for their histopathological, ultrastructural, and apoptotic factor changes in the kidney. Results. ACF- induced heart failure is associated with histopathological signs of congestion and glomerular and tubular atrophy, as well as nuclear and cellular degeneration in the kidney. In parallel, overexpression of proapoptotic Bax protein, release of cytochrome C from the outer mitochondrial membrane into cell cytoplasm, and nuclear transfer of activated caspase 3 indicate apoptotic events. This was confirmed by electron microscopic findings of apoptotic signs in the kidney such as swollen mitochondria and degenerated nuclei in renal tubular cells. Conclusions. This study provides morphological evidence of renal injury during heart failure which may be due to caspase-mediated apoptosis via overexpression of proapoptotic Bax protein, subsequent mitochondrial cytochrome C release, and final nuclear transfer of activated caspase 3, supporting the notion of a cardiorenal syndrome

Chronic Naltrexone Therapy Is Associated with Improved Cardiac Function in Volume Overloaded Rats

Purpose: Myocardial opioid receptors were demonstrated in animals and humans and seem to colocalize with membranous and sarcolemmal calcium channels of the excitation-contraction coupling in the left ventricle (LV). Therefore, this study investigated whether blockade of the cardiac opioid system by naltrexone would affect cardiac function and neurohumoral parameters in Wistar rats with volume overload-induced heart failure. Methods: Volume overload in Wistar rats was induced by an aortocaval fistula (ACF). Left ventricular cardiac opioid receptors were identified by immunohistochemistry and their messenger ribonucleic acid (mRNA) as well as their endogenous ligand mRNA quantified by real-time polymerase chain reaction (RT-PCR). Following continuous delivery of either the opioid receptor antagonist naltrexone or vehicle via minipumps (n = 5 rats each), hemodynamic and humoral parameters were assessed 28 days after ACF induction. Sham-operated animals served as controls. Results: In ACF rats mu-, delta-, and kappa-opioid receptors colocalized with voltage-gated L-type Ca2+ channels in left ventricular cardiomyocytes. Chronic naltrexone treatment of ACF rats reduced central venous pressure (CVP) and left ventricular end-diastolic pressure (LVEDP), and improved systolic and diastolic left ventricular functions. Concomitantly, rat brain natriuretic peptide (rBNP-45) and angiotensin-2 plasma concentrations which were elevated during ACF were significantly diminished following naltrexone treatment. In parallel, chronic naltrexone significantly reduced mu-, delta-, and kappa-opioid receptor mRNA, while it increased the endogenous opioid peptide mRNA compared to controls. Conclusion: Opioid receptor blockade by naltrexone leads to improved LV function and decreases in rBNP-45 and angiotensin-2 plasma levels. In parallel, naltrexone resulted in opioid receptor mRNA downregulation and an elevated intrinsic tone of endogenous opioid peptides possibly reflecting a potentially cardiodepressant effect of the cardiac opioid system during volume overload

Pathological alterations in liver injury following congestive heart failure induced by volume overload in rats

Heart failure has emerged as a disease with significant public health implications. Following progression of heart failure, heart and liver dysfunction are frequently combined in hospitalized patients leading to increased morbidity and mortality. Here, we investigated the underlying pathological alterations in liver injury following heart failure. Heart failure was induced using a modified infrarenal aortocaval fistula (ACF) in male Wistar rats. Sham operated and ACF rats were compared for their morphometric and hemodynamic data, for histopathological and ultrastructural changes in the liver as well as differences in the expression of apoptotic factors. ACF-induced heart failure is associated with light microscopic signs of apparent congestion of blood vessels, increased apoptosis and breakdown of hepatocytes and inflammatory cell inifltration were observed. The glycogen content depletion associated with the increased hepatic fibrosis, lipid globule formation was observed in ACF rats. Moreover, cytoplasmic organelles are no longer distinguishable in many ACF hepatocytes with degenerated fragmented rough endoplasmic reticulum, shrunken mitochondria and heavy cytoplasm vacuolization. ACF is associated with the upregulation of the hepatic TUNEL-positive cells and proapoptotic factor Bax protein concomitant with the mitochondrial leakage of cytochrome C into the cell cytoplasm and the transfer of activated caspase 3 from the cytoplasm into the nucleus indicating intrinsic apoptotic events. Taken together, the results demonstrate that ACF-induced congestive heart failure causes liver injury which results in hepatocellular apoptotic cell death mediated by the intrinsic pathway of mitochondrial cytochrome C leakage and subsequent transfer of activated caspase 3 into to the nucleus to initiate overt DNA fragmentation and cell death

Checkpoints are blind to replication restart and recombination intermediates that result in gross chromosomal rearrangements

Replication fork inactivation can be overcome by homologous recombination, but this can cause gross chromosomal rearrangements that subsequently missegregate at mitosis, driving further chromosome instability. It is unclear when the chromosome rearrangements are generated and whether individual replication problems or the resulting recombination intermediates delay the cell cycle. Here we have investigated checkpoint activation during HR-dependent replication restart using a site-specific replication fork-arrest system. Analysis during a single cell cycle shows that HR-dependent replication intermediates arise in S phase, shortly after replication arrest, and are resolved into acentric and dicentric chromosomes in G2. Despite this, cells progress into mitosis without delay. Neither the DNA damage nor the intra-S phase checkpoints are activated in the first cell cycle, demonstrating that these checkpoints are blind to replication and recombination intermediates as well as to rearranged chromosomes. The dicentrics form anaphase bridges that subsequently break, inducing checkpoint activation in the second cell cycle

Adaptive changes of the cardiac opioid system due to volume induced heart failure

Adaptive Veränderungen des kardialen Opioidsystems im Verlauf einer chronischen kongestiven Herzinsuffizienz wurden bisher nur marginal erforscht. In dieser Arbeit sollte daher zunächst die exakte Lokalisation der Opioidrezeptoren δ (DOR) und κ (KOR) im Myokard gesunder und herzinsuffizienter Ratten untersucht werden. Im linken Ventrikel gesunder Ratten konnten Opioidrezeptor-spezifische Membran-Bindungsstellen aller drei Opioidrezeptoren mittels Radioligand-Rezeptor-Bindungsstudien nachgewiesen werden. Im nächsten Schritt wurden DOR und KOR auf Kardiomyozyten des LV immunhistochemisch dargestellt. Es zeigte sich eine Kolokalisation der ORs mit dem plasmamembranständigen Dihydropyridin-Rezeptor (Cav1.2) und dem intrazellulären sarkoplasmatischen Ryanodin-Rezeptor. Eine Erweiterung dieser Arbeit stellte die Untersuchung adaptiver Veränderungen des kardialen Opioidsystems im Verlauf einer chronischen kongestiven Herzinsuffizienz dar. Dazu wurde mittels einer infrarenalen, aortokavalen Fistel (ACF) eine biventrikuläre Volumenüberladung induziert. 28 ± 2 Tage nach der ACF-Induktion zeigten die Tiere eindeutige morphometrische und hämodynamische Charakteristika einer CHF. Anschließend wurden die linken Ventrikel der insuffizienten Herzen erneut immunhistochemisch auf Veränderungen der Opioidrezeptor-Expression untersucht. Dabei konnte eine signifikante relative Zunahme der optisch integrierten Dichte der DOR- und KOR-spezifischen Immunfluoreszenz festgestellt werden. Um eine Modulation des kardialen Opioidsystems im Verlauf einer CHF zu bestätigen, wurden Western Blot Untersuchungen durchgeführt. Darin stellte sich eine Hochregulation der DOR und KOR auf Proteinebene heraus. Außerdem kam es zu einer vermehrten Expression der endogenen Opioidvorläuferpeptide Proenkephalin und Prodynorphin auf Proteinebene. Diese Ergebnisse lassen einen Einfluss des kardialen Delta- und Kappa-Opioidsystems auf pathophysiologische Vorgänge im Rahmen der Progression einer CHF vermuten.Knowledge about changes in the cardiac opioid system in congestive heart failure (CHF) is scarce. As such, this project investigated the cellular localization of the opioid receptors δ (DOR) and κ (KOR) in left ventricular (LV) myocardium. Opioid receptor (OR) binding sites were detected by radioligand binding. In addition, DOR and KOR localization was determined by double immunofluorescence confocal analysis in the left ventricle of male Wistar rats. DOR and KOR were colocalized with L-type Ca2+ -channels (Cav 1.2) as well as with intracellular ryanodine receptors (RyR) of the sarcoplasmic reticulum. In each rat an infrarenal aortocaval fistula (ACF) was created, and 28 ± 2 days afterwards the extent of CHF was examined by hemodynamic and morphometric measurements. Adaptive changes in DOR and KOR and its endogenous ligands, the precursor peptides proenkephalin (PENK) and prodynorphin (PDYN) during CHF were examined via RT-PCR and Western blot. Following the ACF severe congestive heart failure developed in all rats and was accompanied by up- regulation of DOR/ KOR and PENK/PDYN on mRNA as well as receptor proteins representing consecutive adaptations. These findings may suggest that the cardiac opioid system possesses the ability to play a regulatory role in the pathophysiological development of heart failure

Data_Sheet_1_Identification of glucocorticoid receptors as potential modulators of parasympathetic and sympathetic neurons within rat intracardiac ganglia.PDF

BackgroundEmerging evidences indicate that glucocorticoid receptors (GR) play a regulatory role in cardiac function, particularly with regard to the autonomic nervous system. Therefore, this study aimed to demonstrate the expression and the precise anatomical location of GR in relation to the parasympathetic and sympathetic innervations of the heart.MethodsThe present study used tissue samples from rat heart atria to perform conventional reverse-transcriptase polymerase chain reaction (RT-PCR), Western blot, and double immunofluorescence confocal analysis of GR with the neuronal markers vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP) as well as the mineralocorticoid receptor (MR).ResultsDouble immunofluorescence labeling revealed that GRs were co-expressed with VAChT in parasympathetic principal neuronal somata and nerve terminals innervating atrium. Also, GR colocalized with the sympathetic neuronal marker TH in a cluster of small intensely fluorescent (SIF) cells, on intracardiac nerve terminals and in the atrial myocardium. GR immunoreactivity was scarcely identified on CGRP-immunoreactive sensory nerve terminals. Approximately 20% of GR immunoreactive neuronal somata co-localized with MR. Finally, conventional RT-PCR and Western blot confirmed the presence of GR and MR in rat heart atria.ConclusionThis study provides evidence for the existence of GR predominantly on cardiac parasympathetic neurons and TH-immunoreactive SIF cells suggesting a functional role of cardiac GR on cardiovascular function by modulation of the cardiac autonomic nervous system.</p

Image_1_Identification of glucocorticoid receptors as potential modulators of parasympathetic and sympathetic neurons within rat intracardiac ganglia.TIF

BackgroundEmerging evidences indicate that glucocorticoid receptors (GR) play a regulatory role in cardiac function, particularly with regard to the autonomic nervous system. Therefore, this study aimed to demonstrate the expression and the precise anatomical location of GR in relation to the parasympathetic and sympathetic innervations of the heart.MethodsThe present study used tissue samples from rat heart atria to perform conventional reverse-transcriptase polymerase chain reaction (RT-PCR), Western blot, and double immunofluorescence confocal analysis of GR with the neuronal markers vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP) as well as the mineralocorticoid receptor (MR).ResultsDouble immunofluorescence labeling revealed that GRs were co-expressed with VAChT in parasympathetic principal neuronal somata and nerve terminals innervating atrium. Also, GR colocalized with the sympathetic neuronal marker TH in a cluster of small intensely fluorescent (SIF) cells, on intracardiac nerve terminals and in the atrial myocardium. GR immunoreactivity was scarcely identified on CGRP-immunoreactive sensory nerve terminals. Approximately 20% of GR immunoreactive neuronal somata co-localized with MR. Finally, conventional RT-PCR and Western blot confirmed the presence of GR and MR in rat heart atria.ConclusionThis study provides evidence for the existence of GR predominantly on cardiac parasympathetic neurons and TH-immunoreactive SIF cells suggesting a functional role of cardiac GR on cardiovascular function by modulation of the cardiac autonomic nervous system.</p

Data_Sheet_2_Identification of glucocorticoid receptors as potential modulators of parasympathetic and sympathetic neurons within rat intracardiac ganglia.PDF

BackgroundEmerging evidences indicate that glucocorticoid receptors (GR) play a regulatory role in cardiac function, particularly with regard to the autonomic nervous system. Therefore, this study aimed to demonstrate the expression and the precise anatomical location of GR in relation to the parasympathetic and sympathetic innervations of the heart.MethodsThe present study used tissue samples from rat heart atria to perform conventional reverse-transcriptase polymerase chain reaction (RT-PCR), Western blot, and double immunofluorescence confocal analysis of GR with the neuronal markers vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP) as well as the mineralocorticoid receptor (MR).ResultsDouble immunofluorescence labeling revealed that GRs were co-expressed with VAChT in parasympathetic principal neuronal somata and nerve terminals innervating atrium. Also, GR colocalized with the sympathetic neuronal marker TH in a cluster of small intensely fluorescent (SIF) cells, on intracardiac nerve terminals and in the atrial myocardium. GR immunoreactivity was scarcely identified on CGRP-immunoreactive sensory nerve terminals. Approximately 20% of GR immunoreactive neuronal somata co-localized with MR. Finally, conventional RT-PCR and Western blot confirmed the presence of GR and MR in rat heart atria.ConclusionThis study provides evidence for the existence of GR predominantly on cardiac parasympathetic neurons and TH-immunoreactive SIF cells suggesting a functional role of cardiac GR on cardiovascular function by modulation of the cardiac autonomic nervous system.</p

Heart and lung morphological changes following ACF-induced congestive heart failure.

<p>Increased heart and lung weight indices (<b>A, B, D</b>) but not body weight (<b>C</b>) following ACF-induced congestive heart failure. Note, that heart and lung weight indices were significantly increased at 28 days of ACF rats (n = 10) compared to those of sham operated controls (n = 5) (heart index: ACF 6.5±1.6 versus Controls 3.9±0.2; lung index: ACF 6.9±1.4 versus Controls 3.8±0.3; p<0.01, Student t-test) (<b>D</b>). Data show means ± SD.</p