Rat Heart Atria Research Articles

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

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.

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.

Differential expressions and functions of phosphodiesterase enzymes in different regions of the rat heart

Phosphodiesterase enzymes (PDEs) are responsible for the adjustment of cyclic nucleotide levels. Alterations in PDE expressions in different tissues cause conflicts between functional and clinical effects of PDE inhibitors. Therefore, the aim of this study was to investigate the gene and protein expressions and the functional role of PDEs in atrium and ventricle of rat heart. The expressions of PDEs were examined in cardiac intact tissues and enzymatically isolated cells. The effects of PDE1–5 inhibitors (vinpocetine, EHNA, milrinone, rolipram, sildenafil, and IBMX) on basal and isoprenaline-stimulated contractions and sinus rate were recorded in the isolated spontaneously beating right atrium and electrically stimulated left papillary muscles. The mRNA and protein levels of PDEs were significantly different in atrial and ventricular intact tissues and isolated myocytes. Atrial contractions were increased with vinpocetine while suppressed by EHNA, milrinone, rolipram, sildenafil and IBMX. Milrinone, sildenafil and IBMX increased the heart rate whereas vinpocetine caused negative chronotropy. Papillary muscle contractions have been increased only with the vinpocetine and IBMX. Both the expression and the action of PDE-1–5 show atrial and ventricular differences. Therefore, these differences should be taken into account in the experimental or therapeutic approaches of the heart.

C-kit Positive Cardiac Outgrowth Cells Demonstrate Better Ability for Cardiac Recovery Against Ischemic Myopathy.

ObjectiveResident cardiac stem cells are expected to be a therapeutic option for patients who suffer from severe heart failure. However, uncertainty remains over whether sorting cells for c-kit, a stem cell marker, improves therapeutic outcomes.Materials and methodsCardiac outgrowth cells cultured from explants of rat heart atrium were sorted according to their positivity (+) or negativity (−) for c-kit. These cells were exposed to hypoxia for 3 d, and subsequently harvested for mRNA expression measurement. The cell medium was also collected to assess cytokine secretion. To test for a functional benefit in animals, myocardial infarction (MI) was induced in rats, and c-kit+ or c-kit− cells were injected. The left ventricular ejection fraction (LVEF) was measured for up to 4 weeks, after which the heart was harvested for biological and histological analyses.Results and conclusionExpression of the angiogenesis-related genes, VEGF and ANGPTL2, was significantly higher in c-kit+ cells after 3 d of hypoxic culture, although we found no such difference prior to hypoxia. Secretion of VEGF and ANGPTL2 was greater in the c-kit+ group than in the c-kit− group, while hypoxia tended to increase cytokine expression in both groups. In addition, IGF-1 was significantly increased in the c-kit+ group, consistent with the relatively low expression of cleaved-caspase 3 revealed by western blot assay, and the relatively low count of apoptotic cells revealed by histochemical analysis. Administration of c-kit+cells into the MI heart improved the LVEF and increased neovascularization. These results indicate that c-kit+cells may be useful in cardiac stem cell therapy.

Metrazoline, BU224, BU226 and BU239 act on isolated rat heart atria via I2-imidazoline receptors. Are the ATP-sensitive potassium (KATP) channels also involved?

Metrazoline, BU224, BU226 and BU239 act on isolated rat heart atria via I2-imidazoline receptors. Are the ATP-sensitive potassium (KATP) channels also involved?

Changes of rate and force of contraction of isolated rat heart atria by imidazoline derivatives: RS 45041-190, BU224, BU239 and 2-BFI

Changes of rate and force of contraction of isolated rat heart atria by imidazoline derivatives: RS 45041-190, BU224, BU239 and 2-BFI

The ino- and chronotropic effects of imidazoline drugs on isolated rat heart atria – are I1- imidazoline receptors involved?

The ino- and chronotropic effects of imidazoline drugs on isolated rat heart atria – are I1- imidazoline receptors involved?

Developmental expression of δ‐opioid receptors during maturation of the parasympathetic, sympathetic, and sensory innervations of the neonatal heart: Early targets for opioid regulation of autonomic control

Evidence is accumulating regarding the local opioid regulation of heart function. However, the exact anatomical location of δ-opioid receptors (DORs) and expression during maturation of the autonomic and sensory innervations of the neonatal heart is unknown. Therefore, we aimed to characterize target sites for opioids in neonatal rat heart intracardiac ganglia at postnatal day (P)1, P7 and adulthood (P56-P84). Rat heart atria were subjected to reverse-transcriptase polymerase chain reaction, Western blot, radioligand binding, and immunofluorescence confocal analysis of DORs with the neuronal markers vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP), and substance P (SP). Our results demonstrated DOR mRNA, protein, and binding sites that gradually increased from P1 toward adulthood. Immunofluorescence confocal microscopy showed DOR co-localized with VAChT in large-diameter principal neurons, TH-immunoreactive (IR) small intensely fluorescent (SIF) catecholaminergic cells, and CGRP- or SP-IR afferent nerve terminals arborizing within intracardiac ganglia and atrial myocardium. Co-expression of DOR with VAChT-IR neurons was observed from the first day of birth (P1). In contrast, DORs on TH-IR SIF cells or CGRP-IR fibers were not observed in intracardiac ganglia of P1, but rather in P7 rats. The density of nerve fibers in atrial myocardium co-expressing DORs with different neuronal markers increased from neonatal age toward adulthood. In summary, the enhanced DOR expression parallel to the maturation of cardiac parasympathetic, sympathetic, and sensory innervation of the heart suggests that the cardiac opioid system is an important regulator of neonatal and adult heart function through the autonomic nervous system.

Testing conception of engagement of imidazoline receptors in effects of imidazoline agents on isolated rat heart atria

Testing conception of engagement of imidazoline receptors in effects of imidazoline agents on isolated rat heart atria

Identification of μ‐ and κ‐opioid receptors as potential targets to regulate parasympathetic, sympathetic, and sensory neurons within rat intracardiac ganglia

Recent interest has been focused on the opioid regulation of heart performance; however, specific allocation of opioid receptors to the parasympathetic, sympathetic, and sensory innervations of the heart is scarce. Therefore, the present study aimed to characterize such specific target sites for opioids in intracardiac ganglia, which act as a complex network for the integration of the heart's neuronal in- and output. Tissue samples from rat heart atria were subjected to RT-PCR, Western blot, radioligand-binding, and double immunofluorescence confocal analysis of mu (M)- and kappa (K)-opioid receptors (ORs) with the neuronal markers vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP), and substance P (SP). Our results demonstrated MOR- and KOR-specific mRNA, receptor protein, and selective membrane ligand binding. By using immunofluorescence confocal microscopy, MOR and KOR immunoreactivity were colocalized with VAChT in large-diameter parasympathetic principal neurons, with TH-immunoreactive small intensely fluorescent (SIF) cells, and on nearby TH-IR varicose terminals. In addition, MOR and KOR immunoreactivity were identified on CGRP- and SP-IR sensory neurons throughout intracardiac ganglia and atrial myocardium. Our findings show that MOR and KOR are expressed as mRNA and translated into specific receptor proteins on cardiac parasympathetic, sympathetic, and sensory neurons as potential binding sites for opioids. Thus, they may well play a role within the complex network for the integration of the heart's neuronal in- and output.

Myogenic differentiation in atrium‐derived adult cardiac pluripotent cells and the transcriptional regulation of GATA4 and myogenin on ANP promoter

We established cardiac pluripotent stem-like cells from the left atrium (LA-PCs) of adult rat hearts. These cells could differentiate not only into beating myocytes but also into cells of other lineages, including adipocytes and endothelial cells in the methylcellulose-based medium containing interleukin-3 (IL-3), interleukin-6 (IL-6), and stem cell factor (SCF). In particular, IL-3 and SCF contributed to the differentiation into cardiac troponin I-positive cells. Notably, small population of LA-PCs coexpressed GATA4 and myogenin, which are markers specific to cardiomyocytes and skeletal myocytes, respectively, and could differentiate into both cardiac and skeletal myocytes. Therefore, we investigated the involvement of these two tissue-specific transcription factors in the cardiac transcriptional activity. Coexpression of GATA4 and myogenin synergistically activated GATA4-specific promoter of the atrial natriuretic peptide gene. This combinatorial function was shown to be dependant on the GATA site, but independent of the E-box. The results of chromatin immunoprecipitation and electrophoretic mobility shift assays suggested that myogenin bound to GATA4 on the GATA elements and the C-terminal Zn-finger domain of GATA4 and the N-terminal region of myogenin were required for this synergistic activation of transcription. Taken together, these two transcription factors could be involved in the myogenesis of LA-PCs.

Chronic atropine administration diminishes the contribution of vasoactive intestinal polypeptide to heart rate regulation

Vasoactive intestinal polypeptide (VIP) is implicated in the modulation of vagal effects on the heart rate. In this study, the impact of acute and chronic atropine administration on VIP levels in rat heart atria was investigated in relation to heart rate in the course of vagus nerves stimulation. Anaesthetised control and atropinised (10 mg/kg/day for 10 days) rats pretreated with metipranolol and phentolamine that were either given or not a single dose of atropine were subjected to bilateral vagus nerve stimulation (30 min: 0.7 mA, 20 Hz, 0.2 ms). VIP concentrations in the atria were determined after each stimulation protocol. In control rats with or without single atropine administration, the heart rate upon vagal stimulation was higher than in atropinised animals with or without single atropine dose, respectively. VIP concentrations in the control atria were significantly decreased after the stimulation; the decrease was comparable both in the absence and presence of a single dose of atropine. Compared to controls, VIP levels were significantly decreased after chronic atropine treatment and they were not further reduced by vagal stimulation and single atropine administration. Administration of VIP antagonist completely abolished the differences in the heart rate upon vagal stimulation between control and atropinised groups. In conclusion, the data indicate that chronic atropine administration affects VIP synthesis in rat heart atria and consequently it modifies the heart rate regulation.

Molecular and Electrophysiological Differences in the L-Type Ca<sup>2+</sup> Channel of the Atrium and Ventricle of Rat Hearts

Many pathological conditions induce electrical remodeling, possibly through intracellular Ca2+ overload, but the currently available L-type Ca2+ channel blockers may be detrimental because of their global negative inotropic effects. To determine whether the L-type Ca2+ channel is identical throughout the heart, the distribution of the mRNAs and proteins comprising the L-type Ca2+ channel and its electrophysiological properties were analyzed in rat atria and ventricles. The mRNA of alpha2delta-2 (Cacna2d2) was more abundantly expressed in the atrium (approximately 5-fold) than in the ventricle. In contrast, alpha1C (Cacna1c) (Cav1.2) mRNA was significantly less abundant in the atrium. The level of the alpha1C (Cacna1c) (Cav1.2) protein was decreased (approximately 0.5-fold) and that of alpha2 delta-1 (Cacna2d1) was increased (approximately 2-fold) in the atrium compared with the ventricle. Although the peak ICa,L density showed no significant differences, voltage dependence of inactivation and activation of the current showed a more depolarized shift in the atrium than in the ventricle. These results indicate that in the rat heart the L-type Ca2+ channel differs between the atrium and ventricle with regard to gene expression and electrophysiological properties.

Pharmacological characterisation of sodium channels in sinoatrial node pacemaking in the rat heart

Blockade of sodium channels located in the sinoatrial node can slow diastolic depolarisation rate, recorded in vitro. The objective was therefore to determine whether these blockers could slow heart rate in vivo. The heart rate was firstly measured in spontaneously beating, isolated rat heart atria in the presence of different voltage gated sodium channel blockers. Tetrodotoxin and lidocaine slightly reduced heart rate whereas KC 12291 and R 56865, which mainly interact with the persistent component of the sodium current, concentration dependently and potently induced bradycardia. In the pithed rat, tetrodotoxin induced statistically significant decreases heart rate, maximal effects were: − 32.2 ± 6.1 beat per min. KC 12291 and R 56865 dose-dependently induced bradycardia (Δ heart rate obtained, − 55.1 ± 5.2 beat per min, P < 0.05, and − 71.9 ± 8.5 bpm, P < 0.05, respectively). In conclusion, voltage gated sodium channel blockers rather selective for the persistent current, exert a potent bradycardic effect in the rat.

Expression of Slow Skeletal Myosin Binding C-protein in Normal Adult Mammalian Heart

Myosin binding C-protein exists in three main isoforms in striated muscle. While expression of cardiac muscle type C-protein is detected in skeletal muscle during early fetal development, there have not so far been any reports of the expression of the skeletal muscle isoforms of this protein in either developing or adult vertebrate heart. The present study demonstrates slow skeletal muscle type C-protein in moderate amount in right atrium and interatrial septum of adult human, rabbit, rat and bovine hearts using both immunocytochemical and immunoblotting procedures.

The importance of the prolonged postnatal period in determination of heart atrial muscarinic receptor and β-adrenoceptor densities

Our previous work indicated that hyperstimulation of muscarinic receptors brought about profound changes not only in the density of the muscarinic receptors, but also of the β-adrenoceptors both in vivo and in vitro. Now we try to identify the changes in receptor densities when the neuronal input from the autonomic nerves is disrupted, i.e. when neonatal rats were injected with 6-hydroxydopamine (6-OHDA; able to destroy sympathetic nerves). Although the interruption of neuronal input was successful as indicated by norepinephrine uptake, there was no change in receptor densities in the rat heart atria when 6-OHDA was applied in the first postnatal week only. When we repeated the 6-OHDA treatment consequently (on 14 th, 21 st and 28 th postnatal day) there were clear increases of both receptor types (muscarinic receptors rise to 139% of control, β-adrenoceptors to 134% of control). The atrial muscarinic receptor number was increased (to 146% of control) also when we combined the first week 6-OHDA application with another factor (when the pups abide with mother till the 56th day). The simple non-weaning of animals has no effect on the receptor densities. We can therefore conclude that the first week application of 6-OHDA was insufficient in changing the receptor number and that only repeated 6-OHDA administration was able to change them. Similarly, combination with other factor: non-weaning led to the muscarinic receptors increase. Our results can indicate the importance of the prolonged postnatal period for the heart muscarinic and β-adrenergic receptor number determination.

Distribution of vasoactive intestinal polypeptide in the rat heart: effect of guanethidine and capsaicin

Vasoactive intestinal polypeptide (VIP) is believed to coexist with acetylcholine in postganglionic parasympathetic neurones. However, the presence of VIP in extrinsic nerves and/or other types of intrinsic cardiac neurones has not been excluded. The aim of our study was to examine the distribution and origin of VIP-ergic innervation in the rat heart atria using immunocytochemistry and radioimmunoassay (RIA) combined with two types of denervation: sympathectomy, which was produced by guanethidine treatment and sensory denervation achieved by capsaicin administration. In whole-mount preparations of the intact atria, VIP-immunoreactive (IR) nerve fibres and ganglionic cells were found, the latter being much more numerous in the left atria (LA) than in the right ones. Some of VIP-IR nerve fibres forming bundles appeared to be extrinsic in origin. VIP-IR concentrations determined by RIA in the intact rats were significantly higher in the LA than in the right ones (p < 0.01). However, no changes in VIP-IR levels were found in either atrium after both guanethidine and capsaicin treatment protocols, thus indicating that VIP-immunoreactivity is not associated with either sympathetic or sensory innervation. In conclusion, the ganglionated plexus of the rat atria may comprise at least 3 different neuronal populations expressing VIP-positivity: 1. extrinsic preganglionic parasympathetic fibres, 2. intrinsic postganglionic parasympathetic neurones and 3. intrinsic local circuit neurones that do not express a cholinergic phenotype.

Effect of captopril on vasoactive intestinal peptide levels in the hyperthyroid rat heart atria

Effect of captopril on vasoactive intestinal peptide levels in the hyperthyroid rat heart atria

Vasoactive intestinal polypeptide and calcitonin gene-related peptide in the developing rat heart atria

Vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI) and calcitonin gene-related peptide (CGRP)-LI concentrations were determined in the developing rat heart atria using radioimmunoassay. Peptide levels were analysed on postnatal days 1, 10, 25, 45, 60, and 85 (P1–P85) separately in the right (RA) and left atria (LA). No sex differences were revealed at any age examined. VIP-LI has been already detected in both atria at P1 in concentrations comparable to values at P10. In the RA, VIP-LI levels increased significantly between days P10 and P25, remained high at P45 and then declined. In the LA, VIP-LI concentrations did not differ from those in the RA on days P1, P10, P25, and P45. However, regional differences were found at P60 and P85, when the peptide levels were significantly higher in the LA than in the RA. The postnatal changes in CGRP-LI concentrations were comparable in both atria with similar values at P1 and P85. After birth, CGRP levels decreased gradually till P45, then they increased till P60 and declined again at P85. The results demonstrate that there is an asymmetry in the postnatal development of the atrial VIP-LI and CGRP-LI concentrations. VIP-LI levels reached their maximum at P25, whereas CGRP-LI levels at P60. Relatively high peptide concentrations in neonatal atria and their variations during development might be related to diverse trophic functions of VIP and CGRP.

Vasoactive Intestinal Polypeptide in Rat Heart Atria: the Effect of Hyperthyroidism

The effects of transient and sustained hyperthyroidism on vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI) levels were studied in the heart atria of developing and adult rats. Newborn rats were divided into 5 groups. Neo-T animals were treated with thyroxine (T4) during postnatal days 1-8 and sacrificed at the age of 60 days. Neo-S rats were treated with T4 during postnatal days 1-60 and sacrificed one day later. Adult-1 and Adult-2 animals received T4 during days 52-60 and were sacrificed 5-6 days and 1 day later, respectively. Control animals were injected with saline. VIP-LI concentrations were determined in extracts from the left and right atria separately. In Neo-S and Adult-2 rats, spontaneous heart rate, the weight of both atria and total T4 serum levels were significantly enhanced, while their body weight was decreased. The ratio atria weight to body weight was significantly increased in all groups except for Adult-1 animals. Hyperthyroidism led to a significant decrease in VIP-LI levels in both atria of Neo-S and Neo-T rats. Hyperthyroidism induced in adult rats also decreased VIP-LI levels in both atria. However, this change was only transient. In conclusion, our data have provided new evidence that hyperthyroidism induced during the early neonatal period interferes with the development of VIP-ergic innervation in rat atria. The period of the first few postnatal days seems to be essential for this effect, since VIP-LI concentrations in 60-day-old animals did not significantly differ between Neo-S and Neo-T atria.