Martina Novakova

The detection of the non-M2 muscarinic receptor subtype in the rat heart atria and ventricles

Mammal heart tissue has long been assumed to be the exclusive domain of the M2 subtype of muscarinic receptor, but data supporting the presence of other subtypes also exist. We have tested the hypothesis that muscarinic receptors other than the M2 subtype are present in the heart as minor populations. We used several approaches: a set of competition binding experiments with pirenzepine, AFDX-116, 4-DAMP, PD 102807, p-F-HHSiD, AQ-RA 741, DAU 5884, methoctramine and tripinamide, blockage of M1 muscarinic receptors using MT7 toxin, subtype-specific immunoprecipitation experiments and determination of phospholipase C activity. We also attempted to block M1–M4 receptors using co-treatment with MT7 and AQ-RA 741. Our results show that only the M2 subtype is present in the atria. In the ventricles, however, we were able to determine that 20% (on average) of the muscarinic receptors were subtypes other than M2, with the majority of these belonging to the M1 subtype. We were also able to detect a marginal fraction (6 ± 2%) of receptors that, based on other findings, belong mainly to the M5 muscarinic receptors. Co-treatment with MT7 and AQ-RA 741 was not a suitable tool for blocking of M1–M4 receptors and can not therefore be used as a method for M5 muscarinic receptor detection in substitution to crude venom. These results provide further evidence of the expression of the M1 muscarinic receptor subtype in the rat heart and also show that the heart contains at least one other, albeit minor, muscarinic receptor population, which most likely belongs to the M5 muscarinic receptors but not to that of the M3 receptors.

Regulation of Adrenoceptor and Muscarinic Receptor Gene Expression after Single and Repeated Stress

Although stress is tightly connected with elevated levels of catecholamines, stress effects on target structures of catecholamine action—adrenoceptors (ARs)—has not been deeply studied yet. Similarly, very little is known about changes of muscarinic receptors (MRs) during stress. We determined changes in these receptors in the individual parts of the heart (right atria and ventricles) of animals (rats and mice) exposed to a single and repeated immobilization stress. Changes of tissue catecholamines, β2‐AR gene expression, protein levels, and binding sites were determined in rat right ventricles, and changes in β1‐, β2‐, and β3‐AR gene expression were followed in murine right atria. Tissue catecholamines were elevated, while β2‐AR mRNA levels and β2‐AR proteins and binding were decreased, in rat right ventricles. In murine right atria, β1‐ and β2‐AR gene expression was elevated, while β3‐AR mRNA levels and M2‐MR were reduced. Taken together, our data show that interaction of AR and MR is important for the organism coping with stress and that different heart regions reveal distinct reactions to stress.

Sexual dimorphism in stress-induced changes in adrenergic and muscarinic receptor densities in the lung of wild type and corticotropin-releasing hormone-knockout mice

We tested the hypothesis that single and repeated immobilization stress affect densities of α1-adrenoceptor (α1-AR) and β-AR subtypes, muscarinic receptors (MR), adenylyl cyclase activity (AC) and phospholipase C activity (PLC) in lungs of male and female wild type (WT) and corticotropin-releasing hormone gene (CRH-knockout (KO)) disrupted mice. We found sex differences in the basal levels of α1-AR subtypes (females had 2–3 times higher density of receptors than males) and MR (males had twice the density found in females). In marked contrast, β-AR subtype densities did not differ between sexes. CRH gene disruption decreased all three studied receptors in intact mice (to 20–50% of WT) in both sexes (except β1-AR in females). Stress induced sexually dimorphic responses, while all α1-AR subtypes decreased in females (to 30% of control approximately), only α1A-AR level diminished (about 50%) in males. β1-AR decreased in males (to about 40%) but remained stable in females. β2-AR diminished in females (to about 20–60%) and also in males (to about 30–60%). MR decreased in both sexes (approximately to 50%). AC activity diminished in males (to < 50%) while PLC activity was not changed. In CRH-KO mice, the stress response was severely diminished. Paradoxically, the receptor response to stress was less affected by CRH-KO in males than in females. AC activity did not change in CRH-KO mice. In conclusion, in mice the stress reaction is sexually dimorphic and an intact hypothalamo-pituitary–adrenocortical system is required for the normal reaction of pulmonary adrenergic and MR to stress.

Repeated Immobilization Stress Increases Expression of β3-Adrenoceptor in the Left Ventricle and Atrium of the Rat Heart

Stress is a contributor of many cardiovascular diseases. Positive inotropic and chronotropic effects of catecholamines are regulated via β-adrenergic receptors (ARs). Many reports exist concerning changes of cardiac β1 - and β2 -ARs in stress, but only a few deal with modulation of cardiac β3 -AR. Our aim was to analyze the expression and binding sites of β1 -, β2 - and β3 -ARs and adenylyl cyclase activity in the left ventricle, and β3 -AR expression and binding in the left atrium of rats exposed to acute and chronic immobilization stress (IMO). The concentration of noradrenaline in the ventricle decreased, while adrenaline increased, especially after repeated IMO. The mRNA and protein levels, and binding sites of β3 -subtype significantly rose following chronic IMO, while all parameters for β2 -AR dropped after single and repeated exposure. Similarly, the mRNA levels and binding sites for β3 -subtype increased in the left atrium as a consequence of chronic IMO. The rise in β3 -subtypes and a drop in β2 -subtypes resulted in inhibition of adenylyl cyclase activity within the left ventricle. Taken together, among other factors, up-regulation of β3 -AR could represent an adaptation mechanism, which might be related to altered physiological function of the left ventricle and atrium during prolonged emotional stress and might serve cardioprotective function during catecholamine overload.

Decrease in heart adrenoceptor gene expression and receptor number as compensatory tool for preserved heart function and biological rhythm in M(2) KO animals.

Muscarinic receptors (MR) are main cardioinhibitory receptors. We investigated the changes in gene expression, receptor number, echocardiography, muscarinic/adrenergic agonist/antagonist changes in heart rate (HR) and HR biorhythm in M2 KO mice (mice lacking the main cardioinhibitory receptors) in the left ventricle (LV) and right ventricle (RV). We hypothesize that the disruption of M2 MR, key players in parasympathetic bradycardia, would change the number of receptors with antagonistic effects on the heart (β1- and β2-adrenoceptors, BAR), while the function of the heart would be changed only marginally. We have found changes in LV, but not in RV: decrease in M3 MR, β1- and β2-adrenoceptor gene expressions that were accompanied by a decrease in MR and BAR receptor binding. No changes were found both in LV systolic and diastolic function as assessed by echocardiography (e.g., similar LV end-systolic and end-diastolic diameter, fractional shortening, mitral flow characteristics, and maximal velocity in LV outflow tract). We have found only marginal changes in specific HR biorhythm parameters. The effects of isoprenaline and propranolol on HR were similar in WT and KO (but with lesser extent). Atropine was not able to increase HR in KO animals. Carbachol decreased the HR in WT but increased HR in KO, suggesting the presence of cardiostimulatory MR. Therefore, we can conclude that although the main cardioinhibitory receptors are not present in the heart, the function is not much affected. As possible mechanisms of almost normal cardiac function, the decreases of both β1- and β2-adrenoceptor gene expression and receptor binding should be considered.

Gene expression of adrenoceptors in the hearts of cold-acclimated rats exposed to a novel stressor.

Changes in the heart rate and force of contraction are regulated by catecholamines via adrenoceptors (AR). In this work, we measured gene expression of AR in left heart atria and ventricles in rats exposed to cold stress and in cold‐acclimated rats exposed to a novel stressor (immobilization). We found a significant increase in β3‐AR in left ventricle of rats exposed to acute (1 day) and long‐term (28 days) cold, but no changes in β1‐ and β2‐AR mRNA levels. However, single immobilization significantly decreased β2‐AR mRNA levels both in left atria and ventricles compared to acute cold stress. Application of a novel stressor (immobilization) to previously cold‐acclimated animals did not show decrease of β2‐AR mRNA levels as seen in intact animals. Moreover, β1‐ and β2‐AR did not show any significant changes. Surprisingly, the most prominent changes in the heart were detected for α1B‐AR gene expression. We found decreased levels of α1B‐AR mRNA in the heart of rats exposed to cold and immobilization. We also found that exposure of cold‐acclimated rats to immobilization is responsible for additional decrease of α1B‐AR mRNA levels in heart. It seems that while β‐AR undergoes adaptation, α1B‐AR is probably prepared to modulate heart functions. Proposed mechanism of β‐AR adaptation needs to be elucidated. Thus, we have shown that gene expression of different AR subtypes in the heart is regulated differently by various stressors. A protective role of β2‐, β3‐AR, and α1B‐AR in the process of heart adaptation to chronic stress exposure is proposed.

Heart Adrenoceptor Gene Expression and Binding Sites in the Human Failing Heart

Adrenergic regulation of the heart function is well documented by many studies. Catecholamines act through α1‐, β1‐, β2‐, and β3‐adrenoceptors (ARs) in the heart. There are many findings about the changes of β1‐ and β2‐AR in heart failure (HF). On the other hand, the role of other AR subtypes is not clear yet. We focused on determining how HF could affect gene expression and specific ligand binding to α1A‐, α1B‐, α1D‐, β1‐, β2‐, and β3‐AR. Hearts from 11 patients with HF subjected to transplantation were investigated. As a control, corresponding parts from hearts not suitable for transplantation were used. We have found significantly higher mRNA levels of α1A‐, α1B‐,β1‐, and β2‐AR in the left ventricle of failing hearts compared to the levels in controls. β3‐AR mRNA levels in the left ventricle of failing hearts were not changed. No changes in mRNA levels of all receptors studied in other cardiac areas were found. On the other hand, binding studies showed a substantial decrease in left ventricles of failing hearts in all α1‐AR subtypes and in β1‐ and β2‐AR. However, the binding to β3‐AR was not changed. Our results suggest that α1‐AR changes might be part of a compensatory mechanism, by which the heart suffering from the HF tries to secure its function, and it could be hypothesized that ineffective β3‐AR regulation might be involved in development of HF. According to our knowledge, this is the first report about the β3‐AR binding in HF.

Heart ventricles specific stress-induced changes in β-adrenoceptors and muscarinic receptors.

The left and right ventricles fulfill different role in heart function. Here we compare chamber specific changes in local catecholamine concentrations; gene expression and the receptor protein amount of all three β-adrenoceptors (β-AR) in rat right heart ventricles exposed to acute (1 session) and repeated (7 sessions) immobilization stress (IMMO) vs. previously observed changes in left ventricles. Density of muscarinic receptors as main cardio-inhibitive receptors was also measured. In the right ventricles, noradrenaline and adrenaline were increased. No β1-AR changes were observed, in spite of the increased sympathetic activity. On the other hand, we have found a decrease of β2-AR gene expression (reduction to 30%) after 7 IMMO and protein (to 59%) after 1 IMMO. β3-AR gene expression was increased after 7 IMMO. Muscarinic receptor density was not changed. When comparing correlation in left and right ventricles, there was strong correlation between adrenaline and β2-AR gene expression, protein and β3-AR gene expression in the left ventricles while only correlation between adrenaline and β2-AR mRNA and protein in the right ventricles was found. Our results show that maintenance of cardiac homeostasis under stress conditions are to a great extent achieved by a balance between different receptors and also by a balanced receptor changes in left vs. right ventricles. Taken together, decrease of cardio-stimulating β2-AR represents a new important mechanism by which β2-AR contributes to the heart physiology.