Altered cardiovascular responses in mice lacking the M(1) muscarinic acetylcholine receptor.
ABSTRACT Although the M(2) muscarinic acetylcholine receptor (mAChR) is the predominant functional mAChR subtype in the heart, some responses of the cardiovascular system to acetylcholine (ACh) may be mediated by other mAChR subtypes. The potential effect of M(1) mAChR on heart function was investigated using M(1) knockout (M(1)-KO) mice. In vivo cardiodynamic analysis showed that basal values of heart rate (HR), developed left ventricular pressure (DLVP), left ventricular dP/dt(max) (LV dP/dt(max)), and mean blood pressure (MBP) were similar between wild-type (WT) and M(1)-KO mice. Injection of the putative M(1)-selective agonist 4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium (McN-A-343) produced an increase in LV dP/dt(max), DLVP, HR, and MBP in WT mice but did not affect hemodynamic function in the M(1)-KO mice. The stimulatory effect of McN-A-343 in WT mice was blocked by pretreatment with propranolol, indicating that stimulation of the M(1) mAChRs on sympathetic postganglionic neurons evoked release of catecholamines. Intravenous injection of ACh in both WT and M(1)-KO mice caused atrioventricular conduction block, without a significant change in the frequency of atrial depolarization, or atrial fibrillation. Immunoprecipitation and reverse transcriptase-polymerase chain reaction failed to detect the expression of M(1) mAChR in cardiac tissue from WT mice. The carbachol-induced increase of phospholipase C activity in cardiac tissues was not different between WT and M(1)-KO mice. These results demonstrate that 1) activation of M(1) mAChR subtype on sympathetic postganglionic cells results in catecholamine-mediated cardiac stimulation, 2) M(1) mAChR is not expressed in mouse heart, and 3) administration of ACh to mice induces arrhythmia.
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ABSTRACT: The atrioventricular node (AVN) is a vital component of the pacemaker-conduction system of the heart, co-ordinating conduction of electrical excitation from cardiac atria to ventricles and acting as a secondary pacemaker. The electrical behaviour of the AVN is modulated by vagal activity via activation of muscarinic potassium current, IKACh. However, it is not yet known if this response exhibits 'fade' or desensitization in the AVN, as established for the heart's primary pacemaker--the sinoatrial node. In this study, acute activation of IKACh in rabbit single AVN cells was investigated using whole-cell patch clamp at 37 °C. 0.1-1 μM acetylcholine (ACh) rapidly activated a robust IKACh in AVN myocytes during a descending voltage-ramp protocol. This response was inhibited by tertiapin-Q (TQ; 300 nM) and by the M2 muscarinic ACh receptor antagonist AFDX-116 (1 μM). During sustained ACh exposure the elicited IKACh exhibited bi-exponential fade (τf of 2.0 s and τs 76.9 s at -120 mV; 1 μM ACh). 10 nM ET-1 elicited a current similar to IKACh, which faded with a mono-exponential time-course (τ of 52.6 s at -120 mV). When ET-1 was applied following ACh, the ET-1 activated response was greatly attenuated, demonstrating that ACh could desensitize the response to ET-1. For neither ACh nor ET-1 was the rate of current fade dependent upon the initial response magnitude, which is inconsistent with K+ flux mediated changes in electrochemical driving force as the underlying mechanism. Collectively, these findings demonstrate that TQ sensitive inwardly rectifying K+ current in cardiac AVN cells, elicited by M2 muscarinic receptor or ET-1 receptor activation, exhibits fade due to rapid desensitization.Biochemical and Biophysical Research Communications 06/2012; 423(3):496-502. · 2.28 Impact Factor
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ABSTRACT: It has been nearly a century since Otto Loewi discovered that acetylcholine (ACh) release from the vagus produces bradycardia and reduced cardiac contractility. It is now known that parasympathetic control of the heart is mediated by ACh stimulation of G(i/o)-coupled muscarinic M2 receptors, which directly activate G protein-coupled inwardly rectifying potassium (GIRK) channels via Gβγ resulting in membrane hyperpolarization and inhibition of action potential (AP) firing. However, expression of M2R-GIRK signaling components in heterologous systems failed to recapitulate native channel gating kinetics. The missing link was identified with the discovery of regulator of G protein signaling (RGS) proteins, which act as GTPase-activating proteins to accelerate the intrinsic GTPase activity of Gα resulting in termination of Gα- and Gβγ-mediated signaling to downstream effectors. Studies in mice expressing an RGS-insensitive Gα(i2) mutant (G184S) implicated endogenous RGS proteins as key regulators of parasympathetic signaling in heart. Recently, two RGS proteins have been identified as critical regulators of M2R signaling in heart. RGS6 exhibits a uniquely robust expression in heart, especially in sinoatrial (SAN) and atrioventricular nodal regions. Mice lacking RGS6 exhibit increased bradycardia and inhibition of SAN AP firing in response to CCh as well as a loss of rapid activation and deactivation kinetics and current desensitization for ACh-induced GIRK current (I(KACh)). Similar findings were observed in mice lacking RGS4. Thus, dysregulation in RGS protein expression or function may contribute to pathologies involving aberrant electrical activity in cardiac pacemaker cells. Moreover, RGS6 expression was found to be up-regulated in heart under certain pathological conditions, including doxorubicin treatment, which is known to cause life-threatening cardiotoxicity and atrial fibrillation in cancer patients. On the other hand, increased vagal tone may be cardioprotective in heart failure where acetylcholinesterase inhibitors and vagal stimulation have been proposed as potential therapeutics. Together, these studies identify RGS proteins, especially RGS6, as new therapeutic targets for diseases such as sick sinus syndrome or other maladies involving abnormal autonomic control of the heart.Frontiers in Physiology 01/2012; 3:95.
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ABSTRACT: Fear conditioning in animals has been used extensively to model clinical anxiety disorders. While individual animals exhibit marked differences in their propensity to undergo fear conditioning, the physiologically relevant mediators have not yet been fully characterized. Here, we demonstrate that C57BL/6 inbred mouse strain subjected to a regimen of chronic social defeat stress (CSDS) can be separated into susceptible and resistant subpopulations that display different levels of fear responses in an auditory fear conditioning paradigm. Susceptible mice had significantly more c-Fos protein expression in neurons of the basolateral amygdala (BLA) following CSDS and showed exaggerated conditioned fear responses, while there were no significant differences between groups in innate anxiety- and depressive-like behaviors. Through the use of conditional brain-derived neurotrophic factor (BDNF) knockout strategies, we find that elevated BLA BDNF level following fear conditioning training is a key mediator contributing to determine the levels of conditioned fear responses. Our results also show that relative to susceptible mice, resistant mice had a much faster recovery from conditioned stimuli-induced cardiovascular and corticosterone responses. Systemic administration of norepinephrine reuptake inhibitor atomoxetine increased c-Fos protein expression in BLA neurons following fear conditioning training and promoted the expression of conditioned fear in resistant mice. Conversely, administration of β-adrenergic receptor antagonist propranolol reduced fear conditioning training-induced c-Fos protein expression in BLA neurons and reduced conditioned fear responses in susceptible mice. These findings reveal a novel role for the BDNF signaling within the BLA in mediating individual differences in autonomic, neuroendocrine and behavioral reactivity to fear conditioning.Experimental Neurology 01/2014; · 4.65 Impact Factor