Electrophysiological effects of 5-hydroxytryptamine on isolated human atrial myocytes, and the influence of chronic beta-adrenoceptor blockade.
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ABSTRACT: 5-Hydroxytryptamine (5-HT) has been postulated to play a proarrhythmic role in the human atria via stimulation of 5-HT4 receptors. The aims of this study were to examine the effects of 5-HT on the L-type Ca2+ current (ICaL) action potential duration (APD), the effective refractory period (ERP) and arrhythmic activity in human atrial cells, and to assess the effects of prior treatment with beta-adrenoceptor antagonists. Isolated myocytes, from the right atrial appendage of 27 consenting patients undergoing cardiac surgery who were in sinus rhythm, were studied using the whole-cell perforated patch-clamp technique at 37 degrees C. 5-HT (1 nm-10 microm) caused a concentration-dependent increase in ICaL, which was potentiated in cells from beta-blocked (maximum response to 5-HT, Emax=299+/-12% increase above control) compared to non-beta-blocked patients (Emax=220+/-6%, P<0.05), but with no change in either the potency (log EC50: -7.09+/-0.07 vs -7.26+/-0.06) or Hill coefficient (nH: 1.5+/-0.6 vs 1.5+/-0.3) of the 5-HT concentration-response curve. 5-HT (10 microm) produced a greater increase in the APD at 50% repolarisation (APD50) in cells from beta-blocked patients (of 37+/-10 ms, i.e. 589+/-197%) vs non-beta-blocked patients (of 10+/-4 ms, i.e. 157+/-54%; P<0.05). Both the APD90 and the ERP were unaffected by 5-HT. Arrhythmic activity was observed in response to 5-HT in five of 17 cells (29%) studied from beta-blocked, compared to zero of 16 cells from the non-beta-blocked patients (P<0.05). In summary, the 5-HT-induced increase in calcium current was associated with a prolonged early plateau phase of repolarisation, but not late repolarisation or refractoriness, and the enhancement of these effects by chronic beta-adrenoceptor blockade was associated with arrhythmic potential.
Project description:BackgroundWith aging, the human atrium invariably develops amyloid composed of ANP (atrial natriuretic peptide) and BNP (B-type natriuretic peptide). Preamyloid oligomers are the primary cytotoxic species in amyloidosis, and they accumulate in the atrium during human hypertension and a murine hypertensive model of atrial fibrillation susceptibility. We tested the hypothesis that preamyloid oligomers derived from natriuretic peptides cause cytotoxic and electrophysiological effects in atrial cells that promote arrhythmia susceptibility and that oligomer formation is enhanced for a mutant form of ANP linked to familial atrial fibrillation.MethodsOligomerization was assessed by Western blot analysis. Bioenergic profiling was performed using the Seahorse platform. Mitochondrial dynamics were investigated with immunostaining and gene expression quantitated using quantitative reverse transcription polymerase chain reaction. Action potentials and ionic currents were recorded using patch-clamp methods and intracellular calcium measured using Fura-2.ResultsOligomer formation was markedly accelerated for mutant ANP (mutANP) compared with WT (wild type) ANP. Oligomers derived from ANP, BNP, and mutANP suppressed mitochondrial function in atrial HL-1 cardiomyocytes, associated with increased superoxide generation and reduced biogenesis, while monomers had no effects. In hypertensive mice, atrial cardiomyocytes displayed reduced action potential duration and maximal dV/dT of phase 0, with an elevated resting membrane potential, compared with normotensive mice. Similar changes were observed when atrial cells were exposed to oligomers. mutANP monomers produced similar electrophysiological effects as mutANP oligomers, likely due to accelerated oligomer formation, while ANP and BNP monomers did not. Oligomers decreased Na+ current, inward rectifier K+ current, and L-type Ca++ current, while increasing sustained and transient outward K+ currents, to account for these effects.ConclusionsThese findings provide compelling evidence that natriuretic peptide oligomers are novel mediators of atrial arrhythmia susceptibility. Moreover, the accelerated oligomerization by mutANP supports a role for these mediators in the pathophysiology of this mutation in atrial fibrillation.
Project description:Black patients may be less responsive to beta-blockers than whites. Genetic variants in the beta1-adrenergic receptor (beta1-AR) associated with lesser response to beta-blockers are more common in blacks than in whites. The purpose of this study was to determine whether ethnic differences in response to beta-blockade can be explained by differing distributions of functional genetic variants in the beta1-AR.We measured sensitivity to beta-blockade by the attenuation of exercise-induced tachycardia in 165 patients (92 whites), who performed a graded bicycle exercise test before and 2.5 h after oral atenolol (25 mg). We determined heart rate at rest and at three exercise levels from continuous ECG recordings and calculated the area under the curve. We also measured plasma atenolol concentrations and determined genotypes for variants of the beta1-AR (Ser49Gly, Arg389Gly) and alpha2C-AR (del322-325). The effects of ethnicity, genotype, and other covariates on the heart rate reduction after atenolol were estimated in multiple regression analyses.Atenolol resulted in a greater reduction in exercise heart rate in whites than in blacks (P=0.006). beta1-AR Arg389 (P=0.003), but not the alpha2C-AR 322-325 insertion allele (P=0.31), was independently associated with a greater reduction in heart rate area under the curve. Ethnic differences in sensitivity to atenolol remained significant (P=0.006) after adjustment for beta1-AR and alpha2C-AR genotypes.Ethnic differences in sensitivity to the beta1-blocker atenolol persist even after accounting for different distributions of functional genetic beta1-AR variants, suggesting that additional, as yet unidentified factors contribute to such ethnic differences.
Project description:Background and purposeReperfusion therapy is the standard of care for ischaemic stroke; however, there is a need to identify new therapeutic targets able to ameliorate cerebral damage. Neutrophil β1 adrenoceptors (β1AR) have been linked to neutrophil migration during exacerbated inflammation. Given the central role of neutrophils in cerebral damage during stroke, we hypothesize that β1AR blockade will improve stroke outcomes.Experimental approachRats were subjected to middle cerebral artery occlusion-reperfusion to evaluate the effect on stroke of the selective β1AR blocker metoprolol (12.5 mg·kg-1 ) when injected i.v. 10 min before reperfusion.Key resultsMagnetic resonance imaging and histopathology analysis showed that pre-reperfusion i.v. metoprolol reduced infarct size. This effect was accompanied by reduced cytotoxic oedema at 24 h and vasogenic oedema at 7 days. Metoprolol-treated rats showed reduced brain neutrophil infiltration and those which infiltrated displayed a high proportion of anti-inflammatory phenotype (N2, YM1+ ). Additional inflammatory models demonstrated that metoprolol specifically blocked neutrophil migration via β1AR and excluded a significant effect on the glia compartment. Consistently, metoprolol did not protect the brain in neutrophil-depleted rats upon stroke. In patients suffering an ischaemic stroke, β1AR blockade by metoprolol reduced circulating neutrophil-platelet co-aggregates.Conclusions and implicationsOur findings describe that β1AR blockade ameliorates cerebral damage by targeting neutrophils, identifying a novel therapeutic target to improve outcomes in patients with stroke. This therapeutic strategy is in the earliest stages of the translational pathway and should be further explored.
Project description:BackgroundConsiderable evidence suggests that calcium/calmodulin-dependent protein kinase II (CaMKII) overactivity plays a crucial role in the pathophysiology of heart failure (HF), a condition characterized by excessive β-adrenoceptor (β-AR) stimulation. Recent studies indicate a significant cross talk between β-AR signaling and CaMKII activation presenting CaMKII as a possible downstream mediator of detrimental β-AR signaling in HF. In this study, we investigated the effect of chronic β-AR blocker treatment on CaMKII activity in human and experimental HF.Methods and resultsImmunoblot analysis of myocardium from end-stage HF patients (n=12) and non-HF subjects undergoing cardiac surgery (n=12) treated with β-AR blockers revealed no difference in CaMKII activity when compared with non-β-AR blocker-treated patients. CaMKII activity was judged by analysis of CaMKII expression, autophosphorylation, and oxidation and by investigating the phosphorylation status of CaMKII downstream targets. To further evaluate these findings, CaMKIIδC transgenic mice were treated with the β1-AR blocker metoprolol (270 mg/kg*d). Metoprolol significantly reduced transgene-associated mortality (n≥29; P<0.001), attenuated the development of cardiac hypertrophy (-14±6% heart weight/tibia length; P<0.05), and strongly reduced ventricular arrhythmias (-70±22% premature ventricular contractions; P<0.05). On a molecular level, metoprolol expectedly decreased protein kinase A-dependent phospholamban and ryanodine receptor 2 phosphorylation (-42±9% for P-phospholamban-S16 and -22±7% for P-ryanodine receptor 2-S2808; P<0.05). However, this was paralled neither by a reduction in CaMKII autophosphorylation, oxidation, and substrate binding nor a change in the phosphorylation of CaMKII downstream target proteins (n≥11). The lack of CaMKII modulation by β-AR blocker treatment was confirmed in healthy wild-type mice receiving metoprolol.ConclusionsChronic β-AR blocker therapy in patients and in a mouse model of CaMKII-induced HF is not associated with a change in CaMKII activity. Thus, our data suggest that the molecular effects of β-AR blockers are not based on a modulation of CaMKII. Directly targeting CaMKII may, therefore, further improve HF therapy in addition to β-AR blockade.
Project description:Chronic β-adrenoceptor antagonist (β-blocker) treatment in patients is associated with a potentially anti-arrhythmic prolongation of the atrial action potential duration (APD), which may involve remodelling of repolarising K(+) currents. The aim of this study was to investigate the effects of chronic β-blockade on transient outward, sustained and inward rectifier K(+) currents (I(TO), I(KSUS) and I(K1)) in human atrial myocytes and on the expression of underlying ion channel subunits. Ion currents were recorded from human right atrial isolated myocytes using the whole-cell-patch clamp technique. Tissue mRNA and protein levels were measured using real time RT-PCR and Western blotting. Chronic β-blockade was associated with a 41% reduction in I(TO) density: 9.3 ± 0.8 (30 myocytes, 15 patients) vs 15.7 ± 1.1 pA/pF (32, 14), p < 0.05; without affecting its voltage-, time- or rate dependence. I(K1) was reduced by 34% at -120 mV (p < 0.05). Neither I(KSUS), nor its increase by acute β-stimulation with isoprenaline, was affected by chronic β-blockade. Mathematical modelling suggested that the combination of I(TO)- and I(K1)-decrease could result in a 28% increase in APD(90). Chronic β-blockade did not alter mRNA or protein expression of the I(TO) pore-forming subunit, Kv4.3, or mRNA expression of the accessory subunits KChIP2, KChAP, Kvβ1, Kvβ2 or frequenin. There was no reduction in mRNA expression of Kir2.1 or TWIK to account for the reduction in I(K1). A reduction in atrial I(TO) and I(K1) associated with chronic β-blocker treatment in patients may contribute to the associated action potential prolongation, and this cannot be explained by a reduction in expression of associated ion channel subunits.
Project description:Slow-onset adaptive changes that arise from sustained antidepressant treatment, such as enhanced adult hippocampal neurogenesis and increased trophic factor expression, play a key role in the behavioral effects of antidepressants. alpha(2)-Adrenoceptors contribute to the modulation of mood and are potential targets for the development of faster acting antidepressants. We investigated the influence of alpha(2)-adrenoceptors on adult hippocampal neurogenesis. Our results indicate that alpha(2)-adrenoceptor agonists, clonidine and guanabenz, decrease adult hippocampal neurogenesis through a selective effect on the proliferation, but not the survival or differentiation, of progenitors. These effects persist in dopamine beta-hydroxylase knock-out (Dbh(-/-)) mice lacking norepinephrine, supporting a role for alpha(2)-heteroceptors on progenitor cells, rather than alpha(2)-autoreceptors on noradrenergic neurons that inhibit norepinephrine release. Adult hippocampal progenitors in vitro express all the alpha(2)-adrenoceptor subtypes, and decreased neurosphere frequency and BrdU incorporation indicate direct effects of alpha(2)-adrenoceptor stimulation on progenitors. Furthermore, coadministration of the alpha(2)-adrenoceptor antagonist yohimbine with the antidepressant imipramine significantly accelerates effects on hippocampal progenitor proliferation, the morphological maturation of newborn neurons, and the increase in expression of brain derived neurotrophic factor and vascular endothelial growth factor implicated in the neurogenic and behavioral effects of antidepressants. Finally, short-duration (7 d) yohimbine and imipramine treatment results in robust behavioral responses in the novelty suppressed feeding test, which normally requires 3 weeks of treatment with classical antidepressants. Our results demonstrate that alpha(2)-adrenoceptors, expressed by progenitor cells, decrease adult hippocampal neurogenesis, while their blockade speeds up antidepressant action, highlighting their importance as targets for faster acting antidepressants.
Project description:Background Intravenous high-dose glucagon is a recommended antidote against beta-blocker poisonings, but clinical effects are unclear. We therefore investigated hemodynamic effects and safety of high-dose glucagon with and without concomitant beta-blockade. Methods and Results In a randomized crossover study, 10 healthy men received combinations of esmolol (1.25 mg/kg bolus+0.75 mg/kg/min infusion), glucagon (50 µg/kg), and identical volumes of saline placebo on 5 separate days in random order (saline+saline; esmolol+saline; esmolol+glucagon bolus; saline+glucagon infusion; saline+glucagon bolus). On individual days, esmolol/saline was infused from -15 to 30 minutes. Glucagon/saline was administered from 0 minutes as a 2-minute intravenous bolus or as a 30-minute infusion (same total glucagon dose). End points were hemodynamic and adverse effects of glucagon compared with saline. Compared with saline, glucagon bolus increased mean heart rate by 13.0 beats per minute (95% CI, 8.0-18.0; P<0.001), systolic blood pressure by 15.6 mm Hg (95% CI, 8.0-23.2; P=0.002), diastolic blood pressure by 9.4 mm Hg (95% CI, 6.3-12.6; P<0.001), and cardiac output by 18.0 % (95% CI, 9.7-26.9; P=0.003) at the 5-minute time point on days without beta-blockade. Similar effects of glucagon bolus occurred on days with beta-blockade and between 15 and 30 minutes during infusion. Hemodynamic effects of glucagon thus reflected pharmacologic glucagon plasma concentrations. Glucagon-induced nausea occurred in 80% of participants despite ondansetron pretreatment. Conclusions High-dose glucagon boluses had significant hemodynamic effects regardless of beta-blockade. A glucagon infusion had comparable and apparently longer-lasting effects compared with bolus, indicating that infusion may be preferable to bolus injections. Registration Information URL: https://www.clinicaltrials.gov; Unique identifier: NCT03533179.
Project description:Background and purposeIn cardiac myocytes, cyclic AMP (cAMP) produced by both β1 - and β2 -adrenoceptors increases L-type Ca2+ channel activity and myocyte contraction. However, only cAMP produced by β1 -adrenoceptors enhances myocyte relaxation through phospholamban-dependent regulation of the sarco/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2). Here we have tested the hypothesis that stimulation of β2 -adrenoceptors produces a cAMP signal that is unable to reach SERCA2 and determine what role, if any, phosphodiesterase (PDE) activity plays in this compartmentation.Experimental approachThe cAMP responses produced by β1 -and β2 -adrenoceptor stimulation were studied in adult rat ventricular myocytes using two different fluorescence resonance energy transfer (FRET)-based biosensors, the Epac2-camps, which is expressed uniformly throughout the cytoplasm of the entire cell and the Epac2-αKAP, which is targeted to the SERCA2 signalling complex.Key resultsSelective activation of β1 - or β2 -adrenoceptors produced cAMP responses detected by Epac2-camps. However, only stimulation of β1 -adrenoceptors produced a cAMP response detected by Epac2-αKAP. Yet, stimulation of β2 -adrenoceptors was able to produce a cAMP signal detected by Epac2-αKAP in the presence of selective inhibitors of PDE2 or PDE3, but not PDE4.Conclusion and implicationsThese results support the conclusion that cAMP produced by β2 -adrenoceptor stimulation was not able to reach subcellular locations where the SERCA2 pump is located. Furthermore, this compartmentalized response is due at least in part to PDE2 and PDE3 activity. This discovery could lead to novel PDE-based therapeutic treatments aimed at correcting cardiac relaxation defects associated with certain forms of heart failure.
Project description:AimsMethyltransferase like 3 (METTL3) plays a crucial role in cardiovascular diseases, but its involvement in atrial fibrillation (AF) remains unclear. The study aims to explore the relationship between METTL3 and AF in atrial myocytes.Methods and resultsThe protein level of METTL3 was evaluated in left atrial appendages (LAAs) from patients with persistent AF and in experimental AF models. cAMP-responsive element modulator (CREM) transgenic mice and CaCl2-acetylcholine (ACh)-injected mice were used as AF mice models. Methyltransferase like 3 was globally and atrial conditionally deleted in vivo to assess its role in AF. Confocal fluorescence microscopy was employed to examine calcium handling in atrial myocytes. Methylated RNA immunoprecipitation sequencing was performed to identify the downstream target genes of METTL3. Methyltransferase like 3 protein and RNA N6-methyladenosine (m6A) modification levels were significantly reduced in the LAAs of patients with AF and experimental AF models. Genetic inhibition of METTL3 promoted the development of AF in CREM transgenic mice and CaCl2-ACh-injected mice. Knockdown of METTL3 in atrial myocytes resulted in enhanced calcium handling. Reduced METTL3 levels increased SR Ca2+-ATPase Type 2a activity by up-regulating protocadherin gamma subfamily A, 10. Decreased METTL3 protein in atrial myocytes was attributed to down-regulation of cAMP-responsive element-binding protein 1/ubiquitin-specific peptidase 9 X-linked axis.ConclusionOur study established the pathophysiological role of METTL3 involved in the development of AF and provided a potential mechanism-based target for its treatment.
Project description:In the heart, Na(+) is a key modulator of the action potential, Ca(2+) homeostasis, energetics, and contractility. Because Na(+) currents and cotransport fluxes depend on the Na(+) concentration in the submembrane region, it is necessary to accurately estimate the submembrane Na(+) concentration ([Na(+)]sm). Current methods using Na(+)-sensitive fluorescent indicators or Na(+) -sensitive electrodes cannot measure [Na(+)]sm. However, electrophysiology methods are ideal for measuring [Na(+)]sm. In this article, we develop patch-clamp protocols and experimental conditions to determine the upper bound of [Na(+)]sm at the peak of action potential and its lower bound at the resting state. During the cardiac cycle, the value of [Na(+)]sm is constrained within these bounds. We conducted experiments in rabbit ventricular myocytes at body temperature and found that 1) at a low pacing frequency of 0.5 Hz, the upper and lower bounds converge at 9 mM, constraining the [Na(+)]sm value to ∼9 mM; 2) at 2 Hz pacing frequency, [Na(+)]sm is bounded between 9 mM at resting state and 11.5 mM; and 3) the cells can maintain [Na(+)]sm to the above values, despite changes in the pipette Na(+) concentration, showing autoregulation of Na(+) in beating cardiomyocytes.