Presence Of Isoprenaline Research Articles

Cardiomyocytes from female compared to male mice have larger ryanodine receptor clusters and higher calcium spark frequency.

Sex differences in cardiac physiology are receiving increased attention as it has become clear that men and women have different aetiologies of cardiac disease and require different treatments. There are experimental data suggesting that male cardiomyocytes exhibit larger Ca2+ transients due to larger Ca2+ sparks and a higher excitation-contraction coupling gain; in addition, they exhibit a larger response to adrenergic stimulation with isoprenaline (ISO). Here, we studied whether there are sex differences relating to structural organization of the transverse tubular network and ryanodine receptors (RyRs). Surprisingly, we found that female cardiomyocytes exhibited a higher spark frequency in a range of spark magnitudes. While overall RyR expression and phosphorylation were the same, female cardiomyocytes had larger but fewer RyR clusters. The density of transverse t-tubules was the same, but male cardiomyocytes had more longitudinal t-tubules. The Ca2+ transients were similar in male and female cardiomyocytes under control conditions and in the presence of ISO. The synchrony of the Ca2+ transients was similar between sexes as well. Overall, our data suggest subtle sex differences in the Ca2+ influx and efflux pathways and their response to ISO, but these differences are balanced, resulting in similar Ca2+ transients in field-stimulated male and female cardiomyocytes. The higher spark frequency in female cardiomyocytes is related to the organization of RyRs into larger, but fewer clusters. KEY POINTS: During a heartbeat, the force of contraction depends on the amplitude of the calcium transient, which in turn depends on the amount of calcium released as calcium sparks through ryanodine receptors in the sarcoplasmic reticulum. Previous studies suggest that cardiomyocytes from male compared to female mice exhibit larger calcium sparks, larger sarcoplasmic reticulum calcium release and greater response to adrenergic stimulation triggering a fight-or-flight response. In contrast, we show that cardiomyocytes from female mice have a higher spark frequency during adrenergic stimulation and similar spark morphology. The higher spark frequency is related to the organization of ryanodine receptors into fewer, but larger clusters in female compared to male mouse cardiomyocytes. Despite subtle sex differences in cardiomyocyte structure and calcium fluxes, the differences are balanced, leading to similar calcium transients in cardiomyocytes from male and female mice.

Mass spectrometry captures biased signalling and allosteric modulation of a G-protein-coupled receptor

G-protein-coupled receptors signal through cognate G proteins. Despite the widespread importance of these receptors, their regulatory mechanisms for G-protein selectivity are not fully understood. Here we present a native mass spectrometry-based approach to interrogate both biased signalling and allosteric modulation of the β1-adrenergic receptor in response to various ligands. By simultaneously capturing the effects of ligand binding and receptor coupling to different G proteins, we probed the relative importance of specific interactions with the receptor through systematic changes in 14 ligands, including isoprenaline derivatives, full and partial agonists, and antagonists. We observed enhanced dynamics of the intracellular loop 3 in the presence of isoprenaline, which is capable of acting as a biased agonist. We also show here that endogenous zinc ions augment the binding in receptor–Gs complexes and propose a zinc ion-binding hotspot at the TM5/TM6 intracellular interface of the receptor–Gs complex. Further interrogation led us to propose a mechanism in which zinc ions facilitate a structural transition of the intermediate complex towards the stable state.

A case study: High resolution optical mapping of a 19 year old male victim of sudden cardiac death during a marathon

Fifty thousand athletes suffer sudden cardiac death every year due to ventricular fibrillation (VF). Most sudden cardiac deaths of young patients have unexplained origin due to the absence of structural or electrical disease. Purkinje network is essential for a normal heart function, however, its role during electrical pathologies remains unclear. A case study to investigate the electrophysiological properties and the Purkinje network role in a 19 years old male after suffering a fatal sudden cardiac arrest during a marathon event. A coronary-perfused left ventricular preparation was imaged using high resolution optical mapping of epicardial and endocardial surfaces using a voltage-sensitive dye, Di-4-ANEPPS. Activation time, repolarization time and action potential duration were assessed during paced activity through the His bundle and endocardial and epicardial surfaces. Conduction system activation origins were identified as distant endocardial origins during His bundle pacing. Short-coupled S1S2S3S4 pacing was used to determine effective refractory period (ERP). Pacing protocols were repeated in the presence of isoprenaline (ISO) at 100 nM, 500 nM and 1 μM to mimic stress conditions. No changes in total activation time are observed during myocardial pacing cycle lengths of 667ms in control (63ms) and under ISO (up to 64ms). In contrary, His-ventricle delay during conduction system pacing was shorted from 61ms in control to 42ms, 44ms and 16ms under 100 nM, 500 nM and 1 μM of ISO respectively. Under His pacing, local APD gradients are observed near Purkinje-muscle junctions under ISO (from 3ms/mm and 3 ms/mm to 40ms/mm and 41ms/mm). ERP under His pacing was 55ms shorter than endocardial pacing. 82% of the ectopy origins observed have less than 4 mm distance from the PMJs. Repeated focal activations were observed during VF and 76% of them had less than 4 mm distance from PMJs. No structural abnormalities were identified under 9.4 T MRI. Conduction system electrical abnormalities play a role in arrhythmogenicity and maintenance of VF in this patient during sudden cardiac death.

Pilot Study on Acute Effects of Pharmacological Intraperitoneal L-Homoarginine on Homeostasis of Lysine and Other Amino Acids in a Rat Model of Isoprenaline-Induced Takotsubo Cardiomyopathy.

L-Arginine:glycine amidinotransferase (AGAT) catalyzes the formation of L-homoarginine (hArg) and L-ornithine (Orn) from L-arginine (Arg) and L-lysine (Lys): Arg + Lys ↔ hArg + Orn; equilibrium constant KhArg. AGAT also catalyzes the formation of guanidinoacetate (GAA) and Orn from Arg and glycine (Gly): Arg + Gly ↔ GAA + Orn; equilibrium constant KGAA. In humans, pharmacological hArg is metabolized to Lys. Low circulating and low excretory concentrations of hArg are associated with worse outcomes and mortality in the renal and cardiovascular systems. The metabolism and pharmacology of hArg have been little investigated. In the present study, we investigated the effects of pharmacological hArg (i.p., 0, 20, 220, 440 mg/kg at time point 0 min) on amino acids homeostasis in a rat model of isoprenaline-induced takotsubo cardiomyopathy (i.p., 50 mg/kg at time point 15 min). We measured by gas chromatography-mass spectrometry free and proteinic amino acids, as well as the polyamines putrescine and spermidine in the heart, lung, kidney, and liver of ten rats sacrificed at various time points (range, 0 to 126 min). hArg administration resulted in multiple changes in the tissue contents of several free and proteinic amino acids, as well as in the putrescine-spermidine molar ratio, an indicator of polyamines catabolism. Our results suggest that Lys and Arg are major metabolites of pharmacological hArg. Kidneys and heart seem to play a major metabolic role for hArg. Circulating Lys does not change over time, yet there is a considerable interchange of free Lys between organs, notably kidney and heart, during the presence of isoprenaline in the rats (time range, 15 to 90 min). Antidromic changes were observed for KhArg and KGAA, notably in the heart in this time window. Our study shows for the first time that free hArg and sarcosine (N-methylglycine) are positively associated with each other. The acute effects of high-dosed hArg administration and isoprenaline on various amino acids and on AGAT-catalyzed reaction in the heart, lung, kidney, and liver are detailed and discussed.

B-PO05-013 A CASE STUDY: HIGH RESOLUTION OPTICAL MAPPING OF A 19 YEAR OLD MALE VICTIM OF SUDDEN CARDIAC DEATH DURING A MARATHON

Sudden cardiac death due to ventricular fibrillation impacts 50 000 athletes per year. The most common finding at autopsy amongst young adults is sudden cardiac death with unexplained origins due to the absence of structural cardiac disease. A case study to investigate the electrophysiological properties in a male of 19 years old after suffering a fatal sudden cardiac arrest during a marathon event. A coronary-perfused left ventricular preparation was imaged using high resolution optical mapping of both epicardial and endocardial surfaces using a voltage-sensitive dye, Di-4-ANEPPS. Activation time, repolarization time and action potential duration were assessed during paced activity through the His bundle and endocardial and epicardial surfaces. Conduction system activation origins were identified as distant endocardial origins during His bundle pacing. Short-coupled S1S2S3S4 pacing was used to determine effective refractory period (ERP). Pacing protocols were repeated in the presence of isoprenaline (ISO) at 100nM, 500nM and 1μM to mimic stress conditions. No structural abnormalities were identified at the macroscopic level. At pacing cycle lengths of 667ms, a total myocardial activation time of 63ms was observed. Negligible changes were found in the presence of isoprenaline at all tested concentrations (up to 64ms). However, the latency of activation of conduction system activation origins following His pacing was shortened by ISO from 61ms to 42ms, 44ms and 16ms for 100nM, 500nM and 1μM. At short cycle length (250ms) His pacing, considerable AT latency reduction from 146ms to 43ms was observed under ISO at 100nM, but insensitive to further increases of ISO concentration. Local APD and RT gradients at His pacing origins were greatly enhanced by ISO (from 3ms/mm & 3ms/mm to 40ms/mm & 41 ms/mm) at short cycle lengths. The ERP of the His was up to 55ms shorter than endocardial pacing. Origins of ectopic activity coincided with His paced responses and frequency increased with ISO. The cardiac conduction system showed a greater dynamic range of excitability under short cycle length and isoprenaline stresses compared to the myocardium and likely underlie local repolarization gradients at Purkinje termini.

IOCBIO Sparks detection and analysis software

Analysis of calcium sparks in cardiomyocytes can provide valuable information about functional changes of calcium handling in health and disease. As a part of the calcium sparks analysis, sparks detection and characterization is necessary. Here, we describe a new open-source platform for automatic calcium sparks detection from line scan confocal images. The developed software is tailored for detecting only calcium sparks, allowing us to design a graphical user interface specifically for this task. The software enables detecting sparks automatically as well as adding, removing, or adjusting regions of interest marking each spark. The results of the analysis are stored in an SQL database, allowing simple integration with statistical tools. We have analyzed the performance of the algorithm using a large set of synthetic images with varying spark sizes and noise levels and also compared the analysis results with results obtained by software established in the field. The use of our software is illustrated by an analysis of the effect of isoprenaline (ISO) on spark frequency, amplitude, and spatial and temporal characteristics. For that, cardiomyocytes from C57BL/6 mice were used. We demonstrated an increase in spark frequency, tendency of having larger spark amplitudes, sparks with a longer duration, and occurrence of multiple sparks from the same site in the presence of ISO. We also show that the duration and the width of sparks with the same amplitude were similar in the absence and presence of ISO. The software was released as an open source repository and is available for free use and collaborative development.

Iocbio Sparks Detection and Analysis Software

Analysis of calcium sparks in cardiomyocytes can provide valuable information about functional changes of calcium handling in health and disease. As a part of the calcium sparks analysis, sparks detection and characterization is necessary. Here, we describe a new open-source platform for automatic calcium sparks detection from line scan confocal images. The developed software is tailored for detecting only calcium sparks, allowing us to design a graphical user interface specifically for this task. The software enables detecting sparks automatically as well as adding, removing, or adjusting regions of interest marking each spark. The results of the analysis are stored in an SQL database, allowing simple integration with statistical tools. We have analyzed the performance of the algorithm using a large set of synthetic images with varying spark sizes and noise levels and also compared the analysis results with results obtained by software established in the field. The use of our software is illustrated by an analysis of the effect of isoprenaline (ISO) on spark frequency, amplitude, and spatial and temporal characteristics. For that, cardiomyocytes from C57BL/6 mice were used. We demonstrated an increase in spark frequency, tendency of having larger spark amplitudes, sparks with a longer duration, and occurrence of multiple sparks from the same site in the presence of ISO. We also show that the duration and the width of sparks with the same amplitude were similar in the absence and presence of ISO. The software was released as an open source repository and is available for free use and collaborative development.

Inotropic interventions do not change the resting state of myosin motors during cardiac diastole.

When striated (skeletal and cardiac) muscle is in its relaxed state, myosin motors are packed in helical tracks on the surface of the thick filament, folded toward the center of the sarcomere, and unable to bind actin or hydrolyze ATP (OFF state). This raises the question of whatthe mechanism is that integrates the Ca2+-dependent thin filament activation, making myosin heads available for interaction with actin. Here we test the interdependency of the thin and thick filament regulatory mechanisms in intact trabeculae from the rat heart. We record the x-ray diffraction signals that mark the state of the thick filament during inotropic interventions (increase in sarcomere length from 1.95 to 2.25 µm and addition of 10-7 M isoprenaline), which potentiate the twitch force developed by an electrically paced trabecula by up to twofold. During diastole, none of the signals related to the OFF state of the thick filament are significantly affected by these interventions, except the intensity of both myosin-binding protein C- and troponin-related meridional reflections, which reduce by 20% in the presence of isoprenaline. These results indicate that recruitment of myosin motors from their OFF state occurs independently and downstream from thin filament activation. This is in agreement with the recently discovered mechanism based on thick filament mechanosensing in which the number of motors available for interaction with actin rapidly adapts to the stress on the thick filament and thus to the loading conditions of the contraction. The gain of this positive feedback may be modulated by both sarcomere length and the degree of phosphorylation of myosin-binding protein C.

Late sodium current inhibitors to treat exercise-induced obstruction in hypertrophic cardiomyopathy: an in vitro study in human myocardium.

Background and PurposeIn 30–40% of hypertrophic cardiomyopathy (HCM) patients, symptomatic left ventricular (LV) outflow gradients develop only during exercise due to catecholamine‐induced LV hypercontractility (inducible obstruction). Negative inotropic pharmacological options are limited to β‐blockers or disopyramide, with low efficacy and tolerability. We assessed the potential of late sodium current (INaL)‐inhibitors to treat inducible obstruction in HCM.Experimental ApproachThe electrophysiological and mechanical responses to β‐adrenoceptor stimulation were studied in human myocardium from HCM and control patients. Effects of INaL‐inhibitors (ranolazine and GS‐967) in HCM samples were investigated under conditions simulating rest and exercise.Key ResultsIn cardiomyocytes and trabeculae from 18 surgical septal samples of patients with obstruction, the selective INaL‐inhibitor GS‐967 (0.5 μM) hastened twitch kinetics, decreased diastolic [Ca2+] and shortened action potentials, matching the effects of ranolazine (10μM). Mechanical responses to isoprenaline (inotropic and lusitropic) were comparable in HCM and control myocardium. However, isoprenaline prolonged action potentials in HCM myocardium, while it shortened them in controls. Unlike disopyramide, neither GS‐967 nor ranolazine reduced force at rest. However, in the presence of isoprenaline, they reduced Ca2+‐transient amplitude and twitch tension, while the acceleration of relaxation was maintained. INaL‐inhibitors were more effective than disopyramide in reducing contractility during exercise. Finally, INaL‐inhibitors abolished arrhythmias induced by isoprenaline.Conclusions and ImplicationsRanolazine and GS‐967 reduced septal myocardium tension during simulated exercise in vitro and therefore have the potential to ameliorate symptoms caused by inducible obstruction in HCM patients, with some advantages over disopyramide and β‐blockers.

Is rate–pressure product of any use in the isolated rat heart? Assessing cardiac ‘effort’ and oxygen consumption in the Langendorff‐perfused heart

New Findings What is the central question of this study? Rate–pressure product (RPP) is commonly used as an index of cardiac ‘effort’. In canine and human hearts (which have a positive force–frequency relationship), RPP is linearly correlated with oxygen consumption and has therefore been widely adopted as a species‐independent index of cardiac work. However, given that isolated rodent hearts demonstrate a negative force–frequency relationship, its use in this model requires validation. What is the main finding and its importance? Despite its widespread use, RPP is not correlated with oxygen consumption (or cardiac ‘effort’) in the Langendorff‐perfused isolated rat heart. This lack of correlation was also evident when perfusions included a range of metabolic substrates, insulin or β‐adrenoceptor stimulation. Langendorff perfusion of hearts isolated from rats and mice has been used extensively for physiological, pharmacological and biochemical studies. The ability to phenotype these hearts reliably is, therefore, essential. One of the commonly used indices of function is rate–pressure product (RPP); a rather ill‐defined index of ‘work’ or, more correctly, ‘effort’. Rate–pressure product, as originally described in dog or human hearts, was shown to be correlated with myocardial oxygen consumption (MV˙O2). Despite its widespread use, the application of this index to rat or mouse hearts (which, unlike the dog or human, have a negative force–frequency relationship) has not been characterized. The aim of this study was to examine the relationship between RPP and MV˙O2 in Langendorff‐perfused rat hearts. Paced hearts (300–750 beats min−1) were perfused either with Krebs–Henseleit (KH) buffer (11 mm glucose) or with buffer supplemented with metabolic substrates and insulin. The arteriovenous oxygen consumption (MV˙O2) was recorded. Metabolic status was assessed using 31P magnetic resonance spectroscopy and lactate efflux. Experiments were repeated in the presence of isoprenaline and in unpaced hearts where heart rate was increased by cumulative isoprenaline challenge. In KH buffer‐perfused hearts, MV˙O2 increased with increasing heart rate, but given that left ventricular developed pressure decreased with increases in rate, RPP was not correlated with MV˙O2, lactate production or phosphocreatine/ATP ratio. Although the provision of substrates or β‐adrenoceptor stimulation changed the shape of the RPP–MV˙O2 relationship, neither intervention resulted in a positive correlation between RPP and oxygen consumption. Rate–pressure product is therefore an unreliable index of oxygen consumption or ‘cardiac effort’ in the isolated rat heart.

H2S induces vasoconstriction of rat cerebral arteries via cAMP/adenylyl cyclase pathway

Hydrogen sulfide (H2S), traditionally known for its toxic effects, is now involved in regulating vascular tone. Here we investigated the vasoconstrictive effect of H2S on cerebral artery and the underlying mechanism. Sodium hydrosulfide (NaHS), a donor of H2S, concentration-dependently induced vasoconstriction on basilar artery, which was enhanced in the presence of isoprenaline, a β-adrenoceptor agonist or forskolin, an adenylyl cyclase activator. Administration of NaHS attenuated the vasorelaxant effects of isoprenaline or forskolin. Meanwhile, the NaHS-induced vasoconstriction was diminished in the presence of 8B-cAMP, an analog of cAMP, but was not affected by Bay K-8644, a selective L-type Ca2+ channel agonist. These results could be explained by the revised effects of NaHS on isoprenaline-induced cAMP elevation and forskolin-stimulated adenylyl cyclase activity. Additionally, NaHS-induced vasoconstriction was enhanced by removing the endothelium or in the presence of L-NAME, an inhibitor of nitric oxide synthase. L-NAME only partially attenuated the effect of NaHS which was given together with forskolin on the pre-contracted artery. In conclusion, H2S induces vasoconstriction of cerebral artery via, at least in part, cAMP/adenylyl cyclase pathway.

0406: Effects of FGF23 and Klotho on adult rat cardiomyocytes in culture

The bone derived hormone fibroblast growth factor 23 (FGF23) and its coreceptor Klotho represent a novel endocrine axis regulating mineral metabolism in health and disease. FGF23-Klotho signalling inhibits renal phosphate reabsorption and activation of vitamin D, and reduces secretion of parathyroid hormone. Serum levels of FGF23 rise in chronic kidney disease (CKD). In contrast, tissue expression of Klotho decreases in parallel with CKD progression and reaches low or undetectable levels in end stage renal disease. Numerous studies identify elevated FGF23 as a predictor of adverse clinical outcome. In particular, elevated FGF23 has recently been associated with greater risks of major cardiovascular events and mortality. However, there have been very few studies that have attempted to address the direct effects of FGF23 on myocardium. Moreover whether Klotho is involved in FGF23 – mediated actions on cardiomyocytes is still unclear. In this context, we investigate the role of FGF23 and Klotho in adult rat ventricular myocytes (ARVMs). Using video-edge-detection, epifluorescent microscopy and an Ionoptix® system, performed in isolated cardiomyocytes subjected to FGF23 or Klotho alone, or in association, we showed that FGF23 increases cell size and cell shortening in ARVMs, and induces arrhythmia in the presence of Isoprenaline. In addition Klotho prevents FGF23 effects on adult cardiomyocytes. Indeed, ARVMs subjected to Klotho showed marked protection from FGF23-induced hypertrophic responses and from FGF23- induced arrhythmias in the presence of Isoprenaline. Altogether these preliminary data provide a direct evidence of the role FGF23 in adult cardiomyocytes and suggest that Klotho may have a beneficial effect in preventing adverse cardiovascular outcomes in patients with or without CKD.

SERCA2 activity is involved in the CNP‐mediated functional responses in failing rat myocardium

Myocardial C-type natriuretic peptide (CNP) levels are increased in heart failure. CNP can induce negative inotropic (NIR) and positive lusitropic responses (LR) in normal hearts, but its effects in failing hearts are not known. We studied the mechanism of CNP-induced NIR and LR in failing hearts and determined whether sarcoplasmatic reticulum Ca(2+) ATPase2 (SERCA2) activity is essential for these responses. Contractility, cGMP levels, Ca(2+) transient amplitudes and protein phosphorylation were measured in left ventricular muscle strips or ventricular cardiomyocytes from failing hearts of Wistar rats 6 weeks after myocardial infarction. CNP increased cGMP levels, evoked a NIR and LR in muscle strips, and caused phospholamban (PLB) Ser(16) and troponin I (TnI) Ser(23/24) phosphorylation in cardiomyocytes. Both the NIR and LR induced by CNP were reduced in the presence of a PKG blocker/cGMP analogue (Rp-8-Br-Pet-cGMPS) and the SERCA inhibitor thapsigargin. CNP increased the amplitude of the Ca(2+) transient and increased SERCA2 activity in cardiomyocytes. The CNP-elicited NIR and LR were not affected by the L-type Ca(2+) channel activator BAY-K8644, but were abolished in the presence of isoprenaline (induces maximal activation of cAMP pathway). This suggests that phosphorylation of PLB and TnI by CNP causes both a NIR and LR. The NIR to CNP in mouse heart was abolished 8 weeks after cardiomyocyte-specific inactivation of the SERCA2 gene. We conclude that CNP-induced PLB and TnI phosphorylation by PKG in concert mediate both a predictable LR as well as the less expected NIR in failing hearts.

Popdc knock out results in sick sinus syndrome

Purpose: The Popeye domain containing (Popdc) family of three novel muscle-restricted genes (Popdc1-3) are evolutionarily conserved and expressed prominently within the cardiac conduction system and the purpose of this study was to delineate the role of Popdc1 in sinus node pacemaking using transgenic mice lacking the Popdc1 gene (Popdc1-/-). Methods: Male Popdc1-/- mice (C57BL/6J background) of 5.5 months age were used. The study followed "Principles of laboratory animal care" (NIH Publication no. 85-23 revised 1985) and is in accordance with the United Kingdom Animals (Scientific Procedures) Act, 1986. The ECG in conscious animals was recorded via telemetry at rest, during and after physiological stress (swim) or catecholamine treatment. In addition, extracellular potentials were recorded from isolated sinus node preparations. Results: At 5.5 months, Popdc1-/- mice showed reduced body weight compared to wild type (WT) mice (27±2 vs. 32±1 g; P<0.05). Popdc1-/- mice displayed no obvious pathological cardiac phenotype at rest, but developed an unusual bradycardia with sinus pauses in response to swim stress or catecholamine treatment. In isolated sinus node preparations from Popdc1-/- mice, the intrinsic sinus rate was not significantly different from that in preparations from WT mice (363±16 vs. 387±20 bpm). β-adrenergic stimulation with the agonist isoprenaline (5 nM) induced a positive chronotropic effect (456±18 bpm) in WT preparations, but this effect was blunted (378±9 bpm; P<0.05 vs. WT) in Popdc1-/- preparations. In the presence of isoprenaline (5, 15 and 50 nM), extrasystoles, bursts and sinus pauses were observed, but these three phenotypes were either more prevalent or occurred exclusively in Popdc1-/- preparations. This abberant pacemaking in the Popdc-/- mice could be the result of a disturbance of either the voltage- or Ca2+-clock pacemaker mechanisms. Blocking the pacemaker current I(f) (major component of voltage-clock) with 2 mM Cs(+) induced a bradycardia that was equally pronounced in the WT (265±25 vs. 385±20 bpm at control; P<0.05) and Popdc1-/- (220±14 vs. 355±5 bpm at control; P<0.05) preparations, indicating no change in I(f). However, isoprenaline (50 nM) failed to evoke extrasystoles, bursts and sinus pauses when the Ca2+-clock was rendered dysfunctional with 2 μM ryanodine suggesting a role for the Ca2+-clock in the isoprenaline induced bradycardia phenotype. Conclusions: Popdc1-/- is essential for stress-mediated modulation of cardiac pacemaking. Ca2+-clock proteins are likely mediators of the β-adrenergic stimulation induced sinus bradycardia phenotype.

Voltammetric determination of 6-mercaptopurine using a multiwall carbon nanotubes paste electrode in the presence of isoprenaline as a mediator

In this study, we describe the application of multiwall carbon nanotubes paste electrode (MWCNTPE) in the presence of isoprenaline (ISPT) as the suitable mediator for voltammetric determination of 6-mercaptopurine (6-MP) in aqueous buffer solution (pH 4.0). The results indicate that the electrode is efficient in terms of its electrocatalytic activity for the oxidation of 6-MP in the presence of ISPT as a mediator. Using chronoamperometry, the catalytic reaction rate constant was calculated at 1.68×10−3μM−1s−1. The catalytic peak current was linearly dependent on 6-mercaptopurine concentration in the range of 0.5–900μM 6-MP. The detection limit (3σ) obtained by linear sweep voltammetry (LSV) was 0.1μM. Finally, the proposed method was also examined as a selective, simple and precise electrochemical sensor for the determination of 6-MP in real samples such as urine and tablet.

The effect of ligustrazine on L-type calcium current, calcium transient and contractility in rabbit ventricular myocytes

Ethnopharmacological relevanceLigustrazine, the biologically active ingredient isolated from a popular Chinese medicinal plant, Ligusticum chuanxiong Hort. (Umbelliferae), has been used effectively to treat ischemic heart diseases, cerebrovascular and thrombotic vascular diseases since the 1970s. Materials and methodsAt present, the effect of ligustrazine on L-type calcium current (ICa-L) of ventricular myocytes remains controversial. In this study, we use the whole-cell patch-clamp techniques and video-based edge detection and dual excitation fluorescence photomultiplier systems to study the effects of ligustrazine on ICa-L, and calcium transient and contractility in rabbit ventricular myocytes in the absence and presence of isoprenaline (ISO). ResultsLigustrazine (5μM) in low concentration did not affect ICa-L (P>0.05), higher concentrations of this drug (10, 20, 40, 80μM) inhibited ICa-L in a concentration-dependent manner and reduced ICa-L by 9.6±2.9%, 21.0±4.3%, 33.9±4.3%, and 51.6±7.3%, respectively. Under normal conditions, ligustrazine (40μΜ) reduced baseline of fura-2 fluorescence intensities (FFI, 340/380 ratio), namely diastolic calcium concentration, changes in FFI (ΔFFI, 340/380 ratio) and maximal velocity of Ca2+ rise and decay (340/380ratio/ms) by 6.3%, 26.1%, 25.2%, and 26.5%, and decreased sarcomere peak shorting (PS) and maximal velocity of shorting and relengthening by 36.4%, 31.9%, and 25.0%, respectively. Similarly, ligustrazine (40μM) reduced baseline FFI, ΔFFI, and maximal velocity of Ca2+ rise and decay by 14.1%, 51.1%, 35.2%, and 41.1%, and reduced sarcomere PS and maximal velocity of shorting and relengthening by 38.6%, 50.0% and 39.1%, respectively, in the presence of ISO. ConclusionsLigustrazine not only significantly inhibits ICa-L in a concentration-dependent manner but also suppressed calcium transient and contraction in the absence and presence of ISO.

Molecular Dynamics Simulations of the Effect of the G-Protein and Diffusible Ligands on the β2-Adrenergic Receptor

G-protein-coupled receptors have extraordinary therapeutic potential as targets for a broad spectrum of diseases. Understanding their function at the molecular level is therefore essential. A variety of crystal structures have made the investigation of the inactive receptor state possible. Recently released X-ray structures of opsin and the β 2-adrenergic receptor (β 2AR) have provided insight into the active receptor state. In addition, we have contributed to the crystal structure of an irreversible agonist–β 2 adrenoceptor complex. These extensive studies and biophysical investigations have revealed that agonist binding leads to a low-affinity conformation of the active state that is suggested to facilitate G-protein binding. The high-affinity receptor state, which promotes signal transduction, is only formed in the presence of both agonist and G-protein. Despite numerous crystal structures, it is not yet clear how ligands tune receptor dynamics and G-protein binding. We have now used molecular dynamics simulations to elucidate the distinct impact of agonist and inverse agonist on receptor conformation and G-protein binding by investigating the influence of the ligands on the structure and dynamics of a complex composed of β 2AR and the C-terminal end of the Gα s subunit (GαCT). The simulations clearly showed that the agonist isoprenaline and the inverse agonist carazolol influence the ligand-binding site and the interaction between β 2AR and GαCT differently. Isoprenaline induced an inward motion of helix 5, whereas carazolol blocked the rearrangement of the extracellular part of the receptor. Moreover, in the presence of isoprenaline, β 2AR and GαCT form a stable interaction that is destabilized by carazolol.

Effects of tetraethylammonium, 4-aminopyridine and bretylium on cardiovascular tissues from normo- and hypertensive rats.

Our objective was to test whether potassium-channel blockade is a potential positive inotropic mechanism for heart failure. Thus we studied the effects of tetraethylammonium, 4-aminopyridine and bretylium on left ventricular action potentials, left ventricular contractility in the absence and presence of hypertrophy, and on isolated blood vessels from Wistar Kyoto normotensive rats (WKY) and spontaneously hypertensive rats (SHRs). Tetraethylammonium at 10(-3)-10(-2) M, 4-aminopyridine at 10(-4)-10(-3) M and bretylium at 10(-6)-10(-4) M prolonged the action potentials of the WKY left ventricular strip. Similar concentrations of tetraethylammonium, 4-aminopyridine and bretylium augmented the peak force, prolonged the contractions, and did not cause arrhythmias in the absence or presence of isoprenaline on left ventricular strips from 12-month-old WKY. The 12-month-old SHR has hypertrophy of the left ventricle with reduced contractility and prolongation of relaxation. The effects of tetraethylammonium and bretylium were similar on WKY and SHR, whereas the effects of 4-aminopyridine were reduced on SHR left ventricular contractility, which suggests that the function of the transient outward-blocking potassium channel may be impaired in hypertrophy. Bretylium at < or = 10(-4) M had no effect on the portal vein, intralobar or mesenteric arteries. Tetraethylammonium and 4-aminopyridine at > or = 10(-5) M increased the duration or amplitude, or both, of the portal vein contractions. Tetraethylammonium at > or = 10(-2) M and 4-aminopyridine at > or = 3 x 10(-4) M contracted the mesenteric artery, and 4-aminopyridine also contracted the intralobar pulmonary artery. In summary, we have demonstrated that the action potential prolonging effects of potassium-channel blockade is associated with a positive inotropic effect on the rat left ventricle. The non-specific blockers, tetraethylammonium and 4-aminopyridine, do not have potential as positive inotropes in heart failure because of their widespread effects, including vasoconstriction. The potential of bretylium and some of the newer selective potassium-channel blockers as positive inotropes requires further evaluation.

Genetic disruption of G proteins, Gi2α or Goα, does not abolish inotropic and chronotropic effects of stimulating muscarinic cholinoceptors in atrium

Classically, stimulation of muscarinic cholinoceptors exerts negative inotropic and chronotropic effects in the atrium of mammalian hearts. These effects are crucial to the vagal regulation of the heart beat. This effect is assumed to be mediated via GTP binding (G) proteins, because they can be abolished by Pertussis toxin. However, it is unknown which G proteins are involved. We studied contractility in isolated left or right atrium from genetically manipulated mice with deletion of one of two G proteins, either of the alpha subunit of G(i2) protein (G(i2)alpha) or of the alpha subunit of G(o) protein (G(o)alpha). Preparations were stimulated with carbachol alone or after pretreatment with the beta-adrenoceptor agonist isoprenaline. For comparison, the effects of carbachol on L-type Ca(2+)-channels in isolated ventricular cardiomyocytes were studied. The negative inotropic and chronotropic effects of carbachol alone or in the presence of isoprenaline were identical in atria from knockout or wild-type mice. However, the effect of carbachol on isoprenaline-activated L-type Ca(2+)-channel in isolated ventricular cardiomyocytes was greatly attenuated in both types of knockout mice studied. These data imply that there is either redundancy of G proteins for signal transduction or that Pertussis toxin-sensitive proteins other than G(i2)alpha and G(o)alpha mediate the vagal stimulation in the atrium. Moreover, different G proteins mediate the effect of carbachol in ventricle compared with atrium.

Oestrogen changed cardiomyocyte contraction and β‐adrenoceptor expression in rat hearts subjected to ischaemia–reperfusion

Women with functional ovaries have a lower cardiovascular risk than men and postmenopausal women. However, oestrogen replacement therapy remains controversial. This study examined the effect of ovarian hormone deficiency and oestrogen replacement on ventricular myocyte contractile function and expression of beta-adrenoceptors (beta-ARs). Female Sprague-Dawley rats were subjected to bilateral ovariectomy (OVX) or sham operation (Sham). A subgroup of OVX rats received oestrogen (E2) replacement (40 microg kg(-1) day(-1)) for 4 weeks. Cardiomyocyte shortening was evaluated in basal conditions and in the presence of isoprenaline (ISO). The expression of beta-ARs was assessed by Western blotting. The presence of lactate dehydrogenase (LDH) activity in the coronary effluent was determined. Ovariectomy promoted body weight gain associated with reduced serum E2 and uterine weight, all of which were abolished by treatment with E2. Ovariectomy increased the amplitude of both basal and ISO-stimulated contractions, increased LDH release, upregulated beta1-AR expression and downregulated beta2-AR expression, all of which were restored by treatment with E2. A beta1-AR antagonist, CGP20712A, but not a beta2-AR antagonist, ICI118,551, significantly decreased the amplitude of ventricular myocyte shortening. Oestrogen decreased cardiomyocyte contraction and the expression of beta1-AR, and increased expression of beta2-AR, and all these effects were abolished by the E2 receptor antagonist, ICI182,780. These data suggest that oestrogen plays a cardioprotective role in female rat hearts subjected to ischaemia-reperfusion injury, and the effects of oestrogen are associated with decreased cardiomyocyte contraction and expression of beta1-AR, and increased expression of beta2-AR.