Positive Chronotropic Effects Research Articles

Levosimendan increases the phosphorylation state of phospholamban in the isolated human atrium

Levosimendan (up to 10 µM) given alone failed to increase force of contraction in isolated electrically stimulated (1 Hz) left atrial (LA) preparations from wild-type mice. Only in the additional presence of 0.1 µM rolipram, an inhibitor of the activity of phosphodiesterase IV, levosimendan increased force of contraction in LA and increased the phosphorylation state of phospholamban at amino acid serine 16. Levosimendan alone increased the beating rate in isolated spontaneously beating right atrial preparations from mice and this effect was potentiated by rolipram. The positive inotropic and the positive chronotropic effects of levosimendan in mouse atrial preparations were attenuated by 10 µM propranolol. Finally, we studied the contractile effects of levosimendan in isolated electrically stimulated (1 Hz) right atrial preparations from the human atrium (HAP), obtained during cardiac surgery. We detected concentration-dependent positive inotropic effects of levosimendan alone that reached plateau at 1 µM levosimendan in HAP (n = 11). Levosimendan shortened time of tension relaxation in HAP. Cilostamide (1 µM), an inhibitor of phosphodiesterase III, or propranolol (10 µM) blocked the positive inotropic effect of levosimendan in HAP. Levosimendan (1 µM) alone increased in HAP the phosphorylation state of phospholamban. In conclusion, we present evidence that levosimendan acts via phosphodiesterase III inhibition in the human atrium leading to phospholamban phosphorylation and thus explaining the positive inotropic effects of levosimendan in HAP.

Myocardial perfusion recovery induced by an α-calcitonin gene-related peptide analogue

BackgroundEndogenous calcitonin gene-related peptide (CGRP) induces cardioprotective effects through coronary vasodilation. However, the systemic administration of CGRP induces peripheral vasodilation and positive chronotropic and inotropic effects. This study aims to examine the net effect on coronary perfusion of the systemically administered α-calcitonin gene-related peptide analogue, SAX, in rats during myocardial infarction. MethodsForty Sprague-Dawley rats underwent myocardial infarction. Following left anterior descending artery occlusion, [99mTc]Tc-sestamibi was administered to determine the myocardial perfusion before treatment. Twenty minutes, 24 and 48 h after [99mTc]Tc-sestamibi injection, the rats were treated with either SAX or placebo. Final infarct size was determined three weeks later by [99mTc]Tc-sestamibi SPECT/CT scan. ResultsThirty-one rats survived the surgery and 20 completed the follow-up SPECT/CT scan (SAX n = 12; Placebo n = 8). At baseline, there was no difference in size of perfusion defect between the groups (P = .88), but at follow-up the SAX group had improved myocardial recovery compared to the placebo group (P = .04), corresponding to a relative perfusion recovery of 55% in SAX-treated rats. ConclusionThe CGRP analogue, SAX, has a cardioprotective effect in this rat model of myocardial infarction, improving myocardial perfusion recovery after chronic occlusion of the coronary artery.

Human macrophages directly modulate iPSC-derived cardiomyocytes at healthy state and congenital arrhythmia model in vitro.

The electrophysiological regulation of cardiomyocytes (CMs) by the cardiac macrophages (MΦs) has been recently described as an unconventional role of MΦs in the murine heart. Investigating the molecular and physiological modulation of CM by MΦ is critical to understand the novel mechanisms behind cardiac disorders from the systems perspective and to develop new therapeutic approaches. Here, we developed an in vitro direct coculture system to investigate the cellular and functional interaction between human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and monocyte-derived MΦs both in healthy-state and congenital arrhythmia disease model associated with SCN5A ion channel mutations. Congenital arrhythmia patient-derived (P) and healthy individual-derived control (C) monocytes and derived MΦs exhibited distinct M1- and M2-like polarization-related gene expression pattern. The iPSC-CMs and MΦs formed direct membrane contacts in cocultures demonstrated by time-lapse imaging, scanning electron microscopy, and immunolabeling. The intracellular Ca2+ transients were observed in iPSC-CMs and MΦs when in contact with each other. Interestingly, the C-MΦs in direct contact with C-CMs significantly accelerated the contraction rates, demonstrating the positive chronotropic effect of MΦs on healthy cardiac cultures. Furthermore, the MΦs carrying the SCN5A gene mutation significantly enhanced the arrhythmic events in both C-CMs and P-CMs, implying that the sodium channel mutation in the MΦ is important for the CM function. Importantly, when C-MΦs were coupled to tachycardic P-CMs, the contraction frequency drastically decreased, and rhythmicity enhanced implicating the amelioration of the disease phenotype in vitro. Consequently, our results indicated the functional regulatory role of MΦs on human iPSC-CM contractility by membrane contacts in a physiologically relevant in vitro coculture model of both steady-state and arrhythmia. Our findings could serve as a valuable source for the development of effective immunoregulatory therapies for cardiac arrhythmia in the future.

Positive chronotropic action of HCN channel antagonism in human collecting lymphatic vessels.

Spontaneous action potentials precede phasic contractile activity in human collecting lymphatic vessels. In this study, we investigated the expression of hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels in human collecting lymphatics and by pharmacological inhibition ex vivo tested their potential role in controlling contractile function. Spontaneous and agonist‐evoked tension changes of isolated thoracic duct and mesenteric lymphatic vessels—obtained from surgical patients with informed consent—were investigated by isometric myography, and ivabradine, ZD7288 or cesium were used to inhibit HCN. Analysis of HCN isoforms by RT‐PCR and immunofluorescence revealed HCN2 to be the predominantly expressed mRNA isoform in human thoracic duct and mesenteric lymphatic vessels and HCN2‐immunoreactivity confirmed protein expression in both vessel types. However, in functional experiments ex vivo the HCN inhibitors ivabradine, ZD7288, and cesium failed to lower contraction frequency: conversely, all three antagonists induced a positive chronotropic effect with concurrent negative inotropic action, though these effects first occurred at concentrations regarded as supramaximal for HCN inhibition. Based on these results, we conclude that human collecting vessels express HCN channel proteins but under the ex vivo experimental conditions described here HCN channels have little involvement in regulating contraction frequency in human collecting lymphatic vessels. Furthermore, HCN antagonists can produce concentration‐dependent positive chronotropic and negative inotropic effects, which are apparently unrelated to HCN antagonism.

Effects of Herbal Medicines on the Prevention and Treatment of Contact Dermatitis: A Systematic Review.

Contact dermatitis (CD) is a common inflammatory disease of the skin. CD has a complex and multifaceted treatment, and one of the main components of CD treatment is inflammation management. We summarized the clinical trials exploring the effects of herbal medicine on patients with CD. A systematic review was performed by searching four databases. Clinical trials in English investigating the effect of herbal medicines on CD prevention and treatment published from 2010 to 2020 were reviewed. This study was conducted based on the PRISMA guidelines. Nine clinical trials examining the effects of herbs on CD were identified. A total of 450 patients participated in these studies. Herbal medicines used in the reviewed studies were in various forms. Almost all the studies showed that these herbs were useful in preventing and treating CD. Regarding the side effects of the herbal medicines, few complications such as erythema and papules as well as positive chronotropic effects were reported. Evidence showed that herbal medicines were effective in preventing and treating CD by reducing the inflammation level and increasing antioxidant defense. However, the number of clinical trials was low to draw definitive conclusions. Moreover, the studies were not homogeneous and differed regarding methodology, evaluation tools, and quality. Also, in these studies, the interactions and safety of the herbal medicines were not considered adequately. Therefore, well-designed evidence is required to draw definitive conclusions in this regard.

Electrocardiography effects of Abelmoschus esculentus fruit extract and its isolated compounds using isolated frog heart perfusion

This study evaluated the electrocardiograph effects of Abelmoschus esculentus and its isolated compounds on Rana temporaria (frog) heart. Cardiovascular disease is one of the major causes of mortality which contribute to about 57 million deaths as disclosed by WHO in 2002. There have been many therapeutic approaches to reduce the death rate from cardiovascular disease which include the use of herbal drugs from plants. The whole fruit of Abelmoschus esculentus (Okra) fruit has been used for culinary purposes in Africa. Medicinal properties of Abelmoschus esculentus reported in literature include its use as anti-diabetics, antimicrobial, diuretic agent, and for plasma replacement. However, there have been little or no report on the cardiovascular activities of the extract or isolated compounds from A. esculentus. A. esculentus (Okra) fruits were collected, identified, dried, and powdered. The powdered was extracted using cold maceration with 100% methanol for 72 hours. The crude extract was subjected to repeated fractionation to isolate four (4) compounds. The cardiovascular activities of the crude extract and its isolated compounds were investigated using isolated heart perfusion technique on Rana temporaria (frog) heart. The effects of the extract and isolated compounds on the rate (frequency) and force of contraction (amplitude) of the heart beat were evaluated. The isolated compounds were subjected to spectroscopic analysis using ultraviolent (UV), nuclear magnetic resonance (NMR) (1D and 2D experiments), and mass spectrometry. The compounds were elucidated as quercetin glycoside, quercetin diglycoside, urs-12-ene-3-O-β-D-glucopyranoside and 3-hydroxy-2,3-dihydroimidazo [1,5-a] pyridin-8(5H)-one-5-β-glucopyranoside (esculentoside). The extract and the isolated compounds showed positive inotropic effect on the heart (increase in amplitude of the heart) while they all also possess negative chronotropic effect (decrease in frequency) except compound 4 which possess positive chronotropic effect. This study isolated three different classes of compounds from A. esculentus namely Flavonoids (Quercetin glycoside), Triterpene (Ursolic acid) and pyridine-imidazole (esculentoside). This study also demonstrated that A. esculentus extract and compounds isolated from it has negative chronotropic effects and also possess positive inotropic effects on the heart of R. temporaria.

Exposure to Doxorubicin Modulates the Cardiac Response to Isoproterenol in a Sex‐Dependent Manner

IntroductionSubclinical cardiotoxicity occurs in cancer patients who receive low doses of anthracyclines such as doxorubicin (DOX), which can develop into overt cardiomyopathy when triggered by other cardiovascular stressors. Female rodents are protected from DOX‐induced cardiac dysfunction in experimental studies. However, the sex‐related differences in DOX‐induced increased susceptibility to later‐onset cardiovascular stressors are yet to be determined. Therefore, in the present study, we determined the effects of a low dose of DOX on the cardiac response to catecholamine stress induced by isoproterenol (ISO) in male and female mice. Since sex hormones are believed to mediate the sexually dimorphic response to DOX, we determined whether gonadectomy is able to reverse the observed response.MethodsFive‐week old intact and gonadectomized male and female C57Bl/6 mice were administered saline or DOX (4 mg/kg/week for 5 weeks by intraperitoneal injections). After a 5‐week recovery period, mice were administered daily subcutaneous injections of ISO (10 mg/kg) or saline for 14 days. Cardiac function was measured by echocardiography before, immediately after the first dose of ISO (stress echo), and following 14 days of daily ISO injections. Hearts were harvested and weighed at the end of the experiment.ResultsStress echo shows that the positive chronotropic effect of ISO was blunted in DOX‐treated male and female mice, while the positive inotropic effect is maintained. Following 14 days of daily ISO injections, saline‐treated male mice exhibited a hypertrophic response to ISO, indicated by an increase in the heart weight‐to‐tibia length, while DOX‐treated male mice did not. In addition, ISO caused more reduction of cardiac output in DOX‐treated male mice than that in saline‐treated male mice. On the other hand, both saline‐treated and DOX‐treated female groups exhibited a hypertrophic response to ISO. ISO treatment for 14 days did not cause significant reduction in cardiac output in either saline‐treated or DOX‐treated female mice. Similar experiments were performed in a cohort of gonadectomized male and female mice. Intriguingly, sexually dimorphic responses are observed in gonadectomized mice similar to those observed in intact mice.ConclusionExposure to DOX modulates the cardiac response to isoproterenol in a sex‐dependent manner. DOX treatment abrogates the adaptive hypertrophic response to ISO in male mice, making them more susceptible to declining cardiac output. Deprivation of sex hormones by gonadectomy did not reverse this sexual dimorphism, suggesting that other mechanisms are implicated. Further research is warranted to determine the mechanisms of this sexual dimorphism.

Frequency-Dependent Properties of the Hyperpolarization-Activated Cation Current, If, in Adult Mouse Heart Primary Pacemaker Myocytes.

A number of distinct electrophysiological mechanisms that modulate the myogenic spontaneous pacemaker activity in the sinoatrial node (SAN) of the mammalian heart have been investigated extensively. There is agreement that several (3 or 4) different transmembrane ionic current changes (referred to as the voltage clock) are involved; and that the resulting net current interacts with direct and indirect effects of changes in intracellular Ca2+ (the calcium clock). However, significant uncertainties, and important knowledge gaps, remain concerning the functional roles in SAN spontaneous pacing of many of the individual ion channel- or exchanger-mediated transmembrane current changes. We report results from patch clamp studies and mathematical modeling of the hyperpolarization-activated current, If, in the generation/modulation of the diastolic depolarization, or pacemaker potential, produced by individual myocytes that were enzymatically isolated from the adult mouse sinoatrial node (SAN). Amphotericin-mediated patch microelectrode recordings at 35 °C were made under control conditions and in the presence of 5 or 10 nM isoproterenol (ISO). These sets of results were complemented and integrated with mathematical modeling of the current changes that take place in the range of membrane potentials (−70 to −50 mV), which corresponds to the ‘pacemaker depolarization’ in the adult mouse SAN. Our results reveal a very small, but functionally important, approximately steady-state or time-independent current generated by residual activation of If channels that are expressed in these pacemaker myocytes. Recordings of the pacemaker depolarization and action potential, combined with measurements of changes in If, and the well-known increases in the L-type Ca2+ current, ICaL, demonstrated that ICaL activation, is essential for myogenic pacing. Moreover, after being enhanced (approximately 3-fold) by 5 or 10 nM ISO, ICaL contributes significantly to the positive chronotropic effect. Our mathematical model has been developed in an attempt to better understand the underlying mechanisms for the pacemaker depolarization and action potential in adult mouse SAN myocytes. After being updated with our new experimental data describing If, our simulations reveal a novel functional component of If in adult mouse SAN. Computational work carried out with this model also confirms that in the presence of ISO the residual activation of If and opening of ICaL channels combine to generate a net current change during the slow diastolic depolarization phase that is essential for the observed accelerated pacemaking rate of these SAN myocytes.

Metabolomics analysis delineates the therapeutic effects of hydroethanolic extract of Cucumis sativus L. seeds on hypertension and isoproterenol-induced myocardial infarction

Cucumis sativus L., widely cultivated as an edible vegetable. Its seeds are well reputed for cardiovascular preventive properties. However, the mechanisms underlying for cardiovascular protection of C. sativus are still unidentified. Therefore, this study utilized a metabolomics approach to investigate putative mechanisms of C. sativus seeds in myocardial infarction (MI) and in vitro models of vasoconstriction, atrium, and invasive blood pressure measurement. Results showed that Cu.EtOH extract showed a vasorelaxant response with potent hypotensive effect in normotensive rats and L-NAME induced hypertension. Cu.EtOH caused a negative inotropic and positive chronotropic effect on the atrium. Cu.EtOH protected the animals from ISO-induced myocardial infarction (MI) interventions in left ventricular thickness, cardiomyocyte hypertrophy, mRNA gene expression, and biochemical assays. The metabolomics data suggested that Cu.EtOH mainly affected amino acid metabolism, BCAA degradation, ketone bodies degradation, and oxidative stress. Our study showed that Cu.EtOH suppressed inflammation with a strong anti-myocardial infarction impact. Additionally, our findings indicated Cu.EtOH reverted the amino acid metabolism, BCAA, and ketone bodies degradation. The findings show the antihypertensive mechanism of Cu.EtOH may include the modulation of endothelium-derived relaxing factor (EDRF) produced from nitric oxide (NO) and is connected with vascular endothelial function. C. sativus seeds, in particular, played a pivotal role in the treatment of myocardial and vascular disorders by enhancing the EDRF mechanism, energy generation, and antioxidant capacity. In summary, our findings showed the mechanistic insights on the therapeutic potential of C. sativus seeds for cardiovascular disorders.

Maintained barostatic regulation of heart rate in digesting snakes (Boa constrictor).

When snakes digest large meals, heart rate is accelerated by withdrawal of vagal tone and an increased non-adrenergic-non-cholinergic tone that seems to stem from circulating blood-borne factors exerting positive chronotropic effects. To investigate whether this tonic elevation of heart rate impairs the ability for autonomic regulation of heart during digestion, we characterised heart rate responses to pharmacological manipulation of blood pressure in the snake Boa constrictor through serial injections of sodium nitroprusside and phenylephrine. Both fasting and digesting snakes responded with a robust tachycardia to hypotension induced by sodium nitroprusside, with digesting snakes attaining higher maximal heart rates than fasting snakes. Both fasting and digesting snakes exhibited small reductions of the cardiac chronotropic response to hypertension, induced by injection of phenylephrine. All heart rate changes were abolished by autonomic blockade with the combination of atropine and propranolol. The digesting snakes retained the capacity for compensatory heart rate responses to hypotension, despite their higher resting values, and the upward shift of the barostatic response curve enables snakes to maintain the cardiac limb of barostatic regulation for blood pressure regulation.

Influence of miR-221/222 on cardiomyocyte calcium handling and function

BackgroundCardiovascular disease is the leading cause of death worldwide. Cardiac electrical remodeling including altered ion channel expression and imbalance of calcium homeostasis can have detrimental effects on cardiac function. While it has been extensively reported that miR-221/222 are involved in structural remodeling, their role in electrical remodeling still has to be evaluated. We previously reported that subunits of the L-type Ca2+ channel (LTCC) are direct targets of miR-221/222. Furthermore, HL-1 cells transfected with miR-221 or -222 mimics showed a reduction in LTCC current density while the voltage-dependence of activation was not altered. The aim of the present study was to determine the influence of miR-221/222 on cardiomyocyte calcium handling and function.ResultsTransient transfection of HL-1 cells with miR-221/222 mimics led to slower depolarization-dependent Ca2+ entry and increased proportion of non-responding cells. Angiotensin II-induced Ca2+ release from the SR was not affected by miR-221/222. In miR-222-transfected neonatal cardiomyocytes the isoprenaline-induced positive inotropic effect on the intracellular Ca2+ transient was lost and the positive chronotropic effect on spontaneous beating activity was strongly reduced. This could have severe consequences for cardiomyocytes and could lead to a reduced contractility and systolic dysfunction of the whole heart.ConclusionsThis study adds a new role of miR-221/222 in cardiomyocytes by showing the impact on β-adrenergic regulation of LTCC function, calcium handling and beating frequency. Together with the previous report that miR-221/222 reduce GIRK1/4 function and LTCC current density, it expands our knowledge about the role of these miRs on cardiac ion channel regulation.

Cardiac microtissues from human pluripotent stem cells recapitulate the phenotype of long-QT syndrome

BackgroundHuman induced pluripotent stem cells (hiPSCs) and their derivative cardiomyocytes (hiPSC-CMs) have been successfully used to study the electrical phenotype of cardiac ion channel diseases. However, strategies to mature CMs and more comprehensive systems recapitulating the heart complexity are required to advance our ability to capture adult phenotypes. MethodsWe differentiated wild-type (WT) and long QT syndrome type 1 (LQT1) hiPSCs into CMs, endothelial cells and cardiac fibroblasts. The three cell types were combined to form three-dimensional (3D) spheroids, termed “cardiac microtissues” (cMTs) and the electrophysiological properties were measured using 96-well multi-electrode arrays. ResultsLQT1 cMTs displayed prolonged field potential duration compared to WT controls, thus recapitulating the typical feature of LQTS. Isoprenaline caused a positive chronotropic effect on both LQT1 and WT cMTs. The 96-well multi-electrode array format proved suitable to detect electrical changes directly in the 3D tissues. Conclusions3D hiPSC cMTs are a scalable tool that can be used to identify LQT electrical hallmarks and drug responses. We anticipate this tool can be adopted by pharmaceutical companies to screen cardio-active compounds.

Positive chronotropic effect caused by transthoracic ultrasound in heart of rats.

Pulsed ultrasound can produce chronotropic and inotropic effects on the heart with potential therapeutic applications. Fourteen 3-month-old female rats were exposed transthoracically to 3.5-MHz 2.0-MPa peak rarefactional pressure amplitude ultrasonic pulses of increasing 5-s duration pulse repetition frequency (PRF) sequences. An increase in the heart rate was observed following each PRF sequence: an ∼50% increase after the 4-5-6 Hz sequence, an ∼57% increase after the 5-6-7 Hz sequence, and an ∼48% increase after the 6-7-8 Hz sequence. Other cardiac parameters showed a normal or indicated a compensatory decrease at 3 and 15 min post-ultrasound compared to control.

Hif-1α is not required for the development of cardiac adrenergic control in zebrafish (Danio rerio).

Adrenergic regulation, acting via the sympathetic nervous system, provides a major mechanism to control cardiac function. It has recently been shown that hypoxia inducible factor-1α (Hif-1α) is necessary for normal development of sympathetic innervation and control of cardiac function in the mouse. To investigate whether this may represent a fundamental trait shared across vertebrates, we assessed adrenergic regulation of the heart in wild-type and Hif-1α knockout (hif-1α -/- ) zebrafish (Danio rerio). Wild-type and hif-1α -/- zebrafish larvae (aged 4 and 7 days postfertilisation) exhibited similar routine heart rates within a given age group, and β-adrenergic receptor blockade with propranolol universally reduced heart rate to comparable levels, indicating similar adrenergic tone in both genotypes. In adult fish, in vivo heart rate measured during anaesthesia was identical between genotypes. Treatment of spontaneously beating hearts in vitro with adrenaline revealed a similar positive chronotropic effect and similar maximum heart rates in both genotypes. Tyrosine hydroxylase immunohistochemistry with confocal microscopy demonstrated that the bulbus arteriosus (outflow tract of the teleost heart) of adult fish was particularly well innervated by sympathetic nerves, and nerve density (as a percentage of bulbus arteriosus area) was similar between wild-types and hif-1α -/- mutants. In summary, we did not find any evidence that adrenergic cardiac control was perturbed in larval or adult zebrafish lacking Hif-1α. We conclude that Hif-1α is not essential for the normal development of cardiovascular control or adult sympathetic cardiac innervation in zebrafish, although it is possible that it plays a redundant or auxiliary role.

Comparative characteristics of changes in central hemodynamics during early recovery after different exercise regimes

The cardiovascular system is one of the most important functional systems of the body, which determine the level of physical performance of the body. Insufficient study of the response of the circulatory system to the combination of strength training with endurance exercises requires more detailed comparative studies of the impact of dynamic and static loads on the indicators of central hemodynamics. Accordingly, the aim of our study was to study the characteristics of the reaction of the cardiovascular system in the period of early recovery after dosed exercise of a dynamic and static nature. The study examined the response of the central hemodynamics of young men in the period of early recovery after dynamic loading (Martine functional test) and static loading (holding on the stand dynamometer DS-200 force with a power of 50% of maximum standing force). The change in circulatory system parameters was recorded using a tetrapolar thoracic impedance rheoplethysmogram on a computerized diagnostic complex “Cardio +”. It is established that the dynamic load in the period of early recovery does not cause a significant positive chronotropic effect, leads to a decrease in vascular resistance of blood flow, to an increase in pulse blood pressure. The increase in cardiac output is mainly due to the increase in stroke volume, which indicates a fairly high functional reserves of the heart. It is revealed that under conditions of static loading the reaction of central hemodynamics and the course of early recovery are radically different from the changes of indicators under dynamic loading. In persons with a normodynamic type of reaction to dynamic load, there are no significant changes in the minute volume of blood at a similar volume of active muscle mass static load. Meeting the metabolic needs of working skeletal muscles and compensating for the oxygen debt is realized by increasing the total peripheral vascular resistance and increasing systolic blood pressure in the postpartum period. The physiological meaning of this phenomenon is to maintain a sufficient level of venous return of blood to ensure the pumping function of the heart.

Human histamine H2 receptors can initiate cardiac arrhythmias in a transgenic mouse

Histamine is known to lead to arrhythmias in the human heart. A mouse model to mimic these effects has hitherto not been available but might be useful to study the mechanism(s) of H2-histamine receptor-induced arrhythmias and may support the search for new antiarrhythmic drugs. In order to establish such a model in mice, we studied here the incidence of cardiac arrhythmias under basal and under stimulated conditions in atrial and ventricular preparations from mice that overexpressed the human H2-histamine receptors in a cardiac-specific way (H2-TG) in comparison with their wild-type (WT) littermate controls. We had shown before that histamine exerted concentration and time-dependent positive inotropic and positive chronotropic effects only in cardiac preparations from H2-TG and not from WT. We noted under basal conditions (no drug addition) that right atrial preparations from H2-TG exhibited more spontaneous arrhythmias than right atrial preparations from WT. These arrhythmias in H2-TG could be blocked by the H2-histamine receptor antagonist cimetidine. In a similar fashion, histamine and dimaprit (an agonist at H2 and not H1-histamine receptors) more often induced arrhythmias in right atrial preparations from H2-TG than from WT. To understand better the signal transduction mechanism(s) involved in these arrhythmias, we studied partially depolarized left atrial preparations. In these preparations, a positive inotropic effect of histamine was still present in the additional presence of 44 mM potassium ions (used to block sodium channels) in H2-TG but not WT and this positive inotropic effect could be blocked by cimetidine and this is consistent with the involvement of calcium ion channels in the contractile and thus might mediate also the arrhythmogenic effects of histamine in H2-TG. However, compounds reported to release histamine from cells and thereby leading to arrhythmias in humans, namely morphine, ketamine, and fentanyl, failed to induce a more pronounced positive inotropic effect in atrial preparations from H2-TG compared to WT, arguing against an involvement of histamine release in their proarrhythmic side effects in patients. Measuring left ventricular contractility in isolated retrogradely perfused hearts (Langendorff mode), we detected under basal conditions (no drug application) more spontaneous arrhythmias in hearts from H2-TG than from WT. In summary, we noted that overexpression of human H2-histamine receptors in a novel transgenic animal model can lead to arrhythmias. We suggest that this model might be useful to understand the mechanism(s) of histamine-induced cardiac arrhythmias in humans better in a molecular way and may be of value to screen novel antiarrhythmic drugs.

Beta-1 adrenergic receptors modulate pacemaker activity of mouse sino-atrial myocytes through L-type Cav1.3 channels

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Doctoral program in Biology and Biotechnologies b1- and b2- adrenergic receptors (ARs) are co-expressed in different regions of the heart. The b2/ b1 expression ratio is higher in the sino-atrial node (SAN) than in atria and ventricles, but the specific contribution of either type of receptor to modulation of pacemaker activity is still not well established. Specific stimulation of b2-ARs in rabbit SAN myocytes is associated with a positive shift of the pacemaker "funny" current (If) activation curve. However, previous studies showed that L-type Cav1.3 channels play an important role in the generation of cardiac pacemaker activity by contributing to the diastolic depolarization (DD) in SAN myocytes. Since Cav1.3 channels are positively regulated by b-ARs activation2, we investigated which is the main b-ARs isoform that modulates Cav1.3-mediated ICaL andthe pacemaker activity of SAN myocytes. To address this point, we recorded spontaneous activity and Cav1.3-mediated ICaL from mouse SAN myocytes. We found that the positive chronotropic effect of the non-selective b-AR agonist isoproterenol (ISO, 0.1mM) was decreased by the b1-ARs antagonist CGP-20712 (0.3mM) and the b2-ARs antagonist ICI-118,551 (1mM) by -18% and -9%, respectively. Perfusion of CGP-20712 strongly reduced the positive chronotropic effect induced by ISO. Finally, we recorded Cav1.3-mediated L-type currents in presence of the b1-ARs antagonist. CGP-20712 reduced the basal Cav1.3-mediated ICaL. Furthermore, the increase in Cav1.3-mediated ICaL by isoproterenol was abolished during b1-ARs inhibition by CGP-20712. In conclusion, these preliminary data show that b1- and b2-ARs differently modulate the spontaneous activity of mouse SAN myocytes. In addition, b1-ARs play a predominant role in the adrenergic regulation of L-type Cav1.3 channels to increase pacemaker activity. Future studies will be performed to clarify the role of b2-ARs antagonist on Cav1.3-mediated ICaL and the functional relationships between b-ARs and Cav1.3 channels.

PDE4B and PDE4D differentially regulate cardiac pacemaker activity

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Agence Nationale de la Recherche (ANR) Background Heart rate (HR) is generated by spontaneous electrical activity in the sinoatrial node (SAN) and is modulated by the autonomic nervous system. During sympathetic stimulation, the activation of β-adrenergic receptors (βAR) increases cAMP levels, leading to positive chronotropic effect. Among the 6 cardiac cAMP-PDE families, PDE4 is critical for controlling excitation-contraction coupling (ECC) during β-stimulation in atrial and ventricular myocytes. PDE4 may also be important for automaticity. 3 genes encode for PDE4 in the heart (pde4a, 4b, 4d). We propose to investigate their respective contribution to the regulation of pacemaker activity. Methods Total PDE activity in mouse SAN was determined as the cAMP-hydrolytic activity measured in the absence of PDE inhibitor and the fraction corresponding to PDE4 activity was assessed by including the PDE4 inhibitor Ro-20-1724. The in vitro pacemaker activity was assessed by measuring spontaneous Ca2+ transients in Fluo4-loaded-SAN tissue from wild-type (WT) and PDE4KO mice. Images were obtained using confocal microscopy. Telemetry EKG was recorded in conscious mice in control (CTRL) conditions, after pharmacological denervation with atropine (1 mg/kg) and propranolol (2 mg/kg) and after β-stimulation with isoproterenol (ISO, 1.5 mg/kg). HR variability was evaluated by calculating the SDNN (standard deviation of RR intervals) parameter. Results Ro-20-1724 (10 µM) increased beating rate of intact SAN and increased PKA-phosphorylation of key ECC actors (ryanodine receptor, phospholamban and contractile proteins). PDE4 activity was found to account for 60% of total cAMP-PDE activity in SAN. PDE4A, 4B and 4D isoforms were found to be expressed in mouse SAN. In PDE4BKO SAN, the effect of ISO on SAN beating rate was higher than in WT. Ablation of PDE4D induced decreased beating rate in CTRL and ISO conditions and increased Ca2+ spark frequency compared to WT SAN. In vivo, PDE4BKO and PDE4DKO mice displayed increased resting HR during day and night. HR variability was decreased in PDE4BKO, but not in PDE4DKO mice during the day, and decreased in both genotypes at night compared to WT mice. After atropine + propranolol denervation, the rhythmic phenotype was only maintained in PDE4BKO but not in PDE4DKO mice. The response to β-AR stimulation with ISO was higher in PDE4BKO than in PDE4DKO. In addition, under ISO we observed an increased number of premature beats and atrioventricular blocks in PDE4DKO, but not in PDE4BKO mice. Conclusion PDE4B and PDE4D differentially regulate cardiac pacemaker activity. While PDE4B clearly controls intrinsic SAN automaticity, PDE4D might be important for ANS-mediated regulation of HR and conduction.

Histamine can be Formed and Degraded in the Human and Mouse Heart.

Histamine is metabolized by several enzymes in vitro and in vivo. The relevance of this metabolism in the mammalian heart in vivo is unclear. However, histamine can exert positive inotropic effects (PIE) and positive chronotropic effects (PCE) in humans via H2-histamine receptors. In transgenic mice (H2-TG) that overexpress the human H2 receptor in cardiomyocytes but not in wild-type littermate mice (WT), histamine induced PIE and PCE in isolated left or right atrial preparations. These H2-TG were used to investigate the putative relevance of histamine degrading enzymes in the mammalian heart. Histidine, the precursor of histamine, increased force of contraction (FOC) in human atrial preparations. Moreover, histamine increased the phosphorylation state of phospholamban in human atrium. Here, we could detect histidine decarboxylase (HDC) and histamine itself in cardiomyocytes of mouse hearts. Moreover, our data indicate that histamine is subject to degradation in the mammalian heart. Inhibition of the histamine metabolizing enzymes diamine oxidase (DAO) and monoamine oxidase (MAO) shifted the concentration response curves for the PIE in H2-TG atria to the left. Moreover, activity of histamine metabolizing enzymes was present in mouse cardiac samples as well as in human atrial samples. Thus, drugs used for other indication (e.g. antidepressants) can alter histamine levels in the heart. Our results deepen our understanding of the physiological role of histamine in the mouse and human heart. Our findings might be clinically relevant because we show enzyme targets for drugs to modify the beating rate and force of the human heart.

PDE4B and PDE4D differentially regulate cardiac pacemaker activity

During sympathetic stimulation, the activation of β-adrenergic receptors (βAR) increases cAMP levels, leading to positive chronotropic effect. Among the 6 cardiac cAMP-PDE families, PDE4 is critical for controlling excitation-contraction coupling (ECC) during β-stimulation in atrial and ventricular myocytes. PDE4 may also be important for automaticity. Three genes encode for PDE4 in the heart (pde4a, 4b, 4d). To investigate the respective contribution of PDE4 isoforms to the regulation of pacemaker activity. Total PDE activity in mouse SAN was determined as the cAMP-hydrolytic activity measured in the absence of PDE inhibitor and the fraction corresponding to PDE4 activity was assessed by including the PDE4 inhibitor Ro-20-1724. The in vitro pacemaker activity was assessed by measuring spontaneous Ca 2+ transients in Fluo4-loaded-SAN tissue from Wild-type (WT) and PDE4KO using confocal microscopy. Telemetry EKG was recorded in conscious mice in control (CTRL) conditions, after pharmacological denervation (atropine + propranolol) and after isoproterenol (ISO). PDE4 activity represents 60% of total cAMP-PDE activity in SAN. PDE4A, 4B and 4D isoforms are expressed in mouse SAN. In PDE4BKO, the effect of ISO on SAN beating rate was higher than in WT. Ablation of PDE4D induced decreased beating rate in CTRL and ISO conditions and increased Ca 2+ spark frequency compared to WT. In vivo, PDE4BKO and PDE4DKO mice displayed increased resting HR during day and night. After pharmacological denervation, the rhythmic phenotype was only maintained in PDE4BKO but not in PDE4DKO. The response to ISO was higher in PDE4BKO than in PDE4DKO. In addition, ISO provoked more premature beats and atrioventricular blocks in PDE4DKO than in PDE4BKO mice. PDE4B controls intrinsic SAN automaticity and PDE4D might be important for autonomic nervous system-mediated regulation of HR and conduction.