Rate Of Diastolic Depolarization Research Articles

Characterization of the cellular components of mouse collecting lymphatic vessels reveals that lymphatic muscle cells are the innate pacemaker cells regulating lymphatic contractions.

Collecting lymphatic vessels (cLVs) exhibit spontaneous contractions with a pressure-dependent frequency, but the identity of the lymphatic pacemaker cell is still debated. By analogy to pacemakers in the GI and lower urinary tracts, proposed cLV pacemaker cells include interstitial cells of Cajal like cells (ICLC) or the lymphatic muscle (LMCs) cells themselves. Here we combined immunofluorescence and scRNAseq analyses with electrophysiological methods to examine the cellular constituents of the mouse cLV wall and assess whether any cell type exhibited morphological and functional processes characteristic of pacemaker cells: a continuous if not contiguous network integrated into the electrical syncytium; spontaneous Ca2+ transients; and depolarization-induced propagated contractions. We employed inducible Cre (iCre) mouse models routinely used to target these specific cell populations including: c-kitCreER T2 to target ICLC; PdgfrβCreER T2 to target pericyte-like cells; PdgfrαCreER ™ to target CD34+ adventitial cells and ICLC; and Myh11CreER T2 to target LMCs directly. These specific inducible Cre lines were crossed to the fluorescent reporter ROSA26mT/mG, the genetically encoded Ca2+ sensor GCaMP6f, and the light-activated cation channel rhodopsin2 (ChR2). c-KitCreER T2 labeled both a sparse population of LECs and round adventitial cells that responded to the mast cell activator compound 48-80. PdgfrβCreER T2 drove recombination in both adventitial cells and LMCs, limiting its power to discriminate a pericyte-specific population. PdgfrαCreER ™ labeled a large population of interconnected, oak leaf-shaped cells primarily along the adventitial surface of the vessel. Of these cells, only LMCs consistently, but heterogeneously, displayed spontaneous Ca2+ events during the diastolic period of the contraction cycle, and whose frequency was modulated in a pressure-dependent manner. Optogenetic depolarization through the expression of ChR2 under control of Myh11CreER T2 , but not PdgfrαCreER ™ or c-KitCreER T2 , resulted in propagated contractions upon photo-stimulation. Membrane potential recordings in LMCs demonstrated that the rate of diastolic depolarization significantly correlated with contraction frequency. These findings support the conclusion that LMCs, or a subset of LMCs, are responsible for mouse cLV pacemaking.

Modulation of Spontaneous Action Potential Rate by Inositol Trisphosphate in Myocytes from the Rabbit Atrioventricular Node.

The atrioventricular node (AVN) is a key component of the cardiac conduction system and takes over pacemaking of the ventricles if the sinoatrial node fails. IP3 (inositol 1,4,5 trisphosphate) can modulate excitability of myocytes from other regions of the heart, but it is not known whether IP3 receptor (IP3-R) activation modulates AVN cell pacemaking. Consequently, this study investigated effects of IP3 on spontaneous action potentials (APs) from AVN cells isolated from rabbit hearts. Immunohistochemistry and confocal imaging demonstrated the presence of IP3-R2 in isolated AVN cells, with partial overlap with RyR2 ryanodine receptors seen in co-labelling experiments. In whole-cell recordings at physiological temperature, application of 10 µM membrane-permeant Bt3-(1,4,5)IP3-AM accelerated spontaneous AP rate and increased diastolic depolarization rate, without direct effects on ICa,L, IKr, If or INCX. By contrast, application via the patch pipette of 5 µM of the IP3-R inhibitor xestospongin C led to a slowing in spontaneous AP rate and prevented 10 µM Bt3-(1,4,5)IP3-AM application from increasing the AP rate. UV excitation of AVN cells loaded with caged-IP3 led to an acceleration in AP rate, the magnitude of which increased with the extent of UV excitation. 2-APB slowed spontaneous AP rate, consistent with a role for constitutive IP3-R activity; however, it was also found to inhibit ICa,L and IKr, confounding its use for studying IP3-R. Under AP voltage clamp, UV excitation of AVN cells loaded with caged IP3 activated an inward current during diastolic depolarization. Collectively, these results demonstrate that IP3 can modulate AVN cell pacemaking rate.

IP3R1 underlies diastolic ANO1 activation and pressure-dependent chronotropy in lymphatic collecting vessels.

Pressure-dependent chronotropy of murine lymphatic collecting vessels relies on the activation of the Ca2+-activated chloride channel encoded by Anoctamin 1 (Ano1) in lymphatic muscle cells. Genetic ablation or pharmacological inhibition of ANO1 results in a significant reduction in basal contraction frequency and essentially complete loss of pressure-dependent frequency modulation by decreasing the rate of the diastolic depolarization phase of the ionic pacemaker in lymphatic muscle cells (LMCs). Oscillating Ca2+ release from sarcoendoplasmic reticulum Ca2+ channels has been hypothesized to drive ANO1 activity during diastole, but the source of Ca2+ for ANO1 activation in smooth muscle remains unclear. Here, we investigated the role of the inositol triphosphate receptor 1 (Itpr1; Ip3r1) in this process using pressure myography, Ca2+ imaging, and membrane potential recordings in LMCs of ex vivo pressurized inguinal-axillary lymphatic vessels from control or Myh11CreERT2;Ip3r1fl/fl (Ip3r1ismKO) mice. Ip3r1ismKO vessels had significant reductions in contraction frequency and tone but an increased contraction amplitude. Membrane potential recordings from LMCs of Ip3r1ismKO vessels revealed a depressed diastolic depolarization rate and an elongation of the plateau phase of the action potential (AP). Ca2+ imaging of LMCs using the genetically encoded Ca2+ sensor GCaMP6f demonstrated an elongation of the Ca2+ flash associated with an AP-driven contraction. Critically, diastolic subcellular Ca2+ transients were absent in LMCs of Ip3r1ismKO mice, demonstrating the necessity of IP3R1 activity in controlling ANO1-mediated diastolic depolarization. These findings indicate a critical role for IP3R1 in lymphatic vessel pressure-dependent chronotropy and contractile regulation.

The funny current If is essential for the fight-or-flight response in cardiac pacemaker cells.

The sympathetic nervous system fight-or-flight response is characterized by a rapid increase in heart rate, which is mediated by an increase in the spontaneous action potential (AP) firing rate of pacemaker cells in the sinoatrial node. Sympathetic neurons stimulate sinoatrial myocytes (SAMs) by activating β adrenergic receptors (βARs) and increasing cAMP. The funny current (If) is among the cAMP-sensitive currents in SAMs. If is critical for pacemaker activity, however, its role in the fight-or-flight response remains controversial. In this study, we used AP waveform analysis, machine learning, and dynamic clamp experiments in acutely isolated SAMs from mice to quantitatively define the AP waveform changes and role of If in the fight-or-flight increase in AP firing rate. We found that while βAR stimulation significantly altered nearly all AP waveform parameters, the increase in firing rate was only correlated with changes in a subset of parameters (diastolic duration, late AP duration, and diastolic depolarization rate). Dynamic clamp injection of the βAR-sensitive component of If showed that it accounts for ∼41% of the fight-or-flight increase in AP firing rate and 60% of the decrease in the interval between APs. Thus, If is an essential contributor to the fight-or-flight increase in heart rate.

The effective use of blebbistatin to study the action potential of cardiac pacemaker cells of zebrafish (Danio rerio) during incremental warming.

Blebbistatin potently inhibits actin-myosin interaction, preventing contractile activity of excitable cells including cardiac myocytes, despite electrical excitation of an action potential (AP). We collected intracellular microelectrode recordings of pacemaker cells located in the sinoatrial region (SAR) of the zebrafish heart at room temperature and during acute warming to investigate whether or not blebbistatin inhibition of contraction significantly alters pacemaker cell electrophysiology. Changes were evaluated based on 16 variables that characterized the AP waveform. None of these AP variables nor the spontaneous heart rate were significantly modified with the application of 10 μM blebbistatin when recordings were made at room temperature. Compared with the control group, the blebbistatin-treated group showed minor changes in the rate of spontaneous diastolic depolarization (P = 0.027) and the 50% and 80% repolarization (P = 0.008 and 0.010, respectively) in the 26°C–29°C temperature bin, but not at higher temperatures. These findings suggest that blebbistatin is an effective excitation-contraction uncoupler that does not appreciably affect APs generated in pacemaking cells of the SAR and can, therefore, be used in zebrafish cardiac studies.

Dexmedetomidine Exerts a Negative Chronotropic Action on Sinoatrial Node Cells Through the Activation of Imidazoline Receptors.

Dexmedetomidine (DEX), an α2-adrenoreceptor (α2-AR) and imidazoline receptor agonist, is most often used for the sedation of patients in the intensive care unit. Its administration is associated with an increased incidence of bradycardia; however, the precise mechanism of DEX-induced bradycardia has yet to be fully elucidated. This study was undertaken to examine whether DEX modifies pacemaker activity and the underlying ionic channel function through α2-AR and imidazoline receptors. The whole-cell patch-clamp techniques were used to record action potentials and related ionic currents of sinoatrial node cells in guinea pigs. DEX (≥10 nM) reduced sinoatrial node automaticity and the diastolic depolarization rate. DEX reduced the amplitude of hyperpolarization-activated cation current (If or Ih) the pacemaker current, even within the physiological pacemaker potential range. DEX slowed the If current activation kinetics and caused a significant shift in the voltage dependence of channel activation to negative potentials. In addition, efaroxan, an α2-AR and imidazoline I1 receptor antagonist, attenuated the inhibitory effects of DEX on sinoatrial node automaticity and If current activity, whereas yohimbine, an α2-AR-selective antagonist, did not. DEX did not affect the current activities of other channels, including rapidly and slowly activating delayed rectifier K+ currents (IKr and IKs), L-type Ca2+ current (ICa,L), Na+/Ca2+ exchange current (INCX), and muscarinic K+ current (IK,ACh). Our results indicate that DEX, at clinically relevant concentrations, induced a negative chronotropic effect on the sinoatrial node function through the downregulation of If current through an imidazoline I1 receptor other than the α2-AR in the clinical setting.

CAMP-Dependent Signaling Restores AP Firing in Dormant SA Node Cells via Enhancement of Surface Membrane Currents and Calcium Coupling.

Action potential (AP) firing rate and rhythm of sinoatrial nodal cells (SANC) are controlled by synergy between intracellular rhythmic local Ca2+ releases (LCRs) (“Ca2+ clock”) and sarcolemmal electrogenic mechanisms (“membrane clock”). However, some SANC do not fire APs (dormant SANC). Prior studies have shown that β-adrenoceptor stimulation can restore AP firing in these cells. Here we tested whether this relates to improvement of synchronization of clock coupling. We characterized membrane potential, ion currents, Ca2+ dynamics, and phospholamban (PLB) phosphorylation, regulating Ca2+ pump in enzymatically isolated single guinea pig SANC prior to, during, and following β-adrenoceptor stimulation (isoproterenol) or application of cell-permeant cAMP (CPT-cAMP). Phosphorylation of PLB (Serine 16) was quantified in the same cells following Ca2+ measurement. In dormant SANC LCRs were small and disorganized at baseline, membrane potential was depolarized (−38 ± 1 mV, n = 46), and ICaL, If, and IK densities were smaller vs SANC firing APs. β-adrenoceptor stimulation or application of CPT-cAMP led to de novo spontaneous AP generation in 44 and 46% of dormant SANC, respectively. The initial response was an increase in size, rhythmicity and synchronization of LCRs, paralleled with membrane hyperpolarization and small amplitude APs (rate ∼1 Hz). During the transition to steady-state AP firing, LCR size further increased, while LCR period shortened. LCRs became more synchronized resulting in the growth of an ensemble LCR signal peaked in late diastole, culminating in AP ignition; the rate of diastolic depolarization, AP amplitude, and AP firing rate increased. ICaL, IK, and If amplitudes in dormant SANC increased in response to β-adrenoceptor stimulation. During washout, all changes reversed in order. Total PLB was higher, but the ratio of phosphorylated PLB (Serine 16) to total PLB was lower in dormant SANC. β-adrenoceptor stimulation increased this ratio in AP-firing cells. Thus, transition of dormant SANC to AP firing is linked to the increased functional coupling of membrane and Ca2+ clock proteins. The transition occurs via (i) an increase in cAMP-mediated phosphorylation of PLB accelerating Ca2+ pumping, (ii) increased spatiotemporal LCR synchronization, yielding a larger diastolic LCR ensemble signal resulting in an earlier increase in diastolic INCX; and (iii) increased current densities of If, ICaL, and IK.

Desmin is essential for the structure and function of the sinoatrial node: implications for increased arrhythmogenesis.

Our objective was to investigate the effect of desmin depletion on the structure and function of the sinoatrial pacemaker complex (SANcl) and its implication in arrhythmogenesis. Analysis of mice and humans (SANcl) indicated that the sinoatrial node exhibits high amounts of desmin, desmoplakin, N-cadherin, and β-catenin in structures we call "lateral intercalated disks" connecting myocytes side by side. Examination of the SANcl from an arrhythmogenic cardiomyopathy model, desmin-deficient (Des-/-) mouse, by immunofluorescence, ultrastructural, and Western blot analysis showed that the number of these lateral intercalated disks was diminished. Also, electrophysiological recordings of the isolated compact sinoatrial node revealed increased pacemaker systolic potential and higher diastolic depolarization rate compared with wild-type mice. Prolonged interatrial conduction expressed as a longer P wave duration was also observed in Des-/- mice. Upregulation of mRNA levels of both T-type Ca2+ current channels, Cav3.1 and Cav3.2, in the Des-/- myocardium (1.8- and 2.3-fold, respectively) and a 1.9-fold reduction of funny hyperpolarization-activated cyclic nucleotide-gated K+ channel 1 could underlie these functional differences. To investigate arrhythmogenicity, electrocardiographic analysis of Des-deficient mice revealed a major increase in supraventricular and ventricular ectopic beats compared with wild-type mice. Heart rate variability analysis indicated a sympathetic predominance in Des-/- mice, which may further contribute to arrhythmogenicity. In conclusion, our results indicate that desmin elimination leads to structural and functional abnormalities of the SANcl. These alterations may be enhanced by the sympathetic component of the cardiac autonomic nervous system, which is predominant in the desmin-deficient heart, thus leading to increased arrhythmogenesis.NEW & NOTEWORTHY The sinoatrial node exhibits high amounts of desmin and desmoplakin in structures we call "lateral intercalated disks," connecting side-by-side adjacent cardiomyocytes. These structures are diminished in desmin-deficient mouse models. Misregulation of T-type Ca2+ current and hyperpolarization-activated cyclic nucleotide-gated K+ channel 1 was proved along with prolonged interatrial conduction and cardiac autonomic nervous system dysfunction.

The effect of forskolin on membrane clock and calcium clock in the hypoxic/reoxygenation of sinoatrial node cells and its mechanism.

In this study, we investigated the effect of forskolin (FSK, a selective adenylate cyclase agonist) on the automatic diastolic depolarization of sinus node cells (SNC) with hypoxia/reoxygenation (H/R) injury. The SNC of the newborn rat was randomly assigned into the control group, the H/R (H/R injury) group, or the H/R + FSK (H/R injury + FSK treatment) group. Patch-clamp was performed to record the action potential and electrophysiological changes. The cellular distribution of intracellular calcium concentration was analyzed by fluorescence staining. Compared with the control cells, spontaneous pulsation frequency (SPF) and diastolic depolarization rate (DDR) of H/R cells were reduced from 244.3 ± 10.6 times/min and 108.7 ± 7.8mV/s to 130.5 ± 7.6 times/min and 53.4 ± 6.5mV/s, respectively. FSK significantly increased SPF and DDR of H/R cells to 208.3 ± 8.3 times/min and 93.2 ± 8.9mV/s (n = 15, both p < 0.01), respectively. H/R reduced the current densities of If, ICa,T and inward INCX, which were significantly increased by 10μM FSK treatment (n = 15, p < 0.01). Furthermore, reduced expression of HCN4 and NCX1.1 channel protein were significantly increased by FSK. Inhibitor studies showed that both SQ22536 (a selective adenylate cyclase inhibitor) and H89 (a selective protein kinases A [PKA] inhibitor) blocked the effects of FSK on SPF and DDR. H/R causes pacemaker dysfunction in newborn rat sinoatrial node cells leading to divergence of the DD and the slow of spontaneous APs, which change can be dramatically reversed by FSK through increasing INCX and If current in H/R injury.

Differential effects of class I antiarrhythmic drugs on the guinea pig pulmonary vein myocardium: Inhibition of automatic activity correlates with blockade of a diastolic sodium current component

The effects of class I antiarrhythmic drugs on the automaticity of isolated guinea pig pulmonary vein myocardia were investigated using microelectrode and voltage clamp methods. All of the drugs examined reduced the maximum rate of rise of automatic action potentials. The firing frequency and rate of diastolic depolarization were decreased by aprindine, flecainide and propafenone, but not by cibenzoline, disopyramide and pilsicainide, which correlated with blockade of the sodium current component induced by ramp depolarization mimicking the diastolic depolarization. In conclusion, class I antiarrhythmic drugs which block the diastolic sodium current component inhibit the automaticity of the pulmonary vein myocardium.

Cardiac Overexpression of Adenylyl Cyclase Type VIII Augments Function of the Coupled Oscillatory System and Action Potential Firing Rate of Sinoatrial Nodal Cells

We have shown recently that the intrinsic heart rate (IHR) in vivo (measured during double autonomic blockade) in mice with cardiac-specific overexpression of adenylyl cyclase type VIII(AC VIII) is 30% higher than in wild type littermates (WT). We studied single sinoatrial nodal cell (SANC)to elucidate how AC VIII overexpression affects a coupled-clock system that drives spontaneous action potential (AP) firing in these cells. Spontaneous local Ca2+ releases (LCRs, confocal line-scan microscopy), generated via spontaneous local activation of ryanodine receptors (RyR) of the intracellular Ca2+ oscillator-sarcoplasmic reticulum (SR), occurred earlier in diastole in TgAC8 SANC, accelerating the diastolic AP ignition process that governs the rate of spontaneous diastolic depolarization. This led to an increased rate of AP generation by the ensemble of cell surface membrane ionic current oscillators. In permeabilized (saponin) SANC, Ca2+ signal of individual LCR's, and that of the LCR ensemble were increased in TgAC8 vs WT. Double immunostaining of phosphorylated and total RyR revealed increased phosphorylation of RyR at Ser2808 and Ser2030, and of PLB at Ser16 sites in TgAC8 vs WT SANC. RNA-seq analysis showed an upregulation of several PDEs (including PDE1a, PDE3a, PDE4b, PDE4c, PDE4d, PDE7b and PDE8a), ion channels (Cacna1c, Cacnb2, Kcnb1, Kcnd3, Kcnn1, Kcnq1, Scn1b, Scn4a; Kcnj3 however was downregulated), ATPase Na+/K+ transporters (ATP1a1 and ATP2b1) and calcium cycling proteins (STIM1 and RYR2) were upregulated in TgAC8 vs WT (notably, SIN and PLM were downregulated in TgAC8 vs WT). Thus, cardiac-specific overexpression of AC VIII in SANC increases IHR in vivo via impacting upon both transcriptional and posttranslational proteins modification of the Ca2+-cAMP-PKA-dependent components of the coupled-oscillator system intrinsic to SANC that controls their spontaneous AP firing rate.

Sorafenib Suppresses Basal Spontaneous Beating of Rabbit Sinoatrial Node Cells (SANC) through Inhibition of Vascular Endothelial Growth Factor Receptor 1 (VEGFR1)

Sarcoplasmic reticulum generated local subsarcolemmal Ca2+ releases (LCRs) in SANC appear during diastolic depolarization and activate an inward Na+-Ca2+ exchange current accelerating the diastolic depolarization rate and thus increasing spontaneous SANC firing. Sorafenib, multitargeted tyrosine kinase inhibitor treating renal, liver and thyroid malignances, inhibits VEGFR, platelet-derived growth factor-receptor and rapidly accelerated fibrosarcoma (RAF) kinases. Sorafenib treatment is associated with myocardial infarction and increased incidence of atrial fibrillation that could be linked to suppression of cardiac pacemaker function. The goal of the present study is to determine whether sorafenib affects spontaneous firing of SANC and, if so, define specific downstream targets effected by sorafenib. Expression of VEGFR1, VEGFR2 and PLCγ (assessed by RT-qPCR) in rabbit sinoatrial node was comparable to that of β1-adrenergic receptors. Sorafenib: (1) markedly decreased the LCR size and number (confocal microscopy, Ca2+ indicator Fluo-3); (2) prolonged the LCR period (interval between action potential-induced Ca2+ transient and subsequent LCR); (3) in a time-dependent manner decreased and then abrogated spontaneous firing of single isolated SANC. A selective pan-inhibitor of VEGFR1/2/3 (PTK787/ZK222584) also decreased spontaneous SANC beating rate by ∼50%, suggesting that VEGFR1/2 could be one of major targets of sorafenib. The selective VEGFR2 inhibitor ZM-323881, however, suppressed spontaneous firing in only ∼18% of SANC suggesting that VEGFR1, but not VEGFR2 signaling, is linked to SANC automaticity. Phosphoinositide-3-kinase (PI3K) and phospholipase C (PLC) are downstream targets of VEGFR. While selective PI3K inhibitor PI-103 was lacking effects, PLC inhibitor U-73122 markedly decreased the LCR size, number, extended the LCR period and prolonged the spontaneous SANC cycle length. Our results strongly suggest that activation of VEGFR1-PLC pathway is involved in regulation of normal automaticity of cardiac pacemaker cells.

Inhibitory Effects of Cyclopiazonic Acid on the Pacemaker Current in Sinoatrial Nodal Cells

Objective: The spontaneous action potential of isolated sinoatrial node (SAN) cells is regulated by a coupled-clock system of two clocks: the calcium clock and membrane clock. However, it remains unclear whether calcium clock inhibitors have a direct effect on the membrane clock. The purpose of this study was to investigate the direct effect of cyclopiazonic acid (CPA), a selective calcium clock inhibitor, on the function of the membrane clock of SAN cells. Methods: at SAN cells were isolated by trypsinization and identified based on morphology and electrophysiology. If and HCN currents were recorded via patch clamp technique. The expression of the HCN channel protein was determined by Western blotting analysis. Results: The diastolic depolarization rate of spontaneous action potentials and the current densities of If were reduced by exposure to 10 μM CPA. The inhibitory effect of CPA was concentration-dependent with an IC50 value of 16.3 μM and a Hill coefficient of 0.98. The effect of CPA on If current was also time-dependent, and the If current amplitude was partially restored after washout. Furthermore, the steady-state activation curve of the If current was shifted to a negative potential, indicating that channel activation slowed down. Finally, the protein expression of HCN4 in HEK293 cells was markedly downregulated by CPA. Conclusions: These results indicate that the direct inhibition effect of CPA on the If current in SAN cells is both concentration- and time-dependent. The underlying mechanisms may involve slowing down steady-state activation and the downregulation of pacemaker channel protein expression.

Hypokalaemia: Clinical Signifi cance and Role in the Mechanisms Underlying Cardiac Arrhythmogenesis

Hypokalaemia is the most common electrolyte abnormality seen in cardiac patients, which represents a side effect of diuretic therapy or results from the overactivation of the renin-angiotensinaldosterone system and the sympathetic nervous system in heart failure. Hypokalaemia is known to contribute to an increased risk of ventricular arrhythmias whose mechanism is based on the dynamic interplay of the provocative trigger and the vulnerable substrate. A premature ectopic impulse acts as a provocative trigger, whereas the vulnerable substrate is created by the structural and functional myocardial changes that favour the impulse circulation within a closed conducting pathway, thus perpetuating the re-entrant activation. The premature ectopic impulse that serves as the initiating event for arrhythmia can be generated due to abnormal automaticity or triggered activity. Hypokalaemia facilitates abnormal automaticity by increasing the rate of spontaneous diastolic depolarisation in Purkinje fi bres, which then start to exhibit pacemaker activity that interferes with the regular activations set by the sinoatrial node. The triggered activity is attributable to the early and delayed afterdepolarisations in cardiac myocytes. The early afterdepolarisations are typically precipitated by an excessive lengthening of the cardiac action potential duration that results from the inhibition of the repolarising K+ currents in the setting of hypokalaemia. The delayed afterdepolarisations are related to calcium overload in cardiac cells, which is provoked by hypokalaemia via inhibition of the Na+-K+ ATPase on myocyte sarcolemma. This translates to the increased intracellular Na+ levels, which in turn activate the reverse mode of the Na+-Ca2+ exchange, leading to increased cytosolic Ca2+ concentration. With regard to creating a vulnerable substrate for re-entry, hypokalaemia is known to induce a non-uniform increase in the action potential duration in different myocardial regions, which amplifi es spatial heterogeneities in the recovery of ventricular excitability during the fi nal repolarisation phase. This sets favourable conditions for a unidirectional conduction block upon premature ectopic activation, which initiates the impulse propagation around a small area of refractory cardiac tissue. In addition, hypokalaemia slows cardiac conduction by inducing hyperpolarisation of the myocyte sarcolemma that results in markedly increased excitation threshold. The induced conduction delay in the re-entrant circuit then allows suffi cient time for recovery from refractoriness in the cardiac cells ahead of the excitation wavefront, which sustains re-entrant activation. The risk of ventricular tachyarrhythmia becomes particularly high when hypokalaemia is combined with the administration of cardiac glycosides or class III antiarrhythmic agents.

The Effects of α1-Adrenoreceptors’ Activation in the Interatrial Septum of Newborn and Adult Rats

Bioelectrical properties of various developing fragments of the heart undergo significant changes during pre- and postnatal ontogeny. However, the general pattern of the heart’s structure and the bioelectrical features of various parts of the “mature” heart are specifically determined in the early stages of embryogenesis. Specifically, the presence of myocardium with a phenotype, which is similar to a pacemaker, was revealed in the interatrial septum (IAS) of the mammalian heart using histological methods. However, the electrical activity in this structure remains understudied. The aim of the present study was to examine the capability of the IAS to generate action potentials spontaneously and the effects of adrenergic stimulation on the bioelectrical activity of the IAS. For this purpose, we recorded the resting and action potentials in the multicellular isolated perfused preparations of the IAS and left atrium from the rat heart at the end of day 1 and at day 60 of postnatal life using the standard microelectrode technique. We found that α1-adrenomimetic phenylephrine (PE) modified the patterns of action potentials in the samples of IAS and the atrial myocardium from the animals of both ages. In the resting samples without any electrical stimulation, PE induced the generation of action potential in the IAS but not in the atrium because of oscillations in the level of cytoplasmic calcium. Application of the inhibitor of hyperpolarization activated current (If) ZD 7288 led to a decrease in the rate of slow diastolic depolarization of action potential in the IAS pacemaker cells, a decrease in the frequency, and the appearance of train activity. Thus, in newborn and adult rats, the IAS myocardium generates spontaneous electrical activity. A necessary condition for the occurrence of pacemaker activity is adrenergic stimulation. The capability to generate spontaneous activity is probably associated with the presence of pacemaker current If in the IAS cardiomyocytes.

Ano1 mediates pressure-sensitive contraction frequency changes in mouse lymphatic collecting vessels.

Lymphatic collecting vessels exhibit spontaneous contractions with a pressure-dependent contraction frequency. The initiation of contraction has been proposed to be mediated by the activity of a Ca2+-activated Cl- channel (CaCC). Here, we show that the canonical CaCC Anoctamin 1 (Ano1, TMEM16a) plays an important role in lymphatic smooth muscle pacemaking. We find that isolated murine lymphatic muscle cells express Ano1, and demonstrate functional CaCC currents that can be inhibited by the Ano1 inhibitor benzbromarone. These currents are absent in lymphatic muscle cells from Cre transgenic mouse lines targeted for Ano1 genetic deletion in smooth muscle. We additionally show that loss of functional Ano1 in murine inguinal-axillary lymphatic vessels, whether through genetic manipulation or pharmacological inhibition, results in an impairment of the pressure-frequency relationship that is attributable to a hyperpolarized resting membrane potential and a significantly depressed diastolic depolarization rate preceding each action potential. These changes are accompanied by alterations in action potential shape and duration, and a reduced duration but increased amplitude of the action potential-induced global "Ca2+ flashes" that precede lymphatic contractions. These findings suggest that an excitatory Cl- current provided by Ano1 is critical for mediating the pressure-sensitive contractile response and is a major component of the murine lymphatic action potential.

Preclinical Evaluation of Hydro Alcoholic Extract of fruits of Cascabela thevetia for its Cardio Toxic Activity

Cascabela thevetia is a medicinally active plant used to treat different diseases. The aim of the present study was to pharmacological investigation of the hydro alcoholic extract of the fruit kernel of Cascabela thevetia for its cardiotoxic activity. In this study the fruit kernel was extracted with 70% ethanol by using soxhlet apparatus and 12% yield was obtained. The phytochemical screening of the extract revealed the presence of alkaloids, steroids, saponins, flavonoids and tannins. In this study male Albino Wister rat weighing about 150-250 gm were used. Different pre-estimated doses of Acetylcholine (Ach), Adrenaline (Adr) and hydroalcoholic extract of Cascabela thevetia fruit kernel (CT) were used to find out the effect on heart from the dose response curve. Acetylcholine when applied to heart it produced parasympathomimetic action and decreased the rate of diastolic depolarization (negative tropic action). Adrenaline (sympathomimetic) produced both positive inotropic and chronotropic activity. But CT showed unique cardiac activity in the isolated rat heart, when applied low to high dose in geometrical propagation manner, first it produced negative chronotropic action followed by negative inotropic action. But that same negative tropic activity has been blocked by using adrenaline along with CT. Atropine along with CT became unable to antagonize the activity of CT unlike acetylcholine. Therefore it can conclude that Cascabela thevetia is a potent cardiotoxic drug in moderate to higher doses, help to understand the purpose behind selecting this fruit as abusive and suicidal cases.

Unique Ca2+-Cycling Protein Abundance and Regulation Sustains Local Ca2+ Releases and Spontaneous Firing of Rabbit Sinoatrial Node Cells

Spontaneous beating of the heart pacemaker, the sinoatrial node, is generated by sinoatrial node cells (SANC) and caused by gradual change of the membrane potential called diastolic depolarization (DD). Submembrane local Ca2+ releases (LCR) from sarcoplasmic reticulum (SR) occur during late DD and activate an inward Na+/Ca2+ exchange current, which accelerates the DD rate leading to earlier occurrence of an action potential. A comparison of intrinsic SR Ca2+ cycling revealed that, at similar physiological Ca2+ concentrations, LCRs are large and rhythmic in permeabilized SANC, but small and random in permeabilized ventricular myocytes (VM). Permeabilized SANC spontaneously released more Ca2+ from SR than VM, despite comparable SR Ca2+ content in both cell types. In this review we discuss specific patterns of expression and distribution of SR Ca2+ cycling proteins (SR Ca2+ ATPase (SERCA2), phospholamban (PLB) and ryanodine receptors (RyR)) in SANC and ventricular myocytes. We link ability of SANC to generate larger and rhythmic LCRs with increased abundance of SERCA2, reduced abundance of the SERCA inhibitor PLB. In addition, an increase in intracellular [Ca2+] increases phosphorylation of both PLB and RyR exclusively in SANC. The differences in SR Ca2+ cycling protein expression between SANC and VM provide insights into diverse regulation of intrinsic SR Ca2+ cycling that drives automaticity of SANC.

Basal Spontaneous Firing of Rabbit Sinoatrial Node Cells Is Regulated by Dual Activation of PDEs (Phosphodiesterases) 3 and 4.

Spontaneous firing of sinoatrial node cells (SANCs) is regulated by cAMP-mediated, PKA (protein kinase A)-dependent (cAMP/PKA) local subsarcolemmal Ca2+ releases (LCRs) from RyRs (ryanodine receptors). LCRs occur during diastolic depolarization and activate an inward Na+/Ca2+ exchange current that accelerates diastolic depolarization rate prompting the next action potential. PDEs (phosphodiesterases) regulate cAMP-mediated signaling; PDE3/PDE4 represent major PDE activities in SANC, but how they modulate LCRs and basal spontaneous SANC firing remains unknown. Real-time polymerase chain reaction, Western blot, immunostaining, cellular perforated patch clamping, and confocal microscopy were used to elucidate mechanisms of PDE-dependent regulation of cardiac pacemaking. PDE3A, PDE4B, and PDE4D were the major PDE subtypes expressed in rabbit SANC, and PDE3A was colocalized with α-actinin, PDE4D, SERCA (sarcoplasmic reticulum Ca2+ ATP-ase), and PLB (phospholamban) in Z-lines. Inhibition of PDE3 (cilostamide) or PDE4 (rolipram) alone increased spontaneous SANC firing by ≈20% (P<0.05) and ≈5% (P>0.05), respectively, but concurrent PDE3+PDE4 inhibition increased spontaneous firing by ≈45% (P<0.01), indicating synergistic effect. Inhibition of PDE3 or PDE4 alone increased L-type Ca2+ current (ICa,L) by ≈60% (P<0.01) or ≈5% (P>0.05), respectively, and PLB phosphorylation by ≈20% (P>0.05) each, but dual PDE3+PDE4 inhibition increased ICa,L by ≈100% (P<0.01) and PLB phosphorylation by ≈110% (P<0.05). Dual PDE3+PDE4 inhibition increased the LCR number and size (P<0.01) and reduced the SR (sarcoplasmic reticulum) Ca2+ refilling time (P<0.01) and the LCR period (time from action potential-induced Ca2+ transient to subsequent LCR; P<0.01), leading to decrease in spontaneous SANC cycle length (P<0.01). When RyRs were disabled by ryanodine and LCRs ceased, dual PDE3+PDE4 inhibition failed to increase spontaneous SANC firing. Basal cardiac pacemaker function is regulated by concurrent PDE3+PDE4 activation which operates in a synergistic manner via decrease in cAMP/PKA phosphorylation, suppression of LCR parameters, and prolongation of the LCR period and spontaneous SANC cycle length.

ParamAP: Standardized Parameterization of Sinoatrial Node Myocyte Action Potentials

Sinoatrial node myocytes act as cardiac pacemaker cells by generating spontaneous action potentials (APs). Much information is encoded in sinoatrial AP waveforms, but both the analysis and the comparison of AP parameters between studies is hindered by the lack of standardized parameter definitions and the absence of automated analysis tools. Here we introduce ParamAP, a standalone cross-platform computational tool that uses a template-free detection algorithm to automatically identify and parameterize APs from text input files. ParamAP employs a graphic user interface with automatic and user-customizable input modes, and it outputs data files in text and PDF formats. ParamAP returns a total of 16 AP waveform parameters including time intervals such as the AP duration, membrane potentials such as the maximum diastolic potential, and rates of change of the membrane potential such as the diastolic depolarization rate. ParamAP provides a robust AP detection algorithm in combination with a standardized AP parameter analysis over a wide range of AP waveforms and firing rates, owing in part to the use of an iterative algorithm for the determination of the threshold potential and the diastolic depolarization rate that is independent of the maximum upstroke velocity, a parameter that can vary significantly among sinoatrial APs. Because ParamAP is implemented in Python 3, it is also highly customizable and extensible. In conclusion, ParamAP is a powerful computational tool that facilitates quantitative analysis and enables comparison of sinoatrial APs by standardizing parameter definitions and providing an automated work flow.