Generation of cardiomyocytes from human-induced pluripotent stem cells resembling atrial cells with ability to respond to adrenoceptor agonists.

Atrial fibrillation (AF) is the most common chronic arrhythmia presenting a heavy disease burden. We report a new approach for generating cardiomyocytes (CMs) resembling atrial cells from human-induced pluripotent stem cells (hiPSCs) using a combination of Gremlin 2 and retinoic acid treatment. More than 40% of myocytes showed rod-shaped morphology, expression of CM proteins (including ryanodine receptor 2, α-actinin-2 and F-actin) and striated appearance, all of which were broadly similar to the characteristics of adult atrial myocytes (AMs). Isolated myocytes were electrically quiescent until stimulated to fire action potentials with an AM profile and an amplitude of approximately 100 mV, arising from a resting potential of approximately -70 mV. Single-cell RNA sequence analysis showed a high level of expression of several atrial-specific transcripts including NPPA, MYL7, HOXA3, SLN, KCNJ4, KCNJ5 and KCNA5. Amplitudes of calcium transients recorded from spontaneously beating cultures were increased by the stimulation of α-adrenoceptors (activated by phenylephrine and blocked by prazosin) or ß-adrenoceptors (activated by isoproterenol and blocked by CGP20712A). Our new approach provides human AMs with mature characteristics from hiPSCs which will facilitate drug discovery by enabling the study of human atrial cell signalling pathways and AF. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Timing mechanisms to control heart rhythm and initiate arrhythmias: roles for intracellular organelles, signalling pathways and subsarcolemmal Ca2.

Rhythms of electrical activity in all regions of the heart can be influenced by a variety of intracellular membrane bound organelles. This is true both for normal pacemaker activity and for abnormal rhythms including those caused by early and delayed afterdepolarizations under pathological conditions. The influence of the sarcoplasmic reticulum (SR) on cardiac electrical activity is widely recognized, but other intracellular organelles including lysosomes and mitochondria also contribute. Intracellular organelles can provide a timing mechanism (such as an SR clock driven by cyclic uptake and release of Ca2+, with an important influence of intraluminal Ca2+), and/or can act as a Ca2+ store involved in signalling mechanisms. Ca2+ plays many diverse roles including carrying electric current, driving electrogenic sodium-calcium exchange (NCX) particularly when Ca2+ is extruded across the surface membrane causing depolarization, and activation of enzymes which target organelles and surface membrane proteins. Heart function is also influenced by Ca2+ mobilizing agents (cADP-ribose, nicotinic acid adenine dinucleotide phosphate and inositol trisphosphate) acting on intracellular organelles. Lysosomal Ca2+ release exerts its effects via calcium/calmodulin-dependent protein kinase II to promote SR Ca2+ uptake, and contributes to arrhythmias resulting from excessive beta-adrenoceptor stimulation. A separate arrhythmogenic mechanism involves lysosomes, mitochondria and SR. Interacting intracellular organelles, therefore, have profound effects on heart rhythms and NCX plays a central role. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Inhibition of adenylyl cyclase 1 by ST034307 inhibits IP3-evoked changes in sino-atrial node beat rate.

Atrial arrhythmias, such as atrial fibrillation (AF), are a major mortality risk and a leading cause of stroke. The IP3 signalling pathway has been proposed as an atrial-specific target for AF therapy, and atrial IP3 signalling has been linked to the activation of calcium sensitive adenylyl cyclases AC1 and AC8. We investigated the involvement of AC1 in the response of intact mouse atrial tissue and isolated guinea pig atrial and sino-atrial node (SAN) cells to the α-adrenoceptor agonist phenylephrine (PE) using the selective AC1 inhibitor ST034307. The maximum rate change of spontaneously beating mouse right atrial tissue exposed to PE was reduced from 14.5% to 8.2% (p = 0.005) in the presence of 1 µM ST034307, whereas the increase in tension generated in paced left atrial tissue in the presence of PE was not inhibited by ST034307 (Control = 14.2%, ST034307 = 16.3%; p > 0.05). Experiments were performed using isolated guinea pig atrial and SAN cells loaded with Fluo-5F-AM to record changes in calcium transients (CaT) generated by 10 µM PE in the presence and absence of 1 µM ST034307. ST034307 significantly reduced the beating rate of SAN cells (0.34-fold decrease; p = 0.003) but did not inhibit changes in CaT amplitude in response to PE in atrial cells. The results presented here demonstrate pharmacologically the involvement of AC1 in the downstream response of atrial pacemaker activity to α-adrenoreceptor stimulation and IP3R calcium release.

Endolysosomal calcium release and cardiac physiology.

Calcium ions play a central role in determining the timing and magnitude of the pumping action of heart muscle in a process which couples electrical activity of action potentials to muscle contraction. Regulation of this excitation-contraction coupling is achieved by Ca2+ signalling mechanisms that include activation of Ca2+ mobilising agents which influence the movement of Ca2+ between intracellular membrane-bound compartments. Research discussed here concerns endolysosomes, which play diverse signalling roles throughout the body. In the heart, a population of endolysosomes is strategically placed close to two other important membrane bound organelles, sarcoplasmic reticulum (SR) and mitochondria. In each case this proximity provides a structural basis for highly localised Ca2+ signalling in nanodomains between endolysosomes and the organelle. Ca2+ is released from endolysosomes via at least two varieties of two-pore domain channels (TPCs) in mammalian cardiac cells, TPC1 determining the interaction with mitochondria, while TPC2 controls the influence on SR. Ca2+ release via both TPC1 and TPC2 is enhanced by the Ca2+ mobilising agent, nicotinic acid adenine dinucleotide phosphate (NAADP) which is synthesised in the heart primarily by CD38. In normal physiology, NAADP plays an important regulatory role in which Ca2+ is released from endolysosomes via TPC2 channels into a nanodomain next to SR, and an amplification mechanism resulting from Ca2+ activation of CaMKII enhances SR Ca2+ uptake by the enzyme SERCA to increase the amplitude of the Ca2+ transient accompanying action potentials. A separate mechanism underlies pathology associated with reperfusion after ischaemia, when NAADP-mediated endolysosomal calcium release via TPC1 acts on nearby mitochondria resulting in abnormal SR Ca2+ release and extreme disruption to the normal excitation-contraction coupling process, causing muscle damage. There are different roles for PKA in the two pathways dependant on TPC1 or TPC2. Oxidising conditions during reperfusion following ischaemia promote disulphide bond formation in PKAIalpha causing accumulation of PKAI holoenzyme in endolysosomes and cardioprotective inhibition of TPC1 channels. In the case of TPC2, PKAII actions are thought to enhance NAADP synthesis by CD38 therefore promoting the endolysosomal influence on SR Ca2+. Excessive activation of this pathway leads to cardiac arrhythmias and hypertrophy.

Emerging Evidence for cAMP-calcium Cross Talk in Heart Atrial Nanodomains Where IP3-Evoked Calcium Release Stimulates Adenylyl Cyclases.

Calcium handling is vital to normal physiological function in the heart. Human atrial arrhythmias, eg. atrial fibrillation, are a major morbidity and mortality burden, yet major gaps remain in our understanding of how calcium signaling pathways function and interact. Inositol trisphosphate (IP3) is a calcium-mobilizing second messenger and its agonist-induced effects have been observed in many tissue types. In the atria IP3 receptors (IR3Rs) residing on junctional sarcoplasmic reticulum augment cellular calcium transients and, when over-stimulated, lead to arrhythmogenesis. Recent studies have demonstrated that the predominant pathway for IP3 actions in atrial myocytes depends on stimulation of calcium-dependent forms of adenylyl cyclase (AC8 and AC1) by IP3-evoked calcium release from the sarcoplasmic reticulum. AC8 shows co-localisation with IP3Rs and AC1 appears to be nearby. These observations support crosstalk between calcium and cAMP pathways in nanodomains in atria. Similar mechanisms also appear to operate in the pacemaker region of the sinoatrial node. Here we discuss these significant advances in our understanding of atrial physiology and pathology, together with implications for the identification of potential novel targets and modulators for the treatment of atrial arrhythmias.

IP3-mediated Ca2+ release regulates atrial Ca2+ transients and pacemaker function by stimulation of adenylyl cyclases.

Inositol trisphosphate (IP3) is a Ca2+-mobilizing second messenger shown to modulate atrial muscle contraction and is thought to contribute to atrial fibrillation. Cellular pathways underlying IP3 actions in cardiac tissue remain poorly understood, and the work presented here addresses the question whether IP3-mediated Ca2+ release from the sarcoplasmic reticulum is linked to adenylyl cyclase activity including Ca2+-stimulated adenylyl cyclases (AC1 and AC8) that are selectively expressed in atria and sinoatrial node (SAN). Immunocytochemistry in guinea pig atrial myocytes identified colocalization of type 2 IP3 receptors with AC8, while AC1 was located in close vicinity. Intracellular photorelease of IP3 by UV light significantly enhanced the amplitude of the Ca2+ transient (CaT) evoked by electrical stimulation of atrial myocytes (31 ± 6% increase 60 s after photorelease, n = 16). The increase in CaT amplitude was abolished by inhibitors of adenylyl cyclases (MDL-12,330) or protein kinase A (H89), showing that cAMP signaling is required for this effect of photoreleased IP3. In mouse, spontaneously beating right atrial preparations, phenylephrine, an α-adrenoceptor agonist with effects that depend on IP3-mediated Ca2+ release, increased the maximum beating rate by 14.7 ± 0.5%, n = 10. This effect was substantially reduced by 2.5 µmol/L 2-aminoethyl diphenylborinate and abolished by a low dose of MDL-12,330, observations which are again consistent with a functional interaction between IP3 and cAMP signaling involving Ca2+ stimulation of adenylyl cyclases in the SAN pacemaker. Understanding the interaction between IP3 receptor pathways and Ca2+-stimulated adenylyl cyclases provides important insights concerning acute mechanisms for initiation of atrial arrhythmias.NEW & NOTEWORTHY This study provides evidence supporting the proposal that IP3 signaling in cardiac atria and sinoatrial node involves stimulation of Ca2+-activated adenylyl cyclases (AC1 and AC8) by IP3-evoked Ca2+ release from junctional sarcoplasmic reticulum. AC8 and IP3 receptors are shown to be located close together, while AC1 is nearby. Greater understanding of these novel aspects of the IP3 signal transduction mechanism is important for future study in atrial physiology and pathophysiology, particularly atrial fibrillation.

Calcium Signaling in the Heart.

The aim of this chapter is to discuss evidence concerning the many roles of calcium ions, Ca2+, in cell signaling pathways that control heart function. Before considering details of these signaling pathways, the control of contraction in ventricular muscle by Ca2+ transients accompanying cardiac action potentials is first summarized, together with a discussion of how myocytes from the atrial and pacemaker regions of the heart diverge from this basic scheme. Cell signaling pathways regulate the size and timing of the Ca2+ transients in the different heart regions to influence function. The simplest Ca2+ signaling elements involve enzymes that are regulated by cytosolic Ca2+. Particularly important examples to be discussed are those that are stimulated by Ca2+, including Ca2+-calmodulin-dependent kinase (CaMKII), Ca2+ stimulated adenylyl cyclases, Ca2+ stimulated phosphatase and NO synthases. Another major aspect of Ca2+ signaling in the heart concerns actions of the Ca2+ mobilizing agents, inositol trisphosphate (IP3), cADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate, (NAADP). Evidence concerning roles of these Ca2+ mobilizing agents in different regions of the heart is discussed in detail. The focus of the review will be on short term regulation of Ca2+ transients and contractile function, although it is recognized that Ca2+ regulation of gene expression has important long term functional consequences which will also be briefly discussed.

Pnmt-Derived Cardiomyocytes: Anatomical Localization, Function and Future Perspectives.

In this mini-review, we provide an overview of phenylethanolamine-N-methyl transferase (Pnmt)-derived cardiomyocytes (PdCMs), a recently discovered cardiomyocyte subpopulation. We discuss their anatomical localization, physiological characteristics, possible function, and future perspectives. Their unique distribution in the heart, electrical activity, Ca2+ transient properties, and potential role in localized adrenergic signaling are discussed.

Modernized Classification of Cardiac Antiarrhythmic Drugs.

BACKGROUND: Among his major cardiac electrophysiological contributions, Miles Vaughan Williams (1918-2016) provided a classification of antiarrhythmic drugs that remains central to their clinical use. METHODS: We survey implications of subsequent discoveries concerning sarcolemmal, sarcoplasmic reticular, and cytosolic biomolecules, developing an expanded but pragmatic classification that encompasses approved and potential antiarrhythmic drugs on this centenary of his birth. RESULTS: We first consider the range of pharmacological targets, tracking these through to cellular electrophysiological effects. We retain the original Vaughan Williams Classes I through IV but subcategorize these divisions in light of more recent developments, including the existence of Na+ current components (for Class I), advances in autonomic (often G protein-mediated) signaling (for Class II), K+ channel subspecies (for Class III), and novel molecular targets related to Ca2+ homeostasis (for Class IV). We introduce new classes based on additional targets, including channels involved in automaticity, mechanically sensitive ion channels, connexins controlling electrotonic cell coupling, and molecules underlying longer-term signaling processes affecting structural remodeling. Inclusion of this widened range of targets and their physiological sequelae provides a framework for a modernized classification of established antiarrhythmic drugs based on their pharmacological targets. The revised classification allows for the existence of multiple drug targets/actions and for adverse, sometimes actually proarrhythmic, effects. The new scheme also aids classification of novel drugs under investigation. CONCLUSIONS: We emerge with a modernized classification preserving the simplicity of the original Vaughan Williams framework while aiding our understanding and clinical management of cardiac arrhythmic events and facilitating future developments in this area.

Transverse cardiac slicing and optical imaging for analysis of transmural gradients in membrane potential and Ca2+ transients in murine heart.

KEY POINTS: A robust cardiac slicing approach was developed for optical mapping of transmural gradients in transmembrane potential (Vm ) and intracellular Ca2+ transient (CaT) of murine heart. Significant transmural gradients in Vm and CaT were observed in the left ventricle. Frequency-dependent action potentials and CaT alternans were observed in all ventricular regions with rapid pacing, with significantly greater incidence in the endocardium than epicardium. The observations demonstrate the feasibility of our new approach to cardiac slicing for systematic analysis of intrinsic transmural and regional gradients in Vm and CaT. ABSTRACT: Transmural and regional gradients in membrane potential and Ca2+ transient in the murine heart are largely unexplored. Here, we developed and validated a robust approach which combines transverse ultra-thin cardiac slices and high resolution optical mapping to enable systematic analysis of transmural and regional gradients in transmembrane potential (Vm ) and intracellular Ca2+ transient (CaT) across the entire murine ventricles. The voltage dye RH237 or Ca2+ dye Rhod-2 AM were loaded through the coronary circulation using a Langendorff perfusion system. Short-axis slices (300 µm thick) were prepared from the entire ventricles (from the apex to the base) by using a high-precision vibratome. Action potentials (APs) and CaTs were recorded with optical mapping during steady-state baseline and rapid pacing. Significant transmural gradients in Vm and CaT were observed in the left ventricle, with longer AP duration (APD50 and APD75 ) and CaT duration (CaTD50 and CaTD75 ) in the endocardium compared with that in the epicardium. No significant regional gradients were observed along the apico-basal axis of the left ventricle. Interventricular gradients were detected with significantly shorter APD50 , APD75 and CaTD50 in the right ventricle compared with left ventricle and ventricular septum. During rapid pacing, AP and CaT alternans were observed in most ventricular regions, with significantly greater incidence in the endocardium in comparison with epicardium. In conclusion, these observations demonstrate the feasibility of our new approach to cardiac slicing for systematic analysis of intrinsic transmural and regional gradients in Vm and CaT in murine ventricular tissue.

Synthesis of the Ca2+-mobilizing messengers NAADP and cADPR by intracellular CD38 enzyme in the mouse heart: Role in ß-adrenoceptor signaling.

Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP-ribose (cADPR) are Ca2+-mobilizing messengers important for modulating cardiac excitation-contraction coupling and pathophysiology. CD38, which belongs to the ADP-ribosyl cyclase family, catalyzes synthesis of both NAADP and cADPR in vitro However, it remains unclear whether this is the main enzyme for their production under physiological conditions. Here we show that membrane fractions from WT but not CD38-/- mouse hearts supported NAADP and cADPR synthesis. Membrane permeabilization of cardiac myocytes with saponin and/or Triton X-100 increased NAADP synthesis, indicating that intracellular CD38 contributes to NAADP production. The permeabilization also permitted immunostaining of CD38, with a striated pattern in WT myocytes, whereas CD38-/- myocytes and nonpermeabilized WT myocytes showed little or no staining, without striation. A component of ß-adrenoreceptor signaling in the heart involves NAADP and lysosomes. Accordingly, in the presence of isoproterenol, Ca2+ transients and contraction amplitudes were smaller in CD38-/- myocytes than in the WT. In addition, suppressing lysosomal function with bafilomycin A1 reduced the isoproterenol-induced increase in Ca2+ transients in cardiac myocytes from WT but not CD38-/- mice. Whole hearts isolated from CD38-/- mice and exposed to isoproterenol showed reduced arrhythmias. SAN4825, an ADP-ribosyl cyclase inhibitor that reduces cADPR and NAADP synthesis in mouse membrane fractions, was shown to bind to CD38 in docking simulations and reduced the isoproterenol-induced arrhythmias in WT hearts. These observations support generation of NAADP and cADPR by intracellular CD38, which contributes to effects of ß-adrenoreceptor stimulation to increase both Ca2+ transients and the tendency to disturb heart rhythm.

Optogenetic Control of Heart Rhythm by Selective Stimulation of Cardiomyocytes Derived from Pnmt+ Cells in Murine Heart.

In the present study, channelrhodopsin 2 (ChR2) was specifically introduced into murine cells expressing the Phenylethanolamine n-methyltransferase (Pnmt) gene, which encodes for the enzyme responsible for conversion of noradrenaline to adrenaline. The new murine model enabled the identification of a distinctive class of Pnmt-expressing neuroendocrine cells and their descendants (i.e. Pnmt+ cell derived cells) within the heart. Here, we show that Pnmt+ cells predominantly localized to the left side of the adult heart. Remarkably, many of the Pnmt+ cells in the left atrium and ventricle appeared to be working cardiomyocytes based on their morphological appearance and functional properties. These Pnmt+ cell derived cardiomyocytes (PdCMs) are similar to conventional myocytes in morphological, electrical and contractile properties. By stimulating PdCMs selectively with blue light, we were able to control cardiac rhythm in the whole heart, isolated tissue preparations and single cardiomyocytes. Our new murine model effectively demonstrates functional dissection of cardiomyocyte subpopulations using optogenetics, and opens new frontiers of exploration into their physiological roles in normal heart function as well as their potential application for selective cardiac repair and regeneration strategies.

High resolution structural evidence suggests the Sarcoplasmic Reticulum forms microdomains with Acidic Stores (lysosomes) in the heart.

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) stimulates calcium release from acidic stores such as lysosomes and is a highly potent calcium-mobilising second messenger. NAADP plays an important role in calcium signalling in the heart under basal conditions and following ß-adrenergic stress. Nevertheless, the spatial interaction of acidic stores with other parts of the calcium signalling apparatus in cardiac myocytes is unknown. We present evidence that lysosomes are intimately associated with the sarcoplasmic reticulum (SR) in ventricular myocytes; a median separation of 20 nm in 2D electron microscopy and 3.3 nm in 3D electron tomography indicates a genuine signalling microdomain between these organelles. Fourier analysis of immunolabelled lysosomes suggests a sarcomeric pattern (dominant wavelength 1.80 µm). Furthermore, we show that lysosomes form close associations with mitochondria (median separation 6.2 nm in 3D studies) which may provide a basis for the recently-discovered role of NAADP in reperfusion-induced cell death. The trigger hypothesis for NAADP action proposes that calcium release from acidic stores subsequently acts to enhance calcium release from the SR. This work provides structural evidence in cardiac myocytes to indicate the formation of microdomains between acidic and SR calcium stores, supporting emerging interpretations of NAADP physiology and pharmacology in heart.

Contributions of cardiac "funny" (f) channels and sarcoplasmic reticulum Ca2+ in regulating beating rate of mouse and guinea pig sinoatrial node.

The aim of this study was to investigate the effects on spontaneous beating rate of mouse atrial preparations following selective block of cardiac "funny" (f) channels, I(f), and/or suppression of sarcoplasmic reticulum (SR) function in the absence and presence of ß-adrenoceptor stimulation. ZD7288 [to block I(f)] caused a substantial reduction (222 ± 13 bpm) in beating rate from 431 ± 14 to 209 ± 14 bpm, ryanodine alone (to block SR Ca(2+) release) reduced beating rate by 105 ± 11 bpm, with subsequent addition of ZD7288 further reducing rate by 57 ± 9 bpm. Cyclopiazonic acid (CPA) alone (to inhibit Ca(2+) reuptake by the SR) reduced beating rate by 148 ± 13 bpm with subsequent addition of ZD7288 further reducing rate by 79 ± 12 bpm. In additional experiments measuring Ca(2+) transients in the SA node region using Rhod-2, effects of ivabradine and ZD7288 on rate were again substantially reduced after CPA. Effects of CPA alone on rate developed much more slowly than effects on Ca(2+) transient amplitude. ZD7288, ivabradine, and CPA reduced the slope and maximum response of the log(concentration)-response curves for effects of isoprenaline on beating rate. Very little response to isoprenaline remained after treatment with CPA followed by ZD7288. Similar changes in isoprenaline log(concentration)-response curves were seen in guinea pig preparations. These observations are consistent with a role for Ca(2+) released from the SR in regulating I(f) and therefore beating rate of SA node preparations; there appear to be additional contributions of SR-derived Ca(2+) to effects of ß-adrenoceptor stimulation on beating rate that are independent of I(f).

Two-pore Channels (TPC2s) and Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) at Lysosomal-Sarcoplasmic Reticular Junctions Contribute to Acute and Chronic ß-Adrenoceptor Signaling in the Heart.

Ca(2+)-permeable type 2 two-pore channels (TPC2) are lysosomal proteins required for nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca(2+) release in many diverse cell types. Here, we investigate the importance of TPC2 proteins for the physiology and pathophysiology of the heart. NAADP-AM failed to enhance Ca(2+) responses in cardiac myocytes from Tpcn2(-/-) mice, unlike myocytes from wild-type (WT) mice. Ca(2+)/calmodulin-dependent protein kinase II inhibitors suppressed actions of NAADP in myocytes. Ca(2+) transients and contractions accompanying action potentials were increased by isoproterenol in myocytes from WT mice, but these effects of ß-adrenoreceptor stimulation were reduced in myocytes from Tpcn2(-/-) mice. Increases in amplitude of L-type Ca(2+) currents evoked by isoproterenol remained unchanged in myocytes from Tpcn2(-/-) mice showing no loss of ß-adrenoceptors or coupling mechanisms. Whole hearts from Tpcn2(-/-) mice also showed reduced inotropic effects of isoproterenol and a reduced tendency for arrhythmias following acute ß-adrenoreceptor stimulation. Hearts from Tpcn2(-/-) mice chronically exposed to isoproterenol showed less cardiac hypertrophy and increased threshold for arrhythmogenesis compared with WT controls. Electron microscopy showed that lysosomes form close contacts with the sarcoplasmic reticulum (separation ∼ 25 nm). We propose that Ca(2+)-signaling nanodomains between lysosomes and sarcoplasmic reticulum dependent on NAADP and TPC2 comprise an important element in ß-adrenoreceptor signal transduction in cardiac myocytes. In summary, our observations define a role for NAADP and TPC2 at lysosomal/sarcoplasmic reticulum junctions as unexpected but major contributors in the acute actions of ß-adrenergic signaling in the heart and also in stress pathways linking chronic stimulation of ß-adrenoceptors to hypertrophy and associated arrhythmias.

Hydroxychloroquine reduces heart rate by modulating the hyperpolarization-activated current If: Novel electrophysiological insights and therapeutic potential.

BACKGROUND: Bradycardic agents are of interest for the treatment of ischemic heart disease and heart failure, as heart rate is an important determinant of myocardial oxygen consumption. OBJECTIVES: The purpose of this study was to investigate the propensity of hydroxychloroquine (HCQ) to cause bradycardia. METHODS: We assessed the effects of HCQ on (1) cardiac beating rate in vitro (mice); (2) the "funny" current (If) in isolated guinea pig sinoatrial node (SAN) myocytes (1, 3, 10 µM); (3) heart rate and blood pressure in vivo by acute bolus injection (rat, dose range 1-30 mg/kg), (4) blood pressure and ventricular function during feeding (mouse, 100 mg/kg/d for 2 wk, tail cuff plethysmography, anesthetized echocardiography). RESULTS: In mouse atria, spontaneous beating rate was significantly (P < .05) reduced (by 9% ± 3% and 15% ± 2% at 3 and 10 µM HCQ, n = 7). In guinea pig isolated SAN cells, HCQ conferred a significant reduction in spontaneous action potential firing rate (17% ± 6%, 1 µM dose) and a dose-dependent reduction in If (13% ± 3% at 1 µM; 19% ± 2% at 3 µM). Effects were also observed on L-type calcium ion current (ICaL) (12% ± 4% reduction) and rapid delayed rectifier potassium current (IKr) (35% ± 4%) at 3 µM. Intravenous HCQ decreased heart rate in anesthetized rats (14.3% ± 1.1% at 15mg/kg; n = 6) without significantly reducing mean arterial blood pressure. In vivo feeding studies in mice showed no significant change in systolic blood pressure nor left ventricular function. CONCLUSIONS: We have shown that HCQ acts as a bradycardic agent in SAN cells, in atrial preparations, and in vivo. HCQ slows the rate of spontaneous action potential firing in the SAN through multichannel inhibition, including that of If.

The importance of Ca(2+)-dependent mechanisms for the initiation of the heartbeat.

Mechanisms underlying pacemaker activity in the sinus node remain controversial, with some ascribing a dominant role to timing events in the surface membrane ("membrane clock") and others to uptake and release of calcium from the sarcoplasmic reticulum (SR) ("calcium clock"). Here we discuss recent evidence on mechanisms underlying pacemaker activity with a particular emphasis on the many roles of calcium. There are particular areas of controversy concerning the contribution of calcium spark-like events and the importance of I(f) to spontaneous diastolic depolarisation, though it will be suggested that neither of these is essential for pacemaking. Sodium-calcium exchange (NCX) is most often considered in the context of mediating membrane depolarisation after spark-like events. We present evidence for a broader role of this electrogenic exchanger which need not always depend upon these spark-like events. Short (milliseconds or seconds) and long (minutes) term influences of calcium are discussed including direct regulation of ion channels and NCX, and control of the activity of calcium-dependent enzymes (including CaMKII, AC1, and AC8). The balance between the many contributory factors to pacemaker activity may well alter with experimental and clinical conditions, and potentially redundant mechanisms are desirable to ensure the regular spontaneous heart rate that is essential for life. This review presents evidence that calcium is central to the normal control of pacemaking across a range of temporal scales and seeks to broaden the accepted description of the "calcium clock" to cover these important influences.

Cytosolic calcium ions exert a major influence on the firing rate and maintenance of pacemaker activity in guinea-pig sinus node.

The sino-atrial node (SAN) provides the electrical stimulus to initiate every heart beat. Cellular processes underlying this activity have been debated extensively, especially with regards to the role of intracellular calcium. We have used whole-cell application of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), a rapid calcium chelator, to guinea pig isolated SAN myocytes to assess the effect of rapid reduction of intracellular calcium on SAN cell electrical activity. High-dose (10 mM) BAPTA induced rapid and complete cessation of rhythmic action potential (AP) firing (time to cessation 5.5 ± 1.7 s). Over a range of concentrations, BAPTA induced slowing of action potential firing and disruption of rhythmic activity, which was dose-dependent in its time of onset. Exposure to BAPTA was associated with stereotyped action potential changes similar to those previously reported in the presence of ryanodine, namely depolarization of the most negative diastolic potential, prolongation of action potentials and a reduction in action potential amplitude. These experiments are consistent with the view that cytosolic calcium is essential to the maintenance of rhythmic pacemaker activity.

Pak1 is required to maintain ventricular Ca²âº homeostasis and electrophysiological stability through SERCA2a regulation in mice.

BACKGROUND: Impaired sarcoplasmic reticular Ca(2+) uptake resulting from decreased sarcoplasmic reticulum Ca(2+)-ATPase type 2a (SERCA2a) expression or activity is a characteristic of heart failure with its associated ventricular arrhythmias. Recent attempts at gene therapy of these conditions explored strategies enhancing SERCA2a expression and the activity as novel approaches to heart failure management. We here explore the role of Pak1 in maintaining ventricular Ca(2+) homeostasis and electrophysiological stability under both normal physiological and acute and chronic ß-adrenergic stress conditions. METHODS AND RESULTS: Mice with a cardiomyocyte-specific Pak1 deletion (Pak1(cko)), but not controls (Pak1(f/f)), showed high incidences of ventricular arrhythmias and electrophysiological instability during either acute ß-adrenergic or chronic ß-adrenergic stress leading to hypertrophy, induced by isoproterenol. Isolated Pak1(cko) ventricular myocytes correspondingly showed aberrant cellular Ca(2+) homeostasis. Pak1(cko) hearts showed an associated impairment of SERCA2a function and downregulation of SERCA2a mRNA and protein expression. Further explorations of the mechanisms underlying the altered transcriptional regulation demonstrated that exposure to control Ad-shC2 virus infection increased SERCA2a protein and mRNA levels after phenylephrine stress in cultured neonatal rat cardiomyocytes. This was abolished by the Pak1-knockdown in Ad-shPak1-infected neonatal rat cardiomyocytes and increased by constitutive overexpression of active Pak1 (Ad-CAPak1). We then implicated activation of serum response factor, a transcriptional factor well known for its vital role in the regulation of cardiogenesis genes in the Pak1-dependent regulation of SERCA2a. CONCLUSIONS: These findings indicate that Pak1 is required to maintain ventricular Ca(2+) homeostasis and electrophysiological stability and implicate Pak1 as a novel regulator of cardiac SERCA2a through a transcriptional mechanism.