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.

Flecainide reduces Ca(2+) spark and wave frequency via inhibition of the sarcolemmal sodium current.

AIMS: Ca(2+) waves are thought to be important in the aetiology of ventricular tachyarrhythmias. There have been conflicting results regarding whether flecainide reduces Ca(2+) waves in isolated cardiomyocytes. We sought to confirm whether flecainide inhibits waves in the intact cardiomyocyte and to elucidate the mechanism. METHODS AND RESULTS: We imaged spontaneous sarcoplasmic reticulum (SR) Ca(2+) release events in healthy adult rat cardiomyocytes. Variation in stimulation frequency was used to produce Ca(2+) sparks or waves. Spark frequency, wave frequency, and wave velocity were reduced by flecainide in the absence of a reduction of SR Ca(2+) content. Inhibition of I(Na) via alternative pharmacological agents (tetrodotoxin, propafenone, or lidocaine) produced similar changes. To assess the contribution of I(Na) to spark and wave production, voltage clamping was used to activate contraction from holding potentials of -80 or -40 mV. This confirmed that reducing Na(+) influx during myocyte stimulation is sufficient to reduce waves and that flecainide only causes Ca(2+) wave reduction when I(Na) is active. It was found that Na(+)/Ca(2+)-exchanger (NCX)-mediated Ca(2+) efflux was significantly enhanced by flecainide and that the effects of flecainide on wave frequency could be reversed by reducing [Na(+)](o), suggesting an important downstream role for NCX function. CONCLUSION: Flecainide reduces spark and wave frequency in the intact rat cardiomyocyte at therapeutically relevant concentrations but the mechanism involves I(Na) reduction rather than direct ryanodine receptor (RyR2) inhibition. Reduced I(Na) results in increased Ca(2+) efflux via NCX across the sarcolemma, reducing Ca(2+) concentration in the vicinity of the RyR2.

Ca(2+)-stimulated adenylyl cyclases regulate the L-type Ca(2+) current in guinea-pig atrial myocytes.

Ca(2+)-stimulated adenylyl cyclases (ACs) have recently been shown to play important roles in pacemaking in the sino-atrial node. Here we present evidence that Ca(2+)-stimulated ACs are functionally active in guinea-pig atrial myocytes. Basal activity of an AC in isolated atrial myocytes was demonstrated by the observations that MDL 12,330A (10 µm), an AC inhibitor, reduced L-type Ca(2+) current (I(CaL)) amplitude, while inhibition of phosphodiesterases with IBMX (100 µm) increased I(CaL) amplitude. Buffering of cytosolic Ca(2+) by exposure of myocytes to BAPTA-AM (5 µm) reduced I(CaL) amplitude, as did inhibition of Ca(2+) release from the sarcoplasmic reticulum with ryanodine (2 µm) and thapsigargin (1 µm). [Ca(2+)]i-activated calmodulin kinase II (CaMKII) inhibition with KN-93 (1 µm) reduced I(CaL), but subsequent application of BAPTA-AM further reduced I(CaL). This effect of BAPTA-AM, in the presence of CaMKII inhibition, demonstrates that there is an additional Ca(2+)-modulated pathway (not dependent on CaMKII) that regulates I(CaL) in atrial myocytes. The effects of BAPTA could be reversed by forskolin (10 µm), a direct stimulator of all AC isoforms, which would restore cAMP levels. In the presence of BAPTA-AM, the actions of IBMX were reduced. In addition, inclusion of cAMP in the patch electrode in the whole-cell configuration prevented the effects of BAPTA. These effects are all consistent with a role for Ca(2+)-stimulated AC in the regulation of atrial myocyte I(CaL).

NAADP influences excitation-contraction coupling by releasing calcium from lysosomes in atrial myocytes.

In atrial myocytes, the sarcoplasmic reticulum (SR) has an essential role in regulating the force of contraction as a consequence of its involvement in excitation-contraction coupling (ECC). Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca(2+) mobilizing messenger that acts to release Ca(2+) from an acidic store in mammalian cells. The photorelease of NAADP in atrial myocytes increased Ca(2+) transient amplitude with no effect on accompanying action potentials or the L-type Ca(2+) current. NAADP-AM, a cell permeant form of NAADP, increased Ca(2+) spark amplitude and frequency. The effect on Ca(2+) spark frequency could be prevented by bafilomycin A1, a vacuolar H(+)-ATPase inhibitor, or by disruption of lysosomes by GPN. Bafilomycin prevented staining of acidic stores with LysoTracker red by increasing lysosomal pH. NAADP-AM also produced an increase in the lysosomal pH, as detected by a reduction in LysoSensor green fluorescence. These effects of NAADP were associated with an increase in the amount of caffeine-releasable Ca(2+) in the SR and may be regulated by ß-adrenoceptor stimulation with isoprenaline. These observations are consistent with a role for NAADP in regulating ECC in atrial myocytes by releasing Ca(2+) from an acidic store, which enhances SR Ca(2+) release by increasing SR load.

Regulation of L-type calcium channel and delayed rectifier potassium channel activity by p21-activated kinase-1 in guinea pig sinoatrial node pacemaker cells.

Phosphorylation of ion channels plays an important role in the regulation of cardiac function, but signaling mechanisms controlling dephosphorylation are not well understood. We have tested the hypothesis that p(21)-activated kinase-1 (Pak1), a serine-threonine protein kinase regulated by Ras-related small G proteins, regulates sinoatrial node (SAN) ion channel activity through a mechanism involving protein phosphatase 2A. We report a novel role of Pak1-mediated signaling in attenuating isoproterenol-induced enhancement of L-type Ca(2+) current (I(CaL)) and delayed rectifier potassium current (I(K)) in guinea pig SAN pacemaker cells. We demonstrate that in guinea pig SAN: (1) there is abundant expression of endogenous Pak1 in pacemaker cells; (2) expression of constitutively active Pak1 depresses isoproterenol-induced upregulation of I(CaL) and I(K); (3) inhibition of protein phosphatase 2A increases the enhancement of I(K) and I(CaL) by isoproterenol in Ad-Pak1-infected cells; (4) protein phosphatase 2A coimmunoprecipitates with endogenous Pak1 in SAN tissue; and (5) expression of constitutively active Pak1 suppresses the chronotropic action of isoproterenol on pacemaker activity of intact SAN preparations. In conclusion, our data demonstrate that a Pak1 signaling pathway exists in cardiac pacemaker cells and that this novel pathway plays a role in the regulation of ion channel activity.