If and SR Ca release both contribute to pacemaker activity in canine sinoatrial node cells

Increasing evidence suggests that cardiac pacemaking is the result of two sinoatrial node (SAN) cell mechanisms: a ‘voltage clock’ and a Ca²⁺ dependent process, or ‘Ca²⁺ clock.’ The voltage clock initiates action potentials (APs) by SAN cell membrane potential depolarization from inward currents, of which the pacemaker current (I_f) is thought to be particularly important. A Ca²⁺ dependent process triggers APs when sarcoplasmic reticulum (SR) Ca²⁺ release activates inward current carried by the forward mode of the electrogenic Na⁺/Ca²⁺ exchanger (NCX). However, these mechanisms have mostly been defined in rodents or rabbits, but are unexplored in single SAN cells from larger animals. Here, we used patch-clamp and confocal microscope techniques to explore the roles of the voltage and Ca²⁺ clock mechanisms in canine SAN pacemaker cells. We found that ZD7288, a selective I_f antagonist, significantly reduced basal automaticity and induced irregular, arrhythmia-like activity in canine SAN cells. In addition, ZD7288 impaired but did not eliminate the SAN cell rate acceleration by isoproterenol. In contrast, ryanodine significantly reduced the SAN cell acceleration by isoproterenol, while ryanodine reduction of basal automaticity was modest (∼ 14%) and did not reach statistical significance. Importantly, pretreatment with ryanodine eliminated SR Ca²⁺ release, but did not affect basal or isoproterenol-enhanced I_f. Taken together, these results indicate that voltage and Ca²⁺ dependent automaticity mechanisms coexist in canine SAN cells, and suggest that I_f and SR Ca²⁺ release cooperate to determine baseline and catecholamine-dependent automaticity in isolated dog SAN cells.     

Differential effects of ivabradine and ryanodine on pacemaker activity in canine sinus node and purkinje fibers

Effects of Ivabradine and Ryanodine on Cardiac Pacemakers . Introduction: It is generally accepted that at least 2 major mechanisms contribute to sinus node (SN) pacemaking: a membrane voltage (mainly I_f) clock and a calcium (Ca) clock (localized submembrane sarcoplasmic reticulum Ca²⁺ release during late diastolic depolarization). The aim of this study was to compare the contributions of each mechanism to pacemaker activity in SN and Purkinje fibers (PFs) exhibiting normal or abnormal automaticity. Methods and Results: Conventional microelectrodes were used to record action potentials in isolated spontaneously beating canine SN and free running PF in control and in the presence of 0.1 μM isoproterenol. Ryanodine (0.1–3 μM) and ivabradine (3 μM) were used to inhibit sarcoplasmic reticulum Ca²⁺ release or I_f, respectively. To induce automaticity at low membrane potentials, PFs were superfused with BaCl₂. In SN, ivabradine reduced the rate whereas ryanodine had no effect. Isoproterenol significantly accelerated automatic rate, which was decreased by ivabradine and ryanodine. In normally polarized PFs, ryanodine had no effects on the automatic rate in the absence or presence of isoproterenol, whereas ivabradine inhibited both control and isoproterenol‐accelerated automaticity. In PF depolarized with BaCl₂, ivabradine decreased BaCl₂‐induced automatic rate while ryanodine had no effect. Conclusion: In canine SN, I_f contributes to both basal automaticity and β‐adrenergic‐induced rate acceleration while the ryanodine‐inhibited Ca clock appears more involved in β‐adrenergic regulation of pacemaker rate. In PF, normal automaticity depends mainly on I_f. Inhibition of basal potassium conductance results in high automatic rates at depolarized membrane potentials with SN‐like responses to inhibition of membrane and Ca clocks. (J Cardiovasc Electrophysiol, Vol. 23, pp. 650–655, June 2012)     

Regulation of basal and reserve cardiac pacemaker function by interactions of cAMP-mediated PKA-dependent Ca cycling with surface membrane channels

Decades of intensive research of primary cardiac pacemaker, the sinoatrial node, have established potential roles of specific membrane channels in the generation of the diastolic depolarization, the major mechanism allowing sinoatrial node cells to generate spontaneous beating. During the last three decades, multiple studies made either in the isolated sinoatrial node or sinoatrial node cells have demonstrated a pivotal role of Ca²⁺ and, specifically Ca²⁺ release from sarcoplasmic reticulum, for spontaneous beating of cardiac pacemaker. Recently, spontaneous, rhythmic local subsarcolemmal Ca²⁺ releases from ryanodine receptors during late half of the diastolic depolarization have been implicated as a vital factor in the generation of sinoatrial node cell spontaneous firing. Local Ca²⁺ releases are driven by a unique combination of high basal cAMP production by adenylyl cyclases, high basal cAMP degradation by phosphodiesterases and a high level of cAMP-mediated PKA-dependent phosphorylation. These local Ca²⁺ releases activate an inward Na⁺–Ca²⁺ exchange current which accelerates the terminal diastolic depolarization rate and, thus, controls the spontaneous pacemaker firing. Both the basal primary pacemaker beating rate and its modulation via β-adrenergic receptor stimulation appear to be critically dependent upon intact RyR function and local subsarcolemmal sarcoplasmic reticulum generated Ca²⁺ releases. This review aspires to integrate the traditional viewpoint that has emphasized the supremacy of the ensemble of surface membrane ion channels in spontaneous firing of the primary cardiac pacemaker, and these novel perspectives of cAMP-mediated PKA-dependent Ca²⁺ cycling in regulation of the heart pacemaker clock, both in the basal state and during β-adrenergic receptor stimulation.     

Diastolic transient inward current in long QT syndrome type 3 is caused by Ca overload and inhibited by ranolazine

Long QT syndrome variant 3 (LQT-3) is a channelopathy in which mutations in SCN5A, the gene coding for the primary heart Na⁺ channel alpha subunit, disrupt inactivation to elevate the risk of mutation carriers for arrhythmias that are thought to be calcium (Ca²⁺)-dependent. Spontaneous arrhythmogenic diastolic activity has been reported in myocytes isolated from mice harboring the well-characterized ΔKPQ LQT-3 mutation but the link to altered Ca²⁺ cycling related to mutant Na⁺ channel activity has not previously been demonstrated. Here we have investigated the relationship between elevated sarcoplasmic reticulum (SR) Ca²⁺ load and induction of spontaneous diastolic inward current (I_TI) in myocytes expressing ΔKPQ Na⁺ channels, and tested the sensitivity of both to the antianginal compound ranolazine. We combined whole-cell patch clamp measurements, imaging of intracellular Ca²⁺, and measurement of SR Ca²⁺ content using a caffeine dump methodology. We compared the Ca²⁺ content of ΔKPQ^+/− myocytes displaying I_TI to those without spontaneous diastolic activity and found that I_TI induction correlates with higher sarcoplasmic reticulum (SR) Ca²⁺. Both spontaneous diastolic I_TI and underlying Ca²⁺ waves are inhibited by ranolazine at concentrations that preferentially target I_NaL during prolonged depolarization. Furthermore, ranolazine I_TI inhibition is accompanied by a small but significant decrease in SR Ca²⁺ content. Our results provide the first direct evidence that induction of diastolic transient inward current (I_TI) in ΔKPQ^+/− myocytes occurs under conditions of elevated SR Ca²⁺ load.     

Expression of the L-type calcium channel in human heart failure

L-type calcium channels play an important role in excitation-contraction coupling. After cardiomyocyte depolarization L-type calcium channels open and Ca ²⁺ ions enter the cell. These small Ca ²⁺ inward currents trigger calcium release from the junctional sarcoplasmic reticulum, a process called calcium-induced calcium release. Subsequently, the cytosolic Ca ²⁺ concentration rises rapidly to levels that initiate contraction. In heart failure calcium-induced calcium release is disturbed, and in this review we focus on the L-type calcium channel and its contribution to this defective excitation-contraction coupling.     

Local control of Ca-induced Ca release in mouse sinoatrial node cells

Emerging evidence from large animal models implicates Ca²⁺ regulation, particularly intracellular sarcoplasmic reticulum (SR) Ca²⁺ release, as essential for sinoatrial node (SAN) automaticity. However, despite the apparent importance of SR Ca²⁺ release to SAN cell function it is uncertain how SR Ca²⁺ release is controlled in SAN cells from mouse. Understanding mouse SAN SR Ca²⁺ release mechanism will allow improved understanding of results in studies on SAN from genetic mouse models of Ca²⁺ homeostatic proteins. Here we investigated the functional relationship between sarcolemmal Ca²⁺ influx and SR Ca²⁺ release at the level of single SAN cell, using simultaneous patch-clamp current recording and high resolution confocal Ca²⁺ imaging techniques. In mouse SAN cells, both Ca²⁺ channel currents and triggered SR Ca²⁺ transients displayed bell-shaped, graded function with the membrane potential. Moreover, the gain function for Ca²⁺-induced Ca²⁺ release (CICR) displayed a monotonically decreasing function with strong voltage dependence, consistent with a “local control” mechanism for CICR. In addition, we observed numerous discrete Ca²⁺ sparks at the voltage range of diastolic depolarization, in sharp contrast to the much lower frequency of sparks observed at resting potentials. We concluded that the “local control” mechanism of CICR is responsible for both local Ca²⁺ release during diastolic depolarization and the synchronized Ca²⁺ transients observed during action potential in SAN cells.     

Inositol trisphosphate promotes Na-Ca exchange current by releasing calcium from sarcoplasmic reticulum in cardiac myocytes

An early inward tail current evoked by membrane depolarization (from -80 to -40 mV) sufficient to activate sodium but not calcium current was studied in single voltage-clamped ventricular myocytes isolated from guinea pig hearts. Like forward-mode Na-Ca exchange, this early inward tail current required [Na+]o and [Ca2+]i and is thought to follow earlier reverse-mode Na-Ca exchange that triggers Ca2+ release from sarcoplasmic reticulum. The dependence of the early inward tail current on [Ca2+]i was supported by the ability of small (+10 mV) and large (+80 mV) voltage jumps from -40 mV to decrease and increase, respectively, the size of early inward tail currents evoked by subsequent voltage steps from -80 to -40 mV. As expected, tetrodotoxin selectively inhibited the early inward tail current but not the late inward tail current that followed voltage jumps to +40 mV test potentials. Although tetrodotoxin also blocked the fast Na+ current, replacement of extracellular Na+ by Li+ sustained the fast Na+ current. However, Li+, which does not support Na-Ca exchange, reversibly suppressed both the early and late inward tail currents. Inhibitors (ryanodine and caffeine) and promoters (intracellularly dialyzed inositol 1,4,5-trisphosphate) of sarcoplasmic reticulum Ca2+ release decreased and increased, respectively, the magnitude of the early inward tail current. The results substantiate the hypothesis that Ca2+ release from the sarcoplasmic reticulum participates in early Na-Ca exchange current and demonstrate that inositol 1,4,5-trisphosphate, by releasing Ca2+ from the sarcoplasmic reticulum, can promote Na-Ca exchange across the plasma membrane.     

STIM1–Ca2+ signaling modulates automaticity of the mouse sinoatrial node

Cardiac pacemaking is governed by specialized cardiomyocytes located in the sinoatrial node (SAN). SAN cells (SANCs) integrate voltage-gated currents from channels on the membrane surface (membrane clock) with rhythmic Ca²⁺ release from internal Ca²⁺ stores (Ca²⁺ clock) to adjust heart rate to meet hemodynamic demand. Here, we report that stromal interaction molecule 1 (STIM1) and Orai1 channels, key components of store-operated Ca²⁺ entry, are selectively expressed in SANCs. Cardiac-specific deletion of STIM1 in mice resulted in depletion of sarcoplasmic reticulum (SR) Ca²⁺ stores of SANCs and led to SAN dysfunction, as was evident by a reduction in heart rate, sinus arrest, and an exaggerated autonomic response to cholinergic signaling. Moreover, STIM1 influenced SAN function by regulating ionic fluxes in SANCs, including activation of a store-operated Ca²⁺ current, a reduction in L-type Ca²⁺ current, and enhancing the activities of Na⁺/Ca²⁺ exchanger. In conclusion, these studies reveal that STIM1 is a multifunctional regulator of Ca²⁺ dynamics in SANCs that links SR Ca²⁺ store content with electrical events occurring in the plasma membrane, thereby contributing to automaticity of the SAN.Sinus rhythm of the heart is set by specialized cardiomyocytes located in the sinoatrial node (SAN). These cardiomyocytes (SANCs) lack a resting membrane potential but generate a sinus impulse after spontaneous diastolic depolarization triggers an action potential (AP). Automaticity is achieved in the SANCs by the simultaneous activation of diastolic currents during membrane depolarization and the spontaneous release of Ca²⁺ from internal stores (1–3). Several recent studies show that maintenance of sarcoplasmic reticulum (SR) Ca²⁺ stores is critically important for SAN automaticity, as is evident from genetic studies involving patients and mice that have leaky SR Ca²⁺ stores (4). Mutations in the ryanodine receptor (RYR2) or calsequestrin genes that result in spontaneous Ca²⁺ release cause catecholamiergic polymorphic ventricular tachycardia (CPVT) (5, 6). In addition to ventricular arrhythmias, these patients also develop sinus node dysfunction and bradycardia, which frequently requires permanent pacemaker insertion. Computational studies further reinforce the idea that SAN dysfunction results from leaky RYR2-containing Ca²⁺ stores (5). These studies emphasize the importance of Ca²⁺ signaling in the automaticity of SANCs. Given the emerging role of store-operated Ca²⁺ entry (SOCE) in excitable cells, we asked here whether stromal interaction molecule 1 (STIM1) plays a major role in regulating the Ca²⁺ signaling and automaticity of SANCs.SANs are structurally and functionally heterogeneous, exhibiting differences in shape and size that correspond to differences in electrophysiological features (7). It is believed that this heterogeneity is required to establish regional zones within the SAN for impulse generation by pacemakers. Under resting conditions, clusters of SANCs serve as the dominant pacemaker by firing APs at rates faster than subsidiary pacemakers located in adjacent aspects of the SAN and atria. However, under different chronotropic conditions, as occurs with autonomic stimulation, the dominant pacemaking sites can shift to different regions within the SAN, where subsidiary pacemakers can slow or speed up the heart rate (HR) (8). Dominant pacemakers are established by differences in ion channel distribution that create regional differences in electrophysiological properties such as the maximal diastolic potential (MDP) and the rate of diastolic depolarization. In addition, local intracellular factors also contribute to pacemaking by integrating currents generated at the plasma membrane with the rhythmic release of Ca²⁺ from the SR. Given the importance of Ca²⁺ dynamics to diastolic depolarization of the SAN, we hypothesize that multiple mechanisms must be available to replenish internal Ca²⁺ stores. Refilling internal Ca²⁺ stores in SANCs is known to involve Ca²⁺ entry via voltage-gated Ca²⁺ channels (9). Here we propose that Ca²⁺ store refilling also requires SOCE.In nonexcitable cells such as lymphocytes, the molecular mechanisms underlying SOCE have been well characterized and shown to require STIM1, a single-pass endoplasmic reticulum (ER) membrane protein that serves as the sensor of SR/ER Ca²⁺ store content. When Ca²⁺ stores are depleted, STIM1 molecules in the SR/ER membrane interact with plasma membrane Ca²⁺ channels, such as Orai1, to initiate Ca²⁺ entry into the cytoplasm and consequent refilling of internal Ca²⁺ stores via SR/ER Ca²⁺ pumps. SOCE is therefore an attractive candidate to link the Ca²⁺ and membrane clocks that underlie SANC automaticity. Roles for STIM1-dependent SOCE have been suggested in the heart (10–14). Here, we show that STIM1 is selectively expressed in the SAN where it activates Ca²⁺ entry via Orai1 channels and thereby modulates the Ca²⁺ signals required for pacemaking activity of the SAN. These findings build on an emerging theme, that STIM1 is a multifunctional signaling molecule, to show that STIM1 regulates several aspects of Ca²⁺ signaling in SANCs.     

Membrane depolarization causes a direct activation of G protein-coupled receptors leading to local Ca2+ release in smooth muscle

Membrane depolarization activates voltage-dependent Ca²⁺ channels (VDCCs) inducing Ca²⁺ release via ryanodine receptors (RyRs), which is obligatory for skeletal and cardiac muscle contraction and other physiological responses. However, depolarization-induced Ca²⁺ release and its functional importance as well as underlying signaling mechanisms in smooth muscle cells (SMCs) are largely unknown. Here we report that membrane depolarization can induce RyR-mediated local Ca²⁺ release, leading to a significant increase in the activity of Ca²⁺ sparks and contraction in airway SMCs. The increased Ca²⁺ sparks are independent of VDCCs and the associated extracellular Ca²⁺ influx. This format of local Ca²⁺ release results from a direct activation of G protein-coupled, M₃ muscarinic receptors in the absence of exogenous agonists, which causes activation of Gq proteins and phospholipase C, and generation of inositol 1,4,5-triphosphate (IP₃), inducing initial Ca²⁺ release through IP₃ receptors and then further Ca²⁺ release via RyR2 due to a local Ca²⁺-induced Ca²⁺ release process. These findings demonstrate an important mechanism for Ca²⁺ signaling and attendant physiological function in SMCs.     

The ryanodine receptor leak: how a tattered receptor plunges the failing heart into crisis

It has been persuasively shown in the last two decades that the development of heart failure is closely linked to distinct alterations in Ca²⁺ cycling. A crucial point in this respect is an increased spontaneous release of Ca²⁺ out of the sarcoplasmic reticulum during diastole via ryanodine receptors type 2 (RyR2). The consequence is a compromised sarcoplasmic reticulum Ca²⁺ storage capacity, which impairs systolic contractility and possibly diastolic cardiac function due to Ca²⁺ overload. Additionally, leaky RyR2 are more and more regarded to potently induce proarrhythmic triggers. Elimination of spontaneously released Ca²⁺ via RyR2 in diastole can cause a transient sarcolemmal inward current and hence delayed after depolarisations as substrate for cardiac arrhythmias. In this article, the pathological role and consequences of the SR Ca²⁺-leak and its regulation are reviewed with a main focus on protein kinase A and Ca²⁺-calmodulin-dependent kinase II. We summarise clinical consequences of “leaky RyR2” as well as possible therapeutic strategies in order to correct RyR2 dysfunction and discuss the significance of the available data.     

SR Ca store refill—a key factor in cardiac pacemaking

This study presents a theoretical analysis of the role of store Ca²⁺ uptake on sinoatrial node (SAN) cell pacemaking. Two mechanisms have been shown to be involved in SAN pacemaking, these being: 1) the membrane oscillator model where rhythm generation is based on the interaction of voltage-dependent membrane ion channels and, 2) the store oscillator model where cyclical release of Ca²⁺ from intracellular Ca²⁺ stores depolarizes the membrane through activation of the sodium-calcium exchanger (NCX). The relative roles of these oscillators in generation and modulation of pacemaker rate have been vigorously debated and have many consequences. The main new outcomes of our study are: 1) uptake of Ca²⁺ by intracellular Ca²⁺ stores increases the maximum diastolic potential (MDP) by reducing the cytosolic Ca²⁺ concentration [Ca²⁺]_c and hence decreasing the NCX current; 2) this hyperpolarization enhances recruitment of key pacemaker currents (e.g. the hyperpolarization-activated HCN current (I_f) and T-type Ca²⁺ current (I_T-Ca)); 3) the resultant enhanced Ca²⁺ entry during the pacemaker depolarization increases [Ca²⁺]_c causing advancement of the store Ca²⁺ release cycle and increased NCX current. In overview, the novel feature of our study is an investigation of the role of store Ca²⁺ uptake on SAN pacemaking. This occurs during the early diastolic period and causes enhanced I_f, I_T-Ca and store release (and hence I_NCX) during the later diastolic period. There is thus a symbiotic interaction between the two pacemaker “clocks” over the entire diastolic period, this providing robust and highly malleable SAN pacemaking. Accounting for store Ca²⁺ uptake also provides insight into hitherto unexplained SAN behaviour, as we exemplify for the sinus bradycardia exhibited in catecholaminergic polymorphic ventricular tachycardia (CPVT).     

Electrophysiological effects of amrinone on the automaticity and membrane current system of the rabbit sinoatrial node cells

Summary To elucidate the physiological role of phosphodiesterase (PDE) in cardiac pacemaker cells, we studied the electrophysiological effects of amrinone, an inhibitor of PDE type III, on the spontaneous action potential (AP) and membrane currents, using small preparations (0.2 × 0.2 × 0.1mm) of rabbit sinoatrial (SA) node cells. Amrinone (0.1–1.0mM) progressively increased the AP amplitude, maximal rate of depolarization, and spontaneous firing frequency, shortened the AP duration, and made the threshold potential more negative. In voltage-clamp experiments using double microelectrode techniques, 0.1mM amrinone increased the Ca²⁺ current (I _Ca) obtained on step depolarization from –40 to –10mV by 25.86% ± 4.6% (P < 0.05,n = 6), the delayed rectifier K⁺ current (I _K) tail obtained on repolarization from 10 to –60mV by 22.8% ± 4.7% (P < 0.05,n = 6), and the hyperpolarization-activated inward current (I _h) at –90mV by 19.5% ± 7.3% (P < 0.05,n = 6), respectively. Amrinone did not affect the slope factors of either the inactivation curve forI _Ca (f curve) or the activation curve for the delayed rectifierI _K (p curve). These results suggest that this PDE III inhibitor exerts a positive chronotropic action by enhancing the availability and the conductance of all the tested membrane currents in rabbit SA node cells.     

L-type Ca current in ventricular cardiomyocytes

L-type Ca²⁺ channels are mediators of Ca²⁺ influx and the regulatory events accompanying it and are pivotal in the function and dysfunction of ventricular cardiac myocytes. L-type Ca²⁺ channels are located in sarcolemma, including the T-tubules facing the sarcoplasmic reticulum junction, and are activated by membrane depolarization, but intracellular Ca²⁺-dependent inactivation limits Ca²⁺ influx during action potential. I_CaL is important in heart function because it triggers excitation-contraction coupling, modulates action potential shape and is involved in cardiac arrhythmia. L-type Ca²⁺ channels are multi-subunit complexes that interact with several molecules involved in their regulations, notably by β-adrenergic signaling. The present review highlights some of the recent findings on L-type Ca²⁺ channel function, regulation, and alteration in acquired pathologies such as cardiac hypertrophy, heart failure and diabetic cardiomyopathy, as well as in inherited arrhythmic cardiac diseases such as Timothy and Brugada syndromes.     

Sustained inward current during pacemaker depolarization in mammalian sinoatrial node cells

Several time- and voltage-dependent ionic currents have been identified in cardiac pacemaker cells, including Na(+) current, L- and T-type Ca(2+) currents, hyperpolarization-activated cation current, and various types of delayed rectifier K(+) currents. Mathematical models have demonstrated that spontaneous action potentials can be reconstructed by incorporating these currents, but relative contributions of individual currents vary widely between different models. In 1995, the presence of a novel inward current that was activated by depolarization to the potential range of the slow diastolic depolarization in rabbit sinoatrial (SA) node cells was reported. Because the current showed little inactivation during depolarizing pulses, it was called the sustained inward current (I(st)). A similar current is also found in SA node cells of the guinea pig and rat and in subsidiary pacemaker atrioventricular node cells. Recently, single-channel analysis has revealed a nicardipine-sensitive, 13-pS Na(+) current, which is activated by depolarization to the diastolic potential range in guinea pig SA node cells. This channel differs from rapid voltage-gated Na(+) or L-type Ca(2+) channels both in unitary conductance and gating kinetics. Because I(st) was observed only in spontaneously beating SA node cells, ie, it was absent in quiescent cells dissociated from the same SA or atrioventricular node, an important role of I(st) for generation of intrinsic cardiac automaticity was suggested.     

Termination of Ca2+ release by a local inactivation of ryanodine receptors in cardiac myocytes

In heart, a robust regulatory mechanism is required to counteract the regenerative Ca²⁺-induced Ca²⁺ release from the sarcoplasmic reticulum. Several mechanisms, including inactivation, adaptation, and stochastic closing of ryanodine receptors (RyRs) have been proposed, but no conclusive evidence has yet been provided. We probed the termination process of Ca²⁺ release by using a technique of imaging local Ca²⁺ release, or “Ca²⁺ spikes”, at subcellular sites; and we tracked the kinetics of Ca²⁺ release triggered by L-type Ca²⁺ channels. At 0 mV, Ca²⁺ release occurred and terminated within 40 ms after the onset of clamp pulses (0 mV). Increasing the open-duration and promoting the reopenings of Ca²⁺ channels with the Ca²⁺ channel agonist, FPL64176, did not prolong or trigger secondary Ca²⁺ spikes, even though two-thirds of the sarcoplasmic reticulum Ca²⁺ remained available for release. Latency of Ca²⁺ spikes coincided with the first openings but not with the reopenings of L-type Ca²⁺ channels. After an initial maximal release, even a multi-fold increase in unitary Ca²⁺ current induced by a hyperpolarization to −120 mV failed to trigger additional release, indicating absolute refractoriness of RyRs. When the release was submaximal (e.g., at +30 mV), tail currents did activate additional Ca²⁺ spikes; confocal images revealed that they originated from RyRs unfired during depolarization. These results indicate that Ca²⁺ release is terminated primarily by a highly localized, use-dependent inactivation of RyRs but not by the stochastic closing or adaptation of RyRs in intact ventricular myocytes.     

Myocardial Phosphodiesterases and Regulation of Cardiac Contractility in Health and Cardiac Disease

Phosphodiesterase (PDE) inhibitors are potent cardiotonic agents used for parenteral inotropic support in heart failure. Contractile effects of these agents are mediated through cAMP-protein kinase A-induced stimulation of I _Ca2+ which ultimately results in increased Ca²⁺-induced sarcoplasmic reticulum Ca²⁺ release. A number of additional effects such as increases in sarcoplasmic reticulum Ca²⁺ stores, stimulation of reverse mode Na⁺–Ca²⁺ exchange, direct or cAMP-mediated effects on sarcoplasmic reticulum ryanodine receptor, stimulation of the voltage-sensitive sarcoplasmic reticulum Ca²⁺ release mechanism, as well as A₁ adenosine receptor blockade could contribute to positive inotropic responses to PDE inhibitors. Moreover, some PDE inhibitors exhibit Ca²⁺ sensitizer properties as they could increase the affinity of troponin C Ca²⁺-binding sites as well as reduce Ca²⁺ threshold for thin myofilament sliding and facilitate cross-bridge cycling. Inotropic responses to PDE inhibitors are significantly reduced in cardiac disease, an effect largely attributed to downregulation of cAMP-mediated signalling due to sustained sympathetic activation. Four PDE isoenzymes (PDE1, PDE2, PDE3 and PDE4) are present in myocardial tissue of various mammalian species, of which PDE3 and PDE4 are particularly involved in regulation of cardiac myocyte contraction. PDE cAMP-hydrolysing activity is preserved in compensated cardiac hypertrophy but significantly reduced in animal models of heart failure. However, clinical studies have not revealed any changes in distribution profile as well as kinetic and regulatory properties of myocardial PDEs in failing human hearts. A reduction of PDE inhibitors-induced contractile responses in heart failure has therefore been ascribed to reduced cAMP synthesis due to uncoupling of adenylyl cyclase from β-adrenoreceptor. In cardiac myocytes, PDEs are targeted to distinct subcellular compartments by scaffolding proteins such as myomegalin, mAKAP and β-arrestins. Over subcellular microdomains, cAMP hydrolysis by PDE3 and PDE4 allows to control the activity of local pools of protein kinase A and therefore the extent of protein kinase A-mediated phosphorylation of cellular proteins.     

L-type Ca channel facilitation mediated by H2O2-induced activation of CaMKII in rat ventricular myocytes

The Ca²⁺-dependent facilitation (CDF) of L-type Ca²⁺ channels, a major mechanism for force-frequency relationship of cardiac contraction, is mediated by Ca²⁺/CaM-dependent kinase II (CaMKII). Recently, CaMKII was shown to be activated by methionine oxidation. We investigated whether oxidation-dependent CaMKII activation is involved in the regulation of L-type Ca²⁺ currents (I_Ca,L) by H₂O₂ and whether Ca²⁺ is required in this process. Using patch clamp, I_Ca,L was measured in rat ventricular myocytes. H₂O₂ induced an increase in I_Ca,L amplitude and slowed inactivation of I_Ca,L. This oxidation-dependent facilitation (ODF) of I_Ca,L was abolished by a CaMKII blocker KN-93, but not by its inactive analog KN-92, indicating that CaMKII is involved in ODF. ODF was not affected by replacement of external Ca²⁺ with Ba²⁺ or presence of EGTA in the internal solutions. However, ODF was abolished by adding BAPTA to the internal solution or by depleting sarcoplasmic reticulum (SR) Ca²⁺ stores using caffeine and thapsigargin. Alkaline phosphatase, β-iminoadenosine 5′-triphosphate (AMP-PNP), an autophosphorylation inhibitor autocamtide-2-related inhibitory peptide (AIP), or a catalytic domain blocker (CaM-KIINtide) did not affect ODF. In conclusion, oxidation-dependent facilitation of L-type Ca²⁺ channels is mediated by oxidation-dependent CaMKII activation, in which local Ca²⁺ increases induced by SR Ca²⁺ release is required.     

The role of ryanodine receptors and consequences of their alterations during cardiac insufficiency

Congestive heart failure (CHF) is a leading cause of death. Although changes to other components contribute, it is generally agreed that much of the contractile deficit is due to reduced Ca²⁺ homeostasis that includes alterations in Ca²⁺ current and action potential characteristics, together with reduced Ca²⁺ transient amplitude. CHF is also associated with progressive skeletal muscle dysfunction. In both cardiac and skeletal muscles, the global increase in myoplasmic Ca²⁺ during depolarization, or Ca²⁺ transient, appears to consist of the summation of large numbers of local, unitary Ca²⁺ release events (ie, Ca²⁺ sparks) resulting from the activity of a cluster of ryanodine receptors (RyRs) (ie, RyR1 or RyR2 in skeletal and cardiac muscles, respectively). RyR2 channels from failing hearts have been shown to be hyperphosphorylated by protein kinase A, leading to dissociation of FK506-binding protein 12.6 and altered RyR2 channel function. After reviewing the alterations occurring in cardiomyocytes, the present report summarizes the intrinsic alterations of Ca²⁺ homeostasis in rat extensor digitorum longus skeletal muscle. They include a weaker and prolonged Ca²⁺ transient that could be attributed to both a lower synchronization of the individual Ca²⁺ sparks and a lower synchronization of these events triggered upon depolarization. As in cardiac muscle, these alterations in sarcoplasmic reticulum function are associated with protein kinase A-induced hyperphosphorylation of RyR1 and a concomitant reduction in FK506-binding protein 12. These specific alterations in RyR1-dependent Ca²⁺ release could play a significant role in the specific force decrements in skeletal muscle as well as in the remodelling that occurs secondary to CHF.     

Effects of prostaglandin F_（2α） on small intestinal interstitial cells of Cajal

AIM: To explore the role of prostaglandin F_2α (PGF_2α)) on pacemaker activity in interstitial cells of Cajal (ICC) from mouse small intestine.METHODS: In this study, effects of PGF_2α in the cultured ICC cells were investigated with patch clamp technology combined with Ca²⁺ image analysis.RESULTS: Externally applied PGF_2α (10 μmol/L) produced membrane depolarization in current-clamp mode and increased tonic inward pacemaker currents in voltage-clamp mode. The application of flufenamic acid (a non-selective cation channel inhibitor) or niflumic acid (a Cl^- channel inhibitor) abolished the generation of pacemaker currents but only flufenamic acid inhibited the PGF_2α-induced tonic inward currents. In addition, the tonic inward currents induced by PGF_2α were not inhibited by intracellular application of 5’-[-thio]diphosphate trilithium salt. Pretreatment with Ca²⁺ free solution, U-73122, an active phospholipase C inhibitor, and thapsigargin, a Ca²⁺-ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker currents and suppressed the PGF_2α-induced tonic inward currents. However, chelerythrine or calphostin C, protein kinase C inhibitors, did not block the PGF_2α-induced effects on pacemaker currents. When recording intracellular Ca²⁺ ([Ca²⁺]_i) concentration using fluo-3/AM, PGF_2α broadly increased the spontaneous [Ca²⁺]_i oscillations.CONCLUSION: These results suggest that PGF_2α can modulate pacemaker activity of ICC by acting non-selective action channels through phospholipase C-dependent pathway via [Ca²⁺]i regulation     

Carvedilol analog modulates both basal and stimulated sinoatrial node automaticity

The membrane voltage clock and calcium (Ca²⁺) clock jointly regulate sinoatrial node (SAN) automaticity. VK-II-36 is a novel carvedilol analog that suppresses sarcoplasmic reticulum (SR) Ca²⁺ release but does not block the β-receptor. The effect of VK-II-36 on SAN function remains unclear. The purpose of this study was to evaluate whether VK-II-36 can influence SAN automaticity by inhibiting the Ca²⁺ clock. We simultaneously mapped intracellular Ca²⁺ and membrane potential in 24 isolated canine right atriums using previously described criteria of the timing of late diastolic intracellular Ca elevation (LDCAE) relative to the action potential upstroke to detect the Ca²⁺ clock. Pharmacological interventions with isoproterenol (ISO), ryanodine, caffeine, and VK-II-36 were performed after baseline recordings. VK-II-36 caused sinus rate downregulation and reduced LDCAE in the pacemaking site under basal conditions (P < 0.01). ISO induced an upward shift of the pacemaking site in SAN and augmented LDCAE in the pacemaking site. ISO also significantly and dose-dependently increased the sinus rate. The treatment of VK-II-36 (30 μmol/l) abolished both the ISO-induced shift of the pacemaking site and augmentation of LDCAE (P < 0.01), and it suppressed the ISO-induced increase in sinus rate (P = 0.02). Our results suggest that the sinus rate may be partly controlled by the Ca²⁺ clock via SR Ca²⁺ release during β-adrenergic stimulation.