Pernicious attrition and inter-RyR2 CICR current control in cardiac muscle

In cardiac muscle cells, ryanodine receptor (RyR) mediated Ca^2 + release from the sarcoplasmic reticulum (SR) drives the contractile apparatus. Spontaneous bouts of inter-RyR Ca^2 + induced Ca^2 + release (CICR) generate an elemental unit of SR Ca^2 + release called a spark. Sparks are localized events that terminate soon after they begin. The local control of sparks is not clearly understood. In this article, we review the potential regulatory role that the changing single RyR Ca^2 + current may play. Moreover, we aggregate RyR data into a working scheme of inter-RyR CICR current control of sparks and a potential inter-RyR CICR termination mechanism that we call pernicious attrition. This article is part of a Special Issue entitled “Calcium Signaling in Heart”.     

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.     

Sarcoplasmic Reticulum Function in Murine Ventricular Myocytes Overexpressing SR CaATPase

To examine the effects of the overexpression of sarcoplasmic reticulum (SR) CaATPase on function of the SR and Ca²⁺homeostasis, we measured [Ca²⁺]_itransients (fluo-3), and L-type Ca²⁺currents (I_Ca,L), Na/Ca exchanger currents (I_Na/Ca), and SR Ca²⁺content with voltage clamp in ventricular myocytes isolated from wild type (WT) mice and transgenic (SRTG) mice. The amplitude of [Ca²⁺]_itransients was insignificantly increased in SRTG myocytes, while the diastolic [Ca²⁺]_itended to be lower. The initial and terminal declines of [Ca²⁺]_itransients were significantly accelerated in SRTG myocytes, implying a functional upregulation of the SR CaATPase. We examined the functional contribution of only the SR CaATPase to the initial and the terminal phase of the decline of [Ca²⁺]_i, by abruptly inhibiting Na/Ca exchange with a rapid switcher device. The rate of [Ca²⁺] decline mediated by the SR CaATPase was increased by 40% in SRTG compared with WT myocytes. The function of the L-type Ca²⁺channel was unchanged in SRTG myocytes, while INa/Ca density was slightly (10%) decreased. Measured SR Ca²⁺content was significantly increased by 29% in SRTG myocytes. Thus, overexpression of SR CaATPase markedly accelerates the decline of [Ca²⁺]_itransients, and induces an increase in SR Ca²⁺content, with some downregulation of the Na/Ca exchanger.     

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.     

SR-targeted CaMKII inhibition improves SR Ca²+ handling, but accelerates cardiac remodeling in mice overexpressing CaMKIIδC

Cardiac myocyte overexpression of CaMKIIδ_C leads to cardiac hypertrophy and heart failure (HF) possibly caused by altered myocyte Ca²⁺ handling. A central defect might be the marked CaMKII-induced increase in diastolic sarcoplasmic reticulum (SR) Ca²⁺ leak which decreases SR Ca²⁺ load and Ca²⁺ transient amplitude. We hypothesized that inhibition of CaMKII near the SR membrane would decrease the leak, improve Ca²⁺ handling and prevent the development of contractile dysfunction and HF. To test this hypothesis we crossbred CaMKIIδ_C overexpressing mice (CaMK) with mice expressing the CaMKII-inhibitor AIP targeted to the SR via a modified phospholamban (PLB)-transmembrane-domain (SR-AIP). There was a selective decrease in the amount of activated CaMKII in the microsomal (SR/membrane) fraction prepared from these double-transgenic mice (CaMK/SR-AIP) mice. In ventricular cardiomyocytes from CaMK/SR-AIP mice, SR Ca²⁺ leak, assessed both as diastolic Ca²⁺ shift into SR upon tetracaine in intact myocytes or integrated Ca²⁺ spark release in permeabilized myocytes, was significantly reduced. The reduced leak was accompanied by enhanced SR Ca²⁺ load and twitch amplitude in double-transgenic mice (vs. CaMK), without changes in SERCA expression or NCX function. However, despite the improved myocyte Ca²⁺ handling, cardiac hypertrophy and remodeling was accelerated in CaMK/SR-AIP and cardiac function worsened. We conclude that while inhibition of SR localized CaMKII in CaMK mice improves Ca²⁺ handling, it does not necessarily rescue the HF phenotype. This implies that a non-SR CaMKIIδ_C exerts SR-independent effects that contribute to hypertrophy and HF, and this CaMKII pathway may be exacerbated by the global enhancement of Ca transients.     

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.     

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).     

Leaky RyR2 trigger ventricular arrhythmias in Duchenne muscular dystrophy

Patients with Duchenne muscular dystrophy (DMD) have a progressive dilated cardiomyopathy associated with fatal cardiac arrhythmias. Electrical and functional abnormalities have been attributed to cardiac fibrosis; however, electrical abnormalities may occur in the absence of overt cardiac histopathology. Here we show that structural and functional remodeling of the cardiac sarcoplasmic reticulum (SR) Ca²⁺ release channel/ryanodine receptor (RyR2) occurs in the mdx mouse model of DMD. RyR2 from mdx hearts were S-nitrosylated and depleted of calstabin2 (FKBP12.6), resulting in “leaky” RyR2 channels and a diastolic SR Ca²⁺ leak. Inhibiting the depletion of calstabin2 from the RyR2 complex with the Ca²⁺ channel stabilizer S107 (“rycal”) inhibited the SR Ca²⁺ leak, inhibited aberrant depolarization in isolated cardiomyocytes, and prevented arrhythmias in vivo. This suggests that diastolic SR Ca²⁺ leak via RyR2 due to S-nitrosylation of the channel and calstabin2 depletion from the channel complex likely triggers cardiac arrhythmias. Normalization of the RyR2-mediated diastolic SR Ca²⁺ leak prevents fatal sudden cardiac arrhythmias in DMD.     

Termination of calcium-induced calcium release by induction decay: An emergent property of stochastic channel gating and molecular scale architecture

Calcium-induced calcium release (CICR) is an inherently regenerative process due to the Ca^2 +-dependent gating of ryanodine receptors (RyRs) in the sarco/endoplasmic reticulum (SR) and is critical for cardiac excitation–contraction coupling. This process is seen as Ca^2 + sparks, which reflect the concerted gating of groups of RyRs in the dyad, a specialised junctional signalling domain between the SR and surface membrane. However, the mechanism(s) responsible for the termination of regenerative CICR during the evolution of Ca^2 + sparks remain uncertain. Rat cardiac RyR gating was recorded at physiological Ca^2 +, Mg^2 + and ATP levels and incorporated into a 3D model of the cardiac dyad which reproduced the time-course of Ca^2 + sparks, Ca^2 + blinks and Ca^2 + spark restitution. Model CICR termination was robust, relatively insensitive to the number of dyadic RyRs and automatic. This emergent behaviour arose from the rapid development and dissolution of nanoscopic Ca^2 + gradients within the dyad. These simulations show that CICR does not require intrinsic inactivation or SR calcium sensing mechanisms for stability and cessation of regeneration that arises from local control at the molecular scale via a process we call ‘induction decay’.     

A full range of mouse sinoatrial node AP firing rates requires protein kinase A-dependent calcium signaling

Recent perspectives on sinoatrial nodal cell (SANC)^? function indicate that spontaneous sarcoplasmic reticulum (SR) Ca²⁺ cycling, i.e. an intracellular “Ca²⁺ clock,” driven by cAMP-mediated, PKA-dependent phosphorylation, interacts with an ensemble of surface membrane electrogenic molecules (“surface membrane clock”) to drive SANC normal automaticity. The role of AC-cAMP-PKA-Ca²⁺ signaling cascade in mouse, the species most often utilized for genetic manipulations, however, has not been systematically tested. Here we show that Ca²⁺ cycling proteins (e.g. RyR2, NCX1, and SERCA2) are abundantly expressed in mouse SAN and that spontaneous, rhythmic SR generated local Ca²⁺ releases (LCRs) occur in skinned mouse SANC, clamped at constant physiologic [Ca²⁺]. Mouse SANC also exhibits a high basal level of phospholamban (PLB) phosphorylation at the PKA-dependent site, Serine16. Inhibition of intrinsic PKA activity or inhibition of PDE in SANC, respectively: reduces or increases PLB phosphorylation, and markedly prolongs or reduces the LCR period; and markedly reduces or accelerates SAN spontaneous firing rate. Additionally, the increase in AP firing rate by PKA-dependent phosphorylation by β-adrenergic receptor (β-AR) stimulation requires normal intracellular Ca²⁺ cycling, because the β-AR chronotropic effect is markedly blunted when SR Ca²⁺ cycling is disrupted. Thus, AC-cAMP-PKA-Ca²⁺ signaling cascade is a major mechanism of normal automaticity in mouse SANC.     

Flecainide inhibits arrhythmogenic Ca waves by open state block of ryanodine receptor Ca release channels and reduction of Ca spark mass

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is linked to mutations in the cardiac ryanodine receptor (RyR2) or calsequestrin. We recently found that the drug flecainide inhibits RyR2 channels and prevents CPVT in mice and humans. Here we compared the effects of flecainide and tetracaine, a known RyR2 inhibitor ineffective in CPVT myocytes, on arrhythmogenic Ca²⁺ waves and elementary sarcoplasmic reticulum (SR) Ca²⁺ release events, Ca²⁺ sparks. In ventricular myocytes isolated from a CPVT mouse model, flecainide significantly reduced spark amplitude and spark width, resulting in a 40% reduction in spark mass. Surprisingly, flecainide significantly increased spark frequency. As a result, flecainide had no significant effect on spark-mediated SR Ca²⁺ leak or SR Ca²⁺ content. In contrast, tetracaine decreased spark frequency and spark-mediated SR Ca²⁺ leak, resulting in a significantly increased SR Ca²⁺ content. Measurements in permeabilized rat ventricular myocytes confirmed the different effects of flecainide and tetracaine on spark frequency and Ca²⁺ waves. In lipid bilayers, flecainide inhibited RyR2 channels by open state block, whereas tetracaine primarily prolonged RyR2 closed times. The differential effects of flecainide and tetracaine on sparks and RyR2 gating can explain why flecainide, unlike tetracaine, does not change the balance of SR Ca²⁺ fluxes. We suggest that the smaller spark mass contributes to flecainide's antiarrhythmic action by reducing the probability of saltatory wave propagation between adjacent Ca²⁺ release units. Our results indicate that inhibition of the RyR2 open state provides a new therapeutic strategy to prevent diastolic Ca²⁺ waves resulting in triggered arrhythmias, such as CPVT.     

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.     

K201 (JTV-519) alters the spatiotemporal properties of diastolic Ca<Superscript>2+</Superscript> release and the associated diastolic contraction during β-adrenergic stimulation in rat ventricular cardiomyocytes

K201 has previously been shown to reduce diastolic contractions in vivo during β-adrenergic stimulation and elevated extracellular calcium concentration ([Ca²⁺]_o). The present study characterised the effect of K201 on electrically stimulated and spontaneous diastolic sarcoplasmic reticulum (SR)-mediated Ca²⁺ release and contractile events in isolated rat cardiomyocytes during β-adrenergic stimulation and elevated [Ca²⁺]_o. Parallel experiments using confocal microscopy examined spontaneous diastolic Ca²⁺ release events at an enhanced spatiotemporal resolution. 1.0 μmol/L K201 in the presence of 150 nmol/L isoproterenol (ISO) and 4.75 mmol/L [Ca²⁺]_o significantly decreased the amplitude of diastolic contractions to ~16% of control levels. The stimulated free Ca²⁺ transient amplitude was significantly reduced, but stimulated cell shortening was not significantly altered. When intracellular buffering was taken into account, K201 led to an increase in action potential-induced SR Ca²⁺ release. Myofilament sensitivity to Ca²⁺ was not changed by K201. Confocal microscopy revealed diastolic events composed of multiple Ca²⁺ waves (2–3) originating at various points along the cardiomyocyte length during each diastolic period. 1.0 μmol/L K201 significantly reduced the (a) frequency of diastolic events and (b) initiation points/diastolic interval in the remaining diastolic events to 61% and 71% of control levels respectively. 1.0 μmol/L K201 can reduce the probability of spontaneous diastolic Ca²⁺ release and their associated contractions which may limit the propensity for the contractile dysfunction observed in vivo.     

Luminal Ca2+ controls termination and refractory behavior of Ca2+-induced Ca2+ release in cardiac myocytes

Despite extensive research, the mechanisms responsible for the graded nature and early termination of Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) in cardiac muscle remain poorly understood. Suggested mechanisms include cytosolic Ca2+-dependent inactivation/adaptation and luminal Ca2+-dependent deactivation of the SR Ca2+ release channels/ryanodine receptors (RyRs). To explore the importance of cytosolic versus luminal Ca2+ regulatory mechanisms in controlling CICR, we assessed the impact of intra-SR Ca2+ buffering on global and local Ca2+ release properties of patch-clamped or permeabilized rat ventricular myocytes. Exogenous, low-affinity Ca2+ buffers (5 to 20 mmol/L ADA, citrate or maleate) were introduced into the SR by exposing the cells to "internal" solutions containing the buffers. Enhanced Ca2+ buffering in the SR was confirmed by an increase in the total SR Ca2+ content, as revealed by application of caffeine. At the whole-cell level, intra-SR [Ca2+] buffering dramatically increased the magnitude of Ca2+ transients induced by I(Ca) and deranged the smoothly graded I(Ca)-SR Ca2+ release relationship. The amplitude and time-to-peak of local Ca2+ release events, Ca2+ sparks, as well as the duration of local Ca2+ release fluxes underlying sparks were increased up to 2- to 3-fold. The exogenous Ca2+ buffers in the SR also reduced the frequency of repetitive activity observed at individual release sites in the presence of the RyR activator Imperatoxin A. We conclude that regulation of RyR openings by local intra-SR [Ca2+] is responsible for termination of CICR and for the subsequent restitution behavior of Ca2+ release sites in cardiac muscle.     

Dynamic local changes in sarcoplasmic reticulum calcium: Physiological and pathophysiological roles

Evidence obtained in recent years indicates that, in cardiac myocytes, release of Ca²⁺ from the sarcoplasmic reticulum (SR) is regulated by changes in the concentration of Ca²⁺ within the SR. In this review, we summarize recent advances in our understanding of this regulatory role, with a particular emphasis on dynamic and local changes in SR [Ca²⁺]. We focus on five important questions that are to some extent unresolved and controversial. These questions concern: (1) the importance of SR [Ca²⁺] depletion in the termination of Ca²⁺ release; (2) the quantitative extent of depletion during local release events such as Ca²⁺ sparks; (3) the influence of SR [Ca²⁺] refilling on release refractoriness and the propensity for pathological Ca²⁺ release; (4) dynamic changes in SR [Ca²⁺] during propagating Ca²⁺ waves; and (5) the speed of Ca²⁺ diffusion within the SR. With each issue, we discuss data supporting alternative viewpoints, and we identify fundamental questions that are being actively investigated. We conclude with a discussion of experimental and computational advances that will help to resolve controversies. This article is part of a special issue entitled "Local Signaling in Myocytes."     

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.     

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.     

Excitation–contraction coupling in human heart failure examined by action potential clamp in rat cardiac myocytes

The effect of the loss of the notch in the human action potential (AP) during heart failure was examined by voltage clamping rat ventricular myocytes with human APs and recording intracellular Ca²⁺ with fluorescent dyes. Loss of the notch resulted in about a 50% reduction in the initial phase of the Ca²⁺ transient due to reduced ability of the l-type Ca²⁺ channel to trigger release. The failing human AP increased non-uniformity of cytosolic Ca²⁺, with some cellular regions failing to elicit Ca²⁺-induced Ca²⁺ release from the sarcoplasmic reticulum. In addition, there was an increase in the occurrence of late Ca²⁺ sparks. Monte-Carlo simulations of spark activation by l-type Ca²⁺ current supported the idea that the decreased synchrony of Ca²⁺ spark production associated with the loss of the notch could be explained by reduced Ca²⁺ influx from open Ca²⁺ channels. We conclude that the notch of the AP is critical for efficient and synchronous EC coupling and that the loss of the notch will reduce the SR Ca²⁺ release in heart failure, without changes in (for example) SR Ca²⁺-ATPase uptake.     

Interactions between calcium waves and action potential-induced calcium transients in guinea pig myocytes

Summary In isolated cardiac muscle, spontaneous Ca²⁺ release from the sarcoplasmic reticulum (SR) occurs and is propagated as a wave by a regenerative Ca²⁺-induced Ca²⁺ release mechanism. We have already reported that this wave is followed by a refractory period. The aim of this study is to investigate whether such a refractory period could also inactivate Ca²⁺ release from the SR triggered by an action potential. Myocytes were enzymatically isolated from guinea pig ventricles and loaded with acetoxymethylester form of fura-2 (fura-2 AM). The membrane potential was recorded with a conventional microelectrode technique, and spatio-temporal changes in fura-2 fluorescence were recorded using a digital TV system. After perfusion with potassium-free Tyrode solution, interactions between fluorescence transients due to propagating waves and action potential-induced fluorescence transients were observed. In this study, the action potentialinduced fluorescence transients could be detected in the next video frame after the propagation of the waves and showed gradual restitution of the transients. In addition, the sum of the fluorescence transients triggered by an action potential and the fluorescence transients due to the waves did not show significant change whenever the preceding waves were propagating. These results show that the interaction between the action potential-induced Ca²⁺ release and the calcium wave-induced Ca²⁺ release from the SR have the following characteristics: (1) For the action potentialinduced Ca²⁺ release, no absolute refractory period was observed 33 msec after the calcium wave. This suggests that the calcium waves can be reset by the action potential. (2) Regardless of whether the two release mechanisms are different, both share a common compartment of Ca²⁺ storage.