Carvedilol: Molecular and Cellular Basis for Its Multifaceted Therapeutic Potential

Carvedilol is a unique cardiovascular drug of multifaceted therapeutic potential. Its major molecular targets recognized to date are membrane adrenoceptors (β₁,β₂, and α₁), reactive oxygen species, and ion channels (K⁺ and Ca²⁺). Carvedilol provides prominent hemodynamic benefits mainly through a balanced adrenoceptor blockade, which causes a reduction in cardiac work in association with peripheral vasodilation. This drug assures remarkable cardiovascular protection through its antiproliferative/atherogenic, antiischemic, antihypertrophic, and antiarrhythmic actions. These actions are a consequence of its potent antioxidant effects, amelioration of glucose/lipid metabolism, modulation of neurohumoral factors, and modulation of cardiac electrophysiologic properties. The usefulness of carvedilol in the treatment of hypertension, ischemic heart disease, and congestive heart failure is based on a combination of hemodynamic benefits and cardiovascular protection.     

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.     

A Review of HNS‐32: A Novel Azulene‐l‐Carboxamidine Derivative with Multiple Cardiovascular Protective Actions

HNS‐32 [N¹,N¹‐dimethyl‐N²‐(2‐pyridylmethyl)‐5‐isopropyl‐3,8‐dimethylazulene‐1‐carboxamidine] (CAS Registry Number: 186086‐10‐2) is a newly synthesized azulene derivative. Computer simulation showed that its three dimensional structure is similar to that of the class Ib antiarrhythmic drugs, e.g., lidocaine or mexiletine. HNS‐32 potently suppressed ventricular arrhythmias induced by ischemia due to coronary ligation and/or ischemia‐reperfusion in dogs and rats. In the isolated dog and guinea pig cardiac tissues, HNS‐32 had negative inotropic and chronotropic actions, prolonged atrial‐His and His‐ventricular conduction time and increased coronary blood flow. In the isolated guinea pig ventricular papillary muscle, HNS‐32 decreased maximal rate of action potential upstroke (V?_max) and shortened action potential duration (APD). These findings suggest that HNS‐32 inhibits inward Na⁺ and Ca²⁺ channel currents. In the isolated pig coronary and rabbit conduit arteries, HNS‐32 inhibited both Ca²⁺ channel‐dependent and ‐independent contractions induced by a wide variety of chemical stimuli. HNS‐32 is a potent inhibitor of protein kinase C (PKC)‐mediated constriction of cerebral arteries. It is likely to block both, Na⁺ and Ca²⁺ channels expressed in cardiac and vascular smooth muscles. These multiple ion channel blocking effects are largely responsible for the antiarrhythmic and vasorelaxant actions of HNS‐32. This drug may represent a novel approach to the treatment of arrhythmias.     

Intracellular pH regulation in heart

Intracellular pH (pH_i) is an important modulator of cardiac excitation and contraction, and a potent trigger of electrical arrhythmia. This review outlines the intracellular and membrane mechanisms that control pH_i in the cardiac myocyte. We consider the kinetic regulation of sarcolemmal H⁺, OH⁻ and HCO₃⁻ transporters by pH, and by receptor-coupled intracellular signalling systems. We also consider how activity of these pH_i effector proteins is coordinated spatially in the myocardium by intracellular mobile buffer shuttles, gap junctional channels and carbonic anhydrase enzymes. Finally, we review the impact of pH_i regulatory proteins on intracellular Ca²⁺ signalling, and their participation in clinical disorders such as myocardial ischaemia, maladaptive hypertrophy and heart failure. Such multiple effects emphasise the fundamental role that pH_i regulation plays in the heart.     

Skeletal muscle stem cells propagated as myospheres display electrophysiological properties modulated by culture conditions

In cardiac regenerative therapy, transplantation of stem cells to form new myocardium is limited by their inability to integrate into host myocardium and conduct cardiac electrical activity. It is now hypothesized that refining cell sorting could upgrade the therapeutic result. Here we characterized a subpopulation of skeletal muscle stem cells with respect to their electrophysiological properties. The aim of our study was to determine whether electrophysiological parameters are compatible with cardiac function and can be influenced by culture conditions. Low-adherent skeletal muscle stem cells were isolated from the hind legs of 12-20 week old mice. After 6 days of culture the cells were analysed using patch-clamp techniques and RT-PCR, and replated in different media for skeletal muscle or cardiac differentiation. The cells generated action potentials (APs) longer than skeletal muscle APs, expressed functional cardiac Na⁺ channels (~ 46% of the total channel fraction), displayed fast activating and inactivating L-type Ca²⁺ currents, possibly conducted through cardiac channels and did not show significant Cl⁻ conductance. Moreover, a fraction of cells expressed muscarinic acetylcholine receptors. Conditioning the cells for skeletal muscle differentiation resulted in upregulation of skeletal muscle-specific Na⁺ and Ca²⁺ channel expression, shortening of AP duration and loss of functional cardiac Na⁺ channels. Cardiomyogenic conditions however, promoted the participation of cardiac Na⁺ channels (57% of the total channel fraction). Nevertheless the cells retained properties of myoblasts such as the expression of nicotinic acetylcholine receptors. We conclude that skeletal muscle stem cells display several electrophysiological properties similar to those of cardiomyocytes. Culture conditions modulated these properties but only partially succeeded in further driving the cells towards a cardiac phenotype. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".     

Modulation of sarcoplasmic reticulum Ca<Superscript>2+</Superscript> cycling in systolic and diastolic heart failure associated with aging

Hypertension, atherosclerosis, and resultant chronic heart failure (HF) reach epidemic proportions among older persons, and the clinical manifestations and the prognoses of these worsen with increasing age. Thus, age per se is the major risk factor for cardiovascular disease. Changes in cardiac cell phenotype that occur with normal aging, as well as in HF associated with aging, include deficits in ß-adrenergic receptor (ß-AR) signaling, increased generation of reactive oxygen species (ROS), and altered excitation–contraction (EC) coupling that involves prolongation of the action potential (AP), intracellular Ca²⁺ (Ca _i ²⁺ ) transient and contraction, and blunted force- and relaxation-frequency responses. Evidence suggests that altered sarcoplasmic reticulum (SR) Ca²⁺ uptake, storage, and release play central role in these changes, which also involve sarcolemmal L-type Ca²⁺ channel (LCC), Na⁺–Ca²⁺ exchanger (NCX), and K⁺ channels. We review the age-associated changes in the expression and function of Ca²⁺ transporting proteins, and functional consequences of these changes at the cardiac myocyte and organ levels. We also review sexual dimorphism and self-renewal of the heart in the context of cardiac aging and HF.     

Characterization of ionic currents and electrophysiological properties of goldfish somatotropes in primary culture

Growth hormone release in goldfish is partly dependent on voltage-sensitive Ca²⁺ channels but somatotrope electrophysiological events affecting such channel activities have not been elucidated in this system. The electrophysiological properties of goldfish somatotropes in primary culture were studied using the whole-cell and amphotericin B-perforated patch-clamp techniques. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) of identified somatotropes was measured using Fura-2/AM dye. Goldfish somatotropes had an average resting membrane potential of −78.4 ± 4.6 mV and membrane input resistance of 6.2 ± 0.2 GΩ. Voltage steps from a holding potential of −90 mV elicited a non-inactivating outward current and transient inward currents at potentials more positive than 0 and −30 mV, respectively. Isolated current recordings indicate the presence of 4-aminopyridine- and tetraethylammonium (TEA)-sensitive K⁺, tetrodotoxin (TTX)-sensitive Na⁺, and nifedipine (L-type)- and ω-conotoxin GVIA (N-type)-sensitive Ca²⁺ channels. Goldfish somatotropes rarely fire action potentials (APs) spontaneously, but single APs can be induced at the start of a depolarizing current step; this single AP was abolished by TTX and significantly reduced by nifedipine and ω-conotoxin GVIA. TEA increased AP duration and triggered repetitive AP firing resulting in an increase in [Ca²⁺]_i, whereas TTX, nifedipine and ω-conotoxin GVIA inhibited TEA-induced [Ca²⁺]_i pulses. These results indicate that in goldfish somatotropes, TEA-sensitive K⁺ channels regulate excitability while TTX-sensitive Na⁺ channels together with N- and L-type Ca channels mediates the depolarization phase of APs. Opening of voltage-sensitive Ca²⁺ channels during AP firing leads to increases in [Ca²⁺]_i.     

Trimebutine maleate has inhibitory effects on the voltage-dependent Ca2+ inward current and other membrane currents in intestinal smooth muscle cells

We examined effects of trimebutine maleate on the membrane currents of the intestinal smooth muscle cells by using the tight-seal whole cell clamp technique. Trimebutine suppressed the Ba²⁺ inward current through voltage-dependent Ca²⁺ channels in a dose-dependent manner. The inhibitory effect of trimebutine on the Ba²⁺ inward current was not use-dependent. It shifted the steady-state inactivation curve to the left along the voltage axis. Trimebutine also had inhibitory effects on the other membrane currents of the cells, such as the voltage-dependent K⁺ current, the Ca²⁺-activated oscillating K⁺ current and the acetylcholine-induced inward current. These relatively non-specific inhibitory effects of trimebutine on the membrane currents may explain, at least in part, the dual actions of the drug on the intestinal smooth muscle contractility, i.e. inhibitory as well as excitatory.     

Association of beta-catenin with the alpha-subunit of neuronal large-conductance Ca2+-activated K+ channels

The association of Ca²⁺-activated K⁺ channels with voltage-gated Ca²⁺ channels at the presynaptic active zones of hair cells, photoreceptors, and neurons contributes to rapid repolarization of the membrane after excitation. Ca²⁺ channels have been shown to bind to a large set of synaptic proteins, but the proteins interacting with Ca²⁺-activated K⁺ channels remain unknown. Here, we report that the large-conductance Ca²⁺-activated K⁺ channel of the chicken's cochlear hair cell interacts with β-catenin. Yeast two-hybrid assays identified the S10 region of the K⁺ channel's α-subunit and the ninth armadillo repeat and carboxyl terminus of β-catenin as necessary for the interaction. An antiserum directed against the α-subunit specifically coprecipitated β-catenin from brain synaptic proteins. β-Catenin is known to associate with the synaptic protein Lin7/Velis/MALS, whose interaction partner Lin2/CASK also binds voltage-gated Ca²⁺ channels. β-Catenin may therefore provide a physical link between the two types of channels at the presynaptic active zone.     

Internal pH,Na+, and Ca2+ regulation by trimetazidine during cardiac cell acidosis

Summary The effects of trimetazidine were studied on plasma membrane structures of cardiac cells which control excitability, as well as on cardiac cells that were cultured in normal physiologic conditions and after intracellular acidification.When cardiac cells were kept in normal physiologic conditions, trimetazidine at concentrations ranging from 10^–8 to 3.10^–4 M interacted neither directly nor indirectly with the major ionic transporter systems of cardiac cells, such as ionic channels (Na⁺, K⁺), ATPase, Na⁺/H⁺, and Na⁺/Ca²⁺ exchange systems.Under acid-load conditions trimetazide acts in a dose- and time-dependent manner, in limiting the accumulation of Na⁺ and Ca²⁺ inside cardiac cells and depressing intracellular cell acidosis.It is proposed that trimetazidine plays a key role in limiting the intracellular accumulation of protons that is responsible for cell acidosis during ischemia.Trimetazidine, in protecting cardiac cells against accumulation of protons, limits accumulation of Na⁺ and Ca²⁺.     

Control of cardiac excitability and arrhythmias by microRNAs

Recent studies revealing the important roles of microRNAs（miRNAs） in regulating expression of ion channel genes have opened up a research field for extending and deepening our investi- gation into the cardiac excitability and the associated arrhythmogenesis.Cardiac excitability,the fundamental property of the cardiac myocytes,defines the cardiac conduction,repolarization,automaticity,intracellular calcium handling,and their regional heterogeneity. Our previous and ongoing studies and the work from other laboratories have demonstrated the significant involvement of miRNAs in regulating every aspects of cardiac excitability.We have found earlier that the muscle-specific miRNA miR-1 boosts up the arrhythmogenic potential through targeting gap junction channel connexin 43 in myocardial infarction.A subsequent study revealed that miR-1 can also cause arrhythmias by impairing Ca²⁺ handling by targeting phosphatase.We then identified another muscle-specific miRNA miR-133 promotes abnormal QT prolongation by repressing HERG K⁺ channel expression in diabetic cardiomyopathy. Subsequently,we discovered that both miR- 1 and miR-133 are involved in the reexpression of pacemaker channels HCN2/HCN4 to enhance abnormal automaticity in cardiac hypertrophy.Recently, we further identified miR-328 as an important determinant for atrial fibrillation（AF） and the associated adverse atrial electrical remodeling via targeting L-type Ca²⁺ channels.While all the above-mentioned miRNAs are proarrhythmic,we have newly identified for the first time a natural antiarrhythmic miRNA miR-26.We found that all three members of the miR-26 family is downregulated in their expression in AF tissues and this downregulation increases AF vulnerability as a result of removal of an endogenous antiarrhythmic factor.miR-26 downregulation shortens atrial action potential favoring AF by increasing inward rectifier K⁺ current（IK1） density. This is caused by an upregulation of Kir2.1 K⁺ channel su     

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.     

The short-long-short-short sequence and polymorphic ventricular tachycardias storm

We described a case of a patient who developed repetitive episodes of polymorphic ventricular tachycardias with a stereotypical pattern of initiation. A short-long-short-short (S-L-S-S) cardiac cycle sequence preceded all episodes and was considered to be the underlying initiative mechanism for these fatal arrhythmic events. In the patient, the paroxysmal atrial fibrillation was responsible for S-L-S-S sequence. It had been suggested that the electrophysiological mechanism by which the S-L-S-S cardiac sequence induces ventricular tachyarrhythmias was reentrant excitation, not early afterdepolarization and triggered activity. Early attempts to restore and maintain sinus rhythm by administration of antiarrhythmic drug with amiodarone, the patient experienced no atrial fibrillation and ventricular tachycardia recurrence.     

Crystallographic basis for calcium regulation of sodium channels

Voltage-gated sodium channels underlie the rapid regenerative upstroke of action potentials and are modulated by cytoplasmic calcium ions through a poorly understood mechanism. We describe the 1.35 Å crystal structure of Ca²⁺-bound calmodulin (Ca²⁺/CaM) in complex with the inactivation gate (DIII-IV linker) of the cardiac sodium channel (Na_V1.5). The complex harbors the positions of five disease mutations involved with long Q-T type 3 and Brugada syndromes. In conjunction with isothermal titration calorimetry, we identify unique inactivation-gate mutations that enhance or diminish Ca²⁺/CaM binding, which, in turn, sensitize or abolish Ca²⁺ regulation of full-length channels in electrophysiological experiments. Additional biochemical experiments support a model whereby a single Ca²⁺/CaM bridges the C-terminal IQ motif to the DIII-IV linker via individual N and C lobes, respectively. The data suggest that Ca²⁺/CaM destabilizes binding of the inactivation gate to its receptor, thus biasing inactivation toward more depolarized potentials.     

Role of mitochondrial dysfunction in cardiac glycoside toxicity

Cardiac glycosides, which inhibit the plasma membrane Na⁺ pump, are one of the four categories of drug recommended for routine use to treat heart failure, yet their therapeutic window is limited by toxic effects. Elevated cytoplasmic Na⁺ ([Na⁺]_i) compromises mitochondrial energetics and redox balance by blunting mitochondrial Ca²⁺ ([Ca²⁺]_m) accumulation, and this impairment can be prevented by enhancing [Ca²⁺]_m. Here, we investigate whether this effect underlies the toxicity and arrhythmogenic effects of cardiac glycosides and if these effects can be prevented by suppressing mitochondrial Ca²⁺ efflux, via inhibition of the mitochondrial Na⁺/Ca²⁺ exchanger (mNCE). In isolated cardiomyocytes, ouabain elevated [Na⁺]_i in a dose-dependent way, blunted [Ca²⁺]_m accumulation, decreased the NADH/NAD + redox potential, and increased reactive oxygen species (ROS). Concomitant treatment with the mNCE inhibitor CGP-37157 ameliorated these effects. CGP-37157 also attenuated ouabain-induced cellular Ca²⁺ overload and prevented delayed afterdepolarizations (DADs). In isolated perfused hearts, ouabain's positive effects on contractility and respiration were markedly potentiated by CGP-37157, as were those mediated by β-adrenergic stimulation. Furthermore, CGP-37157 inhibited the arrhythmogenic effects of ouabain in both isolated perfused hearts and in vivo. The findings reveal the mechanism behind cardiac glycoside toxicity and show that improving mitochondrial Ca²⁺ retention by mNCE inhibition can mitigate these effects, particularly with respect to the suppression of Ca²⁺-triggered arrhythmias, while enhancing positive inotropic actions. These results suggest a novel strategy for the treatment of heart failure.     

Future of antiarrhythmic drugs

PURPOSE OF REVIEW: This article briefly summarizes the principal mechanisms of action of contemporary antiarrhythmic agents, delineates their limitations in the treatment of cardiac arrhythmias, and discusses why there is a need for new cardiac antiarrhythmic drugs. RECENT FINDINGS: In recent years, the limited efficacy and proarrhythmic potential of classic antiarrhythmic drugs have focused attention on nonpharmacologic approaches to treatment of cardiac arrhythmias. Despite the current success of ablative therapy and implantable defibrillators, the need is still pressing for new antiarrhythmic drugs. Evolving knowledge about the molecular mechanisms of cardiac arrhythmias provides innovative strategies for discovering new cardiac antiarrhythmic drugs. Some of these have already led to the development of new compounds on the verge of clinical use, and others hold great promise for future drug development. SUMMARY: Cardiac arrhythmias are associated with significant morbidity and mortality in developed countries. Antiarrhythmic drug therapy was traditionally the mainstay of arrhythmia treatment; however, the inefficacy of drug treatment and the potential that antiarrhythmic drugs can provoke life-threatening arrhythmias have generated interest in new approaches to antiarrhythmic drug development. Improved understanding of the cellular and molecular basis of cardiac arrhythmias holds the promise of identifying novel approaches for the treatment of cardiac arrhythmias. These approaches may target traditional and newly discovered cardiac ion channels, as well as new molecular and signaling pathways that modulate arrhythmic substrates.     

Calexcitin: A signaling protein that binds calcium and GTP, inhibits potassium channels, and enhances membrane excitability

A previously uncharacterized 22-kDa Ca²⁺-binding protein that also binds guanosine nucleotides was characterized, cloned, and analyzed by electrophysiological techniques. The cloned protein, calexcitin, contains two EF-hands and also has homology with GTP-binding proteins in the ADP ribosylation factor family. In addition to binding two molecules of Ca²⁺, calexcitin bound GTP and possessed GTPase activity. Calexcitin is also a high affinity substrate for protein kinase C. Application of calexcitin to the inner surface of inside-out patches of human fibroblast membranes, in the presence of Ca²⁺ and the absence of endogenous Ca²⁺/calmodulin kinase type II or protein kinase C activity, reduced the mean open time and mean open probability of 115 ± 6 pS K⁺ channels. Calexcitin thus appears to directly regulate K⁺ channels. When microinjected into molluscan neurons or rabbit cerebellar Purkinje cell dendrites, calexcitin was highly effective in enhancing membrane excitability. Because calexcitin translocates to the cell membrane after phosphorylation, calexcitin could serve as a Ca²⁺-activated signaling molecule that increases cellular excitability, which would in turn increase Ca²⁺ influx through the membrane. This is also the first known instance of a GTP-binding protein that binds Ca²⁺.     

Functional interaction with filamin A and intracellular Ca2+ enhance the surface membrane expression of a small-conductance Ca2+-activated K+ (SK2) channel

For an excitable cell to function properly, a precise number of ion channel proteins need to be trafficked to distinct locations on the cell surface membrane, through a network and anchoring activity of cytoskeletal proteins. Not surprisingly, mutations in anchoring proteins have profound effects on membrane excitability. Ca²⁺-activated K⁺ channels (K_Ca2 or SK) have been shown to play critical roles in shaping the cardiac atrial action potential profile. Here, we demonstrate that filamin A, a cytoskeletal protein, augments the trafficking of SK2 channels in cardiac myocytes. The trafficking of SK2 channel is Ca²⁺-dependent. Further, the Ca²⁺ dependence relies on another channel-interacting protein, α-actinin2, revealing a tight, yet intriguing, assembly of cytoskeletal proteins that orchestrate membrane expression of SK2 channels in cardiac myocytes. We assert that changes in SK channel trafficking would significantly alter atrial action potential and consequently atrial excitability. Identification of therapeutic targets to manipulate the subcellular localization of SK channels is likely to be clinically efficacious. The findings here may transcend the area of SK2 channel studies and may have implications not only in cardiac myocytes but in other types of excitable cells.Small-conductance Ca²⁺-activated K⁺ (SK or K_Ca2) channels are highly unique in that they are gated solely by changes in intracellular Ca²⁺ (Ca²⁺_i) concentration. Hence, the channels function to integrate changes in Ca²⁺ concentration with changes in membrane potentials. SK channels have been shown to be expressed in a wide variety of cells (1–3) and mediate afterhyperpolarizations following action potentials in neurons (1, 4, 5). We have previously documented the expression of several isoforms of SK channels in human and mouse atrial myocytes that mediate the repolarization phase of the atrial action potentials (6, 7). We further demonstrated that SK2 (K_Ca2.2) channel knockout mice are prone to the development of atrial arrhythmias and atrial fibrillation (AF) (8). Conversely, a recent study by Diness et al. suggests that inhibition of SK channels may prevent AF (9). Together, these studies underpin the importance of the precise control for the expression of these ion channels in atria and their potential to serve as a future therapeutic target for AF.Current antiarrhythmic agents target the permeation and gating properties of ion channel proteins; however, increasing evidence suggests that membrane localization of ion channels may also be pharmacologically altered (10). Furthermore, a number of disorders have been associated with mistrafficking of ion channel proteins (11, 12). We have previously demonstrated the critical role of α-actinin2, a cytoskeletal protein, in the surface membrane localization of cardiac SK2 channels (13, 14). Specifically, we demonstrated that cardiac SK2 channel interacts with α-actinin2 cytoskeletal protein via the EF hand motifs in α-actinin2 protein and the helical core region of the calmodulin (CaM) binding domain (CaMBD) in the C terminus of SK2 channel. Moreover, direct interactions between SK2 channel and α-actinin2 are required for the increase in cell surface localization of SK2 channel.Here, to further define the functional interactome of SK2 channel in the heart, we demonstrate the role of filamin A (FLNA), another cytoskeletal protein, in SK2 channel surface membrane localization. In contrast to α-actinin2 protein, FLNA interacts not with the C terminus, but with the N terminus of the cardiac SK2 channel. FLNA is a scaffolding cytoskeletal protein with two calponin homology domains that has been shown to be critical for the trafficking of a number of membrane proteins (15–19). Our data demonstrate that FLNA functions to enhance membrane localization of SK2 channels. Moreover, using live-cell imaging, we demonstrate the critical roles of Ca²⁺_i on the membrane localization of SK2 channel when the channels are coexpressed with α-actinin2, but not FLNA. A decrease in Ca²⁺_i results in a significant decrease in SK2 channel membrane localization. Our findings may have important clinical implications. A rise in Ca²⁺_i—for example, during rapid pacing or atrial tachyarrhythmias—is predicted to increase the membrane localization of SK2 channel and result in the abbreviation of the atrial action potentials and maintenance of the arrhythmias.     

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’.