Atomistic mechanisms of the regulation of small-conductance Ca2+-activated K+ channel (SK2) by PIP2.

Small-conductance Ca2+-activated K+ channels (SK, KCa2) are gated solely by intracellular microdomain Ca2+. The channel has emerged as a therapeutic target for cardiac arrhythmias. Calmodulin (CaM) interacts with the CaM binding domain (CaMBD) of the SK channels, serving as the obligatory Ca2+ sensor to gate the channels. In heterologous expression systems, phosphatidylinositol 4,5-bisphosphate (PIP2) coordinates with CaM in regulating SK channels. However, the roles and mechanisms of PIP2 in regulating SK channels in cardiomyocytes remain unknown. Here, optogenetics, magnetic nanoparticles, combined with Rosetta structural modeling, and molecular dynamics (MD) simulations revealed the atomistic mechanisms of how PIP2 works in concert with Ca2+-CaM in the SK channel activation. Our computational study affords evidence for the critical role of the amino acid residue R395 in the S6 transmembrane segment, which is localized in propinquity to the intracellular hydrophobic gate. This residue forms a salt bridge with residue E398 in the S6 transmembrane segment from the adjacent subunit. Both R395 and E398 are conserved in all known isoforms of SK channels. Our findings suggest that the binding of PIP2 to R395 residue disrupts the R395:E398 salt bridge, increasing the flexibility of the transmembrane segment S6 and the activation of the channel. Importantly, our findings serve as a platform for testing of structural-based drug designs for therapeutic inhibitors and activators of the SK channel family. The study is timely since inhibitors of SK channels are currently in clinical trials to treat atrial arrhythmias.

Mechanoelectric coupling and arrhythmogenesis in cardiomyocytes contracting under mechanical afterload in a 3D viscoelastic hydrogel.

The heart pumps blood against the mechanical afterload from arterial resistance, and increased afterload may alter cardiac electrophysiology and contribute to life-threatening arrhythmias. However, the cellular and molecular mechanisms underlying mechanoelectric coupling in cardiomyocytes remain unclear. We developed an innovative patch-clamp-in-gel technology to embed cardiomyocytes in a three-dimensional (3D) viscoelastic hydrogel that imposes an afterload during regular myocyte contraction. Here, we investigated how afterload affects action potentials, ionic currents, intracellular Ca2+ transients, and cell contraction of adult rabbit ventricular cardiomyocytes. We found that afterload prolonged action potential duration (APD), increased transient outward K+ current, decreased inward rectifier K+ current, and increased L-type Ca2+ current. Increased Ca2+ entry caused enhanced Ca2+ transients and contractility. Moreover, elevated afterload led to discordant alternans in APD and Ca2+ transient. Ca2+ alternans persisted under action potential clamp, indicating that the alternans was Ca2+ dependent. Furthermore, all these afterload effects were significantly attenuated by inhibiting nitric oxide synthase 1 (NOS1). Taken together, our data reveal a mechano-chemo-electrotransduction (MCET) mechanism that acutely transduces afterload through NOS1-nitric oxide signaling to modulate the action potential, Ca2+ transient, and contractility. The MCET pathway provides a feedback loop in excitation-Ca2+ signaling-contraction coupling, enabling autoregulation of contractility in cardiomyocytes in response to afterload. This MCET mechanism is integral to the individual cardiomyocyte (and thus the heart) to intrinsically enhance its contractility in response to the load against which it has to do work. While this MCET is largely compensatory for physiological load changes, it may also increase susceptibility to arrhythmias under excessive pathological loading.

Nitrosylation of cardiac CaMKII at Cys290 mediates mechanical afterload-induced increases in Ca2+ transient and Ca2+ sparks.

Cardiac mechanical afterload induces an intrinsic autoregulatory increase in myocyte Ca2+ dynamics and contractility to enhance contraction (known as the Anrep effect or slow force response). Our prior work has implicated both nitric oxide (NO) produced by NO synthase 1 (NOS1) and calcium/calmodulin-dependent protein kinase II (CaMKII) activity as required mediators of this form of mechano-chemo-transduction. To test whether a single S-nitrosylation site on CaMKIIδ (Cys290) mediates enhanced sarcoplasmic reticulum Ca2+ leak and afterload-induced increases in sarcoplasmic reticulum (SR) Ca2+ uptake and release, we created a novel CRISPR-based CaMKIIδ knock-in (KI) mouse with a Cys to Ala mutation at C290. These CaMKIIδ-C290A-KI mice exhibited normal cardiac morphometry and function, as well as basal myocyte Ca2+ transients (CaTs) and ß-adrenergic responses. However, the NO donor S-nitrosoglutathione caused an acute increased Ca2+ spark frequency in wild-type (WT) myocytes that was absent in the CaMKIIδ-C290A-KI myocytes. Using our cell-in-gel system to exert multiaxial three-dimensional mechanical afterload on myocytes during contraction, we found that WT myocytes exhibited an afterload-induced increase in Ca2+ sparks and Ca2+ transient amplitude and rate of decline. These afterload-induced effects were prevented in both cardiac-specific CaMKIIδ knockout and point mutant CaMKIIδ-C290A-KI myocytes. We conclude that CaMKIIδ activation by S-nitrosylation at the C290 site is essential in mediating the intrinsic afterload-induced enhancement of myocyte SR Ca2+ uptake, release and Ca2+ transient amplitude (the Anrep effect). The data also indicate that NOS1 activation is upstream of S-nitrosylation at C290 of CaMKII, and that this molecular mechano-chemo-transduction pathway is beneficial in allowing the heart to increase contractility to limit the reduction in stroke volume when aortic pressure (afterload) is elevated. KEY POINTS: A novel CRISPR-based CaMKIIδ knock-in mouse was created in which kinase activation by S-nitrosylation at Cys290 (C290A) is prevented. How afterload affects Ca2+ signalling was measured in cardiac myocytes that were embedded in a hydrogel that imposes a three-dimensional afterload. This mechanical afterload induced an increase in Ca2+ transient amplitude and decay in wild-type myocytes, but not in cardiac-specific CaMKIIδ knockout or C290A knock-in myocytes. The CaMKIIδ-C290 S-nitrosylation site is essential for the afterload-induced enhancement of Ca2+ transient amplitude and Ca2+ sparks.

Complex electrophysiological remodeling in postinfarction ischemic heart failure.

Heart failure (HF) following myocardial infarction (MI) is associated with high incidence of cardiac arrhythmias. Development of therapeutic strategy requires detailed understanding of electrophysiological remodeling. However, changes of ionic currents in ischemic HF remain incompletely understood, especially in translational large-animal models. Here, we systematically measure the major ionic currents in ventricular myocytes from the infarct border and remote zones in a porcine model of post-MI HF. We recorded eight ionic currents during the cell's action potential (AP) under physiologically relevant conditions using selfAP-clamp sequential dissection. Compared with healthy controls, HF-remote zone myocytes exhibited increased late Na+ current, Ca2+-activated K+ current, Ca2+-activated Cl- current, decreased rapid delayed rectifier K+ current, and altered Na+/Ca2+ exchange current profile. In HF-border zone myocytes, the above changes also occurred but with additional decrease of L-type Ca2+ current, decrease of inward rectifier K+ current, and Ca2+ release-dependent delayed after-depolarizations. Our data reveal that the changes in any individual current are relatively small, but the integrated impacts shift the balance between the inward and outward currents to shorten AP in the border zone but prolong AP in the remote zone. This differential remodeling in post-MI HF increases the inhomogeneity of AP repolarization, which may enhance the arrhythmogenic substrate. Our comprehensive findings provide a mechanistic framework for understanding why single-channel blockers may fail to suppress arrhythmias, and highlight the need to consider the rich tableau and integration of many ionic currents in designing therapeutic strategies for treating arrhythmias in HF.

A viscoelastic Eshelby inclusion model and analysis of the Cell-in-Gel system.

We develop a viscoelastic generalization of the elastic Eshelby inclusion solution, where the inclusion and surrounding matrix are two different viscoelastic solids and the inclusion's eigenstrain is a time-periodic oscillatory input. The solution exploits the Correspondence Principle of Linear Viscoelasticity and a Discrete Fourier Transform to efficiently capture the steady-state oscillatory behavior of the 3-D mechanical fields. The approach is illustrated here in the context of the recently-developed in vitro Cell-in-Gel system, where an isolated live cardiomyocyte (the inclusion) is paced to contract periodically within a soft hydrogel (the matrix), for the purpose of studying the effect of mechanical load on biochemical signals that regulate contractility. The addition of viscoelasticity improves the fidelity of our previous elastic Eshelby inclusion analysis of the Cell-in-Gel system by accounting for the time-varying fields and the resulting hysteresis and dissipated mechanical energy. This mathematical model is used to study the parametric sensitivities of the relative stiffness of the inclusion, the inclusion's aspect ratio (slenderness), and the cross-link density of the hydrogel matrix.

Mechano-electric and mechano-chemo-transduction in cardiomyocytes.

Cardiac excitation-contraction (E-C) coupling is influenced by (at least) three dynamic systems that couple and feedback to one another (see Abstract Figure). Here we review the mechanical effects on cardiomyocytes that include mechano-electro-transduction (commonly referred to as mechano-electric coupling, MEC) and mechano-chemo-transduction (MCT) mechanisms at cell and molecular levels which couple to Ca2+ -electro and E-C coupling reviewed elsewhere. These feedback loops from muscle contraction and mechano-transduction to the Ca2+ homeodynamics and to the electrical excitation are essential for understanding the E-C coupling dynamic system and arrhythmogenesis in mechanically loaded hearts. This white paper comprises two parts, each reflecting key aspects from the 2018 UC Davis symposium: MEC (how mechanical load influences electrical dynamics) and MCT (how mechanical load alters cell signalling and Ca2+ dynamics). Of course, such separation is artificial since Ca2+ dynamics profoundly affect ion channels and electrogenic transporters and vice versa. In time, these dynamic systems and their interactions must become fully integrated, and that should be a goal for a comprehensive understanding of how mechanical load influences cell signalling, Ca2+ homeodynamics and electrical dynamics. In this white paper we emphasize current understanding, consensus, controversies and the pressing issues for future investigations. Space constraints make it impossible to cover all relevant articles in the field, so we will focus on the topics discussed at the symposium.

Altered K+ current profiles underlie cardiac action potential shortening in hyperkalemia and ß-adrenergic stimulation.

Hyperkalemia is known to develop in various conditions including vigorous physical exercise. In the heart, hyperkalemia is associated with action potential (AP) shortening that was attributed to altered gating of K+ channels. However, it remains unknown how hyperkalemia changes the profiles of each K+ current under a cardiac AP. Therefore, we recorded the major K+ currents (inward rectifier K+ current, IK1; rapid and slow delayed rectifier K+ currents, IKr and IKs, respectively) using AP-clamp in rabbit ventricular myocytes. As K+ may accumulate at rapid heart rates during sympathetic stimulation, we also examined the effect of isoproterenol on these K+ currents. We found that IK1 was significantly increased in hyperkalemia, whereas the reduction of driving force for K+ efflux dominated over the altered channel gating in case of IKr and IKs. Overall, the markedly increased IK1 in hyperkalemia overcame the relative decreases of IKr and IKs during AP, resulting in an increased net repolarizing current during AP phase 3. ß-Adrenergic stimulation of IKs also provided further repolarizing power during sympathetic activation, although hyperkalemia limited IKs upregulation. These results indicate that facilitation of IK1 in hyperkalemia and ß-adrenergic stimulation of IKs represent important compensatory mechanisms against AP prolongation and arrhythmia susceptibility.

ß-adrenergic regulation of late Na+ current during cardiac action potential is mediated by both PKA and CaMKII.

Late Na+ current (INaL) significantly contributes to shaping cardiac action potentials (APs) and increased INaL is associated with cardiac arrhythmias. ß-adrenergic receptor (ßAR) stimulation and its downstream signaling via protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) pathways are known to regulate INaL. However, it remains unclear how each of these pathways regulates INaL during the AP under physiological conditions. Here we performed AP-clamp experiments in rabbit ventricular myocytes to delineate the impact of each signaling pathway on INaL at different AP phases to understand the arrhythmogenic potential. During the physiological AP (2 Hz, 37 °C) we found that INaL had a basal level current independent of PKA, but partially dependent on CaMKII. ßAR activation (10 nM isoproterenol, ISO) further enhanced INaL via both PKA and CaMKII pathways. However, PKA predominantly increased INaL early during the AP plateau, whereas CaMKII mainly increased INaL later in the plateau and during rapid repolarization. We also tested the role of key signaling pathways through exchange protein activated by cAMP (Epac), nitric oxide synthase (NOS) and reactive oxygen species (ROS). Direct Epac stimulation enhanced INaL similar to the ßAR-induced CaMKII effect, while NOS inhibition prevented the ßAR-induced CaMKII-dependent INaL enhancement. ROS generated by NADPH oxidase 2 (NOX2) also contributed to the ISO-induced INaL activation early in the AP. Taken together, our data reveal differential modulations of INaL by PKA and CaMKII signaling pathways at different AP phases. This nuanced and comprehensive view on the changes in INaL during AP deepens our understanding of the important role of INaL in reshaping the cardiac AP and arrhythmogenic potential under elevated sympathetic stimulation, which is relevant for designing therapeutic treatment of arrhythmias under pathological conditions.

Multimodal SHG-2PF Imaging of Microdomain Ca2+-Contraction Coupling in Live Cardiac Myocytes.

RATIONALE: Cardiac myocyte contraction is caused by Ca(2+) binding to troponin C, which triggers the cross-bridge power stroke and myofilament sliding in sarcomeres. Synchronized Ca(2+) release causes whole cell contraction and is readily observable with current microscopy techniques. However, it is unknown whether localized Ca(2+) release, such as Ca(2+) sparks and waves, can cause local sarcomere contraction. Contemporary imaging methods fall short of measuring microdomain Ca(2+)-contraction coupling in live cardiac myocytes. OBJECTIVE: To develop a method for imaging sarcomere level Ca(2+)-contraction coupling in healthy and disease model cardiac myocytes. METHODS AND RESULTS: Freshly isolated cardiac myocytes were loaded with the Ca(2+)-indicator fluo-4. A confocal microscope equipped with a femtosecond-pulsed near-infrared laser was used to simultaneously excite second harmonic generation from A-bands of myofibrils and 2-photon fluorescence from fluo-4. Ca(2+) signals and sarcomere strain correlated in space and time with short delays. Furthermore, Ca(2+) sparks and waves caused contractions in subcellular microdomains, revealing a previously underappreciated role for these events in generating subcellular strain during diastole. Ca(2+) activity and sarcomere strain were also imaged in paced cardiac myocytes under mechanical load, revealing spontaneous Ca(2+) waves and correlated local contraction in pressure-overload-induced cardiomyopathy. CONCLUSIONS: Multimodal second harmonic generation 2-photon fluorescence microscopy enables the simultaneous observation of Ca(2+) release and mechanical strain at the subsarcomere level in living cardiac myocytes. The method benefits from the label-free nature of second harmonic generation, which allows A-bands to be imaged independently of T-tubule morphology and simultaneously with Ca(2+) indicators. Second harmonic generation 2-photon fluorescence imaging is widely applicable to the study of Ca(2+)-contraction coupling and mechanochemotransduction in both health and disease.

Ca2+-activated Cl- current is antiarrhythmic by reducing both spatial and temporal heterogeneity of cardiac repolarization.

The role of Ca2+-activated Cl- current (ICl(Ca)) in cardiac arrhythmias is still controversial. It can generate delayed afterdepolarizations in Ca2+-overloaded cells while in other studies incidence of early afterdepolarization (EAD) was reduced by ICl(Ca). Therefore our goal was to examine the role of ICl(Ca) in spatial and temporal heterogeneity of cardiac repolarization and EAD formation. Experiments were performed on isolated canine cardiomyocytes originating from various regions of the left ventricle; subepicardial, midmyocardial and subendocardial cells, as well as apical and basal cells of the midmyocardium. ICl(Ca) was blocked by 0.5mmol/L 9-anthracene carboxylic acid (9-AC). Action potential (AP) changes were tested with sharp microelectrode recording. Whole-cell 9-AC-sensitive current was measured with either square pulse voltage-clamp or AP voltage-clamp (APVC). Protein expression of TMEM16A and Bestrophin-3, ion channel proteins mediating ICl(Ca), was detected by Western blot. 9-AC reduced phase-1 repolarization in every tested cell. 9-AC also increased AP duration in a reverse rate-dependent manner in all cell types except for subepicardial cells. Neither ICl(Ca) density recorded with square pulses nor the normalized expressions of TMEM16A and Bestrophin-3 proteins differed significantly among the examined groups of cells. The early outward component of ICl(Ca) was significantly larger in subepicardial than in subendocardial cells in APVC setting. Applying a typical subepicardial AP as a command pulse resulted in a significantly larger early outward component in both subepicardial and subendocardial cells, compared to experiments when a typical subendocardial AP was applied. Inhibiting ICl(Ca) by 9-AC generated EADs at low stimulation rates and their incidence increased upon beta-adrenergic stimulation. 9-AC increased the short-term variability of repolarization also. We suggest a protective role for ICl(Ca) against risk of arrhythmias by reducing spatial and temporal heterogeneity of cardiac repolarization and EAD formation.

Mechano-chemo-transduction in cardiac myocytes.

The heart has the ability to adjust to changing mechanical loads. The Frank-Starling law and the Anrep effect describe exquisite intrinsic mechanisms the heart has for autoregulating the force of contraction to maintain cardiac output under changes of preload and afterload. Although these mechanisms have been known for more than a century, their cellular and molecular underpinnings are still debated. How does the cardiac myocyte sense changes in preload or afterload? How does the myocyte adjust its response to compensate for such changes? In cardiac myocytes Ca2+ is a crucial regulator of contractile force and in this review we compare and contrast recent studies from different labs that address these two important questions. The 'dimensionality' of the mechanical milieu under which experiments are carried out provide important clues to the location of the mechanosensors and the kinds of mechanical forces they can sense and respond to. As a first approximation, sensors inside the myocyte appear to modulate reactive oxygen species while sensors on the cell surface appear to also modulate nitric oxide signalling; both signalling pathways affect Ca2+ handling. Undoubtedly, further studies will add layers to this simplified picture. Clarifying the intimate links from cellular mechanics to reactive oxygen species and nitric oxide signalling and to Ca2+ handling will deepen our understanding of the Frank-Starling law and the Anrep effect, and also provide a unified view on how arrhythmias may arise in seemingly disparate diseases that have in common altered myocyte mechanics.

Potassium currents in the heart: functional roles in repolarization, arrhythmia and therapeutics.

This is the second of the two White Papers from the fourth UC Davis Cardiovascular Symposium Systems Approach to Understanding Cardiac Excitation-Contraction Coupling and Arrhythmias (3-4 March 2016), a biennial event that brings together leading experts in different fields of cardiovascular research. The theme of the 2016 symposium was 'K+ channels and regulation', and the objectives of the conference were severalfold: (1) to identify current knowledge gaps; (2) to understand what may go wrong in the diseased heart and why; (3) to identify possible novel therapeutic targets; and (4) to further the development of systems biology approaches to decipher the molecular mechanisms and treatment of cardiac arrhythmias. The sessions of the Symposium focusing on the functional roles of the cardiac K+ channel in health and disease, as well as K+ channels as therapeutic targets, were contributed by Ye Chen-Izu, Gideon Koren, James Weiss, David Paterson, David Christini, Dobromir Dobrev, Jordi Heijman, Thomas O'Hara, Crystal Ripplinger, Zhilin Qu, Jamie Vandenberg, Colleen Clancy, Isabelle Deschenes, Leighton Izu, Tamas Banyasz, Andras Varro, Heike Wulff, Eleonora Grandi, Michael Sanguinetti, Donald Bers, Jeanne Nerbonne and Nipavan Chiamvimonvat as speakers and panel discussants. This article summarizes state-of-the-art knowledge and controversies on the functional roles of cardiac K+ channels in normal and diseased heart. We endeavour to integrate current knowledge at multiple scales, from the single cell to the whole organ levels, and from both experimental and computational studies.

Potassium channels in the heart: structure, function and regulation.

This paper is the outcome of the fourth UC Davis Systems Approach to Understanding Cardiac Excitation-Contraction Coupling and Arrhythmias Symposium, a biannual event that aims to bring together leading experts in subfields of cardiovascular biomedicine to focus on topics of importance to the field. The theme of the 2016 symposium was 'K+ Channels and Regulation'. Experts in the field contributed their experimental and mathematical modelling perspectives and discussed emerging questions, controversies and challenges on the topic of cardiac K+ channels. This paper summarizes the topics of formal presentations and informal discussions from the symposium on the structural basis of voltage-gated K+ channel function, as well as the mechanisms involved in regulation of K+ channel gating, expression and membrane localization. Given the critical role for K+ channels in determining the rate of cardiac repolarization, it is hardly surprising that essentially every aspect of K+ channel function is exquisitely regulated in cardiac myocytes. This regulation is complex and highly interrelated to other aspects of myocardial function. K+ channel regulatory mechanisms alter, and are altered by, physiological challenges, pathophysiological conditions, and pharmacological agents. An accompanying paper focuses on the integrative role of K+ channels in cardiac electrophysiology, i.e. how K+ currents shape the cardiac action potential, and how their dysfunction can lead to arrhythmias, and discusses K+ channel-based therapeutics. A fundamental understanding of K+ channel regulatory mechanisms and disease processes is fundamental to reveal new targets for human therapy.

Electrophysiological Determination of Submembrane Na(+) Concentration in Cardiac Myocytes.

In the heart, Na(+) is a key modulator of the action potential, Ca(2+) homeostasis, energetics, and contractility. Because Na(+) currents and cotransport fluxes depend on the Na(+) concentration in the submembrane region, it is necessary to accurately estimate the submembrane Na(+) concentration ([Na(+)]sm). Current methods using Na(+)-sensitive fluorescent indicators or Na(+) -sensitive electrodes cannot measure [Na(+)]sm. However, electrophysiology methods are ideal for measuring [Na(+)]sm. In this article, we develop patch-clamp protocols and experimental conditions to determine the upper bound of [Na(+)]sm at the peak of action potential and its lower bound at the resting state. During the cardiac cycle, the value of [Na(+)]sm is constrained within these bounds. We conducted experiments in rabbit ventricular myocytes at body temperature and found that 1) at a low pacing frequency of 0.5 Hz, the upper and lower bounds converge at 9 mM, constraining the [Na(+)]sm value to ∼9 mM; 2) at 2 Hz pacing frequency, [Na(+)]sm is bounded between 9 mM at resting state and 11.5 mM; and 3) the cells can maintain [Na(+)]sm to the above values, despite changes in the pipette Na(+) concentration, showing autoregulation of Na(+) in beating cardiomyocytes.

CaMKII-dependent phosphorylation of RyR2 promotes targetable pathological RyR2 conformational shift.

Diastolic calcium (Ca) leak via cardiac ryanodine receptors (RyR2) can cause arrhythmias and heart failure (HF). Ca/calmodulin (CaM)-dependent kinase II (CaMKII) is upregulated and more active in HF, promoting RyR2-mediated Ca leak by RyR2-Ser2814 phosphorylation. Here, we tested a mechanistic hypothesis that RyR2 phosphorylation by CaMKII increases Ca leak by promoting a pathological RyR2 conformation with reduced CaM affinity. Acute CaMKII activation in wild-type RyR2, and phosphomimetic RyR2-S2814D (vs. non-phosphorylatable RyR2-S2814A) knock-in mouse myocytes increased SR Ca leak, reduced CaM-RyR2 affinity, and caused a pathological shift in RyR2 conformation (detected via increased access of the RyR2 structural peptide DPc10). This same trio of effects was seen in myocytes from rabbits with pressure/volume-overload induced HF. Excess CaM quieted leak and restored control conformation, consistent with negative allosteric coupling between CaM affinity and DPc10 accessible conformation. Dantrolene (DAN) also restored CaM affinity, reduced DPc10 access, and suppressed RyR2-mediated Ca leak and ventricular tachycardia in RyR2-S2814D mice. We propose that a common pathological RyR2 conformational state (low CaM affinity, high DPc10 access, and elevated leak) may be caused by CaMKII-dependent phosphorylation, oxidation, and HF. Moreover, DAN (or excess CaM) can shift this pathological gating state back to the normal physiological conformation, a potentially important therapeutic approach.

KN-93 inhibits IKr in mammalian cardiomyocytes.

Calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93 is widely used in multiple fields of cardiac research especially for studying the mechanisms of cardiomyopathy and cardiac arrhythmias. Whereas KN-93 is a potent inhibitor of CaMKII, several off-target effects have also been found in expression cell systems and smooth muscle cells, but there is no information on the KN93 side effects in mammalian ventricular myocytes. In this study we explore the effect of KN-93 on the rapid component of delayed rectifier potassium current (IKr) in the ventricular myocytes from rabbit and guinea pig hearts. Our data indicate that KN-93 exerts direct inhibitory effect on IKr that is not mediated via CaMKII. This off-target effect of KN93 should be taken into account when interpreting the data from using KN93 to investigate the role of CaMKII in cardiac function.

Oxidation of ryanodine receptor (RyR) and calmodulin enhance Ca release and pathologically alter, RyR structure and calmodulin affinity.

Oxidative stress may contribute to cardiac ryanodine receptor (RyR2) dysfunction in heart failure (HF) and arrhythmias. Altered RyR2 domain-domain interaction (domain unzipping) and calmodulin (CaM) binding affinity are allosterically coupled indices of RyR2 conformation. In HF RyR2 exhibits reduced CaM binding, increased domain unzipping and greater SR Ca leak, and dantrolene can reverse these changes. However, effects of oxidative stress on RyR2 conformation and leak in myocytes are poorly understood. We used fluorescent CaM, FKBP12.6, and domain-peptide biosensor (F-DPc10) to measure, directly in cardiac myocytes, (1) RyR2 activation by hydrogen peroxide (H2O2)-induced oxidation, (2) RyR2 conformation change caused by oxidation, (3) CaM-RyR2 and FK506-binding protein (FKBP12.6)-RyR2 interaction upon oxidation, and (4) whether dantrolene affects 1-3. H2O2 was used to mimic oxidative stress. H2O2 significantly increased the frequency of Ca(2+) sparks and spontaneous Ca(2+) waves, and dantrolene almost completely blocked these effects. H2O2 pretreatment significantly reduced CaM-RyR2 binding, but had no effect on FKBP12.6-RyR2 binding. Dantrolene restored CaM-RyR2 binding but had no effect on intracellular and RyR2 oxidation levels. H2O2 also accelerated F-DPc10-RyR2 association while dantrolene slowed it. Thus, H2O2 causes conformational changes (sensed by CaM and DPc10 binding) associated with Ca leak, and dantrolene reverses these RyR2 effects. In conclusion, in cardiomyocytes, H2O2 treatment markedly reduces the CaM-RyR2 affinity, has no effect on FKBP12.6-RyR2 affinity, and causes domain unzipping. Dantrolene can correct domain unzipping, restore CaM-RyR2 affinity, and quiet pathological RyR2 channel gating. F-DPc10 and CaM are useful biosensors of a pathophysiological RyR2 state.

A computational modelling approach combined with cellular electrophysiology data provides insights into the therapeutic benefit of targeting the late Na+ current.

KEY POINTS: The ventricular action potential plateau is a phase of high resistance, which makes ventricular myocytes vulnerable to small electrical perturbations. We developed a computationally based model of GS-458967 interaction with the cardiac Na+ channel, informed by experimental data recorded from guinea pig isolated single ventricular myocytes. The model predicts that the therapeutic potential of GS-458967 derives largely from the designed property of significant potent selectivity for INaL. ABSTRACT: Selective inhibition of the slowly inactivating or late Na(+) current (INaL) in patients with inherited or acquired arrhythmia syndrome may confer therapeutic benefit by reducing the incidence of triggers for arrhythmia and suppressing one component of arrhythmia-promoting cardiac substrates (e.g. prolonged refractoriness and spatiotemporal dispersion of action potential duration). Recently, a novel compound that preferentially and potently reduces INaL, GS-458967 (IC50 for block of INaL = 130 nM) has been studied. Experimental measurements of the effects of GS-458967 on endogenous INaL in guinea pig ventricular myocytes demonstrate a robust concentration-dependent reduction in action potential duration (APD). Using experimental data to calibrate INaL and the rapidly activating delayed rectifier K(+) current, IKr, in the Faber-Rudy computationally based model of the guinea pig ventricular action potential, we simulated effects of GS-458967 on guinea pig ventricular APD. GS-458967 (0.1 µM) caused a 28.67% block of INaL and 12.57% APD shortening in experiments, while the model predicted 10.06% APD shortening with 29.33% block of INaL. An additional effect of INaL block is to reduce the time during which the membrane potential is in a high resistance state (i.e. the action potential plateau). To test the hypothesis that targeted block of INaL would make ventricular myocytes less susceptible to small electrical perturbations, we used the computational model to test the degree of APD prolongation induced by small electrical perturbations in normal cells and in cells with simulated long QT syndrome. The model predicted a substantial dose-dependent reduction in sensitivity to small electrical perturbations as evidenced by action potential duration at 90% repolarization variability in the presence of GS-458967-induced INaL block. This effect was especially potent in the 'disease setting' of inherited long QT syndrome. Using a combined experimental and theoretical approach, our results suggest that INaL block is a potent therapeutic strategy. This is because reduction of INaL stabilizes the action potential waveform by reducing depolarizing current during the plateau phase of the action potential. This reduces the most vulnerable phase of the action potential with high membrane resistance. In summary, by reducing the sensitivity of the myocardial substrate to small electrical perturbations that promote arrhythmia triggers, agents such as GS-458967 may constitute an effective antiarrhythmic pharmacological strategy.