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The heart has intrinsic abilities to autoregulate contractile force in response to mechanical load. Recent experimental studies show that cardiomyocytes have mechano-chemo-transduction (MCT) mechanisms that form a closed feedback loop in the excitation-Ca2+ signaling-contraction (E-C) coupling. This closed feedback loop enables autoregulation of contraction in response to mechanical load changes. Here, we develop the first autoregulatory E-C coupling model that couples electrophysiology, Ca2+ signaling, force development and contraction, and MCT feedback. The model recapitulates the experimental data showing that the mechanical load on cardiomyocytes during contraction increases the L-type Ca2+ current, action potential duration, sarcoplasmic reticulum (SR) Ca2+ content, and SR Ca2+ release, giving rise to increased cytosolic Ca2+ transient (MCT-Ca2+ gain) and enhanced contraction. The model also makes non-trivial predictions on the autoregulation of contraction with moderate MCT-Ca2+ gain under a range of physiological load changes, but arrhythmogenic discordant alternans with excessive MCT-Ca2+ gain under pathological overload. more...

Polydiacetylenes (PDAs) are conjugated polymers that are well known for their colorimetric transition from blue to red with the application of energetic stimulus. Sensing platforms based on polymerized diacetylene surfactant vesicles and other structures have been widely demonstrated for various colorimetric biosensing applications. Although less studied and utilized, the transition also results in a change from a non-fluorescent to a highly fluorescent state, making polydiacetylenes useful for both colorimetric and fluorogenic sensing applications. Here, we focus on the characterization and optimization of polydiacetylene vesicles to tune their sensitivity for fluorogenic sensing applications. Particularly, we look at how the structure of the diacetylene (DA) hydrocarbon tail and headgroup affect the self-assembled vesicle size and stability, polymerization kinetics, and the fluorogenic, blue to red phase transition. Longer DA acyl tails generally resulted in smaller and more stable vesicles. The polymerization kinetics and the blue to red transition were a function of both the DA acyl tail length and structure of the headgroup. Decreasing the acyl tail length generally led to vesicles that were more sensitive to energetic stimuli. Headgroup modifications had different effects depending on the structure of the headgroup. Ethanolamine headgroups resulted in vesicles with potentially increased stimuli responsivity. The lower energy stimulus to induce the chromatic transition was attributed to an increase in headgroup hydrogen bonding and polymer backbone strain. Boronic-acid headgroup functionalization led to vesicles that were generally unstable, only weakly polymerized, and unable to fully transform to the red phase due to strong polar, aromatic headgroup interactions. This work presents the design of PDA vesicles in the context of biosensing platforms and includes a discussion of the past, present, and future of PDA biosensing. more...

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. more...

Cardiac arrhythmias are associated with various forms of heart diseases. Ventricular arrhythmias present a significant risk for sudden cardiac death. Atrial fibrillations predispose to blood clots leading to stroke and heart attack. Scientists have been developing patch-clamp technology to study ion channels and action potentials (APs) underlying cardiac excitation and arrhythmias. Beyond the traditional patch-clamp techniques, innovative new techniques were developed for studying complex arrhythmia mechanisms. Here we review the recent development of methods including AP-Clamp, Dynamic Clamp, AP-Clamp Sequential Dissection, and Patch-Clamp-in-Gel. These methods provide powerful tools for researchers to decipher how the dynamic systems in excitation-Ca2+ signaling-contraction feedforward and feedback to control cardiac function and how their dysregulations lead to heart diseases. more...

Introduction: Given the direct association with malignant ventricular arrhythmias, cardiotoxicity is a major concern in drug design. In the past decades, computational models based on the quantitative structure-activity relationship have been proposed to screen out cardiotoxic compounds and have shown promising results. The combination of molecular fingerprint and the machine learning model shows stable performance for a wide spectrum of problems; however, not long after the advent of the graph neural network (GNN) deep learning model and its variant (e.g., graph transformer), it has become the principal way of quantitative structure-activity relationship-based modeling for its high flexibility in feature extraction and decision rule generation. Despite all these progresses, the expressiveness (the ability of a program to identify non-isomorphic graph structures) of the GNN model is bounded by the WL isomorphism test, and a suitable thresholding scheme that relates directly to the sensitivity and credibility of a model is still an open question. Methods: In this research, we further improved the expressiveness of the GNN model by introducing the substructure-aware bias by the graph subgraph transformer network model. Moreover, to propose the most appropriate thresholding scheme, a comprehensive comparison of the thresholding schemes was conducted. Results: Based on these improvements, the best model attains performance with 90.4% precision, 90.4% recall, and 90.5% F1-score with a dual-threshold scheme (active: 30μM). The improved pipeline (graph subgraph transformer network model and thresholding scheme) also shows its advantages in terms of the activity cliff problem and model interpretability. more...

Photoplethysmography (PPG) signal is potentially suitable in atrial fibrillation (AF) detection for its convenience in use and similarity in physiological origin to electrocardiogram (ECG). There are a few preceding studies that have shown the possibility of using the peak-to-peak interval of the PPG signal (PPIp) in AF detection. However, as a generalized model, the accuracy of an AF detector should be pursued on the one hand; on the other hand, its generalizability should be paid attention to in view of the individual differences in PPG manifestation of even the same arrhythmia and the existence of sub-types. Moreover, a binary classifier for atrial fibrillation and normal sinus rhythm is not convincing enough for the similarity between AF and ectopic beats. In this study, we project the atrial fibrillation detection as a multiple-class classification and try to propose a training pipeline that is advantageous both to the accuracy and generalizability of the classifier by designing and determining the configurable options of the pipeline, in terms of input format, deep learning model (with hyperparameter optimization), and scheme of transfer learning. With a rigorous comparison of the possible combinations of the configurable components in the pipeline, we confirmed that first-order difference of heartbeat sequence as the input format, a 2-layer CNN-1-layer Transformer hybridR model as the learning model and the whole model fine-tuning as the implementing scheme of transfer learning is the best combination for the pipeline (F1 value: 0.80, overall accuracy: 0.87)R. more...

[This corrects the article DOI: 10.1016/j.isci.2022.104667.].

The mammalian heart beats incessantly with rhythmic mechanical activities generating acids that need to be buffered to maintain a stable intracellular pH (pHi) for normal cardiac function. Even though spatial pHi non-uniformity in cardiomyocytes has been documented, it remains unknown how pHi is regulated to match the dynamic cardiac contractions. Here, we demonstrated beat-to-beat intracellular acidification, termed pHi transients, in synchrony with cardiomyocyte contractions. The pHi transients are regulated by pacing rate, Cl-/HCO3 - transporters, pHi buffering capacity, and β-adrenergic signaling. Mitochondrial electron-transport chain inhibition attenuates the pHi transients, implicating mitochondrial activity in sculpting the pHi regulation. The pHi transients provide dynamic alterations of H+ transport required for ATP synthesis, and a decrease in pHi may serve as a negative feedback to cardiac contractions. Current findings dovetail with the prevailing three known dynamic systems, namely electrical, Ca2+, and mechanical systems, and may reveal broader features of pHi handling in excitable cells. more...

The patch clamp technique underwent continual advancement and developed numerous variants in cardiac electrophysiology since its introduction in the late 1970s. In the beginning, the capability of the technique was limited to recording one single current from one cell stimulated with a rectangular command pulse. Since that time, the technique has been extended to record multiple currents under various command pulses including action potential. The current review summarizes the development of the patch clamp technique in cardiac electrophysiology with special focus on the potential applications in integrative physiology. more...

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. more...

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. more...

The heart has two intrinsic mechanisms to enhance contractile strength that compensate for increased mechanical load to help maintain cardiac output. When vascular resistance increases the ventricular chamber initially expands causing an immediate length-dependent increase of contraction force via the Frank-Starling mechanism. Additionally, the stress-dependent Anrep effect slowly increases contraction force that results in the recovery of the chamber volume towards its initial state. The Anrep effect poses a paradox: how can the cardiomyocyte maintain higher contractility even after the cell length has recovered its initial length? Here we propose a surface mechanosensor model that enables the cardiomyocyte to sense different mechanical stresses at the same mechanical strain. The cell-surface mechanosensor is coupled to a mechano-chemo-transduction feedback mechanism involving three elements: surface mechanosensor strain, intracellular Ca2+ transient, and cell strain. We show that in this simple yet general system, contractility autoregulation naturally emerges, enabling the cardiomyocyte to maintain contraction amplitude despite changes in a range of afterloads. These nontrivial model predictions have been experimentally confirmed. Hence, this model provides a new conceptual framework for understanding the contractility autoregulation in cardiomyocytes, which contributes to the heart's intrinsic adaptivity to mechanical load changes in health and diseases. more...

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. more...

BackgroundRapid delayed rectifier K+ current (IKr) and late Na+ current (INaL) significantly shape the cardiac action potential (AP). Changes in their magnitudes can cause either long or short QT syndromes associated with malignant ventricular arrhythmias and sudden cardiac death.MethodsPhysiological self AP-clamp was used to measure INaL and IKr during the AP in rabbit and porcine ventricular cardiomyocytes to test our hypothesis that the balance between IKr and INaL affects repolarization stability in health and disease conditions.ResultsWe found comparable amount of net charge carried by IKr and INaL during the physiological AP, suggesting that outward K+ current via IKr and inward Na+ current via INaL are in balance during physiological repolarization. Remarkably, IKr and INaL integrals in each control myocyte were highly correlated in both healthy rabbit and pig myocytes, despite high overall cell-to-cell variability. This close correlation was lost in heart failure myocytes from both species. Pretreatment with E-4031 to block IKr (mimicking long QT syndrome 2) or with sea anemone toxin II to impair Na+ channel inactivation (mimicking long QT syndrome 3) prolonged AP duration (APD); however, using GS-967 to inhibit INaL sufficiently restored APD to control in both cases. Importantly, INaL inhibition significantly reduced the beat-to-beat and short-term variabilities of APD. Moreover, INaL inhibition also restored APD and repolarization stability in heart failure. Conversely, pretreatment with GS-967 shortened APD (mimicking short QT syndrome), and E-4031 reverted APD shortening. Furthermore, the amplitude of AP alternans occurring at high pacing frequency was decreased by INaL inhibition, increased by IKr inhibition, and restored by combined INaL and IKr inhibitions.ConclusionsOur data demonstrate that IKr and INaL are counterbalancing currents during the physiological ventricular AP and their integrals covary in individual myocytes. Targeting these ionic currents to normalize their balance may have significant therapeutic potential in heart diseases with repolarization abnormalities. Visual Overview: A visual overview is available for this article. more...

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. β-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 β-adrenergic stimulation of IKs represent important compensatory mechanisms against AP prolongation and arrhythmia susceptibility. more...

The Frank-Starling law and Anrep effect describe two intrinsic mechanisms that regulate contraction force in the heart. Based on recent advancements and the historical literature, we propose new perspectives and address several critical issues in this review. (a) The Frank-Starling mechanism and Anrep effect are dynamically linked and act synergistically. (b) An open question is how cardiomyocytes sense mechanical load and transduce to biochemical signals (called mechano-chemo-transduction or MCT) to regulate contraction in response to load changes. (c) One research focus is to identify various mechanosensors and decipher their downstream MCT pathways. (d) Innovative experimental techniques engage different mechanosensors that detect different local strain and stress in the cell architecture. (e) Closed-loop MCT feedback in the dynamic excitation-Ca2+ signaling-contraction system enables autoregulation of contraction in response to physiological load changes. (f) However, pathological overload such as volume and pressure overload lead to excessive MCT-Ca2+ gain, cardiac remodeling, and heart diseases. [ABSTRACT FROM AUTHOR] more...

BackgroundHeart failure (HF) is characterized by electrophysiological remodeling resulting in increased risk of cardiac arrhythmias. Previous reports suggest that elevated inward ionic currents in HF promote action potential (AP) prolongation, increased short-term variability of AP repolarization, and delayed afterdepolarizations. However, the underlying changes in late Na+ current (INaL), L-type Ca2+ current, and NCX (Na+/Ca2+ exchanger) current are often measured in nonphysiological conditions (square-pulse voltage clamp, slow pacing rates, exogenous Ca2+ buffers).MethodsWe measured the major inward currents and their Ca2+- and β-adrenergic dependence under physiological AP clamp in rabbit ventricular myocytes in chronic pressure/volume overload-induced HF (versus age-matched control).ResultsAP duration and short-term variability of AP repolarization were increased in HF, and importantly, inhibition of INaL decreased both parameters to the control level. INaL was slightly increased in HF versus control even when intracellular Ca2+ was strongly buffered. But under physiological AP clamp with normal Ca2+ cycling, INaL was markedly upregulated in HF versus control (dependent largely on CaMKII [Ca2+/calmodulin-dependent protein kinase II] activity). β-Adrenergic stimulation (often elevated in HF) further enhanced INaL. L-type Ca2+ current was decreased in HF when Ca2+ was buffered, but CaMKII-mediated Ca2+-dependent facilitation upregulated physiological L-type Ca2+ current to the control level. Furthermore, L-type Ca2+ current response to β-adrenergic stimulation was significantly attenuated in HF. Inward NCX current was upregulated at phase 3 of AP in HF when assessed by combining experimental data and computational modeling.ConclusionsOur results suggest that CaMKII-dependent upregulation of INaL in HF significantly contributes to AP prolongation and increased short-term variability of AP repolarization, which may lead to increased arrhythmia propensity, and is further exacerbated by adrenergic stress. more...

FRET-based biosensor experiments in adult cardiomyocytes are a powerful way of dissecting the spatiotemporal dynamics of the complicated signaling networks that regulate cardiac health and disease. However, although much information has been gleaned from FRET studies on cardiomyocytes from larger species, experiments on adult cardiomyocytes from mice have been difficult at best. Thus the large variety of genetic mouse models cannot be easily used for this type of study. Here we develop cell culture conditions for adult mouse cardiomyocytes that permit robust expression of adenoviral FRET biosensors and reproducible FRET experimentation. We find that addition of 6.25 µM blebbistatin or 20 µM (S)-nitro-blebbistatin to a minimal essential medium containing 10 mM HEPES and 0.2% BSA maintains morphology of cardiomyocytes from physiological, pathological, and transgenic mouse models for up to 50 h after adenoviral infection. This provides a 10-15-h time window to perform reproducible FRET readings using a variety of CFP/YFP sensors between 30 and 50 h postinfection. The culture is applicable to cardiomyocytes isolated from transgenic mouse models as well as models with cardiac diseases. Therefore, this study helps scientists to disentangle complicated signaling networks important in health and disease of cardiomyocytes. more...

Late Na+ current (INaL) significantly contributes to shaping cardiac action potentials (APs) and increased INaL is associated with cardiac arrhythmias. β-adrenergic receptor (βAR) 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. βAR 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 βAR-induced CaMKII effect, while NOS inhibition prevented the βAR-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. more...

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. more...

Small-conductance Ca2+-activated K+ (SK) channels regulate the excitability of cardiomyocytes by integrating intracellular Ca2+ and membrane potentials on a beat-to-beat basis. The inextricable interplay between activation of SK channels and Ca2+ dynamics suggests the pathology of one begets another. Yet, the exact mechanistic underpinning for the activation of cardiac SK channels remains unaddressed. Here, we investigated the intracellular Ca2+ microdomains necessary for SK channel activation. SK currents coupled with Ca2+ influx via L-type Ca2+ channels (LTCCs) continued to be elicited after application of caffeine, ryanodine or thapsigargin to deplete SR Ca2+ store, suggesting that LTCCs provide the immediate Ca2+ microdomain for the activation of SK channels in cardiomyocytes. Super-resolution imaging of SK2, Cav1.2 Ca2+ channel, and ryanodine receptor 2 (RyR2) was performed to quantify the nearest neighbor distances (NND) and localized the three molecules within hundreds of nanometers. The distribution of NND between SK2 and RyR2 as well as SK2 and Cav1.2 was bimodal, suggesting a spatial relationship between the channels. The activation mechanism revealed by our study paved the way for the understanding of the roles of SK channels on the feedback mechanism to regulate the activities of LTCCs and RyR2 to influence local and global Ca2+ signaling. more...

BackgroundElectrophysiological remodeling and increased susceptibility for cardiac arrhythmias are hallmarks of heart failure (HF). Ventricular action potential duration (APD) is typically prolonged in HF, with reduced repolarization reserve. However, underlying K+ current changes are often measured in nonphysiological conditions (voltage clamp, low pacing rates, cytosolic Ca2+ buffers).Methods and resultsWe measured the major K+ currents (IKr, IKs, and IK1) and their Ca2+- and β-adrenergic dependence in rabbit ventricular myocytes in chronic pressure/volume overload-induced HF (versus age-matched controls). APD was significantly prolonged only at lower pacing rates (0.2-1 Hz) in HF under physiological ionic conditions and temperature. However, when cytosolic Ca2+ was buffered, APD prolongation in HF was also significant at higher pacing rates. Beat-to-beat variability of APD was also significantly increased in HF. Both IKr and IKs were significantly upregulated in HF under action potential clamp, but only when cytosolic Ca2+ was not buffered. CaMKII (Ca2+/calmodulin-dependent protein kinase II) inhibition abolished IKs upregulation in HF, but it did not affect IKr. IKs response to β-adrenergic stimulation was also significantly diminished in HF. IK1 was also decreased in HF regardless of Ca2+ buffering, CaMKII inhibition, or β-adrenergic stimulation.ConclusionsAt baseline Ca2+-dependent upregulation of IKr and IKs in HF counterbalances the reduced IK1, maintaining repolarization reserve (especially at higher heart rates) in physiological conditions, unlike conditions of strong cytosolic Ca2+ buffering. However, under β-adrenergic stimulation, reduced IKs responsiveness severely limits integrated repolarizing K+ current and repolarization reserve in HF. This would increase arrhythmia propensity in HF, especially during adrenergic stress. more...

BackgroundIntracellular pH (pHi) is critical to cardiac excitation and contraction; uncompensated changes in pHi impair cardiac function and trigger arrhythmia. Several ion transporters participate in cardiac pHi regulation. Our previous studies identified several isoforms of a solute carrier Slc26a6 to be highly expressed in cardiomyocytes. We show that Slc26a6 mediates electrogenic Cl-/HCO3- exchange activities in cardiomyocytes, suggesting the potential role of Slc26a6 in regulation of not only pHi, but also cardiac excitability.Methods and resultsTo test the mechanistic role of Slc26a6 in the heart, we took advantage of Slc26a6 knockout (Slc26a6-/- ) mice using both in vivo and in vitro analyses. Consistent with our prediction of its electrogenic activities, ablation of Slc26a6 results in action potential shortening. There are reduced Ca2+ transient and sarcoplasmic reticulum Ca2+ load, together with decreased sarcomere shortening in Slc26a6-/- cardiomyocytes. These abnormalities translate into reduced fractional shortening and cardiac contractility at the in vivo level. Additionally, pHi is elevated in Slc26a6-/- cardiomyocytes with slower recovery kinetics from intracellular alkalization, consistent with the Cl-/HCO3- exchange activities of Slc26a6. Moreover, Slc26a6-/- mice show evidence of sinus bradycardia and fragmented QRS complex, supporting the critical role of Slc26a6 in cardiac conduction system.ConclusionsOur study provides mechanistic insights into Slc26a6, a unique cardiac electrogenic Cl-/HCO3- transporter in ventricular myocytes, linking the critical roles of Slc26a6 in regulation of pHi, excitability, and contractility. pHi is a critical regulator of other membrane and contractile proteins. Future studies are needed to investigate possible changes in these proteins in Slc26a6-/- mice. more...

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. more...

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. more...

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. more...

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. more...

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. more...

RationaleCardiac 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.ObjectiveTo develop a method for imaging sarcomere level Ca(2+)-contraction coupling in healthy and disease model cardiac myocytes.Methods and resultsFreshly 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.ConclusionsMultimodal 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. more...

Isolation of high quality cardiomyocytes is critically important for achieving successful experiments in many cellular and molecular cardiology studies. Methods for isolating cardiomyocytes from the murine heart generally are time-sensitive and experience-dependent, and often fail to produce high quality cells. Major technical difficulties can be related to the surgical procedures needed to explant the heart and to cannulate the vessel to mount onto the Langendorff system before in vitro reperfusion can begin. During this period, transient hypoxia and ischemia may damage the heart, resulting in low yield and poor quality of cells, especially for heart disease models that have fragile cells. We have developed novel in vivo cannulation methods to minimize hypoxia and ischemia, and fine-tuned the entire protocol to produce high quality ventricular myocytes. The high cell quality has been confirmed using important structural and functional criteria such as morphology, t-tubule structure, action potential morphology, Ca2+ signaling, responsiveness to beta-adrenergic agonist, and ability to have robust contraction under mechanically loaded condition. Together these assessments show the preservation of the cardiac excitation-contraction machinery in cells isolated using this technique. The in vivo cannulation method enables consistent isolation of high-quality cardiomyocytes, even from heart disease models that were notoriously difficult for cell isolation using traditional methods. more...

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. more...

Weak electric fields guide cell migration, known as galvanotaxis/electrotaxis. The sensor(s) cells use to detect the fields remain elusive. Here we perform a large-scale screen using an RNAi library targeting ion transporters in human cells. We identify 18 genes that show either defective or increased galvanotaxis after knockdown. Knockdown of the KCNJ15 gene (encoding inwardly rectifying K(+) channel Kir4.2) specifically abolishes galvanotaxis, without affecting basal motility and directional migration in a monolayer scratch assay. Depletion of cytoplasmic polyamines, highly positively charged small molecules that regulate Kir4.2 function, completely inhibits galvanotaxis, whereas increase of intracellular polyamines enhances galvanotaxis in a Kir4.2-dependent manner. Expression of a polyamine-binding defective mutant of KCNJ15 significantly decreases galvanotaxis. Knockdown or inhibition of KCNJ15 prevents phosphatidylinositol 3,4,5-triphosphate (PIP3) from distributing to the leading edge. Taken together these data suggest a previously unknown two-molecule sensing mechanism in which KCNJ15/Kir4.2 couples with polyamines in sensing weak electric fields. more...

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. more...

In February 2014, a group of scientists convened as part of the University of California Davis Cardiovascular Symposium to bring together experimental and mathematical modelling perspectives and discuss points of consensus and controversy on the topic of sodium in the heart. This paper summarizes the topics of presentation and discussion from the symposium, with a focus on the role of aberrant sodium channels and abnormal sodium homeostasis in cardiac arrhythmias and pharmacotherapy from the subcellular scale to the whole heart. Two following papers focus on Na(+) channel structure, function and regulation, and Na(+)/Ca(2+) exchange and Na(+)/K(+) ATPase. The UC Davis Cardiovascular Symposium is 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 focus on Na(+) in the 2014 symposium stemmed from the multitude of recent studies that point to the importance of maintaining Na(+) homeostasis in the heart, as disruption of homeostatic processes are increasingly identified in cardiac disease states. Understanding how disruption in cardiac Na(+)-based processes leads to derangement in multiple cardiac components at the level of the cell and to then connect these perturbations to emergent behaviour in the heart to cause disease is a critical area of research. The ubiquity of disruption of Na(+) channels and Na(+) homeostasis in cardiac disorders of excitability and mechanics emphasizes the importance of a fundamental understanding of the associated mechanisms and disease processes to ultimately reveal new targets for human therapy. more...

This paper is the third in a series of reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation-contraction coupling and arrhythmias: Na(+) channel and Na(+) transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on cardiac Na(+)/Ca(2+) exchange (NCX) and Na(+)/K(+)-ATPase (NKA). While the relevance of Ca(2+) homeostasis in cardiac function has been extensively investigated, the role of Na(+) regulation in shaping heart function is often overlooked. Small changes in the cytoplasmic Na(+) content have multiple effects on the heart by influencing intracellular Ca(2+) and pH levels thereby modulating heart contractility. Therefore it is essential for heart cells to maintain Na(+) homeostasis. Among the proteins that accomplish this task are the Na(+)/Ca(2+) exchanger (NCX) and the Na(+)/K(+) pump (NKA). By transporting three Na(+) ions into the cytoplasm in exchange for one Ca(2+) moved out, NCX is one of the main Na(+) influx mechanisms in cardiomyocytes. Acting in the opposite direction, NKA moves Na(+) ions from the cytoplasm to the extracellular space against their gradient by utilizing the energy released from ATP hydrolysis. A fine balance between these two processes controls the net amount of intracellular Na(+) and aberrations in either of these two systems can have a large impact on cardiac contractility. Due to the relevant role of these two proteins in Na(+) homeostasis, the emphasis of this review is on recent developments regarding the cardiac Na(+)/Ca(2+) exchanger (NCX1) and Na(+)/K(+) pump and the controversies that still persist in the field. more...

This paper is the second of a series of three reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation-contraction coupling and arrhythmias: Na(+) channel and Na(+) transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on Na(+) channel function and regulation, Na(+) channel structure and function, and Na(+) channel trafficking, sequestration and complexing. more...

Toward developing engineered cartilage for the treatment of cartilage defects, achieving relevant functional properties before implantation remains a significant challenge. Various chemical and mechanical stimuli have been used to enhance the functional properties of engineered musculoskeletal tissues. Recently, Ca(2+)-modulating agents have been used to enhance matrix synthesis and biomechanical properties of engineered cartilage. The objective of this study was to determine whether other known Ca(2+) modulators, digoxin and adenosine triphosphate (ATP), can be employed as novel stimuli to increase collagen synthesis and functional properties of engineered cartilage. Neocartilage constructs were formed by scaffold-free self-assembling of primary bovine articular chondrocytes. Digoxin, ATP, or both agents were added to the culture medium for 1 h/day on days 10-14. After 4 weeks of culture, neocartilage properties were assessed for gross morphology, biochemical composition, and biomechanical properties. Digoxin and ATP were found to increase neocartilage collagen content by 52-110% over untreated controls, while maintaining proteoglycan content near native tissue values. Furthermore, digoxin and ATP increased the tensile modulus by 280% and 180%, respectively, while the application of both agents increased the modulus by 380%. The trends in tensile properties were found to correlate with the amount of collagen cross-linking. Live Ca(2+) imaging experiments revealed that both digoxin and ATP were able to increase Ca(2+) oscillations in monolayer-cultured chondrocytes. This study provides a novel approach toward directing neocartilage maturation and enhancing its functional properties using novel Ca(2+) modulators. more...

Variability is inherent in any population, regardless whether the population comprises humans, plants, biological cells, or manufactured parts. Is the variability beneficial, detrimental, or inconsequential? This question is of fundamental importance in manufacturing, agriculture, and bioengineering. This question has no simple categorical answer because research shows that variability in a population can have both beneficial and detrimental effects. Here we ask whether there is a certain level of variability that can maximize benefit to the population as a whole. We answer this question by using a model composed of a population of individuals who independently make binary decisions; individuals vary in making a yes or no decision, and the aggregated effect of these decisions on the population is quantified by a benefit function (e.g. accuracy of the measurement using binary rulers, aggregate income of a town of farmers). Here we show that an optimal variance exists for maximizing the population benefit function; this optimal variance quantifies what is often called the "right mix" of individuals in a population. more...

β-Adrenergic stimulation differentially modulates different K(+) channels and thus fine-tunes cardiac action potential (AP) repolarization. However, it remains unclear how the proportion of I Ks, I Kr, and I K1 currents in the same cell would be altered by β-adrenergic stimulation, which would change the relative contribution of individual K(+) current to the total repolarization reserve. In this study, we used an innovative AP-clamp sequential dissection technique to directly record the dynamic I Ks, I Kr, and I K1 currents during the AP in guinea pig ventricular myocytes under physiologically relevant conditions. Our data provide quantitative measures of the magnitude and time course of I Ks, I Kr, and I K1 currents in the same cell under its own steady-state AP, in a physiological milieu, and with preserved Ca(2+) homeostasis. We found that isoproterenol treatment significantly enhanced I Ks, moderately increased I K1, but slightly decreased I Kr in a dose-dependent manner. The dominance pattern of the K(+) currents was I Kr > I K1 > I Ks at the control condition, but reversed to I Kr more...

To locate the biosensor peptide DPc10 bound to ryanodine receptor (RyR) Ca(2+) channels, we developed an approach that combines fluorescence resonance energy transfer (FRET), simulated-annealing, cryo-electron microscopy, and crystallographic data. DPc10 is identical to the 2460-2495 segment within the cardiac muscle RyR isoform (RyR2) central domain. DPc10 binding to RyR2 results in a pathologically elevated Ca(2+) leak by destabilizing key interactions between the RyR2 N-terminal and central domains (unzipping). To localize the DPc10 binding site within RyR2, we measured FRET between five single-cysteine variants of the FK506-binding protein (FKBP) labeled with a donor probe, and DPc10 labeled with an acceptor probe (A-DPc10). Effective donor positions were calculated from simulated-annealing constrained by both the RyR cryo-EM map and the FKBP atomic structure docked to the RyR. FRET to A-DPc10 was measured in permeabilized cardiomyocytes via confocal microscopy, converted to distances, and used to trilaterate the acceptor locus within RyR. Additional FRET measurements between donor-labeled calmodulin and A-DPc10 were used to constrain the trilaterations. Results locate the DPc10 probe within RyR domain 3, ?35 Å from the previously docked N-terminal domain crystal structure. This multiscale approach may be useful in mapping other RyR sites of mechanistic interest within FRET range of FKBP. more...

Cardiomyocytes contract against a mechanical load during each heartbeat, and excessive mechanical stress leads to heart diseases. Using a cell-in-gel system that imposes an afterload during cardiomyocyte contraction, we found that nitric oxide synthase (NOS) was involved in transducing mechanical load to alter Ca(2+) dynamics. In mouse ventricular myocytes, afterload increased the systolic Ca(2+) transient, which enhanced contractility to counter mechanical load but also caused spontaneous Ca(2+) sparks during diastole that could be arrhythmogenic. The increases in the Ca(2+) transient and sparks were attributable to increased ryanodine receptor (RyR) sensitivity because the amount of Ca2(+) in the sarcoplasmic reticulum load was unchanged. Either pharmacological inhibition or genetic deletion of nNOS (or NOS1), but not of eNOS (or NOS3), prevented afterload-induced Ca2(+) sparks. This differential effect may arise from localized NO signaling, arising from the proximity of nNOS to RyR, as determined by super-resolution imaging. Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) and nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) also contributed to afterload-induced Ca(2+) sparks. Cardiomyocytes from a mouse model of familial hypertrophic cardiomyopathy exhibited enhanced mechanotransduction and frequent arrhythmogenic Ca(2+) sparks. Inhibiting nNOS and CaMKII, but not NOX2, in cardiomyocytes from this model eliminated the Ca2(+) sparks, suggesting mechanotransduction activated nNOS and CaMKII independently from NOX2. Thus, our data identify nNOS, CaMKII, and NOX2 as key mediators in mechanochemotransduction during cardiac contraction, which provides new therapeutic targets for treating mechanical stress-induced Ca(2+) dysregulation, arrhythmias, and cardiomyopathy. more...

Introduction Hyperkalemia is commonly encountered clinically in various physiological and pathological conditions including vigorous physical exercise, ischemia, kidney failure, hemolysis, endocrine diseases, and side effects of the pharmacological therapy (Weiss [...], 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.sup.+] channels. However, it remains unknown how hyperkalemia changes the profiles of each [K.sup.+] current under a cardiac AP. Therefore, we recorded the major [K.sup.+] currents (inward rectifier [K.sup.+] current, [I.sub.K1]; rapid and slow delayed rectifier [K.sup.+] currents, [I.sub.Kr] and [I.sub.Ks], respectively) using AP-clamp in rabbit ventricular myocytes. As [K.sup.+] may accumulate at rapid heart rates during sympathetic stimulation, we also examined the effect of isoproterenol on these [K.sup.+] currents. We found that [I.sub.K1] was significantly increased in hyperkalemia, whereas the reduction of driving force for [K.sup.+] efflux dominated over the altered channel gating in case of [I.sub.Kr] and [I.sub.Ks]. Overall, the markedly increased [I.sub.K1] in hyperkalemia overcame the relative decreases of [I.sub.Kr] and [I.sup.Ks] during AP, resulting in an increased net repolarizing current during AP phase 3. [beta]-adrenergic stimulation of Ife also provided further repolarizing power during sympathetic activation, although hyperkalemia limited [I.sub.Ks] upregulation. These results indicate that facilitation of [I.sub.K1] in hyperkalemia and [beta]-adrenergic stimulation of [I.sub.Ks]. represent important compensatory mechanisms against AP prolongation and arrhythmia susceptibility. Key words: hyperkalemia, sympathetic stimulation, cellular electrophysiology, heart, potassium channels, action potential voltage-clamp, physical exercise, arrhythmia. On sait que l&apos;hyperkaliemie se produit dans diverses situations, y compris pendant l&apos;exercice physique vigoureux. Dans le creur, l&apos;hyperkaliemie est associee avec une diminution de la duree du potentiel d&apos;action (PA), qui est attribuee a des canaux [K.sup.+] dont les proprietes de<>sont alterees. Toutefois, on ne sait toujours pas comment l&apos;hyperkaliemie entrame des variations dans le profil de chacun des courants [K.sup.+] a la base du PA cardiaque. Par consequent, nous avons enregistre les principaux courants [K.sup.+] (courant a rectification entrante ([I.sub.K1]); courants a rectification rapide et lente ([I.sub.Kr] et [I.sub.Ks], respectivement)) a l&apos;aide de la technique de clampage du PA dans des myocytes ventriculaires de lapin. Comme les ions [K.sup.+] peuvent s&apos;accumuler a des frequences cardiaques elevees pendant une stimulation sympathique, nous avons aussi etudie l&apos;effet de l&apos;isoproterenol sur ces courants [K.sup.+]. Nous avons observe qu&apos;[I.sub.K1] etait nettement augmente en hyperkaliemie, tandis que la diminution de la force motrice de l&apos;efflux de [K.sup.+] dominait comparativement au defaut de<>des canaux dans le cas d&apos;[I.sub.Kr] et d&apos;[I.sub.Kr]. Dans l&apos;ensemble, l&apos;augmentation marquee d&apos;[I.sub.K1] en hyperkaliemie parvenait a contrer la diminution relative d&apos;[I.sub.Kr] et d&apos;[I.sub.Ks] pendant le PA, entrainant une augmentation nette des courants de repolarisation pendant la phase 3 du PA. La stimulation [beta]-adrenergique d&apos;[I.sub.Ks] fournissait aussi une puissance de repolarisation supplementaire pendant l&apos;activation sympathique, meme si l&apos;hyperkaliemie limitait la regulation a la hausse d&apos;[I.sub.Ks]. Ces resultats montrent que la facilitation d&apos;[I.sub.K1] en hyperkaliemie et la stimulation [beta]-adrenergique d&apos;[I.sub.K2] representent des modes d&apos;action compensatoires importants contre l&apos;augmentation de la duree du PA et la susceptibilite aux arythmies. [Traduit par la Redaction] Mots-cles : hyperkaliemie, stimulation sympathique, electrophysiologie cellulaire, creur, canaux potassiques, voltage-clamp du potentiel d&apos;action, exercice physique, arythmie. more...

The objective of this work is to examine the contribution of late Na(+) current (INa,L) to the cardiac action potential (AP) and arrhythmogenic activities. In spite of the rapidly growing interest toward this current, there is no publication available on experimental recording of the dynamic INa,L current as it flows during AP with Ca(2+) cycling. Also unknown is how the current profile changes when the Ca(2+)-calmodulin dependent protein kinase II (CaMKII) signaling is altered, and how the current contributes to the development of arrhythmias. In this study we use an innovative AP-clamp Sequential Dissection technique to directly record the INa,L current during the AP with Ca(2+) cycling in the guinea pig ventricular myocytes. First, we found that the magnitude of INa,L measured under AP-clamp is substantially larger than earlier studies indicated. CaMKII inhibition using KN-93 significantly reduced the current. Second, we recorded INa,L together with IKs, IKr, and IK1 in the same cell to understand how these currents counterbalance to shape the AP morphology. We found that the amplitude and the total charge carried by INa,L exceed that of IKs. Third, facilitation of INa,L by Anemone toxin II prolonged APD and induced Ca(2+) oscillations that led to early and delayed afterdepolarizations and triggered APs; these arrhythmogenic activities were eliminated by buffering Ca(2+) with BAPTA. In conclusion, INa,L contributes a significantly large inward current that prolongs APD and unbalances the Ca(2+) homeostasis to cause arrhythmogenic APs. more...