INNOVATIVE TECHNIQUES AND NEW INSIGHTS: Studying cardiac ionic currents and action potentials in physiologically relevant conditions.

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.

Las arritmias cardiacas están asociadas a diferentes tipos de enfermedad cardiaca. Las arritmias ventriculares constituyen un alto riesgo de muerte súbita. La fibrilación auricular predispone a coágulos sanguíneos que pueden producir accidentes cerebrovasculares e infarto miocárdico. Los científicos han desarrollado la técnica de patch-clamp para estudiar los canales iónicos y los potenciales de acción (PAs), que constituyen la base de la excitación y las arritmias cardiacas. Además de las clásicas técnicas de patch-clamp, se desarrollaron técnicas innovativas para estudiar los mecanismos complejos de las arritmias. En este trabajo, describimos diferentes métodos recientemente desarrollados tales como AP-clamp ("clampeo" del PA), Dynamic Clamp ("clampeo" dinámico), AP-Clamp Sequential Dissection, (disección secuencial del "clampeo" del AP), y Patch-Clamp-in-Gel (Patch clamp en gel). Estos métodos constituyen herramientas poderosas para descifrar cómo los sistemas dinámicos que constituyen la excitación-las señales de Ca2+ y la contracción, se retroalimentan para controlar la función cardiaca y cómo sus alteraciones llevan a la enfermedad cardiaca.

MapperPlus: Agnostic clustering of high-dimension data for precision medicine.

One of the goals of precision medicine is to classify patients into subgroups that differ in their susceptibility and response to a disease, thereby enabling tailored treatments for each subgroup. Therefore, there is a great need to identify distinctive clusters of patients from patient data. There are three key challenges to three key challenges of patient stratification: 1) the unknown number of clusters, 2) the need for assessing cluster validity, and 3) the clinical interpretability. We developed MapperPlus, a novel unsupervised clustering pipeline, that directly addresses these challenges. It extends the topological Mapper technique and blends it with two random-walk algorithms to automatically detect disjoint subgroups in patient data. We demonstrate that MapperPlus outperforms traditional agnostic clustering methods in key accuracy/performance metrics by testing its performance on publicly available medical and non-medical data set. We also demonstrate the predictive power of MapperPlus in a medical dataset of pediatric stem cell transplant patients where a number of cluster is unknown. Here, MapperPlus stratifies the patient population into clusters with distinctive survival rates. The MapperPlus software is open-source and publicly available.

On QSAR-based cardiotoxicity modeling with the expressiveness-enhanced graph learning model and dual-threshold scheme.

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: <1µM; non-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.

A training pipeline of an arrhythmia classifier for atrial fibrillation detection using Photoplethysmography signal.

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.

Erratum: Modeling cardiomyocyte mechanics and autoregulation of contractility by mechano-chemo-transduction feedback.

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

Modeling cardiomyocyte mechanics and autoregulation of contractility by mechano-chemo-transduction feedback.

The heart pumps blood into circulation against vascular resistance and actively regulates the contractile force to compensate for mechanical load changes. Our experimental data show that cardiomyocytes have a mechano-chemo-transduction (MCT) mechanism that increases intracellular Ca 2 + transient to enhance contractility in response to increased mechanical load. This study advances the cardiac excitation- Ca 2 + signaling-contraction (E-C) coupling model on conceptual and technical fronts. First, we developed analytical and computational models to perform 3-dimensional mechanical analysis of cardiomyocytes contracting in a viscoelastic medium under mechanical load. Next, we proposed an MCT feedback loop in the E-C coupling dynamic system to shift the feedforward paradigm of cardiac E-C coupling to an autoregulation model. Our combined modeling and experimental studies reveal that MCT enables autoregulation of E-C coupling and contractility in single cardiomyocytes, which underlies the heart's intrinsic autoregulation in compensatory response to load changes in order to maintain the stroke volume and cardiac output.

Exploring the Coordination of Cardiac Ion Channels With Action Potential Clamp Technique.

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.

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.

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.

Emergence of Mechano-Sensitive Contraction Autoregulation in Cardiomyocytes.

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.

Balance Between Rapid Delayed Rectifier K+ Current and Late Na+ Current on Ventricular Repolarization: An Effective Antiarrhythmic Target?

BACKGROUND: Rapid 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. METHODS: Physiological 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. RESULTS: We 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. CONCLUSIONS: Our 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.

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.

Early functional alterations in membrane properties and neuronal degeneration are hallmarks of progressive hearing loss in NOD mice.

Presbycusis or age-related hearing loss (ARHL) is the most common sensory deficit in the human population. A substantial component of the etiology stems from pathological changes in sensory and non-sensory cells in the cochlea. Using a non-obese diabetic (NOD) mouse model, we have characterized changes in both hair cells and spiral ganglion neurons that may be relevant for early signs of age-related hearing loss (ARHL). We demonstrate that hair cell loss is preceded by, or in parallel with altered primary auditory neuron functions, and latent neurite retraction at the hair cell-auditory neuron synapse. The results were observed first in afferent inner hair cell synapse of type I neurites, followed by type II neuronal cell-body degeneration. Reduced membrane excitability and loss of postsynaptic densities were some of the inaugural events before any outward manifestation of hair bundle disarray and hair cell loss. We have identified profound alterations in type I neuronal membrane properties, including a reduction in membrane input resistance, prolonged action potential latency, and a decrease in membrane excitability. The resting membrane potential of aging type I neurons in the NOD, ARHL model, was significantly hyperpolarized, and analyses of the underlying membrane conductance showed a significant increase in K+ currents. We propose that attempts to alleviate some forms of ARHL should include early targeted primary latent neural degeneration for effective positive outcomes.

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.

Enhanced Depolarization Drive in Failing Rabbit Ventricular Myocytes: Calcium-Dependent and ß-Adrenergic Effects on Late Sodium, L-Type Calcium, and Sodium-Calcium Exchange Currents.

BACKGROUND: Heart 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). METHODS: We measured the major inward currents and their Ca2+- and ß-adrenergic dependence under physiological AP clamp in rabbit ventricular myocytes in chronic pressure/volume overload-induced HF (versus age-matched control). RESULTS: AP 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). ß-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 ß-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. CONCLUSIONS: Our 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.

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.

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.

Coupling of SK channels, L-type Ca2+ channels, and ryanodine receptors in cardiomyocytes.

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.

Altered Repolarization Reserve in Failing Rabbit Ventricular Myocytes: Calcium and ß-Adrenergic Effects on Delayed- and Inward-Rectifier Potassium Currents.

BACKGROUND: Electrophysiological 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 RESULTS: We measured the major K+ currents (IKr, IKs, and IK1) and their Ca2+- and ß-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 ß-adrenergic stimulation was also significantly diminished in HF. IK1 was also decreased in HF regardless of Ca2+ buffering, CaMKII inhibition, or ß-adrenergic stimulation. CONCLUSIONS: At 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 ß-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.