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, I_K1; rapid and slow delayed rectifier K⁺ currents, I_Kr and I_Ks, 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 I_K1 was significantly increased in hyperkalemia, whereas the reduction of driving force for K⁺ efflux dominated over the altered channel gating in case of I_Kr and I_Ks. Overall, the markedly increased I_K1 in hyperkalemia overcame the relative decreases of I_Kr and I_Ks during AP, resulting in an increased net repolarizing current during AP phase 3. β-Adrenergic stimulation of I_Ks also provided further repolarizing power during sympathetic activation, although hyperkalemia limited I_Ks upregulation. These results indicate that facilitation of I_K1 in hyperkalemia and β-adrenergic stimulation of I_Ks represent important compensatory mechanisms against AP prolongation and arrhythmia susceptibility.

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.

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 < I K1 < I Ks following β-adrenergic stimulation. We systematically determined the changes in the relative contribution of I Ks, I Kr, and I K1 to cardiac repolarization during AP at different adrenergic states. In conclusion, the β-adrenergic stimulation fine-tunes the cardiac AP morphology by shifting the power of different K(+) currents in a dose-dependent manner. This knowledge is important for designing antiarrhythmic drug strategies to treat hearts exposed to various sympathetic tones.

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.

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.

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.

Late Na⁺ current (I_NaL) significantly contributes to shaping cardiac action potentials (APs) and increased I_NaL 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 I_NaL. However, it remains unclear how each of these pathways regulates I_NaL 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 I_NaL at different AP phases to understand the arrhythmogenic potential. During the physiological AP (2 Hz, 37 °C) we found that I_NaL had a basal level current independent of PKA, but partially dependent on CaMKII. βAR activation (10 nM isoproterenol, ISO) further enhanced I_NaL via both PKA and CaMKII pathways. However, PKA predominantly increased I_NaL early during the AP plateau, whereas CaMKII mainly increased I_NaL 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 I_NaL similar to the βAR-induced CaMKII effect, while NOS inhibition prevented the βAR-induced CaMKII-dependent I_NaL enhancement. ROS generated by NADPH oxidase 2 (NOX2) also contributed to the ISO-induced I_NaL activation early in the AP. Taken together, our data reveal differential modulations of I_NaL by PKA and CaMKII signaling pathways at different AP phases. This nuanced and comprehensive view on the changes in I_NaL during AP deepens our understanding of the important role of I_NaL in reshaping the cardiac AP and arrhythmogenic potential under elevated sympathetic stimulation, which is relevant for designing therapeutic treatment of arrhythmias under pathological conditions.

Background

Rapid delayed rectifier K⁺ current (I_Kr) and late Na⁺ current (I_NaL) 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 I_NaL and I_Kr during the AP in rabbit and porcine ventricular cardiomyocytes to test our hypothesis that the balance between I_Kr and I_NaL affects repolarization stability in health and disease conditions.

Results

We found comparable amount of net charge carried by I_Kr and I_NaL during the physiological AP, suggesting that outward K⁺ current via I_Kr and inward Na⁺ current via I_NaL are in balance during physiological repolarization. Remarkably, I_Kr and I_NaL 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 I_Kr (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 I_NaL sufficiently restored APD to control in both cases. Importantly, I_NaL inhibition significantly reduced the beat-to-beat and short-term variabilities of APD. Moreover, I_NaL 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 I_NaL inhibition, increased by I_Kr inhibition, and restored by combined I_NaL and I_Kr inhibitions.

Conclusions

Our data demonstrate that I_Kr and I_NaL 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.

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.

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.