Kornél Kistamás

Dynamics of the late Na + current during cardiac action potential and its contribution to afterdepolarizations

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.

Calcium and Excitation-Contraction Coupling in the Heart

Cardiac contractility is regulated by changes in intracellular Ca concentration ([Ca2+]i). Normal function requires that [Ca2+]i be sufficiently high in systole and low in diastole. Much of the Ca needed for contraction comes from the sarcoplasmic reticulum and is released by the process of calcium-induced calcium release. The factors that regulate and fine-tune the initiation and termination of release are reviewed. The precise control of intracellular Ca cycling depends on the relationships between the various channels and pumps that are involved. We consider 2 aspects: (1) structural coupling: the transporters are organized within the dyad, linking the transverse tubule and sarcoplasmic reticulum and ensuring close proximity of Ca entry to sites of release. (2) Functional coupling: where the fluxes across all membranes must be balanced such that, in the steady state, Ca influx equals Ca efflux on every beat. The remainder of the review considers specific aspects of Ca signaling, including the role of Ca buffers, mitochondria, Ca leak, and regulation of diastolic [Ca2+]i.

Contribution of ion currents to beat-to-beat variability of action potential duration in canine ventricular myocytes

Although beat-to-beat variability (short-term variability, SV) of action potential duration (APD) is considered as a predictor of imminent cardiac arrhythmias, the underlying mechanisms are still not clear. In the present study, therefore, we aimed to determine the role of the major cardiac ion currents, APD, stimulation frequency, and changes in the intracellular Ca2+ concentration ([Ca2+]i) on the magnitude of SV. Action potentials were recorded from isolated canine ventricular cardiomyocytes using conventional microelectrode techniques. SV was an exponential function of APD, when APD was modified by current injections. Drug effects were characterized as relative SV changes by comparing the drug-induced changes in SV to those in APD according to the exponential function obtained with current pulses. Relative SV was increased by dofetilide, HMR 1556, nisoldipine, and veratridine, while it was reduced by BAY K8644, tetrodotoxin, lidocaine, and isoproterenol. Relative SV was also increased by increasing the stimulation frequency and [Ca2+]i. In summary, relative SV is decreased by ion currents involved in the negative feedback regulation of APD (ICa, IKs, and IKr), while it is increased by INa and Ito. We conclude that drug-induced effects on SV should be evaluated in relation with the concomitant changes in APD. Since relative SV was decreased by ion currents playing critical role in the negative feedback regulation of APD, blockade of these currents, or the beta-adrenergic pathway, may carry also some additional proarrhythmic risk in addition to their well-known antiarrhythmic action.

Experimentally-Based Computational Investigation into Beat-To-Beat Variability in Ventricular Repolarization and Its Response to Ionic Current Inhibition.

Beat-to-beat variability in repolarization (BVR) has been proposed as an arrhythmic risk marker for disease and pharmacological action. The mechanisms are unclear but BVR is thought to be a cell level manifestation of ion channel stochasticity, modulated by cell-to-cell differences in ionic conductances. In this study, we describe the construction of an experimentally-calibrated set of stochastic cardiac cell models that captures both BVR and cell-to-cell differences in BVR displayed in isolated canine action potential measurements using pharmacological agents. Simulated and experimental ranges of BVR are compared in control and under pharmacological inhibition, and the key ionic currents determining BVR under physiological and pharmacological conditions are identified. Results show that the 4-aminopyridine-sensitive transient outward potassium current, Ito1, is a fundamental driver of BVR in control and upon complete inhibition of the slow delayed rectifier potassium current, IKs. In contrast, IKs and the L-type calcium current, ICaL, become the major contributors to BVR upon inhibition of the fast delayed rectifier potassium current, IKr. This highlights both IKs and Ito1 as key contributors to repolarization reserve. Partial correlation analysis identifies the distribution of Ito1 channel numbers as an important independent determinant of the magnitude of BVR and drug-induced change in BVR in control and under pharmacological inhibition of ionic currents. Distributions in the number of IKs and ICaL channels only become independent determinants of the magnitude of BVR upon complete inhibition of IKr. These findings provide quantitative insights into the ionic causes of BVR as a marker for repolarization reserve, both under control condition and pharmacological inhibition.

Disulfide-activated protein kinase G Iα regulates cardiac diastolic relaxation and fine-tunes the Frank-Starling response

The Frank–Starling mechanism allows the amount of blood entering the heart from the veins to be precisely matched with the amount pumped out to the arterial circulation. As the heart fills with blood during diastole, the myocardium is stretched and oxidants are produced. Here we show that protein kinase G Iα (PKGIα) is oxidant-activated during stretch and this form of the kinase selectively phosphorylates cardiac phospholamban Ser16—a site important for diastolic relaxation. We find that hearts of Cys42Ser PKGIα knock-in (KI) mice, which are resistant to PKGIα oxidation, have diastolic dysfunction and a diminished ability to couple ventricular filling with cardiac output on a beat-to-beat basis. Intracellular calcium dynamics of ventricular myocytes isolated from KI hearts are altered in a manner consistent with impaired relaxation and contractile function. We conclude that oxidation of PKGIα during myocardial stretch is crucial for diastolic relaxation and fine-tunes the Frank–Starling response.

Cytosolic calcium changes affect the incidence of early afterdepolarizations in canine ventricular myocytes.

This study was designed to investigate the influence of cytosolic Ca(2+) levels ([Ca(2+)]i) on action potential duration (APD) and on the incidence of early afterdepolarizations (EADs) in canine ventricular cardiomyocytes. Action potentials (AP) of isolated cells were recorded using conventional sharp microelectrodes, and the concomitant [Ca(2+)]i was monitored with the fluorescent dye Fura-2. EADs were evoked at a 0.2 Hz pacing rate by inhibiting the rapid delayed rectifier K(+) current with dofetilide, by activating the late sodium current with veratridine, or by activating the L-type calcium current with BAY K8644. These interventions progressively prolonged the AP and resulted in initiation of EADs. Reducing [Ca(2+)]i by application of the cell-permeant Ca(2+) chelator BAPTA-AM lengthened the AP at 1.0 Hz if it was applied alone, in the presence of veratridine, or in the presence of BAY K8644. However, BAPTA-AM shortened the AP if the cells were pretreated with dofetilide. The incidence of the evoked EADs was strongly reduced by BAPTA-AM in dofetilide, moderately reduced in veratridine, whereas EAD incidence was increased by BAPTA-AM in the presence of BAY K8644. Based on these experimental data, changes in [Ca(2+)]i have marked effects on APD as well as on the incidence of EADs; however, the underlying mechanisms may be different, depending on the mechanism of EAD generation. As a consequence, reduction of [Ca(2+)]i may eliminate EADs under some, but not all, experimental conditions.

Expression of anti-Mullerian hormone receptor on the appendix testis in connection with urological disorders

The female internal sex organs develop from the paramesonephric (Mullerian) duct. In male embryos, the regression of the Mullerian duct is caused by the anti-Mullerian hormone (AMH), which plays an important role in the process of testicular descent. The physiological remnant of the Mullerian duct in males is the appendix testis (AT). In our previous study, we presented evidence for the decreased incidence of AT in cryptorchidism with intraoperative surgery. In this report, the expression of the anti-Mullerian hormone receptor type 2 (AMHR2), the specific receptor of AMH, on the AT was investigated in connection with different urological disorders, such as hernia inguinalis, torsion of AT, cysta epididymis, varicocele, hydrocele testis and various forms of undescended testis. The correlation between the age of the patients and the expression of the AMHR2 was also examined. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry were used to detect the receptors mRNA and protein levels, respectively. We demonstrate that AMHR2 is expressed in the ATs. Additionally, the presence of this receptor was proven at the mRNA and protein levels. The expression pattern of the receptor correlated with neither the examined urological disorders nor the age of the patients; therefore, the function of the AT remains obscure.

Sarcolemmal Ca2+-entry through L-type Ca2+ channels controls the profile of Ca2+-activated Cl− current in canine ventricular myocytes

Ca(2+)-activated Cl(-) current (ICl(Ca)) mediated by TMEM16A and/or Bestrophin-3 may contribute to cardiac arrhythmias. The true profile of ICl(Ca) during an actual ventricular action potential (AP), however, is poorly understood. We aimed to study the profile of ICl(Ca) systematically under physiological conditions (normal Ca(2+) cycling and AP voltage-clamp) as well as in conditions designed to change [Ca(2+)]i. The expression of TMEM16A and/or Bestrophin-3 in canine and human left ventricular myocytes was examined. The possible spatial distribution of these proteins and their co-localization with Cav1.2 was also studied. The profile of ICl(Ca), identified as a 9-anthracene carboxylic acid-sensitive current under AP voltage-clamp conditions, contained an early fast outward and a late inward component, overlapping early and terminal repolarizations, respectively. Both components were moderately reduced by ryanodine, while fully abolished by BAPTA, but not EGTA. [Ca(2+)]i was monitored using Fura-2-AM. Setting [Ca(2+)]i to the systolic level measured in the bulk cytoplasm (1.1μM) decreased ICl(Ca), while application of Bay K8644, isoproterenol, and faster stimulation rates increased the amplitude of ICl(Ca). Ca(2+)-entry through L-type Ca(2+) channels was essential for activation of ICl(Ca). TMEM16A and Bestrophin-3 showed strong co-localization with one another and also with Cav1.2 channels, when assessed using immunolabeling and confocal microscopy in both canine myocytes and human ventricular myocardium. Activation of ICl(Ca) in canine ventricular cells requires Ca(2+)-entry through neighboring L-type Ca(2+) channels and is only augmented by SR Ca(2+)-release. Substantial activation of ICl(Ca) requires high Ca(2+) concentration in the dyadic clefts which can be effectively buffered by BAPTA, but not EGTA.

Role of Gap Junction Channel in the Development of Beat-to-Beat Action Potential Repolarization Variability and Arrhythmias

The short-term beat-to-beat variability of cardiac action potential duration (SBVR) occurs as a random alteration of the ventricular repolarization duration. SBVR has been suggested to be more predictive of the development of lethal arrhythmias than the action potential prolongation or QT prolongation of ECG alone. The mechanism underlying SBVR is not completely understood but it is known that SBVR depends on stochastic ion channel gating, intracellular calcium handling and intercellular coupling. Coupling of single cardiomyocytes significantly decreases the beat-to-beat changes in action potential duration (APD) due to the electrotonic current flow between neighboring cells. The magnitude of this electrotonic current depends on the intercellular gap junction resistance. Reduced gap junction resistance causes greater electrotonic current flow between cells, and reduces SBVR. Myocardial ischaemia (MI) is known to affect gap junction channel protein expression and function. MI increases gap junction resistance that leads to slow conduction, APD and refractory period dispersion, and an increase in SBVR. Ultimately, development of reentry arrhythmias and fibrillation are associated post-MI. Antiarrhythmic drugs have proarrhythmic side effects requiring alternative approaches. A novel idea is to target gap junction channels. Specifically, the use of gap junction channel enhancers and inhibitors may help to reveal the precise role of gap junctions in the development of arrhythmias. Since cell-to-cell coupling is represented in SBVR, this parameter can be used to monitor the degree of coupling of myocardium.

Systolic [Ca2+]i regulates diastolic levels in rat ventricular myocytes.

For the heart to function as a pump, intracellular calcium concentration ([Ca2+]i) must increase during systole to activate contraction and then fall, during diastole, to allow the myofilaments to relax and the heart to refill with blood. The present study investigates the control of diastolic [Ca2+]i in rat ventricular myocytes. We show that diastolic [Ca2+]i is increased by manoeuvres that decrease sarcoplasmic reticulum function. This is accompanied by a decrease of systolic [Ca2+]i such that the time‐averaged [Ca2+]i remains constant. We report that diastolic [Ca2+]i is controlled by the balance between Ca2+ entry and Ca2+ efflux during systole. The results of the present study identify a novel mechanism by which changes of the amplitude of the systolic Ca transient control diastolic [Ca2+]i.