Markéta Bébarová

Advances in patch clamp technique: towards higher quality and quantity.

The patch clamp technique, developed in late 1970s, started a new period of experimental cardiac electrophysiology enabling measurement of ionic currents on isolated cardiomyocytes down to the level of single channels. Since that time, the technique has been substantially improved by development of several upgraded modifications providing so far unavailable data (e.g. action potential clamp, dynamic clamp, high-resolution scanning patch clamp), or facilitating the patch clamp technique by increasing its efficiency (planar patch clamp, automated patch clamp). The current review summarizes the leading new patch clamp based techniques used in cardiac cellular electrophysiology, their principles and prominent related papers.

Effect of ethanol on action potential and ionic membrane currents in rat ventricular myocytes

Aim:  Even though alcohol intoxication is often linked to arrhythmias, data describing ethanol effect on cardiac ionic channels are rare. In addition, ethanol is used as a solvent of hydrophobic compounds in experimental studies. We investigated changes of the action potential (AP) configuration and main ionic membrane currents in rat cardiomyocytes under 20–1500 mm ethanol.

Arrhythmogenesis in Brugada syndrome: Impact and constrains of current concepts

Brugada syndrome (BrS), an inherited arrhythmogenic disease first described in 1992, is characterized by ST segment elevations on the electrocardiogram in the right precordium and by a high occurrence of arrhythmias including the life-threatening ventricular tachycardia/fibrillation. Knowledge of the underlying mechanisms of formation of arrhythmogenic substrate in BrS is essential, namely for the risk stratification of BrS patients and their therapy which is still restrained almost exclusively to the implantation of cardioverter/defibrillator. In spite of many crucial findings in this field published within recent years, the final consistent view has not been established so far. Hence, BrS described 20 years ago remains an actual topic of both clinical and experimental studies. This review presents an overview of the current knowledge related to the pathogenesis of BrS arrhythmogenic substrate, namely of the genetic basis of BrS, functional consequences of mutations related to BrS, and arrhythmogenic mechanisms in BrS.

Addictive drugs, arrhythmias, and cardiac inward rectifiers

In many addictive drugs including alcohol and nicotine, proarrhythmic effects were reported. This review provides an overview of the current knowledge in this field (with a focus on the inward rectifier potassium currents) to promote the lacking data and appeal for their completion, thus, to improve understanding of the proarrhythmic potential of addictive drugs.

The acceleration of cardiomyogenesis in embryonic stem cells in vitro by serum depletion does not increase the number of developed cardiomyocytes

The differentiation of pluripotent embryonic stem (ES) cells into various lineages in vitro represents an important tool for studying the mechanisms underlying mammalian embryogenesis. It is a key technique in studies evaluating the molecular mechanisms of cardiomyogenesis and heart development and also in embryotoxicology. Herein, modest modifications of the basic protocol for ES cell differentiation into cardiomyocytes were evaluated in order to increase the yield and differentiation status of developed cardiomyocytes. Primarily, the data show that ES cell cultivation in the form of non-adherent embryoid bodies (EBs) for 5 days compared to 8 days significantly improved cardiomyogenic differentiation. This is illustrated by the appearance of beating foci in the adherent EBs layer at earlier phases of differentiation from day 10 up to day 16 and by the significantly higher expression of genes characteristic of cardiomyogenic differentiation (sarcomeric alpha actinin, myosin heavy chain alpha and beta, myosin light chain 2 and 7, and transcriptional factor Nkx2.5) in EBs cultivated under non-adherent conditions for 5 days. The ratio of cardiomyocytes per other cells was also potentiated in EBs cultivated in non-adherent conditions for only 5 days followed by cultivation in adherent serum-free culture conditions. Nevertheless, the alteration in the percentage of beating foci among these two tested cultivation conditions vanished at later phases and also did not affect the total number of cardiomyocytes determined as myosin heavy chain positive cells at the end of the differentiation process on day 20. Thus, although these modifications of the conditions of ES cells differentiation may intensify cardiomyocyte differentiation, the final count of cardiomyocytes might not change. Thus, serum depletion was identified as a key factor that intensified cardiomyogenesis. Further, the treatment of EBs with N-acetylcysteine, a reactive oxygen species scavenger, did not affect the observed increase in cardiomyogenesis under serum depleted conditions. Interestingly, a mild induction of the ventricular-like phenotype of cardiomyocytes was observed in 5-day-old EBs compared to 8-day-old EBs. Overall, these findings bring crucial information on the mechanisms of ES cells differentiation into cardiomyocytes and on the establishment of efficient protocols for the cardiomyogenic differentiation of ES cells. Further, the importance of determining the absolute number of formed cardiomyocyte-like cells per seeded pluripotent cells in contrast to the simple quantification of the ratios of cells is highlighted.

Inward rectifying potassium currents resolved into components: modeling of complex drug actions

Inward rectifier potassium currents (IKir,x) belong to prominent ionic currents affecting both resting membrane voltage and action potential repolarization in cardiomyocytes. In existing integrative models of electrical activity of cardiac cells, they have been described as single current components. The proposed quantitative model complies with findings indicating that these channels are formed by various homomeric or heteromeric assemblies of channel subunits with specific functional properties. Each IKir,x may be expressed as a total of independent currents via individual populations of identical channels, i.e., channels formed by the same combination of their subunits. Solution of the model equations simulated well recently observed unique manifestations of dual ethanol effect in rat ventricular and atrial cells. The model reflects reported occurrence of at least two binding sites for ethanol within IKir,x channels related to slow allosteric conformation changes governing channel conductance and inducing current activation or inhibition. Our new model may considerably improve the existing models of cardiac cells by including the model equations proposed here in the particular case of the voltage-independent drug-channel interaction. Such improved integrative models may provide more precise and, thus, more physiologically relevant results.

P1078Correlation between magnitude of cardiac ionic currents and cell membrane capacity: always positive?

Introduction: The magnitude of cardiac ionic currents is generally considered as being directly proportional to the membrane area of investigated cell which may be determined by measurement of the cell membrane capacity. Therefore, the measured values of ionic currents are usually converted to the current density (by dividing measured current by estimated cell membrane capacity) to reduce differences among cells caused by their varying membrane area. This strategy is asserted in patch clamp studies on cardiomyocytes and, in most cases, also on cell lines heterogeneously expressing cardiac ionic channels. Our recent pilot data has shown that the correlation may be absent or even inverse in some cases which may lead to misinterpretation of the evaluated data. Purpose: We aimed to investigate the assumed correlation between the magnitude of cardiac ionic currents (absolute value) and membrane capacity of respective cells. Methods: Experiments were performed by the whole cell patch clamp technique on enzymatically isolated rat atrial myocytes (two inward rectifier potassium currents were investigated, namely IK1 and acetylcholine-sensitive current IK(Ach); at 23 ? 1?C) and on Chinese hamster ovary (CHO) cells transiently expressing human slowly activated delayed rectifier (IKs) channels (KCNQ1, KCNE1, Yotiao; at 37?C). Results: The significance test indicated that the Pearson’s coefficients of correlation (r) between the magnitude of inward component of IK1 or of acetylcholine-induced IK(Ach) and the capacity of respective rat atrial myocytes were significant (r = 0.79 and 0.64, n = 27 and 20, P ? 0.001 and 0.002, respectively). Surprisingly, analogical significant correlation was not apparent in the case of inward component of constitutively active IK(Ach) measured in rat atrial cells (r = 0.17, n = 56). In the case of CHO cells, the magnitude of expressed human IKs and the capacity of CHO cells did not significantly correlate as well. Furthermore, even negative correlation was indicated, i.e. the higher was the cell capacity, the smaller was this current (r = -0.22, n = 18). Conclusions: We proved the expected significant positive correlation between the magnitude of big ionic currents, namely IK1 and acetylcholine-induced IK(Ach), and the membrane area of rat atrial myocytes. On the contrary, no significant correlation was apparent in the case of small constitutively active IK(Ach). Importantly, even negative (but insignificant) correlation was observed in the case of human IKs transiently expressed in CHO cells. Hence, the usually used conversion of the current magnitude to the current density using the membrane capacity of respective cell should be used with caution, namely in the case of small ionic currents in cardiomyocytes and, especially, in the case of currents measured in cell lines. The cause of missing or even inverse proportionality between membrane current and capacity remains to be clarified.