Norbert Szentandrássy

Endocardial versus epicardial differences in L-type calcium current in canine ventricular myocytes studied by action potential voltage clamp

OBJECTIVES The aim of the present study was to assess and compare the dynamics of L-type Ca(2+) current (I(Ca,L)) during physiologic action potential (AP) in canine ventricular cardiomyocytes of epicardial (EPI) and endocardial (ENDO) origin. METHODS I(Ca,L) was recorded on cells derived from the two regions of the heart using both AP voltage clamp and conventional whole cell voltage clamp techniques. RESULTS AP voltage clamp experiments revealed that the decay of I(Ca,L) is monotonic during endocardial AP, whereas the current is double-peaked (displaying a second rise) during epicardial AP. The amplitude of the first peak was significantly greater in ENDO (-4.6+/-0.8 pA/pF) than in EPI cells (-2.8+/-0.3 pA/pF). Application of epicardial APs as command pulses to endocardial cells yielded double-peaked I(Ca,L) profiles, and increased the net charge entry carried by I(Ca,L) during the AP from 0.187+/-0.059 to 0.262+/-0.056 pC/pF (n=5, P<0.05). No differences were observed in current densities and inactivation kinetics of I(Ca,L) between EPI and ENDO cells when studied under conventional voltage clamp conditions. Nisoldipine shortened action potentials and eliminated the dome of the epicardial AP. CONCLUSION I(Ca,L) was shown to partially inactivate before and deactivate during phase-1 repolarization and reopening of these channels is responsible for the formation of the dome in canine EPI cells. The transmural differences in the profile of I(Ca,L) could be well explained with differences in AP configuration.

A Multiscale Investigation of Repolarization Variability and Its Role in Cardiac Arrhythmogenesis

Enhanced temporal and spatial variability in cardiac repolarization has been related to increased arrhythmic risk both clinically and experimentally. Causes and modulators of variability in repolarization and their implications in arrhythmogenesis are however not well understood. At the ionic level, the slow component of the delayed rectifier potassium current (I(Ks)) is an important determinant of ventricular repolarization. In this study, a combination of experimental and computational multiscale studies is used to investigate the role of intrinsic and extrinsic noise in I(Ks) in modulating temporal and spatial variability in ventricular repolarization in human and guinea pig. Results show that under physiological conditions: i), stochastic fluctuations in I(Ks) gating properties (i.e., intrinsic noise) cause significant beat-to-beat variability in action potential duration (APD) in isolated cells, whereas cell-to-cell differences in channel numbers (i.e., extrinsic noise) also contribute to cell-to-cell APD differences; ii), in tissue, electrotonic interactions mask the effect of I(Ks) noise, resulting in a significant decrease in APD temporal and spatial variability compared to isolated cells. Pathological conditions resulting in gap junctional uncoupling or a decrease in repolarization reserve uncover the manifestation of I(Ks) noise at cellular and tissue level, resulting in enhanced ventricular variability and abnormalities in repolarization such as afterdepolarizations and alternans.

Activation of transient receptor potential vanilloid-3 inhibits human hair growth.

In the current study, we aimed at identifying the functional role of transient receptor potential vanilloid-3 (TRPV3) ion channel in the regulation of human hair growth. Using human organ-cultured hair follicles (HFs) and cultures of human outer root sheath (ORS) keratinocytes, we provide the first evidence that activation of TRPV3 inhibits human hair growth. TRPV3 immunoreactivity was confined to epithelial compartments of the human HF, mainly to the ORS. In organ culture, TRPV3 activation by plant-derived (e.g., eugenol, 10-1,000 μM) or synthetic (e.g., 2-aminoethoxydiphenyl borate, 1-300 μM) agonists resulted in a dose-dependent inhibition of hair shaft elongation, suppression of proliferation, and induction of apoptosis and premature HF regression (catagen). Human ORS keratinocytes also expressed functional TRPV3, whose stimulation induced membrane currents, elevated intracellular calcium concentration, inhibited proliferation, and induced apoptosis. Of great importance, these effects on ORS keratinocytes were all mediated by TRPV3, as small interfering RNA-mediated silencing of TRPV3 effectively abrogated the cellular actions of the above agonists. These findings collectively support the concept that TRPV3 signaling is a significant player in human hair growth control. Therefore, TRPV3 and the related intracellular signaling mechanism might function as a promising target for pharmacological manipulations of clinically relevant hair growth disorders.

Reverse rate dependency is an intrinsic property of canine cardiac preparations

AIMS Class III antiarrhythmic agents exhibit reverse rate-dependent lengthening of the action potential duration (APD). In spite of the several theories developed so far to explain this reverse rate dependency (RRD), its mechanism has not yet been clarified. The aim of the present work was to further elucidate the mechanisms responsible for reverse rate-dependent drug effects. METHODS AND RESULTS Action potentials were recorded from multicellular canine ventricular preparations and isolated cardiomyocytes, at cycle lengths (CLs) varying from 0.3 to 5 s, using conventional sharp microelectrodes. APD was either modified by applying inward and outward current pulses, or by superfusion of agents known to lengthen and shorten APD. Net membrane current (I(m)) was calculated from action potential waveforms. The hypothesis that RRD may be implicit in the relationship between I(m) and APD was tested by numerical modelling. Both drug-induced lengthening (by veratrine, BAY-K 8644, dofetilide, and BaCl(2)) and shortening (by lidocaine and nicorandil) of action potentials displayed RRD, i.e. changes in APD were greater at longer than at shorter CL. A similar dependency of effect on CL was found when repolarization was modified by injection of inward or outward current pulses. I(m) measured at various points during repolarization was inversely proportional to APD and to CL. Model simulations showed that RRD is expected as a consequence of the non-linearity of the relationship between I(m) and APD. CONCLUSION RRD of APD modulation is shared, although with differences in magnitude, by interventions of very different nature. RRD can be interpreted as a consequence of the relationship between I(m) and APD and, as such, is expected in all species having positive APD-CL relationship. This implies that the development of agents prolonging APD with direct rate dependency, or even completely devoid of RRD, may be difficult to achieve.

Effects of thymol on calcium and potassium currents in canine and human ventricular cardiomyocytes

Concentration‐dependent effects of thymol (1–1000 μM) was studied on action potential configuration and ionic currents in isolated canine ventricular cardiomyocytes using conventional microelectrode and patch clamp techniques. Low concentration of thymol (10 μM) removed the notch of the action potential, whereas high concentrations (100 μM or higher) caused an additional shortening of action potential duration accompanied by progressive depression of plateau and reduction of Vmax. In the canine cells L‐type Ca current (ICa) was decreased by thymol in a concentration‐dependent manner (EC50: 158±7 μM, Hill coeff.: 2.96±0.43). In addition, thymol (50–250 μM) accelerated the inactivation of ICa, increased the time constant of recovery from inactivation, shifted the steady‐state inactivation curve of ICa leftwards, but voltage dependence of activation remained unaltered. Qualitatively similar results were obtained with thymol in ventricular myocytes isolated from healthy human hearts. Thymol displayed concentration‐dependent suppressive effects on potassium currents: the transient outward current, Ito (EC50: 60.6±11.4 μM, Hill coeff.: 1.03±0.11), the rapid component of the delayed rectifier, IKr (EC50: 63.4±6.1 μM, Hill coeff.: 1.29±0.15), and the slow component of the delayed rectifier, IKs (EC50: 202±11 μM, Hill coeff.: 0.72±0.14), however, K channel kinetics were not much altered by thymol. These effects on Ca and K currents developed rapidly (within 0.5 min) and were readily reversible. In conclusion, thymol suppressed cardiac ionic channels in a concentration‐dependent manner, however, both drug‐sensitivities as well as the mechanism of action seems to be different when blocking calcium and potassium channels.

Inhibition of TASK-3 (KCNK9) channel biosynthesis changes cell morphology and decreases both DNA content and mitochondrial function of melanoma cells maintained in cell culture.

TASK-3 channel overexpression was shown to facilitate the survival of malignantly transformed cells, possibly by providing greater hypoxia tolerance through a still unknown mechanism. Although it has been suggested previously that TASK-3 channels are expressed in the mitochondrial membranes, their role here remains elusive. In this study, a transient transfection of TASK-3 knockdown melanoma cell cultures was produced to show the significance of TASK-3 expression. Reduction of the TASK-3 protein biosynthesis induced characteristic changes in cell morphology, reduced the amount of DNA and decreased metabolic activity and mitochondrial function of melanoma cells when compared with control. These findings indicate that TASK-3 channel expression and function is indispensable for the proliferation and/or survival of the melanoma cells, as they seem to contribute to their mitochondrial functions. The significance is that, in this study, we have shown that TASK-3 channels are expressed in the mitochondria of melanoma malignum cells, and they are essential for maintaining cellular integrity and viability. The TASK-3 knockdown melanoma cell line had altered morphology, reduced DNA content, decreased metabolic activity and impaired mitochondrial function. These data indicate that TASK-3 channels are functionally present in the mitochondria of the melanoma cells, and their function is essential for the survival of these cells, thus TASK-3 channels may be the possible targets of future anticancer therapy.

The Na+/Ca2+ exchange blocker SEA0400 fails to enhance cytosolic Ca2+ transient and contractility in canine ventricular cardiomyocytes

AIMS This study was designed to evaluate the effects of the Na(+)/Ca(2+) exchange (NCX) inhibitor SEA0400 on Ca(2+) handling in isolated canine ventricular myocytes. METHODS AND RESULTS Intracellular Ca(2+) ([Ca(2+)](i)) transients, induced by either field stimulation or caffeine flush, were monitored using Ca(2+) indicator dyes. [Ca(2+)](i)-dependent modulation of the inhibitory effect of SEA0400 on NCX was characterized by the changes in Ni(2+)-sensitive current in voltage-clamped myocytes. Sarcoplasmic reticulum (SR) Ca(2+) release and uptake were studied in SR membrane vesicles. Gating properties of single-ryanodine receptors were analysed in lipid bilayers. Ca(2+) sensitivity of the contractile machinery was evaluated in chemically skinned myocytes. In myocytes paced at 1 Hz, neither diastolic [Ca(2+)](i) nor the amplitude of [Ca(2+)](i) transients was significantly altered by SEA0400 up to the concentration of 1 microM, which was shown to inhibit the exchange current. The blocking effect of SEA0400 on NCX decreased with increasing [Ca(2+)](i), and it was more pronounced in reverse than in forward mode operation at every [Ca(2+)](i) examined. The rate of decay of the caffeine-induced [Ca(2+)](i) transients was decreased significantly by 1 microM SEA0400; however, this effect was only a fraction of that observed with 10 mM NiCl(2). Neither SR Ca(2+) release and uptake nor cell shortening and Ca(2+) sensitivity of the contractile proteins were influenced by SEA0400. CONCLUSION The lack of any major SEA0400-induced shift in Ca(2+) transients or contractility of myocytes can well be explained by its limited inhibitory effect on NCX (further attenuated by elevated [Ca(2+)](i) levels) and a concomitant reduction in Ca(2+) influx due to the predominantly reverse mode blockade of NCX and suppression of L-type Ca(2+) current.

Action potential clamp fingerprints of K+ currents in canine cardiomyocytes: their role in ventricular repolarization

Aim:  The aim of the present study was to give a parametric description of the most important K+ currents flowing during canine ventricular action potential.

Effects of β‐adrenoceptor stimulation on delayed rectifier K+ currents in canine ventricular cardiomyocytes

BACKGROUND AND PURPOSE While the slow delayed rectifier K+ current (IKs) is known to be enhanced by the stimulation of β‐adrenoceptors in several mammalian species, phosphorylation‐dependent regulation of the rapid delayed rectifier K+ current (IKr) is controversial.

Effect of thymol on kinetic properties of Ca and K currents in rat skeletal muscle

BackgroundThymol is widely used as a general antiseptic and antioxidant compound in the medical practice and industry, and also as a stabilizer to several therapeutic agents, including halothane. Thus intoxication with thymol may occur in case of ingestion or improper anesthesia. In the present study, therefore, concentration-dependent effects of thymol (30–600 micro-grams) were studied on calcium and potassium currents in enzymatically isolated rat skeletal muscle fibers using the double vaseline gap voltage clamp technique.ResultsThymol suppressed both Ca and K currents in a concentration-dependent manner, the EC50 values were 193 ± 26 and 93 ± 11 μM, with Hill coefficients of 2.52 ± 0.29 and 1.51 ± 0.18, respectively. Thymol had a biphasic effect on Ca current kinetics: time to peak current and the time constant for inactivation increased at lower (100–200 μM) but decreased below their control values at higher (600 μM) concentrations. Inactivation of K current was also significantly accelerated by thymol (200–300 μM). These effects of thymol developed rapidly and were partially reversible. In spite of the marked effects on the time-dependent properties, thymol caused no change in the current-voltage relationship of Ca and K peak currents.ConclusionsPresent results revealed marked suppression of Ca and K currents in skeletal muscle, similar to results obtained previously in cardiac cells. Furthermore, it is possible that part of the suppressive effects of halothane on Ca and K currents, observed experimentally, may be attributed to the concomitant presence of thymol in the superfusate.