Although late sodium current (I Na-late) has long been known to contribute to plateau formation of mammalian cardiac action potentials, lately it was considered as possible target for antiarrhythmic drugs. However, many aspects of this current are still poorly understood. The present work was designed to study the true profile of I Nalate in canine and guinea pig ventricular cells and compare them to I Na-late recorded in undiseased human hearts. I Na-late was defined as a tetrodotoxin-sensitive current, recorded under action potential voltage clamp conditions using either canonic-or self-action potentials as command signals. Under action potential voltage clamp conditions the amplitude of canine and human I Na-late monotonically decreased during the plateau (decrescendoprofile), in contrast to guinea pig, where its amplitude increased during the plateau (crescendo profile). The decrescendo-profile of canine I Na-late could not be converted to a crescendo-morphology by application of ramplike command voltages or command action potentials recorded from guinea pig cells. Conventional voltage clamp experiments revealed that the crescendo I Na-late profile in guinea pig was due to the slower decay of I Na-late in this species. When action potentials were recorded from multicellular ventricular preparations with sharp microelectrode, action potentials were shortened by tetrodotoxin, which effect was the largest in human, while smaller in canine, and the smallest in guinea pig preparations. It is concluded that important interspecies differences exist in the behavior of I Na-late. At present canine myocytes seem to represent the best model of human ventricular cells regarding the properties of I Na-late. These results should be taken into account when pharmacological studies with I Na-late are interpreted and extrapolated to human. Accordingly, canine ventricular tissues or myocytes are suggested for pharmacological studies with I Na-late inhibitors or modifiers. Incorporation of present data to human action potential models may yield a better understanding of the role of I Na-late in action potential morphology, arrhythmogenesis, and intracellular calcium dynamics. with physiological and pathological significance recognized long ago [1-3], its pathophysiological role in LQT3 [4] and heart failure [5-8] has been emphasized only in the last decades. I Na-late-as an inward current-contributes to plateau formation and is responsible for largely

The role of transient receptor potential melastatin 4 (TRPM4) channels has been frequently tested using their inhibitor 9-phenanthrol in various cardiac preparations; however, the selectivity of the compound is uncertain. Therefore, in the present study, the concentration-dependent effects of 9-phenanthrol on major ionic currents were studied in canine isolated ventricular cells using whole-cell configuration of the patch-clamp technique and 10 mM BAPTA-containing pipette solution to prevent the Ca-dependent activation of TRPM4 channels. Transient outward (I), rapid delayed rectifier (I), and inward rectifier (I) K currents were suppressed by 10 and 30 μM 9-phenanthrol with the blocking potency for I < I < I and partial reversibility. L-type Ca current was not affected up to the concentration of 30 μM. In addition, a steady outward current was detected at voltages positive to -40 mV in 9-phenanthrol, which was larger at more positive voltages and larger 9-phenanthrol concentrations. Action potentials were recorded using microelectrodes. Maximal rate of depolarization, phase-1 repolarization, and terminal repolarization were decreased and the plateau potential was depressed by 9-phenanthrol (3-30 μM), congruently with the observed alterations of ionic currents. Significant action potential prolongation was observed by 9-phenanthrol in the majority of the studied cells, but only at 30 μM concentration. In conclusion, 9-phenanthrol is not selective to TRPM4 channels in canine ventricular myocardium; therefore, its application as a TRPM4 blocker can be appropriate only in expression systems but not in native cardiac cells.

Transient receptor potential melastatin 4 (TRPM4) plays an important role in many tissues, including pacemaker and conductive tissues of the heart, but much less is known about its electrophysiological role in ventricular myocytes. Our earlier results showed the lack of selectivity of 9-phenanthrol, so CBA ((4-chloro-2-(2-chlorophenoxy)acetamido) benzoic acid) was chosen as a new, potentially selective inhibitor. Goal: Our aim was to elucidate the effect and selectivity of CBA in canine left ventricular cardiomyocytes and to study the expression of TRPM4 in the canine heart. Experiments were carried out in enzymatically isolated canine left ventricular cardiomyocytes. Ionic currents were recorded with an action potential (AP) voltage-clamp technique in whole-cell configuration at 37 °C. An amount of 10 mM BAPTA was used in the pipette solution to exclude the potential activation of TRPM4 channels. AP was recorded with conventional sharp microelectrodes. CBA was used in 10 µM concentrations. Expression of TRPM4 protein in the heart was studied by Western blot. TRPM4 protein was expressed in the wall of all four chambers of the canine heart as well as in samples prepared from isolated left ventricular cells. CBA induced an approximately 9% reduction in AP duration measured at 75 and 90% of repolarization and decreased the short-term variability of APD90. Moreover, AP amplitude was increased and the maximal rates of phase 0 and 1 were reduced by the drug. In AP clamp measurements, CBA-sensitive current contained a short, early outward and mainly a long, inward current. Transient outward potassium current (Ito) and late sodium current (INa,L) were reduced by approximately 20 and 47%, respectively, in the presence of CBA, while L-type calcium and inward rectifier potassium currents were not affected. These effects of CBA were largely reversible upon washout. Based on our results, the CBA induced reduction of phase-1 slope and the slight increase of AP amplitude could have been due to the inhibition of Ito. The tendency for AP shortening can be explained by the inhibition of inward currents seen in AP-clamp recordings during the plateau phase. This inward current reduced by CBA is possibly INa,L, therefore, CBA is not entirely selective for TRPM4 channels. As a consequence, similarly to 9-phenanthrol, it cannot be used to test the contribution of TRPM4 channels to cardiac electrophysiology in ventricular cells, or at least caution must be applied.

Within computational models of neurons and myocytes, Markov models describe the underlying voltage-dependent dynamics of ion channels. The Markov model topology is traditionally fixed and chosen based on experimental insights and intuition. This human intuition breaks down, however, when recapitulating large amounts of experimental structural and functional data. We present a rigorous, systematic method for enumeration of biophysically plausible nonisomorphic, rooted topologies and for evaluation of the best topologies that recapitulate a range of experimental data. Markov model topologies are evaluated on their ability to recapitulate experimental channel dynamics using cloud computing. The model evaluation routine prevents overfitting by tracking relative training and validation errors and penalizes stiff model solutions. Due to the significant computational resources required, the code was containerized to utilize cloud computing resources. Containerization allows usage of a wide range of machines without preinstalling required packages. It also enables convenient distribution of the software for widespread use in the channel community. Biophysical constraints including maximum degree of vertices and eliminating long-range state connections reduced over 113 million nonisomorphic graphs to 218 thousand possible Markov topologies. Model topologies are clustered according to various graph theoretic measures such as the mean shortest path, degree of the root node, and number of edges. Graphs with a greater number of edges generally better reproduce canonical channel dynamics with more stable solutions. Including measures to prevent overfitting reduces optimization time and directs computational resources to more promising candidate models. The routine ensures models produced using this systematic approach have the highest fidelity to experimental data with the appropriately complex topology. These ''efficient'' Markov models are particularly valuable when scaling up to modeling complex modulated channel dynamics, such as drug and subunit interactions, at the cellular and tissue scales.