Pharmacological Modulation and (Patho)Physiological Roles of TRPM4 Channel-Part 1: Modulation of TRPM4.

Transient receptor potential melastatin 4 is a unique member of the TRPM protein family and, similarly to TRPM5, is Ca2+-sensitive and permeable to monovalent but not divalent cations. It is widely expressed in many organs and is involved in several functions by regulating the membrane potential and Ca2+ homeostasis in both excitable and non-excitable cells. This part of the review discusses the pharmacological modulation of TRPM4 by listing, comparing, and describing both endogenous and exogenous activators and inhibitors of the ion channel. Moreover, other strategies used to study TRPM4 functions are listed and described. These strategies include siRNA-mediated silencing of TRPM4, dominant-negative TRPM4 variants, and anti-TRPM4 antibodies. TRPM4 is receiving more and more attention and is likely to be the topic of research in the future.

Late Na+ Current Is [Ca2+]i-Dependent in Canine Ventricular Myocytes.

Enhancement of the late sodium current (INaL) increases arrhythmia propensity in the heart, whereas suppression of the current is antiarrhythmic. In the present study, we investigated INaL in canine ventricular cardiomyocytes under action potential voltage-clamp conditions using the selective Na+ channel inhibitors GS967 and tetrodotoxin. Both 1 µM GS967 and 10 µM tetrodotoxin dissected largely similar inward currents. The amplitude and integral of the GS967-sensitive current was significantly smaller after the reduction of intracellular Ca2+ concentration ([Ca2+]i) either by superfusion of the cells with 1 µM nisoldipine or by intracellular application of 10 mM BAPTA. Inhibiting calcium/calmodulin-dependent protein kinase II (CaMKII) by KN-93 or the autocamtide-2-related inhibitor peptide similarly reduced the amplitude and integral of INaL. Action potential duration was shortened in a reverse rate-dependent manner and the plateau potential was depressed by GS967. This GS967-induced depression of plateau was reduced by pretreatment of the cells with BAPTA-AM. We conclude that (1) INaL depends on the magnitude of [Ca2+]i in canine ventricular cells, (2) this [Ca2+]i-dependence of INaL is mediated by the Ca2+-dependent activation of CaMKII, and (3) INaL is augmented by the baseline CaMKII activity.

Electrophysiological Effects of the Transient Receptor Potential Melastatin 4 Channel Inhibitor (4-Chloro-2-(2-chlorophenoxy)acetamido) Benzoic Acid (CBA) in Canine Left Ventricular Cardiomyocytes.

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.

Ion current profiles in canine ventricular myocytes obtained by the "onion peeling" technique.

The profiles of ion currents during the cardiac action potential can be visualized by the action potential voltage clamp technique. To obtain multiple ion current data from the same cell, the "onion peeling" technique, based on sequential pharmacological dissection of ion currents, has to be applied. Combination of the two methods allows recording of several ion current profiles from the same myocyte under largely physiological conditions. Using this approach, we have studied the densities and integrals of the major cardiac inward (ICa, INCX, INa-late) and outward (IKr, IKs, IK1) currents in canine ventricular cells and studied the correlation between them. For this purpose, canine ventricular cardiomyocytes were chosen because their electrophysiological properties are similar to those of human ones. Significant positive correlation was observed between the density and integral of ICa and IKr, and positive correlation was found also between the integral of ICa and INCX. No further correlations were detected. The Ca2+-sensitivity of K+ currents was studied by comparing their parameters in the case of normal calcium homeostasis and following blockade of ICa. Out of the three K+ currents studied, only IKs was Ca2+-sensitive. The density and integral of IKs was significantly greater, while its time-to-peak value was shorter at normal Ca2+ cycling than following ICa blockade. No differences were detected for IKr or IK1 in this regard. Present results indicate that the positive correlation between ICa and IKr prominently contribute to the balance between inward and outward fluxes during the action potential plateau in canine myocytes. The results also suggest that the profiles of cardiac ion currents have to be studied under physiological conditions, since their behavior may strongly be influenced by the intracellular Ca2+ homeostasis and the applied membrane potential protocol.

Mexiletine-like cellular electrophysiological effects of GS967 in canine ventricular myocardium.

Enhancement of the late Na+ current (INaL) increases arrhythmia propensity in the heart, while suppression of the current is antiarrhythmic. GS967 is an agent considered as a selective blocker of INaL. In the present study, effects of GS967 on INaL and action potential (AP) morphology were studied in canine ventricular myocytes by using conventional voltage clamp, action potential voltage clamp and sharp microelectrode techniques. The effects of GS967 (1 µM) were compared to those of the class I/B antiarrhythmic compound mexiletine (40 µM). Under conventional voltage clamp conditions, INaL was significantly suppressed by GS967 and mexiletine, causing 80.4 ± 2.2% and 59.1 ± 1.8% reduction of the densities of INaL measured at 50 ms of depolarization, and 79.0 ± 3.1% and 63.3 ± 2.7% reduction of the corresponding current integrals, respectively. Both drugs shifted the voltage dependence of the steady-state inactivation curve of INaL towards negative potentials. GS967 and mexiletine dissected inward INaL profiles under AP voltage clamp conditions having densities, measured at 50% of AP duration (APD), of -0.37 ± 0.07 and -0.28 ± 0.03 A/F, and current integrals of -56.7 ± 9.1 and -46.6 ± 5.5 mC/F, respectively. Drug effects on peak Na+ current (INaP) were assessed by recording the maximum velocity of AP upstroke (V+max) in multicellular preparations. The offset time constant was threefold faster for GS967 than mexiletine (110 ms versus 289 ms), while the onset of the rate-dependent block was slower in the case of GS967. Effects on beat-to-beat variability of APD was studied in isolated myocytes. Beat-to-beat variability was significantly decreased by both GS967 and mexiletine (reduction of 42.1 ± 6.5% and 24.6 ± 12.8%, respectively) while their shortening effect on APD was comparable. It is concluded that the electrophysiological effects of GS967 are similar to those of mexiletine, but with somewhat faster offset kinetics of V+max block. However, since GS967 depressed V+max and INaL at the same concentration, the current view that GS967 represents a new class of drugs that selectively block INaL has to be questioned and it is suggested that GS967 should be classified as a class I/B antiarrhythmic agent.

Pharmacological Modulation and (Patho)Physiological Roles of TRPM4 Channel-Part 2: TRPM4 in Health and Disease.

Transient receptor potential melastatin 4 (TRPM4) is a unique member of the TRPM protein family and, similarly to TRPM5, is Ca2+ sensitive and permeable for monovalent but not divalent cations. It is widely expressed in many organs and is involved in several functions; it regulates membrane potential and Ca2+ homeostasis in both excitable and non-excitable cells. This part of the review discusses the currently available knowledge about the physiological and pathophysiological roles of TRPM4 in various tissues. These include the physiological functions of TRPM4 in the cells of the Langerhans islets of the pancreas, in various immune functions, in the regulation of vascular tone, in respiratory and other neuronal activities, in chemosensation, and in renal and cardiac physiology. TRPM4 contributes to pathological conditions such as overactive bladder, endothelial dysfunction, various types of malignant diseases and central nervous system conditions including stroke and injuries as well as in cardiac conditions such as arrhythmias, hypertrophy, and ischemia-reperfusion injuries. TRPM4 claims more and more attention and is likely to be the topic of research in the future.

Late sodium current and calcium homeostasis in arrhythmogenesis.

The cardiac late sodium current (INa,late) is the small sustained component of the sodium current active during the plateau phase of the action potential. Several studies demonstrated that augmentation of the current can lead to cardiac arrhythmias; therefore, INa,late is considered as a promising antiarrhythmic target. Fundamentally, enlarged INa,late increases Na+ influx into the cell, which, in turn, is converted to elevated intracellular Ca2+ concentration through the Na+/Ca2+ exchanger. The excessive Ca2+ load is known to be proarrhythmic. This review describes the behavior of the voltage-gated Na+ channels generating INa,late in health and disease and aims to discuss the physiology and pathophysiology of Na+ and Ca2+ homeostasis in context with the enhanced INa,late demonstrating also the currently accessible antiarrhythmic choices.

Late Sodium Current Inhibitors as Potential Antiarrhythmic Agents.

Based on recent findings, an increased late sodium current (INa,late) plays an important pathophysiological role in cardiac diseases, including rhythm disorders. The article first describes what is INa,late and how it functions under physiological circumstances. Next, it shows the wide range of cellular mechanisms that can contribute to an increased INa,late in heart diseases, and also discusses how the upregulated INa,late can play a role in the generation of cardiac arrhythmias. The last part of the article is about INa,late inhibiting drugs as potential antiarrhythmic agents, based on experimental and preclinical data as well as in the light of clinical trials.

Late sodium current in human, canine and guinea pig ventricular myocardium.

Although late sodium current (INa-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 INa-late in canine and guinea pig ventricular cells and compare them to INa-late recorded in undiseased human hearts. INa-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 INa-late monotonically decreased during the plateau (decrescendo-profile), in contrast to guinea pig, where its amplitude increased during the plateau (crescendo profile). The decrescendo-profile of canine INa-late could not be converted to a crescendo-morphology by application of ramp-like command voltages or command action potentials recorded from guinea pig cells. Conventional voltage clamp experiments revealed that the crescendo INa-late profile in guinea pig was due to the slower decay of INa-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 INa-late. At present canine myocytes seem to represent the best model of human ventricular cells regarding the properties of INa-late. These results should be taken into account when pharmacological studies with INa-late are interpreted and extrapolated to human. Accordingly, canine ventricular tissues or myocytes are suggested for pharmacological studies with INa-late inhibitors or modifiers. Incorporation of present data to human action potential models may yield a better understanding of the role of INa-late in action potential morphology, arrhythmogenesis, and intracellular calcium dynamics.