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

Abstract

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.

Differential effects of TRPM4 channel inhibitors on Guinea pig urinary bladder smooth muscle excitability and contractility: Novel 4‐chloro‐2‐[2‐(2‐chloro‐phenoxy)‐acetylamino]‐benzoic acid (CBA) versus classical 9‐phenanthrol

“…Instead, CBA displayed either no or weak inhibition on spontaneous, 20 mM KCl‐induced, and EFS‐induced contractions (Figures 1‐3). The modest CBA‐induced inhibition observed at the highest concentrations likely reflects non‐selectivity 36 . In contrast, 9‐phenanthrol strongly inhibited guinea pig UBSM contractility 3,13,15 .…”

“…Of note, for 20 mM KCl‐induced contractions, we observed an enhancement in the frequency for the highest CBA concentrations, most likely due to non‐selectivity. As CBA has been recently shown to inhibit transient outward K + current and late Na + current in ventricular myocytes, 36 this compound may lack optimal selectivity.…”

“…The modest CBA-induced inhibition observed at the highest concentrations likely reflects non-selectivity. 36 In contrast, 9-phenanthrol strongly inhibited guinea pig UBSM contractility. 3,13,15 In our experiments, the maximum concentration of DMSO to dissolve CBA was 0.3% (v/v).…”

“…As CBA has been recently shown to inhibit transient outward K + current and late Na + current in ventricular myocytes, 36 * of genetic animal models, whether global or inducible conditional, will be extremely useful as has been the case for elucidating the roles of TRPM4 channels in the heart, taste buds, brain, and pancreas. 3 8 ,42-4 6 Unfortunately, the specific TRPM4 knockout mouse or rat strains have not yet been made available to us.…”

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

“…The cardiac Kv11.1 (hERG) channel showed only <5% inhibition of dofetilide binding in the presence of 10 µM CBA. On the contrary, in canine left ventricular cells, 10 µM CBA reduced transient outward K + and late Na + currents by 20 and 47%, respectively [131]. Apart from being a TRPM4 blocker, CBA acts as a chemical chaperone that reduces TRPM4 degradation by the endoplasmic reticulum-associated process [78].…”