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 (I_to) and late sodium current (I_Na,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 I_to. 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 I_Na,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.

中文翻译：

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瞬时受体电位 Melastatin 4 Channel Inhibitor 4-chloro-2-(2-chlorophenoxy)acetamido) 苯甲酸 (CBA) 在犬左心室心肌细胞中的电生理效应

瞬时受体电位褪黑素 4 (TRPM4) 在许多组织中发挥重要作用，包括心脏起搏器和传导组织，但对其在心室肌细胞中的电生理作用知之甚少。我们早期的结果表明 9-phenanthrol 缺乏选择性，因此选择 CBA（(4-chloro-2-(2-chlorophenoxy)acetamido) 苯甲酸）作为一种新的、具有潜在选择性的抑制剂。目标：我们的目的是阐明 CBA 在犬左心室心肌细胞中的作用和选择性，并研究 TRPM4 在犬心脏中的表达。在酶促分离的犬左心室心肌细胞中进行实验。在 37°C 下，在全细胞配置中使用动作电位 (AP) 电压钳技术记录离子电流。在移液管溶液中使用了 10 mM BAPTA，以排除 TRPM4 通道的潜在激活。AP 是用传统的尖锐微电极记录的。CBA 以 10 µM 的浓度使用。通过蛋白质印迹研究心脏中TRPM4蛋白的表达。TRPM4 蛋白在犬心脏的所有四个腔室壁以及从分离的左心室细胞制备的样品中表达。₉₀ . 此外，AP 振幅增加，0 相和 1 相的最大速率被药物降低。在 AP 钳位测量中，CBA 敏感电流包含短的、早期的外向电流，主要是长的内向电流。在 CBA 存在下，瞬时外向钾电流 (I _to ) 和迟发钠电流 (I _Na,L ) 分别降低了约 20% 和 47%，而 L 型钙电流和内向整流器钾电流不受影响。CBA 的这些影响在洗脱后在很大程度上是可逆的。根据我们的结果，CBA 诱导阶段 1 斜率的降低和 AP 幅度的轻微增加可能是由于 I_到. AP 缩短的趋势可以通过在平台期 AP 钳记录中看到的内向电流的抑制来解释。CBA 减少的这种内向电流可能是 I _Na,L，因此，CBA 对 TRPM4 通道并不完全有选择性。因此，与 9-phenanthrol 类似，它不能用于测试 TRPM4 通道对心室细胞心脏电生理的贡献，或者至少必须谨慎使用。