Knockdown of the TRPM4 channel alters cardiac electrophysiology and hemodynamics in a sex- and age-dependent manner in mice.

TRPM4 is a calcium-activated, voltage-modulated, nonselective ion channel widely expressed in various cells and tissues. TRPM4 regulates the influx of sodium ions, thus playing a role in regulating the membrane potential. In the heart, TRPM4 is expressed in both cardiomyocytes and cells of the conductive pathways. Clinical studies have linked TRPM4 mutations to several cardiac disorders. While data from experimental studies have demonstrated TRPM4's functional significance in cardiac physiology, its exact roles in the heart have remained unclear. In this study, we investigated the role of TRPM4 in cardiac physiology in a newly generated Trpm4 knockdown mouse model. Male and female Trpm4 knockdown (Trpm4-/- ) and wild-type mice of different ages (5- to 12- week-old (young) and 24-week-old or more (adult)) were characterized using a multimodal approach, encompassing surface electrocardiograms (ECG), echocardiography recordings, ex vivo ECGs in isolated heart, endocardial mappings, Western blots, and mRNA quantifications. The assessment of cardiac electrophysiology by surface ECGs revealed no significant differences between wild-type and Trpm4-/- young (5- to 12-week-old) mice of either sex. Above 24 weeks of age, adult male Trpm4-/- mice showed reduced heart rate and increased heart rate variability. Echocardiography revealed that only adult male Trpm4-/- mice exhibited slight left ventricular hypertrophic alterations compared to controls, illustrated by alterations of the mitral valve pressure halftime, the mitral valve E/A ratio, the isovolumetric relaxation time, and the mitral valve deceleration. In addition, an assessment of the right ventricular systolic function by scanning the pulmonary valve highlighted an alteration in pulmonary valve peak velocity and pressure in adult male Trpm4-/- mice. Endocardial mapping recordings showed that applying 5 µM of the new TRPM4 inhibitor NBA triggered a third-degree atrioventricular block on 40% of wild-type hearts. These results confirm the key role of TRPM4 in the proper structure and electrical function of the heart. It also reveals differences between male and female animals that have never been reported. In addition, the investigation of the effects of NBA on heart function confirms the role of TRPM4 in atrioventricular conduction.

Species-Specific Effects of Cation Channel TRPM4 Small-Molecule Inhibitors.

Background: The Transient Receptor Potential Melastatin member 4 (TRPM4) gene encodes a calcium-activated non-selective cation channel expressed in several tissues. Mutations in TRPM4 have been reported in patients with different types of cardiac conduction defects. It is also linked to immune response and cancers, but the associated molecular mechanisms are still unclear. Thus far, 9-phenanthrol is the most common pharmacological compound used to investigate TRPM4 function. We recently identified two promising aryloxyacyl-anthranilic acid compounds (abbreviated CBA and NBA) inhibiting TRPM4. However, all aforementioned compounds were screened using assays expressing human TRPM4, whereas the efficacy of mouse TRPM4 has not been assessed. Mouse models are essential to investigate ion channel physiology and chemical compound efficacy. Aim: In this study, we performed comparative electrophysiology experiments to assess the effect of these TRPM4 inhibitors on human and mouse TRPM4 channels heterologously expressed in TsA-201 cells. Methods and Results: We identified striking species-dependent differences in TRPM4 responses. NBA inhibited both human and mouse TRPM4 currents when applied intracellularly and extracellularly using excised membrane patches. CBA inhibited human TRPM4, both intracellularly and extracellularly. Unexpectedly, the application of CBA had no inhibiting effect on mouse TRPM4 current when perfused on the extracellular side. Instead, its increased mouse TRPM4 current at negative holding potentials. In addition, CBA on the intracellular side altered the outward rectification component of the mouse TRPM4 current. Application of 9-phenanthrol, both intracellularly and extracellularly, inhibited human TRPM4. For mouse TRPM4, 9-phenanthrol perfusion led to opposite effects depending on the site of application. With intracellular 9-phenanthrol, we observed a tendency towards potentiation of mouse TRPM4 outward current at positive holding potentials. Conclusion: Altogether, these results suggest that pharmacological compounds screened using "humanised assays" should be extensively characterised before application in vivo mouse models.

Deletion of Trpm4 Alters the Function of the Nav1.5 Channel in Murine Cardiac Myocytes.

Transient receptor potential melastatin member 4 (TRPM4) encodes a Ca2+-activated, non-selective cation channel that is functionally expressed in several tissues, including the heart. Pathogenic mutants in TRPM4 have been reported in patients with inherited cardiac diseases, including conduction blockage and Brugada syndrome. Heterologous expression of mutant channels in cell lines indicates that these mutations can lead to an increase or decrease in TRPM4 expression and function at the cell surface. While the expression and clinical variant studies further stress the importance of TRPM4 in cardiac function, the cardiac electrophysiological phenotypes in Trpm4 knockdown mouse models remain incompletely characterized. To study the functional consequences of Trpm4 deletion on cardiac electrical activity in mice, we performed perforated-patch clamp and immunoblotting studies on isolated atrial and ventricular cardiac myocytes and surfaces, as well as on pseudo- and intracardiac ECGs, either in vivo or in Langendorff-perfused explanted mouse hearts. We observed that TRPM4 is expressed in atrial and ventricular cardiac myocytes and that deletion of Trpm4 unexpectedly reduces the peak Na+ currents in myocytes. Hearts from Trpm4-/- mice presented increased sensitivity towards mexiletine, a Na+ channel blocker, and slower intraventricular conduction, consistent with the reduction of the peak Na+ current observed in the isolated cardiac myocytes. This study suggests that TRPM4 expression impacts the Na+ current in murine cardiac myocytes and points towards a novel function of TRPM4 regulating the Nav1.5 function in murine cardiac myocytes.

Pathological activation of CaMKII induces arrhythmogenicity through TRPM4 overactivation.

TRPM4 is a Ca2+-activated nonselective cation channel involved in cardiovascular physiology and pathophysiology. Based on cellular experiments and numerical simulations, the present study aimed to explore the potential arrhythmogenicity of CaMKII-mediated TRPM4 channel overactivation linked to Ca2+ dysregulation in the heart. The confocal immunofluorescence microscopy, western blot, and proximity ligation assay (PLA) in HL-1 atrial cardiomyocytes and/or TRPM4-expressing TSA201 cells suggested that TRPM4 and CaMKII proteins are closely localized. Co-expression of TRPM4 and CaMKIIδ or a FRET-based sensor Camui in HEK293 cells showed that the extent of TRPM4 channel activation was correlated with that of CaMKII activity, suggesting their functional interaction. Both expressions and interaction of the two proteins were greatly enhanced by angiotensin II treatment, which induced early afterdepolarizations (EADs) at the repolarization phase of action potentials (APs) recorded from HL-1 cells by the current clamp mode of patch clamp technique. This arrhythmic change disappeared after treatment with the TRPM4 channel blocker 9-phenanthrol or CaMKII inhibitor KN-62. In order to quantitatively assess how CaMKII modulates the gating behavior of TRPM4 channel, the ionomycin-permeabilized cell-attached recording was employed to obtain the voltage-dependent parameters such as steady-state open probability and time constants for activation/deactivation at different [Ca2+]i. Numerical simulations incorporating these kinetic data into a modified HL-1 model indicated that > 3-fold increase in TRPM4 current density induces EADs at the late repolarization phase and CaMKII inhibition (by KN-62) completely eliminates them. These results collectively suggest a novel arrhythmogenic mechanism involving excessive CaMKII activity that causes TRPM4 overactivation in the stressed heart.

Hypoxia Enhances Endothelial Intercellular Adhesion Molecule 1 Protein Level Through Upregulation of Arginase Type II and Mitochondrial Oxidative Stress.

Hypoxia plays a crucial role in the pathogenesis of cardiovascular diseases. Mitochondrial enzyme arginase type II (Arg-II) is reported to lead to endothelial dysfunction and enhance the expression of endothelial inflammatory adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). In this study, we investigate the role of Arg-II in hypoxia-induced endothelial activation and the potential underlying mechanisms. Exposure of the human endothelial cells to hypoxia induced a time-dependent increase in Arg-II, HIF1α, HIF2α, and ICAM-1 protein level, whereas no change in the protein level of VCAM-1 and E-selectin was observed. Similar effects were obtained in cells treated with a hypoxia mimetic Dimethyloxaloylglycine (DMOG). Silencing HIF1α, but not HIF2α, reversed hypoxia-induced upregulation of Arg-II. Moreover, silencing Arg-II prevented the ICAM-1 upregulation induced by hypoxia or DMOG. Furthermore, the endothelial cells incubated under hypoxic condition or treated with DMOG or hypoxia enhanced monocyte adhesion, which was inhibited by silencing Arg-II. Lastly, silencing Arg-II prevented hypoxia-induced mitochondrial superoxide production in endothelial cells, and hypoxia-induced ICAM-1 upregulation was reversed by mitochondrial electron transport inhibitor rotenone. These data demonstrate that hypoxia enhances ICAM-1 protein level and monocyte-endothelial interaction through HIF1α-mediated increase in Arg-II protein level on leading to increased mitochondrial reactive oxygen species production. These effects of hypoxia on endothelial cells may play a key role in cardiovascular diseases. Our results suggest that Arg-II could be a promising therapeutic target to prevent hypoxia-induced vascular damage/dysfunction.