Aims Cardiac arrhythmias are a major factor in the occurrence of morbidity and sudden death in patients with cardiovascular disease. Disturbances of Ca2+ homeostasis in the heart contribute to the initiation and maintenance of cardiac arrhythmias. Extrasystolic increases in intracellular Ca2+ lead to delayed afterdepolarizations and triggered activity, which can result in heart rhythm abnormalities. It is being suggested that the Ca2+-activated nonselective cation channel TRPM4 is involved in the aetiology of triggered activity, but the exact contribution and in vivo significance are still unclear. Methods and results In vitro electrophysiological and calcium imaging technique as well as in vivo intracardiac and telemetric electrocardiogram measurements in physiological and pathophysiological conditions were performed. In two distinct Ca2+-dependent proarrhythmic models, freely moving Trpm4−/− mice displayed a reduced burden of cardiac arrhythmias. Looking further into the specific contribution of TRPM4 to the cellular mechanism of arrhythmias, TRPM4 was found to contribute to a long-lasting Ca2+ overload-induced background current, thereby regulating cell excitability in Ca2+ overload conditions. To expand these results, a compound screening revealed meclofenamate as a potent antagonist of TRPM4. In line with the findings from Trpm4−/− mice, 10 µM meclofenamate inhibited the Ca2+ overload-induced background current in ventricular cardiomyocytes and 15 mg/kg meclofenamate suppressed catecholaminergic polymorphic ventricular tachycardia-associated arrhythmias in a TRPM4-dependent manner. Conclusion The presented data establish that TRPM4 represents a novel target in the prevention and treatment of Ca2+-dependent triggered arrhythmias.

Ca 2+ activated non-selective (CAN) cation channels have been described in cardiomyocytes since the advent of the patch clamp technique. It has been hypothesized that this type of ion channel contributes to the triggering of cardiac arrhythmias. TRPM4 is to date the only molecular candidate for a CAN cation channel in cardiomyocytes. Its significance for arrhythmogenesis in living animals remains, however, unclear. In this study, we have tested whether increased expression of wild-type (WT) TRPM4 augments the risk of arrhythmias in living mice. Overexpression of WT TRPM4 was achieved via tail vein injection of adeno-associated viral vector serotype 9 (AAV9) particles, which have been described to be relatively cardiac specific in mice. Subsequently, we performed ECG-measurements in freely moving mice to determine their in vivo cardiac phenotype. Though cardiac muscle was transduced with TRPM4 viral particles, the majority of viral particles accumulated in the liver. We did not observe any difference in arrhythmic incidents during baseline conditions. Instead, WT mice that overexpress TRPM4 were more vulnerable to develop premature ventricular ectopic beats during exercise-induced β-adrenergic stress. Conduction abnormalities were rare and not more frequent in transduced mice compare to WT mice. Taken together, we provide evidence that overexpression of TRPM4 increases the susceptibility of living mice to stress-induced arrhythmias.

Cardiac arrhythmias pose a major threat to a patient's health, yet prove to be often difficult to predict, prevent and treat. A key mechanism in the occurrence of arrhythmias is disturbed Ca2+ homeostasis in cardiac muscle cells. As a Ca2+‐activated non‐selective cation channel, TRPM4 has been linked to Ca2+‐induced arrhythmias, potentially contributing to translating an increase in intracellular Ca2+ concentration into membrane depolarisation and an increase in cellular excitability. Indeed, evidence from genetically modified mice, analysis of mutations in human patients and the identification of a TRPM4 blocking compound that can be applied in vivo further underscore this hypothesis. Here, we provide an overview of these data in the context of our current understanding of Ca2+‐dependent arrhythmias. image

Cardiac conduction disorders are a common cause of lethal cardiac arrhythmias and can be caused by mutations in ion channels. Several studies suggest that mutations in the Transient Receptor Potential subfamily M member 4 (TRPM4) are responsible for hereditary forms of cardiac conduction disorders. The majority of mutations that are described in TRPM4 lead to gain-of-function of channel activity. Analysis of the first identified gain-of-function mutation, p.E7K in a cellular overexpression system suggests that attenuated deSUMOylation of the protein, leads to impaired endocytosis and consequently an increase in the number of functional channels in the plasma membrane. To test the idea that an increased number of TRPM4 channels leads to cardiac arrhythmias, we created a functional overexpression model of the TRPM4 channel in living mice. To this end, we overexpressed TRPM4 in mice using adeno-associated virus serotype 9 (AAV9) particles, which has been described as the most cardiotropic of AAV serotypes. Overexpression of TRPM4 was achieved via tail vein injection of AAV9 particles. Subsequently, we performed telemetric ECG-measurements in freely-moving mice to determine their in vivo cardiac phenotype. In baseline conditions, the heart rate and ECG-parameters were similar between TRPM4-overexpressing and WT mice. Additionally, the number of arrhythmic incidents in resting mice was not different between TRPM4-overexpressing mice and WT mice. Instead, WT mice overexpressing TRPM4 exhibited more premature ventricular ectopic beats during exercise-induces β-adrenergic stress. Conduction abnormalities were rare and was not increased in mice overexpressing TRPM4. Taken together, mice overexpressing TRPM4 were more prone to develop premature ventricular ectopic beats during exercise-induced β-adrenergic stress.

Rationale: TRPM4 is a Ca 2+ -activated non-selective cation channel that is abundantly expressed in the heart. Mutations in the Trpm4 gene are associated with human cardiac conduction disorders, including Progressive Familial Heart Block type I (PFHBI) and Brugada Syndrome. However, the mechanistic role and in vivo significance of TRPM4 in the triggering of cardiac arrhythmias is still completely unclear. Objective: To investigate the role of TRPM4 during pathological Ca 2+ handling in the heart. Methods and results: Using three in vivo pro-arrhythmic assays, we found that Trpm4 -/- mice show a reduced arrhythmic burden compared to control mice. First, aconitine intoxication resulted in severe cardiac arrhythmias, both in WT and Trpm4 -/- animals, but Trpm4 -/- mice developed significantly less ventricular ectopic beats (VEBs) and showed a lower arrhythmic score. Second, during ischemia-reperfusion, induced by 30 min of LAD ligation, significantly more WT animals developed arrhythmias compared to Trpm4 -/- mice. Third, catecholaminergic polymorphic ventricular tachycardia (CPVT) mice, carrying mutations in RyR2, were subjected to a stress test. Significantly more RyR +/R2474S -Trpm4 +/+ animals developed arrhythmias compared to RyR +/R2474S -Trpm4 -/- mice. Conclusion: Our data establish that TRPM4 represents a novel target in the prevention and treatment of cardiac arrhythmias.