TRPM4 in Cardiac Electrical Activity

Cardiovascular Research (2015) 108,21–30 REVIEW doi:10.1093/cvr/cvv213

TRPM4 in cardiac electrical activity

Romain Guinamard1*, Patrice Bouvagnet2, Thomas Hof1, Hui Liu3, Christophe Simard1, and Laurent Salle´ 1

1Groupe Signalisation, Electrophysiologie et Imagerie des Le´sions d’Ische´mie–Reperfusion Myocardique, EA4650, Universite´ de Caen Basse—Normandie, Sciences D, Esplanade de la Paix, CS 2 3 14032, 14032 Caen Cedex 5, France; EA 4173, Universite´ Lyon 1 and Hoˆpital du Nord-Ouest, Lyon, France; and Department of Anatomy, Hainan Medical College, Haikou, Hainan 571101, China Downloaded from https://academic.oup.com/cardiovascres/article/108/1/21/266658 by guest on 30 September 2021

Received 16 December 2014; revised 13 July 2015; accepted 31 July 2015; online publish-ahead-of-print 13 August 2015

+ Abstract TRPM4 forms a non-selective cation channel activated by internal Ca2 . Its functional expression was demonstrated in cardiomyocytes of several mammalian species including humans, but the channel is also present in many other tissues. The recent characterization of the TRPM4 inhibitor 9-phenanthrol, and the availability of transgenic mice have helped to clarify the role of TRPM4 in cardiac electrical activity, including diastolic depolarization from the sino-atrial node cells in mouse, rat, and rabbit, as well as action potential duration in mouse cardiomyocytes. In rat and mouse, pharmacological inhibition of TRPM4 prevents cardiac ischaemia–reperfusion injuries and decreases the occurrence of arrhythmias. Several studies have identified TRPM4 mutations in patients with inherited cardiac diseases including conduction blocks and Brugada syndrome. This review identifies TRPM4 as a significant actor in cardiac electrophysiology. ------Keywords TRPM4 † Calcium-activated non-selective cation channel † Cardioprotection † Brugada † Arrhythmias

1. Introduction 2. TRPM4 expression in the heart, 2+ ACa -activated non-selective monovalent cationic current (NSCCa), with biophysics, and regulatory + no permeability for Ca2 and a single-channel conductance close to 25 pS, properties was described in the early 2000s in cardiomyocytes from adult mice, rats, 1–3 4–6 and humans. Identifying this additional cardiac NSCCa sparked new 2.1 TRPM4 expression profile in the heart investigations, which were further boosted by the concomitant cloning TRPM4 mRNA is present in a large variety of tissues from human, of the transient receptor potential melastatin 4 (TRPM4) gene in these mouse, and rat, the heart being among the major TRPM4 expressing tis- species.7–9 This gene encodes an ion channel protein (TRPM4) whose cur- sues.7,8,25,26 Several studies evaluated TRPM4 mRNA or protein ex- rent properties resemble the aforementioned NSC current and is thus Ca pression in specific parts of the heart, as collected in Table 1. TRPM4 considered as its molecular counterpart. mRNA and/or protein is expressed in nodal and conductive tissue as Three recent developments further highlighted the multiple roles of shown in the mouse sino-atrial node (SAN) and bovine bundle TRPM4 current in the heart: first, the identification of the TRPM4 in- branch.14,30 It is also detected in the atrium, including in humans,2,27 hibitor 9-phenanthrol, which helped unmask the effects of TRPM4 cur- and in the ventricle.14,21,29 Interestingly, several studies reported a sig- rent modulation on small mammals heart functions10; secondly, the nificant weaker expression in ventricles than in atria in rat, mouse, and generation of Trpm4 null mice to study the impact of gene disrup- bovine.14,19,28,31 At the functional level, TRPM4 current has been re- tion11,12; and thirdly, the discovery by positional cloning that TRPM4 corded in rodent as well as in human cardiomyocytes.2,3,19,28,31,33 In mutations are associated with cardiac conduction block and Brugada addition to cardiomyocytes, Trpm4 mRNA has also been detected in syndrome in humans.13 – 15 These contemporary studies provide a por- rat ventricular fibroblasts and arterial smooth muscle cells and human trait of the role(s) of TRPM4 both in cardiac physiology and in patho- atrial fibroblasts.23,34,35 As discussed subsequently, cardiac TRPM4 ex- physiology, as reviewed previously.16– 18 In addition to influencing pression profile can be modified by human TRPM4 gene mutations13 – cardiac development and remodelling19,20 or regulating cardiac con- 15,36 or cardiac diseases such as hypertrophy in rat.28 tractility,21 TRPM4 participates in cardiac electrical activity. This review will sharply focus on this last contribution by collecting existing data. Note that in addition to its role(s) in cardiac activity, TRPM4 is im- 2.2 TRPM4 biophysical properties plicated in other physiological functions such as the immune response, The biophysical and regulatory properties of TRPM4 were described in insulin secretion, breathing activity, smooth muscle contraction, arterial the early of 2000s7,25,37 and have been reviewed extensively.16,38 The constriction, and cell death.16,22 – 24 Trpm4 gene is located on human chromosome 19 and encodes a

* Corresponding author. Tel: +33 2 31 56 51 39; fax: +33 2 31 56 54 53, Email: [email protected] Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2015. For permissions please email: [email protected]. 22 R. Guinamard et al.

Table 1 Cardiac expression of TRPM4 mRNA, protein, and current

Species Heart SAN Atrium Ventricle Conductive tissue References ...... Human mRNA Launay et al.7 mRNA Nilius et al.25 mRNA, current Guinamard et al.2 mRNA, protein Zhang et al.27 mRNA mRNA mRNA (higher) Kruse et al.13 Rat mRNA mRNA (lower) Guinamard et al.28 Current Guinamard et al.28 Protein Protein Piao et al.29 Current Zhainazarov3 Downloaded from https://academic.oup.com/cardiovascres/article/108/1/21/266658 by guest on 30 September 2021 Mouse mRNA Murakami et al.8 mRNA Nilius et al.25 mRNA Jang et al.26 mRNA mRNA mRNA (lower) Demion et al.19 mRNA, protein, current Demion et al.30 Demion et al.30 Protein Protein (lower) Mathar et al.31 Current Simard et al.32 Current Mathar et al.21 Bovine Protein Protein (lower) Protein Liu et al.14

Reported currents were recorded from freshly isolated cardiomyocytes. Higher and lower indicate the level of mRNA or protein expression compared with other parts of the heart.

1214-amino-acid protein,7 whose tertiary structure features six trans- myocytes, indicating that the current can activate in an intact cell with 2+ 45 membrane domains. The functional channel is a homotetramer. physiological [Ca ]i. TRPM4 has calmodulin-binding sites, Walker B motifs, ATP, and phos- phatidyl inositol 4,5-bisphosphate (PIP2) binding sites, a glycosylation site and putative phosphorylation sites for protein kinase A (PKA) 3. Tools to unmask TRPM4 and protein kinase C (PKC).16,38 –40 contributions to cardiac electrical TRPM4-transfected HEK-293 cells develop an ionic current with a activity linear unitary current–voltage relationship and a conductance between 20 and 25 pS,7,34 similar to endogenous TRPM4 currents recorded 3.1 Pharmacological inhibitors in cardiomyocytes (Figure 1). The channel is equally permeable to 46 + + 2+ Flufenamic acid inhibits TRPM4 (IC50 ¼ 3 mmol/L), but this promis- Na and K ,butnotpermeabletoCa (permeability sequence: 47 + + + + + cuous drug also affects a variety of other channels. In addition, Na K . Cs . Li »Ca2 ).7,41 Channel open probability TRPM4 is inhibited by glibenclamide (10 mmol/L),30 spermine increases with membrane depolarization, resulting in an outwardly rec- (IC ¼ 61 mmol/L),37 and quinine (IC from 113 to 450 mmol/L),48 tifying current at the whole-cell level.7,25 50 50 but again, these drugs also inhibit other channels.38

More recently, 9-phenanthrol was shown to inhibit TRPM4 (IC50 ¼ 2.3 Regulatory properties of the TRPM4 17 mmol/L).10 It can be applied to the inside or the outside of the mem- current brane and most probably acts directly on the channel as it is effective in 10 The intracellular effector PKC as well as PIP2 and oxygen species such cell-free patches. 9-Phenanthrol has been tested on other channels, 1,42 – 44 as H2O2 increases TRPM4 activity. Conversely, TRPM4 activity is including various transient receptor potential (i.e. TRP) channels inhibited by internal adenine nucleotides such as ATP and ADP.37 (including TRPC3, TRPC6, TRPM5, and TRPM7), and so far has shown 2+ 10,49 – 51 Ca activates the TRPM4 channel with an EC50 up to 370 mmol/L a specificity for TRPM4 within this family. Nevertheless, high when measured in the inside-out configuration, but from 20 to concentrations of 9-phenanthrol (0.1 mmol/L) inhibit the delayed out- + + 0.5 mmol/L in the whole-cell configuration after overexpression in ward rectifier K current and the voltage-gated Ca2 current in mouse cell lines.7,42 This discrepancy unmasks that TRPM4 sensitivity to cardiomyocytes, whereas lower concentrations do not.32 Further- 2+ 42,43 2 Ca is modulated by regulators such as calmodulin and PIP2. more, a recent study showed that 9-phenanthrol inhibits the Cl chan- Inside-out recordings of TRPM4 current from human atrial cardiomyo- nel TMEM16A over a concentration range that was previously 52 cytes revealed an EC50 at 21 mmol/L with channel openings recorded associated with TRPM4-specific effects. In addition to modulating 2 even at 0.1 mmol/L (Figure 1). This EC50 is probably lower in intact these ion channels, 9-phenanthrol also inhibits bovine heart cells, similar to heterologous TRPM4 in HEK cells25,37 and thus cyclic-AMP-dependent PKA catalytic subunit, although these data 2+ TRPM4 might be able to activate at physiological [Ca ]i. Accordingly, were collected only using biochemical assays in vitro and were never whole-cell recordings in mouse ventricular cardiomyocytes exhibit a confirmed in cellular models.53 As described subsequently, comparing + Ca2 -transient-activated current attributed to TRPM4 that was absent the effect of 9-phenanthrol in wild-type (WT) vs. knock-out animals has from Trpm4 knock-out mice.21 In addition, unitary TRPM4 currents are been a valuable complement to confirm that TRPM4 is the primary tar- detectable in cell-attached patches from dedifferentiated rat ventricular get of 9-phenanthrol. TRPM4 in cardiac electrical activity 23

knock-out second strain develops cardiac hypertrophy and exhibits ECG perturbations that include conduction blocks, sinus pauses, and enhanced ectopic atrial activity.19 This discrepancy between the phenotypes of the two strains is not yet understood. However, experi- mentsconductedonmulticellularpreparationsorisolated cardiomyocytes from these mice have given valuable data regarding the role of TRPM4 in cardiac electrical activity, as described in the following sections. Although pharmacological approaches reveal the direct effects of ion channels on electrical activity, transgenic (knock-out) animals can reveal more integrated functions of ion channels in situ, but their pheno-

types can be more difficult to interpret due to developmental Downloaded from https://academic.oup.com/cardiovascres/article/108/1/21/266658 by guest on 30 September 2021 modifications or compensatory effects. From the standpoint of cardiac + + + electrophysiology, major currents (voltage-gated Ca2 ,K , and Na + currents) as well as Ca2 transients are not affected in Trpm4 knock- out mice, which indicate that the global cardiac channel pattern is not strongly modified so the model remains useful to unmask TRPM4 contributions to electrical activity.21 Note that the newly designed cardiac-specific Trpm4 knock-out mice may give valuable results in the future by excluding extracardiac impact of TRPM4 disruption.20 It is already known that this mouse does not show ECG variations in basal conditions, compared with WT.20

3.3 Contribution of human genetics Exploration of TRPM4 function in electrical cardiac activity beneﬁted from mutational screening of patients with cardiac disturbances. Car- diac dysfunction in patients with TRPM4 mutations does not necessar- ily indicate, by itself, that TRPM4 is directly implicated in cardiac electrical activity because developmental or regulatory modiﬁcations can occur, such as those in transgenic animals. However, as described subsequently, it was a valuable complement to animal models and in vitro experiments.

Figure 1 Biophysical properties of TRPM4. (A) TRPM4 current/ 4. TRPM4 in the control of voltage relationship in inside-out membrane patches from human atrial cardiomyocytes with 140 mmol/L NaCl in the pipette and membrane potential in cardiac cells 140 mmol/L (black squares) or 14 mmol/L (open circles) NaCl in 4.1 Modulation of diastolic depolarization the bath. Vm corresponds to the membrane potential. Equilibrium po- + tentials are indicated for Na and Cl2 for the experiment with and beating rate in the SAN 14 mmol/L NaCl in the bath. Permeability ratios compared with Sinus rhythm, at the onset of cardiac activity, arises from spontaneous + + 2+ 2 Na permeability are indicated for K ,Ca ,andCl .(B)Cardiac action potential (AP) generation in the sino-atrial pacemaker cells. 2+ TRPM4 open probability (Po) as a function of [Ca ]i.Upperinset + These cells express a specific pattern of ion channels and transporters shows inside-out recordings with various [Ca2 ] (V ¼ +40 mV). c i m that produce a spontaneous diastolic depolarization (DD), which and o correspond to the close and open states. (C) Cardiac TRPM4 2 causes membrane potential trajectory to pass the activation threshold open probability (Po) as a function of voltage (see Guinamard et al. 2+ for details). of T-type and L-type Ca currents (ICa,T; ICa,L). The DD is mediated by the hyperpolarization-activated cyclic nucleotide-gated ion channel + (HCN)54,55 and an inward Ca2 -activated current evoked by local + Ca2 releases from the sarcoplasmic reticulum.56 In mouse SAN, the + + 3.2 Trpm4 knock-out mice main component of the Ca2 -activated current is due to the Na / + + Two groups had developed Trpm4 knock-out mice from the 129/SvJ Ca2 exchanger, but an additional Ca2 -activated current can be re- + and C57bl/6J strains.11,12 The two groups first reported altered im- corded in the presence of Li .57 This last current conducts monovalent + + mune response, and mice were later used to evaluate cardiovascular cations (Na and K ), as observed in rabbit SAN cells.58 Because func- roles of TRPM4.19,31 Surprisingly, the cardiac phenotypes of these tional TRPM4 single-channel currents were reported in mouse isolated two Trpm4 knock-out mice differ one from the other. The first one SAN cells30 and two different mutations in the TRPM4 gene have been does not develop a specific cardiac phenotype under basal conditions reported in patients with bradycardia,36 TRPM4 was considered as a and its electrocardiogram (ECG) remains normal. However, the in- candidate to support this current. crease in contractility in response to b-adrenergic stimulation is en- In freely beating isolated right atrial preparations from mouse, rat, hanced in this first Trpm4 knock-out strain.21 In contrast, the and rabbit, which retain the SAN, 9-phenanthrol reversibly reduces 24 R. Guinamard et al.

the beating rate with an IC50 matching TRPM4 inhibition. It has no effect generation by inﬂuencing the DD (Figure 2). Because the experiments when the experiments are conducted in Trpm4 knock-out mice iso- were performed on isolated atria, it excludes any participation of exter- lated atrium (Figure 2),59 which indicates that 9-phenanthrol acts via nal regulatory processes such as modiﬁcations of neuro-hormonal reg- Trpm4 inhibition and not by modulating other ion channel targets in ulations by 9-phenanthrol. Because isolated right atria have a lower this preparation. Moreover, in rabbit SAN cells, 9-phenanthrol reduces beating rate than the heart rate of conscious animals (300 vs. the slope of the DD.59 Thus TRPM4 participates in sinus rhythm 500 b.p.m.), 9-phenanthrol may have elucidated a phenomenon that is absent in vivo. Unfortunately, no data are available yet regarding in vivo sinus rhythm regulation by pharmacological modulation of TRPM4. However, a recent study using telemetric ECG recordings in conscious and unrestrained mice showed a higher incidence of sinus pauses in Trpm4 knock-out mice than in littermate controls, which is 19

consistent with a contribution of TRPM4 in sinus activity in vivo. Downloaded from https://academic.oup.com/cardiovascres/article/108/1/21/266658 by guest on 30 September 2021 2+ The precise level of [Ca ]i reached during the local release in SAN cells is not well known. A computer simulation study indicates that 2+ [Ca ]i variations of the order of 1 mmol/L would be sufficient to activate TRPM4 and thus augment the DD slope in SAN cells.60 Interestingly, the effect of 9-phenanthrol is inversely proportional to initial beating rate: the lower the initial beating rate, the stronger its reduction by 9-phenanthrol.59 These data suggest that TRPM4 would activate only during bradycardia. In addition, these in vitro results may explain the lack of disparity in the heart rates measured in vivo in WT vs. Trpm4 knock-out mice,19,21 as well as in cardiac-specific Trpm4 knock-out mice.20 Indeed, these mice are not spontaneously bradycar- dic and thus do not express TRPM4 activity. In vivo, isoprenaline infusion increases the heart rate slightly more in Trpm4 knock-out mouse than in WT controls.21 However, that effect is not reproduced in isolated right atria from those animals, suggesting that the difference in vivo does not occur through a direct regulation of cardiac TRPM4 activity by b-adrenergic stimulation.21 The stronger effects of 9-phenanthrol at low beating rate further suggest that the channel may have a stronger influence in humans than in mice because the resting heart rate is around 60 b.p.m. instead of 500 b.p.m. Bradycardia was reported in three patients with Y790H TRPM4 mutations.36 It is tempting to speculate that bradycardia may be due to a decrease in TRPM4 expression in these patients. Unfortu- nately, biophysical and expression properties of this mutant were not evaluated. Moreover, the meta-analysis of data from patients with TRPM4 mutations13 – 15,36 does not provide any persuasive results regarding a relation between TRPM4 expression level and heart rate. Thelackofclaritymaybeduetothesmallcohortsizeofpatients with such mutations. 4.2 Modulation of the AP in cardiomyocytes Figure 2 Effect of TRPM4 inhibition on cardiac pacing and diastolic TRPM4 participates in both atrial and ventricular APs in mouse. Intra- depolarization in SAN. (A) An intracellular microelectrode impaling an cellular microelectrode recordings on isolated atria revealed that isolated right atrium (RA) is schematized on the left panel. Spontan- eous beating rate as a function of time is reported for an isolated 9-phenanthrol reduces AP duration without affecting the resting mem- 33 mouse atrium. There is no significant difference between beating rates brane potential or AP amplitude (Figure 3). The effect was preserved + + from Trpm4 / (black line) and Trpm42/2 (dotted line) mice. Appli- in the presence of H-89, a PKA inhibitor, excluding a side effect of cation of 9-phenantrhol produces a reduction in the beating rate in 9-phenanthrol on this kinase. As the 9-phenanthrol-induced AP short- + + Trpm4 / but not in Trpm42/2 mouse atrium. (B) Representative ening did not occur in atria from Trpm4 knock-out mice, we can be con- APs from experiments presented in (A), in control and with fident that 9-phenanthrol effects on other channels are not responsible 59 9-phenanthrol (see Hof et al. for details). (C) Preparation including for this phenomenon.33,52,61 Moreover, a comparison of APs recorded an isolated right atrium which was opened to unmask the SAN is sche- on isolated atria from WT and Trpm4 knock-out mice revealed a 25% matized on the left panel. The right panel reported the AP recorded shortened AP induced by gene disruption.33 This AP shortening in from rabbit SAN in control or under the superfusion of 9-phenanthrol Trpm4 knock-out mice was confirmed by patch-clamp experiments 1025 mol/L. (D) Schematized SAN cells AP with theoretical reversal on atrial isolated cardiomyocytes and shown to be independent of potentials (EX) for ions. Main ionic currents involved in each phase of 2+ + 19 2+ voltage-gated Ca or K channel modulation. AP are indicated. [Ca ]i variations are indicated under AP (see Hof et al.59 for details). The role of TRPM4 in ventricular AP remains incompletely understood. Intracellular microelectrode recordings from papillary muscles TRPM4 in cardiac electrical activity 25

+ repolarization, while Ca2 channels are still open.21 In contrast, intracellular microelectrode recordings in the ventricular wall of the C57bl/ 6J mice did not reveal any effect of 9-phenanthrol superfusion on AP parameters, even though this drug reduced AP duration in the atria.33 In addition, APs recorded in isolated ventricular cardiomyocytes were indistinguishable in WT and Trpm4 knock-out mice.19 Whether this discrepancy between studies is due to mice strain (129/SvJ vs. C57bl/6J), preparation (papillary muscle vs. ventricular wall), or technical approaches (intracellular microelectrode vs. patch clamp) remains to be determined to reconcile these results. Three properties of TRPM4 current explain its sole inﬂuence on car-

diomyocyte AP duration: (i) TRPM4 open probability increases with Downloaded from https://academic.oup.com/cardiovascres/article/108/1/21/266658 by guest on 30 September 2021 depolarization1 – 3,7,25,30,33 and thus emerges as the AP begins; (ii) 2+ 1 – 3,7,30,33 TRPM4 is activated by [Ca ]i, which increases during AP; + + and (iii) TRPM4 is equally permeable for Na and K ions,7 predicting a reversal potential of the TRPM4 current between 0 and 220 mV. Ac- cordingly, TRPM4 conducts inward current at negative membrane potentials, which favours depolarization, and outward current at positive membrane potentials. Thus, TRPM4 activation tends to drive the membrane potential close to 0 mV, which prolongs the plateau phase of the cardiac AP (Figure 3). Data from ventricular cardiomyocytes indicate + that variations in AP duration modify the driving force for Ca2 entry and thus impact excitation–contraction coupling and mechanical activity.21 TRPM4 participation in the cardiomyocyte AP has only been evaluated in mouse.19,21,33 The role(s) of TRPM4 in cardiomyocyte AP may be different in humans as the plateau phase of the AP is more promin- ent compared with mouse.62,63

5. TRPM4 in cardiac diseases Figure 3 Involvement of TRPM4 in cardiomyocyte AP. (A) Prepar- 5.1 Hypoxia–reoxygenation arrhythmias ation including an isolated right atrium (RA), an intracellular micro- TRPM4 was suspected to participate in electrical perturbations follow- electrode, and a generator to stimulate the preparation are ing cardiac ischaemia and reperfusion episodes because of its sensitivity schematized on the left panel. Effect of TRPM4 inhibition or disruption + to Ca2 and ATP.64,65 Myocardial ischaemia (thus hypoxia) reduces on atrial AP is illustrated on the right panel. Black trace: stimulated AP + + + 2 recorded from a Trpm4 / mouse-isolated atrium using intracellular ATP levels leading to [Ca ]i accumulation, a phenomenon exacer- 2+ microelectrode under superfusion of physiological solution. Grey bated at the first time of reperfusion. This [Ca ]i overload is in part dotted trace: application of the TRPM4 inhibitor 9-phenanthrol pro- responsible for triggered activity that arises before the end of AP repo- duces a shortening of the AP without affecting other parameters. larization, which has been dubbed the early afterdepolarization Black dotted trace: AP recorded from a Trpm42/2 mouse is shorter (EAD).66 – 68 +/+ 33 than that from the Trpm4 mouse (see Simard et al. for details). In a model of hypoxia–reoxygenation on isolated mouse interventri- (B) Upper panel: schematized cardiac AP with theoretical reversal po- cular septum, EADs were observed under reoxygenation, but both tentials (EX) for ions. Main ionic currents involved in each phase of the TRPM4 inhibitors 9-phenanthrol and flufenamic acid reduced their oc- AP are indicated. Black line: theoretical AP without TRPM4. Dotted 32 + currence. A significant effect of 9-phenanthrol was observed at con- line: theoretical AP after TRPM4 activation. [Ca2 ] variations are in- i centrations starting from 1025 M, a concentration that does not dicated under AP. Lower panel: schematized cardiomyocyte indicating + 2+ the impact of Na and Ca channels opening on TRPM4 activity. SR, modulate voltage-gated K or Ca channels in cardiomyocytes, but sarcoplasmic reticulum; DVm, membrane depolarization; RyR, ryano- it was not tested on Trpm4 knock-out mice to definitely exclude other dine receptor. targets. Note that the cardioprotective effect of 9-phenanthrol was also observed on hypoxia-induced arrhythmias.32 Interestingly, in a mouse model of ischaemic heart failure induced by ligation of the left anterior descending (LAD) artery, there was a trend in Trpm4 knock-out mice derived from the 129/SvJ strain revealed a showing fewer ventricular ectopic beats in Trpm4 knock-out mouse.69 10% shortened AP, compared with WT. Further, using patch-clamp re- Perhaps, due to the low number of animal subjects (six WT and four + cordings, the authors unmasked a Ca2 -activated whole-cell current in knock-outs), the difference did not reach significance, according isolated ventricular cardiomyocytes from WT animals that disappeared to the conventional statistical hypothesis tests. However, if these in Trpm4 knock-out.21 AP shortening in these Trpm4 knock-out mice data can be replicated and the effect confirmed, then these in vivo 2+ 2+ results in an enhanced Ca influx through L-type Ca channel be- results would be consistent with the in vitro experiment, showing + cause of the increased driving force for Ca2 influx due to earlier that the pharmacological inhibition of TRPM4 reduced ventricular 26 R. Guinamard et al. hypoxia-induced arrhythmias.32 In the LAD artery model, the authors when expressed in HEK-293 cells. Although this may indicate that a re- also reported a higher survival rate in the Trpm4 knock-out group a duction in TRPM4 functional expression could result in the Brugada week after surgery; however, mice died from left ventricular rupture syndrome, two other mutations (T873I and L1075P) showed no signifi- or pulmonary oedema but not from arrhythmias.69 cant variation in TRPM4 current, compared with WT.15 More perplex- Another study evaluated the long-term effect of 9-phenanthrol ing, the mutation G844D identified in a familial case with conduction when applied under pre-conditioning conditions in isolated rat heart.70 block was also found in the cohort of patients with Brugada syndrome, It produced a cardioprotective effect on contractile function, limited but this particular mutation was demonstrated to lead to a TRPM4 gain tissue damage, and proved to be prophylactic against hypoxia– of function in HEK-293 cells.14,15 reoxygenation-induced ventricular arrhythmias.70 Note that induction Although the relation between TRPM4 mutation and inherited of ischaemia–reperfusion for 40 min in rats before lethal anaesthesia cardiac diseases is established, several points remain to be clarified: increased the expression level of Trpm7 mRNA, but not that of (i) heterologous expression in CHO or HEK-293 cells revealed that 71

Trpm4 mRNA. Thus, regarding TRPM4, the effect of ischaemia– several mutations modified TRPM4 protein and current expression, Downloaded from https://academic.oup.com/cardiovascres/article/108/1/21/266658 by guest on 30 September 2021 reperfusion most likely occurs through modulation of channel activity but this remains to be confirmed in cardiac cells; (ii) modelling the im- rather than gene expression. pact of increasing or decreasing the TRPM4 current may provide one Altogether, these data indicate that, even if the pathway is not clearly part of the mechanism, but the contribution of TRPM4 in cardiomyo- identified yet, TRPM4 is involved in hypoxia and hypoxia–reoxygena- cytes is not totally understood and, even worse, not described in the tion injuries, including arrhythmias, and thus TRPM4 might be an inter- conductive tissue; and (iii) the potential impact of TRPM4 mutations esting new target for cardioprotection. on cardiac development and global cardiovascular system, as shown in Trpm4 knock-out mice, has to be evaluated. 5.2 Inherited cardiac diseases Note that Trpm4 knock-out mice exhibit multilevel conduction 19 Genetic testing for inherited cardiac arrhythmias identified a number of blocks, including first- and second-degree AVBs. Although second- genes, several of those encoding ion channels.72,73 It most often leads degree AVB is most likely due to paroxysmal parasympathetic over- to complex interpretations as mutations of the same ion channel can be drive, first-degree AVB is not and thus is probably attributable to related to several pathologies and, reciprocally, a single pathology can changes in ion channel expression. These results suggest that either be attributed to the mutation of several types of ion channels. TRPM4 increase or decrease in TRPM4 expression may lead to conduction participates in this complex scheme.18 The TRPM4 mutations known to blocks. In contrast, it can also be due to different roles of TRPM4 in be related to cardiac electrical perturbations are reported in Table 2 human and mouse conduction. and schematized in Figure 4. 5.3 Cardiac hypertrophy 5.2.1 Conduction blocks Electrical activity is modified by cardiac hypertrophy. In the ECG, A correlation between TRPM4 and human disease was first discovered hypertrophy prolongs the QT interval, modifies the cardiac axis, and in a family with a cardiac bundle branch conduction block transmitted may lead to cardiac arrest.76,77 At the cellular level, it prolongs the ven- with an autosomal dominant inheritance.13 An E7K mutation was iden- tricular AP, which is attributable to the modified expression level of a + tified in this family on the TRPM4 gene. Later, other mutations were variety of ion channels including Ca2 channels and HCN77 – 79 and in- found in unrelated families with similar autosomal dominant isolated creases the incidence of afterdepolarizations.80 TRP channels—includ- conduction blocks.14,36 These mutations were found in a quarter ing TRPC1, TRPC3, and TRPC6—have also been associated with the of the right bundle branch blocks and a tenth of the atrioventricular development of cardiac hypertrophy.81 blocks (AVBs).36 The impact of TRPM4 mutations on cardiac conduc- Besides TRPCs, the overexpression of a TRPM4-like current was ob- tion is consistent with the high level of TRPM4 expression detected served in an in vitro model of cardiomyocyte dedifferentiation exhibiting in bovine conductive tissue.14 In four of these mutants, TRPM4 cell hypertrophy1 and in the in vivo model of the spontaneously hyper- properties were evaluated after heterologous expression in CHO or tensive rat (SHR) which develops cardiac hypertrophy.28 The mRNA HEK-293 cells. None of the mutations affects biophysical (single- levels similarly showed elevated Trpm4 expression in SHR myocytes channel conductance and ion selectivity) or regulatory (sensitivity to compared with normotensive controls. Whether this overexpression + voltage and Ca2 ) properties, but an increase in the amplitude of the participates in the prolongation of the QT interval and ventricular AP whole-cell current was detected for all.13,14 This was attributed to an in SHR28,78 remains to be determined. It might also participate in the increase in the number of channel proteins expressed at the cell mem- hypertrophy-induced EADs similar to what was observed in the mouse brane, due to a deregulation of small ubiquitin modifier conjugation model of hypoxia–reoxygenation-induced EADs,32 but that remains to (SUMOylation) involved in channel recycling.13,14 be evaluated. TRPM4 also modulates cardiac development and hypertrophy and, 5.2.2 Brugada by so, may impact electrical activity. Indeed, Trpm4 knock-out mice ex- Cohorts of patients with Brugada syndrome were screened for TRPM4 hibit cardiac hypertrophy not due to cardiomyocyte hypertrophy but mutations. Although a first study did not observe any mutation within to an increase in the cardiomyocyte number.46 Cardiac hypertrophy 27 patients,36 a second one found one mutation over 51 patients.74 A in Trpm4 knock-out mice might also develop secondarily to chronic third study reported 11 different mutations of the gene in 20 of the 247 hypertension as these animals have elevated blood pressures due to in- patients who did not carry any mutation on SCN5A gene,15 another creased catecholamine secretion.45 However, the newly designed major gene responsible for Brugada syndrome.75 Among these muta- cardiac-specific Trpm4 knock-out mice revealed an increased angioten- tions, K914X results in a non-functional channel, as the protein is trun- sin II-stimulated hypertrophy, compared with WT, consistent with the cated.15 Another one, P779R, decreases whole-cell TRPM4 current idea that TRPM4 is involved in this hypertrophy, directly at the cardiac compared with WT without variations of single-channel properties level.20 These aspects have to be evaluated in humans; however, none TRPM4 in cardiac electrical activity 27

Table 2 TRPM4 mutations in patients with perturbations of cardiac electrical activity

Mutant Protein location ECG Whole-cell current: ratio Protein expression level Effect References perturbation mutation/WT at membrane ...... E7K Putative CaM PFHBI 2 Increase Attenuated Kruse et al.13 binding site deSUMOylation Q131H Putative CaM RBBB, BrS n.d. n.d. n.d. Stallmeyer binding site et al.36 Duthoit et al.74 R144W N-terminus BrS n.d. n.d. n.d. Liu et al.15 R164W N-terminus RBBB 4.5 n.d. Altered SUMOylation Liu et al.14 35 Q293R N-terminus RBBB n.d. n.d. n.d. Du et al. Downloaded from https://academic.oup.com/cardiovascres/article/108/1/21/266658 by guest on 30 September 2021 A432T N-terminus RBBB 4.2 n.d. n.d. Liu et al.14 RBBB Stallmeyer et al.36 BrS Liu et al.15 G555R N-terminus BrS n.d. n.d. n.d. Liu et al.15 G582S N-terminus AVB n.d. n.d. n.d. Stallmeyer et al.36 F773I TM1-2 linker BrS n.d. n.d. n.d. Liu et al.15 P779R TM2 BrS 0.4 No change n.d. Liu et al.15 Y790H TM2 AVB bradycardia n.d. n.d. n.d. Stallmeyer et al.36 G844D ABC motif RBBB 7.5 Increase n.d. Liu et al.14 RBBB Stallmeyer et al.36 BrS Liu et al.15 Q854R TM2-3 linker BrS n.d. n.d. n.d. Liu et al.15 T873I TM3 BrS 0.8 No change n.d. Liu et al.15 K914R Putative AVB n.d. n.d. n.d. Stallmeyer SUMOylation site et al.36 K914X Putative BrS 0.2 Decrease Non-functional Liu et al.15 SUMOylation site channel P970S P-loop RBBB n.d. n.d. n.d. Stallmeyer et al.36 L1075P C-terminus BrS 1.2 Increase n.d. Liu et al.15 P1204L C-terminus BrS n.d. n.d. n.d. Liu et al.15

Brs, Brugada syndrome; RBBB, right bundle branch conduction block; PFHBI, progressive familial heart block type I.

of the existing reports (at present) indicates that TRPM4 mutations in Until now, patients with TRPM4 mutations were not reported with humans affect cardiac development.13 – 15,36,74 any phenotype other than electrical perturbations, whereas knock-out mice develop cardiac hypertrophy due to hyperplasia19 and persistent hypertension attributed to an increased catecholamine release from the adrenal gland.31 In addition, Trpm4 knock-out mice also have 6. Unanswered questions and future altered regulations in the immune system.11,12,82 It is not clear whether developments patients, at least with the K914X mutation, developed such dysfunction—even if this possibility was evaluated. Anyway, the small number 6.1 Species dependence of TRPM4 role in of patients and the heterogeneity of their symptoms are major obsta- cardiac physiology and limitations of animal cles to elucidate TRPM4 channelopathies in cardiology. models TRPM4 has been detected in cardiac tissue from all mammalian species 6.2 TRPM4 and partner proteins SUR1, examined, with relative expression between cardiac areas comparable TRPC3 among species (Table 1). But because cardiac properties (heart TRPM4 activity/expression/trafﬁcking has to be evaluated regarding its rhythm and AP parameters) show variations among species, the results ability to interact with partner proteins.83 Among these, co-expression obtained in small mammals must be considered cautiously for implica- of SUR1 and TRPM4 in COS-7 cells leads to the formation of SUR1– tions regarding human cardiology. However, ECG perturbations re- TRPM4 heteromers84 that increase TRPM4 sensitivity to ATP and glib- lated to TRPM4 mutations already demonstrate that this channel enclamide, two known regulators of both TRPM430,37 and SUR1. It also + plays a role in humans.13 –15,36,74 increases the afﬁnity of TRPM4 for Ca2 and calmodulin.84 Inhibition of 28 R. Guinamard et al.

compared with ventricular cardiomyocytes and the plateau membrane potential during the AP does not exceed 0 mV.92 These data, collectively, may be of great importance for electrical cardiac perturbations as the conductive tissue is known to be a major source of arrhythmias.93 –95 Unfortunately, despite these compelling indicators, no data are available yet to substantiate the direct involvement of TRPM4 in conductive tissue. 6.4 TRPM4 as a target to correct cardiac perturbations As TRPM4 is implicated in cardiac electrical activity and its perturbations, it appears as a new target in the design of antiarrhythmic drugs.

However, the widespread distribution of TRPM4 among tissues might Downloaded from https://academic.oup.com/cardiovascres/article/108/1/21/266658 by guest on 30 September 2021 be inconvenient for the use of such drugs because of many potential side effects. Because the heart is one of the most highly expressing tissues with regard to TRPM4 mRNA, one can argue that it will be a major target, which would potentially minimize side effects. In that sense, note that, until now, the sole human pathologies related to TRPM4 mutations are perturbations of cardiac electrical activity. It confirms that the heart would probably be one of the first targets of TRPM4 modu- Figure 4 TRPM4 mutations and cardiac conduction diseases. Sche- lators. However, as TRPM4 has several roles in cardiac electrical activ- matized TRPM4 subunit with identified mutations related to cardiac ity, we are far from fully predicting the impact of its modulation on conduction disease. For clarity sake, only mutations with a functional global cardiac electrical activity. Most probably, this will depend on sev- study are indicated. Numbers under mutations correspond to the ra- eral parameters such as beating rate or cause of arrhythmias (ischaemia, tio of mutant to WT current (asterisk, significantly different from mutations, hypertrophy, and so on). In addition, one should keep in WT). mind that TRPM4 may have an important role in generation and control of respiratory rhythm and breathing;96,97 thus any drug targeting cardiac TRPM4 could affect respiration in parallel. this complex protects against neuroinflammation in the context of Nevertheless, the development of new pharmacological compounds to 85 subarachnoid haemorrhage in humans and rats, suggesting that specifically target TRPM4 would provide essential tools to further pro- SUR1-TRPM4 inhibition by glibenclamide is an efficient pathway to pre- gress in the understanding of its role in heart as well as in other tissues. vent brain injuries following ischaemic stroke.86 The SUR1–TRPM4 complex is still in debate as another group was not able to detect such coupling.87 However, it may occur in heart, as 7. Conclusion 88 SUR1 and SUR2 are expressed. Association of these subunits with Kir Altogether, these data pointed TRPM4 as an actor in cardiac electro- proteins forms ATP-sensitive potassium channels (KATP). The expres- physiology. Although our understanding of its roles is incomplete, par- sion of SUR1 in cardiac tissue in which TRPM4 is also present leaves ticularly for inherited cardiac diseases, recent progress is valuable and open the possibility of their association that remains to be explored. predicts further developments. TRPM4 has to be included in mathem- The physiological impact of the SUR1–TRPM4 association may atical models of cardiac AP and electrical propagation. In relation with counteract KATP effects. Indeed, SUR1-Kir6.2 and SUR2A-Kir6.2 as data obtained at the cell level, it will contribute to the understanding of 84 – 86,88 well as SUR1–TRPM4 are inhibited by internal ATP. Under direct involvement of TRPM4 in controlling membrane potential of car- conditions of metabolic stress, such as that induced by ischaemia, the diac cells. TRPM4 should also be considered when developing antiar- reduction of internal ATP levels may naturally lead to KATP activation, + rhythmic drugs as it might be an interesting target or responsible for which could exert a cardioprotective effect by generating outward K side effects. current that stabilizes the resting membrane potential and shortens the AP.89 In contrast, SUR1–TRPM4 might also be activated, which would Acknowledgement + produce mainly an inward Na current in favour of cell depolarization We thank Christopher Del Negro for editing the manuscript. and AP prolongation. TRPC3 might also be a TRPM4 partner in heart. Indeed, both pro- Conflict of interest: none declared. teins can interact when co-expressed in HEK-293 cells.90 Both channels are expressed in cardiomyocytes, and their co-expression increases Funding 28,91 during ventricular hypertrophy. T.H. and C.S. are recipients of a fellowship from the French Ministe`re de l’Enseignement et de la Recherche. R.G. and L.S. are employed by the 6.3 TRPM4 in the conductive tissue French Ministe`re de l’Enseignement et de la Recherche. The team of P.B. Several observations strongly suggest that TRPM4 is implicated in car- was supported by a grant (Projet de Recherche Clinique 2008) and by diac conduction along the His and Purkinje tract: (i) immunolabelling foundation Renaud Febvre. data that revealed a high TRPM4 protein expression in bovine conductive tissue14;(ii)TRPM4 mutations occur in patients with cardiac con- References + 13,14 1. Guinamard R, Rahmati M, Lenfant J, Bois P. 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