Role of TRPM4 ion channel in pediatric arrhythmic syndromes

Abstract

TRPM4 is a member of transient receptor potential (TRP) ion channel group which greatly contributes to cardiomyocyte function and physiology. It controls the dynamic stabilization of calcium level and is involved in sinus node regulation and cardiac conduction. Most of pathogenic variants in TRPM4 gene are associated with conduction disorders and various types of arrhythmic syndromes presenting in children and early adolescents. In addition, TRPM4 gain- and loss-of-function variants are discussed in connection to Brugada syndrome and long QT-syndrome. In this review we summarize current knowledge on the role of TRPM4 variants in pediatric arrhythmic syndromes, discuss molecular mechanisms of TRPM4 dysfunction, provide clinical illustrations and case presentations underlining the role of TRPM4 in molecular pathogenesis of arrhythmic syndromes presented in young age.

Key Words: Transient receptor potential channels; Children; Pathophysiology; Arrhythmia; Cardiac conduction

Core Tip: TRPM4 ion channel plays a crucial role in cardiovascular system and myocardial physiology. Mutations in TRPM4 are linked to cardiac conduction disorders and arrhythmic syndromes in children. Current knowledge on the role of TRPM4 variants in pediatric arrhythmic syndromes are summarized along with a discussion of molecular mechanisms of this ion channel dysfunction. Clinical illustrations underscoring the role of TRPM4 in arrhythmic syndromes manifested in childhood are presented.

INTRODUCTION

TRPM4 belongs to the large and diverse family of transient receptor potential (TRP) channels, which are expressed in both excitable and non-excitable tissues and are involved in various cellular responses from neuronal bursting activity to fluid secretion. TRPM4 channels play a crucial role in the wide range of physiological processes, including but not limited to cardiac rhythm, immune response and insulin secretion. The importance of TRPM4 in the heart physiology is illustrated by the fact that majority of pathogenic variants of the TRPM4 gene are associated with conduction and arrhythmic disorders such as progressive conduction block, atrioventricular block, Brugada syndrome and long QT syndrome.

TRP ION CHANNELS

The history of TRP channels dates back to 1975[1] when an archetypal TRP protein was cloned from fruit flies. Now members of the large family of TRP channels are found in all eukaryotes. TRP channels are very diverse in cation selectivity and activation mechanisms[2]. The family of TRP channels, which includes over 50 members, is subdivided into seven major subfamilies basing on their sequential and topological similarities. The subfamilies include TRPC (Canonical), TRPV (Vanilloid), TRPM (Melastatin), TRPA (Ankyrin), TRPN (NompC, i.e. No mechanoreceptor potential C), TRPP (Polycystin) and TRPML (MucoLipin) channels[3,4]. Presently, 28 genes of the TRP family have been described in mammals, including 27 genes expressed in humans[4,5].

TRP channels belong to a superfamily of P-loop channels[6] along with voltage-dependent potassium, sodium and calcium ion channels[7]. Over four hundreds of 3D aligned cryo-electron microscopy (cryoEM) structures of P-loop channels, including 88 TRP channels, are currently available in the database www.plic3da.com. A TRP channel represents a homo-tetramer of four subunits where one subunit includes six transmembrane segments. In most excitable cells such as neurons and cardiomyocytes the influx of cations into the cytoplasm due to TRP opening facilitates action potentials[8]. In non-excitable types of the cells, TRP channels trigger voltage-dependent calcium channels through membrane depolarization along with chloride and potassium channels, and through modulating transcription and translation, influences many cellular events, such as cellular contraction, migration and secretion[4]. Expression of TRP channels are mainly detected in endocrine system, immune and nervous systems, genitourinary, respiratory systems and gastrointestinal tract as well as in cardiovascular system so that the channels are involved in many physiological and pathological processes[2,9].

The subfamily of TRPM channels includes eight members (TRPM1-TRPM8), forming four pairs categorized basing on their sequence similarity[10]. TRPM channels serve as Ca²⁺ sensors involved in many key cellular processes such as cardiac rhythm and immunity, arterial pressure regulation, mineral homeostasis and various types of reception such as taste reception, photoreception and thermo-reception. Mutations in TRPM genes cause several inherited human diseases including ocular, neurological, cardiac, intestinal and kidney disorders depending on the type of the channel. Preclinical studies in animal models highlighted TRPM channels as promising new therapeutic targets[11,12].

TRPM4 ION CHANNELS

A distinctive feature of the TRPM4 and TRPM5 pair of channels is their calcium impermeability[13,14]. TRPM4 is widely expressed in cardiovascular system and can be activated by the intracellular accumulation of Ca²⁺[15,16]. In parallel, TRPM4 greatly regulates intracellular Ca²⁺ homeostasis. By influencing the membrane potential TRPM4 modifies excitability of cells[7,17] participating in various physiological processes such as immune cell response, insulin release, arterial constriction and cardiomyocyte conductivity[3]. In the cardiovascular system, TRPM4 channels play a key role in Ca²⁺ homeostasis and Ca²⁺-dependent cellular events, most importantly, in excitation-contraction coupling, action potential propagation and contraction of cardiomyocytes and vascular smooth muscle cells[18].

There are 3 splice variants of TRPM4: A, b, c[7,15,19]. TRPM4b is the largest and most widely expressed variant. TRPM4 channels are permeable for monovalent cations with the following selectivity: Na⁺ > K⁺ > Cs⁺ > Li⁺, but they are not permeable for Ca²⁺[7,20,21]. TRPM4 channels do not demonstrate voltage-dependent and calcium-dependent inactivation, they activate in 2-4 seconds with the peak current reached at approximately 10 seconds[7]. The maximal amplitudes for TRPM4 mediated inward and outward currents are observed at -80 and +80 mV, respectively[7].

Several 3D structures of TRPM4 in different ligand-bound states have been obtained using cryoEM[22-25]. All these structures represent the closed channel state, while the open-state conformation is still elusive[22-25]. In contrast to voltage-gated sodium channels, in TRPM4 the sliding voltage-sensing helix S4 in the voltage-dependent domain has only two positively charged residues; therefore TRPM4 demonstrates much weaker voltage sensitivity than classical voltage-gated ion channels[3,26].

Cytosolic N- and C-terminal regions of TRPM4 contain functionally important domains (Figure 1), including the C-terminal coiled coil domain involved in the channel tetramerization, and two ATP-binding cassette motifs in the N-terminal nucleotide-binding domain mediating TRPM4 inhibition.

Figure 1 Overall architecture of the hTRPM4 subunit. A: A schematic representation of the major structural components in hTRPM4; the N-terminus has four melastatin regions and Pre-S1 region; the transmembrane domain contains six helices (S1-S6); helices S5, S6 and P-loop form pore module; the C-terminus contains transient receptor potential and C-terminal domain helices; alpha-helices are represented by cylinders and beta-strands are indicated by arrows; variants are numbered: 1-E7K; 2-R164W; 3-D198G; 4-A432T; 5-V441M; 6-R499W; 7-G582S; 8-T677I; 9-P779R; 10-R819C; 11-G844D; 12-T873I; 13-K914X; 14-V921I; 15-R964S; 16-L1075P; 17-K1140X; B: 3D model of the hTRPM4 subunit (PDB ID: 5WP6). MHR1-4: Four melastatin regions; TM: Transmembrane; TRP: Transient receptor potential; CTD: C-terminal domain.

TRPM4 is activated by increased intracellular Ca²⁺ [(Ca²⁺)i] with Kd values ranging from 0.4 mkM[7] to 9.8 mkM[27], while membrane depolarization modulates the channel activity[7]. Functional experiments show that the TRPM4 channels open at the late repolarization phase of action potential (AP) when (Ca²⁺)i is in the high sub-micromolar to micromolar range and the channels remain open even near the maximum diastolic potential[28] providing an impact in AP prolongation and abnormal diastolic depolarizations at the very end of AP in case of pathology.

TRPM4 influences cardiac development and remodeling, modulating cardiac contractility and overall electrical activity in the heart[17]. TRPM4 is expressed in different types of cardiac cells including contractile atrial and ventricular cardiomyocytes, sinoatrial node (SAN), conductive myocyte, and atrial and ventricular stromal cells[29]. The level of the TRPM4 gene expression in Purkinje fibers is the highest among other human cardiac tissues[30] suggesting a significant contribution of the channels to the AP regulation in these cells[31].

In the SAN, intracellular Ca²⁺ oscillations largely determine cardiac automaticity. When voltage-dependent L-type Ca²⁺ channels Cav1.3 and Cav1.2 are activated during initial diastolic depolarization (DD) phase in SAN cell, the influx of the extracellular Ca²⁺ into the cytoplasm triggers sarcoplasmic Ca²⁺ release via the ryanodine receptors[32]. This cytoplasmic rise in (Ca²⁺)i activates the Na⁺-Ca²⁺ exchanger protein, which mediates Ca²⁺ efflux via exchange for Na⁺ inflow. This leads to an inward Na⁺ current through HCN4 that contributes to the membrane depolarization and increase the pace-making DD rate[33]. It is suggested that TRPM4 channel contributes to the inward Na⁺ current during the DD phase and has an impact on the cardiac automaticity through inward driving force[34,35]. In rodents inhibition of TRPM4 channels by 9-phenanthrol reduces cardiac rate, supporting the hypothesis of DD acceleration via TRPM4 upon decreased heart rate[34,36]. Overall, the TRPM4 channels regulate cardiac automaticity by modulating depolarization slope and (Ca²⁺)i in pacemaker cells.

TRPM4 expressed in atrial cardiomyocytes is involved in atrial electrophysiology both in rodents and humans[14,37]. In Trpm4 KO mice the duration of atrial AP was shorter and 9-phenanthrol further shortened it compared to WT animal cells[37]. Importantly, the channel is also responsive to shear stress induced by the IP3 receptor-mediated Ca²⁺ release in atrial cells[38]. Of note, TRPM4 may contribute to fibroblasts grow and differentiation in human and mice, thus, TRPM4 channel may be involved in atrial tissue fibrosis[39]. Recently, TRPM4 channels were shown to be a component of focal adhesions between fibroblasts and extracellular matrix[40] and it is reasonable to suggest a role of TRPM4 in atrial fibrillation through both electrophysiological mechanisms and matrix component modification[41].

The system of terminal Purkinje fibers (PFs) propagates electrical impulses to the ventricular cardiomyocytes, facilitating excitation-contraction cycle in ventricular tissue. The TRPM4 channels are highly represented in PFs, as compared to other myocardial chambers[30], suggesting their central role in PFs electrical functioning[31]. The propagation failure of PFs fallowed by TRPM4 channels overexpression was demonstrated both in silico and in vitro[31,42]. Importantly, optical mapping of PFs during mechanical stimulation demonstrated a clear interconnection with TRPM4 channels activation supporting TRPM4 role in PFs-associated arrhythmogenesis[43]. Taking into account the very high expression of TRPM4 in PFs compared to other myocardial tissues, its importance in cardiac conduction and impulse propagation seems undoubted underscoring the need for further pharmacological and physiological research focused on TRPM4 to prevent cardiac arrhythmias.

In contrast to atrial and septal conductive myocardium, the role of TRPM4 channels in the ventricular tissue remains enigma, especially taking into account their relatively low expression level compared to supraventricular tissues[44]. The participation of TRPM4 channels in AP duration of canine ventricular cardiomyocytes has been confirmed only recently[45]. In addition, their expression and Ca²⁺-activated non-selective cation current are greatly enhanced in the ventricular cardiomyocytes after pressure overload[46]. Moreover, a prolongation QT interval was observed in connection to increased TRPM4 activity[46]. It is considered that TRPM4 channel may also be involved in proving β-adrenergic inotropic effect on the ventricular myocardium[47]. Hence, overall data on TRPM4 expression and function in ventricular myocytes are much scarce compared to the data regarding its function in the SAN and conductive cardiomyocytes, leaving potential role of TRPM4 in non-PF associated arrhythmic events open.

TRPM4 ION CHANNELS IN THE HEART PATHOPHYSIOLOGY

TRPM4 channels translate any variation of intracellular Ca²⁺ into the voltage variation, thus affecting cardiac contractility and overall electrical activity of the heart. Accordingly, TRPM4 dysfunctions are associated with various cardiac disorders and gain-of-function (GoF) and loss-of-function (LoF) mutations in the TRPM4 gene are associated with various types of cardiomyocyte dysfunction. In particular, TRPM4 channels are involved in the heart arrhythmogenicity, mainly with conduction abnormalities that are often present in childhood of both inherited and acquired origins. In Table 1 we summarized available data of TRPM4 variants that have been electrophysiologically studied[30,48-53].

Table 1 TRPM4 variants electrophysiologically studied.

Substitution	Variant	Location	Effect	Underlying mechanism	Protein expression level on membrane	Current density	Ca2+ dependence	Voltage-dependence	Electrophysiological results	Classification	ECG morphology; phenotype	Ref.
p.E7K	c.19G>A	N-terminus. Putative CaM binding site	GoF	Impaired SUMOylation (attenuated deSUMOylation). Impaired endocytosis Enhanced interaction of PIP2 and TRPM4 protein (increased risk of generating triggered activities)	↑	↑			Prolonged AP (due to facilitated open state condition). Depolarizing shifts of the resting membrane potential (depending on the channel density or maximal activity). Insensitivityto PIP2 depletion	Pathogenic	PFHB1B, RBBB, progressive conduction block	[30,49]
p. R164W	c.490C>T	N-terminus	GoF		No changes	↑	No changes		No effect on SUMOylation stimulation. Dynamitin-insensitive	Pathogenic	PFHB1B	[48]
p.D198G		Putative CaM binding site			No changes	No changes				Uncertain significance		[50,51]
p.A432T	c.1294G>A	N-terminus	GoF	Likely to be caused by protein misfolding and retention in the endoplasmic reticulum	↓	↓	No changes		Cotransfection experiments of TRPM4 mutants with Ubc9 showed an increase of current density (stimulating effect on SUMOylation). Dynamitin-insensitive. Lowering the incubation temperature of cells expressing the variant to 28 °C for 24 hours increased their expression both at the total level (A432T 78% ± 3% of WT); and at the cell surface level (A432T 72% ± 8% of WT). A significant increase in the current density of A432T was observed (599 ± 104 pA/pF at 28 °C vs 230 ± 27 pA/pF at 37 °C), whereas WT showed no significant change (639 ± 101 pA/pF at 28 °C vs 553 ± 74 pA/pF at 37 °C;P > 0.05)	Pathogenic	PFHB1A. PFHB1B. Atrioventricular block. Brugada syndrome	[49]
p.A432T/G582S		N-terminus	LoF	Likely to be caused by protein misfolding and retention in the endoplasmic reticulum	↓	No changes			Lowering the incubation temperature of cells expressing the variant to 28 °C for 24 hours increased their expression both at the total level (A432T/G582S 69% ± 5% of WT) and at the cell surface level (A432T/G582S 65% ± 7% of WT)
p.V441M			LoF			↓						[49]
p.R499W			LoF			↓						[48]
p.G582S	c.1744G>A	N-terminus	GoF	Direct SUMOylation of TRPM4 is unlikely to be linked to the increased expression and gain of function of the variant	↑	↑ (171% ± 20% of WT)				Pathogenic	PFHB1A. PFHB1B. Brugada syndrome	[48]
p.T677I		N-terminus			No changes	No changes				Uncertain significance
p. P779R	c.2336C>G	TM2			↓	↓	↓	V1/2 ↑		Uncertain significance	Brugada syndrome. Right bundle brunch block
p.G844D	c.2531G>A	ABC-motif	GoF		No changes	↑	No changes		Cotransfection experiments of TRPM4 mutants with Ubc9 showed an increase of current density (stimulating effect on SUMOylation). Dynamitin-insensitive	Pathogenic	PFHB1B right bundle-branch block. Brugada syndrome. Long QT syndrome	[48]
p.T873I	c.2618C>T	TM3			↑		↓	No changes		Uncertain significance		[48]
p.K914X	c.2740A>T	Putative SUMOylation site		Non-functional channel	↓	No current	↓	No changes		Likely benign	PFHB1B right bundle-branch block	[52]
p.V921I	c.2761G>A	TM4-TM5			No changes	No significant changes				Likely benign	PFHB1B	[49,53]
p.L1075P	c.3224T>C	C-terminus			↑	↓	↓	No changes		Uncertain significance	PFHB1B

AP: Action potential; GoF: Gain-of-function mutation; LoF: Loss-of-function mutation; PFHBIA: Progressive familial conduction block type IA; PFHBIB: Progressive familial conduction block type IB; RBBB: Right bundle branch block; ECG: Electrocardiography.

First, genetic variants in the TRPM4 gene were described in large families with isolated Progressive Familial Heart Block type I[30,54]. Later TRPM4 variants were associated with isolated cardiac conduction disease, right bundle branch block, and Brugada phenomenon and syndromes[55]. Beyond, the role of TRPM4 variants in genetically determined congenital atrio-ventricular conduction block presented in early childhood was reported[48].

In spite of the fact that majority of TRPM4 variants are associated with Brugada syndrome and conduction defects, phenotype-genotype correlations in TRPM4 variants remain ambiguous. Thus, both LoF and GoF variants were reported in clinical syndromes with overlapping phenotypes. In addition, Gualandi et al[56] identified a double heterozygosity for pathogenic mutations in SCN5A and TRPM4 in a Brugada syndrome patient. Mutations G219E and T160M in the KCNQ1 and TRPM4 genes, respectively, were detected in a 37-year-old patient with long QT syndrome[57]. Surprisingly, both TRPM4 GoF and LoF mutations can be associated with similar phenotypes. All the above suggests that the TRPM4 gene variants may have a modulating effect in case of digenic inheritance. These findings highlight the importance of identifying all genetic variants in spite of detected a pivotal mutation in well-known causal genes for a proper understanding of the genetic nature of the disease and an adequate assessment of therapeutic approaches and prognosis.

TRPM4 VARIANTS IN PEDIATRIC PATIENTS

Here we illustrate two clinical cases of pediatric patients with an early manifestation of cardiac conduction disorders, as well as decreased contractility and cardiac chamber dilation possibly associated with TRPM4 variants. The structural modeling of the variants along with analysis of biophysical consequences may suggest a possible link between respective mutations and rare clinical phenotype observed in children.

Сase presentation 1

The patient was first observed by cardiologist at the age of 5.5 years due to episodes of sustained atrial tachycardia with the heart rate 160-180 beats per minute (bpm), incomplete right bundle branch block and variable atrioventricular (AV) conduction. Areas of sinus rhythm with heart rate 85-154 bpm were occasionally simultaneously registered (Figure 2). Echocardiography revealed chambers dilatation with ejection fraction (EF) of left ventricle 26%. A family history was unremarkable with no history of syncope and sudden cardiac death. Bisoprolol therapy was initiated without conduction worsening with temporal effect on the heart rate (120-130 bpm). Heart failure medication included diuretics and ACE inhibitors. Of note, at the moments of achieving rhythm control the patient demonstrated a tendency towards normalization of chamber sizes and EF. However, after several years of follow up no sinus rhythm was registered, and symptoms of left ventricular dysfunction progressed despite medication. From 6 years old the patient maintained at atrial tachycardia with consistently high heart rate (120-180 bpm), left ventricular EF kept reduced to 20%-25% especially during the periods of high heart rate. Several attempts of antiarrhythmic therapy with various beta-blockers (metoprolol tartrate, carvedilol, anaprilin) and ivabradine had no sufficient effect. At the age of 14 the patient was referred to our clinic with complaints on dizziness, fatigue and palpitations. According to Holter ECG data, the patient had constant atrial tachycardia up to 200 bpm with no sinus rhythm; creatine kinase and troponin levels were within normal range. Cardiac magnetic resonance imaging excluded structural abnormalities and acute inflammation but demonstrated in the intramural interventricular septum fibrosis. Endomyocardial biopsy showed no signs of myocarditis. Due to clinical symptoms and failure of antiarrhythmic therapy the radiofrequency ablation (RFA) of atrial arrhythmia was performed. After leveling the atrial ectopic activity (series of applications in the crista terminus area), a stable nodal rhythm with a heart rate of 70 bpm was obtained. Endo-electrophysiological study revealed a recovery time of second-order pacemaker to be 3000 milliseconds. Atropine infusion resulted in junctional rhythm with heart rate of 105 bpm, and the second-order pacemaker recovery time 800 millisecond, time for AV Wenckebach point was 220 bpm. Thus, the patient was diagnosed with sick sinus syndrome. Within the next several years, atrial tachycardia did not progress, and the patient presented a constant junctional rhythm with heart rate 39-101 bpm (average heart rate 62 bpm during the day and 47 during the night). The duration of rhythm pauses ranged from 1800 millisecond to 2976 millisecond (Figure 3). Given the absence of clear indications such as complaints, symptomatic bradycardia, clinically significant rhythm pauses and signs of arrhythmogenic myocardial dysfunction, it was decided to refrain from the pacemaker implantation. After 4.5 months post RFA a complete normalization of the heart chambers and contractility was observed and within next 2-years of follow-up the patient did not report any recurrence of symptoms.

Figure 2 Electrocardiography during sinus rhythm with heart rate 85-154 bpm.

Figure 3 The duration of rhythm pauses ranged from 1800 milliseconds to 2976 milliseconds. А: Pectoral leads; B: Standard leads.

Knowing the early debut of atrial arrhythmias, sick sinus syndrome and AV conduction defects the patient underwent genetic testing. A target gene panel including 54 genes potentially associated with various arrhythmogenic syndromes (ABCC9, AKAP9, ANK2, CACNA1C, CACNA2D1, CACNB2, CALM1, CASQ2, CAV3, CTNNA3, DES, DPP6, DSC2, DSG2, DSP, EMD, GJA1, GJA5, GPD1 L, HCN4, JUP, KCNA5, KCND3, KCNE1, KCNE5, KCNE2, KCNE3, KCNH2, KCNJ2, KCNJ5, KCNJ8, KCNQ1, LMNA, NKX2-5, NOS1AP, NPPA, PKP2, PLN, PRKAG2, RANGRF, RYR2, SCN1B, SCN2B, SCN3B, SCN4B, SCN5A, SCN10A, SLMAP, SNTA1, TGFB3, TMEM43, TRDN, TRPM4, TECRL) was used with the Illumina MiSeq platform followed by results verification by Sanger sequencing (Supplementary material). The genetic testing revealed a missense variant in TRPM4 gene (hg38, Chr19: 49200722, rs749078579, NM 017636.4: C.C2890A: p.Arg964Ser) in a heterozygous state (Table 2).

Table 2 Genetic variants identified in patients.

Patients	Gene	GRCh38	Nucleotide change	Amino acid change	db SNP, MAF%	CADD	SIFTcat	PolyPhenCat
Patient 1	TRPM4	Chr19:49200722,	NM 017636.4: C.C2890A	p.Arg964Ser	rs749078579, 0.001592%	26	Deleterious	PD
Patient 2	TRPM4	Chr19:49210799	NM 017636.4: C.A3418T	p.Lys1140Ter	No data	45	NA	NA
Patient 2	MYPN	Chr10: 68201958	NM 032578.4: C.A3623T	p.Asp1208Val	No data	32	Deleterious	PD

CADD: Combined annotation dependent depletion (GRCh38-v1.6); NA: Not available; PD: Probably damaging.

In the cryoEM structure of calcium- and decavanadate-bound channel (PDB ID: 5wp6) arginine R964 in at the extracellular part of P-loop helix P1 forms an inter-subunit salt bridge with D984 and donates an H-bond to Q977. These contacts between P-loop residues, which are present in all four subunits of the homotetrameric channel, stabilize the folding of P-loops that harbor the selectivity filter residues Q977 and G976. Mutation R964S should affect stability of the P-loop domain leading to the possibility that the destabilized selectivity filter (Figure 4) could permeate, besides Na⁺, other cations.

Figure 4 TRPM4 cryoEM structure. A: Extracellular view of the TRPM4 cryoEM structure (PDB ID: 5wp6). Four identical subunits are differently colored to highlight intersubunit contacts of the four R964 residues, which stabilize the folding of P-loops that harbor the channel selectivity-filter; arginine R964 in P-loop helix P1 and glycine G976 in the selectivity filter are shown by spherical atoms; B: Close-up views of 3D-aligned AlphaFold 3 (AF3) models of the wildtype channel TRPM4 (green) and R964S mutant channel (brown); mutation R964S eliminates the salt bridge that stabilizes an intersubunit contact between helix P1 and a linker connecting the selectivity filter and helix S6; C: Close-up view of the selectivity filter region in the AF3 models; elimination of the intersubunit contact (B) cases a noticeable widening of the selectivity-filter region that likely affects the ion selectivity of the channel.

Similar substitution was earlier described by Dong and co-authors with another amino acid substitution (hg19, chr 19: 49703979, TRPM4: NM 017636.4: C.2455C>T, p.R964C). This variant was found in a 10-year-old girl with high degree AV block and a family history of syncope, conductive disorders and sudden cardiac death[58]. However, neither atrial tachycardia nor chamber dilation reduced contractility in this case. The reason of the significantly different phenotypes associated with variants R964S (in our case) and R964C (in the previously published case[58]) is unclear. It may be due to various effects of the serine and cysteine substitutions on the stability of P-loops and hence the ion selectivity of the mutant channels, or due to other genetic or environmental causes. However, the damaging effect of this substitution along with typical phenotype and conductive abnormalities allow considering this variant causative and pathogenic. Of importance, apart from the sinus nodus and AV conduction disturbances, the patient presented primary with atrial tachycardia and reduced left ventricular (LV) contractility. In spite the fact that the latter was considered as arrhythmia-induced cardiac remodeling especially taking into account its full resolution followed by heart rhythm control, this further underlines the potential role of TRPM4 channel not only in cardiac conduction, but also in cardiomyocyte contractility.

Сase presentation 2

A 3-month-old male infant with the absence of family history of cardiac disorders or sudden cardiac death was hospitalized due to signs of heart failure and sinus tachycardia of 176 bpm. Echocardiography showed marked LV dilation and severely reduced ejection fraction (LVEF-21% when normal range is 55%-75%, LVEDD 46 mm; Z-score +9.8) (Figure 5A). Coronary angiography ruled out congenital coronary artery anomalies and there were no signs of active infection. Serological and PCR tests for common cardiotropic viruses (herpesviridae, enterovirus, parvovirus B19) were negative but cardiac and heart failure markers were significantly increased (troponin I level 148.1 pg/mL. with normal range < 40 pg/mL; NT-proBNP 18425 pg/mL). Electrocardiography (ECG) demonstrated diffuse repolarization abnormalities with inverted T waves (Figure 5B), and Holter ECG showed no significant arrhythmias or conduction disturbances. Genetic testing was performed using the same NGS target panel as for case 1 and revealed a stop codon variant detected in TRPM4 gene (hg38, Chr19: 49210799, NM 017636.4: C.A3418T: p.Lys1140Ter) in a heterozygous state. In addition, a new missense variant in MYPN gene (hg38, Chr10: 68201958, NM 032578.4: C.A3623T: p.Asp1208Val) was detected in a heterozygous state. This variant has no data of frequency and according to various prediction analysis tools can be suggested as damaging. After initiation of heart failure therapy (captopril, carvedilol, digoxin, furosemide, and spironolactone) the patient showed clinical improvement with a significant reduction in NT-proBNP and improved LV systolic function. Over the following 12 months after initial presentation at 3 months of age the patient demonstrated progressive recovery with normalization of LV systolic function (LVEF 62%) and significant improvement of myocardial architecture (LVEDD 24.5 mm; Z-score +1.96) under continued therapy; by 2 years of age ECG showed normalization of T waves and repolarization patterns (Figure 5C and D). While contemporary diagnostic possibilities do not allow to fully exclude viral-mediated myocardial injury or ischemic and microvascular alteration during perinatal period as triggering factors, the role of LoF TRPM4 variant in myocardial maladaptation and dysfunction seems possible, especially in combination with another variant of unknown significance in MYPN. Taking into account the distal location of the truncating stop-codon in C-terminal cytoplasmic end beyond transmembrane part of the protein, potential deleterious effect of this variant can involve protein-protein interactions with anchoring proteins, integration with cytoskeleton and membrane localization signals (Figures 1 and 4).

Figure 5 Echocardiography and Electrocardiography findings: A and B: At 3 months of age: Left ventricular dilation with spherical configuration and diffuse repolarization abnormalities, including T-wave inversion on electrocardiography (ECG); C and D: At 2 years of age: Normalization of left ventricular size (LVEDD 24.5 mm; Z-score +1.96) and resolution of T-wave inversion with normalized repolarization patterns on ECG.

As illustrated in these two clinical cases, TRPM4 channels play crucial role in proper heart functioning, affecting the key features of cardiac tissue. The impact of TRPM4 to heart automaticity and excitability has been shown using in vivo and in vitro models[31,34,37]. In contrast, the influence of TRPM4 on cardiac contractility remains controversial. In TRPM4-deficient mice, myocardium demonstrates an increased inotropic response both in vitro and in vivo[59]. Repolarization in Trpm4(-/-) ventricular myocytes was significantly decreased, resulting in an increased driving force for the L-type Ca²⁺ current during AP, leading to altered sarcomeric contractility[59]. Medert et al[60] showed that the relevance of TRPM4 for cardiac contractility depends on TRPM4 expression levels and on the disease status. They also demonstrated that increased β-adrenergic inotropy was observed in mice on a 129/SvJ genetic background, but not on the C57Bl/6N background[60]. However, the precise role of TRPM4 channels in cardiac contractility in mice model remains unclear.

The two presented cases underline the potential role of TRPM4 channel not only in cardiac conduction and impulse generation, but also in myocardial inotropic force and contractility – the functions, which potentially can be attributed to TRPM4 expression not only in myocytes but also in stromal and smooth muscle cells. Of note, in both cases the decreased contractility almost fully resolved under favorable conditions considering TRPM4 channel as an important actor of myocardial adaptation to stress. Along with data available on TRPM4-accosiated myocardial response on pressure overload and ischemic injury, this potentially might open a new direction of pharmacological research aiming on TRPM4 modulation and cardiac contractility.

CONCLUSION

Nowadays, it became indisputable that the TRPM4 gene is an important participant in cardiac electrical activity and is associated with cardiac conduction abnormalities often presented in childhood: Isolated Progressive Familial Heart Block type I, with Brugada syndrome, LQT syndrome. Genetic testing of probands, cascading screening of relatives and their dynamic follow-up are of a high clinical importance in cases of TRPM4-associated rhythm disorders in order to stratify risk, determine treatment traits, and timely offer pacemaker implantation. In addition, variants in TRPM4 gene can aggravate the phenotype of the disease with the background of main causative mutations underscoring the possibility of digenic inheritance. Further studies on TRPM4 function apart from its involvement in AP will allow to expand its role in myocardial physiology. With that, TRPM4 channel has a potential to become a novel therapeutic target to effectively prevent and treat cardiac arrhythmias and modulate cardiac contractility.

Currently, several small molecules are used to investigate the potential of TRPM4 as a therapeutic target[26]. Molecules such as 9-phenanthrol, flufenamic acid, anthranilic acid derivatives CBA and NBA benzoic acid can serve as promising candidates for drug development[26,61]. A binding site for small molecule inhibitors targeting human TRPM4 has recently been identified[62] becoming a huge step forward in the drug development for the diseases associated with TRPM4.