Deletion of Trpm4 alters the function and expression of NaV1.5 channel in murine cardiac myocytes

Transient receptor potential melastatin member 4 (TRPM4) encodes a Ca+ -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 block 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 immunohistochemistry studies on isolated atrial and ventricular cardiac myocytes and surface, pseudo and intracardiac ECGs either in vivo or on 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 the myocytes. Hearts from Trpm4-/- mice presented increased sensitivity towards mexiletine, a Na+ channel blocker, and slower intraventricular conduction, consistent with the reduction of peak Na+ current observed in the isolated cardiac myocytes. This reduction in Na+ current is explained by the observed decrease in protein expression of NaV1.5 in Trpm4-/- mice. This study suggests that Trpm4 expression impacts Na+ current in murine cardiac myocytes and points towards a novel function of Trpm4 regulating the NaV1.5 expression in murine cardiac myocytes.


Introduction
The cardiac Ca 2+ -activated non-selective cation (NSca) currents were first measured in cultured rat neonatal myocytes in the early 1980s [1]. The molecular iden-tity of these current components remained largely unknown until a member of TRPM family, TRPM4b, was cloned [2][3][4][5], which was found to share the biophysical properties of a native NSca current from human atrial myocytes [6]. TRPM4 belongs to the transient receptor potential (TRP) ion channels superfamily, comprising mostly large Ca 2+ -permeable cation channels that are expressed in different tissue types and are activated by a broad spectrum of physicochemical stimuli under varying cellular conditions. TRPM4 and TRPM5 are the only members of the TRP family that are not permeable to divalent cations such as Ca 2+ or Mg 2+ . Instead, TRPM4 is activated by an increase in intracellular Ca 2+ , and voltage further modulates its gating, resulting in an outwardly rectifying current [7][8][9]. This distinctive biophysical feature of TRPM4 could allow Na + entry at negative potentials leading to membrane depolarization, and K + efflux at positive potentials facilitating membrane repolarization. In human hearts, the expression of TRPM4 has been detected in Purkinje fibers, atria, and ventricles [10]. Clinical relevance of TRPM4 emerged from the identification of the first mutation in patients with hereditary cardiac conduction slowing disorders. This mutation caused the replacement of a glutamate residue at position 7 with lysine (p.E7K), which led to an increase in protein function and surface expression [10]. To date, more than 25 variants of TRPM4 are associated with conduction disorders and Brugada syndrome [11][12][13][14][15]. However, phenotype-genotype correlations with TRPM4 variants remain ambiguous as both gainand loss-of-function were reported to have overlapping clinical phenotypes. In one of our recent works, we found that some TRPM4 mutations altered the protein degradation rate, which led to either increased or decreased stability of the protein at the membrane [13]. How the altered degradation rates are related to the clinical phenotypes of the respective mutations is still unclear. In mouse heart, the role of TRPM4 has Deletion of Trpm4 alters the function and expression of Na V 1.
Previous studies have proposed the hypothesis that TRPM4 may functionally affect the activity of other cardiac ion channels, such as the main cardiac voltage-gated Na + channel Na V 1.5 [12,24]. Since TRPM4 carries an inward current close to resting membrane potential and an outward current at depolarized potentials, increasing or decreasing TRPM4 expression may critically influence the availability of Na V 1.5 channels.
In the present study, we evaluated whether TRPM4 indeed has any crosstalk with Na V 1.5 in cardiac cells and tissues. We investigated the consequences of Trpm4 deletion on cardiac electrical activity in mice. We electrophysiologically characterized individual isolated cardiac myocytes from atria and ventricles and observed a decrease in the functional expression of Na V 1.5 due to Trpm4 deletion. We further demonstrated the impact of Trpm4 deletion with pseudo-and intracardiac ECGs on Langendorff-perfused explanted hearts. This study provides the first evidence of Trpm4 directly impacting Na + current in murine cardiac myocytes.

2) Isolation of atrial and ventricular myocyte isolation
Mice were anesthetized with 200 mg/kg ketamine and 20 mg/kg xylazine i.p. and sacrificed by cervical dislocation, after which the heart was rapidly excised, cannulated, and mounted on a Langendorff column for retrograde perfusion at 37°C. The heart was rinsed free of blood for 5 mins with nominally Ca 2+ -free solution containing (mmol/L): 135 NaCl, 4 KCl, 1.2 MgCl 2 , 1.2 NaH 2 PO 4 , 10 HEPES, 11 glucose, pH 7.4 (NaOH).
Next, the heart was perfused with the same solutions with 50 µM Ca 2+ and collagenase type II (1 mg/mL, CLS-2, Worthington, NJ, USA) for 15 minutes. Following digestion, the atria and ventricles were separated and transferred to nominal 100 µM Ca 2+ solution. For atrial myocytes isolation, only the apex of right atrium was excised and minced into small pieces. The cells were dispersed by gentle pipetting with a fire-polished glass pasteur pipette. The ventricle was transferred to above-mentioned buffer with 100 µM Ca 2+ , minced into small pieces to liberate single ventricular myocytes by gentle pipetting, and filtered through a 100 µm nylon mesh. For electrophysiology studies, isolated myocytes were washed 3 times and the calcium concentration was progressively raised to 1 mmol/L within ~30 minutes. Then the cell suspension was placed on a gently rotating shaker at room temperature 22-25ºC until use, within 6 hours after isolation.

3) Single-cell myocyte electrophysiology Data acquisition and analysis
Patch-clamp experiments were performed on isolated myocytes in voltage-or current-clamp mode using MultiClamp 700B and VE-2 controlled by Clampex 10 via a digidata 1332A or 1440A series, respectively. Data were exported to IGOR PRO (WaveMetrics, Lake Oswego, OR, USA) for analysis. Patch electrodes were pulled from borosilicate glass capillaries (World Precision Instruments, Germany GmbH) and had a resistance between 2 -5 MΩ when filled with internal solutions. All experiments were conducted at room temperature (22 -25°C).

Voltage clamp recordings
Peak sodium currents (I Na ) were measured in wholecell configuration using an internal solution (mmol/L) 60 CsCl, 70 Cs-Aspartate, 1 CaCl 2 , 1 MgCl 2 , 10 HEPES, 11 EGTA and 5 Na 2 ATP, pH 7.2 (CsOH). Isolated myo-cytes were bathed in solution containing (mmol/L) 10 NaCl, 120 N-methyl-D-glutamine (NMDG-Cl), 1.8 CaCl 2 , 1.2 MgCl 2 , 5 CsCl, 10 HEPES, and 5 glucose, pH 7.4 (CsOH) along with 10 µM CoCl 2 , and 10 µM nifedipine to inhibit Ca 2+ currents. Peak I Na was measured from a holding potential of -110 mV following steps of 5 mV from -130 to +35 mV with a cycle length of 5 s. Current densities (pA/pF) were calculated by dividing the peak current amplitude by cell capacitance. Series resistance and cell membrane capacitance were compensated for 80%. Voltage dependence of activation was determined from the I/V relationship by normalizing peak I Na to driving force and plotting normalized conductance vs. membrane voltage (V m ). Voltage dependence of steady-state inactivation was obtained by plotting the normalized peak current (25 ms test pulse to -20 mV after a 500 ms conditioning pulse) vs. V m . Voltage dependence of activation and inactivation curves were fitted with the Boltzmann function (( , where V 1/2 is the half-maximal voltage of (in)activation and k is the slope factor. I K1 was measured using the same internal and external solutions as those used for action potential recordings mentioned above without amphotericin B. Na + channels were blocked with 50 µM tetrodotoxin (TTX) and Ca 2+ channels were blocked with 3 mM cobalt chloride (CoCl 2 ) added to the external solution. I K1 barium-sensitive current was calculated by subtracting potassium current recorded after perfusion of the extracellular solution containing 100 µM barium chloride (BaCl 2 ) to potassium current recorded before application of BaCl 2 .

4) Pseudo-and intracardiac ECG recordings on Langendorff-perfused hearts
Mice were anesthetized with 200 mg/kg ketamine and 20 mg/kg xylazine i.p. and sacrificed by cervical dislocation, after which the heart was rapidly excised. Hearts were retrogradely perfused on a Langedorff system using a modified Krebs-Henseleit buffer (KHB) containing (mmol/L) 116.5 NaCl 2 , 25 NaHCO 3 , 4.7 KCl, 1.2 KH 2 PO 4 , 1.2 MgSO 4 , 11.1 glucose, 1.5 CaCl 2 , 2 Na-pyruvate and bubbled with 95% O 2 and 5% CO 2 at 37°C. The perfusion pressure was maintained at 70 mmHg. Pseudo-ECGs were recorded using a pair of thin silver electrodes, with the negative end at the root of aorta and the positive on the apex of the ventricles. The data were acquired using Powerlab bioamplifiers and were low-pass filtered at 5 kHz and high-pass filtered at 10 kHz and sampled at 1 k/sec. For the initial 10 mins, the heart was perfused with KHB buffer followed by 40 µg/mL mexiletine or vehicle control (methanol) dissolved in KHB. Pseudo-ECGs were continuously monitored throughout the experiment and later analyzed using LabChart Pro V.8 (AD Instruments, Australia). The shape of the P wave, QRS complex, and QT region were defined as previously reported [26]. To quantify the ECG perturbation by mexiletine, we defined the QRS duration 2 minutes before the start of the mexiletine perfusion as pre-mexiletine and 20 minutes after the start of mexiletine perfusion as post-mexiletine. We also studied atrioventricular (AV) and intra-ventricular (IV) conduction delays using 1.1F (EPR-800, Millar Instruments, Houston, TX) octapolar intracardiac catheter inserted through the right atrium (RA) and advanced into the right ventricle (RV) on the explanted Langendorff-perfused heart. Proper catheter position was verified by visualization of at least six intracardiac electrocardiograms at the level of right atria and ventricle overlaid by the P wave and QRS complex from the pseudo-ECGs on the same explanted heart. A representative ECG trace from a typical intracardiac recording configuration is shown in Figure 5A. To calculate the conduction delay, we measured the time delay between the first derivative negative peaks from different electrodes (CH 1-6) of the catheter. The distance between each electrode on the catheter is 1 mm. AV delay was measured between CH 6 and 5 (1 mm distance), while IV delay was measured between CH 4 and 1 (3 mm distance).

5) RNA preparation and real-time quantitative RT-PCR
RNA was extracted from isolated ventricular and atrial myocytes. For atrial myocytes, the left and right atria were separated and pooled from three mice for each side. RNA isolation was performed as reported previously [27]. PCR amplification was performed with TaqMan gene expression assay probes for mouse TRPM4 (Mm01205532-m1), Na V 1.5 (Mm01342518-m1), and GAPDH(Mm99999915-g1). Relative quantification was performed using the comparative threshold (CT) method (ΔΔCT) after determining the CT values for the reference (GAPDH) and the target genes (TRPM4 and Na V 1.5) for each sample set.

7) Statistical analyses
Data are presented as mean ± SEM. Statistical significance of differences between two means were determined using Student's t-test if n ≥ 5. P values ≤ 0.05 were accepted as significant and represented as # in respective figure panels. The experimenter and person analyzing the data were blindfolded for genotype of the mice. The number of mice and the number of cells used for each experiment are represented as N and n, respectively, in the figure legends.

I. Deletion of Trpm4 affects the mouse cardiac action potential
Previous studies have reported the expression of Trpm4 in atrial and ventricular tissues from mouse heart [16,17,28]. To evaluate the expression of Trpm4 uniquely in isolated myocytes from different compartments of the heart, we quantified protein mRNA expression using freshly isolated atrial and ventricular myocytes. In line with the reported studies, we observed Trpm4 protein ( Figure 1A and B) and mRNA ( Figure 1C) expression in atrial and ventricular cells. Trpm4 -/mice [28] showed a reduction of the 130 kDa Trpm4 signal both in atria and ventricles. Since a strong expression of Trpm4 has been previously reported in colon, we used it as a positive control in WT. Expectedly, this signal was reduced in Trpm4 -/- (Figure1A).
To determine the functional consequences of Trpm4 deletion on cardiac electrical activity at the cellular level, we recorded cardiac action potential (AP) from freshly isolated right atrial and ventricular cardiomyocytes from WT and Trpm4 -/mice ( Figure 2). In AP recordings from atrial cardiomyocytes, we did not observe any difference in upstroke velocity or action potential duration (APD) among the groups (Figure 2A, and 2B). However, the resting membrane potential (RMP) of atrial cardiomyocytes from Trpm4 -/was significantly depolarized by ~1 mV when compared to the WT ( Figure 2B). Since in cardiac myocytes I K1 is one of the major components for the RMP, we compared the I K1 currents and did not observe any significant alterations in the peak currents (p > 0.05, Supplementary 1). We also measured APs in ventricular cardiomyocytes ( Figure 2C). Here, we observed a decrease in the upstroke velocity in Trpm4 -/mice (p = 0.03); whereas neither APDs nor the RMP were altered ( Figure 2D).

II. Deletion of Trpm4 affects Na + current in atrial and ventricular myocytes
To study the ionic mechanism contributing to the decrease in the upstroke velocity of the action potential recordings from ventricular myocytes, we measured Na + current using a voltage step protocol in whole-cell configuration. In ventricular cardiomyocytes, the peak Na + current density was reduced by 30% in Trpm4 -/mice compared to WT ( Figure 3A and B, Table 1). Although our AP recordings from atrial myocytes did not reveal any alteration in the upstroke velocity, the peak Na + current in atrial myocytes was reduced by 25% in Trpm4 -/compared to WT ( Figure 3D and E, Table 1). In addition, the membrane capacitance of atrial myocytes from Trpm4 -/was reduced by 20% in both peak Na + current and I K1 recordings. (Table 1, Supplementary 1, B). This decrease in the membrane capacitance did not influence the reduction in the peak Na + current observed in atrial myocytes from Trpm4 -/mice (Supplementary 2). To further assess any alterations in the biophysical properties of the peak Na + current, we measured the V 1/2 of steady-state activation and inactivation. We observed no differences in V 1/2 of steady-state inactivation and activation between WT and Trpm4 -/ventricular cardiomyocytes (Figure 3 C, Table 1). In atrial myocytes, however, we observed a significant depolarizing shift of 4 mV in the V 1/2 of steady-state activation in Trpm4 -/mice compared to WT, while the steady-state inactivation was not altered (Figure 3 F, Table 1). Since this reduction of peak Na + current was not previously reported in this mouse line [16], we recently generated in our laboratory a new Trpm4 -/mouse strain on a C57BL/6J background (Supplementary 3 and 4) by targeting Trpm4 exon 10 instead of the exon 15-16 [25]. We measured peak Na + current in WT and Trpm4 -/ventricular myocytes and observed a similar decrease of 25% in peak Na + current in theseTrpm4 -/cells (Supplementary 5).

Figure 2. Action potential measurement in isolated mouse atrial and ventricular myocytes.
Representative traces of AP recorded in WT (black) and

III. Trpm4 -/mouse hearts display an increased sensitivity towards the Na + channel inhibitor mexiletine
Since our data indicated an alteration in Na + current density in atrial and ventricular Trpm4 -/cardiomyocytes, we next investigated the consequences of Trpm4 deletion on the whole heart by in vivo surface ECGs in deeply anesthetized mice. We did not observe any significant alteration in the ECG parameters apart from the heart rate ( Table 2). The heart rate in Trpm4 -/was markedly reduced to 280 ± 19 bpm compared to WT (361 ± 20 bpm; p = 0.02). Further investigations of the electrical properties of Trpm4 -/mice were conducted using pseudo ECG recordings on Langendorff-perfused explanted mouse hearts. Such explanted mouse hearts allow perfusion of different drugs while alterations in electrical proper-  ties can be tracked in real-time. Figure 4A shows an example of a two-lead pseudo-ECG and the analyzed parameters on a perfused explanted WT mouse heart beating spontaneously. In pseudo-ECGs recorded from control buffer perfused hearts, the P duration was significantly increased in Trpm4 -/compared to WT (p = 6*10 -5 ) ( Figure 4B, Table 2). Other ECG parameters including PR interval, QT, QTc and HR were not altered by Trpm4 deletion. QRS duration, however, showed a non-significant trend for broadening in Trpm4 -/hearts (p = 0.06) ( Figure 4C, Table  2). To assess whether Trpm4 deletion affected QRS duration, we challenged the explanted hearts with 40 µg/ mL mexiletine, a Na + channel blocker [29]. Mexiletine broadened the QRS duration in both genotypes (Figure 4D). However, QRS duration from Trpm4 -/hearts presented a two-fold increase in mexiletine sensitivity compared to WT (QRS broadening (%): 102 ± 18 vs. 44 ± 7, p = 0.02) ( Figure 4E). The QRS broadening observed in our recordings was exclusively mexiletine-dependent, as methanol (vehicle control) did not have any effect on the QRS duration ( Figure 4D).

IV. Intra-ventricular conduction is slower in Trpm4 -/mouse heart than in WT
To investigate any potential conduction delay due to the reduction of peak Na + current in Trpm4 -/mice, we performed intracardiac ECG measurements on the spontaneously beating explanted hearts using intracardiac catheters with eight electrodes. In most explanted hearts, we could acquire electrical activity from at least six electrodes, one from the right atrium and five from the right ventricle. Pseudo-ECGs were simultaneously acquired, allowing the correct identification of atrial (A) and ventricular (V) peaks in intracardiac ECGs ( Figure 5A). The AV delay measured between the A and V signal at an electrode distance of 1 mm did not reveal any difference between the genotypes. However, the intra-ventricular (IV) conduction time from the right ventricle measured at an electrode distance of 3 mm was markedly slower in Trpm4 -/than in WT hearts (0.95 ± 0.1 vs. 0.3 ± 0.07 ms, p = 0.002) ( Figure 5C).

V. Knockdown of Trpm4 alters Na V 1.5 expression in mouse ventricles
To further investigate the reduction of peak Na + current observed in isolated atrial and ventricular myocytes, we quantified the expression of Scn5a encoding the cardiac Na + channel Na V 1.5. mRNA expression levels of Na V 1.5 in isolated atrial or ventricular cardiac myocytes ( Figure 6A) did not differ between WT or Trpm4 -/-. However, the total protein expression of Na V 1.5 in whole ventricular tissue was significantly reduced by 50% in Trpm4 -/compared to WT (Figure 6B and C). In atrial tissue, we observed a slight decrease in the total Na V 1.5 expression level that we could not statistically validate ( Figure 6B and C).

Discussion
In the present study, we compared the functional expression of Trpm4 in freshly isolated murine atrial and ventricular myocytes and studied the consequences of Trpm4 deletion on mouse heart electrical activity.
Here we observed an unexpected decrease in Na V 1.5 expression and function in Trpm4-deficient mouse hearts.
Since the reports in which TRPM4 was identified as the prime molecular candidate for NSca [2,4], functional expression of this channel has been detected in different parts of the heart such as the sino-atrial node, atria, and very low levels in ventricles [6,11,17,20,28,30]. In this study, we quantified the expression of Trpm4 in isolated myocytes from the atria and ventricles. The cardiac myocytes from right atria, and both the ven tricles displayed a stronger Trpm4 protein expression signal; these signals were abolished in Trpm4 -/hearts. The functional role of this channel in cardiac electrical activity remains unclear. Trpm4 is suggested to prolong early APDs [16,18], which has been shown using the generic Trpm4 inhibitor 9-phenanthrol [31,32] and in Trpm4 -/mice [33][34][35]. In our perforated-patch AP recordings on isolated myocytes, however, we did not observe any changes in the APDs, while the upstroke velocity in Trpm4 -/ventricular myocytes was significantly slower compared to WT. The fast kinetics of Na V 1.5 underlie the upstroke velocity of a cardiac AP. We, therefore, compared any alteration in the functional expression of Na V 1.5 due to Trpm4 deletion in myocytes from atria and ventricles. Indeed, the peak Na + currents conducted by Na V 1.5 in cardiac myocytes were reduced by 25 and 30% in atria and ventricles, respectively, in Trpm4 -/mice compared to WT. The decrease in peak Na + current correlated with a 50% decrease in Na V 1.5 protein expression in ventricles, while in atria 8 Table 2. Summary of surface and pseudo-ECG parameters from anesthetized mice. we could not statistically validate any change in Na V 1.5 expression. However, in atrial myocytes, our study and others reported stronger Trpm4 expression compared to ventricles [6,17,18,28], the impact of Trpm4 deletion is more complex. In addition to a decrease in the peak Na + current, biophysical properties of the Na + current were also altered with a depolarizing shift of 4 mV in steady-state activation. The RMP of atrial myocytes from Trpm4 -/mice was depolarized by 1 mV; however, we did not observe any alteration in I K1 in atrial myocytes from Trpm4 -/mice. The combined effects of reduced peak Na + currents, altered biophysical properties, and depolarized RMP in Trpm4 -/could reduce the total availability of Na V 1.5 for activation. Na V 1.5-mediated Na + current plays a critical role in excitability and conduction velocity in cardiac tissue [36,37]. The strong functional reduction in Na V 1.5 expression in cardiac myocytes observed in our study was expected to reflect on the whole-heart electrical activity by slowing atrial and ventricular depolariza-tion [38]. Indeed, in our pseudo-ECG recordings on explanted hearts, P duration was longer, and QRS duration shows a trend toward broadening in Trpm4 -/hearts. Challenging the explanted hearts with the Na + channel blocker mexiletine confirmed the broadening of QRS in Trpm4 -/hearts, which were more sensitive towards mexiletine than WT hearts. In addition to slower depolarization, Trpm4 -/hearts also presented significant slowing of intraventricular conduction in the right ventricles compared to WT as observed in intracardiac electrocardiograms. These cellular and whole-heart electrophysiology findings are consistent with a functional reduction of Na V 1.5 in Trpm4-deficient hearts.
In addition, surface ECGs from anesthetized Trpm4 -/mice showed a lower heart rate than those from WT mice. Although a direct decrease in heart rate was not observed in the previous studies, the slowing of electrical propagation and sinus pauses were more evident in Trpm4 -/mice than in WT [17]. Moreover, in this study, the mice were freely moving and the baseline heart rate was ~500 bpm, while in our case under anesthesia, the heart rate was ~300 bpm. This somehow unmasked the effect of Trpm4 deletion on the heart rate. A previous study has shown that, in mouse sinoatrial node, Trpm4 activates at lower heart rates, thus acting as an accelerator and increasing the heart rate to counteract bradycardia [39]. However, we did not observe any heart rate change in spontaneously beating Trpm4 -/hearts in pseudo-ECGs, where the basal heart rate is even lower (~220 bpm) ( Table 2). The effect on heart rate in Trpm4 -/mouse surface ECGs is either due to anesthesia or a functional consequence of Trpm4 deletion in the sino-atrial node, which needs to be cautiously explored in future studies. Although cardiac phenotypes in Trpm4 -/mice have been characterized earlier by different groups, none of these studies observed any alteration in peak Na + currents or Na V 1.5 protein expression [16,17]. This discrepancy between our and other studies could be partially explained by different study designs (isolated myocytes vs. whole tissues, perforated-patch clamp vs. microelectrode / whole-cell recordings). A recent review [40] highlighted that the difference in cardiac phenotype observed in Trpm4 -/mice could be strain-dependent. In this study, we showed a similar reduction of peak Na + current due to Trpm4 deletion in ventricular myocytes from two different mouse lines ( Figure 3 and Supplementary 5). Furthermore, we validated the reduction of peak Na + current in whole heart using pseudo-ECGs on mexiletine-perfused explanted hearts and protein expression studies.
To the best of our knowledge, this study provides the first evidence of a functional interaction between Trpm4 and Na V 1.5 in cardiac tissue. Na V 1.5 contributes to the fast upstroke in the depolarization phase of a cardiac action potential, while Trpm4-mediated current has been found in the repolarization phase [16][17][18][19]. It is intriguing how seemingly distinct depolarizing and repolarizing current components can interact. Yet, a similar concept of ion channel co-regulation has been observed recently for Na V 1.5 and K ir 2.1 [41][42][43]. The authors showed Na V 1.5, and K ir 2.1 modulate each other's function and expression within a macromolecular complex through their respective PDZ-binding domains to regulate cardiac excitability. Another study shows the interaction between hERG and Na V 1.5 even at the level of mRNA transcripts [44]. The authors demonstrate an interaction of the transcripts of hERG1a, hERG1b, and Na V 1.5, which regulate each other's expression at the membrane. Although our study has not addressed whether Trpm4 and Na V 1.5 directly or indirectly interact at mRNA or protein level, we observed a change in the protein expression and function of Na V 1.5 due to Trpm4 deletion without any alteration in Scn5a mRNA expression, suggesting a possible interaction and regulation at the post-translational and membrane trafficking levels. This supports our hypothesis that Trpm4 and Na V 1.5 are interacting protein partners and important for stabilization at the membrane. Nevertheless, future studies to discern possible mechanisms and to determine the level and other partners in the Trpm4-Na V 1.5 interactions are warranted. The importance of TRPM4 in cardiac electrical activity is highlighted by mutations in its gene linked to conduction disorders such as right bundle branch block, Brugada syndrome, and atrio-ventricular block [24,40].
Although the first mutation reported in human TRPM4 was a gain-of-function mutation linked to progressive heart block, several later studies reported both gainand loss-of-function mutations in TRPM4 related to different cardiac conduction disorders [11][12][13]45]. Until now, the mechanisms underlying the genotype-phenotype correlation in TRPM4 mutations remains unclear.
Our study suggests a molecular interaction between TRPM4 and Na V 1.5, brings in a newer perspective on the possible implications of different TRPM4 mutations found in humans with cardiac conduction disorders. We propose that alterations in TRPM4 expression due to mutations could critically affect the availability of Na V 1.5 at the membrane. Thus, an increase or decrease in TRPM4 expression may eventually lead to conduction delays, which are indeed observed in TRPM4 mutation carriers.
In summary, we observed a decrease in Na V 1.5 expression and function in Trpm4-deficient mouse hearts. While the molecular mechanisms underlying this observation are not yet fully understood, one can speculate that the observed overlapping clinical phenotype found in patients with mutations in either SCN5A or TRPM4 may be explained by the co-regulation of Na V 1.5 expression by the channel TRPM4. Future research addressing this intriguing hypothesis is required.