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

Abstract

Transient receptor potential melastatin member 4 (TRPM4) encodes a Ca²⁺-activated, non-selective cation channel that is functionally expressed in several tissues, including the heart. Pathogenic mutants in TRPM4 have been reported in patients with inherited cardiac diseases, including conduction blockage and Brugada syndrome. Heterologous expression of mutant channels in cell lines indicates that these mutations can lead to an increase or decrease in TRPM4 expression and function at the cell surface. While the expression and clinical variant studies further stress the importance of TRPM4 in cardiac function, the cardiac electrophysiological phenotypes in Trpm4 knockdown mouse models remain incompletely characterized. To study the functional consequences of Trpm4 deletion on cardiac electrical activity in mice, we performed perforated-patch clamp and immunoblotting studies on isolated atrial and ventricular cardiac myocytes and surfaces, as well as on pseudo- and intracardiac ECGs, either in vivo or in Langendorff-perfused explanted mouse hearts. We observed that TRPM4 is expressed in atrial and ventricular cardiac myocytes and that deletion of Trpm4 unexpectedly reduces the peak Na⁺ currents in myocytes. Hearts from Trpm4^-/- mice presented increased sensitivity towards mexiletine, a Na⁺ channel blocker, and slower intraventricular conduction, consistent with the reduction of the peak Na⁺ current observed in the isolated cardiac myocytes. This study suggests that TRPM4 expression impacts the Na⁺ current in murine cardiac myocytes and points towards a novel function of TRPM4 regulating the Na_v1.5 function in murine cardiac myocytes.

Keywords: SCN5A; TRPM4; cardiac conduction disorder; channelosome; intracardiac ECG; mexiletine.

Figure 1

Expression of Trpm4 in mouse heart models. (A) Representative immunoblots of Trpm4 protein expression from right (RA) and left (LA) atrium, ventricles, isolated ventricular myocytes (v.myocytes), and colon tissue. (B) Quantification of the immunoblot showing protein expression in the atria (N = 10), ventricles (N = 11), and v.myocytes (N = 4). (C) Quantification of Trpm4 mRNA using RT-qPCR. The Ct values were normalized to GAPDH (N = 4). Note: #: p < 0.05, WT (circle, black) vs. Trpm4^−/− (square, red).

Figure 2

Action potential measurement in isolated mouse atrial and ventricular myocytes. Representative traces of AP recorded in WT (black) and Trpm4^−/− (red) mice either from (A) right atrial myocytes (N = 5, n = 19) or (C) ventricular myocytes (N = 5, n = 14). Average values for AMP (peak amplitude), RMP, V_max in WT (circle, black), and Trpm4^−/− (square, red) either from (B) right atrial myocytes or (D) ventricular myocytes. Note: #: p < 0.05, WT vs. Trpm4^−/−.

Figure 3

Na⁺ current recordings from isolated cardiomyocytes. Representative Na⁺ current traces recorded from myocytes in WT (black) or Trpm4^−/− (red) mice, either from ventricular (N = 5, n = 9 WT, n = 12 Trpm4^−/−) (A) or atrial (N = 5, n = 20 WT, n = 22 Trpm4^−/−) tissue (D), using a voltage step protocol as shown in inset. Na⁺ current–voltage relationships in WT and Trpm4^−/− mice from either ventricular (B) or atrial (E) cardiomyocytes. (C,F) Voltage dependence of activation (open square or circle) and inactivation (filled in square or circle) fitted with Boltzmann equation in WT and Trpm4^−/− mice from either ventricular or atrial cardiomyocytes, respectively. Note: #: p < 0.05, WT vs. Trpm4^−/− at a given voltage).

Figure 4

Pseudo-electrocardiogram recordings of WT and Trpm4^−/− explanted hearts. (A) Representative pseudo-ECG trace from WT heart highlighted for different ECG parameters, considered for further comparative studies. Average P (B) and QRS (C) durations recorded from either WT (N = 19: circle, black) or Trpm4^−/− (N = 22: square, red) hearts. (D) Representative ECG traces before and after perfusion with mexiletine. (E) QRS durations calculated when perfused with either buffer or mexiletine (Mex.) in WT (N = 7, black) and Trpm4^−/− (N = 11, red) mice or perfused with the vehicle control, methanol (N = 6, blue). (F) The degree of broadening of QRS due to mexiletine perfusion compared between the genotypes as % inhibition (WT: circle, black and Trpm4^−/−: square, red). Note: #: p < 0.05, WT vs. Trpm4^−/−, ns- not significant.

Figure 5

Intracardiac electrocardiogram recordings on WT and Trpm4^−/− explanted hearts. (A) Representative pseudo (P.ECG) and intracardiac (IC.ECG) ECG trace from a WT heart. A and V respectively represent atrial and ventricle signals from the intracardiac catheter recorded in different electrode channels (CH 1-7). Conduction delays either between the atria and ventricle (AV delay, CH 6-5) or intraventricular (IV delay, CH 4-1) were measured as the time delay between respective channel signals. Representative trace showing signal derivatives from CH 1 and 4 to measure IV delay (B). Average AV delay (C) and IV delay (D), compared between the genotypes (WT: circle, black and Trpm4^−/−: square, red). Note: N = 6, #: p < 0.05, WT vs. Trpm4^−/−; ns = not significant.

Figure 6

Expression of Na_v1.5 in mouse atria and ventricles. (A) Representative immunoblots showing Na_v1.5 expression from atria and ventricles, either from WT or Trpm4^−/− mice. (B) Quantification of the immunoblots showing the total Na_v1.5 expression in atria (N = 6; p = 0.63) and ventricles (N = 13; p = 0.20): WT (circle, black) or Trpm4^−/− (square, red). (C) Na_v1.5 mRNA quantification using RT-qPCR from atrial and ventricular tissue; WT (circle, black) or Trpm4^−/− (square, red). The Ct values were corrected with GAPDH (N = 4; p = 0.87 and 0.89 for atria and ventricles, respectively).

Figure 7

Co-immunoprecipitation of Na_v1.5 and TRPM4 in HEK-293 cells. (A) Left panel: Input signals confirming the expression of Na_v1.5 with or without FLAG tag and TRPM4. The alpha subunit of the Na/K pump was used as a loading control. Right panel: In the immunoprecipitated (IP) fraction, the signal was observed only in the presence of FLAG-Na_v1.5 and TRPM4. (B) Left panel: Input signals confirming the expression of Na_v1.5 with or without FLAG tag and TRPM5. Right panel: In the immunoprecipitated fraction, no signal was observed for TRPM5, even though the IP of FLAG-Na_v1.5 was successful.