Biophysical properties of NaV1.5 channels from atrial-like and ventricular-like cardiomyocytes derived from human induced pluripotent stem cells

Abstract

Generating atrial-like cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) is crucial for modeling and treating atrial-related diseases, such as atrial arrythmias including atrial fibrillations. However, it is essential to obtain a comprehensive understanding of the electrophysiological properties of these cells. The objective of the present study was to investigate the molecular, electrical, and biophysical properties of several ion channels, especially Na_V1.5 channels, in atrial hiPSC cardiomyocytes. Atrial cardiomyocytes were obtained by the differentiation of hiPSCs treated with retinoic acid (RA). The quality of the atrial specification was assessed by qPCR, immunocytofluorescence, and western blotting. The electrophysiological properties of action potentials (APs), Ca²⁺ dynamics, K⁺ and Na⁺ currents were investigated using patch-clamp and optical mapping approaches. We evaluated mRNA transcript and protein expressions to show that atrial cardiomyocytes expressed higher atrial- and sinoatrial-specific markers (MYL7, CACNA1D) and lower ventricular-specific markers (MYL2, CACNA1C, GJA1) than ventricular cardiomyocytes. The amplitude, duration, and steady-state phase of APs in atrial cardiomyocytes decreased, and had a shape similar to that of mature atrial cardiomyocytes. Interestingly, Na_V1.5 channels in atrial cardiomyocytes exhibited lower mRNA transcripts and protein expression, which could explain the lower current densities recorded by patch-clamp. Moreover, Na⁺ currents exhibited differences in activation and inactivation parameters. These differences could be explained by an increase in SCN2B regulatory subunit expression and a decrease in SCN1B and SCN4B regulatory subunit expressions. Our results show that a RA treatment made it possible to obtain atrial cardiomyocytes and investigate differences in Na_V1.5 channel properties between ventricular- and atrial-like cells.

Figure 1

Characterization of specific-markers of vCMs and aCMs. (A) qPCR analysis of several cardiomyocyte genes implicated in cellular excitability (SCN5A, CACNA1C, CACNA1D, GJA1) and contraction (TNNT2, MYL2, MYL7). The analysis of exon 25 of SCN5A mRNA covered all isoforms, including the adult and neonatal isoforms. (B) Fluorescence images showing immunolabeling of cardiac TNNT2, MYL7, MYL2, ACTN1 (α-actinin), GJA1 (connexin 43), and nuclei (DAPI, cyan) (scale bar: 40 µm). Immunofluorescence images were acquired using Zeiss LSM780 confocal microscope, processed with ZEN software (Zeiss), and adapted with ImageJ software version 1.54f (NIH, Bethesda, MD, USA). (C) Western blot analysis of the expression of several excitation–contraction coupling proteins and ion channels in vCMs and aCMs. All images of cropped blot section were exposed with an optimal time to observe protein bands. All cropped blot sections were delimited by black lines. Cropped strain-free blots showing total proteins served as loading control. Original blots are presented in Suppl. Fig. S4. Top panel, middle panel, and bottom panel, respectively, refer to “Blot 1”, Blot 2” and “Blot 3″ in Suppl. Fig. S4. Cropped section areas are indicated in Suppl. Fig. S4 by red lines. Immunoblot images were adapted with ImageJ software and arranged with Microsoft Powerpoint software version microsoft 365 (Microsoft, Redmond, WA, USA).

Figure 2

Characterization of the cardiac electrical activity, Ca²⁺ homeostasis and atrial-specific K⁺ currents in vCMs and aCMs. (A) Superposed APs recorded in current-clamp mode at 1 Hz. The dashed line represents 0 mV. The inset represents a magnification of the depolarizing phase of the action potential. The 3 ms scale bar represents the duration of the injected current pulse. (B, C, D) Box and whiskers summarizing the resting membrane potential (B), the overshoot (C), and the APD at 90% repolarization (D). (E, H) Representative optical action potentials (OAPs, E) and Ca²⁺ transients (H) simultaneously recorded in CM monolayers using RH237 and Rhod-2, respectively. The vertical dashed lines represent a stimulation at 1 Hz. (F, I) Representative activation maps at 1 Hz. The left symbol (□) indicates the position of the stimulating electrodes and the right symbol (■) indicates the position of the recordings. (G, J) Box and whiskers summarizing AP conduction (G) and Ca²⁺ propagation (J) velocities (CV and CaPV). (K) Representative K⁺ current densities recorded in voltage-clamp mode before and after the perfusion of 0.1 mmol/L and 1 mmol/L 4-AP, an inhibitor of voltage-gated potassium channels. The dashed line represents zero current. The inset corresponds to the patch-clamp protocol to record K⁺ currents. (L) Normalized I/V obtained after subtracting the K⁺ currents recorded under 4-AP perfusion from the K⁺ currents measured without 4-AP. The resulting trace corresponds to the 4-AP-sensitive K⁺ currents.

Figure 3

Biophysical properties of L-type voltage-gated Ca²⁺ channels in vCMs and aCMs. (A) Representative Ca²⁺ current densities recorded in voltage-clamp mode. The dashed line represents zero current. (B) Normalized intensity/voltage relationships (I/V). (C) Steady-state activation and inactivation of Ca²⁺ currents. The patch-clamp protocols used are inserted as insets in the graph for activation (right) and inactivation (top).

Figure 4

Biophysical properties of voltage-gated Na⁺ channels in vCMs and aCMs. (A) Representative Na⁺ current densities recorded in voltage-clamp mode. The dashed line represents zero current. (B) Normalized intensity/voltage relationships (I/V). (C) Steady-state activation and inactivation of Na⁺ currents. (D, E) Box and whiskers summarizing the half-activation and half-inactivation potentials (D) and the K slope factor (E). (F) Recovery from inactivation. (G) Box and whiskers summarizing the recovery time constants. (H) The time constants of fast inactivation decay plotted as a function of voltage. Inset shows the percentage of I/I_max as a function of time.

Figure 5

Characterization of the regulatory β-subunits of the Na_V1.5 channel. (A) β-subunit expression in vCMs normalized to the expression of SCN1B. (B) β-subunit expression in aCMs normalized to the expression of SCN1B. All β-subunits (SCN1B, SCN2B, SCN3B, and SCN4B) of the Na_V1.5 channel were evaluated by qPCR. (C) Comparison of the expression of β-subunits between vCMs and aCMs.