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

Figures

• 

  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…

• 

  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…

• 

  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/Imax as a function of time.

• 

  Figure 5. Characterization of the regulatory β-subunits of the NaV1.5 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 NaV1.5 channel were evaluated by qPCR. (C) Comparison of the expression of β-subunits between vCMs and aCMs.