A distinct de novo expression of Nav1.5 sodium channels in human atrial fibroblasts differentiated into myofibroblasts

Abstract

Fibroblasts play a major role in heart physiology. They are at the origin of the extracellular matrix renewal and production of various paracrine and autocrine factors. In pathological conditions, fibroblasts proliferate, migrate and differentiate into myofibroblasts leading to cardiac fibrosis. This differentiated status is associated with changes in expression profile leading to neo-expression of proteins such as ionic channels. The present study investigates further electrophysiological changes associated with fibroblast differentiation focusing on the activity of voltage-gated sodium channels in human atrial fibroblasts and myofibroblasts. Using the patch clamp technique we show that human atrial myofibroblasts display a fast inward voltage gated sodium current with a density of 13.28 ± 2.88 pA pF(-1) whereas no current was detectable in non-differentiated fibroblasts. Quantitative RT-PCR reveals a large amount of transcripts encoding the Na(v)1.5 α-subunit with a fourfold increased expression level in myofibroblasts when compared to fibroblasts. Accordingly, half of the current was blocked by 1 μm of tetrodotoxin and immunocytochemistry experiments reveal the presence of Na(v)1.5 proteins. Overall, this current exhibits similar biophysical characteristics to sodium currents found in cardiac myocytes except for the window current that is enlarged for potentials between -100 and -20 mV. Since fibrosis is one of the fundamental mechanisms implicated in atrial fibrillation, it is of great interest to investigate how this current could influence myofibroblast properties. Moreover, since several Na(v)1.5 mutations are related to cardiac pathologies, this study offers a new avenue on the fibroblasts involvement of these mutations.

Figure 1. Appearance of a fast inward voltage gated sodium current with human atrial fibroblast differentiation

A, cells were stained after 7 (left panel) or 12 (right panel) days of culture with anti-fibronectin (green labelling) and anti-α-SMA (red labelling) antibodies. B, Western blot, representative of 3 experiments, performed with protein preparations obtained from human atrial fibroblasts at 5 and 15 days of culture using antibodies to VGSC and β-actin. C, percentage of cells that exhibit a voltage gated transient inward current after 3–7 days (D3–D7, n = 7), 8–12 days (D8–D12, n = 12) or 13–17 days (D13–D17, n = 22) of culture. An example of current after 15 days of culture is shown (top panel).

Figure 2. Whole-cell properties of VGSCs recorded in human atrial fibroblasts

A, representative traces of whole cell currents recorded after 5 (D5), 10 (D10) or 15 (D15) days of fibroblast culture using the protocol shown on top. B, current density–voltage relationship recorded after 5 (circles, n = 5), 10 (squares, n = 3) and 15 (triangles, n = 8) days of culture. Currents were elicited as described in A.

Figure 3. Biophysical properties of VGSCs recorded in human atrial myofibroblasts after 15 days in culture

A, voltage dependence of activation (filled circles, n = 8) and inactivation (open circles, n = 5) fitted with a Boltzmann function (see Methods). Sodium conductance (G) was calculated from the I–V curve experiments (Figure 2A). This conductance was normalized to maximum conductance (G_max) obtained during the I–V curve and plotted versus imposed membrane potential. Steady state inactivation was assayed as described in Methods and plotted versus membrane pre-pulse potential. B, the overlap of activation and inactivation of sodium channels defines a range of voltages (window) where the channels could be partially activated without being fully inactivated. The availability of channels at a stable potential is calculated as the product of the activation and the inactivation Boltzmann functions (Huang et al. 2010). C, fast (filled circles) and slow (open circles) inactivation time constants as a function of membrane potential. The decay phases of currents elicited as described in Fig. 2A (n = 8) were fitted with a double exponential to estimate the open state inactivation time constants.

Figure 4. mRNA expression levels of VGSC subunits in human atrial fibroblasts and myofibroblasts

Relative quantities versus GAPDH of Na_vα (A) and β (B) subunit transcripts evaluated by real-time reverse transcription polymerase chain reaction (RT-qPCR) using mRNA extract from human atrial fibroblasts after 3–7 (fibroblasts) or 13–17 (myofibroblasts) days of culture.

Figure 5. Molecular identity of the VGSC α-subunit recorded in human atrial myofibroblasts

A, representative example of the effect of TTX at 1 μm on fibroblast sodium current. Currents were elicited by a standard test pulse from a holding potential of –120 mV to a test pulse of −20 mV for 50 ms. TTX was perfused until current stabilization and washout with a control perfusion to check reversibility. B, effects of 1 μm TTX (n = 4) on the voltage gated sodium current evoked as described in A on myofibroblasts (grey bars) or HEK293 cells transiently transfected with Na_v1.5 +β₁ subunits (white bar). C, fibroblasts (5 days of culture; D5) and myofibroblasts (15 days of culture; D15) observed in fluorescence. Cells were stained with anti-Na_v1.5 antibody and the nuclei were stained using TO-PRO-3. Scale bar corresponds to 20 μm.