Molecular identity of the late sodium current in adult dog cardiomyocytes identified by Nav1.5 antisense inhibition

Abstract

Late Na(+) current (I(NaL)) is a major component of the action potential plateau in human and canine myocardium. Since I(NaL) is increased in heart failure and ischemia, it represents a novel potential target for cardioprotection. However, the molecular identity of I(NaL) remains unclear. We tested the hypothesis that the cardiac Na(+) channel isoform (Na(v)1.5) is a major contributor to I(NaL) in adult dog ventricular cardiomyocytes (VCs). Cultured VCs were exposed to an antisense morpholino-based oligonucleotide (Na(v)1.5 asOligo) targeting the region around the start codon of Na(v)1.5 mRNA or a control nonsense oligonucleotide (nsOligo). Densities of both transient Na(+) current (I(NaT)) and I(NaL) (both in pA/pF) were monitored by whole cell patch clamp. In HEK293 cells expressing Na(v)1.5 or Na(v)1.2, Na(v)1.5 asOligo specifically silenced functional expression of Na(v)1.5 (up to 60% of the initial I(NaT)) but not Na(v)1.2. In both nsOligo-treated controls and untreated VCs, I(NaT) and I(NaL) remained unchanged for up to 5 days. However, both I(NaT) and I(NaL) decreased exponentially with similar time courses (tau = 46 and 56 h, respectively) after VCs were treated with Na(v)1.5 asOligo without changes in 1) decay kinetics, 2) steady-state activation and inactivation, and 3) the ratio of I(NaL) to I(NaT). Four days after exposure to Na(v)1.5 asOligo, I(NaT) and I(NaL) amounted to 68 +/- 6% (mean +/- SE; n = 20, P < 0.01) and 60 +/- 7% (n = 11, P < 0.018) of those in VCs treated by nsOligo, respectively. We conclude that in adult dog heart Na(v)1.5 sodium channels have a "functional half-life" of approximately 35 h (0.69tau) and make a major contribution to I(NaL).

Fig. 1.

Potency of antisense oligonucleotide (asOligo) to silence Na_v1.5 expression in HEK293 cell line stably expressing Na_v1.5. A: representative raw traces of transient Na⁺ current (I_NaT) were recorded at different membrane potentials (V_m) in control conditions [nonsense oligonucleotide (nsOligo)] and in the presence of Na_v1.5 asOligo. B: average data for peak I_NaT-voltage (V) relationship 5 days after nsOligo or Na_v1.5 asOligo delivery. Solid lines show theoretical I-V curves fitted in accordance with Eq. 3. asOligo reduced the maximum I_NaT conductance (G_max) from 12.5 × 10⁻³ to 5.1 × 10⁻³ nS/pF (P < 0.05, F-test) without changes in steady-state activation (SSA) parameters (see Table 2). *Statistically significant (P < 0.05) differences in data points. C: average experimental data of steady-state inactivation (SSI) along with their theoretical Boltzmann fits (Eq. 2, solid and dashed lines). D: asOligo did not affect I_NaT decay time course evaluated by the double-exponential fit (Eq. 1). Shown are data points of 2 time constants (τ_fast, τ_slow) at different V_m, pooled from 9 or 10 cells. E: I_NaT decreases in exponential manner in response to asOligo delivery. Shown are data points along with the linear regression (solid line for nsOligo) and a single-exponential fit (asOligo, Eq. 4). Exponential time constant (τ) value is indicated. Data points in B–E are means ± SE. A and C, insets: voltage-clamp protocols. V_h, holding potential; V_p, prepulse. Statistically significant difference (P < 0.05) in B and E was evaluated by ANOVA followed by Bonferroni's post hoc test. Detailed statistics for all SSA and SSI parameters of the theoretical fits shown in B and C are presented in Table 2. Equations 1–4 are given in materials and methods.

Fig. 2.

Cell culture model and intracellular delivery of oligonucleotides. A: only slight changes in shape (rounded edges) of dog ventricular cardiomyocytes (VCs) were evident after 5 days of culture. 10 μm/div, distance between calibration dots. B: fluorescein-tagged oligo uptake by cardiomyocytes was monitored by confocal microscopy. Confocal images of live cardiomyocytes loaded with fluorescein-tagged oligo are shown. Optical slices were 0.5 μm (Zeiss Axiovert 100, Bio-Rad MRC 1024, excitation/emission wavelength 488/522 nm).

Fig. 3.

I_NaT remains unchanged in cultured adult dog cardiomyocytes. A: representative raw current trace recorded in freshly isolated VCs (left) and after 5 days in culture (right) at different V_m. B: peak I_NaT-V relationship obtained in freshly isolated cells and cells cultured for 5 days. Theoretical curves fit to I-V (Eq. 3, solid and dashed lines) were not statistically different. C: SSI data points along with the fit to a Boltzmann function (Eq. 2, solid and dashed lines, respectively). No statistically significant difference was found when SSI curves were compared. D: decay time constants of I_NaT (double-exponential fit, Eq. 1) were evaluated at the different V_m. E: maximum density of I_NaT remained unchanged in these conditions. Data in B–D are means ± SE and were pooled from 5–8 cells. Detailed statistics for all SSA and SSI parameters of the theoretical fits shown in B and C are presented in Table 2. B and C, insets: voltage-clamp protocols.

Fig. 4.

Cultured adult dog cardiomyocytes represent a useful model to study I_NaL. A: data points of I_NaL-V relationships for freshly isolated and cultured cells along with their theoretical fits (Eq. 3, solid and dashed lines). Inset: typical examples of raw I_NaL traces recorded in fresh and cultured cells are shown superimposed. There was no statistical difference between the data points or theoretical fit of the I-V curves. B and C: density and SSI of I_NaL remain unchanged in cultured cells. C: data points of SSI are shown along with their theoretical fits (Eq. 2, solid and dashed lines). Inset: our voltage-clamp protocol. D: decay time course of I_NaL was evaluated by the 2-exponential fit (Eq. 1) and remained unchanged in the cultured cells. All data are means ± SE. Detailed statistics for all SSA and SSI parameters of the theoretical fits shown in A and C are presented in Table 2.

Fig. 5.

asOligo effectively knocks down Na_v1.5 expression in adult dog cardiomyocytes. A: asOligo caused decrease in peak I_NaT. Representative raw traces of I_NaT were recorded at different membrane potentials in control conditions (nsOligo, right) and with asOligo (left). B: peak I_NaT-V relationship obtained in cells treated with nsOligo (control) and asOligo. *Statistically significant differences in data points (P < 0.05) compared at different V_m. Solid (nsOligo) and dashed (asOligo) lines show theoretical curves of I-V (Eq. 3) fitted to data points. asOligo significantly reduced G_max from 1.39 to 1.01 pS/pF (P < 0.05, F-test). C: asOligo did not affect the SSI of I_NaT. Shown are data points of SSI along with their theoretical fits (Eq. 2; solid lines nsOligo and dashed lines asOligo; see Table 2 for SSI parameters). D: asOligo did not affect the decay time course of I_NaT. The two time constants, τ_fast and τ_slow (double-exponential fit, Eq. 1), vs. membrane and the relative contribution of τ_slow (k_slow) are given at top and bottom, respectively. E: time course of I_NaT decrease in response to Na_v1.5 knockdown by asOligo. Data points are shown along with the linear regression (solid line for nsOligo) and exponential fit (asOligo). Time constant is indicated; n is the number of cardiomyocytes. The initial level of I_NaT (100%) was 47.3 ± 9 pA/pF (n = 25). Statistically significant difference (P) in A and E was evaluated by ANOVA followed by Bonferroni's post hoc test. Detailed statistics for all SSA and SSI parameters of the theoretical fits shown in A and C are presented in Table 2. Bars in B and D and points in A and C represent means ± SE.

Fig. 6.

Molecular identity of I_NaL in dog ventricular cardiomyocytes assessed by asOligo specific to mRNA encoding Na_v1.5. A: I_NaL density is statistically significantly reduced by the asOligo (P < 0.001). Inset: superimposed representative raw I_NaL traces recorded in the presence of nsOligo and asOligo, respectively. B: knockdown of SCN5A gene by asOligo leads to the parallel decrease of both I_NaT and I_NaL, as I_NaL-to-I_NaT ratio remains unchanged. C: asOligo does not affect the fine structure of I_NaL decay evaluated by the 2-exponential fit (Eq. 1). D: statistical analysis of the time course of I_NaL density changes in response to asOligo. Data points are shown along with the linear regression (solid line for nsOligo) and the exponential fit (asOligo). The time constant is indicated; the initial level (100%) of I_NaL current density was 0.223 ± 0.025 pA/pF (n = 18). Statistically significant difference (P) in A and E was evaluated by ANOVA followed by Bonferroni's post hoc test. Detailed statistics for all SSA and SSI parameters of the theoretical fits shown in A and C are presented in Table 2. Bars in A–C and points in D represent means ± SE.

Fig. 7.

Theoretical evaluation of the fine structure of I_NaL determined by Na_v1.5 in adult dog cardiomyocytes. Parameters (time constants and densities) for the idealized, 2-exponential decay time course of I_NaL were assigned from the averaged experimental data measured in cultured cardiomyocytes treated with nsOligo (density 0.276 pA/pF at 200 ms, τ₁ = 48.6, τ₂ = 502 ms, k₂ = 0.268) or asOligo (0.132 pA/pF, τ₁ = 38.8, τ₂ = 497 ms, k₂ = 0.305). Currents generated by the burst (I_BM in A) and late scattered openings (I_LSM in B) were obtained from Eqs. 5 and 6 (results), respectively, and the total I_NaL (C) was obtained as the sum of these two. The difference currents between nsOligo and asOligo-treated cells are shown as shaded areas.