Post-transcriptional silencing of SCN1B and SCN2B genes modulates late sodium current in cardiac myocytes from normal dogs and dogs with chronic heart failure

Abstract

The emerging paradigm for Na⁺ current in heart failure (HF) is that its transient component (I_NaT) responsible for the action potential (AP) upstroke is decreased, whereas the late component (I_NaL) involved in AP plateau is augmented. Here we tested whether Na_vβ₁- and Na_vβ₂-subunits can modulate I_NaL parameters in normal and failing ventricular cardiomyocytes (VCMs). Chronic HF was produced in nine dogs by multiple sequential coronary artery microembolizations, and six dogs served as a control. I_Na and APs were measured by the whole cell and perforated patch-clamp in freshly isolated and cultured VCMs, respectively. I_NaL was augmented with slower decay in HF VCMs compared with normal heart VCMs, and these properties remained unchanged within 5 days of culture. Post-transcriptional silencing SCN1B and SCN2B were achieved by virally delivered short interfering RNA (siRNA) specific to Na_vβ₁ and Na_vβ₂. The delivery and efficiency of siRNA were evaluated by green fluorescent protein expression, by the real-time RT-PCR, and Western blots, respectively. Five days after infection, the levels of mRNA and protein for Na_vβ₁ and Na_vβ₂ were reduced by >80%, but mRNA and protein of Na_v1.5, as well as I_NaT, remained unchanged in HF VCMs. Na_vβ₁-siRNA reduced I_NaL density and accelerated I_NaL two-exponential decay, whereas Na_vβ₂-siRNA produced an opposite effect in VCMs from both normal and failing hearts. Physiological importance of the discovered I_NaL modulation to affect AP shape and duration was illustrated both experimentally and by numerical simulations of a VCM excitation-contraction coupling model. We conclude that in myocytes of normal and failing dog hearts Na_vβ₁ and Na_vβ₂ exhibit oppositely directed modulation of I_NaL.

the emerging paradigm for Na⁺ current (I_Na) in chronic heart failure (HF) is that its transient component (I_NaT) responsible for the action potential (AP) upstroke and excitation propagation is decreased, whereas the late component (I_NaL) involved in AP plateau is augmented (21, 23, 25, 42, 45, 48). Molecular mechanisms of these HF-related I_Na alterations are still understudied. Numerous studies indicate a possible role of Na_vβ auxiliary subunits to modulate Na⁺ channel (NaCh) expression and function (27), but implications of Na_vβ in I_NaL modulation have not been studied in detail, especially in HF. Our previous studies in a canine chronic HF model showed that the protein level of the main NaCh isoform expressed in the heart, Na_v1.5, underlying I_NaL (18), is reduced but remains unchanged for Na_vβ₁- and Na_vβ₂-subunits, making these β-subunits relatively upregulated (48). Thus an intriguing possibility could be that differential expression of Na_v1.5 and Na_vβ-subunits in normal and failing hearts can contribute, at least in part, to I_NaL alterations observed in HF.

In the present study using a combination of experimental and numerical modeling approaches, we tested a hypothesis that Na_vβ₁ and Na_vβ₂ can modulate I_NaL in ventricular cardiomyocytes (VCMs) isolated from adult failing dog hearts. We performed post-transcriptional silencing of SCN1B and SCN2B genes by the sequence-directed RNA interference using short interfering RNA (siRNA). We took advantage of the established adult dog VCMs culture model that preserves I_NaL and I_NaT over 5 days, i.e., sufficient time for gene silencing and membrane protein turnover (18). We found that silencing of SCN1B and SCN2B genes significantly lessens or enhances I_NaL, respectively. Physiological significance of these β-subunits–related modulations of I_NaL was illustrated by the concomitant AP changes both experimentally and in silico.

MATERIALS AND METHODS

HF Model

The study conforms to the Guidelines for Care and Use of Laboratory Animals published by the National Institutes of Health and was approved by the Animal Care and Use Committee of the Henry Ford Health System. Chronic HF that is similar by a vast array of functional and pathophysiological parameters (35) to that in humans was produced in nine dogs by multiple sequential coronary artery microsphere embolizations as previously described (36). Six normal dogs serve as a control. At the time of harvesting the heart (∼3 mo after last embolization), left ventricular (LV) ejection fraction was ∼25%.

Cell Culture and Transfection

Midmyocardial VCMs were enzymatically isolated from the apical LV midmyocardial slices as reported previously (21). The yield of viable rod-shaped, Ca²⁺-tolerant VCMs varied from 40% to 70%. VCMs were cultured for 5 days [the time frame required for the gene silencing (18)], as described previously (23). During this time, VCMs manifested slightly rounded edges, i.e., well-known characteristics for the cultured adult cardiomyocytes (9, 31). Adenovirus-delivered siRNAs did not further affect morphology of VCMs (Supplemental Fig. S1). We added 10 μl of 5 × 10⁸ virus particle/ml directly to the medium of culture dish containing ∼15 × 10⁴ cells and cultured them further during the next 4 days (5 days total). Effectiveness of transfection was visually monitored in individual cardiomyocytes (Nikon Diaphot 200) by green fluorescent protein (GFP) fluorescence (Fig. 2A, inset, and Supplemental Fig. S1).

Fig. 2.

Post-transcriptional silencing of Na_vβ₁- or Na_vβ₂-subunits expression does not affect the whole cell transient sodium current, I_NaT, in patch-clamped cultured left ventricular cardiac myocytes from dogs with chronic HF. A: representative raw traces of I_NaT were recorded at different membrane potentials (V_m) in cells 5 days after infection with the virus containing control nonsilencing siRNA (control, left), β₁-siRNA (middle), or β₂-siRNA (right). Image of cells expressing green fluorescent protein (GFP) is shown at left inset. B: average data for peak I_NaT-voltage relationship obtained in cultured cells for 5 days with virally delivered control siRNA, Na_vβ₁-siRNA, and Na_vβ₂-siRNA. Solid lines show theoretical curves fit to current-voltage (I-V) to evaluate steady-state activation (SSA) parameters (Supplemental Eq. S2, solid dashed and dotted lines, respectively). C: average experimental data of steady-state inactivation (SSI) data points along with the fit to a Boltzmann function (Supplemental Eq. S3, solid, dashed, and dotted lines, respectively). No statistically significant difference was found when theoretical SSI curves were compared (F test). D: decay time constants of I_NaT (double exponential fit; Supplemental Eq. S1) were evaluated at the different membrane potentials. E: maximum density of I_NaT remained unchanged in these conditions. Data in B–D represents means ± SE and were pooled from 5 to 8 cells. Detailed statistics for all SSA and SSI parameters of the theoretical fits shown in B and C are presented in Table 1. Voltage-clamp protocols are shown in B and C, insets. V_h, holding potential; V_p, prepulse.

siRNA Design

Double-stranded hairpin siRNAs corresponding to the previously sequenced dog Na_vβ₁ and Na_vβ₂ (GenBank DQ061859 and AY263393, respectively) were designed as recommended (8). The details and sequences of silencing and nonsilencing control siRNAs used in this study are given in Supplemental Table S1. A Blast search for these sequences against the GenBank database was performed and did not reveal matches with any other gene.

Real-time PCR and Western Blot Analysis

To quantify mRNA levels, we used the fluorescence-based (cyber green) kinetic real-time PCR performed using 7500 Fast Real-Time PCR System Applied Biosystems sequence detection system. The relative mRNA abundance expressed as arbitrary units was calculated using the expression levels of all transcripts normalized to GAPDH mRNA. This value was then normalized to mRNA levels measured from VCMs transfected with the nonsilencing siRNA for the appropriate transcript (2^−ΔΔCt method). The sets of primers are given in Supplemental Table S2. Membrane protein preparations were obtained from ∼3 × 10⁶ myocytes as previously described (Ref. 48; Supplemental Material for details). Antibody was detected with Western blot chemoluminescence reagent (NEN Life Science Product). Western blots were considered specific if the peptide epitope reduced the band intensity. The resulting images of Western blots were scanned, and relative densities of bands were quantified using SigmaGel or ImageJ software, which includes a background subtraction algorithm. The primary polyclonal anti-Na_vβ₁ and ant-Na_vβ₂ antibodies were obtained from Cell Applications (San Diego, CA) and Alomone Labs (Jerusalem, Israel), respectively. Representative full Western blots with these antibodies are shown in the Supplemental Fig. S2. Polyclonal calsequestrin antibody (Abcam) was used for the protein loading control.

Electrophysiology

I_Na and AP were recorded using a conventional whole cell and perforated patch-clamp techniques, respectively. The details of a double exponential fit to decay kinetics of the I_NaT and I_NaL as well as evaluation of the steady state activation (SSA) and steady state inactivation (SSI) voltage dependency is given in the Supplemental Material (Supplemental Equations S1, S2, and S3, respectively).

Numerical AP Model

To address physiological significance of I_NaL modulation by β-subunits, we used a modified excitation-contraction (E-C) coupling model of normal and failing canine ventricular myocyte developed previously by Winslow et al. (46). Our model modification to include I_NaL equations has been recently published (44).

All experimental procedures and numerical modeling details (including numerical values of parameters used in the present study) are available in the Supplemental Material (expanded Supplemental Methods section).

Statistical Analysis

Statistics are reported as means ± SE with n representing the number of cells. Multiple comparisons between treatment groups were made using one-way ANOVA followed by the Bonferroni's post hoc test. The significance of changes of theoretical SSA and SSI fits was evaluated with an F test (StatMost, Data-Most, Salt Lake City, UT) for tabulated values predicted by the model (Supplemental Eqs. S2 and S3) at a confidence level of 0.95. Differences for both experimental data and model predictions were considered statistically significant for P < 0.05.

RESULTS

Efficacy of siRNAs to Silence SCN1B and SCN2B Genes

The efficacy of virally delivered siRNAs to silence concomitant SCN1B and SCN2B genes in cultured VCMs was assessed by changes in both mRNA transcript levels and membrane protein levels of Na_vβ₁ and Na_vβ₂ (Fig. 1). The expression of the reporter gene GFP was controlled both visually and by RT-PCR (Fig. 2A and Supplemental Fig. S1). We found that after 4 to 5 days of culture, 100% of cells expressed GFP confirming successful viral transfection. Level of mRNA transcripts encoding Na_vβ₁ and Na_vβ₂ (Fig. 1A) dramatically decreased in VCMs treated with the corresponding siRNA compared with those treated with control nonsilencing siRNA. The expression of the main pore forming cardiac subunit Na_v1.5, or other auxiliary β-subunits, not targeted by the siRNA, remained unchanged in these cells (Fig. 1A). Parallel to mRNA, membrane protein levels for both Na_vβ₁ (Fig. 1C) and Na_vβ₂ (Fig. 1D) were also dramatically reduced in response to the siRNA treatment. These results confirm efficacy and specificity of the virally delivered siRNAs to silence SCN1B and SCN2B genes in cultured adult dog VCMs.

Fig. 1.

Efficacy of virally delivered Na_vβ₁-short interfering RNA (siRNA) and Na_vβ₂-siRNA to post-transcriptionally silence SCN1B and SCN2B genes in ventricular cardiac myocytes from dogs with chronic heart failure (HF). A: SCN5A, SCN1B, SCN2B, and SCN3B mRNA expression levels detected by the real-time PCR and normalized to GAPDH mRNA and to the respective mRNA levels detected in cells transfected with nonsilencing siRNA (2^−ΔΔCt method) for each gene. *,**Significantly reduced mRNA level compared with other genes in the group (P < 0.05, ANOVA followed by Bonferroni's post hoc test). Data gathered from 4 to 7 transfections. B and C, top: representative Western blot of Na_vβ₁ (SCN1B) and Na_vβ₂ (SCN2B) subunits (bottom bands) and housekeeping protein calsequestrin (CSQ; top bands) for normalization in cells treated with virally delivered siRNA. Control stands for nonsilencing siRNA. B and C, bottom: bar graphs, in arbitrary units (AU), of the relative protein density, which demonstrates dramatic reduction of Na_vβ₁ or Na_vβ₂ in response to the transfection with concomitant siRNA when normalized to the CSQ, and then normalized to the quantity of the immunoreactive protein in cells transfected with nonsilencing siRNA. Membrane proteins were isolated from at least 3 transfections for each condition (n is shown at the bars). Control in B and C stands for the control, nonsilencing siRNA. *,**Protein levels that were significantly reduced compared with the control (P < 0.05, P < 0.05, ANOVA followed by Bonferroni's post hoc test). In this and in the rest of the figures Na_vβ₁ and Na_vβ₂ were abbreviated to β₁ and β₂, respectively.

Post-transcriptional Silencing of SCN1B and/or SCN2B Genes Does Not Affect I_NaT

We tested whether reduced expression of Na_vβ₁ or Na_vβ₂ can affect I_NaT. Figure 2A shows representative family of current traces at different depolarization steps to evaluate the current voltage (I-V) relationship in cells transfected with control nonsilencing siRNA (Fig. 2, left) and with siRNAs designed to silence either SCN1B (Fig. 2, middle) or SCN2B (Fig. 2, right) genes. We found that I-V, SSA (Fig. 2B), SSI, (Fig. 2C), two-exponential decay kinetics (Fig. 2D), and density for I_NaT (Fig. 2E) remained unchanged in VCMs with silenced SCN1B or SCN2B genes, respectively. Statistical data for SSA and SSI parameters for these experiments are given in Table 1.

Table 1.

Voltage dependence of SSA and SSI for I_Na in ventricular cardiomyocytes from normal dogs and dogs with chronic heart failure

	SSI Parameters			SSA Parameters
Conditions	V1/2A, mV	kA, mV	n	V1/2G, mV	kG, mV	n
Normal heart, INaT
Control siRNA	−82.0 ± 0.6	−5.9 ± 0.8	7	−41.3 ± 0.1	5.2 ± 0.4	5
Navβ1-siRNA	−81.9 ± 0.7	−6.2 ± 0.6	5	−40.9 ± 1.2	5.9 ± 0.2	5
Navβ2-siRNA	−82.1 ± 1.8	−6.1 ± 1.1	5	−40.7 ± 1.8	6.0 ± 0.8	5
Failing heart, INaT
Control siRNA	−81.6 ± 1.7	−6.7 ± 0.7	11	−40.3 ± 1.5	6.4 ± 0.4	10
Navβ1-siRNA	−82.2 ± 1.4	−5.9 ± 0.9	9	−40.2 ± 1.1	6.1 ± 0.3	10
Navβ2-siRNA	−81.9 ± 1.5	−6.4 ± 0.7	11	−39.9 ± 1.7	5.6 ± 0.4	9
Normal heart, INaL
Control siRNA	−81.7 ± 0.5	−6.2 ± 0.5	7	−37.8 ± 1.2	6.1 ± 0.8	5
Navβ1-siRNA	−80.2 ± 1.6	−6.4 ± 0.4	5	−40.1 ± 0.8	5.3 ± 0.4	5
Navβ2-siRNA	−80.9 ± 1.4	−6.2 ± 0.8	5	−41.0 ± 0.7	5.1 ± 0.6	5
Failing heart, INaL
Control siRNA	−80.2 ± 0.8	−6.3 ± 0.5	15	−38.5 ± 1.9	5.4 ± 0.7	11
Navβ1-siRNA	−82.0 ± 1.2	−6.1 ± 0.4	19	−39.2 ± 3.1	5.6 ± 0.9	6
Navβ2-siRNA	−81.6 ± 1.2	−6.6 ± 0.4	16	−39.1 ± 1.1	5.6 ± 0.5	12

Values are means ± SE; n, cell number. Steady-state inactivation and steady-state activation (SSI and SSA) parameters were obtained from Na⁺ current (I_Na) data fit to the Supplemental Eqs. S2 and S3, respectively. We did not find statistically significant differences between parameters within the experimental groups evaluated by the ANOVA followed by Bonferroni's post hoc test. siRNA, short interfering RNA; I_NaT, transient Na⁺ component; I_NaL, late Na⁺ component; V_1/2G and k_G, midpoint and the slope of the respective Boltzmann function underlying the NaCh SSA; V_1/2A an k_A, midpoint and slope of the relationship of SSI parameters.

Modulation of I_NaL by Na_vβ₁ and Na_vβ₂ Subunits

At first we tested whether primary cell culture affects the established paradigm that I_NaL is augmented and slower in failing compared with normal hearts (25, 42, 45). It is evident from Fig. 3, B–D, that within 5 days culture I_NaL remained robust in both VCMs from normal and failing hearts, and the paradigm was preserved. Moreover, our infection procedure did not alter I_NaL parameters in failing VCMs when freshly isolated and treated with virus containing nonsilencing siRNA were compared (Fig. 3, B–D). In contrast with I_NaT, we found a significant but opposite change in I_NaL in response to reduced expression of Na_vβ₁ or Na_vβ₂ by the siRNAs (Fig. 3). As evident from the figure, in VCMs isolated from both normal and failing hearts, reduction of Na_vβ₁ expression resulted in I_NaL decay acceleration and density reduction. In contrast, I_NaL decay was delayed and density was increased in VCMs with reduced levels of Na_vβ₂. Figure 4A shows I-V relationship for these experiments. Clearly, reduction of Na_vβ₁ or Na_vβ₂ expression reduced or increased density of I_NaL within the wide range of membrane potentials including potentials corresponding to the AP plateau, respectively. SSA (Fig. 4A) and SSI (Fig. 4B) parameters remained unchanged in these conditions (statistical data are given in Table 1).

Fig. 3.

Na_vβ₁ and Na_vβ₂ subunits modulate late Na⁺ current (I_NaL) decay kinetics and density in ventricular cardiac myocytes from both normal dogs and from dogs with chronic heart failure. Shown are results of post-transcriptional silencing of SCN1B and SCN2B genes with the corresponding siRNAs. A: representative raw traces of I_NaL were recorded in cells from the normal (top) and failing (bottom) hearts cultured for 5 days after transfection with the control nonsilencing siRNA (control) and with Na_vβ₁-siRNA or Na_vβ₂-siRNA (indicated by the arrows), respectively. Exponential fits (Supplemental Eq. S1) are shown by solid lines and are superimposed with the experimental traces. Bottom, inset: voltage protocol. Statistical data for current density (B), and decay time constants (C and D) evaluated by the double exponential fit (Supplemental Eq. S1) in freshly isolated myocytes and after 5 days in culture of normal hearts, in freshly isolated myocytes of failing dog hearts and after 5 days in culture infected by the nonsilencing (ns-siRNA) or silencing siRNA, respectively, are shown. There was no statistically significant difference between I_NaL parameters when freshly isolated and cultured myocytes were compared for both normal and failing hearts. All I_NaL parameters remain significantly different when normal and failing hearts were compared for both freshly isolated and cultured cells infected by the ns-siRNA (*, §). Infection of myocytes of normal and failing heart with adenovirus containing Na_vβ₁- or Na_vβ₂-siRNA caused statistically significant (**, ¶) reduction or increase (†, ‡) in the I_NaL density (B) or decay time constants for both normal and failing hearts, respectively. Statistical significance, P < 0.05, was evaluated by the ANOVA followed by Bonferroni's post hoc test. Data represent means ± SE; cell numbers are indicated at the bars.

Fig. 4.

Post-transcriptional silencing of SCN1B and SCN2B genes does not affect the SSA and SSI of I_NaL in ventricular cardiac myocytes from normal dogs (NH) and dogs with chronic heart failure (HF). A and B: I_NaL-voltage relationship in cultured cells from NH and HF transfected with nonsilencing siRNA (control), with Na_vβ₁-siRNA and Na_vβ₂-siRNA, respectively. Solid lines show theoretical curves of SSA (Supplemental Eq. S2). In response to Na_vβ₁-siRNA and Na_vβ₂-siRNA a normalized maximum Na⁺ conductance (G_max) was significantly (P < 0.05) reduced (2.08 ± 0.07, n = 5, NH; 3.56 ± 0.03, n = 5, HF) or increased (3.45 ± 0.04, n = 4, NH; 6.56 ± 0.03, n = 12, HF), in comparison with control (2.67 ± 0.05, n = 5, NH; 5.37 ± 0.04 pS/pF, n = 11, HF), respectively. C and D: data points of SSI for I_NaL along with the Boltzmann function fit (solid lines, Supplemental Eq. S3) for NH and HF, respectively. Detailed statistical comparison of all SSA and SSI parameters are given in Table 1. Data represent means ± SE pooled from 5 to 8 cells. *Statistical significant difference (P < 0.05, ANOVA followed by Bonferroni's post hoc test) compared with control in A. B and D insets shows voltage-clamp protocols.

Na_vβ₁ and Na_vβ2 Subunits Modulate AP Duration

We recorded APs in response to different stimulation rates (0.25, 0.5, and 1.0 Hz) in VCMs treated with control nonsilencing siRNA and with siRNAs directed to silence either SCN1B or SCN2B genes. Figure 5A shows representative traces of APs recorded in these conditions. Statistical data on APD modulation by SCN1B and SCN2B genes is shown in Fig. 5B. Reduced expression of Na_vβ₁ caused significant shortening of APD at all pacing rates tested. In contrast, reduction of Na_vβ₂ expression significantly increased APD. Because ion currents change during primary culture of adult VCMs (30, 31) (see Study Limitations for inherent constrains of electrophysiological studies of the cultured VCMs), the effect of I_NaL modulation by the β-subunits on APs of the ventricular myocytes of the failing heart needs to be further evaluated. For this purpose we used E-C coupling model of canine ventricular myocytes (46) with new formulations of I_NaL (44), in which we included experimentally measured I_NaL modulation by β-subunits (the model details are given in the Supplement Material). Figure 6 shows simulated AP traces and simultaneous I_NaL traces in control and after silencing of Na_vβ₁ or Na_vβ₂ subunits in VCMs of both normal and failing hearts at a stimulation rate of 1.0 Hz, respectively. Simulated APs become notably shortened or prolonged in response to I_NaL changes caused by the reduction of the Na_vβ₁ or Na_vβ₂ subunit expression, respectively.

Fig. 5.

Effects of post-transcriptional silencing of Na_vβ₁ and Na_vβ₂ subunits on action potential (AP) of cardiac myocytes from dogs with chronic HF. A: superimposed typical AP traces recorded at stimulation rate 0.25 Hz in cells transfected with control nonsilencing siRNA (control), with Na_vβ₁-siRNA and Na_vβ₂-siRNA, respectively. B: statistical data for AP duration at 90% (APD₉₀) of repolarization assessed at 3 different stimulation rates of 0.25, 0.5, and 1.0 Hz. Data represent means ± SE pooled from 5 to 15 cells obtained from at least 3 different transfections after 5 days in culture. *Statistical significant difference (P < 0.05, ANOVA followed by Bonferroni's post hoc test) compared with control.

Fig. 6.

Numerical model simulations of dog ventricular myocyte APs (A and B) and simultaneous I_NaL dynamics (C and D) from normal and failing hearts (normal vs. HF, respectively) in control and in response to SCN1B or SCN2B gene silencing. All simulations presented in the figure are based on the respective changes of I_NaL parameters measured experimentally by patch-clamp (see Supplemental Material for the modeling details).

DISCUSSION

Result Summary and Possible Importance

For the first time, we report a highly effective RNA interference-based method for post-transcriptional silencing SNCB1 and/or SNCB2 genes in cultured adult cardiac myocytes isolated from the left ventricles of normal dogs and dogs with chronic HF. The Na_vβ₁-siRNA and Na_vβ₂-siRNA used in this study were virally delivered and caused ∼80% reduction of expression of SNCB1 and/or SNCB2 genes assessed by the mRNA transcript levels and the membrane protein content. The whole cell transient current, I_NaT, was not affected by the reduced expression of either Na_vβ₁ or Na_vβ₂ but caused a significant loss-of-function or gain-of-function of I_NaL, respectively. Post-transcriptional SNCB1 and/or SNCB2 gene silencing effects on I_NaL were similar for normal and failing hearts. Our experiments and in silico simulations predict notable AP duration changes within modulation range of I_NaL by both β-subunits measured experimentally, thus indicating that this modulation could be a physiologically important mechanism of AP regulation in both normal and failing myocardium (Figs. 5 and 6). We also simulated I_NaL dynamics underlying the AP changes (Fig. 6, C and D). Please note that simulated I_NaL profile during APs well corresponds to the respective experimentally measured I_NaL profile reported previously in canine VCMs (3).

Many prior studies have demonstrated an important role of I_NaL. It was shown that I_NaL reduction by 50–60% by tetrodotoxin (TTX), saxitoxin, counter-current injection, or a specific I_NaL blocker ranolazine, i.e., in the range similar to Na_vβ₁-siRNA-related modulation (Figs. 3 and 4), causes significant physiological effects in freshly isolated human and dog HF VCMs (21, 41, 42, 44), specifically 1) reduced AP duration and duration variability, 2) eliminated EADs, 3) improved single cell contractility, and 4) reduced diastolic Ca²⁺ accumulation. Therefore, in this regard overall I_NaL decrease produced by silencing SCN1B reported in the present study is expected to be physiological significant. However, silencing SCN2B gene causes overall I_NaL increase, i.e., similar to that reported for HF (Figs. 3, 4, and 5; Refs. 41, 44, and 45). Therefore, targeting SCN1B but not SCN2B gene with the aim to modulate I_NaL in HF is likely to be considered as a plausible target for the future gene therapy.

Cultured Adult Cardiomyocytes as a Model to Study NaCh Regulation

Primary cultures of cardiomyocytes are a useful model for broad reasons: 1) it produces a substantial amount of viable homogeneous population of terminally differentiated cells free of extracellular matrix and neurohumoral factors and 2) it is suitable for patch-clamp experiments and molecular biology approaches. As we have recently reported, NaCh expression underlying both I_NaT and I_NaL remains robust over 5 days in culture in VCMs of the normal dog (18). This time frame is sufficient to silence genes of interest causing a significant NaCh protein level reduction (18). In the present study, we show that HF-related differences in I_NaL remained during the primary VCMs culture. Indeed I_NaL density was larger (Fig. 3B) and inactivation kinetics was slower (Fig. 3, C and D) in VCMs from failing compared with normal heart. Therefore, the primary culture of adult dog cardiomyocytes is a suitable model to study molecular mechanisms of I_NaL regulation in normal and failing VCMs.

Molecular Mechanisms of I_NaL Modulation by Na_vβ₁ and Na_vβ₂ Subunits

The effects of the β-subunits on Na_v1.5 and other Na_vs were extensively studied in the past both in the heterologous expression systems and, recently, in the native heart tissues although with contrasting results (7, 14, 19, 27). Our study was not aimed to solve these controversies but rather to assess whether Na_vβ₁ or Na_vβ₂ modulates I_NaL in the native NaCh environment of VCMs isolated from both normal and failing hearts. Below we discuss three important questions of the molecular mechanisms of I_NaL modulation by the β-subunits.

Which Na_v underlies I_NaL in the heart and, therefore, is targeted by the SCN1B and SCN2B silencing?

Using antisense morpholino oligonucleotides we have recently reported that Na_v1.5 is the major (at least 60%) contributor to the I_NaL in adult cardiac myocytes from normal dogs (18). Although the major target in our study is likely Na_v1.5, the neuronal NaCh isoforms (Na_v1.1, Na_v1.2, Na_v1.3) reported in the heart (11, 15, 16) may also contribute to I_NaL. Indeed it has been shown that low doses of TTX, which preferentially blocks neuronal NaCh, reduce AP duration in cardiac Purkinje fibers (6) and block the persistent non-inactivating I_NaP current in the rat (13, 37). Single channel studies revealed multiple conductance levels of NaCh also pointing to a possible contribution of the different (neuronal) NaCh isoforms to the I_NaL in the rat (29) and in humans (our unpublished data). Recently it has been reported that the I_NaP, produced by Na_v1.1, can be reduced (2) or increased by the Na_vβ₁ (39).

2) What are manifestations of I_NaL modulation by SCN1B and SCN2B?

A recent study has shown that SCN1B gene silencing caused increased I_NaP as a result of increased expression of SCN5A in the SCN1B-null mice (14)_. The biophysical properties of I_NaP recorded in the SCB1-null mice, however, have not been fully examined, which makes it difficult to compare them with well-known properties of the slowly inactivating I_NaL reported in a variety of species including humans, dogs, guinea pig, rabbit, and rat (26). To explain the augmented I_NaP in SCN1B-null mice, Lopez-Santiago et al. (14) suggest a transcriptional regulation mechanism. Their suggestion is based on the fact that in the absence of Na_vβ₁ the transcription/translation of SCN5A in these hearts appears to be deregulated leading to the Na_v1.5 protein upregulation. Contrary to these results, another recent study has shown that the post-transcriptional silencing of SCN1B by siRNA reduces mRNA and protein levels of Na_v1.5 in neonatal rat VCMs (7). Accordingly, the I_NaL component is expected to be decreased but has not been assessed by Deschenes et al. (7). Different results could reflect acute (siRNA) versus long-term (SCN1B-null mice) effects, compensatory effects, or, possibly, age effects (newborn vs. older animals), as well as species-dependent effects. Furthermore, regulation of NaCh can also depend on the state of disease. Indeed, recently it has been shown that Na_v1.5 expression and inactivation modulation by the Na_vβ₁ is dependent on the state of tubulin cytoskeleton polymerization (4), which is reportedly increased in failing human hearts and in HF animal models (12).

3) How do Na_vβ₁ or Na_vβ₂ subunit interactions with NaCh produce observed I_NaL changes?

Direct interaction between cytoplasmic COOH terminus (CT) domain of Na_v1.1 with Na_vβ₁ and Na_vβ₃ has recently been demonstrated (39) and thus offers possible molecular mechanisms for I_NaL modulation by Na_vβ₁ found here (if similar interactions exist for Na_v1.5). The role of the CT to regulate Na_v1.5 inactivation has been recently suggested via the Ca²⁺-calmodulin-dependent interaction with the III-IV linker, responsible for the initial fast inactivation (1, 33), and has been elucidated for I_NaL regulation (20). It has been also suggested that the Na_vβ₁ subunit modulates Na_v1.5 inactivation via interaction with the microtubule cytoskeleton (4).

Although the role of Na_vβ₂ for the function of Na_v still is not clear, the present study is the first to our knowledge to demonstrate Na_vβ₂ effect on I_NaL in the native cardiac cells. Initially, Na_vβ₂ has been implemented in intercellular adhesion and recruitment of a cytoskeleton protein ankyrin to the plasma membrane at sites to cell-to-cell contact (17). Accordingly, a direct role of Na_vβ₂ subunit on the gating of Na_v was not suggested. NaCh protein has direct attachments to the submembrane cytoskeleton via ankyrin (1) and can be related to the cytoskeleton-dependent effects on its gating (5, 43). Recently, we reported that heterologously expressed Na_vβ₂ did not reveal any effect on I_NaL produced by human Na_v1.5 (19) that can be explained by a lack of coassembly of the Na_v1.5 α-subunit with the Na_vβ₂ subunit (49, 50). Accordingly, for the first time here we report evidence of Na_vβ₂ to modulate I_NaL in adult dog VCMs.

The NaCh protein consists of the main pore forming α-subunit surrounded by the covalently bound Na_vβ₁ and Na_vβ₃ as well as disulfide-linked Na_vβ₂ and Na_vβ₄ subunits (27). The Na_vβ₃ and Na_vβ₄ subunits are highly homologous with their counterparts Na_vβ₁ and Na_vβ₂, respectively, but may have a distinct and opposite function for NaCh gating (2, 10, 28, 32, 47). Therefore, besides the aforementioned direct effects on NaCh, the deprivation of Na_vβ₁ or Na_vβ₂ subunits from this multiprotein complex may enhance effects of their counterparts. Indeed, heterologous coexpression of Na_vβ₄ with neuronal Na_v1.1 can induce I_NaP (2) or can modulate Na_v1.5-related I_NaL (28). The same idea could be extended for Na_vβ₃ subunit, which may lead to faster inactivation of NaCh (10, 32), and contribute to Na_vβ₁ silencing effects on I_NaL in myocytes from HF reported here. Therefore, elucidation of interactive effects of Na_vβ₃ and Na_vβ₄ on I_NaL in failing heart merits consideration in future studies.

Physiological Significance

As mentioned above, I_NaL contribution into HF mechanisms has been demonstrated in experiments where correction of I_NaL in failing cardiomyocytes resulted in 1) rescue of normal repolarization (Figs. 5 and 6), 2) decrease beat-to-beat APD variability, and 3) improvement of Ca²⁺ handling and contractility (22, 25, 41, 42, 44). Accordingly, I_NaL has emerged as a novel target for cardioprotection to treat the failing heart (24, 26, 34). The fact that I_NaT is decreased but I_NaL is increased in HF (21, 23, 25, 42, 45, 48) suggests that not all NaCh must be equally targeted. Class I antiarrhythmic drugs that block I_NaT are proarrhythmic in HF because they slow conduction, thus worsening conduction problems (38), and facilitate the development of re-entry. Accordingly, new strategies for treatment must be taken into account considering the aforementioned constraint: the new type of smart drug or modulator should preferentially reduce I_NaL but not affect I_NaT (40). Therefore, the results of the present study suggest that silencing SCN1B but not SCN2B could be a plausible mechanism to modulate I_NaL in HF with the aim to improve both contractility and rhythm.

Study Limitations

Besides being a suitable model for the patch-clamp and molecular biology studies discussed above, the primary culture of adult VCMs has its limitations. A major limitation arises from the notion that over the culturing period VCMs undergo changes in their morphology and protein expression levels including those of ion channels (31) that, in turn, change their AP shape and duration. Another possibility to affect AP determinants is that post-transcriptional silencing of Na_vβ may affect other off-target ion channel expression. We found L-type Ca²⁺ channels remained unaffected (Supplemental Fig. S3). On the other hand, recently it has been shown that siRNA induced silencing of Na_vβ₁ caused reduction in expression of some potassium channels, which may affect repolarization process, besides I_NaL modulation (7). Accordingly, although cell culture well suits the purpose to specifically test modulation of I_NaL by Na_vβ₁ and Na_vβ₂ subunits (luckily the density of I_NaL does not change in control cell cultures), our results in cultured VCMs with regard to AP changes (Fig. 5) should be treated with caution. For example, although the direction of change of AP duration in the cultured cells is the same as expected from the physiological role of I_NaL to support the AP plateau given unaltered I_CaL (Supplemental Fig. S3), the specific values of the change may not necessarily report those in intact myocardium. Therefore, our respective experimental data with APs in cell culture play only a supportive role in the present study to illustrate that cultured cells maintain electrical excitability due to the robust I_NaT and still generate cardiac-like (yet changed) APs. To overcome this limitation and to illustrate physiological relevance of I_NaL modulation by Na_vβ₁ and Na_vβ₂, we used an E-C coupling model (Fig. 6) to simulate respective AP changes in both normal and failing VCMs. Another limitation of the study is that roles of Na_vβ₃ and Na_vβ₄ subunits in I_NaL modulation were not evaluated. Elucidation of interactive effects of Na_vβ₃ and Na_vβ₄ on I_NaL awaits future studies.

Conclusion

Based on our results with a highly effective RNA interference-based method for post-transcriptional silencing SNCB1 and/or SNCB2 genes in intact ventricular cardiac myocytes, we conclude that Na_vβ₁ and Na_vβ₂ exhibit oppositely directed modulation of I_NaL in ventricular myocytes of normal and failing dog hearts. We illustrate importance of this modulation to affect AP duration and shape both experimentally (Fig. 5) and in silico (Fig. 6).

GRANTS

This study was supported by National Heart, Lung, and Blood Institute Grants HL-53819 and HL-074238, by a grant-in-aid from the American Heart Association (0350472Z; to A. Undrovinas), and, in part, by the Intramural Research Program of the National Institute on Aging (to V. A. Maltsev; the numerical modeling part).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).