DataSheet1.pdf

<p>In cardiomyocytes, the voltage-gated transient outward potassium current (I_to) is responsible for the phase-1 repolarization of the action potential (AP). Gain-of-function mutations in KCND3, the gene encoding the I_to carrying K_V4.3 channel, have been associated with Brugada syndrome (BrS). While the role of I_to in the pro-arrhythmic mechanism of BrS has been debated, recent studies have suggested that an increased I_to may directly affect cardiac conduction. However, the effects of an increased I_to on AP upstroke velocity or sodium current at the cellular level remain unknown. We here investigated the consequences of K_V4.3 overexpression on Na_V1.5 current and consequent sodium channel availability. We found that overexpression of K_V4.3 protein in HEK293 cells stably expressing Na_V1.5 (HEK293-Na_V1.5 cells) significantly reduced Na_V1.5 current density without affecting its kinetic properties. In addition, K_V4.3 overexpression decreased AP upstroke velocity in HEK293-Na_V1.5 cells, as measured with the alternating voltage/current clamp technique. These effects of K_V4.3 could not be explained by alterations in total Na_V1.5 protein expression. Using computer simulations employing a multicellular in silico model, we furthermore demonstrate that the experimentally observed increase in K_V4.3 current and concurrent decrease in Na_V1.5 current may result in a loss of conduction, underlining the potential functional relevance of our findings. This study gives the first proof of concept that K_V4.3 directly impacts on Na_V1.5 current. Future studies employing appropriate disease models should explore the potential electrophysiological implications in (patho)physiological conditions, including BrS associated with KCND3 gain-of-function mutations.</p

Mass Spectrometry-Based Identification of Native Cardiac Nav1.5 Channel α Subunit Phosphorylation Sites

Cardiac voltage-gated Na⁺ (Nav) channels are key determinants of action potential waveforms, refractoriness and propagation, and Nav1.5 is the main Nav pore-forming (α) subunit in the mammalian heart. Although direct phosphorylation of the Nav1.5 protein has been suggested to modulate various aspects of Nav channel physiology and pathophysiology, native Nav1.5 phosphorylation sites have not been identified. In the experiments here, a mass spectrometry (MS)-based proteomic approach was developed to identify native Nav1.5 phosphorylation sites directly. Using an anti-NavPAN antibody, Nav channel complexes were immunoprecipitated from adult mouse cardiac ventricles. The MS analyses revealed that this antibody immunoprecipitates several Nav α subunits in addition to Nav1.5, as well as several previously identified Nav channel associated/regulatory proteins. Label-free comparative and data-driven phosphoproteomic analyses of purified cardiac Nav1.5 protein identified 11 phosphorylation sites, 8 of which are novel. All the phosphorylation sites identified except one in the N-terminus are in the first intracellular linker loop, suggesting critical roles for this region in phosphorylation-dependent cardiac Nav channel regulation. Interestingly, commonly used prediction algorithms did not reliably predict these newly identified <i>in situ</i> phosphorylation sites. Taken together, the results presented provide the first <i>in situ</i> map of basal phosphorylation sites on the mouse cardiac Nav1.5 α subunit

Mass Spectrometry-Based Identification of Native Cardiac Nav1.5 Channel α Subunit Phosphorylation Sites

Cardiac voltage-gated Na⁺ (Nav) channels are key determinants of action potential waveforms, refractoriness and propagation, and Nav1.5 is the main Nav pore-forming (α) subunit in the mammalian heart. Although direct phosphorylation of the Nav1.5 protein has been suggested to modulate various aspects of Nav channel physiology and pathophysiology, native Nav1.5 phosphorylation sites have not been identified. In the experiments here, a mass spectrometry (MS)-based proteomic approach was developed to identify native Nav1.5 phosphorylation sites directly. Using an anti-NavPAN antibody, Nav channel complexes were immunoprecipitated from adult mouse cardiac ventricles. The MS analyses revealed that this antibody immunoprecipitates several Nav α subunits in addition to Nav1.5, as well as several previously identified Nav channel associated/regulatory proteins. Label-free comparative and data-driven phosphoproteomic analyses of purified cardiac Nav1.5 protein identified 11 phosphorylation sites, 8 of which are novel. All the phosphorylation sites identified except one in the N-terminus are in the first intracellular linker loop, suggesting critical roles for this region in phosphorylation-dependent cardiac Nav channel regulation. Interestingly, commonly used prediction algorithms did not reliably predict these newly identified <i>in situ</i> phosphorylation sites. Taken together, the results presented provide the first <i>in situ</i> map of basal phosphorylation sites on the mouse cardiac Nav1.5 α subunit

Mass Spectrometry-Based Identification of Native Cardiac Nav1.5 Channel α Subunit Phosphorylation Sites

Cardiac voltage-gated Na⁺ (Nav) channels are key determinants of action potential waveforms, refractoriness and propagation, and Nav1.5 is the main Nav pore-forming (α) subunit in the mammalian heart. Although direct phosphorylation of the Nav1.5 protein has been suggested to modulate various aspects of Nav channel physiology and pathophysiology, native Nav1.5 phosphorylation sites have not been identified. In the experiments here, a mass spectrometry (MS)-based proteomic approach was developed to identify native Nav1.5 phosphorylation sites directly. Using an anti-NavPAN antibody, Nav channel complexes were immunoprecipitated from adult mouse cardiac ventricles. The MS analyses revealed that this antibody immunoprecipitates several Nav α subunits in addition to Nav1.5, as well as several previously identified Nav channel associated/regulatory proteins. Label-free comparative and data-driven phosphoproteomic analyses of purified cardiac Nav1.5 protein identified 11 phosphorylation sites, 8 of which are novel. All the phosphorylation sites identified except one in the N-terminus are in the first intracellular linker loop, suggesting critical roles for this region in phosphorylation-dependent cardiac Nav channel regulation. Interestingly, commonly used prediction algorithms did not reliably predict these newly identified <i>in situ</i> phosphorylation sites. Taken together, the results presented provide the first <i>in situ</i> map of basal phosphorylation sites on the mouse cardiac Nav1.5 α subunit

Variable levels of fibrosis in <i>Scn5a</i>^+/− mice.

<p><b>A.</b> Sirius red staining of ventricle from 85 week-old WT, mildly and severely affected <i>Scn5a</i>^+/− mice. Fibrosis appears in red. A score of 0 was attributed to the WT mouse shown, 1 to the mild <i>Scn5a</i>^+/− mouse and respectively 2 and 3 to left and right severe <i>Scn5a</i>^+/− mice. <b>B. </b><i>Atf3</i> and <i>Egr1</i> mRNA ventricular levels (in arbitrary units) in WT (open bars), mildly (grey bars) and severely (black bars) affected <i>Scn5a</i>^+/− mice as a function of age. ***, p<0.001 <i>versus</i> WT and mild.</p

Decreased Na_v1.5 expression in <i>Scn5a</i>^+/− mice.

<p><b>A.</b> Representative Western blots showing the expression levels of Na_v1.5 in WT mice and <i>Scn5a</i>^+/− mice (mean age = 18±1 weeks) with a mild or a severe phenotype. Protein loading was controlled by anti-actin immunoblotting. <b>B.</b> Quantification of Na_v1.5 expression in WT mice (open bars) and <i>Scn5a</i>^+/− mice with mild (grey bars) and severe (black bars) phenotype was performed on Western blots from 10 mice in each group by normalizing the intensities of the Na_v1.5 bands to the actin bands. ***, p<0.001 <i>versus</i> WT; †, p<0.05 <i>versus</i> mild <i>Scn5a</i>^+/− mice. <b>C.</b> Confocal images of Na_v1.5 in ventricular cardiomyocytes isolated from WT mice (left panel) and <i>Scn5a</i>^+/− mice with mild (middle panel) and severe (right panel) phenotype. Scale bar, 20 µm. In <i>Scn5a</i>^+/− images presented, a WT cardiomyocyte is also included in the image frame (yellow arrows) to illustrate the clear difference in WT <i>versus Scn5a</i>^+/− intercalated disc staining intensities.</p

Variable degrees of ventricular conduction defects in <i>Scn5a</i>^+/− mice.

<p><b>A.</b> Representative lead I ECGs from 10 week-old wild-type (WT) mice and <i>Scn5a</i>^+/− mice with mild and severe conduction defects. Scale bar, 100 ms. <b>B.</b> Distribution of QRS interval duration with corresponding Gaussian fits in 10 week-old WT mice (white bars) and <i>Scn5a</i>^+/− mice with mild (grey bars) and severe (black bars) phenotype. <b>C.</b> Effects of age (X-axis) on QRS interval duration (Y-axis) in WT mice (open symbols) and <i>Scn5a</i>^+/− mice with mild (filled circles) and severe (filled triangles) phenotype. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009298#pone-0009298-t001" target="_blank">Table 1</a> for statistics.</p

Variable effects of Na⁺ channel blockade in <i>Scn5a</i>^+/− mice and <i>SCN5A</i>-mutated patients.

<p>Ajmaline-induced increase in QRS interval (Y-axes) in 10 week- and >53 week-old WT (n = 11 and n = 10; open bars), mildly (n = 10 and n = 10; grey bars) and severely (n = 11 and n = 10; black bars) affected <i>Scn5a</i>^+/− mice (left panel), and in young (6–31 years; mean = 22 years) and older (41–61 years; mean = 50 years) non-mutated (n = 18 and n = 17, open bars) and <i>SCN5A</i>-mutated patients with either mild (n = 12 and n = 11; grey filled bars) or severe (n = 7 and n = 12; black filled bars) QRS interval prolongation (right panel). *, ***, p<0.05 and p<0.001 respectively <i>versus</i> WT mice or non-mutated patients. †, p<0.05 <i>versus Scn5a</i>^+/− mice or <i>SCN5A</i>-mutated patients with a mild phenotype. The difference between old mildly affected mice and old WT mice did not reach significance (p = 0.07). Same comment for the older patients.</p

Moderate ionic remodeling in <i>Scn5a</i>^+/− mice.

<p>Percentage of variation in ventricular expression (Y-axes) of 46 genes encoding ion channel subunits (ch) and connexins (Cx) in <i>Scn5a</i>^+/− mice with mild (grey bars; n = 5) and severe (black bars; n = 7) phenotype <i>versus</i> WT mice (n = 10). Sub, subunits. *, **, ***, p<0.05, p<0.01 and p<0.001 respectively <i>versus</i> WT; †, †††, p<0.05 and p<0.001 respectively <i>versus</i> mild phenotype.</p

Variable I_Na densities in <i>Scn5a</i>^+/− single ventricular cardiomyocytes.

<p><b>A.</b> Representative I_Na traces (protocol in <i>inset</i>) obtained from ventricular myocytes in a 12 week-old WT mouse and <i>Scn5a</i>^+/− mice with mild and severe phenotype. Horizontal bar, 2 ms; vertical bar, 40 pA/pF. <b>B.</b> I_Na density in myocytes from WT and <i>Scn5a</i>^+/− mice with mild and severe phenotype (4 mice in each group, 4 to 6 cells for each mouse). **, p<0.01 <i>versus</i> WT mice.</p