Rat Rem2 shRNAs did not change VGCC peak currents in hippocampal neurons.

<p><i>A,</i> VGCC currents from control neurons and neurons transfected with rRem2-targeted shRNAs. There was no change in current density at either 4 d post-transfection (N = 11, p = 0.24) or 10 d post-transfection (N = 5–7, p = 0.48). <i>B,</i> Rem2 mRNA relative values in hippocampal neuron culture was measured at 1, 4, 8 and 14 DIV (N = 5–9) with PCR quantitative analysis. The relative value was normalized to the value of 1 DIV.</p

shRNA did not reduce endogenous Rem2 measured by immunocytochemistry in hippocampal neurons.

<p><i>A</i> and <i>C,</i> Exemplar images of neurons transfected with GFP or GFP-rRem2; or co-transfected with GFP and control vector <i>versus</i> GFP and a pool of rRem2 shRNAs. Left column, expressed GFP; right column, rRem2 detected with antibody against Rem2 and visualized with secondary antibody Cy3. Scale bar, 20 µm. <i>B</i> and <i>D,</i> Summarized GFP and Cy3 fluorescent levels indicated with mean gray value. Neurons overexpressing rRem2 (n = 41) showed significantly stronger fluorescent signal than control neurons (N = 15) (p = 0.0003). There was not significantly difference between neurons transfected with rRem2 shRNA (N = 51) and control plasmid (N = 48) (p = 0.25).</p

Rat Rem2 shRNAs reduced mEPSCs frequency in hippocampal neurons.

<p><i>A,</i> Exemplar recording of mEPSCs from neurons transfected with rRem2-targeted shRNAs or the control plasmid. <i>B,</i> and <i>C,</i> Summarized mEPSCs frequency and amplitude 4 (N = 6) and 10 d (N = 12) after transfection, showing that the mEPSCs frequency was reduced significantly at 10 d after transfection of shRNAs compared with control transfection (p = 0.006). There was no change in amplitude (p = 0.55).</p

GFP-hRem2, which was insensitive to rRem2-targeted shRNAs, could not rescue the reduction in mEPSCs frequency induced by shRNAs.

<p><i>A,</i> and <i>B</i>, immunoblot for GFP of lysates of untransfected HEK cells or cells transfected with GFP, GFP-rRem2 plus either rRem2 shRNAs or empty vector, and GFP-hRem2 plus either rRem2 shRNAs or empty vector. The rRem2-targeted shRNAs reduced GFP-rRem2 expression (N = 4, p = 0.03). <i>C,</i> VGCC currents recorded from cultured hippocampal neurons 3 d after transfection with GFP-hRem2 (N = 8) or GFP (N = 7). GFP-hRem2 reduced VGCC currents (p = 0.03). <i>D,</i> mEPSC frequency recorded at 4 d (N = 8) and 10 d (N = 16–21) after transfection with empty vector, rRem2-targeted shRNAs, or rRem2-targeted shRNAs together with GFP-hRem2. Transfection with rRem2-targeted shRNAs reduced mEPSCs frequency at 10 d compared to control (p = 0.02). Co-transfection with GFP-hRem2 did not rescue the effect of rRem2-targeted shRNAs (p = 0.12).</p

Over-expression of Rem2 reduced VGCCs without changes in mEPSCs in cultured hippocampal neurons.

<p><i>A,</i> Exemplar VGCCs were recorded from neurons transfected with GFP-rRem2 or GFP. The currents were induced by a ramp protocol −80 mV to +50 mV over 500 ms after a 50 ms step to −80 mV from a holding potential of −70 mV. <i>B,</i> Summarized VGCC currents recorded 4 (N = 7) and 10 d (N = 8) after Rem2 over-expression, showing VGCCs decreased markedly at 4 d (p = 0.03) and there was not significant difference at 10 d (p = 0.1). <i>C,</i> Exemplar recording of mEPSCs from neurons transfected with GFP-rRem2 or GFP only. <i>D,</i> and <i>E</i>, mEPSCs showed no change in mEPSCs frequency or amplitude at 4 d post-transfection (N = 9–10; frequency p = 0.11; amplitude p = 0.53) or 10 d post-transfection (N = 15; frequency p = 0.19; amplitude p = 0.36).</p

Summary of electrophysiological data in the adult rat cardiomyocytes.

<p>Summary of electrophysiological data in the adult rat cardiomyocytes.</p

A drug-induced long-QT syndrome patient with the p.E409Q variant in SNTA1.

<p>(A) A 12-lead resting electrocardiogram (ECG) from the proband during the post-arrest period showed markedly prolonged QT intervals. The QTc was 562 ms, and the notched T wave was noted. (B) The QTc interval was normalized and the T wave notch disappeared 2 months after aborted sudden cardiac death. (C) Pedigree of the family. An arrow is the proband with p.E409 variant. (D) Sequence conservation across species for A390V and E409Q versus normal in SNTA1. SCA, sudden cardiac arrest; and VF, ventricular fibrillation.</p

Electrophysiological data of Na_V1.5 in HEK293T cells coexpressing PMCA4b, nNOS, and either WT or SNTA1 mutants.

<p>(A) Representative traces of inward Na⁺ current for the 3 groups tested. (B) I-V curve. (C) Activation (G/Gmax). (D) Inactivation (I/Imax). (E) Recovery (P2/P1).</p

Syntrophin mutation and late sodium current.

<p>A schematic diagram shows variants in SNTA1 lead to disrupted binding to PMCA4b and released inhibition of nNOS, resulting in increased <i>I</i>_Na-L through S-nitrosylation of Na_V1.5.</p

Late Na⁺ current in adult rat cardiomyocyte infected with adenoviruses expressing either the WT or one of the two <i>SNTA1</i> mutants.

<p>(A) Representative late Na⁺ currents in with WT and the <i>SNTA1</i> variants (Fig 5A). (B) E409Q-SNTA1 significantly increased <i>I</i>_Na-L compared to that of WT-SNTA1. * P<0.05 versus WT-SNTA1. <i>I</i>_Na indicates sodium current; TTX, tetrodotoxin. Mean and standard error of mean (SEM) are shown in the graph.</p