We report for the first time abnormalities in cardiac ventricular electrophysiology in a genetically modified murine model lacking the Scn3b gene (Scn3b−/−). Scn3b−/− mice were created by homologous recombination in embryonic stem (ES) cells. RT-PCR analysis confirmed that Scn3b mRNA was expressed in the ventricles of wild-type (WT) hearts but was absent in the Scn3b−/− hearts. These hearts also showed increased expression levels of Scn1b mRNA in both ventricles and Scn5a mRNA in the right ventricles compared to findings in WT hearts. Scn1b and Scn5a mRNA was expressed at higher levels in the left than in the right ventricles of both Scn3b−/− and WT hearts. Bipolar electrogram and monophasic action potential recordings from the ventricles of Langendorff-perfused Scn3b−/− hearts demonstrated significantly shorter ventricular effective refractory periods (VERPs), larger ratios of electrogram duration obtained at the shortest and longest S1–S2 intervals, and ventricular tachycardias (VTs) induced by programmed electrical stimulation. Such arrhythmogenesis took the form of either monomorphic or polymorphic VT. Despite shorter action potential durations (APDs) in both the endocardium and epicardium, Scn3b−/− hearts showed ΔAPD90 values that remained similar to those shown in WT hearts. The whole-cell patch-clamp technique applied to ventricular myocytes isolated from Scn3b−/− hearts demonstrated reduced peak Na+ current densities and inactivation curves that were shifted in the negative direction, relative to those shown in WT myocytes. Together, these findings associate the lack of the Scn3b gene with arrhythmic tendencies in intact perfused hearts and electrophysiological features similar to those in Scn5a+/− hearts.

The understanding of internalization pathways of lipo- or polyplexes is crucial for engineering successful reagents for nonviral gene transfection. A known inhibitor of fluid phase endocytosis (FPE), rottlerin, was used to quantify the contribution of this pathway by flow cytometric and fluorescence assays. Rottlerin was shown to be a specific inhibitor of transfection by polyethylene imine (PEI-25)/DNA complexes, leading to a decrease in the amount of transfected HeLa and CHO-K1 cells and a decrease in the expression of enhanced green fluorescent protein (EGFP) reporter gene by up to 50%. Experiments using fluorescently labeled polyplexes result in a decrease of uptake by up to 40%. Additionally, rottlerin does not cross-inhibit clathrin- and caveolin-mediated endocytotic pathways of internalization, consistent with direct uptake inhibition by rottlerin. Nonspecific effects as a result of toxicity were ruled out by control experiments at concentrations where rottlerin inhibition was specific. These findings suggest that for CHO-K1 and HeLa cells, internalization of PEI-25/DNA complexes by FPE plays a decisive role in gene transfection. The establishment of an additional pathway that is independent of clathrin- and caveolin-mediated endocytotic uptake may have an impact on the design of future reagents of nonviral gene therapy and investigations of the uptake pathways and intracellular trafficking involved.

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Ventricular arrhythmogenesis in long QT 3 syndrome (LQT3) involves both triggered activity and re-entrant excitation arising from delayed ventricular repolarization. Effects of specific L-type Ca 2+ channel antagonism were explored in a gain-of-function murine LQT3 model produced by a ∆KPQ 1505-1507 deletion in the SCN5A gene. Monophasic action potentials (MAPs) were recorded from epicardial and endocardial surfaces of intact, Langendorff-perfused Scn5a+/∆ hearts. In untreated Scn5a+/∆ hearts, epicardial action potential duration at 90% repolarization (APD 90 ) was 60.0 ± 0.9 ms compared with 46.9 ± 1.6 ms in untreated wild-type (WT) hearts (P < 0.05; n = 5). The corresponding endocardial APD 90 values were 52.0 ± 0.7 ms and 53.7 ± 1.6 ms in Scn5a+/∆ and WT hearts, respectively (P > 0.05; n = 5). Epicardial early afterdepolarizations (EADs), often accompanied by spontaneous ventricular tachycardia (VT), occurred in 100% of MAPs from Scn5a+/∆ but not in any WT hearts (n = 10). However, EAD occurrence was reduced to 62 ± 7.1%, 44 ± 9.7%, 10 ± 10% and 0% of MAPs following perfusion with 10 nM, 100 nM, 300 nM and 1 µM nifedipine, respectively (P < 0.05; n = 5), giving an effective IC 50 concentration of 79.3 nM. Programmed electrical stimulation (PES) induced VT in all five Scn5a+/∆ hearts (n = 5) but not in any WT hearts (n = 5). However, repeat PES induced VT in 3, 2, 2 and 0 out of 5 Scn5a+/∆ hearts following perfusion with 10 nM, 100 nM, 300 nM and 1 µM nifedipine, respectively. Patch clamp studies in isolated ventricular myocytes from Scn5a+/∆ and WT hearts confirmed that nifedipine (300 nM) completely suppressed the inward Ca 2+ current but had no effect on inward Na + currents. No significant effects were seen on epicardial APD 90 , endocardial APD 90 or ventricular effective refractory period in Scn5a+/∆ and WT hearts following perfusion with nifedipine at 1 nM, 10 nM, 100 nM, 300 nM and 1 µM nifedipine concentrations. We conclude that L-type Ca 2+ channel antagonism thus exerts specific anti-arrhythmic effects in Scn5a+/∆ hearts through suppression of EADs.

AimIn contrast to extensive reports on the roles of Nav1.5 α-subunits, there have been few studies associating the β-subunits with cardiac arrhythmogenesis. We investigated the sino-atrial and conduction properties in the hearts of Scn3b−/− mice.MethodsThe following properties were compared in the hearts of wild-type (WT) and Scn3b−/− mice: (1) mRNA expression levels of Scn3b, Scn1b and Scn5a in atrial tissue. (2) Expression of the β3 protein in isolated cardiac myocytes. (3) Electrocardiographic recordings in intact anaesthetized preparations. (4) Bipolar electrogram recordings from the atria of spontaneously beating and electrically stimulated Langendorff-perfused hearts.ResultsScn3b mRNA was expressed in the atria of WT but not Scn3b−/− hearts. This was in contrast to similar expression levels of Scn1b and Scn5a mRNA. Immunofluorescence experiments confirmed that the β3 protein was expressed in WT and absent in Scn3b−/− cardiac myocytes. Lead I electrocardiograms from Scn3b−/− mice showed slower heart rates, longer P wave durations and prolonged PR intervals than WT hearts. Spontaneously beating Langendorff-perfused Scn3b−/− hearts demonstrated both abnormal atrial electrophysiological properties and evidence of partial or complete dissociation of atrial and ventricular activity. Atrial burst pacing protocols induced atrial tachycardia and fibrillation in all Scn3b−/− but hardly any WT hearts. Scn3b−/− hearts also demonstrated significantly longer sinus node recovery times than WT hearts.ConclusionThese findings demonstrate, for the first time, that a deficiency in Scn3b results in significant atrial electrophysiological and intracardiac conduction abnormalities, complementing the changes in ventricular electrophysiology reported on an earlier occasion.

1. Flecainide and quinidine exert contrasting pro-arrhythmic and anti-arrhythmic effects in mouse hearts containing the loss-of-function, Scn5a(+/-), and the gain-of-function, Scn5a(+/DeltaKPQ), mutations in their sodium channel alpha-subunits. 2. The following properties were accordingly compared in wild-type and Scn3b(-/-) hearts modelling modifications in the beta-subunit, before and after introduction of either agent: (i) ventricular arrhythmogenecity and effective refractory periods (VERP) in response to programmed electrical stimulation (PES); (ii) monophasic action potential waveforms recorded from the left ventricular epicardium and endocardium; (iii) action potential durations (APD) obtained from the monophasic action potentials; and (iv) critical intervals derived from the APD and VERP values. 3. Ventricular tachycardia was induced by PES in 11 out of 15 Scn3b(-/-) hearts and 0 out of 17 wild-type hearts. This incidence was reduced to three out of eight Scn3b(-/-) hearts but increased to three out of eight wild-type hearts with flecainide. 4. Arrhythmogenic incidence was reduced to two out of eight Scn3b(-/-) hearts and remained at 0 out of eight wild-type hearts in the presence of quinidine. 5. Ventricular effective refractory periods were prolonged and endocardial and epicardial APD shortened, resulting in negative critical intervals in both Scn3b(-/-) and wild-type hearts treated by either flecainide or quinidine. Nevertheless, endocardial APD remained consistently longer than epicardial APD, leaving similar, positive endocardial-epicardial, differences, DeltaAPD, in treated and untreated Scn3b(-/-) and wild-type hearts. 6. It is concluded that both flecainide and quinidine exert anti-arrhythmogenic effects in Scn3b(-/-) hearts, doing so through modifying VERP rather than DeltaAPD, in contrast to their differing effects in Scn5a(+/-) and Scn5a(+/DeltaKPQ) hearts.