BACKGROUND Cardiac sodium channel β-subunit mutations have been associated with several inherited cardiac arrhythmia syndromes. OBJECTIVE To identify and characterize variations in SCN1Bb associated with Brugada (BrS) and sudden infant death syndromes (SIDS). METHODS AND RESULTS Patient 1 was a 44-y/o male with an ajmaline-induced Type-1 ST-segment elevation in V1 and V2 supporting the diagnosis of BrS. Patient 2 was a 62-y/o female displaying a coved-type BrS ECG who developed cardiac arrest during fever. Patient 3 was a 4-m/o female SIDS case. All known exons and intron borders of BrS and SIDS susceptibility genes were amplified and sequenced in both directions. A R214Q variant was detected in exon 3A of SCN1Bb (Navβ1B) in all three probands, but not in any other gene previously associated with BrS or SIDS. R214Q was identified in 4 of 807 ethnically-matched healthy controls (0.50%). Wild type (WT) and mutant genes were expressed in TSA201 cells and studied using whole-cell patch-clamp and co-immunoprecipitation techniques. Co-expression of SCN5A/WT+SCN1Bb/R214Q resulted in peak sodium channel current (INa) 56.5% smaller compared to SCN5A/WT+SCN1Bb/WT ( n=11–12, p<0.05 ). Co-expression of KCND3/WT+SCN1Bb/R214Q induced a Kv4.3 current (Ito) 70.6% greater compared with KCND3/WT+SCN1Bb/WT(n=10–11, p<0.01). Co-immunoprecipitation indicated structural association between Navβ1B and Nav1.5 and Kv4.3. CONCLUSION Our results suggest that R214Q variation in SCN1Bb is a functional polymorphism that may serve as a modifier of the substrate responsible for Brugada or SIDS phenotypes via a combined loss of function of INa and gain of function of Ito.

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The standard methods for toxicity testing using rodent models cannot keep pace with the increasing number of chemicals in our environment due to time and resource limitations. Hence, there is an unmet need for fast, sensitive, and costeffective alternate models to reliably predict toxicity. As part of Tox21 Phase III's effort, a 90-compound library was created and made available to researchers to screen for neurotoxicants using novel technology and models. The chemical library was evaluated in zebrafish in a dose-range finding test for embryo-toxicity (ie, mortality or morphological alterations induced by each chemical). In addition, embryos exposed to the lowest effect level and nonobservable effect level were used to measure the internal concentration of the chemicals within the embryos by bioanalysis. Finally, considering the lowest effect level as the highest testing concentration, a functional assay was performed based on locomotor activity alteration in response to light-dark changes. The quality control chemicals included in the library, ie, negative controls and replicated chemicals, indicate that the assays performed were reliable. The use of analytical chemistry pointed out the importance of measuring chemical concentration inside embryos, and in particular, in the case of negative chemicals to avoid false negative classification. Overall, the proposed approach presented a good sensitivity and supports the inclusion of zebrafish assays as a reliable, relevant, and efficient screening tool to identify, prioritize, and evaluate chemical toxicity.

Background: The rapid delayed rectifier K⁺ current (I_Kr), carried by the hERG protein, is one of the main repolarising currents in the human heart and a reduction of this current increases the risk of ventricular fibrillation. α1-adrenoceptors (α1-AR) activation reduces I_Kr but, despite the clear relationship between an increase in the sympathetic tone and arrhythmias, the mechanisms underlying the α1-AR regulation of the hERG channel are controversial. Thus, we aimed to investigate the mechanisms by which α1-AR stimulation regulates I_Kr. Methods: α1-adrenoceptors, hERG channels, auxiliary subunits minK and MIRP1, the non PIP₂-interacting mutant D-hERG (with a deletion of the 883-894 amino acids) in the C-terminal and the non PKC-phosphorylable mutant N-terminal truncated-hERG (NTK-hERG) were transfected in HEK293 cells. Cell membranes were extracted by centrifugation and the different proteins were visualized by Western blot. Potassium currents were recorded by the patch-clamp technique. I_Kr was recorded in isolated feline cardiac myocytes. Results: Activation of the α1-AR reduces the amplitude of I_hERG and I_Kr through a positive shift in the activation half voltage, which reduces the channel availability at physiological membrane potentials. The intracellular pathway connecting the α1-AR to the hERG channel in HEK293 cells includes activation of the Gαq protein, PLC activation and PIP₂ hydrolysis, activation of PKC and direct phosphorylation of the hERG channel N-terminal. The PKC-mediated I_Kr channel phosphorylation and subsequent I_Kr reduction after α1-AR stimulation was corroborated in feline cardiac myocytes. Conclusions: These findings clarify the link between sympathetic nervous system hyperactivity and I_Kr reduction, one of the best characterized causes of torsades de pointes and ventricular fibrillation.

Diabetic patients have a higher incidence of cardiac arrhythmias, including ventricular fibrillation and sudden death, and show important alterations in the electrocardiogram, most of these related to the repolarization. In myocytes isolated from diabetic hearts, the transient outward K+ current (Ito) is the repolarizing current that is mainly affected. Type 1 diabetes alters Ito at 3 levels: the recovery of inactivation, the responsiveness to physiologic regulators, and the functional expression of the channel. Diabetes slows down Ito recovery of inactivation because it triggers the switching from fast-recovering Kv4.x channels to the slow-recovering Kv1.4. Diabetic animals also have decreased responsiveness of Ito towards the sympathetic nervous system; thus, the diabetic heart develops a resistance to its physiologic regulator. Finally, diabetes impairs support of various trophic factors required for the functional expression of the channel and reduces Ito amplitude by decreasing the amount of Kv4.2 and Kv4.3 proteins.

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