T he cardiac action potential (AP) is initiated by the Na + channel Na V 1.5, an established key element for cardiac excitability and impulse propagation. The importance of Na V 1.5 is exemplified by the myriad of cardiac disorders caused by hundreds of mutations identified in SCN5A, the gene coding for Na V 1.5.1 For some SCN5A mutation carriers, cardiac conduction slowing or block, secondary to reduced Na + channel function, predisposes them to ventricular arrhythmias and sudden cardiac death. Editorial see p 132 Clinical Perspective on p 160The cardiac Na + channel is composed of a 220-kDa α-subunit, Na V 1.5, constituting the pore of the channel, which is known to associate with four ≈30-kDa β-subunits. Recent studies have demonstrated that many proteins interact with and regulate Na V 1.5. 2 The physiological relevance of these interactions, however, is poorly understood, mainly due to a lack of in vivo studies. Many protein-protein interaction motifs for these regulatory proteins are located at the C-terminus of Na V 1.5. 2 In particular, we have previously demonstrated that Na V 1.5 associates with the dystrophinsyntrophin multiprotein complex (DMC) in cardiac cells.3 InBackground-Sodium channel Na V 1.5 underlies cardiac excitability and conduction. The last 3 residues of Na V 1.5 (Ser-IleVal) constitute a PDZ domain-binding motif that interacts with PDZ proteins such as syntrophins and SAP97 at different locations within the cardiomyocyte, thus defining distinct pools of Na V 1.5 multiprotein complexes. Here, we explored the in vivo and clinical impact of this motif through characterization of mutant mice and genetic screening of patients. Methods and Results-To investigate in vivo the regulatory role of this motif, we generated knock-in mice lacking the SIV domain (∆SIV). ∆SIV mice displayed reduced Na V 1.5 expression and sodium current (I Na ), specifically at the lateral myocyte membrane, whereas Na V 1.5 expression and I Na at the intercalated disks were unaffected. Optical mapping of ∆SIV hearts revealed that ventricular conduction velocity was preferentially decreased in the transversal direction to myocardial fiber orientation, leading to increased anisotropy of ventricular conduction. Internalization of wild-type and ΔSIV channels was unchanged in HEK293 cells. However, the proteasome inhibitor MG132 rescued ΔSIV I Na , suggesting that the SIV motif is important for regulation of Na V 1.5 degradation. A missense mutation within the SIV motif (p.V2016M) was identified in a patient with Brugada syndrome. The mutation decreased Na V 1.5 cell surface expression and I Na when expressed in HEK293 cells. Conclusions-Our results demonstrate the in vivo significance of the PDZ domain-binding motif in the correct expression of Na V 1.5 at the lateral cardiomyocyte membrane and underline the functional role of lateral Na V 1.5 in ventricular conduction. Recherche 1087, L'Institut du Thorax, Nantes, France (R.R.); Centre National de la Recherche Scientifique Unité Mixte de Recherche 6291, Nantes, France...
To elucidate the mechanisms of glomerulonephritis, including Goodpasture’s syndrome, mouse models are used that use heterologous Abs against the glomerular basement membrane (GBM) with or without preimmunization with foreign IgG from the same species. These studies have revealed the requirement of either FcγR or complement, depending on the experimental model used. In this study, we provide evidence that both FcγR and complement are obligatory for a full-blown inflammation in a novel attenuated passive model of anti-GBM disease. We demonstrate that administration of subnephritogenic doses of rabbit anti-GBM Abs followed by a fixed dose of mouse mAbs to rabbit IgG, allowing timing and dosing for the induction of glomerulonephritis, resulted in reproducible complement activation via the classical pathway of complement and albuminuria in wild-type mice. Because albuminuria was absent in FcR-γ-chain−/− mice and reduced in C3−/− mice, a role for both FcγR and complement is postulated. Because C1q−/− and C4−/− mice lacking a functional classical and lectin pathway did develop albuminuria, we suggest involvement of the alternative pathway of complement. Anti-GBM glomerulonephritis occurs acutely following the administration of mouse anti-rabbit IgG, and proceeds in a chronic fashion dependent on both FcγR and complement. This novel attenuated model allows elucidating the relative contribution of different mediator systems of the immune system to the development of renal injury, and also provides a platform for the assessment of different treatment protocols and evaluation of drugs that ultimately may be beneficial for the treatment of anti-GBM mediated glomerulonephritides.
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