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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Transient receptor potential melastatin member 4 (TRPM4) encodes a Ca2+-activated, non-selective cation channel that is functionally expressed in several tissues, including the heart. Pathogenic mutants in TRPM4 have been reported in patients with inherited cardiac diseases, including conduction blockage and Brugada syndrome. Heterologous expression of mutant channels in cell lines indicates that these mutations can lead to an increase or decrease in TRPM4 expression and function at the cell surface. While the expression and clinical variant studies further stress the importance of TRPM4 in cardiac function, the cardiac electrophysiological phenotypes in Trpm4 knockdown mouse models remain incompletely characterized. To study the functional consequences of Trpm4 deletion on cardiac electrical activity in mice, we performed perforated-patch clamp and immunoblotting studies on isolated atrial and ventricular cardiac myocytes and surfaces, as well as on pseudo- and intracardiac ECGs, either in vivo or in Langendorff-perfused explanted mouse hearts. We observed that TRPM4 is expressed in atrial and ventricular cardiac myocytes and that deletion of Trpm4 unexpectedly reduces the peak Na+ currents in myocytes. Hearts from Trpm4−/− mice presented increased sensitivity towards mexiletine, a Na+ channel blocker, and slower intraventricular conduction, consistent with the reduction of the peak Na+ current observed in the isolated cardiac myocytes. This study suggests that TRPM4 expression impacts the Na+ current in murine cardiac myocytes and points towards a novel function of TRPM4 regulating the Nav1.5 function in murine cardiac myocytes.

Hyperacute rejection of pig xenografts transplanted in humans is caused by endothelial cell binding of pre-formed xenoreactive antibodies (XAb) and activation of the classical pathway of complement. Human XAb mainly consist of anti-Galalpha1 3Gal antibodies, which occur in IgM, IgG and IgA classes. Whereas IgM anti-Galalpha1 3Gal antibodies have an established role in hyperacute rejection, the potential role of IgG XAb in this process is still controversial. The aim of the present study was to assess the specificity and functional properties of IgG and IgM XAb. Both classes were present in all human plasma samples tested, with a high inter-individual variability. Levels of IgG XAb did not correlate with levels of IgM XAb. Binding to Galalpha1 3Gal is strongly correlated with binding to the pig cell line PK15, both for IgG and for IgM, pointing to Galalpha1 3Gal as the major antigen recognized. Both purified IgM and IgG induced C3 deposition on PK15 cells and complement-dependent cytotoxicity in a dose-dependent way. The combination of IgG and IgM XAb resulted in an additive effect on cytotoxicity. Affinity-purified IgG anti-Galalpha1 3Gal antibodies were 22 times less potent than IgM in induction of cytotoxicity. These results indicate a quantitative, but not a qualitative, difference between IgM and IgG anti-pig antibodies concerning their complement-activating properties. Therefore, both classes of XAb are of importance in the pathogenesis of hyperacute rejection, and the relative importance of each class may differ considerably between individual patients, depending on the ratio of IgG and IgM XAb present in serum.

Background: In cardiac ventricular muscle cells, the presence of voltage-gated sodium channels Na v 1.5 at the lateral membrane depends in part on the interaction between the dystrophin–syntrophin complex and the Na v 1.5 C-terminal PDZ-domain-binding sequence Ser-Ile-Val (SIV motif). α1-Syntrophin, a PDZ-domain adaptor protein, mediates the interaction between Na v 1.5 and dystrophin at the lateral membrane of cardiac cells. Using the cell-attached patch-clamp approach on cardiomyocytes expressing Na v 1.5 in which the SIV motif is deleted (ΔSIV), sodium current (I Na ) recordings from the lateral membrane revealed a SIV-motif-independent I Na . Since immunostaining has suggested that Na v 1.5 is expressed in transverse (T-) tubules, this remaining I Na might be carried by channels in the T-tubules. Of note, a recent study using heterologous expression systems showed that α1-syntrophin also interacts with the Na v 1.5 N-terminus, which may explain the SIV-motif independent I Na at the lateral membrane of cardiomyocytes. Aim: To address the role of α1-syntrophin in regulating the I Na at the lateral membrane of cardiac cells. Methods and Results: Patch-clamp experiments in cell-attached configuration were performed on the lateral membranes of wild-type, α1-syntrophin knockdown, and ΔSIV ventricular mouse cardiomyocytes. Compared to wild-type, a reduction of the lateral I Na was observed in myocytes from α1-syntrophin knockdown hearts. Similar to ΔSIV myocytes, a remaining I Na was still recorded. In addition, cell-attached I Na recordings from lateral membrane did not differ significantly between non-detubulated and detubulated ΔSIV cardiomyocytes. Lastly, we obtained evidence suggesting that cell-attached patch-clamp experiments on the lateral membrane cannot record currents carried by channels in T-tubules such as calcium channels. Conclusion: Altogether, these results suggest the presence of a sub-pool of sodium channels at the lateral membrane of cardiomyocytes that is independent of α1-syntrophin and the PDZ-binding motif of Na v 1.5, located in membrane domains outside of T-tubules. The question of a T-tubular pool of Na v 1.5 channels, however, remains open.

In two fully mismatched kidney transplantation models, donor-derived LPS-DexDC induce a donor-specific T-cell hyporesponse. However, in this setting allograft survival was not improved, suggesting an important role for T cells with indirect alloreactivity. Understanding the underlying mechanism involved in the rejection process will improve the development of a cell-based immunotherapy.

The Cre/lox system is a potent technology to control gene expression in mouse tissues. However, cardiac-specific Cre recombinase expression alone can lead to cardiac alterations when no loxP sites are present, which is not well understood. Many loxP-like sites have been identified in the mouse genome that might be Cre sensitive. One of them is located in the Dmd gene encoding dystrophin, a protein important for the function and stabilization of voltage-gated calcium (Cav1.2) and sodium (Nav1.5) channels, respectively. Here, we investigate whether Cre affects dystrophin expression and function in hearts without loxP sites in the genome. In mice expressing Cre under the alpha-myosin heavy chain (MHC-Cre) or Troponin T (TNT-Cre) promoter, we investigated dystrophin expression, Nav1.5 expression, and Cav1.2 function. Compared to age-matched MHC-Cre− mice, dystrophin protein level was significantly decreased in hearts from MHC-Cre+ mice of more than 12-weeks-old. Quantitative RT-PCR revealed decreased mRNA levels of Dmd gene. Unexpectedly, calcium current (ICaL), but not Nav1.5 protein expression was altered in those mice. Surprisingly, in hearts from 12-week-old and older TNT-Cre+ mice, neither ICaL nor dystrophin and Nav1.5 protein content were altered compared to TNT-Cre−. Cre recombinase unpredictably affects cardiac phenotype, and Cre-expressing mouse models should be carefully investigated before experimental use.