Biology subjects

Rougier, J.-S.

Publications and source records attributed to Rougier, J.-S..

3 recordsLinked to original sources

bioRxiv2019

Single-molecule localization of Nav1.5 reveals different modes of reorganization at the lateral membrane and T-tubules of cardiomyocytes

Mutations in the gene encoding the sodium channel Nav1.5 cause various cardiac arrhythmias. This variety may arise from different determinants of Nav1.5 expression between cardiomyocyte domains. At the lateral membrane and T-tubules, Nav1.5 localization and function remain insufficiently characterized. We used novel single-molecule localization microscopy (SMLM) and modeling to define nanoscale features of Nav1.5 localization and distribution at the lateral membrane, groove, and T-tubules in wild-type, dystrophin-deficient (mdx) mice, and mice expressing C-terminally truncated Nav1.5 ({Delta}SIV). We show that Nav1.5 organizes as distinct clusters in the groove and T-tubules which density and distribution partially depend on SIV and dystrophin. We found that overall reduction in Nav1.5 expression in mdx and {Delta}SIV cells results in a non-uniform redistribution with Nav1.5 being specifically reduced at the groove of {Delta}SIV and increased in T-tubules of mdx cardiomyocytes. Nav1.5 mutations may therefore site-specifically affect Nav1.5 localization and distribution depending on site-specific interacting proteins.

physiology↗

bioRxiv2019

A distinct pool of Nav1.5 channels at the lateral membrane of murine ventricular cardiomyocytes

BackgroundIn cardiac ventricular muscle cells, the presence of voltage-gated sodium channels Nav1.5 at the lateral membrane depends in part on the interaction between the dystrophin-syntrophin complex and the Nav1.5 C-terminal PDZ-domain-binding sequence Ser-Ile-Val (SIV motif). 1-Syntrophin, a PDZ-domain adaptor protein, mediates the interaction between Nav1.5 and dystrophin at the lateral membrane of cardiac cells. Using the cell-attached patch-clamp approach on cardiomyocytes expressing Nav1.5 in which the SIV motif is deleted ({Delta}SIV), sodium current (INa) recordings from the lateral membrane revealed an SIV-motif-independent INa. Since immunostainings have suggested that Nav1.5 is expressed in transverse (T-) tubules, this remaining INa might be conducted by channels in the T-tubules. Of note, a recent study using heterologous expression systems showed that 1-syntrophin also interacts with the Nav1.5 N-terminus, which may explain the SIV-motif independent INa at the lateral membrane of cardiomyocytes.\n\nAimTo address the role of 1-syntrophin in regulating the INa at the lateral membrane of cardiac cells.\n\nMethods and resultsPatch-clamp experiments in cell-attached configuration were performed on the lateral membranes of wild-type, 1-syntrophin knock-down, and {Delta}SIV ventricular mouse cardiomyocytes. Compared to wild-type, a reduction of the lateral INa was observed in myocytes from 1-syntrophin knockdown hearts. However, similar to {Delta}SIV myocytes, a remaining INa was still recorded. In addition, cell-attached INa recordings from lateral membrane did not differ significantly between non-detubulated and detubulated {Delta}SIV cardiomyocytes. Lastly, we obtained evidence suggesting that cell-attached patch-clamp experiments on the lateral membrane cannot record currents conducted by channels in T-tubules such as calcium channels.\n\nConclusionAltogether, these results suggest the presence of a sub-pool of sodium channels at the lateral membrane of cardiomyocytes that is independent of 1-syntrophin and the PDZ-binding motif of Na 1.5, located in membrane domains outside of T-tubules. The question of a T-tubular pool of Nav1.5 channels however remains open.

physiology↗

bioRxiv2019

Dystrophin and calcium current are decreased in cardiomyocytes expressing Cre enzyme driven by αMHC but not TNT promoter

BackgroundThe Cre/lox system is a potent technology to control gene expression in mouse tissues. However, cardiac alterations following cardiac-specific Cre enzyme expression in non-loxP-flanked genome heart have been reported. Recently, many loxP like sites have been identified in the wild-type mouse genome. Interestingly one of them is localized in the Dmd gene encoding the dystrophin protein known to be crucial for stabilization of cardiac voltage-gated ion channels Nav1.5 and Cav1.2. AimHere, we studied the potential alteration of dystrophin expression in adult alpha-myosin heavy chain (MHC)-Cre mice, which are extensively used for cardiac-specific recombination, and investigated Troponin T (TNT)-Cre mice as a potential alternative. MethodsCardiac-specific MHC-Cre and TNT-Cre mouse lines expressing Cre recombinase under the control of the cardiac-specific alpha-myosin-heavy chain, and rat cardiac troponin T2 promoter respectively were used. Western blots, quantitative RT-PCR, immunostainings, and patch-clamp experiments were performed to characterize MHC-Cre and TNT-Cre mouse hearts and cardiomyocytes. ResultsDystrophin protein level was decreased in hearts from 12-week-old MHC-Cre+ mice compared to MHC-Cre-. Reduction of dystrophin was more pronounced with age. No significant difference was observed between 8-week-old MHC-Cre+ mice and MHC-Cre-. Immunostainings performed on cardiac sections showed reduced dystrophin signal at the lateral membrane of MHC-Cre+ cardiomyocytes. Quantitative RT-PCR showed decreased mRNA levels of Dmd gene encoding dystrophin. Finally, patch-clamp experiments showed a significant decrease in calcium current (ICaL) in adult MHC-Cre+ cardiomyocytes compared to MHC-Cre-. Neither dystrophin nor ICaL was reduced in adult TNT-Cre+ mouse hearts compared to TNT-Cre-. ConclusionIn contrary to TNT-Cre+ mice, the sole expression of Cre recombinase can alter the cardiac phenotype of MHC-Cre+ mice. Thus, researchers should include the "Cre-only" condition as control condition when designing experiments with Cre mouse strains.

physiology↗