This study provides the first evidence that the secretome from EAT promotes myocardial fibrosis through the secretion of adipo-fibrokines such as Activin A.

Rationale:The cardiac sodium channel Na v 1.5 plays a key role in excitability and conduction. The 3 last residues of Na v 1.5 (Ser-Ile-Val) constitute a PDZ-domain binding motif that interacts with the syntrophin-dystrophin complex. As dystrophin is absent at the intercalated discs, Na v 1.5 could potentially interact with other, yet unknown, proteins at this site.Objective: The aim of this study was to determine whether Na v 1.5 is part of distinct regulatory complexes at lateral membranes and intercalated discs. Methods and Results:Immunostaining experiments demonstrated that Na v 1.5 localizes at lateral membranes of cardiomyocytes with dystrophin and syntrophin. Optical measurements on isolated dystrophin-deficient mdx hearts revealed significantly reduced conduction velocity, accompanied by strong reduction of Na v 1.5 at lateral membranes of mdx cardiomyocytes. Pull-down experiments revealed that the MAGUK protein SAP97 also interacts with the SIV motif of Na v 1.5, an interaction specific for SAP97 as no pull-down could be detected with other cardiac MAGUK proteins (PSD95 or ZO-1). Furthermore, immunostainings showed that Na v 1.5 and SAP97 are both localized at intercalated discs. Silencing of SAP97 expression in HEK293 and rat cardiomyocytes resulted in reduced sodium current (I Na ) measured by patch-clamp. The I Na generated by Na v 1.5 channels lacking the SIV motif was also reduced. Finally, surface expression of Na v 1.5 was decreased in silenced cells, as well as in cells transfected with SIV-truncated channels. Conclusions:These data support a model with at least 2 coexisting pools of Na v 1.5 channels in cardiomyocytes: one targeted at lateral membranes by the syntrophin-dystrophin complex, and one at intercalated discs by SAP97. (Circ Res. 2011;108:294-304.) Key Words: sodium channel Ⅲ Na v 1.5 Ⅲ MAGUK proteins Ⅲ SAP97 Ⅲ dystrophin T he cardiac sodium channel Na v 1.5 initiates the cardiac action potential, thus playing a key role in cardiac excitability and impulse propagation. The physiological importance of this channel is illustrated by numerous cardiac pathologies caused by hundreds of mutations identified in SCN5A, the gene encoding Na v 1.5. 1 The Na v 1.5 channel is composed of one 220-kDa ␣-subunit that constitutes a functional channel, and 30-kDa ␤-subunits. In addition to these accessory ␤-subunits, several proteins have been shown to regulate and interact with Na v 1.5. 1,2 In most cases, the physiological relevance of these interactions is poorly understood, mainly because of a lack of appropriate animal models. Many of the interacting proteins bind to the C terminus of Na v 1.5, where several protein-protein interaction motifs are located. 1,2 We have shown that the ubiquitin-protein ligase Nedd4-2 binds the PY motif of Na v 1.5 and reduces the sodium current (I Na ) in HEK293 cells by promoting its internalization. 3 We have also demonstrated that Na v 1.5 associates with the dystrophin-syntrophin multiprotein complex (DMC) in cardiac cells. 4 In dystrophin-deficient mice (m...

Cardiac myocytes are characterized by distinct structural and functional entities involved in the generation and transmission of the action potential and the excitation-contraction coupling process. Key to their function is the specific organization of ion channels and transporters to and within distinct membrane domains, which supports the anisotropic propagation of the depolarization wave. This review addresses the current knowledge on the molecular actors regulating the distinct trafficking and targeting mechanisms of ion channels in the highly polarized cardiac myocyte. In addition to ubiquitous mechanisms shared by other excitable cells, cardiac myocytes show unique specialization, illustrated by the molecular organization of myocyte-myocyte contacts, e.g., the intercalated disc and the gap junction. Many factors contribute to the specialization of the cardiac sarcolemma and the functional expression of cardiac ion channels, including various anchoring proteins, motors, small GTPases, membrane lipids, and cholesterol. The discovery of genetic defects in some of these actors, leading to complex cardiac disorders, emphasizes the importance of trafficking and targeting of ion channels to cardiac function. A major challenge in the field is to understand how these and other actors work together in intact myocytes to fine-tune ion channel expression and control cardiac excitability.

Abstract-Membrane-associated guanylate kinase (MAGUK) proteins are major determinants of the organization of ion channels in the plasma membrane in various cell types. Here, we investigated the interaction between the MAGUK protein SAP97 and cardiac Kv4.2/3 channels, which account for a large part of the outward potassium current, I to , in heart. We found that the Kv4.2 and Kv4.3 channels C termini interacted with SAP97 via a SAL amino acid sequence. SAP97 and Kv4.3 channels were colocalized in the sarcolemma of cardiomyocytes. In CHO cells, SAP97 clustered Kv4.3 channels in the plasma membrane and increased the current independently of the presence of KChIP and dipeptidyl peptidase-like protein-6. Suppression of SAP97 by using short hairpin RNA inhibited I to in cardiac myocytes, whereas its overexpression by using an adenovirus increased I to . Kv4.3 channels without the SAL sequence were no longer regulated by Ca 2ϩ /calmodulin kinase (CaMK)II inhibitors. In cardiac myocytes, pull-down and coimmunoprecipitation assays showed that the Kv4 channel C terminus, SAP97, and CaMKII interact together, an interaction suppressed by SAP97 silencing and enhanced by SAP97 overexpression. In HEK293 cells, SAP97 silencing reproduced the effects of CaMKII inhibition on current kinetics and suppressed Kv4/CaMKII interactions. In conclusion, SAP97 is a major partner for surface expression and CaMKII-dependent regulation of cardiac Kv4 channels. Key Words: potassium channels Ⅲ cardiac myocytes Ⅲ MAGUK proteins Ⅲ calcium/calmodulin-dependent protein kinase Ⅲ dipeptidyl peptidase-like protein 6 I n the heart, the transient outward potassium current, I to , is among the main repolarizing currents that contribute to the early repolarization phase of the action potential and to its adaptation to changes in cardiac cycle length. 1,2 During cardiac diseases, I to is often altered, thus contributing to the risk of cardiac arrhythmias. 3,4 There is a general consensus that voltage-dependent Kv4.2 and Kv4.3 channels are the main molecular determinants of cardiac I to . [5][6][7][8] These ␣ subunits tether with several partners to form functional channels. The best-known partner of Kv4 channels is the Kv channel-interacting protein KChIP. 9 This protein assembles with the N terminus of the pore-forming Kv4 ␣ subunit and acts as a chaperone, regulating the channels surface expression and electrophysiological properties. 9,10 Dipeptidyl peptidase-like protein 6 (DPPX) is another subunit that regulates the activation and inactivation properties of Kv4 channels. 11,12 Membrane-associated guanylate kinase (MAGUK) proteins are important partners for the organization of several ion channels. 13,14 The MAGUK protein SAP97 is abundantly expressed in myocardium and interacts with voltagedependent Shaker channels Kv1.5 15,16 and K ir channels. 17 As in other tissues, SAP97 may regulate the targeting of cardiac ion channels in the sarcolemma. Indeed, in neonatal rat myocytes, SAP97 overexpression causes the clustering and immobilization of Kv1.5 ...

Atrial myocytes are continuously exposed to mechanical forces including shear stress. However, in atrial myocytes, the effects of shear stress are poorly understood, particularly with respect to its effect on ion channel function. Here, we report that shear stress activated a large outward current from rat atrial myocytes, with a parallel decrease in action potential duration. The main ion channel underlying the increase in current was found to be Kv1.5, the recruitment of which could be directly observed by total internal reflection fluorescence microscopy, in response to shear stress. The effect was primarily attributable to recruitment of intracellular pools of Kv1.5 to the sarcolemma, as the response was prevented by the SNARE protein inhibitor N-ethylmaleimide and the calcium chelator BAPTA. The process required integrin signaling through focal adhesion kinase and relied on an intact microtubule system. Furthermore, in a rat model of chronic hemodynamic overload, myocytes showed an increase in basal current despite a decrease in Kv1.5 protein expression, with a reduced response to shear stress. Additionally, integrin beta1d expression and focal adhesion kinase activation were increased in this model. This data suggests that, under conditions of chronically increased mechanical stress, the integrin signaling pathway is overactivated, leading to increased functional Kv1.5 at the membrane and reducing the capacity of cells to further respond to mechanical challenge. Thus, pools of Kv1.5 may comprise an inducible reservoir that can facilitate the repolarization of the atrium under conditions of excessive mechanical stress.trafficking | cardiomyocytes | potassium current

Membrane lipid composition is a major determinant of cell excitability. In this study, we assessed the role of membrane cholesterol composition in the distribution and function of Kv1.5-based channels in rat cardiac membranes. In isolated rat atrial myocytes, the application of methyl-β-cyclodextrin (MCD), an agent that depletes membrane cholesterol, caused a delayed increase in the Kv1.5-based sustained component, I kur , which reached steady state in ∼7 min. This effect was prevented by preloading the MCD with cholesterol. MCD-increased current was inhibited by low 4-aminopyridine concentration. Neonatal rat cardiomyocytes transfected with Green Fluorescent Protein (GFP)-tagged Kv1.5 channels showed a large ultrarapid delayed-rectifier current (I Kur ), which was also stimulated by MCD. In atrial cryosections, Kv1.5 channels were mainly located at the intercalated disc, whereas caveolin-3 predominated at the cell periphery. A small portion of Kv1.5 floated in the low-density fractions of step sucrose-gradient preparations. In live neonatal cardiomyocytes, GFP-tagged Kv1.5 channels were predominantly organized in clusters at the basal plasma membrane. MCD caused reorganization of Kv1.5 subunits into larger clusters that redistributed throughout the plasma membrane. The MCD effect on clusters was sizable 7 min after its application. We conclude that Kv1.5 subunits are concentrated in cholesterol-enriched membrane microdomains distinct from caveolae, and that redistribution of Kv1.5 subunits by depletion of membrane cholesterol increases their current-carrying capacity.

Cholesterol is an important determinant of cardiac electrical properties. However, underlying mechanisms are still poorly understood. Here, we examine the hypothesis that cholesterol modulates the turnover of voltage-gated potassium channels based on previous observations showing that depletion of membrane cholesterol increases the atrial repolarizing current I Kur. Whole-cell currents and single-channel activity were recorded in rat adult atrial myocytes (AAM) or after transduction with hKv1.5-EGFP. Channel mobility and expression were studied using fluorescence recovery after photobleaching (FRAP) and 3-dimensional microscopy. In both native and transduced-AAMs, the cholesterol-depleting agent M␤CD induced a delayed (Ϸ7 min) increase in I Kur; the cholesterol donor LDL had an opposite effect. Single-channel recordings revealed an increased number of active Kv1.5 channels upon M␤CD application. Whole-cell recordings indicated that this increase was not dependent on new synthesis but on trafficking of existing pools of intracellular channels whose exocytosis could be blocked by both N-ethylmaleimide and nonhydrolyzable GTP analogues. Rab11 was found to coimmunoprecipitate with hKv1.5-EGFP channels and transfection with Rab11 dominant negative (DN) but not Rab4 DN prevented the M␤CD-induced I Kur increase. Three-dimensional microscopy showed a decrease in colocalization of Kv1.5 and Rab11 in M␤CD-treated AAM. These results suggest that cholesterol regulates Kv1.5 channel expression by modulating its trafficking through the Rab11-associated recycling endosome. Therefore, this compartment provides a submembrane pool of channels readily available for recruitment into the sarcolemma of myocytes. This process could be a major mechanism for the tuning of cardiac electrical properties and might contribute to the understanding of cardiac effects of lipid-lowering drugs.cardiac myocytes ͉ ion channels ͉ membrane lipids ͉ trafficking ͉ channel recycling

Background-Brugada syndrome (BrS) is caused mainly by mutations in the SCN5A gene, which encodes the ␣-subunit of the cardiac sodium channel Na v 1.5. However, Ϸ20% of probands have SCN5A mutations, suggesting the implication of other genes. MOG1 recently was described as a new partner of Na v 1.5, playing a potential role in the regulation of its expression and trafficking. We investigated whether mutations in MOG1 could cause BrS. Methods and Results-MOG1 was screened by direct sequencing in patients with BrS and idiopathic ventricular fibrillation.A missense mutation p.Glu83Asp (E83D) was detected in a symptomatic female patient with a type-1 BrS ECG but not in 281 controls. Wild type (WT)-and mutant E83D-MOG1 were expressed in HEK Na v 1.5 stable cells and studied using patch-clamp assays. Overexpression of WT-MOG1 alone doubled sodium current (I Na ) density compared to control conditions (PϽ0.01). In contrast, overexpression of mutant E83D alone or E83DϩWT failed to increase I Na (PϽ0.05), demonstrating the dominant-negative effect of the mutant. Microscopy revealed that Na v 1.5 channels failed to properly traffic to the cell membrane in the presence of the mutant. Silencing endogenous MOG1 demonstrated a 54% decrease in I Na density. Conclusions-Our results support the hypothesis that dominant-negative mutations in MOG1 can impair the trafficking of Na v 1.5 to the membrane, leading to I Na reduction and clinical manifestation of BrS. Moreover, silencing MOG1 reduced I Na , demonstrating that MOG1 is likely to be important in the surface expression of Na v 1.5 channels. All together, our data support MOG1 as a new susceptibility gene for BrS. (Circ Cardiovasc Genet. 2011;4:261-268.)