scholarly journals Distinct CASK domains control cardiac sodium channel membrane expression and focal adhesion anchoring

2019 ◽  
Author(s):  
Adeline Beuriot ◽  
Catherine A. Eichel ◽  
Gilles Dilanian ◽  
Florent Louault ◽  
Dario Melgari ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Sodium Channel ◽  
Sodium Current ◽  
Adaptor Proteins ◽  
Functional Domain ◽  
Membrane Expression ◽  
Glycoprotein Complex ◽  
Cardiac Sodium Channel ◽  
Dystrophin Glycoprotein Complex ◽  
Dystrophin Glycoprotein

ABSTRACTMembrane-associated guanylate kinase (MAGUK) proteins function as adaptor proteins to mediate the recruitment and scaffolding of ion channels in the plasma membrane in various cell types. In the heart, the protein CASK (Calcium/CAlmodulin-dependent Serine protein Kinase) negatively regulates the main cardiac sodium channel, NaV1.5, which carries the sodium current (INa) by preventing its anterograde trafficking. CASK is also a new member of the dystrophin-glycoprotein complex, and like syntrophin, binds to the C-terminal domain of the channel. Here we show that both L27B and GUK domains are required for the negative regulatory effect of CASK on INa and NaV1.5 surface expression and that the HOOK domain is essential for interaction with the cell adhesion dystrophin-glycoprotein complex. Thus, the multi-modular structure of CASK potentially provides the ability to control channel delivery at adhesion points in cardiomyocyte.SUMMARYSequential functional domain deletion approach identifies three critical domains of CASK in cardiomyocytes. CASK binds the cell adhesion dystrophin-glycoprotein complex through HOOK domain and inhibits NaV1.5 channel membrane expression by impeding trafficking through L27B and GUK domains.

Abstract 15925: Localization of Nav1.5 at the Intercellular Junctions Resolved by Diffraction-Unlimited Microscopy in Three Dimensions and by Correlative Light-Electron Microscopy

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Alejandra Leo-Macias ◽  
Esperanza Agullo-Pascual ◽  
Eli Rothenberg ◽  
Mario Delmar
Keyword(s):  
Electron Microscopy ◽  
Sodium Channel ◽  
Ventricular Myocytes ◽  
Sodium Current ◽  
Intercellular Junctions ◽  
Three Dimensions ◽  
Macromolecular Complex ◽  
Adult Heart ◽  
Mechanical Coupling ◽  
Cardiac Sodium Channel

Sodium current amplitude, kinetics and regulation depend on the properties of the pore-forming protein (mostly NaV1.5 in adult heart) and on the specific molecular partners with which the channel protein associates. The composition of the voltage-gated sodium channel macromolecular complex is location-specific; yet, the exact position of NaV1.5 in the subcellular landscape of the intercalated disc (ID), remains unclear. We implemented diffraction unlimited microscopy (direct stochastic optical reconstruction microscopy, or “dSTORM”) to localize the pore-forming subunit of the cardiac sodium channel NaV1.5 with a resolution of 20nm on the XY plane. In isolated adult ventricular myocytes, NaV1.5 was found in distinct semi-circular clusters. When the entire population of clusters within a 500 nm window from the ID was considered (more than 350 individual clusters analyzed), 75% of them localized to N-cadherin rich sites. NaV1.5-distal clusters were found at an average 313±15 nm from the cell end. Introducing an astigmatic lens in the light path allowed us to solve cluster location in three dimensions, at resolutions of 20 nm in XY and 40 nm in the z plane. Three-dimensional images confirmed the preferential localization at or near N-cadherin plaques, and further suggested that NaV1.5 arrives to the membrane via N-cadherin-anchored paths, most likely microtubules. In additional experiments, we developed a novel approach to correlate the image of NaV1.5 clusters by dSTORM with the cellular ultrastructure as resolved by electron microscopy on the same sample. This “correlative light-electron microscopy” method confirmed the preference of NaV1.5 clusters at sites of mechanical coupling. Overall, we provide the first ultrastructural description of NaV1.5 at the cardiac ID and its relation with the major electron-dense domains of the adult heart. Our data support a model by which microtubule-mediated delivery of NaV1.5 anchors at N-cadherin-rich sites, likely “mixed junctions” also containing desmosomal molecules (such as plakophilin-2; see Cerrone et al; Circulation 129:1092-1103, 2014) and connexin43. These findings have major implications to the understanding of sodium current disruption in diseases affecting the integrity of the ID.

scholarly journals Contraction-Induced Loss of Plasmalemmal Electrophysiological Function Is Dependent on the Dystrophin Glycoprotein Complex

2021 ◽  
Vol 12 ◽  
Author(s):  
Cory W. Baumann ◽  
Angus Lindsay ◽  
Sylvia R. Sidky ◽  
James M. Ervasti ◽  
Gordon L. Warren ◽  
...  
Keyword(s):  
M Wave ◽  
Glycoprotein Complex ◽  
Dystrophic Mouse ◽  
Dystrophin Deficiency ◽  
Torque Loss ◽  
Dystrophin Glycoprotein Complex ◽  
The Many ◽  
Dystrophin Glycoprotein ◽  
Mouse Lines

Weakness and atrophy are key features of Duchenne muscular dystrophy (DMD). Dystrophin is one of the many proteins within the dystrophin glycoprotein complex (DGC) that maintains plasmalemmal integrity and cellular homeostasis. The dystrophin-deficient mdx mouse is also predisposed to weakness, particularly when subjected to eccentric (ECC) contractions due to electrophysiological dysfunction of the plasmalemma. Here, we determined if maintenance of plasmalemmal excitability during and after a bout of ECC contractions is dependent on intact and functional DGCs rather than, solely, dystrophin expression. Wild-type (WT) and dystrophic mice (mdx, mL172H and Sgcb−/− mimicking Duchenne, Becker and Limb-girdle Type 2E muscular dystrophies, respectively) with varying levels of dystrophin and DGC functionality performed 50 maximal ECC contractions with simultaneous torque and electromyographic measurements (M-wave root-mean-square, M-wave RMS). ECC contractions caused all mouse lines to lose torque (p<0.001); however, deficits were greater in dystrophic mouse lines compared to WT mice (p<0.001). Loss of ECC torque did not correspond to a reduction in M-wave RMS in WT mice (p=0.080), while deficits in M-wave RMS exceeded 50% in all dystrophic mouse lines (p≤0.007). Moreover, reductions in ECC torque and M-wave RMS were greater in mdx mice compared to mL172H mice (p≤0.042). No differences were observed between mdx and Sgcb−/− mice (p≥0.337). Regression analysis revealed ≥98% of the variance in ECC torque loss could be explained by the variance in M-wave RMS in dystrophic mouse lines (p<0.001) but not within WT mice (R2=0.211; p=0.155). By comparing mouse lines that had varying amounts and functionality of dystrophin and other DGC proteins, we observed that (1) when all DGCs are intact, plasmalemmal action potential generation and conduction is maintained, (2) deficiency of the DGC protein β-sarcoglycan is as disruptive to plasmalemmal excitability as is dystrophin deficiency and, (3) some functionally intact DGCs are better than none. Our results highlight the significant role of the DGC plays in maintaining plasmalemmal excitability and that a collective synergism (via each DGC protein) is required for this complex to function properly during ECC contractions.

Abstract 15657: Regulation of Cardiac Regeneration by Hippo Pathway and Dystrophin Glycoprotein Complex

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Yuka Morikawa ◽  
James F Martin
Keyword(s):  
Hippo Pathway ◽  
Myocardial Damage ◽  
Cardiac Regeneration ◽  
Myocardial Cell ◽  
Damage Area ◽  
Glycoprotein Complex ◽  
Dystrophin Glycoprotein Complex ◽  
Heart Size ◽  
The Hippo Pathway ◽  
Dystrophin Glycoprotein

Regeneration of the mammalian heart is limited in adults. In rodents, endogenous regenerative capacity exists during development and in neonate but is rapidly repressed after birth. We are elucidating the mechanisms responsible for regenerative repression and applying this knowledge to reactivate cardiac regeneration in adult hearts. We have previously shown that the Hippo pathway is responsive for regenerative repression, however, the molecular and cellular mechanism responsible remain unclear. The Hippo pathway controls heart size by repressing myocardial cell proliferation during development. By deleting Salv, a modulator of Hippo pathway, we found myocardial damage in the postnatal and adult heart was repaired anatomically and functionally. This heart repair occurred primarily through proliferation of preexisting cardiomyocyte. We observed that cardiomyocytes in border the zone protrude and fill the damage area during Hippo-mediated cardiac regeneration and thus preventing formation of fibrotic scars. The molecular analysis identified components of dystrophin glycoprotein complex (DGC) as downstream targets of Hippo pathway. The DGC anchors the cytoskeleton and extracellular matrix and is involved in cell migration. The studies using the muscular dystrophy mouse model, mdx, reveals that DGC is required for endogenous cardiac regeneration and cardiomyocyte protrusion. Taken together, we show that cardiomyocyte protrusion is an essential process for cardiac regeneration and the Hippo pathway regulates it through regulating DGC. Our studies provide insights into the mechanisms leading to repair of damaged hearts from endogenous cardiomyocytes and novel information into DGC function.

Abstract 1503: Knockdown of Mog1 Expression Reduces Sodium Currents by Reducing the Trafficking of Cardiac Sodium Channel Na V 1.5 to the Cell Membrane

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Susmita Chakrabarti ◽  
Sandro Yong ◽  
Shin Yoo ◽  
Ling Wu ◽  
Qing Kenneth Wang
Keyword(s):  
Sodium Channel ◽  
Ventricular Myocytes ◽  
Sodium Current ◽  
Expression System ◽  
Heart Association ◽  
Hek293 Cells ◽  
Similar Reduction ◽  
Mammalian Expression ◽  
Ventricular Cells ◽  
Cardiac Sodium Channel

The cardiac sodium channel (Na v 1.5) plays a significant role in cardiac physiology and leads to cardiac arrhythmias and sudden death when mutated. Modulation of Na v 1.5 activity can also arise from changes to accessory subunits or proteins. Our laboratory has recently reported that MOG1, a small protein that is highly conserved from yeast to humans, is a co-factor of Na v 1.5. Increased MOG1 expression has been shown to increase Na v 1.5 current density. In adult mouse ventricular myocytes, these two proteins were found to be co-localized at the intercalated discs. Here, we further characterize the regulatory role of MOG1 using the RNA interference technique. Sodium current was recorded in voltage-clamp mode from a holding potential of −100 mV and activated to −20 mV. In 3-day old mouse neonatal ventricular cells transfected with siRNA against mouse MOG1 decreased sodium current densities (pA/pF) compared to control or scramble siRNA treated cells (−10.2±3.3, n=11 vs. −165±16, n=20 or −117.9±11.7, n=11). A similar reduction in sodium current was observed in mammalian expression system consisting of HEK293 cells stably expressing human Na v 1.5, by transfecting siRNAs against either human or mouse MOG1 (−41.7±8.3, n=7 or, −82.6±9.6, n=7 vs. −130.6±11.5, n=7; −111.5±8.5, n=7, respectively). Immunocytochemistry revealed that the expression of MOG1 and Na v 1.5 were decreased in both HEK and neonatal cells when compared to scramble siRNAs or control groups. These results show that MOG1 is an essential co-factor for Na v 1.5 by way of a channel trafficking. Such interactions between MOG1 and Na v 1.5 suggest that early localization of MOG1 on the membrane of neonatal cardiomyocytes may be necessary for proper localization and the distribution of Na v 1.5 during cardiac development. This research has received full or partial funding support from the American Heart Association, AHA National Center.

scholarly journals Impact of sarcoglycan complex on mechanical signal transduction in murine skeletal muscle

2006 ◽  
Vol 290 (2) ◽  
pp. C411-C419 ◽  
Author(s):  
Elisabeth R. Barton
Keyword(s):  
Skeletal Muscle ◽  
Mechanical Load ◽  
Eccentric Contraction ◽  
Eccentric Contractions ◽  
Normal Muscle ◽  
Glycoprotein Complex ◽  
Dystrophin Glycoprotein Complex ◽  
Severe Pathology ◽  
Murine Skeletal Muscle ◽  
Dystrophin Glycoprotein

Loss of the dystrophin glycoprotein complex (DGC) or a subset of its components can lead to muscular dystrophy. However, the patterns of symptoms differ depending on which proteins are affected. Absence of dystrophin leads to loss of the entire DGC and is associated with susceptibility to contractile injury. In contrast, muscles lacking γ-sarcoglycan (γ-SG) display little mechanical fragility and still develop severe pathology. Animals lacking dystrophin or γ-SG were used to identify DGC components critical for sensing dynamic mechanical load. Extensor digitorum longus muscles from 7-wk-old normal (C57), dystrophin- null ( mdx), and γ-SG-null ( gsg−/−) mice were subjected to a series of eccentric contractions, after which ERK1/2 phosphorylation levels were determined. At rest, both dystrophic strains had significantly higher ERK1 phosphorylation, and gsg−/− muscle also had heightened ERK2 phosphorylation compared with wild-type controls. Eccentric contractions produced a significant and transient increase in ERK1/2 phosphorylation in normal muscle, whereas the mdx strain displayed no significant proportional change of ERK1/2 phosphorylation after eccentric contraction. Muscles from gsg−/− mice had no significant increase in ERK1 phosphorylation; however, ERK2 phosphorylation was more robust than in C57 controls. The reduction in mechanically induced ERK1 phosphorylation in gsg−/− muscle was not dependent on age or severity of phenotype, because muscle from both young and old (age 20 wk) animals exhibited a reduced response. Immunoprecipitation experiments revealed that γ-SG was phosphorylated in normal muscle after eccentric contractions, indicating that members of the DGC are modified in response to mechanical perturbation. This study provides evidence that the SGs are involved in the transduction of mechanical information in skeletal muscle, potentially unique from the entire DGC.

scholarly journals Processing and Assembly of the Dystrophin Glycoprotein Complex

Traffic ◽  
2006 ◽  
Vol 8 (3) ◽  
pp. 177-183 ◽  
Author(s):  
Michael J. Allikian ◽  
Elizabeth M. McNally
Keyword(s):  
Glycoprotein Complex ◽  
Dystrophin Glycoprotein Complex ◽  
Dystrophin Glycoprotein

scholarly journals The Dystrophin Glycoprotein Complex Regulates the Epigenetic Activation of Muscle Stem Cell Commitment

2018 ◽  
Vol 22 (5) ◽  
pp. 755-768.e6 ◽  
Author(s):  
Natasha C. Chang ◽  
Marie-Claude Sincennes ◽  
Fabien P. Chevalier ◽  
Caroline E. Brun ◽  
Melanie Lacaria ◽  
...  
Keyword(s):  
Stem Cell ◽  
Muscle Stem Cell ◽  
Glycoprotein Complex ◽  
Dystrophin Glycoprotein Complex ◽  
Cell Commitment ◽  
Dystrophin Glycoprotein

scholarly journals Caveolin-3: A Causative Process of Chicken Muscular Dystrophy

Biomolecules ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1206
Author(s):  
Tateki Kikuchi
Keyword(s):  
Muscular Dystrophy ◽  
Missense Mutation ◽  
Ubiquitin Ligase ◽  
Core Protein ◽  
Ubiquitin Protein Ligase ◽  
Glycoprotein Complex ◽  
Dystrophin Glycoprotein Complex ◽  
Slow Twitch ◽  
Fast Twitch ◽  
Dystrophin Glycoprotein

The etiology of chicken muscular dystrophy is the synthesis of aberrant WW domain containing E3 ubiquitin-protein ligase 1 (WWP1) protein made by a missense mutation of WWP1 gene. The β-dystroglycan that confers stability to sarcolemma was identified as a substrate of WWP protein, which induces the next molecular collapse. The aberrant WWP1 increases the ubiquitin ligase-mediated ubiquitination following severe degradation of sarcolemmal and cytoplasmic β-dystroglycan, and an erased β-dystroglycan in dystrophic αW fibers will lead to molecular imperfection of the dystrophin-glycoprotein complex (DGC). The DGC is a core protein of costamere that is an essential part of force transduction and protects the muscle fibers from contraction-induced damage. Caveolin-3 (Cav-3) and dystrophin bind competitively to the same site of β-dystroglycan, and excessive Cav-3 on sarcolemma will block the interaction of dystrophin with β-dystroglycan, which is another reason for the disruption of the DGC. It is known that fast-twitch glycolytic fibers are more sensitive and vulnerable to contraction-induced small tears than slow-twitch oxidative fibers under a variety of diseased conditions. Accordingly, the fast glycolytic αW fibers must be easy with rapid damage of sarcolemma corruption seen in chicken muscular dystrophy, but the slow oxidative fibers are able to escape from these damages.

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