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  • 1
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    Online Resource
    Mechanosensitive Gene Regulation by Myocardin-Related Transcription Factors Is Required for Cardiomyocyte Integrity in Load-Induced Ventricular Hypertrophy
    Ovid Technologies (Wolters Kluwer Health) ; 2018
    In:  Circulation Vol. 138, No. 17 ( 2018-10-23), p. 1864-1878
    Details
    In: Circulation, Ovid Technologies (Wolters Kluwer Health), Vol. 138, No. 17 ( 2018-10-23), p. 1864-1878
    Abstract: Hypertrophic cardiomyocyte growth and dysfunction accompany various forms of heart disease. The mechanisms responsible for transcriptional changes that affect cardiac physiology and the transition to heart failure are not well understood. The intercalated disc (ID) is a specialized intercellular junction coupling cardiomyocyte force transmission and propagation of electrical activity. The ID is gaining attention as a mechanosensitive signaling hub and hotspot for causative mutations in cardiomyopathy. Methods: Transmission electron microscopy, confocal microscopy, and single-molecule localization microscopy were used to examine changes in ID structure and protein localization in the murine and human heart. We conducted detailed cardiac functional assessment and transcriptional profiling of mice lacking myocardin-related transcription factor (MRTF)-A and MRTF-B specifically in adult cardiomyocytes to evaluate the role of mechanosensitive regulation of gene expression in load-induced ventricular remodeling. Results: We found that MRTFs localize to IDs in the healthy human heart and accumulate in the nucleus in heart failure. Although mice lacking MRTFs in adult cardiomyocytes display normal cardiac physiology at baseline, pressure overload leads to rapid heart failure characterized by sarcomere disarray, ID disintegration, chamber dilation and wall thinning, cardiac functional decline, and partially penetrant acute lethality. Transcriptional profiling reveals a program of actin cytoskeleton and cardiomyocyte adhesion genes driven by MRTFs during pressure overload. Indeed, conspicuous remodeling of gap junctions at IDs identified by single-molecule localization microscopy may partially stem from a reduction in Mapre1 expression, which we show is a direct mechanosensitive MRTF target. Conclusions: Our study describes a novel paradigm in which MRTFs control an acute mechanosensitive signaling circuit that coordinates cross-talk between the actin and microtubule cytoskeleton and maintains ID integrity and cardiomyocyte homeostasis in heart disease.
    Type of Medium: Online Resource
    ISSN: 0009-7322 , 1524-4539
    URL: Article
    DOI: 10.1161/CIRCULATIONAHA.117.031788
    Language: English
    Publisher: Ovid Technologies (Wolters Kluwer Health)
    Publication Date: 2018
    detail.hit.zdb_id: 1466401-X
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  • 2
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    Online Resource
    Sodium Channel Remodeling in Subcellular Microdomains of Murine Failing Cardiomyocytes
    Ovid Technologies (Wolters Kluwer Health) ; 2017
    In:  Journal of the American Heart Association Vol. 6, No. 12 ( 2017-12-02)
    Details
    In: Journal of the American Heart Association, Ovid Technologies (Wolters Kluwer Health), Vol. 6, No. 12 ( 2017-12-02)
    Abstract: Cardiac sodium channel (NaV1.5) dysfunction contributes to arrhythmogenesis during pathophysiological conditions. Nav1.5 localizes to distinct subcellular microdomains within the cardiomyocyte, where it associates with region‐specific proteins, yielding complexes whose function is location specific. We herein investigated sodium channel remodeling within distinct cardiomyocyte microdomains during heart failure. Methods and Results Mice were subjected to 6 weeks of transverse aortic constriction (TAC; n=32) to induce heart failure. Sham–operated on mice were used as controls (n=20). TAC led to reduced left ventricular ejection fraction, QRS prolongation, increased heart mass, and upregulation of prohypertrophic genes. Whole‐cell sodium current (I Na ) density was decreased by 30% in TAC versus sham–operated on cardiomyocytes. On macropatch analysis, I Na in TAC cardiomyocytes was reduced by 50% at the lateral membrane (LM) and by 40% at the intercalated disc. Electron microscopy and scanning ion conductance microscopy revealed remodeling of the intercalated disc (replacement of [inter‐]plicate regions by large foldings) and LM (less identifiable T tubules and reduced Z‐groove ratios). Using scanning ion conductance microscopy, cell‐attached recordings in LM subdomains revealed decreased I Na and increased late openings specifically at the crest of TAC cardiomyocytes, but not in groove/T tubules. Failing cardiomyocytes displayed a denser, but more stable, microtubule network (demonstrated by increased α‐tubulin and Glu‐tubulin expression). Superresolution microscopy showed reduced average Na V 1.5 cluster size at the LM of TAC cells, in line with reduced I Na . Conclusions Heart failure induces structural remodeling of the intercalated disc, LM, and microtubule network in cardiomyocytes. These adaptations are accompanied by alterations in Na V 1.5 clustering and I Na within distinct subcellular microdomains of failing cardiomyocytes.
    Type of Medium: Online Resource
    ISSN: 2047-9980
    URL: Article
    DOI: 10.1161/JAHA.117.007622
    Language: English
    Publisher: Ovid Technologies (Wolters Kluwer Health)
    Publication Date: 2017
    detail.hit.zdb_id: 2653953-6
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  • 3
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    Structural and Functional Characterization of a Na v 1.5-Mitochondrial Couplon
    Ovid Technologies (Wolters Kluwer Health) ; 2021
    In:  Circulation Research Vol. 128, No. 3 ( 2021-02-05), p. 419-432
    Details
    In: Circulation Research, Ovid Technologies (Wolters Kluwer Health), Vol. 128, No. 3 ( 2021-02-05), p. 419-432
    Abstract: The cardiac sodium channel Na V 1.5 has a fundamental role in excitability and conduction. Previous studies have shown that sodium channels cluster together in specific cellular subdomains. Their association with intracellular organelles in defined regions of the myocytes, and the functional consequences of that association, remain to be defined. Objective: To characterize a subcellular domain formed by sodium channel clusters in the crest region of the myocytes and the subjacent subsarcolemmal mitochondria. Methods and Results: Through a combination of imaging approaches including super-resolution microscopy and electron microscopy we identified, in adult cardiac myocytes, a Na V 1.5 subpopulation in close proximity to subjacent subsarcolemmal mitochondria; we further found that subjacent subsarcolemmal mitochondria preferentially host the mitochondrial NCLX (Na + /Ca 2+ exchanger). This anatomic proximity led us to investigate functional changes in mitochondria resulting from sodium channel activity. Upon TTX (tetrodotoxin) exposure, mitochondria near Na V 1.5 channels accumulated more Ca 2+ and showed increased reactive oxygen species production when compared with interfibrillar mitochondria. Finally, crosstalk between Na V 1.5 channels and mitochondria was analyzed at a transcriptional level. We found that SCN5A (encoding Na V 1.5) and SLC8B1 (which encode Na V 1.5 and NCLX, respectively) are negatively correlated both in a human transcriptome data set (Genotype-Tissue Expression) and in human-induced pluripotent stem cell-derived cardiac myocytes deficient in SCN5A . Conclusions: We describe an anatomic hub (a couplon) formed by sodium channel clusters and subjacent subsarcolemmal mitochondria. Preferential localization of NCLX to this domain allows for functional coupling where the extrusion of Ca 2+ from the mitochondria is powered, at least in part, by the entry of sodium through Na V 1.5 channels. These results provide a novel entry-point into a mechanistic understanding of the intersection between electrical and structural functions of the heart.
    Type of Medium: Online Resource
    ISSN: 0009-7330 , 1524-4571
    URL: Article
    DOI: 10.1161/CIRCRESAHA.120.318239
    RVK:
    XA 10000
    Language: English
    Publisher: Ovid Technologies (Wolters Kluwer Health)
    Publication Date: 2021
    detail.hit.zdb_id: 1467838-X
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  • 4
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    Online Resource
    Missense Mutations in Plakophilin-2 Cause Sodium Current Deficit and Associate With a Brugada Syndrome Phenotype
    Ovid Technologies (Wolters Kluwer Health) ; 2014
    In:  Circulation Vol. 129, No. 10 ( 2014-03-11), p. 1092-1103
    Details
    In: Circulation, Ovid Technologies (Wolters Kluwer Health), Vol. 129, No. 10 ( 2014-03-11), p. 1092-1103
    Abstract: Brugada syndrome (BrS) primarily associates with the loss of sodium channel function. Previous studies showed features consistent with sodium current ( I Na ) deficit in patients carrying desmosomal mutations, diagnosed with arrhythmogenic cardiomyopathy (or arrhythmogenic right ventricular cardiomyopathy). Experimental models showed correlation between the loss of expression of desmosomal protein plakophilin-2 (PKP2) and reduced I Na . We hypothesized that PKP2 variants that reduce I Na could yield a BrS phenotype, even without overt structural features characteristic of arrhythmogenic right ventricular cardiomyopathy. Methods and Results— We searched for PKP2 variants in the genomic DNA of 200 patients with a BrS diagnosis, no signs of arrhythmogenic cardiomyopathy, and no mutations in BrS-related genes SCN5A, CACNa1c, GPD1L , and MOG1. We identified 5 cases of single amino acid substitutions. Mutations were tested in HL-1–derived cells endogenously expressing Na V 1.5 but made deficient in PKP2 (PKP2-KD). Loss of PKP2 caused decreased I Na and Na V 1.5 at the site of cell contact. These deficits were restored by the transfection of wild-type PKP2, but not of BrS-related PKP2 mutants. Human induced pluripotent stem cell cardiomyocytes from a patient with a PKP2 deficit showed drastically reduced I Na . The deficit was restored by transfection of wild type, but not BrS-related PKP2. Super-resolution microscopy in murine PKP2-deficient cardiomyocytes related I Na deficiency to the reduced number of channels at the intercalated disc and increased separation of microtubules from the cell end. Conclusions— This is the first systematic retrospective analysis of a patient group to define the coexistence of sodium channelopathy and genetic PKP2 variations. PKP2 mutations may be a molecular substrate leading to the diagnosis of BrS.
    Type of Medium: Online Resource
    ISSN: 0009-7322 , 1524-4539
    URL: Article
    DOI: 10.1161/CIRCULATIONAHA.113.003077
    Language: English
    Publisher: Ovid Technologies (Wolters Kluwer Health)
    Publication Date: 2014
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  • 5
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    Single-Molecule Localization of the Cardiac Voltage-Gated Sodium Channel Reveals Different Modes of Reorganization at Cardiomyocyte Membrane Domains
    Ovid Technologies (Wolters Kluwer Health) ; 2020
    In:  Circulation: Arrhythmia and Electrophysiology Vol. 13, No. 7 ( 2020-07)
    Details
    In: Circulation: Arrhythmia and Electrophysiology, Ovid Technologies (Wolters Kluwer Health), Vol. 13, No. 7 ( 2020-07)
    Abstract: Mutations in the gene encoding the cardiac voltage-gated sodium channel Na v 1.5 cause various cardiac arrhythmias. This variety may arise from different determinants of Na v 1.5 expression between cardiomyocyte domains. At the lateral membrane and T-tubules, Na v 1.5 localization and function remain insufficiently characterized. Methods: We used novel single-molecule localization microscopy and computational modeling to define nanoscale features of Na v 1.5 localization and distribution at the lateral membrane, the lateral membrane groove, and T-tubules in cardiomyocytes from wild-type (N=3), dystrophin-deficient ( mdx ; N=3) mice, and mice expressing C-terminally truncated Na v 1.5 (ΔSIV; N=3). We moreover assessed T-tubules sodium current by recording whole-cell sodium currents in control (N=5) and detubulated (N=5) wild-type cardiomyocytes. Results: We show that Na v 1.5 organizes as distinct clusters in the groove and T-tubules which density, distribution, and organization partially depend on SIV and dystrophin. We found that overall reduction in Na v 1.5 expression in mdx and ΔSIV cells results in a nonuniform redistribution with Na v 1.5 being specifically reduced at the groove of ΔSIV and increased in T-tubules of mdx cardiomyocytes. A T-tubules sodium current could, however, not be demonstrated. Conclusions: Na v 1.5 mutations may site-specifically affect Na v 1.5 localization and distribution at the lateral membrane and T-tubules, depending on site-specific interacting proteins. Future research efforts should elucidate the functional consequences of this redistribution.
    Type of Medium: Online Resource
    ISSN: 1941-3149 , 1941-3084
    URL: Article
    DOI: 10.1161/CIRCEP.119.008241
    Language: English
    Publisher: Ovid Technologies (Wolters Kluwer Health)
    Publication Date: 2020
    detail.hit.zdb_id: 2425487-3
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  • 6
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    Abstract 15925: Localization of Nav1.5 at the Intercellular Junctions Resolved by Diffraction-Unlimited Microscopy in Three Dimensions and by Correlative Light-Electron Microscopy
    Ovid Technologies (Wolters Kluwer Health) ; 2014
    In:  Circulation Vol. 130, No. suppl_2 ( 2014-11-25)
    Details
    In: Circulation, Ovid Technologies (Wolters Kluwer Health), Vol. 130, No. suppl_2 ( 2014-11-25)
    Abstract: 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.
    Type of Medium: Online Resource
    ISSN: 0009-7322 , 1524-4539
    URL: Article
    DOI: 10.1161/circ.130.suppl_2.15925
    Language: English
    Publisher: Ovid Technologies (Wolters Kluwer Health)
    Publication Date: 2014
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