Differences in the Structural Characteristics of Adult Guinea Pig and Rat Cardiomyocytes during Their Adaptation and Maintenance in Long-Term Cultures: Confocal Microscopy Study

Horackova, Magda; Byczko, Zenobia

doi:10.1006/excr.1997.3775

Cited by 30 publications

(22 citation statements)

References 24 publications

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“…Our results show that there was no morphological dedifferentiation of ARVM in culture because under our culture conditions ARVMs maintained a rodshaped morphology for up to 4 days (Fig. 3A) for both uninfected and shCTR-infected cells (21). The cell capacitance recorded from control (78.15 Ϯ 5.51 pF (n ϭ 16)) and SAP97-silenced (78.77 Ϯ 4.6 pF, n ϭ 27) cells was not significantly different (p ϭ not significant) after 3 days in culture.…”

Section: Sap97mentioning

confidence: 57%

Regulation of Cardiac Inward Rectifier Potassium Current (IK1) by Synapse-associated Protein-97

Vaidyanathan

Taffet

Vikstrom

et al. 2010

Journal of Biological Chemistry

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Synapse-associated protein-97 (SAP97) is a membrane-associated guanylate kinase scaffolding protein expressed in cardiomyocytes. SAP97 has been shown to associate and modulate voltage-gated potassium (Kv) channel function. In contrast to Kv channels, little information is available on interactions involving SAP97 and inward rectifier potassium (Kir2.x) channels that underlie the classical inward rectifier current, I K1 . To investigate the functional effects of silencing SAP97 on I K1 in adult rat ventricular myocytes, SAP97 was silenced using an adenoviral short hairpin RNA vector. Western blot analysis showed that SAP97 was silenced by ϳ85% on day 3 post-infection. Immunostaining showed that Kir2.1 and Kir2.2 co-localize with SAP97. Co-immunoprecipitation (co-IP) results demonstrated that Kir2.x channels associate with SAP97. Voltage clamp experiments showed that silencing SAP97 reduced I K1 whole cell density by ϳ55%. I K1 density at ؊100 mV was ؊1.45 ؎ 0.15 pA/picofarads (n ‫؍‬ 6) in SAP97-silenced cells as compared with ؊3.03 ؎ 0.37 pA/picofarads (n ‫؍‬ 5) in control cells. Unitary conductance properties of I K1 were unaffected by SAP97 silencing. The major mechanism for the reduction of I K1 density appears to be a decrease in Kir2.x channel abundance. Furthermore, SAP97 silencing impaired I K1 regulation by ␤ 1 -adrenergic receptor (␤1-AR) stimulation. In control, isoproterenol reduced I K1 amplitude by ϳ75%, an effect that was blunted following SAP97 silencing. Our co-IP data show that ␤1-AR associates with SAP97 and Kir2.1 and also that Kir2.1 co-IPs with protein kinase A and ␤1-AR. SAP97 immunolocalizes with protein kinase A and ␤1-AR in the cardiac myocytes. Our results suggest that in cardiac myocytes SAP97 regulates surface expression of channels underlying I K1 , as well as assembles a signaling complex involved in ␤1-AR regulation of I K1 .In the heart, inward rectifier potassium current (I K1 ) plays a key role in stabilizing the resting membrane potential, determining the excitation threshold, and initiating the final repolarization phase of the action potential. Inward rectifier potassium channel subfamily 2 members (Kir2.1, Kir2.2, and Kir2.3 (Kir2.x) 3 ) are the molecular correlates of I K1 in the heart (1, 2). In contrast to other cardiac ion channels, relatively little information is available on protein-protein interactions involving Kir2.x channels that are important for their function. We have shown recently that Kir2.3 is regulated by synapse-associated protein 97 (SAP97) in a heterologous expression system, but it is not known if cardiac Kir2.x channels are regulated in a similar manner in situ (3).SAP97 belongs to the membrane-associated guanylate kinase family of proteins and is ubiquitously expressed in the heart (4). The membrane-associated guanylate kinase family of proteins are involved in the trafficking and assembly of proteins into macromolecular signaling complexes (5). They have protein-protein interaction domains such as PDZ (PSD-95, disclarge, ZO-1) domains, guanylate k...

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Section: Sap97mentioning

confidence: 57%

Regulation of Cardiac Inward Rectifier Potassium Current (IK1) by Synapse-associated Protein-97

Vaidyanathan

Taffet

Vikstrom

et al. 2010

Journal of Biological Chemistry

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“…This regularly involves remodeling of the intricate t-tubular network of adult cardiomyocytes. 36 Because the complex intracellular architecture of cardiomyocytes is likely to play an important role in compartmentation of cAMP signaling, our experimental strategy aimed at preserving the native structure of the adult cell. To be able to work with freshly isolated cells, we have introduced HCN2-camps into cardiomyocytes by creating transgenic mice expressing the sensor in a cardiomyocyte specific manner.…”

Section: Discussionmentioning

confidence: 99%

Cyclic AMP Imaging in Adult Cardiac Myocytes Reveals Far-Reaching β ₁ -Adrenergic but Locally Confined β ₂ -Adrenergic Receptor–Mediated Signaling

Nikolaev

Bünemann

Schmitteckert

et al. 2006

Circulation Research

319

291

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Abstract-␤ 1 -and ␤ 2 -adrenergic receptors (␤ARs) are known to differentially regulate cardiomyocyte contraction and growth. We tested the hypothesis that these differences are attributable to spatial compartmentation of the second messenger cAMP. Using a fluorescent resonance energy transfer (FRET)-based approach, we directly monitored the spatial and temporal distribution of cAMP in adult cardiomyocytes. We developed a new cAMP-FRET sensor (termed HCN2-camps) based on a single cAMP binding domain of the hyperpolarization activated cyclic nucleotide-gated potassium channel 2 (HCN2). Its cytosolic distribution, high dynamic range, and sensitivity make HCN2-camps particularly well suited to monitor subcellular localization of cardiomyocyte cAMP. We generated HCN2-camps transgenic mice and performed single-cell FRET imaging on freshly isolated cardiomyocytes. Whole-cell superfusion with isoproterenol showed a moderate elevation of cAMP. Application of various phosphodiesterase (PDE) inhibitors revealed stringent control of cAMP through PDE4ϾPDE2ϾPDE3. The ␤ 1 AR-mediated cAMP signals were entirely dependent on PDE4 activity, whereas ␤ 2 AR-mediated cAMP was under control of multiple PDE isoforms. ␤ 1 AR subtype-specific stimulation yielded Ϸ2-fold greater cAMP responses compared with selective ␤ 2 -subtype stimulation, even on treatment with the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) (⌬FRET, 17.3Ϯ1.3% [␤ 1 AR] versus 8.8Ϯ0.4% [␤ 2 AR]). Treatment with pertussis toxin to inactivate G i did not affect cAMP production. Localized ␤ 1 AR stimulation generated a cAMP gradient propagating throughout the cell, whereas local ␤ 2 AR stimulation did not elicit marked cAMP diffusion. Our data reveal that in adult cardiac myocytes, ␤ 1 ARs induce far-reaching cAMP signals, whereas ␤ 2 AR-induced cAMP remains locally confined. by the endogenous agonists noradrenaline and adrenaline represents the strongest mechanism to increase cardiac chronotropy and inotropy. 1 The mammalian heart contains 3 ␤AR subtypes: the ␤ 1 -, the ␤ 2 -, and the ␤ 3 AR. The ␤ 1 -and ␤ 2 AR subtypes dominate the cardiac response to adrenergic stimulation. Both are expressed in cardiomyocytes, couple primarily to G s , and mediate cAMP formation, whereas coupling of the ␤ 2 AR to G i has been described in several animal species and in failing human cardiomyocytes. [2][3][4] The second messenger cAMP then leads to activation of protein kinase A (PKA), which phosphorylates key regulators of the cardiac excitation/contraction machinery, including the L-type Ca 2ϩ channel, phospholamban, the ryanodine receptor, and troponin T and I. However, selective stimulation of these 2 receptor subtypes elicits different physiological responses. ␤ 1 AR stimulation, but not ␤ 2 AR stimulation, seems to induce cardiomyocyte hypertrophy. 5 Transgenic mice with cardiomyocyte-specific overexpression of the ␤ 1 AR develop progressive cardiac hypertrophy and heart failure, whereas ␤ 2 AR transgenic mice do not show such abnormalities. 6,7 Isolated cardio...

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“…Such protrusions are characteristic of cells attempting to es-tablish a connection with other cells to form an association that are seen in cells undergoing differentiation. This process is facilitated by the polymerization of actin that forms filamentous or F-actin giving rise to polymorphic shapes of the cells with pseudopod-like protrusions (35). Formation of such structural configuration was evaluated by rhodamine-conjugated phalloidin staining for F-actin and visualized by fluorescence microscopy as described (36).…”

Section: Synergistic Induction Of Saa By Il-1 and Il-6 -mentioning

confidence: 99%

Activation of Sp1 and Its Functional Co-operation with Serum Amyloid A-activating Sequence Binding Factor in Synoviocyte Cells Trigger Synergistic Action of Interleukin-1 and Interleukin-6 in Serum Amyloid A Gene Expression

Ray

Schatten

Ray

1999

Journal of Biological Chemistry

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The serum amyloid A (SAA) protein has been implicated in the progression and pathogenesis of rheumatoid arthritis through induction of collagenase activity in synovial fibroblast cells that line the joint tissues. We demonstrate that SAA is synergistically induced in synovial cells by interleukin (IL)-1 and IL-6 that are present at significantly high level in the synovial fluid of arthritis patients. These cytokines induced phenotypic changes in synovial cells, promoting protrusion and increased cellular contact. Induction of SAA under this condition is mediated by promoter elements located between ؊254 and ؊226, which contains binding sites for transcription factors Sp1 and SAA activating sequence binding factor (SAF). Mutation of these sequences abolishes SAA promoter response to IL-1 and IL-6.

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Differences in the Structural Characteristics of Adult Guinea Pig and Rat Cardiomyocytes during Their Adaptation and Maintenance in Long-Term Cultures: Confocal Microscopy Study

Cited by 30 publications

References 24 publications

Regulation of Cardiac Inward Rectifier Potassium Current (IK1) by Synapse-associated Protein-97

Regulation of Cardiac Inward Rectifier Potassium Current (IK1) by Synapse-associated Protein-97

Cyclic AMP Imaging in Adult Cardiac Myocytes Reveals Far-Reaching β ₁ -Adrenergic but Locally Confined β ₂ -Adrenergic Receptor–Mediated Signaling

Activation of Sp1 and Its Functional Co-operation with Serum Amyloid A-activating Sequence Binding Factor in Synoviocyte Cells Trigger Synergistic Action of Interleukin-1 and Interleukin-6 in Serum Amyloid A Gene Expression

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Differences in the Structural Characteristics of Adult Guinea Pig and Rat Cardiomyocytes during Their Adaptation and Maintenance in Long-Term Cultures: Confocal Microscopy Study

Cited by 30 publications

References 24 publications

Regulation of Cardiac Inward Rectifier Potassium Current (IK1) by Synapse-associated Protein-97

Regulation of Cardiac Inward Rectifier Potassium Current (IK1) by Synapse-associated Protein-97

Cyclic AMP Imaging in Adult Cardiac Myocytes Reveals Far-Reaching β 1 -Adrenergic but Locally Confined β 2 -Adrenergic Receptor–Mediated Signaling

Activation of Sp1 and Its Functional Co-operation with Serum Amyloid A-activating Sequence Binding Factor in Synoviocyte Cells Trigger Synergistic Action of Interleukin-1 and Interleukin-6 in Serum Amyloid A Gene Expression

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Cyclic AMP Imaging in Adult Cardiac Myocytes Reveals Far-Reaching β ₁ -Adrenergic but Locally Confined β ₂ -Adrenergic Receptor–Mediated Signaling