“Orphan” Connexin43 in Plakophilin-2 Deficient Hearts Revealed by Volume Electron Microscopy
Previous studies revealed an abundance of functional Connexin43 (Cx43) hemichannels consequent to loss of plakophilin-2 (PKP2) expression in adult murine hearts. The increased Cx43-mediated membrane permeability is likely responsible for excess entry of calcium into the cells, leading to an arrhythm...
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| Format: | Article |
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Frontiers Media S.A.
2022-03-01
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| Series: | Frontiers in Cell and Developmental Biology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fcell.2022.843687/full |
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| author | Chantal J. M. van Opbergen Joseph Sall Chris Petzold Kristen Dancel-Manning Mario Delmar Feng-Xia Liang |
| author_facet | Chantal J. M. van Opbergen Joseph Sall Chris Petzold Kristen Dancel-Manning Mario Delmar Feng-Xia Liang |
| author_sort | Chantal J. M. van Opbergen |
| collection | DOAJ |
| description | Previous studies revealed an abundance of functional Connexin43 (Cx43) hemichannels consequent to loss of plakophilin-2 (PKP2) expression in adult murine hearts. The increased Cx43-mediated membrane permeability is likely responsible for excess entry of calcium into the cells, leading to an arrhythmogenic/cardiomyopathic phenotype. The latter has translational implications to the molecular mechanisms of inheritable arrhythmogenic right ventricular cardiomyopathy (ARVC). Despite functional evidence, visualization of these “orphan” (i.e., non-paired in a gap junction configuration) Cx43 hemichannels remains lacking. Immuno-electron microscopy (IEM) remains an extremely powerful tool to localize, with nanometric resolution, a protein within its native structural landscape. Yet, challenges for IEM are to preserve the antigenicity of the molecular target and to provide access for antibodies to reach their target, while maintaining the cellular/tissue ultrastructure. Fixation is important for maintaining cell structure, but strong fixation and vigorous dehydration (as it is routine for EM) can alter protein structure, thus impairing antigen-antibody binding. Here, we implemented a method to combine pre-embedding immunolabeling (pre-embedding) with serial block-face scanning electron microscopy (SBF-SEM). We utilized a murine model of cardiomyocyte-specific, Tamoxifen (TAM) activated knockout of PKP2. Adult hearts were harvested 14 days post-TAM, at this time hearts present a phenotype of concealed ARVC (i.e., an arrhythmogenic phenotype but no overt structural disease). Thick (200 µm) vibratome slices were immunolabelled for Cx43 and treated with nanogold or FluoroNanogold, coupled with a silver enhancement. Left or right ventricular free walls were dissected and three-dimensional (3D) localization of Cx43 in cardiac muscle was performed using SBF-SEM. Reconstructed images allowed us to visualize the entire length of gap junction plaques, seen as two parallel, closely packed strings of Cx43-immunoreactive beads at the intercalated disc. In contrast, in PKP2-deficient hearts we observed bulging of the intercellular space, and entire areas where only one of the two strings could be observed, indicating the presence of orphan Cx43. We conclude that pre-embedding and SBF-SEM allowed visualization of cardiac Cx43 plaques in their native environment, providing for the first time a visual complement of functional data indicating the presence of orphan Cx43 hemichannels resulting from loss of desmosomal integrity in the heart. |
| first_indexed | 2024-04-14T01:11:47Z |
| format | Article |
| id | doaj.art-02fb3793e1a549d89a48b5314d9a8268 |
| institution | Directory Open Access Journal |
| issn | 2296-634X |
| language | English |
| last_indexed | 2024-04-14T01:11:47Z |
| publishDate | 2022-03-01 |
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| spelling | doaj.art-02fb3793e1a549d89a48b5314d9a82682022-12-22T02:21:00ZengFrontiers Media S.A.Frontiers in Cell and Developmental Biology2296-634X2022-03-011010.3389/fcell.2022.843687843687“Orphan” Connexin43 in Plakophilin-2 Deficient Hearts Revealed by Volume Electron MicroscopyChantal J. M. van Opbergen0Joseph Sall1Chris Petzold2Kristen Dancel-Manning3Mario Delmar4Feng-Xia Liang5The Leon H. Charney Division of Cardiology, New York University Grossman School of Medicine, New York, NY, United StatesMicroscopy Laboratory, Division of Advanced Research Technologies, New York University Grossman School of Medicine, New York, NY, United StatesMicroscopy Laboratory, Division of Advanced Research Technologies, New York University Grossman School of Medicine, New York, NY, United StatesMicroscopy Laboratory, Division of Advanced Research Technologies, New York University Grossman School of Medicine, New York, NY, United StatesThe Leon H. Charney Division of Cardiology, New York University Grossman School of Medicine, New York, NY, United StatesMicroscopy Laboratory, Division of Advanced Research Technologies, New York University Grossman School of Medicine, New York, NY, United StatesPrevious studies revealed an abundance of functional Connexin43 (Cx43) hemichannels consequent to loss of plakophilin-2 (PKP2) expression in adult murine hearts. The increased Cx43-mediated membrane permeability is likely responsible for excess entry of calcium into the cells, leading to an arrhythmogenic/cardiomyopathic phenotype. The latter has translational implications to the molecular mechanisms of inheritable arrhythmogenic right ventricular cardiomyopathy (ARVC). Despite functional evidence, visualization of these “orphan” (i.e., non-paired in a gap junction configuration) Cx43 hemichannels remains lacking. Immuno-electron microscopy (IEM) remains an extremely powerful tool to localize, with nanometric resolution, a protein within its native structural landscape. Yet, challenges for IEM are to preserve the antigenicity of the molecular target and to provide access for antibodies to reach their target, while maintaining the cellular/tissue ultrastructure. Fixation is important for maintaining cell structure, but strong fixation and vigorous dehydration (as it is routine for EM) can alter protein structure, thus impairing antigen-antibody binding. Here, we implemented a method to combine pre-embedding immunolabeling (pre-embedding) with serial block-face scanning electron microscopy (SBF-SEM). We utilized a murine model of cardiomyocyte-specific, Tamoxifen (TAM) activated knockout of PKP2. Adult hearts were harvested 14 days post-TAM, at this time hearts present a phenotype of concealed ARVC (i.e., an arrhythmogenic phenotype but no overt structural disease). Thick (200 µm) vibratome slices were immunolabelled for Cx43 and treated with nanogold or FluoroNanogold, coupled with a silver enhancement. Left or right ventricular free walls were dissected and three-dimensional (3D) localization of Cx43 in cardiac muscle was performed using SBF-SEM. Reconstructed images allowed us to visualize the entire length of gap junction plaques, seen as two parallel, closely packed strings of Cx43-immunoreactive beads at the intercalated disc. In contrast, in PKP2-deficient hearts we observed bulging of the intercellular space, and entire areas where only one of the two strings could be observed, indicating the presence of orphan Cx43. We conclude that pre-embedding and SBF-SEM allowed visualization of cardiac Cx43 plaques in their native environment, providing for the first time a visual complement of functional data indicating the presence of orphan Cx43 hemichannels resulting from loss of desmosomal integrity in the heart.https://www.frontiersin.org/articles/10.3389/fcell.2022.843687/fullarrhythmogenic right ventricular cardiomyopathyplakophilin-2serial block-face scanning electron microscopyvolume electron microscopyconnexin43 hemichannelsnanogold |
| spellingShingle | Chantal J. M. van Opbergen Joseph Sall Chris Petzold Kristen Dancel-Manning Mario Delmar Feng-Xia Liang “Orphan” Connexin43 in Plakophilin-2 Deficient Hearts Revealed by Volume Electron Microscopy Frontiers in Cell and Developmental Biology arrhythmogenic right ventricular cardiomyopathy plakophilin-2 serial block-face scanning electron microscopy volume electron microscopy connexin43 hemichannels nanogold |
| title | “Orphan” Connexin43 in Plakophilin-2 Deficient Hearts Revealed by Volume Electron Microscopy |
| title_full | “Orphan” Connexin43 in Plakophilin-2 Deficient Hearts Revealed by Volume Electron Microscopy |
| title_fullStr | “Orphan” Connexin43 in Plakophilin-2 Deficient Hearts Revealed by Volume Electron Microscopy |
| title_full_unstemmed | “Orphan” Connexin43 in Plakophilin-2 Deficient Hearts Revealed by Volume Electron Microscopy |
| title_short | “Orphan” Connexin43 in Plakophilin-2 Deficient Hearts Revealed by Volume Electron Microscopy |
| title_sort | orphan connexin43 in plakophilin 2 deficient hearts revealed by volume electron microscopy |
| topic | arrhythmogenic right ventricular cardiomyopathy plakophilin-2 serial block-face scanning electron microscopy volume electron microscopy connexin43 hemichannels nanogold |
| url | https://www.frontiersin.org/articles/10.3389/fcell.2022.843687/full |
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