Publicação
Heart ischemia gives a second life to Cx43: Novel roles ascribed to Cx43 that go beyond gap junction intercellular communication
| Resumo: | A well-balanced intercellular communication between the different cells within the myocardium is vital for the maintenance of cardiac homeostasis and function. In the heart, connexin43 (Cx43)-containing gap junction (GJ) channels localized at the intercalated discs (IDs) ensure the passage of small molecules between adjacent cardiomyocytes, contributing for the synchronized heart beating. At longer distances, intracardiac communication rely on extracellular vesicles (EVs), which may also contain Cx43 channels at their surface. Besides, mounting evidence associates Cx43 with myriad channel-independent functions, including cell adhesion, differentiation and gene expression. Although abnormal expression and heterogeneous redistribution of Cx43 have been implicated in several cardiac disorders, including myocardial infarction and heart failure, the underlying mechanisms and regulatory proteins remain largely unknown. Hence, we applied unbiased proteomics to map the Cx43 interactome in rat hearts, in basal conditions and following ischemia and ischemia/reperfusion (I/R). Our results demonstrate that Cx43 binds to a large number of unforeseen proteins involved in various biological processes, such as intracellular trafficking, metabolism and transcription. Grounded on our proteomic data, we identified the interaction of Cx43 with the endocytic regulators Eps15 homology domain-containing proteins (EHDs). Our results showed that EHD1 participated in the internalization and lateralization of Cx43 in vitro. Moreover, the association between Cx43 and EHDs was increased in the ischemic heart, implicating EHDs in the pathological remodeling of Cx43 channels. Despite considerable progress in the definition of EV biogenesis mechanisms, selective EV protein sorting remains obscure, particularly under pathological conditions. Hence, we used Cx43 as a model substrate, which enabled the identification of ubiquitin as the signal driving the sorting of EV-Cx43. In addition, the presence of Cx43 in EVs determined the selective incorporation of miRNAs into EVs and facilitated the delivery of doxorubicin to tumor cells, with a concomitant reduced cardiotoxicity in vivo, ascribing a role for Cx43 in targeted EV-cell communication. Finally, we showed that the expression of Cx43 significantly impacted on the cellular transcriptome, which was correlated with the presence of Cx43 channels at the inner nuclear membrane. Strikingly, ischemia downregulated both the release of Cx43 in EVs, as well as the amount of Cx43 in the nucleus, suggesting that non-canonical functions of Cx43, beyond GJ-mediated intercellular communication, are compromised under pathological conditions. Overall, the results presented in this thesis contribute to a better understanding of the channel-dependent and -independent functions of Cx43 in the heart, together with the identification of the binding partners regulating some of these functions. These data may bring new perspectives for the design of novel diagnostic and therapeutic strategies for cardiovascular disorders. |
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| Autores principais: | Marques, Tânia Sofia Martins |
| Assunto: | connexin43 gap junctions intercellular communication extracellular vesicles nuclei acute myocardial ischemia heart failure |
| Ano: | 2020 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso embargado |
| Instituição associada: | Universidade de Coimbra |
| Idioma: | inglês |
| Origem: | Estudo Geral - Universidade de Coimbra |
| Resumo: | A well-balanced intercellular communication between the different cells within the myocardium is vital for the maintenance of cardiac homeostasis and function. In the heart, connexin43 (Cx43)-containing gap junction (GJ) channels localized at the intercalated discs (IDs) ensure the passage of small molecules between adjacent cardiomyocytes, contributing for the synchronized heart beating. At longer distances, intracardiac communication rely on extracellular vesicles (EVs), which may also contain Cx43 channels at their surface. Besides, mounting evidence associates Cx43 with myriad channel-independent functions, including cell adhesion, differentiation and gene expression. Although abnormal expression and heterogeneous redistribution of Cx43 have been implicated in several cardiac disorders, including myocardial infarction and heart failure, the underlying mechanisms and regulatory proteins remain largely unknown. Hence, we applied unbiased proteomics to map the Cx43 interactome in rat hearts, in basal conditions and following ischemia and ischemia/reperfusion (I/R). Our results demonstrate that Cx43 binds to a large number of unforeseen proteins involved in various biological processes, such as intracellular trafficking, metabolism and transcription. Grounded on our proteomic data, we identified the interaction of Cx43 with the endocytic regulators Eps15 homology domain-containing proteins (EHDs). Our results showed that EHD1 participated in the internalization and lateralization of Cx43 in vitro. Moreover, the association between Cx43 and EHDs was increased in the ischemic heart, implicating EHDs in the pathological remodeling of Cx43 channels. Despite considerable progress in the definition of EV biogenesis mechanisms, selective EV protein sorting remains obscure, particularly under pathological conditions. Hence, we used Cx43 as a model substrate, which enabled the identification of ubiquitin as the signal driving the sorting of EV-Cx43. In addition, the presence of Cx43 in EVs determined the selective incorporation of miRNAs into EVs and facilitated the delivery of doxorubicin to tumor cells, with a concomitant reduced cardiotoxicity in vivo, ascribing a role for Cx43 in targeted EV-cell communication. Finally, we showed that the expression of Cx43 significantly impacted on the cellular transcriptome, which was correlated with the presence of Cx43 channels at the inner nuclear membrane. Strikingly, ischemia downregulated both the release of Cx43 in EVs, as well as the amount of Cx43 in the nucleus, suggesting that non-canonical functions of Cx43, beyond GJ-mediated intercellular communication, are compromised under pathological conditions. Overall, the results presented in this thesis contribute to a better understanding of the channel-dependent and -independent functions of Cx43 in the heart, together with the identification of the binding partners regulating some of these functions. These data may bring new perspectives for the design of novel diagnostic and therapeutic strategies for cardiovascular disorders. |
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