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Canadian Journal of Cardiology
Basic Research| Volume 37, ISSUE 10, P1593-1606, October 2021

A Novel Role of Claudin-5 in Prevention of Mitochondrial Fission Against Ischemic/Hypoxic Stress in Cardiomyocytes

Published:April 07, 2021DOI:https://doi.org/10.1016/j.cjca.2021.03.021

      Abstract

      Background

      Downregulation of claudin-5 in the heart is associated with the end-stage heart failure. However, the underlying mechanism ofclaudin-5 is unclear. Here we investigated the molecular actions of claudin-5 in perspective of mitochondria in cardiomyocytes to better understand the role of claudin-5 in cardioprotection during ischemia.

      Methods

      Myocardial ischemia/reperfusion (I/R; 30 min/24 h) and hypoxia/reoxygenation (H/R; 24 h/4 h) were used in this study. Confocal microscopy and transmission electron microscope (TEM) were used to observe mitochondrial morphology.

      Results

      Claudin-5 was detected in murine heart tissue and neonatal rat cardiomyocytes (NRCM). Its protein level was severely decreased after myocardial I/R or H/R. Confocal microscopy showedclaudin-5 presented in the mitochondria of NRCM. H/R-induced claudin-5 downregulation was accompanied by mitochondrial fragmentation. The mitofusin 2 (Mfn2) expressionwas dramatically decreased while the dynamin-related protein (Drp) 1 expression was significantly increased after H/R. The TEM indicatedH/R-induced mitochondrial swelling and fission. Adenoviral claudin-5 overexpression reversed these structural disintegration of mitochondria. The mitochondria-centered intrinsic pathway of apoptosis triggered by H/R and indicated by the cytochrome c and cleaved caspase 3 in the cytoplasm of NRCMs was also reduced by overexpressing claudin-5. Claudin-5 overexpression in mouse heart also significantly decreased cleaved caspase 3 and the infarct size in ischemic heart with improved systolic function.

      Conclusion

      We demonstrated for the first time the presence of claudin-5 in the mitochondria in cardiomyocytes and provided the firm evidence for the cardioprotective role of claudin-5 in the preservation of mitochondrial dynamics and cell fate against hypoxia- or ischemia-induced stress.

      Résumé

      Contexte

      Une régulation négative de la claudine-5 dans le cœur est associée à l'insuffisance cardiaque au stade terminal. Cependant, le mécanisme sous-jacent de la claudine-5 n'est pas clair. Nous avons étudié les mécanismes d'actions moléculaires de la claudine-5 dans les mitochondries des cardiomyocytes afin de mieux comprendre le rôle de la claudine-5 dans la cardioprotection pendant l'ischémie.

      Méthodes

      L'ischémie/reperfusion (I/R; 30 min/24 h) du myocarde et l'hypoxie/régénération (H/R; 24 h/4 h) ont été appliquées lors de cette étude. La microscopie confocale et la microscopie électronique en transmission (MET) ont été utilisées pour l'observation de la morphologie des mitochondries.

      Résultats

      La claudine-5 a été détectée dans le tissu cardiaque murin et dans les cardiomyocytes néonataux de rat (CNR). Son niveau d'expression protéique était sévérement diminué après une I/R du myocarde ou une H/R. La microscopie confocale a montré que la claudine-5 était présente dans les mitochondries des CNR. La régulation négative de la claudine-5 induite par l'H/R s'est accompagnée d'une fragmentation mitochondriale. Le niveau d'expression de la mitofusine-2 (Mfn2) a considérablement diminué tandis que celui de la protéine de type dynamine-1 (Drp1) a augmenté de manière significative après l'H/R. La MET a mis en èvidence le gonflement et la fission des mitochondries induits par l'H/R. La surexpression de la claudine-5 par infection adénovirale a inversé cette désintégration structurelle des mitochondries. La voie intrinsèque de l'apoptose au niveau mitochondrial, déclenchée par l'H/R et indiquée par l'expression du cytochrome c et de la caspase-3 clivée dans le cytoplasme des CNR, a aussi été réduite lors de la surexpression de la claudine-5. La surexpression de la claudine-5 dans le cœur de souris a également diminué de manière significative la caspase-3 à l'état clivé et la taille de l'infarctus dans le cœur ischémique, avec une amélioration de la fonction systolique.

      Conclusion

      Nous avons démontré pour la première fois la présence de la claudine-5 dans les mitochondries des cardiomyocytes et fourni des preuves solides du rôle cardioprotecteur de la claudine-5 dans la préservation de la dynamique mitochondriale et du devenir des cellules opposées au stress induit par l'hypoxie ou l'ischémie.
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