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Phosphorylation in Novel Mitochondrial Creatine Kinase Tyrosine Residues Render Cardioprotection against Hypoxia/Reoxygenation Injury
OBJECTIVE: Ischemic cardiomyopathy (ICM) is the leading cause of heart failure. Proteomic and genomic studies have demonstrated ischemic preconditioning (IPC) can assert cardioprotection against ICM through mitochondrial function regulation. Considering IPC is conducted in a relatively brief period,...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Korean Society of Lipidology and Atherosclerosis
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8159762/ https://www.ncbi.nlm.nih.gov/pubmed/34095014 http://dx.doi.org/10.12997/jla.2021.10.2.223 |
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author | Park, Nammi Marquez, Jubert Garcia, Maria Victoria Faith Shimizu, Ippei Lee, Sung Ryul Kim, Hyoung Kyu Han, Jin |
author_facet | Park, Nammi Marquez, Jubert Garcia, Maria Victoria Faith Shimizu, Ippei Lee, Sung Ryul Kim, Hyoung Kyu Han, Jin |
author_sort | Park, Nammi |
collection | PubMed |
description | OBJECTIVE: Ischemic cardiomyopathy (ICM) is the leading cause of heart failure. Proteomic and genomic studies have demonstrated ischemic preconditioning (IPC) can assert cardioprotection against ICM through mitochondrial function regulation. Considering IPC is conducted in a relatively brief period, regulation of protein expression also occurs very rapidly, highlighting the importance of protein function modulation by post-translational modifications. This study aimed to identify and analyze novel phosphorylated mitochondrial proteins that can be harnessed for therapeutic strategies for preventing ischemia/reperfusion (I/R) injury. METHODS: Sprague-Dawley rat hearts were used in an ex vivo Langendorff system to simulate normal perfusion, I/R, and IPC condition, after which the samples were prepared for phosphoproteomic analysis. Employing human cardiomyocyte AC16 cells, we investigated the cardioprotective role of CKMT2 through overexpression and how site-directed mutagenesis of putative CKMT2 phosphorylation sites (Y159A, Y255A, and Y368A) can affect cardioprotection by measuring CKMT2 protein activity, mitochondrial function and protein expression changes. RESULTS: The phosphoproteomic analysis revealed dephosphorylation of mitochondrial creatine kinase (CKMT2) during ischemia and I/R, while preserving its phosphorylated state during IPC. CKMT2 overexpression conferred cardioprotection against hypoxia/reoxygenation (H/R) by increasing cell viability and mitochondrial adenosine triphosphate level, preserving mitochondrial membrane potential, and reduced reactive oxygen species (ROS) generation, while phosphomutations, especially in Y368, nullified cardioprotection by significantly reducing cell viability and increasing ROS production during H/R. CKMT2 overexpression increased mitochondrial function by mediating the proliferator-activated receptor γ coactivator-1α/estrogen-related receptor-α pathway, and these effects were mostly inhibited by Y368A mutation. CONCLUSION: These results suggest that regulation of quantitative expression and phosphorylation site Y368 of CKMT2 offers a unique mechanism in future ICM therapeutics. |
format | Online Article Text |
id | pubmed-8159762 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Korean Society of Lipidology and Atherosclerosis |
record_format | MEDLINE/PubMed |
spelling | pubmed-81597622021-06-04 Phosphorylation in Novel Mitochondrial Creatine Kinase Tyrosine Residues Render Cardioprotection against Hypoxia/Reoxygenation Injury Park, Nammi Marquez, Jubert Garcia, Maria Victoria Faith Shimizu, Ippei Lee, Sung Ryul Kim, Hyoung Kyu Han, Jin J Lipid Atheroscler Original Article OBJECTIVE: Ischemic cardiomyopathy (ICM) is the leading cause of heart failure. Proteomic and genomic studies have demonstrated ischemic preconditioning (IPC) can assert cardioprotection against ICM through mitochondrial function regulation. Considering IPC is conducted in a relatively brief period, regulation of protein expression also occurs very rapidly, highlighting the importance of protein function modulation by post-translational modifications. This study aimed to identify and analyze novel phosphorylated mitochondrial proteins that can be harnessed for therapeutic strategies for preventing ischemia/reperfusion (I/R) injury. METHODS: Sprague-Dawley rat hearts were used in an ex vivo Langendorff system to simulate normal perfusion, I/R, and IPC condition, after which the samples were prepared for phosphoproteomic analysis. Employing human cardiomyocyte AC16 cells, we investigated the cardioprotective role of CKMT2 through overexpression and how site-directed mutagenesis of putative CKMT2 phosphorylation sites (Y159A, Y255A, and Y368A) can affect cardioprotection by measuring CKMT2 protein activity, mitochondrial function and protein expression changes. RESULTS: The phosphoproteomic analysis revealed dephosphorylation of mitochondrial creatine kinase (CKMT2) during ischemia and I/R, while preserving its phosphorylated state during IPC. CKMT2 overexpression conferred cardioprotection against hypoxia/reoxygenation (H/R) by increasing cell viability and mitochondrial adenosine triphosphate level, preserving mitochondrial membrane potential, and reduced reactive oxygen species (ROS) generation, while phosphomutations, especially in Y368, nullified cardioprotection by significantly reducing cell viability and increasing ROS production during H/R. CKMT2 overexpression increased mitochondrial function by mediating the proliferator-activated receptor γ coactivator-1α/estrogen-related receptor-α pathway, and these effects were mostly inhibited by Y368A mutation. CONCLUSION: These results suggest that regulation of quantitative expression and phosphorylation site Y368 of CKMT2 offers a unique mechanism in future ICM therapeutics. Korean Society of Lipidology and Atherosclerosis 2021-05 2021-01-19 /pmc/articles/PMC8159762/ /pubmed/34095014 http://dx.doi.org/10.12997/jla.2021.10.2.223 Text en Copyright © 2021 The Korean Society of Lipid and Atherosclerosis. https://creativecommons.org/licenses/by-nc/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Original Article Park, Nammi Marquez, Jubert Garcia, Maria Victoria Faith Shimizu, Ippei Lee, Sung Ryul Kim, Hyoung Kyu Han, Jin Phosphorylation in Novel Mitochondrial Creatine Kinase Tyrosine Residues Render Cardioprotection against Hypoxia/Reoxygenation Injury |
title | Phosphorylation in Novel Mitochondrial Creatine Kinase Tyrosine Residues Render Cardioprotection against Hypoxia/Reoxygenation Injury |
title_full | Phosphorylation in Novel Mitochondrial Creatine Kinase Tyrosine Residues Render Cardioprotection against Hypoxia/Reoxygenation Injury |
title_fullStr | Phosphorylation in Novel Mitochondrial Creatine Kinase Tyrosine Residues Render Cardioprotection against Hypoxia/Reoxygenation Injury |
title_full_unstemmed | Phosphorylation in Novel Mitochondrial Creatine Kinase Tyrosine Residues Render Cardioprotection against Hypoxia/Reoxygenation Injury |
title_short | Phosphorylation in Novel Mitochondrial Creatine Kinase Tyrosine Residues Render Cardioprotection against Hypoxia/Reoxygenation Injury |
title_sort | phosphorylation in novel mitochondrial creatine kinase tyrosine residues render cardioprotection against hypoxia/reoxygenation injury |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8159762/ https://www.ncbi.nlm.nih.gov/pubmed/34095014 http://dx.doi.org/10.12997/jla.2021.10.2.223 |
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