title: Chymase mediates injury and mitochondrial damage in cardiomyocytes during acute ischemia/reperfusion in the dog.
abstract: Cardiac ischemia and reperfusion (I/R) injury occurs because the acute increase in oxidative/inflammatory stress during reperfusion culminates in the death of cardiomyocytes. Currently, there is no drug utilized clinically that attenuates I/R injury in patients. Previous studies have demonstrated degranulation of mast cell contents into the interstitium after I/R. Using a dog model of I/R, we tested the role of chymase, a mast cell protease, in cardiomyocyte injury using a specific oral chymase inhibitor (CI). 15 adult mongrel dogs had left anterior descending artery occlusion for 60 min and reperfusion for 100 minutes. 9 dogs received vehicle and 6 were pretreated with a specific CI. In vivo cardiac microdialysis demonstrated a 3-fold increase in interstitial fluid chymase activity in I/R region that was significantly decreased by CI. CI pretreatment significantly attenuated loss of laminin, focal adhesion complex disruption, and release of troponin I into the circulation. Microarray analysis identified an I/R induced 17-fold increase in nuclear receptor subfamily 4A1 (NR4A1) and significantly decreased by CI. NR4A1 normally resides in the nucleus but can induce cell death on migration to the cytoplasm. I/R caused significant increase in NR4A1 protein expression and cytoplasmic translocation, and mitochondrial degradation, which were decreased by CI. Immunohistochemistry also revealed a high concentration of chymase within cardiomyocytes after I/R. In vitro, chymase added to culture HL-1 cardiomyocytes entered the cytoplasm and nucleus in a dynamin-dependent fashion, and promoted cytoplasmic translocation of NR4A1 protein. shRNA knockdown of NR4A1 on pre-treatment of HL-1 cells with CI significantly decreased chymase-induced cell death and mitochondrial damage. These results suggest that the beneficial effects of an orally active CI during I/R are mediated in the cardiac interstitium as well as within the cardiomyocyte due to a heretofore-unrecognized chymase entry into cardiomyocytes.
authors.affiliation: Birmingham Veteran Affairs Medical Center and Division of Cardiovascular Disease, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Junying
authors.initials: J
authors.lastname: Zheng
authors.affiliation: Birmingham Veteran Affairs Medical Center and Division of Cardiovascular Disease, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Chih-Chang
authors.initials: CC
authors.lastname: Wei
authors.affiliation: Teijin Institute for Bio-Medical Research, Teijin Pharma Ltd, Tokyo, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Hase
authors.affiliation: Birmingham Veteran Affairs Medical Center and Division of Cardiovascular Disease, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Ke
authors.initials: K
authors.lastname: Shi
authors.affiliation: Birmingham Veteran Affairs Medical Center and Division of Cardiovascular Disease, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Cheryl R
authors.initials: CR
authors.lastname: Killingsworth
authors.affiliation: Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Silvio H
authors.initials: SH
authors.lastname: Litovsky
authors.affiliation: Birmingham Veteran Affairs Medical Center and Division of Cardiovascular Disease, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Pamela C
authors.initials: PC
authors.lastname: Powell
authors.affiliation: Teijin Institute for Bio-Medical Research, Teijin Pharma Ltd, Tokyo, Japan.
authors.forename: Tsunefumi
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: Wake Forest University, Winston Salem, North Carolina, United States of America.
authors.forename: Carlos M
authors.initials: CM
authors.lastname: Ferrario
authors.affiliation: Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Andras
authors.initials: A
authors.lastname: Rab
authors.affiliation: Department of Biostatistics University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Inmaculada
authors.initials: I
authors.lastname: Aban
authors.affiliation: Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: James F
authors.initials: JF
authors.lastname: Collawn
authors.affiliation: Birmingham Veteran Affairs Medical Center and Division of Cardiovascular Disease, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Louis J
authors.initials: LJ
authors.lastname: Dell'Italia
mesh_terms: D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D053818:Chymases / Q000037:antagonists & inhibitors / Q000502:physiology*; D003593:Cytoplasm / Q000378:metabolism; D004285:Dogs; D004705:Endocytosis; D007511:Ischemia / Q000473:pathology; D008407:Mast Cells / Q000201:enzymology; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000201:enzymology*; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1 / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010447:Peptide Hydrolases / Q000737:chemistry; D015427:Reperfusion Injury; D014168:Transferrin / Q000378:metabolism; D019210:Troponin I / Q000097:blood; D014947:Wounds and Injuries / Q000378:metabolism*
chemical_list: D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D014168:Transferrin; D019210:Troponin I; D010447:Peptide Hydrolases; D053818:Chymases; D020780:Matrix Metalloproteinase 9
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