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.
Highlights
Following an acute myocardial infarction, reperfusion of the myocardium is a required event after the ischemic insult for salvaging viable myocardium
The heart rate, systolic and diastolic blood pressures in vehicle- and chymase inhibitor (CI)-treated dogs were not affected during the 5 days of CI pretreatment, but the systolic and diastolic pressures did decrease with CI at the end of I/R protocol compared to the vehicle-treated dogs (Table S1)
The mRNA levels of the two genes, nuclear receptor subfamily 4A1 (NR4A1) and activating transcription factor 3 (ATF3), were validated by real-time room temperature (RT)-PCR (Figure 4b) and the results indicated that NR4A1 and ATF3 were elevated more than 15- and 30-fold, respectively, in the vehicle treated I/R LV compared to the vehicle treated non-I/R and normal LV (Figure 4b)
Summary
Following an acute myocardial infarction, reperfusion of the myocardium is a required event after the ischemic insult for salvaging viable myocardium. Reperfusion causes further cardiomyocyte injury and death [1]. Limiting reperfusion injury is an important objective to improve myocardial salvage and currently there is no effective therapy for this in the clinical setting. Resident mast cell (MC) degranulation is an early event in ischemia/reperfusion (I/R) injury. MCs normally exist in the myocardium in an intact form but become activated in response to acute stress, rapidly releasing enzymes and hormonal mediators into the cardiac interstitium [2]. We utilize a dog model to test whether using a specific oral chymase inhibitor [12] in the interstitial fluid (ISF) space attenuates cardiomyocyte injury during I/R
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