Conditional knock out of cytosolic thioredoxin (Trx) in cardiomyocytes impairs cardiac function and exacerbates ischemia-reperfusion injury

Abstract Introduction Coronary heart disease with myocardial infarction (MI) is a leading cause of mortality in the world despite timely reperfusion of occluded coronary artery. Myocardial damage due to restoration of blood flow, known as reperfusion injury account for about 50% of final MI size. The role of Trx in protection against myocardial ischemia reperfusion has been reported; however, these studies were mostly performed using ex vivo isolated mouse/rat heart with retrograde perfusion or with injected rhTrx. However, the role of loss of Trx specifically in the myocytes using a conditional deletion has never been reported. Here, we determined whether loss of Trx in myocytes would promote ischemia-reperfusion injury, even if the endocardial endothelial cells express normal levels of Trx. Purpose We investigated the effect of specific deletion of Trx in cardiomyocytes on base-line cardiac function and cardiac function in ischemia-reperfusion of heart using conditional knockout of Trx (aMHC-Trx-KO) in the cardiomyocytes with normal levels of Trx in the endothelial cells to determine specifc contribution of Trx in cardiac function in I/R. Methods A Trx floxed mouse line was created with introduction of loxP sites flanking the mouse Trx gene. Further, the conditional deletion of Trx in cardiomyocytes was produced by breeding homozygous Trx-floxed (Trxfl/fl) mice with aMHC-Cre mice strain. The resulting aMHC-Trx-KO mice was identified by genotyping and conditional deletion of Trx in myocytes was achieved by injecting Tomoxifen (75mg/Kg in oil). The aMHC-Trx-KO mice were subjected to 30 minutes of ischemia by ligation of left descending coronary artery(LAD) and reperfusion was performed by release of slipknot and allowing blood flow of 60 minutes before sacrificing animals. The animal protocol was approved by the IACUC of the Texas Tech University Health Sciences Center, Lubbock. Echocardiography on animals was performed using Vevo 3100 (Visual Sonic). Statistical analysis was performed by Prism 8.0 (GraphPad). Results Deletion of Trx caused significant cardiac dysfunction in aMHC-Trx-KO mice compared to Trxfl/fl mice without treatment with tomoxifen. The ejection fraction (EF%, 52.82 vs. 73.83) and fractional shortening (FS%, 41.42 vs.25.89) were significantly reduced. Further, these mice subjected to I/R showed exacerbated cardiac dysfunction with significant decrease in heart rate and did not survive the reperfusion. We also found significant mitochondrial structural abnormalities and mitochondrial dysfunction in aMHC-Trx-KO mice compared to non-transgenic or Trxfl/fl mice without tomoxifen treatment. Conclusion Deletion of Trx in cardiomyocyte causes significant myocardial dysfunction. Further, Trx is required to protect against myocardial dysfunction in ischemia-reperfusion injury. Our data also suggest that cardiac mitochondria is specifically impacted by loss of Trx deletion.Figure-1