Wei Li1, David Kennedy2, Zhili Shao3, Xi Wang4, Andre Klaassen Kamdar5, Malory Weber3, Kayla Mislick3, Kathryn Kiefer3, Rommel Morales3, Brendan Agatisa-Boyle3, Diana M Shih6, Srinivasa T Reddy6, Christine S Moravec7, W H Wilson Tang8. 1. Department of Biomedical Sciences, Joan C. Edwards School of Medicine, Marshall University, WV, United States. 2. Department of Medicine, University of Toledo, OH, United States. 3. Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, OH, United States. 4. Department of Medicine, Stanford University School of Medicine, CA, United States. 5. Department of Medicine, University of Minnesota, MN, United States. 6. Department of Medicine, Division of Cardiology, University of California at Los Angeles, Los Angeles, CA, United States. 7. Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, OH, United States. 8. Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, OH, United States; Department of Cardiovascular Medicine, Heart and Vascular Institute, Cleveland Clinic, OH, United States; Center for Clinical Genomics, Cleveland Clinic, OH, United States. Electronic address: tangw@ccf.org.
Abstract
BACKGROUND: Mitochondrial oxidation is a major source of reactive oxygen species (ROS) and mitochondrial dysfunction plays a central role in development of heart failure (HF). Paraoxonase 2 deficient (PON2-def) mitochondria are impaired in function. In this study, we tested whether PON2-def aggravates HF progression. METHODS AND RESULTS: Using qPCR, immunoblotting and lactonase activity assay, we demonstrate that PON2 activity was significantly decreased in failing hearts despite increased PON2 expression. To determine the cardiac-specific function of PON2, we performed heart transplantations in which PON2-def and wild type (WT) donor hearts were implanted into WT recipient mice. Beating scores of the donor hearts, assessed at 4 weeks post-transplantation, were significantly decreased in PON2-def hearts when compared to WT donor hearts. By using a transverse aortic constriction (TAC) model, we found PON2 deficiency significantly exacerbated left ventricular remodeling and cardiac fibrosis post-TAC. We further demonstrated PON2 deficiency significantly enhanced ROS generation in heart tissues post-TAC. ROS generation was measured through dihydroethidium (DHE) using high-pressure liquid chromatography (HPLC) with a fluorescent detector. By using neonatal cardiomyocytes treated with CoCl2 to mimic hypoxia, we found PON2 deficiency dramatically increased ROS generation in the cardiomyocytes upon CoCl2 treatment. In response to a short CoCl2 exposure, cell viability and succinate dehydrogenase (SDH) activity assessed by MTT assay were significantly diminished in PON2-def cardiomyocytes compared to those in WT cardiomyocytes. PON2-def cardiomyocytes also had lower baseline SDH activity. By using adult mouse cardiomyocytes and mitochondrial ToxGlo assay, we found impaired cellular ATP generation in PON2-def cells compared to that in WT cells, suggesting that PON2 is necessary for proper mitochondrial function. CONCLUSION: Our study suggests a cardioprotective role for PON2 in both experimental and human heart failure, which may be associated with the ability of PON2 to improve mitochondrial function and diminish ROS generation.
BACKGROUND: Mitochondrial oxidation is a major source of reactive oxygen species (ROS) and mitochondrial dysfunction plays a central role in development of heart failure (HF). Paraoxonase 2 deficient (PON2-def) mitochondria are impaired in function. In this study, we tested whether PON2-def aggravates HF progression. METHODS AND RESULTS: Using qPCR, immunoblotting and lactonase activity assay, we demonstrate that PON2 activity was significantly decreased in failing hearts despite increased PON2 expression. To determine the cardiac-specific function of PON2, we performed heart transplantations in which PON2-def and wild type (WT) donor hearts were implanted into WT recipient mice. Beating scores of the donor hearts, assessed at 4 weeks post-transplantation, were significantly decreased in PON2-def hearts when compared to WT donor hearts. By using a transverse aortic constriction (TAC) model, we found PON2 deficiency significantly exacerbated left ventricular remodeling and cardiac fibrosis post-TAC. We further demonstrated PON2 deficiency significantly enhanced ROS generation in heart tissues post-TAC. ROS generation was measured through dihydroethidium (DHE) using high-pressure liquid chromatography (HPLC) with a fluorescent detector. By using neonatal cardiomyocytes treated with CoCl2 to mimic hypoxia, we found PON2 deficiency dramatically increased ROS generation in the cardiomyocytes upon CoCl2 treatment. In response to a short CoCl2 exposure, cell viability and succinate dehydrogenase (SDH) activity assessed by MTT assay were significantly diminished in PON2-def cardiomyocytes compared to those in WT cardiomyocytes. PON2-def cardiomyocytes also had lower baseline SDH activity. By using adult mouse cardiomyocytes and mitochondrial ToxGlo assay, we found impaired cellular ATP generation in PON2-def cells compared to that in WT cells, suggesting that PON2 is necessary for proper mitochondrial function. CONCLUSION: Our study suggests a cardioprotective role for PON2 in both experimental and humanheart failure, which may be associated with the ability of PON2 to improve mitochondrial function and diminish ROS generation.
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