Hyperglycemia-Induced Apoptosis in Mouse Myocardium

Mitochondrial Cytochrome c–Mediated Caspase-3 Activation Pathway

  1. Lu Cai1,
  2. Wei Li4,
  3. Guangwu Wang1,
  4. Luping Guo1,
  5. Youchun Jiang1 and
  6. Y. James Kang123
  1. 1Department of Medicine, University of Louisville, Louisville, Kentucky
  2. 2Department of Pharmacology and Toxicology, University of Louisville, Louisville, Kentucky
  3. 3Jewish Hospital Heart and Lung Institute, Louisville, Kentucky
  4. 4Department of Hemapoietic Disorders, the First Teaching Hospital, Norman Bethune University of Medical Sciences, Changchun, People’s Republic of China

    Abstract

    Diabetic cardiomyopathy is related directly to hyperglycemia. Cell death such as apoptosis plays a critical role in cardiac pathogenesis. Whether hyperglycemia induces myocardial apoptosis, leading to diabetic cardiomyopathy, remains unclear. We tested the hypothesis that apoptotic cell death occurs in the diabetic myocardium through mitochondrial cytochrome c–mediated caspase-3 activation pathway. Diabetic mice produced by streptozotocin and H9c2 cardiac myoblast cells exposed to high levels of glucose were used. In the hearts of diabetic mice, apoptotic cell death occurred as detected by terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) assay. Correspondingly, caspase-3 activation as determined by enzymatic assay and mitochondrial cytochrome c release detected by Western blotting analysis were observed. Supplementation of insulin inhibited diabetes-induced myocardial apoptosis as well as suppressed hyperglycemia. To explore whether apoptosis in diabetic hearts is related directly to hyperglycemia, we exposed cardiac myoblast H9c2 cells to high levels of glucose (22 and 33 mmol/l) in cultures. Apoptotic cell death was detected by TUNEL assay and DAPI nuclear staining. Caspase-3 activation with a concomitant mitochondrial cytochrome c release was also observed. Apoptosis or activation of caspase-3 was not observed in the cultures exposed to the same concentrations of mannitol. Inhibition of caspase-3 with a specific inhibitor, Ac-DEVD-cmk, suppressed apoptosis induced by high levels of glucose. In addition, reactive oxygen species (ROS) generation was detected in the cells exposed to high levels of glucose. These results suggest that hyperglycemia directly induces apoptotic cell death in the myocardium in vivo. Hyperglycemia-induced myocardial apoptosis is mediated, at least in part, by activation of the cytochrome c–activated caspase-3 pathway, which may be triggered by ROS derived from high levels of glucose.

    Footnotes

    • Address correspondence and reprint requests to Dr. Lu Cai or Dr. Y. James Kang, Department of Medicine, University of Louisville, 511 S. Floyd St., MDR 535, E-mail: lcai1{at}hotmail.com

      Received for publication 2 May 2001 and accepted in revised form 26 February 2002.

      DAB, 3,3′ -diaminobenzidine; DCFDA, carboxy-dichlorodihydrofluorescein diacetate; DMEM, Dulbecco’s modified Eagle’s medium; DTT, dithiothreitol; FBS, fetal bovine serum; HRP, horseradish peroxidase; MTT, microculture tetrazolium; PMSF, phenylmethylsulfonyl fluoride; ROS, reactive oxygen species; STZ, streptozotocin; TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling.

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