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(Circulation. 2001;103:1984.)
© 2001 American Heart Association, Inc.

Basic Science Reports

Serpin Protein CrmA Suppresses Hypoxia-Mediated Apoptosis of Ventricular Myocytes

Rhonna M. Gurevich, MSc; Kelly M. Regula, BSc; Lorrie A. Kirshenbaum, PhD

From the Institute of Cardiovascular Sciences, St Boniface General Hospital Research Centre, and the Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada.

Correspondence to Dr Lorrie A. Kirshenbaum, Institute of Cardiovascular Sciences, St Boniface General Hospital Research Centre, Room 3016, 351 Taché Avenue, Winnipeg, Manitoba, Canada, R2H 2A6. E-mail Lorrie{at}sbrc.umanitoba.ca

Background—In this study, we ascertain whether caspase 8 activation and mitochondrial defects underlie apoptosis of ventricular myocytes during hypoxia. As an approach to circumvent the potential shortcomings surrounding the limited permeability and short half-life of the synthetic peptide inhibitors designed to block caspase activation, we constructed a replication-defective adenovirus encoding the serpin caspase inhibitor protein CrmA to ensure efficient and continual inhibition of caspase 8 activity during chronic hypoxia.

Methods and Results—In contrast to normoxic cells, oxygen deprivation of postnatal ventricular myocytes for 24 hours resulted in a 9-fold increase (P<0.05) in apoptosis as determined by Hoechst 33258 staining and nucleosomal DNA laddering. Moreover, hypoxia provoked a 1.5-fold increase (P<0.01) in caspase 8–like activity. Furthermore, hypoxia provoked perturbations to mitochondria consistent with the mitochondrial death pathway, including permeability transition pore (PT) opening, loss of mitochondrial membrane potential (_m), and cytochrome c release. Importantly, CrmA suppressed caspase 8 activity, PT pore changes, loss of _m, and apoptosis but had no effect on hypoxia-mediated cytochrome c release. Furthermore, Bongkrekic acid, an inhibitor of PT pore, prevented hypoxia-induced PT pore changes, loss of _m, and apoptosis but had no effect on hypoxia-mediated cytochrome c release.

Conclusions—To our knowledge, we provide the first direct evidence for the operation of CrmA as an antiapoptotic factor in ventricular myocytes during prolonged durations of hypoxia. Furthermore, our data suggest that perturbations to mitochondria including PT pore changes and _m loss are caspase-regulated events that appear to be separable from cytochrome c release.

Key Words: myocytes • apoptosis • hypoxia • gene therapy

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