© 1999 American Heart Association, Inc.
Cellular Biology |
Regulation of Bcl-2 Family Proteins During Development and in Response to Oxidative Stress in Cardiac Myocytes
Association With Changes in Mitochondrial Membrane Potential
From the National Heart and Lung Institute, Cardiac Medicine Section (S.A.C., P.H.S.), and the Division of Biomedical Sciences, Molecular Pathology Section (A.C.), Imperial College School of Medicine, London, UK.
Correspondence to Angela Clerk, PhD, Division of Biomedical Sciences, Molecular Pathology Section, Imperial College School of Medicine, Sir Alexander Fleming Building, South Kensington, London SW7 2AZ, United Kingdom. E-mail s.a.cook{at}ic.ac.uk
Abstract—Cardiac myocyte
apoptosis is potentially important in many cardiac disorders.
In other cells, Bcl-2 family proteins and mitochondrial dysfunction are
probably key regulators of the apoptotic response. In the
present study, we characterized the regulation of
antiapoptotic (Bcl-2, Bcl-xL) and proapoptotic (Bad,
Bax) Bcl-2 family proteins in the rat heart during development and in
oxidative stress–induced apoptosis. Bcl-2 and Bcl-xL were
expressed at high levels in the neonate, and their expression was
sustained during development. In contrast, although Bad and Bax were
present at high levels in neonatal hearts, they were barely
detectable in adult hearts. We confirmed that
H2O2 induced cardiac myocyte cell death,
stimulating poly(ADP-ribose) polymerase proteolysis (from 2 hours),
caspase-3 proteolysis (from 2 hours), and DNA fragmentation (from 8
hours). In unstimulated neonatal cardiac myocytes, Bcl-2 and Bcl-xL
were associated with the mitochondria, but Bad and Bax were
predominantly present in a crude cytosolic fraction. Exposure of
myocytes to H2O2 stimulated rapid translocation
of Bad (<5 minutes) to the mitochondria. This was followed by the
subsequent degradation of Bad and Bcl-2 (from 
30 minutes). The
levels of the mitochondrial membrane marker cytochrome oxidase remained
unchanged. H2O2 also induced translocation of
cytochrome c from the mitochondria to the cytosol within
15 to 30 minutes, which was indicative of mitochondrial dysfunction.
Myocytes exposed to H2O2 showed an early loss
of mitochondrial membrane potential (assessed by
fluorescence-activated cell sorter
analysis) from 15 to 30 minutes, which was partially restored
by 1 hour. However, a subsequent irreversible loss of mitochondrial
membrane potential occurred that correlated with cell death. These data
suggest that the regulation of Bcl-2 and mitochondrial function are
important factors in oxidative stress–induced cardiac myocyte
apoptosis.
Key Words: myocyte • mitochondrial membrane potential • apoptosis • oxidative stress • Bcl-2
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