Abstract
Oxidative stress promotes cardiac myocyte apoptosis through the mitochondrial death pathway. Since Bcl-2 family proteins are key regulators of apoptosis, we examined the effects of H2O2 on the expression of principal Bcl-2 family proteins (Bcl-2, Bcl-xL, Bax, Bad) in neonatal rat cardiac myocytes. Protein expression was assessed by immunoblotting. Bcl-2, Bax, and Bad were all down-regulated in myocytes exposed to 0.2 mm H2O2, a concentration that induces apoptosis. In contrast, although Bcl-xL levels initially declined, the protein was re-expressed from 4-6 h. Bcl-xL mRNA was up-regulated from 2 to 4 h in neonatal rat or mouse cardiac myocytes exposed to H2O2, consistent with the re-expression of protein. Four different untranslated first exons have been identified for the Bcl-x gene (exons 1, 1B, 1C, and 1D, where exon 1 is the most proximal and exon 1D the most distal to the coding region). All were detected in mouse or rat neonatal cardiac myocytes, but exon 1D was not expressed in adult mouse hearts. In neonatal mouse or rat cardiac myocytes, H2O2 induced the expression of exons 1B, 1C, and 1D, but not exon 1. These data demonstrate that the Bcl-x gene is selectively responsive to oxidative stress, and the response is mediated through distal promoter regions.
Highlights
Cardiac myocytes, the contractile cells of the heart, are terminally differentiated cells, which withdraw from the cell cycle in the perinatal period
H2O2 Promotes Down-regulation of Bcl-2 Family Proteins in Cardiac Myocytes—We examined the effects of different concentrations of H2O2 (2, 0.2, and 0.02 mM) on the expression of four principal Bcl-2 family proteins, which are expressed in cardiac myocytes: Bcl-2, Bcl-xL, Bax, and Bad. 0.2 mM H2O2 results in myocyte death associated with features of apoptosis [4], whereas higher concentrations induce non-apoptotic cell death. 0.02 mM H2O2 is non-toxic, but has effects on gene expression
The levels of Bax and Bad were decreased in myocytes exposed to 0.2 mM H2O2 (Fig. 2, E and F), albeit to a lesser extent than in myocytes exposed to 2 mM H2O2 (Fig. 2, A and B)
Summary
The contractile cells of the heart, are terminally differentiated cells, which withdraw from the cell cycle in the perinatal period. Bcl-xL mRNA was up-regulated from 2 to 4 h in neonatal rat or mouse cardiac myocytes exposed to H2O2, consistent with the re-expression of protein.
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