Abstract
Cardiac hypertrophy is a complex process involving the coordinated actions of many genes. In a high throughput screen designed to identify transcripts that are actively translated during cardiac hypertrophy, we identified a number of genes with established links to hypertrophy, including those coding for Sp3, c-Jun, annexin II, cathepsin B, and HB-EGF, thus showing the general utility of the screen. Focusing on a candidate transcript that has not been previously linked to hypertrophy, we found that protein levels of the tumor suppressor PTEN (phosphatase and tensin homologue on chromosome ten) were increased in the absence of increased messenger RNA levels. Increased PTEN expression by recombinant adenovirus in cultured neonatal rat primary cardiomyocytes caused cardiomyocyte apoptosis as evidenced by increased caspase-3 activity and cleaved poly(A)DP-ribose polymerase. Expression of PTEN was also able to block growth factor signaling through the phosphatidylinositol 3,4,5-triphosphate pathway. Surprisingly, expression of a catalytically inactive PTEN mutant led to cardiomyocyte hypertrophy, with increased protein synthesis, cell surface area, and atrial natriuretic factor expression. This hypertrophy was accompanied by an increase in Akt activity and improved cell viability in culture.
Highlights
Cardiac hypertrophy is an adaptive response of the heart to both intrinsic and extrinsic stimuli
In a high throughput screen designed to identify transcripts that are actively translated during cardiac hypertrophy, we identified a number of genes with established links to hypertrophy, including those coding for Sp3, c-Jun, annexin II, cathepsin B, and HB-EGF, showing the general utility of the screen
To determine if any molecular markers of hypertrophy were activated, we examined whether AdH123YPTEN-infected cells express atrial natriuretic factor (ANF), a readily identified marker of cardiomyocyte hypertrophy and stress, which accumulates in a perinuclear ring
Summary
Reagents—All cell culture media were from Life Technologies, Inc. unless otherwise stated. PTEN Phosphatase Assays—Duplicate 100-mm plates seeded with 1.96 ϫ 106 cardiomyocytes were either mock infected or infected with AdlacZ, AdWTPTEN, or AdH123YPTEN at an m.o.i. ϭ 10 ifu/cell. Twenty-four h following infection (or mock treatment) cardiomyocytes transduced with AdlacZ, AdWTPTEN, or AdH123YPTEN were incubated for 6 h in leucine-free RPMI 1640 supplemented with 5. Akt Assay—Duplicate 100-mm plates seeded with 1.96 ϫ 106 cardiomyocytes were either mock infected or infected with AdlacZ, AdWTPTEN, or AdH123YPTEN at an m.o.i. ϭ 10 infectious units/cell. For a positive control mock infected cells were treated with 10 nm of IGF-1 for 15 min prior to harvest. Cells were mock infected or infected with AdlacZ, AdWTPTEN, or AdH123YPTEN at an m.o.i. of 10 ifu/cell for 1 h at 37 °C, allowed to recover for 12 h in 1:1, placed into 100 l of DMEM/F-12 without phenol red. All samples were done in triplicate, and data were analyzed by Student’s t test
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