Abstract 18407: Nucleolar Stress is an Early Response to Stress in Cardiac Cells: Role of the Nucleolar Proteins Nucleostemin and Nucleophosmin
Introduction: Nucleolar integrity is essential to maintain ribosome biogenesis, control cell proliferation, and the cellular stress response. Nucleolar stress, characterized by loss of nucleolar integrity, has not been described in the cardiac context. Previously we demonstrated a role for the nucleolar protein nucleostemin (NS) in the cardiac response to pathological insult. NS is known to interact with nucleophosmin (NPM), a marker of nucleolar stress with cytoprotective properties. Doxorubicin (DOX) induces nucleolar stress in non cardiac cells. In this study, we show that nucleolar stress is an early event in doxorubicin mediated cardiotoxicity as measured by the dynamic behavior of NS and NPM in vitro and in vivo.
Methods: Neonatal rat cardiomyocytes (NRCM) and cardiac progenitor cells (CPC) were treated with 1μM DOX, 0.05 μg/ml Actinomycin D (ActD) or DMSO. NS and NPM were knocked down in CPC by specific shRNA expressing vectors. NPM function was ablated in NRCM by the inhibitor NSC348883. In vivo, 7 day old mice received a single dose of DOX 15 mg/kg or saline control and sacrificed 24h later. Confocal microscopy, WB, qRT-PCR and FACS were employed in this study to assess NS and NPM localization, protein and transcript levels, and cell death respectively.
Results: DOX and ActD induced NS and NPM delocalization into the nucleoplasm within 4h in NRCM and 6h in CPC. In NRCM, nucleolar disruption was associated with suppression of new 45S pre-rRNA synthesis. At 24h, NS protein levels decreased by 13% and 34% in NRCM and CPC respectively. Cell death significantly increased at 16h in NRCM and at 24h in CPC, suggesting that nucleolar stress precedes DOX induced cytotoxity. Moreover, downregulation of NS and NPM function significantly increased DOX-induced cell death both in NRCM and CPC. We obtained similar results in hearts from DOX treated mice, observing a 20% decrease in NS protein levels and 1.7 fold reduction in pre-rRNA 45S transcripts.
Conclusion: Our results are the first demonstration of nucleolar stress in cardiac cells as well as associating DOX toxicity with impairment of nucleolar function. Our findings point to preservation of nucleolar integrity as a new strategy for protection against DOX mediated cardiomyopathy and other cellular stresses.
- © 2010 by American Heart Association, Inc.