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INNOVATIVE METHODOLOGY

In situ dynamically monitoring the proteolytic function of the ubiquitin-proteasome system in cultured cardiac myocytes

¹Cardiovascular Research Institute and ⁶Department of Laboratory Medicine and Pathology, University of South Dakota; ⁵Cardiovascular Research Institute, South Dakota Health Research Foundation, Sioux Falls, South Dakota 57105; ²Department of Pathophysiology, Guangzhou Medical College, Guangzhou, Guangdong; ³Department of Pathophysiology, Wuhan University, Wuhan, Hubei; and ⁴Cardiovascular Research Institute, Nanjing Medical University, Nanjing, Jiangsu, China

Submitted 23 December 2003 ; accepted in final form 9 April 2004

The ubiquitin-proteasome system (UPS) is responsible for turnover of most cellular proteins in eukaryotes. Protein degradation by the UPS serves quality control and regulatory functions. Proteasome inhibition showed great promise in effectively treating cancer and restenosis. UPS dysfunction in cardiac hypertrophy and failure has recently been suspected but remains to be investigated. A system capable of monitoring dynamic changes in proteolytic function of the UPS in cardiac myocytes in situ would no doubt benefit significantly efforts to decipher the pathogenic significance of UPS dysfunction in the heart and to evaluate the effect of proteasome inhibition on cardiac myocytes. We successfully established such a system in cultured cardiac myocytes by delivering and expressing a modified green fluorescence protein (GFPu) gene using recombinant adenoviruses. GFPu contains a ubiquitination signal sequence fused to the COOH terminus. Fluorescence microscopy and Western blots revealed that protein abundance of modified green fluorescent protein (GFPu), but not wild-type green fluorescent protein, in cultured cardiac myocytes was incrementally increased when function of the proteasomes was inhibited in various degrees by specific inhibitors. The increase in GFPu protein levels and fluorescence intensity is paralleled by a decrease in the in vitro peptidase activity of the proteasomes. Our results demonstrate that GFPu can be used as a surrogate marker to monitor dynamic changes in proteolytic function of the UPS in cardiac myocytes in situ. Application of this novel system reveals that moderate levels of H₂O₂, a reactive oxygen species generator, impair proteolytic function of the UPS in cultured cardiac myocytes.

protein degradation; proteasome inhibition; adenoviruses; tissue culture; rat

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