Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex
Hui-Hua Li, … , Da-Zhi Wang, Cam Patterson
Hui-Hua Li, … , Da-Zhi Wang, Cam Patterson
Published October 15, 2004
Citation Information: J Clin Invest. 2004;114(8):1058-1071. https://doi.org/10.1172/JCI22220.
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Article Cardiology

Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex

Abstract

Calcineurin, which binds to the Z-disc in cardiomyocytes via α-actinin, promotes cardiac hypertrophy in response to numerous pathologic stimuli. However, the endogenous mechanisms regulating calcineurin activity in cardiac muscle are not well understood. We demonstrate that a muscle-specific F-box protein called atrogin-1, or muscle atrophy F-box, directly interacts with calcineurin A and α-actinin-2 at the Z-disc of cardiomyocytes. Atrogin-1 associates with Skp1, Cul1, and Roc1 to assemble an SCFatrogin-1 complex with ubiquitin ligase activity. Expression of atrogin-1 decreases levels of calcineurin A and promotes its ubiquitination. Moreover, atrogin-1 attenuates agonist-induced calcineurin activity and represses calcineurin-dependent transactivation and NFATc4 translocation. Conversely, downregulation of atrogin-1 using adenoviral small interfering RNA (siRNA) expression enhances agonist-induced calcineurin activity and cardiomyocyte hypertrophy. Consistent with these cellular observations, overexpression of atrogin-1 in hearts of transgenic mice reduces calcineurin protein levels and blunts cardiac hypertrophy after banding of the thoracic aorta. These studies indicate that the SCFatrogin-1 ubiquitin ligase complex interacts with and represses calcineurin by targeting calcineurin for ubiquitin-mediated proteolysis, leading to inhibition of cardiac hypertrophy in response to pathologic stimuli.

Authors

Hui-Hua Li, Vishram Kedar, Chunlian Zhang, Holly McDonough, Ranjana Arya, Da-Zhi Wang, Cam Patterson

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Figure 1

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Molecular interaction of atrogin-1 with α-actinin-2 and calcineurin A (C...
Molecular interaction of atrogin-1 with α-actinin-2 and calcineurin A (CnA). (A) Yeast 2-hybrid analysis of atrogin-1 interaction with α-actinin-2 (left) and calcineurin A (right). pGBKT7 and pACT2 are empty plasmids. pGAD-T7 vector expresses a T antigen–GAL4 activation domain fusion that interacts with p53 (positive control reaction). (B) In vitro interactions of atrogin-1 with α-actinin-2 and calcineurin A in GST pull-down assays. The ability of α-actinin-2 (top) or calcineurin A (bottom) expressed in COS-7 cells to be retained by GST or a GST–atrogin-1 fusion protein was analyzed by immunoblotting after binding reactions. (C) COS-7 cells were transfected with Xpress-tagged (Xp) atrogin-1 and GFP-tagged calcineurin A or HA-tagged α-actinin-2 expression plasmids as indicated. Equal amounts of cell extract were immunoprecipitated with the Xpress antibody and analyzed by immunoblotting with antibodies directed against Xpress (to detect atrogin-1) and HA (to detect α-actinin-2, top) or against GFP (to detect calcineurin A, bottom). WCE, whole cell extract. (D) Endogenous protein interactions were examined in cardiomyocyte cell lysates that were immunoprecipitated with preimmune serum or anti–atrogin-1 antibody and analyzed by immunoblotting with antibodies to detect α-actinin (top), calcineurin A (middle), and atrogin-1 (bottom). (E) Direct protein interactions were detected by incubation of GST–atrogin-1 or GST (1 μg) with 1 μg of recombinant calcineurin A or α-actinin-2 proteins. Mixtures were precipitated with anti-GST antibody and analyzed by immunoblotting with antibodies against α-actinin, calcineurin A, and GST. (F) Coimmunostaining analysis of endogenous atrogin-1, calcineurin A, and α-actinin-2 in neonatal cardiomyocytes. The overlay shows that atrogin-1 colocalizes with both α-actinin (top) and calcineurin A (middle).