P491Loss of the ubiquitin ligase Atrogin1/MAFbx (Muscle atrophy F-box) causes age-related cardiomyopathy and premature death

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Abstract

Purpose

The maintenance of cardiomyocyte (CM) size and performance is ensured by the balance between protein synthesis and degradation. Atrogin-1/MAFbx is a E3 ubiquitin-ligase specifically expressed by muscle cells. The role of MAFbx in the heart is debated. Several papers demonstrated that MAFbx mediates regression of cardiac hypertrophy by calcineurin degradation. However, a recent study suggests that inhibition of MAFbx prevents hypertrophy induced by pressure overload. The aim of our study is to address the role of MAFbx in CM homeostasis using the experimental model of MAFbx KO mice.

Methods

Hearts from six and sixteen month (mo.) old MAFbx KO mice were compared with age- and sex-matched controls. Echocardiography was performed to assess heart function. Cryosections were analyzed by immunofluorescence and electron microscopy (EM). Apoptosis was evaluated by TUNEL stain. Quantitative RT-PCR and WB were used to investigate markers of endoplasmic/sarcoplasmic reticulum (ER) stress (CHOP, Atf4, GADD34), protein synthesis and autophagy (LC3, p62).

Results

MAFbx KO mice have shorter life span than controls. Six mo. MAFbx KO mice show no alterations in cardiac morphology and histology, as compared to controls. However, sixteen mo. MAFbx KO hearts appear abnormal in shape with grossly dilated left atria, rounded ventricles, increased sphericity index and increased heart/body weight ratio. During aging MAFbx KO hearts develop left ventricle (LV) cardiac fibrosis which results in impaired diastolic relaxation. Aged MAFbx KO hearts show RV hypertrophy (CM area, KO:279.85 ± 1.52 vs C:230.82 ± 1.5, in µm2), increased capillary/CM ratio and fetal gene reactivation. TUNEL stain analysis demonstrates increased apoptosis in the LV of aged MAFbx KO mice. EM analysis shows mitochondrial alterations and dilated ER/SR cisternae in aged MAFbx KO LV CMs. Up-regulation of markers of ER stress, impairment of the autophagic flux and activation of the Unfolded Protein Response (UPR) are detected in aged MAFbx KO hearts. Aged MAFbx KO hearts also show accumulation of several critical components of autophagy and of signaling pathways controlling protein homeostasis.

Conclusions

MAFbx KO mice develop an age-dependent restrictive cardiomyopathy. MAFbx is required for normal autophagy and its absence triggers a defect in autophagy flux with activation of UPR that causes apoptotic cell death and fibrosis.

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