Purpose: Control of cardiomyocyte (CM) proteostasis operated by the ubiquitin/proteasome (UPS) and autophagy/lysosome systems is fundamental for heart adaptation to both physiologic and pathologic stresses. Reduced efficiency of either UPS or autophagy/lysosome system occurs during ageing and has been associated to cardiomyopathies. Atrogin-1 is a muscle specific ubiquitin ligase, targeting for degradation signalling proteins involved in cardiac hypertrophy. However, the role of Atrogin-1 in CM biology and its involvement in the molecular mechanism of cardiac dysfunction, are largely unexplored. Methods: We analyzed hearts from Atrogin-1 knock-out (KO) mice from 6 mo. onwards until death, and compared it to that of age- and sex-matched controls. Functional, immunofluorescence and electron microscopy analyses were performed. Markers of ER/SR stress and autophagy/lysosome systems were investigated by RTqPCR and WB. In vivo pulsed SILAC proteomics and bioinformatics, Co-IP, in vitro assays and in vivo viral silencing were performed to identify novel targets of Atrogin-1. Results: By using in vivo and in vitro assays we identified a novel target of Atrogin-1, the ESCRTIII protein CHMP2B, that plays a fundamental role in autophagy. Failure to degrade CHMP2B in Atrogin-1 KO mice caused autophagy impairment, accumulation of intracellular protein aggregates, activation of the unfolded protein response and subsequent CM apoptosis, all of which increased progressively during ageing. The alterations in cellular proteostasis resulted in cardiomyopathy with a restrictive pattern, characterized by myocardial remodelling with interstitial fibrosis, diastolic dysfunction (Edt, KO: 20.7±2.7 vs WT: 27.8±5.7, in msec), arrhythmias and secondary LA and ventricular remodelling, as well as CM hypertrophy (CM area, KO: 289.34±2.23 vs WT: 236.77±1.64, in μm2). Aged Atrogin-1 KO mice had reduced tolerance to treadmill exercise compared to controls, and shortened life span (KO: 17±1 vs WT: 24±1.2, in mo.). In vivo reduction of CHMP2B protein level in the KO mice restored normal autophagy and protected CMs from cell death resulting from CHMP2B proteotoxicity. Conclusions: Our data highlight the importance of regulated proteolysis in the heart and show that the loss of Atrogin-1 per se is sufficient to cause cardiac damage, which evolves into cardiomyopathy when protein quality control becomes less efficient, as occurring in aging. Such cardiomyopathy represents a novel model of proteotoxic myocardial remodelling and will be useful to determine the mechanism of impaired proteostasis to CM damage.

Autophagy is crucial in the turnover of cell components both in constitutive conditions and in response to starvation. Defects of this degradative system play a role in various diseases, but little is known about autophagy in muscular dystrophies. Mutations in any of the three genes coding for collagen VI cause several muscle diseases in humans, including Ullrich congenital muscular dystrophy (UCMD), Bethlem myopathy (BM) and Congenital myosclerosis. Collagen VI null (Col6a1−/−) mice display an early onset myopathic phenotype characterized by organelle defects, mitochondrial dysfunction and spontaneous apoptosis, leading to myofiber degeneration. We found that persistence of abnormal organelles and apoptosis are caused by defective autophagy. Indeed, skeletal muscles of Col6a1−/− mice displayed an impairment of autophagic flux, which matched the lower induction of Beclin 1 and Bnip3 and the lack of autophagosomes after starvation. Notably, reactivation of autophagy by genetic, dietary and pharmacological approaches restored myofiber survival and ameliorated the dystrophic phenotype of Col6a1−/− mice. Furthermore, muscle biopsies from patients affected by UCMD and BM showed reduced levels of Beclin 1 and Bnip3. These findings indicate that defective activation of the autophagic machinery plays a pathogenic role in congenital muscular dystrophies.