34P Investigating sarcomeric changes in Cullin-3 knockout muscles.

Defective protein degradation is one of the primary contributors in the pathogenesis of neuromuscular diseases. Muscle cells rely on the ubiquitin-proteasome system for the degradation of most muscle proteins via the addition of the ubiquitin 'recognition' tag onto the cellular targets by E3-ligases such as Cullin-3 (Cul3). Our work has shown that loss of Cul3 in muscle cells in vitro impairs their differentiation, while its deletion in mouse skeletal muscles results in postnatal lethality marked with severe myopathy. Cul3 knockout (KO) muscles showed reduced fibers size and revealed positive Gomori Trichrome stain protein aggregates; resembling of nemaline bodies found in nemaline myopathy (NM) patients. Additionally, the proteome analysis revealed unexpected accumulation of non-muscle alpha-actinins 1 and 4 within the tissue. We further explored the nature of these protein aggregates in Cul3 KO diaphragms and tongues. However, immunofluorescence analyses of KO muscles showed no evidence of alpha-actinin containing aggregates. Analysis of electron microscopic images also revealed that aggregates in Cul3 KO muscles did not originate from expanded Z-discs or thin filament structures, typically associated with nemaline bodies. However, we did observe changes to the sarcomere organization. Specifically, significant increases in Z-disc widths and sarcomere lengths were found in KO muscles. In addition, Cul3 KO muscles showed deregulation of sarcomeric and intermediate filament protein levels, such as myosin, sarcomeric alpha-actinin, desmin or filamin C. Despite histological analyses of Cul3 KO muscles sharing similarities with features of NM patients features, the nature of these protein aggregates remains elusive. Nevertheless, Cul3 KO muscles show accumulation of non-muscle actinins, altered sarcomere structure, and deregulation of sarcomeric protein levels. [ABSTRACT FROM AUTHOR]