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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]

Loss of cullin-3 in skeletal muscle (Cul3-skmKO) leads to postnatal death in mice caused by respiratory distress. Muscles appeared underdeveloped, and muscle mass was reduced by approximately 50% compared to controls owing to reduced hypertrophy of developing muscles. Our histological analyses using Gomori trichrome stained diaphragms found aggregates in Cul3-skmKO that are reminiscent of nemaline bodies, characteristic for nemaline myopathy. We also performed proteome and enrichment analyses of diaphragm and tongue muscles. These results indicated a reduction of mitochondrial metabolism and energy generation as well as sarcomerogenesis in Cul3-skmKO, while proteins related to reactive oxygen species metabolic processes are deregulated. The latter finding suggests that Cul3-skmKO may undergo metabolic stress, leading to ROS production and induction of cell death. We used immune-blot analyses to study changes to mitochondrial and sarcomeric proteins, and markers for apoptosis and necrosis in Cul3-skmKO and control diaphragms. We also performed ultrastructural analyses using electron microscopy to identify the nature of the pathological protein aggregates, quantify mitochondrial content and size, and correlate changes to the biochemical analyses. Our ultrastructural analyses revealed unchanged mitochondrial content and sizes between Cul3-skmKO and controls. We observed electron dense material in Cul3-skmKO diaphragms that could stem from cells undergoing cell death, giving rise to Gomori trichrome positive aggregates. However, levels of apoptotic and necrotic marker proteins remained unchanged when normalized to sarcomeric myosin or decreased when normalized to β-actin. Biochemical analysis of electron transport chain and sarcomeric proteins confirmed data from our proteome analyses when normalized to β-actin. Conversely, when normalized to sarcomeric myosin, no differences could be identified. This suggests a decreased contribution of muscle tissues to the total tissue sample analysed by immunoblots in skmKO, perhaps reflective of the earlier identified loss in overall muscle mass. Further studies are required to identify if metabolic stress leading to production of reactive oxygen species may be involved the origin and development of the observed cell death in Cul3-skmKO muscles. [ABSTRACT FROM AUTHOR]