Understanding the Role of ZAKβ in the Maintenance and Regulation of Skeletal Muscle Function

Leucine-zipper and sterile-alpha-motif kinase β (ZAKβ) is a MAPKKK and novel skeletal muscle protein first identified as part of a protein complex found at the z-disc. Loss of ZAKβ has been implicated in a muscle myopathy, whereby human patients exhibit skeletal muscle weakness and fibre atrophy, suggesting a pivotal role in muscle maintenance. To elucidate its function, a mouse ZAK-/- line has been characterised in detail in combination with proteomics and in vivo modelling. Visibly, ZAK-/- mice show a much milder phenotype compared to the human condition. In ZAK-/- mice, histopathological data from muscle regeneration, ageing, sex, and overload conditions reveals both age and activity as drivers of this condition. ZAK-/- mice exhibit an increase in the percentage of slow fibres and centralised nuclei in tonically active muscles, consistent with similar observations in patients. We also observe an accumulation of the large actin cross-linking protein FLNC and the Chaperone Assisted Selective Autophagy (CASA)-associated protein BAG3, suggesting inefficient protein turnover. Overexpression of ZAKβ in adult muscle fibres is sufficient to induce fibre growth. We performed a phosphoproteomics assay to identify putative substrates of ZAKβ kinase activity. This assay identified a significant number of proteins associated with adhesion and cytoskeletal organisation at the z-disc and costamere. The presence of both SYNPO2 and FLNC in this screen suggested a role for ZAKβ in the protein turnover mechanism of cytoskeletal adhesion factors. Mechanical overloading of hind limb skeletal muscle was associated with the exacerbation of FLNC aggregates suggesting that ZAKβ signalling is necessary for FLNC turnover throughout the physiological response to increased mechanical stress. We suggest a role for ZAKβ in the regulation of z-disc and costameric protein complexes, integral for the mediation of the hypertrophic response following mechanical stress, and that mislocalisation of FLNC and BAG3 underlies the pathogenic mechanisms of the ZAK-deficiency.