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

Myofibrillar myopathy hallmarks associated with ZAK deficiency

The ZAK gene encodes two functionally distinct kinases, ZAKα and ZAKβ. Homozygous loss of function mutations affecting both isoforms causes a congenital muscle disease. ZAKβ is the only isoform expressed in skeletal muscle and is activated by muscle contraction and cellular compression. The ZAKβ substrates in skeletal muscle or the mechanism whereby ZAKβ senses mechanical stress remains to be determined. To gain insights into the pathogenic mechanism, we exploited ZAK-deficient cell lines, zebrafish, mice and a human biopsy. ZAK-deficient mice and zebrafish show a mild phenotype. In mice, comparative histopathology data from regeneration, overloading, ageing and sex conditions indicate that while age and activity are drivers of the pathology, ZAKβ appears to have a marginal role in myoblast fusion in vitro or muscle regeneration in vivo. The presence of SYNPO2, BAG3 and Filamin C (FLNC) in a phosphoproteomics assay and extended analyses suggested a role for ZAKβ in the turnover of FLNC. Immunofluorescence analysis of muscle sections from mice and a human biopsy showed evidence of FLNC and BAG3 accumulations as well as other myofibrillar myopathy markers. Moreover, endogenous overloading of skeletal muscle exacerbated the presence of fibres with FLNC accumulations in mice, indicating that ZAKβ signalling is necessary for an adaptive turnover of FLNC that allows for the normal physiological response to sustained mechanical stress. We suggest that accumulation of mislocalized FLNC and BAG3 in highly immunoreactive fibres contributes to the pathogenic mechanism of ZAK deficiency

ZAK beta is activated by cellular compression and mediates contraction-induced MAP kinase signaling in skeletal muscle

Mechanical inputs give rise to p38 and JNK activation, which mediate adaptive physiological responses in various tissues. In skeletal muscle, contraction-induced p38 and JNK signaling ensure adaptation to exercise, muscle repair, and hypertrophy. However, the mechanisms by which muscle fibers sense mechanical load to activate this signaling have remained elusive. Here, we show that the upstream MAP3K ZAK beta is activated by cellular compression induced by osmotic shock and cyclic compression in vitro, and muscle contraction in vivo. This function relies on ZAKO's ability to recognize stress fibers in cells and Z-discs in muscle fibers when mechanically perturbed. Consequently, ZAK-deficient mice present with skeletal muscle defects characterized by fibers with centralized nuclei and progressive adaptation towards a slower myosin profile. Our results highlight how cells in general respond to mechanical compressive load and how mechanical forces generated during muscle contraction are translated into MAP kinase signaling.Peer reviewe