P.162Novel Kbtbd13R408C-knockin mouse model phenocopies NEM6 myopathy

Nemaline myopathy patients with mutations in KBTBD13 (NEM6) display a peculiar impaired muscle relaxation, which compromises contractility and daily-life activities. Recently, we established that the origin of the impaired relaxation kinetics is myogenic by measuring the relaxation rate using transcranial magnetic stimulation. The majority of NEM6 patients harbor the Dutch founder mutation (c.1222C>T, p.Arg408Cys). To study how KBTBD13R408C affects muscle structure and function, we engineered a Kbtbd13R408C-knockin mouse model. Kbtbd13R408C-knockin mice are born at mendelian ratios and have a normal life span. Histological evaluation revealed the presence of nemaline bodies - a hallmark feature of NEM – and hypertrophy of slow-twitch fibers, a hallmark feature of NEM6. Intact muscle mechanics assays in soleus muscle revealed impaired relaxation kinetics in Kbtbd13R408C-knockin mice compared to Kbtbd13R408C-wildtype littermates. Low-angle x-ray diffraction studies in intact soleus muscle revealed that the actin layer line 6 spacing is reduced in Kbtbd13R408C-knockin mice compared to wild-type littermates, which indicates that the left-handed pitch of the thin filament helix is shorter in Kbtbd13R408C-knockin mouse muscle. Furthermore, the spacing of the actin subunit repeat (27 Å) was reduced in Kbtbd13R408C-knockin mice; when converted to thin filament stiffness, this indicates that the thin filament is stiffer in Kbtbd13R408C-knockin mice. These findings are in line with data from low angle x-ray diffraction studies that we performed in muscle fibers isolated from biopsies of NEM6 patients harboring the KBTBD13R408C mutation. Modeling studies suggest that thin filament stiffening contributes to the impaired relaxation kinetics. Thus, the Kbtbd13R408C-knockin mouse model phenocopies NEM6 myopathy both structurally and functionally. Hence, this model enables us to further decipher the pathomechanism underlying impaired muscle relaxation in NEM6 and provides opportunities to test therapeutic strategies.