FP.02 Mutation in KBTBD13 causes stiffening of thin filaments in skeletal muscle

Abstract

Nemaline myopathy is caused by mutations in genes encoding thin filament proteins. Nemaline myopathy-type 6 (NEM6) is caused by mutations in <i>KBTBD13</i>. NEM6 patients display muscle weakness and slow kinetics of muscle relaxation. Whether KBTBD13 plays a role in thin filament structure/function, and whether such role can explain the NEM6 phenotype, is incompletely understood. Therefore, we developed a KBTBD13R408C–knockin (KI) mouse model. In line with the patients' phenotype, KI mice exhibit nemaline rods and slow relaxation kinetics. We used low-angle x-ray diffraction to study whether structural changes in the myofilaments can explain the contractile phenotype of KI mice. Intact soleus and extensor digitorum longus muscles (EDL) were dissected from wt and KI mice. Low angle x-ray diffraction experiments were conducted at the BioCAT beamline, Argonne National Laboratories. Muscles were mounted between a force transducer and a fixed hook, exposed to the x-ray beam during relaxed and maximal tetanic conditions. Images were recorded with 15ms acquisition time using a Pilatus 3xM1 detector set at 1.26 meter distance from the muscle. Results show that in relaxed soleus muscles the actin monomer spacing (27A) and myosin backbone spacing (28A) are shorter in KI compared to wt mice (2.7329 vs 2.7343nm; 2.8764nm vs 2.8797nm). Upon maximal tetanic activation, which was comparable between KI and wt muscles, the 27A spacing increased less in KI compared to wt muscles, indicating increased stiffness. Upon maximal tetanic activation, the 28A spacing increased in wt muscles, but this increase was comparable to that in KI mice. EDL muscle of KI mice showed no differences in actin monomer or myosin backbone spacings. Results indicate that thin filament stiffness, but not thick filament stiffness, is increased in KI mice, and that this increase is muscle-type specific. Sarcomere modeling shows that the increased stiffness of thin filaments contributes to slow relaxation kinetics.