The loss of slow skeletal muscle isoform of troponin T in spindle intrafusal fibers explains the pathophysiology of Amish nemaline myopathy

KEY POINTS: The pathogenic mechanism and the neuromuscular reflex-related phenotype (e.g. tremors accompanied by clonus) of Amish nemaline myopathy, as well as of other recessively inherited TNNT1 myopathies, remain to be clarified. The truncated slow skeletal muscle isoform of troponin T (ssTnT) encoded by the mutant TNNT1 gene is unable to incorporate into myofilaments and is degraded in muscle cells. By contrast to extrafusal muscle fibres, spindle intrafusal fibres of normal mice contain a significant level of cardiac TnT and a low molecular weight splice form of ssTnT. Intrafusal fibres of ssTnT-knockout mice have significantly increased cardiac TnT. Rotarod and balance beam tests have revealed abnormal neuromuscular co-ordination in ssTnT-knockout mice and a blunted response to a spindle sensitizer, succinylcholine. The loss of ssTnT and a compensatory increase of cardiac TnT in intrafusal nuclear bag fibres may increase myofilament Ca2+ -sensitivity and tension, impairing spindle function, thus identifying a novel mechanism for the development of targeted treatment. ABSTRACT: A nonsense mutation at codon Glu180 of TNNT1 gene causes Amish nemaline myopathy (ANM), a recessively inherited disease with infantile lethality. TNNT1 encodes the slow skeletal muscle isoform of troponin T (ssTnT). The truncated ssTnT is unable to incorporate into myofilament and is degraded in muscle cells. The symptoms of ANM include muscle weakness, atrophy, contracture and tremors accompanied by clonus. An ssTnT-knockout (KO) mouse model recapitulates key features of ANM such as atrophy of extrafusal slow muscle fibres and increased fatigability. However, the neuromuscular reflex-related symptoms of ANM have not been explained. By isolating muscle spindles from ssTnT-KO and control mice aiming to examine the composition of myofilament proteins, we found that, in contrast to extrafusal fibres, intrafusal fibres contain a significant level of cardiac TnT and the low molecular weight splice form of ssTnT. Intrafusal fibres from ssTnT-KO mice have significantly increased cardiac TnT. Rotarod and balance beam tests revealed impaired neuromuscular co-ordination in ssTnT-KO mice, indicating abnormality in spindle functions. Unlike the wild-type control, the beam running ability of ssTnT-KO mice had a blunted response to a spindle sensitizer, succinylcholine. Immunohistochemistry detected ssTnT and cardiac TnT in nuclear bag fibres, whereas fast skeletal muscle TnT was detected in nuclear chain fibres, and cardiac α-myosin was present in one of the two nuclear bag fibres. The loss of ssTnT and a compensatory increase of cardiac TnT in nuclear bag fibres would increase myofilament Ca2+ -sensitivity and tension, thus affecting spindle activities. This mechanism provides an explanation for the pathophysiology of ANM, as well as a novel target for treatment.