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Abstract：
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.
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.
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.
摘要：
阿米什人线虫肌病的致病机制和神经肌肉反射相关表型（例如伴有阵抗的震颤），以及其他隐性遗传性TNNT1肌病，还有待澄清。由突变体TNNT1基因编码的截短的肌钙蛋白T（ssTnT）慢骨骼肌同工型无法掺入肌丝，并在肌细胞中降解。与外用肌纤维相比，正常小鼠的纺锤体融合纤维含有显着水平的心脏TnT和低分子量的ssTnT剪接形式。ssTnT敲除小鼠的融合内纤维具有显著增加的心脏TnT。旋转杆和平衡木测试显示，ssTnT敲除小鼠的神经肌肉协调异常，对纺锤体敏化剂的反应迟钝，琥珀酰胆碱.杂合核袋纤维中ssTnT的丢失和心脏TnT的代偿性增加可能会增加肌丝Ca2敏感性和张力，削弱主轴功能，从而确定了开发靶向治疗的新机制。
TNNT1基因密码子Glu180的无义突变导致阿米什人线虫肌病（ANM），一种具有婴儿致命性的隐性遗传性疾病。TNNT1编码肌钙蛋白T(ssTnT)的慢骨骼肌同工型。截短的ssTnT不能整合到肌丝中并且在肌肉细胞中降解。ANM的症状包括肌肉无力，萎缩,挛缩和震颤伴随着阵阵风。ssTnT敲除（KO）小鼠模型概括了ANM的关键特征，例如脱脂时慢肌纤维的萎缩和增加的易疲劳性。然而,ANM的神经肌肉反射相关症状尚未得到解释.通过从ssTnT-KO和对照小鼠中分离肌肉纺锤体，旨在检查肌丝蛋白的组成，我们发现,与外用纤维相反，融合内纤维含有大量的心脏TnT和ssTnT的低分子量剪接形式。来自ssTnT-KO小鼠的融合内纤维具有显著增加的心脏TnT。旋转杆和平衡木测试显示ssTnT-KO小鼠的神经肌肉协调受损，指示主轴功能异常。与野生型对照不同,ssTnT-KO小鼠的束运行能力对纺锤体敏化剂有迟钝的反应，琥珀酰胆碱.免疫组织化学检测到核袋纤维中的ssTnT和心脏TnT，而在核链纤维中检测到快速骨骼肌TnT，心脏α-肌球蛋白存在于两个核袋纤维之一中。核袋纤维中ssTnT的丢失和心脏TnT的代偿性增加会增加肌丝Ca2敏感性和张力，从而影响纺锤体的活动。这种机制为ANM的病理生理学提供了解释，以及新的治疗靶点。
TNNT1基因密码子Glu180的无义突变导致阿米什人线虫肌病（ANM），一种具有婴儿致命性的隐性遗传性疾病。TNNT1编码肌钙蛋白T(ssTnT)的慢骨骼肌同工型。截短的ssTnT不能整合到肌丝中并且在肌肉细胞中降解。ANM的症状包括肌肉无力，萎缩,挛缩和震颤伴随着阵阵风。ssTnT敲除（KO）小鼠模型概括了ANM的关键特征，例如脱脂时慢肌纤维的萎缩和增加的易疲劳性。然而,ANM的神经肌肉反射相关症状尚未得到解释.通过从ssTnT-KO和对照小鼠中分离肌肉纺锤体，旨在检查肌丝蛋白的组成，我们发现,与外用纤维相反，融合内纤维含有大量的心脏TnT和ssTnT的低分子量剪接形式。来自ssTnT-KO小鼠的融合内纤维具有显著增加的心脏TnT。旋转杆和平衡木测试显示ssTnT-KO小鼠的神经肌肉协调受损，指示主轴功能异常。与野生型对照不同,ssTnT-KO小鼠的束运行能力对纺锤体敏化剂有迟钝的反应，琥珀酰胆碱.免疫组织化学检测到核袋纤维中的ssTnT和心脏TnT，而在核链纤维中检测到快速骨骼肌TnT，心脏α-肌球蛋白存在于两个核袋纤维之一中。核袋纤维中ssTnT的丢失和心脏TnT的代偿性增加会增加肌丝Ca2敏感性和张力，从而影响纺锤体的活动。这种机制为ANM的病理生理学提供了解释，以及新的治疗靶点。
