Deficiency of slow skeletal muscle troponin T causes atrophy of type I slow fibres and decreases tolerance to fatigue

Abstract

Key pointsr Loss of slow skeletal muscle troponin T (ssTnT) due to a nonsense mutation in codon Glu 180 causes a lethal form of recessively inherited nemaline myopathy.r We studied the phenotypes of partial and total loss of ssTnT in the diaphragm and soleus muscles of Tnnt1 gene targeted mice.r ssTnT deficiency resulted in significant decreases in other slow fibre-specific myofilament proteins whereas fast fibre-specific myofilament proteins were increased correspondingly.r ssTnT deficiency produced significantly smaller type I slow fibres and compensatory growth of type II fast fibres with significantly reduced contractile force and tolerance to fatigue.r ssTnT-deficient soleus muscle exhibited a significant numbers of small-sized central nuclei type I fibres, indicating regeneration.r The data support the critical role of ssTnT in skeletal muscle function and the causal effect of its loss on the pathogenesis of nemaline myopathy. AbstractThe total loss of slow skeletal muscle troponin T (ssTnT encoded by TNNT1 gene) due to a nonsense mutation in codon Glu 180 causes a lethal form of recessively inherited nemaline myopathy (Amish nemaline myopathy, ANM). To investigate the pathogenesis and muscle pathophysiology of ANM, we studied the phenotypes of partial and total loss of ssTnT in Tnnt1 gene targeted mice. An insertion of neomycin resistance cassette in intron 10 of Tnnt1 gene caused an approximately 60% decrease in ssTnT protein expression whereas cre-loxP-mediated deletion of exons 11-13 resulted in total loss of ssTnT, as seen in ANM muscles. In diaphragm and soleus muscles of the knockdown and knockout mouse models, we demonstrated that ssTnT deficiency resulted in significantly decreased levels of other slow fibre-specific myofilament proteins whereas fast fibre-specific myofilament proteins were increased correspondingly. Immunohistochemical studies revealed that ssTnT deficiency produced significantly smaller type I slow fibres and compensatory growth of type II fast fibres. Along with the slow fibre atrophy and the changes in myofilament protein isoform contents, ssTnT deficiency significantly reduced the tolerance to fatigue in soleus muscle. ssTnT-deficient soleus muscle also contains significant numbers of small-sized central nuclei type I fibres, indicating active regeneration. The data provide strong support for the essential role of ssTnT in skeletal muscle function and the causal effect of its loss in the pathology of ANM. This observation further supports the hypothesis that the function of slow fibres can be restored in ANM patients if a therapeutic supplement of ssTnT is achieved.

Carbonic Anhydrase III Is Expressed in Mouse Skeletal Muscles Independent of Fiber Type-Specific Myofilament Protein Isoforms and Plays a Role in Fatigue Resistance

“…The development of Tnnt1 -KO mice in C57BL/6 strain was reported previously (Wei et al, 2014). The genotypes of the mice were verified by PCR.…”

“…The expression of CAIII was examined in soleus muscle of WT and Tnnt1 -KO myopathy mice (Johnston et al, 2000; Jin et al, 2003; Wei et al, 2014). Western blot and glycerol-SDS-gel demonstrate that the loss of slow TnT in Tnnt1 -KO mice caused a drastic loss of slow type I fibers in soleus muscle indicated by the diminished levels of slow TnI as compared to WT controls (Figures 11A,C), reflecting a switch to more fast fiber content.…”

“…Mimicking a recessively inherited lethal disease originally found in the Amish caused by a nonsense mutation at codon Glu 180 (Johnston et al, 2000; Jin et al, 2003; Wei et al, 2014), the loss of slow TnT in Tnnt1 -KO mice caused a drastic loss of type I slow fiber contents and a switch to more fast fiber contents in soleus muscle with significantly decreased resistance to fatigue (Wei et al, 2014). The maintained level of CAIII in Tnnt1 -KO soleus muscle not only demonstrates that the expression of CAIII is not restricted to slow fibers in the soleus muscle but also suggests an adaptation to sustain fatigue tolerance.…”

“…As previously reported (Wei et al, 2014), cross sections of mouse soleus muscle were blocked in phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBS-T) and 1% BSA at room temperature for 30 min. Endogenous peroxidase was inactivated by incubation with 1% H 2 O 2 in PBS-T at room temperature for 10 min.…”

“…TNNT1 myopathies are featured by loss of slow twitch muscle fibers and presented with severe muscle atrophy, weakness and failure of respiratory muscle (Johnston et al, 2000; Jin et al, 2003; Amarasinghe et al, 2016). Mouse models of TNNT1 myopathy reproduced the slow muscle atrophy and degeneration phenotypes and showed a significant loss of fatigue resistance of soleus and diaphragm muscles (Feng et al, 2009; Wei et al, 2014). …”