Potential cytotoxicity of truncated slow skeletal muscle troponin T ( ssTnT ) in a loss of function TNNT1 myopathy mouse model

Figures

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  Fig. 1. Construction of ANM‐KI mice. (A) ANM‐KI gene targeting construct to insert the nonsense mutation at codon Glu180. A unique EcoRI restriction enzyme cutting site is engineered after the p.Glu180* stop codon as a specific marker for identification of the Tnnt1 pGlu180* allele. The positions of primers used for RT‐PCR detection of ANM‐KI mutant mRNA are indicated. (B) The representative agarose gel from repeated analyses shows the Tnnt1 p.Glu180* mutation mRNA is readily detectable in ANM‐KI homozygote soleus muscle using RT‐PCR. Expression of the mutant mRNA was confirmed with presence of the engineered EcoRI cutting site. (C) The representative western blot from repeated analyses confirmed at protein level the loss of intact ssTnT in soleus muscle of ANM‐KI mice with the expression of cardiac TnT (cTnT) indicating active muscle regeneration.

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  Fig. 2. More severe functional loss of ANM‐KI mouse soleus muscle than that caused by ssTnT‐KO. (A) Ex vivo contractility studies showed that ANM‐KI mouse soleus muscle had maximum muscle weight‐normalized twitch force similar to that of WT control. (B) Tetanic contraction fatigue test showed impaired fatigue resistance of ANM‐KI mouse soleus muscle similar to that of ssTnT‐KO soleus muscle. (C) The post fatigue recovery of ANM‐KI soleus muscle tetanic force production was, however, slower and less than that of ssTnT‐KO. Values are presented as mean ± SE. n = 6 in WT, 4 in ssTnT‐KO, and 3 in ANM‐KI groups. *P < 0.05 for ANM‐KI vs WT; & P < 0.05 for ssTnT‐KO vs WT; # P < 0.05 in ANM‐KI vs ssTnT‐KO in two‐way ANOVA test with mean comparison using Bonferroni test.

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  Fig. 3. ANM‐KI mouse soleus muscle shows human ANM‐like slow‐to‐fast fiber type switch. (A) The twitch contractile and relaxation times of superfused ANM‐KI soleus muscle were both significantly shorter than those of WT control, indicating a switch to more fast type fibers in ANM‐KI soleus muscles. Values are presented as mean ± SE. n = 6 for WT and 3 for ANM‐KI. *P < 0.05 vs WT by Student's t‐test. (B) Western blots of ANM‐KI mouse soleus muscle showed higher levels of the fast isoforms of skeletal muscle TnT (fsTnT) and Tnl (fsTnI). Densitometry quantification of the TnI blot found that the percentage of ssTnI in total TnI decreased more in ANM‐KI than that in ssTnT‐KO soleus muscles. Values are presented as mean ± SE. n = 5 for ssTnT‐KO and n = 4 for ANM‐KI groups. *P < 0.05 vs ssTnT‐KO in Student's t‐test. (C) SDS gel and western blot of total protein extracts from frozen muscle sections of two ANM patients showed the complete loss of ssTnT. The levels of slow skeletal muscle TnI (ssTnI) were decreased in comparison to the control sample, indicating reduced slow fiber contents. The expression of cardiac TnT (cTnT) was detectable, reflecting active muscle regeneration.

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  Fig. 4. ANM‐KI reproduces human ANM‐like myopathic changes with adaptive fast fiber hypertrophy more severe than that caused by ssTnT‐KO. (A) At 2 months old, slow‐fiber‐rich soleus muscles of ANM‐KO and ANM‐KI mice both showed atrophy of slow fibers identified by type I MHC stain using mAb FA2. ANM‐KI soleus muscle showed more hypertrophy of fast type fibers with decreased total fiber number than that of ANM‐KO soleus muscle. The images were taken using an automated microscope (BZ‐X810, Keyence, Itasca, IL, USA) and from multiple photos stitched using BZ‐H4 software to cover the entire cross section of the mouse soleus muscles. (B) Frozen sections of two ANM patient muscles stained with Hematoxylin & Eosin and immunohistochemistry using mAb FA2 against MHC I and mAb CT3 against slow TnT and cardiac TnT in comparison with a control human muscle sample. As ssTnT is absent in ANM patient muscle (Fig. 3C), the positive stain of mAb CT3 represents the expression of cardiac TnT. The results demonstrate that the preserved type I slow fibers include MHC I(+),cTnT(−) fibers with larger cross sectional area (CSA) (indicated by the blue arrows) and MHC I(+),cTnT(+) fibers with smaller CSA…

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  Fig. 5. Expression of cardiac TnT in adult ANM‐KI and ssTnT‐KO mouse soleus muscles indicating active regeneration. (A) Representative western blot from repeated analyses using mAb CT3 that recognizes both ssTnT and cardiac TnT showed that the expression of cardiac TnT (cTnT) was detected in adult ANM‐KI mouse soleus muscle at a higer level than that in ssTnT‐KO soleus muscle, indicating a more active muscle regeneration. (B) Consistent with the expression of cardiac TnT detected in western blot (Fig. 3C), cardiac TnT‐positive slow fibers (MHC I‐positive) are present in the two ANM patient samples. The CSA of MHC I(+),cardiac TnT(+) fibers is smaller than that of MHC I(+),cardiac TnT(−) fibers in the ANM patient muscles. Values are presented as mean ± SE. n = 3 fields of normal and each of the patient slides. *P < 0.05 vs normal control in Student's t‐test.

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  Fig. 6. ANM‐KI and ssTnT‐KO produced different transcriptomic changes in young adult mouse soleus muscles. (A) Soleus muscle mRNA expression profiles of ANM‐KI (n = 3) and ssTnT‐KO (n = 3) mice were compared with that of WT control (n = 3). The heatmaps show fold changes (Log2FC) in the two myopathic models vs the WT control. 215 common changes were found in the gene expression profiles of ANM‐KI and ssTnT‐KO soleus muscle whereas ANM‐KI soleus muscle showed unique changes in the expression of other 215 genes and ssTnT‐KO soleus muscle showed unique changes in other 340 genes, indicating significantly different pathological and adaptive changes in these two TNNT1 myopathy animal models. Comparison between the mRNA expression profiles of ANM‐KI and ssTnT‐KO groups found 21 statistically significant changes. (B) Gene ontology analysis of the 21 genes identified Cyp26b1 as an outstanding change (which has an increase in ssTnT‐KO but a decrease in ANM‐KI soleus muscles (A). (C) Representative western blot from repeated analyses verified at protein level the differential expressions of Cyp26b1 mRNA in slow fiber‐rich quadratus femoris (QF) muscle of WT, ssTnT‐KO and ANM‐KI mice in comparison…

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  Fig. 7. More decrease in the total number of muscle fibers and more fast fiber hypertrophy in aging ANM‐KI soleus muscle than that in ssTnT‐KO control, indicating progressive skeletal muscle remodeling with age. (A) Immunohistochemistry staining of soleus muscle cryosections using mAb FA2 against type I slow MHC and mAb CT3 against slow and cardiac TnT showed more advanced increases in cross‐sectional area of fast fibers (negatively stained fibers) in aging ANM‐KI mouse soleus muscles as compared to that of ssTnT‐KO and WT controls. The images were taken using an automated microscope (BZ‐X810, Keyence, Itasca, IL, USA) and from multiple photos stitched using BZ‐H4 software to cover the entire cross section of the mouse soleus muscles. It is worth noting that the significant fast fiber hypertrophy in ANM‐KI soleus muscle resulted in large total muscle cross‐sectional areas in contrast to the atrophic ssTnT‐KO soleus muscles. (B) Quantification of muscle fiber numbers showed that soleus muscles of aging ANM‐KI mice had decreased total fiber numbers vs that of WT control while a similar trend was seen in the ssTnT‐KO muscle. The significant change in ANM‐KI soleus muscles is mainly from the…

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  Fig. 8. Development of fiber loss, slow fiber atrophy, or fast fiber hypertrophy from absences in neonatal ANM‐KI mouse muscles. (A) Representative immunohistological staining from repeated analyses of soleus muscle sections from 3‐day‐old mice using mAb FA2 against type I MHC found no slow fiber atrophy or loss in neonatal ANM‐KI samples as compared to age‐matched WT control, whereas 14–21‐day‐old ANM‐KI mouse soleus muscles showed notable loss of total and slow fibers with fast fiber hypertrophy vs WT control. The images in the two middle panels were taken using an automated microscope (BZ‐X810, Keyence, Itasca, IL, USA) and from multiple photos stitched using BZ‐H4 software to cover the entire cross section of the mouse soleus muscles. (B) Western blots of abdominal rectus muscles from three 4‐day‐old ANM‐KI mice detected significantly more cardiac TnT (cTnT) than age‐matched WT control, suggesting a possible compensation for the loss of ssTnT function in neonatal slow‐fiber‐rich muscles.