Hypertrophic cardiomyopathy-linked Tnnc1 variants are associated with distinct myopathic changes in slow skeletal muscle of mice

Figures

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  Fig. 1. Soleus muscle obtained from cTnC-A8V-/- mice has reduced specific force at the highest tested stimulation frequency. Soleus muscles were isolated from 7—7.5-month-old WT A8V+/+, A8V+/-, and A8V-/- mice. In (A) absolute force (N/g) and (B) specific force (N/cm2) is shown for soleus muscles over increasing frequencies. In (C) % specific Force (N/cm2)-A8V variant is plotted. Muscle fatigability is shown in (D) as maximal force by 20 s intervals, (E) normalized force (SF20s – N/cm2), (F) maximal force at 20 s intervals (MF20s) fatigue index (%), and (G) specific force at 20 s intervals (SF20s) fatigue index (%). Soleus muscles were obtained from different mice at these numbers; n = 5 (+/+), n = 4 (+/-), and n = 5 (-/-) for all data presented in this figure. Significance was determined using a Kruskal–Wallis test since comparing 3 groups. In (B and D) the means ± SEM were compared for each time point or frequency value. Differences were considered significant if p < 0.05 and denoted * p < 0.05

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  Fig. 2. The cTnC-C84Y+/- variant does not impact specific force in intact mouse soleus muscle. Soleus muscles were isolated from 7 – 7.5-month-old WT C84Y+/+ and C84Y+/- mice. In (A) absolute force (N/g) and (B) specific force (N/cm2) is shown for soleus muscles over increasing frequencies. In (C) % specific force (N/cm2)-C84Y mutant is plotted. Muscle fatigability is shown in (D) as maximum force by 20 s intervals, (E) normalized force (SF20s – N/cm2), (F) maximal force at 20 s intervals (MF20s) fatigue index (%), and (G) specific force at 20 s intervals (SF20s) fatigue index (%). Soleus muscles were obtained from different mice at these numbers; n = 4 (+/+) and n = 4 (+/-) and used to generate all data presented in this figure. Significance was determined using a two-tailed Mann–Whitney test since comparing two groups and data presented as means ± SEM

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  Fig. 3. The presence of cTnC variants alters abundance of type I fibers in A8V-/- mouse soleus muscle. Soleus muscles were isolated from the 7 - 7.5-month-old WT A8V+/+, A8V+/-, and A8V-/- and C84Y+/+ and C84Y+/- mice and fiber typing was performed using immunostaining to detect expression of fast and slow MyHC isoforms. (A) Representative, merged immunofluorescence images (left-most column), and individual channels (continuing left-to-right) show type I fibers (blue), type IIa fibers (green), and type IIb fibers (red) and cell boundaries stained using wheat germ agglutinin (WGA; yellow). Unstained fibers were reported as type IIx. In several A8V+/- panels a fluorescent artifact outside of the muscle was cropped out for visual purposes. Images were obtained at 10X magnification. (B) Percentages of fiber types are shown for A8V+/+, A8V+/-, and A8V-/- soleus muscles. (C) Percentages of fiber types are shown for C84Y+/+ and C84Y+/- soleus muscles. Soleus muscles were obtained from different mice and n = 4 for all genotypes except n=5 for A8V-/-. Significance was determined using a Kruskal-Wallis test followed by the Dunn’s multiple comparisons test for each individual fiber type for the A8V…

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  Fig. 4. The cTnC-A8Vmutant mouse soleus muscle exhibits both type I and IIa fiber atrophy. The distribution of fiber sizes is plotted as percent of fibers for soleus muscles from WT A8V+/+, A8V+/-, and A8V-/- mice. The distribution of cross-sectional areas (µm2) is shown for soleus muscle from WT and A8V mutant mice in (A) for type I and (B) for type IIa fibers. Bars show average values and individual values are also shown. The cross-sectional areas of all measured fibers (C) type I fibers (D) type IIa fibers, and (E) shows all fiber types. Chi-square (χ2) tests were performed to assess atrophic changes (reduced CSA) in the soleus muscles in two designated categories small (300–1100 μm2) versus large (1101–1900 μm2) and results are shown in (F) for type I fibers and (G) type IIa fibers. Differences were considered significant * if p < 0.05. Significance was determined by the Kruskal–Wallis test for each fiber size category (4A-B) and data presented as mean ± SEM. For (4C-E) one-way ANOVA was used followed by the Tukey’s multiple comparisons test and p values obtained are reported in Table 2. For (4F-G) Chi square contingency plots were analyzed using the Fisher’s exact test. For all…

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  Fig. 5. The cTnC-C84Y+/- mouse soleus muscle exhibits type I fiber hypertrophy. The distribution of fiber sizes is plotted as percent of fibers for soleus muscles from WT C84Y+/+ and C84Y+/- mice. The distribution of cross-sectional areas (µm2) is shown for soleus muscle from WT and C84Y mutant mice in (A) for type I and (B) for type IIa fibers. Bars show average values and individual values are also shown. The cross-sectional areas of all fibers measured (C) type I fibers (D) type IIa fibers, and (E) shows all fiber types. Chi-square (χ2) tests were performed to assess atrophic changes (reduced CSA) in the soleus muscles in two designated categories small (300–1100 μm2) versus large (1101–1900 μm2) for (F) type I fibers and (G) type IIa fibers. Differences were considered significant * if p < 0.05. Significance was determined by a two-tailed Mann–Whitney test (4A-B). For (4C-E) an unpaired two-tailed t-test was used and p values reported in Table 2. Data is presented as mean ± SEM and individual values shown. For (4F-G) Chi square contingency plots were analyzed using the Fisher’s exact test. For all genotypes assessed, n = 4 was used with soleus muscles obtained from different mice

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  Fig. 6. Histological evaluation of soleus muscle reveals that cTnC-A8V mice have increased central nucleation and mild regeneration. In (A) transverse sections of soleus muscle obtained from WT (A8V+/+), A8V+/-, A8V-/-, WT (C84Y+/+), and C84Y+/- mice was stained with Hematoxylin and O Red Oil. Bar = 50 μm, 10 × magnification. (B) The percentage of central nucleated fibers is compared to the total number of fibers in soleus muscles from WT (A8V+/+), A8V+/-, WT (C84Y+/+), and C84Y+/- mice. (C) Shows immunofluorescence images of embryonic myosin heavy chain (eMHC) staining (red) with cell borders labeled with wheat germ agglutinin (WGA). (D) Shows quantification of the eMHC+ fibers for all the groups. (E) The % Area Fraction is plotted comparing total lipid area/total muscle area. (F) O Red Oil positive staining is shown in soleus muscle. Significance was determined for A8V groups using a Kruskal–Wallis followed by the Dunn's multiple comparisons test and for the C84Y groups a two-tailed Mann–Whitney test was used. Data was plotted as mean ± SEM and individual values shown. Differences were considered significant and marked * if p < 0.05 with * indicating p < 0.01 and ** indicating p < 0.001