Sarcomeric deficits underlie MYBPC1-associated myopathy with myogenic tremor

Figures

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  Figure 1. Generation and physiological characterization of the MYBPC1 E248K KI mouse model. (A) Schematic representation of the sMyBP-C domain structure and location of the E248K variant in the M-motif; white and gray ovals denote immunoglobulin and fibronectin-III domains, dark gray and light gray horizontal rectangles indicate the Pro/Ala and M-motif, and colored rectangles depict spliced sequences. (B) Approximately 15% of heterozygous KI mice failed to survive through weaning but showed similar survival trends as WT past 3 weeks of age; survival chart: n = 295 WT (151 male and 144 female) and n = 154 heterozygous KI (80 male and 74 female). The overall survival bar-graph shows: n = 97 WT (52 male and 45 female) and n = 65 heterozygous KI (37 male and 28 female). (C) Both male and female heterozygous KI mice were significantly smaller compared with WT littermates through 24 weeks of age; n = 22 WT (10 male and 12 female) and n = 24 heterozygous KI (13 male and 11 female). (D) During the first 15 days of postnatal life, average tremor value at rest was significantly higher in heterozygous KI pups compared with WT; n = 14 WT (4 male and 10 female) and n = 9 heterozygous KI (6 male and 3…

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  Figure 2. Behavioral and phenotypic assessment of heterozygous E248K KI mice. (A) Young adult heterozygous KI mice performed significantly fewer rearing events and spent less time rearing in an open field assay compared with WT littermates; all ages, n = 7 WT (2 male and 5 female) and n = 8 heterozygous KI (2 male and 6 female). (B) Young adult heterozygous KI mice exhibited significantly more foot slips and increased time freezing or time immobile in an ataxia box assay compared with WT littermates; all ages, n = 8 WT (5 male and 3 female) and n = 15 heterozygous KI (8 male and 7 female). (C) Young adult heterozygous KI mice displayed significantly decreased overall grip and body strength during an inverted hang assay compared with WT littermates, as evidenced by significantly less time on the grid; 4 weeks, n = 12 WT (7 male and 5 female) and n = 11 heterozygous KI (6 male and 5 female); 6 weeks, n = 13 WT (8 male and 5 female) and n = 13 heterozygous KI (7 male and 6 female); and 8 weeks, n = 17 WT (9 male and 8 female) and n = 13 heterozygous KI (7 male and 6 female). (D) Plethysmography analysis revealed significant deficits in peak inspiratory flow for heterozygous KI animals at 2…

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  Figure 3. Biochemical analysis of heterozygous KI muscles at 4 weeks. (A–C) Representative immunoblots and quantification of the percent expression of major myofibrillar proteins in heterozygous KI EDL (A), soleus (B), and diaphragm (C) muscles indicated statistically unaltered protein expression at 4 weeks of age. All values are compared with WT, which was set to 100% following normalization to Hsp90 that was used as a loading control. All blots were processed in parallel. Individual data points represent the number of biological samples evaluated for each protein obtained from both male (M) and female (F) mice, as indicated in the dot plots. Statistical significance was calculated with a normalized 2-tailed Student’s t test.

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  Figure 4. Functional and histological evaluation of heterozygous KI EDL muscles at 4 weeks. (A–C) Heterozygous KI EDL muscle exhibited significantly reduced peak isometric force production (A) that persisted after normalization for the decrease in mass (B), and reduced contraction and relaxation velocities at higher frequencies (C); n = 8 WT (7 male and 1 female) and n = 7 heterozygous KI (5 male and 2 female). Statistical significance was calculated with repeated measures 1-way ANOVA (A and C) or Mann-Whitney U (B) tests. (D) Representative cross-sectional images of WT and heterozygous KI EDL muscles stained for laminin (membrane marker; teal pseudocolor). Whole muscle CSA of heterozygous KI EDL muscles was significantly decreased compared with WT; n = 4 WT (2 male and 2 female) and n = 4 heterozygous KI (2 male and 2 female). Data points represent 3 cross-sectional measurements per muscle along the longitudinal axis of the tissue to account for innate variation between the middle and ends. Statistical significance was calculated with 1-tailed Student’s t test. Scale bars: 500 µm, inset 50 µm. (E) Plotting of fiber size distribution indicated that heterozygous KI EDL muscles contained…

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  Figure 5. Functional alterations in heterozygous KI EDL myofibers at 4 weeks of age. (A–F) Single, permeabilized KI EDL myofibers exhibited decreased passive tension (A), maximal Ca2+-activated force and tension (B), Ca2+ sensitivity of force (D), loaded shortening velocity (E), and power output (F), independent of any change in the rate of tension redevelopment (max ktr) (C). For D, 3 pCa50 values were calculated for each myofiber, one from the tension-pCa curve and the other 2 from the 2 x-intercepts of the linearized tension-pCa fits (44); n = 7 WT fibers obtained from 5 male and 2 female animals and n = 9 heterozygous KI fibers obtained from 4 male and 5 female animals. Statistical significance was determined via a 2-tailed Student’s t test (A, C, D, and F) or 2-way ANOVA (B). #P < 0.05, *P < 0.01.

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  Figure 6. Structural evaluation of heterozygous KI EDL muscles at 4 weeks of age. (A) sMyBP-C assumed its typical distribution at the C-zone of A-bands in both WT and heterozygous KI EDL muscles; however, bundles of split myofibrils and misaligned sarcomeres were observed in heterozygous KI muscles; n = 4 WT (2 male and 2 female) and n = 4 heterozygous KI (2 male and 2 female). Scale bar: 5 µm. (B) Electron micrographs of longitudinal sections of WT EDL muscles confirmed the presence of properly organized and aligned sarcomeres with intact internal membranes forming typical triads (open arrowheads) at the level of A/I junctions. In contrast, heterozygous KI muscles exhibited less compact Z-discs and frequent Z-disc streaming; less defined A-, I-, and M-bands; enlarged and more abundant mitochondria (asterisks); and fragmented and/or misaligned internal membranes (closed arrowheads); n = 4 WT (2 male and 2 female) and n = 4 heterozygous KI (2 male and 2 female). Scale bar: 500 nm. (C and D) Cross-sectional electron micrographs of WT and heterozygous KI EDL muscles displayed normal hexagonal arrays of myosin filaments (C); however, the CSA of individual thick filaments and the interfilament…

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  Figure 7. Structural evaluation of heterozygous KI soleus and diaphragm muscles at 4 weeks of age. (A) sMyBP-C exhibited its expected localization at the C-zone of the A-band in WT and heterozygous KI soleus muscles; however, similar to 4-week heterozygous KI EDL muscle, bundles of split myofibrils and misaligned sarcomeres were observed in the latter; n = 4 WT (2 male and 2 female) and n = 4 heterozygous KI (2 male and 2 female). Scale bar: 5 µm. (B) Electron micrographs of longitudinal sections of WT soleus muscles revealed the presence of structurally intact and registered sarcomeres with orderly internal membranes forming typical triads (open arrowheads) at the level of A/I junctions. In contrast, heterozygous KI soleus and diaphragm muscles displayed less compact Z-discs and frequent Z-disc streaming; hazy A-, I-, and M-bands; enlarged and more abundant mitochondria (asterisks); and fragmented and/or misaligned internal membranes (closed arrowheads); n = 4 WT (2 male and 2 female) and n = 4 heterozygous KI (2 male and 2 female). Scale bar: 500 nm. (C and D) Cross-sectional electron micrographs of WT and heterozygous KI soleus muscles contained typical hexagonal arrays of myosin…