A1603P and K1617del, Mutations in β-Cardiac Myosin Heavy Chain that Cause Laing Early-Onset Distal Myopathy, Affect Secondary Structure and Filament Formation In Vitro and In Vivo

Fig. 1. Mutant constructs. (a) A molecular model to show how GST is fused to the start of the coiled coil, in the GST–LMM constructs. Yellow: β-sheet, purple: α-helix, blue: C-terminus of GST and linker. (b) The positions of the two mutations studied here and the positions of the two 15-heptad constructs with respect to the full-length myosin, as well as the LMM construct used here, with amino acid numbers as indicated. Nominal positions of the four “skip” residues are shown. (c) Partial sequences of β-MHC, showing the WT sequence, and the mutant sequences for A1603P and K1617del underneath. The heptad assignments for WT are indicated as shown and positions of mutations as indicated. (d) SDS-PAGE gels of purified 15 heptad (15H) constructs. (e) SDS-PAGE gels of purified GST–LMM constructs and purified LMM constructs after GST has been proteolytically removed.

Fig. 2. CD spectra and gel filtration for each pair of WT and mutant 15H constructs. (a) The mean spectra for each pair of WT and mutant constructs (n = 3) performed at a temperature of 10 °C. (b) The 222-nm value for each construct (± S.D.). (c) The 222/208 ratio for each pair of WT and mutant constructs (mean ± S.D). (d) Results from gel filtration for each of the four constructs performed at room temperature.

Fig. 3. CD spectra and thermal melting curves for WT LMM and the mutant constructs. (a) CD spectra (at 10 °C) for WT and the two mutant LMM constructs. The average spectra for five experiments are shown. (b) The measured 222-nm values for WT and the two mutant LMM constructs. * and *** indicate a significant difference from WT, with p < 0.05 and p < 0.0001, respectively (n = 5). (c) The average thermal melting curves for each WT and mutant constructs (minimum of two experiments). The % folded values are normalized values such that fraction folded is 100% at 10 °C and 0% at 80 °C, for easier comparison. The Tm for WT is ~ 48 °C, and that for K1617del and A1603P is ~ 50 °C. (d) The solubility of WT and mutant GST–LMM constructs. Mean values ± S.E.M. are shown. n = 3.

Fig. 4. Effects of mutations on polymer formation by GST–LMM constructs that have the GST tag still attached. (a) Low-magnification electron micrographs of negatively stained WT and the two mutant LMM constructs (A1603P and K1617del). The scale bar is 500 nm. (b) Gallery of negatively stained filaments of WT and the two mutant LMM constructs (A1603P and K1617del). White arrowheads indicate GST moieties projecting from the filaments. (c) 1:1 mixtures of WT:A1603P and WT:K1617del. The scale bar for panels b and c is 50 nm. (d) Filament lengths and widths for GST–LMM constructs. Individual values are shown, together with the mean ± S.D. Length measurements were only made when the ends of the filaments were clearly identifiable and filaments were not bundled. For widths, n = 34 (WT), n = 24 (A1603P), n = 11 (WT:A1603P), n = 4 (K1617del) and n = 18 (WT:1617del). For lengths, n = 34 (WT); n = 24 (A1603P), n = 11 (wt:A1603P), n = 4 (K1617del) and n = 18 (WT:K1617del). Significant differences between WT and each of the other types of filament are indicated by asterisks: ** p < 0.01, *** p < 0.001, **** p << 0.001.

Fig. 5. Incorporation of eGFP–MHC constructs into sarcomeres in cultured skeletal muscle myotubes formed by C2C12 cells. (a) Fluorescent images of skeletal muscle myotubes expressing eGFP–MHC constructs (green in merged image) co-stained for the Z-disc protein α-actinin (magenta in merged image). (b) Fluorescence images of skeletal muscle myotubes expressing GFP–MHC constructs (green in merged image) co-stained for the M-line protein myomesin (magenta in merged image). (c) Western blots for untransfected skeletal muscle myotubes (UT) and myotubes expressing WT and mutant eGFP–MHC constructs probed with anti-GFP. Gel lanes are approximately equally loaded, as shown by the blot probed with anti-α-tubulin (as a loading control).

Fig. 6. Mutant isoforms of eGFP–MHC affect incorporation into muscle sarcomeres in cultured skeletal muscle myotubes formed by C2C12 cells. (a) Images of individual myofibrils within cultured skeletal muscle myotubes expressing eGFP–MHC constructs as shown and co-stained for skeletal myosin using the A4.1025 antibody. (b) Example line profiles for MHC (magenta) and eGFP–MHC (green) across five sarcomeres from the images shown in panel a. (c) Mean fluorescence intensity profiles for eGFP–MHC organization across a single sarcomere, for WT and each mutant. The mean plot profile was calculated from measurements of at least 12 myofibrils (with 2–5 sarcomeres measured individually per myofibril) from different myotubes (n = 24, WT; n = 12, A1603P; n = 13, K1617del). Positions of the peak intensity values and the minimum intensity value (at the M-line) are indicated for WT eGFP–MHC. (d) The ratio between the mean peak intensity values (as in panel c) and the mean minimum intensity value at the M-line. (e) The mean peak values (averaged for both peaks, either side of the M-line) for WT eGFP–MHC and each of the mutants. (f) The mean peak–peak distance between peak maxima either side of the…

Fig. 7. Mutations in eGFP–MHC affect incorporation into sarcomeres in adult rat cardiomyocytes. (a) Representative images of single adult rat cardiomyocytes to show the variation in incorporation of eGFP–MHC into the sarcomere and/or aggregates. Green: eGFP–MHC, magenta: anti-myosin heavy-chain antibody (anti-MHC, A4.1025). Similar MOIs (multiplicity of infection) were used for each adenovirus for each eGFP–MHC construct. (b) Typical images of a small number of sarcomeres shown at higher magnification to demonstrate how mutant eGFP–MHC isoforms can have a reduced incorporation (c) The plot shows the results of a quantitative analysis, to estimate the overall percentage of cells that either show integrated, partially integrated or aggregated eGFP–MHC in the adult cardiomyocytes for WT eGFP–MHC and each mutant. Mean values for three separate experiments are shown, with the mean ± S.D. for each construct tested (n = 3). Significant differences from the WT results are indicated by asterisks (* p < 0.05, ** p < 0.001, *** p < 0.0001). (d) SDS PAGE gel of cardiomyocytes that express the eGFP–MHC constructs or were not infected with adenovirus (UT). Two bands attributable to the endogenous MHC…

Fig. 8. Results from MD simulation of the composite model coiled-coil and mutant models. (a) The distance between the helices (Dcom) as a function of residue position for WT (black), A1603P (red) and K1617del (green). Error bars represent the SD during the simulation. (b) Relative solvent accessibility (RSA) for side-chain atoms in each residue. Low values indicate solvent exclusion and thereby highlight the core residues; upper and lower plots show the RSA values for chains A and B, respectively; color scheme as per part a. Error bars are omitted for clarity. (c) Heptad length, the distance between Cα atoms in residues at positions i and i + 7, along chain A (upper) and chain B (lower). (d) Inter-heptad angle, the angle between the lines linking Cα atoms in residues at positions (i – 7), (i) and (i, i + 7), for chain A (upper) and chain B (lower). RMSD for all Cα atoms compared to the initial structure for WT (e), A1603P (f), and K1617del (g). The plots are divided by color into the different structure clusters. Example structures for the most heavily populated clusters (> 10%) are shown beneath. The position of the residue being investigated (or K1616 in the case of K1617del) is shown…