Probing relaxed myosin states in hypertrophic cardiomyopathy by second harmonic-generation microscopy

Figures

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  Figure 1:. pSHG sensitivity on myosin conformation in psoas skeletal muscle. A) Optical scheme of the SHG microscopy setup. The excitation beam's optical path is shown in red, while the emission path is in purple. Abbreviations: λ/4, quarter-wave plate; BPF, bandpass filter; PMT, photomultiplier. B) SHG image of a demembranated rabbit psoas fibre. Bright bands correspond to sarcomeric A bands. Scale bar: 20 μm. The cyan square highlights a representative region of interest (ROI) selected for acquisition (13 × 13 μm). C) Selected ROI shown in panel B illuminated at different polarization angle. D) Representative SHG polarization curves in rigor, relaxation, and after exposure to a high concentration of Mavacamten (Mava; 50 μM). Circles represent raw data, and the continuous lines indicate the best fit of Eq. 1, yielding a best-fit parameter of γRigor=0.68 in rigor, γRelax=0.36 in relax, and γMava=0.27 with Mavacamten. E) Graph illustrating γ values among the Rigor state, Relax state, and after exposure of Mavacamten (Mava) in rabbit skinned psoas fibers. Data were obtained from 6 psoas fibres, with multiple ROIs collected across each strip, producing 35 data points for the rigor state, 24…

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  Figure 2:. pSHG sensitivity on demembranated and intact cardiac preparations. A) Representative SHG image of a demembranated mouse ventricular wall strip. Borders between individual cells are visible. Scale bar: 20 μm. The cyan square highlights a representative region of interest (ROI) selected for acquisition (13 × 13 μm). B) Graph illustrating γ values among the Rigor state, Relax state, and after exposure of Mavacamten (Mava) in mouse skinned cardiac samples. Data were obtained from 5 skinned cardiac samples dissected from 3 mice. Multiple ROIs were collected across each strip, producing 18 data points for the rigor state, 18 for relaxation, and 18 for Mavacamten. Data are reported as mean ± S.E.M. A one-way repeated measures ANOVA was performed with a Tukey post-hoc correction. C) Comparison of γ values in mouse demembranated versus intact cardiac preparations. The graph shows γ values measured in both skinned (open circles) and intact (filled circles) multicellular cardiac preparations. Data were collected first in a relax solution and subsequently after the addition of 10 μM Mavacamten. For intact preparations, stimulation was applied at a frequency of 0.1 Hz, with imaging performed…

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  Figure 3:. pSHG in R403Q-MYH7 mutation A) SHG image of a sample from a wild-type (WT) minipig, and B) sample from a minipig carrying the MYH7-R403Q hypertrophic mutation. Cyan squares highlight representative regions of interest (ROIs) selected for acquisition (13 × 13 μm). A marked difference is evident between healthy and pathological tissue, with the mutated tissue exhibiting a high degree of structural disarray. Scale bars: 20 μm. C) Pharmacological manipulation of skinned preparations from WT and R403Q minipigs. The graph shows γ values obtained under different conditions: in Relax solution, after exposure to 100% 2-deoxyATP (dATP), and following treatment with 10 μM Mavacamten (Mava). Data were obtained from 7 skinned cardiac samples dissected from 3 WT minipig and 11 skinned cardiac samples dissected from 3 R403Q-MYH7 minipig. Multiple ROIs were collected across each strip, producing 45 and 76 data points for the relax state, 77 and 103 for 2-deoxyATP, and 76 and 91 for Mavacamten in WT and R403Q-MYH7, respectevely. Data are reported as mean ± S.E.M. A two-way repeated measures ANOVA was performed with a Tukey post-hoc correction. D) Estimation of the fraction of ON state myosin…

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  Figure 4:. X-ray diffraction in R403Q-MYH7 mutation. A) Representative X-ray diffraction patterns from wild-type (WT) and R403Q minipig ventricular strips in relaxing solution. B) Top row: Graphs comparing changes in M3 reflection intensity (IM3) between WT and R403Q samples under different conditions. The first two graphs show intensity differences between Relaxed and 2-deoxyATP (dATP) treated samples (WT and R403Q, respectively) and between Relaxed and Mavacamten (Mava) treated samples. The third and fourth graphs illustrate ΔIM3, comparing the changes in IM3 between WT and R403Q samples with 2-deoxyATP and Mavacamten treatments. Bottom row: Graphs comparing the spacing of the M6 meridional reflection (SM6) between WT and R403Q samples under different conditions. The first two graphs display shifts in SM6 between Relaxed and 2-deoxyATP treated samples and between Relaxed and Mavacamten treated samples for both groups. The third and fourth graphs show ΔSM6, comparing shifts in SM6 between WT and R403Q with 2-deoxyATP and Mavacamten treatments. Data were obtained from > 15 skinned cardiac samples dissected from 2 WT minipig and 12 skinned cardiac samples dissected from 2 R403Q-MYH7 minipigs.

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  Figure 5:. ATPase activity in R403Q mutation. A) Representative traces of NADH absorbance from wild-type (WT) minipig preparations (left column) and preparations from minipigs carrying the MYH7-R403Q hypertrophic mutation (right column) under different conditions: Relax, 100% 2-deoxyATP (dATP), and 10 μM Mavacamten (Mava). B) Graph illustrating ATP consumption across different sample conditions: in Relaxing solution, after exposure to 100% 2-deoxyATP, and following treatment with 10 μM Mavacamten. Data were obtained from 17 skinned cardiac samples dissected from 3 WT minipig and 21 skinned cardiac samples dissected from 3 R403Q-MYH7 minipig. Each preparation is associated with a data point in the three conditions. Data are reported as mean ± S.E.M. A mixed effects ANOVA was performed with a Tukey post-hoc correction.