Probing relaxed myosin states in hypertrophic cardiomyopathy by second harmonic-generation microscopy
- Department of Experimental and Clinical Medicine, University of Florence, Italy
- Illinois Institute of Technology, United States
- College of Basic Medical Sciences, Dalian Medical University, China
- National Institute of Optics (INO) - CNR, Italy
- School of Physics and Astronomy, University of Glasgow, United Kingdom
- Department of Statistics, Computer Science and Applications "G. Parenti", University of Florence, Italy
- School of Medicine and College of Engineering, University of Washington, United States
- Institute of Clinical Physiology (IFC) - CNR, Italy
Figures
Figure 1 with 2 supplements
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 fiber. 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). The right column shows selected ROI illuminated at different polarization angles. (C) Representative SHG polarization curves in rigor, after the exposure to a high concentration of 2-deoxyATP (dATP, 100%), in 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 Equation 1, yielding a best-fit parameter of γRigor = 0.68 in rigor, γdATP = 0.53 with dATP, γRelax = 0.36 in relax, and γMava = 0.27 with Mavacamten. (D) Graph illustrating γ values among the Rigor state, after exposure of dATP, Relax state, and after exposure of Mavacamten (Mava) in rabbit skinned psoas fibers. Data were obtained from 9 psoas fibers, with multiple ROIs collected across each strip, producing 35 data points for the Rigor state, 22 for dATP, 43 for relaxation, and 50 for Mavacamten. Data are reported as mean ± SEM. On comparing Rigor vs. dATP, Relax, and Mava, we included conditions as fixed effects, whereas on pairwise comparisons between dATP, Relax, and Mava, the strip was also added as a fixed effect. (E) Graph illustrating γ values in relaxing solution and in relaxing solution supplemented with 5% dextran in rabbit skinned psoas fibers. Data were obtained from 3 psoas fibers, with multiple ROIs collected across each strip, producing 30 data points for Relax state and 30 data points for dextran. On comparing Relax vs. 5% dextran, we included condition as a fixed effect along with strip. (F) Graph illustrating γ values in relaxing conditions at three different temperatures (10, 15, and 20 °C) in rabbit skinned psoas fibers. Data were obtained from 5 psoas fibers, with multiple ROIs collected across each strip, producing 35, 41, and 48 data points at 10, 15, and 20 °C, respectively. On comparing the effect of temperature, we included temperature as a fixed effect along with strip. For each comparison, we reported the p-value associated with the Wald test of the specific coefficient. In each experiment, we adopted the Bonferroni correction to evaluate the significance of the p-values.
Figure 1—figure supplement 1
Passive force measurements in psoas and minipig myofibrils.
The graph shows passive force recorded in myofibrils initially exposed to an ATP-containing solution (pre-dATP), followed by exposure to a 100% 2-deoxyATP solution (dATP), and subsequently returned to the ATP-containing solution (post-dATP), in both psoas myofibrils (average sarcomere length: 2.77±0.07 µm) and minipig myofibrils (average sarcomere length: 2.20±0.08 µm). Statistical analysis was performed using two-way ANOVA followed by Tukey’s post hoc test.
Figure 1—figure supplement 2
Rabbit psoas myofibrils.
Graph illustrating γ values among the Rigor state (23 data points), Relax state (24 data points), and after exposure of Mavacamten (Mava) (24 data points), in rabbit skinned psoas myofibrils. Data are reported as mean ± SEM. A one-way repeated measures ANOVA was performed with a Tukey post-hoc correction.
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, after exposure to 2-deoxyATP (dATP), Relax state, and after exposure to Mavacamten (Mava) in mouse skinned cardiac samples. Data were obtained from 14 skinned cardiac samples dissected from 5 mice. Multiple ROIs were collected across each strip, producing 24 data points for the R Rigor state, 32 for dATP, 86 for relaxation, and 52 for Mavacamten. Data are reported as mean ± SEM. On comparing Rigor vs. dATP, Relax, and Mava, we included conditions as fixed effects, while on pairwise comparisons between dATP, Relax, and Mava, the strip was also added as a fixed effect. (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 during the long diastolic phase. Data were obtained from five skinned cardiac samples dissected from three mice and six intact trabeculae dissected from three mice. Multiple ROIs were collected across each strip, producing 55 data points for the Relax state and 52 for Mavacamten in skinned preparation while 30 data points for the resting state and 30 for Mavacamten were obtained from intact preparations. Data are reported as mean ± SEM. On comparing Relax/Resting vs. Mava, a stratified analysis was performed in the two preparation groups (Skinned and Intact). We included condition as a fixed effect, along with strip. To compare the two preparations in the two conditions (Relax/Resting vs. Mava), we considered a mixed-effect model with preparation as a fixed effect. For each comparison, we reported the p-value associated with the Wald test of the specific coefficient. In each experiment, we adopted the Bonferroni correction to evaluate the significance of the p-values.
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 Rigor, in Relax solution, after exposure to 100% 2-deoxyATP (dATP), and following treatment with 10 µM Mavacamten (Mava). Data were obtained from 11 skinned cardiac samples dissected from 4 WT minipigs and 11 skinned cardiac samples dissected from 3 R403Q-MYH7 minipigs. Multiple ROIs were collected across each strip, producing 33 data points for the R Rigor state in WT samples only. For WT and R403Q-MYH7 samples, respectively, 45 and 76 data points were obtained for the Relax state, 77 and 103 for 2-deoxyATP, and 76 and 91 for Mavacamten. Data are reported as mean ± SEM. On comparing Rigor vs. Relax, dATP, and Mava we included conditions as fixed effects. Pairwise comparisons between dATP, Relax, and Mava were performed on each group (WT and R403Q) using mixed-effect models with condition and strip as fixed effects. The comparison between genotypes on each condition (dATP, Relax, and Mava) was performed using mixed-effect models with genotype as fixed effects. The likelihood ratio test was performed to test the interaction between genotype and condition. For each comparison, we reported the p-value associated with the Wald test of the specific coefficient. In each experiment, we adopted the Bonferroni correction to evaluate the significance of the p-values.
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 dATP 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 dATP-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 dATP 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.
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% dATP, 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 ± SEM. Pairwise comparisons between Relax, dATP, and Mava were performed on each group (WT and R403Q) using mixed-effect models with condition and strip as fixed effects. The comparison between genotypes on each condition (Relax, dATP, and Mava) was performed using mixed-effect models with genotype as fixed effects. The likelihood ratio test was performed to test the interaction between genotype and condition. For each comparison, we reported the p-value associated with the Wald test of the specific coefficient. In each experiment, we adopted the Bonferroni correction to evaluate the significance of the p-values.
Figure 6
Comparison of γ values among different species under distinct chemically modulated conditions.
Comparison of γ values measured in skinned preparations from rabbit psoas muscle, mouse cardiac muscle, and minipig cardiac muscle under the following conditions: Rigor solution (A), Relax solution with 100% 2-deoxy-ATP (dATP) (B), Relax solution (C), and Relax solution after treatment with high concentrations of Mavacamten (50 µM in psoas, 10 µM in mouse and minipig preparations; Mava) (D). Data are presented as mean ± SEM. For each condition (Rigor, dATP, Relax, and Mava), the species effect was evaluated considering mixed models with species as fixed effects.
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Probing relaxed myosin states in hypertrophic cardiomyopathy by second harmonic-generation microscopy
eLife 14:RP107730.
https://doi.org/10.7554/eLife.107730.3
