No Difference in Myosin Kinetics and Spatial Distribution of the Lever Arm in the Left and Right Ventricles of Human Hearts

Figures

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  Figure 1. Changes of conformation of a cross-bridge during the contractile cycle are reflected by changes of anisotropy of the myosin lever arm (red line). The kinetics of orientation changes of a lever arm is a characteristic property of muscle. The instrument used to measure anisotropy is shown in Figure S1. Fluorescent LC sits on a lever arm of a cross-bridge (red rectangle). LC orientation is indicated by the magenta arrow; (A) XB is originally free of actin. Lower and upper 50 KDa domains of myosin XB (gray and blue, respectively) are separated. The lever arm is facing down. Steady State Fluorescence Anisotropy (SSFA) of the lever arm is low. (B) The anisotropy of LC+LV (rigor) complex (apo form of ventricular XB is bound to actin filament). Lower and upper 50 KDa domains of XB are closed. The anisotropy assumes intermediate value (SSFA = 0.135); (C) Interaction between XB containing ADP and actin. Lower and upper 50 KDa domains of XB are closed the anisotropy of a complex assumes the highest value (SSFA = 0.201). This is consistent with recent report (Wulf et al., 2016). The lower plot in each anisotropy graph is a residual. X2 indicates goodness of non-linear fit to a thin black…

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  Figure 2. (A) A diffraction limited laser beam (green ellipsoid) is focused on an isolated myocyte sitting on a coverslip. A confocal microscope sees only the Observational Volume (OV, outlined by a broken line). The A-band (red) is the only region where the interaction between actin and myosin occurs. (B) Fluorescence is collected from the OV shown here in 3D as a green sphere imbedded in the A-band. It is projection of the confocal aperture on the image plane. The diameter of the OV (0.5 μm) is equal to the diameter of the confocal pinhole (50 μm) divided by the magnification of the objective (100x). Myosin within the A-band is fluorescently labeled (red). The I-bands are non-fluorescent (blue). Thick black lines are the H-zones; thin black lines are the Z-bands. The myocyte contracts (i.e., develops normal force), but does not shorten because it is cross-linked (see Methods); (C) One in approximately one thousand myosin molecules within the thick filament is labeled at LC Cys 174 and Cys 181. The transition dipole moments the dye of two labeled myosins are marked in magenta. Emission dipoles of the dye attached to these two cysteines point in the same direction. Labeling of only 1 in a…

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  Figure 3. The time course of intensities of fluorescent light from myosin. Fluorescent intensities are polarized parallel (red) and perpendicular (black) with respect to the axis of the myocyte. Polarization of fluorescence is shown in blue. The average polarization of fluorescence was 0.400 ± 0.158. The signal from the OV of a left ventricle in the presence of MgADP.

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  Figure 4. Conformational transitions of 6 XBs during contraction of a ventricle. Red line indicates change of anisotropy. (A) Binding of cross-bridges to a thin filament of a ventricle is characterized by the rate constant kB; (B) transition from apoenzyme to holoenzyme form of myosin is characterized by the rate constant kADP; (C) dissociation of a cross-bridge from a thin filament characterized by the rate constant kDISS.

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  Figure 5. A typical autocorrelation function of the lever arm fluctuations of contracting cardiac myosin in HF RV. Filled circles are the data. Red line is non-linear fit to analytical solution described in Mettikolla et al. (2011).

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  Figure 6. Spatial distribution of lever arm angles of cross-bridges of contracting HF ventricles. The orientation of ~6 cross-bridges in 27 different preparations of LV and 26 preparations of RV Data shows no difference in FWHM. Bars are data. Red line is the best fit.