Fig 1.
The coordination of the GFP moiety (green) and its emission dipole moment (red arrow) with myosin S1 consisting of a heavy chain (blue and black), ELC (silver), and RLC (red).
The portion of the lever-arm in the heavy chain appearing in black is the α-helix segment associated with the lever-arm orientation and depicted by the black arrow. The insert shows the time-resolved coordinates for GFP chromophore emission dipole moment in red and lever-arm helix in blue corresponding to spherical polar angles (β’,α’) and (β,α) defined relative to a fiber fixed frame.
Fig 2.
The fiber frame xz plane in Fig 1 lies in the microscope focal plane and in the lab frame as shown.
Lab and fiber frames are related to each other by a rotation through χ. Images of the heart shown subsequently relate to the lab frame like the image of the test pattern.
Fig 3.
Myosin S1 consisting of a heavy chain, ELC (blue), and RLC (yellow) performing a powerstroke with a lever-arm rotation while strongly actin bound.
The arc subtended by, Φ, the angle a single lever-arm helix rotates in sequential images defines a sequence of chords on a circle of diameter L equal to the lever-arm length indicated. Chord length is step-size, d, given by Eq 3. The ELC N-terminal extension, shown actin bound at the end of the powerstroke, plays a major role in the proposed mechanism for strain sensing in the myosin.
Fig 4.
The Sypro Ruby stained and immunoblotted SDS-PAGE gels.
The Sypro Ruby gel band intensities establish ZRLC content in a zebrafish adult heart extract relative to a known amount of in vitro expressed HCRLC (panel a). These samples produce calibrated blot intensities for HCRLC and ZRLC under a standardized protein immunoblotting protocol using the RLC antibody as described in Methods and previously [11]. The standards are compared to immunoblots from HCRLC-GFP and ZRLC in WT and TgGFP(+) embryos (panel b) to measure relative fractions of HCRLC-GFP and ZRLC content (ZRLCrep, Eq 1). The relative amount of ZRLC in WT and TgGFP(+) embryo hearts confirmed ZRLC content removed (ZRLCrem, Eq 2) by their comparison to control β-actin expression (panel c).
Fig 5.
Comparison of heart rate in Beats Per Minute (BPM), Shortening Fraction (SF), and cyclical ventricle volume changes for WT and TgGFP(+) embryos in the presence (blue) and absence (black) of PTU treatment.
Error bars show standard deviation for n embryos.
Fig 6.
A single frame (left) and an averaged image of the heart (right) running at 50–60 bpm.
Fig 7.
Panels a-c. In vivo cardiac myosin active cycle from auxotonic (force developing), through near-isometric (maximum force), to detachment (relaxation) phases. Error bars indicate standard deviation of the mean for 15 or 24 cardiac muscle fibers from 8 or 6 embryos and corresponding to 1251 or 1436 single photoactivated myosin coordinates from relaxed and active hearts, respectively. Dotted vertical lines in panels a & b define the boundaries between step-size frequencies computed from areas under the curves for nominal steps of 0, 3, 5, and 8 nm (nominal step-size corresponds to the in vivo measured step-sizes of 0, 2, 4, and 6 nm as indicated on the x-axis legend). Areas at step-size boundaries are split equally between adjoining step-sizes. Panel d. Simulated (blue) and observed (black and same as in panel c) relaxed cardiac (ca) myosin step-size distribution. Simulation is based on dispersion of thick filament structure surmised from the atomic model of Alamo et al. [27] as described in METHODS. In vivo relaxed skeletal (sk) myosin step-size from zebrafish embryo trunk muscle (red) is shown for comparison [31].
Fig 8.
Myosin flux through the 4-pathway network contrasting 3 phases of muscle contraction in the beating heart.
Myosin begins and ends detached from actin and with ATP bound in the contraction cycle. The green box with dashed line boundaries group the strong actomyosin bound states. Blue vector v at the end of the myosin lever-arm is positive net force on, and positive velocity of, the thick filament in units where amplitudes are equal. Fluxes through the network, fi, differ depending on contraction phases (values in Table 1). Measured values for fi are in green while computed values are indicated in black. Four pathways cross from beginning to end of the contraction cycle. The top pathway populated by flux f1 executes an 8 nm step-size. The middle pathway populated by flux f3 executes a 3 nm step-size. It releases Pi while weakly actin bound without doing work. The bottom pathway populated by flux f4 is branched and executes 5 or 5+3 nm step-sizes. The branch from the bottom pathway is populated by flux f7 and executes the 5+3 nm step-size. Strain sensitivity is modeled with mechanisms in two subpathways within the shaded regions. The upper mechanism is populated by fluxes f2 and f8 from the 8 and 3 nm steps when the ELC N-terminus binds actin for actomyosin in rigor. The taut (blue line), intermediate (green curve), and slack (red wave) ELC N-terminus for muscle in near-isometric, auxotonic, or unloaded phases have high, modest, or zero strain when net force v is zero in isometric, intermediate in auxotonic, or high in unloaded phase. The linear (blue) actin bound ELC N-terminus is proposed to inhibit ATP binding by lowering active site accessibility for ATP at the small arrow near the myosin head. Inhibited ATP binding extends actomyosin attachment time indicated by the clock icon and quantitated in our single myosin measurements as a 0 length step. The lower mechanism is populated by flux f6 from the 5 nm step with ADP bound. Near-isometric, auxotonic, or unloaded phases have high, intermediate, or zero strain (of an unspecified myosin element) when net force is zero, intermediate, or high. Strain lowers ADP release rate. Short duration ADP rate inhibition flux, f7, leaves to continue with the 3 nm step. Long duration ADP rate inhibition flux, f6 − f7, continues with the 0 length step. For either the ATP accessibility or ADP release rate mediated mechanisms (top or bottom strain sensing mechanisms), low net force inhibits myosin cycling by extending the time myosin is strongly actin bound by >2Δt.
Table 1.
a. Cardiac myosin flux through in vivo active cycle in 3 phases.
Table 2.
a. Step probabilities.
Table 3.
Myosin flux through the 4-pathway network in a zebrafish embryo beating heart.