Cardiac myosin filaments are directly regulated by calcium

Figures

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  Figure 1.. Effects of the inhibitor (MYK-7660) on the chemo-mechanical activity of different sarcomere systems. (a) The concentration-dependent steady-state ATPase activity (moles ATP used per second per mole of myosin S1 heads) of bovine cardiac myosin subfragment S1 with actin (open symbol) and with RTFs at pCa 6 (closed symbols). Inhibition (IC50 = 9 ± 3 μM) is specific to the RTF-S1 system, implicating that the compound inhibits the ATPase activity by shutting down the RTF system and not through actin and myosin. (b) Concentration-dependent transient kinetics of the Ca2+ release rate (s−1) from the RTF system. The AC50 of the increase in the Ca2+ release rate is measured as 3 ± 0.5 μM. (c) The concentration-dependent relative maximum force of permeabilized porcine myocardium. The IC50 of force inhibition is 3 ± 1 μM. IC50 and Hill slope parameters to the fits are given in Table S1. Lines in all plots are Hill fits to the data. (d) Relative force-pCa curve of permeabilized porcine myocardium in the absence or presence of 100 μM inhibitor (MYK-7660) during x-ray diffraction experiments. There is no detectable force increase above baseline generated by the myocardium in the presence of the…

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  Figure S1.. Thin filament-based x-ray reflections in the presence and absence of inhibitor (MYK-7660). (a and b) The intensity of the sixth-order actin-based layer line (a) and the third-order troponin meridional reflection (b) at pCa 4.5 normalized to its value at pCa 8 (C: control; I: inhibitor).

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  Figure 2.. Myosin heads move radially away from the thick filament backbone. (a) Representative x-ray diffraction patterns from permeabilized porcine myocardium in different Ca2+ in the absence of force. (b) Change of equatorial intensity ratio (Δ I1,1/I1,0) at different Ca2+concentrations in the presence (red) and absence (black) of inhibitor (MYK-7660). (c) The radius of the average mass of myosin heads (Rm) at different Ca2+ concentrations in the presence (red) and absence (black) of inhibitor. Ca2+ shifts the distribution of myosin heads away from the thick filament backbone towards the thin filaments. Data are expressed as mean ± SEM (n = 12 in the control group and n = 11 in the inhibitor group).

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  Figure 3.. Thick filament structure changes in the presence of Ca2+. (a and b) The intensity of first-order myosin-based layerline (a) and third-order of myosin-based meridional reflection (b) in different concentrations of Ca2+ in the presence (red) and absence (black) of inhibitor (MYK-7660). (c and d) The intensity (c) and spacing (d) of the sixth-order of myosin-based meridional reflection in different Ca2+ concentrations in the presence (red) and absence (black) of inhibitor. Ca2+ reduces the proportion of myosin heads in ordered states on the thick filament and induces structural changes in the thick filament backbone. Data are expressed as mean ± SEM (n = 12 in the control group and n = 11 in the inhibitor group).

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  Figure 4.. The biochemical SRX states of myosin in reconstituted STF are modulated by Ca2+. (a) Normalized myosin SRX population (expressed as a fraction of the initial %SRX at pCa 8; black symbols) and normalized basal myosin ATPase activity (expressed as [ATPase value − ATPase value at pCa 8]; grey symbols) in STF reconstituted from bovine cardiac full-length myosin (solid symbols) and bovine cardiac myosin S1-subfragment (open symbols) at different Ca2+ concentrations. (b) Normalized myosin SRX population in STF formed from full-length myosin at 30 mM KCl (0 mM free Mg2+, dark grey; and 2 mM free Mg2+, black) and 150 mM KCl (open circles and solid symbols) at different Ca2+ concentrations. Ca2+ destabilizes the biochemical myosin SRX state(s) only when assembled into thick filaments. (c) Normalized basal myosin ATPase activity (expressed as [ATPase value − ATPase value at pCa 8]; in STF reconstituted from bovine cardiac full-length myosin (red), bovine cardiac HMM (black), and subfragment-S1 (grey), and in recombinant human cardiac 2-hep (green) and 25-hep HMM (blue) at different Ca2+ concentrations. All data are expressed as mean ± SEM (n ≥ 9 from three independent experiments that used…

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  Figure 5.. Ca2+-mediated dual filament regulation mechanism in cardiac muscle. A schematic cartoon of the cardiac thin (thin grey bar) and thick filament (thick grey bar; not drawn to scale) showing the organization of the regulated thin filament and myosin. (a) In the absence of Ca2+, the thin and thick filaments in resting muscle are in the deactivated (off) states, and individual myosin heads may be in either the off (orange) or the on (green) state(s). (b) Ca2+ (red) can independently bind to the thick and thin filament and activate them simultaneously. Ca2+-mediated regulation of the thick filament destabilizes the myosin heads from the off states (orange) to the on states (red heads), along with the already on heads (green) which allows the swinging of the S2-subfragment that facilitates myosin heads to bind to actin.