Orthophosphate increases the efficiency of slow muscle-myosin isoform in the presence of omecamtiv mecarbil

Figures

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  Fig. 1. Effect of OM on isometric force development. Force development following T-jump (from 1 to 12 °C) in control (a) and in the presence of 1 μM OM (b) on an activated fibre (pCa 4.5). Traces show the length change per half-sarcomere (upper trace) and the force (lower trace). The artefact in the traces marks the transition to the test temperature drop. The horizontal line in the lower panels indicates zero force. Fibre length, 4.5 mm; length of the segment under the striation follower, 0.8 mm; average sarcomere length within the segment, 2.3 µm; CSA, 3000 µm2; pCa, 4.5. Dependence of the isometric force T0 (c) and of the rate of force development rTD (d) on the concentration of OM. Lines, simulation with model of Fig. 6. Data are mean values ± SEM from eight fibres. Source data are provided as a Source Data file.

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  Fig. 2. Effect of OM on the relevant mechanical parameters determined by stiffness measurements. Sample records of the force response (middle trace) to a step length change (upper trace) imposed at the isometric plateau of an activated fibre (pCa 4.5) in control (a) and in the presence of 1 μM OM (b). Lower traces indicate zero force. c T1 relations in control (filled circles) and in the presence of 1 μM OM (open circles) from the same fibre as (a) and (b). Dashed lines are 1st order regression equations fitted to the data. Same fibre as in Fig. 1. d Dependence of T0 (left ordinate, circles) and half-sarcomere stiffness (right ordinate, triangles) on the concentration of OM. Circles are the same data as in Fig. 1c. Continuous line, model simulation of T0–[OM] relation; dashed line, mean value of k0 in the whole range of OM concentrations. e Dependence of half-sarcomere strain (Y0) on OM-modulated isometric force (T0). Dotted line, linear regression on data points. f Dependence of cross-bridge stiffness (e0) on the concentration of OM. Continuous line, value in the absence of OM. Data points in d–f are mean values ± SEM from eight fibres. Source data are provided as a Source Data file.

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  Fig. 3. Effect of OM on myofilament Ca2+ sensitivity for motor attachment. a T0-pCa relation in control (filled circles) and in the presence of 1 μM OM (open circles). Continuous (control) and dashed (1 μM OM) lines are calculated by fitting Hill equation T0 = T0,45/( 1+ 10n(pCa−pCa50)) to data. The best fit parameters are listed in Table 2. b Dependence of the stiffness of the motor array (e0, left ordinate) and of the fraction of attached motors (β, right ordinate) on [Ca2+]. β in control at saturating [Ca2+] is assumed 0.4719. Continuous (control) and dashed (1 μM OM) lines are calculated by fitting the Hill’s sigmoidal equation to data. c Dependence of the strain per motor (s0, left ordinate) and of the force per motor (F0, right ordinate) on [Ca2+]. d Dependence of the difference (ΔT0) between the fitted T0-pCa relation in 1 μM OM (dashed line in a) and in control (continuous line in a) on [Ca2+]; dashed line indicates zero difference. In all panels, [Ca2+] is expressed in pCa units. Data in a–c are mean values ± SEM from four fibres. Source data are provided as a Source Data file.

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  Fig. 4. Effect of Pi on the isometric mechanical parameters with and without OM. Filled circles and continuous lines, control; open circles and dashed lines, in the presence of 1 μM OM. a Pi dependence of the isometric force (T0) expressed in units relative to the value without added Pi (T0,1). b Rate of force development (rTD). c Stiffness of the motor array (e0) relative to the value without added Pi (e0,1). d Motor strain (s0, left ordinate) and motor force (F0, right ordinate). In each panel: symbols, experimental data; lines, model simulation. Data are mean values ± SEM from three fibres. Source data are provided as a Source Data file.

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  Fig. 5. Effect of OM on the rate of ATP hydrolysis in isometric contraction and its modulation by Pi. a Sample records from an activated fibre (pCa 4.5) of the time course of force and light absorbance (in absorbance units, a.u.) in the absence (left) and in the presence (right) of 1 μM OM. The dotted line on the absorbance trace is the 1st order regression equation fitted to the trace after force has attained a steady value. The slope of the regression is used as a measure of the ATP hydrolysis rate (kcat). b Dependence on OM of T0 (circles and dashed line) and kcat (triangles and continuous line). Values are relative to the isometric values in the absence of OM (98 ± 17 kPa and 0.055 ± 0.007 mM s−1, respectively). c Dependence on Pi of the isometric force (T0) relative to the value without added Pi (T0,1) in the absence (filled circles and continuous line) and in the presence (open circles and dashed line) of 1 μM OM. d Dependence on Pi of kcat relative to the value without added Pi (kcat,1). Symbols and lines as in (c). e Tension cost of the isometric contraction (ET). Symbols and lines as in (c). ET is normalized by the value in the absence of OM and no added Pi. b–e symbols,…

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  Fig. 6. Chemo-mechanical cycle of the myosin motor. The green-highlighted part is the original model37; the pink-highlighted part is the branched pathway promoted by OM. Myosin catalytic domain is green for the motors without OM (M) and magenta and brown for the OM-bound motors (MOM) before and after the isomerization step (step 11), respectively. The working stroke in the attached head is indicated by the tilting of the light chain domain (the lever arm, blue) towards the Z-line. The system of linear differential equation for the calculation of state transitions is reported in Supplementary Note 2.

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  Fig. 7. Estimate of myofilament compliance and motor strain. T1 relations from a fibre in control (a) and in the presence of 1 μM OM (b) at pCa 4.5 (circles) and 6.59 (squares). The relations are obtained by plotting the extreme force attained at the end of the length step, T1, versus the step size. Lines in a and b are 1st order regression equations fitted to the data (pCa 4.5, continuous; pCa 6.59, dashed). The abscissa intercept of each line is the average strain of the half-sarcomere before the length step (Y0). Fibre length, 4.2 mm; segment length under the striation follower, 0.77 mm, average segment sarcomere length, 2.34 µm, test temperature, 12.2 °C. c Relations of Y0 versus T0 at different pCa determined in control (filled circles) and in the presence of 1 μM OM (open circles); mean values ± SEM from four fibres. The lines are linear regressions on the pooled data for forces >30 kPa in control (continuous) and in the presence of OM (dashed). The slope and the ordinate intercept of the linear regressions are listed in Table 1. d Relations of half-sarcomere strain (Y0) versus the stiffness of the myosin motors at different pCa (e0) in control (filled circles) and in the presence…