Stress-dependent activation of myosin in the heart requires thin filament activation and thick filament mechanosensing

Figures

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  Fig. 1.. Temperature dependence of RLC N- and C-lobe orientation in relaxed trabeculae with unphosphorylated RLC. (A) Orientation of E (black) and BC (red) probes in the C- and N-lobe of RLC, respectively, in the blocked (orange) and free (yellow) head in the human β-cardiac myosin IHM (PDB 5TBY, essential light chains not shown for clarity), with respect to the filament axis (dashed line). (B) <P2> for E (black; mean ± SEM, n = 3) and BC (red; mean ± SEM, n = 3) probes in the absence (open circles) and in the presence (filled circles) of 3% Dextran T-500 in relaxed trabeculae at pCa 9 at 2.1 µm SL, fitted with Boltzmann curves (SI Appendix, Table S1 and Materials and Methods). Filled and open diamonds: values of <P2> (mean ± SEM, n = 3) at 26 °C and pCa 4.7 at the plateau of contraction in the presence (active isometric force per CSA [T0] = 84 ± 7 kPa; mean ± SEM, n = 6) and in the absence of Dextran (T0 = 78 ± 5 kPa; mean ± SEM, n = 6), respectively. Before activation, SL was set at 2.1 µm. (C) Mean angle (θME) and dispersion (σME) of the maximum entropy orientation distribution for E and BC probes in the presence of Dextran before RLC phosphorylation under relaxing conditions (pCa 9)…

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  Fig. 2.. Temperature dependence of RLC N- and C-lobe orientation in relaxed trabeculae after RLC phosphorylation. (A) <P2> for E (black; mean ± SEM, n = 4) and BC (red; mean ± SEM, n = 5) probes before (filled triangles) and after (open triangles) RLC phosphorylation in relaxed trabeculae at pCa 9.0 at 2.1 µm SL in the presence of 3% Dextran, fitted with Boltzmann curves (SI Appendix, Table S1). Filled and open inverted triangles: values of <P2> at 26 °C and pCa 4.7 (mean ± SEM, n = 3) before (T0 = 84 ± 7 kPa; mean ± SEM, n = 6) and after RLC phosphorylation (T0 = 65 ± 6 kPa; mean ± SEM, n = 6), respectively. (B) Mean angle (θME) and dispersion (σME) of the maximum entropy orientation distribution for E and BC probes in the presence of Dextran under relaxing conditions at 32 °C before RLC phosphorylation (pink) and after RLC phosphorylation at 32 °C (violet) and 5 °C (blue) and during active contraction at 26 °C (green). Bars are mean ± SEM.

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  Fig. 3.. Effect of SL on RLC N- and C-lobe orientation in relaxed trabeculae. (A) Trabecular length change relative to the initial length (L0) in relaxed trabeculae at pCa 9.0 ([Ca2+] = 1 nM); the SL in micrometers shown above the trace was measured before the stretch staircase and calculated after each stretch from the relation in SI Appendix, Fig. S3. Passive force response and <P2> for E and BC probes were recorded in single trabeculae before (black) and after (gray) RLC phosphorylation. Values of <P2> during full calcium activation for the two probes (E, triangles; BC, circles) are shown in the box. Temperature 26 °C, 3% Dextran T-500. Dependence of passive force (B) and <P2> (C) on SL, recorded at the initial length and at 5 s after each stretch, before (black) and after (gray) RLC phosphorylation; triangles, E probe (mean ± SEM, n = 4); circles, BC probe (mean ± SEM, n = 5). Dashed line in B is the curve fitted to all data using the function F = exp(a + b × SL + c × SL2) (a = 87.5, b = −88.9 µm−1, c = 22.5 µm−2). Dashed lines in C denote relaxed <P2> values at short SL; active <P2> values are shown in the box (symbols as in A).

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  Fig. 4.. The effect of SL on RLC-lobe orientation in trabeculae activated at submaximal [Ca2+]. (A) Relative trabecular length change (ΔL/L0) and SL in µm calculated from the relation in SI Appendix, Fig. S3. Force and <P2> for E and BC probes during the stretch/release staircase recorded in single trabeculae at different [Ca2+] before and after RLC phosphorylation and in the presence of Blebbistatin 25 µM, as shown in the legend. Dashed lines: <P2> values in relaxing conditions (pCa 9) before (black) and after (gray) RLC phosphorylation. Values of <P2> for the two probes (E, triangles; BC, circles) during full calcium activation before (black) and after (gray) RLC phosphorylation are shown in the box (same as in Fig. 2A). Temperature, 26 °C; 3% Dextran T-500. (B) Traces at the time marked (asterisk) in A are shown on an expanded time scale; dashed lines indicate pre-stretch values. The dependence of force (C) and <P2> (D) on SL recorded before the stretch staircase and at 5 s after each stretch; triangles, E probe (mean ± SEM, n = 4); circles, BC probe (mean ± SEM, n = 4); same color code as in A. Data in the presence of 25 µM Blebbistatin (purple) are mean ± SD, n = 2. Dashed line in C…

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  Fig. 5.. RLC phosphorylation enhances SA in partially activated cardiac trabeculae. (A) Relative trabecular length change and SL changes in micrometers (calculated from SI Appendix, Fig. S3), FSA, and <P2> changes (Δ<P2>) for E and BC probes with respect to their relaxed values (pCa 9.0) during the stretch staircase. (B) Traces at the time marked (asterisk) in A shown on an expanded time scale. Dashed lines mark the zero.

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  Fig. 6.. The dependence of the RLC-lobe orientation on filament stress. Dependence of the relative change in probe orientation (R) on passive (circles, pCa 9.0) and active filament stress in the presence of calcium (squares, pCa 7.00; triangles, pCa 6.60; inverted triangles, pCa 6.48; and diamonds, pCa 6.34) in cardiac trabeculae before (black) and after (orange) RLC phosphorylation. Filled symbols, E probe (mean ± SEM, n = 4); open symbols, BC probe (mean ± SEM, n = 5). Short dashed line marks the zero. Red line, linear regression on the data at pCa 9 with 95% confidence bands [slope = (1.2 ± 0.2) × 10−3 pN−1; intercept = 0.002 ± 0.005]. Orange line: linear regression on the data at pCa < 9.0 with 95% confidence bands [slope = (8.7 ± 0.4) × 10−3 pN−1; intercept = 0.059 ± 0.006]. In gray, dependence of R on passive force in the presence of 25 µM Blebbistatin (crossed circles) and on active force (circles; mean ± SEM, n = 5) in the pCa range 9 to 4.7 for the skeletal RLC E probe in muscle fibers, calculated from figure 5 in Fusi et al. (5). Dashed and solid gray lines, linear regressions on passive [slope = (2.5 ± 0.1) × 10−3 pN−1; intercept = −0.042 ± 0.009] and active [slope = (2.5 ±…

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  Fig. 7.. A working hypothesis for the changes in the conformation of myosin motors in the C-zone of the thick filament induced by RLC phosphorylation and stretch. (A) In the relaxed cardiac trabecula (pCa 9 to 7), folded helical myosin motors (black) have RLC N- and C-lobes in IHM orientations (magenta and yellow circles, respectively). Folded nonhelical (gray) motors have only the N-lobe in IHM orientations and the C-lobe in conformational equilibrium (magenta wedges). MyBP-C (white circles) in the C-zone may link to actin or tether folded helical motors. The thin filament (dark gray) is fully switched off. Z, Z-disk. (B) Physiological levels of RLC phosphorylation (star) release only a few motors from the folded conformation (light gray) and favor the formation of MyBP-C links with actin. (C) The thin filament is partially activated by calcium (pCa > 7) and by MyBP-C links, allowing myosin motors to bind to actin and generate force (green). (D) The stretch (ΔL) triggers the stress-dependent activation of the myosin motors and may promote the formation of additional MyBP-C links that further increase the activation of the thin filament, allowing more force-generating motors to bind to…