Subsarcomeric regulation of thin and thick filaments in skeletal muscle myofibrils

Fig. 1.. Spatial distributions of TnC and RLC orientations in thin- and thick-filament zones in rigor and relaxed myofibrils. (A) Schematic of a sarcomere highlighting thin (I, A) and thick (P, C, D) filament zones. Sarcomere ends, Z-band (Z); sarcomere midpoint, M-band (M). (B) Phase contrast (PC) and Fluorescence Polarization Microscopy (FPM) images of myofibrils exchanged with RLC-E, TnC-C, and TnC-E probes in rigor conditions in the absence of calcium (pCa 9). Yellow lines mark the sarcomere ends (Z-bands). (Scale bar, 2 µm.) One-dimensional distributions of <P2> along the myofibril axis are shown below the polarized images. (C) Average distribution of <P2> (mean ± SE) for the RLC-E probe (black) along the thick filament and for the TnC-C (cyan) and TnC-E (magenta) probes along the thin filament in single sarcomeres, in Rigor-pCa 9 (empty circles), Rigor-pCa 4.7 (filled circles), and in Relaxing conditions at pCa 9 (triangles) at 17 °C. RLC-E data: Rigor-pCa 9, sarcomere length (SL) = 2.47 ± 0.02 µm (n = 9 myofibrils); Rigor-pCa 4.7, SL = 2.59 ± 0.03 µm (n = 10); Relax-pCa 9, SL = 2.58 ± 0.03 µm (n = 4). TnC-C data: Rigor-pCa 9, SL = 2.63 ± 0.01 µm (n = 14); Rigor-pCa 4.7, SL = 2.67 ±…

Fig. 2.. Effect of temperature and osmotic compression on the spatial distributions of RLC orientations in relaxed skeletal myofibrils. (A) Spatial distribution of <P2> (mean ± SE) for the RLC-E probe along the thick filament in the relaxed sarcomere (pCa 9.0) at 12 °C (n = 4 myofibrils), 17 °C (n = 4), 21 °C (n = 6), 27 °C (n = 4), and 30 °C (n = 4), in the absence of Dextran; sarcomere length = 2.55 ± 0.04 µm. Solid lines, simulated distribution of <P2> for the RLC-E probe using the model described in SI Appendix, Supporting information Text; vertical dashed lines, thick filament tips; green rectangles, C-zone. (B) Average <P2> values (pooled data and mean ± SE) in P, C, and D zones of the thick filament used to simulate the spatial distribution of <P2> along the thick filament shown in A. (C) Spatial distribution of <P2> (mean ± SE) for the RLC-E probe in the absence (empty circles, same data as in A) and in the presence (filled circles) of 4% (w/v) Dextran T-500, at 17 °C (n = 5), 21 °C (n = 6), and 30 °C (n = 6); sarcomere length = 2.52 ± 0.04 µm. Solid and dashed lines, simulated distribution of <P2> in the presence and absence of Dextran, respectively. (D) Average <P2> values in P,…

Fig. 3.. Sarcomere length-dependent activation of the myosin motors in C and D zones of the thick filament in relaxed skeletal myofibrils. (A) Fluorescent images of a single skeletal myofibril exchanged with the RLC-E probe mounted between the puller (Left) and cantilever (Right) in relaxing solution (pCa 9.0) at the initial resting length (Top) and after stretches of 16% (Middle) and 32% (Bottom) of the initial length. Temperature = 30 °C, 4% (w/v) Dextran T-500. The yellow dashed line marks the resting position of the cantilever (zero force). (Scale bar, 10 µm.) (B) Passive force-sarcomere length relationship in single myofibrils without probes (Control, gray; n = 8 myofibrils) and myofibrils exchanged with RLC-E (black; n = 10), TnC-E (magenta; n = 14), and TnC-C (cyan; n = 10) probes. (C) Top panels, schematics of the sarcomere at the three myofibril lengths in (A). Lower panel, average distributions of <P2> (mean ± SE; n = 7) for the RLC-E probe along the thick filament in the relaxed sarcomere (pCa 9.0) at the three sarcomere lengths. Solid and dashed red lines, simulated <P2> distributions for the RLC-E probe along the thick filament at short and long sarcomere lengths; dashed…

Fig. 4.. Spatial distributions of RLC and TnC orientations in the sarcomere of Ca2+-activated myofibrils. (A) Time course of force and (B) sarcomere length (SL) in single skeletal myofibrils activated at time zero at pCa = 6.6 (filled symbols) and pCa = 4.7 (empty symbols), in the absence (black, mean ± SE, n = 15) and in the presence (magenta, mean ± SE, n = 17) of 20 µM Para-Nitro Blebbistatin (PNB). Temperature = 30 °C. Magenta dashed lines mark the zero force and the average SL before activation in the presence of PNB. (C) Spatial distribution of <P2> for the RLC-E probe along the thick filament (circles) and model simulations (lines) before activation (pCa 9) and at the steady state of activation at pCa 6.6 and 4.7 (n = 6 myofibrils for pCa 4.7, n = 5 for pCa 9 and 6.6). (D) <P2> values for the RLC-E probe (pooled data and mean ± SE) in P-, C-, and D-zones of the thick filament used to simulate the spatial distribution of <P2> in C. (E and F) Spatial distributions of <P2> for TnC-C and TnC-E probes along thin filaments (circles) and model simulations (lines) at pCa 9, 6.6, and 4.7 (n = 6 myofibrils). Model parameters are shown in SI Appendix, Table S3. Yellow square, I-band at 2.5 µm…

Fig. 5.. Effect of PNB on the spatial distribution of RLC and TnC probes in the sarcomere of calcium-activated myofibrils. Spatial distributions of <P2> for RLC-E (A), TnC-C (B), and TnC-E (C) probes at pCa 9/6.6/4.7 in the presence of the myosin inhibitor PNB (20 µM) (n = 6 myofibrils for each probe) and at pCa 4.7 in the absence of PNB (filled black circles, as shown in Fig. 4). Yellow squares, I-band width at 2.8 µm sarcomere length. Short-dashed lines, model simulations of probe orientation distribution in the presence of PNB. Solid black lines, model simulations of data in the absence of PNB at pCa 4.7. Model parameters are shown in SI Appendix, Table S3.

Fig. 6.. Zonal activation of thin and thick filaments in the sarcomere of skeletal muscle. Schematic of myosin motor conformations in P, C, D zones of the thick filament and thin filament activation in I- and A-bands in the half-sarcomere in relaxed, rigor, and calcium-activated myofibrils. (A) In the absence of calcium (pCa 9) and under zero filament force the thin filament is in the blocked-OFF state, and the folded-OFF state of myosin is enhanced in the C-zone of the thick filament. Constitutively ON motors are shown in gray. (B) Stretching of the titin links at long sarcomere lengths (≥3.3 µm, pCa 9) causes an increase in the passive force applied on the thick filament associated with conformational changes in D-type and C-type titin super-repeats, in D- and C-zones, respectively, and loss of MyBP-C links in the C-zone. Folded myosin motors are released from the OFF state in D- and C-zones. (C) Under rigor conditions and zero filament force in the presence of calcium, myosin motors are strongly bound to actin, inducing the transition of the thin filament to the Open state (red) in the A-band and to a partially activated state (yellow) in the I-band. (D) At intermediate calcium…