Cooling intact and demembranated trabeculae from rat heart releases myosin motors from their inhibited conformation

Figures

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  Figure S1.. Equatorial and meridional intensity distributions from the same trabecula either quiescent or in the diastolic phase between beats. (A and B) Equatorial (A) and meridional (B) intensity distributions (black; stimulation frequency, 1 Hz; purple, quiescent). Temperature, 27°C. The steplike appearance of the equatorial reflections is due to the horizontal 8× binning of the detector pixels before readout as explained in Materials and methods. Total exposure time, 40 ms. a.u., arbitrary unit.

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  Figure S2.. Reversibility of the effect of temperature on the structure of the thick filament. (A and B) Meridional intensity distributions at 27°C before (black trace) and after (green trace) high (39°C, red trace) and low temperature (10°C, blue trace) in one intact (A) and one demembranated trabecula in the presence of Dextran (B). Total exposure time, 40 ms. a.u., arbitrary unit.

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  Figure S3.. Temperature and pH dependence of the orientation of the myosin regulatory light chain in relaxed trabeculae. Orientation of a bifunctional rhodamine probe cross-linking helices B and C in the N-lobe of the regulatory light chain of myosin (BC probe; Park-Holohan et al., 2021), expressed by the order parameter <P2>, in a cardiac trabecula incubated in relaxing solution with pH set at 7.1 at 23°C (black) and at constant pH = 7.1 (red), in the absence (empty symbols and dashed lines; noDex) or in the presence (filled symbols and continuous lines; Dex) of 3% Dextran (wt/vol). Lines are Boltzmann fits to the data.

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  Figure 1.. X-ray diffraction patterns in isolated trabeculae. (A–D) 2-D x-ray diffraction pattern in intact quiescent trabeculae (A and C) and in relaxed demembranated trabeculae in the presence of 3% Dextran (B and D). Temperature, 38–39°C (A and B) and 7–9°C (C and D). FReLoN detector; sample-to-detector distance, 1.6 m. Data added from n = 4 intact or demembranated trabeculae, respectively. Total exposure time, 160 ms. (E) Schematic of the cardiac sarcomere, delimited by Z-bands (black), contains overlapping actin (dark gray) and myosin filaments (white). MyBP-C–containing C-zone, cyan; layers of myosin motors (horizontal lines on myosin filament). M-band, myosin filament midpoint. (F and G) Meridional intensity distributions at 38–39°C (F) and 7–9°C (G) from the intact and demembranated trabeculae in A and C (magenta line), and B and D (black line), respectively. Data normalized by the main peak of the T1 reflection at 38–39°C for both intact and demembranated trabeculae. Continuous and dashed horizontal lines indicate orders of the shorter (higher-angle, H) and longer (lower-angle, L) myosin periodicities at high temperature (Table S1), respectively.

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  Figure 2.. Effect of cooling on the equatorial reflections. (A) Equatorial intensity distributions (perpendicular to the trabecular axis) from one intact trabecula at five temperatures. Magenta, 39°C; orange, 27°C; green, 22°C; cyan, 17°C; blue, 7°C. (B) Corresponding profiles in one demembranated trabecula in the presence of 3% Dextran. Total exposure time, 40 ms. Vertical continuous and dashed lines, position of 1,0 and 1,1 reflections in intact trabecula at 38°C, respectively. (C and D) Spacing of 1,0 reflection (d10) and ratio of equatorial intensities (I11/I10). Gray circles and line, intact trabeculae; empty circles and dashed black line, demembranated trabeculae in the absence of Dextran; black circles and continuous line, demembranated trabeculae in the presence of 3% Dextran. Mean ± SE; n = 5 intact trabeculae and n = 4 demembranated trabeculae. Gray square in C and D, diastolic value in intact trabeculae paced at 1 Hz (T = 26.4°C; data from Brunello et al., 2020); gray diamond in D, peak force value in intact trabeculae (T = 26.4°C; data from Brunello et al., 2020); black diamond in D, maximal calcium activation ([Ca2+] = 20 µM; 3% Dextran; T = 27°C; from Brunello et al., 2020).…

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  Figure 3.. Effect of cooling on the first myosin layer line (ML1). (A) Axial profiles (parallel to the meridional axis) of the mixed myosin (ML1) and actin (AL1) layer line reflection in intact trabeculae at five relevant temperatures. Magenta, 39°C; orange, 28°C; green, 22°C; cyan, 16°C; blue, 7°C. Data added from n = 5 intact trabeculae. (B) Corresponding profiles in demembranated trabeculae in the presence of Dextran. Data added from n = 4 demembranated trabeculae. Vertical continuous and dashed line, ML1 and AL1 spacings, respectively, as estimated in Brunello et al. (2020). (C) Intensity of ML1 reflection (IML1). Gray circles and line, intact trabeculae; empty circles and dashed black line, demembranated trabeculae in the absence of Dextran; black circles and continuous line, demembranated trabeculae in the presence of 3% Dextran. Mean ± SE; n = 5 intact trabeculae and n = 4 demembranated trabeculae. Data for intact trabeculae are normalized by the IML1 value at 39°C. Data for demembranated trabeculae are normalized by the IML1 value at 38°C in the presence of Dextran. a.u., arbitrary unit.

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  Figure 4.. Effect of cooling on the M3 reflection. (A) Axial profiles of the M3 reflection (parallel to the trabecular axis) in intact trabeculae at five relevant temperatures. (B) Corresponding profiles in demembranated trabeculae in the presence of Dextran. Color code and number of trabeculae as in Fig. 3, A and B; gray trace in B is the axial profile of M3 reflection during maximal calcium activation (pCa 4.7; T = 27°C) scaled with respect to its axial profile at pCa 9.0 at 27°C; data from Brunello et al., 2020. Vertical lines mark the position of the three component peaks of the M3 reflection in intact trabeculae at 39°C (continuous line, la; dashed line, ma; dashed-dotted line, ha) and the additional lower angle peak (star) that was only visible at selected temperatures below 20°C and is not considered to be part of the M3 reflection. (C and D) M3 spacing (SM3; C) and intensity (IM3; D). Data in D are corrected by the radial width and normalized by the value at 39°C and 38°C in intact trabeculae and in demembranated trabeculae in the presence of Dextran, respectively. Gray circles and line, intact trabeculae; empty circles and dashed black line, demembranated trabeculae in the absence…

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  Figure S4.. Effect of cooling on the component peaks of the M3 reflection and the star peak. (A–D) Spacing (A) and fractional intensity in intact (B) and demembranated trabeculae in the absence (C) and presence (D) of Dextran. Diamonds, star peak; inverted triangles, la peak; squares, ma peak; triangles, ha peak. Gray and pink symbols, intact trabeculae; black and purple symbols, demembranated trabeculae in the presence (filled symbols) or in the absence (empty symbols) of Dextran. Mean ± SE; n = 5 intact trabeculae and n = 4 demembranated trabeculae. Red symbols in A and B, diastolic value in intact trabeculae paced at 1 Hz; T = 26.4°C; data added from n = 6 trabeculae. Green symbols in A, C, and D, demembranated trabeculae in the presence of 3% Dextran at full calcium activation, pCa 4.7; T = 27°C; data from Brunello et al., 2020. (E) Cross-meridional width of the M3 reflection (wM3). Gray symbols, intact trabeculae; black symbols, demembranated trabeculae in the presence (filled symbols) or absence (empty symbols) of Dextran. Mean ± SE; n = 5 intact trabeculae and n = 4 demembranated trabeculae.

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  Figure 5.. Effect of cooling on the M6 reflection. (A) Axial profiles of the M6 reflection in intact trabeculae at five relevant temperatures. (B) Corresponding profiles in demembranated trabeculae in the presence of Dextran. Color code and number of trabeculae as in Fig. 3, A and B. Vertical dashed lines mark the position of the two component peaks of the reflection in intact trabeculae at 39°C. (C and D) M6 spacing (SM6; C) and intensity (IM6; D). Data in D are corrected by the radial width and normalized by the value at 38°C in intact trabeculae and in demembranated trabeculae in the presence of Dextran. Gray circles and line, intact trabeculae; empty circles and dashed black line, demembranated trabeculae in the absence of Dextran; black circles and continuous line, demembranated trabeculae in the presence of 3% Dextran. Mean ± SE; n = 5 intact trabeculae and n = 4 demembranated trabeculae. Gray square and diamond in C, diastolic and systolic peak force values, respectively, in intact trabeculae paced at 1 Hz (T = 26.4°C; data from Brunello et al., 2020); black diamond in C, maximal calcium activation ([Ca2+] = 20 µM; 3% Dextran; T = 27°C; data from Brunello et al., 2020). a.u.,…

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  Figure S5.. Effect of cooling on the M1 reflection. (A and B) Axial profiles of the M1 and T1 reflections from intact trabeculae (A) and demembranated trabeculae in the presence of dextran (B). Magenta, 39/38°C; orange, 28/26°C; green, 22°C; cyan, 16/18°C; blue, 7/9°C in intact and demembranated trabeculae, respectively. Vertical continuous and dashed lines, position of the two main peaks of the M1 reflection, and dashed-dotted line, average position of the T1 reflection in intact trabeculae at 39°C. (C–F) M1 spacing (SM1; C), total intensity (IM1; D), and spacing (E) and fractional intensity (F) of its two component peaks (ha, triangles; la, inverted triangles). Data added from n = 5 intact trabeculae and n = 4 demembranated trabeculae. Gray symbols, intact trabeculae; black symbols, demembranated trabeculae in the presence (filled symbols) or absence (empty symbols) of Dextran. Red symbols, diastolic values in intact trabeculae paced at 1 Hz; T = 26.4°C; data added from n = 6 trabeculae. Data in D normalized by the value at 39/38°C in intact trabeculae and in demembranated trabeculae in the presence of Dextran. a.u., arbitrary unit.

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  Figure S6.. Effect of cooling on the T1 reflection. (A and B) T1 spacing (ST1; A), total intensity (IT1; B). Gray symbols, intact trabeculae; black symbols, demembranated trabeculae in the presence (filled symbols) or absence (empty symbols) of Dextran. Data added n = 5 intact and n = 4 demembranated trabeculae. Red square in A, diastolic value in intact trabeculae paced at 1 Hz; T = 26.4°C; data added from n = 6 trabeculae. Intensities normalized by the value at 39/38°C in intact trabeculae and in demembranated trabeculae in the presence of Dextran.

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  Figure S7.. Effect of cooling on the M2 reflection. (A and B) Axial profiles of the M2 reflection from intact trabeculae (A) and demembranated trabeculae in the presence of Dextran (B). Magenta, 39/38°C; orange, 27/26°C; green, 22°C; cyan, 16/18°C; blue, 9/7°C in intact and demembranated trabeculae, respectively. Vertical dotted and dashed-dotted line, position of the two main peaks of M2L; continuous and dashed lines, position of the two main peaks of M2H in intact trabeculae at 39°C. (C–F) M2L spacing (SM2L; C) and total intensity (IM2L; D) and M2H spacing (E) and total intensity (F). Gray circles and line, intact trabeculae; empty circles and dashed black line, demembranated trabeculae no Dextran; black circles and continuous line, demembranated trabeculae in the presence of Dextran. Data added from n = 5 intact trabeculae and n = 4 demembranated trabeculae. Red square in C and E, diastolic value in intact trabeculae paced at 1 Hz; T = 26.4°C; data added from n = 6 trabeculae. Intensities in D and F are normalized by IM2H at 39/38°C in intact trabeculae and in demembranated trabeculae in the presence of Dextran. a.u., arbitrary unit.