Myosin and tropomyosin-troponin complementarily regulate thermal activation of muscles

Abstract

Contraction of striated muscles is initiated by an increase in cytosolic Ca2+ concentration, which is regulated by tropomyosin and troponin acting on actin filaments at the sarcomere level. Namely, Ca2+-binding to troponin C shifts the "on-off" equilibrium of the thin filament state toward the "on" state, promoting actomyosin interaction; likewise, an increase in temperature to within the body temperature range shifts the equilibrium to the on state, even in the absence of Ca2+. Here, we investigated the temperature dependence of sarcomere shortening along isolated fast skeletal myofibrils using optical heating microscopy. Rapid heating (25 to 41.5°C) within 2 s induced reversible sarcomere shortening in relaxing solution. Further, we investigated the temperature-dependence of the sliding velocity of reconstituted fast skeletal or cardiac thin filaments on fast skeletal or β-cardiac myosin in an in vitro motility assay within the body temperature range. We found that (a) with fast skeletal thin filaments on fast skeletal myosin, the temperature dependence was comparable to that obtained for sarcomere shortening in fast skeletal myofibrils (Q10 ∼8), (b) both types of thin filaments started to slide at lower temperatures on fast skeletal myosin than on β-cardiac myosin, and (c) cardiac thin filaments slid at lower temperatures compared with fast skeletal thin filaments on either type of myosin. Therefore, the mammalian striated muscle may be fine-tuned to contract efficiently via complementary regulation of myosin and tropomyosin-troponin within the body temperature range, depending on the physiological demands of various circumstances.

Figure S1.

Fluctuation analysis for the average length of five sarcomeres along a fast skeletal myofibril at rest. (A) Snapshot of phase-contrast image sequence of two rabbit psoas myofibrils immersed in relaxing solution (pCa 9). Pink arrows indicate six consecutive Z-disks analyzed in B; i.e., the average length of five sarcomeres was analyzed. Imaging was performed at 30 fps. Scale bar, 5 µm. (B) Time course of changes in the average SL. (C) Histogram showing the variance of SL. Average SL, 2.16 ± 0.017 µm (mean ± SD), indicating that the precision is 17 nm under the present experimental setting.

Figure 1.

Thermal activation of fast skeletal myofibrils immersed in relaxing solution. (A) Schematic illustration of the experimental system. A rabbit psoas myofibril was immersed in relaxing solution on a coverslip. Temperature was directly increased by an IR laser beam (λ = 1,455 nm), which elicits sarcomere shortening as a function of the distance from the heat source (as shown by the red-yellow gradient; see the color bar on the right). (B) Changes in temperature (ΔT) generated by IR-laser irradiation. Three levels of gradients were adjusted by neutral density filters: 40%, 25%, and 15% for high, medium, and low powers, respectively. Data expressed as mean ± SEM. Error bars are <0.2°C (within plots). n = 3 for all power levels. (C) Left: Phase-contrast images of a skeletal myofibril in the relaxing solution (pCa 9). Top, middle, and bottom show images before, during, and 1 s after heating for 2 s, respectively. Sarcomeres within the yellow-outlined rectangle were analyzed. Pink and blue arrows in each image indicate the positions of Z-disks along a myofibril analyzed. Imaging was performed at 30 fps. Right: Intensity profiles of four sarcomeres along a myofibril shown left. Starting point on the x-axis (i.e., 0 µm) indicates the position of the Z-disk shown by a pink arrow before heating. The heat source was ∼28 µm away from the myofibril. ΔT was 17.1°C (cf. temperature gradient for high power in B). (D) Relationship of the temperature during heating versus changes in sarcomere length (ΔSL). ΔSL was calculated as SL before heating minus SL during heating. Each plot in the graph indicates the average shortening of sequentially connected four to six sarcomeres along a myofibril. n = 20, 10, and 11 myofibrils at 31.6 ± 0.1°C, 38.9 ± 0.3°C, and 41.5 ± 0.2°C, respectively. Data expressed as mean ± SEM. P is determined by Dunnett’s multiple comparison test. *P < 0.05. NS, not statistically significant. (E) Arrhenius plot for ΔSL. ΔSL is expressed in logarithm. Average values from D were used. ΔSL = 1.08 × 10²⁶ exp (−1.65 × 10⁵/RT) (r² = 1.00). T, absolute temperature. R, gas constant. r², coefficient of determination. Experiments were performed at 25 ± 1°C.

Figure 2.

Thermal activation of F-actin, fast skeletal thin filaments, and cardiac thin filaments on fast skeletal myosin in an in vitro motility assay. (A) Schematic illustration showing the experimental system. Actin filaments (F-actin) or reconstituted skeletal or cardiac thin filaments interacted with skeletal or β-cardiac myosin treated on the glass coverslip in the absence (pCa 9) or presence (pCa 5) of Ca²⁺ (as shown on right). Temperature was increased by IR laser irradiation (from the baseline temperature of 23°C up to 40°C), inducing filament movements in various directions. The periods of heating were 2 and 10 s for skeletal and β-cardiac myosin, respectively. (B) Relationship between temperature and the sliding velocity of F-actin (black open circles), skeletal thin filaments (red closed circles), or cardiac thin filaments (blue closed circles) on skeletal myosin at pCa 9. n = 786, 684, and 665 for F-actin, skeletal thin filaments, and cardiac thin filaments, respectively. Data expressed as mean ± SEM. (C) Same as in B at pCa 5. n = 1,453, 1,296, and 1,442 for F-actin, skeletal thin filaments, and cardiac thin filaments, respectively. (D) Graph comparing the sliding velocity ratios at pCa 9/pCa 5 for F-actin, skeletal thin filaments, and cardiac thin filaments over the range of temperature. Data expressed as mean ± SEM. (E) Arrhenius plot for the sliding velocity of F-actin, skeletal thin filaments, and cardiac thin filaments at pCa 9. T, absolute temperature. Average values from B were used. Sliding velocity (V) is expressed in logarithm. F-actin: V = 1.09 × 10⁶ exp (−2.85 × 10⁴/RT) (r² = 0.95). Skeletal thin filaments: V = 2.30 × 10²⁹ exp (−1.68 × 10⁵/RT) (r² = 0.98). Cardiac thin filaments: V = 1.27 × 10¹⁷ exp (−9.43 × 10⁴/RT) (r² = 0.97). Q₁₀, 1.4, 8.2, and 3.3 for F-actin, skeletal thin filaments and cardiac thin filaments, respectively. (F) Same as in E for F-actin, skeletal thin filaments, and cardiac thin filaments at pCa 5. Average values from C were used. F-actin: V = 8.15 × 10⁵ exp (−2.73 × 10⁴/RT) (r² = 0.97). Skeletal thin filaments: V = 2.11 × 10 exp (−1.76 × 10⁴/RT) (r² = 0.80). Cardiac thin filaments: V = 3.86 × 10⁴exp (−1.94 × 10⁴/RT) (r² = 0.90). Q₁₀, 1.4, 1.2, and 1.3 for F-actin, skeletal thin filaments and cardiac thin filaments, respectively. Data obtained below 32°C (shown in the grey region) were not employed for the fitting in E and F. See Tables S1, S2, and S3 for details.

Figure S2.

Temperature gradients in an in vitro motility assay. Changes in temperature (ΔT) generated by IR-laser irradiation were measured based on the changes in fluorescence intensity of rhodamine-labeled actin filaments. Three levels of gradients were adjusted by using neutral density filters: 60%, 50%, and 15% for high, medium, and low powers, respectively. Data are expressed as mean ± SEM. Error bars, <0.2°C (within plots). n = 3 for all power levels.

Figure 3.

Thermal activation of F-actin, fast skeletal thin filaments, and cardiac thin filaments on β-cardiac myosin in an in vitro motility assay. (A) Relationship between temperature and the sliding velocity of F-actin (black open squares), skeletal thin filaments (red closed squares), and cardiac thin filaments (blue closed squares) on β-cardiac myosin at pCa 9. Temperature was increased from the baseline of 23°C by IR-laser irradiation for 10 s. n = 545, 354, and 720 for F-actin, skeletal thin filaments, and cardiac thin filaments, respectively. Data expressed as mean ± SEM. (B) Same as in A at pCa 5. n = 931, 1,121, and 872 for F-actin, skeletal thin filaments, and cardiac thin filaments, respectively. (C) Graph comparing the sliding velocity ratios at pCa 9/pCa 5 for F-actin and skeletal thin filament over the range of temperature. Data expressed as mean ± SEM. (D) Arrhenius plot for the sliding velocity of F-actin, skeletal thin filaments, and cardiac thin filaments at pCa 9. T, absolute temperature. Average values from A were used. Sliding velocity (V) is expressed in logarithm. F-actin: V = 5.25 × 10¹⁷ exp (−1.00 × 10⁵/RT) (r² = 0.98). Skeletal thin filaments: V = 1.43 × 10⁴⁶ exp (−2.72 × 10⁵/RT) (r² = 0.95). Cardiac thin filaments: V = 2.00 × 10²² exp (−1.30 × 10⁵/RT) (r² = 0.88). Q₁₀, 3.5, 30, and 5.1 for F-actin, skeletal thin filaments, and cardiac thin filaments, respectively. (E) Same as in D for F-actin, skeletal thin filaments, and cardiac thin filaments at pCa 5. Average values from B were used. F-actin: V = 1.57 × 10¹⁶ exp (−9.09 × 10⁴/RT) (r² = 0.99). Skeletal thin filaments: V = 6.57 × 10¹³ exp (−7.66 × 10⁴/RT) (r² = 0.99). Cardiac thin filaments: V = 3.76 × 10⁶ exp (−3.37 × 10⁴/RT) (r² = 0.89). Q₁₀, 3.1, 2.6, and 1.5 for F-actin, skeletal thin filaments, and cardiac thin filaments, respectively. Data obtained below 32°C (shown in the gray region) were not employed for the fitting in D and E. See Tables S4, S5, and S6 for details.

Figure S3.

Fraction of mobile filaments upon IR laser irradiation at pCa 9. (A) Relationship between temperature and the fraction of mobile skeletal thin filaments (red closed circles) or cardiac thin filaments (blue closed circles) on skeletal myosin. Regression analysis was performed based on the Hill equation (see Materials and methods). The T₅₀ values were 32.5°C and 28.5°C for skeletal and cardiac thin filaments, respectively. (B) Relationship between temperature and the fraction of mobile skeletal thin filaments (red closed squares) or cardiac thin filaments (blue closed squares) on β-cardiac myosin. The T₅₀ values were 34.1°C and 30.3°C for skeletal and cardiac thin filaments, respectively. See Table S7 for details.

Figure 4.

Proposed complementary effects of myosin and regulatory proteins on the temperature dependence of sarcomeric activation in fast skeletal versus cardiac muscle. Left: Regulation of the thin filament state by Ca²⁺ (top) or heating (bottom). Ca²⁺ binding to TnC shifts the thin filament state from off to on (top). Heating induces Ca²⁺-independent thermal activation of thin filaments via partial dissociation of Tm–Tn from F-actin (or weakening of the binding of Tm–Tn to F-actin), resulting in sliding movements of thin filaments in an in vitro motility assay (sliding velocity faster with fast skeletal Tm–Tn on fast skeletal myosin [red line] than with cardiac Tm–Tn on cardiac myosin [blue line]; see Figs. 2 and 3). Right: Complementary relationship between temperature and Tm–Tn regulation (left) or ATPase cycle of myosin (right) that causes Ca²⁺-independent thermal activation observed in the present study. Curved arrows indicate the rates of attachment or detachment of myosin to actin. The present as well as previous studies (Chandy et al., 1999; Houmeida et al., 2010; Risi et al., 2017; Heeley et al., 2002, 2006, 2019) suggest that the inhibitory effect of Tm–Tn on the thin filament state (i.e., Tm–Tn regulation) against a rise of temperature is weaker in cardiac muscle (blue line) than in fast skeletal muscle (red line). Therefore, an increase in temperature to within the body temperature range activates thin filaments to a greater magnitude in fast skeletal muscle (high Q₁₀) than in cardiac muscle (low Q₁₀). The ATPase cycle of fast skeletal myosin (red line) is faster than that of β-cardiac myosin (blue line) (Walklate et al., 2016), which results in a relative loss of recruitable heads in fast skeletal muscle at temperatures lower than body temperature. Accordingly, an increase in temperature to the body temperature range activates myosin to a greater magnitude in cardiac muscle (high Q₁₀) than in fast skeletal muscle (low Q₁₀). This way, the temperature dependence of striated muscle contraction is regulated by Tm–Tn and myosin in a complemental manner.