Crossbridge properties investigated by fast ramp stretching of activated frog muscle fibres

Abstract

Very fast ramp stretches at 9.5-33 sarcomere lengths s(-1) (l0 s(-1)) stretching speed, 16-25 nm per half-sarcomere (nm hs(-1)) amplitude were applied to activated intact frog muscle fibres at tetanus plateau, during the tetanus rise, during the isometric phase of relaxation and during isotonic shortening. Stretches produced an almost linear tension increase above the isometric level up to a peak, and fell to a lower value in spite of continued stretching, indicating that the fibre became suddenly very compliant. This suggests that peak tension (critical tension, P(c)) represents the tension at which crossbridges are forcibly detached by the stretch. The ratio of P(c) to the isometric tension at tetanus plateau (P0) was 2.37 +/- 0.12 (S.E.M.). This ratio did not change significantly at lower tension (P) during the tetanus rise but decreased with time during the relaxation and increased with speed during isotonic shortening. At tetanus plateau P(c) occurred when sarcomere elongation attained a critical length (L(c)) of 10.98 +/- 0.13 nm hs(-1), independently of the stretching speed. L(c) remained constant during the tetanus rise but decreased on the relaxation and increased during isotonic shortening. Length-clamp experiments on the relaxation showed that the lower values of P(c)/P ratio and L(c), were both due to the slow sarcomere stretching occurring during this phase. Our data show that P(c) can be used as a measure of crossbridge number, while L(c) is a measure of crossbridge mean extension. Accordingly, for a given tension, crossbridges on the isometric relaxation are fewer than during the rise, develop a greater individual force and have a greater mean extension, while during isotonic shortening crossbridges are in a greater number but develop a smaller individual force and have a smaller extension.

Figure 1. Force response of a single intact muscle fibre to a fast ramp stretch applied at tetanus plateau

The peak tension reached during the stretch represents the tension necessary to break the attached crossbridges. The arrow on the sarcomere length signal indicates the time of the tension peak. Broken vertical line and short break on the record indicate the start and the end of the fast time base recording. The vertical dotted line shows the end of the isometric phase of relaxation after which sarcomere length along the fibre starts to become inhomogeneous as indicated by the great shortening occurring beyond this point. Time elapsed during the break, 44 ms. Stretching speed: 14.7 l₀ s⁻¹.

Figure 2. Critical tension as a function of relative tension during the tetanus rise (A) and relative plateau tensions in presence of various BDM concentrations (B)

Different symbols represent different experiments from 10 (tetanus rise) and 3 (BDM data) fibres. The lines on the graphs represent the direct proportionality. The slight deviation from the linearity at low tensions on the tetanus rise, is probably due to the small sarcomere shortening occurring during this phase, which tends to increase the critical tension. The deviation is not present on BDM data since no sarcomere shortening was occurring during the stretch application.

Figure 3. Critical tension as a function of relative tension during isotonic shortening, and during relaxation in normal and length-clamped conditions

Open symbols refer to data on the isometric relaxation (5 fibres). Half-filled symbols refer to experiments in which sarcomere length was clamped during relaxation. Length-clamp eliminated the deviation from linearity present on the isometric relaxation. For clarity only one experiment is plotted, but similar results were obtained in all the four experiments carried out. The isotonic data (6 fibres, filled symbols) were fitted (broken line) by the linear equation: P_c= 0.362 + 0.64 P/P₀. The intercept of this equation on the ordinate (zero isotonic tension), shows that P_c is 0.36 the plateau value. This means that at V_max, attached crossbridges are still 36% of crossbridges attached at plateau of an isometric tetanus. Different symbols represent different experiments.

Figure 4. Critical length at various relative tensions during tetanus rise, isotonic shortening, isometric and length–clamp relaxation

L_c is constant during the tetanus rise (□), while at low tensions it increases during the shortening (▴) and decreases during relaxation (○). This last effect is not present when relaxation data are obtained under length-clamp conditions (half-filled circles). Mean and s.e.m. from 6 (isotonic), 10 (rise), 5 (relaxation) and 4 (relaxation in length-clamp) experiments.

Figure 5. Effect of sarcomere length-clamp on the force response to the stretch on the tetanus relaxation

Continuous lines, length-clamp; short broken lines, isometric contraction from the same fibre. Records in length clamp are shifted upward for clarity. Note the slow sarcomere lengthening occurring during the isometric phase of relaxation and its disappearance upon length-clamping. Critical force and critical length are both increased by the length-clamp procedure. The broken vertical line indicates the change from the slow to the fast sampling. Length clamp starts at the vertical arrow and ends 0.5 ms before the stretch.