The effect of length on the relationship between tension and intracellular [Ca2+] in intact frog skeletal muscle fibres

Abstract

1. The relationship between tension and intracellular calcium concentration ([Ca2+]i) in intact frog skeletal muscle fibres was determined at two fibre lengths, corresponding to mean sarcomere lengths (SL) of 2.2 and 2.9 micron. Tension and [Ca2+]i were recorded during the slow decline of tension following stimulation in the presence of cyclopiazonic acid (CPA), a sarcoplasmic reticulum Ca2+-uptake pump inhibitor. [Ca2+]i was estimated by injecting the K+ salt form of the fluorescent dye fura-2 into the fibres. Experimental temperature was 3.0 C. 2. At a SL of 2.2 micron, where thick and thin filaments fully overlap, the [Ca2+]i corresponding to 50 % tension generation ([Ca2+]50) was 1.09 +/- 0.02 microM (mean +/- S.E.M., n = 61 contractions). At a SL of 2.9 micron, where overlap is approximately 50 %, the [Ca2+]50 was significantly lower, 0.69 +/- 0. 02 microM (n = 22 contractions). This is in agreement with previous results from skinned fibres. 3. The relationship between tension and [Ca2+]i was very steep, as reported previously from experiments at a SL of 2.2 micron in which the membrane permeant acetoxymethyl ester form of fura-2 was used. The fall in tension from 90 to 10 % occurred in 0.12 +/- 0.01 pCa units (mean +/- S.E.M., n = 61) for a SL of 2.2 micron and 0.17 +/- 0.01 pCa units (n = 22) for a SL of 2.9 micron, corresponding to Hill coefficients of 15.4 and 10.9, respectively. 4. We conclude that the increase in sensitivity of tension to [Ca2+] that occurs in skinned skeletal muscle fibres upon stretch also occurs in intact fibres, that the steepness of the relation between tension and [Ca2+]i in intact fibres reported previously cannot be attributed to the use of the acetoxymethyl ester form of fura-2 to report [Ca2+]i, and that the steepness decreases as myofilament overlap decreases.

Figure 1. Fura-2 ratio and tension records at mean sarcomere lengths (SL) of 2.2 μm and 2.9 μm from a representative fibre

The records were obtained in the order A, B, C and then D, with 10 min rest between each set. A, fura-2 ratio (upper trace) and tension (lower trace) records resulting from pairs of stimuli, with 50 ms separation between stimuli within a pair and 120 s rest between pairs. SL was 2.9 μm, temperature was 3.0 °C. The time calibration bar applies to all traces in all panels of the figure. B, records as in A except that SL has been reduced to 2.2 μm and contractions are separated by 60 s. C, repeat at SL = 2.9 μm. D, repeat at SL = 2.2 μm. Fibre 970625.

Figure 2. Tension plotted against [Ca²⁺]_i at mean sarcomere lengths (SL) of 2.2 μm and 2.9 μm from a representative fibre

The tension records shown in Fig. 1 are normalized to 1.0 for each individual contraction and plotted here against [Ca²⁺] values calculated from the corresponding fura-2 ratio records. All contractions with tension relaxation times (90 to 10 %) between 10 and 30 s are superimposed. This includes the final 3 contractions at SL = 2.9 μm from Fig. 1A, the final 5 contractions at SL = 2.2 μm from Fig. 1B the final 2 contractions at SL = 2.9 μm from Fig. 1C and the final 5 contractions at SL = 2.2 μm from Fig. 1D for a total of 10 plots at SL = 2.2 μm and 5 plots at SL = 2.9 μm. Note that the plots cluster by SL, with those from SL = 2.9 μm falling to the left (lower [Ca²⁺]_i) of those from SL = 2.2 μm. Temperature was 3.0 °C. Fibre 970625.