SUMMARY1. The effects of decreasing pH from 7 40 to 6-20 on the tension developed by direct activation of the myofilaments and by Ca2+ release from the sarcoplasmic reticulum were studied comparatively in segments of single cells of skeletal muscle (frog semitendinosus) and cardiac muscle (rat ventricle) from which the sarcolemma had been removed by micro-dissection (skinned muscle cells).2. The concentration of free Ca2+ in the solutions was buffered with ethylene glycol-bis (/1-aminoethylether N,N'-tetraacetic acid (EGTA). 4. The pH optimum for loading the sarcoplasmic reticulum of skinned fibres from skeletal muscle decreased when the pCa (-log [free Ca2+]) in the loading solution decreased. The optimum was pH 7 40-700 for a loading at pCa 7 75, pH 7-00-6'60 at pCa 7*00 and pH 6-60-6-20 at pCa 6-00.5. The pH optimum for loading the sarcoplasmic reticulum of skinned cardiac cells with a solution at pCa 7-75 was about pH 7 40 as in skeletal muscle fibres. But the cardiac sarcoplasmic reticulum could not be loaded with a [free Ca2+] much higher than pCa 7*75 because a higher [free Ca2+] triggered a Ca2+-induced release of Ca2+ from the sarcoplasmic reticulum.6. The pH optimum of about 7*40 for the loading of the cardiac sarcoplasmic reticulum was also optimum for the Ca2+-induced release of Ca2+ from it. 7. It was concluded that the effects of acidosis on the cardiac sarcoplasmic reticulum accentuate the depressive action of decreasing pH on the myofilaments. This may explain the pronounced depression of contractility observed during acidosis in cardiac muscle. In contrast, a moderate acidosis causes an effect on skeletal muscle sarcoplasmic reticulum that could compensate for the depressive action on the myofilaments, which is, in addition, less pronounced than in cardiac muscle.

Ca2+ to a critical level at which it released a fraction of the Ca2+ it contained. Each contraction was followed by a re-sequestration of Ca2+, the kinetics of which conditioned the duration of the cycles.6.

No abstract

Annals New York A c a d e m y of Sciences muscle fibers, and (2) the apparent stability constant recently described by Allen and Blinks'" for Ca-EGTA (2.51 X lo6 M-' at pH 7.00). which seems the most likely to be correct.5 The correspondence between the free [Ca2+] values obtained with the different apparent stability constants is variable with the total [EGTA] used. FIGURE 2 shows the correspondence in the presence of 5 x M total EGTA and FIGURE 3 shows the correspondence in the presence of 4 x 10-:' M total EGTA. Finally, very little EGTA binds to Mg'+; an apparent stability constant of 4.00 X 10' M-' for the Mg-EGTA complex' was used in the computer program.In all experiments, free [Mg'+] was 3.16 x M, [MgATP2-] was 3.16 X M, pH was 7.00 + 0.01 buffered with 20 mM imidazole, ionic strength was adjusted to 0.160 M by appropriate additions of KCI (taking the small binding of K + to ATP into account), 7 mM glucose was added and the temperature was maintained at 22 r 0.2" C by the temperature-controlled stage of the microscope. The dimensions of the preparations were measured on the cells stretched to a sarcomere length of 2 . 3 * 0.1 p m , which was the sarcomere length used for the experiments. Data were expressed as mean and standard deviation (SD), compared with Student's t-test and judged as significantly different when p was < 0.05. RESULTS Firsf Expcrimmtal Profocwl Used to Drmori.s~ratt~ u Cti'+-lnduc r dRelecise of C(i2+ from the Surcwplrismic RcticulurnThe first experimental protocol used to demonstrate a CaY+-induced release of Ca2+ from the SR is shown in FIGURE 1 for a skinned cell from the rabbit atrium. A slight increase of the free [Caz+] in the presence of a low total [EGTA] of 5 x M resulted in the induction of cyclic contractions. The same increase of free [Ca'+] in the presence of M total EGTA resulted in cyclic contractions of smaller amplitude. The cyclic contractions disappeared completely in the presence of 2 x M total EGTA. The decrease in the amplitude of the cyclic contractions when total [EGTA] was increased without modifying the free [Caz+] suggested that these cyclic contractions were generated by a Ca2+ sink within the cell that competed for Ca'+ with the Ca-EGTA buffer. This Ca2+ sink was not the mitochondria because the cyclic contractions induced by a free [Ca'+] lower than 5 x M ruthenium red or 5 x M a~i d e .~ Since the atrial tissue generally does not contain transverse tubules," the Ca2+ sink could not be within sealed-over transverse tubules as observed in skinned skeletal muscle fiber^.^ But the cyclic contractions were inhibited by 10-2M caffeine, which partially emptied the S R of Ca2+, and were absent in skinned cells in which the SR was destroyed by the detergent Brij 58 or the surfactant d e o x y c h~l a t e .~ It was concluded, therefore, that the cellular Ca2+ sink generating the cyclic contractions was the SR. However, the observation of cyclic contractions does not by itself demonstrate a Ca2+-induced release of Ca2+ from the SR. The Ca'+-induced release of Ca...

A a S TRA C T During partial Ca 2+ activation, skinned cardiac cells with sarcoplasmic reticulum destroyed by detergent developed spontaneous tension oscillations consisting of cycles (0.1-1 Hz) of rapid decrease of tension corresponding to the yield of some sarcomeres and slow redevelopment of tension corresponding to the reshortening of these sarcomeres. Such myofilament-generated tension oscillations were never observed during the full activation induced by a saturating [free Ca 2+] or during the rigor tension induced by decreasing [MgATP] in the absence of free Ca z+ or when the mean sarcomere length (SL) of the preparation was > 3.10/gin during partial Ca 2+ activation. A stiff parallel elastic element borne by a structure that could be digested by elastase hindered the study of the SL--active tension diagram in 8-13-p.m-wide skinned ceils from the rat ventricle, but this study was possible in 2-7-/.tm-wide myofibril bundles from the frog or dog ventricle. During rigor the tension decreased linearly when SL was increased from 2.35 to 3.80 p.m. During full Ca 2+ activation the tension decreased by < 20% when SL was increased from 2.35 to -3.10/~m. During partial Ca 2+ activation the tension increased when SL was increased from 2.35 to 3.00/,m. From this observation of an apparent increase in the sensitivity of the myofilaments to Ca z+ induced by increasing SL during partial Ca 2+ activation, a model was proposed that describes the tension oscillations and permits the derivation of the maximal velocity of shortening (Vmax). Vmax was increased by increasing [free Ca 2+] or decreasing [free Mg 2+] but not by increasing SL.