reply: We appreciate the interest expressed by Drs. Barclay and Loiselle (2) in our recent paper (11). We used N-benzyl-p-toluene sulfonamide (BTS) to inhibit cross-bridge force production and myosin ATPase-dependent ATP splitting (measured with analytical biochemistry). BTS has been shown to inhibit cross-bridge force production and ATP splitting to comparable extents while not affecting the shape of Ca2+ transients during contractions, suggesting that BTS does not affect sarcoplasmic reticulum (SR)-Ca2+ release or Ca2+ pumping. Under conditions of near maximal force (2 s continuous isometric contraction), cross bridges accounted for more than 50% of ATP splitting, which was consistent with the literature. Surprisingly, however, using the same experimental approach, our results indicated that cross bridges accounted for only 20% of the total ATP splitting during submaximal contractions (∼35% of maximal isometric force; 2–10 s continuous contractions). We suggested that ion pumps, primarily Ca2+-ATPase, accounted for the remaining ATP utilization, although this was not directly measured. We provided calculations based on stated assumptions, as well as other evidence from the literature in support of this explanation.
Barclay and Loiselle (2) express skepticism regarding our suggested high relative contribution of Ca2+-pumping based on: i) heat measurements in fatigued muscles and muscles exposed to dantrolene (to inhibit SR Ca2+ release); ii) estimates of Ca2+ release per stimulus pulse; and iii) belief that our ATP splitting per cross bridge would be only one-quarter the value previously assumed. We address each of these points.
i) Heat production occurs in proportion to the amount of ATP split, and when heat measurements are used in combination with the stretch technique, extrapolation to zero force allows for the partitioning of heat into tension-dependent (cross bridges) and tension-independent (primarily SR Ca2+-ATPase) components. Barclay (1) and Wendt and Barclay (9) used the stretch technique to study mouse extensor digitorum longus (EDL) muscles and demonstrated that the heat associated with the tension-independent component amounted to 35% of total in fatigued muscles and 42% in dantrolene-treated muscles (control = 33%), respectively. These findings appear to be in disagreement with our estimate of Ca2+ pumping during submaximal contractions. It should be noted, however, that the heat measurements were performed during short contraction intervals (0.2–0.5 s), whereas our measurements (using BTS) were performed during longer periods (2–10 s). Earlier studies have shown that the percent contribution of the non-tension-dependent components to ATP splitting increases as contraction duration is prolonged in mouse EDL muscles (4). Another potential factor to consider is that we used BTS, whereas the cited studies employed the stretch technique. However, why BTS and stretch would yield fairly concordant results during conditions of maximal force, but not during submaximal force, is unclear.
ii) Barclay and Loiselle (2) suggest that the amount of Ca2+ released per pulse (action potential) would result in a much lower Ca2+ pumping rate than we estimate. The actual amount of total Ca2+ released and the activity of the Ca2+ pump cannot be directly measured in intact muscle fibers. Estimates of these parameters generally depend on modeling. Based on the Ca2+ release values given in the cited study (at 16°C), one can calculate that ATP splitting by the Ca2+ pump would range from 1.6 to 17.3 mM ATP during the submaximal 2-s contraction (we estimate 21.4 mM). Clearly, the estimated Ca2+ release (and hence Ca2+ pump) values will depend on the model employed with its inherent assumptions.
iii) Barclay and Loiselle (2) suggest that if cross bridges account for only 20% of energy use, then the rate of ATP splitting per cross bridge must be one-quarter the value previously assumed. However, they do not state what the assumed value is. Indeed, the value varies markedly depending on the conditions studied (e.g., contraction duration, type of contraction, temperature, fiber type, etc.). If we assume that at 35% of maximal isometric force only 35% of the cross bridges are functioning and a myosin subfragment I concentration of 150 μM (7), then ATP splitting per cross bridge would amount to 65, 34, and 21 s−1 during the 2-, 5-, and 10-s contractions, respectively, at 30°C. These values are similar to or higher than those reported in the literature for isometric contractions after adjusting for temperature (assuming a Q10 of 2.5 for myosin ATPase) (3, 5, 6, 8, 10).
We find the skepticism expressed by Barclay and Loiselle and the exchange of views in these letters to be refreshing and hope that this exchange will stimulate physiologists to take a more active interest in the field of muscle energetics. Ultimately, however, independent verification in the form of hard data collected during relevantly designed experiments will be required to settle the issue.
- Copyright © 2007 the American Physiological Society