Interplay of Troponin- and Myosin-Based Pathways of Calcium Activation in Skeletal and Cardiac Muscle: The Use of W7 as an Inhibitor of Thin Filament Activation  Bishow B. Adhikari, Kuan Wang  Biophysical Journal  Volume 86, Issue 1, Pages 359-370 (January 2004) DOI: 10.1016/S0006-3495(04)74112-0 Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 1 Schematic of instrument and an example of raw data obtained during calcium activation of ATPase and tension. (A) Single skeletal fiber or a small bundle of cardiac papillary fibers is mounted between two tweezers, one attached to a length controller and the other to a force transducer, inside a cuvette. Solution of varying pCa can be pumped around the sample using a peristaltic pump and a pCa gradient maker that mixes solutions of low and high calcium. For fluorescence measurements, excitation light is focused on the sample, and two detectors, one of each for the signal and the background, are used to collect the resulting fluorescence with appropriate excitation and emission filters. Sarcomere length is measured from diffraction patterns from a He-Ne laser. (B) Raw data of NADH fluorescence (ATPase) and tension that are used to construct two calcium activation curves of the same single rabbit psoas fiber. Biophysical Journal 2004 86, 359-370DOI: (10.1016/S0006-3495(04)74112-0) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 2 The reversible inhibition of isometric tension of rabbit psoas fibers by W7 at pCa 4.0. (A) A single fiber, initially under relaxation, was activated at pCa 4.0 and subjected to increasing and decreasing concentrations of W7 before another final relaxation. The numbers above the trace indicate the concentration of W7 in μM. (B) Another fiber was treated with 300μM W7 after activation, which resulted in tension levels approaching the relaxing value, and then treated with 50, 20, and 10μM W7 with a corresponding recovery of tension. Subsequently, when the fiber was treated with relaxing solution the tension returned to relaxing values. (C) The same fiber from B after 10min relaxation was activated first in 300μM W7; then without W7; then finally relaxed. Biophysical Journal 2004 86, 359-370DOI: (10.1016/S0006-3495(04)74112-0) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 3 Isometric tension and stiffness at pCa 4.0 of rabbit psoas fibers as a function of W7 concentration. Tension was normalized to values obtained without W7. The data were obtained from four experiments at 20°C (solid symbols) and three experiments at 5°C (unfilled symbols). The solid lines are drawn to fit the tension data using an equation of a hyperbola with half-maximal values (KI-Tension) of 22μM at 20°C and 32μM at 5°C. Inset shows the proportionality between tension and stiffness (2000, 440, and 96Hz) at the indicated W7 concentrations at 20°C. Biophysical Journal 2004 86, 359-370DOI: (10.1016/S0006-3495(04)74112-0) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 4 The reversible inhibition of calcium activation of ATPase and tension of single rabbit psoas fibers by W. The pCa curves of ATPase (unfilled symbols) and tension (solid symbols) from two adjacent sections (I and II) of a single fiber are plotted against the solution pCa. Multiple activation cycles, each preceded by a period of relaxation, were carried on two adjacent sections of a single fiber in the presence of the indicated concentrations of W7. The activation sequences were 100μM (I-Cycle 1) and 0μM (I-Cycle 2) in the first fiber section, and 300μM (II-Cycle 1), 20μM (II-Cycle 2), and 0μM (II-Cycle 3) in the second fiber section. The data were normalized to the curves at 0μM W7 in each set. The fitted values of midpoint (pK) and slope (n, in parentheses) are given next to each curve. An example of ATPase/force-pCa curves from an untreated fiber is shown for comparison. The brackets (l for leading component and t for trailing component) indicate the components of ATPase that are not accompanied by significant levels of tension. Biophysical Journal 2004 86, 359-370DOI: (10.1016/S0006-3495(04)74112-0) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 5 The reversible inhibition of calcium activation of ATPase and tension by W7 in mouse heart fibers. All activation curves were obtained sequentially from the same sample, after relaxation of muscle between each activation, in the following order: without W7 (Cycle 1), with 100μM W7 (Cycle 2), with 25μM W7 (Cycle 3), and finally again after washing W7 (Cycle 4). The data were normalized to the curves obtained in the first activation cycle without W7 (Cycle 1). The fitted values of pK and n (in parentheses) are given next to each curve. Biophysical Journal 2004 86, 359-370DOI: (10.1016/S0006-3495(04)74112-0) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 6 The inhibition of calcium activation of tension and ATPase of a rabbit psoas fiber by BDM and by BDM and W7. All activation curves were obtained sequentially in the same fiber, after relaxation of sample between each activation, in the following order: 30mM BDM (Cycle 1), 10mM BDM plus 50μM W7 (Cycle 2), 10mM BDM (Cycle 3), and finally without BDM and W7 (Cycle 4; note that the fiber broke near the maximum activation). The data were normalized to the curves without BDM and W7 (Cycle 4; l over bracket indicates the leading component of ATPase as in Fig. 4). Fitted values of pK and n (in brackets) are given next to each curve. Biophysical Journal 2004 86, 359-370DOI: (10.1016/S0006-3495(04)74112-0) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 7 Enhancement of inhibition by the combination of 50μM W7 and 10mM BDM in single fibers from rabbit psoas muscle. Average ATPase and tension-pCa curves are plotted for 50μM W7, 10mM BDM, 50μM W7, and 10mM BDM in A. The pCa curves of the combined W7 and BDM data are compared with that of the predicted curves based on sums of each inhibition curves in B. The fitted values of pK and n (in parentheses) for the ATPase and tension curves are given next to each curve. Note the significant drops in cooperativity and maximal values that lead to higher sensitivities when W7 and BDM are combined. The data were obtained from individual activations in 3–4 fibers for each condition. Biophysical Journal 2004 86, 359-370DOI: (10.1016/S0006-3495(04)74112-0) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 8 Relative tension cost (ATPase/tension) during activation in the presence of W7 and BDM. The ATPase/tension ratios are plotted against the solution pCa for activations of skeletal and cardiac fibers at the indicated concentrations of W7 and BDM. (A) W7 inhibition in skeletal fibers; (B) W7 inhibition in cardiac fibers; and (C) BDM inhibition in skeletal fibers. Biophysical Journal 2004 86, 359-370DOI: (10.1016/S0006-3495(04)74112-0) Copyright © 2004 The Biophysical Society Terms and Conditions