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Effect of pH, phosphate, and ADP on relaxation of myocardium after photolysis of diazo 2 S. J. SIMNETT, E. C. JOHNS, S. LIPSCOMB, I. P. MULLIGAN, AND C. C. ASHLEY University Laboratory of Physiology, Oxford OX1 3PT, United Kingdom muscle; heart; calcium; guinea pig; cross bridges is poorly understood. Studies of relaxation of the intact heart are complicated by the geometry of the organ, the auxotonicity of relaxation, and the beat-to-beat modulation of the inotropic state because of changes in the degree of ventricular filling and catecholamine stimulation. We have studied cardiac myofilaments in isolation from other cellular components to determine the kinetics of myofilament relaxation and to examine how these kinetics are altered by changes in the concentration of the byproducts of ATP hydrolysis. During ischemia there is a decrease in cardiac output and a change in the rates of relaxation and activation. These changes, caused by a combination of alterations in the functions of the cellular membranes and the myofilaments, are brought about, at least in part, by increases in the intracellular concentrations of metabolites such as ADP and Pi and by a decrease in the intracellular pH (pHi ) (2, 3, 14, 16, 18). It has been proposed that the fall in pHi plays a major role in this decline of contractile function in intact cardiac tissue. However, the attenuation of force in hypoxic conditions, and those mimicking ischemia, cannot be wholly attributed to an increasing acidity, because the majority of CARDIAC MUSCLE RELAXATION the decline in contractile force occurs before the onset of acidosis (2, 10, 17). The time course of the increase in Pi concentration ([Pi]) is, however, very similar to that of force degradation (10, 17). Much effort has been made to elucidate the effect of these metabolites on individual cellular processes such as Ca21 uptake by the sarcoplasmic reticulum (SR). However, little is known about any effects of these metabolites on myofibrillar relaxation. Until recently, relaxation was induced by moving surface membranepermeabilized preparations (skinned fibers) into solutions containing relatively high concentrations of Ca21 buffers such as EGTA. These protocols produce relaxations that are dependent on, and limited by, diffusion and equilibration of the buffers. Use of the photolabile caged Ca21 chelator diazo 2 circumvents this problem. The affinity of diazo 2 for Ca21 rapidly changes after exposure to ultraviolet light (1). Initially, the Ca21 affinity is low [dissociation constant (Kd ) 5 2.2 µM]; it increases on photolysis (Kd 5 0.073 µM), producing a rapid decrease in the free Ca21 concentration within a skinned muscle fiber (4, 25, 30–32, 35). In this study we have used this photolysis method to investigate the effect of increases in the concentrations of metabolites, which accumulate during ischemia and hypoxia (MgADP, Pi, and H1 ), on the rate of myofilament relaxation. The chemically skinned trabecular preparation, in which the cellular membranes have been rendered nonfunctional, permits effects on the myofilaments (including possible changes in the Ca21 affinity of troponin C) to be examined separately from changes in the SR and surface membrane. This study provides insight into changes in the cardiac myofilament relaxation processes that occur during ischemia. Additionally, information is provided about the control of cross-bridge transitions when the free Ca21 is rapidly (#2 ms) removed from the muscle system. METHODS Guinea pig preparation. Female albino Dunkin-Hartley guinea pigs (200–300 g) were killed by cervical dislocation in accordance with institutional guidelines. Their hearts were removed and placed in an iced, oxygenated modified Tyrode solution (5.0 mM HEPES; pH 7.35; see Solutions). Trabeculae were dissected from the right ventricle and placed in a bath of light paraffin oil maintained at 8°C. Trabeculae with a uniform diameter of 100–200 µm and a length of 2–3 mm were selected, and aluminum T clips were attached at either end. The trabeculae were transferred to the photolysis apparatus and suspended between two stainless steel hooks (100 µm in diameter). The trabeculae were skinned by immersion in a ‘‘skinning’’ solution (see Solutions) for 30 min at 12°C and then transferred to a ‘‘relaxing’’ solution. The length of each trabecula 0363-6135/98 $5.00 Copyright r 1998 the American Physiological Society H951 Downloaded from http://ajpheart.physiology.org/ by 10.220.33.2 on June 18, 2017 Simnett, S. J., E. C. Johns, S. Lipscomb, I. P. Mulligan, and C. C. Ashley. Effect of pH, phosphate, and ADP on relaxation of myocardium after photolysis of diazo 2. Am. J. Physiol. 275 (Heart Circ. Physiol. 44): H951–H960, 1998.— The aim of this study was to examine the effect of the metabolites H1, ADP, and Pi on the rate of cardiac relaxation. We used guinea pig right ventricular trabeculae that had been chemically skinned, allowing the myofilaments to be studied in isolation. Laser-flash photolysis of the caged Ca21 chelator diazo 2, causing a rapid fall in intracellular Ca21, enabled investigation of relaxation independently of the rate of Ca21 diffusion. On the photolysis of diazo 2, the trabeculae relaxed biphasically with exponential rate constants (k1 and k2 ) of 10.07 and 4.23 s21, respectively, at 12°C and 18.35 and 2.52 s21, respectively, at a nominal 20°C. Increasing the concentration of both protons (pH 7.2–6.8) and MgADP (0.5–3.4 mM) slowed the two phases of the relaxation transients. Raising the concentration of Pi from the control level of 1.36 mM to 15.2 mM increased the rate of both phases, with relaxation becoming monoexponential at 19.4 mM Pi (with a k of 20.31 s21 at 12°C). Cardiac muscle was compared with skeletal muscle under identical conditions; in cardiac muscle 19.4 mM Pi increased the rate of relaxation, whereas in skeletal muscle this concentration of Pi slowed relaxation. We conclude that the mechanism of relaxation differs between cardiac and skeletal muscle. This study is a direct demonstration of the effects of ATP metabolites on cardiac myofilament processes during relaxation. H952 CAGED CA21 CHELATOR AND RELAXATION Fig. 1. Protocol for photolysis procedure for a single trabecular relaxation transient. Once steady-state force has been reached, trough containing diazo 2 is lowered pneumatically, leaving trabeculae suspended in air; 400 ms later laser is fired. This produces a 20-ns pulse of light with a wavelength of 347 nm (,100 mJ), which causes photolysis of diazo 2, rapid uptake of Ca21, and relaxation of trabeculae. Temperature 5 12°C; trabecular diameter 5 145 µm; maximum steady-state force (Pmax ) 5 20 mN · mm22. Length 5 3.5 mm. length of the preparation was exposed to the laser pulse and was optimized each time. Solutions. The composition of the experimental solutions was calculated using a program written in Fortran 77, using equilibrium constants from Smith and Martell (37). Adjustments were made to take into account the experimental temperatures employed (12°C in all but one set of experiments). The program corrected the ionic strength (to 0.15 M) using potassium propionate. The pH was set to an appropriate value (7.2 except in the experiments investigating the effect of pH) by the addition of a small amount of 5 M potassium hydroxide. All solutions contained 60 mM N, Nbis[2-hydroxyethyl]-2-aminoethanesulfonic acid (BES), 5 mM MgATP, and 1 mM free Mg21. EGTA (10 mM) was added to the relaxing and activating (pCa 4.5) solutions except the zeroEGTA relaxing solution. Creatine phosphokinase (15 U/ml) and creatine phosphate (10 mM) were added to all solutions except those containing added ADP. Contaminant Pi was assessed by a phosphate assay kit available from Sigma (Poole, UK). The method of Jaworek and Welsch (15) was employed to assess contaminant ADP levels. Leupeptin (0.5 mM) was added to the relaxing solution to prevent deterioration of the fibers by Ca21-activated proteases. The skinning solution was prepared by the addition of 1% (vol/vol) Triton X-100 to relaxing solution. All reagents were obtained from Sigma, except for BES, which was obtained from Calbiochem (Cambridge, UK), and were of analytical grade. The modified (no Ca21 ) Tyrode solution (pH 7.35) was oxygenated and contained (in mM) 134 NaCl, 5.4 KCl, 1.2 MgSO4, 11.1 glucose, and 5.0 HEPES. Omission of Ca21 from the Tyrode solution enhanced the viability of the trabeculae after dissection. Curve fitting. The relaxation transients were fitted with two exponential processes, k1 and k2, using the curve-fitting program P.Fit (Biosoft, Cambridge) and the equation Y 5 A exp(2k1 x) 1 B exp(2k2 x) 1 C (1) where Y is force, X is time, and A and B represent the relative proportions of the fast phase (rate constant k1 ) and slow phase (rate constant k2 ) processes, respectively. Both the individual and mean curves were well fitted by a double exponential. Mean relaxation transients were obtained by normalizing and then averaging all of the individual transients. To ensure the closest possible fit, the data were fitted several times using different initial settings for the parameters. The fitting process did not take the standard deviation of each point into consideration; therefore, the standard errors given for the curve-fitting parameters are for the fitting process only. The standard error of any point from the mean curve was ,3%. The constant C expresses the remnant (% tension) after the curve-fitting process has been completed. Diazo 2 solutions. Diazo 2 was initially a gift from Drs. R. Y. Tsien and S. R. Adams (University of California, San Diego); it was later custom synthesized by Molecular Probes (Eugene, OR). The experimental diazo 2 solution contained 2 mM diazo 2 and had no added EGTA. The Ca21 concentration of the diazo 2 solution was adjusted to produce activations that were ,95% of Pmax by adding 10-µl aliquots of 20 mM CaCl2. Effect of pH on diazo 2 kinetics. Ca21 kinetics at different pH values were investigated by monitoring the decrease in relative fluorescence of the Ca21 indicator fluo 3 after photolysis of 400 µM diazo 2. The change in relative fluorescence of fluo 3 (excitation wavelength 488 nm, emission wavelength 525 nm) was followed using a laser-scanning confocal microscope (LSCM; MRC-600, BioRad) with a time resolution of 2 ms. The LSCM was equipped with a flash lamp; both pieces of Downloaded from http://ajpheart.physiology.org/ by 10.220.33.2 on June 18, 2017 was adjusted to produce passive force; sarcomere length was ,2.2 µm (measured using a light microscope). Frog muscle preparation. Rana temporaria were obtained from Blades Biological (Edenbridge, UK) and stored at 2°C in shallow water, without food, for a maximum period of a week. Frogs were stunned and killed by cervical dislocation according to institutional guidelines. Single muscle fibers were dissected from the semitendinosus muscle and placed in a bath of light mineral oil at 8°C. The fibers were treated as the trabeculae except that they were chemically skinned for 12 min, as opposed to 30 min, at 12°C. Apparatus and experimental protocol. The apparatus designed by Ferenczi (11) allows rapid changes of the solution that bathes each muscle fiber. The laser-flash photolysis technique and the triggering and recording instrumentation were essentially the same as those described previously by that author (11). Experiments were carried out at 12°C, except during the study investigating temperature effects, when a nominal value of 20°C was selected. Initially, a steady-state activation was reached at 90–100% of the maximal activation (maximum steady-state force, Pmax ) achieved in a pCa 4.5 solution containing 10 mM EGTA (see Solutions). The preparation was transferred to a relaxing solution and then to the diazo 2 solution via a second relaxing solution that contained no Ca21 buffer (zero-EGTA solution) for ,60 s. The experimental procedure for photolysis of diazo 2 in a single trabecula and the resultant, typical relaxation transient are shown in Fig. 1. The trough containing the diazo 2 solution was lowered to leave the fiber suspended in air; 400 ms later the laser was fired. There was a decline of ,5% in developed force before the laser fired. The photolysis of diazo 2 caused a rapid chelation of Ca21 and a very fast relaxation (Fig. 1). Usually two or three relaxation transients could be obtained from the same preparation without apparent deterioration of the Pmax value; however, we limited ourselves to two per trabeculae as a precaution against any damage. The full H953 CAGED CA21 CHELATOR AND RELAXATION equipment were controlled by a Macintosh Ilex (Apple Computers) equipped with a NB-M10 16A/D-D/A board (National Instruments). The composition of the solutions was 122 mM KCl, 10 mM NaCl, 1 mM MgCl2, 10 mM HEPES, and 20 µM fluo 3. The initial ‘‘free’’ Ca21 was calculated as 66 nM, and the final free Ca21 after 70% diazo 2 photolysis was calculated as 5 nM. The experiments were performed at three different pH values, 6.5, 7.0, and 7.5 (see Fig. 10, A, B, and C, respectively). The decline in relative fluorescence fitted closely to a single exponential curve (with rates of 0.3, 0.6, and 0.44 ms21, respectively). Both decreasing and increasing the pH from 7.0 slowed the uptake of Ca21 by diazo 2 after photolysis. RESULTS Trabecular relaxation rates after photolysis of diazo 2 and effect of temperature. The rate of relaxation of skinned trabeculae from the guinea pig was investigated using the caged Ca21 chelator diazo 2 (Fig. 1). After the photolysis of diazo 2, the trabeculae relaxed with an average half-time of 72.4 6 2.6 ms (mean 6 SE; n 5 8) at 12°C. The mean relaxation transient was well fitted with a double exponential curve fit (Fig. 2) with the fitting values shown in Table 1. Ideally, these experiments would have been performed at the body temperature of the guinea pig (37.2–40°C). However, to enable .70% photolysis to occur at 2 mM diazo 2, the trabeculae were photolysed in air rather than in the experimental trough (25, 31). After removal from the trough, the temperature of a fiber suspended in air will decrease rapidly toward the dew point because of evaporation (11). This would result in an increase in the ionic strength of the solutions within the fiber and result in a decline in maximal force production in the 400 ms between the trough dropping and the laser firing. Thus the majority of experiments were carried out at the dew point of the laboratory (12°C) with little or no evaporation, cooling, or change in ionic strength occurring before photolysis. In some experiments the trough temperature was set to 20°C and the relaxation transient on photolysis was investigated. Given that some evaporative loss and cooling would have occurred, a 5% decline in Pmax was observed. The average relaxation transient at a nominal 20°C (see Fig. 2) was well fitted by double exponential processes with the parameters shown in Table 1. Comparison of the parameters indicated that increasing the temperature greatly increased the rate and proportion of the fast phase (k1 ), whereas the rate of the slow phase (k2 ) decreased only slightly. The mean half-time of relaxation decreased to 53.4 6 1.84 (SE) ms (n 5 8) with the increase in temperature to a nominal 20°C. It has been shown that muscle activation is cooperative after photolysis of the caged Ca21 molecules nitr-5 or DM-nitrophen (4). Figure 3 shows the half-time of Table 1. Curve-fitting parameter values for relaxation transient in guinea pig skinned trabeculae Control 20°C pH 6.8 0.5 mM MgADP 0.8 mM MgADP 3.4 mM MgADP k1 , s21 6 SEM A, % 6 SEM k2 , s21 6 SEM B, % 6 SEM C, % 6 SEM n 10.07 6 0.3 18.35 6 0.25 3.94 6 0.64 6.69 6 0.12 6.00 6 0.16 2.36 6 0.05 49.2 6 2.84 92.29 6 1.01 57.4 6 0.65 80.09 6 2.12 58.87 6 2.68 70.6 6 2.58 4.23 6 0.12 2.52 6 0.39 0.68 6 0.01 0.64 6 0.23 1.91 6 0.09 0.53 6 0.07 50.82 6 2.91 15.1 6 0.63 45.5 6 0.55 27.5 6 2.68 45.19 6 2.57 29.21 6 1.71 0.32 6 0.07 21.85 6 0.66 1.98 6 1.2 24.21 6 4.49 21.05 6 0.24 23.35 6 1.03 8 8 8 7 7 7 Parameter values (n 5 no. of trabeculae) were used to fit relaxation transients with double exponential curves. SE were derived from curve fit of mean data; they are not SE from mean transients themselves. A and B, relative proportions of processes k1 and k2 ; C, remnant (% tension) after curve-fitting procedure has been completed (see Eq. 1). Downloaded from http://ajpheart.physiology.org/ by 10.220.33.2 on June 18, 2017 Fig. 2. Average relaxation transients and their double exponential curve fits (dashed lines) at 12°C (control, n 5 8) and nominal 20°C (n 5 8) from skinned trabeculae, showing effect of temperature on speed of relaxation on photolysis of 2 mM diazo 2. Average transients were well fitted with double exponential curve fits; parameters are shown in Table 1. Average trabecular diameter 5 151.7 6 10.7 (SE) µm (n 5 14). Fig. 3. Graph of half-time of relaxation against postphotolysis force level in skinned trabeculae expressed as percentage of Pmax (value was obtained at saturating Ca21 levels). Each trabecula was activated to 90–100% of Pmax and then relaxed to differing degrees by varying energy of laser output. Graph was fitted with a linear regression line (P.Fit) with the equation y 5 0.0065x 1 62.2; r 5 20.038, r2 5 0.001. Dashed lines, 95% confidence limits. Each data point is an individual experimental transient; 2 mM diazo was used in each case. H954 CAGED CA21 CHELATOR AND RELAXATION Fig. 4. Effect of Pi on rate of relaxation of skinned trabeculae from guinea pig after photolysis of diazo 2. Transients were well fitted with double exponential curve fits (dashed lines) with constants shown in Table 2. Average trabecular diameter 5 165 6 19.6 (SE) µm (n 5 25). Control level of Pi 5 1.36 mM. Fig. 5. Effect of Pi on half-time of skinned trabecular relaxation. Each data point is average of at least 5 values; points are fitted with a parabolic curve. increases in [Pi] (from 1 to 5.5 mM) there is a decline in the half-time of relaxation [from 63 6 4.2 (SE) (n 5 7) to 44 6 4.3 (SE) ms (n 5 6)]; however, further increases in Pi have little effect on the half-time (Fig. 5). The relaxation transients were well fitted with double exponential curve fits (shown by the dashed lines in Fig. 4). Attempts were made to fit the curves with a single exponential curve, but the results did not give a good fit as judged by both the sum of the squares and by eye. However, the mean relaxation transient at 19.4 mM Pi was an exception. The results of the curve-fitting procedure show that the initial effect of Pi was to increase k1. Further additions of Pi produced no other change in k1 but increased the proportion of the relaxation associated with it (A). This occurred at the expense of k2, and the relaxation was eventually characterized by a single exponential. Thus raising the concentration of Pi produced a general increase in the effect of the fast phase of trabecular relaxation, until at 19.4 mM the relaxation became monoexponential. The effect of further Pi addition (.19.4 mM) on the relaxation transients could not be investigated—the degradation in Pmax was such that the relaxation transients were too small to be analyzed accurately at this ionic strength. Effect of Pi on rate of relaxation of single skinned skeletal muscle fibers. In contrast to its effect on the relaxation rate of skinned trabeculae seen here, it was shown previously that in semitendinosus muscle skinned fibers from the frog Pi produces a gradual slowing of the relaxation transient (32). To compare the effect of Pi on the relaxation rate in the two muscle types, the experiment was repeated in frog semitendinosus muscle fibers using the same solutions and conditions as for the cardiac experiments. The results (Fig. 6) confirm that in frog skeletal muscle Pi slows the relaxation rate. This indicates that the differing effect of Pi in these cardiac and skeletal muscle preparations is caused by biological differences as opposed to variations in the experimental conditions (e.g., ionic strength or pH). The relaxation transients were well fitted with Downloaded from http://ajpheart.physiology.org/ by 10.220.33.2 on June 18, 2017 relaxation of fully activated fibers (90–100% of Pmax ) relaxed to differing levels by variation of the laser beam energy. The graph indicates that, over the range investigated, the rate of relaxation is constant and independent of the degree of relaxation (Fig. 3). Thus cardiac relaxation does not appear to be cooperative, or at least the step(s) is not rate limiting, unlike the activation process in muscle (4). We have previously shown (4) that, in single frog fibers relaxed by diazo 2 photolysis, the rate of relaxation is also independent of the degree of prephotolysis force. Finally, experiments used the Ca21 indicator fluo 3 together with 2 mM diazo 2 in single myocytes (20); it was shown that the free Ca21 declined rapidly and uniformly from an assumed free Ca21 concentration of ,2 µM to 100 nM on 70% diazo 2 photolysis, as was suggested by initial simulations (Fig. 3 in Ref. 25). Recent time-resolved measurements indicate that there is a ,1% sarcomere length change in single frog skinned fibers on diazo 2 photolysis producing .80% relaxation from Pmax with a half-time of 67.4 6 4.2 (SE) ms at 5°C (B. Hoskins, S. Lipscomb, P. J. Griffiths, and C. C. Ashley, unpublished observations), implying that the relaxation transient is a good measure of the deactivation of the force-generating unit (cross bridges). Effect of Pi on rate of relaxation of skinned cardiac trabeculae. Increasing total [Pi] to 5.67 mM produced a decline in Pmax of 30.6 6 1.8 (SE)%, (n 5 7; data not shown). The relationship between the decline in tension and log [Pi] is linear and is consistent with previous reports (14, 18, 28). [Pi] in the control solutions was determined to be 1.36 mM (0.36 mM H2PO4 1 at pH 7.2 as the principal species, 0.58 mM HPO22 4 calculated from the solution program). The precise origin of this Pi (ATP or creatine phosphate) was not investigated. The effect of Pi on the rate of trabecular relaxation after the photolysis of diazo 2 is shown in Fig. 4. Elevating [Pi] caused an increase in the rate of relaxation. The effect of Pi is twofold: with relatively small H955 CAGED CA21 CHELATOR AND RELAXATION double exponential curves; the parameters are shown in Table 2. The constants indicate that, in skeletal muscle, Pi slows the rate of k1 with a slight decrease in A. Effect of ADP on rate of relaxation of skinned cardiac trabeculae. An assay to determine the concentration of ADP in the experimental solutions (see METHODS ) showed the concentration in the control solution to be 0.05 mM. The levels in the high-ADP solutions were all found to be within 65.0% error of the assay. The concentration of MgADP (the active species) in each solution was calculated using the solution program (see METHODS ). Raising the concentration of MgADP to 3.4 mM caused an increase in Pmax of 80.2 6 1.0 (SE)% (n 5 7) at 3.4 mM MgATP; this is consistent with previous reports (14). Addition of MgADP produced a significant slowing of the trabecular relaxation rate, after photolysis of diazo 2, at all concentrations investigated (see Fig. 7). Comparisons of the half-times (Fig. 8) show that for each subsequent increase of the MgADP concentration there is a significant difference from the previous one, and the relationship between half-time and concentration of MgADP showed a saturation, with a half- Fig. 7. Effect of MgADP on rate of skinned trabecular relaxation after photolysis of 2 mM diazo 2. Average transients were well fitted with double exponential curve fits (dashed lines) with parameters shown in Table 1. Average trabecular diameter 5 145.6 6 15.2 (SE) µm (n 5 29). maximal effect at an MgADP concentration of 1.5 mM. The mean tension transients (Fig. 7) were well fitted with double exponential curve fits; the parameters used for the curve-fitting procedure (Table 1) reveal that MgADP decreases the speed of the fast phase (k1 ). Effect of pH on rate of relaxation of skinned cardiac trabeculae. Decreasing the pH of the experimental solutions from 7.2 to 6.8 reduced the maximal activated tension to 66.77 6 1.92 (SE)% (n 5 8) of the control; this is consistent with previous reports (18). A decrease in pH caused a marked decline in the rate of relaxation after the photolysis of diazo 2. The halftime of the mean relaxation transient at 12°C increased from 72.4 6 2.6 (SE) ms (n 5 8) at pH 7.2 to 260.9 6 25.1 (SE) ms (n 5 10) at pH 6.8 (Fig. 9). The mean transients were well fitted with double exponential curves (dashed lines in Fig. 9); the rate constants are shown in Table 1. The rate constants show that there is a decrease in both the fast (k1 ) and slow (k2 ) phases of the relaxations, with little alteration in the percentage of the relaxations associated with each phase (A and B, respectively). One explanation for the results seen in Fig. 9 is that there may be a change in the kinetics of Ca21 uptake by diazo 2 with decreasing pH. However, the results Table 2. Curve-fitting parameter values for relaxation transient in skinned cardiac and skeletal muscle Control 5.67 mM Pi 15.2 mM Pi 19.4 mM Pi Frog control Frog 19.4 mM Pi k1 , s21 6 SEM A, % 6 SEM k2 , s21 6 SEM B, % 6 SEM C, % 6 SEM n 13.39 6 0.60 22.86 6 0.14 22.37 6 1.08 20.31 6 1.25 22.61 6 0.29 16.1 6 0.06 81.68 6 6.62 63.48 6 1.01 78.42 6 6.10 101 6 2.4 93.40 6 0.62 81.5 6 0.16 5.48 6 0.78 5.82 6 0.13 7.79 6 1.01 24.2 6 6.65 39.3 6 1.01 27.36 6 6.08 1.65 6 0.21 1.29 6 0.02 10.3 6 0.46 22.3 6 0.12 0.41 6 0.16 20.11 6 0.09 0.11 6 0.28 20.30 6 0.17 0.11 6 0.45 20.48 6 0.10 7 6 5 7 6 6 Parameter values (n 5 no. of muscle fibers or trabeculae) were used to fit relaxation transients with double exponential curves. SE were derived from curve fit of mean data; they are not SE from mean transients themselves. Downloaded from http://ajpheart.physiology.org/ by 10.220.33.2 on June 18, 2017 Fig. 6. Effect of 19.4 mM Pi on rate of relaxation of single skinned semitendinosus muscle fibers from frog on photolysis of 2 mM diazo 2. Transients were well fitted with double exponential curve fits (dashed lines) with constants shown in Table 2. Slowing of relaxation rate seen in this skeletal muscle preparation is in contrast to acceleration of relaxation rate seen in trabecular preparation (Fig. 4). Experiments were carried out using same solutions as those used for the trabecular experiments in Fig. 4, i.e., ionic strength 5 0.15 mM and pH 5 7.2. Temperature 5 12°C; average fiber diameter 5 102.4 6 10.8 (SE) µm; and average fiber length 5 ,3 mm (n 5 12). H956 CAGED CA21 CHELATOR AND RELAXATION Downloaded from http://ajpheart.physiology.org/ by 10.220.33.2 on June 18, 2017 Fig. 8. Effect of increasing MgADP concentration [MgADP] on speed of skinned trabecular relaxation after photolysis of 2 mM diazo 2. Each point is average of at least 7 points (6SE). Half-maximal activation occurs at MgADP concentration of 1.5 mM. Points are fitted to a parabola. reported in Fig. 10 indicate that the changes in diazo 2 kinetics are not significant over the time scale of these relaxation transients (see METHODS ). DISCUSSION Trabecular relaxation rates after photolysis of diazo 2 and effect of temperature. After the photolysis of diazo 2, the half-time of relaxation of skinned trabeculae from the guinea pig was 72.4 6 2.6 (SE) ms at 12°C and 53.4 6 1.8 (SE) ms at 20°C. These relaxation rates are significantly faster than those of electrically stimulated guinea pig trabeculae (half-time 5 ,276 ms at 20°C; J. C. Kentish, personal communication). This suggests that in intact preparations under normal conditions the transfer of cross bridges from an activated to a relaxed state is not a rate-limiting factor for relaxation and Fig. 10. Relative decrease in fluorescence of 20 µM fluo 3 after photolysis of 400 µM diazo 2 at 3 pH values, 6.5 (A), 7.0 (B), and 7.5 (C). Photolysis occurred at time 0. Data were fitted with a single exponential curve fit (solid line). Fig. 9. Effect of decreasing pH on relaxation of skinned trabeculae from guinea pig after photolysis of 2 mM diazo 2. Average transients were well fitted with double exponential curve fits (dashed lines) with parameters shown in Table 1. Temperature 5 12°C; average trabecular diameter 5 171.5 6 10.7 (SE) µm (n 5 16). would imply that the activity of the SR Ca21 pump plays the major role in relaxation, as suggested by Luo and colleagues (23). It is usually hypothesized that during relaxation, cross bridges enter weakly bound states via the same pathway as that followed in actively contracting muscle. However, if the rate of relaxation after photolysis of diazo 2 (rate constants of 10 s21 and 4 s21 in skinned trabeculae at 12°C) is compared with the rate of CAGED CA21 CHELATOR AND RELAXATION two exponentials. Increasing the temperature increased k1 and the proportion of the relaxation associated with this phase. At body temperature myofilament relaxation may well be monoexponential. It was impossible to test this theory, because if the temperature of the fiber was the body temperature of a guinea pig (,38°C) there would be a very large degradation of force during the 400 ms in which the trabecula is suspended in air before the laser firing. It is not possible to conduct the experiments with the fibers in solution when the diazo 2 concentration is 2 mM, because the ‘‘optical filter’’ of the diazo 2 (molar extinction coefficient 5 22,000 M21 ·cm21 at 370 nm) surrounding the fiber results in an inadequate degree of diazo 2 photolysis. Photolysis of 2 mM diazo 2 in single myocytes under paraffin oil is certainly possible (9). Any uncertainties about the validity of inferring that the kinetic behavior of skinned trabeculae is the same as that of equivalent unskinned preparations were previously dispelled by Saeki and colleagues (34), who showed that the cross-bridge dynamics of skinned and intact trabeculae from the ferret were not significantly different at 20°C. Effect of Pi on rate of relaxation of skinned cardiac trabeculae. The assay of the phosphate concentration in the experimental solutions found the level of Pi in the control solutions, presumably caused by the breakdown of either creatine phosphate or ATP, to be very similar to that found in myocytes under normal conditions (,1 mM; Refs. 14 and 17). Thus any effects of this contaminant Pi on the myofilaments in the control conditions will be a good approximation of those in intact muscle cells under normal metabolic conditions. The highest [Pi] investigated here (19.4 mM) is similar to that reached within 2 min of cardiac ischemia (16). The results showing that Pi increased the rate of trabecular relaxation were unexpected because in skeletal muscle it decreases the rate of relaxation (32). One set of experiments (effect of 19.4 mM Pi on the speed of relaxation) was repeated in frog semitendinosus skinned muscle fibers, using the same experimental solutions and conditions as for the cardiac experiments. It was revealed that the observed discrepancy between cardiac and skeletal systems was not caused by experimental variations, e.g., the ionic strength or pH of the solutions, but was the result of real differences between the muscle types. It has previously been shown that cardiac tissue is more sensitive to Pi than skeletal muscle is (14, 18); the slope of the graph of cardiac force against log [Pi] is twice as steep as that of skeletal muscle (18). The reason for this discrepancy has not yet been fully elucidated. The model of Pate and Cooke (33) predicts that the slope of the relationship is proportional to the fraction of cross bridges in the main force-generating cross-bridge state (AM · ADP in their model), suggesting that in cardiac muscle there are more forcegenerating cross bridges. However, to account for the observed difference in the slope of the force-log [Pi] relationship between cardiac and skeletal muscle fibers there would have to be twice as many cross bridges in Downloaded from http://ajpheart.physiology.org/ by 10.220.33.2 on June 18, 2017 relaxation of rigor cross bridges after photorelease of caged ATP in the absence of Ca21 (,0.45 s21 in guinea pig trabeculae; Ref. 5), then it is apparent that the process of relaxation is distinct from that of the equivalent transition during relaxation from rigor. This is despite the fact that both processes apparently involve the binding of ATP and transfer of cross bridges to the nonforce, weakly bound state. Our result suggests that the mechanism by which cross-bridge force decays when Ca21 is rapidly removed from the thin filament is either inherently different from cross-bridge detachment during sustained muscle contraction or that the same pathway is differently regulated (I. P. Mulligan, R. E. Palmer, S. J. Simnett, S. Lipscomb, and C. C. Ashley, unpublished observations). The average relaxation transient and the individual transients were well fitted with two exponential processes. The rate constants of the two phases were 10.07 and 4.23 s21 (Table 1); these are consistent with reports of the relaxation rate initiated by diazo 2 photolysis in single skinned ventricular myocytes from the rat at pH 7.0–7.1 and 20–21°C (30, 38). In frog skeletal muscle, the relaxation transient after the photolysis of diazo 2 can also be well fitted to two exponential processes; however, the rate constant of each phase is slightly greater than that in cardiac muscle at the same temperature (31). Although the two rate constants of the faster and slower processes are not greatly different, they suggest the possibility that there are two populations of cross bridges and that they relax (pass to a nonforce, weakly bound state) by two distinct pathways. It has previously been suggested that the ratelimiting step in the cross-bridge cycle is the dissociation of ADP from the attached cross bridges (33, 36); it is possible that one of the rate constants represents cross bridges passing through this cross-bridge transition. The other rate constant may represent cross bridges in states before the isomerization step [AM8ADP · Pi to AM · ADP · Pi, where A is actin and M is myosin, in the model of Pate and Cooke (33)] relaxing through the reversal of this or via alternative reaction steps to the relaxed state. From comparisons of the control relaxation transients, it is apparent that the rate of relaxation varied somewhat between different sets of experiments. However, in each set of experiments the experimental conditions (e.g., control or 0.5 mM MgADP) were selected randomly and in most cases it was sufficient to obtain two relaxation transients under different conditions from each fiber. Comparisons of these individual transients always produced the same results as the mean results, regardless of the order in which they were performed. Increasing the temperature of the trabeculae to a nominal 20°C accelerated the relaxation rate by 35%. It was not possible to measure the temperature of the trabeculae at the time of photolysis, but from the results of Ferenczi (11) it would be expected that the fiber temperature decreased by 2–3°C in the 400 ms between the trough being lowered and the laser firing. Again, the relaxation transients were closely fitted by H957 H958 CAGED CA21 CHELATOR AND RELAXATION bridge states and resulting in the observed increase in isometric tension. An increase in MgADP caused a decrease in the speed of skinned trabecular relaxation after the photolysis of diazo 2 (4, 35). This was a graded effect, with the relaxation half-time increasing with each addition of MgADP (to concentrations well above physiological levels); it is consistent with the effect of raised MgADP concentrations on the relaxation of skinned skeletal muscle fibers following diazo 2 photolysis (R.E. Palmer, personal communication). There are at least two possible mechanisms by which MgADP may be acting. It could, by end product inhibition, decrease the rate of MgADP release from the several AM · ADP states, or it could compete with ATP for the myosin nucleotide binding site, acting as a competitive inhibitor. The situation regarding competitive inhibition appears to be inconclusive. Cooke and Pate (8) found that, in skeletal muscle, the effect of MgADP on maximum unloaded shortening velocity was consistent with a competitive inhibition of the MgATP for the nucleotide binding site. This contrasts with the results of Lipscomb and colleagues (21), who investigated the effect of altered MgATP concentrations on the slowing of force relaxation produced by a rise in MgADP. This group reported that, in frog skeletal muscle, increasing the MgATP levels from 5 to 15 mM did not affect the slowing of relaxation produced by MgADP on photolysis of diazo 2. It was concluded that MgADP and MgATP binding are noncompetitive at the single nucleotide binding site identified by crystallography (21). Investigations by Lu and colleagues (22) into the effect of caged ADP release on isometric force were inconclusive as to the mechanism by which MgADP was acting. If MgADP was acting as a competitive inhibitor, it would be expected that the relationship of inverse of the velocity of relaxation (represented by half-time) against the concentration of MgADP would be a straight line. However, as shown in Fig. 8, the data points are best fitted by a parabolic curve, suggesting that MgADP is acting as a partial competitive inhibitor. It is possible that the mechanism of action of MgADP is dependent on the degree of force being produced by the cross bridges, i.e., whether they are in an isometric or isotonic situation. Increases in the concentration of MgADP are known to occur during ischemia in the heart; the concentration rises from a resting level of ,0.05 mM up to a maximum of 1–2 mM (16). The buildup of MgADP, although not as large as that of Pi, would cause a change in contractile function. MgADP has two favorable effects; it increases both Pmax and the apparent Ca21 sensitivity of the myofilaments. These properties of MgADP would tend to increase the inotropic state of the heart. However, offsetting this, MgADP decreases the maximal speed of both contraction and relaxation. Therefore, the presence of MgADP would tend to decrease cardiac output. The importance of any pathological MgADP buildup is debatable, because levels do not rise significantly in the first 5–10 min of ischemia—the time Downloaded from http://ajpheart.physiology.org/ by 10.220.33.2 on June 18, 2017 the force-generating state in cardiac muscle as in skeletal muscle. Another possibility is that the crossbridge cycle Pi release step is closer to equilibrium in cardiac tissue and hence is more susceptible to perturbation by changes in steady-state [Pi]. The data from the curve fitting of the relaxation transients reveal that the addition of small concentrations of Pi increases the speed of k1, with a slight decline in the proportion of A. Further additions of Pi caused no further changes in k1, but increased A at the expense of B, until the relaxation became monoexponential at 19.4 mM Pi. This suggests that Pi has a greater effect on the latter part of the tension decline, whereas the initial decay of force is largely unaffected. Also, it implies that increasing [Pi] reduces the number of cross bridges that relax via the slow phase. If relaxation can be described by two exponentials, this suggests that there may be two distinct populations of cross bridges (A and B) that relax by different pathways with rate constants k1 and k2, respectively. Thus the fact that at 19.4 mM Pi the relaxation becomes monoexponential suggests that, at high [Pi], all the cross bridges relax by the same pathway. However, although there is a clear difference between cardiac and skeletal muscle in the effect that raised Pi has on the relaxation transient as judged by the changes in half-times, it may be difficult to make a clear interpretation of this change in terms of the constants k1 and k2. This is because the values of these rate constants are too similar to determine for certain that they represent two distinct populations. Nevertheless, the experimental finding that cardiac and skeletal muscle respond in very different ways to a raised [Pi] under identical conditions remains clear. Whatever the mechanism by which Pi increases the rate of relaxation (positive lusitropic effect), this effect may be beneficial in cardiac ischemia and hypoxia, in which there is a large accumulation of Pi (up to 20 mM, Ref. 16; see also Ref. 6) and energy supply for relaxation is limited. Effect of ADP on rate of relaxation of skinned cardiac trabeculae. The release of MgADP is thought to occur toward the end of the cross-bridge power stroke and to be closely followed by MgATP binding and cross-bridge detachment (or transition into a weakly bound, nonforce-generating state). It has been suggested that this transition (or perhaps the MgADP isomerization step) is the rate-limiting step for cross-bridge cycling and is slow so as to match the ATPase rate. The increase in Pmax with increasing MgADP concentrations seen here is consistent with previous reports, both in cardiac (14) and skeletal (13, 14, 33) muscle. MgADP also increases the apparent Ca21 sensitivity of the myofibrils (14). As the MgADP concentration is increased, the free energy of the AM · ADP state decreases relative to the A · M state, making the release of MgADP (and subsequent cross-bridge cycling) less energetically favorable (33). Thus raised levels of MgADP would be expected to retard cross-bridge dissociation, increasing the population of high force-generating cross- CAGED CA21 CHELATOR AND RELAXATION Protons are thought to be released at several stages of the cross-bridge cycle (see Eq. 2, in which the states within the box are considered to be non-force-generating, weakly bound cross bridges), and thus they could slow relaxation by a mass action (concentration) effect on any one of the transitions with which they are involved. However, it would appear that simple mass action effects of H1 on these steps (ADP release and Pi rebinding) would speed up and not slow down the rate of relaxation (Eq. 2) by enhancing Pi rebinding or ADP release. Metzger and Moss (24), investigating the kinetics of force redevelopment after rapid shortening in skeletal muscle, revealed that pH modulates the forcegenerating step of the cross-bridge cycle in a manner that is not compatible with mass action effects. It has been shown that the maximum ATPase rate is decreased in acidosis (7, 13); any slowing of enzymatic activity may suggest a reduction in the rate of crossbridge cycling. The mechanism of proton action has not been clearly defined. In physiological systems, the two types of Pi are the diprotonated (H2PO2 4 ) and the monoprotonated ) forms. In skeletal muscle it has been sug(HPO22 4 gested (27) that the active form is diprotonated Pi and that the myofibrillar effect of protons is caused by an increase in the proportion of this species. However, this has been disputed by other studies in skeletal and cardiac muscle (10, 18). It is thought that Pi is released as the diprotonated form and is converted to the monoprotonated form with the formation of a hydrogen ion; thus it may be expected that proton effects occur via a shift in this equilibrium. There is controversy as to whether this is true in skeletal muscle (28); in cardiac muscle the action of H1 is thought to be independent of Pi (10, 18, 28). This proposal is confirmed by the results of this study, in which H1 decreased the cardiac relaxation rate and Pi increased it. The effects of Pi, ADP, and H1 on Pmax and Ca21 sensitivity have been documented in skinned cardiac muscle; they are qualitatively the same as in skinned skeletal muscle. Pi and protons decrease Pmax and Ca21 sensitivity, whereas ADP has the opposite effect. The mechanism by which these metabolites affect contractile function is not entirely clear. There have been suggestions that the effect of Pi on Pmax, is a result of a decline in the free energy of hydrolysis of ATP, which is proportional to [ATP]/([ADP] · [Pi]) (33). However, increases in [Pi] reduce Pmax, whereas increases in ADP levels have the opposite effect (8, 14). It is most probable that Pi and ADP act by altering the equilibrium of certain cross-bridge transitions. (2) Downloaded from http://ajpheart.physiology.org/ by 10.220.33.2 on June 18, 2017 during which the majority of decline in contractile function occurs (16). MgADP will, however, play a role in the contractile state of the heart during long periods of ischemia. Effect of pH on rate of relaxation of skinned cardiac trabeculae. It has previously been shown that hydrogen ions produce a slowing of the rate of skeletal muscle relaxation after photolysis of diazo 2 (31). Here it has been shown that acidosis also decreases the relaxation rate of cardiac muscle, with the average half-time for relaxation increasing from 72.4 6 2.6 (SE) ms (n 5 8) at pH 7.2 to 260.9 6 25.1 (SE) ms (n 5 8) at pH 6.8. The demonstrated decline in Pmax is in agreement with previous studies (13, 18). Prior investigations into the rate of relaxation during acidosis in intact preparations have been contradictory. Fry and Poole-Wilson (12) recorded a decline in the speed of guinea pig papillary muscle relaxation with increasing acidosis, whereas in ferret papillary muscle the reverse was observed (3, 29). Allen and colleagues (3) made simultaneous measurements of Ca21 and force during acidosis and found that although the relaxation rate decreased, the Ca21 transient was prolonged. They suggested that this might be the result of a decrease in the level of Ca21 binding to troponin C during acidosis (Ref. 3; see also Ref. 7). It is possible that these effects are caused by changes in phospholamban or other SR proteins, as opposed to direct effects on the myofilaments. This is because the present results indicate a decrease in relaxation rate with decreased pH rather than the increase required if the relaxation rate was determined solely by the Ca21 off-rate from troponin C, assuming that no change in the Ca21 on-rate is associated with the well-described pH-induced decrease in affinity (19). Whatever the reason, it is unlikely that changes in diazo 2 kinetics are causing the observed change in relaxation rate. Although the kinetics are slightly slower at pH 6.5 compared with pH 7.5, they are still greater than ,300 s21 and thus 30-fold faster than the fast phase of the relaxation transient. Thus diazo 2 kinetics are not considered to be rate limiting in these experiments. The trabecular relaxations after the photolysis of diazo 2 were well fitted with double exponential curve fits. Acidosis slows the rate of both the fast and the slow transitions, with little effect on the proportions of the two phases. The lack of shift in the proportions of the two phases suggests that there is no alteration in the distribution of cross bridges. This supports the notion that the decline in steady-state force is caused by a reduction in the amount of force produced by each strongly bound cross bridge. H959 H960 CAGED CA21 CHELATOR AND RELAXATION Experiments with fluo 3 were performed by Drs. W. Zhang, E. Niggli, and H. Oetliker at the University of Bern, Switzerland. This work was supported by grants from the British Heart Foundation (BHF) and the Wellcome Trust; a BHF Intermediate Fellowship was awarded to I. P. Mulligan. Present address of I. P. Mulligan: Dept. of Cardiovasc. Med., John Radcliffe Hosp., Oxford OX3 9DU, UK. Address for reprint requests: C. C. Ashley, Univ. Lab. of Physiology, Parks Rd., Oxford OX1 3PT, UK. Received 26 November 1996; accepted in final form 1 May 1998. 18. 19. 20. REFERENCES 21. 22. 23. 24. 25. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. Downloaded from http://ajpheart.physiology.org/ by 10.220.33.2 on June 18, 2017 1. Adams, S. R., J. P. Kao, and R. Y. Tsien. Biologically useful chelators that take up Ca21 upon illumination. J. Am. Chem. Soc. 111: 210–218, 1989. 2. Allen, D. G., P. G. Morris, C. H. Orchard, and J. S. Pirolo. 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