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Am J Physiol Heart Circ Physiol 287: H2712-H2718, 2004. First published August 26, 2004; doi:10.1152/ajpheart.01067.2003
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Basal myosin light chain phosphorylation is a determinant of Ca2+ sensitivity of force and activation dependence of the kinetics of myocardial force development

M. Charlotte Olsson, Jitandrakumar R. Patel, Daniel P. Fitzsimons, Jeffery W. Walker, and Richard L. Moss

Department of Physiology and University of Wisconsin Cardiovascular Research Center, University of Wisconsin Medical School, Madison, Wisconsin 53706

Submitted 10 November 2003 ; accepted in final form 23 August 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
It is generally recognized that ventricular myosin regulatory light chains (RLC) are ~40% phosphorylated under basal conditions, and there is little change in RLC phosphorylation with agonist stimulation of myocardium or altered stimulation frequency. To establish the functional consequences of basal RLC phosphorylation in the heart, we measured mechanical properties of rat skinned trabeculae in which ~7% or ~58% of total RLC was phosphorylated. The protocol for achieving ~7% phosphorylation of RLC involved isolating trabeculae in the presence of 2,3-butanedione monoxime (BDM) to dephosphorylate RLC from its baseline level. Subsequent phosphorylation to ~58% of total was achieved by incubating BDM-treated trabeculae in solution containing smooth muscle myosin light chain kinase, calmodulin, and Ca2+ (i.e., MLCK treatment). After MLCK treatment, Ca2+ sensitivity of force increased by 0.06 pCa units and maximum force increased by 5%. The rate constant of force development (ktr) increased as a function of Ca2+ concentration in the range between pCa 5.8 and pCa 4.5. When expressed versus pCa, the activation dependence of ktr appeared to be unaffected by MLCK treatment; however, when activation was expressed in terms of isometric force-generating capability (as a fraction of maximum), MLCK treatment slowed ktr at submaximal activations. These results suggest that basal phosphorylation of RLC plays a role in setting the kinetics of force development and Ca2+ sensitivity of force in cardiac muscle. Our results also argue that changes in RLC phosphorylation in the range examined here influence actin-myosin interaction kinetics differently in heart muscle than was previously reported for skeletal muscle.

kinetics of force development; skinned myocardium

MYOSIN REGULATORY LIGHT CHAIN (RLC), a thick filament protein associated with the neck region of the myosin molecule, belongs to the superfamily of EF-hand Ca2+-binding proteins, which includes troponin C (TnC) and calmodulin (CaM) (25). In mammalian muscles, RLC is phosphorylated by Ca2+/CaM-dependent myosin light chain kinase (MLCK) and dephosphorylated by light chain phosphatase (1). In smooth muscle, phosphorylation of RLC is necessary for force production, and the amount of force is related to the level of phosphorylation (16, 40). In skeletal and cardiac muscles, RLC phosphorylation appears to modulate contraction, whereas Ca2+ binding to the regulatory site on TnC is the regulatory switch that activates force production (33). Previous studies have shown that MLCK-induced phosphorylation of RLC 1) increases Ca2+ sensitivity of force in both skinned skeletal (24, 42, 44) and cardiac muscles (8, 29, 30, 41); 2) increases the rate constant of force redevelopment (ktr) in skinned skeletal muscle at low levels of activation (24, 42); and 3) eliminates force-dependent changes in the rate constant of relaxation in skinned skeletal muscle (37). Furthermore, in cardiac muscle, increases in Ca2+ sensitivity of force after treatment with agonists for {alpha}1-adrenoreceptors or endothelin receptors are thought to be mediated by increased phosphorylation of RLC (2, 39).

Under normal physiological conditions, the antagonistic actions of MLCK and RLC phosphatase result in near-steady phosphorylation of 30–40% of total RLC in human (26), pig (32), and rat (12, 31) ventricular myocardium. Because RLC phosphorylation is known to increase Ca2+ sensitivity of force and ktr in skeletal muscle, we hypothesized that the basal phosphorylation of RLC in myocardium contributes to baseline force and ktr in myocardium. To test this idea, we examined the Ca2+ dependencies of force and ktr in rat skinned trabeculae treated with 2,3-butanedione monoxime (BDM) to dephosphorylate RLC (to ~7% of total RLC) and then treated them with MLCK to restore phosphorylation to ~58% of total RLC.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
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Experimental solutions. Chemicals were purchased from Sigma except for CaCl2 (Orion Research), propionic acid (Fluka), creatine phosphate (ICN), ATP (Roche), and CaM (Calbiochem). Solution compositions were calculated using the computer program of Fabiato (11) and the stability constants (corrected to pH 7.0 and 15°C) listed by Godt and Lindley (14). Unless otherwise stated, all solutions contained (in mmol/l) 100 BES, 14.5 creatine phosphate, and 5 DTT. In addition, pCa 9.0 solution contained (in mmol/l) 7 EGTA, 0.02 CaCl2, 5.42 MgCl2, and 4.74 ATP; pCa 4.5 solution contained (in mmol/l) 7 EGTA, 7.01 CaCl2, 5.26 MgCl2 and 4.81 ATP; and preactivating solution contained (in mmol/l) 0.07 EGTA, 5.42 MgCl2 and 4.74 ATP. Ionic strength of all solutions was adjusted to 180 mmol/l using potassium propionate. A range of solutions containing different free Ca2+ concentrations ([Ca2+]free; i.e., pCa 6.0–5.5) for determining Ca2+ sensitivities of force and ktr were prepared by mixing solutions of pCa 9.0 and pCa 4.5. Smooth muscle MLCK (smMLCK) was prepared as described previously (35) and stored at –80°C until used in an experiment.

Preparation of skinned trabeculae. Wistar rats (female, 150–200 g) were anesthetized with inhaled isoflurane (15% isoflurane in mineral oil) using procedures approved by the University of Wisconsin Medical School Institutional Animal Care and Use Committee. The hearts were then rapidly excised from each rat, rinsed, and placed in a dissecting dish filled with modified Ringer solution [containing (in mmol/l) 120 NaCl, 19 NaHCO3, 1.2 Na2HPO4,1.2 MgSO4, 5 KCl, 1 CaCl2, and 10 glucose; pH 7.4 at 22°C] preequilibrated with 95% O2-5% CO2. The right ventricle was cut open, and, when present, unbranched trabeculae (1–3 mm long and 75–250 µm wide) running between the free wall and the atrioventricular valve were dissected free and tied to sticks to hold muscle length fixed during skinning. Before the trabeculae were removed, each heart was exposed to fresh Ringer solution containing 30 mM BDM for 15 min to reduce RLC phosphorylation levels to close to 0%. This concentration of BDM was chosen because preliminary studies showed that lower concentrations failed to reduce RLC phosphorylation to <10%. The trabeculae were then transferred to relaxing solution [containing (in mmol/l) 100 KCl, 20 imidazole, 7 MgCl2, 2 EGTA, 4 ATP, and 5 DTT; pH 7.0 at 4°C] containing 1% Triton X-100. After trabeculae were skinned for ~24 h, they were washed in fresh relaxing solution (1 h) and then stored at –20°C in relaxing solution containing glycerol [50:50 (vol/vol)]. The skinned trabeculae were used in experiments within 1 wk.

Attachment of preparations to experimental apparatus. On the day of an experiment, skinned trabeculae were washed in relaxing solution for 30 min before they were cut free from the sticks and their ends trimmed. The trimmed trabeculae were then transferred to a stainless steel experimental chamber (34) containing pCa 9.0 solution. The ends of each trabecula were attached to the arms of a motor (model 312B, Aurora Scientific) and force transducer (model 403, Aurora Scientific), as described earlier (34). The chamber assembly was then placed on the stage of an inverted microscope (Olympus) fitted with a x40 objective and a CCTV camera (model WV-BL600, Panasonic). Filtered light (transmission >620 nm) from a halogen lamp was used to illuminate the skinned preparations. Bitmap images of the preparations were acquired using an AGP 4X/2X graphics card and associated software (ATI Technologies) and were used to assess mean sarcomere length during the course of each experiment. Changes in force and motor position were sampled (16-bit resolution, DAP5216a, Microstar Laboratories) at 2.0 kHz using SLControl software developed in this laboratory (http://www.slcontrol.com). Data were saved to computer files for later analysis.

Experimental design and protocol. At the start of each experiment, the skinned trabecula was stretched to a mean sarcomere length of ~2.35 µm (15°C). First, steady-state force and ktr were measured simultaneously in Ca2+ activating solution containing either submaximal (pCa 5.8 and 5.7) or saturating (pCa 4.5) [Ca2+]free using the modified multistep protocol of Brenner and Eisenberg (6) described in detail previously (38). These measurements yielded Ca2+ sensitivities of force and ktr in BDM-treated (RLC dephosphorylated) skinned trabeculae. Next, the trabeculae were incubated for 10 min in preactivating solution containing 5 mM BDM (to inhibit force development); for 15 min in preactivating solution containing 5 mM BDM, 67 µM CaCl2, 6 µM CaM, and 0.43 µM smMLCK (to phosphorylate RLC); and then for 30 min (3x solution change) in pCa 9.0 solution (to wash out MLCK). Finally, force-pCa and ktr-pCa relationships were characterized.

Once mechanical measurements were finished, the trabeculae were cut free at the points of attachment, placed in denaturing sample buffer containing 8 M urea, 5% {beta}-mercaptoethanol, 2% Triton X-100, and 2% carrier ampholytes (Pharmalyte pH 4.5–5.4, Amersham Pharmacia), and stored at –80°C until subsequent analysis of RLC phosphorylation by two-dimensional gel electrophoresis using a mini gel system (Bio-Rad). On the day that gels were run, the samples were allowed to warm to room temperature, sonicated for 20 min, and loaded onto polyacrylamide gels cast in capillary tubes (Bio-Rad). The first-dimension IEF tube gels contained 8 mol/l urea, 4% acrylamide-bisacrylamide (30% T:5.4% C), 2% Triton X-100 (pH 4.5–5.4), 2% ampholytes, 0.02% ammonium persulfate, and 0.2% TEMED. The samples were electrofocused at 500 V for 15 min and 750 V for 3,600 V·h. Each IEF tube gel was placed on the top of a 12% SDS-PAGE slab gel and electrophoresed at 150 V (constant voltage) until dye ran off the end of the gel. Gels were then silver stained using methods described previously (43). The percent RLC phosphorylation was quantified by scanning the gels using a densitometer (GDS-8000, UVP Bioimaging Systems) and accompanying software (Labworks UVP Bioimaging Systems).

Data analysis. Cross-sectional areas of skinned trabeculae were calculated by assuming that the trabeculae were cylindrical and by equating the width, measured from video images of the mounted preparations, to diameter. Each Ca2+-activated force (P) at pCa between 6.0 and 5.5 was expressed as a fraction of the maximum Ca2+-activated force (Po) developed by the same preparations at pCa 4.5, i.e., P/Po. To determine the Ca2+ sensitivity of isometric force (pCa50), force-pCa data were fitted with the following Hill equation: P/Po = [Ca2+]/(k+ [Ca2+]), where nH is the slope (Hill coefficient) and k is the Ca2+ concentration for half-maximal activation (pCa50). ktr was determined by linear transformation of the half-time (t1/2) of force recovery [ktr = –ln 0.5 x (t1/2)–1], as described previously (7, 38).

All data are presented as means ± SE. Statistical analysis of the data was done using either paired or unpaired t-tests. P values <0.05 were taken as indicating significant differences.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Ca2+ sensitivity of force in BDM-, sham-, and MLCK-treated rat skinned trabeculae. All trabeculae used in these experiments were first treated with BDM to reduce RLC phosphorylation to near 0% and were then skinned for measurements of contractile properties and subsequent treatment with MLCK to assess the effects of RLC phosphorylation on contractile properties. As a control for MLCK treatment (i.e., incubation in preactivating solution containing Ca2+-CaM-smMLCK), some trabeculae were sham treated with identical solutions lacking Ca2+-CaM-smMLCK. To ensure that sham- and MLCK-treated trabeculae had similar baseline contractile properties, force and ktr were measured at pCa 5.8, 5.7, and 4.5 before the sham or MLCK treatments. Statistical analysis using unpaired t-tests indicated that force and ktr were not significantly different in the trabeculae that were subsequently sham or MLCK treated (Table 1).


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  		Table 1. Force and ktr in control, sham-treated, and MLCK-treated skinned trabeculae

 
Whereas sham treatment did not affect force at pCa 4.5 (41.1 ± 2.7 vs. 40.6 ± 2.7 mN/mm2), treatment with MLCK significantly increased maximal Ca2+-activated force (44.9 ± 3.6 vs. 42.6 ± 3.8 mN/mm2; Table 1). Force-pCa relationships from skinned trabeculae subjected to either sham or MLCK treatments were sigmoidal and were well fit with the Hill equation, as described in MATERIALS AND METHODS (Fig. 1). pCa50, an index of Ca2+ sensitivity of force, was significantly increased by MLCK treatment, from pCa 5.64 ± 0.01 in sham-treated skinned trabeculae to pCa 5.70 ± 0.01 after MLCK treatment. nH, an index of apparent cooperativity in the activation of force, was unchanged by MLCK treatment, i.e., nH was 5.2 ± 0.2 in sham-treated trabeculae and 5.3 ± 0.3 in MLCK-treated trabeculae. Thus MLCK treatment increased maximal Ca2+-activated force and the Ca2+ sensitivity of force in skinned myocardium.



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  		Fig. 1. Effects of sham and myosin light chain kinase (MLCK) treatments on Ca2+ sensitivity of force in rat skinned trabeculae. Force-pCa relationships were obtained after a 15-min incubation in preactivating solution without ({bullet}; 10 sham-treated preparations) or with 67 µM CaCl2, 6 µM calmodulin (CaM), and 0.43 µM smooth muscle MLCK (smMLCK) ({circ}; 11 MLCK-treated preparations). Forces measured at submaximal free Ca2+ concentration ([Ca2+]free) were expressed relative to the maximal force measured at pCa 4.5. The smooth lines were fit using the following Hill equation: P/Po = [Ca2+] nH/(knH + [Ca2+]nH), where P is the force measured at submaximal [Ca2+]free, Po is the force measured at maximal [Ca2+]free (pCa 4.5), nH is the Hill coefficient, and k is the [Ca2+]free required for half-maximal activation (pCa50). The fitted values for nH and pCa50 were 4.82 and pCa 5.64, respectively, in sham-treated trabeculae and 4.83 and pCa 5.70, respectively, in MLCK-treated trabeculae. Data points are means ± SE.

 
Maximal ktr and Ca2+ sensitivity of ktr in sham- and MLCK-treated skinned trabeculae. For all treatment conditions that were studied, ktr increased when [Ca2+]free was increased. To illustrate these activation-dependent changes in ktr, records of force redevelopment at various [Ca2+]free are shown in Fig. 2 for both sham-treated (RLC dephosphorylated) and MLCK-treated trabeculae. Steady-state isometric force at each pCa was normalized to 1.0 to provide better visualization of the activation dependence of the kinetics of force redevelopment. The maximal values of ktr obtained at pCa 4.5 did not differ between sham- and MLCK-treated trabeculae (Table 1), and the ktr-pCa relationships from these two treatment groups were virtually indistinguishable (Fig. 3A). However, this finding does not mean that RLC phosphorylation had no effects on the kinetics of force development. Because RLC phosphorylation changes the relationship between steady-state force and [Ca2+]free (Fig. 1), comparisons of kinetics at similar forces might be more appropriate for assessing possible effects of activation. When ktr (normalized to maximum values in each preparation) was plotted versus steady isometric force (as a fraction of maximal force), ktr increased with force in both sham- and MLCK-treated trabeculae. However, MLCK treatment shifted the ktr-force relationship to the right of the relationship from sham-treated trabeculae, i.e., ktr at submaximal activations were slowed by MLCK treatment (Fig. 3B). Thus when ktr was plotted against pCa, RLC phosphorylation appeared to have no effect on cross-bridge interaction kinetics, but when changes in Ca2+ sensitivity are taken into account, RLC phosphorylation was found to slow ktr at submaximal activations.



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  		Fig. 2. Tension records obtained during measurements of the apparent rate constant of force redevelopment (ktr) in sham or MLCK-treated skinned trabeculae. ktr was measured at submaximal (at pCa 5.8–5.7) and maximal (pCa 4.5) [Ca2+]free after a 15-min incubation of trabeculae in preactivating solution without (A; sham treatment) or with 67 µM CaCl2, 6 µM CaM, and 0.43 µM smMLCK (B; MLCK treatment). The force transients are replotted here relative to the peak steady-state force reached after the step change in muscle length. Po and ktr values at each pCa are in parentheses.

 


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  		Fig. 3. Effects of sham and MLCK treatments on Ca2+ and force-dependent changes in ktr. ktr was measured during submaximal activations (at pCa 5.8–5.7) and during maximal activation (at pCa 4.5) after a 15-min incubation of trabeculae in preactivating solution without ({bullet}; 10 experiments) or with 67 µM CaCl2, 6 µM CaM, and 0.43 µM smMLCK ({circ}; 11 experiments). In each case, ktr values obtained during submaximal activations were expressed relative to the maximal ktr measured in the same trabeculae at pCa 4.5 and were then plotted against pCa (A) or normalized Ca2+-activated force (B) recorded before the release/restretch protocol. Data are means ± SE.

 
As a control, ktr was measured during submaximal and maximal activations in skinned trabeculae before sham or MLCK treatment. Before treatment, all trabeculae displayed a similar increase in ktr from ~3 s–1 at pCa 5.8 to ~12 s–1 at pCa 4.5 (Table 1).

RLC phosphorylation in sham- and MLCK-treated trabeculae. At the end of experiments on sham- and MLCK-treated trabeculae, each preparation was cut free at the points of attachment, placed in urea sample buffer, and stored at –80°C until subsequent analysis of RLC phosphorylation using two-dimensional PAGE/IEF gels. In the examples shown in Fig. 4, a sham-treated trabecula had 6% RLC phosphorylation (top) and a MLCK-treated trabecula had 50% RLC phosphorylation (bottom). Mean values for RLC phosphorylation were 7 ± 1% of total RLC in sham-treated trabeculae and 58 ± 4% of total RLC in MLCK-treated trabeculae (Table 1).



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  		Fig. 4. Myosin regulatory light chain (RLC) phosphorylation in typical sham- and MLCK-treated skinned trabeculae. RLC phosphorylation in trabeculae used for mechanical measurements was determined by two-dimensional SDS-PAGE/IEF of sham-treated and MLCK-treated preparations. In these examples, RLC phosphorylation was 6% of total RLC in the sham-treated trabecula and 50% of total RLC in the MLCK-treated trabecula.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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In this study, the effects of basal levels of RLC phosphorylation on mechanical properties were assessed in cardiac skinned trabeculae in which BDM was used to reduce RLC phosphorylation from basal to very low levels. MLCK was then used to restore phosphorylation to approximately basal levels. Thus mechanical properties were measured before and after treatment in the same skinned trabecula so that each preparation served as its own control. The level of RLC phosphorylation was also measured in each trabecula after completion of the mechanical measurements. Increasing the level of phosphorylation from an average of ~7% to ~58% resulted in an increase in the Ca2+ sensitivity of force, a small but significant increase in maximum force, and slowing of cross-bridge cycling kinetics at similar levels of submaximal activation. Thus it appears that basal level of RLC phosphorylation is an important determinant of the extent and kinetics of force development in mammalian ventricular myocardium.

Effect of BDM on RLC phosphorylation. It is well established that under physiological conditions, the balance between MLCK-induced phosphorylation and phosphatase-induced dephosphorylation of RLC maintains a relatively constant level of RLC phosphorylation during the cardiac cycle. For example, in rat myocardium, ~40% of RLC is in the phosphorylated state (12, 31). To understand the importance of this basal RLC phosphorylation in cardiac contraction, our experimental plan included a protocol to reduce RLC phosphorylation to near zero. This was achieved in our experiments with BDM, a compound that is thought to act as a "chemical phosphatase" (47) or as an agent with "phosphatase-like activity" (4) and has been shown to cause dephosphorylation of RLC in rat myocardium (45, 46). In our studies on rat myocardium, treatment of trabeculae with BDM before they were skinned reduced RLC phosphorylation to an average of 7% of total RLC.

Effect of RLC phosphorylation on Ca2+ sensitivity of force. Our observations that RLC phosphorylation increased both maximal force (by ~5%) and the Ca2+ sensitivity of force (a leftward shift in the force-pCa relationship at all Ca2+ concentrations; pCa50 = 0.06) with no effect on the slope of force-pCa relationship differ somewhat from some previous studies (30, 41). Sweeney and Stull (41) reported that RLC phosphorylation had no significant effect on maximal force in rabbit myocardium and shifted the force-pCa relationship to the left at activations below but not above 50% maximal force, with a concomitant decrease in the slope of the force-pCa relationship. Morano et al. (30) studied Ca2+ sensitivity of force in porcine skinned myocardium by measuring force at maximal and one submaximal [Ca2+] and reported a 4.4% increase in maximal isometric force after MLCK treatment. This increase in maximal force did not reach statistical significance, potentially due to a reported large variability in Ca2+ sensitivity between individual skinned preparations. The basis for the difference in results between studies is not known for certain, but there were significant differences in protocols and preparations. We studied rat skinned trabeculae with initial (post-BDM) phosphorylation levels of ~7% of total RLC, whereas Morano et al. (30) reported that ~46% of RLC in pig myocardium was phosphorylated before treatment with MLCK. Sweeney and Stull (41) reported that the RLC phosphorylation in rabbit skinned psoas muscle and myocardium was 5–10% before MLCK and 60–75% after MLCK treatment. Unlike the present work, both of these earlier studies measured RLC phosphorylation on fibers different from those used in the mechanical studies.

Effects of RLC phosphorylation on activation dependence of ktr. Regardless of RLC phosphorylation levels, ktr in skinned trabeculae varied with the level of activating Ca2+ (or force), increasing as Ca2+ (or force) was increased from submaximal to maximal levels (Fig. 3). These Ca2+- and force-dependent changes in ktr are consistent with previous results from rat living myocardium (3) and rat (13, 48), mouse (38), and guinea pig (36) skinned myocardium. Here, we also show that RLC phosphorylation (to an average of ~58% of total) has no significant effect on the ktr-pCa relationship in rat myocardium compared with trabeculae with near-zero (~7%) RLC phosphorylation. However, the ktr-force relationship was shifted to the right such that MLCK-treated trabeculae redeveloped a given force at slower rate than trabeculae treated just with BDM. This lack of effect of RLC phosphorylation on the ktr-pCa relationship in rat skinned myocardium is consistent with an earlier report by Rossmanith et al. (39) showing that endothelin-induced RLC phosphorylation had no effect on rise time or relaxation time of twitch force in electrically paced myocardial preparations from the rat.

Both the present results and those of Rossmanith et al. (39) differ significantly from the effects of RLC phosphorylation on ktr in skinned skeletal muscle fibers reported earlier by Metzger et al. (24) and Sweeny and Stull (42). In skeletal muscle fibers at intermediate levels of activation, but not at very low or at maximal activations, ktr was greater in MLCK-treated fibers. These groups also found that RLC phosphorylation increased force at low and intermediate [Ca2+] but not at high [Ca2+]. Thus RLC phosphorylation appears to speed the kinetics of myosin-actin interactions in skeletal muscle, whereas in cardiac muscle RLC phosphorylation has no effect on kinetics when expressed as a function of pCa but a slowing of kinetics when ktr is plotted against isometric force as an index of activation.

Possible mechanism of effects of RLC phosphorylation on mechanical properties. The effects of RLC phosphorylation on myocardial force can be explained by an increase in numbers of force-generating cross-bridges, force per cross-bridge, or both. In a simple two-state model (5, 15), the cross-bridges cycle between a force-generating state and a nonforce-generating state, and the process is controlled by the rate constants fapp (for the transition to the force-generating state) and gapp (for the return to the non-force generating state (15). The fraction of cycling cross-bridges in the force-generating state can be calculated as the ratio fapp/(fapp + gapp), and steady-state isometric force (P) is given by P = n x F x fapp/(fapp + gapp), where n is the number of cycling cross-bridges and F is the mean force per cross-bridge (5). On the basis of this model, a RLC phosphorylation-mediated increase in Ca2+ sensitivity of force in cardiac or skeletal muscles could be due to an increase in n or F or fapp/(fapp + gapp). In skeletal muscle, phosphorylation of RLC had no significant effect on either n or F (42) but increased ktr (24, 42). Because ktr = fapp + gapp, a straightforward explanation for an increase in ktr in skeletal muscle would be an increase in fapp, which would also increase the ratio fapp/(fapp + gapp) and hence force. In contrast, our data from cardiac muscle are consistent with a two-state model in which RLC phosphorylation increases maximum force and the Ca2+ sensitivity of force and slows ktr by reducing the value of gapp.

In this regard, the effects of RLC phosphorylation on myocardial contraction are similar to the effects of increased MgADP, which also increases maximum force, increases Ca2+ sensitivity of force, and slows the kinetics of force development (20, 21). MgADP is thought to exert these effects by competing with MgATP for the nucleotide-binding pocket on myosin, thereby slowing cross-bridge detachment (9), relaxation from rigor (22), relaxation from active contraction (19), and overall cycling rate (17). From these results in skeletal muscle, it is tempting to postulate that RLC phosphorylation in cardiac muscle prolongs attached states by influencing MgADP dissociation kinetics. However, high MgADP also slows unloaded shortening velocity (23), whereas the effects of RLC phosphorylation on shortening velocity appear to be minimal (for a review, see Ref. 27). A possible reason for this apparent discrepancy is that the effects of basal RLC phosphorylation on Vmax in striated muscles have yet to be fully characterized, particularly in cardiac muscle preparations. Enzymatically digested single cardiomyocytes may be the most reasonable cellular preparation to utilize, because larger myocardial preparations contain variable amounts of connective tissue, which may complicate the assessment of Vmax. However, several technical issues remain to be resolved with regard to single cardiomyocytes. First, Davis and co-workers (10) have demonstrated a transmural gradient in the level of RLC phosphorylation across the ventricular wall. Investigators would need to pinpoint the origin of the single cardiomyocyte (i.e., epicardium vs. endocardium) to avoid large variations in the basal level of RLC phosphorylation. Second, quantifying the level of RLC phosphorylation in a single cardiomyocyte using conventional electrophoretic techniques (e.g., isoelectric focusing) is technically challenging. Nevertheless, the ultimate experimental goal should involve measuring both Vmax and the level of RLC phosphorylation in the same single cardiomyocyte. It is also worth noting that RLC phosphorylation has been shown to slow the rate of relaxation of active force by up to threefold (37), an observation that is consistent with modulation of cross-bridge detachment by phosphorylation. The detailed mechanisms underlying the effects of RLC phosphorylation on the regulatory and mechanical behavior of striated muscles and how these mechanisms might differ in skeletal and cardiac muscles remain to be fully elucidated.

Physiological significance of RLC phosphorylation. The basal level of RLC phosphorylation is likely to play a major role in defining the Ca2+ sensitivity force under normal physiological condition. Because a large change (from ~7 to ~55%) in the level of RLC phosphorylation produces only a modest change in Ca2+ sensitivity of force (pCa50 = 0.06), one would anticipate a much smaller alteration in Ca2+ sensitivity of force with changes in the level of RLC phosphorylation that occurs in chronic endurance exercise of rats [from ~40 to ~55% (12)] or that observed in old spontaneously hypertensive rats [from ~40 to ~20% (31)] and hibernating European hamsters [from ~45 to ~20%; (28)], respectively. However, it is possible that only modest changes in the level of RLC phosphorylation are required to compensate adverse effects on Ca2+ sensitivity of force that may occur under nonphysiological conditions, e.g., during acidosis, lack of Ca2+ availability, and heart failure.

In summary, the overall influence of RLC phosphorylation on tension development kinetics appears to differ in cardiac and skeletal muscles. In skeletal muscle, the phosphorylation-induced increases in Ca2+ sensitivity of force and in ktr have been interpreted in terms of a structural model in which RLC phosphorylation causes movement of myosin heads and increases the probability of interaction with actin (18, 24, 29). Such a model does not account for an apparent decrease in gapp due to RLC phosphorylation in cardiac muscle. Instead, the slowed kinetics of cross-bridge cycling in cardiac muscle suggests that RLC phosphorylation slows a state transition associated with cross-bridge detachment. This mechanism does not exclude the possibility that RLC phosphorylation increases fapp in cardiac muscle, but the fact that cycling kinetics are slowed suggests that the effects of RLC phosphorylation to reduce gapp are dominant. An additional difference between muscle types may be the level of RLC phosphorylation under basal conditions. The data presented here show that basal phosphorylation of ~50% of RLC in cardiac muscle is an important determinant of Ca2+ sensitivity of force and the kinetics of force development.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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This study was supported by National Heart, Lung, and Blood Institute Grant HL-47053 (to R. L. Moss and J. W. Walker) and by a grant from the American Heart Association, Northland Affiliate (to M. C. Olsson).


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. R. Patel, Dept. of Physiology, Univ. of Wisconsin Medical School, 1300 University Ave., Madison, WI 53706 (E-mail: jrpatel{at}physiology.wisc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
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 RESULTS
 DISCUSSION
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