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J Physiol (2003), 553.2, pp. 457-471
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2003.053694
23Tm on isometric tension and the cross-bridge kinetics in bovine myocardium |
ABSTRACT |
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Tropomyosin (Tm) spans seven actin monomers and contains seven quasi-repeating, loosely similar regions, 1-7. Deletion of regions 2-3 decreases the in vitro sliding speed of synthetic filaments of actin-Tm-Troponin (Tn), and weakens Tm binding to the actin-myosin subfragment 1 (S1) complex (acto-S1). The thin filament was selectively removed from bovine myocardium by gelsolin, and the actin filament was reconstituted, followed by further reconstitution with Tm and Tn. In this reconstitution, full-length Tm (control) was compared with Tm internal deletion mutant
(Resubmitted 19 August 2003; accepted 15 September 2003; first published online 18 September 2003)
Corresponding author M. Kawai: Department of Anatomy and Cell Biology, University of Iowa, Iowa City, IA 52242, USA. Email: masataka-kawai{at}uiowa.edu
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INTRODUCTION |
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The major constituents of the contractile apparatus in striated muscle are the thick and thin filaments. Force is generated when a protrusion from the thick filament, called a cross-bridge, attaches to the thin filament and catalyses ATP hydrolysis. The thin filament is composed of actin, tropomyosin (Tm) and three subunits of the troponin (Tn) molecules. Modification of a portion of the thin filament and examination of how this modification alters its function provide an excellent method of studying the structure-function relationship. However, it has been difficult to extract Tm and to replace it with a mutant Tm, because Tm is a filamentous protein. Recently, both Ishiwata's group (Fujita et al. 1996; Fujita & Ishiwata, 1998) and our group (Fujita et al. 2002; Fujita & Kawai, 2002) have succeeded in removing the thin filament from bovine myocardium and in reconstituting the thin filament with constituent proteins. After reconstitution, sinusoidal analysis was performed to access cross-bridge kinetics. The structure of the reconstituted myocardium was examined by electron microscopy and SDS-PAGE, and its function was measured by isometric tension and cross-bridge kinetics. These methods confirmed that the reconstitution was complete both structurally and functionally. The ability to selectively remove the thin filament and to reconstitute it with component proteins offers a unique possibility to study the role of a specific domain of a thin filament protein by using genetically engineered mutant proteins.
Examination of Tm mutants has been helpful in understanding the regulatory function of the thin filament. Hitchcock-DeGregori and her coworkers (Hitchcock-DeGregori & Varnell, 1990; Hitchcock-DeGregori & An, 1996) found that deletion of region 2 or 3 out of seven quasi-repeating regions does not interrupt Ca2+ regulation. They also found that these regions contribute modestly to Tm-Tn binding to actin. Also, Landis et al. (1999) have found that it is not the length of Tm that is critical for proper regulatory function but, rather, it is the specific regions of Tm that are critical for regulation. A broad internal region of Tm is important for its binding to actin decorated with myosin subfragment one (S1), and the strength of this binding correlates with the retention of physiological Tm-Tn-mediated regulation. These investigators have also studied the function of regions of 2-3 of Tm by deleting these regions (23Tm). Their results showed that Ca2+-dependent regulation of in vitro motility and thin filament-S1-ATPase activity was preserved even with the deletion. However, Tm binding to acto-S1 was weakened by an order of magnitude, and in vitro sliding speed was decreased by 36 %. Based on these observations, they concluded that regions 2 and 3 contribute to stabilizing the myosin-induced 'active state' of the thin filament.
In this report, we have combined the actin filament reconstitution technique with the sinusoidal analysis technique to study the effect of MgATP, inorganic phosphate (Pi) and MgADP concentrations on the apparent rate constants of exponential processes by using Tm isolated from bovine myocardium, recombinant rat Tm and mutant 23Tm. Our results indicate that deletion of regions 2-3 significantly diminishes the force and the stiffness supported by each cross-bridge, increases the cooperativity, but does not affect the Ca2+ sensitivity.
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METHODS |
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Chemicals and solutions
Creatine phosphate (Na2CP), adenosine 5'-triphosphate (Na2H2ATP), and Mops were purchased from Sigma Chemical Co. (St Louis, MO, USA); Triton X-100, CaCO3, Mg(OH)2, NaOH, KOH, K2HPO4, KH2PO4, NaN3 and propionic (Prop) acid were purchased from Fisher Scientific (Hanover Park, IL, USA); ethylene glycol bis (2-aminoethyl ether)-N,N,N',N' tetraacetic acid (H4EGTA) was purchased from Amresco Inc. (Solon, OH, USA); and creatine kinase (CK) was purchased from Boehringer Mannheim (Indianapolis, IN, USA).
The compositions of each solution used are summarized in Table 1. Activating solutions are designated by mSnP, where m represents the millimolar concentration of MgATP2- (S) and n represents that of Pi (P). For example, the 5S8P solution (control activating solution) contained 5 mM MgATP and 8 mM Pi. Other activating solutions were created by mixing two extreme solutions. For the ATP study, 0S8P and 5S8P solutions were made first, and they were mixed appropriately. For the Pi study, 5S0P and 5S32P solutions were made first, and they were mixed appropriately. For the ADP (D) study, 0D and 3D solutions were made first, and they were mixed appropriately. The 00D solution is the same as 0D solution, except that 00D also contained CP and CK. All activating solutions, except for 0D and 3D, contained 15 mM Na2CP and 320 units ml-1 CK (0.64 mg ml-1) to regenerate ATP, and 10 mM NaN3 to inhibit mitochondrial ATPase. In all solutions, pH was adjusted to 7.00, the Mg2+ concentration to 0.5 mM, the total Na to 55 mM, and ionic strength to 200 mM. pCa was 4.66 unless otherwise stated. For the pCa study, 5S8P solution was used as the base solution, and the ratio of CaEGTA:EGTA was adjusted according to the specified pCa value by keeping the total EGTA concentration at 6 mM. EGTA, CaEGTA and Pi were added as neutral K salts; MgATP, MgADP and CP as neutral Na salts; and free ATP as Na2K1.7ATP (neutral salt). Individual concentrations of ionic species were calculated with our computer program assuming multiple equilibria and using the following apparent association constants (log values at pH 7.00): CaEGTA, 6.28; MgEGTA, 1.61; CaATP, 3.70; MgATP, 4.00; CaCP, 1.15; MgCP, 1.30.
Muscle fibres and proteins
Bovine hearts were obtained from a slaughterhouse and immediately cooled with crushed ice. The muscle bundles (~2 mm in diameter and 10 mm in length) were excised from a free and straight portion of the right ventricular myocardium and incubated in the Na skinning solution for 3 h at 0 °C. The Na skinning solution was used to minimize initial contraction. For further skinning, the solution was replaced with K skinning solution and stored overnight at 0 °C. BDM and EGTA were used to minimize force development during preparation. The solution was then replaced with a storage solution that contained 50 % glycerol, but was otherwise the same as K skinning solution, and stored at 0 °C overnight. The solution was then replaced once again, and the muscle bundles were stored in a freezer (-20 °C) without freezing.
Actin was extracted from acetone powder (Kondo & Ishiwata, 1976) from rabbit white skeletal muscles according to the method of Spudich & Watt (1971). The acetone powder was a present from Dr Ishiwata's laboratory. We used rabbit skeletal actin for reconstitution because its sequence is almost identical to bovine cardiac actin with four minor substitutions (cardiac 
skeletal: D2E, E3D, L298M, S357T) (Vandekerckhove & Weber, 1979). Purified G-actin was stored at 0 °C and used within 1 week of purification. Tn was prepared from bovine myocardium as reported previously (Tobacman & Adelstein, 1986).
Tm (90 % 
, 10 % 
) was prepared from bovine myocardium (Tobacman & Adelstein, 1986). This Tm is acetylated as reported by Monteiro et al. (1994), and is hence designated as acetyl-Tm. Ala-Ser--Tm (AS-Tm) was prepared from E. coli by using rat -Tm cDNA. The N-terminal dipeptide (Ala-Ser) was included to compensate for the poor polymerizability of bacterially expressed Tm that lacks N-terminal acetylation (Monteiro et al. 1994). The cDNA encoding AS-Tm was altered using the ExSite mutagenesis kit (Stratagene) (Landis et al. 1999). The mutant constructs included deletions of 231 base pairs (Gln47 to Ser123). AS-23 Tm was prepared from E. coli by using this mutant cDNA. Bovine plasma gelsolin was prepared according to the method of Kurokawa et al. (1990).
Experimental procedure and deduction of kinetic constants
A strip of bovine myocardium (cardiac muscle fibres) was dissected from the skinned muscle bundle, and each end was attached to a stainless steel wire (diameter 210 µm) with nail polish: one wire was attached to a tension transducer and the other wire to a length driver. Fibres were stretched until a small passive tension was detected. At this point, the average sarcomere length was about 1.9-2.1 µm. The length (L0 ~2 mm) of the fibres was determined by measuring the end-to-end distance. The diameter was measured under a dissection microscope (
20). The fibres were chemically skinned further in relaxing solution (Rx) containing 1 % Triton X-100 for 20 min. Triton X-100 was washed out with Rx solution before the experiment began.
Selective removal and reconstitution of the thin filament were performed as described previously (Fujita et al. 1996, 2002; Fujita & Ishiwata, 1998, 1999; Ishiwata et al. 1998). In brief, cardiac muscle fibre was first activated in the control activating solution (5S8P) (Fig. 1A), and this was followed by gelsolin treatment for 30-60 min (Fig. 1B). The actin filament was then reconstituted from G-actin (1 mg ml-1) with four to eight solution changes of 7 min each, and active tension was tested again (Fig. 1C). Tm/Tn reconstitution was then performed in a solution containing 0.3-0.6 mg ml-1 Tm and 0.5-0.6 mg ml-1 bovine Tn (see legend of Table 1 for details) overnight (12-15 h), and active tension was measured again (Fig. 1D). Because it was difficult to control the exact length of the actin (or thin) filament reconstituted, and because tension and stiffness of subsequent experiments depended on the filament length, we normalized all subsequent data to the tension developed after the actin filament reconstitution (Fig. 1C). This method of normalization is also advantageous because it does not depend on the diameter of the preparation, which is another source of a scatter of the data.
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Figure 1. A slow pen trace of isometric tension at each step of removal and reconstitution of the thin filament A, control myocardium; B, after gelsolin treatment for 50 min (G); C, after actin filament reconstitution (Ac); D, after thin filament reconstitution (Tm & Tn) for 12 h; E, MgATP study; F, pCa study; G, Pi study; H, MgADP study; and I, final activation and rigor induction. Control myocardium was first activated in the 5S8P solution that included 5 mM MgATP and 8 mM Pi (pCa 4.66) at 25 °C (A), followed by relaxing solution (Rx). Before activation, myocardium was immersed in the 5S8P solution (pCa 4.66) at 2 °C, but it did not develop tension because of the low temperature. After gelsolin treatment, myocardium was immersed in the 5S8P solution at 25 °C to confirm the removal of thin filament (B). All activations including rigor were performed at 25 °C. Numbers above tension traces indicate the Ca2+ concentration in pCa units (F), or the concentration (mM) of MgATP (E), Pi (G) and MgADP (H). Rg designates the rigor solution (I). | ||
Three muscle models (acetyl-Tm reconstituted, AS-Tm reconstituted and AS-23Tm reconstituted; all in the presence of bovine Tn) were maximally activated in the presence of Ca2+ in a temperature-controlled bath at 25 °C for subsequent experiments. The sinusoidal waveform at 18 discrete frequencies (f: 0.13-100 Hz) was digitally synthesized on a PC with 386 CPU (Industrial Computer Source, San Diego, CA, USA), and applied to the muscle length via a 14-bit D/A converter. The amplitude of the length oscillation was 0.125 %. Tension and length signals were simultaneously digitized by two 16-bit A/D converters at a rate of ~100 kHz, the signals were accumulated and averaged, and complex modulus data Y(f) were calculated as the ratio of the force change to the length change in the frequency domain. The data were corrected using the rigor response as reported previously (Kawai & Brandt, 1980). The complex modulus data were resolved into two exponential processes (B and C) by fitting the data to eqn (1) (Kawai & Brandt, 1980; Wannenburg et al. 2000; Fujita et al. 2002):
Process B Process C
Y(f) = H - B/(1 + b/fi) + C/(1 + c/fi), (1)
where i = 
-1; lowercase letters b and c represent the characteristic frequencies of processes B and C, respectively, and uppercase letters B and C represent their respective magnitudes. H is a constant. 2
b and 2c are the apparent rate constants of the respective processes. Process B is a low-frequency exponential delay (b ~1 Hz) at which muscle generates oscillatory work. Process C is a high frequency exponential advance (c ~6 Hz) at which the muscle absorbs work from the length driver. Stiffness was calculated as Y
H - B + C. This is the stiffness extrapolated to the infinite frequency, and its value is not very different from the stiffness measured at 100 Hz in cardiac muscles. Process B corresponds to 'phase 3', process C corresponds to 'phase 2' and Yè corresponds to 'phase 1' of tension transients in response to a step-length change (Huxley & Simmons, 1971; Heinl et al. 1974). Process A (phase 4), which is normally present in fast twitch skeletal muscles, is absent in myocardium (Saeki et al. 1991; Kawai et al. 1993; Wannenburg et al. 2000). An implicit assumption of the transient analysis in response to a length change (step or sinusoidal) is that the rate constants of elementary steps are strain sensitive (Huxley & Simmons, 1971; Kawai & Brandt, 1980; Halvorson, 1993; Kawai & Zhao, 1993), so that a length change causes a transient instability in the equilibrium of the cross-bridge cycle. A gradual approach to the new steady state is observed as tension transients in step analysis and exponential processes in sinusoidal analysis. In relaxed and rigor muscle fibres, these transients or exponential processes are absent, indicating that the exponential processes are the results of cycling cross-bridges. Details of the sinusoidal analysis technique have previously been published (Kawai & Brandt, 1980).
Statistical analysis
Data are expressed as means ± S.E.M. Data were analysed by Kruskal-Wallis non-parametric test (following Bartlett's test of homogeneity of variance) followed by Newman-Keuls correction for multiple comparisons between means. The confidence interval is 95 %. Statistical comparisons were performed using Prism (version 3.0) software package (GraphPad Software Inc., San Diego, CA, USA).
SDS-PAGE
Myocardium at each step of reconstitution was pooled and dissolved in a sample-diluting buffer (2 % SDS, 25 % glycerol, 5 % -mercaptoethanol, 62.5 mM Tris-HCl, pH 6.8) and heated for 3 min at 90 °C. Five fibres were dissolved in 30 µl diluting buffer and 3 µl was loaded in each lane of the gel. SDS-PAGE was carried out according to the method of Laemmli (1970) with an 8-16 % linear gradient running gel and a 4 % stacking gel (Bio-Rad, Hercules, CA, USA). Protein was stained with Coomassie brilliant blue.
Electron microscopy
The fibres at each step of reconstitution were fixed in a solution that contained 2.5 % glutaraldehyde and 100 mM sodium cacodylate (pH 7.2) overnight at 2 °C. The fibres were then immersed in a solution containing 0.5 % tannic acid for 30 min. The preparations were washed with 100 mM sodium cacodylate (pH 7.2), then postfixed with 1 % OsO4 in the same buffer for 2 h at 2 °C, dehydrated with ethanol and acetone, and embedded in Poly/Bed 812 (Polysciences, Warrington, PA, USA). Sections were stained sequentially with saturated uranyl acetate and 2.6 % lead citrate at 20 °C. The thickness of the longitudinal section was 60 nm and that of the cross-section was 70 nm. Electron micrographs were taken at the Electron Microscope Center of the College of Medicine at the University of Iowa.
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RESULTS |
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Functional reconstitution of the thin filament
Figure 1 shows a slow pen trace record of isometric tension at each step of removal and reconstitution of the thin filament. A cardiac fibre (myocardium) was initially activated by the Ca2+-activating solution (5S8P, pCa 4.66) (Fig. 1A). This tension averaged 51 ± 6 kN m-2 (n = 26). Then at G, the fibre was treated by the extraction solution at 2 °C for 50 min (range: 30-60 min) to selectively remove the thin filament. BDM (40 mM) was used to suppress tension development during gelsolin treatment that requires Ca2+. After gelsolin treatment, 
12 % active tension developed when examined with the 5S8P solution (Fig. 1B). At Ac, the fibre was treated by the actin reconstitution solution (Table 1), which contained 1 mg ml-1 G-actin at 2 °C to reconstitute the actin filament. This solution also contained 80 mM KI, and the solution was replaced every 7 min to avoid actin nucleation. After a total of 28 min (7 min 4) in the actin reconstitution solution, this particular fibre developed ~65 % of the control tension (Fig. 1C). Tension developed by the actin filament averaged 25 ± 4 kN m-2 (n = 25). Relaxation was achieved by immersing fibres in the solution containing 40 mM BDM (Rx) at 2 °C. We noticed that there was a small residual tension after relaxation, averaging 10 ± 5 % (n = 19) of the control tension, as shown in Fig. 1C, but this tension gradually diminished during the subsequent treatment of the myocardium overnight. To reconstitute the thin filament, the actin filament-reconstituted fibres were immersed in the Tm and Tn reconstitution solution (Table 1) for 12 h at 2 °C (Tm & Tn in Fig. 1C). Isometric tension after reconstitution of regulatory proteins (Fig. 1D) was tested in the standard activating solution (5S8P), and it was comparable to that of the control fibres (Fig. 1A). In other words, the reconstitution of Tm and Tn augmented active tension by ~50 % over the tension developed by the actin filament-reconstituted fibres. Therefore, we confirmed that greater tension develops at 25 °C if Tm and Tn are reconstituted, as reported earlier (Fujita et al. 2002; Fujita & Kawai, 2002).
Sinusoidal analysis was performed at this stage (Fig. 1D), and complex moduli are compared in Fig. 2 for three different Tms - acetyl-Tm, AS-Tm and AS-23Tm. This figure demonstrates that all features of the complex modulus - minima and maxima - are present at approximately the same frequency in all the muscle groups we studied. The only difference is that the magnitude of the complex modulus was diminished to ~40 % in the case of AS-23Tm (Fig. 2G and I) compared to acetyl-Tm (Fig. 2A and C) or to AS-Tm (Fig. 2D and F). Note a change in the ordinate in Fig. 2G and I.
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Figure 2. Complex modulus of thin filament-reconstituted myocardium during activation The plots of the complex modulus Y(f) of thin filament-reconstituted myocardium by using acetyl-Tm (A-C), AS-Tm (D-F) and AS- | ||
After the full reconstitution of the thin filament, the MgATP study was performed in the range 0.05-5 mM as shown in Fig. 1E. This was followed by the Ca2+ study in the range pCa 7.0-4.0 (Fig. 1F), the Pi study in the range 0-32 mM (Fig. 1G), and the MgADP study in the range 0-3 mM (Fig. 1H). Finally, the fibre was activated by the standard activating solution (5S8P), followed by rigor induction (Fig. 1I). The reproducibility of the standard tension was adequate (> 80 %) until the end of the Pi study, but the tension diminished somewhat after the MgADP study, as we previously noticed in rabbit psoas fibres. It is possible that ADP has some deteriorating effects on the muscle fibres.
Effect of phosphate (Pi) on exponential process B
The effect of Pi in the range 0-32 mM (added Pi) on exponential process B in the three muscle models was studied as in Fig. 1G to determine the kinetic constants associated with elementary steps 4 and 5 (see eqn (2)):
where A = actin, M = myosin, D = MgADP and P = Pi, and * signifies the second conformational state. These studies were carried out in the maximal Ca2+-activating condition (pCa 4.66) in the presence of a saturating MgATP concentration (5 mM). The complex modulus data were fitted to eqn (1) to obtain the apparent rate constant 2b. 2b was then plotted against the Pi concentration (Fig. 3A) and fitted to eqn (3) to deduce the kinetic constants of elementary steps 4 and 5 of eqn (2) (Kawai & Halvorson, 1991).
where P = [Pi], and the kinetic constants (k4, k-4, K5) are defined in eqn (2). 
= 1 if there is no faster equilibrium to the left of eqn (2). In reality, because there are fast equilibria to the left of eqn (2), takes a value between 0 and 1 (see eqn (6) below and Table 2). In Fig. 3A, it can be seen that when the Pi concentration is increased, the rate constant 2b increases and saturates. The continuous line and squares are from AS-Tm, and the dashed line and circles are from AS-23Tm (mutant Tm). The curves are based on eqn (3) with best fit parameters. From these fittings, k4, k-4 (rate constants of the force-generation step) and K5 (Pi association constant) were deduced, and K4 (= k4/k-4) was calculated for each preparation. The data were then averaged for five to seven preparations and are shown in Table 2 together with S.E.M. The statistical analyses are also included in Table 2. This table demonstrates that the equilibrium constant K4 is indistinguishable between the acetyl-Tm and AS-Tm groups, but it increases 3 times in the AS-23Tm group. Similarly, K5 is indistinguishable between the acetyl-Tm and AS-Tm groups, but it increases 3 times in the AS-23Tm group. The table further demonstrates that the increase in K4 is primarily due to a decrease in k-4. With regard to acetyl-Tm and AS-Tm, there was no statistically significant difference in any of the parameters studied (Table 2).
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Figure 3. Effect of phosphate The rate constant 2 | ||
On the tension vs. Pi concentration plots (Fig. 3B), we found that tension decreased with an increase in the Pi concentration. We also found that the tension of the mutant group was about 40 % that of the control. Similarly, stiffness decreased with an increase in the Pi concentration (Fig. 3C), and the stiffness of the mutant group was about 40 % that of the control group. Isometric tension and stiffness measured in the 5S8P solution (pCa 4.66) during the ATP study (Fig. 1E) are summarized in Table 3. This table demonstrates that both tension and stiffness are indistinguishable between the acetyl-Tm and AS-Tm groups, but they decrease to 40 % in the AS-23Tm group.
Effects of MgATP and MgADP on exponential process C
The effects of MgATP (0.05-5 mM) and MgADP (0-3 mM) on exponential process C in the three muscle models were studied as in Fig. 1E and H to determine the kinetic constants associated with elementary steps 0, 1 and 2 (eqn (4)):
where S = MgATP and D = MgADP. These studies were carried out under the maximal Ca2+-activating condition (pCa 4.66) in the presence of 8 mM Pi. The complex modulus data were fitted to eqn (1) to obtain the apparent rate constant 2c. 2c was then plotted against the MgATP (Fig. 4) or MgADP (Fig. 5) concentration and fitted to eqn (5) to deduce the kinetic constants of elementary steps 0, 1 and 2 of eqn (4) (Kawai & Halvorson, 1989).
where S = [MgATP], D = [MgADP], and the kinetic constants are as defined in eqn (4). In Fig. 4, it can be seen that 2c increased and saturated as the MgATP concentration was increased for AS-Tm, but the increase was not that evident in the case of the mutant Tm. In Fig. 5, it can be seen that the rate constant 2c decreased when the MgADP concentration was increased. From these results, K0 (association constant for MgADP), K1 (association constant for MgATP), k2 and k-2 (rate constants of the ATP isomerization step) were deduced, and K2 (= k2/k-2) was calculated for each preparation. The data were then averaged for six to eight preparations and are shown in Table 2 together with S.E.M. This table demonstrates that both K1 and K2 are indistinguishable between acetyl-Tm- and AS-Tm-reconstituted myocardium. K1 in AS-23Tm is not significantly different from the control, but K2 in AS-23Tm decreases significantly (to 30 % of control). This decrease is primarily due to an increase in k-2. K0 is indistinguishable between acetyl-Tm- and AS-Tm-reconstituted myocardium, but decreases to 30 % in AS-23 Tm. In terms of acetyl-Tm and AS-Tm, there was no significant difference in any of the kinetic constants we studied (Table 2).
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Figure 4. Effect of MgATP The rate constant 2 | ||
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Figure 5. Effect of MgADP The rate constant 2 | ||
Combination of eqns (2) and (4)
By putting eqn (4) to the left of eqn (2), the two cross-bridge schemes can be combined (Kawai & Halvorson, 1991). Here we merge both weakly attached states (AMS, AMDP) and detached states (MS, MDP) and designate them in the detached state (Det), because it is difficult to distinguish these states in our method, which depends on strongly attached cross-bridges. in eqn (3) is then calculated from K1, K2 and S:
The values are calculated for S = 5 mM and listed in Table 2.
Cross-bridge distribution
The cross-bridge distribution in each state was then calculated using eqns (8)-(14) of Zhao & Kawai (1996) and is shown in Fig. 6. In Fig. 6, it can be seen that the distribution of strongly attached cross-bridges was not any different between acetyl-Tm and AS-Tm, and slightly increased (+15 %) in AS-23Tm. Conversely, the distribution of detached cross-bridges was not any different between acetyl-Tm and AS-Tm, and slightly decreased (-15 %) in AS-23Tm. From these results we conclude that the attached cross-bridge number does not decrease in AS-23Tm, supporting the idea that it is the tension per cross-bridge that is diminished in AS-23Tm.
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Figure 6. Cross-bridge distribution Calculated cross-bridge distribution in acetyl-Tm-reconstituted myocardium (open bars), AS-Tm-reconstituted myocardium (black bars) and AS- | ||
pCa-tension relationship
The effect of pCa (pCa = - log[Ca2+]) in the range 7.0-4.0 on tension and stiffness in the three muscle models was studied as in Fig. 1F. Figure 7A shows the pCa-tension relationship and Fig. 7B shows the pCa-stiffness relationship. It can be seen from Fig. 7A that the plot of tension in the mutant group (AS-23 Tm) is steeper than in the control group (AS-Tm), indicating that the cooperativity is greater in the mutant group than in the control group. In contrast, the mid-point, which indicates the Ca2+ sensitivity, is not any different between these two groups. Similarly in the stiffness plot (Fig. 7B), the plot of the mutant group is steeper than that of the control group, but the mid-point is not any different between the two groups. Tension was fitted to eqn (7) (Hill equation), and nH (Hill factor) and pCa50 (half-saturation point) were deduced:
The results are summarized in Table 3. This table demonstrates that the Hill factor was not significantly different between acetyl-Tm- reconstituted (1.74 ± 0.23) and AS-Tm- reconstituted (1.85 ± 0.06) myocardium. pCa50 (Ca2+ sensitivity) was slightly larger in acetyl-Tm- reconstituted (5.40 ± 0.08) than in AS-Tm-reconstituted (5.24 ± 0.04) myocardium. While the Hill factor increased by 1.4 times from AS-Tm (1.85 ± 0.06) to AS-23Tm (2.62 ± 0.15), pCa50 did not change at all from AS-Tm (5.24 ± 0.04) to AS-23Tm (5.25 ± 0.03). In terms of acetyl-Tm and AS-Tm, there was no statistically significant difference in any of the parameters (tension, stiffness, Hill factor) we studied, except for the slight effect on the Ca2+ sensitivity (Table 3).
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Figure 7. pCa-tension and -stiffness relationships Normalized isometric tension (A) and stiffness (B) are plotted against the pCa. AS-Tm, | ||
In some preparations we noticed a small tension even in the pCa 7.0 solution. This is the Ca2+-insensitive tension, and it should be distinguished from the Ca2+-sensitive tension. The Ca2+-insensitive tension averaged 2.0 ± 0.7 % (n = 11) of the control tension for AS-Tm, and 2.3 ± 1.4 % (n = 9) of the control tension for AS-23Tm.
SDS-PAGE
SDS-PAGE was performed to examine the degree of reconstitution and the results are shown in Fig. 8. Lane 1 is molecular mass markers. Lane 2 is control (myocardium without extraction or reconstitution). Lane 3 is gelsolin-treated fibres. In lane 3, actin is significantly decreased. Also, TnT, Tm and TnI are decreased. However, myosin heavy chain (MHC) and myosin light chain (MLC)1 appear to be the same or increased because of a greater loading in lane 3. These results indicate that thin filament proteins were extracted, but thick filament proteins were not extracted. In lane 3, a prominent band below -actinin is gelsolin that was not washed out. Lane 4 is purified G-actin, and lane 5 is actin-reconstituted fibres. Actin in lane 5 was significantly restored compared to lane 3. Lane 6 is purified bovine Tn. TnT and TnI can be seen in this lane. Lane 8 is purified acetyl-Tm, and lane 7 is reconstituted fibres using this Tm together with Tn. Actin, TnT, Tm and TnI can be seen on lane 7. Lane 10 is purified AS-Tm, and lane 9 is reconstituted fibres using this Tm together with Tn. Once again actin, TnT, Tm and TnI can be seen. Lane 12 is AS-23Tm, and lane 11 is reconstituted fibres using AS-23Tm together with Tn. Actin, TnT and residual (normal) Tm can be seen in lane 11. In lane 11, AS-23Tm is superimposed by TnI. In lanes originating from fibres (lanes 2, 3, 5, 7, 9, 11), MHC, -actinin and MLC1 can be recognized.
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Figure 8. SDS-PAGE of cardiac muscle fibres and proteins Lane 1, molecular mass markers (kDa). Lane 2, myocardium without extraction or reconstitution. Lane 3, gelsolin-treated myocardium. Lane 4, purified G-actin. Lane 5, actin-reconstituted myocardium. Lane 6, purified bovine Tn. Lane 7, reconstituted myocardium using acetyl-Tm. Lane 8, purified acetyl-Tm. Lane 9, reconstituted myocardium using AS-Tm. Lane 10, purified AS-Tm. Lane 11, reconstituted myocardium using AS- | ||
Electron microscopy (EM)
Muscle fibres were examined by EM and are shown in Fig. 9 both in the longitudinal section and in the cross-section. Fig. 9A is a control fibre. In the longitudinal section, the thin filament can be seen in the I band region and going into the A band region. In the cross-section, it can be seen that the thick filament is surrounded by the thin filament. Figure 9B is a fibre after gelsolin treatment. The thin filament is significantly diminished, but there may still be some left along the Z line. This residual thin filament is essential for reconstitution of the actin filament. In the cross-section, there is hardly any thin filament left, but there is often empty space where the thin filament existed previously. After reconstitution with purified G-actin, the actin filament reappears (Fig. 9C). In cross-section, the thick filament can be seen surrounded by the actin filament. The thin filament was reconstituted with AS-Tm and bovine Tn in Fig. 9D, and with AS-23Tm and bovine Tn in Fig. 9E. In Fig. 9D and E, the thin filament can be found both in the longitudinal section and in the cross-section.
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Figure 9. Electron microscopic images of myocardium at each step of extraction and reconstitution A, untreated myocardium; B, after gelsolin treatment; C, after actin filament reconstitution; D, after thin filament reconstitution with AS-Tm and bovine Tn; and E, after thin filament reconstitution with AS- | ||
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DISCUSSION |
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Reconstitution technique and its potential
The present study employed gelsolin-mediated thin filament removal, followed by thin filament reconstitution to determine the effects of Tm internal deletion mutant AS-23Tm on cross-bridge function. The reconstitution was examined by SDS-PAGE (Fig. 8), EM (Fig. 9), isometric tension (Fig. 1 and Fig. 3B), complex modulus (Fig. 2), stiffness (Fig. 3C), apparent rate constants (Figs 3A, 4 and 5), and the rate and association constants of the elementary steps of the cross-bridge cycle (Table 2). The results generally indicate that the structural and functional reconstitution was satisfactory in cardiac muscle fibres as reported earlier (Fujita et al. 1996, 2002). A possible shortfall is that the technique is demanding in that it requires freshly prepared actin and extremely well-prepared cardiac muscle fibres. An additional shortfall is that the technique was only partially satisfactory for more commonly used preparations such as rabbit psoas fibres (Funatsu et al. 1994).
Because of the known effects of AS-23Tm on the actin-myosin filament interaction in solution, we anticipated that AS-23Tm would similarly alter the cross-bridge function in a selective and informative manner. Further, we anticipated that insights would be gained by comparing the previous biochemical data with the new physiological and mechanical data. These anticipations are examined and supported by our results. The principle finding is that the deletion decreases the tension generated by each strongly attached cross-bridge, suggesting that Tm modulates this important parameter.
AS-23Tm diminishes the contractility during Ca2+ activation
Hitchcock-DeGregori and colleagues were the first to create Tm internal deletion mutants. They found that the second and third of the seven quasi-repeat regions of Tm could be deleted (AS-23Tm) with relatively minor functional consequences (Hitchcock-DeGregori & Varnell, 1990; Hitchcock-DeGregori & An, 1996). Similarly, Landis et al. (1999) found that thin filament motility was regulated by Ca2+ in the presence of AS-23Tm in an in vitro motility assay using heavy meromyosin as a motor. However, the sliding of AS-23Tm-reconstituted thin filament was abnormal. While 90 % of the control AS-Tm filament moved continuously, only 67 % of AS-23Tm filament moved at 73 % of the speed of control (Landis et al. 1999). We have now found a similar but larger effect in cardiac muscle fibres. Fibres with AS-23Tm generated only about 40 % of the tension of control fibres that consisted of either recombinant Tm (AS-Tm) or muscle Tm (acetyl-Tm) (Table 3). We further found that in the presence of AS-23Tm, strongly attached cross-bridges not only produced sub-normal force but also exhibited sub-normal stiffness. For several reasons, these effects cannot be due to altered Ca2+ binding to Tn. We found that the pCa50 was the same for fibres with AS-23Tm (5.25 ± 0.03) as for fibres with control AS-Tm (5.24 ± 0.04) (Table 3). Also, it was found that AS-23Tm had no effect on Tm-Tn binding to actin in the presence/absence of Ca2+ (Landis et al. 1999). These findings are consistent with the fact that the residues deleted in AS-23Tm do not have a direct contact with Tn (reviewed in Tobacman, 1996).
Can blocking of the actin sites by Tn explain reduced tension and stiffness in AS-23Tm ?
It is important to enquire how the altered structure of AS-23Tm might account for the observed findings. A straightforward effect of AS-23Tm is to increase the Tn density on the thin filament, from one in seven actin molecules to one in five actin molecules (Landis et al. 1999). It has been known for some time that the Tn molecule is relatively large, larger than initially envisaged by Ebashi & Endo (1968), and the globular domain of Tn is approximately two actins long (Takeda et al. 2003). However, the increased Tn density resulting from AS-23Tm is unlikely to produce enough steric interference to explain our results. If each Tn interferes with two actins, then the fraction of actins available for cross-bridge binding is 5/7 = 0.71 in the presence of acetyl-Tm or AS-Tm, and 3/5 = 0.6 in the presence of AS-23Tm, resulting in a 16 % decrease (= 1 - 0.6/0.71). We observed a much larger decrease (60 %) in both tension and stiffness. Furthermore, the effect of short Tm on S1-thin filament binding in solution (Tobacman & Butters, 2000) is not consistent with steric hindrance by Tn in two respects. In the presence of a low S1 concentration (very low binding saturation), binding is weakened to an extent that depends on the region of Tm deleted, but not on the length of the deleted segment. At the higher S1 concentration, S1 binding is much tighter to actin-Tm-Tn than to bare actin in both control and AS-23Tm, because of cooperative activation. Based on these reasons, we conclude that the blockage of the actin sites by Tn cannot explain the reduced tension and stiffness observed in fibres with AS-23Tm.
Two ways to reduce tension and stiffness
What, then, would be the mechanisms to explain the reduced tension and stiffness in myocardium with AS-23Tm? One possibility is that the Tm deletion reduces the number of strongly attached, tension- and stiffness-generating cross-bridges. The alternative possibility is that the mutant Tm reduces tension and stiffness in each strongly attached cross-bridge. To distinguish these two possibilities, we performed sinusoidal analysis and deduced the kinetic constants of elementary steps of the cross-bridge cycle, which consists of six states (Figs 3, 4 and 5, and Table 2). From the equilibrium constants, we calculated the number of cross-bridges in each state (Fig. 6). Our results indicate that the number of strongly attached cross-bridges does not decrease in fibres containing AS-23Tm, but instead slightly increases (Fig. 6, Att). From these results we conclude that, with AS-23Tm, force per cross-bridge is reduced to ~40 % and stiffness per cross-bridge is reduced to ~40 % compared to AS-Tm.
Possible mechanism to reduce tension and stiffness on each cross-bridge with AS-23Tm
Our earlier investigation found that the temperature effect on tension is significantly greater when Tm and Tn are present than when they are absent (Fujita & Kawai, 2002). This temperature effect is caused by stereo-specific and hydrophobic interaction between actin and myosin molecules (Zhao & Kawai, 1994; Murphy et al. 1996; Wang & Kawai, 2001). The increase in the temperature effect therefore implies an increase in stereo-specific and hydrophobic interaction between actin and myosin molecules when Tm, Tn and Ca2+ are present. Consistent with these findings, it was observed that Tm and Tn increased force per cross-bridge in myocardium (Fujita et al. 2002), as well as force and sliding speed in in vitro motility assays (Gordon et al. 1998; Bing et al. 2000a,b; Homsher et al. 2000) at ~25 °C. In solution, Tm and S1 increased each other's actin affinity (including a 104-fold effect of S1 on Tm-actin binding), and Tm internal deletion mutants including 23Tm modified this effect (Landis et al. 1999; Rosol et al. 2000; Tobacman & Butters, 2000). When these lines of evidence are combined, we are bound to conclude that Tm, in the presence of Tn and Ca2+, modifies the actin surface so as to improve the actomyosin interface for increased stereo-specific and hydrophobic interaction. The improved interface results in the increased force, increased stiffness and increased effect of temperature on isometric tension. This modification is partially impaired by AS-23Tm, thereby diminishing the force- and stiffness-generating capability of cross-bridges. That is to say that the stiffness of strongly attached cross-bridges depends on the strength of the stereo-specific interaction between actin and myosin molecules, and this strength is diminished with AS-23Tm.
Effects of substituting Ala-Ser for acetylation at the N-terminus of Tm
Bacterially expressed eukaryotic proteins lack N-terminal acetylation, which for Tm results in N-terminal uncoiling and impaired function (Palm et al. 2003). Because it was reported that the addition of an Ala-Ser at the N-terminus corrects this functional defect (Monteiro et al. 1994), we included this dipeptide in bacterially expressed Tm. Therefore, it was necessary to characterize the effect of substituting Ala-Ser for N-terminal acetylation. Our results indicate that there is no significant difference between acetyl-Tm and AS-Tm in terms of tension, stiffness, Hill factor (nH) and the kinetic constants of the elementary steps when they are fully reconstituted with Tn (Tables 2 and 3). However, there was a small effect of the recombinant protein on the apparent Ca2+ affinity. The pCa50 decreased slightly from 5.40 ± 0.08 to 5.24 ± 0.04, suggesting that the Tm N-terminus might modulate the apparent Ca2+ affinity. The mechanism for this is unclear, but it may relate to the recent finding that a substitution of the Ala-Ser dipeptide for N-acetylation results in a weakening of the Tm end-to-end overlap joint that is stabilized by Tn (Palm et al. 2003). It is known that Tn binds to Tm most tightly by interactions between the N-terminus of TnT and the C-terminus (region 7) of Tm (Phillips et al. 1979; Ishii & Lehrer, 1991; Butters et al. 1993; Fisher et al. 1995; Hammell & Hitchcock-DeGregori, 1996; Hinkle et al. 1999).
Cooperativity
We observed that the Hill coefficient (nH) was 1.85 ± 0.06 for fibres containing AS-Tm and 2.62 ± 0.15 for fibres with AS-23Tm (Table 3), an increase of 1.42 (± 0.09)-fold, which indicates an increase in cooperativity. It is interesting, therefore, that in comparison to AS-Tm, AS-23Tm increases nH by the same factor that it increases the number of Ca2+-binding sites on each thin filament: 7/5 = 1.4. For any multimeric assembly, the maximal possible Hill coefficient equals the number of ligand-binding sites. Our previous investigation in rabbit psoas fibres revealed that the cooperative length of the thin filament is ~4 regulatory units (Ding et al. 2002), corresponding to 28 actin monomers with a length of about 150 nm. If this parameter is not altered in the presence of 23Tm, then the number of Ca2+-binding sites in one cooperative unit increases from 4 to 5.6 (7/5-fold), because only one Ca2+-binding site is physiologically significant per Tn molecule in cardiac muscles. The nH we found is smaller than this; therefore, other factors may also be involved in determining overall cooperativity, including cross-bridge interaction with actin. It has been found that AS-23Tm increases the cooperativity of S1-thin filament binding both in the presence and in the absence of Ca2+ (Tobacman & Butters, 2000), and this mechanism is an additional possibility that could contribute to the increased nH observed (Fig. 6).
Conclusion
We conclude that Tm changes the conformation of actin in the presence of Tn and Ca2+ so as to increase the stereo-specific and hydrophobic interaction between actin and myosin molecules. This modulatory action of Tm is partially impaired in AS-23Tm, resulting in a decrease in tension and stiffness. The cooperativity is increased presumably because of the increased Ca2+ binding per unit length of the thin filament.
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Acknowledgements
We thank Drs Shin'ichi Ishiwata and Hideaki Fujita for providing us with gelsolin and acetone powder; Dr Sathivel Chinathambi for helping us with SDS-PAGE; Dr Jian Shao of the Electron Microscope Facility in the College of Medicine at the University of Iowa for his help in taking the EM images; and Ms Mary Bryant for excellent technical assistance. This work was supported by grants NSF 98-14441 and NIH HL70041 to M.K., and AHA Postdoctoral Fellowship 0320083Z to X.L. Any opinions, findings and conclusions or the contents of this work are solely the responsibility of the authors and do not necessarily reflect the views of the National Science Foundation. Similarly, the contents of this work are solely the responsibility of the authors and do not necessarily represent the official view of National Institutes of Health.
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and continuous line; AS-
and dashed line). Experiments were carried out in the presence of 5 mM MgATP and at pCa 4.66. Curved lines in A are theoretical projections based on eqn (3) and its best-fitted parameters. Tension and stiffness were normalized to the tension generated when the actin filament was reconstituted and activated by the 5S8P solution. The means of 5-7 experiments with S.E.M. error bars are shown.