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Cardiovascular Research 2003 60(3):580-588; doi:10.1016/j.cardiores.2003.09.011
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Copyright © 2003, European Society of Cardiology

Myosin light chain isoforms modify force-generating ability of cardiac myosin by changing the kinetics of actin–myosin interaction

Hiroshi Yamashita*,a, Seiryo Sugiurab, Hideo Fujitaa, So-ichiro Yasudaa, Ryozo Nagaia, Yasutake Saekic, Kenji Sunagawad and Haruo Sugie

aDepartment of Cardiovascular Medicine, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan
bGraduate School of Frontier Sciences, Institute of Environmental Studies, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan
cDepartment of Physiology, Tsurumi University School of Dental Medicine, 2-3-1 Tsurumi, Tsurumi, Yokohama 230-8501, Japan
dDepartment of Cardiovascular Dynamics, National Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka, Japan
eDepartment of Physiology, School of Medicine, Teikyo University, 2-11-1 Kaga, Itabashi, Tokyo 173-0003, Japan

*Corresponding author. Tel.: +81-3-3815-5411x33072; fax: +81-3-3814-0021. Email address: hiroyama-tky{at}umin.ac.jp

Received 1 August 2003; revised 3 September 2003; accepted 9 September 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: To investigate the functional role of myosin light chain (MLC) isoforms in cardiac muscles, we examined the motor function of two different myosins the structure of which differed only in the MLC. Methods: We purified myosin from atria (A-myosin) and ventricles (V-myosin) of young rats, which contained atrial-type and ventricular-type MLCs, respectively, but having identical {alpha}-heavy chain isoform. Actin filament velocity (Vel) was determined in the in vitro motility assay. Average force of myosin molecules (F) was estimated and single events of actin–myosin interaction were recorded with the laser trap technique. Results: Vel was slightly higher in A-myosin than in V-myosin, while actin-activated ATPase activity was not different. F, determined from force versus actin filament length relation, was ~60% higher in V-myosin (3.3 vs. 2.1 pN/µm). The mean duration of isometric force events was longer in V-myosin than in A-myosin (323±13 vs. 294±30 ms, p<0.05), while the amplitudes of unitary displacement and force of a single myosin molecule did not differ between them. Conclusion: The MLC isoform can be a determinant of force-generating ability of cardiac myosin by modulating crossbridge kinetics without affecting the catalytic activity.

KEYWORDS Cardiac myosin; Myosin light chain; ATPase activity; In vitro motility assay; Actin translocating velocity; Laser trap; Unitary displacement; Unitary force


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Cardiac myosin is a hexamer consisting of two myosin heavy chains (MHC), each associated with two myosin light chains (MLC), i.e., the essential light chain (ELC or LC1) and the regulatory light chain (RLC or LC2). In cardiac muscles, there are multiple isoforms in each subunit, and their expression is regulated in tissue-specific manner under various physiological and pathological conditions [1]. For example, there are two MHC isoforms ({alpha}- and β-MHC) to make up three myosin isoforms (V1, V2, and V3) [2]. In rodents, adult ventricular muscle predominantly contains V1 (homodimer of {alpha}-MHC), and the MHC composition shifts from V1 to V3 under mechanical overload [3,4]. Since V3 has the lowest ATPase activity and the highest economy of force production, the isoform shift towards V3 is considered to be the adaptation in response to mechanical load. Studies using in vitro motility assay demonstrated distinct mechanical properties of these isoforms at the molecular level and gave further support to this concept [5,6]. In human ventricular muscles, however, functional roles of the MHC isoforms is controversial. Although recent studies revealed that human ventricular muscles from normal subjects expressed significant amount of {alpha}-MHC at both RNA and protein levels, the net change observed in hypertrophied or failing human ventricles was much smaller compared to the rodent models [7–12].

On the other hand, the MLC isoforms have been shown to change significantly in response to mechanical load in human ventricles, and functional properties also change in parallel with the MLC isoform shift [10,13]. In mammalian adult hearts, ventricular muscle predominantly expresses ventricular-type MLC (VLC1 and VLC2), and atrial muscle expresses atrial-type counterparts (ALC1 and ALC2). The ALC1 is also a major isoform in fetal ventricles [14], but it is re-expressed in hypertrophied ventricles caused by various heart diseases [10,11,15]. Studies using human cardiac tissues showed that ventricular fibers containing even small amount of ALC1 had higher shortening velocity and maximum isometric force compared to control subjects [16]. Although these results suggested possible involvement of the MLC shift in the adaptation of human heart to pathological overload, we should be careful in interpreting these results, because these functional parameters could be influenced by other factors at tissue or cellular levels.

In the present study, we focused on possible roles of the MLC isoforms in cardiac adaptation mechanism. We purified two different myosins from atria and ventricles of young rats. Because these myosins contained identical MHC and different sets of MLCs, these myosins provided us with a good opportunity to dissect the functional role of the MLC isoforms. We characterized motor function of these myosins using the in vitro motility assay and the single motor assay systems, where actin–myosin interaction was reconstituted from isolated proteins. The results showed that myosin molecules containing ventricular-type MLCs had longer duration of force generation and higher average force compared to those with atrial-type MLCs. The ATPase activity, however, was not different between these myosins. The present study constitutes the first evidence of the unique role of MLC as a modulator of motor function at the molecular level and gives support for functional significance of the MLC isoforms in cardiac adaptation.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Proteins
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996). Animals were anesthetized with 5 ml of dimethylether, and hearts were excised rapidly. Ventricular myosin (V-myosin) was purified from ventricles of 4-week-old male Wistar rats (n = 6), as described elsewhere [17]. Atrial myosin (A-myosin) was purified from atria of 8-week-old male Wistar rats, because higher amount of atrial myosin with identical isoform composition was obtained from 8-week-old animals compared to 4-week-old ones. Atrial tissues from four animals were pooled, and myosin was purified in the same way as V-myosin. Six independent samples of both A- and V-myosin were prepared in parallel, and functional assays were performed simultaneously. Immediately before each experiment, inactive myosin molecules binding to actin in an ATP-independent manner were removed as previously described [18]. Actin was prepared from rabbit back muscles by the method of Spudich and Watt [19]. Gelsolin was prepared from bovine plasma by the method of Kurokawa et al. [20]. The MHC isoform composition was analyzed by SDS–PAGE according to Reiser and Kline [21]. The phosphorylation level of LC2 was analyzed by urea polyacrylamide gel electrophoresis [22], and dephosphorylated control samples were treated with acid phosphatase [23]. NEM-myosin was prepared as described [24].

2.2. Actin-activated ATPase activity
Actin-activated ATPase of myosin samples was measured as described by mixing myosin (0.05 mg/ml) with actin (5–35 µmol/l) in an assay buffer (25 mmol/l KCl, 10 mmol/l imidazole, 0.4 mmol/l EGTA, 2 mmol/l MgCl2 and 1 mmol/l DTT, pH 7.0) at 37 °C [18]. The reaction was initiated by adding 2 mmol/l MgATP. Data from two to three independent myosin samples were averaged.

2.3. In vitro motility assay
The in vitro motility assay was performed by the method of Kron and Spudich [25] with some modifications [5]. The experiments were done at 30 °C in two motility assay buffers with different KCl concentrations (20 or 40 mmol/l KCl, 5 mmol/l MgCl2, 1 mmol/l EGTA, 2 mmol/l MgATP, 10 mmol/l DTT, 10 mmol/l MOPS, pH 7.2, and 0.7% methylcellulose, 2.5 mg/ml glucose, 0.1 mg/ml glucose oxydase and 0.02 mg/ml catalase). The velocities of 90–96 moving filaments from four independent preparations were averaged.

2.4. Average force of myosin molecules
The ability of individual myosin molecules to generate force was estimated by the method of Sugiura et al. [26] (Fig. 1A). In brief, 40 µl of myosin sample (0.25 µg/ml) was spread over nitrocellulose-coated surface of the flow cell identical with that used for the in vitro motility assay. Fluorescent actin filaments, each with a gelsolin-coated polystyrene bead (1 µm in diameter) attached to its barbed end, were suspended in a force assay buffer (25 mmol/l KCl, 4 mmol/l MgCl2, 1 mmol/l EGTA, 2 mmol/l MgATP, 25 mmol/l imidazole, 1% 2-mercaptoethanol, 2.5 mg/ml glucose, 0.1 mg/ml glucose oxydase and 0.02 mg/ml catalase, pH 7.5) and introduced into the flow cell. After a bead was captured by the laser trap under microscopic observation, the actin filament was brought into contact with myosin molecules on the bottom of the flow cell. The force generated by multiple myosin molecules on the actin filament was measured as a steady stall force (1–5 s in duration) [26]. The trap stiffness was 0.03–0.06 pN/nm. The experiments were done at 30 °C.


Figure 1
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  		Fig. 1 (A) Schematic diagram of the system for measuring average force of myosin molecules. LT, laser trap; PB, polystyrene bead; FA, fluorescently labeled actin filament; M, myosin molecule; L, length of actin filament interacting with the myosin layer; XL, xenon lamp; SIT, silicon intensified target camera; F, fluorescent image of the bead; B, bright field image of the bead. (B) Schematic drawing of the single motor assay system. LT, laser trap; PB, polystyrene bead; FA, fluorescently labeled actin filament; M, myosin molecule; PD, photodiode detector.

 
2.5. Single motor assay
Using dual-beam laser optical trap system similar to that of Molloy et al. [27], we measured unitary displacement and force generated by a single myosin molecule (Fig. 1B). The laser beam was split into two with rectangular electric pulses (10 kHz) applied to a pair of orthogonally mounted acousto-optic deflectors (AODs) (TS100, Isle Optics, UK) to obtain two independent optical traps. A single fluorescent actin filament, to which a pair of polystyrene beads were attached on both ends with NEM-myosin, was held horizontally by capturing the two beads in the two optical traps. Glass beads (1.7 µm in diameter; Bang Lab., IN) were fixed on the bottom of the flow cell, and their top surfaces were covered with myosin molecules sparsely by applying diluted myosin sample into the flow cell. The flow cell was filled with a unitary event buffer (25 mmol/l KCl, 6 mmol/l MgCl2, 1 mmol/l EGTA, 1 µmol/l MgATP, 25 mmol/l imidazole, 1% 2-mercaptoethanol, 2.5 mg/ml glucose, 0.1 mg/ml glucose oxydase and 0.02 mg/ml catalase, pH 7.5), and the actin filament, made taut between the two traps, was brought into contact with a single myosin molecule on the glass bead to induce interaction between them. Movement of a bead trapped by one of the traps was recorded by a quadrant photodiode detector (Hamamatsu Photonics, Japan). Unitary displacements generated by a myosin molecule were recorded under low trap stiffness (0.03 pN/nm). To record unitary isometric force generated by a single myosin molecule, the output of the quadrant photodiode detector was applied to a feedback circuit driving the two acousto-optic deflectors to control the trap position, so that the bead was held stationary in position. The isometric force was measured as a linear function of the trap displacement. With this feedback system, the trap stiffness was increased to 2.1 pN/nm. The experiments were done at 30 °C.

2.6. Data collection and analysis
Data were sampled at 4 KHz and stored in a personal computer by an A–D converter (MacLab, ADInstruments, Australia) and analyzed off-line. Unitary events were identified by monitoring the root square mean of the data. To estimate the mean duration of unitary events, a single exponential was fit to frequency histograms of the event duration by nonlinear curve-fitting software (Igor, Move Metrics). Data were expressed as mean±S.E.M. A two-tailed unpaired Student's t test was used to determine significant difference between group means and fit parameters. A value of p<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. SDS–PAGE
As shown in the 5–20% gradient SDS–PAGE, A- and V-myosins contained different LC-1 and LC-2 isoforms (Fig. 2A). The MHC isoforms were further separated on the 5% glycerol SDS–PAGE, confirming the earlier report that both A- and V-myosins contained only {alpha}-MHC isoform, while the reference sample from the ventricle of an 8-week-old animal contained both {alpha}- and β-MHC isoforms [28] (Fig. 2B). On urea gels, both ALC-2 in A-myosin and VLC-2 in V-myosin showed a single band identical to that of fully dephosphorylated control myosin treated with acid phosphatase, indicating that both ALC-2 and VLC-2 were dephosphorylated to a similar extent during the purification procedure (data not shown).


Figure 2
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  		Fig. 2 (A) Five to twenty percent gradient SDS–PAGE of A-myosin (A) and V-myosin (V). MHC, myosin heavy chain; ALC1, atrial light chain-1; ALC2, atrial light chain-2; VLC1, ventricular light chain-1; VLC2, ventricular light chain-2. A-myosin contains ALC-1 and ALC-2, while V-myosin contains VLC-1 and VLC-2. (B) Five percent glycerol SDS-PAGE of A-myosin (A) and V-myosin (V) separating the MHC isoforms. Both myosins contain predominantly {alpha}-MHC ({alpha}{alpha}-homodimer). R represents ventricular myosin obtained from an 8-week-old rat containing both {alpha}- and β-MHC as a reference.

 
3.2. Actin-activated ATPase activity
In Fig. 3, actin-activated ATPase activity of A- and V-myosins are plotted as a function of actin concentration. The Vmax (1.23 s–1 for A-myosin and 1.30 s–1 for V-myosin) and Km (18.2 µmol for A-myosin and 14.5 µmol for V-myosin) did not differ significantly between these myosins. These results confirmed the earlier finding that the MLC isoform composition had no significant effect on ATPase activity in solution [14,29,30].


Figure 3
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  		Fig. 3 Actin-activated ATPase activity of A-myosin (open circles) and V-myosin (closed triangles). Curves were fitted by the Michaelis–Menten kinetics.

 
3.3. In vitro motility assay
As shown in Fig. 4, unloaded sliding velocity of actin filament on myosin was slightly higher for A-myosin than for V-myosin in the motility assay buffer with 40 mmol/l KCl (5.9±0.1 vs. 5.1±0.1 µm/s, p<0.01, n = 90–96). However, in the assay buffer with 20 mmol/l KCl, the difference between these myosins was smaller and not statistically significant (4.2±0.1 vs. 4.0±0.1 µm/s, N.S., n = 90–96).


Figure 4
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  		Fig. 4 Actin filament velocity of A-myosin (shaded column) and V-myosin (filled column) in the motility assay buffers with 20 and 40 mmol/l KCl concentration. Values are means±S.E.M. of velocities of smoothly moving filaments from four independent myosin preparations (n = 90–96). **p<0.01 vs. V-myosin.

 
3.4. Average force of myosin molecules
In Fig. 5, steady stall force generated by multiple myosin molecules is plotted as a function of actin filament length. In the present study, we applied both A- and V-myosin samples into the flow cell at the concentration of 0.25 mg/ml, a value known to saturate the nitrocellulose-coated surface of the flow cell with myosin molecules [25,26,31]. Consequently, the number of myosin molecules interacting with unit length of actin filament was considered to be similar between these myosins, and the slope of the linear regression line between the stall force and the actin filament length could serve as average force generated by myosin molecules [26,31]. The slope for V-myosin was ~60% higher than that for A-myosin (3.3 vs. 2.1 pN/µm), indicating that V-myosin could generate ~60% higher average force compared to A-myosin.


Figure 5
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  		Fig. 5 Relationship between the stall force and the actin filament length (L) for A-myosin (open circles) and V-myosin (closed triangles). Linear regression lines are drawn by the least-squares method.

 
3.5. Single motor assay
As shown in Fig. 6A, the bead attached to one end of an actin filament exhibited discrete stepwise movements within the trap under low trap stiffness. Mean amplitude of the steps was not statistically different between A- and V-myosins (13.1±0.7 vs. 14.7±1.3 nm, N.S.). In both myosins, frequency histograms of the displacement amplitude showed broad distribution with two peaks at 8–9 and 15–16 nm (Fig. 7A). This suggests that the amplitude of unitary displacement was the same in both myosins, being 8–9 nm. As shown in Fig. 7B, histograms of displacement duration also exhibited broad distribution and were well fitted to single exponential curves, being consistent with the idea that a first-order kinetic process limited detachment of myosin molecules from actin. The mean duration of the displacement events, estimated from the exponential fit of the duration distribution, was longer in V-myosin than in A-myosin (256±33 vs. 167±5 ms, p<0.01).


Figure 6
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  		Fig. 6 (A) Examples of displacement records of A-myosin (A) and V-myosin (V) under low trap stiffness. Vertical deflections indicate movements of the bead in the direction parallel to the actin filament long axis. Arrows indicate unitary events. (B) Examples of isometric force records of A-myosin (A) and V-myosin (V) with the feedback loop closed. Vertical deflections indicate movement of the trap position, representing force transients.

 

Figure 7
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  		Fig. 7 (A) Frequency histograms showing distribution of the amplitude of displacement in A-myosin (A) and V-myosin (V). Dashed lines represent Gaussian components having peaks at around 8–9 and 15–16 nm in both myosins. (B) Distribution of the duration of displacement events in A-myosin (A) and V-myosin (V). Dashed lines represent single exponential fits of the data.

 
Typical recordings of isometric force events under high trap stiffness with feedback loop closed are shown in Fig. 6B. Mean amplitude of the force events was not significantly different between these myosins (1.51±0.05 vs. 1.41±0.04 pN, N.S.). As with the displacement events, frequency histograms of force amplitude showed broad distribution, but we could identify only a single peak at ~1.4 pN in both myosins (Fig. 8A), suggesting that the unitary force was the same in these myosins. On the other hand, the mean duration of force events, estimated from the exponential fit of the duration distribution, was longer in V-myosin than in A-myosin (323±13 vs. 294±30 ms, p<0.05) (Fig. 8B).


Figure 8
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  		Fig. 8 (A) Frequency histograms showing distribution of the amplitude of force transients in A-myosin (A) and V-myosin (V). Only one peak was evident at ~1.4 pN in both myosins. (B) Distribution of the duration of force transients in A-myosin (A) and V-myosin (V). Dashed lines represent single exponential fits of the data.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
We investigated the functional role of MLC isoforms in cardiac muscles by characterizing the molecular function of two different myosins, the structure of which differed only in MLC. In accordance with previous reports that the catalytic activity of cardiac myosin depended primarily on its MHC composition [3,4,29,30], the actin-activated ATPase activity was not different between these myosins (Fig. 3). The motor activity of the molecule, however, was significantly different between these myosins.

The actin filament velocity was slightly but significantly higher in A-myosin than in V-myosin in the motility assay buffer with 40 mmol/l KCl (Fig. 4). The results were consistent with the reported effects of MLC composition on the actin filament velocity in a similar motility assay and the unloaded shortening velocity of cardiac muscle fibers [32,33]. However, the actin filament velocity was not significantly different between these myosins in the assay buffer with lower ionic strength (20 mmol/l KCl). It suggests that the ADP-releasing step in the actomyosin ATPase reaction, which has been regarded as the rate-limiting step determining the actin filament velocity in the motility assay and the unloaded shortening velocity of cardiac muscle [17,34], may involve electrostatic interaction between actin and myosin. Since the amino-terminal domain of LC1 is postulated to interact with actin [35,36], the difference in the actin filament velocity between A- and V-myosin may be associated with the difference in charge distribution of amino-acid residues in the amino-terminus between ALC1 and VLC1 [37].

The most salient but puzzling finding was that V-myosin generated ~60% higher average force compared to A-myosin without significant changes in the ATPase activity (Fig. 5). In our earlier report, we compared average force of rat V1 and V3 myosins, consisting of {alpha}- and β-MHC homodimers, respectively, in a similar assay and found that the average force was not different between these myosins, while the ATPase activity of V1 was two times higher than that of V3 [26]. These findings, together with the present results, indicate that the catalytic activity of cardiac myosin depends primarily on its MHC isoform composition, while its force-generating ability is largely determined by its MLC isoforms.

In order to dissect the underlying mechanism, we performed single motor assay to find that (1) both unitary displacement and force were equal between these myosins, but (2) the event duration was longer in V-myosin than in A-myosin under both low load (Fig. 7) and isometric conditions (Fig. 8). We interpreted the results by assuming the two-state model of the crossbridge, where each crossbridge is postulated to repeat attachment (force-generating state) and detachment (non-force-generating state) cyclically while hydrolyzing ATP [38]. The fraction of time during which the crossbridge is attached and generating force in each cycle is defined as the duty ratio (f). In this model, time-averaged force of an individual crossbridge (Fave) is given as the product of the unitary force (Funi) and duty ratio (Fave=Funi x f) and considered to be equivalent to the average force estimated in the present study. Since V-myosin had higher average force than A-myosin and Funi was similar between these myosins, V-myosin could have higher f, which could be supported by the longer duration of isometric force generation.

The clear functional differences between the MLC and MHC isoforms may reflect the structure of the molecule. The catalytic site is located on the head domain of MHC, while the MLCs are bound in the light chain-binding domain (LCBD), which is composed of a long {alpha}-helix (the "lever arm") and postulated to play a critical role in transducing the chemical energy of ATP hydrolysis into mechanical work of muscle contraction [39,40]. In this model, the LCBD may amplify the small conformational changes originated in the head domain upon ATP hydrolysis and transmits it to the rod portion of the molecule to produce the power stroke and force. In accordance with the model, MLC removal in skeletal myosin severely depressed the actin filament velocity as well as the force-generating ability of the molecule, while the ATPase activity was not affected [41]. The dominant effect of the MLC isoforms on force generation may support the putative important role of the LCBD in the model.

The significance of MLC isoform shift in cardiac adaptation has not been better recognized as compared to the MHC isoforms. In mitral valve disease patients, the MLC isoforms in atrial muscles shifted from atrial-type (ALC1 and ALC2) to ventricular-type (VLC1 and VLC2) in parallel with the MHC isoform shift from {alpha}- to β-MHC [42]. This isoform shift in the MLC could contribute to the adaptation for increased mechanical load, because the ventricular-type MLCs were related with higher force production. On the other hand, atrial type LC1 (ALC1) was reported to be expressed in hypertrophied ventricles in aortic stenosis and congenital heart disease patients [10,11,15,16]. The expression of ALC1, however, decreased in end-stage failing hearts [43]. It suggests that the expression of ALC1 may be an adaptive response only in the earlier stage of hypertrophy by improving diastolic relaxation by accelerating crossbridge kinetics, which was presented in the higher shortening velocity and shorter time of tension development in hypertrophied human fibers expressing ALC1 [16]. In the advanced stage of hypertrophy or heart failure, expression of VLC1 may increase again to compensate for the depressed contractile function.

In summary, the present study constitutes the first in vitro experimental support for the MLC isoform-based cardiac adaptation mechanism at the molecular level. The experiments, however, were done on rat cardiac myosin consisting predominantly of {alpha}-MHC and could not be immediately extrapolated to the adaptation mechanism in human ventricles containing mainly β-MHC. Further study based on β-MHC is needed to establish the functional significance of MLC isoforms in human hearts, and it will provide molecular basis for the cardiac adaptation in various heart diseases and help find a clue to novel approach to treating heart failure.


   Acknowledgements
 
This study was supported in part by Grant-in-Aids for Scientific Research C(2)-11670661 and C(2)-13670689 from the Ministry of Education, Science and Culture of Japan, Grant-in-Aids from the Vehicle Racing Commemorative Foundation, the Uehara Science Foundation and the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research.


   Notes
 
Time for primary review 22 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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