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J Physiol (2003), 548.1, pp. 167-174
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2002.031922
-cardiac myosin changes the contractile kinetics of cultured mouse myotubes |
ABSTRACT |
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The properties of myosin expressed in muscle are a major determinant of muscle performance. In this study we used a novel approach to examine the functional impact of changes in myosin heavy chain (MHC) isoform expression, as well as the consequences of expressing the mutant MHC implicated in familial hypertrophic cardiomyopathy (FHC). Cultured mouse myoblasts that normally express fast embryonic myosin were untransfected, or stably transfected with a plasmid expressing either wild-type (cWT) or mutant (D778G or G741R)
(Received 4 September 2002; accepted after revision 21 January 2003; first published online 7 February 2003)
Corresponding author S. Calaghan: School of Biomedical Sciences, University of Leeds, Leeds LS2 9NQ, UK. Email: s.c.calaghan{at}leeds.ac.uk
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INTRODUCTION |
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In striated muscle, contraction is initiated by the interaction of the contractile proteins actin and myosin. The properties of the myosin heavy chain (MHC) isoform expressed in muscle are a major determinant of muscle function. MHC expression can switch from fast to slow isoforms, and vice versa, in a sequential and reversible manner, and is modulated by a number of factors including neuromuscular activity, mechanical loading, hormonal influences and age (see Pette & Staron, 2000). For example, in the mammalian heart, hypothyroidism, pressure overload hypertrophy and failure are all associated with increased expression of the slow -MHC isoform (Nadal-Ginard & Mahdavi, 1989).
The role of MHC in determining cardiac performance is underscored by the finding that around half of all cases of the disease familial hypertrophic cardiomyopathy (FHC) are associated with mutations in -MHC (Roberts & Sigwart, 2001). FHC is the most common cause of sudden cardiac death in the young. As well as affecting the heart, these mutations in MHC can also impair skeletal muscle function (Thompson et al. 1997). It is still not clear how mutations of -MHC cause the disease FHC, although evidence suggests that alterations in contractility are the primary trigger leading to cardiac hypertrophy, sarcomeric disarray and increased interstitial fibrosis (Geisterfer-Lowrance et al. 1996; Roberts & Sigwart, 2001). We have shown that two different FHC myosin mutants (D778G and G741R; Harada et al. 1993; Arai et al. 1995) incorporate normally into muscle sarcomeres in cultured mouse myotubes, in agreement with the idea that sarcomeric disarray occurs as a consequence, rather than a cause, of altered contractility (Miller et al. 2000). The pivotal question appears to be whether hypocontractility leads to compensatory hypertrophy or whether hypertrophy develops as a result of the increased energy demand associated with hypercontractility (Moss & Periera, 2000). For example, in vitro assays of motility and ATPase activity have provided evidence that supports both loss (Cuda et al. 1993; Sata & Ikebe, 1996; Roopnarine & Leinwand, 1998) and gain (Palmiter et al. 2000; Tyska et al. 2000; Yamashita et al. 2000) of function for the most widely studied FHC mutation in myosin, R403Q, located in the actin binding site.
In the present study we have used cultured mouse myotubes, which normally express embryonic and fast myosin isoforms, to examine the impact of heterologous expression of the slow -cardiac myosin isoform on contractile kinetics. We have also compared the effect of expression of two FHC mutants (D778G and G741R), located in the converter domain of -cardiac myosin, with the expression of wild-type -cardiac myosin (cWT). Chick and rat myotubes have been used previously to examine Ca2+ handling and contraction (e.g. Saito & Ozawa, 1986; Grouselle et al. 1991), and skinned quail myotubes have been used to determine the effects of troponin T mutations on the Ca2+-sensitivity of force production and unloaded shortening velocity (Sweeney et al. 1998). However, this is the first study to compare directly the contractile properties of myotubes expressing different myosin isoforms, or mutant myosin.
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METHODS |
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Tissue culture and expression of cardiac myosin
cDNA. The two -cardiac myosin mutations were synthesised by site-directed mutagenesis of full-length human -cardiac MHC cDNA (a kind gift from Professor Vosberg, Max Planck Institute, Bad-Nauheim, Germany) and sequence-overlap extension (SOE) PCR. The mutant human -cardiac myosin cDNA was then cloned into an expression vector pCMV5, as described previously (Miller et al. 2000). The -cardiac myosin isoform is the same as the slow myosin isoform expressed in skeletal muscle.
Growth and transfection of the myogenic cell line. Conditionally immortal H2kb-tsA58 myogenic cells (Morgan et al. 1994) were proliferated in growth medium (Dulbecco's modified Eagle medium, DMEM, with Glutamax; GibcoBRL) supplemented with 20 % fetal calf serum (FCS), 2 % chick embryo extract (CEE), 100 µg ml-1 penicillin/streptomycin (PS), and 20 U ml-1 murine recombinant 
-interferon (GibcoBRL) at 33 °C. To differentiate the cells into myotubes, the medium was exchanged for differentiating medium (DMEM Glutamax supplemented with 2 % FCS, 1 % CEE and 100 µg ml-1 PS) and the cells were cultured at 37 °C.
A single 'fast' clone that did not express slow myosin when differentiated into myotubes was used for the transfections described below. To select this clone, several clones were differentiated into myotubes and immunoblotted to determine whether they expressed fast or slow myosin, using antibodies specific for these isoforms (N336 and A4840, respectively; Maggs et al. 2000). This 'fast' clone was stably transfected with either cWT or mutant human -cardiac myosin. Cells were trypsinised and replated the day before transfection. They were split, counted and diluted in DMEM containing 10 % FCS to give 2 
107 cells ml-1. A 27 µg aliquot of the pCMV5 expression vector containing either cWT or mutant -cardiac MHC heavy chain cDNA, together with 3 µg of the pGKneo vector (to enable transfected cells to be selected for by resistance to the antibiotic G418) were added to 0.5 ml of cells in a 0.4 cm electroporation cuvette (Flowgen). After 1 min they were electroporated at 250 V, 150 µF using the EquiBio CellJect electroporator (Flowgen). The cells were then immediately plated out into fresh growth medium in two 20 cm dishes. To select for stable transfectants, the growth medium was replaced 48 h later and supplemented with 1 mg ml-1 G418 (50 µg ml-1 active). After 14-21 days, individual G418-resistant colonies were picked into single wells of a 24-well plate and expanded in the absence of G418 into duplicate 25 cm2 flasks.
Approximately 30 stably transfected clones each for cWT, and the two mutant isoforms, were analysed by SDS-PAGE and Western blotting, using an anti--cardiac myosin antibody A4840 (Maggs et al. 2000). One cWT, one G741R and one D778G mutant myosin-expressing clone, which expressed the -cardiac myosin at similar and relatively high levels, were selected for further analysis. Pig cardiac myosin (Sigma) was used as a standard to estimate the relative expression levels.
Quantification of expression levels. Expression levels of the human -cardiac MHC in stably transfected myotubes were further analysed by SDS-PAGE and Western blotting, using either 5 % acrylamide gels that separated out myosin isoforms (Hughes et al. 1993), or 7.5 % minigels. Equal quantities of protein sample (determined using the Pierce Bradford Assay kit) from myotubes (5-day-old) were loaded into each of the lanes. Known amounts of pig myosin (Sigma) were loaded in one or more control lanes to enable quantification. Proteins were transferred onto a nitrocellulose membrane and probed using the primary anti-myosin antibodies: A4840 or BAD5 (for human -cardiac myosin); F1652 (for embryonic myosin); or A1025 (for both of these myosin isoforms; see Maggs et al. 2000, and references therein). Immunoreactivity was visualised using a peroxidase-based chemiluminescent substrate kit (Pierce Supersignal). Blots were imaged and the intensity of the bands quantified using Scion Image. The intensity of bands from myotube samples was compared with that of the standards to estimate the proportion of the total myosin that was the expressed -cardiac myosin.
Myotube contraction
The contractile properties of 5- to 7-day-old stably transfected myotubes, cultured on laminin-coated coverslips and differentiated as described above, were investigated. The coverslips were placed in a chamber on the stage of a Nikon Diaphot microscope and perfused with Hepes-based bathing solution of the following composition (mM): NaCl 113, KCl 5, Na2HPO4 1, MgSO4.7 H2O 1, CaCl2 1, Hepes 5, glucose 10, sodium acetate 20 and insulin (5 U l-1) at pH 7.4 (22-24 °C). Shortening was measured using a video-edge detection system (Crescent Electronics, UT, USA; see Fig. 2A and B). Myotubes were field stimulated at 1 Hz via platinum electrodes in the bath to determine the effect of cWT and mutant myosin expression on the kinetics of the twitch. Myotubes were partially attached to the coverslip, but were able to shorten. Not every myotube on the coverslip contracted in response to a given stimulus. However, we saw no evidence of a difference in the number of myotubes that contracted in response to stimulation between the four populations of myotubes studied. Due to the nature of the preparation (see Fig. 2A) it was not possible to measure the length of the myotubes accurately. Furthermore, since it was only possible to track the shortening of myotubes from one end using this technique, we cannot present data on the absolute degree of shortening or absolute velocity of shortening. The time from stimulation to peak shortening and the time from peak contraction to half-relaxation were recorded for each contraction. In addition, the maximum velocity of shortening (Vmax) was estimated by differentiation of a Gaussian relationship fitted to the shortening phase of the twitch, which was normalised to the maximum degree of shortening (Clampfit 8, Axon Laboratories).
The relationship between shortening and frequency of stimulation was determined between 2 and 200 Hz. Each myotube was exposed to sequentially increasing frequency of stimulation. Stimulation was applied for a 10 s period and the myotubes were allowed to recover before the next period of stimulation. Due to the sampling frequency of the camera, the profile of individual contractions could not be monitored reliably at stimulation frequencies above 20 Hz, but the maximum level of shortening achieved in a summating and tetanising preparation could be measured with confidence as the peak of the envelope of shortening (Fig. 3A). Shortening-frequency data were fitted to the Boltzmann relationship, and tetanic fusion frequency was determined from the asymptote of the relationships (Microcal Origin 5.0). None of the myotubes displayed significant levels of fatigue during tetani.
Statistical analysis
All data are presented as means ± S.E.M. Comparisons between groups were made by one-way analysis of variance with post hoc analysis by the Student-Newman-Keuls method (Jandel Sigmastat 2.0). P < 0.05 was considered statistically significant.
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RESULTS |
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The myotubes used in this study are the same permanently transfected clones that we have shown previously to differentiate normally (Miller et al. 2000). In the three populations of transfected myotubes, expression levels of -cardiac myosin were similar. Figure 1 shows a representative Western blot of samples from cWT, D778G and G741R myotubes probed with an antibody to -cardiac myosin and to embryonic myosin. The amount of -cardiac myosin expressed as a percentage of the total amount of skeletal myosin (-cardiac plus embryonic myosin) was: 25 % (24.2-25.9 %; n = 2) for cWT, 23 ± 2 % (n = 5) for D778G and 24 ± 5 % (n = 4) for G741R.
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Figure 1. Autoradiograph of two representative blots probed with an antibody to A 100 µg aliquot of total protein was loaded into each lane. Bands represent approximately 3 µg of protein for | ||
We first analysed the contractile kinetics of untransfected myotubes that do not express slow myosin. The myotubes, stimulated at 1 Hz, were found to produce twitches in which the time to peak shortening (ttp) was 133 ± 7 ms (n = 19) and the time to half-relaxation (t0.5) was 90 ± 5 ms (Fig. 2C and D). The maximum velocity of shortening (Vmax), was 15 ± 2 normalised shortening s-1 (Fig. 2E). When the frequency of stimulation was sequentially increased, we predicted that a fused tetanic contraction would be obtained at frequencies greater than 200 Hz (the maximum frequency used in this study; Fig. 3B).
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Figure 2. Shortening recorded at 1 Hz stimulation in untransfected myotubes (UT) and myotubes expressing cWT and mutant A, a video-edge detection system was used to record shortening from one end of each myotube. The image shows representative cWT myotubes; the scale bar is 100 µm. B, representative contraction recorded from a myotube expressing mutant D778G | ||
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Figure 3. Shortening-frequency relationships in untransfected myotubes and myotubes expressing cWT and mutant A, representative recordings of protocol from an untransfected myotube. Fluctuations in length recordings at high (> 20 Hz) stimulation frequencies indicate momentary loss of edge tracking rather than real shortening-lengthening cycles. See Methods for more detail. B, shortening-frequency relationships. Data were fitted to the Boltzmann relationship and tetanic fusion frequency was determined from the asymptote of the relationship. Data are means ± S.E.M. of 8-13 observations. | ||
Next we looked at the effect of heterologous expression of 25 % of slow -cardiac myosin in myotubes that normally only express fast myosin. Heterologous expression of -cardiac myosin in the fast myotubes slowed contractile kinetics. The values for ttp and t0.5 were 18 and 33 % longer, respectively, (P < 0.05) for cWT versus untransfected myotubes (Fig. 2C and D). Vmax was reduced compared with untransfected myotubes (13 ± 1 normalised shortening s-1; n = 13), although this difference was not significant (P > 0.05). Analysis of shortening-frequency relationships (Fig. 3A) showed that the tetanic fusion frequency was markedly reduced from > 200 Hz in untransfected myotubes to 38 Hz in cWT myotubes (Fig. 3B).
In this study, the expression level of both the mutant isoforms of -cardiac MHC was 23-24 % of the total myosin. In contrast to the effect of expression of cWT, expression of D778G mutant -cardiac myosin in the 'fast' myotubes did not slow the contractile kinetics. Both ttp and t0.5 were 24 % shorter (P < 0.05) for D778G compared with cWT, and did not differ from that recorded in untransfected myotubes (P > 0.05; Fig. 2C and D). Vmax was significantly higher (P < 0.05) in D778G myotubes (17 ± 1 normalised shortening s-1; n = 17) than in cWT myotubes (Fig. 2E). Consistent with the increased speed of shortening and relaxation, D778G dramatically increased tetanic fusion frequency (to > 200 Hz) compared with cWT (38 Hz; Fig. 3B).
In contrast to the impact of D778G, expression of the G741R mutant -cardiac myosin had no effect on the contractile kinetics of myotubes at 1 Hz stimulation compared with expression of cWT. During stimulation at 1 Hz, ttp and t0.5 were similar in cWT and G741R myotubes (Fig. 2C and D) and Vmax was identical to that recorded in cWT myotubes (13 ± 1 normalised shortening s-1; n = 20). Although G741R had no significant impact on the contractile kinetics seen following stimulation at 1 Hz, this mutation did increase tetanic fusion frequency to 164 Hz (compared with 38 Hz seen in cWT myotubes; Fig. 3B).
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DISCUSSION |
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In the study presented here, we have examined the impact of heterologous expression of a slow isoform of myosin in 'fast' myotubes and made the first direct comparison of the effect of two different FHC mutations in the converter domain of the myosin head on contractile kinetics. We believe that our technique for studying the contractile properties of transfected myotubes has the following noteworthy advantages. First, we can examine the sole consequences of myosin isoform shifts without factors that may complicate interpretation. Second, we can also rule out the possibility that changes in kinetics are due to poor preparation or isolation of myosin. Third, we used human -cardiac myosin, the isoform found in the heart of patients with FHC. Finally, in myotubes at 5-7 days after differentiation, expression of mutant myosin does not cause sarcomeric disarray, therefore we were able to examine the primary trigger for hypertrophy before any disorganisation of the sarcomere occurs.
In contrast, there are several disadvantages to the methods currently used to study the effect of myosin properties on myosin function in the heart. For example, in studies using actin and myosin in vitro, the quality and quantity of both preparations can have a profound effect on myosin function (Yamashita et al. 2000). Moreover, in preparations such as these, the regulatory elements such as tropomyosin and troponins are usually absent. Other studies in this field have used tissue from the thyroidectomised rat (e.g. Herron & McDonald, 2002), from transgenic mice (e.g. Semsarian et al. 2002) or from patients with FHC (Larsson & Moss, 1993). However, cardiac myocytes from hypothyroid rats also exhibit changes in intracellular Ca2+ handling (Kiss et al. 1994), and tissue from transgenic mice and patients with FHC may have compensatory changes in other proteins besides myosin. Furthermore, sarcomeric disorganisation may also complicate the interpretation of data obtained from the muscle of transgenic animals.
In this study we have shown that expression of 25 % slow -cardiac myosin on a fast myosin background has a marked impact on contractile kinetics. In comparison with untransfected 'fast' myotubes, myotubes containing 25 % -cardiac myosin exhibited an increase in ttp and t0.5 (18 and 33 %, respectively) and a marked reduction in fusion frequency. If there is no interaction between myosins, the impact on function will be quantitatively less than the change in expression level when slow myosin is expressed on a fast myosin background. For example, if -MHC was twice as slow as embryonic myosin, expression of 25 % -MHC should reduce contractile kinetics by 12.5 %. Our data are in agreement with an in vitro study by Harris et al. (1994) and in vivo work in the murine heart by Tardiff et al. (2000), which suggest that the impact of the slower myosin isoform dominates (by placing a load on the faster myosin) when both fast and slow isoforms are present in muscle.
Both D778G and G741R mutations are in the converter domain of myosin. However, we found that whilst the D778G mutant -cardiac myosin dramatically increases contractile kinetics compared with cWT, the impact of the G741R mutation is less marked. The D778G mutant myosin significantly increased contractile kinetics, Vmax and tetanic fusion frequency, whereas the only observed effect of the G741R mutation was an increase in fusion frequency, when compared with cWT myotubes. The frequency at which individual contractions fuse is dependent on the duration and thus the kinetics, of contraction and relaxation. The kinetics of myotube contraction and relaxation, in turn, are dependent on both intracellular Ca2+ handling and the rate of crossbridge cycling. Our data for G741R suggest that determination of tetanic fusion frequency is a more sensitive way to detect changes in kinetics than measurement of the rate of activation and relaxation at one frequency of stimulation. Effectively, as fusion frequency reflects the kinetics of contraction over a wide range of frequencies, this parameter is more likely to highlight a small difference in the properties of the myosin than ttp and t0.5 at 1 Hz stimulation. Since the kinetics of contraction of myotubes expressing 23 % D778G -cardiac myosin are the same as untransfected myotubes, we propose that the speed of the D778G -cardiac myosin is similar to that of the fast endogenous myosin of the myotube.
It is likely that the D778G mutation alters the kinetics of the crossbridge cycle. The rate of relaxation and Vmax of myotubes expressing D778G is higher than that seen in myotubes expressing cWT -cardiac myosin; both of these parameters give an index of the rate of crossbridge detachment. It is reasonable to assume that an increase in the rate of detachment means that crossbridges remain attached to the actin filament for less time, perhaps due to a shorter duty cycle (VanBuren et al. 1995). Although the changes in contractile kinetics of transfected myotubes we have observed could be ascribed to alterations in intracellular Ca2+ handling, we have no evidence to support this. There is very limited data on the effect of myosin mutations on [Ca2+]i handling. It has been shown that cardiac myocytes from 
MHC403/+ mice have reduced sarcoplasmic reticular Ca2+ levels and ryanodine receptor expression (Semsarian et al. 2002). However, it is difficult to extrapolate these data to those of the present study, which were performed on acutely transfected myotubes with mutations in different regions of the myosin molecule. Furthermore, Ca2+ handling in myotubes at 5-7 days after differentiation is likely to be different from that seen in the adult cardiac myocyte (see Bulteau et al. 1998).
To date, the literature concerning the effect of FHC mutations on myosin and muscle function is somewhat diverse, and there is limited data on the D778G and G741R mutations. Interpretation is further complicated by the use of different myosin isoforms (smooth muscle and -cardiac, as well as human -cardiac). Our results are consistent with a recent study of the mutation equivalent to D778G in expressed smooth muscle myosin (Yamashita et al. 2000), which reported a faster actin filament sliding velocity, consistent with an increase in the rate of crossbridge detachment and a decrease in the time that the myosin crossbridge spends attached to actin. The average force per crossbridge was unchanged (Yamashita et al. 2000). The only previous report for the G741R mutation showed that Vmax and isometric force generation was reduced in slow skeletal muscle fibres from patients with FHC (Lankford et al. 1995). This suggests a 'loss of function' for myosin containing this mutation, in contrast to our data, which is consistent with a small 'gain of function' for G741R.
If the behaviour of myosin in vivo and in vitro is dominated by the slowest myosin isoform expressed, then heterologous expression of faster mutant myosins (such as D778G) on the slow human -cardiac myosin background may have limited functional consequences. However, a recent study has shown a clear increase in myocyte power output in rat cardiac myocytes expressing only 12 % -MHC compared with those expressing entirely -MHC (Herron & McDonald, 2002). If there is a marked functional difference between fast and slow myosin (for example, -MHC has 2-3 times faster actin-activated ATPase activity and actin filament sliding velocity than -MHC; Litten et al. 1982; Harris et al. 1994), then the impact of increasing the expression of fast myosin may be sufficient to overcome the potential 'dominating' effect of slow myosin. Furthermore, in the heart of the patient with FHC, expression of mutant myosin may be as high as 41 ± 24 % of the total myosin (Kohler et al. 2002). We would therefore predict that the effect in FHC is more marked than that which we have demonstrated here, where mutant myosin is only expressed at 25 % of the total myosin.
Our data are consistent with the idea that FHC is an 'energy cost' disease (Sweeney et al. 1998; Palmiter et al. 2000; Redwood et al. 2000; Yamashita et al. 2000; Blair et al. 2001). Using different myosin isoforms, contractile velocity has been shown to correspond with tension cost (Bottinelli et al. 1994). The increase in Vmax that we observed in myotubes expressing D778G mutant myosin would increase velocity at each submaximal load, thereby increasing peak power (unless the curvature of the force-velocity relationship was also changed).
In conclusion, our data from myotubes expressing cWT -cardiac MHC support the theory that the behaviour of myosin in vivo is dominated by the slowest myosin isoform expressed. Furthermore, a direct comparison of the contractile consequences of two FHC mutations in the converter domain of myosin suggests that FHC associated with both D778G and G741R is an energy cost disease, but that the phenotype of patients with the D778G mutation is likely to be more severe.
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Acknowledgement
This work was sponsored by the British Heart Foundation.
Author's present address
Gaynor Miller: Department of Physiological Science, UCLA, Los Angeles, USA.
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