Divergent abnormal muscle relaxation by hypertrophic cardiomyopathy and nemaline myopathy mutant tropomyosins
Daniel E. Michele,
Pierre Coutu and
Joseph M. Metzger
Department of Physiology, University of Michigan, Ann Arbor, Michigan 48109-0622
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ABSTRACT
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Mutations in tropomyosin (Tm) have been linked to distinct inherited diseases of cardiac and skeletal muscle, hypertrophic cardiomyopathy (HCM), and nemaline myopathy (NM). How HCM and NM mutations in nearly identical Tm proteins produce the vastly divergent clinical phenotypes of heightened, prolonged cardiac muscle contraction in HCM and skeletal muscle weakness in NM is currently unknown. We report here a direct comparison of the effects of HCM (A63V) and NM (M9R) mutant Tm on membrane-intact myocyte contractile function as assessed by adenoviral gene transfer to fully differentiated cardiac muscle cells. Wild-type, and mutant HCM, and mutant NM proteins were expressed at similar levels in myocytes and incorporated into sarcomeres. Interestingly, HCM mutant Tm produced significantly longer contractions by slowing relaxation, whereas NM mutant Tm produced the opposite effect of accelerated muscle relaxation. We propose slowed relaxation caused by HCM mutant Tm can directly contribute to diastolic dysfunction seen in HCM even without secondary cardiac remodeling. Conversely, hastening of relaxation by NM mutant Tm may shift the force-frequency relationship in skeletal muscle and contribute to muscle weakness seen in NM. Together, these results implicate divergent, abnormal "turning off" of muscle contraction as a cellular basis for the differential pathogenesis of mutant Tm-associated HCM and NM.
tropomyosin; nemaline myopathy; hypertrophic cardiomyopathy; calcium; muscle
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INTRODUCTION
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TROPOMYOSIN (Tm) is a key protein involved in the regulation of striated muscle contraction (9, 35). Tm, by its conformation on the actin filament, relays the signal from the Ca2+ sensor, the troponin complex, to the actin-myosin interactions that ultimately produce force. During a normal muscle contraction, Ca2+ binding to troponin C causes conformational changes within the troponin complex, allowing Tm to move off the weak myosin binding sites on actin. Myosin binding activates the myosin ATPase and causes further translocation of Tm into the groove of the actin filament allowing strong, force-generating interactions between myosin and actin (15, 36). Therefore, the regulation of the kinetics of intact muscle contraction and relaxation is clearly complex, involving the kinetics of the Ca2+ transient, the kinetics of Ca2+ binding and conformational changes within the thin-filament regulatory proteins, and the kinetics of the myosin motor.
The key role of Tm in the regulation of structure and function of striated muscle has been highlighted by the identification of mutations in
-Tm associated with human cardiac and skeletal myopathies. Four mutations, D175N, E180G, K70T, and A63V, in the human TPM1 gene have been associated with hypertrophic cardiomyopathy (HCM) (23, 34, 43). HCM has been linked to mutations in multiple contractile proteins of the cardiac sarcomere including, in addition to
-Tm, the ß-myosin heavy chain and associated light chains, troponin I, and troponin T, myosin binding protein C, and actin (3, 20). One interesting feature of Tm-associated HCM is that although the mutant Tm protein is expressed in skeletal muscle, it does not produce a noted skeletal muscle myopathy (34). In contrast, a mutation in the human
-Tm TPM3 gene, which shares 90% sequence identity with TPM1 Tm but is primarily expressed in slow skeletal muscle fibers, has been associated with a rare skeletal muscle disorder, nemaline myopathy (NM) (13). NM has a diverse clinical presentation, generally first presenting as skeletal muscle hypotonia followed by appearance of nemaline rods within the skeletal muscle fibers (24). NM has also been linked to mutations in nebulin and skeletal muscle actin (25, 27).
Despite the general similarity of the thin filament regulation of cardiac and skeletal muscle contraction (9, 35) and the nearly identical sequence and structure of the Tm proteins produced by the Tm genes mutated in HCM and NM, the striated muscle functional presentations of HCM and NM are markedly divergent. The clinical hallmarks of the HCM heart include a hypercontractile heart with markedly prolonged muscle contractions and diastolic dysfunction, reducing the time and pressure gradients for ventricular filling. To date, most of the functional changes in HCM myocardium have been attributed to the abnormal cardiac hypertrophy and interstitial fibrosis that stiffens the ventricular walls (4). In contrast, NM skeletal muscle is hypocontractile, with muscle weakness in the most severe cases producing a "floppy infant" phenotype (24). Because the sarcomeric structure of NM muscle is disrupted by NM rods, it has been suggested that these structural alterations cause the NM-associated hypotonia. Hypotheses have been proposed that the sarcomeric and organ level structural alterations seen in HCM and NM bring about the differential striated muscle functional phenotypes of HCM and NM. However, we propose here an alternative hypothesis. We hypothesized that differences in how the HCM and NM mutations alter the ability of Tm to regulate muscle contraction in response to dynamic changes in intracellular Ca2+ concentration directly underlie the differences in pathogenesis of HCM and NM. In the present study, the direct effects of expression of an HCM mutation or a NM mutation in
-Tm on the regulation of intact striated muscle function were examined and compared. The results from the present experiments shed insight on the role of Tm in the regulation of muscle contraction through dynamic changes in intracellular Ca2+ and how alterations through HCM or NM mutations in Tm impact the kinetics of muscle contraction. The elucidation of the direct effects of mutations in Tm on intact muscle contraction has implications for understanding the differential striated muscle functional presentation of HCM and NM.
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METHODS
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Isolation of adult cardiac myocytes and adenoviral gene transfer.
The construction and purification of recombinant adenoviral vectors containing the HCM mutation A63V or the NM mutation M8R (analogous to M9R in TPM3 Tm) in the full-length human
-Tm cDNA (TPM1 gene product) with a COOH-terminal FLAG epitope was described previously (1618). Ca2+-tolerant adult cardiac myocytes were isolated enzymatically from female Sprague-Dawley rats (200 g) as previously described and plated (2 x 104 cells) on 18-mm2 laminin-coated coverslips for 2 h in DMEM with 5% FBS and 1% penicillin/streptomycin (P/S) (41). Myocytes were treated for 1 h with viral vectors diluted in serum-free DMEM with P/S at 500 pfu/cell. These titers of viral vectors result in gene transfer to nearly all the cardiac myocytes in the culture dish [9095% by LacZ reporter constructs (30), >85% by TmFLAG expression (18)]. Adult cardiac myocytes were maintained in media 199, HEPES modification (Sigma) supplemented with 0.2 mg/ml BSA, 5 mM reduced glutathione, and P/S. To maintain the excitability of the cultured adult cardiac myocytes for the functional experiments, the coverslips containing the cardiac myocytes were placed in a custom-built electrical field stimulus chamber 12 h post vector treatment, continuously paced at 0.5 Hz. Media was changed every 12 h until 4 days post vector treatment (37).
Expression of NM and HCM mutant Tm in adult cardiac myocytes.
Protein expression was quantified by SDS-PAGE and Western blotting. For Tm, the Tm311 antibody (Sigma) was diluted 1:106 in TBS with 0.05% Tween 20 and 5% nonfat dry milk, followed by a horseradish peroxidase-conjugated IgG antibody and enhanced chemiluminescence (ECL) detection. Myofilament-bound protein was determined by permeabilizing the cardiac myocytes with 0.1% Triton X-100 in relaxing solution and washing the myocytes with fresh relaxing solution as described previously (18). The incorporation of the vector-expressed Tm protein into the sarcomere was visualized with immunofluorescence immunocytochemistry with an M2 anti-FLAG antibody (Sigma) and a Texas Red (Molecular Probes)-conjugated anti-IgG antibody followed by confocal microscopy (18). Electron microscopy was performed on isolated cardiac myocytes as previously described (40).
Intact myocyte contractile function.
Sarcomere shortening in response to field stimulation was used to measure intact myocyte contractile function as described (37). Briefly, the coverslips containing the myocytes formed the bottom of a chamber with platinum stimulation electrodes. Myocytes were stimulated with 5-ms square-wave pulses in supplemented media 199 (described above) at 30°C or 37°C. A 10-mW HeNe laser was focused on the myocytes, and the first-order diffraction line was focused onto a linear position detector (model LSC 5D, United Detector Technology). The signal was amplified and collected at 5,000 Hz on a digital oscilloscope. A viewing screen was used to estimate the initial sarcomere length and the amplitude of sarcomere length change. Ten twitches from each myocyte were averaged. The time from stimulation to maximum shortening (Tpeak), from stimulus to 50% relengthening (TS1/2R), and from maximum shortening to one-half relengthening (T1/2R) were determined. The maximum rates of shortening (-dL/dt) and relengthening (+dL/ dt) were calculated and normalized to the maximum shortening amplitude. Data was analyzed by comparing all groups with one-way ANOVA using a Student-Newman-Keuls post hoc test, with P < 0.05 indicating statistical significance.
Calcium transients.
The myocytes were loaded for 6 min in supplemented media 199 solution containing 5 µM fura-2/AM (from Molecular Probes) and 0.01% of Pluronic F-127 (from Molecular Probes). After three washes, the myocytes remained in supplemented media 199 for the measurements followed by 5 min for deesterification. Fluorescence was measured using a microscope-based high-speed ratio fluorescence spectrometer (model M-40, Photon Technology International) at 37°C. The myocytes were field stimulated at a frequency <0.2 Hz, and the fluorescence ratio (340 nm/380 nm) was sampled at 100 Hz. Ten stimulations were collected and averaged for each studied myocyte. No attempt was made to convert fluorescence ratio into absolute calcium level. The times from stimulus to peak (Tpeak) and to 50% decay (TS1/2R) were recorded. Curve fitting to custom fit functions using Igor software (WaveMetrics) was performed. The decay portion of the curves (from 10% decay) were fitted to single exponential functions (
).
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RESULTS
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Expression of HCM and NM mutant Tm in adult cardiac myocytes.
We have recently characterized a primary culture model system of fully differentiated striated muscle cells that allows the specific replacement of a portion of the endogenous Tm isoforms with human
-Tm by adenoviral-mediated gene transfer (18). In the present study, an HCM mutation or the analogous NM mutation in human TPM1
-Tm was expressed to directly compare the effects of these mutations on the regulatory function of Tm in fully differentiated intact striated muscle cells. Figure 1A shows the expression of wild-type (WT), HCM (A63V), and NM (M9R)
-Tm in adult cardiac myocytes 4 days post gene transfer. The COOH-terminal FLAG epitope on
-Tm results in the expressed
-TmFLAG protein to migrate more slowly (each comigrates with WT
-TmFLAG expressed in HEK-293 cells), allowing direct quantification of each protein. The samples shown in Fig. 1A were collected from myocytes that were detergent permeabilized prior to sampling. These results indirectly indicate that the expressed WT, HCM, and NM
-Tm proteins are binding to the myofilaments, and the amount of expression of myofilament-bound HCM and NM
-Tm is similar to WT. Quantitative analysis of the expression of WT, HCM, and NM
-Tm in adult cardiac myocytes showed no significant changes in isoform expression of endogenous Tm or in total Tm stoichiometry in both intact and Triton X-100-permeabilized myocytes (data not shown). These findings are similar to our previous results expressing these same WT and mutant Tm proteins (1618). Furthermore, isoform expression of other thin-filament proteins were also unchanged (data not shown), as shown previously (1618). This indicates that the only change in myofilament protein expression seen in vector-treated cardiac myocytes is the specific replacement of
4050% of the endogenous Tm with the vector-expressed Tm proteins.
Figure 1, BD, shows representative three-dimensional confocal reconstructions showing the localization of epitope-tagged WT, HCM, and NM mutant
-Tms. Both the HCM and NM mutant Tm proteins are capable of incorporating into the sarcomeres of adult cardiac myocytes similar to WT Tm (16, 17). The periodic immunolabeling of WT, HCM, and NM mutant Tm occurred at a spacing consistent with normal thin filament incorporation. There was no evidence of alteration in gross sarcomere structure (with a resting sarcomere length of
1.8 µm in cultured adult cardiac myocytes, the thin filaments are in overlap). Indeed, in dual-label experiments, expressed Tm colocalized with FITC-labeled phalloidin and localized between Z-disks labeled with
-actinin antibody (not shown and Refs. 1618). Also these data indicate the WT, HCM mutant, and NM mutant Tm incorporate into myofilaments without accumulation in the cytoplasm. This, along with the Western blot data, indicates specific stoichiometric replacement of
40% of the endogenous Tm with mutant Tm protein in the muscle cell myofilaments. Figure 1, BD, also shows representative electron micrographs that indicate this expression of HCM and NM mutant Tm also does not result in direct effects on the ultrastructure of the striated muscle sarcomere (16). This suggests that changes in sarcomere structure seen in HCM or NM result from secondary, long-term consequences in vivo. In addition, any mechanical changes are likely due to the direct effects of the mutant protein on the contractile function of cells and not due to ultrastructural changes within the myocytes. Given that the expression level and incorporation of the HCM and NM mutant proteins are comparable to the WT Tm protein (Fig. 1), we speculate that patients heterozygous for HCM or NM mutations likely have equivalent levels of protein produced by the normal and mutant Tm genes incorporated into the sarcomere.
Stability of intact cell contractile function of cultured cardiac myocytes.
To be able to utilize this model muscle cell system for functional analysis of expressed Tm proteins, it is important to determine the functional stability of these cultured adult cardiac myocytes. We have previously demonstrated that steady-state Ca2+-activated contractile function of rat cardiac myocytes myofilaments does not change in short-term primary culture (30). However, the stability of intact myocyte function has not been demonstrated. Therefore, we systematically measured sarcomere shortening and calcium transients in intact cardiac myocytes over the 4-day primary culture period. As demonstrated in Fig. 2, parameters of cardiac sarcomere shortening are unaffected over the time course (4 days) of these experiments. This functional stability at the level of the sarcomere is also reflected in the stability of the Ca2+ transient kinetics over time in culture (Fig. 2). Taken together with the previously demonstrated stability of cultured cardiac myocytes in steady-state myofilament calcium sensitivity, these data suggest the cultured cardiac myocytes provide a relevant and stable model system with which to study contractile function of expressed Tm proteins. Furthermore, treatment of cultured cardiac myocytes with a reporter gene adenovirus (AdLacZ) at concentrations equivalent to those used for the following functional studies produced no significant effects on either sarcomere shortening or calcium transients (Fig. 2). These data suggest adenoviral infection per se has no deleterious effect on the functional properties of intact cultured cardiac myocytes, at least under these experimental conditions.

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Fig. 2. Assessment of the stability of calcium transient and sarcomere shortening properties in isolated adult cardiac myocytes during short-term primary culture. A and B: representative calcium transient (A) and sarcomere shortening (B) traces of control and AdLacZ-transduced single cardiac myocytes on days 2 and 4 in primary culture. C: summary of the time to peak (Tpeak), time from stimulus to 50% decay (TS1/2R), and decay time constant (tau) for the calcium transient data on days 2, 3, and 4. D: summary of the amplitude, Tpeak, and time from stimulus to 50% relengthening (TS1/2R) for the sarcomere shortening data on days 1, 2, 3, and 4. Note that TS1/2R is measured from stimulus to 50% decay and differs from the calculation of T1/2R in Figs. 4 and 5 (see METHODS). Values are means + SE; n ranged from 2640 myocytes for controls and 3444 for AdLacZ myocytes. *Control day 2 Tpeak different from control day 3, but not day 4 (ANOVA, post hoc test).
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Differential effects of HCM and NM mutant Tm on intact myocyte contractile function.
Figure 3 shows sarcomere shortening at 37°C from single cardiac myocytes treated with vectors driving expression of WT, NM mutant, or HCM mutant Tm. The data shown are normalized to maximal shortening for direct comparisons of the kinetics of myocyte shortening. Most notably, the myocyte expressing HCM Tm shows a markedly prolonged contraction, exclusively resulting from a slowing of myocyte relengthening (relaxation). A summary of the parameters that describe the kinetics of contraction at 37°C of myocytes treated with vectors driving expression of WT, NM mutant, or HCM mutant Tm is shown in Fig. 4. Expression of WT human
TmFLAG in adult cardiac myocytes, which was previously shown to not produce any significant effects on steady-state Ca2+-activated isometric force production in permeabilized cardiac myocytes (1618), did not significantly alter any of the parameters describing the kinetics of intact myocyte contraction. This observation supports the conclusion that adenovirus treatment of adult cardiac myocytes and expression of WT Tm does not directly affect the contractile responses of intact adult cardiac myocytes after 4 days in culture. In addition, this result agrees with our previous findings that treatment of adult cardiac myocytes with adenovirus driving expression of the LacZ reporter gene does not directly affect the free Ca2+ transient or the contractile response of normal intact adult cardiac myocytes (37).

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Fig. 3. Representative tracings of sarcomere shortening measured by laser-based diffraction from representative control myocytes or myocytes expressing WT, NM (M9R), or HCM (A63V) mutant TmFLAG. Myocytes were stimulated with a 5-ms square-wave pulse at the 40 ms time point. Traces shown are the average of 10 traces for each myocyte. Shortening was normalized to maximum shortening to show the relative changes in contractile kinetics.
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Expression of HCM mutant TmFLAG (A63V) in adult cardiac myocytes produced a significant increase (
30%) in the time the myocytes remained contracted (FWHM, time from 50% shortening to 50% relengthening), and a significant increase (
50%) in the time to half-maximal relengthening. This direct effect of HCM Tm on the rate of relaxation is also shown by the significant decrease in the maximal rate of relengthening (+dL/dt). Time to peak shortening and shortening amplitude were not consistently affected (neither parameter was significantly different from WT
TmFLAG), and there was no significant effect on the rate of shortening (-dL/dt). This suggests that HCM Tm does not produce a marked change in cardiac myocyte contractility. These results suggest that the major direct effect of HCM mutant Tm on intact myocyte contraction is the slowing of cardiac myocyte relaxation.
If HCM mutant Tm slows relaxation, does the expression of NM mutant Tm differentially affect the kinetics of muscle cell contraction? Figure 4 shows that although there was a trend toward decreased T1/2R, NM Tm expression did not significantly change the time to half-maximal relaxation or the rate of muscle cell relaxation at 37°C when comparing all four experimental groups. This result suggests that at 37°C NM mutant Tm expression may be able to slightly hasten cardiac myocyte relaxation; however, relative to the much larger effect of HCM mutant Tm, this small change is not statistically significant in a four-group ANOVA comparison.
The normal physiological temperature range for human skeletal muscle, especially for muscles in the extremities, is quite large with values of 30°C documented in vivo (1). Given that muscle relaxation is profoundly temperature sensitive, we speculated that the effects of NM mutant Tm on muscle cell relaxation might be revealed to a greater extent at the lower end of the physiological range of temperatures. Figure 5 shows the summary of sarcomere shortening at 30°C. Rates of shortening were not different among groups, and the time-to-peak shortening (Tpeak) of either mutants was not statistically different from control or WT TmFLAG. There was a small but significant increase in the amplitude of shortening with HCM mutant Tm expression at 30°C. Similar to the effect at 37°C, HCM mutant Tm expression produced the most significant effects on slowing of relaxation [increased T1/2R and decreased maximum relaxation rate (+ dL/dtmax)]. Interestingly, the NM mutant Tm produced a marked and statistically significant (ANOVA) decrease in the time to half-maximal relaxation and a increase in the +dL/dtmax compared with control and WT TmFLAG at 30°C. This result indicates that muscle cell relaxation can be accelerated by the NM mutation in Tm. Thus the rate of relaxation in muscle cells can be markedly and differentially altered by changes in thin filament regulation induced by mutation of Tm associated with cardiac or skeletal muscle myopathies.

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Fig. 5. Summary of the kinetics of myocyte shortening at 30°C in myocytes treated with viral vectors expressing WT TmFLAG, NM (M9R) TmFLAG, and HCM (A63V) TmFLAG. The calculation of the parameters of shortening are described in the METHODS, and the data were analyzed by ANOVA. * Significantly different from the control, TmFLAG, and M9R (P < 0.05). #Significantly different from control, TmFLAG, and A63V TmFLAG (P < 0.05). A63V TmFLAG Tpeak was also significantly different from M9R (P < 0.05) but not WT TmFLAG or control. There were no other significant differences among these groups. Values are means ± SE; n = 28, control; n = 24, AdTmFLAG; n = 26, AdTmFLAG M9R; n = 28, AdTmFLAG A63V.
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DISCUSSION
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A main finding of this report is that HCM and NM mutant Tms have divergent, direct effects on the regulation of the kinetics of intact striated muscle cell relaxation. We propose that the differential clinical phenotypes associated with HCM and NM can in turn be attributed to the divergent effects of the HCM or NM mutant Tm proteins on the regulation of contraction in the absence of, or prior to, secondary muscle remodeling in vivo. The HCM mutant Tm protein is expressed in both cardiac and skeletal muscle but produces a cardiac muscle restricted phenotype of an enlarged, hypercontractile heart (34). In contrast, the NM mutant Tm is expressed in skeletal muscle and produces marked skeletal muscle weakness and a distinct sarcomere pathology: nemaline rods (24). The results of the present study support the hypothesis that the differential effects of mutations in Tm on striated muscle contractile function play an important role in the differential phenotype of HCM and NM.
Implications for HCM pathogenesis.
One advantage of using an in vitro muscle cell system to characterize the effects of mutant contractile proteins is that the direct effect of the mutant protein on contractile function can be examined in the absence of possible compensatory adaptations that may occur in vivo. Therefore, the identification of a relaxation defect directly caused by HCM mutant Tm expression in isolated cardiac myocytes suggests that the HCM mutant Tm protein itself could directly slow the relaxation of the HCM myocardium. This abnormal relaxation of cardiac muscle, termed diastolic dysfunction, is one of the common features of HCM patients (2). Previously, diastolic dysfunction in HCM has been largely attributed to changes in chamber morphology and wall stiffness due to cardiac hypertrophy. However, the data from the present study indicate that the mutations in the contractile proteins themselves may directly cause relaxation abnormalities and diastolic dysfunction in the absence of myocyte hypertrophy or alterations of sarcomere structure (Fig. 1, B and C).
Ca2+ release from the sarcoplasmic reticulum in cardiac muscle normally occurs over the submaximal activation range (8). This suggests that the increases in steady-state contractile Ca2+ sensitivity noted for HCM mutations in Tm (6, 17, 22) may produce hypercontractility. Under the relatively unloaded conditions of shortening in cultured adult cardiac myocytes, the HCM mutant Tm produced no increase in amplitude of shortening at 37°C, and only a modest increase in amplitude of shortening at 30°C without changes in the rate of shortening at either temperature. Thus muscle shortening is not markedly affected by increases in Ca2+ sensitivity caused by HCM mutant Tm and perhaps is rate limited by a step downstream of thin filament activation. Although velocity of shortening was unchanged, it is still possible that HCM Tm proteins in cardiac muscle could produce hypercontractility and increased power output (force x velocity) under loaded conditions similar to those encountered in vivo. Transgenic mice expressing high levels of D175N mutant Tm produce a small increase in Ca2+ sensitivity of contraction but show slight decreases in organ level contractility (positive dP/ dtmax) relative to nontransgenic littermates (22). Because mild pathological changes in heart morphology were evident in these mice, the effect of decreased contractility could be due to the secondary remodeling that occurred in these hearts.
The present results may also indicate why mutations in Tm associated with HCM do not produce a marked skeletal muscle myopathy. The release of Ca2+ in tetanic skeletal muscle contractions achieves free Ca2+ concentrations that nearly maximally activate the contractile apparatus (7). Thus increases in Ca2+ sensitivity caused by HCM mutations (6, 17, 22) will not likely augment maximal force production and do not appear to affect muscle shortening velocity (Figs. 4 and 5). In addition, the slowing of relaxation of skeletal muscle is likely not as important in skeletal muscle as it is in cardiac muscle, because in cardiac muscle the time the myocardium is relaxed is vital for repriming the ventricular chambers for the next contraction. Therefore, although HCM mutant Tm can increase the Ca2+ sensitivity of steady-state contraction in biopsies of skeletal muscle from HCM patients (5), this may not augment normal skeletal muscle physiology enough to produce a noted myopathy.
Implications for mutant Tm-associated NM.
The NM mutation in Tm produces a direct alteration in the kinetics of cardiac myocyte contraction opposite to the effects caused by HCM mutation in Tm. The increased rate of relaxation caused in vitro by NM mutant Tm was demonstrated at 30°C, a temperature in the physiological range for limb muscles in vivo (1). Our finding of a greater effect of the NM mutation at 30°C compared with 37°C correlates with the clinical manifestations of NM, as NM disease is known to be most profound in the limb extremities (13). NM mutant Tm produced no significant change in the twitch amplitude or rate of shortening. This implies that muscle activation is rate limited by components downstream of thin filament activation. The primary and direct effect of NM mutant Tm on intact muscle contraction is to accelerate the "turning off" or relaxation of the striated muscle cell.
Given the direct increase in relaxation kinetics caused by NM mutant Tm (Fig. 5), one might expect changes in motor unit activity as an adaptation to compensate for these effects, i.e., motor unit recruitment and/or changes in frequency to produce tetanus. The acceleration of relaxation caused by NM mutant Tm would be expected to right-shift the force-frequency relationship in skeletal muscle to higher frequencies. In other words, at any given submaximal
-motor neuron stimulation frequency, force output would be decreased, which may in part explain the muscle weakness seen in NM patients. Indirect evidence exists that compensatory changes in motor unit activity and mechanical load/ability of sarcomeres to bear force may be involved in NM pathogenesis. Many NM patients show a progressive fiber type remodeling including type I predominance and type II fiber hypertrophy (39). This could result from changes in muscle motor unit activity. Patients with mutations in the Tm gene TPM3 actually show a mild type II fiber predominance (13, 33). In addition, in an experimental model of Achilles tenotomy in the rat, nemaline rod formation occurs (along with core-like lesions), and development of the nemaline rods is dependent on the muscle being innervated (12). Thus a combination of factors including motor unit activity and muscle mechanical activity under conditions of altered load on the sarcomeres may be important in the development of ultrastructural changes seen in NM in vivo.
Finally, it should be noted the NM mutant Tm gene TPM3 is not normally expressed to significant levels in cardiac muscle. Furthermore, NM patients do not normally have cardiac involvement because many of the genes mutated in NM are restricted to skeletal muscle. Thus the data presented here in cardiac myocytes as a model system of fully differentiated striated muscle cells need to be confirmed in a skeletal muscle cell system. A differentiating quail skeletal muscle myotube system has been previously used to study TnT mutations associated with HCM (32), but this model system is not amenable to antibiotic-selected overexpression of Tm isoforms (31). Nevertheless, there have been reports of NM patients that present with dilated cardiomyopathy (in contrast to HCM), and nemaline rods can and do form in cardiac muscle of these patients. This suggests that cardiac striated muscle may be affected similarly to skeletal muscle if the mutant genes are expressed in heart (11). Indeed, the TPM1 gene, which is the predominant Tm isoform expressed in the human heart, is considered a candidate gene for NM (38).
Implications for the role of Tm in the regulation of striated muscle contraction.
The contractile function of intact striated muscle cells is complex, involving the kinetics of the Ca2+ transient, the kinetics of Ca2+ binding and conformational changes within the thin filament regulatory proteins, and the kinetics of the myosin motor. In this regard, the mechanisms involved in the regulation of relaxation of intact striated muscle are not well understood. Based on recent studies from the phospholamban knockout mouse, the regulation of Ca2+ sequestration into the sarcoplasmic reticulum has been concluded to be the primary mechanism of modulating heart muscle relaxation in response to ß-adrenergic stimulation (14). The present results indicate that modulation of the thin-filament regulatory proteins by specific HCM or NM mutation of Tm can differentially alter the kinetics of striated muscle cell relaxation. Thus the " turning off" of the thin filament regulatory system, independent of direct modulation of the sarcoplasmic reticulum Ca2+-ATPase, is also important in determining the kinetics of muscle cell relaxation.
One hypothesis to explain the effects of HCM and NM mutant Tm on relaxation kinetics is that the presence of the NM and HCM mutation in Tm alters the stability of one of the Ca2+-bound conformations of Tm on the thin filament (29). The stabilization or destabilization of Ca2+-bound states by HCM and NM mutation of Tm, respectively, may feed back to the troponin conformation. In this case, Ca2+ affinity and the off rate of Ca2+ from troponin C (TnC) would be altered. For instance, for HCM mutant Tm, the stabilization of Ca2+-bound conformation of Tm would increase the TnC Ca2+ affinity (17), slow the Ca2+ off rate from TnC, and in turn slow myocyte relaxation (Figs. 4 and 5). However, the mechanism by which this feedback occurs is unclear because although some of the HCM mutations (E180G, D175N) and the NM mutation (M9R) reside in regions important for troponin binding, the A63V and K70T mutations reside outside this region (42). However, the A63V mutant Tm still modulates relaxation kinetics (Figs. 3 and 4), and the A63V and K70T mutant Tm also confer increased Ca2+ sensitivity of steady-state force production (17). Thus this feedback may occur through long-range conformational changes in Tm, Tm-actin binding affinity, or perhaps conformational changes in actin itself.
Finally, the direct modulation of relaxation kinetics by HCM and NM mutation of Tm identifies the myofilament regulatory system as a potential target in treating or preventing contractile dysfunction seen in HCM and NM patients. The forced detachment of myosin from actin during relaxation has been shown to cause a transient rise in the free intracellular Ca2+ concentration (10, 28). This indirectly suggests that the Ca2+ binding and dissociation from the contractile apparatus during relaxation may be involved in buffering a portion of the free Ca2+ during relaxation. This raises the possibility that there could be a direct link between an alteration in a contractile protein that modulates the kinetics of actively contracting muscle, the modulation of intracellular Ca2+ transients, and the induction of Ca2+-activated signaling pathways important for the secondary remodeling of striated muscle, like cardiac hypertrophy for HCM mutations (21, 26). Thus the regulation of thin-filament regulatory protein activation and deactivation by intracellular Ca2+ may represent a new target for therapies designed to treat contractile dysfunction in HCM and NM and possibly other diseases of inappropriate relaxation of muscle, like heart failure (19).
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ACKNOWLEDGMENTS
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We thank Faris Albayya for continued efforts in virus vector preparations.
This work was funded by National Institutes of Health (National Institutes of Health) Training Grants for D. E. Michele and grants from NIH and the American Heart Association to J. M. Metzger. J. M. Metzger is an Established Investigator of the American Heart Association.
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FOOTNOTES
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Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
Address for reprint requests and other correspondence: J. M. Metzger, 7730 Medical Science II, Dept. of Physiology, Univ. of Michigan, 1301 E. Catherine, Ann Arbor, MI 48109-0622 (E-mail: metzgerj{at}umich.edu).
10.1152/physiolgenomics.00099.2001.
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REFERENCES
|
---|
-
Bennett AF. Thermal dependence of muscle function. Am J Physiol Regulatory Integrative Comp Physiol 247: R217R229, 1984.[Abstract/Free Full Text]
-
Betocchi S, Bonow RO, Bacharach SL, Rosing DR, Maron BJ, and Green MV. Isovolumic relaxation period in hypertrophic cardiomyopathy: assessment by radionuclide angiography. J Am Coll Cardiol 7: 7481, 1986.[ISI][Medline]
-
Bonne G, Carrier L, Richard P, Hainque B, and Schwartz K. Familial hypertrophic cardiomyopathy: from mutations to functional defects. Circ Res 83: 580593, 1998.[Abstract/Free Full Text]
-
Bonow RO. Left ventricular diastolic function in hypertrophic cardiomyopathy. Herz 16: 1321, 1991.[ISI][Medline]
-
Bottinelli R, Coviello DA, Redwood CS, Pellegrino MA, Maron BJ, Spirito P, Watkins H, and Reggiani C. A mutant tropomyosin that causes hypertrophic cardiomyopathy is expressed in vivo and associated with an increased calcium sensitivity. Circ Res 82: 106115, 1998.[Abstract/Free Full Text]
-
Bottinelli R, Coviello DA, Redwood CS, Pellegrino MA, Maron BJ, Spirito P, Watkins H, and Reggiani C. A mutant tropomyosin that causes hypertrophic cardiomyopathy is expressed in vivo and associated with an increased calcium sensitivity. Circ Res 82: 106115, 1998.[Abstract/Free Full Text]
-
Caputo C, Edman KA, Lou F, and Sun YB. Variation in myoplasmic Ca2+ concentration during contraction and relaxation studied by the indicator fluo-3 in frog muscle fibres. J Physiol 478: 137148, 1994.[Abstract]
-
Fabiato A. Myoplasmic free calcium concentration reached during the twitch of an intact isolated cardiac cell and during calcium-induced release of calcium from the sarcoplasmic reticulum of a skinned cardiac cell from the adult rat or rabbit ventricle. J Gen Physiol 78: 457497, 1981.[Abstract]
-
Gordon AM, Homsher E, and Regnier M. Regulation of contraction in striated muscle. Physiol Rev 80: 853924, 2000.[Abstract/Free Full Text]
-
Gordon AM and Ridgway EB. Cross-bridges affect both TnC structure and calcium affinity in muscle fibers. Adv Exp Med Biol 332: 183192, 1993.[Medline]
-
Ishibashi-Ueda H, Imakita M, Yutani C, Takahashi S, Yazawa K, Kamiya T, and Nonaka I. Congenital nemaline myopathy with dilated cardiomyopathy: an autopsy study. Hum Pathol 21: 7782, 1990.[ISI][Medline]
-
Karpati G, Carpenter S, and Eisen AA. Experimental core-like lesions and nemaline rods: a correlative morphological and physiological study. Arch Neurol 27: 237251, 1972.[ISI][Medline]
-
Laing NG, Wilton SD, Akkari PA, Dorosz S, Boundy K, Kneebone C, Blumbergs P, White S, Watkins H, and Love DR. A mutation in the alpha tropomyosin gene TPM3 associated with autosomal dominant nemaline myopathy. Nat Genet 9: 7579, 1995. [Published erratum appears in Nat Genet (Jun) 10: 249, 1995.][ISI][Medline]
-
Li L, Desantiago J, Chu G, Kranias EG, and Bers DM. Phosphorylation of phospholamban and troponin I in beta-adrenergic-induced acceleration of cardiac relaxation. Am J Physiol Heart Circ Physiol 278: H769H779, 2000.[Abstract/Free Full Text]
-
McKillop DF and Geeves MA. Regulation of the interaction between actin and myosin subfragment 1: evidence for three states of the thin filament. Biophys J 65: 693701, 1993.[Abstract]
-
Michele DE, Albayya FP, and Metzger JM. A nemaline myopathy mutation in alpha-tropomyosin causes defective regulation of striated muscle force production. J Clin Invest 104: 15751581, 1999.[Abstract/Free Full Text]
-
Michele DE, Albayya FP, and Metzger JM. Direct, convergent hypersensitivity of calcium-activated force generation produced by hypertrophic cardiomyopathy mutant alpha-tropomyosins in adult cardiac myocytes. Nat Med 5: 14131417, 1999.[ISI][Medline]
-
Michele DE, Albayya FP, and Metzger JM. Thin filament protein dynamics in fully differentiated adult cardiac myocytes: toward a model of sarcomere maintenance. J Cell Biol 145: 14831495, 1999.[Abstract/Free Full Text]
-
Minamisawa S, Hoshijima M, Chu G, Ward CA, Frank K, Gu Y, Martone ME, Wang Y, Ross J Jr, Kranias EG, Giles WR, and Chien KR. Chronic phospholamban-sarcoplasmic reticulum calcium ATPase interaction is the critical calcium cycling defect in dilated cardiomyopathy. Cell 99: 313322, 1999.[ISI][Medline]
-
Mogensen J, Klausen IC, Pedersen AK, Egeblad H, Bross P, Kruse TA, Gregersen N, Hansen PS, Baandrup U, and Borglum AD. Alpha-cardiac actin is a novel disease gene in familial hypertrophic cardiomyopathy. J Clin Invest 103: R39R43, 1999.[ISI][Medline]
-
Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, Grant SR, and Olson EN. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell 93: 215228, 1998.[ISI][Medline]
-
Muthuchamy M, Pieples K, Rethinasamy P, Hoit B, Grupp IL, Boivin GP, Wolska B, Evans C, Solaro RJ, and Wieczorek DF. Mouse model of a familial hypertrophic cardiomyopathy mutation in alpha-tropomyosin manifests cardiac dysfunction. Circ Res 85: 4756, 1999.[Abstract/Free Full Text]
-
Nakajima-Taniguchi C, Matsui H, Fujio Y, Nagata S, Kishimoto T, and Yamauchi-Takihara K. Novel missense mutation in cardiac troponin T gene found in Japanese patient with hypertrophic cardiomyopathy. J Mol Cell Cardiol 29: 839843, 1997.[ISI][Medline]
-
North KN, Laing NG, and Wallgren-Pettersson C. Nemaline myopathy: current concepts. The ENMC International Consortium and Nemaline Myopathy. J Med Genet 34: 705713, 1997. [Published erratum appears in J Med Genet (Oct) 34:879, 1997.][ISI][Medline]
-
Nowak KJ, Wattanasirichaigoon D, Goebel HH, Wilce M, Pelin K, Donner K, Jacob RL, Hubner C, Oexle K, Anderson JR, Verity CM, North KN, Iannaccone ST, Muller CR, Nurnberg P, Muntoni F, Sewry C, Hughes I, Sutphen R, Lacson AG, Swoboda KJ, Vigneron J, Wallgren-Pettersson C, Beggs AH, and Laing NG. Mutations in the skeletal muscle alpha-actin gene in patients with actin myopathy and nemaline myopathy. Nat Genet 23: 208212, 1999.[ISI][Medline]
-
Passier R, Zeng H, Frey N, Naya FJ, Nicol RL, McKinsey TA, Overbeek P, Richardson JA, Grant SR, and Olson EN. CaM kinase signaling induces cardiac hypertrophy and activates the MEF2 transcription factor in vivo. J Clin Invest 105: 13951406, 2000.[Abstract/Free Full Text]
-
Pelin K, Hilpela P, Donner K, Sewry C, Akkari PA, Wilton SD, Wattanasirichaigoon D, Bang ML, Centner T, Hanefeld F, Odent S, Fardeau M, Urtizberea JA, Muntoni F, Dubowitz V, Beggs AH, Laing NG, Labeit S, de la Chapelle A, and Wallgren-Pettersson C. Mutations in the nebulin gene associated with autosomal recessive nemaline myopathy. Proc Natl Acad Sci USA 96: 23052310, 1999.[Abstract/Free Full Text]
-
Ridgway EB and Gordon AM. Muscle calcium transient. Effect of post-stimulus length changes in single fibers. J Gen Physiol 83: 75103, 1984.[Abstract]
-
Rosol M, Lehman W, Craig R, Landis C, Butters C, and Tobacman LS. Three-dimensional reconstruction of thin filaments containing mutant tropomyosin. Biophys J 78: 908917, 2000.[Abstract/Free Full Text]
-
Rust EM, Westfall MV, and Metzger JM. Stability of the contractile assembly and Ca2+-activated tension in adenovirus infected adult cardiac myocytes. Mol Cell Biochem 181: 143155, 1998.[ISI][Medline]
-
Sweeney HL and Feng HS. Structure-function analysis of cytoskeletal/contractile proteins in avian myotubes. Methods Cell Biol 52: 275282, 1998.[ISI]
-
Sweeney HL, Feng HS, Yang Z, and Watkins H. Functional analyses of troponin T mutations that cause hypertrophic cardiomyopathy: insights into disease pathogenesis and troponin function. Proc Natl Acad Sci USA 95: 1440614410, 1998.[Abstract/Free Full Text]
-
Tan P, Briner J, Boltshauser E, Davis MR, Wilton SD, North K, Wallgren-Pettersson C, and Laing NG. Homozygosity for a nonsense mutation in the alpha-tropomyosin slow gene TPM3 in a patient with severe infantile nemaline myopathy. Neuromuscul Disord 9: 573579, 1999.[ISI][Medline]
-
Thierfelder L, Watkins H, MacRae C, Lamas R, McKenna W, Vosberg HP, Seidman JG, and Seidman CE. Alpha-tropomyosin and cardiac troponin T mutations cause familial hypertrophic cardiomyopathy: a disease of the sarcomere. Cell 77: 701712, 1994.[ISI][Medline]
-
Tobacman LS. Thin filament-mediated regulation of cardiac contraction. Annu Rev Physiol 58: 447481, 1996.[ISI][Medline]
-
Vibert P, Craig R, and Lehman W. Steric-model for activation of muscle thin filaments. J Mol Biol 266: 814, 1997.[ISI][Medline]
-
Wahr PA, Michele DE, and Metzger JM. Parvalbumin gene transfer corrects diastolic dysfunction in diseased cardiac myocytes. Proc Natl Acad Sci USA 96: 1198211985, 1999.[Abstract/Free Full Text]
-
Wallgren-Pettersson C, Pelin K, Hilpela P, Donner K, Porfirio B, Graziano C, Swoboda KJ, Fardeau M, Urtizberea JA, Muntoni F, Sewry C, Dubowitz V, Iannaccone S, Minetti C, Pedemonte M, Seri M, Cusano R, Lammens M, Castagna-Sloane A, Beggs AH, Laing NG, and de la Chapelle A. Clinical and genetic heterogeneity in autosomal recessive nemaline myopathy. Neuromuscul Disord 9: 564572, 1999.[ISI][Medline]
-
Wallgren-Pettersson C, Rapola J, and Donner M. Pathology of congenital nemaline myopathy. A follow-up study. J Neurol Sci 83: 243257, 1988.[ISI][Medline]
-
Westfall MV, Pasyk KA, Yule DI, Samuelson LC, and Metzger JM. Ultrastructure and cell-cell coupling of cardiac myocytes differentiating in embryonic stem cell cultures. Cell Motil Cytoskeleton 36: 4354, 1997.[ISI][Medline]
-
Westfall MV, Rust EM, Albayya F, and Metzger JM. Adenovirus-mediated myofilament gene transfer into adult cardiac myocytes. Methods Cell Biol 52: 307322, 1997.[ISI][Medline]
-
White SP, Cohen C, and Phillips GN Jr. Structure of co-crystals of tropomyosin and troponin. Nature 325: 826828, 1987.[ISI][Medline]
-
Yamauchi-Takihara K, Nakajima-Taniguchi C, Matsui H, Fujio Y, Kunisada K, Nagata S, and Kishimoto T. Clinical implications of hypertrophic cardiomyopathy associated with mutations in the alpha-tropomyosin gene. Heart 76: 6365, 1996.[Abstract]