Muscle LIM protein (MLP) has been suggested to be an important mediator of mechanical stress in cardiac tissue, but the role that it plays in skeletal muscle remains unclear. Previous studies have shown that it is dramatically upregulated in fast-to-slow fiber-type transformation and also after eccentric contraction (EC)-induced muscle injury. The functional consequences of this upregulation, if any, are unclear. In the present study, we have examined the skeletal muscle phenotype of MLP-knockout (MLPKO) mice in terms of their response to EC-induced muscle injuries. The data suggest that while the MLPKO mice recover completely after EC-induced injury, their torque production lags behind that of heterozygous littermates in the early stages of the recovery process. This lag is accompanied by decreased expression of the muscle regulatory factor MyoD, suggesting that MLP may influence gene expression. In addition, there is evidence of type I fiber atrophy and a shorter resting sarcomere length in the MLPKO mice, but no significant differences in fiber type distribution. In summary, MLP appears to play a subtle role in the maintenance of normal muscle characteristics and in the early events of the recovery process of skeletal muscle to injury, serving both structural and gene-regulatory roles.
- eccentric contractions
- passive tension
the skeletal muscle z disk has been shown to be one of the more important “sensing” and structural elements of the sarcomere (12). In addition to the well-known and primarily structural proteins α-actinin, titin, actin, and nebulin, there is increasing evidence for Z-disk proteins playing an active role in signal transduction by serving as the anchoring site for numerous proteins involved with signaling. Some examples of this include cypher/Z band alternatively spliced postsynaptic density 95/Drosophila disc-large/zonula occludens-1-containing protein (ZASP)/oracle, which binds the COOH terminus of α-actinin in the Z disk in addition to PKC (33), and the filamin-, actinin-, and telethonin-binding protein of the Z-disk of skeletal muscle (FATZ) family, which bind α-actinin, filamin, and telethonin in the Z disk while also potentially regulating calcineurin activity (13). The number and extent of identified interactions among Z-disk proteins is continually growing, and it is clear that many of the proteins and protein interactions at this sarcomeric site traditionally considered “structural” play important roles in the regulation of muscle function.
Muscle LIM protein (MLP), a Z-disk protein with the potential for gene-regulatory responses, was recently identified as being dramatically upregulated in response to eccentric contraction (EC)-induced muscle injury (3, 6). MLP binds the structural proteins β-spectrin, telethonin, nebulin-related protein (N-RAP), zyxin, and α-actinin (11, 14, 18, 21) in the Z disk, suggesting that it may play a linker or scaffolding role. It has also been found in the nucleus during early development (1), in which it is a potent activator of the myogenic regulatory factor MyoD (19). This nuclear activity suggests that MLP may have some transcriptional activity in addition to its structural role, leading to the intriguing possibility that MLP is a striated muscle stress sensor (18). Indeed, there is evidence that MLP translocates between the cytoplasm and the nucleus in response to physical stimuli (10). It is also upregulated after skeletal muscle denervation (1) and during fast-to-slow fiber-type transition (5, 26, 32).
A MLP-knockout model (MLPKO) of this mouse (2) shows severe cardiac hypertrophy, myofibrillar disarray, and a less compliant heart (11, 24). Perhaps as a result of the dramatic cardiac muscle phenotype present in the MLPKO mice, the effect of loss of MLP in skeletal muscle has not been documented as carefully. The few data available suggest that MLPKO mice have somewhat swollen fibers and some defects in neuromuscular transmission and were not able to hold onto a weight for as long as wild-type mice (2). The dramatic rise in MLP expression levels after injury or exercise of skeletal muscle suggests, however, that MLP may play a larger role in this tissue than previously demonstrated. In this study, therefore, we performed a careful analysis of different aspects of the skeletal muscle phenotype of the MLPKO mice, focusing our attention on aspects of skeletal muscle physiology that previous studies have suggested to be influenced by MLP.
We measured fiber-type proportions and fiber areas because MLP expression has been shown to change in response to fiber-type switching, and thus we hypothesized that the absence of MLP would influence fiber-type distribution or perhaps specifically the maintenance of type I fibers. This hypothesis was examined in three different muscles because of their dramatically different fiber-type distributions, use patterns, and architectural properties. The extensor digitorum longus (EDL) and tibialis anterior (TA) are primarily phasic muscles and therefore contain almost exclusively fast fibers (type II), whereas the soleus is a postural muscle and therefore contains a good proportion of slow fibers (type I) in addition to some fast type II fibers. The histological techniques used also allowed us to look for changes in fiber size variability (present with many neuromuscular diseases), evidence of degeneration and/or regeneration, and inflammation, any of which might be possible if, as predicted by previous studies, MLP plays a role in the maintenance of normal skeletal muscle. We also examined the passive mechanical behavior of MLPKO skeletal muscle because previous studies (18, 24) suggested that MLPKO mice have altered passive cardiac mechanics, perhaps because of MLP’s interaction with the titin-capping protein telethonin. Passive muscle characteristics are important for a number of reasons, including important influences on the normal range of motion of a muscle and stretch-based signaling. Our hypothesis was that the absence of MLP would result in a shorter resting sarcomere length and perhaps a stiffer sarcomere, which would indicate that MLP acts in series with passive mechanical structures such as titin.
Finally, previous studies have shown a dramatic upregulation of MLP with EC-induced injury (3). This increase was EC specific, not observed with either passive stretch or isometric contraction, and we therefore hypothesized that MLP plays a role in either the initial injury process (such that upregulation of MLP after injury would be a part of the protective result of repeated EC bouts, possibly by providing structural stability at the Z disk) or in the recovery of muscle from ECs. Upregulation in this case would be a part of the gene expression program involved with rebuilding the muscle after injury. These varied hypotheses reflect the dual role of MLP in the sarcomere and the nucleus, with some aspects (e.g., passive mechanical studies) likely resulting from its structural role in the sarcomere and others (e.g., effect of MLP on ECs) resulting from a combination of both localizations and roles. Although the data presented in this study do not show a dramatic phenotype in the skeletal muscle of MLPKO mice, they do suggest that the role of MLP in skeletal muscle remains worthy of further investigation.
Male and female 2- to 3-mo-old MLPKO mice (average age, 11.7 ± 0.57 wk; average mass, 29.1 ± 1.5 g) were obtained from Dr. Ju Chen (Department of Medicine, University of California, San Diego) and housed four per cage at 20–23°C with a 12:12-h dark-light cycle. Control animals consisted of age-, mass-, and sex-matched heterozygous as well as wild-type mice. No significant differences were found between the heterozygous and wild-type mice in any of the measured parameters, so only the heterozygous values are presented herein because more heterozygous littermates were available for study. All procedures were approved by the University of California, San Diego Animals Subjects Committee.
TA, EDL, and soleus muscles from unexercised male mice and the same muscles from the contralateral leg in exercised male mice were removed and frozen in isopentane cooled with liquid nitrogen. Tissue sections (10 μm) were processed for routine hematoxylin and eosin staining, and myosin ATPase histochemistry was performed according to standard methods using acidic pH 4.3 to identify type I fibers and basic pH 10.2 to identify type II fibers. Types I and II fiber sizes were determined using Metamorph software (Universal Imaging, Downingtown, PA) to record the area stained darkly in either the acidic or basic labeling conditions, respectively, at ×10 magnification and dividing this area by the number of labeled fibers in each section. One section from the midbelly of the muscle was analyzed.
Fiber-type analysis was performed on the TA, EDL, and soleus muscles of heterozygous and MLPKO animals using a modified version of the SDS-PAGE method developed by Talmadge and Roy (28). Either a 55-μm-thick section of the muscle (for muscle fiber-type percentage analysis) or a single-fiber segment (for determination of fiber type after passive mechanics) was dissolved in sample buffer [100 mM dithiothreitol, 2% SDS, 80 mM Tris base, pH 6.8, 10% glycerol, and 1.2% (wt/vol) bromphenol blue], boiled for 2 min, and then either frozen at −80°C or run immediately on the gel. Stacking and resolving gels with a total gel size of 16 × 22 cm and a thickness of 0.75 mm consisted of 4% or 8% acrylamide, respectively. Gels were run at a constant voltage of 275 V for ∼21 h at 4°C and then silver stained according to the manufacturer's protocol (Bio-Rad Laboratories, Hercules, CA). Fiber-type percentage analysis was performed in duplicate or in triplicate and analyzed using NIH Image software (version 1.62). Single-fiber analysis was performed once simply to determine the myosin heavy chain isoform of the mechanically tested fiber.
Passive mechanical experiments were performed on fibers from either the fifth-toe muscle of the EDL or the soleus. These two muscles were selected on the basis of the fiber type and length homogeneity observed within each muscle (4) and the dramatically different fiber type profiles between muscles. Immediately after the animals were killed by cervical dislocation while the animals were under general anesthesia, the hindlimbs were transected proximal to the knee and immediately placed into a mammalian Ringer solution (137 mM NaCl, 5 mM KCl, 24 mM NaH2PO4, 2 mM CaCl2, 1 mM MgSO4, 11 mM glucose, and 10 mg/l curare). Occasionally, the contralateral limb from the exercised animal was used for mechanical experiments; in this case, hindlimbs were transected after the TA muscles were removed and frozen for RNA analysis.
EDL and soleus muscles were quickly removed from the hindlimb and placed for at least 60 min in a relaxing solution containing 59.4 mM imidazole, 86 mM KCH4O3S, 0.13 mM Ca(KCH4O3S)2, 10.8 mM Mg(KCH4O3S)2, 5.5 mM K3EGTA, 1 mM KH2PO4, 5.1 mM Na2ATP, and 50 μM leupeptin (27). While in the relaxing solution, the fifth-toe muscle of the EDL was dissected under magnification, and both this and the soleus muscle were separated into two bundles for storage. These muscles were then pinned to parafilm-coated cork and stored in a solution of 170 mM K+-propionate, 5 mM K3EGTA, 5.3 mM MgCl2, 10 mM imidazole, 21.2 mM Na2ATP, 1 mM NaN3, 2.5 mM glutathione, 50 μM leupeptin, and 50% (vol/vol) glycerol (9) at −20°C for up to 3 wk.
At the time of the experiment, bundles were removed from storage solution and placed in relaxing solution. Single-fiber segments (2–3 mm in length) were carefully dissected and transferred to the mechanical chamber, in which they were tied with 10-0 nylon suture to titanium wires (Aldrich Chemical 26601-9, Milwaukee, WI) secured to a force transducer (model 405A; Aurora Scientific, Aurora, ON, Canada) and a motor (model 318B; Aurora Scientific). The motor was connected to a micromanipulator such that fiber stretching could be controlled by either the motor or the micromanipulator and fiber orientation could be adjusted. Slack length was determined using a combination of visual orientation of the fiber such that it did not have a curved appearance and maintenance of the tension within 0.002 mV (<0.1 mg) of its background level. This method has been shown to be repeatable (15). Under ×10 magnification, the fiber length and diameter in three places were measured by manipulating the fiber in and out of the field of view with a micrometer attached to a micromanipulator. Mean fiber diameter was used to estimate fiber cross-sectional area, assuming the fibers were cylindrical. Fiber length was also determined by transilluminating the fiber with a HeNe laser (Melles Griot, Irvine, CA) to permit measurement of the sarcomere length at slack length and after 0.5-mm stretch. Sarcomere length was determined by projecting the diffraction pattern onto a photodiode array (PDA, model S2048; PerkinElmer Reticon, Fremont, CA) positioned above the fiber. The position of the PDA was calibrated using 2.50-, 3.33-, and 5.00-μm diffraction gratings before the experiment, and first-order diffraction peaks were used to calculate sarcomere length according to standard grating equations. Fiber length was calculated assuming constant sarcomere number using the following formula: The fiber length used in the final analysis was the average of both the visually determined and calculated fiber length, which were within 0.5 mm of each other. The fiber was carefully checked for mechanical integrity such that any discoloration, abrasion, swelling, or disrupted diffraction pattern disqualified the fiber from further analysis. All experiments were performed at room temperature (∼22°C).
Dynamic experiments were performed using LabView software (National Instruments, Austin, TX) to control the motor, record force from the force transducer, and record the diffraction pattern from the PDA. Three stretch protocols consisting of five cycles of stretch and release at 35% strain were imposed on the muscle: low velocity (5%/s strain rate), intermediate velocity (10%/s strain rate), and high velocity (100%/s strain rate). Sarcomere length and force were recorded in real time for all strain rates and analyzed with MatLab software (MathWorks, Natick, MA) for tangent modulus (final slope in the last 5% of each stretch cycle), hysteresis loop area, starting sarcomere length, peak sarcomere length, peak stress, and peak strain. After the cyclical stretch protocol, stress relaxation was recorded by stretching the fiber 20%, 30%, or 40% and then holding at that length for 2 min. MatLab was then used to calculate peak and steady-state stress and to fit an exponential equation to the stress relaxation decay to compare stress relaxation time constants between genotypes.
After the dynamic stretch protocols, muscle fibers were stretched to failure in 2-min intervals and 250-μm increments using the micrometer attached to the motor. Sarcomere length and tension were recorded at the end of each 2-min interval, and the slope of the stress-strain curve (elastic modulus) was determined (15). At the end of the experiment, the fibers were placed in 10 μl of sample buffer and stored at −20°C for fiber-type analysis as described above.
Titin molecular weight determination.
EDL and soleus muscles from MLPKO and heterozygous littermates were removed and frozen in liquid nitrogen. Agarose (1%) gel electrophoresis was used as previously described (30) to evaluate the molecular weight of titin in the samples, which was calibrated using a standard containing rat cardiac muscle and human soleus, two titin isoforms for which the molecular mass had unequivocally been determined.
The exercise apparatus and regimen have been described in detail elsewhere (3). In brief, mice were positioned in a specially designed jig that allowed measurement of ankle dorsiflexion torque and control of ankle position (to control muscle length) via an ergometer (custom-modified model 360B; Aurora Scientific) while the animals were under general anesthesia (2% isoflurane). The anterior compartment, consisting primarily of the TA and EDL muscles, was stimulated at 10 V using sterile subcutaneous 28-gauge needle electrodes (Grass Instruments, Braintree, MA) placed in the vicinity of the right peroneal nerve, ∼0.5 mm under the skin, just lateral to the midline and distal to the knee joint. Optimum stimulation frequency (resulting in a flat and fused isometric torque record) was usually determined to be 200 Hz.
The eccentric bout consisted of 50 eccentric contractions, 1/min, with the footplate forcing 78° plantarflexion at about one fiber length per second, starting 150 ms after the muscle was activated. Before all exercise paradigms, maximal isometric torque was measured as the mean of two isometric contractions. During the EC bout, isometric twitches were recorded every five contractions and needle electrodes were repositioned if necessary to achieve maximal twitch. The contralateral leg underwent only isometric testing, consisting of just enough isometric contractions (∼5) to determine maximum isometric torque without any eccentric contractions.
Mice were subjected to one of two protocols: a preliminary protocol or a more detailed protocol. In the preliminary protocol, the same animals were followed over time as they recovered from the injury protocol. Thus these animals were returned to their cages after the initial exercise bout and then subjected to the isometric testing protocol described above on both the eccentrically exercised and contralateral legs 6, 24, 72, 120, 168, and 216 h (9 days) after the initial bout. After the last time point, these animals were euthanized by cervical dislocation while under anesthesia. In the more detailed protocol, muscles were collected for analysis from a separate set of animals 6, 12, or 24 h after the initial exercise bout with no intermediate testing. TA muscles were removed and frozen in liquid nitrogen-cooled isopentane for further analysis, and animals were euthanized by cervical dislocation while they were under anesthesia. After euthanasia, the heart, lungs, and liver were collected and weighed wet, before being frozen in liquid nitrogen and then lyophilized overnight for dry weight and dry-to-wet ratio determination. In four animals of each genotype, heart rate was measured via three-lead ECG and recorded every 5 min throughout the initial exercise bout while the animals were under anesthesia.
RNA was extracted from the TAs of exercised and contralateral legs using a combination of the standard TRIzol reagent (Invitrogen, Carlsbad, CA) and RNeasy (Qiagen, Valencia, CA) protocols described elsewhere (3). RNA (500 ng) from each sample were reverse transcribed according to the manufacturer's protocol (SuperScript II; Invitrogen). Quantitative real-time PCR was performed with the Cepheid SmartCycler (Sunnyvale, CA) using primers specific to the genes of interest, and the reaction product was quantified by monitoring the fluorescence levels of the intercalating dye SYBR Green (Sigma) and comparing them with a PCR product standard described elsewhere (3). Primers are listed in Table 1. All samples were run at least in duplicate, along with a plasmid standard. Amplification conditions were as follows. An initial hold at 95°C for 5 min was followed by 40 cycles of denaturing at 95°C for 15 s, followed by annealing and/or extension at 66°C [cardiac ankyrin repeat protein (CARP) and MyoD] or 70°C (Ankrd2/Arpp) for 40 s, followed by a melt curve. The success of each reaction was assessed on the basis of observing a single reaction product on an agarose gel and a single peak on the DNA melting temperature curve determined at the end of the reaction.
Comparisons between genotypes were performed with Fisher's protected least significant difference when significant differences were found using ANOVA with StatView software (SAS Institute, Cary, NC). Comparisons between the distribution of fiber types tested between genotypes and between muscles for the single-fiber mechanical analysis were performed using χ2 analysis. P ≤ 0.05 was considered statistically significant, and all values are presented as means ± SE.
Body masses were not significantly different between genotypes. Heart mass, both wet and dry, was significantly higher in the MLPKO animals (P < 0.0001) (Table 2), and the dry-to-wet ratio was significantly lower in the MLPKO animals (P < 0.01), suggesting both hypertrophy and edema or fluid retention in the heart. Lung wet and dry masses were also significantly higher in MLPKO mice (P < 0.05), with no difference observed in the dry-to-wet ratio, suggesting that the increased lung masses were due to increased lung parenchyma rather than to edema. No differences in liver wet, dry, or dry-to-wet ratios were found between genotypes.
Histology, histochemistry, and fiber-type analysis.
No obvious histological abnormalities, such as centralized nuclei, necrosis, increased fiber size variability, abnormal fiber shape, or altered fiber density, were observed in the skeletal muscle of MLPKO mice compared with heterozygous controls using histochemical staining (Fig. 1A). Two-way ANOVA of fiber size determined from myosin ATPase staining by genotype and fiber type revealed a nearly significant effect of genotype (P = 0.0505) (Fig. 1, A and B), with heterozygous fibers being larger than MLPKO fibers. No main effects of fiber size for type I or II fibers or in the interaction term between fiber type and genotype were observed. The fiber size difference also was observed when the cross-sectional area of single fibers used for mechanical analysis was determined. In heterozygous control mice, soleus fibers had a larger cross-sectional area than EDL fibers measured with this technique, but the soleus fibers of the MLPKO mice were significantly smaller than the EDL fibers (Fig. 1C) (P < 0.05). When fiber size was analyzed by fiber type instead of by muscle type, there was a significant interaction between fiber type and genotype, such that type I and IIa fibers were smaller in the MLPKO mice compared with controls (P < 0.05; data not shown). There was no significant difference in fiber-type distribution in the TA, EDL, or soleus muscles between genotypes (Fig. 1D).
Resting sarcomere length was significantly shorter in MLPKO mice compared with heterozygous controls with regard to both the EDL and soleus muscles (Fig. 2A) (P < 0.01), and EDL fibers consistently had a longer resting sarcomere length than soleus fibers in both genotypes (Fig. 2A) (P < 0.0001). There continued to be a difference between the resting sarcomere lengths of MLPKO and control animals when fiber type was taken into account, with heterozygous fibers of all types being significantly longer than MLPKO fibers (P < 0.02), but there was no significant difference in resting sarcomere lengths among fiber types (P > 0.3; data not shown), indicating that something differed between the soleus and the EDL besides fiber-type distribution. The different resting sarcomere lengths between genotypes and muscles translated directly to smaller peak sarcomere length in all dynamic experiments (5%, 10%, and 100%/s cyclical strain rate and all three strains of the stress-relaxation experiment) and steady-state sarcomere length (in the stress-relaxation experiments) in MLPKO mice compared with heterozygous mice and longer sarcomere lengths in the EDL compared with the soleus muscles (P < 0.05; data not shown). No other differences between MLPKO mice and controls were observed. The elastic modulus in the stretch to failure experiment revealed the EDL to be less stiff than the soleus (Fig. 2B) (P < 0.001), and this result was confirmed in the cyclical stretch experiments at all three strain rates examined (Fig. 2C) (P < 0.05). As with the resting sarcomere length data, there was no difference in the elastic modulus between fiber types (P > 0.2; data not shown), suggesting that the differences in the stiffness of the soleus and the EDL fibers were not due to fiber-type differences but rather to intrinsic differences between the two muscles. There were no significant differences between genotypes in the distribution of fiber types tested (P > 0.4), but there was a significant difference in fiber-type distribution between the EDL and the soleus fibers, as expected (P < 0.0001). Agarose gel electrophoresis showed no significant differences in the molecular weight of titin in either the EDL or soleus muscles of the heterozygous control animals compared with the MLPKO mice (EDL, 3,570 ± 41 kDa vs. 3,590 ± 26 kDa, respectively; soleus, 3,400 ± 3 kDa vs. 3,510 ± 30 kDa, respectively).
Injury and recovery after eccentric contractions.
When identical mice were followed over time, no significant differences in the recovery of the heterozygous and MLPKO mice after EC-induced muscle injury were found using two-way ANOVA over time and between genotypes (Fig. 3A). This suggests that any deficits in the response of the MLPKO mice to EC-induced injuries over the course of 9 days were small. Closer inspection, however, revealed that the MLPKO mice appeared to lag behind their heterozygous littermates in the early stages of torque recovery up to 24 h after initial injury. When separate animals were used for each time point, such that any effect of repeated anesthesia and muscle stimulation was removed, a significant deficit was found at the early recovery time points after injury in the MLPKO mice (Fig. 3B) (P < 0.01). There was no significant difference between the genotypes in the initial injury amount as determined using Student's t-test, and previous work has suggested that significant recovery occurs within 24 h after an EC-induced injury bout using this model (Barash IA and Lieber RL, unpublished data; see also Ref. 3). Taken together, both of these findings suggest that the difference between genotypes is due to impaired recovery and not to increased injury. Moreover, this difference was not due to increased physiological stress after injury in the MLPKO animals, because cardiovascular stress as indicated by heart rate measured during the exercise bout was not different between the two genotypes and did not increase throughout the exercise bout (Fig. 3C).
Heterozygous animals showed a robust increase in MyoD expression 6 h after injury, and this response was significantly attenuated in MLPKO mice (Fig. 4A) (P < 0.01). No differences were observed in the expression levels of CARP or Ankrd2/Arpp, both of which increased to the same extent in the exercised legs in both genotypes (Fig. 4, B and C).
MLP in cardiac muscle has been the focus of recent studies (16, 18, 31), but its effect in skeletal muscle has largely been ignored. Herein we present the results of a detailed examination of the skeletal muscle phenotype of MLPKO mice. Although the phenotype is not as dramatic as in cardiac muscle, it suggests that MLP plays a role in both the structure and function of skeletal muscle.
MLPKO mice have a severe dilated cardiomyopathy that manifests within 2 wk after birth (2). Adult animals show the typical symptoms of this disorder observed in humans, including enlarged hearts, systolic dysfunction, ventricular dilation, and eventually heart failure. Because of the potential for the cardiac phenotype to influence the skeletal muscle phenotype as a result of heart failure leading to insufficient peripheral perfusion, we recorded heart, lung, and liver masses, including dry masses and the dry-to-wet ratios, which are inversely related to the percentage of water in the tissue (Table 2). The increased wet heart and lung masses in the MLPKO mice were not surprising and have been reported elsewhere (2). The dry heart and lung masses were both significantly higher than those in control animals, but only the dry-to-wet ratio of the heart was significantly different from that in controls. This indicates that both the heart and lungs of MLPKO mice have increased tissue mass (i.e., dry mass), but that only the heart demonstrates increased water content as shown by a lower dry-to-wet ratio. No difference between genotypes was observed with any of the liver masses. Although the significance of the increased tissue mass of the lungs remains unknown, these results suggest that although the MLPKO animals used in the experiments had cardiomyopathy, they had not yet reached a stage of generalized edema or end-stage heart failure that would have had a large systemic effect on their response to stressful stimuli or on the perfusion and function of tissues, including skeletal muscle. Even if the MLPKOs had some degree of heart failure, it is not likely that this effect would have a significant effect on the differences shown in this investigation. First, although heart failure can lead to skeletal muscle atrophy (8), this occurs only in severe disease, which we do not think was a factor in our study. Second, although impaired cardiac function would be expected to influence the recovery after and the response to exercise, the exercise described in this model did not result in an increase in heart rate even in heterozygous animals (Fig. 3C). This suggests that the exercise in question was only a minor cardiovascular stress, and therefore the animal's response to it was unlikely to have been influenced by mild cardiac disturbances. We cannot completely rule out the possibility, however, that cardiac disease influenced the skeletal muscle phenotype described in this study, perhaps through increasing circulating levels of cytokines such as TNF-α (29).
To obtain a clear picture of the effect of the absence of MLP on skeletal muscle function, we performed a survey of the basic attributes of MLPKO muscle. Interestingly, fiber size of MLPKO muscle showed a trend toward being smaller than that of heterozygous littermates close to statistical significance, both when measured on heterozygous fiber cross sections and when individual fiber cross-sectional area was measured under the microscope during passive mechanical studies (Fig. 1, B and C). Cross-sectional area measurements obtained using these two methods resulted in different data, which is not surprising, given that the measurements obtained during passive mechanics assumed that the fibers were cylindrical when they obviously were not (Fig. 1A). It is significant that both methods of cross-sectional area measurement demonstrated the slowest fibers (soleus fibers, primarily types I and IIa) to be small in MLPKO muscles compared with controls, but no effect on the fastest fibers (EDL fibers, primarily types IIx and IIb).
A previous study (2) mentioned that skeletal muscle fibers of MLPKO mice appeared swollen, although quantitative measurements of fiber size were not reported. We did not observe evidence of swollen or injured fibers in unexercised MLPKO mice. The reduced size of the MLPKO fibers observed in our study suggests that either there is a fiber type trend toward slower fibers in the transgenic animals (not observed) (Fig. 1D) or that the muscles of these animals undergo a degree of atrophy. The larger decrease in fiber size observed in type I compared with type II fibers is consistent with type I fiber atrophy. MLP is upregulated during fast-to-slow fiber-type transitions (26, 32), suggesting that MLP may be involved in the maintenance of slow fibers. The lack of a difference in the fiber-type distribution of the TA, EDL, or soleus muscles between the two genotypes suggests, however, that MLP is not essential for the development of slow fibers.
Mechanically, MLP has been suggested to play a role in stress transduction across muscle cells. Young MLPKO hearts that have not yet developed cardiomyopathy show higher compliance than their wild-type controls (18), although the hearts of older MLPKO animals that have a large amount of fibrosis and scarring are stiffer (24), suggesting that in young animals with only limited complications of heart disease, MLP contributes to the passive tension of cardiac muscle. MLP binds a large array of proteins within the sarcomere, including α-actinin and telethonin/T-Cap (18, 21), and may influence titin, the main protein responsible for passive tension in cardiac and skeletal muscle (20), through these proteins. In our data, we did not observe any direct effect of the absence of MLP on passive tension in skeletal muscle, but we did note a decrease in resting sarcomere length of both the EDL and the soleus in MLPKO animals compared with heterozygous controls (Fig. 2A). Titin is thought to be involved in establishing resting sarcomere length (20), so the effect of MLP may be indirect through titin. Direct measurement of the molecular weight of titin in the MLPKO animals by using agarose gel electrophoresis indicates that this shortened sarcomere length is not due to expression of a shortened version of titin. It remains possible that in skeletal muscle, MLP acts in series with titin, such that the elastic element in MLPKO animals is shorter than in controls.
It is possible that we did not observe the effect of MLP on the modulus of skeletal muscle, because MLP is not as highly expressed in skeletal muscle as in cardiac muscle (26), so the effect may be below the detection limit. Another possibility is that differences in collagen content and extracellular matrix material between types of striated muscle may have confounded our data. Although we did not observe a difference between the stiffness of MLPKO vs. heterozygous controls, we did note a difference between fibers from the EDL and the soleus muscles from both genotypes, with the soleus being stiffer (Fig. 2, B and C). This is consistent with results observed when whole muscles were examined by others (22), but not when isolated myofibrils were tested (23), suggesting that even though we have tested isolated, skinned single fibers, there was still a significant extracellular matrix surrounding the fiber that could have contributed to the passive tension. Consistent with this hypothesis is that no differences in either resting sarcomere length or stiffness were observed between fiber types, suggesting that we detected differences between muscle types independent of fiber type. Any effect of MLP on the passive mechanical behavior of titin therefore might be masked by a greater contribution of collagen, for example, to the measured stiffness. This effect also could have confounded the cardiac mechanical data collected by others.
In addition to the effect on the resting sarcomere length and fiber size, mice lacking MLP showed an impaired response to EC-induced muscle injury. Two experiments were conducted to investigate this response. In the first screening experiment, mice from each genotype were subjected to about 50 ECs and their isometric torque was followed for 9 days after the initial exercise bout. These animals were therefore anesthetized and isometrically tested seven times after the exercise bout. In the second experiment, animals were subjected to the same initial exercise protocol, but separate animals were tested immediately after the EC bout and then killed 6, 12, or 24 h thereafter, for a total of only two bouts of anesthesia and two isometric testing sessions per animal. In the screening experiment, no overall impediment was observed with regard to the recovery in MLPKO mice. There was no statistically significant difference between the responses of heterozygous and MLPKO mice as assessed using repeated-measures ANOVA (Fig. 3A). This indicates that MLP is not necessary for force recovery after EC-induced injuries, at least when examined up to 9 days after the exercise bout. There was, however, a slight indication that MLPKO mice lagged behind their littermates in the early stages of torque recovery up to 24 h after injury. This finding was more obvious during the second experiment, with separate animals tested isometrically at each time point (Fig. 3B). Two-way ANOVA in this case revealed a significant effect of genotype and time, suggesting that MLPKO mice were functionally impaired at the early time points during this experiment.
These data are also supported by gene expression changes after EC-induced injuries. While the absence of MLP had no effect on expression levels of either CARP or Arpp, two proteins known to increase after EC-induced injuries (Fig. 4, B and C) (3), expression of MyoD was significantly decreased 6 h after exercise, the only time point at which it was elevated in control animals (Fig. 4A). Admittedly, this is just a single time point, and it remains possible that peak MyoD expression in the MLPKO mice simply occurs at a different time point than the one analyzed, perhaps earlier than 6 h after exercise; yet, this result is intriguing because of the known MLP effect of increasing MyoD, mrf4, and myogenin function (19). MLP itself is missing a transactivation domain but is thought to serve as a cofactor for these basic helix-loop-helix transcription factors. It is possible, therefore, that during a normal response to muscle injury, MLP stimulates not only MyoD activity but also MyoD expression. Although for a while it was thought that MyoD (and myf5) functioned only during the first stage of development or regeneration and that mrf4 and myogenin functioned only during the later differentiation stage, recent evidence has suggested that mrf4 can in fact act early to stimulate the muscle phenotype and potentially also MyoD expression (17). This is therefore a potential mechanism whereby MLP could influence MyoD expression. Regardless of the reason that MyoD expression is reduced in this model, its reduction would have a profound effect on the regenerative ability of skeletal muscle, because MyoD is a prime player in the early activation of satellite cells (7, 25). Its reduced expression could therefore explain the delayed recovery of the torque response observed in MLPKO mice.
The effect of the absence of MLP on the recovery after EC-induced injuries was small, and it remains possible that if experiments were performed on a muscle that normally expresses a higher level of MLP (i.e., a muscle with a slower fiber-type distribution such as the soleus or the diaphragm), a more robust phenotype would be observed. Much larger phenotypic changes are observed in the cardiac muscle of MLPKO mice (2) than in the skeletal muscle, perhaps reflecting the differential expression of MLP in these tissues.
Our data suggest that MLP plays a subtle but nonetheless important role in the maintenance of normal muscle function, including fiber size, resting sarcomere length, and response to injury. This observation is consistent with the role of MLP as a stress sensor in both cardiac and skeletal muscles.
This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grants AR-40050 and AR-45039 and by the Department of Veterans Affairs.
We are grateful for helpful discussions with Dr. Ilke Lorenzen-Schmidt, Dr. Andrew McCulloch, and Peter Costandi, as well as for the generous gift of the MLPKO mice from Dr. Ju Chen.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2005 the American Physiological Society