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Am J Physiol Regul Integr Comp Physiol 283: R583-R590, 2002. First published May 23, 2002; doi:10.1152/ajpregu.00080.2002
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Vol. 283, Issue 3, R583-R590, September 2002

Diaphragm contractile dysfunction in MyoD gene-inactivated mice

Jessica L. Staib1, Steven J. Swoap2, and Scott K. Powers1,3

1 Departments of Exercise and Sport Sciences and 3 Physiology Center for Exercise Science, University of Florida, Gainesville, Florida 32611; and 2 Department of Biology, Williams College, Williamstown, Massachusetts 01267


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MyoD is one of four myogenic regulatory factors found exclusively in skeletal muscle. In an effort to better understand the role that MyoD plays in determining muscle contractile properties, we examined the effects of MyoD deletion on both diaphragmatic contractile properties and myosin heavy chain (MHC) phenotype. Regions of the costal diaphragm from wild-type and MyoD knockout [MyoD (-/-)] adult male BALB/c mice (n = 8/group) were removed, and in vitro diaphragmatic contractile properties were measured. Diaphragmatic contractile measurements revealed that MyoD (-/-) animals exhibited a significant (P < 0.05) downward shift in the force-frequency relationship, a decrement in maximal specific tension (Po; -33%), a decline in maximal shortening velocity (Vmax; -37%), and concomitant decrease in peak power output (-47%). Determination of MHC isoforms in the diaphragm via gel electrophoresis revealed that MyoD elimination resulted in a fast-to-slow shift (P < 0.05) in the MHC phenotype toward MHC types IIA and IIX in MyoD (-/-) animals. These data indicate that MyoD deletion results in a decrease in diaphragmatic submaximal force generation and Po, along with decrements in both Vmax and peak power output. Hence, MyoD plays an important role in determining diaphragmatic contractile properties.

myogenic regulatory factors; myosin heavy chain; maximal specific tension; maximal shortening velocity; oxidative capacity


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

SKELETAL MUSCLE is a dynamic tissue composed of a variety of muscle fiber types that generate the forces required to breathe, maintain position, and to ambulate. The diverse metabolic, contractile, and protein isoform properties of skeletal muscle allow optimal function for the variety of motor tasks required to meet its functional demands (47). Skeletal muscle fiber types are commonly classified according to the type of myosin heavy chain (MHC) proteins that are expressed (38, 48). Generally, one or more of the four major MHC genes, classified as types I, IIA, IIX, and IIB, are expressed in rodent skeletal muscle fibers (46).

The specific MHC isoform expressed within the skeletal muscle fibers contributes to the contractile characteristics of the fiber. For example, type I fibers generally have high levels of oxidative enzymes and greater endurance, whereas type IIB fibers have high levels of glycolytic enzymes and fatigue more rapidly (22). Single-fiber contractile studies also suggest that the intrinsic force generating properties of a fiber (i.e., specific tension) depend on MHC content (12). Furthermore, it is widely accepted that MHC isoforms correlate closely with the maximal shortening velocity (Vmax) of single muscle fibers (5, 14, 40, 45, 52, 53). Specifically, the presence of type IIB MHC is correlated with a faster shortening velocity, higher power output, and a higher curvature of the force-velocity relationship compared with the presence of type I MHC (5, 18, 40, 50, 52).

Four regulatory proteins found exclusively in skeletal muscle have been identified as important regulators of muscle-specific gene expression and include: MyoD, myogenin, Myf-5, and MRF4 (19, 22, 24, 53). The primary function of these myogenic regulatory factors (MRFs) appears to be involved with determination and development of muscle, including activation of muscle-specific genes. Some evidence suggests that MRFs may also play a more extended role in the maintenance of mature skeletal muscle fiber phenotypes (22, 24, 31, 33, 37, 42, 53, 56).

The continued expression of MyoD mRNA beyond myoblast formation and differentiation in adult muscle suggests that it also functions to control gene expression in the adult. In this regard, MyoD mRNA is expressed primarily in fast muscle of adult rats and mice, and this expression changes with manipulation of muscle fiber type, indicating that MyoD may be involved in regulating fiber type-specific gene expression (24, 49, 56). Furthermore, both MyoD and myogenin have been implicated as regulators of fiber phenotype as MyoD and myogenin mRNA transcripts are preferentially located in fast and slow muscle fibers, respectively (53). Hence, expression of specific MRFs in adult muscle may contribute to the diversity of individual muscle phenotypes as well as to skeletal muscle plasticity (24, 56).

On the basis of correlations between myogenic factors and MHC expression, it has been suggested that the myogenin:MyoD ratio may regulate fiber phenotype (24). Nonetheless, this issue remains controversial. Although some investigations suggest that a connection between the myogenin:MyoD ratio and muscle phenotype exists (22-24), other studies report a limited association between muscle phenotype and these myogenic factors (11, 13, 27, 35, 36, 44). For example, it has been shown that MyoD and myogenin may be directly involved in controlling fiber type-specific gene expression in response to external signals such as hypothyroidism, chronic low-level frequency stimulation, cross-reinnervation, denervation, and hindlimb suspension (9, 11, 15, 35, 60). Although these interventions promote a slow-to-fast myosin transition, no changes in myogenin expression occurred. Collectively, these results question the notion that MyoD and myogenin exclusively control the myosin phenotype of skeletal muscle.

Although MyoD has been identified as an important transcription factor in skeletal muscle and the absence of MyoD impairs expression of type IIB MHC, the impact of MyoD deletion on whole muscle contractile properties is unknown (23, 24, 31, 41, 53, 56). Therefore, this investigation determined the effects MyoD deletion on in vitro contractile performance in the mouse diaphragm. On the basis of observations provided by Seward et al. (49), we anticipated that MyoD deletion would result in a fast-to-slow shift in diaphragmatic MHC phenotype. Therefore, compared with diaphragms from wild-type (WT) animals, we hypothesized that MyoD deletion would alter diaphragmatic contractile properties resulting in a reduction in both maximal specific force production (Po) and Vmax.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and Experimental Design

To test our hypotheses, these experiments examined the in vitro diaphragmatic contractile properties of both WT (BALB/c) and MyoD knockout [MyoD (-/-)] adult (9 mo old) mice. The WT control animals were true WTs generated by separate breeders and were distinguished from MyoD (-/-) by their genetic makeup and coat colors [WT = white; MyoD (-/-) = brown]. Each animal was individually housed, fed mouse chow and water ad libitum, and maintained on a 12:12-h light-dark photoperiod for 6 wk before these experiments. Animals in both WT and MyoD (-/-) groups (n = 8/group) were killed, and their diaphragms were removed for measurement of in vitro contractile properties and selected biochemical measurements.

In Vitro Measurement of Costal Diaphragm Contractile Function

Diaphragmatic strip preparation. Anesthesia was induced by an intraperitoneal injection of pentobarbital sodium (65 mg/kg body wt). After a surgical plane of anesthesia was reached, the diaphragm was quickly excised with the ribs and central tendon attached and placed in a dissecting chamber bath containing Krebs-Hensleit solution aerated with 95% O2-5% CO2 gas. Two adjacent strips of muscle (dimensions ~8 × 4 mm) from the ventral costal diaphragm were cut parallel with the connective tissue fibers retaining a portion of rib and central tendon on each strip to enable the attachment of a clamp. After the two strips were cut, remaining costal diaphragm tissue was carefully trimmed of fat and connective tissue, rinsed free of blood, blotted dry, and then rapidly frozen in liquid nitrogen and stored at -80°C for subsequent biochemical analyses. Specifically, the left posterior costal diaphragm was retained for measurement of citrate synthase (CS) activity and the remainder of the costal diaphragm for MHC analysis.

The two muscle strips designated for contractile measurements were individually suspended vertically in two separate organ baths between two lightweight Plexiglas clamps connected to a force transducer in a jacketed tissue bath (Radnoti, Monrovia, CA) containing Krebs-Hensleit solution. The jacketed tissue bath pH was maintained at 7.4 ± 0.05, the osmolality of the bath was ~290 mosmol/kgH2O, and temperature was maintained at 37 ± 0.5°C for both isometric and isotonic measurements.

Isometric and isotonic contractile properties of each muscle strip were measured simultaneously using the two individual organ baths. Because our experiments required the measurement of a large number of contractile properties, two muscle strips were studied to reduce the metabolic load placed on each muscle. Specifically, one muscle strip was used to measure muscle isometric contractile properties (e.g., force-frequency relationship and muscle fatigue properties), whereas the second muscle strip was used to measure muscle isotonic contractile properties (e.g., muscle-shortening velocities). Isometric muscle force production was monitored using an isometric force transducer (Grass, model FT03, Quincy, MA), whereas isotonic muscle force production was monitored with a combined force and position transducer capable of monitoring both force and shortening velocity (Aurora Instruments, model 300B, Ontario, Canada). Each transducer output was amplified and differentiated by operational amplifiers subjected to analog-to-digital conversion for analysis using a computer-based data-acquisition system (LabVIEW 6i, National Instruments, Houston, TX). The force transducers were calibrated before and after each experiment using calibration weights.

Determination of optimal length-tension relationship. After a 15-min thermoequilibration period in the bath, each strip was field stimulated (modified Grass Instruments S48 stimulator) along its entire length using platinum wire electrodes with a 500-ms train of supramaximal (~120 V) monophasic pulses delivered at 300 Hz. In vitro contractile measurements began with determination of the muscle's optimal length (Lo) for isometric tetanic tension development. The muscle was adjusted to its Lo at which maximal tetanic tension was obtained by systematically adjusting the length of the muscle with a micrometer while evoking single-twitch, isometric contractions. Both isometric and isotonic contractile properties were measured at Lo.

Peak twitch tension, time-to-peak tension, and half relaxation time. Peak isometric twitch tension (Pt) was determined from a series of single pulses (2-ms duration). A computer-based algorithm was used to determine time-to-peak tension (TPT). One-half relaxation time (1/2 RT) was analyzed by the temporal pattern of force decline after a maximal isometric twitch.

Determination of the force-frequency relationship. Maximal isometric tetanic force was measured at 10, 20, 40, 80, 100, 150, 200, 250, and 300 Hz with a supramaximal stimulus train of 250-ms duration. At the completion of all contractile measurements, Lo was measured using calipers while the strips remained suspended between the two Plexiglas clamps.

Peak tetanic tension. Peak isometric tetanic contractions were produced with a supramaximal stimulus train of 250-ms duration (300 Hz). Maximal isometric tetanic tension (Po) was determined from a series of two contractions with a 2-min recovery between measurements to prevent muscle fatigue.

Determination of the force-velocity relationship. The force-velocity relationship was determined by measurement of the force and velocity of muscle shortening at 12 different isotonic loads (150-ms train at 150 Hz) over the range of ~5-80% of Po at 37°C. Force-velocity data were fit to the Hill equation with a least-squares technique (20). Vmax was determined by solving for velocity when force equals zero (10). To assess muscle function after the force-velocity protocol, two final maximal tetanic Po were determined and compared with the maximal tetanic Po before the force-velocity data collection. Peak power was determined by finding the product of force and velocity.

Rate of fatigue and recovery. Muscle fatigue was defined as the rate of decline in muscle force production. The rate of diaphragmatic fatigue development was determined at 37°C by monitoring the decrease in isometric force production over a 30-min contractile protocol. The costal diaphragm strip was stimulated by unfused tetanic contractions using a stimulus train of 30 Hz every 2 s with a train duration of 250 ms. The ratio of the period of muscle contraction to rest (duty cycle) was 12.5%. Tolerance to fatigue was assessed by the percentage of initial force maintained at the end of the 30-min protocol. Recovery from the fatigue protocol was determined by the measurement of three maximal tetanic contractions at 1, 5, and 10 min postfatigue. These times were chosen because our preliminary experiments revealed that the healthy adult mouse diaphragm regains ~80% of initial force-generating capacity within 10 min following this type of fatigue protocol (unpublished data).

Measurement of costal diaphragm cross-sectional area. After the measurements of contractile properties, the strips were removed, blotted dry, and weighed. The total muscle cross-sectional area (CSA) of the in vitro preparation was calculated by the algorithm CSA (cm2) = [wet mass (g)/fiber length (cm) × 1.056 (g/cm3)], where wet mass was the weight of the diaphragm strip, 1.056 g/cm3 was the density of the muscle, and fiber length was expressed in centimeters measured at Lo (26).

Biochemical Analysis

Tissue homogenization for enzyme assay. Each muscle sample from the costal region of the diaphragm (~10 mg) was added to 1 ml of cold 100 mM phosphate buffer (pH = 7.4) in a 3-ml glass homogenization tube. The homogenization process consisted of eight passes of the glass pestle through the homogenate using a low-speed (~50 revolution/min) motorized homogenizer (Eberbach ConTorque, Ann Arbor, MI). At the completion of homogenization, additional cold 100 mM phosphate buffer was added to further dilute the sample (1:101 wt/vol). Homogenates were then centrifuged (3°C; 700 g for 10 min) to remove the insoluble protein from the homogenate. The supernatant was removed and immediately assayed to determine CS activity and protein concentration.

Analysis of CS activity. CS activity was analyzed as a marker of muscle-oxidative capacity using the technique described by Srere (51). Briefly, CS activity was measured indirectly via the reaction of CoASH with DTNB spectrophotometrically detected by a colorimetric change at 412 nm. Each sample was assayed at 25°C in triplicate, and specific activities were normalized to protein concentrations.

Analysis of protein concentration. Protein concentrations in the muscle homogenates were determined using the technique described by Bradford (6).

MHC analysis. Myofibrillar protein was isolated from diaphragmatic samples using techniques described by Baldwin et al. (1). Separation of MHC isoform proteins was performed with the SDS-PAGE technique using a modified procedure described by Talmadge and Roy (54). A 1- to 2-µg sample of myofibrillar protein diluted in sample buffer was loaded into 0.75-mm thick minigels (8% SDS-PAGE separating; 4% SDS-PAGE stacking) and electrophoresed using the Bio-Rad mini-PROTEAN II cell apparatus for ~20 h at 4°C (Bio-Rad Laboratories, Hercules, CA). In each gel, duplicate myofibrillar samples from the diaphragm were run along with lanes containing molecular weight standards as well as control samples of soleus and plantaris muscles. Gels were stained with Rapid Coomassie Blue and subsequently analyzed (Research Products, Mt. Prospect, IL). The relative concentrations of myosin isoforms were determined by scanning the gels with a Gel Doc Chemi Doc 2000 Gel Documentation System (Bio-Rad Laboratories). Each MHC band from the video image was then digitized and analyzed twice for optical density with video-analysis software (Quantity One, BioRad Laboratories).

Statistical Analysis

The experiment was designed to test the hypothesis that loss of MyoD in skeletal muscle would alter muscle contractile properties compared with skeletal muscle with MyoD present. Comparisons between experimental groups [WT vs. MyoD (-/-)] for each dependent variable (Pt, Po, TPT, 1/2 RT, Vmax, peak power, CS, and MHC) were subjected to a Student's t-test. Force-frequency curves between groups were statistically analyzed using a Bonferroni's corrected t-test. Repeated-measures ANOVA was implemented to compare fatigue (2 × 8) and force recovery (2 × 3) indexes (group × time). Significance was established a priori at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Morphometric Characteristics

Animal body weights did not differ between the two experimental groups [WT = 28.25 ± 4.06 g; MyoD (-/-) = 30.64 ± 2.62 g; P > 0.05]. Furthermore, the CSA of the costal diaphragm strips used for contractile property measurements did not differ between groups (P > 0.05).

In Vitro Costal Diaphragm Contractile Measurements

Twitch force development and maximal specific tension. The impact of MyoD deletion on diaphragmatic twitch characteristics and maximal tetanic forces in the MyoD (-/-) and WT animals is depicted in Table 1. Compared with WT, MyoD (-/-) animals exhibited a 33% reduction in Po and a 44% decrement in Pt forces (P < 0.05). Although MyoD (-/-) animals displayed slower 1/2 RT, no differences were observed in TPT compared with their WT counterparts. Although the Pt/Po ratio tended to be lower in MyoD (-/-) animals, these differences were not significant.

                              
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  		Table 1.   Diaphragm isometric contractile characteristics

Force-frequency characteristics. Figure 1 illustrates the relationship between diaphragm force production and muscle stimulation frequency. Compared with WT, MyoD (-/-) animals exhibited a marked reduction (P < 0.05) in diaphragm tension during in vitro stimulation as illustrated by the downward shift in the force-frequency curve (Fig. 1A). Indeed, specific force production was markedly diminished in MyoD (-/-) animals compared with WT at all stimulation frequencies ranging from 10 to 300 Hz. Note, however, when expressed as percent Po, no differences existed between groups (P > 0.05; Fig. 1B).


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  		Fig. 1.   Isometric diaphragm contractile characteristics in wild-type (WT) and MyoD knockout [MyoD (-/-)] animals. A: diaphragm force-frequency characteristics expressed as a function of specific force production. B: diaphragm force-frequency characteristics expressed as a percent of maximal specific tension (% Po). Values are means ± SE; n = 8/group. *P < 0.05.

Force-velocity characteristics and peak power output. To assess the effect of MyoD deletion on isotonic contractile performance, in vitro force-velocity measurements were performed. Diaphragms from the MyoD (-/-) animals displayed a significant downward shift in the force-velocity relationship compared with WT (Fig. 2). Furthermore, as illustrated in Fig. 3A, diaphragmatic Vmax was 37% (P < 0.05) higher in WT compared with MyoD (-/-) animals. Similarly, gene deletion had deleterious effects (-47%; P < 0.05) on the diaphragm's peak power output (Fig. 3B).


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  		Fig. 2.   Diaphragm force-velocity characteristics in WT and MyoD (-/-) animals. Values are means ± SE; n = 8 (WT) or n = 7 [MyoD (-/-)].



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  		Fig. 3.   Isotonic diaphragm contractile characteristics in WT and MyoD (-/-) animals. A: maximal shortening velocity expressed as muscle lengths per second. Values are means ± SE; n = 8/group. *P < 0.05. B: peak power production expressed in mW/Ncm2. Values are means ± SE; n = 8 (WT) or n = 7 [MyoD (-/-)]. *P < 0.05.

Fatigue. Costal diaphragm endurance was evaluated during a 30-min fatigue protocol. Depicted in Fig. 4, no differences existed in the rate of diaphragmatic fatigue development between groups during the fatigue protocol, and no differences existed in the rate of recovery from fatigue (P > 0.05).


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  		Fig. 4.   Diaphragm force production during a 30-min fatigue protocol and during 10 min of recovery in WT and MyoD (-/-) animals. Values are means ± SE; n = 6 (WT) or n = 8 [MyoD (-/-)]. No differences existed (P > 0.05) at any time period.

Biochemical Characteristics

MHC profile. Portions of the costal diaphragm were used for quantification of MHC profiles. Results, summarized in Fig. 5, indicate that MyoD deletion resulted in a significant (P < 0.05) shift from MHC type IIB toward both MHC type IIA (from 30% of the total pool to 42%) and MHC type IIX (33-45%). No differences existed in the percent of type I MHC isoforms between MyoD (-/-) and WT animals.


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  		Fig. 5.   Diaphragm myosin heavy chain (MHC) phenotypes in WT and MyoD (-/-) animals. A: representative SDS-PAGE showing WT and MyoD (-/-) diaphragm MHC profiles. B: histogram comparing densitometric analyses of all MHC SDS-PAGE bands. Values are means ± SE; n = 6 (WT) or n = 8 [MyoD (-/-)]. *P < 0.05. Sol, soleus muscle.

Diaphragmatic oxidative capacity. CS activity was measured as a marker of diaphragmatic oxidative capacity in the costal diaphragm of each group. No significant (P > 0.05) differences in CS activity existed between groups [WT = 84.22 ± 2.50 µmol · g-1 · min-1; MyoD (-/-) = 91.41 ± 3.68 µmol · g-1 · min-1; P > 0.05].


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Overview of Principle Findings

These are the first experiments to examine the effect of MyoD deletion on diaphragmatic contractile function. Our results clearly demonstrate that MyoD is essential for normal diaphragmatic contractile function in adult rodents. Specifically, compared with WT controls, MyoD knockout results in dramatic decreases in diaphragmatic Po, Vmax, and maximal power output. Furthermore, MHC analysis revealed that MyoD deletion results in a shift in diaphragmatic MHC phenotype from MHC type IIB toward the slower types IIA and IIX MHC phenotypes. Interestingly, MyoD deletion did not influence the rate of diaphragmatic fatigue or CS activity. Collectively, these findings support the hypothesis that MyoD knockout results in a decrease in submaximal and maximal specific tension, Vmax, and peak power output in the diaphragm. A brief discussion of these experimental results follows.

MyoD Deletion Impairs Diaphragmatic Maximal Isometric Force Production

Although our results clearly indicate that MyoD deletion results in a large reduction in diaphragmatic specific Po, our data do not reveal the mechanism(s) responsible for this observation. Theoretically, decrements in MyoD (-/-) diaphragmatic Po could be due to 1) compromised cytoskeletal proteins associated with the sarcomere, 2) decreased myofibrillar protein concentration, 3) impaired intracellular Ca2+ handling and excitation-contraction coupling, 4) reduced force production per individual cross bridge, or 5) a combination of these factors. On the basis of previous reports, it appears that MyoD knockout could negatively impact muscle-specific Po by altering at least three of these factors.

First, MyoD (-/-) skeletal muscle has been shown to have altered sarcomere scaffolding (7, 8, 28, 29). Healthy skeletal muscle contains a complex cytoskeletal system that is essential to normal contractile function. A key cytoskeletal protein that is impaired or missing in MyoD (-/-) animals is desmin (7, 8, 28, 29). Desmin is a muscle-specific intermediate filament and is the primary structural protein contained within the extrasarcomeric cytoskeleton theorized to serve in the transmission of mechanical forces longitudinally, laterally, and to propagate mechanochemical signals throughout the myofibers (12, 25, 34, 55). Therefore, any abnormality in desmin production within muscle fibers could result in impaired function of the sarcomere (57).

Several lines of evidence indicate that deletion of MyoD results in impaired expression of desmin in skeletal muscle. First, Sabourin et al. (43) failed to detect desmin in cultures of MyoD-deleted satellite cells. However, using more precise detection methods, Yablonka-Reuveni et al. (61) later found that desmin is expressed in muscle of MyoD-deficient mice but at a lower level than WT counterparts. Moreover, work by White et al. (59) revealed that although MyoD (-/-) myoblasts are able to synthesize desmin, the expressed desmin does not appear to be appropriately organized within the muscle. As such, given that distinct morphological abnormalities have been observed in adult skeletal muscle from MyoD (-/-) mice, it seems possible that MyoD knockout could impair muscle contractile properties via the reduced expression of desmin (28-30).

A second factor, in addition to the thick filaments, the Ca2+-regulatory thin filaments may also contribute to alterations in muscle force production. Troponin and tropomyosin constitute the Ca2+-sensitive switch that regulates the contraction of striated muscle fibers (48). Although myosin generates tension during muscle contraction, actin and Ca2+-regulatory proteins regulate tension generation. Incidentally, it has been shown that MyoD (-/-) mice differ from WT littermates in the response of fast muscle fibers to Ca2+ activation that parallels differences in troponin T isoform expression (32). Thus the basis of altered Ca2+ regulation of contraction in MyoD (-/-) fibers could arise from a disruption in the normal expression of contractile and regulatory protein isoforms in these fibers. Consequently, altered troponin T expression provides a theoretical basis for the disruption in Ca2+ sensitivity during contraction observed in MyoD (-/-) fibers.

MyoD Deletion Reduced Diaphragmatic Vmax

Another important finding in the current investigation is that diaphragmatic Vmax was 37% lower in MyoD (-/-) animals compared with WT. Skeletal muscle-shortening velocity is directly proportional to the rate of cross-bridge cycling and, theoretically, cross-bridge cycling rates are altered by changing attachment rate constants, dissociation constants, or both (16, 17). Studies investigating skeletal muscle Vmax reveal that cross-bridge cycling rates could be influenced by a variety of factors including MHC composition/myosin head binding states, myosin light chain isoforms, and myosin binding protein C (3).

Regarding MHC content, many studies reported a strong correlation between Vmax, maximum actin-activated myosin ATPase activity, and MHC isoform composition (2, 4, 52). Because there is a robust correlation between MyoD and MHC IIB gene expression patterns as well as evidence from in vitro and cell culture experiments suggesting that MyoD activates the MHC IIB gene, we hypothesized that MyoD knockout would lead to a decrease in diaphragmatic Vmax (58). Our data clearly support this hypothesis. However, it is unclear if the decrease in diaphragmatic Vmax observed in the MyoD (-/-) animals was due entirely to the fast-to-slow shift in MHC phenotype. Indeed, it seems possible that a portion of the reduced diaphragmatic Vmax in the MyoD (-/-) animals was due to altered expression of other myofibrillar proteins that also exist in a number of different isoforms (e.g., myosin light chain isoforms, myosin binding protein C, etc.) (3, 40, 47, 52). To date, it is unknown if MyoD deletion alters the expression of myofibrillar proteins involved in the regulation of muscle Vmax; this is an interesting area for future research.

MyoD Deletion Does Not Alter Diaphragmatic Fatigue Properties

Paradoxically, the change in MHC phenotype from fast to slow was not accompanied by a concomitant change in either fatigue tolerance or CS activity (Figs. 4 and 5). This observation is somewhat surprising given that metabolic changes usually accompany myofibrillar changes. The dysregulation between myofibrillar phenotype and metabolic phenotype in these MyoD (-/-) mice adds to a growing body of evidence that indicates myofibrillar phenotype conversion is not always mirrored by a proportional shift in oxidative enzymes during transition toward a slower fiber type (11, 13, 27, 35, 36, 44). Although overall oxidative capacities are generally higher in MHC type I and IIA fibers compared with type IIB, individual oxidative capacities differ across a single MHC fiber population. For example, Powers et al. (39) reported that succinate dehydrogenase activity varies widely within MHC types I, IIA, and IIB fiber types in the rat costal diaphragm. The range of oxidative capacities across the MHC fiber types supports the notion that MHC shifts do not always parallel changes in fiber-oxidative enzyme activities. In fact, it is likely that the oxidative capacity of the diaphragms from both of our experimental groups is determined by the biochemical adaptation to functional demands placed on the muscle and is not simply determined by intrinsic myogenic factors alone (21).

The lack of change in the metabolic phenotype was also unexpected given that the ratio of myogenin:MyoD has been shown to be important in regulating metabolic phenotype in some experimental models (24). Others showed that overexpression of myogenin can lead to an increase in oxidative capacity, and therefore, likely induce fatigue resistance in skeletal muscle of mice (22-24). Consequently, we would have expected the oxidative capacity of these MyoD (-/-) diaphragms, where the ratio of MyoD:myogenin is zero, to increase dramatically. However, both CS activity and measurements of fatigue tolerance suggest no alteration in the metabolic profile of these muscles (Fig. 4). Instead, it is more likely that a delicate balance between repression and activation of the MRFs prevails and an equilibrium is maintained through a highly regulated integrative network of factors rather than a single, direct control (13).

Conclusions

Our results suggest that MyoD is required for the normal function of adult skeletal muscle, and mice missing this gene display distinct muscle phenotypes with impaired contractile characteristics. In the MyoD (-/-) mouse diaphragm, it seems likely that several factors could contribute to the impaired diaphragmatic Po, Vmax, and peak power output. Specifically, deletion of MyoD expression could result in reduced expression of desmin as well as the elimination of the MHC IIB phenotype as both of these proteins have profound influence on skeletal muscle contractile properties. Additional studies are required to elucidate the specific downstream proteins regulated by MyoD and the role that these proteins play in the regulation of muscle contractile function.


    ACKNOWLEDGEMENTS

The authors thank Dr. M. Rudnicki for engineering and donating the transgenic animals necessary to carry out these experiments.


    FOOTNOTES

Address for reprint requests and other correspondence: S. K. Powers, P. O. Box 118206, Center for Exercise Science, Univ. of Florida, Gainesville, FL 32611 (E-mail: spowers{at}hhp.ufl.edu).

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.

May 23, 2002;10.1152/ajpregu.00080.2002

Received 9 February 2002; accepted in final form 17 May 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Baldwin, KM, Cooke DA, and Cheadle WG. Time course adaptations in cardiac and skeletal muscle to different running programs. J Appl Physiol 42: 267-272, 1977.

2.   Barany, M. ATPase activity of myosin correlated with speed of muscle shortening. J Gen Physiol 50: 197-218, 1967.

3.   Bottinelli, R. Functional heterogeneity of mammalian single muscle fibres: do myosin isoforms tell the whole story? Pflügers Arch 443: 6-17, 2001.

4.   Bottinelli, R, Betto R, Schiaffino S, and Reggiani C. Unloaded shortening velocity and myosin heavy chain and alkali light chain isoform composition in rat skeletal muscle fibres. J Physiol 478: 341-349, 1994.

5.   Bottinelli, R, Schiaffino S, and Reggiani C. Force-velocity relations and myosin heavy chain isoform compositions of skinned fibres from rat skeletal muscle. J Physiol 437: 655-672, 1991.

6.   Bradford, MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254, 1976.

7.   Capetanaki, Y, and Milner DJ. Desmin cytoskeleton in muscle integrity and function. Subcell Biochem 31: 463-495, 1998.

8.   Capetanaki, Y, Milner DJ, and Weitzer G. Desmin in muscle formation and maintenance: knockouts and consequences. Cell Struct Funct 22: 103-116, 1997.

9.   Carlsen, H, and Gundersen K. Helix-loop-helix transcription factors in electrically active and inactive skeletal muscles. Muscle Nerve 23: 1374-1380, 2000.

10.   Chow, JW, and Darling WG. The maximum shortening velocity of muscle should be scaled with activation. J Appl Physiol 86: 1025-1031, 1999.

11.   Czernik, PJ, Peterson CA, and Hurlburt BK. Preferential binding of MyoD-E12 versus myogenin-E12 to the murine sarcoma virus enhancer in vitro. J Biol Chem 271: 9141-9149, 1996.

12.   Danowski, BA, Imanaka-Yoshida K, Sanger JM, and Sanger JW. Costameres are sites of force transmission to the substratum in adult rat cardiomyocytes. J Cell Biol 118: 1411-1420, 1992.

13.   Dupont-Versteegden, EE, Houle JD, Gurley CM, and Peterson CA. Early changes in muscle fiber size and gene expression in response to spinal cord transection and exercise. Am J Physiol Cell Physiol 275: C1124-C1133, 1998.

14.   Eddinger, TJ, and Moss RL. Mechanical properties of skinned single fibers of identified types from rat diaphragm. Am J Physiol Cell Physiol 253: C210-C218, 1987.

15.   Eftimie, R, Brenner HR, and Buonanno A. Myogenin and MyoD join a family of skeletal muscle genes regulated by electrical activity. Proc Natl Acad Sci USA 88: 1349-1353, 1991.

16.   Geiger, PC, Cody MJ, Macken RL, and Sieck GC. Maximum specific force depends on myosin heavy chain content in rat diaphragm muscle fibers. J Appl Physiol 89: 695-703, 2000.

17.   Geiger, PC, Cody MJ, and Sieck GC. Force-calcium relationship depends on myosin heavy chain and troponin isoforms in rat diaphragm muscle fibers. J Appl Physiol 87: 1894-1900, 1999.

18.   Greaser, ML, Moss RL, and Reiser PJ. Variations in contractile properties of rabbit single muscle fibres in relation to troponin T isoforms and myosin light chains. J Physiol 406: 85-98, 1988.

19.   Heilig, A, and Pette D. Changes in transcriptional activity of chronically stimulated fast twitch muscle. FEBS Lett 151: 211-214, 1983.

20.   Hill, AV. The heat of shortening and dynamic constants of muscle. Proc R Soc Lond B Biol Sci 126: 136-195, 1938.

21.   Hodge, K, Powers SK, Coombes J, Fletcher L, Demirel HA, Dodd SL, and Martin D. Bioenergetic characteristics of the costal and crural diaphragm in mammals. Respir Physiol 109: 149-154, 1997.

22.   Hughes, SM, Chi MM, Lowry OH, and Gundersen K. Myogenin induces a shift of enzyme activity from glycolytic to oxidative metabolism in muscles of transgenic mice. J Cell Biol 145: 633-642, 1999.

23.   Hughes, SM, Koishi K, Rudnicki M, and Maggs AM. MyoD protein is differentially accumulated in fast and slow skeletal muscle fibres and required for normal fibre type balance in rodents. Mech Dev 61: 151-163, 1997.

24.   Hughes, SM, Taylor JM, Tapscott SJ, Gurley CM, Carter WJ, and Peterson CA. Selective accumulation of MyoD and myogenin mRNAs in fast and slow adult skeletal muscle is controlled by innervation and hormones. Development 118: 1137-1147, 1993.

25.   Huijing, PA. Muscle, the motor of movement: properties in function, experiment and modelling. J Electromyogr Kinesiol 8: 61-77, 1998.

26.   Kandarian, SC, and White TP. Mechanical deficit persists during long-term muscle hypertrophy. J Appl Physiol 69: 861-867, 1990.

27.   Kraus, B, and Pette D. Quantification of MyoD, myogenin, MRF4 and Id-1 by reverse-transcriptase polymerase chain reaction in rat muscles---effects of hypothyroidism and chronic low-frequency stimulation. Eur J Biochem 247: 98-106, 1997.

28.   Lescaudron, L, Creuzet SE, Li Z, Paulin D, and Fontaine-Perus J. Desmin-lacZ transgene expression and regeneration within skeletal muscle transplants. J Muscle Res Cell Motil 18: 631-641, 1997.

29.   Lescaudron, L, Li Z, Paulin D, and Fontaine-Perus J. Desmin-lacZ transgene, a marker of regenerating skeletal muscle. Neuromuscul Disord 3: 419-422, 1993.

30.   Li, Z, Mericskay M, Agbulut O, Butler-Browne G, Carlsson L, Thornell LE, Babinet C, and Paulin D. Desmin is essential for the tensile strength and integrity of myofibrils but not for myogenic commitment, differentiation, and fusion of skeletal muscle. J Cell Biol 139: 129-144, 1997.

31.   Megeney, LA, Kablar B, Garrett K, Anderson JE, and Rudnicki MA. MyoD is required for myogenic stem cell function in adult skeletal muscle. Genes Dev 10: 1173-1183, 1996.

32.   Metzger, JM, Rudnicki MA, and Westfall MV. Altered Ca2+ sensitivity of tension in single skeletal muscle fibres from MyoD gene-inactivated mice. J Physiol 485: 447-453, 1995.

33.   Molkentin, JD, and Olson EN. Defining the regulatory networks for muscle development. Curr Opin Genet Dev 6: 445-453, 1996.

34.   Monti, RJ, Roy RR, Hodgson JA, and Edgerton VR. Transmission of forces within mammalian skeletal muscles. J Biomech 32: 371-380, 1999.

35.   Mozdziak, PE, Greaser ML, and Schultz E. Myogenin, MyoD, and myosin expression after pharmacologically and surgically induced hypertrophy. J Appl Physiol 84: 1359-1364, 1998.

36.   Mozdziak, PE, Greaser ML, and Schultz E. Myogenin, MyoD, and myosin heavy chain isoform expression following hindlimb suspension. Aviat Space Environ Med 70: 511-516, 1999.

37.   Olson, EN. Regulation of muscle transcription by the MyoD family. The heart of the matter. Circ Res 72: 1-6, 1993.

38.   Pette, D, and Staron RS. Cellular and molecular diversities of mammalian skeletal muscle fibers. Rev Physiol Biochem Pharmacol 116: 1-76, 1990.

39.   Powers, SK, Criswell D, Lieu FK, Dodd S, and Silverman H. Exercise-induced cellular alterations in the diaphragm. Am J Physiol Regul Integr Comp Physiol 263: R1093-R1098, 1992.

40.   Reiser, PJ, Moss RL, Giulian GG, and Greaser ML. Shortening velocity in single fibers from adult rabbit soleus muscles is correlated with myosin heavy chain composition. J Biol Chem 260: 9077-9080, 1985.

41.   Rudnicki, MA, Braun T, Hinuma S, and Jaenisch R. Inactivation of MyoD in mice leads to up-regulation of the myogenic HLH gene Myf-5 and results in apparently normal muscle development. Cell 71: 383-390, 1992.

42.   Rudnicki, MA, and Jaenisch R. The MyoD family of transcription factors and skeletal myogenesis. Bioessays 17: 203-209, 1995.

43.   Sabourin, LA, Girgis-Gabardo A, Seale P, Asakura A, and Rudnicki MA. Reduced differentiation potential of primary MyoD-/- myogenic cells derived from adult skeletal muscle. J Cell Biol 144: 631-643, 1999.

44.   Sarbassov, DD, Stefanova R, Grigoriev VG, and Peterson CA. Role of insulin-like growth factors and myogenin in the altered program of proliferation and differentiation in the NFB4 mutant muscle cell line. Proc Natl Acad Sci USA 92: 10874-10878, 1995.

45.   Schiaffino, S, Ausoni S, Gorza L, Saggin L, Gundersen K, and Lomo T. Myosin heavy chain isoforms and velocity of shortening of type 2 skeletal muscle fibres. Acta Physiol Scand 134: 575-576, 1988.

46.   Schiaffino, S, Gorza L, Sartore S, Saggin L, Ausoni S, Vianello M, Gundersen K, and Lomo T. Three myosin heavy chain isoforms in type 2 skeletal muscle fibres. J Muscle Res Cell Motil 10: 197-205, 1989.

47.   Schiaffino, S, and Reggiani C. Myosin isoforms in mammalian skeletal muscle. J Appl Physiol 77: 493-501, 1994.

48.   Schiaffino, S, and Reggiani C. Molecular diversity of myofibrillar proteins: gene regulation and functional significance. Physiol Rev 76: 371-423, 1996.

49.   Seward, DJ, Haney JC, Rudnicki MA, and Swoap SJ. bHLH transcription factor MyoD affects myosin heavy chain expression pattern in a muscle-specific fashion. Am J Physiol Cell Physiol 280: C408-C413, 2001.

50.   Sieck, GC, Han YS, Prakash YS, and Jones KA. Cross-bridge cycling kinetics, actomyosin ATPase activity and myosin heavy chain isoforms in skeletal and smooth respiratory muscles. Comp Biochem Physiol B Biochem Mol Biol 119: 435-450, 1998.

51.   Srere, PA. Citrate synthase. Methods Enzymol 13: 3-5, 1969.

52.   Sweeney, HL, Kushmerick MJ, Mabuchi K, Sreter FA, and Gergely J. Myosin alkali light chain and heavy chain variations correlate with altered shortening velocity of isolated skeletal muscle fibers. J Biol Chem 263: 9034-9039, 1988.

53.   Talmadge, RJ. Myosin heavy chain isoform expression following reduced neuromuscular activity: potential regulatory mechanisms. Muscle Nerve 23: 661-679, 2000.

54.   Talmadge, RJ, and Roy RR. Electrophoretic separation of rat skeletal muscle myosin heavy-chain isoforms. J Appl Physiol 75: 2337-2340, 1993.

55.   Tidball, JG, and Law DJ. Dystrophin is required for normal thin filament-membrane associations at myotendinous junctions. Am J Pathol 138: 17-21, 1991.

56.   Voytik, SL, Przyborski M, Badylak SF, and Konieczny SF. Differential expression of muscle regulatory factor genes in normal and denervated adult rat hindlimb muscles. Dev Dyn 198: 214-224, 1993.

57.   Warrick, HM, and Spudich JA. Myosin structure and function in cell motility. Annu Rev Cell Biol 3: 379-421, 1987.

58.   Wheeler, MT, Snyder EC, Patterson MN, and Swoap SJ. An E-box within the MHC IIB gene is bound by MyoD and is required for gene expression in fast muscle. Am J Physiol Cell Physiol 276: C1069-C1078, 1999.

59.   White, JD, Scaffidi A, Davies M, McGeachie J, Rudnicki MA, and Grounds MD. Myotube formation is delayed but not prevented in MyoD-deficient skeletal muscle: studies in regenerating whole muscle grafts of adult mice. J Histochem Cytochem 48: 1531-1544, 2000.

60.   Witzemann, V, and Sakmann B. Differential regulation of MyoD and myogenin mRNA levels by nerve induced muscle activity. FEBS Lett 282: 259-264, 1991.

61.   Yablonka-Reuveni, Z, Rudnicki MA, Rivera AJ, Primig M, Anderson JE, and Natanson P. The transition from proliferation to differentiation is delayed in satellite cells from mice lacking MyoD. Dev Biol 210: 440-455, 1999.

Am J Physiol Regul Integr Comp Physiol 283(3):R583-R590
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