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Am J Physiol Cell Physiol 282: C113-C124, 2002. First published September 21, 2001; doi:10.1152/ajpcell.00294.2001
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Vol. 282, Issue 1, C113-C124, January 2002

Coregulation of fast contractile protein transgene and glycolytic enzyme expression in mouse skeletal muscle

Patricia L. Hallauer and Kenneth E. M. Hastings

Montreal Neurological Institute and Biology Department, McGill University, Montreal, Quebec, Canada H3A 2B4


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Little is known of the gene regulatory mechanisms that coordinate the contractile and metabolic specializations of skeletal muscle fibers. Here we report a novel connection between fast isoform contractile protein transgene and glycolytic enzyme expression. In quantitative histochemical studies of transgenic mouse muscle fibers, we found extensive coregulation of the glycolytic enzyme glycerol-3-phosphate dehydrogenase (GPDH) and transgene constructs based on the fast skeletal muscle troponin I (TnIfast) gene. In addition to a common IIB > IIX > IIA fiber type pattern, TnIfast transgenes and GPDH showed correlated fiber-to-fiber variation within each fast fiber type, concerted emergence of high-level expression during early postnatal muscle maturation, and parallel responses to muscle under- or overloading. Regulatory information for GPDH-coregulated expression is carried by the TnIfast first-intron enhancer (IRE). These results identify an unexpected contractile/metabolic gene regulatory link that is amenable to further molecular characterization. They also raise the possibility that the equal expression in all fast fiber types observed for the endogenous TnIfast gene may be driven by different metabolically coordinated mechanisms in glycolytic (IIB) vs. oxidative (IIA) fast fibers.

muscle gene regulation; metabolic gene expression; muscle fiber phenotype


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EACH DIFFERENTIATED CELL PHENOTYPE involves multiple, functionally coordinated, cellular specializations. Skeletal muscle provides striking examples of coordinated specializations in terms of contractile function and energy metabolism. Skeletal muscle fibers contain high levels both of contractile proteins and of ATP-producing metabolic enzymes, which together make possible the extremely high energy flux of actively exercising muscle (22, 36). Moreover, muscle fibers are biochemically and physiologically specialized, with different fibers expressing functionally distinct contractile protein isoforms (45, 54) and different relative amounts of oxidative vs. glycolytic metabolic enzymes (21, 43, 44, 51). These specialized features are related to, and are at least partly determined by, motor unit usage patterns and are thought to represent coordinated optimizations of muscle fiber contractile and metabolic properties for differing usage profiles (6, 47, 54). Reporter transgene studies indicate that muscle fiber contractile and metabolic specializations are based largely on gene transcriptional regulation (1, 13, 19, 27, 30, 50, 52), but almost nothing is known of the specific mechanisms that coordinate contractile and metabolic gene expression.

The majority of mammalian muscle fibers fall into one of several distinct contractile/metabolic phenotypes termed fiber types. Slow (type I) fibers express "slow" isoforms of myosin heavy chain and many other contractile proteins (45, 54), high or intermediate levels of oxidative enzymes, and lower levels of glycolytic enzymes than are found in fast fibers (21, 43, 44, 51). Several distinct fast fiber types [IIA, IIB, IIX (also called IID)] exist, which express different fast myosin heavy chain isoforms (45, 54) and have glycolytic enzyme levels that vary from high (in IIB fibers) to low (in IIA fibers) and inversely varying oxidative enzyme levels (21, 43, 51). Thus muscle fibers can be ordered in a metabolic spectrum, a continuum of increasing glycolytic enzyme levels, on which the fiber types fall in a characteristic order: I-IIA-IIX-IIB (51). This order also appears to correspond to a motor unit usage spectrum, with frequency of use decreasing from left to right (20).

Muscle fiber phenotype is plastic. Perturbations of neuromuscular activity patterns or muscle mechanical loading can result in fiber type transformations that involve both contractile protein isoform switches and changes in metabolic enzyme levels (4, 35, 46, 47). In general, such transformations occur in steps between adjacent positions on the I-IIA-IIX-IIB fiber type spectrum. Rightward transformations caused, e.g., by reducing neuromuscular activity or mechanical load, elevate glycolytic enzyme levels (34, 35, 60). Conversely, leftward transformations, e.g., those caused by increasing activity or load, reduce glycolytic enzyme levels (4, 14, 49, 61).

The existence of a characteristic order of the fiber types on the glycolytic spectrum, and the occurrence of coordinated contractile and metabolic changes in fiber type transformation experiments, imply the existence of regulatory mechanisms that integrate metabolic and contractile protein gene expression.

Current knowledge of adult muscle gene regulation mostly concerns mechanisms that operate at the left end of the I-IIA-IIX-IIB fiber type spectrum, in highly active motor units expressing slow contractile protein isoforms and/or high levels of oxidative, and low levels of glycolytic, enzymes (7, 9, 12, 15, 23, 38, 42, 66, 67). Among several activity-regulated factors, at least one, the Ca2+-dependent protein phosphatase calcineurin, plays a role in the expression both of slow isoform contractile protein genes (9, 12, 66) and of a gene related to oxidative metabolism, myoglobin (9), and hence represents a candidate contractile/metabolic coordination mechanism.

Much less is known of regulatory mechanisms at work at the right end of the I-IIA-IIX-IIB fiber type spectrum, which is characterized by less frequent contractile activity, high-level glycolytic gene expression, and fast isoform contractile protein gene expression (6, 20). Several fast isoform contractile protein genes (13, 19, 28, 65) and glycolytic enzyme genes (5, 33, 52, 56) have been studied, but signaling pathways controlling fiber type-related differential expression have yet to be delineated. In the genes encoding creatine kinase and the glycolytic enzyme aldolase A, DNA cis-elements have been identified that differentially affect expression in subsets of IIB fiber-enriched muscles (55, 56, 58). However, the relationship of these elements to fiber type-specific gene expression or contractile activity levels is unknown. An E-box has been implicated in the fiber type-specific expression of the IIB myosin heavy chain gene (65), but it is also known that E-boxes are not involved in high-level expression of the creatine kinase or aldolase A promoters in fast glycolytic (i.e., IIB) fibers (55, 58). Thus research to date has not identified a specific molecular framework for investigating the mechanisms that coordinate fast contractile protein and glycolytic enzyme gene regulation. In the present report we describe a novel and experimentally accessible coordination between fast isoform contractile protein transgene and glycolytic enzyme expression. These findings emerged from our studies of the gene encoding the fast skeletal muscle isoform of the contractile regulatory protein troponin I (TnIfast).

We have previously reported an unexplained behavior of TnIfast gene constructs in transgenic mice, i.e., differential expression among the fast fiber types in a IIB > IIX > IIA pattern (19). This pattern was a departure from the behavior of the endogenous TnIfast gene, which is expressed at equal levels in all fast fiber types. A similar, unexplained, IIB > IIX > IIA pattern has been observed for a variety of other contractile protein transgene constructs (13, 16, 27). In an attempt to relate the IIB > IIX > IIA pattern to biologically significant differences among the fast fiber types, we have analyzed the relationship between TnIfast transgene expression and metabolic enzyme levels, using glycerol-3-phosphate dehydrogenase (GPDH) and succinate dehydrogenase (SDH) as indicators of glycolytic and oxidative expression. Our results reveal a strong link between TnIfast transgene and GPDH expression and show that the common IIB > IIX > IIA expression profile of these genes is part of a remarkably broad pattern of coregulation. Other aspects of coregulation include covariation in expression levels among individual fibers within each of the fast fiber types, parallel emergence of differential expression during early postnatal muscle maturation, and parallel responses to experimental muscle under- or overloading. Our results also show that the IRE, a well-characterized muscle-specific enhancer in the TnIfast gene's first intron, carries the regulatory information for GPDH-coregulated expression.

The extensive similarities between IRE-driven TnIfast transgene expression and GPDH expression imply a novel and intimate gene regulatory relationship. By documenting quantitative coregulation, and by localizing the relevant regulatory information to a small DNA fragment, these findings greatly consolidate the notion of a coordinating mechanism between fast isoform contractile protein genes and glycolytic enzyme genes and open a way to its further characterization. In addition, our findings provide new insight into the previously unexplained IIB > IIX > IIA expression pattern shown by TnIfast and other muscle transgenes, which is now seen to reflect coordinated expression with glycolytic enzyme genes. That this aspect is not evident in the behavior of the endogenous TnIfast gene suggests an unexpected heterogeneity of mechanisms driving high-level gene expression of the TnIfast gene, and likely other genes as well, in the different metabolically specialized fast fiber types.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DNA constructs and transgenic mice. Production of TnILacZ1 transgenic mice has been described previously (19). The TnILacZ1 construct contains 530 bp of upstream DNA, the first exon, first intron, and the first (untranslated) part of the second exon of the quail TnIfast gene, linked to the Escherichia coli LacZ beta -galactosidase (beta -gal) gene and SV40 splicing and polyadenylation sequences. Two independent transgenic lines, 29 and 36, were used. Unless indicated otherwise, data presented are from line 29. We also have produced 3xIRE-tkLacZ transgenic mice (unpublished work). 3xIRE-tkLacZ contains three copies of a 148-bp IRE-containing DNA fragment (68), two upstream and one downstream, of a heterologous reporter gene consisting of the -105 to +57 herpes virus thymidine kinase (tk) promoter (37) linked to the same LacZ/SV40 DNA elements used in TnILacZ1. Two independent lines, 19 and 69, were used. In all cases hemizygous male or female animals >6 wk old were used, unless otherwise indicated.

Histochemistry. Histochemistry was done on serial cross-sections of frozen muscles that were collected on glass coverslips. Sections were of 10-µm or 8-µm thickness for adult or perinatal/juvenile muscle, respectively. beta -Gal histochemistry using X-Gal was as previously described (19), with room temperature incubation for 20 h. GPDH and SDH histochemical reactions were at 37°C for 30 min or, in some cases, 10 min (see below). GPDH reaction conditions (0.1 M sodium phosphate buffer, pH 7.4, 1 mM sodium azide, 0.8 mM phenazine methosulfate, 1.2 mM nitro blue tetrazolium, and 9.3 mM alpha -glycerol phosphate) were based on Dunn and Michel (14). Both cytoplasmic (EC 1.1.1.8) and mitochondrial (EC 1.1.99.5) GPDH enzymes of the glycerol phosphate shuttle may contribute to the activity detected (51). SDH reaction conditions (0.1 M Tris · HCl, pH 7.5, 1 mM sodium azide, 1 mM phenazine methosulfate, 1.5 mM nitro blue tetrazolium, 5 mM EDTA, and 48 mM disodium succinate) were based on the work of Blanco et al. (3) and Nolte and Pette (41). After histochemical reactions, tissue sections were rinsed extensively with water and mounted for microscopy with polyvinyl alcohol resin (Immu-Mount, Shandon).

For each analysis of histochemical stain optical densities, 100-200 contiguous fibers (except where otherwise indicated) were measured microscopically using a digital camera and JAVA software (Jandel) and a standard series of neutral density filters for optical density calibration (19). There was negligible nonspecific background staining for beta -gal, but not for GPDH and SDH. To determine GPDH and SDH staining backgrounds, tissue blanks were prepared by incubating sections in parallel in reagent mixes lacking the specific enzyme substrates, and these backgrounds were subtracted on a fiber-by-fiber basis. Because the same field of fibers in the same orientation was analyzed in substrate-containing and substrate-lacking reactions, this subtraction eliminates both nonspecific background staining and any variation that might be due to slight departures from even illumination across the field. The background subtract was particularly important for GPDH because the tissue nonspecific dehydrogenase backgrounds were higher in more-glycolytic fibers.

We routinely used 30-min reactions for GPDH because this gave stronger signals than shorter reactions, without distorting relative staining intensities. There was a linear relationship between 10- and 30-min-staining optical densities of individual fibers across the whole range of staining intensities observed. Although reaction rates declined during the assay (the 30-min optical densities were 2 rather than 3 times higher than the 10-min optical densities), the relative optical densities of any two fibers were the same in the 10- and 30-min assays, indicating that this reaction falloff was similar in all fibers.

Fiber types were determined by immunohistochemical analysis of serial sections using monoclonal antibodies specific for IIB [BF-F3 (53)], IIA [SC-71 (53)], and type I [A4.840 (64)] myosin heavy chains. (See Ref. 19 for micrographs showing the relationship between TnILacZ1 beta -gal expression and myosin fiber type immunohistochemistry.) Type IIX fibers were identified on the basis of their lack of reaction with these three antibodies. A variable percentage of muscle fibers are hybrid or intermediate types that contain more than one myosin isoform (47). Any IIB/IIX or IIX/IIA intermediate fibers were counted as IIB and IIA fibers, respectively, on the basis of their reaction with the IIB- and IIA-specific antibodies, so the IIX fiber type excludes intermediate types. Hybrid fibers reacting with both type IIA and type I myosin antibodies were typed as IIA fibers. In the case of perinatal muscle, sections also were incubated with antibody N3.36 against the MHCperi/neo isoform of myosin heavy chain (10), and fibers that did not react with the IIB-, IIA-, or I-specific antibodies but that did react with N3.36 were typed as peri/neonatal fast fibers.

Under- and overload experiments. Hindlimb muscles, including the soleus, were underloaded by hindlimb suspension carried out as described by McCarthy et al. (34) for 14 days. Plantaris muscle overload experiments, also of 14 days in duration, were based on bilateral ablation of synergist muscles, the soleus and gastrocnemius (61, 62) as follows. In chloral hydrate-anesthetized mice, the biceps femoris muscle was sectioned along its lateral aspect, following the junction of the peroneus longus muscle and the lateral head of the gastrocnemius muscle. These latter were separated to reveal the soleus muscle, which was removed from tendon to tendon. At the ankle, tendons attaching the lateral and medial heads of the gastrocnemius muscle were isolated from the plantaris tendon, the tibial and plantar nerves, and the saphenous blood vessels. Both gastrocnemius tendons were sectioned, and the entire muscle was removed at the knee. Within each under- or overload experiment, all animals, control and treated, were same-age siblings, and all sections were coprocessed for GPDH or beta -gal histochemistry so as to be directly comparable. Care and use of animals followed guidelines of the Canadian Council for Animal Care under protocols approved by the McGill University Animal Care Committee. Statistical analysis was performed at the VassarStats website (http://faculty.vassar.edu/~lowry/VassarStats.html#menu) created and maintained by Richard Lowry, Department of Psychology, Vassar College, Poughkeepsie, NY.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TnILacZ1 beta -gal expression levels correlate with glycolytic enzyme levels in adult muscle. TnILacZ1 is a TnIfast/beta -gal reporter construct that shows a IIB > IIX > IIA > I fiber type differential expression pattern in transgenic mouse skeletal muscle (19). We investigated the relationship between TnILacZ1 transgene expression and metabolic gene expression by histochemical analysis of serial cryostat sections of hindlimb muscles for TnILacZ1-encoded beta -gal and for marker enzymes of glycolytic or oxidative metabolism. We chose GPDH as the marker glycolytic enzyme because a histochemical stain has been developed and because GPDH levels correlate well with other glycolytic enzymes in skeletal muscle (45). We used SDH as a marker for oxidative enzyme expression levels (14, 51).

Low-magnification views of hindlimb sural muscle sections showed a markedly similar distribution of beta -gal (Fig. 1D) and GPDH (Fig. 1B) staining in different muscles and muscle regions. Staining for both enzymes was strong in most areas of the medial and lateral gastrocnemius muscles (MG and LG in Fig. 1C), very weak in the soleus muscle (S), and intermediate in the plantaris muscle (P) and in the more central portions of the gastrocemius muscle, especially the medial gastrocnemius complex zone (dashed line in Fig. 1C) and the plantaris-adjacent lateral gastrocnemius muscle (PLG). SDH (Fig. 1A) showed a grossly complementary distribution, with high levels in the soleus, plantaris, and adjacent regions of the gastrocnemius, and low levels in the peripheral gastrocnemius. Examination at higher magnification showed a wide range of beta -gal and GPDH expression levels in individual fibers and a striking correspondence of the distributions of the two enzymes (Fig. 1, compare E and F).


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  		Fig. 1.   Histochemical analysis of beta -galactosidase (beta -gal), glycerol-3-phosphate dehydrogenase (GPDH), and succinate dehydrogenase (SDH) expression in TnILacZ1 transgenic mouse hindlimb sural muscles. A-D: low-magnification views of serial tissue sections stained for SDH (A), GPDH (B), and beta -gal (D). Individual muscles and muscle regions are identified in C. MG, medial gastrocnemius; LG, lateral gastrocnemius; P, plantaris; PLG, plantaris-adjacent lateral gastrocnemius; S, soleus. The dashed line outlines the complex zone of the MG. E and F: higher-magnification views of serial sections of the plantaris muscle stained for GPDH (E) and beta -gal (F). The GPDH and TnILacZ1 transgene beta -gal expression patterns are very similar at both low (B and D) and high (E and F) magnification.

For quantitative comparison, optical densities of individual muscle fibers were measured by microdensitometry following histochemical staining of serial sections. In the case of GPDH and SDH, where nonspecific tissue background staining is not negligible, backgrounds, determined by incubation of serial sections in solutions lacking the specific enzyme substrate, were subtracted on a fiber-by-fiber basis. Background-subtracted end-point optical densities may not be strictly proportional to enzyme levels; however, this is immaterial for our purposes, and the only quantitative assumption we make is that a greater net optical density represents more enzyme. Moreover, we found that similar relative net optical densities among muscle fibers were obtained with both standard (30 min) and shorter (10 min) GPDH and SDH reaction times (see MATERIALS AND METHODS). Under such conditions, standard end-point net optical density measures will rank fibers quantitatively in the same order as would be obtained by more laborious directly proportional estimates of enzyme levels based on kinetic analysis of stain development (14, 51). Thus our end-point staining measures provide an efficient means to assess overall quantitative trends.

The scatterplots in Fig. 2 show the relationship between TnILacZ1 beta -gal and GPDH in individual fibers in several muscles. GPDH levels formed a continuum when observed over the whole muscle fiber population, in agreement with previous studies (51). TnILacZ1 beta -gal levels likewise formed a smoothly varying continuum. It is evident from the scatterplot data that beta -gal and GPDH expression levels were strongly correlated (r > 0.85, P < 0.0001; see Fig. 2 legend), as would be expected from the great similarity of their staining patterns seen in Fig. 1. Figure 2 also shows that the different fiber types occupied different but partly overlapping regions of the beta -gal/GPDH distribution. The fiber type expression profiles of beta -gal, GPDH, and SDH are presented as histograms in Fig. 3. Fiber types were determined by myosin heavy chain immunohistochemistry (IIB/IIX hybrids were typed as IIB, and IIX/IIA and IIA/I hybrids were typed as IIA). TnILacZ1 beta -gal expression showed the IIB > IIX > IIA > I pattern we have previously noted (19). GPDH levels also showed a IIB > IIX > IIA > I pattern, while SDH showed a IIA > I ~ IIX > IIB pattern, consistent with previous studies of these enzymes in rat muscle (14, 51).


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  		Fig. 2.   Correlated expression of GPDH and TnILacZ1 beta -gal in individual muscle fibers. beta -Gal and GPDH histochemical staining optical densities of individual muscle fibers of adult TnILacZ1 transgenic mouse muscle were measured by microdensitometry on serial sections. A: extensor digitorum longus (EDL) muscle. B: tibialis anterior (TA) muscle. C: plantaris muscle. In each muscle a patch of 100-200 contiguous fibers was analyzed. Overall correlation coefficients were 0.954, 0.868, and 0.930 for A-C, respectively, and these were all highly significant (P < 0.0001). Fiber types, determined by myosin immunohistochemistry on additional serial sections, are indicated by symbols. Correlation coefficients within each of the fast fiber types are reported in Table 1, along with other similar data. Significant (P < 0.01) within-fiber-type correlations were found for IIB, IIX, and IIA in A and for IIX and IIA in B and C.



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  		Fig. 3.   Fiber type profile of beta -gal, GPDH, and SDH expression in TnILacZ1 transgenic mouse plantaris muscle. Histochemical staining optical densities of the same patch of 147 contiguous fibers were measured for each enzyme by microdensitometry on serial sections, and fiber types were determined on additional serial sections by myosin immunohistochemistry. Histogram bars show means and SD. Both beta -gal and GPDH, but not SDH, show IIB > IIX > IIA differential expression among fast fibers.

Although GPDH and TnILacZ1 beta -gal showed a common IIB > IIX > IIA profile, these enzymes were more closely correlated with each other than either of them was with myosin-based fiber type. Fiber-to-fiber variations in TnILacZ1 beta -gal and GPDH levels were correlated not only between fiber types but also within any given fast fiber type. Within-fiber-type correlations are evident in Fig. 2, particularly in the IIX fiber type, and Table 1 summarizes these and other similar data for several different muscles in two TnILacZ1 transgenic mouse lines. In 39 separate analyses of within-fiber-type variation there was a significant (P < 0.05) positive correlation between beta -gal and GPDH levels in 27 cases (P values with asterisks in Table 1), including 13/13 type IIA and 10/13 type IIX analyses. Moreover, although only relatively few of the IIB correlations achieved statistical significance at the P <=  0.05 confidence level, a positive correlation nonetheless appears to be present, because the correlation coefficient r was positive in 11/13 cases, significantly different (chi 2 = 6.24, P < 0.05) from the equal frequencies of positive and negative values that would be expected in the absence of a correlation. Together, these data clearly show a positive within-fiber-type correlation between TnILacZ1 beta -gal expression and GPDH expression.

                              
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  		Table 1.   Within-fiber-type correlation of GPDH and TnIfast transgene beta -gal expression

Our previous work has shown that, within a given fiber type, there are muscle-to-muscle differences in TnILacZ1 beta -gal mean expression levels. For example, expression is weaker in IIA fibers in the soleus muscle than in IIA fibers in the plantaris or gastrocnemius muscles (19). Figure 4 shows that GPDH likewise showed reduced expression in soleus IIA fibers compared with plantaris IIA fibers and that SDH was expressed at comparable levels. Thus a muscle-specific difference in TnILacZ1 beta -gal expression levels within a single fiber type has a parallel in GPDH expression.


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  		Fig. 4.   beta -Gal, GPDH, and SDH expression in TnILacZ1 transgenic mouse plantaris and soleus muscle IIA fibers. Histochemical stain optical densities for all three enzymes were measured, on serial sections, in the same set of IIA fibers (n = 54 for soleus, n = 39 for plantaris). Plantaris and soleus data were measured on the same sections, so values for any given enzyme are directly comparable. Histogram bars show means and SD. Note that beta -gal and GPDH are expressed at considerably lower levels in soleus than in plantaris IIA fibers and that SDH is expressed at similar levels. All within-enzyme between-muscle differences are significant (P < 0.001, t-test).

Developmental coemergence of TnILacZ1 beta -gal and GPDH high-level expression. The similarity of TnILacZ1 and GPDH expression patterns in adult muscle fibers raised the question whether these patterns arose through similar developmental mechanisms. We have previously shown that TnIfast transgene IIB > IIX > IIA differential expression among the fast fiber types emerges during the first weeks of postnatal life, when the IIB, IIX, and IIA adult fast fiber types themselves arise through differential activation of the IIB, IIX, and IIA myosin heavy chain genes (19). [Before activation of the adult genes, immature fast fibers express the perinatal/neonatal fast myosin heavy chain isoform MHCperi/neo (11).]

We analyzed GPDH and TnILacZ1 beta -gal expression during postnatal muscle development and found a striking correlation between the emergence of high-level expression of the two enzymes (Fig. 5). At postnatal day (PND) 3, GPDH and beta -gal were both expressed at low levels in all fibers (Fig. 5A). During subsequent maturation (Fig. 5, B-D), there was a graded and concerted increase in the range of expression levels for both enzymes. Although expression remained low in some fibers, an increasing fraction of the fiber population expressed both beta -gal and GPDH at higher levels than were observed at PND 3, and there was a progressive and coordinated increase in expression level upper limits for both enzymes. From PND 6 onward, there was a significant positive correlation between beta -gal and GPDH levels (see Fig. 5 legend). These results show that high-level expression of TnILacZ1 beta -gal and GPDH emerges in concert, i.e., at the same time and in the same fibers, during early postnatal muscle maturation.


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  		Fig. 5.   Coemergence of TnILacZ1 beta -gal and GPDH high-level differential expression during postnatal maturation. beta -Gal and GPDH histochemical stain optical densities were measured in individual fibers on serial sections of TnILacZ1 transgenic mouse plantaris muscle at different times of postnatal life [postnatal day (PND) 3-24]. A contiguous patch of 100-200 fibers was analyzed at each time point except at PND 3 (76 fibers) and PND 6 (86 fibers). All samples in the figure were coprocessed for histochemistry so as to be directly comparable. In A-D the axes have the same scales throughout to emphasize the increasing range of beta -gal and GPDH expression levels during postnatal maturation, and all fiber types are depicted as filled circles. E-H: same data as A-D, respectively, but on expanded scales, where appropriate, to show more detail. In addition, fibers are identified by fiber type using the symbols shown in H. Peri/neo, perinatal fast, expressing MHCperi/neo. There is a significant correlation between beta -gal and GPDH expression at PND 6, 9, and 24 (r > 0.57, P < 0.0001), but not at PND 3 (r = 0.086, P = 0.25). At PND 24, there is a significant beta -gal/GPDH correlation within each of the fast fiber types (r > 0.59, P < 0.01).

Fiber type analysis showed that adult patterns were fully established by PND 24 (Fig. 5H). Fast fiber maturation into the adult IIB, IIX, IIA types was complete, both beta -gal and GPDH showed IIB > IIX > IIA expression, and levels of the two enzymes were correlated within each of the fast fiber types (see Fig. 5 legend). At PND 9 (Fig. 5G), resolution of the perinatal fast fiber population into the adult fast fiber types was incomplete, although a subpopulation of fast fibers expressing the adult IIB myosin isoform could be identified. This subpopulation expressed higher mean levels of both beta -gal and GPDH than the remainder of the fast fiber population, identified by expression of the MHCperi/neo myosin isoform. Thus, from its first appearance during fast fiber maturation, the IIB fiber type is marked by comparatively high-level expression of both TnILacZ1 beta -gal and GPDH. At PND 6 (Fig. 5F), no adult fast myosin isoforms were expressed, only the MHCperi/neo isoform, and although overall beta -gal and GPDH expression levels were low, both enzymes were expressed at detectably higher mean levels in the fast fiber population than in the slow fiber population. At PND 3 (Fig. 5E), beta -gal but not GPDH showed preferential expression in fast fibers. This last result identifies an apparent difference between TnILacZ1 beta -gal and GPDH expression, a point we discuss further below (see DISCUSSION). However, regardless of any difference relating to low-level fast/slow differential expression in perinatal (PND 3) muscle, the results in Fig. 5 clearly show a remarkable similarity between TnILacZ1 beta -gal and GPDH in the emergence of high-level differential expression among the adult fast fiber types during postnatal maturation.

TnILacZ1 expression is not consistently correlated with SDH levels. Glycolytic and oxidative enzyme levels in skeletal muscle fibers tend to be inversely related, but this is not a strict, or general, rule. For example, GPDH was at higher levels in IIA fibers than in type I fibers in any muscle, and SDH showed a parallel, not an inverse, relationship (Fig. 3; see also Refs. 14 and 51). Likewise, both enzymes were at higher levels in adult muscle than in neonatal muscle (Fig. 5 and data not shown). In addition, while GPDH was at higher levels in plantaris IIA fibers than in soleus IIA fibers, SDH levels were similar in the two muscles (Fig. 4). In each of these comparisons, we found that TnILacZ1 expression levels were higher in the comparison partner having higher GPDH levels (Figs. 3-5). Thus TnILacZ1 expression showed a consistent positive correlation with glycolytic enzyme levels but did not show a consistent inverse correlation with oxidative enzyme levels. Therefore, in terms of metabolic specialization, TnILacZ1 transgene expression appears to be linked to the glycolytic system per se, and not (inversely) to the oxidative system or to the overall metabolic profile, i.e., glycolytic/oxidative ratio.

Correlated responses of GPDH and TnILacZ1 beta -gal expression to under- and overloading. Glycolytic enzyme levels in muscle respond to use/disuse and changes in mechanical loading. The markedly similar expression profiles of the TnILacZ1 transgene and GPDH both in adult and developing muscle led us to question whether the TnILacZ1 transgene might also respond to under- and overloading. Hindlimb suspension is an underloading model that has previously been shown to elevate glycolytic enzyme (34) and TnIfast protein (8) levels in the soleus muscle. We found that hindlimb suspension of TnILacZ1 transgenic for 14 days also led to increases in beta -gal staining that were evident on inspection (Fig. 6) and confirmed by microdensitometry (Fig. 7A). Moreover, there was a significant correlation between GPDH and beta -gal levels in individual fibers in the hindlimb-suspended soleus as shown in the scatterplot in Fig. 8B, indicating that the fibers that upregulated TnILacZ1 expression to the greatest extent were also the fibers that upregulated GPDH to the greatest extent. We also found that TnILacZ1 beta -gal expression was downregulated in all fiber types in plantaris muscle following 14 days of overload by synergist ablation (Fig. 7B), which has been shown to downregulate glycolytic enzyme expression, including GPDH (14). Scatterplot analysis (Fig. 8, C and D) confirmed that overloading did not perturb the overall beta -gal/GPDH correlation in plantaris muscle, indicating that, at the individual fiber level, decreased beta -gal expression was associated with decreased GPDH expression, and vice versa. Thus TnILacZ1 transgene expression responds to under- and overloading in parallel with glycolytic enzyme genes.


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  		Fig. 6.   Increased expression of TnILacZ1 beta -gal and GPDH in soleus muscle after 14 days of underloading by hindlimb suspension. A and C show control and B and D show underloaded muscles from TnILacZ1 transgenic mice. A and B: beta -gal histochemistry. C and D: GPDH histochemistry. Control and underloaded muscles were coprocessed to permit direct comparisons. Diagrams show muscle outlines: S, soleus; P, plantaris; PLG, plantaris-adjacent lateral gastrocnemius.



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  		Fig. 7.   Quantitation of TnILacZ1 beta -gal response to muscle under- and overloading. After beta -gal histochemistry, optical densities of individual fibers were measured by microdensitometry. A contiguous patch of 100-200 fibers was analyzed in each muscle, and the mean optical density for each fiber type was determined and treated as a single data point. One muscle was analyzed from each of several control (n = 3 and n = 5 in A and B, respectively) and treated (n = 4 and n = 6 in A and B, respectively) animals. Histogram bars show the among-animal means and SD for each fiber type. All control and underloaded (or control and overloaded) muscles were coprocessed for beta -gal histochemistry to permit direct comparisons. All differences in comparisons of control vs. under- or overloaded muscle fiber optical densities were significant (P < 0.01, t-test) except in the case of type I fibers.



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  		Fig. 8.   Correlated expression of GPDH and TnILacZ1 beta -gal in individual fibers in under- and overloaded muscles. GPDH and beta -gal histochemical stain optical densities were determined on serial sections in 100-200 individual fibers forming single contiguous patches in control muscles (A and C), an underloaded soleus muscle (B), and an overloaded plantaris muscle (D). Control and treated muscles were coprocessed so that data are directly comparable. There were significant (P < 0.0001) positive correlations between GPDH and beta -gal: r = 0.401 in A, r = 0.789 in B, r = 0.856 in C, and r = 0.921 in D.

TnIfast IRE enhancer drives GPDH-correlated expression. The TnIfast gene contains an enhancer in the first intron, the IRE (31, 68), that drives much of the gene's regulatory behavior. The IRE, when linked to heterologous promoters, drives gene expression that is activated during myoblast differentiation in transfected muscle cell culture lines and is fast muscle specific in transgenic mice (31, 40, 68, and unpublished observations). We have prepared transgenic mice carrying a heterologous beta -gal reporter construct, 3xIRE-tkLacZ, in which the herpes virus thymidine kinase promoter is driven by three copies of the 148-bp IRE. Like TnILacZ1, 3xIRE-tkLacZ is expressed specifically in skeletal muscle, and in a IIB > IIX > IIA pattern that emerges during early postnatal life (unpublished observations). Examination of within-fiber-type variation by quantitative microdensitometry showed that beta -gal expression correlated with GPDH levels. As shown in Table 1, a positive correlation coefficient r was obtained in 10/12 within-fiber-type comparisons involving two independent 3xIRE-tkLacZ transgenic lines, and in all of these cases the correlation achieved statistical significance at the P < 0.01 level. This result shows that the IRE drives heterologous gene expression in a pattern that correlates with GPDH expression.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our results reveal a novel and very close relationship between glycolytic enzyme and contractile protein transgene regulation in skeletal muscle fibers. Previous studies had identified qualitative relationships between expression of particular myosin isoforms and metabolic enzyme levels (14, 43, 51). Here we show for the first time a quantitative correlation between expression levels of a contractile protein transgene and a metabolic enzyme. Our findings provide both the initial evidence for a novel contractile/metabolic coordinating mechanism and a molecular genetic context for its further study. They also generate new insight into an unexplained IIB>IIX>IIA expression pattern observed for several contractile protein transgene constructs (13, 16, 19, 27), and this has implications for our understanding of the mechanisms that drive high-level gene expression in adult fast muscle fibers.

Similarity of TnIfast transgene and glycolytic enzyme expression profiles. Our results show that the TnIfast transgene construct TnILacZ1 is differentially expressed among muscle fibers at levels that correlate with levels of the glycolytic indicator enzyme GPDH. An inverse correlation with oxidative enzyme (SDH) levels was less complete than the positive correlation with GPDH, indicating that TnILacZ1 expression is linked specifically to glycolytic enzyme levels and not, inversely, to oxidative enzymes or to muscle fiber overall metabolic profiles. In addition to their markedly similar expression patterns in adult muscle fibers, including a IIB > IIX > IIA > I fiber type pattern, TnILacZ1 and GPDH show important regulatory parallels during postnatal development; high-level expression emerges at the same time and in the same fibers during muscle maturation. Moreover, we found that the TnILacZ1 transgene responds to muscle under- and overloading in a manner parallel to that previously established for glycolytic enzymes (14, 34, 61). This extensive coregulation suggests that common, or thoroughly integrated, mechanisms drive expression of TnILacZ1 and GPDH genes. Because GPDH expression is a good indicator of glycolytic gene expression in general (45), TnILacZ1 expression is presumably integrated not uniquely with GPDH but with a metabolic regulatory system controlling many glycolytic enzyme genes. Regulatory information for GPDH-coregulated expression is carried by the TnIfast first-intron enhancer, the IRE.

In our studies we observed only one apparent difference between TnILacZ1 and GPDH expression patterns. TnILacZ1 showed preferential expression in fast fibers in perinatal (PND 3) muscle. This is consistent with the known two-stage developmental emergence of the TnILacZ1 fiber type pattern, namely, 1) a prenatally established preferential expression in fast, as opposed to slow, fibers, and 2) a postnatally induced differential superactivation within the maturing fast fiber population to generate IIB > IIX > IIA expression (19). In contrast, GPDH did not show differential expression in fast and slow fibers at PND 3, suggesting that it does not share the first regulatory mechanism (although it apparently does share the second mechanism, leading to IIB > IIX > IIA expression postnatally). However, other glycolytic enzymes, beta -enolase and aldolase A, are expressed in a two-stage developmental pattern markedly similar to that of TnILacZ1, including a prenatal stage marked by preferential expression in secondary (presumptive fast) muscle fibers (2, 25, 26, 32, 57). Thus each of the several components of the TnILacZ1 overall expression pattern appears to have a counterpart in the regulation of at least some glycolytic enzyme genes. This indicates an extensive and intimate regulatory coordination of fast isoform contractile protein transgene and glycolytic enzyme gene expression.

The similarity of TnILacZ1 and glycolytic enzyme gene expression in skeletal muscle may seem at odds with their different expression patterns in the organism as a whole. The TnILacZ1 transgene, like the endogenous TnIfast gene, is highly specific for skeletal muscle, while glycolytic enzymes, which have a cellular housekeeping function, are expressed broadly. However, several glycolytic enzymes have been shown to be expressed from multiple genes and/or promoters, some of which are muscle specific [e.g., aldolase (24, 52, 59, 63), phosphofructokinase (39), phosphoglyceromutase (5), and enolase (17)]. Thus muscle-specific regulatory mechanisms may drive both contractile protein and high-level glycolytic enzyme gene expression in skeletal muscle. The use of tissue-specific regulatory mechanisms to drive high-level expression of this class of housekeeping genes may facilitate regulatory coordination with tissue-specific genes such as muscle contractile protein genes.

Sarcomeric muscle cells are specialized for the rapid transformation of chemical energy (ATP) into mechanical force and are capable of uniquely high ATP flux (36). ATP flux in active muscle fibers reflects balanced synthesis (by metabolic enzymes including mitochondria) and hydrolysis (by the contractile apparatus and, to a lesser extent, the sarcoplasmic reticulum Ca2+ pump) (22, 36). A need to balance ATP synthesis and hydrolysis would create a selective pressure favoring the evolution of mechanisms, such as those underlying TnILacZ1/GPDH coregulation, that could contribute to the quantitative coordination of contractile protein and metabolic enzyme gene expression in skeletal muscle fibers.

Nature of the coordinating mechanism. In experimental fiber type transformation experiments, changes in metabolic enzyme levels generally precede contractile protein isoform switches (reviewed in Ref. 48), and it has been suggested that glycolytic enzyme changes may have a regulatory role in signaling, through unknown mechanisms, to contractile protein genes (14). Such serial regulation could account for coexpression of TnILacZ1 and glycolytic enzyme genes. However, there is no direct evidence for such regulatory pathways, and it remains possible that TnILacZ1 and glycolytic enzyme genes are independently reacting to common, or integrated, upstream signals.

Cellular fiber type identity is a signal that could potentially coordinate fiber-to-fiber differences in the expression of various genes. However, we do not find that TnILacZ1 or GPDH are expressed at distinct, characteristic levels in each fiber type. Rather, expression levels form smoothly varying continuums within, as well as among, the fast fiber types. The within-fiber-type correlation between TnILacZ1 and GPDH levels indicates that the fiber-to-fiber differences observed in both enzymes represents real variation and cannot be due solely to random measurement errors. Moreover, gradation of TnILacZ1 or GPDH levels within a fiber type does not solely reflect the blending of distinct fiber type-specific levels in hybrid or intermediate fibers, because hybrid fibers usually constitute only a small minority and because variation is clearly evident in our data within the IIX fiber type, which by our typing method does not include hybrid fibers (see MATERIALS AND METHODS). These results indicate that TnILacZ1 and GPDH differential expression among fast fibers is not determined by cellular fiber type identity. Rather, expression levels reflect an intracellular gene regulatory parameter that varies smoothly, rather than in a quantal or saltatory manner, as fiber type does. The molecular nature of the hypothetical smoothly graded signal is unknown, but its activity is likely to be inversely related to muscle fiber contractile activity. Intracellular Ca2+ exposure is a correlate of contractile activity that has been implicated, through calcineurin (9, 12, 66) and protein kinase C (18) pathways, in the high-level expression of oxidative metabolic enzymes and slow isoform contractile protein genes in highly active motor units. It is possible that Ca2+-sensing molecules could also play a role in the relationship between contractile activity and TnILacZ1/glycolytic enzyme gene expression, although in this case the net effect of elevated Ca2+ levels would be to repress rather than augment gene expression.

The within-fiber-type correlation between GPDH and 3xIRE-tkLacZ beta -gal expression shows that the mechanism that drives TnILacZ1 transgene expression in a glycolytic enzyme-correlated pattern operates through the IRE enhancer. Comparison of the IRE enhancer with a well-characterized muscle glycolytic enzyme gene promoter/enhancer, the aldolase A pM promoter (56, 58), reveals little direct sequence similarity. In the absence of conserved sequence features, identification of the relevant cis-elements depends on further detailed functional characterization of the IRE and of aldolase pM (and other glycolytic) promoters/enhancers.

The IRE enhancer and mechanisms of high-level gene expression in adult fast fibers. The IRE is the principal known regulatory element of the TnIfast gene. It drives gene activation during myoblast differentiation in culture (31) and skeletal muscle-specific expression in vivo (40 and unpublished observations). These activities can readily be related to the endogenous TnIfast gene, which is also activated during myoblast differentiation and is skeletal muscle specific (29). On the other hand, the IRE-driven IIB > IIX > IIA pattern does not correspond to the pattern of endogenous TnIfast gene expression, which is equal expression in all fast fiber types (19, 29). A number of other contractile protein transgenes also unexpectedly show IIB > IIX > IIA differential expression among the fast fiber types (13, 16, 27), suggesting that a broadly relevant common mechanism is at work. Our results provide a biological context for understanding the IIB > IIX > IIA pattern by showing that it represents part of a larger pattern of coordinated expression of contractile protein transgenes with glycolytic enzyme genes. Our results further suggest that different mechanisms may drive high-level expression of the endogenous TnIfast gene in the most-glycolytic (IIB) vs. the most-oxidative (IIA) fast fibers.


    ACKNOWLEDGEMENTS

We thank Richard Tsika for sharing expertise regarding the hindlimb-suspension protocol and Stefano Schiaffino for providing antibodies. The A4.840 and N3.36 antibodies, developed by Helen Blau, were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of National Institute of Child Health and Human Development and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA.


    FOOTNOTES

This research was supported by the Medical Research Council of Canada/Canadian Institutes of Health Research.

K. E. M. Hastings is a Killam Scholar of the Montreal Neurological Institute.

Address for reprint requests and other correspondence: K. Hastings, Montreal Neurological Institute and Biology Dept., McGill Univ., 3801 University St., Montreal, Quebec, Canada H3A 2B4 (E-mail: khastings{at}mni.mcgill.ca).

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.

First published September 21, 2001; 10.1152/ajpcell.00294.2001

Received 28 June 2001; accepted in final form 11 September 2001.


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