INTRODUCTION

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PREVIOUS STUDIES involving both humans and rodents clearly show that high resistance-training paradigms of either the concentric or eccentric type induce adaptations in skeletal myosin heavy chain (MHC) isoform expression. These shifts predominantly involve rearrangement in the pattern of expression involving the fast MHC isoforms, i.e., type IIx to IIa in humans and IIb to IIx in rodents (1, 4-6, 10, 12, 13). These findings also suggest that such adaptations can occur after only a few training sessions when evaluated at the mRNA level of analysis (5). Furthermore, in these particular rodent studies, the analyses were performed on muscles normally expressing a high predominance of fast type II fibers (4-6). In contrast, Diffee et al. (10) reported that a resistance-training paradigm of the isometric type appeared to be more effective than one consisting of high-force (90% maximum) isovelocity-concentric contractions in terms of maintaining expression of the type I or slow MHC in a predominantly slow (soleus) muscle undergoing a chronic state of unloading even though the two programs produced similar effects on augmenting muscle mass. In the context of these isomyosin adaptations in response to resistance training, other studies have shown that the model of functional overload, which involves the chronic overloading of a targeted fast muscle by the removal of its synergists, induces the upregulation of both the slow I and fast type IIa MHCs while concomitantly downregulating the two fastest MHCs, i.e., type IIb and IIx (3, 17, 19). Furthermore, the functional-overload model essentially defines the upper limits of the adaptive potential of a fast muscle to transform its structural/functional properties in response to mechanical stimuli. In this model, the mechanical stimulus is chronically imposed on the muscle, and thus it undergoes significantly greater increments in its mass compared with muscles undergoing repetitive resistance-training paradigms (4-6, 10).

The mechanism(s) for such adaptations as muscle enlargement and the upregulation of slower MHC expression remains unknown for all models of mechanical loading. However, the fact that slow isoforms can be upregulated in response to mechanical stress suggests that either a chronic mechanically loaded contractile activity paradigm or one possibly biased to isometric activity may be an important prerequisite stimulus for inducing upregulation of the slower MHC genes. Therefore, the present study was undertaken to test the hypothesis that repetitive isometric contractions, of varying duration and consisting of relatively high frequencies of direct muscle activation, play a central role in inducing transformations in MHC plasticity whereby the slower MHC isoforms become upregulated at the expense of the faster isoforms. Surprisingly, the findings reported herein suggest that, although isometric contractions of high activation frequency induce significant transformations in type IIb to IIx MHC expression and a relatively high degree of muscle enlargement, they are ineffective, based on the experimental paradigms utilized herein, in inducing the fast-to-slow transformations typically seen in models of chronic functional overload.

	METHODS

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Animal care and experimental groups. Female Sprague-Dawley rats (250-260 g; n = 24) were used in this study. All animals were housed individually and given food and water ad libitum. The experiments described were approved by our institutional animal care committee. Animals were randomly allocated into two major groups that underwent different training programs: isometric resistance-training program I (IT-I; n = 12) and isometric resistance-training program II (IT-II; n = 12). In each program, rats were trained for either 6 sessions (6S; n = 6) or 12 sessions (12S; n = 6). This design thus resulted in four groups of six rats per group: IT-I (6S), IT-I (12S), IT-II (6S), and IT-II (12S).

Electrode implantation and training apparatus. Each animal was anesthetized (4.5 mg/kg acepromazine and 75 mg/kg ketamine), and stimulating electrodes were surgically implanted into the left MG and connected to a head plug cemented to the skull of the rat so that the activation pattern of the muscle could be controlled. A sham operation was performed on the right leg, which served as a control. The specific details for these procedures have been described previously (5, 6, 10).

Muscle processing. Approximately 15 h after the last training session, the animals were anesthetized by a lethal injection of pentobarbital sodium (120 mg/kg); the left and right MG muscles were rapidly removed, weighed, and quickly dissected into superficial "white," mixed, and deep "red" regions (4-6); and the samples were quick frozen on dry ice and stored at 70°C for subsequent biochemical analyses. The muscle was partitioned into these regions because previous studies have shown that there are potentially differential adaptations in MHC transformation, particularly in the superficial white (consisting predominantly of types IIb and IIx MHC) and the deep red (consisting predominantly types IIx and IIa MHC) regions (5). In view of previous findings on functionally overloaded skeletal muscle (3, 16, 17), we reasoned that fibers in the red region would be more likely to transform MHC in the direction of the slower MHCs (i.e., types IIa and I) compared with the superficial white region.

Total RNA and protein isolation. Total cellular RNA and total muscle proteins were simultaneously coextracted from frozen muscle samples by using the TRI reagent (Molecular Research Center, Cincinnati, OH) according to the company's protocol, which is based on the method described by Chomcznski (7). Total proteins were separated in the organic phase and subsequently precipitated with isopropanol, washed with guanidine hydrochloride and ethanol, and suspended in 1% SDS (7). Protein concentration was determined by the Bio-Rad protein assay kit, by using gamma globulin as standard. Samples were adjusted to protein concentration of 1 mg/ml with 1% SDS and were stored at 20°C until later analysis for MHC distribution pattern by SDS-PAGE (18). Total RNA was precipitated from the aqueous phase with isopropanol, and after being washed with ethanol, it was dried and suspended in a small volume of 0.5% SDS in Tris-EDTA buffer (10 mM Tris, pH 8.0, and 1 mM EDTA). This method of RNA extraction can yield high-quality undegraded RNA, free of protein and DNA, on the basis of a ratio of optical density at a wavelength of 260 nm to that at 280 nm of ~2 and as tested by agarose gel electrophoresis with ethidium bromide staining, showing both 28S and 18S ribosomal RNA with an intensity ratio of ~2:1 (8). The RNA concentration was determined by optical density at a wavelength of 260 nm (by using a unit of optical density at a wavelength of 260 nm equivalent to 40 µg/ml). The RNA samples were stored frozen at 70°C until being subsequently analyzed for MHC mRNA expression by Northern hybridization.

MHC mRNA analysis. MHC mRNA expression was analysed by using Northern blot hybridization. Because four different probes needed to be tested (types I, IIa, IIx, and IIb), each sample was loaded on four separate gels, ~5 µg per each. Gel and transfer conditions were as explained previously (5, 17). Oligonucleotides complimentary to the 3' untranslated sequences of the MHC mRNAs were purchased from Chemgene (Waltham, MA). The sequences for the oligonucleotides used for MHC types I, IIa, and IIb were as reported by Gustafson et al. (11). For MHC type IIx, we used a 20-base probe complimentary to the 3' nontranslated sequence of a cDNA clone isolated by Schiaffino from rat diaphragm muscle of the following 5'3'sequence: GGTCA CTTTC CTGCT TTGGA (9). The 5' ends of the probes were labeled to 1-2 × 10⁹ counts · min¹ · µg DNA¹ with [-³²P]ATP by using polynucleotide kinase. The blots were prehybridized and then hybridized overnight, washed, and exposed to autoradiographic film with an intensifying screen as described in detail previously (5, 17).

MHC protein analysis. Skeletal MHC isoforms were separated by using a SDS-PAGE technique (18). The gels were run at 275 V for ~22 h under refrigeration. After electrophoresis, the gels were stained for 1 h with Brilliant blue G 250 (Sigma Chemical) and destained with 25% methanol and 10% acetic acid. The separated MHC bands were scanned and quantified by using a Molecular Dynamics densitometer. The peaks of interest representing the distinct MHC isoforms were identified in the digitized densitometric data sets. The area of each peak was determined by integration (Image Quant Software, Molecular Dynamics). The MHC identity on the resulting banding pattern was verified on the basis of Western blotting techniques by using a battery of MHC antibodies as obtained from the laboratory of Dr. S. Schiaffino (data not shown).

Statistical analyses. All data are reported as means ± SE. All statistical analyses were performed by using a computer software package (Prism, GraphPad Software). For each training paradigm, each MHC was analyzed separately by using either a one-way analysis of variance test or a t-test for paired samples, with statistical significance set at the P < 0.05 level.

	RESULTS

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Muscle weight. After 6S, there was no significant change in the trained MG muscle weight undergoing the IT-I program, whereas IT-II caused a 6% increase (P < 0.01) relative to the nontrained muscle (Table 1). 12S of both the IT-I and IT-II training programs increased the MG wet muscle weight by 11 and 13%, respectively, in the stimulated vs. nonstimulated muscles (P < 0.05). These increments in muscle mass are similar in magnitude to previous reports in which both fast and slow rodent skeletal muscles were trained under conditions involving high-force-output paradigms of either the isometric or concentric-isovelocity type (4-6, 10).

MHC expression. In the superficial white region of the MG only the fast types IIb and IIx MHCs were expressed at both the protein and mRNA levels, with the type IIb being the more predominant isoform (i.e., ~75% of the pool), whereas, in the deep red region, all four adult MHCs were abundantly expressed, with the type IIx MHC being the most predominant isoform (e.g., accounting for ~50% of the total MHC pool) (Fig. 1).

View larger version (59K): [in this window] [in a new window]   View larger version (63K): [in this window] [in a new window]   Fig. 1.   Representative myosin heavy chain (MHC) mRNA signals and MHC proteins as separated by SDS-PAGE in both white medial gastrocnemius (A) and red medial gastrocnemius (B) muscles for isometric resistance-training program I (IT# I; 100 Hz) and isometric resistance-training program II (IT# II; 60 Hz) after 12 sessions of training. Also shown is signal after hybridization with 18S rRNA probe. C, control; T, trained.

View larger version (34K): [in this window] [in a new window]   Fig. 2.   MHC isoform protein and mRNA expression in white medial gastrocnemius (A) and red medial gastrocnemius (B) muscles of isometric resistance-training program I (see METHODS). Values are means ± SE bars. 6S, 6 sessions; 12S, 12 sessions. * P < 0.05, trained (TRN) vs. control (CON) (by using paired t-test).

View larger version (34K): [in this window] [in a new window]   Fig. 3.   MHC isoform protein and mRNA expression in white medial gastrocnemius (A) and red medial gastrocnemius (B) muscles of isometric resistance-training program II (see METHODS). Values are means ± SE bars. * P < 0.05, trained vs. control (by using paired t-test).

	DISCUSSION

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Previous studies clearly show that increasing the mechanical stress imposed on skeletal muscle induces significant enlargement of the fiber population and a transformation in MHC phenotype in a subset of the fiber pool (4-6, 10). In animal models, two primary approaches have been used to elevate the mechanical stress imposed on the muscle. The first involves resistance-training programs that are predicated on classic training paradigms used to train human subjects (1, 12, 13). However, the unique feature of the animal model is that, by using stimulating electrodes, the activation pattern of the muscle can be controlled to both regulate the active state of the muscle and ensure that all the motor units in the muscle participate in the contraction response (4-6, 10). In this way one can examine the adaptive response by focusing on different regions of the muscle with known differences in fiber type (15). In these modes of training rodent muscle, there has been relatively good agreement with that reported in human subjects with regard to the degree of enlargement and the modest fast-to-slow transformation in MHC phenotype that are induced (1, 12, 13).

A second approach to loading the muscle and one that is more or less unique to animal models involves the surgical removal of those muscles that act synergistically to the target muscle so that the latter becomes chronically overloaded whenever the animal is in a weight-bearing mode (3, 16, 17). Fast muscles overloaded in this manner typically hypertrophy by 70-100% and undergo significant transformations in MHC phenotype that involve significant downregulation of type IIb MHC and upregulation of the slow type I and fast type IIa MHCs (17). Interestingly, this upregulation of the type I and type IIa MHCs is largely confined to the inner red core of the muscle (16), which typically contains a predominance of fibers that express the type IIa and IIx isoforms (e.g., Fig. 1).

The rationale of the experiments performed in the present study is based on a previous study by Diffee et al. (10) showing that high-force-output contractions performed in the isometric mode are effective in blunting the decrease in type I MHC expression in the unloaded slow-twitch skeletal muscle (10). Our findings in rodents suggest that although the isometric training paradigms used herein can induce the same types of adaptation in MHC phenotype seen with other resistance paradigms involving either a concentric or eccentric contraction mode (10), they are ineffective in providing a stimulus to increase either pretranslational or translational events affecting expression of both the type IIa and type I MHC genes.

Several factors may account for the ineffectiveness of the training programs used herein for inducing increased expression of the slower MHC phenotypes. First, an insufficient number of contractions could have been performed during each training session to elevate those factors involved in increasing pretranslational/translational events for increasing slower myosin isoform gene expression. This could be an important possibility because the functional-overload model typically places a chronic stress on the target muscle that is sufficient to induce all fibers to essentially double their size. Consequently, a greater number of isometric contractions may have to be elicited each day to more fully transform a fast muscle to express a slow(er) MHC phenotype.

Second, the training program, although of sufficient duration to alter regulation of the type IIb and IIx MHCs, may require a longer duration (i.e., many weeks) to accumulate sufficient levels of factors involved in the regulation of these slower MHC genes. In this context, it is curious that chronic electrical stimulation, a model used to globally induce a fast-to-slow muscle phenotype, requires a longer time course of stimulation to induce changes in the sarcomeric genes than it does for transforming changes in either the mitochondrial or sarcoplasmic reticular systems (2). Thus the two resistance programs used herein may have to be extended for many weeks to achieve a further expression of a slower contractile phenotype compared with that induced in the present study.

Third, the activation frequencies (60 and 100 Hz) used herein to stimulate the muscle may have been too high to achieve a more complete fast-to-slow MHC phenotype. Previous studies have provided evidence to suggest that continuous low-frequency-stimulation protocols (~10 Hz) of fast muscles were effective in causing fast-to-slow MHC transitions (2, 15), whereas stimulation of slow muscles at high frequencies, i.e., 100 Hz, caused an opposite type of transformation (2, 15). In the context of these observations, we have recently determined (retrospectively) that both fast- and slow-twitch skeletal muscles, when stimulated isometrically for durations in excess of 4 s per contraction and at frequencies >50 Hz, undergo a progressive fatigue response during the course of a training session (unpublished observations). This observation raises the possibility that repetitive high-frequency contractions of sufficient duration performed in an isometric mode may actually result in an insufficient amount of force being imposed on those moderately slow motor units, i.e., units expressing IIa and IIx MHC and predominating in the red MG, to enable them to achieve a "slower" phenotype.

In conclusion, the findings reported herein suggest that isometric contraction paradigms, when used in the context of a typical resistance-training mode and normally involving a high frequency of stimulation to fully activate the muscle, are not any more effective than conventional high-frequency concentric-eccentric resistance paradigms in terms of inducing upregulation of expression of the two slower isomyosins expressed in adult skeletal muscle. Despite the fact that our results show that these two training paradigms did not induce an increase in type I MHC expression, important information was obtained that is relevant to designing future experiments to address issues concerning the regulation of MHC plasticity. More research is needed to define the type of mechanical stimuli, its frequency of activation, and the amount of contractile output that must be accumulated to induce the type of MHC transformation seen in models of chronic functional overload.