Denervation alters myosin heavy chain expression and contractility of developing rat diaphragm muscle

Departments of Anesthesiology and Physiology and Biophysics, Mayo Clinic and Foundation, Rochester, Minnesota 55905

	ABSTRACT

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We hypothesized that unilateral denervation (DNV) of the rat diaphragm muscle (Dia_m) in neonates at postnatal day 7 (D-7) alters normal transitions of myosin heavy chain (MHC) isoform expression and thereby affects postnatal changes in maximum specific force (P_o) and maximum unloaded shortening velocity (V_o). The relative expression of different MHC isoforms was analyzed electrophoretically. With DNV at D-7, expression of MHC_neo in the Dia_m persisted, and emergence of MHC_2X and MHC_2B was delayed. By D-21 and D-28, relative expression of MHC_2A and MHC_2B was reduced in DNV compared with control (CTL) animals. Expression of MHC_neo also reappeared in adult Dia_m by 2-3 wk after DNV, and relative expression of MHC_2B was reduced. At each age, P_o was reduced and V_o was slowed by DNV, compared with CTL. In CTL Dia_m, postnatal changes in P_o and V_o were associated with an increase in fast MHC isoform expression. In DNV Dia_m, no such association existed. We conclude that, in the Dia_m, DNV induces alterations in both MHC isoform expression and contractile properties, which are not necessarily causally linked.

contractile properties; maturation; specific force; shortening velocity

	INTRODUCTION

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SEVERAL MYOSIN HEAVY CHAIN (MHC) isoforms exist in skeletal muscle, which, in adults, correspond with the histochemical classification of different fiber types (22, 48, 52). During the first four postnatal weeks, there are dramatic transitions in MHC isoform expression in the rat diaphragm muscle (Dia_m) (29, 30, 51, 59). At birth (postnatal day 0; D-0), the MHC_neo isoform is predominantly expressed, but, by postnatal day 28 (D-28), the MHC_neo isoform completely disappears. In contrast, MHC_2X and MHC_2B isoform expression appears in the rat Dia_m only by the end of the second postnatal week.

The mechanism underlying postnatal transitions in MHC isoform expression is unknown. Transition in MHC isoform expression may be affected by changes in the pattern of innervation, circulating thyroid hormones, and/or genetic program (17, 25, 28, 51). Innervation of the rat Dia_m is established by embryological day 18 (25), and a pattern of multiple motoneuron innervation of Dia_m fibers persists until postnatal day 14 (D-14) (1, 41). Therefore, during the first two postnatal weeks, MHC isoform expression in the rat Dia_m fibers can be influenced by more than one motoneuron. Although previous studies have suggested that innervation is not a requirement for the ultimate expression of adult fast MHC isoforms in hindlimb muscles (3, 8, 35, 43), this does not exclude an influence of innervation pattern on the normal postnatal transitions in MHC isoform expression. In the adult rat Dia_m, removal of neural influence by denervation (DNV) results in the coexpression of slow and fast MHC isoforms within single fibers (7, 46, 62). In the present study, we hypothesize that DNV will disrupt the normal postnatal transitions in MHC isoform expression in the rat Dia_m.

Several previous studies have demonstrated that the contractile properties of muscle fibers correspond with MHC isoform expression. For example, fibers expressing MHC_slow or MHC_neo isoforms have slower maximum unloaded shortening velocities (V_o) compared with fibers expressing fast MHC isoforms (39, 40, 49, 50, 58). This explains, at least in part, the slower V_o of the Dia_m during early postnatal development, when there is predominant expression of MHC_neo and MHC_slow isoforms (29, 51, 58). The predominant expression of MHC_neo and MHC_slow isoforms may also explain the lower maximum specific force (force normalized for fiber cross-sectional area, P_o) and greater fatigue resistance of the neonatal Dia_m (20, 21, 29, 51, 58, 59, 64) In the adult rat Dia_m, DNV leads to a dramatic slowing of V_o and a marked reduction in P_o, which are not directly proportional to changes in MHC isoform expression (37). Accordingly, in the present study, we hypothesize that unilateral DNV of the rat Dia_m during early postnatal development also leads to a slowing of V_o and a reduction in P_o and that these effects are independent of altered MHC isoform expression.

	METHODS

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General procedures. Experiments were performed on 39 young and 31 adult male Sprague-Dawley rats. In the younger animal groups, pregnant mothers were received at 14 days gestation, and at the time of birth, neonatal rats from each litter were randomly assigned to one of the six groups: 1) control (CTL) animals studied at D-14 (n = 6); 2) CTL animals studied at postnatal day 21 (D-21; n = 7); 3) CTL animals studied at D-28 (n = 7); 4) DNV animals studied at D-14 (n = 6); 5) DNV animals studied at D-21 (n = 7); and 6) DNV animals studied at D-28 (n = 6). Adult animals were divided into one of four groups: 1) Sham CTL animals (n = 8); 2) DNV animals studied after 1 wk (n = 7); 3) DNV animals studied after 2 wk (n = 8); and 4) DNV animals studied after 3 wk (n = 8). Animals were housed in separate cages under a 12:12-h light-dark cycle. Adult animals and the mothers were fed with Purina rat chow and provided with water ad libitum. Body weights were monitored daily in all groups. Surgical procedures were performed under aseptic conditions, and recovery of animals from surgery was carefully monitored. The Institutional Animal Care and Use Committee of the Mayo Clinic approved all procedures.

Phrenicotomy. In both young and adult groups, unilateral, rather than bilateral, DNV was performed to enhance survival of the animals and to match similar procedures performed in previous studies (23, 37, 61-63). In each of the younger DNV groups, the right phrenic nerve was transected in the neck at D-7. This age was selected because it precedes both the elimination of polyneuronal innervation of the Dia_m (1, 41) and the emergence of MHC_2X and MHC_2B isoform expression (29-31, 51, 59).

In the D-7 rats, surgeries were performed using hypothermic anesthesia. The young rat was placed beneath a shallow layer of ice chips until the righting reflex was lost and spontaneous breathing ceased. The animal was then placed supine on a strip of aluminum foil, cushioned, and surrounded by ice chips. The surgical procedure was completed within 10 min, during which time the animal's body temperature was maintained at 10°C. Adult animals were anesthetized using ketamine (60 mg/kg) and xylazine (2.5 mg/kg) that was administered intramuscularly. In both groups, a midline incision was made over the trachea, and the right phrenic nerve was sectioned in the neck at a point beneath the sternomastoid muscle. A portion of the distal end of the phrenic nerve was removed to prevent re-innervation of the Dia_m, and to minimize neurotrophic effects emanating from the remaining nerve stump. The wound was closed with 6-0 silk sutures, and the surgical wounds were treated topically with Neosporin ointment (containing aerosporin, neomycin, and bacitracin). After surgery, younger animals were gradually rewarmed under an infrared lamp. Using a pair of cotton-tipped applicators, the animal was vigorously but gently manipulated, with occasional pressing on the thoracic and abdominal walls, to promote ventilation. Within 4-8 min, the rat pups fully recovered, and they were then returned to their mother, who readily accepted them for care and feeding. A 95% success rate was achieved using this DNV procedure. Adult animals fully recovered within 1 h after surgery. In previous studies, our laboratory demonstrated that blood-gas levels were normal in the DNV animals, indicating ventilatory compensation for unilateral paralysis of the Dia_m (37).

In vitro measurement of contractile properties. The in vitro preparations for determining isometric and isotonic contractile properties of the Dia_m have been previously described (29, 37, 64). Briefly, at D-14, D-21, and D-28, rat pups were reanesthetized using furane inhalation, whereas adult animals were anesthetized with ketamine (60 mg/kg) and xylazine (2.5 mg/kg). The Dia_m was rapidly excised, and two muscle segments (3- to 4-mm width) were dissected from the right midcostal region, one for isometric measurements and the other for isotonic measurements. The isometric and isotonic experiments were performed simultaneously using two different systems. In both cases, the muscle segments were suspended vertically in glass tissue chambers containing Rees-Simpson solution (pH 7.4) with the following composition (in mM): 135 Na⁺, 5 K⁺, 2 Ca²⁺, 1 Mg²⁺, 120 Cl, 25 HCO₃, and 0.012 d-tubocurarine. The solution was aerated with 95% O₂-5% CO₂ and maintained at either 26°C (isometric) or 15°C (isotonic). The cooler temperature was used in the isotonic studies to improve the accuracy of measurements of the time required for force to redevelop after a step change in muscle length (see below for methods of measuring V_o using the "slack test"). In both cases, the muscle segments were stimulated directly with current pulses (0.5-ms pulse duration) via platinum plate electrodes placed on either side of the muscle. A computer controlled the stimulus pattern. Stimulus intensity was raised until a maximal twitch response was obtained and was then set at 125% of this maximal stimulus intensity (supramaximal stimulation, ~230 mA). During single-pulse stimulation, muscle fiber length was adjusted until a maximal twitch force was obtained. This optimal length (L_o) was measured using digital calipers.

For isometric force measurements, the costal margin origin of muscle fibers was clamped to a steel rod that was fixed to a micromanipulator. The central tendon of the Dia_m segment was glued to a nylon mesh that was then attached to a calibrated force transducer (Grass FT 03). All force responses were displayed on a storage oscilloscope (Nicolet 410), recorded on a chart recorder (Gould TA2000), and digitized at a 1-kHz sampling rate using Lab View software. Subsequently, force was normalized for the cross-sectional area (CSA) of the muscle segment, which was estimated using the following formula

For measurements of V_o, the costal margin origin of fibers was fixed in series with a micromanipulator for length adjustments in establishing L_o. The central tendon of the muscle segment was glued to a small piece of aluminum foil that was then attached to a force transducer (Cambridge Technology, model 300B) via a fine glass pipette. This connection provided a noncompliant attachment of the muscle segment to the force transducer and prevented tearing of the central tendon. The slack test was used to determine V_o (13). In this procedure, muscle length was rapidly shortened in a series of four to six steps, ranging from 5 to 15% of L_o, while the muscle was maximally activated at 75 Hz. During the slack in muscle length (dL), force fell to zero, and the time required for force to redevelop (dt) was then used to calculate V_o (dL/dt; expressed as L_o/s). Because dt was prolonged at 15°C, this lower bath temperature improved the accuracy in measuring V_o.

MHC isoform composition. Myosin was extracted from the muscle segments by scissors mincing in a high-salt solution (in mM: 300 NaCl, 100 NaH₂PO₄, 50 Na₂HPO₄, 1 Na₄P₂O₇, 10 EDTA, pH 6.5) at 4°C for 40 min (4). Extracts were centrifuged, and the supernatants were recovered. Ten microliters of supernatant were diluted (1:10) in a low-salt buffer consisting of 1 mM EDTA and 0.1% 2-mercaptoethanol (vol/vol) and stored overnight at 4°C to allow precipitation of myosin filaments. The filament solution was subsequently centrifuged to form a pellet, which was then dissolved in myosin sample buffer (0.5 M CaCl₂, 10mM NaH₂PO₄), followed by dilution (1:200) in SDS samples buffer [62.5 mM Tris·HCl, 2% (wt/vol) SDS, 10% glycerol, 5% (vol/vol) 2-mercaptoethanol, and 0.001% (wt/vol) bromphenol blue at pH 6.8]. The samples were boiled for 2 min and stored at 80°C. Different MHC isoforms were separated by SDS-PAGE gel electrophoresis. Gel preparation was based on a modification of the procedure by Sugiura (54). A 3.5% acrylamide concentration (pH 6.8) was used in the stacking gel, and the resolving gel (8 × 10 cm in size, 0.75-mm thick, Hoefer SE250) consisted of a gradient of 5-8% acrylamide (pH 8.8) with 25% (vol/vol) glycerol. All samples were run at a constant current of 20 mA/gel until the tracking dye reached the bottom of the gel (~1.75 h). After completion of the gel run, the gels were removed from the plates and silver stained according to the procedure of Oakley et al. (38). The relative expression of different MHC isoforms was then quantified by densitometry.

Statistics. All data are reported as means ± SE. A two-way ANOVA was used to evaluate changes in contractile properties and MHC isoform expression, with age and experimental condition (CTL vs. DNV) used as grouping variables. When appropriate, an unpaired t-test was used as a post hoc analysis to compare CTL and DNV groups. A multiple stepwise linear regression was used to determine the contribution of each MHC isoform to the correlation between postnatal MHC isoform transitions and changes in P_o, and V_o. Statistical significance of group differences and regressions were tested at P < 0.05.

	RESULTS

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During the first four postnatal weeks, rats displayed rapid weight gain, which was not significantly affected by DNV (Table 1). During early postnatal development, there was also a progressive increase in L_o of the Dia_m, which was not significantly affected by DNV (Table 1).

View this table: [in this window] [in a new window]   Table 1.   Body weights and optimal Diam fiber lengths in CTL and DNV rats

DNV-induced alterations in MHC isoform composition of the Dia_m. Postnatal transitions in MHC isoform composition of the Dia_m were altered in the DNV rats. During the first 3 wk after DNV, the relative expression of the MHC_neo isoform in the Dia_m was significantly higher in the DNV rats compared with CTL animals of the corresponding age (P < 0.05; Table 2). By D-28, in the CTL Dia_m, expression of the MHC_neo isoform was completely absent, whereas expression of the MHC_neo isoform persisted in the DNV animals. However, like CTL animals, expression of the MHC_neo isoform in the DNV Dia_m gradually decreased with postnatal development (P < 0.05).

View this table: [in this window] [in a new window]   Table 2.   Effect of DNV on MHC isoform composition of the rat Diam during early postnatal development

After 1 and 2 wk of DNV in the younger animals (i.e., D-14 and D-21), the relative expression of the MHC_slow isoform was lower in the DNV Dia_m compared with the corresponding age-matched CTL (P < 0.05; Table 2). In the D-28 rats, the relative expression of the MHC_slow isoform was comparable between CTL and DNV Dia_m.

The relative expression of the MHC_2A isoform was higher in the DNV Dia_m compared with CTL at D-14 (P < 0.05; Table 2). However, by D-21 and D-28, the relative expression of the MHC_2A isoform was lower in the DNV animals compared with the corresponding age-matched CTL (P < 0.05).

Expression of the MHC_2X isoform appeared only by D-14 in the CTL Dia_m. In contrast, after 1 wk of DNV, there was no expression of the MHC_2X isoform in the D-14 DNV Dia_m (Table 2). Expression of the MHC_2X isoform in the DNV Dia_m was delayed until D-21. In D-21 and D-28 animals, the relative expression of the MHC_2X isoform did not differ between DNV and CTL animals.

Similar to the expression of the MHC_2X isoform, emergence of the MHC_2B isoform did not occur until D-14 in the CTL Dia_m. Expression of the MHC_2B isoform was completely absent in the DNV Dia_m at D-14 (Table 2). At D-21 and D-28, the relative expression of the MHC_2B isoform was significantly lower in the DNV Dia_m compared with corresponding age-matched CTL (P < 0.05; Table 2).

The relative MHC isoform composition of the adult Dia_m was also altered by DNV (P < 0.05; Table 3). Two and three weeks after DNV, expression of the MHC_neo reappeared in the DNV Dia_m. The relative expression of the MHC_slow isoform in the Dia_m increased 1 and 2 wk after DNV compared with CTL animals (P < 0.05; Table 3). By the third week after DNV, the relative expression of the MHC_slow isoform was comparable between DNV and CTL Dia_m. The relative expression of the MHC_2X isoform decreased in the DNV Dia_m after 1 and 2 wk compared with CTL (P < 0.05; Table 3). By the third week after DNV, the relative expression of the MHC_2X isoform was comparable between DNV and CTL Dia_m. The relative expression of the MHC_2B isoform also decreased in the DNV Dia_m compared with CTL (P < 0.05; Table 3). This decrease in the relative expression of the MHC_2B isoform in the DNV Dia_m persisted by the third week after DNV.

View this table: [in this window] [in a new window]   Table 3.   Effect of DNV on MHC isoform composition of the adult rat Diam

DNV-induced alterations in Dia_m P_o and Vo. DNV significantly reduced the P_o of the developing Dia_m at each age (P < 0.05; Fig. 1A). From D-14 to D-28 in CTL animals, P_o increased by ~28% (P < 0.05; Fig. 1A), whereas in the DNV group, P_o remained constant at about one-half that of the CTL muscle. In CTL animals, the increase in P_o from D-14 to D-28 correlated with an increase in the relative expression of adult fast MHC (MHC_2A, MHC_2X and MHC_2B) isoforms (r² = 0.28; P < 0.05; Fig. 2A). In contrast, in the DNV animals, there was no correlation between the increase in adult fast MHC isoform expression and P_o (r² = 0.09, P > 0.05; Fig. 2A).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   The effects of denervation (DNV) on maximum tetanic force (Po) of the diaphragm muscle (Diam) for postnatal days 14-28 (D-14 to D-28; A) and in adults (B). Open bars, values (means ± SE; n = 6-8 for each group) from control (CTL) animals; solid bars, values from animals exposed to unilateral DNV for 1-3 wk.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Correlation between changes in Diam Po and the relative expression of adult fast myosin heavy chain (MHC) isoforms from D-14 to D-28 (A) and in adults (B). , , , Values from CTL animals; , , , values from DNV animals.

In the adult Dia_m, DNV also resulted in a decrease in P_o compared with CTL (P < 0.05; Fig. 1B). This DNV-related reduction in Dia_m P_o was comparable across the three time periods after DNV. There was also no correlation between the DNV-induced reduction in P_o and any change in MHC composition of the Dia_m (r² = 0.01, P > 0.05; Fig. 2B).

In CTL animals, V_o of the Dia_m nearly doubled between D-14 and D-28 (P < 0.05; Fig. 3A). In the DNV Dia_m, V_o was significantly slower than in CTL at each age (P < 0.05; Fig. 3A), and there was no age-related change. The progressive increase in Dia_m V_o in CTL animals correlated with the increase in relative composition of fast MHC isoforms (r² = 0.76; P < 0.05; Fig. 4A). In DNV animals, there was no correlation between Dia_m V_o and the relative composition of fast MHC isoforms (r² = 0.06, P > 0.05; Fig. 4A).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effects of DNV on maximum unloaded shortening velocity (Vo) of the DIAm from D-14 to D-28 (A) and in adults (B). Open bars, values (means ± SE; n = 6-8 for each group) from CTL animals; solid bars, values from animals exposed to unilateral DNV for 1-3 wk.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Correlation between changes in Diam Vo and the relative expression of adult fast MHC isoforms from D-14 to D-28 (A) and in adults (B). , , , Values from CTL animals; , , , values from DNV animals.

In adult animals, DNV was also associated with a slowing of Dia_m V_o, which did not depend on the duration of DNV (P < 0.05; Fig. 3B). This DNV-related slowing of Dia_m V_o was not correlated with any change in MHC isoform composition of the muscle (r² = 0.06, P > 0.05; Fig. 4B).

	DISCUSSION

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The results of the present study support our hypotheses that unilateral DNV 1) alters normal postnatal transitions in MHC isoform expression and 2) causes marked changes in contractile properties of the developing Dia_m. Although the pattern of DNV-induced alterations in MHC isoform expression were generally consistent with a reduction in P_o and a slowing of V_o, there does not appear to be a strong causal relationship between these changes. Unilateral DNV at D-7 did delay the expression of MHC_2X and MHC_2B isoforms and prolonged the expression of MHC_neo, but these alterations were disproportionate to the dramatic changes in Dia_m contractile properties induced by DNV. Thus, although alterations in MHC isoform expression may have contributed in part to the Dia_m contractile changes induced by DNV, other factors must be involved.

Postnatal transitions in MHC isoform expression. Previous studies have suggested that innervation is required for the repression of MHC_neo gene expression (16, 18, 44). This may account for the reexpression of MHC_neo that occurs after DNV in adult skeletal muscle (46). However, other studies have reported that the transition from MHC_neo to adult fast MHC isoform expression does not require intact innervation (3, 8, 35, 43). The results of the present study clearly indicate that intact innervation is not absolutely required for the postnatal transition in MHC isoform expression in the rat Dia_m. However, DNV at D-7 did prolong the expression of MHC_neo in the rat Dia_m, which lends support to a possible suppression of MHC_neo gene expression by factors emanating from phrenic motoneurons. It is possible that postnatal MHC isoform transitions ultimately depend on preprogrammed fiber phenotype (44) and that changes in the pattern of innervation (e.g., polyneuronal to single motoneuron innervation) or removal of neural influence only modulate the timing of this eventual transition.

Influence of DNV on muscle contractile properties. During early postnatal development of the rat Dia_m, there is a progressive increase in P_o and V_o that reaches adult values by ~D-28 (29, 51, 58, 64). The results of the present study clearly demonstrate that unilateral DNV causes a marked reduction in Dia_m P_o and a slowing of V_o, even in the youngest animals (D-14). Furthermore, the subsequent developmental increase in both P_o and V_o was completely blunted. These observations extend and confirm previous results in the adult Dia_m, in which a dramatic reduction in P_o and slowing of V_o was observed after unilateral DNV (33, 37, 61, 63).

In the adult Dia_m, we found that 2 wk of unilateral Dia_m paralysis induced by tetrodotoxin blockade of phrenic nerve action potential propagation also caused changes in P_o and V_o that were comparable to DNV (37, 63). In contrast, 2 wk of unilateral Dia_m paralysis induced by spinal cord hemisection at C₂ resulted in little, if any, change in P_o and V_o (37). It has been suggested that Dia_m adaptations after unilateral DNV result from passive mechanical strain imposed by continued inspiratory-related contractions of the intact contralateral side (27, 53, 60). However, the passive mechanical effects induced by paralysis of the right side of the Dia_m after C₂ spinal cord hemisection are entirely comparable to those induced by unilateral DNV and tetrodotoxin nerve blockade. Yet, the morphometric and contractile adaptations of the Dia_m were quite different across these models. Furthermore, in another study in the rabbit, our laboratory found that the sternal region of the paralyzed Dia_m passively shortened while the midcostal region was passively stretched by the continued inspiratory-related activation of the contralateral side. Despite these differences in passive strain between the two Dia_m regions after DNV, the morphometric and contractile adaptations were comparable (61). Based on these combined results, we conclude that the passive mechanical effects of unilateral paralysis per se do not underlie the contractile changes induced by DNV.

It is more likely that the DNV-induced contractile changes of the Dia_m result from the removal of a neurotrophic influence. It has been reported that injection of nerve extracts can attenuate DNV-induced atrophy of the rat extensor digitorum longus muscle (9). Similarly, it has been shown that ciliary neurotrophic factor can attenuate DNV-induced atrophy of the rat soleus muscle (26). Therefore, it appears that phrenic motoneurons may express a neurotrophic factor that maintains muscle fiber morphometry and possibly contractile properties.

Relationship between muscle contractile properties and MHC isoform expression. In single muscle fibers, a number of studies have demonstrated a relationship between MHC isoform expression and fiber contractile properties (2, 12, 20, 21, 39, 40, 45, 49, 50, 55). Based on the results of these studies, it is well accepted that muscle fibers expressing fast MHC isoforms generate greater V_o than fibers expressing MHC_slow or MHC_neo isoforms. The relationship between MHC isoform expression and P_o of single muscle fibers is more controversial. Some studies have reported no difference in P_o across fibers expressing MHC_slow and fast MHC isoforms (19, 36, 57), whereas other studies have reported that fibers expressing fast MHC isoforms generate greater P_o than fibers expressing MHC_slow (2, 12, 20, 21, 49, 50).

In mixed skeletal muscles, it has been shown that contractile properties correlate with the relative composition of MHC isoforms (5, 15, 29, 42, 58, 59). In the present study, we found that postnatal changes in P_o and V_o in the CTL Dia_m were correlated with the relative proportion of fast MHC isoforms comprising the muscle. These results are consistent with previous observations in the rat Dia_m (29, 51, 58, 59, 64). The postnatal changes in Dia_m, P_o, and V_o were blunted by DNV; yet transitions in MHC isoform expression in the Dia_m still occurred, albeit at a different rate compared with the normal postnatal transitions observed in CTL animals. Thus the DNV-induced changes in Dia_m contractile properties were not correlated with changes in MHC isoform composition.

In several previous studies, it has been reported that experimentally induced changes in muscle contractile properties are consistent with changes in MHC isoform composition, suggesting a cause and effect relationship. For example, after 2 wk of hindlimb suspension in the rat, V_o of the soleus muscle becomes faster, which is consistent with an increase in the relative expression of fast MHC isoforms (15). Similarly, after spinal cord transection at T₁₂-T₁₃, V_o of the cat soleus and medial gastrocnemeus muscles becomes faster, consistent with an increase in the relative expression of fast MHC isoforms (42). Conversely, in response to hypothyroidism, there is a slowing of V_o of the rat plantaris muscle and a decrease in the relative expression of fast MHC isoforms (5). In the rat Dia_m, our laboratory also found a dramatic slowing of V_o in response to hypothyroidism but very little change in the relative expression of fast MHC isoforms (24). In the developing rat Dia_m, our laboratory found that hypothyroidism caused a reduction in P_o and a slowing of V_o, which was consistent with, but directly proportional to, a small decrease in the relative expression of fast MHC isoforms (51). Thus, although alterations in MHC isoform composition may be consistent with contractile changes, the proportionality of these changes may be completely different. This raises important questions as to the actual cause and effect relationship between the concurrent changes in MHC isoform expression and contractile properties.

There are several alternative mechanisms by which DNV might have affected Dia_m contractile properties. Specific force is dependent on the number of cross bridges in parallel, the recruitment of cross bridges in response to elevated intracellular calcium concentration, and cross-bridge cycling kinetics (10, 56). DNV has been shown to influence protein synthesis (6, 32, 34) and fiber cross-sectional area (23, 37, 60-63). Thus an effect on the number of cross bridges in parallel (e.g., MHC protein content per half sarcomere) is a possibility. It is also possible that DNV influenced excitation-contraction coupling by affecting either the elevation in intracellular calcium concentration or the Ca²⁺ sensitivity of force generation (11, 14, 47). Finally, the slowing of V_o clearly reflects an effect of DNV on cross-bridge cycling kinetics. Future studies are needed to elucidate the underlying mechanisms for the DNV-induced alterations in Dia_m contractile properties.

	ACKNOWLEDGEMENTS

We are grateful to Dr. David Megirian for assistance with the experiments and Dr. Y.S. Prakash for comments on the manuscript and assistance with some of the studies.

	FOOTNOTES

This research was supported by National Heart, Lung, and Blood Institute Grants HL-34817 and HL-37680.

Address for reprint requests and other correspondence: G. C. Sieck, Anesthesia Research, Mayo Clinic, Rochester, MN 55905 (E-mail: sieck.gary{at}mayo.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. §1734 solely to indicate this fact.

Received 20 July 1999; accepted in final form 27 April 2000.

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