INTRODUCTION

Top Abstract Introduction METHODS RESULTS DISCUSSION References

THE MOST WIDELY USED ANIMAL model for exploring the pathophysiology of genetic hypertension in humans is the spontaneously hypertensive rat (SHR), which was originally established by Okamoto and Aoki (20) from a colony of normotensive Wistar rats [Wistar-Kyoto (WKY)]. According to Pickar et al. (23), in SHR the development of hypertension occurs in three stages: the rising of blood pressure (4-8 wk), the arrival of blood pressure to peak value (14-18 wk), and the stabilization of blood pressure and the appearance of adaptive changes in various organs (24 wk).

Although there is general agreement that skeletal muscles of hypertensive humans contain a higher percentage of fast-twitch fibers than the normotensive controls (10, 15), the fiber type composition of skeletal muscles from SHRs is the subject of an ongoing controversy. Thus Gray and colleagues (2, 11) have reported that soleus muscles from 6- to 28-wk-old SHRs contain more slow-twitch (type I) and fewer fast-twitch (type II) fibers than the homologous muscles of age-matched WKY rats. In contrast, Benbachir-Lamrini et al. (3, 4) and Lewis et al. (19) have reported that soleus muscles from SHRs aged 4-17 wk contain fewer type I fibers and more type II fibers than the soleus of age-matched WKY rats.

It is important to note that all three groups cited above determined the fiber type composition of soleus muscles from SHR and WKY rats using myosin ATPase-based (2-4, 11, 19) and succinate dehydrogenase-based (2, 11) histochemistry. However, as it has been stated previously, both enzyme-based histochemistry (14) and immunohistochemistry (5) are strategies of limited suitability for studying fiber type composition in transforming muscles, because they cannot convincingly identify fibers coexpressing two or three myosin heavy chain (MHC) isoforms (hybrid fibers). More recently, analysis of muscle proteins from whole muscles and from single muscle fiber segments using SDS-PAGE has become the preferred strategy in research concerned with the effect of various conditions on myosin isoform expression/fiber type composition in skeletal muscle. Indeed, the major advantage of this latter strategy is that it can rapidly detect and quantitate MHC isoform expression in whole muscles as well as in hybrid fibers (21, 22). Furthermore, this strategy allows detection, at the whole muscle or single fiber level, of changes in myosin light chain (MLC) isoforms expression (21, 22, 25) that can also occur in transforming muscles.

In the present study we used the microelectrophoretic methodology to compare the MHC isoform, MLC2 isoform, and fiber type composition of soleus muscles from SHRs and WKY rats, at the three stages of development of hypertension defined by Pickar et al. (23). A secondary aim of the study was to assess whether the reduced specific tetanic force and contractile kinetics parameters reported by Gray and colleagues (2, 7, 12, 13) and Lewis et al. (19) for SHR soleus are due in part to functional alterations of the myofibrillar compartment. For this purpose we used the skinned muscle preparation, which allows direct activation of the myofibrillar compartment in solutions of carefully controlled free calcium concentrations (26, 28, 29).

	METHODS

Top Abstract Introduction METHODS RESULTS DISCUSSION References

Animals. The male normotensive WKY rats and SHRs used in this study were bred at the Baker Medical Research Institute (Melbourne, Victoria, Australia) from stock obtained in 1985 from one of the original breeders of SHR (Y. Yamori) at the Shimane Institute of Health Science. The rats were housed in a temperature-controlled environment (22°C) with a 12:12-h light-dark cycle and had access to food and water ad libitum. Before and on the day of the experiment, systolic blood pressure was measured in conscious rats by tail-cuff plethysmography (IITC Life Science Instruments) while the animals were in a restriction chamber at 27°C (9, 16). The animals used for this study belonged to three age groups, representing the three stages in the development of hypertension (23): 1) the rising phase of blood pressure, before the onset of hypertension (4 wk), 2) the arrival of blood pressure to its peak value at the onset of hypertension (16-18 wk), and 3) the maintenance of blood pressure at elevated levels (24 wk). On the day of the experiment, rats were killed by deep halothane inhalation, and the soleus muscles were quickly removed, blotted dry on filter paper, weighed, and placed under paraffin oil saturated with water. One muscle was used for single fiber analyses and the contralateral muscle was used for preparing whole muscle homogenate.

Preparation of skinned, single muscle fiber segments. Single fibers were isolated randomly under paraffin oil using a dissecting microscope, fine jewelers forceps, and iris scissors, as described in detail elsewhere (26). The paraffin oil plays three important roles: 1) it facilitates the visualization of the single fiber by having a different refractive index, 2) it precludes fiber swelling/fiber-water loss when fiber segments are prepared, and 3) it confers the fiber a quasicircular cross-sectional area through the surface tension exerted on the fiber at the fiber/oil interface. A video camera-monitor system (Olympus) was used to measure the length and the width of the fiber segment in at least three places along its length, while still under paraffin oil. The volume of a fiber segment was calculated assuming it to be a cylinder with a diameter equal to the mean value of the fiber width. Some fiber segments were examined for calcium-activation characteristics as well as MHC composition, whereas others were examined for MHC composition only.

Calcium-activation characteristics of chemically skinned, single muscle fibers. Single fiber segments were mounted, at slack length, under oil, between a force transducer (Sensonor, Horten, Norway) and a pair of fine Barcroft forceps or were incubated directly in SDS solubilizing buffer. In this study, the slack length of a fiber segment was obtained by releasing the slightly stretched fiber segment until no measurable force could be detected (<2.5 µN) and the preparation was just "taut." After mounting, the fiber segment was incubated for 10 min in a relaxing solution (solution A; see composition below) containing 2% Triton, a strategy known to disrupt cellular membranes without affecting the contractile system (27). While in this solution, the average sarcomere length was measured from the diffraction maxima of a HeNe laser beam illuminating the mounted preparation as described previously (28). At slack length, the average sarcomere length of the preparation was 2.65 ± 0.02 µm (n = 45) for fibers from the 16- and 24-wk-old rats. This corresponds to the "optimum" sarcomere length for maximum Ca²⁺ force development in skinned rat muscle fibers from adult rats (28). The "slack" sarcomere length of fibers segments from the 4-wk-old rats was 3.14 ± 0.01 µm (n = 12). Preliminary experiments showed that at this sarcomere length, the maximum Ca²⁺-activated force developed in two fiber preparations from 4-wk-old rats was greater than that produced when the fibers were allowed to shorten to a sarcomere length of 2.7 µm.

The calcium-activation characteristics of single muscle fiber segments were determined from force responses developed at 21 ± 2°C, in a series of strongly buffered Ca²⁺ solutions prepared as described in detail elsewhere (26, 29). All solutions contained (in mM) 117 K⁺, 36 Na⁺, 1 Mg²⁺ (free), 90 HEPES, 8 total ATP, 10 creatine phosphate, and 1 sodium azide, pH 7.10 ± 0.01 at 22°C. The relaxing solution (solution A) contained 50 mM EGTA² (pCa = log [Ca²⁺]  9), the maximally Ca²⁺-activating solution (solution B) contained 49.8 mM CaEGTA² and 0.2 mM excess EGTA² (pCa = 4.3), and the preactivating solution (solution C) contained 49.75 mM hexamethylenediamine tetraacetate (HDTA²) and 0.25 mM EGTA² (pCa  8). A series of solutions with a range of pCa values of 6.5-4.3 was obtained by mixing solutions A and B in different proportions. The osmolality of all solutions was 295 ± 2 mosmol/kg. Before exposure to Ca²⁺-activating solutions, the fiber segment was incubated in solution C. Each fiber preparation was first maximally activated at pCa 4.3 (solution B), then relaxed in solution A (pCa 9), equilibrated in the preactivating solution, and then exposed to a set of 10 activating solutions in decreasing order of pCa values (6.5 to 4.3). In this study, the second maximum Ca²⁺-activated force response was on average 8.19% (±5.74 SD; range 0.4-21%) less than the first response. For calculating the submaximal level of activation in a solution of a particular pCa value, the steady level of force recorded in that solution was divided by the interpolated value of the maximum Ca²⁺-activated force response as described in detail elsewhere (24). This strategy corrects for the small degree of deterioration in force production associated with the repeated activation of the skinned fiber preparation.

The following Ca²⁺-activation characteristics were determined for each individual fiber segment from the isometric force-pCa curves: 1) maximum Ca²⁺-activated force per cross-sectional area (CaF_max/CSA; kN/m²), determined from the amplitude of the first force response of a fiber in the maximally Ca²⁺-activating solution and from its cross-sectional area; 2) pCa₅₀ (representing the pCa where 50% maximal force was produced); and 3) the Hill coefficient n_Ca, which relates to the steepness of the force-pCa curve, described by the theoretical relation

(1)

where P_t is the fraction of maximal force developed by the fiber segment at a given Ca²⁺ concentration. For each force-pCa curve, the values of pCa₅₀ and n_Ca were determined using the nonlinear regression analysis option of the GraphPad Prism software.

SDS-PAGE analysis of MHC and MLC isoforms. MHC isoform analyses were performed on whole muscle homogenates and single muscle fiber segments, whereas MLC isoform analyses were performed on whole muscle homogenates only. Whole muscles were homogenized in six volumes of solution A, and protein concentration was determined using the Bradford protein assay (6). Each homogenate was diluted with SDS-PAGE solubilizing buffer (62.5 mM Tris, 2.3% SDS, 5% -mercaptoethanol, 12.5% glycerol, 13.6% sucrose, 0.01% Bromophenol blue, 0.1 mM phenylmethylsulfonyl fluoride, 2 µM leupeptin, and 1 µM pepstatin) and boiled for 5 min. The single fibers to be analyzed by SDS-PAGE were placed in 12 µl SDS-PAGE solubilizing buffer either immediately after dissection under paraffin oil or after being first examined for contractile characteristics as described above. In both cases, the single muscle fiber segments were incubated in the solubilizing buffer for 24 h at room temperature and then were boiled for 5 min. MHC isoforms in whole muscle homogenates and single muscle fibers were analyzed on 0.75-mm-thick slab gels using the Hoefer Mighty Small gel apparatus and the Laemmli procedure (18) with minor modifications. Briefly, the separating gel contained 6.7 or 7.9% acrylamide, 0.15 or 0.096% bis-acrylamide, 375 or 425 mM Tris pH 8.8, 40% (vol/vol) glycerol, and 0.1 or 0.3% (wt/vol) SDS and was polymerized with ammonium persulfate and N,N,N',N'-tetramethylethylenediamine. The stacking gel contained 4% acrylamide and the running buffer contained 32.5 mM Tris, 288 mM glycine, and 0.1% SDS, and the gels were run for 24 h at 16°C and at constant voltage (100-120 V). A 6-µl sample aliquot containing 0.05 mg/ml muscle protein or 0.025-0.05 nl fiber/µl was applied per electrophoretic well. The gels were stained using Bio-Rad Silver Stain Plus. The MHC isoforms were identified based on data reported in the literature and on SDS-PAGE analysis of MHC isoforms in rat muscles containing one dominant isoform: soleus (MHCI), diaphragm (MHCIID), and levator ani (MHCIIB) (14). The nomenclature used for fiber types (e.g., IIA, IID/X) and for MHC isoforms (e.g., IIa, IId/x) follows that of Pette and Staron (21, 22).

Low-molecular-weight myofibrillar proteins were separated on SDS-PAGE using a separating gel containing 18% acrylamide, 0.09% bis-acrylamide, 0.75 M Tris pH 9.3, 10% glycerol, 0.1% SDS, a stacking gel containing 4% acrylamide, and a running buffer containing 50 mM Tris, 380 mM glycine, and 0.1% SDS. For each homogenate, a 10-µl sample containing 0.15 mg protein/ml was loaded per electrophoretic well. Gels were run at a constant current (10 mA/gel) until the lowest molecular weight marker reached the bottom of the gel (4-5 h) and then were stained with Pharmacia Biotech silver stain. The MLC2 band was identified on the basis of data reported in literature, of the electrophoretic mobility of purified rabbit MLC2 (gift from Dr. T. Walsh, QUT, Australia), and of SDS-PAGE analysis of low-molecular-weight myofibrillar proteins in segments of single muscle fibers containing only one fast or one slow MHC isoform.

Estimation of MHC isoform and MLC2 isoform composition in whole muscle homogenates. The gels for both high- and low-molecular-weight proteins were analyzed using a Molecular Dynamics Personal Densitometer and the volumetric quantitation method, with background correction provided by the ImageQuaNT software version 4.1 (Molecular Dynamics).

View larger version (15K): [in this window] [in a new window]   View larger version (14K): [in this window] [in a new window]   Fig. 1.   A: relationship between OD1MHCi (computed difference between optical density of the protein band corresponding to MHCi isoform and that of the background) expressed in arbitrary units and the total protein concentration in the homogenate sample. B: relationship between OD2MHCi (an optical parameter derived from OD1MHCi as explained in METHODS) expressed in arbitrary units and the total protein concentration in the homogenate sample. , MHCI; , MHCIIa; x, MHCIId/x.

(2)

(3)

(4)

(5)

(6)

	RESULTS

Top Abstract Introduction METHODS RESULTS DISCUSSION References

Physical characteristics. In Table 1 are shown the average systolic blood pressure (BP) readings for the WKY rats and SHRs examined in this study. The 4-wk-old SHRs had a mean BP value of 105.6 mmHg, which was 25% higher than that of the age-matched WKY (84.8 mmHg), but was 28% lower than the mean BP value for the 24-wk-old normotensive rats (146.0 mmHg). In contrast, the more mature SHRs (16 and 24 wk old) had BP values that were 43-56% higher than the BP values for the age-matched WKY and markedly higher than the upper limit for normotensive animals. The highest average BP value for normotensive WKY rats was 146 mmHg for the 24-wk-old animals, which is in full agreement with the highest average BP value of 150 mmHg reported for WKY rats aged 4-52 wk (4). Because the highest BP value recorded in WKY rats in this study was 165 mmHg, all rats that had a BP reading 165 mmHg were regarded as normotensive.

The body weight of SHRs at 4 and 24 wk of age was not significantly different from that of age-matched WKY rats, whereas the 16-wk-old SHRs were slightly (8%) but significantly heavier (P < 0.05) than the age-matched normotensive rats (Table 1). The mean soleus muscle wet weight per body weight values did not change with age in either of the two strains examined. The mean muscle wet weight per body weight values were, however, significantly higher in SHR than in WKY rats at all three ages examined.

MHC isoform composition of whole muscles. Electrophoretic analyses of whole tissue homogenates prepared from WKY rat and SHR soleus muscles revealed three high-molecular-weight protein bands of decreasing electrophoretic mobility, corresponding to slow MHCI, fast MHCIId/x, and fast MHCIIa (Fig. 2). It is important to note for the same amount of protein applied, the SHR soleus sample displayed MHCI, MHCIIa, and MHCIId/x bands whereas the WKY soleus sample displayed only MHCI and MHCIIa. The percentages of the MHC isoforms in muscle homogenates of 4-, 16-, and 24-wk-old SHRs and WKY rats are shown in Table 2.

View larger version (35K): [in this window] [in a new window]   Fig. 2.   Representative electrophoretic profile of MHC isoforms detected in soleus muscles from 24-wk-old Wistar-Kyoto (WKY) rats (lane 2) and spontaneously hypertensive rats (SHRs) (lane 3). Whole tissue homogenates were prepared from WKY rat and SHR soleus muscles, and proteins were analyzed by SDS-PAGE as described in METHODS. Lane 1: laboratory MHC isoform marker containing the 4 MHC isoforms known to be expressed by adult rat hindlimb skeletal muscles: MHCI, fast MHCIIb, fast MHCIId/x, and fast MHCIIa. Amount of protein applied per electrophoretic well: 0.3 µg.

At 4 wk of age, the soleus muscle from SHRs contained a lower proportion of MHCI (14.2%) and a higher proportion of MHCIId/x (+12.5%) than the soleus muscle from the age-matched WKY rats. The proportion of MHCIIa isoform was, however, similar in SHRs and WKY rats.

An increase in age from 4 to 24 wk was accompanied by clear changes in the relative proportion of MHC isoforms in the soleus muscles of both SHRs and WKY rats. The direction of these changes was MHC isoform dependent, with the percentage of MHCI isoform increasing and the percentage of MHCII isoforms decreasing with age in both strains. For all MHC isoforms, the major age-related changes occurred during the rat development from 4 to 16 wk. Thus the proportion of MHCI isoform increased between 4 and 16 wk, by 20.1% in WKY and 15.0% in SHR, but it did not change significantly between 16 and 24 wk of age in either strain. The proportion of MHCIIa decreased significantly (by 18.6%) in WKY between 4 and 16 wk, but did not change in SHR. Between 16 and 24 wk no significant change was detected in the proportion of MHCIIa in either strain.

Finally, the proportion of MHCIId/x in WKY rats decreased from low (1.5% at 4 wk) to nondetectable levels at 16 and 24 wk, whereas in the SHR it decreased from 14.0% at 4 wk to ~2.4% at 16 wk and this level remained unchanged in 24-wk-old rats. In summary, at all ages examined, the soleus muscle from SHRs contained a statistically significant higher proportion of fast MHC isoforms (comprising both MHCIIa and MHCIId/x) and a lower proportion of slow MHC isoform than soleus muscle from age-matched WKY rats.

MLC2 isoform composition of whole muscles. Each myosin molecule contains two regulatory light chains (MLC2) that exist in two isoforms: MLC2f (fast isoform) and MLC2s (slow isoform). In SDS gels, MLC2f has a higher electrophoretic mobility than MLC2s (Fig. 3A). In agreement with the MHC isoform data, soleus muscles from SHRs were found to contain a statistically significant lower proportion of MLC2s and a higher proportion of MLC2f than their WKY counterparts at all ages examined. This can be seen also in Fig. 3. In Fig. 3A are shown side-by-side two representative electrophoretograms of low-molecular-weight proteins produced by SDS-PAGE analyses of SHR and WKY soleus muscle homogenates from 24-wk-old rats, and the bar graph in Fig. 3B illustrates the differences between the calculated values for the ratio MLC2s/MLC2_t (where MLC2_t = MLC2s + MLC2f) in soleus muscles of 4-, 16-, and 24-wk-old SHRs and WKY rats.

View larger version (36K): [in this window] [in a new window]   View larger version (18K): [in this window] [in a new window]   Fig. 3.   A: representative electrophoretogram of low-molecular-mass (<45 kDa) muscle proteins from 24-wk-old WKY rat (lane 1) and SHR (lane 2) soleus muscles. Arrows indicate the bands corresponding to actin, slow MLC2 isoform (MLC2s), and fast MLC2 isoform (MLC2f). B: bar chart for ratio MLC2s/MLC2t (where MLC2t = MLC2s + MLC2f) in soleus muscles of 4-, 16-, and 24-wk-old SHRs (filled bars) and WKY (open bars) rats. # Statistically significant difference between 4-wk-old and adult (16 wk and 24 wk) rats of same strain. * Statistically significant difference between SHR soleus and age-matched WKY rat soleus. Amount of protein applied per electrophoretic well: 3 µg.

Fiber type populations yielded by random dissection. Electrophoretic analyses of MHC isoform composition in muscle homogenates prepared from soleus muscles of SHRs and WKY rats provide very useful information on strain-related differences in MHC expression at the whole muscle level, but give no indication on the types of fibers that make up the muscles compared. To obtain this information, microelectrophoretic analyses of MHC isoform expression were performed in segments of single fibers, randomly dissected from soleus muscles of SHRs and WKY rats. These analyses showed that soleus muscles from 4-, 16-, and 24-wk-old WKY rats and age-matched SHRs contain four fiber types: I (pure slow; containing MHCI), I+IIA (hybrid slow/fast, containing different proportions of MHCI and MHCIIa), IIA (pure fast, containing MHCIIa), and IIA+IID (hybrid fast/fast containing different proportions of MHCIIa and MHCIId/x) (Fig. 4). In agreement with the MHC isoform composition data shown in Table 2, no fibers containing the MHCIIb isoform were detected in any of the muscles examined.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Representative electrophoretograms of MHC isoform composition of fiber types yielded, on random dissection, by soleus muscles of SHRs and WKY rats. Lane 1: laboratory marker containing the 4 MHC isoforms known to be expressed by adult rat hindlimb skeletal muscles: MHCI, fast MHCIIb, fast MHCIId/x, and fast MHCIIa. Lane 2: slow fiber (type I). Lane 3: hybrid slow/fast fiber (type I+IIA). Lane 4: fast fiber (type IIA). Lane 5: hybrid fast/fast fiber (type IIA+IID). Sample size: 0.3 nl fiber per electrophoretic well. Details regarding the preparation of single fibers, their solubilization in SDS-PAGE solubilizing buffer, and their microelectrophoretic analysis by SDS-PAGE are described in METHODS.

In Table 3 are shown, for each strain- and age-fiber group examined, the proportions of each fiber type, expressed as percent of the number of fibers examined per rat (%f_R), the actual number of fibers, and percent of the total number of fibers examined per age group (%f_T). The data presented in Table 3 were generated by analyses of 472 randomly dissected fibers (minimum 10 fibers/rat and 73 fibers/age group). It is important to note that a relatively large number of hybrid fibers were detected among the fiber segments dissected from the soleus muscle of SHRs of all ages and from soleus muscles of 4-wk-old WKY rats.

The proportions of type I and type I+IIA fibers isolated from the soleus muscles of 4-wk-old SHR were, on average, 1.4 and 1.9 times lower than those for 4-wk-old WKY rats. However, the proportion of type IIA fibers isolated from 4-wk-old SHR soleus muscle was 4.9 times greater than that for soleus muscle of age-matched WKY. Moreover, ~13% of the fibers dissected from the 4-wk-old SHR soleus muscles were type IIA+IID fibers, whereas no such fibers were found among the fibers dissected from the age-matched WKY soleus.

An increase in age was found to be accompanied, in the two strains, by an increase in the proportion of slow (type I) fibers and a general decrease in the proportion of fast (type IIA, I+IIA, and IIA+IID) fibers. It is important to note, however, that the proportion of IIA fibers found in the soleus muscle of 24-wk-old SHRs was significantly greater than that in the age-matched WKY rats. Moreover, the 24-wk-old SHR soleus produced a sizable proportion of hybrid fibers (8.1% type IIA+IID and 4.9% I+IIA), whereas no fibers of these two types were detected among the fibers sampled from the soleus muscle of 24-wk-old WKY rats. As seen in Table 3, the most striking difference with respect to the proportion of fiber types produced by soleus muscles from SHRs and WKY rats relates to the significantly larger proportion of fast fibers (pure type IIA and hybrid fast/fast type IIA+IID) and hybrid fibers (types I+IIA and IIA+IID) detected among the fibers sampled from soleus muscles of SHR of all ages examined.

Correlation between MHC isoform composition, MLC2 composition, and fiber type populations. As shown in Fig. 5A, the MHCI composition (%MHCI) of all muscles examined in this study was directly proportional to their relative content of MLC2s (MLC2s/MLC2_t). Furthermore, for all muscles analyzed, there was a tight correlation between the relative content of each MHC isoform detected (%MHCi) in the muscle and the proportion of corresponding fiber type derived from the analysis of single fiber segments obtained by random microdissection of the contralateral soleus muscle (Fig. 5B). When calculating the proportion of fibers corresponding to each MHC isoform (i.e., MHCI, MHCIIa, and MHCIId/x) represented in the muscles examined, the hybrid fibers that expressed two MHC isoforms (i.e., fiber types I+IIA and IIA+IID) were apportioned with equal weights to each of the two respective MHCi pools. The good correlation found in this study between the MHC isoform composition of the whole soleus muscle and the proportion of fiber types produced by single fiber dissection indicates that, when the sample size is sufficiently large, the proportion of fiber types in the sample of dissected fibers is a reasonable approximation of the fiber type composition of the whole muscle.

View larger version (9K): [in this window] [in a new window]   View larger version (10K): [in this window] [in a new window]   Fig. 5.   A: relationship between the proportion of MLC2s isoform (MLC2s/MLC2t) and MHCI isoform (%MHCI) in soleus muscles from SHRs and WKY rats. Data points, representing mean values for 6-8 rats, were obtained from Table 2 and Fig. 3B. B: correlation between relative content of each MHC isoform detected (%MHCi) in soleus muscle and proportion of corresponding fiber type derived from analysis of single fiber segments obtained by random microdissection of the contralateral soleus muscle. In calculating proportion of fibers corresponding to each MHC isoform (i.e., MHCI, MHCIIa, and MHCIId) represented in the muscles examined, hybrid fibers that expressed 2 MHC isoforms (i.e., fiber types I+IIA and IIA+IID) were apportioned with equal weights to each of the 2 respective MHCi pools.

Calcium-activation characteristics of single fibers. Fiber diameter, CaF_max/CSA, sensitivity to Ca²⁺ of the isometric force response (pCa₅₀), and the degree of cooperativity within the myofibrillar compartment with respect to calcium regulation of the contractile response (n_Ca) were determined as described in the methods for individual fiber segments isolated from soleus muscles of 4-, 16-, and 24-wk-old WKY and age-matched SHR before electrophoretic analyses. The results are summarized in Tables 4-6.

As seen in Table 4, for each strain and each fiber type group that allowed a meaningful statistical analysis (at least 3 data points per sample), an increase in rat age was accompanied by a statistically significant increase in fiber diameter. Also, the diameter of type I and type IIA fibers from 16-wk-old SHRs appeared to be slightly but significantly larger than that of age-matched controls. The lack of statistically significant differences between diameter values of other groups of fibers may be due to the low number of fibers in the respective groups.

The increase in age from 4 to 16 wk was accompanied by a statistically significant increase in the CaF_max/CSA developed by type I fibers from the soleus muscle of WKY rats (+35%) and SHR (+57%) (see Table 4). No further significant increase in average CaF_max/CSA was observed for either strain when the rat age increased from 16 to 24 wk. A similar trend was observed for all other fiber type groups.

For each fiber type group, CaF_max/CSA developed by SHR soleus muscle fibers were not significantly different from those recorded for the age-matched WKY at all three ages examined. Assuming that the force per cross bridge does not change, this suggests that there were no differences in the number of cross bridges per one-half sarcomere length per cross-sectional area.

Because SHR soleus contained a larger percentage of MHCIIa than MHCI and yielded a larger proportion of type IIA fibers than the WKY rat soleus (see Tables 2 and 3), it was of interest to examine whether type IIA fibers developed a lower CaF_max/CSA than the type I fibers, which may explain in part the lower specific tetanic force reported in the literature (2, 7, 12, 13, 19) for SHR soleus. The comparison of the pooled data for type I and type IIA fibers from SHR and WKY rat soleus showed that type IIA fibers developed a statistically significantly lower CaF_max/CSA (P < 0.02; Mann-Whitney test) than type I fibers for 4-wk-old rats, but not for the other age groups.

Table 5 shows the summary of the Ca²⁺-sensitivity data. Types IIA and IIA+IID fibers showed a significantly lower sensitivity to Ca²⁺ by a factor of 1.3 (lower pCa₅₀ values by ~0.1) than type I fibers. Interestingly, hybrid (I+IIA) fibers exhibited either pCa₅₀ values characteristic of type I fibers (4- and 16-wk-old WKY rats) or of type IIA (4-wk-old SHRs) fibers. The hybrid fast fibers (type IIA+IID) displayed Ca²⁺ sensitivities similar to those of pure type IIA fibers. For all fiber type groups, no significant differences were observed between the average pCa₅₀ values recorded for single fibers of the same type from soleus muscles of WKY rats and age-matched SHRs. Assuming that the length of the myosin and actin filaments does not change with age, the consistently higher pCa₅₀ values by 0.1 log units noted for all fiber types from 4-wk-old animals of both strains may be related to differences in sarcomere length or interfilament distance (28).

The degree of cooperativity within the myofibrillar compartment with respect to contractile activation is indicated by the value of the Hill coefficient (see METHODS). As shown in Table 6, type I fibers have a lower Hill coefficient than the fibers containing fast MHC isoforms (type IIA and IIA+IID). The hybrid type (I+IIA) fibers displayed Hill coefficients that were characteristic of either type I or type IIA fibers. No significant age-related differences were detected between the n_Ca values for soleus fibers from either strain or between the n_Ca values for soleus fibers from SHRs and WKY rats.

	DISCUSSION

Top Abstract Introduction METHODS RESULTS DISCUSSION References

This is the first study in which soleus muscles from SHRs and normotensive (WKY) rats, at three stages in the development of high blood pressure, were examined with respect to MHC isoform expression (%MHCi), MLC2 isoform expression (MLC2s/MLC2_t), and fiber type populations (%f_T), using electrophoretic analyses of muscle proteins from whole muscle homogenates and segments of randomly dissected single muscle fibers. For all muscles examined, the data produced by these three independent electrophoretic methods were closely correlated (Fig. 5). Calcium-activation properties of the contractile apparatus in electrophoretically typed fibers from soleus muscles of the two strains were also determined using permeabilized single fiber preparations.

There was good agreement between the MHC composition of soleus muscle from WKY rats found in this study by SDS-PAGE and the MHC/fiber type composition of soleus muscle from normotensive rats of similar age, determined by others using histochemical techniques. For example, the MHC composition of soleus muscles from 24-wk-old WKY rats found in this study (MHCI: 89%; MHCIIA: 11%) (Table 2) is in close agreement with the fiber type composition reported by Armstrong and Phelps (1) for 25- to 26-wk-old Sprague-Dawley rats (87% type I fibers, 13% type IIA fibers), by Kovanen (17) for 25-wk-old Wistar rats (90% type I fibers, 10% type IIA fibers), and by Lewis et al. (19) for 12- to 17-wk-old WKY rats (93% type I fibers, 7% type IIA fibers).

A major conclusion from this study is that at all stages in the development of hypertension, soleus muscles of SHRs contain a statistically significantly higher proportion of MHCII and MLC2f isoforms than soleus muscles from age-matched WKY rats. This conclusion was further supported by the data obtained from the fiber type population study, which showed that random sampling of single fiber segments from the SHR soleus produced a higher proportion of fibers expressing MHCII isoforms (type IIA; type I + IIA, and type IIA+IID) than from soleus of age-matched WKY rats. As calculated from the data in Table 3, the average percentages of type I and type I+IIA fibers sampled from the soleus muscle of 4-wk-old SHRs were 1.4 times and 1.9 times lower than those from the age-matched WKY soleus. A similar difference in the proportion of type I and type IIc/IntI (probably type I+IIA) fibers was reported for soleus muscles from 4-wk-old SHRs and WKY rats by Benbachir-Lamrini et al. (3, 4). By comparison, in the present study it was found that soleus muscles from 4-wk-old SHRs produced approximately eight times more fast fibers than the soleus from the age-matched WKY rats. A major contributor to this difference was the subpopulation of hybrid fast/fast (IIA+IID) fibers, which amounted to 13.3% of the total number of fibers examined for this SHR age group, but was not detected in the fiber population dissected from WKY muscle. In the studies of Benbachir-Lamrini et al. (3, 4), the 4-wk-old SHR soleus contained only 1.7-2 times more type IIA fibers than the age-matched WKY soleus and displayed no IIA+IID fibers. We attribute this difference between our and Benbachir-Lamrini's results to the higher sensitivity and resolving power of the microelectrophoretic method particularly with respect to hybrid fibers (5). An increase in age from 4 to 24 wk was accompanied by an increase in the proportion of type I fibers and a decrease in the proportion of type II fibers produced by soleus muscles from both rat strains. As a result of the decrease in fast fibers, the 24-wk-old WKY soleus yielded only 2.5% fast fibers (type IIA), whereas the 24-wk-old SHR soleus yielded ~21% fast fibers (comprising both IIA and IIA+IID fiber types) (Table 3). The higher proportion of fast fibers sampled from SHR soleus throughout maturation observed in this study is in agreement with the data obtained by Benbachir-Lamrini et al. for 8- to 10-wk-old (3) and 12-wk-old (4) animals and by Lewis et al. (19) for 12- to 17-wk-old rats, but strongly disagrees with the data of Atrakchi et al. for 16- to 18-wk old rats (2) and Gray for 24- to 28-wk-old rats (11).

The microelectrophoretic analyses of single muscle fibers in this study have provided valuable new information concerning the presence and the percentage of hybrid fibers (I+IIA and IIA+IID) in soleus muscles from SHRs and WKY rats. As indicated by Hämäläinen and Pette (14) and Bottinelli et al. (5), identification of these fibers can be achieved by microelectrophoretic methodology but not by histochemical or immunohistochemical techniques. In this study it was found that at 4 wk of age, WKY rat soleus yielded almost twice as many hybrid slow/fast fibers (I+IIA) as the SHR soleus; however, this population of hybrid fibers decreased quite dramatically to nondetectable levels with rat maturation. In contrast, maturation in SHRs was accompanied by only a slight decrease in the percentage of type I+IIA fibers sampled from soleus muscle. Furthermore, according to our data, soleus muscles from SHR of all ages produced a sizable proportion of hybrid fast/fast fibers (IIA+IID), ranging from ~13% at 4 wk to ~8% at 24 wk, whereas only a very small proportion of such fibers (~1%) was yielded by soleus of 16-wk-old WKY rats. The presence of hybrid fibers is regarded by Pette and Staron (22) as an indicator of muscle transformation and their MHC composition as an indicator of the direction of fiber type transition. Seen in this context, the size of the hybrid fiber population and the combination of MHC isoforms detected in soleus muscles from SHRs at the three ages examined suggest that in SHR soleus, there is an active process of fiber type transition in the direction IIDIIAI. This process takes place not only in the very young rats but also persists in mature animals. In contrast, WKY rat soleus appears to undergo fiber transition (in the direction IIAI) only in young rats (from 4 to 16 wk of age). Thus, with respect to fiber transformation, the adult SHR soleus resembles more the soleus from 4-wk-old WKY rats than that of the adult WKY rat, suggesting that many fibers of the adult SHR soleus muscle are still subjected to a process of transition.

The higher percentage of fast fibers and hybrid fibers in SHR than in WKY rat soleus muscle is in full agreement with previous reports that skeletal muscles of hypertensive humans contain a higher percentage of fast-twitch fibers than the normotensive controls (10, 15). One can argue that this characteristic may not be related to high blood pressure because it occurred in 4-wk-old SHRs before the onset of hypertension. However, one cannot rule out a causal relationship between hypertension and MHC expression in the soleus muscle because it is clear from the present study that the BP of the 4-wk-old SHR was significantly elevated compared with the age-matched controls (Table 1).

In this study we did not find any statistically significant difference between single muscle fibers of the same type from SHR and WKY soleus muscles with respect to the CaF_max/CSA and the sensitivity to Ca²⁺ of the contractile machinery. Furthermore, the degree of cooperativity of the contractile apparatus and Ca²⁺-regulatory system with respect to Ca²⁺ regulation of the contractile response, expressed by the Hill coefficient, was similar in single fibers of the same type from soleus muscles of SHRs and WKY rats. Nevertheless, the pooled data for type I and type IIA fibers from SHR and WKY rat soleus showed that, in soleus muscles of 4-wk-old animals, there was a clear tendency for the type IIA fibers to develop lower CaF_max/CSA compared with the type I fibers (69.2 ± 15.0 kN/m², n = 12, vs. 100.6 ± 7.6 kN/m², n = 57; P < 0.02, Mann-Whitney). The higher percentage of type IIA fibers and the lower percentage of type I fibers in SHR soleus may explain in part previously reported observations with intact soleus muscles from young SHRs (less than 10 wk old), which developed 15% lower tetanic forces compared with their counterparts from WKY rats (2, 4). The difference in the maximal Ca²⁺-activated specific force between type I and type IIA fibers was not statistically significant for the older rats of either strain.

In summary, the present study demonstrates that although for a given fiber type there are no significant differences between SHR and WKY rat soleus muscles with respect to any of the Ca²⁺-activation properties examined, the soleus muscles from SHRs and WKY rats are at different states of transformation as reflected by major differences in MHC composition and fiber type populations.