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1Department of Medicine, Veterans Affairs Long Beach Healthcare System, Long Beach; Departments of 2Medicine, 3Orthopedic Surgery, and 4Physiology and Biophysics, University of California, Irvine, California
Submitted 1 February 2005 ; accepted in final form 10 April 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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positive pressure; maximum shortening velocity; myosin heavy chain; myofibril injury; muscle atrophy F-box; atrogin-1
Given the background above, there are a number of unresolved issues related to the functional consequences of CMV. For instance, in a previous study, we observed that CMV seemed to produce an increase in Vmax that occurred without commensurate changes in myosin isoform composition as examined at both the heavy and light chain levels (36). Hence, in the present study, we address this issue again in an attempt to either confirm or reject the hypothesis that CMV increases Vmax and alters the shape of the force-velocity relationship in the low-force, high-velocity region. This is an important issue to be resolved because an increase in Vmax without concomitant changes in myosin isoform composition would suggest that other factors such as altered myofibrillar ultrastructure may be responsible for the noted changes in Vmax (5). Additionally, although it has been shown that CMV produces a significant degree of myofibrillar disarray (1, 30, 35), it is unclear what underlying mechanisms are responsible for this phenomenon. In our earlier study (36), we observed that 3 days of CMV dramatically increased the expression of muscle atrophy factor-box (MAF-box; also known as atrogin-1), an E3 ligase in the ubiquitin-proteasome pathway. MAF-box has been shown to play an important role in the development of muscle atrophy under a variety of altered physiological conditions, and, as such, may play a key role in mediating changes in both Po and Vmax induced by CMV.
We addressed these issues in the present study by employing a time-course paradigm whereby the diaphragm underwent 1, 2, and 3 days of CMV. At the functional level, we performed regression analyses to test the hypothesis that increases in Vmax after CMV are correlated to myofibrillar disarray. With respect to MAF-box, we used time-course analyses to determine whether elevations in MAF-box precede or follow the appearance of myofibrillar derangement. This approach is essential for testing the hypothesis that increased MAF-box expression is partially or completely responsible for the development of VIDD.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The Research and Development Subcommittee on animal studies of the Veterans Affairs Long Beach Healthcare System approved the study. We studied 24 adult male New Zealand White rabbits. The animals were assigned randomly into four groups: 1) control; 2) 1-day CMV; 3) 2-day CMV; and 4) 3-day CMV. Six animals were assigned to each group. Pressure-limited ventilation (model 7200ae Nellcor-Puritan Bennett or model 840 Nellcor-Puritan Bennett, Tyco Healthcare, Carlsbad, CA) was applied in the CMV groups. The ventilator inspiratory pressure, inspiratory time, and rate were set as previously described (35, 36). The latter was set sufficiently high to suppress inspiratory efforts, as detected from the inspiratory flow waveform (35). The inspired air was humidified, positive-end expiratory airway pressure was set at 0 cmH2O, and inspired O2 fraction was set to maintain arterial PO2 at greater than 60 Torr. The animals in the CMV groups were euthanized after the completion of a given period of CMV (i.e., 1, 2, or 3 days). The control group was euthanized after the surgical procedure was completed. We did not include a sham control group consisting of sedated animals breathing spontaneously for the respected duration of CMV because we have previously shown that anesthetic or sedative drugs did not have any influence on the effect of mechanical ventilation on diaphragmatic function (35).
The surgery was performed under general anesthesia using aseptic techniques. The initial dose of anesthetics consisted of ketamine hydrochloride (35 mg/kg) and xylazine (5 mg/kg) administered intramuscularly. During the surgical procedure, a maintenance dose of ketamine hydrochloride (2 mg/kg)-xylazine (0.2 mg/kg) was administered intramuscularly every 20 min as needed. The depth of anesthesia was monitored by the absence of jaw tone. The trachea was cannulated with a tracheostomy tube (4 mm ID, 6 cm long) through a tracheostomy incision. The external jugular vein was cannulated for continuous infusion (lactated Ringer, 100 ml·kg–1·day–1) and intravenous medications. The common carotid artery was cannulated for blood pressure (model P23XL Grass, Astro-Med, West Warwick, RI) and for heart rate monitoring and arterial blood sampling, as previously reported (35, 36). A feeding tube was inserted into the stomach via a small incision in the esophagus. Prealbumin measurements were obtained in the controls and animals receiving 3 days of CMV only, because prealbumin half-life is 48 h (19), and, therefore, meaningful changes will be detected at 72 h. After the surgical procedures, the skin was closed in layers, except for the control group.
Animal Monitoring During Mechanical Ventilation
During CMV, continuous intravenous sedation was maintained with diazepam at a loading dose of 4 mg/kg intramuscularly, followed by a continuous intravenous infusion of 2–5 mg/h titrated to limb movement. This diazepam dose was insufficient to suppress the respiratory center. Suppression of the diaphragm was maintained by adjusting the ventilator settings and, if necessary, by administration of additional maintenance doses of a ketamine-xylazine mixture. The following medications were also given: buprenorphine (0.05 mg/kg sc) every 12 h for analgesia, atropine sulfate (0.02 mg/kg sc) every 12 h to reduce bronchial secretions, and penicillin G procaine (300,000 U intramuscularly) every 12 h to prevent infection. To prevent metabolic acidosis, sodium bicarbonate was administered (1–3 meq/h iv), titrated according to arterial pH (37). Electrolytes were maintained, and liquid enteral nutrition (F3978SP, BioServ, Frenchtown, NJ) was delivered through the feeding tube at 25 cal·kg–1·day–1 in six equally divided doses. Blood pressure and heart rate were continuously monitored. The analog signals were displayed in real time on a computer monitor of a data-acquisition system and recorded every 2 h (WinDaq/Pro, Dataq Instruments, Akron, OH). Rectal temperature was continuously monitored and maintained at 36–38°C with a heating blanket. Airway pressure, tidal volume, and flow signals were monitored from the ventilator display screen. Passive ventilation during CMV was monitored from the flow and airway pressure signals for inspiratory efforts. To prevent atelectasis, the lungs were inflated with a sequence of inspiratory pressure to produce tidal volume of 15 ml/kg for five consecutive breaths every 15 min in all animals during the surgical procedure and mechanical ventilation. Passive stretches have not been shown to be the major determinant of morphological adaptation to diaphragm inactivity (43). The animals were positioned in various postures (dorsal, ventral, left and right lateral decubitus) every 4 h. Suctioning of the trachea was performed as needed. The bladder was expressed every 12 h. A physician or a research scientist provided round-the-clock coverage and animal care for the duration of the study. All experimental procedures were in strict accordance with the Animal Welfare Act.
In Vitro Measurements of Diaphragm Contractile Properties
Isometric contractile properties.
At the end of the experiments, the animals were euthanized with an overdose of pentobarbital sodium (100 mg/kg iv). The diaphragm muscle was rapidly excised from the midcostal region, with the insertion of fibers at the ribs and central tendon intact. A diaphragm muscle strip (
5 mm wide) was obtained and mounted vertically between two platinum plate electrodes that covered the entire length of the muscle strip in a 26°C bath containing Rees-Simpson solution (39) through which 95% O2-5% CO2 was continuously aerated, maintaining a pH of 7.40. The rib end of the muscle was clamped, and the central tendon of the muscle was attached to a calibrated lever of a Cambridge system (model 300B, Aurora Scientific) to allow adjustment of muscle length. The muscle was stimulated using 1.5-ms duration of monophasic rectangular pulses delivered via a current amplifier (Mayo Foundation, Engineering Section), which was controlled by a Grass S88 stimulator (Astro-Med). Current intensity was adjusted until maximum tetanic force (at 50 Hz and train of 500 ms) responses were obtained. Thereafter, the stimulus intensity was set at 125% of this value. Muscle preload was adjusted by using the micromanipulator until Lo, the length at which the muscle produces Po, was achieved. Subsequently, Lo was measured using digital calipers. At Lo, peak twitch tension (Pt), time to peak twitch tension, and the time for Pt to relax to one-half Pt were determined from a series of contractions induced by single-pulse stimuli. Isometric and isotonic force generations were determined by use of the Cambridge system, controlled by using customized routine software (LabTech Notebook, Andover, MA), and implemented on a personal computer. The analog-to-digital board (MetraByte DAC-16) sampled the force and length outputs at a frequency of 1,000 Hz/channel. To determine Po, the muscle strip was stimulated by trains of 1 s at 40, 50, 75, and 100 Hz with at least a 2-min interval between each stimulus frequency. Forces were normalized for muscle cross-sectional area, which was estimated by the following formula: muscle mass (g)/[Lo (cm) x muscle density (g/cm3)], where 1.056 g/cm3 was used for muscle density.
Isotonic contractile properties. After isometric force measurements, the muscle strip was allowed to equilibrate for 15 min before measurement of isotonic contractile properties. The force-velocity relationship was determined by using the isotonic mode of the Cambridge system with computer-controlled afterload. The muscle was tested at a minimum of 15 different afterload conditions (1.0–100% Po). The muscle began each contraction at Lo. The shortening velocity at each afterload was determined by calculating the slope of the length vs. time record over successive 10-ms segments. The segment with the greatest slope was used as the velocity of measurement for that specific load. Force-velocity data were then fitted to a linearized form of the Hill equation (17). The least squares technique was used to find the best fit to this equation. This procedure involved an iterative regression routine that began with an initial value for a, and performed 100 successive iterations using a specified incremental factor for a. The computer program identified the best fit for the force-velocity data by determining which values of a and b provided the smallest sum of squares between the measured and predicted velocities. Vmax was determined by using the Hill equation and solving for velocity when force equaled zero. Velocity of contraction, expressed as muscle length (normalized for Lo) per second (ML/s), was plotted against force. Power, the product of force and velocity of contraction, expressed as watts per kilogram of muscle wet weight, was calculated and plotted against force. In addition, the maximum or peak power output was determined.
Determination of Abnormal Myofibrils
A segment of diaphragm muscle strip was obtained for electron microscopy (EM) from four animals in each group. The muscle strip was stretched to 1.5 times the resting excised muscle length (approximate optimal length for muscle force generation) (29) and fixed in 6.5% glutaraldehyde in a 0.1 M sodium cacodylate buffer. The muscle was then cut into 2-mm pieces and reimmersed in the same solution for an additional hour. After fixation, the tissue was washed three times for 10 min each in 0.1 M sodium cacodylate buffer (pH 7.4). The tissue was postfixed in 1.0% OsO4 and 1.0% potassium ferrocyanate in the same buffer for 4 h. The tissues were then washed in three changes of distilled water for 10 min each. Dehydration followed through a graded series of ethanol; then the tissue was infiltrated with propylene oxide twice for 10 min each, 50% araldite-50% propylene oxide for 2 h, and 100% araldite for 24 h and polymerized for 2 days. Sections (1 µm thick) were cut on an LKB Ultratome III with a diamond knife and stained with 0.25% toluidine blue solution. After muscle fibril orientation was determined under light microscopy, blocks were reoriented, and ultrathin sections (50–70 nm) were cut transversely to the muscle fiber axis. The sections were contrasted with uranyl acetate and bismuth subnitrite for transmission EM.
The magnitude of myofibril disassembly was estimated by determining the volume density of normal or abnormal myofibrils and mitochondria per volume of muscle fiber from the EM transverse section samples at a final magnification of x24,000 (25). The pathologist or technician working on the EM images was uninformed about the nature of the study. Ten micrographs were obtained by systematic sampling in one ultrathin section from each block. Two randomly chosen blocks per sample, i.e., a total of 20 micrographs, were analyzed. The micrographs were scanned with a film scanner (Polaroid Sprint Scan 45i, Meyter Instruments, Houston, TX), and the image was subsequently projected on a computer screen. A 140-point square grid was superimposed on the image using image analysis software (Image Pro Plus version 4.0, Media Cybernetics, Silver Spring, MD). Points that fell onto the image were assigned as follows: 1) normal myofibril, 2) abnormal myofibril, defined as disruption of myofibril bundles or disorganized myofibrillar pattern, 3) normal mitochondria, 4) abnormal swollen mitochondria with abnormal cristae, and 5) miscellaneous, which included lipid droplets, vacuoles, intermyofibril space, and nuclei. Volume density of normal or abnormal myofibrils and mitochondria is the number of points in each category, expressed as a percentage of the total number of points in all categories (35).
Contractile Protein and Molecular Analysis
Quantification of contractile protein fractions.
Purified myofibrillar protein from diaphragm muscle homogenates of each sample was denatured and loaded into the wells of 4–15% gradient Tris·HCl polyacrylamide gels (Bio-Rad, Hercules, CA) at a protein concentration of 2 µg/well. Gels were run at room temperature for 2 h and at a voltage of 120 V. Running buffer consisted of 100 mM Tris, 100 mM tricine, and 0.1% SDS (pH 8.3), whereas sample buffer consisted of 100 mM Tris, 5% glycerol, 4% SDS, 0.05% bromophenol blue, and 5% 
-mercaptoethanol. At the completion of electrophoresis, gels were stained for 1 h with R-250 Coomassie blue and then destained by using a 40% methanol-10% acetic acid solution. Gels were then digitally imaged and analyzed with ImageQuant software (Molecular Dynamics, Sunnyvale, CA). We obtained the relative proportions of myosin and actin by measuring the density of each contractile protein band and expressing it as a percentage of the total density of all proteins in that sample.
Discontinuous PAGE separation of MHC isoforms.
Myosin heavy chain (MHC) protein isoforms were separated by using techniques described by us previously (7, 8). The separating gel consisted of 8% acrylamide, 0.16% bis-acrylamide, 30% glycerol, 0.4% SDS, 0.2 M Tris (pH 8.8), and 0.1 M glycine. This solution was degassed for 15 min, and polymerization was then initiated by adding TEMED (0.05% final concentration) and ammonium persulfate (0.1% final concentration) to the separating gel solution. The separating gel was poured, layered with ethyl alcohol, and given 30 min to polymerize. The stacking gel solution contained 4% acrylamide, 0.08% bis-acrylamide, 30% glycerol, 70 mM Tris (pH 6.7), 4 mM EDTA, and 0.4% SDS. This solution was also degassed for 15 min before addition of TEMED (0.05% final concentration) and ammonium persulfate (0.1% final concentration). It was then layered onto the separating gel. The running buffer contained 0.1 M Tris, 0.15 M glycine, and 0.1% SDS. A SG-200 vertical slab gel system (C.B.S. Scientific, Del Mar, CA) was used for electrophoresis. Gels were run for 24 h, at 270 V. MHC protein isoform bands were stained by use of a silver stain kit (Bio-Rad, Richmond, CA). The MHC isoform bands were digitally imaged and quantified with ImageQuant software (Molecular Dynamics).
Determination of MLC isoforms.
Analyses of myosin light chain (MLC) protein isoform composition were performed as described previously (9). Purified myofibrillar protein from each sample was denatured and loaded (5–10 µg) into the well of a 5–20% gradient gel. The gels were run by using a constant current of 50 mA per gel for a period of 1 h. The MLCs were identified by use of known molecular weight markers. The gels were fixed and stained with R-250 Coomassie blue. The MLCs were digitally imaged and quantified via ImageQuant software (Molecular Dynamics). Data for a given essential MLC isoform was expressed relative to the total area for all the essential light chains, that is, the slow MLC1 (sMLC1), fast MLC1 (fMLC1), and fast MLC3 (fMLC3). A similar approach was used for the regulatory MLC isoforms, that is, the slow MLC2 (sMLC2) and fast MLC2 (fMLC2).
Determination of MHC isoforms and MAF-box mRNA using RT coupled with PCR. One microgram of total RNA was reverse transcribed for each muscle sample using the Superscript II RT and a mix of oligo(dT) (100 ng/reaction) and random primers (200 ng/reaction) (Invitrogen, Life Technologies, Carlsbad, CA) in a 20-µl total reaction volume at 42°C for 50 min, according to the provided protocol. At the end of the RT reaction, the tubes were heated at 72°C for 15 min to stop the reaction and then stored at –80°C until used in the PCR reactions for specific mRNA analyses (see below). The sequence for the various MHC primers of rabbit oligonucleotide (Invitrogen, Life Technologies) for 3'-region sense primer and antisense primer, respectively, were 1) MHCslow: GGA TCC CTG GAG CAG GAG AA, and CTT GCA TTG AGG GCA TTC AG; 2) MHC2A: CAC AAA TCT ATC TAA ATT CC, and TCC TTT GCA GTA GGG TAG; 3) MHC2X: ACT GCA AGC CAA GGT GAA AT, and TTA TCT CCC AGA ATC ATA AG; 4) MHC2B: AGA GGC TGA GGA ACA ATC CA, and ACT TGA TGC ACA AGG TAG TG (27); and 5) MAF-box (rat oligonucleotide): CCG TGC ATG GAT GGT CAG TG, and AGA CCG GCT ACT GTG GAA GAG. In each PCR reaction, 18S ribosomal RNA was coamplified with the target cDNA (mRNA) to serve as an internal standard and to allow correction for differences in starting amounts of total RNA. The Classic 18S and Alternate 18S were used for the PCR reaction with MHC and MAF-box, respectively. The PCR conditions for each primer set and 18S competimer were optimized, so that the target mRNA and 18S products were in the linear range. For each specific target mRNA, the RT and PCR reactions were carried out under identical conditions. One microliter of each RT reaction without dilution was used for the PCR amplification. The PCR reactions for MHCslow, MHC2A and MHC2X were performed using 3 mM of MgCl2 and 2 mM of MgCl2 for MHC2B, using standard PCR buffer (Invitrogen, Life Technologies), 0.2 mM dNTP, 1 mM specific primer set, 0.5 µM 18S primer/competimer mix, and 0.75 unit of DNA Taq polymerase in 25 µl total volume. Similar procedures were used for the MAF-box primers except that the MgCl2 concentration was 2 mM. Amplifications were carried out in a Bio-Rad iCycler (model iCycler iQ, Bio-Rad Laboratories). For MHC, the initial denaturing step was 3 min at 96°C, followed by 28 cycles for 1 min at 96°C, 50 s at 50°C, and 1 min at 72°C, and a final step of 3 min at 72°C. The MAF-box PCR began with an initial denaturing step of 2 min at 95°C, followed by 26 cycles of 45 s at 95°C, 1 min at 55°C, 90 s at 72°C, and a final step of 5 min at 72°C. PCR products were separated on a 2.0% agarose gel by electrophoresis and stained with ethidium bromide, and signal quantification was conducted by laser scanning densitometry. In this approach, each specific mRNA signal was normalized to its corresponding 18S. In addition to the diaphragm muscle, we analyzed the MAF-box mRNA of the medial gastrocnemius muscle in four of the animals in the 1-day, 2-day, and 3-day CMV groups, and all of the animals in the control group.
Statistics and Data Analyses
Values are means ± SE unless specifically indicated. A one-way ANOVA (SigmaStat, version 3.0, SPSS Science, Chicago, IL) was used for comparison of variables among groups. When the F value was significant, post hoc analysis was performed using Tukey's test for pairwise multiple comparisons. A linear regression was used to determine the relationship between abnormal myofibrils and Po and Vmax. A similar regression analysis was performed to assess the relationship between MAF-box mRNA levels and the volume density of abnormal myofibrils. Group differences and linear regression were considered significant when P 
0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The stability of the muscle preparation was evaluated by comparing measurements of Po. The initial measurements of Po were made once the muscle strip preparation was properly attached to the Cambridge ergometer system and Lo was determined. The mean values for the control, 1-day, 2-day, and 3-day CMV groups were 25.9, 19.3, 16.9, and 15.4 N/cm2, respectively. Po was again measured just before force-velocity measurements, and the mean values for the four groups were 25.3, 19.1, 17.8, and 15.6 N/cm2 (control, 1-day, 2-day, and 3-day groups, respectively).
Table 2 shows that Lo, time to peak twitch tension, and the time for Pt to relax to one-half Pt were not significantly different among groups, whereas Pt and Po decreased by 39 and 44% after 3 days of CMV, respectively (P < 0.01). Vmax increased progressively from a mean control value of 6.4 ML/s to 6.6, 7.7, and 8.1 ML/s after 1, 2, and 3 days of CMV, respectively (P < 0.02; see also Fig. 1, top). Figure 1 shows the entire force-velocity and force-power relationships for the different groups. When force is expressed as percentage of Po, it is clear that the increases in shortening velocity partially compensated for the loss of force such that the reduction in maximal power was minimized (Fig. 1, bottom).
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As shown in Table 3, CMV did not affect the relative proportions of MHC and actin protein pools (expressed relative to the total pool of contractile proteins). Similarly, the MHC isoforms’ mRNA levels were unaltered (Fig. 2).
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Figure 3 demonstrates that MAF-box mRNA levels of the diaphragm were significantly increased, 355% of control at the earliest time point (i.e., 1 day), and remained elevated at both the 2-day (365%) and 3-day (260%) time points as well. The pairwise comparison of 3-day CMV and control was significant at P = 0.07; however, the degree of elevation was consistent with that observed in our previous study of 2.7-fold of the control value (36). If we combine the MAF-box mRNA data of 3-day CMV of prior and present studies, the difference between the control and the 3-day time point was significant at P < 0.01. Unquestionably, MAF-box mRNA increased early in the course of CMV and remained elevated for the duration of its application.
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Diaphragmatic Ultrastructure Alterations
The volume density of abnormal myofibrils (compared with the control group) was unchanged after 1 day of CMV (see Figs. 4 and 5). However, the presence of abnormal myofibrils became very noticeable after 2 days of CMV (see Figs. 4 and 5; P < 0.01), accounting for 33% of the total myofibrillar volume density at the 3-day time point. On the other hand, the volume densities of normal and abnormal mitochondria did not change over the course of time. There was no significant correlation between MAF-box mRNA levels and the volume densities of abnormal myofibrils (y = 11.4x + 8.0; r = 0.33). This lack of correlation is not surprising given that MAF-box mRNA was upregulated as early as the first day of CMV, whereas significant ultrastructure changes were not detectable until after the second day of CMV.
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As shown in Fig. 6, there was a significant correlation between the volume density of abnormal myofibrils and the loss of Po (r = 0.64; P < 0.01). The correlation between the volume density of abnormal myofibrils and Vmax was even stronger, accounting for 76% of the variance in Vmax (r = 0.87; P < 0.01).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Functional Consequences of CMV and Potential Structural Basis
Previous studies have conclusively shown that CMV produces a rapid and dramatic decrease in the ability of the diaphragm muscle to produce isometric tension. One might assume that this type of ventilator-induced diaphragmatic dysfunction also applies to other loading conditions within the force-velocity relationship. Interestingly, however, the findings of the present study, as well as our previous publication (36), demonstrate that CMV produces a compensatory phenomenon in the low force-high velocity region of the force-velocity relationship. The functional consequences of this compensatory phenomenon (within the context of the force-velocity relationship; see Fig. 1) are to 1) progressively minimize the large force deficit as the muscle moves from high-to-low loading conditions; 2) minimize the loss of maximal power that would be predicted from the large loss in Po; and 3) actually increase Vmax and performance (above control values) under low loading conditions. On the basis of these observations, some might argue that it is inappropriate to use the term VIDD in describing the loss in Po that results from CMV. It should be stressed, however, that the large reduction in Po is so profound that it functionally truncates the force-velocity relationship by 40%, and that performance of the CMV diaphragm only achieves that of the control diaphragm at loads less than 5% of Po. Hence, the findings of this study with respect to the force-velocity relationship represent an important data set that helps to clarify the true functional scope of VIDD.
In a previous study (36), we observed that 3 days of CMV produced a significant reduction in maximal power. In the present study, we again found that the mean maximal power value of the 3-day CMV group was less than that of the control group. However, the degree of change (11%) was not large enough to be statistically significant. If we combine the findings of the present study with those of our previous publication (36), then it is clear that CMV does reduce maximal power. As emphasized above, however, the changes in the low-force, high-velocity region of the force-velocity relationship in the CMV diaphragm muscles help to minimize the degree to which maximal power is reduced.
Since the late 1980s, a number of single-fiber and whole muscle studies (4, 12, 14, 31, 34) attempted to identify the underlying mechanisms responsible for determining maximal shortening velocity, as defined by either extrapolation of the force-velocity relationship or slack tests. Currently, it is commonly accepted that the MHC isoform composition of a fiber or muscle is the primary determinant that accounts for the differences in Vmax between slow and fast muscle. Importantly, however, it should be noted that Riley et al. (32, 33) observed that Vmax or the maximal unloaded shortening velocity (Vo) can be altered under abnormal physiological conditions, independent of changes in myosin isoform composition, and it has been suggested that the increases in Vmax observed by these investigators occurred as a result of altered myofilament ultrastructure. Interestingly, the findings of the present study are somewhat analogous to those of Riley et al. Specifically, we observed an increase in Vmax that appears to be correlated with the degree of abnormal myofibrils and not to changes in myosin isoform composition. Riley et al. suggested that muscle unloading produces myosin-independent changes in Vmax by selectively reducing the thin-filament density. Although we did not specifically address this issue at the EM level, our protein analyses at the whole muscle level demonstrate that there was not a selective reduction in actin-to-myosin ratio. Nevertheless, the collective findings of our studies and those of Riley and coworkers (32, 33) demonstrate that reduced loading or activation of skeletal muscle can produce increases in Vmax that appear to be related to altered ultrastructure and not to changes in myosin isoform composition. Clearly, more detailed analyses are needed to better define the underlying causes of this phenomenon.
As noted above, the most profound functional consequence of CMV is the large reduction in Po. In the present study, we observed that the increased volume density of abnormal myofibrils could account for 40% of the loss of Po (Fig. 6, top). Additionally, Fig. 6 shows a significant reduction in Po at the 1-day time point that occurred in the absence of detectable changes in ultrastructure. Some possible reasons for the absence of a stronger relationship might be as follows. First, determining the loss of abnormal myofibrillar volume density may not accurately describe the full extent of myofibrillar dysfunction (as is probably the case). For instance, we did not examine possible changes in thin-filament density or thin- filament structure as attempted by Riley et al. (32, 33). Second, CMV might alter the excitability and/or excitation-contraction coupling of diaphragm muscle. To our knowledge, the effects of CMV on these two key processes are unknown. It has been shown, however, that mechanical unloading of rodent hindlimb muscles does not alter Ca2+ handling in a manner that can account for the large loss observed in Po as in the present study (18). Clearly more detailed analyses are needed 1) to better delineate the functional cellular or molecular defects; and 2) to isolate the underlying cellular and molecular mechanisms responsible for inducing such functional defects.
Time Course of MAF-Box Expression
With respect to the second consideration above, it is clear from the EM analyses of the present study (35) that one of the primary defects produced by CMV is myofibrillar disarray. The factors responsible for producing this disarray represent an important area of investigation. Currently, it is thought that there are at least three degradatory pathways that might mediate the extent of VIDD; these include 1) increased degradation via oxidative stress (2, 38, 42); 2) the involvement of calcium-activated proteases (38); and 3) the ATP-dependent ubiquitin-proteasome pathway (11, 36).
In skeletal muscle, the majority of intracellular proteolysis is thought to occur through the ubiquitin-proteasome system (23). Muscle proteins destined for degradation by the ubiquitin-proteasome pathway are first covalently linked to a chain of ubiquitin molecules that marks them for proteolysis by the 26S proteasome (15). One key set of enzymes responsible for attaching ubiquitin to protein substrates is the ubiquitin-conjugating enzymes (E3s). Three of these have been identified to play key roles in the activation of proteolysis during muscle atrophy. E3
(also called Ubr1/Ubr2) acts on proteins with basic and large hydrophobic NH2-terminal amino acids (termed the "N-end rule pathway") (21). The other two E3 ligases, MAF-box and muscle ring finger-1 (MuRF-1), were recently discovered in skeletal muscle (3, 16) and shown to be dramatically overexpressed in all forms of muscle wasting studied in rodents, including mechanical unloading (3). Furthermore, after hindlimb denervation, knockout mice lacking these enzymes show reduced muscle atrophy (3).
Currently, there are four key findings that support the hypothesis that MAF-box plays a role in mediating the development of VIDD. First, in a previous study (36), we found that 3 days of CMV produced a large increase in the mRNA levels of MAF-box. A more recent study by Deruisseau et al. (11) examining rat diaphragm muscle also made similar observations. Second, we observed that assisted mechanical ventilation markedly attenuated the loss of diaphragm function that results from CMV. Importantly, we also found in this same study that MAF-box mRNA levels were unaffected in the assisted mechanical ventilation group, i.e., they did not increase above baseline values. Third, in the present study, we found that MAF-box mRNA levels were elevated before the appearance of ultrastructural derangement. Finally, Deruisseau et al. recently observed that CMV produces an increase in the ubiquitin-protein conjugates (both myofibrillar and cytosolic). These correlative findings represent an important set of data necessary for establishing a causal role for MAF-box and the ubiquitin-proteasome pathway in mediating the deleterious effects of CMV. These findings, however, need to be complemented by more mechanistic studies whereby MAF-box and other components of the ubiquitin-proteasome system can either be over- or underexpressed.
The modestly increased MAF-box mRNA in the gastrocnemius does not repudiate the hypothesis on the role of the ubiquitin-proteasome pathway in mediating the development of VIDD. The highly elevated MAF-box mRNA levels in the diaphragm suggest that the diaphragm muscle is more sensitive to alterations associated with disuse than the hindlimb muscle. Whether the increased sensitivity of the diaphragm muscle to disuse is related to its prior activation history is unclear.
Important Technical Considerations and Limitations
Some might question whether the acid-base disturbances observed in the present study might explain some or all of the loss in diaphragmatic function due to CMV. As we have noted previously (35), in a study in which a spontaneous breathing control group was available for the same duration of the study as the CMV group, the acid-base status had no effect on diaphragmatic force. Similarly, in previous studies in which metabolic acidosis was induced, diaphragm muscle force was maintained, provided the pH was greater than 6.8 (10), or when respiratory acidosis was nonexistent (41). Furthermore, metabolic acidosis did not activate the ubiquitin-proteasome pathway (26).
It has been shown that muscle atrophy is associated with the upregulation of both MAF-box and MuRF-1 mRNA levels. Our analyses in the present study were restricted to MAF-box because we have been unable to develop a suitable MuRF-1 oligonucleotide for rabbit muscle. It should be noted, however, that Deruisseau et al. (11) recently reported that mechanical ventilation produced an increase in MuRF-1 mRNA levels in rat diaphragm muscle.
In summary, the force-velocity data of the present study clearly demonstrate that the dysfunction produced by CMV is profound and negatively impacts function throughout 80–90% of the force-velocity relationship. At the structural level, the dysfunction appears to be the result of extensive myofibrillar disarray. Importantly, we observed that CMV produced an upregulation of MAF-box mRNA levels that occurred before the presence of ultrastructural alterations, and this finding provides further support for the idea that the ubiquitin-proteasome pathway is partially or completely responsible for the dysfunction that results from CMV. If this degradatory pathway plays a fundamental role in VIDD, then there are very important clinical implications because critically ill patients are frequently afflicted with conditions such as sepsis, insulin resistance, uremia, and malnutrition (22). Each of these individual conditions is known to accelerate proteolysis through the ubiquitin-proteasome pathway (22, 24).
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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.
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-Cardiac-like myosin heavy chain as an intermediate between MHCIIa and MHCIb in transforming rabbit muscle. Am J Physiol Cell Physiol 274: C595–C602, 1998.This article has been cited by other articles:
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  D. J. Falk, K. C. DeRuisseau, D. L. Van Gammeren, M. A. Deering, A. N. Kavazis, and S. K. Powers Mechanical ventilation promotes redox status alterations in the diaphragm J Appl Physiol, October 1, 2006; 101(4): 1017 - 1024. [Abstract] [Full Text] [PDF]
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 K. C. DeRuisseau, R. A. Shanely, N. Akunuri, M. T. Hamilton, D. Van Gammeren, A. M. Zergeroglu, M. McKenzie, and S. K. Powers Diaphragm Unloading via Controlled Mechanical Ventilation Alters the Gene Expression Profile Am. J. Respir. Crit. Care Med., November 15, 2005; 172(10): 1267 - 1275. [Abstract] [Full Text] [PDF]
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