© 2002 American Thoracic Society
Controlled Mechanical Ventilation Leads to Remodeling of the Rat Diaphragm Respiratory Division, Critical Care Division, McGill University Health Centre; and Respiratory Muscle Biology Group, Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada Correspondence and requests for reprints should be addressed to Dr. Basil J. Petrof, Respiratory Division, Room L411, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, PQ, H3A 1A1 Canada. E-mail: basil.petrof{at}muhc.mcgill.ca
Little is known about the structural response of the diaphragm to controlled mechanical ventilation. We examined effects of this intervention on muscle mass, myosin heavy chain isoforms, and contractile function in the rat diaphragm. Animals were mechanically ventilated for up to 4 days, and comparisons were made with normal control rats as well as spontaneously breathing animals anesthetized for the same duration as the mechanical ventilation group. The diaphragm-to-body weight ratio was significantly reduced in the mechanical ventilation group only. After mechanical ventilation, an increase in hybrid fibers coexpressing both type I (slow) and type II (fast) myosin isoforms was found within the diaphragm, which occurred at the expense of the pure type I fiber population. In contrast, the percentages of type I, type II, and hybrid fibers in the limb muscles (soleus and extensor digitorum longus) did not differ between experimental groups. The optimal length for force production, as well as maximal force-generating capacity of the diaphragm, was also significantly decreased in mechanically ventilated animals. We conclude that even short-term controlled mechanical ventilation produces significant remodeling and functional alterations of the diaphragm, which could impede efforts at discontinuing ventilatory support.
Key Words: respiratory muscles • myosin heavy chain isoforms • hybrid fibers • diaphragm disuse • weaning
Prolonged mechanical ventilation is associated with a high complication rate (1), and difficulties in weaning patients from mechanical ventilation are frequent (reviewed in [2–5]). Respiratory muscle dysfunction appears to play a central role in this problem, and it has been hypothesized that diaphragm disuse may greatly contribute to difficulties in weaning patients from mechanical ventilation (2–5). Controlled mechanical ventilation (CMV) is employed in the management of patients with respiratory failure of different etiologies and is characterized by the complete absence of spontaneous breathing efforts by the patient (4). Two early studies, one in baboons (6) and the other in rats (7), initially reported that CMV leads to a substantial reduction in diaphragm force-generating capacity. Recently, other investigators have confirmed these findings in rats (8) and young piglets (9). However, there has been very little study of the structural changes that occur in the diaphragm after instituting CMV. Several different models have been employed to study the effects of disuse on muscle fiber types and other morphologic characteristics of skeletal muscle (10–15). In the diaphragm, surgical phrenicotomy and pharmacologic blockade of phrenic nerve activity with tetrodotoxin have been employed (16, 17). However, CMV imposes a different form of muscle disuse on the diaphragm, with the latter being not only electrically quiescent but also mechanically unloaded and phasically shortened on an intermittent basis by cyclical lung inflation (4, 5, 18). The diaphragm could be particularly susceptible to disuse changes, as it differs from traditionally studied limb skeletal muscles by its persistent rhythmic activation throughout both wakefulness and sleep without any period of sustained rest (19). Furthermore, to the extent that the intrinsic genetic programming of muscle cells may differ among skeletal muscles (20), this could further contribute to dissimilar responses between the diaphragm and limb muscles. An important aspect of the structural remodeling response to altered levels or patterns of skeletal muscle activity resides in the ability of individual myofibers to modify their myosin heavy chain (MHC) isoform expression profile (reviewed in [21]). The MHC component of the myosin molecule constitutes the predominant protein in skeletal muscle as well as the primary molecular basis for differences in maximum shortening velocity and power output among muscles (22, 23). In addition, type I fibers have been reported to generate lower levels of isometric force than type II fibers within the rat diaphragm (24, 25). Individual myofibers generally express only one MHC isoform to the exclusion of all of the others, and fiber type identification by traditional myofibrillar ATPase histochemistry is largely based on this assumption (21). However, more sophisticated immunohistochemical approaches have revealed that skeletal muscle disuse often leads to an increase in myofibers that coexpress more than one MHC isoform, which are referred to as hybrid fibers (21). The impact of CMV on diaphragm MHC isoform expression has not been reported. Accordingly, the principal objectives of this study were as follows: (1) to determine whether CMV alters the pattern of MHC isoform expression at the individual myofiber level in the rat diaphragm and (2) to evaluate the effects of CMV on diaphragm muscle mass as well as the cross-sectional area of individual diaphragm myofibers expressing either type I MHC, type II MHCs, or both (hybrid fibers). In addition, these parameters were related to measurements of diaphragm contractile function in a subset of animals.
Experimental Groups Pathogen-free 3- to 4-month-old adult male Sprague-Dawley rats (450–550 g) were randomly assigned to one of the following: (1) a CMV group; (2) a spontaneously breathing anesthetized group (ANE), which received the same level of nutritional support and surgical interventions as the CMV group (see below); and (3) a spontaneously breathing control group (CTL), in which no interventions were made before animals were killed. The study was approved by the institutional animal care and use committee.
Pharmacologic and Surgical Preparation In the CMV group, a pediatric ventilator (Babylog 8000; Drager, Lubeck, Germany) equipped with clinical-grade sterile tubing and filters was initially set as follows: frequency = 90 per minute, tidal volume = 5 ml/kg, fractional inspired oxygen = 0.24, and positive end-expiratory pressure = 4 cm H2O. The fractional inspired oxygen and level of ventilation were adjusted as required to maintain acceptable blood gas values. The absence of respiratory muscle effort was confirmed by the lack of triggering of the ventilator (trigger threshold = -0.25 cm H2O) as well as the stable and reproducible shape of the tracheal pressure waveform. All animals were continuously monitored by two physicians trained in critical care medicine who performed alternating 12-hour shifts throughout the duration of each experiment. Additional detail on these methods is provided in the online data supplement.
Immunohistochemical and Morphometric Analysis
Muscle Contractility Analysis
Northern Blot Analysis
Statistical Analysis
Duration of Mechanical Ventilation As shown in Table 1 , there was no significant difference in total protocol duration between the CMV (range, 44–93 hours) and spontaneously breathing ANE (range, 52–93 hours) groups. At the time at which animals were killed, values for blood pressure and arterial blood gases also showed no statistically significant differences between the two groups, although the arterial PCO2 did tend to be higher in the ANE group.
Diaphragm and Hindlimb Muscle Mass There were no significant differences in initial body weight among the three groups. In the ANE and CMV groups, there were small decreases in total body weight over the experimental period amounting to mean changes of 3.5% and 1.7%, respectively. Table 2 shows muscle weight to body weight ratios at the time of animal death. Although the ANE group demonstrated a trend toward decreased diaphragm weight/body weight (mean reduction of 7.5% as compared with CTL), this did not reach statistical significance. On the other hand, a significant decrease in diaphragm weight/body weight was observed in the CMV group, which amounted to a mean reduction of 13.4% compared with CTL. In addition, there were significant and equivalent decreases in muscle weight/body weight for the fast-twitch EDL (but not the soleus) in the ANE and CMV groups, consisting of mean reductions of 16.7% compared with CTL values.
MHC Isoform Expression The mean percentages of type I, type II, and hybrid fibers (with type I/type II coexpression) in the diaphragm for all animals are depicted in Figure 1 . The CTL and ANE group diaphragms did not differ from one another. In contrast, diaphragm myofibers expressing type I MHC in an exclusive fashion were significantly reduced in the CMV group (see Figure 1A). This was associated with a significant increase (approximately fivefold) in the percentage of hybrid fibers in CMV group diaphragms compared with the other two groups (see Figure 1B). Furthermore, there was a strong trend toward a significant relationship between the duration of mechanical ventilation and the percentage of hybrid fibers in the diaphragm, as revealed by a correlation coefficient of 0.85 (p = 0.07). For the two hindlimb muscles (EDL and soleus), there were no significant differences among the three groups of animals in the relative percentages of type I, type II, and hybrid fibers (see Figure 2) . However, as with the diaphragm, some hybrid fibers were observed in both hindlimb muscles even under baseline CTL conditions.
Representative diaphragm tissue sections from the three experimental groups are shown in Figure 3 . Serial sections were reacted with antibodies directed against either type I or type II MHCs, and positive immunoreactivity is indicated by dark staining of the fibers. In the CMV group diaphragm, the same fibers can be seen to stain darkly with both antibodies, thus indicating their identity as hybrid fibers. Such hybrid fibers were infrequent in the ANE and CTL diaphragms. In addition, there was significant muscle fiber atrophy in the diaphragms of ANE as well as CMV animals in comparison with the CTL group. Quantitative analysis of diaphragm muscle fiber cross-sectional area for the three groups is shown in Table 3 . In the ANE group, the mean decreases in fiber cross-sectional area (compared with CTL) amounted to 18.3%, 29.3%, and 21.6%, respectively, for type I, type II, and hybrid fibers. For the CMV group, mean cross-sectional area decreased by 23.2%, 23.7%, and 27.7%, respectively, in type I, type II, and hybrid fibers.
Northern blot analysis was performed in a subset of CMV and CTL animals (n = 3 for each group). For the group as a whole, MHC isoform mRNA levels in the CMV group diaphragms (normalized to 18S ribosomal RNA and expressed as the percentage of CTL diaphragm values obtained from the same blot) were as follows: 170 ± 65% for type IIa, 122 ± 43% for type IIx, 104 ± 23% for type IIb, and 100 ± 22% for type I.
Diaphragm Contractile Function The mean diaphragm strip length at which maximal force was achieved (optimal length) was found to be greater in the CTL (28.7 ± 0.7 mm, n = 4) and ANE (30.4 ± 0.9 mm, n = 2) groups than in the CMV animals (26.8 ± 0.9 mm, n = 3). When CTL and ANE values were pooled (29.3 ± 1.1 mm) and compared with the CMV group, statistical significance was achieved using both nonparametric (p = 0.028) and parametric (p = 0.016) approaches. Diaphragmatic twitch (Pt) and tetanic (Po) force were normalized to muscle strip cross-sectional area. Once again, because the small numbers of animals and the fact that values for CTL (Pt = 7.29 ± 1.67 Newtons [N]/cm2, Po = 18.08 ± 2.13 N/cm2) and ANE (Pt = 8.48 ± 1.23 N/cm2, Po = 17.75 ± 0.18 N/cm2) did not differ from one another, the CTL and ANE groups were pooled for the purposes of statistical analysis. As shown in Figure 4 , maximal twitch and tetanic force production by the diaphragm were significantly reduced in the CMV group using both nonparametric (for Pt and Po, p = 0.028) and parametric (for Pt, p = 0.028; for Po, p = 0.005) methods of comparison. There were no significant differences in contraction time, half-relaxation time, or fatigue resistance (data not shown).
This study evaluated the effects of CMV on diaphragm phenotype at the individual muscle fiber level, through immunodetection of MHC isoform switching. The principal findings of this investigation are as follows: (1) CMV led to a significant increase in hybrid fibers within the diaphragm, which occurred at the expense of "pure" myofibers with exclusive expression of type I MHC; (2) CMV produced a significant decrease in diaphragm mass relative to total body mass, together with a nonselective reduction in the mean cross-sectional area of all fiber types; and (3) even after correction for the level of muscle atrophy, CMV caused major reductions in diaphragm force-generating capacity as well as a decrease in optimal diaphragm muscle length. The major novel finding of our study is that despite a relatively short period of CMV, a significant slow-to-fast MHC isoform shift occurred in the diaphragm. In contrast, there were no changes of this nature in hindlimb muscles from the same animals, which is consistent with previous studies of hindlimb muscle disuse within such a short time frame (11, 26). The fact that only the diaphragm demonstrated these rapid changes in MHC expression after instituting CMV suggests at least two possibilities. First, there could be a greater susceptibility of the diaphragm to disuse, such that the threshold duration of muscle disuse needed to induce MHC isoform switching in the diaphragm is lower than that of limb muscles. This could be plausibly related to the unique functional role of the diaphragm among skeletal muscles. Second, the rather unusual nature of CMV as a form of muscle disuse (i.e., combined presence of reduced electrical activity, mechanical unloading, and intermittent passive shortening) could also serve as a particularly powerful stimulus for MHC transformations in the diaphragm.
Methodologic Considerations Nutritional deprivation has a preferential atrophic effect on fast-twitch fibers (27–29). This could account for the fact that the EDL, but not the slow-twitch soleus, demonstrated a statistically significant loss of muscle mass relative to body weight in the CMV and ANE groups. Along these same lines, in ANE group diaphragms the type II fiber population exhibited a disproportionately large reduction in mean cross-sectional area in comparison with the type I fibers (29.3% versus 18.3%). Although nutritional support was provided in our study, the caloric and protein intakes in CMV and ANE groups were limited to approximately 30% of free-eating CTL values. Unfortunately, it was not feasible to supply greater levels of nutritional support without developing high residual gastric contents with the attendant risk of aspiration. The efficiency of intestinal absorption under our experimental conditions is unknown, and the stress of animal surgery may also have contributed to a state of increased catabolism. Nonetheless, nutritional deprivation per se is unlikely to account for the changes in MHC isoform expression or diaphragm contractile function found in the CMV group. In this regard, a previous study that also used MHC isoform-specific antibodies on serial cross-sections of rat diaphragm did not report any increase in hybrid fibers after nutritional deprivation (30). Similarly, Lewis and colleagues found no increase in type IIc fibers (the equivalent of hybrid fibers by ATPase histochemistry) in malnourished rats (29). These previous investigations are consistent with the fact that hybrid fibers remained at control levels in the ANE rats of our study. In addition to the increase in hybrid fibers, CMV group diaphragms also showed equivalent decreases in type II and type I muscle fiber cross-sectional area (23.7% and 23.2%, respectively), which is more consistent with muscle disuse (31) rather than malnutrition alone. Finally, it should be noted that malnutrition alone does not reduce maximal specific force production by the diaphragm (28, 29). The use of neuromuscular blockade in this study also raises the question of whether the changes in MHC isoform composition of the diaphragm might be attributable to a state of "chemical denervation" of the muscle. We believe this is unlikely for several reasons. First, within the time frame examined in our study, chemical as well as surgical denervation of the diaphragm (16, 17) and hindlimb muscles (13) is associated with a fast-to-slow conversion of MHC isoforms. Therefore, the changes expected with denervation are diametrically opposed to the slow-to-fast MHC isoform shift observed in the diaphragm after CMV. Second, denervation effects related to intravenous administration of doxacurium would be expected to affect hindlimb muscles as well as the diaphragm. In fact, the limb muscles are reportedly more susceptible than the diaphragm to denervation-like effects induced by neuromuscular blocking agents (32). However, we found no evidence of increased MHC isoform switching in hindlimb muscles of the CMV group. Finally, although diaphragm denervation causes a rapid reduction in specific force (16, 17, 33) similar to that found in our study, CMV-induced decreases in diaphragm specific force also occur in the absence of neuromuscular blocking agents (7–9).
Comparison with Previous Studies
Functional and Clinical Implications The alterations in MHC isoform composition found after CMV in our study do not explain major reductions in isometric force production by the diaphragm. In fact, type II fibers in the rat diaphragm reportedly generate higher levels of isometric tension than type I fibers (24, 25). Therefore, factors other than the type I to type II fiber transformation observed in the diaphragm must account for the loss of isometric force production after short-term CMV. Although not apparent by light microscopy in our study, one possible mechanism underlying the reduced force-generating capacity of the diaphragm would be some form of damage at the ultrastructural level. This has been described in other disuse models (31, 33, 35). Because muscle disuse has been reported to increase the susceptibility of myofibers to contraction-induced injury (31), it is also conceivable that occasional "breakthrough" episodes of spontaneous respiration during CMV could contribute to diaphragm injury. Another plausible hypothesis is that CMV could induce a preferential loss or alteration of contractile protein elements (36), perhaps as a direct consequence of the diaphragm remodeling process itself. Other potential effects such as interference with excitation–contraction coupling mechanisms also need to be considered. Clearly, further studies are needed to determine the precise etiology of CMV-induced decreases in diaphragm force production, particularly as such changes would be predicted to greatly impede efforts at weaning patients from mechanical ventilation. Another finding with potential clinical implications is that the optimal length for diaphragm force production was reduced in the CMV group. Similar findings were reported by Le Bourdelles and colleagues after 48 hours of CMV (7), but not by Powers and colleagues (8) for periods of CMV up to 24 hours. A reduction in optimal length provides strong albeit indirect evidence for a loss of sarcomeres in series (37, 38), which could also help to account for the greater loss of global diaphragm mass in the CMV group as compared with the ANE cohort. The most likely stimulus for sarcomere elimination in our study would be a reduction in diaphragm muscle length caused by changes in lung volume during CMV. Hence, one possibility is that the applied ventilator settings led to an increase in end-expiratory lung volume (e.g., through the use of positive end-expiratory pressure or induction of dynamic hyperinflation [39]). Alternatively, it is conceivable that cyclical lung inflation by the ventilator, which leads to intermittent passive shortening of the diaphragm during inspiration (18), serves as a stimulus for sarcomere resorption. Whatever the mechanism, we speculate that such changes might be deleterious in the clinical setting, as alterations in optimal length related to CMV could be inappropriate for the lung volume and associated diaphragm length changes encountered during spontaneous breathing efforts. In summary, we conclude that even short-term (2–4 days) imposition of CMV leads to major remodeling of the rat diaphragm. If also present in humans, such changes could contribute to the difficulties encountered in discontinuing ventilatory support in many patients, particularly after more prolonged periods of mechanical ventilation. Further studies are needed to address the importance of this phenomenon in mechanically ventilated patients. In addition, it will be important to determine how varying degrees of spontaneous respiratory effort and other aspects of the patient-ventilator interaction (4) are able to modify these responses.
: This article is dedicated to the memory of Jun Luo. Supported by grants from the Canadian Cystic Fibrosis Foundation (B.J.P.), the Quebec Pulmonary Association (B.J.P. and S.B.G.), the Canadian Institutes of Health Research (B.J.P. and S.B.G.), the Fonds de la recherche en sante du Quebec (B.J.P.), and a post-doctoral fellowship from the Canadian Lung Association (L.Y.).
This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.org Received in original form February 11, 2002; accepted in final form May 9, 2002
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