To our knowledge, these data are the first report of significant changes in MHC expression with corresponding physiologic alterations in any respiratory muscle of an animal model of emphysema. These changes are qualitatively similar to those reported in severe human emphysema [12, 13]: that is, they too are manifested as a shift toward slower MHC isoforms. Quantitatively, however, the changes are far less marked than the shift demonstrated in humans.
The most studied animal model for the adaptation of respiratory muscle to emphysema has been the hamster with elastase-induced emphysema. The hamster model has probably been chosen for these studies in large part because of the impressive increases in lung volumes that can be created in hamsters, with total lung capacity sometimes reaching 200% of control values. Despite the demonstration of length adaptation [1–4, 6] and fatigue resistance [2, 3, 6] in the diaphragms of emphysematous hamsters, not until very recently could a change in fiber type distribution be demonstrated , and in that study histochemical classification was employed that could not separate out IIx from IIb fibers. Furthermore, no study in emphysematous hamster diaphragm has demonstrated a change in twitch kinetics.
Intratracheal instillation of elastase in rats creates a form of panacinar emphysema that in some ways more closely parallels the human disease than that created by instillation of elastase in hamsters. Changes in lung volumes and compliance in the rat are more similar to those seen in humans [14–18]. An electron microscopic comparison of human emphysema with elastase-induced emphysema in rats revealed remarkably similar pictures of elastin disintegration accompanied by increased collagen deposition and reorganization .
Our results for MHC expression in rat diaphragm from normal and emphysematous rats must be viewed first in the light of previous quantitative studies of MHC expression in normal rat diaphragm that used techniques able to separate out all known isoforms. In 7–9-week-old Sprague-Dawley rats, Kanbara et al.  found by in situ hybridization that 1.0% of fibers expressed only IIb and 8.8% coexpressed the IIx and IIb isoforms. This is in contrast with our RT-PCR result of 21.6% IIb in normals, but immunocytochemical results much more in line with Kanbara's findings. In that study, the values for MHCs I, IIa, and IIx were not markedly different from those reported here for normal rat diaphragm by RT-PCR. By electrophoretic separation, Sugiura et al.  found IIb MHC to represent 6.1% of all MHC protein in young rat diaphragm, and their IId/IIx result of 44.9% is also quite different from our PCR result, while their I and IIa findings are very similar to our PCR results, and their IId/IIx result closely parallels our IIx area percentage immunocytochemistry result. Okumoto et al. reported even less IIb and more IId protein by electrophoresis in 5-month-old rats .
The differences between the findings of these reports and ours might at first glance result from the age of the animals studied, as our rats were significantly older at 13 months. However, it has been reported that there is an age-related transition from fast to slow MHC in rat limb muscles [30, 31] and diaphragm , which would render this explanation unlikely. A potential explanation of our results in relation to Kanbara's would be that there was a systematically greater concentration of IIb mRNA in IIb and IIb/IIx fibers than of other isoforms' mRNAs in their respective fibers. Sugiura et al. and Okumoto et al. used Wistar rats, although this would be unlikely to explain the difference between their findings and ours. In the final analysis, it is difficult to draw any firm conclusions from comparisons between studies addressing mRNA levels and those addressing protein levels; and similarly between techniques measuring protein by gel and those measuring protein by immunocytochemistry.
Although increased activities of citrate synthase  and SDH [2, 3] have usually been demonstrated in the diaphragm in animal models of emphysema, fiber type has generally not been found to be significantly altered [1–3, 7]. Only in one recent paper was a shift in MHC in emphysematous diaphragm demonstrated, but the histochemical technique used precluded the separation of IIb and IIx fibers . Another study has shown increased expression of IIa at the expense of IIx MHC in emphysematous hamster scalene muscle with the use of MHC monoclonal antibodies, but there were none of the expected physiologic changes accompanying the shift .
We demonstrate here a shift from IIb toward IIx in emphysematous rat diaphragm at both the protein and mRNA levels. Although this shift toward a slower isoform is not as marked as that reported in humans [12, 13], in which MHC type I is upregulated and both IIa and IIx are downregulated, there is precedent for restricted MHC adaptation in rats. Termin et al.  for example, have noted that whereas chronically stimulated rabbit fast-twitch muscle results in appreciable increases in slow myosin isoforms, chronic stimulation of rat muscle tends to bring about shifts toward the slower types among the type II isoforms. Other factors that might lead to less impressive MHC shifts in rodent models of emphysema than in humans include the much shorter time course over which the adaptations have a chance to occur, the greater compliance of the rodent thorax to pulmonary hyperexpansion, and differences in the diaphragmatic load created in the human disease in comparison with the animal models that have yet to be fully worked out.
Our finding in the diaphragms of rats with emphysema is very similar to that demonstrated by Sugiura et al. in the diaphragms of chronically swimming rats . We found IIb mRNA expression to decrease by 31% and IIb MHC-determined fiber type to decrease by 35% in emphysema. Sugiura et al. found that after 10 weeks of endurance swimming, IIb protein from costal diaphragm of rats fell by 54%, also without statistically significant changes in the amounts of other MHC isoforms. This suggests that the effects of emphysema on the diaphragm are, as has been suggested by others, at least in part a function of increased workload.
Other studies of MHC adaptation in animal models of increased diaphragmatic work have had conflicting results. Although various treadmill-running protocols have shown increases in the aerobic capacity of the rat diaphragm [32, 34–37], running has generally not been demonstrated to cause consistent changes in the relative expression of MHC isoforms [29, 38]. Although one paper has shown a decrease in type IIb fibers and an increase in type I fibers , and another has shown only a significant decrease in type IIb fibers , a third paper reported a surprising increase in type IIb fibers .
In contrast, inspiratory resistive loading by tracheal narrowing has consistently shown increases in the percentage of type I fibers and the corresponding MHC isoform in the diaphragm [41–43], closely resembling the changes seen in severe human emphysema. Respiratory loading by chronic hypercapnea has also demonstrated similar changes .
Given these findings, one can postulate that shifts toward slower MHC isoforms in diaphragmatic muscle fibers result primarily from some combination of the degree and chronicity of the work performed by the muscle. If this is so, then one could arrange the experimental methods that have been explored that impose increased diaphragmatic work, from least imposed load to greatest imposed load, as follows: running, swimming, emphysema, hypercapnea, tracheal banding. It is possible that in emphysema, the additional impact on diaphragmatic physiology of fiber shortening resulting from pulmonary hyperexpansion [1–6] affects MHC isoform shifts beyond the influence of a pure increase in the workload on the muscle.
In addition to being the first demonstration of a decrease in the expression of the IIb MHC isoform in an animal model of emphysema, this study is the first to demonstrate that such a shift toward slower isoforms in emphysema has a significant physiologic impact on diaphragmatic function in vitro. We show that both twitch speed and fatiguing properties move toward slower-twitch characteristics with this decrease in IIb expression. Given previous work in this area, it is not unexpected that even a shift only between the fast isoforms but away from IIb would result in measurable physiological changes. It has been noted, for example, that IIx fibers have significantly higher SDH activity than IIb fibers [24, 45] and, further, that IIb motor units are significantly more fatigue sensitive than IIx motor units . Schiaffino et al. have also demonstrated, in rat whole muscles, a slower maximum velocity of shortening in muscles made up of predominantly IIx versus IIb fibers . Further, Sant'Ana Pereira et al.  have shown a major difference in actomyosin ATPase activity in rat single IIb and IIx fibers, and Sieck et al.  have shown this in single fibers from rat diaphragm.
Finally, we examined the expression of the SERCA proteins in these animals, because these proteins are responsible for a significant fraction of energy consumption in skeletal muscle, second only to the energy consumed by the ATPase responsible for movement of the myosin head. Further, diaphragmatic fatigue might be related to SERCA function  and, as discussed above, our emphysematous animals showed decreased diaphragm strip fatigability. Previous work has suggested that although SERCA expression in limb muscles seems to respond to increases in functional load by upregulating SERCA 2 (the slow SERCA isoform) and downregulating SERCA 1 (the fast isoform) , similar responses might not occur in diaphragm . Our data showing no difference in the expression of SERCA 1 and SERCA 2 between emphysematous and control animals are consistent with this finding. We did not measure SERCA activity or phospholamban phosphorylation. It is certainly possible that the overall activities of one or both of the SERCA isoforms are, in fact, different between emphysematous and control diaphragm, although the numbers of fibers expressing each isoform are not different.
Because mechanical indices of relaxation reflect the function of SERCA in sequestering calcium in the sarcoplasmic reticulum , we measured half relaxation times on our diaphragmatic strips. It is not surprising that, with no change in SERCA 1 and SERCA 2 expression detected by immunocytochemistry, we found no difference in the relaxation times between control and emphysematous diaphragm strips.