Background In patients with severe chronic obstructive pulmonarydisease, the diaphragm undergoes physiologic adaptations characterizedby an increase in energy expenditure and relative resistanceto fatigue. We hypothesized that these physiologic characteristicswould be associated with structural adaptations consisting ofan increased proportion of less-fatigable slow-twitch musclefibers and slow isoforms of myofibrillar proteins.
Methods We obtained biopsy specimens of the diaphragm from 6patients with severe chronic obstructive pulmonary disease (mean[±SE] forced expiratory volume in one second, 33±4percent of the predicted value; residual volume, 259±25percent of the predicted value) and 10 control subjects. Theproportions of the various isoforms of myosin heavy chains,myosin light chains, troponin, and tropomyosin were determinedby sodium dodecyl sulfate–polyacrylamide-gel electrophoresis.We also used immunocytochemical techniques to determine theproportions of the various types of muscle fibers.
Results The diaphragm-biopsy specimens from the patients hadhigher percentages of slow myosin heavy chain I (64±3vs. 45±2 percent, P<0.001), and lower percentagesof fast myosin heavy chains IIa (29 ± 3 vs. 39 ±2 percent, P = 0.01) and IIb (8±1 vs. 17±1 percent,P<0.001) than the diaphragms of the controls. Similar differenceswere noted when immunohistochemical techniques were used tocompare the percentages of these fiber types in the two groups.In addition, the patients had higher percentages of the slowisoforms of myosin light chains, troponins, and tropomyosin,whereas the controls had higher percentages of the fast isoformsof these proteins.
Conclusions Severe chronic obstructive pulmonary disease increasesthe slow-twitch characteristics of the muscle fibers in thediaphragm, an adaptation that increases resistance to fatigue.
Two decades ago, Roussos and Macklem1 demonstrated that thediaphragm, the major inspiratory muscle, can become fatigued.Although the site of muscular fatigue can occur anywhere inthe motor pathway between the cerebral cortex and the musclefibers themselves, previous investigators have demonstratedthat a particular type of fatigue — i.e., low-frequencyfatigue — occurs at the level of the muscle cell itself.2,3Bellemare and Bigland-Ritchie4 and others5,6,7,8,9 have demonstratedthat various types of exercise can elicit low-frequency diaphragmaticfatigue in normal subjects. Surprisingly, there has been littlemention of exercise-induced low-frequency diaphragmatic fatiguein chronic obstructive pulmonary disease (COPD). Indeed, Polkeyet al.10 recently reported that low-frequency diaphragmaticfatigue did not develop in patients with severe COPD duringtreadmill exercise to "exhaustion."
Bellemare and Grassino11 have shown that the diaphragms of patientswith severe COPD expended more energy — as evaluated bythe diaphragmatic time–tension index — than thediaphragms of normal subjects. We hypothesized that this combinationof a chronic increase in energy expenditure and relative resistanceto low-frequency fatigue (in the diaphragms of patients withsevere COPD) would be associated with profound structural adaptationscharacterized by an increase in slow-twitch muscle characteristics(i.e., increased proportions of less-fatigable slow-twitch fibersand slow isoforms of the myofibrillar proteins).
Methods
Patients and Control Subjects
We obtained biopsy specimens from the costal diaphragm of 6patients with severe COPD (3 men and 3 women) who were undergoinglung-volume–reduction surgery and 10 control subjects(5 men and 5 women). We used two subgroups of controls. Thefirst consisted of four subjects with a mild impairment in pulmonaryfunction who were undergoing resection of solitary pulmonarynodules; in those subjects, biopsies were performed at the timeof surgery. The second type consisted of six brain-dead organdonors in whom biopsies were performed at the time of organharvest before circulatory arrest.
Since the brain-dead control subjects were nonsmokers who hadno history of symptoms or signs of cardiopulmonary or neuromusculardisease before their neurologic catastrophes, we presumed thatthese subjects would have had normal pulmonary-function testsbefore the onset of their neurologic events. Moreover, sincethe interval between the onset of the neurologic events (inthese subjects) and biopsy of the diaphragm was less than 24hours, the myofibrillar protein composition of these diaphragmsshould not have changed during this period.12,13
Informed consent for biopsies in both the patients and the controlswho underwent surgery was obtained from each of these participants,and our protocol was approved by the institutional review boardsof the Philadelphia Veterans Affairs Medical Center and theHospital of the University of Pennsylvania. In contrast, forthe brain-dead organ donors, informed consent was obtained fromthe family of each subject, and this portion of the protocolwas approved by the human-studies committee at Columbia–PresbyterianHospital, New York.
Biopsies
Full-thickness biopsy specimens (approximately 20 to 25 mm by6 to 8 mm; weight, 4 g) were obtained from the same region ofthe right anterior costal diaphragm lateral to the insertionof the phrenic nerve, frozen in isopentane, and then transferredto liquid nitrogen and stored at -70°C until being used.
Overview of Biochemical Determinations
Myosin light chains were analyzed from preparations of purifiedmyosin, whereas purified myofibrils were used for the measurementof all other myofibrillar proteins (myosin heavy chains, troponin,and tropomyosin).
Preparation of Myofibrils and Purification of Myosin
Myofibrils were prepared according to the method of Solaro etal.14 with the addition of a protease-inhibitor set (2 mM sodiumazide, 0.1 mM phenylmethylsulfonyl fluoride, 10 mM of sodiumpyrophosphate, and 1 µg each of leupeptin and pepstatinA per milliliter) in the homogenization buffer. To purify myosin,part of the myofibrillar suspension was dialyzed for five tosix hours against 200 volumes of 20 mM TRIS–hydrochloricacid (pH 7.5), 0.5 M potassium chloride, 1 mM magnesium chloride,and 0.5 mM -mercaptoethanol, and then subjected to a "bath-flow"procedure with a resin of phalloidin-stabilized, actin-coatedSepharose B prepared according to the method of Grandmont-Leblancand Gruda.15 This resin was first equilibrated against the dialysisbuffer and then incubated for 30 minutes at 25°C with themyofibrillar solution in a ratio of 1 mg of myofibril per gramof resin. The resin was washed twice with 10 volumes of dialysisbuffer, and the myosin was then diluted by 2 M potassium chloride.Some trace amounts of actomyosin were then precipitated with35 percent ammonium sulfate, and the myosin was subsequentlyprecipitated from the supernatant by increasing concentrationsof ammonium sulfate (up to 50 percent). Protein concentrationswere determined by Bradford's reaction.
Myofibrils or purified myosin preparations were dialyzed overnightagainst 200 volumes of 100 mM TRIS–hydrochloric acid (pH8.0) and 1 mM -mercaptoethanol. The samples for sodium dodecylsulfate–polyacrylamide-gel electrophoresis (SDS-PAGE)were then prepared by diluting the proteins with Laemmli's bufferby a factor of 4 to 6 and then incubating them at 95°C forfive minutes. SDS-PAGE for myosin heavy chains was performedaccording to the method of Talmadge and Roy,16 which we adaptedfor use with human tissue by increasing the total running timeto 32 hours and decreasing the voltage (i.e., 50 V for 1 hour,100 V for 2 hours, and 220 V for 29 hours at 8°C). The amountof protein loaded per lane was 0.15 µg for silver-stainedgels and 1.5 µg for Coomassie-stained gels.
SDS-PAGE for myosin light chains, troponins, and tropomyosinwas performed according to the method of Laemmli17 on 15 percentpolyacrylamide gels supplemented with 10 percent glycerol; 1.5µg of protein was loaded per lane. Coomassie-stained gelswere scanned on a Pharmacia Ultroscan densitometer (LKB). Thedensitometric signal (i.e., peak absorption) for our 0.75-mm-thickgels was linear up to concentrations of 5 µg of proteinper lane.
Two-Dimensional SDS-PAGE
Two-dimensional SDS-PAGE was performed as described by O'Farrell.18The sample preparation and running conditions were identicalto those described previously.19
Western Blot Analysis
Immunoblotting for myosin heavy chains was performed accordingto the method of Hughes et al.20 The supernatants of all monoclonalantibodies against myosin heavy chains were obtained from theDevelopmental Studies Hybridoma Bank, with the exception ofantibody BF-F3, which was kindly provided by Dr. Stefano Schiaffino.21We used F(ab)2 anti-IgG and anti-IgM, peroxidase-conjugatedgoat antimouse secondary antibodies (Cappel). The bands werevisualized with ECL peroxidase substrate (Amersham) and exposedto Kodak XAR-2 x-ray film.
Immunocytochemical Analysis
The types of fiber were identified by indirect immunofluorescencewith monoclonal antibodies specific for the following myosinheavy chains: NOQ7.54D for type I,22 SC71 for type IIa,22 andBF-F3 for type IIb.21 Our staining protocols have been previouslydescribed.22,23 We determined the proportions of the varioustypes of fiber by counting the fibers on the serial 10-µmsections, each stained with a specific antimyosin antibody.Approximately 200 fibers were counted per cross-section. Weclassified the fibers as type I, IIa, or IIb on the basis ofthe antibody that yielded maximal fluorescence on fluorescenceantibody staining; therefore, our classification method didnot assess the proportions of fibers that may have expressedmore than one myosin heavy chain. The cross-sectional areasof the fibers were determined according to previously describedmethods.24
Statistical Analysis
Quantitative data are described as means ±SE. Group t-testswere used to evaluate the statistical significance of differencesbetween groups.25 Differences were attributed to chance unlessthey were significant at the 0.05 level.
Results
Characteristics of the Study Subjects
The patients did not differ significantly from the control subjectswith respect to age, height, or weight (Table 1). Pulmonary-functionmeasurements were carried out only in the patients and the fourcontrol subjects who underwent surgery. The patients had greaterresidual volume, functional residual capacity, and total lungcapacity than the control subjects, and the controls had highervalues with respect to the forced expiratory volume in one secondand the ratio of the forced expiratory volume in one secondto forced vital capacity.
Table 1. Characteristics of the Patients with Chronic Obstructive Pulmonary Disease and the Control Subjects.
Analyses of Myosin Heavy Chains
SDS-PAGE and Immunoblotting
As shown in Figure 1, SDS-PAGE revealed that isoform IIb iseither absent or markedly diminished in the patients. In contrast,isoform I is more prominent in the patients than in the controls.When immunoblotting was performed with the monoclonal antibodyA4.1025 (Figure 2), the distribution of myosin heavy chainsin both controls and patients was the same as that shown inFigure 1, since this monoclonal antibody recognizes all myosin-heavy-chainisoforms. When monoclonal antibodies A4.951 and A4.974, whichreact specifically with myosin heavy chains I and IIa, respectively,were used, reactions were positive for both controls and patients.Immunoblotting with monoclonal antibody BF-F3, which is specificfor isoform IIb, was strongly positive only in controls andwas either absent or weakly positive in patients.
Figure 1. Representative Coomassie-Stained SDS-PAGE Gels, Showing the Distribution of the I, IIa, and IIb Isoforms of the Myosin Heavy Chain in the Costal Diaphragms of Three Control Subjects and Three Patients with COPD.
A molecular-weight standard (S) is shown at the left.
Figure 2. Western Blot Analyses of the I, IIa, and IIb Isoforms of the Myosin Heavy Chain in the Costal Diaphragms of a Representative Control Subject and a Patient with COPD.
The monoclonal antibodies used are shown.
Quantitation of Myosin-Heavy-Chain Isoforms
We used Coomassie-stained densitograms to quantitate the proportionsof the myosin-heavy-chain isoforms present in diaphragm-biopsyspecimens from control subjects and patients with COPD. Controlsubjects had higher percentages of myosin heavy chains IIa andIIb, whereas patients had a higher percentage of myosin heavychain I (Table 2).
Table 2. Composition of Myosin Heavy Chains and Fiber Types Present in the Costal Diaphragm.
Immunocytochemical Analysis
Qualitative Observations
A comparison of cross-sections of diaphragm from a representativepatient with COPD and a control subject (Figure 3) showed thatthe patient had a higher proportion of type I fibers, a lowerproportion of type IIa fibers, and no evidence of type IIb fibers.In addition, the cross-sectional areas of the fibers from thepatient were smaller.
Figure 3. Immunofluorescent Staining of Serial Sections of Costal Diaphragm from a Representative Control Subject and a Patient with COPD (x270).
Cross-sections were preincubated with the following antibodies: NOQ7.54D antibody in Panels A and B, which is specific for myosin heavy chain I; SC71 antibody in Panels C and D, which is specific for myosin heavy chain IIa; and BF-F3 antibody in Panels E and F, which is specific for myosin heavy chain IIb. SDS-PAGE showed the following distribution of myosin heavy chains I, IIa, and IIb: 45, 41, and 14 percent, respectively, in control specimens and 70, 25, and 5 percent, respectively, in patient specimens.
Quantitative Observations
As shown in Table 2, the patients with COPD had a higher percentageof type I fibers, whereas controls had a higher percentage oftype IIb fibers. Most important, as can be seen from a comparisonof the upper and lower portions of Table 2, the percentage ofeach of the principal types of fibers — I, IIa, and IIb— was markedly similar to the proportion of its correspondingmyosin heavy chain in the costal diaphragm.
As illustrated in Figure 3, some of the fibers in specimensfrom both the patient and the control subject were sectionedobliquely. To eliminate these fibers from our comparisons ofcross-sectional area, we determined the ratio between the majorand minor axes of these elliptical fibers and did not includefibers in which this ratio exceeded 1.25 in our quantitativecalculations. Nonetheless, for the three fiber types, the cross-sectionalarea of the fibers from the patients was 40 to 60 percent lessthan that of fibers from the controls.
Composition of Myosin Light Chains
The purified preparations of myosin from diaphragm-biopsy specimensfrom controls contained six types of myosin light chains (Figure 4A,lane 3): 1sa, 1sb, 1f, 2s, 2f, and 3f ("s" and "f" designateslow and fast isoforms, respectively). Table 3 indicates thatthe patients had significantly higher percentages of myosinlight chains 1sa, 1sb, and 2s, whereas the controls had higherpercentages of 1f and 2f. The two-dimensional gel shown in Figure 4Billustrates these differences in the composition of myosinlight chains more clearly than the one-dimensional gel shownin Figure 4A.
Figure 4. Low-Molecular-Weight Region Identified by One-Dimensional SDS-PAGE (Panel A) and Two-Dimensional SDS-PAGE (Panel B).
Panel A shows the results of silver staining. Lanes 1 and 2 show purified myosin from rat extensor digitorum longus (EDL) and soleus, respectively. Lanes 3 and 4 show purified myosin from a representative control and a patient, respectively; and lanes 5 and 6 show purified myofibrils from the same control and patient. The bands were identified on the basis of the migration of proteins with known molecular weights and fast (f) and slow (s) myosin light chains of purified myosin from rat EDL and soleus, commercial preparations of tropomyosin from rabbit back muscle (Sigma, data not shown), and fast troponin isoforms isolated according to the method of Greaser and Gergely28 from rabbit psoas muscle (data not shown). Panel B shows the results of staining with Coomassie blue.
Table 3. Distribution of Myosin Light Chains and Regulatory Proteins in the Costal Diaphragm.
Regulatory Myofibrillar Proteins
Figure 4A shows the results for a representative patient andcontrol, whereas Table 3 presents a statistical comparison ofthe percentages of different isoforms of tropomyosin and subunitsof troponin in the two groups. As shown in Table 3, controlsubjects had a higher percentage of fast -tropomyosin, whereasthe patients had a higher percentage of slow -tropomyosin. Inaddition, the patients had a higher percentage of troponin Cs,whereas the controls had higher percentages of troponins Tfand If . The groups did not differ significantly with respectto the percentage of troponin Cf .
Discussion
Our results show that the diaphragms of patients with severeCOPD have a higher proportion of type I (slow-twitch) fibersand a lower proportion of type II (fast-twitch) fibers thanthe normal diaphragm. These data are consistent with the hypothesisthat severe COPD transforms fast-twitch fibers to slow-twitchfibers in the diaphragm.
Is Myosin Heavy Chain Iix Expressed in the Human Diaphragm?
Much work regarding adaptations of the diaphragm in COPD hasbeen carried out in rats and hamsters, whose diaphragms andlimb muscle contain a third fast-twitch myosin-heavy-chain isoform,designated IIx.16,21,29 The occurrence of this IIx isoform inthe human diaphragm has not been evaluated. Using SDS-PAGE gelsand immunoblotting, multiple investigators have reported thathuman limb muscle contains only three myosin-heavy-chain isoforms:I, IIa, and IIb.30,31,32 Our results in the diaphragm are consistentwith these observations in limb muscle. Therefore, we and otherworkers have found no evidence of the expression of IIx in humandiaphragm or limb muscle.
Recently, Smerdu et al.33 carried out a series of experimentsin human limb and trunk skeletal muscle using in situ hybridizationtechniques to detect messenger RNA and immunocytochemical techniquesto type muscle fibers according to the expression of myosinheavy chains. They concluded that human muscle contained threeprincipal fiber types (I, IIa, and IIx), which express a singlemyosin-heavy-chain transcript, and two populations of hybridfibers, which express either I and IIa or IIa and IIx myosin-heavy-chaintranscripts. However, no specific antibody exists for the IIxmyosin heavy chain, and thus, the results of their immunocytochemicalfiber typing cannot be accepted unequivocally.21
Also, BF-35 is a monoclonal antibody that stains all fibersexcept IIx.21 This antibody stained all fibers in diaphragm-biopsyspecimens from both control subjects and patients with COPD.We interpret this observation as ruling out the presence ofpure IIx fibers in the human diaphragm. Therefore, we believethat more experimental work is necessary to clarify the statusof the expression of IIx in the human diaphragm.
Finally, with respect to the physiologic consequences of thesupposition that all the IIb fibers in our diaphragm sampleswere really IIx fibers, previous workers have demonstrated thatdifferent fiber types classified on the basis of the compositionof myosin heavy chains have different maximal velocities (withIIb having the highest velocity, IIx and IIa having intermediatevelocity, and I having the lowest velocity) as well as differentresistances to fatigue (with I being the most resistant, followedin descending order by IIa, IIx, and IIb).34 Therefore, evenif one suggests that all type IIb fibers in our human diaphragmswere type IIx, the decreases in type IIx and the increases intype I fibers noted in the specimens from the patients wouldbe consistent with our concept that severe COPD elicits an increasein slow-twitch characteristics of the human diaphragm.
Relation between the Severity of Copd and the Changes in the Fiber Types Present
The data of Sanchez et al.35,36 indicate that moderate COPDis associated with atrophy of both type I and type II fibers,with no change in the relative proportions of these types offibers. However, our patients with COPD had more severe disease,manifested by greater abnormalities in spirometric measurementsand appreciably greater hyperinflation. Our results suggestthat only the diaphragms of patients with severe COPD show theswitch from fast-twitch fibers to slow-twitch fibers.
Adaptations in Myosin Light Chains and Regulatory Proteins
Billeter et al.37 have shown that in human skeletal muscle,fiber types IIa and IIb exclusively express myosin light chains1f, 2f, and 3f. Similarly, fast forms of troponin and tropomyosinlargely occur in type II fibers, whereas the slow isoforms ofthese proteins predominate in type I fibers.38,39 Therefore,in view of these previous studies, the decreases in fast isoformsof both myosin light chains (i.e., 1f and 2f ) and regulatoryproteins (i.e., -tropomyosin and troponins Tf and If) that weobserved in the diaphragm-biopsy specimens from patients withCOPD can be explained by the reduction in type II fibers inthese diaphragms.
Differential Adaptations of Diaphragm and Limb Muscles
Jakobsson and coworkers40 demonstrated that the proportion oftype I fibers is decreased in the quadriceps muscle of patientswith severe COPD. Therefore, the limb muscles and the diaphragmappear to adapt in a qualitatively different manner to severeCOPD. Importantly, we recently concluded that severe congestiveheart failure elicits similar differential adaptations in diaphragmand limb muscles.19
We believe that the difference in the relative activity betweenthe diaphragm and limb muscle accounts for the above-noted differencesin adaptations. First, the work of Bellemare and Grassino11indicates that the diaphragmatic time–tension index —a measure of diaphragmatic energy expenditure — is greatlyincreased, even during breathing at rest in patients with severeCOPD. Second, Mancini et al.41 have shown that this index isgreater in patients with congestive heart failure than in controlsubjects during both rest and exercise. Therefore, the diaphragmsof both patients with severe COPD and patients with congestiveheart failure can be viewed as undergoing constant moderateexercise, and the adaptations that we noted in the diaphragmsof our patients resemble those elicited by endurance trainingin limb muscles.42,43
By contrast, patients with severe COPD and congestive heartfailure have a sedentary lifestyle, and the muscles of theirarms and legs are therefore less active than those of normalsubjects. In response to this decreased activity, the limb musclesadapt in ways that resemble those elicited by deconditioning:the proportion of slow-twitch fibers is decreased, and the proportionof fast-twitch fibers is increased.44
Conclusions
Our data show that in patients with severe COPD, the proportionof slow-twitch fibers in the diaphragm increases, whereas theproportion of fast-twitch fibers decreases. There is also anincrease in the slow myofibrillar-protein isoforms and a decreasein the fast isoforms. These adaptations may render the diaphragmmore resistant to fatigue.
Supported by a Veterans Affairs Merit Review Grant (to Dr. Levine).
We are indebted to Drs. Alfred P. Fishman, Peter T. Macklem,H. Lee Sweeney, Kevin McCully, Neal Rubinstein, and RebeccaHoffman for useful discussion; to Dr. Donna Mancini for thediaphragm-biopsy specimens from the brain-dead organ donors;and to Taitan Nguyen, B.S.E., for assistance in the preparationof the manuscript.
Source Information
From the Pulmonary and Critical Care Divisions, Philadelphia Veterans Affairs Medical Center, Allegheny University of the Health Sciences, and the University of Pennsylvania (S.L.); and the Division of Thoracic Surgery (L.K.) and the Pennsylvania Muscle Institute (S.L., J.L., B.T.), University of Pennsylvania — all in Philadelphia.
Address reprint requests to Dr. Levine at the Pulmonary and Critical Care Division (111P), Veterans Affairs Medical Center, University and Woodland Aves., Philadelphia, PA 19104.
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-mercaptoethanol, and then subjected to a "bath-flow" procedure with a resin of phalloidin-stabilized, actin-coated Sepharose B prepared according to the method of Grandmont-Leblanc and Gruda.15 This resin was first equilibrated against the dialysis buffer and then incubated for 30 minutes at 25°C with the myofibrillar solution in a ratio of 1 mg of myofibril per gram of resin. The resin was washed twice with 10 volumes of dialysis buffer, and the myosin was then diluted by 2 M potassium chloride. Some trace amounts of actomyosin were then precipitated with 35 percent ammonium sulfate, and the myosin was subsequently precipitated from the supernatant by increasing concentrations of ammonium sulfate (up to 50 percent). Protein concentrations were determined by Bradford's reaction. 
-tropomyosin, whereas the patients had a higher percentage of slow 
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