Contractility and myosin heavy chain isoform patterns in developing tracheal muscle

ELSEVIER Respiration Physiology 98 (1994) 101-110

RESPIRATION PHYSIOLOGY

Contractility and myosin heavy chain isoform patterns in developing tracheal muscle

D.A. Roepke, S.L. Griffith, R.A. Meiss, R.A. Rhoades, C.S. Packer*

Department qf Physiology and Biophysics. Indiana University School of Medicole. 635 Barnhill Drive. Indianapolis. IN 46202-5120. USA

Accepted 16May 1994

Abstract Changes in airway smooth muscle reactivity with development may be caused by either modification of the excitation-contraction coupling system or alteration of the contractile apparatus. The mechanism responsible for the reported changes in reactivity was addressed in this study by examining airway smooth muscle contractility and myosin heavy chain isoform patterns as a function of post-neonatal development. Changes in length and force, in response to supramaximal electrical stimulation, were recorded simultaneously as functions of time for tracheal smooth muscle (TSM) strips from 8-week-old and 25-week-old male rabbits. Both the passive and active length-tension (E-T) curves as well as the force-velocity (F-V) curves for the two age groups of rabbit TSM were not significantly different indicating no changes in contractililty during post-neonatal development in rabbits. This conclusion is surprising in light of reports of myosin heavy chain (MHC) isoform shifts in porcine trachealis during comparable periods of development. Therefore, MHC isoform ratios were compared by sodium dodecyl sulfate- polyacrylimide gel electrophoresis for tracheal smooth muscle from male rabbits of 8 and 25 weeks of age. Unlike the reported MHC isoform shifts in the pig tracheal muscle, the rabbit trachealis showed no difference in MHC isoform ratios between the two age groups compared in this study. In conclusion, no changes occur in contractility or MHC isoform patterns during post-neonatal development of rabbit tracheal smooth muscle. Therefore, reported changes in airway muscle reactivity are likely due to changes in receptors or in second messenger systems rather than to changes in the contractile apparatus.

Keywords: Airways, smooth muscle; Contractility, airway smooth muscle; Development; Mammals, rabbit; Muscle, smooth

I. Introduction

Changes in airway smooth muscle reactivity to various agonists during development have been repor ted for several different species. H u m a n infants are repor ted to have

* Corresponding author. Tel.: 317-274-7772; Fax: 317-274-3318.

0034-5687/94/$7.00 © 1994 Elsevier Science B.V. All rights reserved SSD1 0 0 3 4 - 5 6 8 7 ( 9 4 ) 0 0 0 5 4 - 4

102 D.A. Roepke et al. / Respiratkm Physioh~gV 98 (1994~ 101-110

greater airway reactivity than older children in response to various agonists (Mont- gomery and Tepper, 1989). Indeed, many children experience asthma (i.e. airway hyper- reactivity that includes a component of bronchcoconstriction) which is "outgrown" with maturation. Rabbit tracheal smooth muscle (TSM) reactivity to a variety of agonists changes with development. Newborn rabbit TSM shows a greater reactivits, to nicotine, acetylcholine (ACh) and histamine than does adult rabbit TSM. In fact, by the time the rabbit is an adult, histamine causes relaxation in TSM precontracted with ACh (Hayashi and Toda, 1980). Conversely, it has been reported that TSM from preterm sheep requires higher concentrations of either ACh or K + to generate tension of a similar magnitude to that developed in adult sheep in response to the same reagent (Panitch et al., 1989). The porcine airway also exhibits a progressive decline in airway reactivity to histamine and carbachol during post-natal development. Furthermore, airway reactivity is not fully maximal until birth, as fetal airway reactivity is less than that of suckling pigs to specific agonists (Sparrow and Mitchell, 1990). These findings from several species would suggest that the developmental maturation of the post-natal lung involves a decline in airway reactivity.

Reactivity of the TSM in response to agonists strongly influences the function of the trachea by determining the state of contraction (Koslo et al., 1986). Changes in TSM tone have been demonstrated to influence compliance, resistance to deformation, air- flow properties and pressure-volume relationships (Koslo e t a l . , 1986; Olsen et al., 1967; Penn et al., 1988), and these changes have a clear clinical implication (Bhutani et al., 1968).

Whether changes in reactivity and isometric force development are due to alterations at the excitation-contraction coupling level or are due to developmental modification at the level of the contractile apparatus is unknown. Modification of contractility at the contractile apparatus level with development has been demonstrated in vascular (Duckies and Banner, 1984) and gallbladder (Denehy and Ryan, 1986) smooth muscle. However, no complete study of shortening behavior and of the force-velocity relationship has been reported for TSM. Isometric force production is only one parameter of contractility and perhaps is not the best index of contractility changes in muscle of organs in which changes in lumen diameter have profound physiological importance (Packer and Stephens, 1985a and b). In addition, while muscle force production may be unaltered, shortening ability and/or velocity of shortening may be changed (Barany, 1967), An understanding of the F-V relationship enables speculation about crossbridge activity since maximum velocity of shortening (V ...... ) is an index of crossbridge cycling rate and of actomyosin ATPase activity (Barany, 1967; Packer, 1990). To eliminate stimulus level variability and to ensure maximum steady state responses, electrical field stimulation can be used.

There is considerable evidence for a cause-and-effect relationship between shifts in myosin heavy chain (MHC) isoform patterns and shifts in the shortening velocities of cardiac and skeletal muscles (Litten et al., 1982; Pagani, 1984; Sweeney et al., 1986). Furthermore, significant evidence has been found for shifts in MHC gene expression during striated muscle development (Wade and Kedes, 1989). Mohammad and Sparrow (1988) have reported shifts in MHC isoforms (200 and 204 kDa proteins) during post-neonatal development in porcine tracheal muscle. These investigators speculated

D.A. Roepke et al. / Respiration Physiology 98 (1994) 101-I i0 103

that airway smooth muscle shortening velocity would also change with development. However, no evidence for this speculation has been provided thus far. The use of tracheal smooth muscle in the study of airway smooth muscle MHC isoforms in general is appropriate due to the notable lack of difference in myosin content and in the muscle MHC isoform ratios between the trachea, bronchi and bronchioles within specific age groups in humans (Sparrow and Mitchell, 1990). The purpose of this study was to determine (l) whether rabbit tracheal smooth muscle contractility is altered during post-neonatal development, and (2) whether or not changes in MHC isoform patterns correlate with any changes found in contractility of rabbit tracheal smooth muscle.

2. Methods

Male 8- and 25-week-old New Zealand white rabbits were anesthetized with sodium pentobarbital (50 mg/kg body weight) by intraperitoneal injection. Then whole tracheae from immediately caudal to the cricoid cartilage to just within the thoracic cavity were removed. Each trachea was placed in ice-cold (4°C) Krebs-Henseleit solution (115 mM NaC1, 25 mM NaHCO3, 1.38 mM NaH2PO 4, 2.51 mM KC1, 2.46 mM MgSO4, 1.91 mM CaC12 and 5.56 mM dextrose).

Parallel-fibered smooth muscle strips of approximately 2-3 mm in length were cut from the tracheae. The strips were mounted horizontally between platinum plate elec- trodes in a muscle bath through which Krebs-Henseleit solution, gassed with a mix- ture of 95 ° o 02/5°o CO 2 (in a separate reservoir to minimize disturbance of measure- ments) and maintained at a constant temperature of 37 ° C, was circulated. The muscle strips were attached at one end to a stainless steel lever (Cambridge Instruments 300 H Dual Mode Servo), and at the other end to a sensitive photo-electric force transducer.

Changes in length and force were recorded simultaneously as functions of time by a strip chart recorder (Gould 2600S) for the rabbit TSM in response to direct muscle membrance depolarization via supramaximal electrical field stimulation (30 volts, 60 Hz, alternating polarity for 10 s). After an equilibration period of one hour during which the muscle was stimulated at a minimal resting tension every 5 min, passive and active L-T data were obtained. Changes in length were achieved by manipulation of the servo-motor lever. All F-V data was collected with the muscle strips stimulated while fixed at an optimal initial length for maximum tension development (1o) which was determined for each individual muscle strip from the L-T data collected for that strip. An instantaneous load-clamping device was utilized to obtain maximal shortening and velocity of shortening for a variety of loads.

Maximal isometric force (Po) data was normalized for tissue cross-sectional area (N/cm2; calculated from the tissue length and weight). This method of normalization has been used in previous developmental studies (Denehy and Ryan, 1986; Panitch et al., 1989; Seidel and Allen, 1979). Other force data was normalized by calculating it as a fraction of Po. All shortening data was calculated as a fraction of optimal length. The maximum slopes of the active shortening phases of contractions with loads less than (Po) gave the maximum velocities of shortening against the various loads. The

104 D.A. Roepke et al. / Respiration Physiolog3' 98 (1994) 101-110

velocity data was normalized to lo/s. Maximum velocities at zero load (Vma 0 for the rabbit TSM were calculated by finding the mean antilog of the intercept values of linear regressions for extrapolations of the semilogarithmic plots of the exponential portions of the F-V curves (Packer and Stephens, 1985a and b). Mean passive and active L-T curves, mean F-V curves and mean Po and Vm~ X values for the younger age group of rabbit TSM were compared with the mean passive and active L-T curves, mean F-V curves and Po and V ..... values of the TSM of the older age group.

M H C isoforms were separated by sodium dodecyl sulfate-polyacrylimide gel electrophoresis (SDS-PAGE). Excess connective tissue of the serosa and mucosa adher- ent to the tracheal muscle was dissected away. The tracheal muscle was frozen with liquid nitrogen and pulverized. The powdered muscle was then acetone-dried and dissected in a low vacuum. Eight samples were produced for each age group. Each sample from the 8-week-old group consisted of frozen powdered muscle from the tracheae of 3-4 rabbits while each sample from the 25-week-old group consisted of frozen, powdered muscle from tracheae of 2 rabbits. 650 ug of each sample was dissolved in 100 ul of gel dissociation media (200 mM Tris, 3°0 SDS, 10 mM DTT and 0.1 ° o bromophenol blue at a pH of 8.0). The dissolved sample was heated at 100 ~C for 30 min to cause dissociation of the proteins. Samples were then sedimented at 3200 rpm. for 5 min (Eppendorf centrifuge 3200, Brinkmann) and 45ul of the super- natant was applied to 5°; acrylimide/0.75°,o bis slab gels using the running buffer system of Porzio and Pearson (1977). Bovine serum albumin (BSA) standards, as well as heavy molecular weight standards, in SDS gel dissociation medium were heated at 100 °C for one minute and applied to lanes in the gels alongside the samples. The gels were subjected to electrophoresis for approximately 5 hours at 10 °C at a constant voltage of 300 V. Subsequently, the gels were stained with Coomassie Brilliant Blue R250 overnight and then destained for 1 hour with a 10°,; methanol/5%,,o acetic acid solution. Relative amounts of the rabbit tracheal smooth muscle M H C isoform bands were determined by quantitative densitometric scanning (BIO-RAD, model 620 video densitometer) and digitized data was analyzed with the BIO-RAD 1-D Analyst soft- ware on an IBM PC using known concentrations of BSA as the standard. Integration of the peaks produced data in the form of Optical Density multiplied by millimeters (OD x mm). The densitometric analysis was repeated 5 times for each of the 16 samples (8 samples of each age group) and the five results were averaged to give a mean concentration & t h e M H C isoform bands for each sample. These values were then utilized to obtain mean values for each age group and these means were compared with an unpaired Student's t-test.

3. Results

A representative tracing of the effect of varying TSM strip length on tension development is shown in Fig. 1. The muscle strip was set at an initial short length result- ing in a small amount of passive tension. The muscle was then maximally stimulated and active tension developed. Following complete relaxation back to the passive resting tension, the TSM strip was stretched to a new longer length and an increase in passive

D.A. Roepke et al. / Respiration Physiology 98 (1994) I01-I 10 105

0 -

Q,5-

l o -

o Z 1.5-

2.0-

2.5-

4 5 -

40 -

3 5 -

30 -

2 s -

I

i i i I i i i i ,i i i i D- ,t 10 rain ------I

TIME (rain)

Fig. 1. Example of raw length-tension data obtained from a rabbit tracheal smooth muscle strip. An initial length of about 1.25 mm was set (upper panel) so that there was some passive resting force or tension (i.e. the strip was not slack;lower panel) and then, supra-maximally stimulated via electrical field stimulation and the active force produced at that length was recorded (lower panel). The muscle strip was stretched an additional 0.1-0.2 mm (upper panel) between contractions while the muscle was at rest approximately every 5 rain. The instantaneous increase in passive force and the active force produced at each new longer length were recorded. Data from experiments of this kind were used to produce the L-T curves shown in Fig. 2.

t ens ion was measured . T h e n the musc le was maximal ly s t imula ted again and increased active t ens ion resulted. This pro tocol was repea ted unt i l fur ther s t re tching of the prepa- ra t ion resul ted in high pass ive t ens ion bu t little or no active tens ion . D a t a of the k ind p resen ted in this example was used to p roduce the m e a n leng th- tens ion (E-T) curves p resen ted in Fig. 2 and some of the m e a n paramete r s p resen ted in Tab le 1 were ob t a ined f rom indiv idua l L-T curves.

The m e a n va lues for the two age groups of rabbi t T S M strip character is t ics are c o m p a r e d in Tab le 1. There were no differences in op t imal length (lo) no r in cross- sect ional area ( C S A ) of the musc le strips ut i l ized from 8-week-old and 25-week-old rabb i t T S M ( P > 0.05). Tab le 1 also shows tha t there is no signif icant difference be tween the Po values for the 8-week-old rabbi t T S M (n = 6) as c o m p a r e d with the 25-week- old rabbi t T S M (n = 5), n o r was there any difference be tween the m e a n values for V m a x

for the rabbi t T S M from the younger (n = 4) and older (n = 8) age groups ( P > 0.05). The m e a n pass ive L-T curves for the 8-week-old (n = 8) and 25-week-old (n = 50)

rabbi t T S M are p resen ted in Fig. 2A. The two curves are no t significantly different

A.Passive Tension B, Active Tension

1.50-

1 . 00 - z _o

0 . 5 0 -

i i

0.50 0 .50 '1 !

0.75 1.00

MUSCLE LENGTH ( i l l O)

• 8-week-old Rabbit TSM

• 2 5 - w e e k - o l d Rabbit TSM

,l i i

0.75 1.00

MUSCLE LENGTH (1/I o )



106 D.A. Roepke et al. /Re.spiration Physiology 98 (19941 l O l - l lO

Fig. 2. Panel A: Comparison of mean _+ SE passive tracheal muscle L-T curves for 8-week-old 01 - 8) and 25-wcek-old (n = 5) rabbits. There is no difference between the two curves (P> 0.05). Panel B: Comparison of mean + SE active L-T curves for TSM from 8-week-old (n = 8) and 25-week-old (n = 5) rabbits. There is no difference between the two curves (P> 0.05).

( P > 0.05). T h i s is a lso t rue for the m e a n ac t ive L - T c u r v e s for r a b b i t T S M , w h i c h are

p r e s e n t e d in Fig. 2B. T h e m e a n F - V c u r v e s for the 8 -week-o ld (n = 6) a n d 2 5 - w e e k - o l d

(n = 16) r a b b i t T S M are s h o w n in Fig. 3. A s in the c a s e for the L - T r e l a t i o n s h i p s , n o

d i f fe rence w a s f o u n d b e t w e e n the m e a n F - V c u r v e s for the two age g r o u p s ( P > 0.05).

T h e M H C i s o f o r m r a t i o s are p r e s e n t e d in T a b l e 2. T h e r e is n o s ign i f ican t d i f f e rence

b e t w e e n t he M H C 2 0 0 / M H C 2 o 4 r a t i o s for the 8 - w e e k - o l d (n = 8) a n d 25 -week -o ld 01 = 81

r a b b i t T S M ( P > 0.05). T h e r e was , h o w e v e r , a s ign i f ican t d e c r e a s e in the p r o p o r t i o n o f

n o n - m u s c l e m y o s i n to m u s c l e m y o s i n b e t w e e n the ages o f 8 -weeks a n d 2 5 - w e e k s in the

r a b b i t T S M P ( < 0 . 0 1 ) .

Table I Rabbit tracheal smooth muscle strip characteristics

1. (mm) *CSA (ram 2) P. (mN/mm 2) V ..... (l.,'sec)

8 week 1.97 +_ 0.12 0.0111 +_ 0.0004 0.566 + 0.096 0.149 + 0.053 (n = 6) (n = 8) (n = 6) (n = 4)

25 week 1.95 _+ 0.07 0.01 l 1 _+ 0.0009 0.497 _+ 0.048 0.246 + 0.(143 01 = 5) 07 = 10) 01 - 5 ) (n = 8 )

* Cross-sectional area.

D.A. Roepke et al. / Respiration Physiology 98 (I 994) 101-i 10 107

0 .150 -

• 8 - w e e k - o l d Rabbit TSM

• 2 5 - w e e k - o l d Rabbit TSM

.9 v o _z z 0 . 1 0 0 - uJ I--

0 (n

>- I-

0 . 0 5 0 - ,..i tu >

0.50 1.00

M U S C L E A F T E R L O A D (P/Po)

Fig. 3. Compar i son of mean +_ SE F-V curves for T S M from the 8-week-old (n - 6) and 25-week-old (n = 14) rabbits. The two curves are super imposible ( P > 0.05).

Table 2

M H C isoform rat ios for rabbi t t racheal smooth muscle

MHC2oo Non-musc le myosin MHC2o4 Muscle myosin

8 week 1.00 +_ 0.15 *0.24 _+ 0.04

01 - 8) 25 wcek 0.88 _+ 0.08 0.15 + 0.01 (t~ = 8)

* Indicates a value that is significantly greater than the other value in the same column (P<O.001).

4 . D i s c u s s i o n

The fact that the passive L-T curves for the two age groups of rabbit TSM are the same indicates that there is no change in compliance between the ages of 8-weeks and 25-weeks in the rabbit TSM. This suggests that there is no change in the relative proportions of elastic components and contractile machinery in the rabbit TSM.

There is no difference in the maximum force-generating abilities of the TSM of the two age groups of rabbits. This indicates that there is no change in the numbers of force-generating sites (crossbridges) per cross-sectional area of tissue for the TSM during the stages of development studied.

108 D.A. Roepke et al. / Respiration Physiology 98 (1994) lO l - l lO

The maximum velocity of shortening (V ...... ) remained constant from the age of 8-weeks to 25-weeks in the rabbit TSM, which suggests that crossbridge cycling rate and actomyosin ATPase activity are unaltered during this period of development. This conclusion is based on the fact that for zero external load, any change that might occur in the velocity of shortening would have to be attributed to a change in the activity of those structures and enzymes responsible for the mobility of the myosin heads and thin filaments of the contractile apparatus (Barany, 1967; Warshaw et al., 1988).

The mechanical data show that there was no functional change in tracheal smooth muscle contractility in rabbits during post-neonatal development. This conclusion is not consistent with the prediction that there would be changes in contractility with development based on the results of Mohammad and Sparrow (1988) which indicated changes in the MHC isoforms during post-neonatal development of the TSM in the pig. However, the fact that no difference was found in the TSM MHC2oo/MHC:<~4 ratios between the two age groups of rabbits studied is consistent with the contractility findings, but fails to resolve the conflict between contractility data presented here and predictions based on the porcine MHC isoform report by Mohammad and Sparrow (1988). While the fact that the MHC isoform patterns of rabbit TSM did not change during development is consistent with the lack of change in contractility during the same period, there is no evidence in this consistency to support any contentions that there may be a cause-and-effect correlation between myosin heavy chain isoform patterns and velocities of muscle shortening in smooth muscle. Indeed, smooth muscle may be unlike striated muscles (Litten et al., 1982; Packer, 1990; Packer et al., 1991 ; Pagani, 1984; Sweeney et al., 1986) in this regard. More recent work by Sparrow and Mitchell (1990) indicates that the airway smooth muscle MHC isoform ratios are not significantly different between neonate and adult humans.

Interestingly, Murphy et al. (1991) have revealed insignificant changes in MHC isoform patterns of similar age groups of swine (4 weeks and 10 weeks), although differences in concentration-response curves to KC1 were found between the two age groups. These researchers suggest that there is a significant change in ability to develop contractile force that occurs with development but this change cannot be explained by a change in MHC isoforms.

The significant decrease in the proportion of non-muscle myosin to muscle myosin in the rabbit TSM suggests that there is some increase in the relative amount of contractile machinery with development. This is not surprising given the increase in absolute muscle mass and lumen diameter with growth and development. It must be noted that Mohammad and Sparrow (1988) and Sparrow and Mitchell (1990) were unable to identify non-muscle myosin in the human and porcine trachea even by radio-immunoblotting, although it was well identified in the human bronchi and bronchioles. This raises the question of the physiological significance of this finding in the rabbit TSM. Apparently myosin heavy chain isoform expression does not affect contractile function in airway muscle.

In conclusion, because there are no functional (biomechanical) changes in active contractility, changes in reactivity of the rabbit TSM that occur during the period of development between the two experimental age groups studied are likely due to changes in the excitation-contraction coupling mechanism.

D.A. Roepke et al. / Respiration Ph)'siology 98 (1994) 101-110 109

Acknowledgement

Supported by the Canadian Heart Foundation and NIH grant number T35 HL7584. Thanks are due to Marlene (King) Brown for expert typing of this manuscript.

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Contractility and myosin heavy chain isoform patterns in developing tracheal muscle