Camp. Biochem. Physiol. Vol. 77A, No. 2, pp. 377-382, 1984 0300-9629/84 $3.00 + 0.00 Printed in Great Britain 6 1984 Pergamon Press Ltd

MECHANICAL PROPERTIES OF MUSCLES FROM XENOPUS

BOREALIS FOLLOWING MAINTENANCE IN ORGAN CULTURE

M. J. N. MCDONAGH*

Department of Physiology, University of Bristol, UK

(Received 19 April 1983)

Abstract-l. The mechanical properties of sartorii muscles from the toad Xenopus borealis were studied in organ cultures lasting up to 26 days.

2. Isometric twitch and tetanic tensions diminished in force reaching half their initial values after 15 days in culture. In contrast, twitch time to peak time to half relaxation, twitch-tetanus ratio and muscle weight-body weight ratio, were unchanged.

3. Maximum isotonic shortening velocity (V,,,) declined, with a half time similar to that of the isometric force.

4. The fall in isometric tension is probably due to a breakdown of activation in some fibres. The change in V,_ could be due to a loss of functional sarcomeres in series with the tendons,

INTRODUCTION

The effects of denervation of muscles in situ have been followed, but in addition to nerve loss the muscles are subject to other factors, for example the strains imposed by the activity (or denervation) of other muscles and the influence of other physiological systems via the intact blood supply. By following the changes in isolated muscles it should be possible to examine the effects of denervation in a controlled environment.

Harris and Miledi (1972) described a technique for culturing amphibian muscle. They found that frog muscles could be kept for up to 2 months without major changes in ultra structure. Glycogen granules were seen in abundance. Nuclei, mitochondria and sarcoplasmic reticulum had their usual organisation. Pinocytotic vesicles similar to those seen in fresh muscles were observed, presumably indicating the persistence of active exchange of material between muscle and extracellular fluid. These authors studied the electrophysiological characteristics of the muscles during the first 2 weeks of culture, and found that resting and action potentials were near normal during this period although neuromuscular transmission failed after 5-7 days.

In the present experiments the mechanical pro- perties of muscles kept under culture conditions similar to those of Harris and Miledi (1972) were investigated in order to see if mechanical function is also preserved. There is a suggestion from work on mammals that mechanical function may not be as well preserved as electrophysiological function. Purves and Sackmann (1974) working on cultured rat diaphragm muscles found that the muscle twitch became weaker after 224 days, whereas the resting membrane potential was well maintained for 9 days.

A preliminary report of this work has appeared

*Present address: MRC Muscle Group, Department of Physiology and Pharmacology, Medical School, Queen’s Medical Centre, Clifton Boulevard, Nottingham NC7 2UH, UK. Telephone : 0602 700111.

in the Proceedings of the Physiological Society (McDonagh, 1982).

METHODS

Dissection and culture technique

Toads (Xenopus borealis) weighing 7734 g were used because, unlike frogs, they fed themselves readily. Well fed animals are essential for long survival of muscles in culture (Harris and Miledi, 1972). The physiological properties of Xenopus borealis muscle are indistinguishable for those of the more commonly used (Lannergren, 1979) Xenopus laeuis (M. J. N. McDonagh, unpublished observations). The details of the culture methods were very similar to those described by Harris and Miledi (1972). but briefly the sartorius muscle was dissected out under aseptic conditions inside a laminar flow operating bench. The animal was stunned, decapitated and pithed. It was then submerged for 5 set in 70% alcohol in order to kill bacteria on the skin surface. The skin covering the legs was cut around the waist and pulled off. Fresh sterile scissors were then used for the subcutaneous dissection. The superficial fascia along the lateral edge of the muscle was cut through and a 50 mm piece of cotton was tied to the distal tendon of the muscle which was then freed from its insertion, The muscle was lifted clear of the leg using blunt dissection. Short lengths of cotton were tied to the medial and lateral sides of the connective tissue plate to which the proximal end of the muscle is attached. The plate was then cut free from the body.

After dissection the muscle was tied to a glass gibbet. The gibbet and muscle were then placed in a Pyrex glass chamber holding 90 ml of sterile culture medium (Fig. 1, see figure legend for further details). Once the muscle and gibbet had been inserted, the top of the chamber was plugged with sterile non-absorbent cotton wool and covered with a loosely fitting lid of aluminium foil. The chambers containing the muscles were kept at room temperature (between 18 and 21°C) in a clean cabinet.

The totally defined culture medium 199 (Morgan et al., 1950) was adapted for use with frog tissues by altering the inorganic ionic concentrations to those usual for frog Ringer solution (Harris and Miledi, 1972). This left the organic constituents at half their normal concentrations. Harris (1968) found that this did not alter the survival time and physiological activities of the muscles. The final ionic con- centrations of the culture media were NaCI, 94 mM ; KCI, 2.7 mM ; CaCI,, 2.0 mM ; MgSO,, 3.0 mM ; Na,HPO,, 0.5 mM ;

377

M. J. N. MCDONAGH

-1 I : IO. The muscle was attached 1~3 the long arm tbf the Ic\er, Weights were attached to the short arm of ihc le!or by mean\ of a 670-mm-long, 2-mm-dia elastic thread. The long Ieber ratio and the compliant load attachrncnt reduced the equ- valent mass of the rotating syhtern to IN mg.

i 50mm

Fig. 1. Glass culture chamber A: non-absorbent cotton wool covered with aluminium foil lid. B: glass gibbet. C: Sartorius

muscle. D and E : outlet and inlet pipes.

NaH2P0,. 3.6 mM; NaHCO,, 2.0 mM; pH 7.6; osmotic pressure, 219 mosmols ; ionic strength, 0.12. Other meta- bolites added to the medium were insulin (0.1 IUjml), glucose (22 mM) and glutamine (2 mM). The antibiotic chloram- phenicol (5 &m!) and the fungicide funigizone (2 yg/ml) were also added.

At the end of the culture period a muscle was removed from the chamber and gibbet, and its proximal end was attached to a Perspex holder. Holder and muscle were then immersed in a glass chamber (Blinks, 1965) full of oxygenated eulture media at 20 ‘C. The distal end of the muscle was attached to a force transducer (Statham. Type U.F.1 4 oz. resonant frequency 450 Hz). The output of the transducer was amplified and then displayed on an oscilloscope. Isotonic tetanic concentrations were also measured using a length transducer (Tl transducer, Washington Instruments) modified to use a reversed-biased diode as the sensor. This gave an improved signal/noise ratio, linearity and rate of response. The length transducer included a 65-mm-long dural lever of mass 348 mg and lever ratio

The muscles were sfimulatcd with / mwc l3ulscs whilst immersed in culture media. In initial experiments the addition of rt-tubocurarine (1.5 x IO ’ M) did not affect the tension produced by supramaximal stimulation. These pulses \\erz provided by a stimulator capahlc ofdellvcring up to 400 mA. Current flowed parallel to the long axis of the muscle between four IO-mm-dia hoops of platinum wire fixed at right-angles to this axis. Pulse frequency and pulp duration wcrc controlled by a gated pulse generalor and Digitimcr. Twitches and tetani were evoked using twuze m;lximal voltages with the muscle at a length at which twitch force wab maximal. Tetanic stimulation consisted of trains of these pulses at 125 per set for 400 msec. Twitches and tetani bet-e given every 2 min during both lhe isometric and is(+tortic measurements. With this rest interval fatlguc was not a problem.

RESlILTS

The progress of denervation induced changes in the mechanical properties of Xerq~us sartorius muscles was investigated by taking measurements from muscles after periods in culture ranging from 4 to 26 days. Figure 2 shows typical myograms of isometric twitches and tetani from a control muscle and from a muscle removed from culture after 26 days.

Table 1 summarises the values of the mechanical measurements taken from uncultured toad sartorius muscles. Also included for comparison are the similar results obtained by Lannergren (1979) using single fibres and by Ridge and Thomson i 1980) using whole Xrnopus muscle. The data indicate that the main fibre type present is Lannergren’s Type 2 fibre.

The muscles were weighed after the mechanical measurements were taken (i.e. at the end of the culture period). The ratio muscle weight~body weight (mgjg) showed no change with time in cutture. The value of 2.94 1.0 (mean&SD) for cultured muscles was the same as the value of 2.9kO.8 (mean& SD) obtained for control muscles. As is common with denervated amphibian twitch muscle, fibrillation was not oh- served in any of the cultured muscles.

Changes in twitch and tetanic tension are illustrated in Fig. 3, which shows that within 15 days twitch and

Table f. Xmopus twitch muscle: normal values of mechanical propertic\

r, (IilXC)

This work

MCUl 32.x 31.5 124 26X 0.44 I.95 SEM 1.7 2.1 18.8 24 0.04 If7

3 1s 1s 15 14 14 4

Lannergren (1979)

Type 2 iibre 34.3 34.8 * * 0.61 .i? Type 4 fibre 117 151 * * 0.21 I.1

Ridge and Thomson (1980) 39.6 34.8 * * 0.37

The table showa the mechanical properrres ofnormal uncultured X~nopus twitch muscle Vafucs from the literature

we included for comparison. Measurements in these experiments and in Lannergren‘s work were taken ~1 20 C.

Ridge and Thomson’s results were at WC. Rzdge and Thomson’s results arc from whole CSICIIW- d~giiorum

Longus IV muscles. Lannergren’s results are from single fibres.

Abbreviations: r,. twitch time to peak; T,,,. time to half relaxation from peak twitch tension: 7,. rwltch (cnsion : ‘/;, tctanic tension: V,,,,,. velocity of unloaded shortening.

*Data comparisons inappropriate.

.-Data not available.

a

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Muscle force in organ culture

b

100 mN AIUrnN 10 msec

d

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50 msec 20 msec

Fig. 2. Isometric myograms : A and C from a control muscle; B and D from a muscle culture for 26 days. A and B twitches. C and D tetani (stimulation at 125 Hz). Records have been retouched for clarity.

tetanic tension had dropped to 50% of its original value. Twitch-tetanus ratio and time to peak tension (Fig. 4) show no progressive change with time in culture (see figure legends). This was also true of time to half relaxation. Most values are within one standard deviation of the mean control value at time zero. This is in complete contrast to the tension data shown in Fig. 3.

Measurements of isotonic tetanic contractions were also made at a series of loads. Figure 5 shows isotonic myograms and below force-velocity curves taken after 0, 13 and 22 days in culture. The maximum velocity of contraction showed a progressive reduction with time in culture, as can be seen from Fig. 6 (open circles). Velocities at all other loads were similarly reduced. This contrasts with the lack of change in the time course of the isometric twitch and the twitch-tentanus ratio.

The tetanic force of isometric contractions was reduced to around 20 N towards the end of the culture period (Fig. 3). The tetanic force of such a muscle is indicated in brackets at 20 days in Fig. 6. However, there were muscles cultured for short periods which had similar low tensions, for example at 5 days (Fig. 6). Despite its low tension this muscle had a similar velocity to a muscle at 6 days with a tetanic force of 100 N. This indicates that the progressive reduction in the maximum velocity of contraction of the cultured muscle is not due to an interaction between the low tetanic tension of the cultured muscles and the inertia of the recording system. The contribution of inertia was checked in the following way. Whole fresh sartorius muscles were divided longitudinally into strips which had tetanic tensions similar to that of cultured muscles. The maximum velocity of contraction of these strips was measured. The measured maximum velocities of the weaker strips was reduced in a systematic way as a

result of the inertia of the lever system. A plot of V,,,.., of strip/V,,, of whole muscle vs tetanic tension enabled an inertial correction factor to be calculated. This has been applied to the points (tilled circles) in Fig. 6, and it can be seen that inertial effects do not explain the reduction of contraction velocity in culture.

DISCUSSION

According to Harris and Miledi (1972) muscles cultured under conditions similar to those described in these experiments appeared normal when examined under the electron microscope (see Introduction). It is, therefore, unlikely that the loss of muscle force de- scribed here is associated with gross structural changes in the muscles such as loss of myofibrils. Part of the loss of tension could be due to a reduction of the muscle action potential. Harris and Miledi (1972) found a re- duction of the action potential from 128 mV to 95 mV in muscles from poorly fed animals. However, this did not occur in well fed animals as those used in my experiments. It is also unlikely that the loss of force could be due to failure of neuromuscular transmission in any remaining nerve endings because, as curariz- ation showed, the pulse width used was sufficient for direct stimulation of the muscle fibres. The fact that the time to peak and time to half relaxation of the twitch were unchanged suggests that the time constants of calcium release and reuptake by the sarcoplasmic reticulum were unaltered. The most likely cause of the loss of force is progressive all or none failure of action potential propagation in the sarcolemma or in the t-tubules.

In the present experiments the time course of twitch contractions of amphibian muscle did not change following denervation and culture in vitro. This result contrasts with the findings in mammals. In the rat both

3x0 M. J. N. MC.DONAGH

1 I I I I I 1 0 5 10 I5 20 25 30

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I I I I I I 1 0 5 10 15 20 25 30

Days in Culture

Fig. 3. Above: twitch tension changes in cultured muscle. The linear regression coefficient of the points (including 0 days). r = -0.77 (P <: 0.01). The point at 0 days represents the mean of 15 control muscles. The bar indicates 1 SD. Each of the other points represents one muscle. Figures 4 and 5 are organised in a similar way.

Below: tetanic tension changes, stimulation at 125 Hz. r = -0.71 (P < 0.01).

E.D.L. and soleus muscle showed an increase in time to et al., 1981) that the increase in time to peak found in peak and time to half relaxation following denervation denervated mammalian muscle is due to the reduced in oitw (Finol et al., 198 1). These changes were virtually amplitude and prolongation of the muscle action complete after 4 days. Assuming a QIo for the process potential which occurs at the same time. The fact that of 2-2.5, equivalent changes at 20°C might be expected an equivalent change in muscle action potential does to be complete after 13-18 days in my experiments. No not occur in cultured amphibian muscle (Harris and such changes were found. It has been suggested (Finol Miledi, 1972) could well explain the lack of change in

Muscle force in organ culture 381

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Fig. 4. Above: time to peak twitch tension of cultured muscles. r = -0.15 (P > 0.1).

Below: twitch-tetanus ratios of cultured muscles. r = -0.03 (P > 0.1). These variables show no progressive change

during culture.

the time course of the twitch found in the present experiments. The time course of the twitch does change in cultured mammalian muscle (McDonagh, 1980).

If all or none failure of action potential propagation in the sarcolemma or t-tubules is occurring in the cultured muscle, this would fit well with the finding that there is a progressive reduction of maximum velocity of shortening but no change in the time course of isometric contractions. If the all-or-none failure affected individual half-sarcomeres the number of active sarcomeres in series would be reduced with a consequent reduction in muscle shortening velocity. Inactive half-sarcomeres would increase the compli- ance of the muscle, thus tending to increase time to peak of the twitch and to decrease twitch-tetanus ratio. This was not observed, so any such inactive half- sarcomeres must be stiffer than those of normal non- active muscle. This could occur if they were in rigor. An

Fig. 6. Maximum isotonic contraction velocity of culture muscle.

0, control values (the mean of these is shown(O) k 1 SD); 0, uncorrected points (each point represents one muscle); 0, points corrected for inertia (r = -0.840, P i 0.01). The figures in brackets are the tetanic forces of two muscles (see

text).

Sartorius Isotonic Recordings

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Fig. 5. Above: isotonic myograms from control sartorius muscle contracting against a load equal to 25% of isometric tetanic value.

Below: force velocity curves of cultured muscle. Results from three different muscles. Load expressed as percentage of isometric tetanic force.

0, control muscle (muscle length, 22 mm) ; 0, after 13 days in culture (muscle length, 20.5 mm); A, after 22 days in

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altcrtxttt~c explanation is that there IS a structural reduction in the number of sarcomcres in series in the cultured muscle. This might arise because muscles were not fully stretched to body length while kept in culture. Williams and Goldspink (1978) have shown in the mouse soleus that immobilisation in the shortened position leads to a reduction in sarcomerc number and an increase in sarcomere length. To resolve these

qttestions it would be interesting to measure sarcomere number, and sarcomere length. in ~~rn~hibiail muscles cultured undrr stretched and unstretched conditions.

.4(,krlr~~~lrdyrn1~,~1f The author thanks the Muscular

Dystrophy Group ofGreat Britain for support and for a post- graduate scholarship during this work.

Blinks 1. R. (1965) Convenient apparatus for recording contractions of isolated heart muscle. J. ~ppl. P/z,KC)/. 203, 755.-759.

Final H. J.. Lewis D. M. and Owens R. (19X 1) The effects of