Judith Jones, Julian Emmanuel, Hazel Sutherland, Malcom J. Jackson(1), Jonathan C.
Jarvis, and Stanley Salmons
Department of Human Anatomy and Cell Biology and (1) Department of Medicine, University of Liverpool, U.K.
Abstract
Induction of fatigue resistance is essential to the use of skeletal muscle in cardiac assistance.
Cardiac patterns of work present a considerable challenge to a skeletal muscle, and the current practice of escalating the amount of stimulation progressively to an operational level of use is believed to minimize damage to muscle fibres. Although such a concept has analogies in exercise training regimes, the assumption that it is also true under the radically different conditions of electrical stimulation has never been tested, and the rate of escalation used has no firm scientific basis in either theory or experiment. The study presented here begins to address this gap in our knowledge. We show that prestimulation of rabbit ankle dorsiflexor muscles with a non-damaging pattern of activity does render the muscles less susceptible to the damaging effects of a more intensive regime, and that protection develops maximally between 7 and 14 days of prestimulation. In the context of cardiac assistance, confirmation of the protective effect of prestimulation opens the way to the systematic design of protocols that preserve the integrity of the muscle graft while ensuring that the patient receives the benefits of surgery at the earliest possible post-operative stage.
Key words: muscle damage, chronic stimulation, conditioning, protection, rabbit.
BAM 7(1): 39-44, 1997
There is growing interest in new surgical approaches to the treatment of end-stage heart failure, based on transfer- ring the latissimus dorsi muscle into the chest as a pedicled skeletal muscle graft. Such applications depend on adap- tive transformation, or 'conditioning', of the skeletal mus- cle by chronic s t i m u l a t i o n so that it acquires fatigue-resistant properties [1, 31, 34]. One version of this operation, which has been carried out in 500-600 patients worldwide, is cardiomyoplasty, in which the muscle is configured as a wrap around the ventricles of a failing heart. However, enthusiasm for this operation has been tempered by reports of long-term fibrofatty tissue replace- ment in the grafted muscle, both in experimental animals [2, 6, 22, 30] and in man [11, 18, 26]. A systematic study of damage sustained by sheep latissimus dorsi muscle, with interventions that represented different stages in the cardiomyoplasty procedure, showed that ligation of perfo- rating branches of the intercostal arteries, which supply vessels in the distal part of the muscle, and mobilization of the muscle with reattachment at a reduced length, were both potentially damaging to the muscle, particularly when
combined with stimulation [12]. This study also confirmed that a small, but significant, level of damage was sustained even when the muscle was stimulated in situ.
The actual mechanisms responsible for the damage in- duced by stimulation have never been established, and may be mechanical or metabolic in nature (for reviews see [32]).
It seems likely that a major factor is the disparity between the level of work demanded of the muscle by chronic stimulation and its capacity for generating ATP on a con- tinuous basis through oxidative metabolism. Such damage would be more serious in the early stages of stimulation, when transformation is as yet incomplete. It would also be exacerbated by any vascular insufficiency that resulted from mobilization of the muscle, which is indeed sug- gested by the work cited above [12].
If the initial trigger for stimulation-induced damage is metabolic overload of the muscle, then stimulation with a less intensive pattern should be less harmful. This predic- tion was confirmed in an earlier study from this laboratory in which muscles were subjected to continuous stimulation at a series of constant frequencies; damage was markedly
less at the lower frequencies [20, 21].
In the conditioning protocol that has been widely adopted for clinical cardiomyoplasty [5], stimulation commences with a single impulse per burst, and the number of impulses per burst and the frequency of the bursts is increased progressively over a period of 8 weeks. This practice is believed to minimize stimulation-induced muscle damage, an intuitively reasonable assumption that has, however, never been tested formally. Exercise studies in both ani- mals and man have shown that the susceptibility of a muscle to exercise-induced damage can be reduced by a prior bout of exercise [3,4,7,10]. The question is whether an analogous protective effect can be produced under the more artificial conditions of electrical neuromuscular stimulation, and whether a pattern that itself causes mini- mal damage might protect the muscle from the challenge of a more demanding regime.
In the present study we have incorporated the knowledge gained in our previous studies of stimulation-induced mus- cle damage [19, 20] into a simple two-stage protocol.
Prestimulation was conducted at a continuous low fre- quency of 2.5 Hz, a pattern which would not, of itself, cause significant damage. The muscles were then subjected to a stimulation regime (10 Hz continuously for 9 days) that we have previously found to produce histological manifesta- tions of fibre damage [19]. Using a quantitative morpho- logical technique, we show that a period of stimulation with a non-damaging pattern does indeed have a significant cytoprotective effect.
Methods Prestimulation
The study was carried out on 12 New Zealand White rabbits of either sex, with body weights in the range 2.5-3.8 kg. These were treated in accordance with the United Kingdom Government's Animals (Scientific Procedures) Act of 1986. Preanaesthetic medication with atropine sul- phate (Sigma Chemical Co. Ltd.; 3 mg kg" ) and diazepam (Roche Products Ltd.; 5 mg kg" ) given subcutaneously, was followed by a single anaesthetic dose of fen- tanyl/fluanisone (Hypnorm, Janssen Pharmaceutica, fen- tanyl citrate 0.315 mg ml" and fluanisone 10 mg ml" ; 0.3 ml kg" ) given intramuscularly. Full aseptic precautions were observed.
The implantable stimulators used in this study were of a new design, based on gate-array technology [36]. Each stimulator was capable of generating 12 different patterns, including an 'off condition, any of which could be selected remotely after implantation by using a stroboscope to transmit a sequence of light pulses through the skin. The low-profile stimulators were placed under the skin of the flank, and the electrode leads were guided subcutaneously to the left hind limb. One electrode was secured in the lateral head of gastrocnemius deep to the common peroneal nerve; the other was placed more distally on the surface of the muscle. This arrangement placed the nerve in a stimu- lating current field without subjecting it to physical contact
or compromising its blood supply. The devices were capa- ble of delivering impulses of 3.2 V amplitude and 200 |Lis duration, an intensity that is sufficient to produce su- pramaximal stimulation of muscles supplied by the com- mon peroneal nerve, and in particular the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles. Al- though these muscles had not been disturbed surgically, a quiescent postoperative interval of at least 7 days was allowed for any residual systemic effects of anaesthesia or surgery to subside. Stimulation was then initiated by the appropriate sequence of light pulses, and maintained con- tinuously at 2.5 Hz for 24 hours per day for 2 days (n = 5), 7 days (n = 5), or 14 days (n = 5). This pattern was chosen on the basis of previous work [20], which showed that continuous stimulation at 2.5 Hz was not damaging to TA, and produced only minimal damage in EDL. At the end of the prestimulation period, another sequence of light pulses was used to switch the stimulators to a continuous 10 Hz pattern, which was maintained for 9 days in every case; we and others [23] have found that this period corresponds to the maximum development of histological damage.
We did not have enough stimulators of the new type for all of the experiments, so in each of 5 animals we implanted two stimulators of a previous design [16] that could be switched between an 'off condition and a fixed frequency - in this case 2.5 Hz or 10 Hz. The stimulators were switched differentially by covering one of them while delivering a single light pulse to the other.
Terminal procedures
At the termination of each experiment, the rabbit was killed by an overdose of Sagatal (60 mg ml" sodium pentobarbitone, approximately 2 ml kg" ) delivered intra- venously. For morphometric assessment of damage, the left TA and EDL muscles were immediately excised and full-width blocks were cut from the widest part of the muscles. Right TA and EDL muscles served as unstimu- lated controls. All blocks were orientated on cork discs and frozen in isopentane pre-cooled in liquid nitrogen vapour.
Quantification of damage
Transverse cryostat sections of 10 (im thickness were cut and stained by the regressive haematoxylin and eosin tech- nique. The quantitative morphology technique has been described previously [19]. In brief, the muscle section was overlaid by a grid of 1 mm squares. Every third 1 mm square was brought into register with an eye-piece graticule containing a square 10 by 10 grid, and the per- centage of intersections falling on damaged muscle fibres, equivalent to the percentage volume of damaged tissue, was determined. Damage was defined as invasion of a fibre by mononuclear cells, vacuolization of cytoplasm, the presence of internal nuclei or hypercontraction; it is recog- nized that some of the fibres that fall within this definition would not necessarily be damaged irreversibly. The per- centage volume of endomysial tissue separating the fibres was determined in the same way.
Statistics
Because this type of data is not always normally distrib- uted, results from the 2-day, 7-day and 14-day pre-stimu- lated muscles and their unstimulated controls were analysed by the Kruskal-Wallis non-parametric ANOVA test. Differences between the stimulated muscles and their controls were analyzed a posteriori by Dunn's Multiple Comparisons Test. All results are expressed as mean ± SEM.
Results
Prestimulation
The 3 groups of experimental muscles were prestimu- lated at 2.5 Hz for 2 d, 7d, and 14 d; all were then challenged with a 9-day period of stimulation at 10 Hz.
These groups will be referred to as 2d-prestimulated, 7d- prestimulated and 14d-prestimulated.
No damage was detected in any of the contralateral control muscles. In the 2d-prestimulated TA muscles, dam- aged tissue occupied 8.6 ± 2.7% of the tissue volume (n = 5); in the 7d-prestimulated TA muscles, the damage amounted to 4.7 ± 1.8% (n = 5); and in the 14d-prestimu- lated TA muscles, the damage was not significantly differ- ent from zero (0.9 ± 0.5%, n = 5). The EDL muscles showed a similar response: damage represented 8.3 ± 1.0%
(n = 5) of the tissue volume in the 2d-prestimulated EDL muscles, 3.3 ± 0.6% (n = 5) in the 7d-prestimulated EDL muscles, and was not significantly different from zero in the 14d-prestimulated EDL muscles (0.8 ± 0.4%, n = 5).
These results are illustrated in Figure 1.
12.0-
O) CO
1
o>i
10.0- 8.0-
2d 7d 14d
Pra-fttimulation (days)
Figure 1. The volume percentage of damage induced in tibialis anterior (TA) and extensor digitorum Ion- gus (EDL) muscles of the rabbit by chronic indirect stimulation for 9 d at 10 Hz after prestimulation for 2, 7, 14 d at 2.5 Hz. Significant differences from control are denoted *** (P < 0.001) and * (P <
0.05). N.S. = not significantly different to control.
The percentage volume occupied by endomysial tissue increased significantly in all stimulated muscles, but no difference was observed between the experimental groups (Figure 2).
Discussion
We show here for the first time that the damaging conse- quences of stimulation can be ameliorated, or even averted, if the muscle is initially exposed to a period of stimulation with a non-damaging pattern. Prestimulation for 2 days did not appear to have an appreciable protective effect, but prestimulation for longer periods produced a progressive increase in protection, to the extent that the longest period used in the study (14 days) was sufficient to prevent damage entirely.
It was established previously that the pattern of prestimu- lation used in these experiments was associated with a very low level of damage in EDL, and no detectable damage in TA [20], It is conceivable that some of the damage ob- served after challenging the muscle with 9 days' stimula- tion at 10 Hz resulted from the prestimulation regime, but it seems unlikely because the damage observed was actu- ally greater for the shorter periods of prestimulation.
The level of damage observed in the TA muscle pres- timulated for 2 days was higher than we have observed in previous studies involving only stimulation at 10 Hz for 9 days [19, 20]. In those studies, moreover, EDL was con- sistently more susceptible to damage than TA, whereas in the present study the damage in the 2d-prestimulated group
3
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-OO
0 0>
3
1
0)O)
i
4)<5 o.
Control 2d 7d 14d
Pro-stimulation (days)
Figure 2. The volume percentage of endomysial tissue in the muscles of Figure 1. The three stimulated groups do not differ from each other in this respect, but in each case there is significantly more non- muscle tissue between the fibres than in the control muscles. (Symbols as for Figure 1.)
of animals was similar in these two muscles. As we have noted before [19], damage is a highly variable phenome- non and we should not be too surprised to find a difference between different studies in the overall level of damage.
The apparent lack of a differential susceptibility between TA and EDL is harder to explain, but it is possible that even 2 days of prestimulation had a slight protective effect which was more pronounced in the EDL muscles, reducing their level of damage to that of TA.
The question arises as to whether prestimulation initiates events along the same pathway as ischaemic precondition- ing, the effect of single or repeated short ischaemic epi- sodes on the ability of myocardium to survive prolonged ischaemia and reperfusion without damage [24, 27, 38].
Protection in this case appears to be associated with the expression of heat shock proteins (HSPs) and other stress- related proteins [8, 9]. A similar phenomenon may occur in skeletal muscle, since exercise is reported to produce a marked rise in HSP 70 and other stress proteins in limb muscles [29, 35]. However, this expression of stress pro- teins occurs within hours of the triggering event [35]. In the present experiments even 2 days of prestimulation was without obvious benefit. It seems that the duration of prestimulation needs to be between 7 and 14 days for a protective effect to be observed. This points away from a role of HSPs and other stress-related proteins in the effects of prestimulation demonstrated here.
An alternative possibility is that the protective influence of prestimulation is related to the early stages of transfor- mation, which include an increase in capillary density [15]
and changes in the activities of enzymes involved in key metabolic pathways (reviewed in [28]). A recent study of the effects of chronic stimulation at 2.5 Hz on rabbit skeletal muscle included a description of changes in en- zymes of metabolism [25]. These changes were less ex- treme, and developed at a slower rate, than the changes associated with stimulation at 10 Hz [14]. In Table 1, which has been drawn from the earlier data, it is possible to make a direct comparison between the changes induced by chronic stimulation at 2.5 Hz and 10 Hz after 2 weeks,
corresponding to the maximum period of prestimulation used in this study. At the lower frequency of stimulation most of the enzymes showed relatively modest changes at this stage, with one exception: there was a striking increase in the activity of hexokinase. An increase of this type probably facilitates the entry into the cells of large amounts of glucose [37], which appears to be utilized during this phase of the response both for synthesizing glycogen and for generating energy by aerobic glycolysis [13]. This and more recent evidence [33] suggests strongly that the han- dling of exogenous glucose plays a pivotal role in the early stages of the response of a muscle to stimulation.
The early changes have significant functional conse- quences: after only 2 weeks of stimulation at 2.5 Hz the muscle can sustain cardiac levels of work without fatigue [17]. Thus the prestimulated muscle is better adapted to meet the metabolic challenge posed by a more intensive regime.
In conclusion, these experiments provide formal confir- mation that prestimulation with a non-damaging pattern of activity renders a muscle less susceptible to the potentially damaging effects of a sudden, substantial increase in de- mand. The nature of the changes responsible for this cyto- protective effect remains to be established, although the time course is suggestive of a metabolic adaptation. The way is now open for the systematic design of protocols that produce the maximum protection of the muscle in the shortest possible time. In the context of providing cardiac assistance from skeletal muscle, progress in this area will ensure that the patient receives the benefits of a highly invasive procedure at an earlier post-operative stage.
Acknowledgements
The authors gratefully acknowledge the contribution to this work of Mrs M Hastings and Miss AJ Craven and grant support from the British Heart Foundation and the Engi- neering and Physical Sciences Research Council. Stimula- tors were developed as part of an ongoing collaboration with Dr DJ Hitchings and his colleagues at the University of Staffordshire.
Table 1. Activities of enzymes of metabolic significance following 2 weeks of continuous stimulation at 10 Hz or 2.5 Hz, expressed as a percentage ( SEM) of control values.
Stimulation frequency
10 Hz 2.5 Hz
HK
418 44 493 106
LDH
88 12 87 4
CPK
82 107
6 7
CS
344 101
35 25
SDH
400 59 127 24
HAD
382 162
49 29
KACAT
699 90 155 10
GOT
291 31 130 9
HK = hexokinase; LDH = lactate dehydrogenase; CPK = creatine phosphokinase; CS = citrate synthase; SDH = succinate dehydrogenase; HAD = p-hydroxyacyl CoA dehydrogenase; KACAT = 3-ketoacyl CoA transferase; GOT = glutamate- oxaloacetate transaminase. Data from Henriksson et al 1986 [14] and Mayne et al 1996 [25].
Address correspondence to:
Professor Stanley Salmons, Department of Human Anat- omy and Cell Biology, University of Liverpool, Liverpool L69 3BX, U.K., tel. +151-794 5496, fax +151-794 5517, Email s.salmons@liverpool.ac.uk.
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