Layout 1 Electrical stimulation in science and medicine Eur J Transl Myol 35 (3) 14058, 2025 doi: 10.4081/ejtm.2025.14058 What is a paradigm? A formal definition could be: ‘A generally accepted set of assumptions and concepts that constitute a way of viewing reality in an intellectual discipline’. Put more simply, it’s a theory that seems to explain everything. By their very nature, paradigms are firmly entrenched, and overturning them is no easy matter. Historically, the alter- native theory has often been accepted only after the death of its discoverer. I therefore consider myself fortunate in having had a hand in disproving two paradigms within my scientific lifetime. These form the subject of this article. The first of these paradigms is the notion of ‘terminal dif- ferentiation’. This states that once cells have differentiated fully into their adult state they are incapable of expressing their genome further. In the 1960s, I quizzed two professors about this, both of them members of the Royal Society. Both asserted that this was an indisputable fact. The second paradigm is the notion of chemotrophic in- fluence. This will take more explanation, and I will first show how I was introduced to it. After I had graduated in Physics at Imperial College Lon- don, and pursued a postgraduate degree in Electronics and Communication, I decided that my real interests lay in the life sciences. I therefore enrolled, with three others, in the two-year Master’s course in Physiology at University Col- lege London. During the second year we were assigned to the wonderful Ricardo Miledi as tutor, who set us the task of writing essays on topics that were at the cutting edge of physiology. The subject of one of these was a paper by A.J. Buller, J.C. Eccles, and R.M. Eccles.1 Let’s place this in context. As long ago as 1874, Ranvier described ‘white’ and ‘red’ muscles in animals and noticed that the ‘white’ muscles contracted and relaxed more quickly than the ‘red’ muscles. Using these contractile speeds we can refer to these broad types of skeletal muscle as ‘fast’ and ‘slow’. Nearly a cen- tury later it was shown that the nerves supplying fast mus- cles carry brief, high-frequency bursts of impulses, whereas those supplying slow muscles conduct prolonged low- frequency trains.2,3 These different patterns interested Jack Eccles, who was al- ready well-known for his contributions to neurophysiology (he shared the Nobel Prize in Physiology or Medicine in 1963). At that time Arthur Buller was working in his lab; he had become Professor and Head of the Department of Physiology at Bristol when I first met him. As he explained it to me, Eccles wanted to see if the type of muscle in- fluenced the firing patterns of the respective motor neurons. For these experiments they cut and cross-anastomosed the motor nerves, so that each type of muscle would be rein- nervated by the other’s nerve. The effects of this cross-re- innervation were studied some weeks later. They failed to demonstrate any difference in the firing pat- terns of the neurons. But during one of these laborious ex- periments – at about three o’clock in the morning – Eccles suggested that they had a look at the contractile speed of the muscles instead. To their surprise a remarkable change had occurred; the fast muscle had become slower-contract- ing and the slow muscle had become faster-contracting. This finding was the subject of the paper that Miledi gave us to study. After considering possible explanations the au- thors suggested that the muscles had responded to the in- fluence of ‘quickening’ and ‘slowing’ chemical trophic factors transported to them along their motor nerves. There was no direct evidence for such factors – no more than there had been for other postulated substances from the past, the basis of previously discredited paradigms such as aether, caloric fluid, and phlogiston. However, in fairness it was a perfectly reasonable proposition. Paul Weiss had demon- strated axonal flow,4 so there was a candidate mechanism Abstract The discovery that skeletal muscle can respond adaptively to use, even to the extent of re- expressing its genome, overturned two paradigms and led to new insights into gene regulation and a variety of clinical applications. Key Words: muscle, fast, slow, stimulation, implantable device, adaptation, gene expression, paradigm. Eur J Transl Myol 35 (3) 14058, 2025 doi: 10.4081/ejtm.2025.14058 Paradigm shifts: how electrical stimulation opened up new avenues in science and medicine Stanley Salmons Emeritus Professor, University of Liverpool, Liverpool, United Kingdom. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (CC BY-NC 4.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. - 288 - Electrical stimulation in science and medicine Eur J Transl Myol 35 (3) 14058, 2025 doi: 10.4081/ejtm.2025.14058 for the transport of such substances, and this was reinforced by the discovery of neurotrophins such as Nerve Growth Factor that had other functions.5 At all events, the convincing arguments in the paper, and the standing of its principal author, led to its widespread ac- ceptance. We had a new paradigm for the differentiation of skeletal muscle into fast and slow types. This, however, would not form the subject of my own re- search. At least, not yet. University of Birmingham I got married soon after my graduation from University College London, and as my wife was studying medicine at the University of Birmingham Medical School, I looked for research positions there. I accepted a research fellowship in the Department of Anatomy. The aim of the project was to study muscle activity in freely-moving primates, using ra- diotelemetry. This was ideally suited to my background in physics and electronics as well as physiology. The original objective was to record electromyographic signals (EMG) but I judged that muscle force should also be measured, as the two are not closely correlated under all conditions. For this I developed the buckle transducer, which allowed force to be measured without cutting the tendon.6,7 The principle is illustrated in Figure 1. Tension in the tendon is recorded by strain gauges incorporated into the frame or the cross- piece. The associated circuit design was challenging. Primates are adept at removing devices attached to them. Such devices would also require connections to be made through the skin, with the accompanying risk of infection. To avoid these problems, it would be essential to implant the device. But if we were to observe the normal locomotive behaviour of the animals, data would need to be transmitted over several metres, and the implant would have to work for several weeks. The only way to manage this with a small, battery- operated device was to use a pulse technique. I decided to record muscle tension by modulating pulse width, while the integrated EMG would modulate the pulse frequency. This had the advantage of ensuring that the most detailed infor- mation was collected at times of maximum activity. This was the 1960s, before the era of integrated circuits. It was possible to buy a single gate (the devices in your smart- phone have many millions of gates) but the current drain of such a circuit was about 10 mA, which was prohibitive. Fortunately, this was the time of the Space Race, and min- iature discrete components had become available. These typically included transistors of the size of a match head and resistors 4 mm long and 1 mm in diameter. I designed much of the circuitry around these components. However I remained dissatisfied with the overall current drain, and I began looking for a better circuit that could be triggered to generate a single pulse. A change in semiconductor tech- nology provided the solution. Briefly, junction transistors consisted of a sandwich of semiconductor layers into which n-type and p-type doping substances had been diffused. The semiconductor used at that time was germanium, and the transistors had a ‘pnp’ configuration. When silicon transistors first appeared they had an ‘npn’ configuration. I was interested to find a circuit that combined transistors of both types and this presented new possibilities to me. I constructed a circuit of this kind that was designed to deliver a single pulse. The circuit misbehaved. Instead of generating a single pulse it generated an unending sequence of pulses. Because of the importance of minimising current drain I al- ways measured it by having a multimeter in the circuit, and I noticed that the needle of the meter was not moving. I turned up the sensitivity. At the maximum sensitivity the needle moved, indicating a current drain of only 21 µA. I stared at it. Lesson 1: If an experiment yields an unanticipated result, pay close attention: this could be your big break! The writer and scientist Isaac Asimov put it well: The most exciting phrase to hear in science, the one that heralds new discoveries, is not ‘Eureka!’ but ‘That’s funny.’ The significance struck me like a bolt of lightning. As a gen- erator of single pulses the circuit was worthless, but it would make a fantastic stimulator! I knew exactly what experiment I wanted to do with this circuit. In fact Buller must have had something similar in mind when he wrote: “It would be very difficult technically to arrange artificial stimulation of several weeks duration so that there was a prolonged transposition of these char- acteristic frequencies of action, independently of nerve cross-union.” My newly discovered circuit would make this difficult task feasible! I was a member of The Biological Engineer- ing Society and the International Society on Biotelemetry, and took electronics journals and newspapers, so I was conversant with the techniques I’d need to use to minia- turise the device. I had also conducted animal experiments during my Physiology degree. There was just one prob- lem: although I was surgically skilled I had never con- ducted aseptic procedures, those in which the animals recovered from anaesthesia. There was, however, some- one in the Department who had. Dr Gerta Vrbovà had joined the Department of Anatomy not long before. I had read her papers, in which she inves- tigated what would happen if the impulse traffic reaching the slow soleus muscle were abolished, which was achieved by transecting the spinal cord and dividing the muscle ten- - 289 - Figure 1. Principle of the buckle transducer. Electrical stimulation in science and medicine Eur J Transl Myol 35 (3) 14058, 2025 doi: 10.4081/ejtm.2025.14058 don. Under these conditions, the muscle became faster con- tracting. The observation was suggestive of an influence of impulse activity on muscle properties, but it was criticized on the grounds that the muscle was rendered severely atro- phic by this procedure, and the results could therefore be influenced by contraction of adjacent fast muscles. Propo- nents of the chemotrophic explanation remained unmoved. The experiment I wanted to do was the opposite of silencing a slow muscle; I wanted to impose a postural type of low- frequency pattern on a fast muscle. I remember putting the suggestion to her, not in her room but leaning on one of the wide windowsills of the corridor that led to the main lecture theatre. She had two objections. First, these muscles would not be electrically silent, so we would be superimposing impulse activity of the slow-muscle type over the top of ex- isting fast muscle activity. To this I responded that the effect of a continuous low-frequency pattern of activity should surely overcome that of intermittent, and possibly in- frequent, bursts. Her second objection was that the animals in her experiments had been paraplegic and therefore inca- pable of much movement; it would be difficult to maintain stimulation in an intact, conscious animal in any conven- tional way. In answer to this I told her that I believed I could construct a totally implantable stimulator, and this would make it possible to conduct the experiment in a freely mov- ing animal. She shrugged and said we could try it. It took six months of development. I reduced the current drain from the initial 21 µA to just 6 µA. I would base the design on existing mercury (later zinc-air) batteries of the type used in hearing aids. But whereas in hearing aids they would last for just 36 hours, in my device they would last for several months. Initially I soldered miniature compo- nents to a flexible printed circuit, on which I had etched the required pattern electrochemically. This could then be rolled up, the components interdigitating. Later I purchased a min- iature spot-welder, and assembled the circuit in three di- mensions (Figure 2). John Powell, our skilled departmental workshop technician, turned a hollow bullet-shaped device out of medical quality stainless steel, 30mm long by 8 mm diameter. It was in two halves, and I embedded the circuit in resin in one half and placed the batteries in the other. When the halves were clipped together the stimulator began to work. For the leads I obtained pacemaker wire, which I insulated with thin-walled silicone rubber tubing. I swelled these with xylene, threaded the wire through them, then al- lowed them to contract again. The electrodes were medical quality stainless steel, welded to the ends. These were placed near the common peroneal nerve of a rabbit to stim- ulate muscles in the front compartment of one hind limb, and delivered pulses of 0.5 msec duration at a frequency of 10 Hz. The first experiment served only to show that the rabbit would not tolerate a device placed subcutaneously. In order to place it in the peritoneal cavity I encapsulated it in sil- icone rubber into which I incorporated a piece of Dacron mesh. The mesh was included when we sewed up the peri- toneal wall, thus preventing the device from migrating and strangulating the intestine with the leads. The remaining issue was a tendency for the muscle contractions, which could be observed and palpated externally, to weaken or even cease entirely the moment the skin was closed. This required one or the other electrode to be moved. It was a problem I solved later. The device enabled the long-term effects of stimulation to be studied in a freely-moving animal unencumbered by leads. We demonstrated it at the meeting of the Physiolog- ical Society held at the National Institute for Medical Re- search, Mill Hill, London (now the Crick Institute in Central London). In those days it was permitted to exhibit animals and we had taken a cage with a large white rabbit that had the stimulator implanted. The device itself was also dis- played. It attracted a lot of attention. When we conducted the terminal experiment on the first rabbit there was never any doubt about the result. After only a few weeks of stimulation the fast tibialis anterior and ex- tensor digitorum muscles clearly contracted and relaxed more slowly. Thus encouraged we embarked on a series. In the course of this series I made some changes to the ex- perimental procedure. In the measurements of twitch con- traction I was concerned about the resting tension of the muscle, since overstretching can itself extend the duration of contraction and, especially, relaxation. I felt it was not enough to test this by prodding the tendon with a finger. I therefore repeated the measurements with the muscle lengthened and shortened by 2 mm from the expected op- timum. The contractions were recorded on 35 mm film, and as I was doing all the analysis, I could select only measurements obtained with the optimum settings. I also felt it was important to measure the rate of rise of tetanic tension, because under these conditions the muscle was fully activated. All this time I was still working on my original PhD proj- ect, which had some techniques in common. Nevertheless, the stimulation experiments were clearly a better vehicle for a PhD thesis. The Introduction was just one page, but I wrote an extensive Discussion, based on a survey of all the literature that involved changes in muscle use. It struck me that wherever changes were observed in the contractile speed of a muscle it could be explained in terms of a change in the impulse activity reaching it. The thesis con- cluded: - 290 - Figure 2. Implantable stimulator circuit. Electrical stimulation in science and medicine Eur J Transl Myol 35 (3) 14058, 2025 doi: 10.4081/ejtm.2025.14058 ‘A slow time course of relaxation is appropriate to the maintenance of a smooth contraction for pro- longed periods. Furthermore a given tension can be maintained with less frequent activation than would be required of a fast muscle. As a result, the rate of utilisation of chemical energy by a postural muscle is low enough to be supported by a predominantly oxidative metabolism. Slowness, it seems, enables a postural muscle to be continuously active by elim- inating the need for short-term energy reserves and the recovery periods required to replenish them. I would therefore suggest that the speed:activity re- lationship represents an adaptive response which en- ables a skeletal muscle to perform the type of activity demanded of it.’8 My PhD was awarded in 1968. I shall always be grateful to the memory of my supervisor, Professor Eric Ashton, for allowing me the freedom to pur- sue this new and productive research avenue. It was fortu- itous for me, however, in the sense that our main collaborator, Dr Charles Oxnard, had left for a senior post in Australia, and not long afterwards the primate colony was disbanded, ending any possibility of conducting the experi- ments he’d intended. Meanwhile I was appointed to a Sto- thert Research Fellowship of the Royal Society, and Eric Ashton became a valued colleague rather than a supervisor. When it came to writing up the work for publication Vrbovà suggested that we combine her earlier experiments with the present ones. I agreed, but it did present problems. Lesson 2: Regardless of the seniority of any others involved, never allow a paper on which you are an author to be published unless you have had the opportunity to read, and if necessary revise, the content. There’s a corollary to that, of course, if you are the principal author: Lesson 3: Always give every coauthor on your paper, however junior, the chance to read, and if necessary revise, the content. A person can fairly be expected to be called upon to defend a paper on which they are a coauthor, and they can hardly do so if they’ve never seen it. Those principles were as important for me then as they are now. The 1959 paper was eventually published with all my alterations.9 Harvard Medical School, Boston Shortly after this I attended the 8th International Conference on Medical and Biological Engineering, at Chicago. While there I made contacts that led to the offer of an NSF Fel- lowship with Professor Elwood Henneman, at Harvard Medical School. Working with Henneman was one of the most formative experiences of my career and he became a role model for me. My colleague Peter Clamann and I would invari- ably meet him for a sandwich lunch in his room, where we would discuss, among other things, recent papers, current progress and the detailed design of the next ex- periment. If we arrived as he was concluding a meeting he would introduce each of us to the departing visitor. At the 1st Neuroscience Conference in Washington he in- troduced me to anyone he happened to be speaking to. Wherever possible I have done the same with all my jun- ior colleagues. In addition to being a solicitous senior colleague he was esteemed as a careful, systematic scientist. Henneman sin- gle-handedly shifted the paradigm that motor neurons were recruited at random, or by alternation (‘rotation’) of active motor units. Instead he showed, in a series of ex- periments notable for their elegance and rigour, that motor units are recruited in an orderly and predictable way.10-12 This observation fitted perfectly with my own studies, as it suggested that the resultant hierarchy of impulse activity would then be reflected in the properties of the correspon- ding muscles. During this time, I redesigned the stimulator, which now fitted into a stainless steel capsule that screwed together, with terminals that allowed the leads to be changed more easily; this device could therefore be reused indefinitely. I also redesigned the electrodes and the surgical technique for implanting them. Pacemaker wire was flexible, but not sufficiently flexible in the space available in a rabbit, so in- stead I adopted fine, multistrand Cooner wire. I made a sim- ple tool with which to fashion a loop at the end of the lead. The elongated loop was drawn through the muscle tissue underlying the common peroneal nerve inside a hypoder- mic needle inserted from the opposite side. The other lead was located in the conductive space above the nerve. Each was mounted on a small piece of Dacron™ velour so that it could be secured with a couple of 5/0 sutures. This ar- rangement ensured that the current field always passed through the common peroneal nerve, and was confined to it. After this we never had any problems with loss of stim- ulation after closing the skin or during voluntary move- ments of the hind limb. We could now stimulate reliably for 24 h/day for many months, and a new ‘batteryectomy’ op- eration allowed us to extend the duration of stimulation still further. While I was in Boston, I forged a collaboration with Frank Sréter and John Gergely at the Boston Biomedical Research Institute in the city. They brought to bear a range of tech- niques with which we could study biochemical changes in the stimulated fast muscles. The recent discovery of myosin light chains was of particular interest, as these were dis- tinctly different in fast and slow muscle. They were visual- ised using SDS gel electrophoresis. We were excited to see that after four weeks of chronic low-frequency stimulation the light chain pattern typical of fast muscles had changed to one that contained two additional bands corresponding to those found in slow muscle. This result was published in Nature.13 After longer periods of stimulation the fast light chains progressively disappeared, and after ten weeks the stimulated fast muscle exhibited only light chains of the - 291 - Electrical stimulation in science and medicine Eur J Transl Myol 35 (3) 14058, 2025 doi: 10.4081/ejtm.2025.14058 slow muscle type14,15 (Figure 3). In the soleus muscle of the rabbit a fast component comprises about 4.5% of the light chain fraction, but a soleus muscle stimulated for ten weeks was homogeneously slow15 (Figure 3). If stimulation was stopped after six weeks the original fast light chain complement was re-established.16 Corresponding changes were seen in the myosin heavy chains.15 Fast muscles subjected to chronic low-frequency stimulation acquired a light meromyosin paracrystal stain- ing pattern identical to that of slow muscle.13,17 Nτ-methyl- histidine, a normal component of the heavy chain of fast, but not slow, muscle, disappeared completely following stimulation. It was restored following cessation of stimula- tion.16 Stimulated fast muscle that was stained for myofi- brillar adenosine triphosphatase with alkali incubation showed the histochemical appearance of slow muscle.18 Although biochemical changes had been observed in enzymes of metabolism in response to endurance exercise19 and chronic stimulation20 these were of a quantitative na- ture. Our demonstration of qualitative changes in myosin light and heavy chains provided the first evidence that a fully differentiated muscle could re-express its genome. We had overturned the first paradigm, namely the notion of ‘ter- minal differentiation’. The second paradigm, the ‘chemotrophic theory’, proved to be a more difficult undertaking. The results obtained with the implantable stimulator were themselves too clear-cut to be challenged. But the chemotrophic theory was well es- tablished, and the thesis that impulse activity is responsible for the differentiation of fast and slow muscles was not widely accepted. Rather, arguments were advanced to ex- plain the results in terms of chemotrophism. There were, in fact, ten such explanations and I set about disproving all ten. A full account of these experiments may be found else- where.21 Here I will describe how just three of those key objections could be dismissed. Generalized degeneration and regeneration Those who raised the first of these objections asserted that chronic low-frequency stimulation was damaging, and fast muscles suffered wholesale degeneration followed by gen- eration of new muscle fibres of the slow type. The follow- ing studies addressed this proposition. In my own laboratory rabbit muscles, which are more or less completely transformed to the slow type after 6 weeks of continuous stimulation at 10 Hz, were histologically normal at all intermediate stages, showing no evidence of widespread degeneration. Our electron microscopy studies, described below, would have been impossible had damage featured significantly in the muscles. We also looked at developmental isoforms of myosin. During nor- mal development, muscles change from embryonic to neonatal to fast isoforms, or from embryonic to neonatal to slow isoforms. Regenerating muscle fibres would there- fore be expected to exhibit recapitulation of these devel- opmental isoforms. But the transition during chronic stimulation involved neither the neonatal isoforms of myosin light chains nor those of myosin heavy chains15, nor did it involve recapitulation of embryonic isoforms.22 Explanations based on damage or selective proliferation of slow fibres were also incompatible with the observation that normal physiological and biochemical properties were resumed after cessation of stimulation, and the customary mosaic histochemical appearance restored, with no ev- idence of fibre type grouping.23 Yet perhaps the most persuasive evidence that damage was not a prerequisite for type transformation was that it could be seen taking place within existing adult fibres. Partially transformed fibres showed myofibrillar adenosine triphos- phatase staining that was intermediate in staining inten- sity18. Gel electrophoresis performed on single fibres taken from transforming muscle revealed the simultaneous pres- ence of myosin light chain isoforms of both the fast and the slow type.24 Using immunogold electron microscopy we could show that, during transformation, fast and slow MHCs were present at the sarcomere level in the same mus- cles.25 The decline of fast and the increase of slow myosin heavy chains (MHCs) could also be visualized in individual fibres by immunogold staining with silver enhancement26 (Figure 4). The inescapable conclusion is that the transfor- mation of type induced by chronic electrical stimulation is a distinct phenomenon, and any contribution from damage is quite incidental, not obligatory. Species-specificity The original stimulation experiments were conducted on rabbits and initially it was claimed that the phenomenon was unique to that species. To date, however, similar results have been demonstrated in rat, cat, dog, goat, sheep, pig, and man (references at Salmons, 2018).21 The basic mech- anisms of adaptation therefore appear to be the same in all mammalian species. However, larger animals have slower- contracting muscles and they normally activate them at lower frequencies; lower frequencies of stimulation are therefore sufficient to elicit the same extent of adaptive - 292 - Figure 3. SDS-polyacrylamide gel electrophoresis of myosin purified from tibialis anterior muscle after var- ious periods of stimulation. neo: neonatal myosin from 3-day-old rabbit leg muscle. 70sol: myosin prepared from adult soleus muscle after stimulation for 70 days. Electrical stimulation in science and medicine Eur J Transl Myol 35 (3) 14058, 2025 doi: 10.4081/ejtm.2025.14058 change. Conversely smaller animals have faster-contracting muscles and full adaptation calls for more demanding re- gimes of activity.26,27 Cross-reinnervation and stimulation By the early 1970s there had been many studies of the ef- fects of cross-reinnervation on fast and slow muscles, and proponents of the chemotrophic theory were still inclined to regard the effect of chronic stimulation as a separate phe- nomenon, one that was quite distinct from that of cross-re- innervation. I had conceived of a way of testing this idea some years be- fore, but back then I felt that the stimulation pattern needed to be optimised further before embarking on it. By 1975, however, we had built up a good deal of experience with the stimulation technique and its effects on physiological properties, myosin composition, and histochemical changes in metabolic enzymes. Those effects were so complete I felt the time was right to go ahead. The experiment consisted of two parts. In the first part, I stimulated the anterior tibial muscles in 2 rabbits continu- ously for 5 months. The results, which could now be based on a battery of physiological and biochemical measures, were clear cut. A long-term change in activity without changing the innervating nerve produced effects greater than cross-reinnervation in every respect. The second part of the experiment placed stimulation in opposition to cross-reinnervation. It involved two groups of rabbits, operated in matched pairs. In both groups, I cross-anastomosed the motor nerve of the fast tibialis an- terior muscle to the nerve supplying the slow soleus mus- cle. This was a classical cross-reinnervation procedure al- though to avoid any pull on the nerves and to eliminate the possibility of reinnervation by the original nerve I per- formed the cross in one direction only. Electrode leads were implanted in both groups. Eight weeks later a stim- ulator was connected to the leads in the experimental group, delivering impulses at 10 Hz to the cross-reinner- vated soleus muscle in an approximation of its original physiological activity. The leads in the control group were dummies (Figure 5d). The surgical procedure is worth describing in more detail. The original cross-reinnervation experiments were con- ducted with muscles in the posterior compartment of the hind limb, muscles such as flexor digitorum longus, whose nerve is not too dissimilar in size to that of the soleus mus- cle (less than 1 mm in diameter). The stimulation aspect of the proposed experiment, however, meant that I had to use the common peroneal nerve, which is many times larger - 293 - Figure 4. Thin serial cross-sections of rabbit tibialis an- terior muscle after stimulation for 2 weeks, stained by the immunogold technique with silver intensification. (A) Antibody specific for fast myosin (B) Antibody spe- cific for slow myosin. In two fibres (arrows), A-bands that stain lightly for fast myosin also stain heavily for slow myosin, evidence of transformation in progress within intact fibres. Scale bar = 50 µm. Figure 5. Schematic diagram illustrating the experimen- tal models described in the text. (a) Cross-reinnervation interpreted as a chemotrophic process. (b) Cross-rein- nervation interpreted as an adaptive process. (c) Chronic stimulation. (d) Cross-reinnervation combined with chronic stimulation. F, fast muscle characteristics; ‘F’, nerve that normally supplies a fast muscle; S, slow muscle characteristics; ‘S’, nerve that normally supplies a slow muscle. Electrical stimulation in science and medicine Eur J Transl Myol 35 (3) 14058, 2025 doi: 10.4081/ejtm.2025.14058 and carries a sizeable sensory component. This nerve runs under the biceps femoris muscle and dips below a slip of the gastronemius muscle. At this point it divides into be- tween 4 and 13 branches and I familiarised myself thor- oughly with the destination of all of them. Under a dissecting microscope I could now reliably isolate the motor branch innervating the fast tibialis anterior muscle, using fine watchmaker forceps and handling only the perineural connective tissue between the branches. The nerve to the slow soleus muscle was exposed using a lateral approach, shown to me by A.R. Luff. The two nerves were then cut and anastomosed gently using a single loop of 9/0 or 10/0 nylon. This suture material is used in ophthalmic surgery and is equivalent in diameter to just a few red blood cells, so it would present a minimal barrier to regenerating axons. The material is not easy to handle, however; draughts had to be avoided, and I polished brand new watchmaker forceps with jeweller’s rouge to prevent the suture from clinging to their surface. Again the results were clear cut. Cross-reinnervation alone closely reproduced the results published previously by others. However, under conditions in which cross-reinner- vation was not accompanied by a change in activity, be- cause stimulation had been used to maintain the original level of activity, no change in characteristics could be ob- served. Such a detailed complementarity of effect would be very hard to explain other than in terms of an identical un- derlying mechanism.14 I think this paper was the only one I have written for a major publication that was accepted immediately as a full-length Nature article, with not even a request to deal with referees’ comments. I remember Professor Alan M. Kelly of the Uni- versity of Pennsylvania saying to me, ‘That paper was the nail in the coffin of the chemotrophic theory.’ The second paradigm had indeed been shifted. Electron microscopy Examining the effects of chronic stimulation at the ultra- structural level Dennis Gale and I recorded an increase in the Z-line width, from that typical of fast muscle (39 nm) to that of slow muscle (78 nm).28 As we later discovered, these measurements correspond closely to the overlap of actin filaments in the mammalian Z-line.29 We also ob- served a marked increase in the volume of mitochondria.28 Using stereological techniques, Brenda Eisenberg and I generated a time course for these changes, together with changes in the T-system, over periods from 6 h to 24 weeks of stimulation.30 Interestingly, mitochondrial volume in- creased several-fold over the first 3 weeks of stimulation, then fell rapidly after 7 weeks, although it still remained well above the levels typical of slow muscle. This complete time course agreed with changes in enzymes of oxidative metabolism.31,32 Together with single fibre studies of me- tabolites, the changes shed light on adaptations that enabled chronically stimulated muscles to sustain a continuous high level of ATP utilization.33 This explained the remarkable fa- tigue-resistance of stimulated muscles, the bioenergetic cor- relates of which were studied in dogs using in vivo31 P-NMR spectroscopy.34 Reversibility of the effects of stimulation We had already made some observations of the restoration of fast muscle characteristics following cessation of stimu- lation, including myosin light chain composition, myosin ATPase, Nτ-methylhistidine, and Ca2+-uptake by sar- coplasmic reticulum.16 In further experiments we estab- lished the time course of recovery, dealing comprehensively with ultrastructural,35 and physiological, histochemical, and metabolic characteristics23,36 These studies established the ‘first-in, last out’ nature of the response to stimulation, which we’ll revisit below. Early events – the need for remote switching We had shown that chronic stimulation of fast muscles re- sulted in a change in the expression of myosin isoforms.13- 15 We had also demonstrated changes in the ratio, as well as the amount, of phosphorylase kinase isozymes.37 Others had documented changes in the amount and molecular type of proteins involved in calcium transport and binding.38,39 The events that gave rise to these changes were, of course, of great interest, and this posed a need to examine changes at the very earliest stages of type trans- formation. We therefore needed to make sure that the ef- fects of stimulation could be dissociated completely from the consequences of anaesthesia and surgery. The implantable stimulator worked continuously from the moment of assembly and therefore as soon as it was im- planted. Clearly there was a need to switch it on or off re- motely after implantation. Initially I tried a miniature capillary switch which, placed subcutaneously, could be op- erated by a magnet. Later I developed an optical switch that formed part of a redesigned stimulator which could be trig- gered remotely through the fur, skin, and subcutaneous tis- sues of the subject by a single discharge from an electronic flash gun (known in North America as a strobe)40. This was totally reliable and also more convenient to use. Later still these devices evolved further, making use of integrated cir- cuitry.41,42 With these modifications we could now allow days or even weeks to elapse for recovery after surgery, and could then activate the implanted device and study the ef- fects of stimulation on its own, which we did for periods ranging from 6 hours to 24 weeks. We will now look at the results. Regulatory events The redifferentiation of skeletal muscle in response to a change in activity provides a valuable experimental model in which to investigate the regulation and coordination of expression of the large number of genes involved. Our ultrastructural studies had already revealed signs con- sistent with mobilisation of transcriptional and trans- lational activity.43 We looked at the basis for these changes, first studying events in the polyamine pathway. The activities of ornithine decarboxylase and S-adenosyl- L-methionine decarboxylase increased markedly between 18 and 48 h of stimulation. These changes in enzyme ac- tivities were followed, between 3 and 11 days, by a - 294 - Electrical stimulation in science and medicine Eur J Transl Myol 35 (3) 14058, 2025 doi: 10.4081/ejtm.2025.14058 sequence of phasic elevations in the concentrations of pu- trescine, spermidine and spermine.44 The phosphorylation pattern of cytoplasmic proteins and the activity of cyclic AMP-dependent protein kinase changed significantly by 12 days of stimulation and became indistinguishable from those of slow muscle by 3 to 4 weeks.44 To examine early stages in the expression of myosin, Northern blots of mRNA were hybridized with cDNA probes specific to the fast myosin heavy chain gene. These showed greatly reduced hybridization in fast muscles that had been stimulated for as little as 7 days. This indicated that changes in the expression of the corresponding genes were initiated much earlier than might have been sup- posed.45 We used cDNA probes to study the expression of four genes: Carbonic Anhydrase (CAIII), fast Myosin Heavy Chain (MHCf), α-actin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). During 21 days of stimulation the mRNA for CAIII, which is specific for slow muscle fibres, rose significantly, whereas that of MHCf fell markedly. GAPDH mRNA declined steadily to levels typi- cal of slow muscle, which is less dependent on anaerobic glycolysis. There were early changes in actin mRNA, sug- gestive of coordination with other contractile proteins dur- ing the fast-to-slow transition.46 Focusing more on myosin, we constructed a time course for the reciprocal changes in fast and slow myosin heavy chains during 4 days to 6 weeks of stimulation and 4 to 12 days of recov- ery after cessation of stimulation.47 In another study this was extended to myosin light chains.48 We thus had clear evidence that for several classes of muscle protein, the re- sponse to chronic stimulation involves regulatory events at a pre-translational level. The adaptive response of mitochondrial and cytoplasmic enzymes to chronic stimulation calls for coordinated reg- ulation of genes located within nuclear chromatin and mi- tochondrial DNA. In two extensive studies with Sandy Williams we demonstrated reciprocal changes in the ex- pression of aldolase and mitochondrial cytochrome b,49 and changes in nuclear genes that encode mitochondrial proteins.50 A definitive pathway for all these phenomena has yet to emerge, although a number of candidate pathways have been described.51,52 It seems unlikely, however, that adap- tive changes are produced by a single pathway. For exam- ple, under conditions of chronic stimulation there is a monophasic decline in enzymes of glycolysis,20,32 but the response of mitochondrial volume and enzymes of oxi- dative metabolism is biphasic.30,32 In the latter case, we showed that the secondary, declining phase coincides with the transition from fast to slow MHCs15 and fails to occur under stimulating conditions that do not produce that tran- sition.53,54 This is strongly suggestive of a linkage between metabolic changes and myosin isoforms. Other signalling pathways may be responsible for coordinating expression of enzymes encoded by nuclear and mitochondrial DNA.50,55,56 Changes in metabolites33,57 point to inter- actions that may be adaptive but may or may not take place at the gene level. We can no doubt anticipate further progress in this complex and rapidly developing field. Damage – again Earlier I provided a number of lines of evidence that en- abled us to comprehensively dismiss the notion that the re- sponse to chronic stimulation was the result of damage. It therefore came as a surprise when Maier and his colleagues published evidence of damage, amounting to as much as 25% of the muscle, in stimulated muscles. They went on to attribute at least part of the conversion to slow characteris- tics to a population of satellite cells from which new slow muscle fibers were formed.58,59 Why had they observed such serious damage when we had not? In our laboratory the way the electrodes are placed and the use of a constant low voltage confines the stimulating current field to the common peroneal nerve. I understand that the practice in the other group was to increase the stim- ulating voltage during the initial reduction in palpable con- tractions. Firstly, this is unnecessary, because the adaptive response takes place independently of force development during this period.60 Secondly, it is highly undesirable, be- cause of potential spread of the stimulating current to the powerful plantar flexor muscles. Muscles of the anterior compartment are then subjected to simultaneous stretch and stimulation, potentially causing the damage that results from eccentric work. Although this provided a ready explanation for these au- thors’ findings, it was important to seek a more definitive answer because their reported damage occurred at the very stage when the regulatory events underlying gene re-ex- pression are known to occur. As indicated in the previous section, these phenomena are the subject of considerable research interest. If extensive damage were present, it would alter entirely our perception of observations in this period. It was therefore important to confirm that, in our hands at least, muscle damage was not a factor. We therefore decided to put the issue on a thoroughly quan- titative basis, using statistically valid sampling protocols and multivariate analysis to take into account variation within the cross-section and length of the muscle as well as between muscles and experimental subjects, and we did this for a variety of stimulation patterns.61-63 We found that the volume percentage of degenerating fibers was 3.4% to 3.8% (mean ± SD) for continuous stimulation, and 1.0% ± 1.0% for intermittent stimulation. Because the tibialis anterior muscle in the rabbit contains no more than 5% of slow (type 1) fibres, fast-to-slow type transformation based on degen- eration would have to involve at least 95% of the fibres in this muscle. In our hands, damage affected 3%–4% of these fibres, so it cannot account for the observed transformation. While we can therefore dismiss the conclusions of Maier et al., their observations should serve as a warning to others of the potential damage that can result from excessive levels of stimulation. The threshold hypothesis Let us return to the original paper of Buller, Eccles, and Ec- cles.1 Why, after careful discussion, did they discount the idea that impulse activity was responsible for the differen- tiation of fast and slow muscle, proposing instead the che- - 295 - Electrical stimulation in science and medicine Eur J Transl Myol 35 (3) 14058, 2025 doi: 10.4081/ejtm.2025.14058 motrophic theory? Their problem was understandable, for there appeared to be a sharp differentiation of muscle into fast-contracting and slow-contracting types. As we now know, the situation is even more complex: muscle fibres show continuous variation in some parameters (such as enzymes of metabolism) but discontinuous variation in others (such as myosin light and heavy chains). The latter is well illustrated by the mosaic histochemical and immuno- histochemical appearance of sections stained to demon- strate myofibrillar ATPase and myosin. How can this be a consequence of motor neurone firing patterns, which are inherently variable? I presented a new concept to address this problem at a con- ference on the plasticity of muscle held in Konstanz, 1979. This was published in the book of proceedings.64 It postu- lates that some components of muscle (such as enzymes in- volved in metabolism) respond continuously in a more or less linear way to increased activity. But others, such as the myosin proteins (with their important influence on the speed of contraction) respond along an S-shaped curve. This, as a physicist would immediately perceive, creates a threshold and hysteresis. To elaborate, it explains the sta- bility of the fast and slow fibre types, because for these properties to change, the level of activity must rise above a threshold or fall substantially below it, and intermediate values are not open to them. The explanation is consistent with the orderly sequence of changes brought about both by chronic stimulation and endurance exercise65 and the ‘first-in, last-out’ nature of the changes induced by stimu- lation of a fast muscle and following recovery after cessa- tion of stimulation.23,35,36 This is illustrated schematically in Figure 6. The horizontal axis denotes the time course of stimulation and recovery and the vertical axis indicates the adaptive response. There is an initial response from more readily inducible phenomena such as the increase in oxi- dative capacity. This is indicated in the Figure by a notional lower threshold. With a further increase in activity the upper threshold is crossed, initiating transcriptional events asso- ciated with the synthesis of myosin isoforms of the slow muscle type. Clearly these two thresholds will be crossed in the reverse order when the level of activity declines. After the talk Arthur Buller clapped me on the shoulder and said one word, ‘Brilliant!’, a significant accolade. Others evidently also embraced the idea because the or- derly sequence of changes brought about by chronic stim- ulation and the threshold notion soon came into general use, together with the notion of adaptation, which I had described in my PhD thesis and early publications twenty years before. University of Liverpool In 1987 I took up the Chair of Medical Cell Biology at the University of Liverpool. Jonathan Jarvis and Caroline Mayne (now Munro) took on the logistical challenges of moving the lab and all its activities to its new home, a task they managed to perfection. Clinical applications At an early stage, physiological observations of the changes induced by chronic stimulation revealed a remarkable in- crease in the fatigue resistance of the muscle (Figure 7), and we had studied the underlying biology. This knowledge put me in a good position to make a contribution to emerging clinical applications of electrical stimulation. Initially, I became involved in the society DIENST, and I was invited to become President. I accepted the invitation, provided I could change the name! It became the ISTS (In- ternational Society for Therapeutic Stimulation) and at- tracted members from the USA, France, UK, Germany, Austria, Sweden, Hungary, Switzerland, and Japan, as well as the original strong contingent from Finland. In time the society was supplanted by the very successful IFESS (In- ternational Functional Stimulation Society), on which I served as a Board Member. - 296 - Figure 6. The threshold hypothesis. Figure 7. Fatigue resistance of stimulated muscle. The response to a series of tetani, consisting of 500 ms trains of impulses at 25 impulses/s delivered every 1.25 s. The tension developed by the control muscle (lower trace) declines rapidly after an initial phase of post-tetanic po- tentiation. The stimulated muscle (upper trace), which shows the slight post-teanic depression characteristic of slow-contracting muscle, can sustain this taxing regime almost indefinitely without decrement. Electrical stimulation in science and medicine Eur J Transl Myol 35 (3) 14058, 2025 doi: 10.4081/ejtm.2025.14058 Every application poses its own functional requirements, and these must be taken into account when designing the corresponding stimulation regime.66 Stimulation of the diaphragm One focus was on ventilatory insufficiency, such as that caused by injuries to the cervical spine. Studies were al- ready ongoing in which electrical stimulation was used to activate the diaphragm or abdominal muscles to improve tidal volume67-69. Such stimulation is unphysiological, re- sulting inevitably in muscle fatigue. The practice was to avoid this by starting with a prolonged period of intermit- tent stimulation. At a conference on the subject in Hamburg I proposed an alternative initial regime grounded on my basic studies. In response to these suggestions a period of low-frequency conditioning was tried in 2 patients. Satis- factory resistance to diaphragmatic fatigue was achieved in just 3 weeks instead of 6, and both patients were independ- ent of the mechanical ventilator by 2 months (Baer, personal communication). Stimulation of paralysed muscle Electrical stimulation of muscles in human subjects who had been paralysed by stroke or spinal cord injury was al- ready being employed – commercially in some cases – to restore the forces and coordinated movements needed for posture and movement.70-72 Just as in the previous applica- tion there is a risk that it will result in premature onset of fatigue. Again this can be avoided by preceding the func- tional stimulation with a less challenging program of stim- ulation which can be progressively escalated to adapt the muscles, rendering them more fatigue-resistant. In this way periods of grasping and manipulating (upper limb) and standing and walking (lower limb) can be extended safely.66 Artificial sphincters With suitable prestimulation an artificial sphincter can be created from skeletal muscle to treat patients with fecal in- continence.73,74 I advised Norman Williams on possible pro- tocols. Pedicled muscle grafts could also be used to provide better management of voiding in patients fitted with a stoma.75,76 Following encouraging meetings with Moshe Kon and John Barker we developed a continent stoma in the pig77,78, work that demonstrates a strong potential for clinical application. Cardiac assistance A major commitment in my research group was the use of skeletal muscle to augment cardiac muscle impaired by dis- ease. This started with a letter, published in 1975 in re- sponse to a paper in which an attempt had been made to use diaphragm muscle for cardiac assist. In the letter I pointed out that the potential for such an application had been un- derestimated because it took no account of adaptive change.79 Some six years later the letter was picked up by John Macoviak, a young surgeon working in the laboratory of Dr Larry W. Stephenson, then in Philadelphia. This began a twenty-year collaboration, during which my lab be- came the British Heart Foundation Skeletal Muscle Assist Research Group (Figure 8). Normal skeletal muscles cannot sustain cardiac work, a re- gime that greatly exceeds the functional demands habitually placed on them. But a suitable conditioning regime renders a pedicled graft of skeletal muscle capable of augmenting cardiac function. Our muscle of choice was the latissimus dorsi muscle, which could be mobilised without seriously impairing the patient’s mobility and transferred into the tho- racic cavity. Our approach was to configure the muscle as an auxiliary ventricle, stimulated to operate in counterpul- sation with the heart. The expanding skeletal muscle ven- tricle would then reduce the load of the patient’s weakened heart during cardiac systole, and in cardiac diastole it would contract, assisting the systemic and coronary circulations (Figure 9).66 - 297 - Figure 8. The research group in 1992. Figure 9. Schematic of a skeletal muscle ventricle. Electrical stimulation in science and medicine Eur J Transl Myol 35 (3) 14058, 2025 doi: 10.4081/ejtm.2025.14058 The accompanying research program included such de- tailed studies as the effect of varying the frequency and duty cycle of stimulation used for conditioning (reviewed in Sal- mons 2009),66 flow patterns that revealed the importance of travelling vortices in the heart and in single-conduit skeletal muscle ventricles80,81, and fresh insights into the blood supply of the latissimus dorsi muscle.82,83 These studies sup- ported steady progress in surgical application, to the point where a skeletal muscle ventricle was still delivering effec- tive cardiac assistance after implantation for four years in a dog84. Progress was also being made with a single conduit version of the skeletal muscle ventricle in the pig81 and we showed that this could provide assistance at least equal to that available from an intra-aortic balloon pump.85 The idea of using an adaptively conditioned latissimus dorsi muscle was picked up by cardiac surgeons and used in a technique they termed cardiomyoplasty, in which the graft was wrapped around the patient’s heart. Some two thousand cases were operated. There were a number of reasons why cardiomyoplasty disappointed initial expectations (for re- view see Salmons 2008).86 Unfortunately, this high-profile failure also spelled the end of the skeletal muscle ventricle work, which had, and still has, greater potential. Stimulation of denervated muscle As mentioned above, there was already a good deal of re- search and commercial activity involved in the pursuit of electrical stimulation as a solution to mobility problems caused by stroke or spinal cord injury. However it was Hel- mut Kern who interested me in a more difficult problem: muscles inactivated by nerve damage, such as that caused by injuries of the brachial plexus or the cauda equina. At first I was skeptical, but I was won over by his preliminary results with an intensive program of stimulation. As a result we embarked on the EU-RISE program, which involved a number of European research groups with the following main clinical objectives: i) Restoration of muscle mass. For cosmetic reasons and to improve skin cushioning; ii) Re- storation of muscle force. For standing up and standing of short duration, with associated improvements in local blood flow, skin condition, bone density, and general cardiovas- cular fitness; iii) Improved fatigue resistance. For standing of longer duration. The challenges have been discussed elsewhere.87 Early re- sults were encouraging, affording clear evidence that some patients would benefit in fitness, appearance, and self-es- teem.88 As before my group performed a variety of experi- ments to provide the necessary knowledge base.89-92 These long-term experiments were made possible by a remarkable implantable stimulator, developed by the Vienna group, that met the demanding specification for remote operation, power delivery, and operating lifetime yet was small enough to be implanted into a rabbit.93 Stimulation of denervated muscles in animals was not new, of course, but to reflect the actual clinical conditions to be encountered our experiments included stimulation of mus- cles with long-standing denervation94. These experiments revealed both the benefits and the limitations of the tech- nique, and stressed the therapeutic advantages of introduc- ing a program of stimulation during the initial, nondegenerative phase of the muscle response to nerve or root injury. Conclusions This journey began with an urge to address, and overturn, two existing paradigms. In the process we established the adaptive capacity of skeletal muscle and developed an un- derstanding of the underlying phenomena. The resultant clinical applications show how this remarkable property can provide a workable basis for developing new therapeutic modalities. List of abbreviations EMG, electromyographic signals. SDS, sodium dodecyl sulphate. CAIII, carbonic anhydrase. MHC, myosin heavy chain. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. IFESS, International Functional Electrical Stimulation So- ciety. Acknowledgements I would like to thank my collaborators and those who passed through my research group over the years. Their names are recorded as authors in the relevant references. In particular I must pay tribute to Dr (now Professor) Jonathan C. Jarvis and Dr Hazel Sutherland, who played a crucial role in accomplishing much of the work described here. Conflict of interest The author declares no potential conflict of interest, and confirms accuracy. Ethics approval and consent to participate Not applicable. Availability of data and materials All data generated or analyzed during this study are in- cluded in this published article. Corresponding author Stanley Salmons, Emeritus Professor, University of Liver- pool, Liverpool, United Kingdom. ORCID ID: 0000-0002-1392-9869 E-mail: ssalmons242@gmail.com References 1. Buller AJ, Eccles JC, Eccles RM. Interactions between motoneurons and muscles in respect of the characteristic speeds of their responses. J Physiol 1960;150:417-439. - 298 - mailto:ssalmons242@gmail.com Electrical stimulation in science and medicine Eur J Transl Myol 35 (3) 14058, 2025 doi: 10.4081/ejtm.2025.14058 2. Eccles JC, Eccles RM, Lundberg A. The action poten- tials of the alpha motoneurones supplying fast and slow muscles. J Physiol 1958;142:275-291. 3. Vrbová G. Changes in the motor reflexes produced by tenotomy. J Physiol 1963;166:241-250. 4. Weiss P, Hiscoe HB. Experiments on the mechanism of nerve growth. Journal of Experimental Zoology 1948; 107:315-396. 5. Levi-Montalcini R, Angeletti PU. Nerve Growth Factor. Physiol Rev 1968;48:534-569. 6. Salmons S. Fig. 6.52 Principle of the buckle transducer. In: Cobbold RSC, editor. Transducers for Biomedical Measurements: Principles and Applications: Wiley; 1974. p 184-185. 7. Salmons S. In vivo tendon tension and bone strain measurement and correlation. J Biomech 1975;8:87. 8. Salmons S. The development and use of implantable electronic devices to investigate the influence of activity on the contractile speed of mammalian skeletal muscle [Ph.D. Thesis]. Birmingham: University of Birming- ham; 1968. 9. Salmons S, Vrbová G. The influence of activity on some contractile characteristics of mammalian fast and slow muscles. J Physiol 1969;201:535-549. 10. Henneman E, Somjen G, Carpenter DO. Functional sig- nificance of cell size in spinal motoneurones. J Neuro- physiol 1965;28:560–580. 11. Henneman E, Somjen G, Carpenter DO. Excitability and inhibitability of motoneurones of different sizes. J Neurophysiol 1965;28:599–620. 12. Henneman E, Clamann HP, Gillies JD, Skinner RD. Rank order of motoneurons within a pool: law of com- bination. J Neurophysiol 1974;37:1338–1349. 13. Sréter FA, Gergely J, Salmons S, Romanul F. Synthesis by fast muscle of myosin light chains characteristic of slow muscle in response to long-term stimulation. Nat New Biol 1973;241:17-19. 14. Salmons S, Sréter FA. Significance of impulse activity in the transformation of skeletal muscle type. Nature 1976;263:30–34. 15. Brown WE, Salmons S, Whalen RG. The sequential re- placement of myosin subunit isoforms during muscle type transformation induced by long term electrical stimulation. J Biol Chem 1983;258:14686-14692. 16. Sréter FA, Elzinga M, Mabuchi K, Salmons S, Luff AR. The Nt-methylhistidine content of myosin in stimulated and cross-reinnervated skeletal muscles of the rabbit. FEBS Letters 1975;57:107-111. 17. Sréter FA, Romanul FCA, Salmons S, Gergely J. The effect of a changed pattern of activity on some biochem- ical characteristics of muscle. In: Milhorat AT, editor. Exploratory Concepts in Muscular Dystrophy II. Vol- ume No. 333, International Congress Series. Amster- dam: Excerpta Medica; 1974. p 338-343. 18. Romanul FCA, Sréter FA, Salmons S, Gergely J. The effect of a changed pattern of activity on histochemical characteristics of muscle fibres. In: Milhorat AT, editor. Exploratory Concepts in Muscular Dystrophy II. Vol- ume No. 333, Intern. Congress Series. Amsterdam: Ex- cerpta Medica; 1974. p 344-348. 19. Holloszy JO, Booth FW. Biochemical adaptations to en- durance exercise in muscle. Annu Rev Physiol 1976;38: 273-291. 20. Pette D, Smith ME, Staudte HW, Vrbová G. Effects of long-term electrical stimulation on some contractile and metabolic characteristics of fast rabbit muscle. Pflugers Arch 1973;338:257-272. 21. Salmons S. The adaptive response of skeletal muscle: what is the evidence? Muscle & Nerve 2018;57:531- 541. 22. Hoffman RK, Gambke B, Stephenson LW, Rubinstein NA. Myosin transitions in chronic stimulation do not involve embryonic isozymes. Muscle Nerve 1985;8: 796-805. 23. Brown JMC, Henriksson J, Salmons S. Restoration of fast muscle characteristics following cessation of chronic stimulation: physiological, histochemical and metabolic changes during slow-to-fast transformation. Proc Biol Sci 1989;235:321-46. 24. Pette D, Schnez U. Coexistence of fast and slow type myosin light chains in single muscle fibres during trans- formation as induced by long term stimulation. FEBS Letters 1977;83:128-30. 25. Franchi LL, Murdoch A, Brown WE, et al. Subcellular localisation of newly incorporated myosin in rabbit fast skeletal muscle undergoing stimulation-induced type transformation. J Muscle Res Cell Motil 1990;11: 227-39. 26. Jarvis JC, Mokrusch T, Kwende MMN, et al. Fast-to- slow transformation in stimulated rat muscle. Muscle Nerve 1996;19:1469-75. 27. Mayne CN, Mokrusch T, Jarvis JC, Gilroy SJ, Salmons S. Stimulation-induced expression of slow muscle myo- sin in a fast muscle of the rat: evidence of an un- restricted adaptive capacity. FEBS Letters 1993;327: 297-300. 28. Salmons S, Gale DR, Sréter FA. Ultrastructural aspects of the transformation of muscle fibre type by long term stimulation: changes in Z-discs and mitochondria. J Anat 1978;127:17-31. 29. Squire J. The structural basis of muscular contraction. New York: Plenum Press; 1981. 30. Eisenberg BR, Salmons S. The reorganisation of sub- cellular structure in muscle undergoing fast-to-slow type transformation: a stereological study. Cell Tissue Res 1981;220:449-471. 31. Chi MM-Y, Hintz CS, Henriksson J, Salmons S, Hel- lendahl RP, Park JL, et al. Chronic stimulation of mam- malian muscle: enzyme changes in individual fibers. Am J Physiol Cell Physiol 1986;251:C633-C642. 32. Henriksson J, Chi MM-Y, Hintz CS, et al. Chronic stim- ulation of mammalian muscle: changes in enzymes of six metabolic pathways. Am J Physiol Cell Physiol 1986;251:C614-C632. 33. Henriksson J, Salmons S, Chi MM-Y, Hintz CS, Lowry OH. Chronic stimulation of mammalian mus- cle: changes in metabolite concentrations in individual fibers. Am J Physiol Cell Physiol 1988;255:C543– C551. 34. Clark BJ, III, Acker MA, McCully K, et al. In vivo 31P- - 299 - Electrical stimulation in science and medicine Eur J Transl Myol 35 (3) 14058, 2025 doi: 10.4081/ejtm.2025.14058 NMR spectroscopy of chronically stimulated canine skeletal muscle. Am J Physiol Cell Physiol 1988;254: C258-266. 35. Eisenberg BR, Brown JMC, Salmons S. Restoration of fast muscle characteristics following cessation of chronic stimulation. The ultrastructure of slow-to-fast transformation. Cell Tissue Res 1984;238:221-230. 36. Salmons S. On the reversibility of stimulation-induced muscle transformation. In: Pette D, editor. The Dynamic State of Muscle Fibres. Berlin: Walter de Gruyter; 1990. p 401-414. 37. Lawrence JC, Jr., Krsek JA, Salsgiver WJ, et al. Phos- phorylase kinase isozymes in normal and electrically stimulated skeletal muscles. Am J Physiol Cell Physiol 1986;250:C84-9. 38. Heilmann C, Pette D. Molecular transformations in sar- coplasmic reticulum of fast-twitch muscle by electro- stimulation. Eur J Biochem 1979;93:437-446. 39. Leberer E, Seedorf U, Pette D. Neural control of gene expression in skeletal muscle. Calcium-sequestering proteins in developing and chronically stimulated rabbit skeletal muscles. Biochem J 1986;239:295-300. 40. Brown J, Salmons S. Percutaneous control of an im- plantable muscle stimulator via an optical link. J Biomed Engin 1981;3:206-208. 41. Salmons S, Jarvis JC. A simple optical switch for im- plantable devices. Med Biol Eng Comput 1991;29: 554-556. 42. Jarvis JC, Salmons S. A family of neuromuscular stim- ulators with optical transcutaneous control. J Med Engin Technol 1991;15:53-57. 43. Joplin RE, Franchi LL, Salmons S. Changes in the size and synthetic activity of nuclear populations in chron- ically stimulated rabbit skeletal muscle. J Anat 1987;155:39-50. 44. Mastri C, Salmons S, Thomas GH. Early events in the response of fast skeletal muscle to chronic low- frequency stimulation: polyamine biosynthesis and pro- tein phosphorylation. Biochem J 1982;206:211-219. 45. Brown WE, Salmons S, Pullen L, Brownson C. Gene re-expression in stimulated fast skeletal muscle II. Early changes in myosin heavy chain mRNA. J Muscle Res Cell Motil 1987;8:81. 46. Brownson C, Isenberg H, Brown W, et al. Changes in skeletal muscle gene transcription induced by chronic stimulation. Muscle Nerve 1988;11:1183-1189. 47. Brownson C, Little P, Jarvis JC, Salmons S. Reciprocal changes in myosin isoform mRNAs of rabbit skeletal muscle in response to the initiation and cessation of chronic electrical stimulation. Muscle Nerve 1992;15: 694-700. 48. Brownson C, Little P, Mayne CN, Jet al. Reciprocal changes in myosin isoform expression in rabbit fast skeletal muscle resulting from the application and re- moval of chronic electrical stimulation. In: el Haj A, ed- itor. Molecular Biology of Muscle. ISBN 0 948601 35 3 ed. Volume no. 46, Society for Experimental Biology Symposium. Colchester, U.K.: The Company of Biolo- gists Ltd.; 1992. p 301-310. 49. Williams RS, Salmons S, Newsholme EA, Mellor J. Ac- tivity-induced regulation of aldolase gene expression in skeletal muscle (abstract). Abstracts, American Heart Association 58th Scientific Sessions, Washington, DC 1985. 50. Williams RS, Salmons S, Newsholme EA, et al Regu- lation of nuclear and mitochondrial expression by con- tractile activity in skeletal muscle. J Biol Chem 1986; 261:376-380. 51. Bassel-Duby R, Olson EN. Signaling pathways in skeletal muscle remodeling. Annu Rev Biochem 2006; 75:19-37. 52. Schiaffino S, Sandri M, Murgia M. Activity-dependent signaling pathways controlling muscle diversity and plasticity. Physiology (Bethesda) 2007;22:269-278. 53. Mayne CN, Sutherland H, Jarvis JC, et al. Induction of a fast-oxidative phenotype by chronic muscle stimula- tion: histochemical and metabolic studies. Am J Physiol Cell Physiol 1996;270:C313-320. 54. Sutherland H, Jarvis JC, Kwende MMN, et al. The dose-related response of rabbit fast muscle to long-term low-frequency stimulation. Muscle Nerve 1998;21: 1632-1646. 55. Williams RS, Garcia Moll M, Mellor J, Salmons S, Har- lan W. Adaptation of skeletal muscle to increased con- tractile activity. Expression of nuclear genes encoding mitochondrial proteins. J Biol Chem 1987;262:2764- 2767. 56. Hood DA, Zak R, Pette D. Chronic stimulation of rat skeletal muscle induces coordinate increases in mito- chondrial and nuclear mRNAs of cytochrome-c-oxidase subunits. Eur J Biochem 1989;179:275-280. 57. Salmons S, Jarvis JC, Mayne CN, et al. Changes in ATP, phosphocreatine and 16 metabolites in rabbit muscles stimulated for up to 96 hours. Am J Physiol Cell Physiol 1996;271:C1167-1171. 58. Maier A, Gambke B, Pette D. Degeneration-regenera- tion as a mechanism contributing to the fast to slow con- version of chronically stimulated fast-twitch rabbit muscle. Cell Tissue Res 1986;244:635-643. 59. Maier A, Gorza L, Schiaffino S, Pette D. A combined histochemical and immunohistochemical study on the dynamics of fast-to-slow fiber transformation in chron- ically stimulated rabbit muscle. Cell Tissue Res 1988; 254:59-68. 60. Mayne CN, Jarvis JC, Salmons S. Dissociation between metabolite levels and force fatigue in the early stages of stimulation-induced transformation of mammalian skeletal muscle. Basic Appl Myol 1991;1:63–70. 61. Lexell J, Jarvis JC, Downham DY, Salmons S. Quanti- tative morphology of stimulation–induced damage in rabbit fast–twitch muscles. Cell Tissue Res 1992;269: 195–204. 62. Lexell J, Jarvis JC, Downham DY, Salmons S. Stimu- lation-induced damage in rabbit fast-twitch skeletal muscles: a quantitative morphological study of the in- fluence of pattern and frequency. Cell Tissue Res 1993;273:357-362. 63. Lexell J, Jarvis JC, Downham DY, Salmons S. Stimu- lation-induced muscle damage. Basic Appl Myol 1994; 4:59-66. - 300 - Electrical stimulation in science and medicine Eur J Transl Myol 35 (3) 14058, 2025 doi: 10.4081/ejtm.2025.14058 64. Salmons S. The response of skeletal muscle to different patterns of use - some new developments and concepts. In: Pette D, editor. Plasticity of Muscle. Berlin: Walter de Gruyter; 1980. p 387-399. 65. Salmons S, Henriksson J. Invited review: The adaptive response of skeletal muscle to increased use. Muscle Nerve 1981;4:94-105. 66. Salmons S. Adaptive change in electrically stimulated muscle: a framework for the design of clinical protocols (Invited review). Muscle Nerve 2009;40:918-935. 67. Glenn WW, Phelps ML, Elefteriades JA, et al. Twenty years of experience in phrenic nerve stimulation to pace the diaphragm. Pacing Clin Electrophysiol 1986; 9:780-784. 68. Elefteriades JA, Hogan JF, Handler A, Loke JS. Long- term follow-up of bilateral pacing of the diaphragm in quadriplegia. N Engl J Med 1992;326:1433-1434. 69. Baer GA, Talonen PP, Shneerson JM, et al. Phrenic nerve stimulation for central ventilatory failure with bi- polar and four-pole electrode systems. Pacing Clin Elec- trophysiol 1990;13:1061-1072. 70. Kralj A, Bajd T. Functional electrical stimulation. Standing and walking after spinal cord injury. Florida: CRC Press, Inc; 1989. 208 p. 71. Peckham PH, Keith MW, Kilgore KL. Restoration of upper extremity function in tetraplegia. Top Spinal Cord Inj Rehabil 1999;5:33-43. 72. Horch KW, Kipke DR, editors. Neuroprosthetics: Theory and Practice (Series on Bioengineering & Bio- medical Engineering—Vol. 8). River Edge, NJ: World Scientific Publishing Co; 2017. 73. Baeten C, Spaans F, Fluks A. An implanted neuromus- cular stimulator for faecal continence following pre- viously implanted gracilis muscle: report of a case. Dis Colon Rectum 1988;31:134-137. 74. Williams NS, Hallan RI, Koeze TH, Watkins ES. Con- struction of a neorectum and neoanal sphincter follow- ing previous proctocolectomy. Br J Surg 1989;76: 1191-1194. 75. Zonnevijlle ED, Somia NN, Stremel RW, et al. Sequen- tial segmental neuromuscular stimulation: an effective approach to enhance fatigue resistance. Plast Reconstr Surg 2000;105:667-673. 76. Bardoel JW, Stadelmann WK, Tobin GR, et al. Use of the rectus abdominis muscle for abdominal stoma sphincter construction: an anatomical feasibility study. Plast Reconstr Surg 2000;105:589-595. 77. Russold MF, Ramnarine I, Ashley Z, et al. Practical and effective stomal sphincter creation: evaluation in pigs. Dis Colon Rectum 2010;53:467-474. 78. Salmons S, Russold MF, Ramnarine I, Ashley Z, Su- therland H, Jarvis JC. Can you make a sphincter out of skeletal muscle? Physiology News 2010;79:23-25. 79. Salmons S. On the feasibility of using diaphragm mus- cle as a myocardial substitute. Med Biol Eng 1975;13: 608-609. 80. Shortland AP, Jarvis JC, Salmons S. Haemodynamic considerations in the design of a skeletal muscle ven- tricle. Med Biol Eng Comput 2003;41:529-535. 81. Capoccia M, Sutherland H, Salmons S, Jarvis JC. The hemodynamic function of intrathoracic skeletal muscle ventricles after recovery from surgery in pigs. Artif Org 2002;26:235-237. 82. Woo EB-C, Jarvis JC, Hooper TL, Salmons S. Avoiding ischemia in latissimus dorsi muscle grafts: electrical prestimulation versus vascular delay. Ann Thorac Surg 2002;73:1927-1932. 83. Salmons S, Tang ATM, Jarvis JC, Degens H, Hastings M, Hooper TL. Morphological and functional evidence, and clinical importance, of vascular anastomoses in the latissimus dorsi muscle of the sheep. J Anat 1998;193: 93-104. 84. Thomas GA, Hammond RL, Greer K, et al. Functional assessment of skeletal muscle ventricles after pumping for up to four years in circulation. Ann Thorac Surg 2000;70:1281-1289; discussion 1290. 85. Ramnarine IR, Capoccia M, Ashley Z, et al. Counter- pulsation from the skeletal muscle ventricle and the in- traaortic balloon pump in the normal and failing circulations. Circulation 2006;114 (Suppl):I-10-15. 86. Salmons S. Cardiac assistance from skeletal muscle: a reappraisal. Eur J Cardiothorac Surg 2008;35:204-213. 87. Salmons S, Ashley Z, Sutherland H, et al. Functional electrical stimulation of denervated muscles: basic is- sues. Artif Org 2005;29:199-202. 88. Kern H, Salmons S, Mayr W, et al. Recovery of long- term denervated human muscles induced by electrical stimulation. Muscle Nerve 2005;31:98-101. 89. Ashley Z, Sutherland H, Lanmuller H, et al. Determina- tion of the chronaxie and rheobase of denervated limb muscles in conscious rabbits. Artif Org 2005;29:212- 215. 90. Ashley Z, Sutherland H, Lanmuller H, et al. Atrophy, but not necrosis, in rabbit skeletal muscle denervated for periods up to one year Am J Physiol Cell Physiol 2007;292:C440-451. 91. Ashley Z, Sutherland H, Russold MF, et al. Therapeutic stimulation of denervated muscles: the influence of pat- tern. Muscle Nerve 2008;38:875-886. 92. Salmons S, Jarvis JC. Functional electrical stimulation of denervated muscles: an experimental evaluation. Artif Org 2008;32:597-603. 93. Lanmüller H, Ashley Z, Unger E, et al. Implantable de- vice for long-term electrical stimulation of denervated muscles in rabbits. Med Biol Eng Comput 2005;43:535- 540. 94. Ashley Z, Salmons S, Boncompagni S, et al. Effects of chronic electrical stimulation on long-term denervated muscles of the rabbit hind limb. J Muscle Res Cell Motil 2007;28:203-217. Disclaimer All claims expressed in this article are solely those of the author and do not necessarily represent those of his affili- ated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this ar- ticle or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. Submitted: 4 June 2025. Accepted: 24 June 2025. Early access: 6 August 2025. - 301 -