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Dual amperometric microelectrodes were used to measure local pO2 and perfusion at multiple sites in the fast-twitch tibialis anterior muscles of anaesthetized rabbits. Six muscles were stimulated continuously at 10, 5, or 2.5 Hz. For all three frequencies, perfusion declined to about 50% of resting levels and recovered after stimulation. These changes corresponded to a rise followed by a fall in extracellular pO2. The highest levels of pO2 were reached during stimulation at 10 Hz. Eight muscles were stimulated tetanically at 100 Hz for 200 ms with duty cycles that were varied between 1.3 and 20.0%. Perfusion rose to 8.7 +/- 2.0 ml s(-1) 100 g(-1) at a duty cycle of 5% and declined with further increases in duty cycle. pO2 was depressed for duty cycles less than 10% but rose above resting levels at higher duty cycles. It is suggested that the paradoxical combination of elevated pO2 and depressed perfusion is attributable to stimulation conditions that exceed the oxygen transport capacity of a fast muscle.

The elevated intramuscular pressure (IMP) associated with sustained muscle contraction can affect blood flow, and could influence the long-term viability of functional skeletal muscle grafts. We therefore examined the relationship between force, peak IMP and blood flow in the tibialis anterior muscle of the anaesthetized rabbit. During isometric contractions. IMP was related linearly to force, and only the slope of the relationship varied between animals. During isotonic contractions, however, the highest values of IMP were found at the lowest force levels, and IMP appeared to be related to the amount and speed of shortening. During repeated isometric contractions, the ratio of IMP to force varied with time, stimulation pattern and subject. Mean blood flow did not differ appreciably between repetitive isometric contractions at duty cycles of 10-40%, and was unrelated to integrated pressure, integrated force, or depth from the surface. We conclude: (1) that IMP is unlikely to affect mean blood flow during cyclic activity that has a duty cycle less than 40%; and (2) that the clinical use of IMP as a predictor of muscle force appears to be justified only for single isometric contractions, and needs to be interpreted cautiously when contractions involve shortening or fatigue.

Background: In the clinical application of transformed skeletal muscle to cardiac assistance there is evidence that the latissimus dorsi muscle (LDM) wrap can undergo atrophy, which would prevent it from providing a sustained functional improvement. Possible causes are ischaemia and degeneration related to the conditioning process. We studied the nutritional and structural changes occurring under different stimulation regimes with the aim of improving the conditioning protocol., Methods: Microelectrodes were used to measure regional perfusion and oxygenation in the rabbit LDM during mobilisation and subsequent repeated contraction. Group A muscles (n = 10) were conditioned for 6 weeks at 10 Hz, Group B muscles (n = 10) for 2 weeks at 2.5 Hz. Each muscle was then mobilised and tested in a hydraulic apparatus which recorded the pressure generated in a closed circuit., Results: Muscles of Group A and Group B demonstrated transformation of fibre type, with a predominance of type I (62 +/- 4%) fibres in Group A and type IIa (68 +/- 9%) fibres in Group B. There was no evidence of muscle degeneration. After 10 min of fatigue testing the pressure produced was 53 +/- 5% of initial values in Group A and 51 +/- 8% in Group B, compared to 8 +/- 1% in the control group (P < 0.001). Maximum rate of relaxation was faster in Group B than in Group A (46 +/- 3% vs. 36 +/- 3% of control muscle, P < 0.05). Mobilisation resulted in a decrease in the distal perfusion of the control muscles (P < 0.05) and PO2 decreased by 8.7 +/- 1.7 mmHg during a fatigue test, which resulted in rapid loss of contractile function to 46 +/- 1% of the initial value within 1 min. In both Groups A and B the perfusion of all regions of the muscles both before and after mobilisation was greater than that of controls. During the same fatigue test, the PO2 of the distal regions was maintained and the contractile function fell more slowly to between 70 and 80% of initial values within 1 min., Conclusion: We showed that ischaemia in the distal region of the control LDM could result from mobilisation and repeated contraction. Muscle transformation improved perfusion and prevented a fall in tissue PO2 during a sustained series of contractions. Muscles that were conditioned at 2.5 Hz shared the improved perfusion of the fully transformed muscle, but had faster relaxation characteristics. Short periods of in situ conditioning prior to mobilisation may help to avoid ischaemic changes in distal parts of the LDM while achieving fatigue resistance in the grafted muscle at an earlier postoperative stage.

Background: The fatigue resistance and power-to-weight ratio of skeletal muscle that has been conditioned by electrical stimulation makes cardiac assistance from a graft of such muscle a realistic prospect. A skeletal muscle must be surgically reconfigured to act on the circulating blood, but little is known about the power losses that accompany such interventions. We investigated in acute experiments the hydraulic performance of approximately cylindrical pumps made from sheep latissimus dorsi (LD) muscles, having first characterized the performance of each muscle in situ., Methods and Results: Force-length and force-velocity relations were measured in situ for LD that had received either 8 weeks of stimulation at 2 Hz or no chronic stimulation. Two sizes of skeletal muscle ventricle (SMV) were formed from the same muscles, and their hydraulic performance was measured. The hydraulic performance was also calculated from the linear data, models of the force-length and force-velocity curves, and a description of the stress distribution within the SMV wall. The model predicted well the isovolumetric function of the ventricles and the optimum afterload but overestimated the flow and therefore the power. In conditioned ventricles the performance was particularly poor because of the slow contractile properties of the muscles., Conclusions: If SMVs are to pump effectively against the arterial impedance, the pressure drop caused by flow (or the internal resistance) should be lower than that of the ventricles we constructed. Progress can be made through refinement of surgical technique and stimulation protocols that generate faster fatigue-resistant muscles.

The effects of repetitive muscle contraction on energy state and tension production were studied in rabbit tibialis anterior/extensor digitorum longus muscles that had been subjected to 90 days of continuous indirect electrical stimulation at 10 Hz. Anesthetized chronically stimulated and control rabbits were challenged with 15 min of stimulation at 4 and 15 tetani/min. Pi-to-phosphocreatine (PCr) ratio (Pi/PCr) was measured in vivo before, during, and after acute stimulation by 31P-magnetic resonance spectroscopy, and tension was recorded at the same time. Although Pi/PCr was low at rest, it was significantly higher in chronically stimulated muscle than in control muscle (0.20 +/- 0.02 vs. 0.05 +/- 0.01, P < 0.05). Stimulation of control muscle for 15 min at both 4 and 15 tetani/min induced a significant rise in Pi/PCr, whereas the same conditions in chronically stimulated muscle did not produce any significant departure from initial levels. The tension produced by control muscle fell to 93 +/- 3% of its initial value during stimulation at 4 tetani/min and to 61 +/- 7% at 15 tetani/min, respectively. In chronically stimulated muscle, on the other hand, tension was potentiated above its initial level at both stimulation rates (135 +/- 15 and 138 +/- 11%, respectively) and remained significantly elevated throughout each trial. The ability of chronically stimulated muscle to sustain high levels of activity with minimal perturbations in Pi/PCr or decrement in tension is attributable to cellular adaptations that include a well-documented increase in oxidative capacity.

This study was designed to gain an insight into mechanisms by which cytochrome c gene expression is enhanced by increased contractile activity in skeletal muscle. When rat tibialis anterior muscles were stimulated (10 Hz, 0.25 ms) for 0, 2, 6, 12, or 24 h or 2, 5, 9, or 13 days (n = 4 for each time point), cytochrome c protein (enzyme-linked immunosorbent assay) and mRNA (Northern blot analysis) concentrations started to increase by 9 days, and this was associated with concurrent decreases in cytochrome c mRNA-protein interaction (RNA gel mobility shift assay). We found that the decreased RNA-protein interaction in the stimulated muscle extract was restored by ultracentrifugation (150,000 g, 1 h) in the supernatant fraction. The 150,000 g pellet fraction of stimulated muscle was capable of inhibiting the RNA-protein interaction in control tibialis anterior muscles. These results provide evidence of an inhibitory factor that is responsible for decreasing RNA-protein interaction in the 3'-untranslated region of cytochrome c mRNA in continuously stimulated muscle.

The capacity of skeletal muscle to oxidize fatty acids increases with endurance training. The oxidation of long-chain fatty acids occurs in mitochondria and is initiated by a carnitine-dependent transport step in which three enzymes help fatty acyl groups enter the matrix compartment. The purpose of this study was to determine whether pretranslational regulation of one of these three enzymes, carnitine palmitoyltransferase II (CPT II), as estimated from the level of CPT II mRNA, plays a role in the doubling of CPT activity in skeletal muscle of rats subjected to daily 2-h bouts of running on treadmills (P. A. Móle, L. B. Oscai, and J. O. Holloszy. J. Clin. Invest. 50: 2323-2330, 1971). After 100 min/day of running on motor-driven treadmills for 2 wk, CPT II mRNA in the plantaris muscle was unchanged when normalized per unit of extracted RNA but was 50% higher (P < 0.05) over sedentary controls when normalized per unit of muscle wet weight. To test whether additional contractile activity would make CPT II mRNA even higher, continuous indirect electrical stimulation was imposed on the tibialis anterior muscles. After 9 days of chronic stimulation, CPT II mRNA was 63, 221, and 137% greater than control (P < 0.001) when normalized to extracted RNA, muscle wet weight, and whole muscle, respectively, compared with the muscle in the control rats. These data indicate that pretranslational regulation of CPT II occurs in response to increased contractile activity in skeletal muscle.

Glucose transport and phosphorylation are the first steps in the utilization of extracellular glucose by skeletal muscle. We have examined the relationships between proteins mediating these steps in single fibers of identified type dissected from rabbit skeletal muscle. The level of the glucose transporter isoform GLUT4, measured by immunoblotting, varied among fibers by a factor of 20 (slow oxidative > fast oxidative glycolytic > fast glycolytic). In fibers from the tibialis anterior muscle, GLUT4 was correlated (r2 = 0.75) with the activity of malate dehydrogenase, an enzyme representative of oxidative energy metabolism. In these fibers a strong correlation (r2 = 0.70) was also observed between GLUT4 and hexokinase activity. GLUT1 levels were barely detectable, regardless of fiber type. To investigate the possible role of muscle activity in controlling the expression of transporters, tibialis anterior muscles were activated by chronic electrical stimulation of the peroneal nerves. GLUT1 levels increased after 1 day of stimulation to a plateau that was severalfold higher than the level in non-stimulated cells. Hexokinase activity and the GLUT4 level changed in parallel: both were increased by approximately 2.5-fold after 1 day and by 14-fold after 21 days. Thus, while both GLUT1 and GLUT4 were regulated by muscle activity, only GLUT4 expression was coordinated with the expression of hexokinase.

Changes in myosin heavy chain (MHC) mRNAs were studied in rabbit fast-twitch muscles during continuous electrical stimulation at 10 Hz for periods up to 3 weeks, and during the first 12 days of the recovery process that followed cessation of 6 weeks' stimulation. Two cDNA probes were used to detect MHC mRNAs specific to fast- and slow-twitch skeletal muscle in RNase protection assays and Northern- and slot-blot analyses. The isolation and base sequence of one of these probes, corresponding to the MHC gene expressed in soleus (slow-twitch), is described. At an early stage of the response to stimulation, fast MHC mRNA was replaced by slow MHC mRNA. During recovery, this process occurred in reverse but took longer. The time course of recovery was slightly faster in tibialis anterior than in extensor digitorum longus. The changes in mRNAs during both stimulation and recovery reflected changes in the corresponding muscle proteins.

The apparatus described in this communication enables the force-velocity relationship to be determined for whole rabbit muscles in vivo and their resistance to fatigue to be assessed at specified rates of external work. The ergometer generates constant-velocity motion, controlling a force of up to 50 N, over a range of velocity up to 500 mm/s and a distance of 20 mm. This distance corresponds to the range of shortening of the rabbit tibialis anterior muscle from full plantar flexion to full dorsiflexion of the foot, equivalent to approximately 28% fiber shortening. Activated muscles can be allowed to shorten at constant velocity from any point on their isometric force trajectory. Cyclic releases for fatigue testing can be made at rates up to 30 releases/min over a period of 6-8 h. The timing of the release and return strokes of the ergometer is under the control of a digital programmer that also synchronizes the delivery of activating stimuli to the muscle nerve and trigger signals to the recording equipment. An electrohydraulic design was chosen because it is simpler to engineer than an electromagnetic actuator, is reliable in continuous cyclic use, and can be assembled, at least in part, from available industrial components.

Transformation of fast-twitch into slow-twitch skeletal muscle was induced in adult rabbits by chronic low-frequency stimulation and studied at the ultrastructural level. With the use of stereological techniques, a time course was established for changes in mitochondrial volume, sarcotubular system, and Z-band thickness for periods of stimulation ranging from 6 h to 24 weeks. T-tubules, terminal cisternae, and sarcoplasmic reticulum decreased at an early stage and reached levels typical of slow muscle after only 2 weeks of stimulation. Transformation of Z-band structure took place between 1 1/2 and 3 weeks after the onset of stimulation. Mitochondrial volume increased several fold over the first 3 weeks of stimulation, and fell rapidly after 7 weeks, although it still remained above the levels typical of slow muscle. Although there was no sign of degradation and regeneration of the muscle fibers themselves, considerable structural reorganization was evident at the subcellular level after 1 week of stimulation. The fibers passed through a less well organized transitional stage in which fibers could not be assigned to a normal ultrastructural category. After 3 weeks all of the stimulated fibers could be assigned to the normal slow-twitch category although some subcellular irregularities persisted even after 24 weeks. The ultrastructural alterations are discussed in relation to functional and biochemical changes in the whole muscle.

The latissimus dorsi muscles of six dogs were made fatigue resistant by chronic electrical conditioning. Once the muscles were conditioned, oxygen consumption was measured during periods of exercise. The ratio of the tension developed to oxygen consumed during moderate stimulation (300 msec on) for the control and the electrically conditioned muscles was 16.3 +/- 3.5 and 36.5 +/- 6.7 kg-sec/ml oxygen, respectively. During intense stimulation (800 msec on) the ratio was 12.6 +/- 2.1 and 54.2 +/- 8.9 kg-sec/ml oxygen, respectively. Thus the conditioned muscle was able to develop and maintain tension with a considerably reduced oxygen expenditure. The increased efficiency of the conditioned muscle helps to explain its increased resistance to fatigue and the ability of pumping chambers constructed from electrically preconditioned skeletal muscle to perform sustained cardiac type work.

An increase in mitochondrial biogenesis in mammalian cells requires a coordinated increase in the expression of a number of nuclear genes that encode mitochondrial proteins. To examine the regulatory mechanisms involved, we used specific anti-sense RNA probes to estimate the cellular concentrations of mRNA transcripts of two such nuclear genes in rabbit tibialis anterior muscles subjected in vivo to 10-21 days of indirect electrical stimulation. The unstimulated contralateral muscle in the same animals provided a base line for comparison. Change in expression of mitochondrial proteins was assessed in terms of the enzymatic capacity of citrate synthase and cytochrome oxidase, which increased 2.1-fold after 10 days and 5.5- and 4.1-fold, respectively, after 21 days of stimulation. As a proportion of total cellular RNA, messenger RNA encoding subunit beta of F1-ATPase increased 2.2-fold over control levels after 10 days and 2.3-fold after 21 days; mRNA encoding subunit VIC of cytochrome oxidase increased 1.3-fold and 1.9-fold over control levels after stimulation for 10 and 21 days, respectively. These changes were not attributable to nonspecific effects of stimulation on all mRNA transcripts, since aldolase A mRNA decreased to 26% of control levels after 21 days of stimulation. Furthermore, mRNA transcripts from these nuclear genes encoding mitochondrial proteins did not increase to the same extent as mRNA transcripts of mitochondrial genes such as cytochrome b, which increased 5.9-fold after 21 days of stimulation. We conclude that the increase in mitochondrial biogenesis induced by electrical stimulation of skeletal muscle is supported by pretranslational regulation of expression of nuclear genes encoding mitochondrial proteins. There are, however, indications that translational or post-translational regulatory events may also be involved.

Continuous stimulation of a rabbit fast muscle at 10 Hz changes its physiological and biochemical parameters to those of a slow muscle. These transformations include the replacement of myosin of one type by myosin of another type. Two hypotheses could explain the cellular basis of these changes. First, if fibers were permanently programmed to be fast or slow, but not both, a change from one muscle type to another would involve atrophy of one fiber type accompanied by de novo appearance of the other type. Alternatively, preexisting muscle fibers could be changing from the expression of one set of genes to the expression of another. Fluorescein-labeled antibodies against fast (AF) and slow (AS) muscle myosins of rabbits have been prepared by procedures originally applied to chicken muscle. In the unstimulated fast peroneus longus muscle, most fibers stained only with AF; a small percentage stained only with AS; and no fibers stained with both antibodies. In stimulated muscles, most fibers stained with both AF and AS; with increasing time of stimulation, there was a progressive decrease in staining intensity with AF and a progressive increase in staining intensity with AS within the same fibers. These results are consistent with a theory that individual preexisting muscle fibers can actually switch from the synthesis of fast myosin to the synthesis of slow myosin.

Implantable electronic stimulators were used to subject fast-twitch tibialis anterior and extensor digitorum longus muscles of adult rabbits to a chronically increased level of use. Stimulation was discontinued after 6 weeks and physiological, histochemical and biochemical properties of the muscles were examined at intervals over the ensuing 20 weeks. Previous work had shown that 6 weeks of stimulation was sufficient to bring about a substantial transformation of type in fast-twitch muscles, which then exhibited much of the character of muscles of the slow-twitch type. The present experiments showed that these stimulation-induced changes were completely reversible. The time-course of reversion was such that the muscles had recovered their original fast properties by about 12 weeks after the cessation of stimulation. The contractile characteristics and post-tetanic potentiation typical of fast muscle returned rapidly, in only 3-4 weeks, and over the same period the proportion of histochemical type 1 fibres declined from about 70% to control levels. Changes in fatigue-resistance, capillary density and enzyme activity followed a more prolonged time-course; in particular, the decline in the activity of enzymes of oxidative metabolism corresponded closely to that already established for the mitochondrial volume fraction. Reacquisition of fast properties was not accompanied by any changes in specific force-generating capacity. Observations from these experiments and from a related morphological study fit into a 'first-in, last-out' pattern for the response to stimulation and recovery. The slow-to-fast reversion that takes place during the recovery period provides a further opportunity for testing causal associations within the events underlying type transformation. It has important consequences for therapeutic applications that make use of the fatigue-resistant character of chronically stimulated muscle.

Increased contractile activity of skeletal muscle augments the volume fraction and enzymatic capacity of mitochondria and suppresses the enzymatic capacity of several cytoplasmic enzymes of glycolysis. To examine the biochemical mechanisms underlying these effects, we measured the concentrations of cytochrome b mRNA and aldolase A mRNA in tibialis anterior muscles of adult rabbits that had been stimulated via the motor nerve to contract continuously at 10 Hz for 5 or 21 days; these were compared with the corresponding levels in the unstimulated limbs of the same animals. After 21 days of stimulation aldolase mRNA had fallen to one-fourth of control levels, while cytochrome b mRNA had increased by 5-fold. A reduction in aldolase mRNA was already evident after only 5 days of stimulation, whereas the level of cytochrome b mRNA was not elevated at this stage. Mitochondrial DNA was unchanged after 5 days but had increased by 4-fold after 21 days. We conclude that contractile activity in skeletal muscle produces reciprocal changes in the expression of these two genes at a transcriptional level but via different regulatory mechanisms. Enhancement of the expression of the mitochondrial cytochrome b gene appears to be proportional to the increase in its copy number and may not, therefore, depend upon changes in transcriptional or translational efficiency. The reduction in aldolase A mRNA occurs at an earlier stage in the response to contractile activity and is probably mediated by a reduced transcriptional efficiency.

The changes which follow cross reinnervation of mammalian fast and slow twitch muscles may reflect a capacity of skeletal muscle to respond adaptively to different functional requirements. This interpretation is supported by experiments in which long-term electrical stimulation was used both to reproduce and to oppose the effects of cross reinnervation.

Clinical experience reviewed elsewhere in this issue supports the use of electrical stimulation of the diaphragm via the phrenic nerve in selected patients in whom ventilation is inadequate, intermittent or absent. This paper represents a muscle biologist's view of some of the fundamental problems posed by this technique. It reviews the long-term changes that take place in skeletal muscles in response to chronic stimulation, and it indicates how these may be exploited in order to make electrophrenic respiration more effective and more acceptable both to the patient and to the clinical support team.

1. The time course of contraction and relaxation in the isometric twitch of a rabbit soleus muscle becomes more rapid following tenotomy and spinal cord section. This increase in speed could be prevented by long-term electrical stimulation at frequencies of 5 or 10/sec. It was not prevented by stimulation at frequencies of 20 or 40/sec.2. Long-term electrical stimulation of fast rabbit and cat muscles at a frequency of 10/sec had a slowing effect on the time course of contraction and relaxation.3. It is concluded that the almost continuous low frequency discharge of motoneurones innervating postural muscles plays an important part in establishing and maintaining the slow time course of contraction of these muscles. The characteristically different speeds of contraction of fast and slow striated muscles can in this way be related to the different patterns of impulse activity which they normally receive.