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Fast muscles of the rat hind limb were stimulated continuously at 10 or 20 Hz for periods of 55-61 days by means of an implantable neuromuscular stimulator. Gel electrophoresis clearly demonstrated the presence in stimulated muscles of slow myosin light and heavy chains, although fast isoforms were still present in all cases. Thus, contrary to previous reports, induction of slow myosin isoforms does occur in this, as in other, mammalian species. The time course of the response to stimulation appears to be more extended than that seen in the rabbit.

The aim of this study was to determine whether muscle fibre degeneration brought about by chronic low-frequency electrical stimulation was related to the pattern and frequency of stimulation. Rabbit fast-twitch muscles, tibialis anterior and extensor digitorum longus, were stimulated for 9 days with pulse trains ranging in frequency from 1.25 Hz to 10 Hz. Histological data from these muscles were analysed with multivariate statistical techniques. At the lower stimulation frequencies there was a significantly lower incidence of degenerating muscle fibres. Fibres that reacted positively with an antineonatal antibody were most numerous in the sections that revealed the most degeneration. The dependence on frequency was generally similar for the two muscles, but the extensor digitorum longus muscles showed more degeneration than the tibialis anterior at every frequency. Muscles subjected to 10 Hz intermittent stimulation showed significantly less degeneration than muscles stimulated with 5 Hz continuously, although the aggregate number of impulses delivered was the same. The incidence of degeneration in the extensor digitorum longus muscles stimulated at 1.25 Hz was indistinguishable from that in control, unstimulated muscles; for the tibialis anterior muscles, this was also true for stimulation at 2.5 Hz. We conclude that damage is not an inevitable consequence of electrical stimulation. The influence of pattern and frequency on damage should be taken into account when devising neuromuscular stimulation regimes for clinical use.

We review here various ways in which cardiac assistance might be derived from a patient's own skeletal muscle. Calculations based on experimental data and optimistic estimates of the efficiency of the energy conversions involved suggest that the continuous assist available would be limited to about 2 litres a minute if a muscle were used to energise an electromechanical device. It would be more efficient to couple the energy mechanically or hydraulically, but these approaches still pose problems of anatomical placement, muscle attachment, fluid leakage, and cost. Unless these issues can be addressed, the use of skeletal muscle as an internal power source for mechanical circulatory assist devices will remain an unworkable concept. Configurations that couple skeletal muscle contraction directly to the circulation would be more efficient and less costly. In terms of the energy available, a skeletal muscle ventricle could be designed to provide a continuous partial assist of 1-2 l/min, with flows of up to 8 l/min sustainable for limited periods. Such an approach offers new possibilities for the surgical treatment of chronic cardiac failure.

The purpose of this study was to examine the contention that stimulation-induced damage, resulting in degeneration with subsequent regeneration, plays a major role in the transformation of fibre type brought about by chronic electrical stimulation. Data from histological and histochemical sections of 9-day-stimulated rabbit fast-twitch muscles were analysed with multivariate statistical techniques. Fibre degeneration and regeneration varied non-systematically between sample areas at any given cross-sectional level. In the extensor digitorum longus muscle, but not in the tibialis anterior, there was more degeneration in proximal than in distal portions of the muscle. The extensor digitorum longus muscle consistently showed more degeneration than the tibialis anterior muscle. Degeneration was less extensive for an intermittent pattern of stimulation that delivered half the aggregate number of impulses of continuous stimulation. Degeneration and regeneration varied markedly between individual rabbits in each of the groups. Sections that revealed the most degeneration and regeneration also had more fibres that reacted positively with an anti-neonatal antibody. Rigorous analysis of different sources of variation has helped to explain apparent conflicts in the literature. The incidence of muscle fibre damage in the stimulated tibialis anterior muscle is low, showing that the contribution of degenerative-regenerative phenomena to fibre type conversion in this muscle is insignificant.

There has been no reliable evidence that the actions of anabolic steroids extend to limb muscles. In this study, female rabbits were treated with anabolic steroid (nandrolone decanoate) or arachis oil placebo for 4 weeks or 12 weeks. After 12 weeks, tibialis anterior muscles of treated animals showed highly significant increases in wet weight (38%), twitch tension (66%), maximum isometric tetanic tension (48%), maximum cross-sectional area (27%), and specific tension (17%). Fiber type composition showed a significant trend toward a less oxidative metabolic character. The experiments provided clear physiological and morphological evidence of a steroid-induced hypertrophy that was not attributable to fluid retention or changes in body weight. Of the muscles examined, the myotrophic effect was confined to the tibialis anterior muscle; extensor digitorum longus, plantaris, and soleus muscles showed no significant response. The work establishes an experimental model for the responses of limb muscles to anabolic compounds.

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.

Chronic indirect electrical stimulation of adult mammalian skeletal muscle brings about a transformation from the fast-twitch to the slow-twitch type. Underlying this transformation there is a sequence of profound changes in the expression of proteins involved in all the major molecular systems of the muscle. These include qualitative changes in the expression of myosin light and heavy chain isoforms. The time course of these changes has been studied in some detail at the protein level, both during chronic stimulation and during the recovery process that follows the cessation of stimulation. Here we report on the use of cDNA probes to study corresponding changes in myosin heavy chain (MHC) and light chain (MLC) mRNAs in rabbit fast-twitch muscles during continuous electrical stimulation at 10 Hz and during the first 12 days of recovery after cessation of 6 weeks of stimulation. At an early stage of the response to stimulation, fast MHC mRNA is replaced by slow MHC mRNA. During recovery this process occurs in reverse but takes longer. Broadly similar changes are seen for MLC mRNAs, although the time course is somewhat different. These experiments contribute to a growing body of evidence that many of the protein changes induced by chronic stimulation are the result of regulatory events that take place at a pre-translational level.

Skeletal muscle ventricles (SMV) were constructed from canine left latissimus dorsi muscle. The animals were divided into three groups: group A (n = 5), SMVs rested 4 weeks without electrical conditioning; group B (n = 6), SMVs rested 4 weeks and then electrically conditioned for 6 weeks; group C (n = 5), SMVs rested 18 weeks without electrical conditioning. At the end of each protocol, the SMVs were acutely tested by connecting them to a mock-circulation device. The SMVs in group C developed stroke work at physiologic preloads superior to any previously reported, as high as 194% of left ventricular stroke work at afterloads of 80 mmHg. The SMVs in group B developed work outputs equivalent to 53% of the left ventricle, which is still more than four times that of the right ventricle. The results show that it is possible to harvest sufficient work from skeletal muscle ventricles to fully replace cardiac function at physiologic preloads.

Immunogold labelling was used to study the distribution of newly synthesized slow muscle myosin (SM) at the ultrastructural level as it replaced fast muscle myosin (FM) in rabbit muscles undergoing stimulation-induced type transformation. Control fast muscle was labelled strongly with antibody to FM and control slow muscle with antibody to SM; label was confined to the A-band. Well-defined differences in the distribution of label within the A-band suggested that the monoclonal antibodies used corresponded to epitopes on different parts of the myosin molecule; this was confirmed by Western blots of subfragments prepared from FM and SM. After 4 weeks of continuous stimulation at 10 Hz, fibres of the tibialis anterior muscle reacted with antibodies to both isoforms; after 6 weeks, labelling was obtained only with antibody to SM. After a 7-week period of stimulation and 3 further weeks of recovery, fibres again reacted with both antibodies. In all positively-labelled sections, the distribution of gold particles was characteristic of the antibody and independent of the origin or history of the fibres. This observation supports the conclusion that newly synthesized myosin is capable of being incorporated throughout the length and cross-section of the A-band.

Latissimus dorsi pedicle grafts (LDPGs) were wrapped around the heart in eight dogs. In four dogs, the LDPGs were stimulated chronically; the remaining four dogs served as unstimulated controls. Right-sided cardiac filling pressures were normal in all dogs when measured 4 months after graft application. Mean tension generated by the viable LDPGs was 153 +/- 49.9 g. LDPGs contracting in synchrony with the heart did not increase cardiac output. In one dog, the aortic pressure changed from 140/100 to 155/85 mm Hg during synchronous contraction of the LDPG. Three dogs were placed on cardiopulmonary bypass, and their hearts were placed in fibrillation. The LDPGs were then stimulated at a burst frequency of 85 Hz and contracted vigorously. Under these conditions, the left ventricular pressure increased by an average of 15 mm Hg with each LDPG contraction; however, the mean aortic pressure was virtually unchanged. Left ventricular and aortic pressures of 125/20 and 125/65 mm Hg, respectively, could be obtained with manual compression of the fibrillating heart. This study indicates that although LDPGs can be made to contract chronically and in synchrony with the heart, they do not necessarily augment left ventricular performance.

During the fibre-type transformation induced by chronic electrical stimulation of rabbit fast-twitch muscle, replacement of the fast forms of the two classes of myosin light chain by their slow isoforms occurs asynchronously. Studies of total cellular myosin light chains and of the slow-to-fast transition now justify the conclusion that the asynchrony is due to switching between the expression of fast and slow genes for the two light chain classes at sequential stages of the transformation process.

Skeletal muscle ventricles (SMVs) were constructed from canine latissimus dorsi and connected to a totally implantable mock circulation device. The SMVs, stimulated by an implantable pulse generator, pumped continuously for up to 8 weeks in free-running beagle dogs. Systolic pressures produced by the SMVs, initially of 139 +/- 7.2 mmHg and after 1 month of continuous pumping of 107 +/- 7 mmHg, were comparable to normal physiologic pressures in the adult beagles (114 +/- 21 mmHg). After 2 weeks of continuous pumping, the mean stroke work of the SMVs was 0.4 X 10(6) ergs, a performance that compares favorably with the animal's cardiac ventricles. This study shows that canine skeletal muscle which has not received prior training or electrical conditioning can perform sustained work at the high levels needed for an auxiliary cardiovascular pump. It might be possible eventually to use such muscle pumps in humans to assist the failing circulation and to provide support in children with certain types of congenital heart defects.

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.

When fast-twitch skeletal muscles of the adult rabbit are subjected to continuous low-frequency activity by electrical stimulation of the corresponding motor nerves, the fibers undergo an ultrastructural transformation, so that after 6 weeks they have acquired an appearance typical of slow-twitch fibers. In the present study, stimulation was discontinued at this stage in order to follow the reverse transformation, in which the fibers recovered their original morphological characteristics under conditions of normal endogenous activity. Stereological techniques were used to assess the time course of this process over a period of 20 weeks in terms of fiber cross-sectional area, extent of T-system, thickness of the Z-band, and volume fraction of mitochondria in the fiber core. Fibers of transformed muscles were smaller than those of control muscles, but the differences were no longer evident after 9 weeks of recovery. After 2 weeks the T-system was still of limited extent, as is characteristic of slow-twitch fibers; it increased toward the amount typical of fast-twitch fibers between 2 and 4 weeks, and had reached its full extent by 12 weeks. The wide Z-bands characteristic of slow-twitch fibers were retained for 4 weeks, but the thickness had begun to decrease by 8 weeks and recovery was complete by 12 weeks. The mitochondrial volume did not increase during recovery, in contrast to the large increases which had been observed to take place between 2 and 6 weeks during the fast-to-slow transformation. Overall, the recovery of fast-twitch ultrastructural characteristics was complete, but followed a more extended time course, and involved less myofibrillar disruption at an intermediate stage, than the original fast-to-slow transformation.

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.

Earlier investigations involving chronic muscle stimulation have shown that skeletal muscle cells possess a much greater metabolic plasticity than had previously been recognized. We have described more fully the time course for the changes in different enzyme systems in single fibres of rabbit fast-twitch tibialis anterior (TA) muscles after periods of continuous stimulation of up to 10 weeks. After 2-5 wk every fibre shows higher levels of many oxidative enzymes than any control fibre; in some cases these levels are 2-10 times higher (well above any found even in the control soleus, a slow-twitch muscle). Citrate synthase, hexokinase and 3-oxoacid CoA-transferase are representatives of this group of enzymes. Other enzymes, such as malate dehydrogenase and amino acid aminotransferases also increase dramatically, but peak single fibre levels do not reach much above the highest in controls. These differential effects confirm at the single fibre level that chronic stimulation can alter mitochondrial composition. According to their staining reaction for myofibrillar ATPase, TA fibres are approximately 25% type IIA, and 75% type IIB, but by 5 wk these are converted to a mixture of type I, IIA and IIC fibres. At 5 wk, levels of glycolytic and high-energy phosphate transfer enzymes had decreased by 80% or more, and seemed to be adjusted to levels appropriate to their (new) ATPase type. This is in contrast to many enzymes of oxidative metabolism, which increase without synchronization with fibre type change. Determinations of metabolite concentrations in individual fibres from muscles freeze-clamped after varying periods of stimulation gave results which differ strikingly from data for acute stimulation. The findings reinforce our previous view that the high levels of ATP utilization engendered by chronic stimulation of muscle elicit a matching response in ATP production through a series of profound adaptations. Some of these are never encountered under the less extreme conditions of endurance exercise. Such features add to the interest and value of the chronic stimulation model as a means of studying the metabolic plasticity of muscle.

This study was designed to evaluate the role of vitamin D sufficiency, as reflected in serum 25-hydroxyvitamin D (25-OHD) concentrations, on serum minerals and bone mineralization in very premature infants. Seventy-two infants (mean +/- SD gestation 30.1 +/- 2.5 weeks, mean +/- SD birth weight 1178 +/- 278 gm) were observed serially for the first 3 months of life. Mean serum calcium and phosphorus values, but not magnesium, remained low prior to 12 weeks. The percentage of infants with moderate to severe hypomineralization was 75% at 3 weeks, 55% at 6 weeks, 54% at 9 weeks, and 15% at twelve weeks. Low serum calcium and phosphorus values, high alkaline phosphatase activity, and moderate-severe hypomineralization were more frequent in infants weighing less than 1000 gm and in those with lower mineral intake. With a 400 IU vitamin D supplement, 45% of infants could maintain an initially normal serum 25-OHD concentration or increase low concentrations, whereas 55% had falling or persistently low (less than or equal to 15 ng/ml) 25-OHD concentrations. Birth weight and mineral intakes were comparable in these two groups, yet the group with the lower serum 25-OHD concentration had lower serum calcium and higher alkaline phosphatase values, and a higher percentage of moderate to severe hypomineralization. Regardless of birth weight, mineral intake, or 25-OHD concentration, increases in serum calcium and phosphorus values and in mineralization were seen at postconception term (12 weeks in most infants, nine weeks in those weighing 1250 to 1600 gm). At 12 weeks of age, but not before, serum 25-OHD concentration was directly correlated with serum calcium (r = 0.47, P less than 0.01) and serum phosphorus (r = 0.47, P less than 0.01) and inversely correlated with alkaline phosphatase values (r = -0.71, P less than 0.01). Mineral availability and 25-OHD sufficiency both appear to be important and to act synergistically, with neither totally compensating for the other.

Because the efficiency of vitamin D absorption or hepatic uptake and 25-hydroxylation appears decreased in very premature infants, the routine use of 25-hydroxycholecalciferol (25-OHD3) supplementation has been suggested. Absorption studies of a 3 micrograms/kg orally administered dose of 25-OHD3 showed peak serum 25-hydroxyvitamin D2 and -vitamin D3 (25-OHD) concentrations at 4 to 8 hours similar in timing but of lesser magnitude to those seen in adults. Administration of 1 microgram/kg birth weight/day of 25-OHD3 corrected moderately low, but not very low serum (25-OHD) concentrations, and 2 micrograms/kg BW/day resulted in rapid and sustained increase in serum 25-OHD. Administration of 800 IU ergocalciferol (D2) also produced significantly higher serum 25-OHD concentrations than those in infants given 400 IU vitamin D2, but increases in serum 25-OHD were more gradual than in infants given 25-OHD3. In treatment trials with infants weighing less than 1500 gm, those given 800 IU D2, compared with those given 400 IU D2, had higher serum calcium concentrations and less frequent moderate or severe hypomineralization. Infants given 2 micrograms/kg BW 25-OHD3 had a significant increase in serum phosphorus values, but a decrease in serum calcium and magnesium concentrations, and parathyroid hormone also was suppressed to low normal values. The frequency of moderate to severe hypomineralization remained the same as in infants given 400 IU D2. In a subgroup of infants, serum 1,25-dihydroxyvitamin D was elevated over adult values, both in infants given 25-OHD3 (68.5 +/- 8.4 pg/ml) and in infants given vitamin D2 (60 +/- 6.7 pg/ml). Serum vitamin D concentrations were undetectable in four of six infants receiving 25-OHD3, but were elevated (5 to 31 ng/ml) in four infants receiving vitamin D2. Although 800 to 1000 IU D2 can be recommended as routine vitamin D supplementation in very premature infants fed standard formula, the use of 25-OHD3 requires further study.

Ten infants with rapidly developing and severe episodes of hypoxaemia (15-120 s duration) were studied. In infants over 2 months old most episodes occurred when awake, after a sudden noxious stimulus. In younger infants frequent yet undetected episodes occurred during sleep and feeding. Arterial PO2 fell below 20 mm Hg within 20 s, and loss of consciousness, sometimes with convulsions, occurred after 30 s. Clinical observations, measurements of respiratory movements, air flow, oesophageal pressure, external oblique surface electromyogram, and, in two cases, chest fluoroscopy and microlaryngoscopy documented episodes of no inspiratory flow but continued expiratory activity at low lung volume with partial or complete glottic closure. In five infants, episodes continued despite tracheostomy or an indwelling nasotracheal tube. No intracardiac shunt could be demonstrated and the rapid fall in arterial PO2 was attributed to lack of ventilation at a maximum expiratory position in the presence of a rapid recirculation time. In five infants tested there was a low proportion of phosphatidylcholine in the tracheal aspirate. In one infant audible expiratory braking (grunting) was present for most of the awake time. This previously unrecognised mechanism for severe hypoxaemia may be one cause of neurodevelopmental damage and sudden death in infants and young children.

The purpose of this study was to evaluate mechanisms of mineral homeostasis and mineralization in term infants with recommended vitamin D intakes. Infants fed human milk (nine), cow milk-based formula (11), or soy-based formula (11) were studied at 2 weeks and at 2, 4, 6, 9, and 12 months of age. While receiving 400 IU of vitamin D, all infants maintained serum vitamin D concentrations higher or equal to normal adult concentrations, and serum 25-hydroxyvitamin D levels were maintained at or above normal adult levels. Serum 1,25-dihydroxyvitamin D concentrations were higher than those of adults in the formula-fed but not in the human milk-fed infants. Serum calcium and phosphorus values were similar in all groups; however, urine phosphorus excretion and urine calcium excretion were adjusted to intakes. Serum parathyroid hormone values were normal in all groups. Bone mineral content significantly increased with age similarly in all groups; however, an apparent plateau occurred at 6 months of age in all groups. Bone width steadily increased with age. Taken as a whole, these data suggest that the vitamin D-sufficient term infant fed human milk, cow milk-based formula, or the soy-based formula studied can regulate mineral metabolism within acceptable physiologic limits to attain similar levels of serum minerals and bone mineral content.

Skeletal muscle ventricles (SMV) were constructed from canine latissimus dorsi muscle and chronically stimulated to pump fluid within a totally implantable mock circulation device. They were stimulated to contract at either 95/min (N = 7) or 54/min (N = 4). The preload was chronically maintained at 20 mmHg. After 1 month of continuous pumping the SMV were capable of generating a stroke work of 0.134 X 10(6) ergs and power output of 0.021 watts, representing 61% and 42% of the stroke work and power output of the native canine right ventricle, respectively.

The adaptive response of mammalian fast-twitch skeletal muscle to long-term low-frequency stimulation involves coordinated changes in the expression of a large number of genes and an increase in the synthesis of proteins and nucleic acids. Morphological correlates of these changes were sought in a qualitative and quantitative study of nuclear populations that included autoradiography at both light and electron microscopic levels. Stimulation-induced changes in biosynthetic activity were found to be supported by increases in the numbers of both non-muscle and muscle nuclei, and myonuclear counts were significantly increased in relation to sarcoplasmic volume. Moreover, the chronically stimulated muscle fibres showed ultrastructural signs consistent with mobilisation of transcriptional and translational activity.

Latissimus dorsi skeletal muscle ventricles were constructed in six beagles. They first underwent a period of vascular delay and of electrical preconditioning over several weeks. The skeletal muscle ventricles were then connected to a totally implantable mock circulation that allowed for the chronic measurement of pressures and flows produced by the muscle. The skeletal muscle ventricles were actuated by stimulation of the motor nerve with an implanted generator that delivered brief pulse trains. The skeletal muscle ventricles pumped continuously against an afterload of 80 mm Hg with a preload of 40 to 50 mm Hg at a rate of 54 times per minute. At initiation of pumping, systolic pressure was 135 +/- 24 mm Hg and flow was 464 +/- 116 ml/min. After 2 weeks of continuous pumping, the systolic pressure was 104 +/- 1 mm Hg and continuous flow was 206 +/- 16 ml/min. Two of the skeletal muscle ventricles pumped continuously for 5 and 9 weeks, respectively. At the end of that time one was still capable of generating pressure up to 205 mm Hg and the other, 160 mm Hg. These results suggest that a chronic auxiliary skeletal muscle ventricle is a feasible approach to the treatment of end-stage cardiac failure.

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.

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.

Chronic stimulation converts skeletal muscle of mixed fiber type to a uniform muscle made up of type I, fatigue-resistant fibers. Here, the bioenergetic correlates of fatigue resistance in conditioned canine latissimus dorsi are assessed with in vivo phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy. After chronic electrical stimulation, five dogs underwent 31P-NMR spectroscopic and isometric tension measurements on conditioned and contralateral control muscle during stimulation for 200, 300, 500, and 800 ms of an 1,100-ms duty cycle. With stimulation, phosphocreatine (PCr) fell proportional to the degree of stimulation in both conditioned and control muscle but fell significantly less in conditioned muscle at all but the least intense stimulation period (200 ms). Isometric tension, expressed as a tension time index per gram muscle, was significantly greater in the conditioned muscle at the two longest stimulation periods. The overall small change in PCr and the lack of a plateau in tension observed in the conditioned muscle are similar to that seen in cardiac muscle during increased energy demand. This study indicates that the conditioned muscle's markedly enhanced resistance to fatigue is in part the result of its increased capacity for oxidative phosphorylation.

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.

Skeletal muscle undergoes profound changes in morphological, physiological, and biochemical character when subjected to prolonged periods of increased use. Although increased use may be brought about in a variety of ways, the results show consistent features. In particular, endurance exercise and chronic stimulation differ only in degree: the properties which change in response to exercise are also those which change at an early stage of stimulation; the properties which are resistant to change under exercise conditions change only after prolonged stimulation. There is therefore a hierarchy of stability in the properties of skeletal muscle which is revealed in its response to changing functional demands. The adaptive potential of muscle provides a logical framework for understanding neural influences on the emergence of fiber types during muscle development. It is also relevant to the study of pathological conditions which may involve a sustained departure from normal postural and locomotor patterns of activity.

Serum 1,25(OH)2D concentrations were measured in serial serum samples from 19 premature infants of 29.6 +/- 1.3 weeks gestation and 1,129 +/- 159 g birthweight. 1,25(OH)2D was always normal or elevated and mean concentrations increased with age (adult, 55.2 +/- 13; infants, 1-2 weeks, 81.5 +/- 37.7 pg/mg; 3 weeks, 65 +/- 21; 6 weeks, 90.0 +/- 17.3; 9 weeks, 99.0 +/- 25.1; 12 weeks, 103.3 +/- 26.6 pg/ml). No correlation was seen with 25-OHD. Infants given 800 IU D2 supplements had lower 1,25(OH)2D levels than infants given 400 IU D2. Breast fed infants had initially higher 1,25(OH)2D levels; however, this was not sustained. These preliminary data suggest that premature infants regulate 1,25(OH)2D production similar to more mature infants and children. Whether the premature infant has a normal gastrointestinal and/or bone responsiveness to 1,25(OH)2D and whether these elevated 1,25(OH)2D concentrations are "adequately elevated" requires further study.

The criteria which serve to distinguish the slow from the fast type of mammalian skeletal muscle include differences at the ultrastructural level, in particular differences in Z disc morphology and mitochondrial content. If a fast muscle is subjected to sustained low frequency impulse activity similar to that normally received by a slow muscle, its physiological and biochemical properties become indistinguishable from those of a slow muscle. In the present study we have examined the fine structure of fast muscles stimulated in this way. The thickness of Z discs was significantly greater than that of control fast muscles and indistinguishable from that of slow muscles. Mitochondrial profiles, seen only infrequently in control fast muscles, were abundant in their stimulated counterparts. The regulatory influence of impulse activity on the differentiation of skeletal muscle thus extends to some of its most characteristic ultrastructural features.

The mRNA products of four genes, carbonic anhydrase III (CAIII), fast myosin heavy chain (MHCf), actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were assayed by Northern and slot-blot analysis in rabbit tibialis anterior and extensor digitorum longus muscles that were subjected to electrical stimulation for periods of up to 21 days. Marked changes in mRNA levels were seen for all four genes. The mRNA for CAIII, which is specific for type 1 fibers, rose significantly, whereas the MHCf mRNA fell markedly in the stimulated muscles. Changes in GAPDH mRNA were consistent with a reduced dependence on anaerobic glycolysis as an energy source. Actin mRNA levels were noticeably depressed in the early stages of stimulation. Thus for several classes of muscle protein, the response to chronic low-frequency stimulation appears to involve changes at the level of gene transcription.

Fourteen very low birthweight infants (mean +/- SD 1,070 +/- 180 g and 29.3 +/- 1.9 weeks gestation) fed their own mother's milk were clinically followed until 3-4 months of age with frequent measurements of serum calcium, phosphorus, magnesium, 25-hydroxyvitamin D (25-OHD), parathyroid hormone, alkaline phosphatase, and albumin, and urine calcium, phosphorus, and magnesium. These infants were matched for birthweight and gestation with 14 infants (1,075 +/- 152 g and 29.0 +/- 1.7 weeks) who had been similarly followed during concomitant studies of infants fed standard formula (Similac 20 cal/oz). Urine phosphorus was markedly lower in the breast milk-fed group from initiation of feedings, and serum phosphorus became significantly lower at and after 6 weeks of age. The fall in serum phosphorus was accompanied by a marked calciuria. Parathyroid hormone was suppressed in the breast milk-fed group, although serum calcium was not elevated and did not differ from formula-fed infants. A high incidence of moderate-severe hypomineralization on radiographs was seen in both breast milk- and formula-fed groups. Six of 14 breast-fed infants required phosphorus supplementation at 8-10 weeks of age because of significant hypophosphatemia, hypercalciuria, and hypomineralization. These infants differed from those not requiring phosphorus supplements by being smaller at birth but not of lower gestation, and having persistently low serum 25-OHD at and after 6 weeks of age.