Hazel Sutherland

Fast-to-slow transformation in stimulated rat muscle.

Several previous studies have failed to demonstrate changes due to chronic stimulation in contractile speed of innervated fast rat muscles, and it has been suggested that the adaptive capacity of skeletal muscle in this species is limited. We have reassessed this contention. Fast muscles of the rat hind limb were stimulated continuously at 10 or 20 Hz for 55–61 days. The maximum shortening velocity of the extensor digitorum longus muscles was reduced to 50% of the control value. The proportion of type 1 fibers increased from 4% in control muscle to 34% in stimulated muscles and there was a corresponding reduction in type 2B/D fibers. The proportion of type 2A fibers after stimulation was similar to that in control muscles. These results, taken together with our published analyses of myosin isoform composition of these muscles, show that the mechanisms that control gene expression in response to activity are not exclusive to larger mammals.

The dose-related response of rabbit fast muscle to long-term low-frequency stimulation

Rabbit tibialis anterior muscles were stimulated continuously at 2.5 Hz, 5 Hz, or 10 Hz for 10 months. The resulting adaptive transformation was dose‐related for contractile speed, myosin isoform composition, and enzyme activities. The “fast‐oxidative” state produced by stimulation at 2.5 Hz was stable: even after 10 months, 84% of the fibers were of type 2A. Absence of a secondary decline in oxidative activity in these muscles provided strong evidence of a causal link between myosin transitions and metabolic adaptation. Significant fiber loss occurred only after prolonged stimulation at 10 Hz. The myosin isoform composition of individual muscles stimulated at 5 Hz resembled that of muscles stimulated at either the lower or the higher frequency, behavior consistent with a threshold for fiber type change. In clinical applications such as cardiomyoplasty, muscles could be used more effectively by engineering their properties to combine speed and power of contraction with the necessary resistance to fatigue.

Ochronotic osteoarthropathy in a mouse model of alkaptonuria, and its inhibition by nitisinone

Background Alkaptonuria (AKU) is a rare metabolic disease caused by deficiency of homogentisate 1,2 dioxygenase, an enzyme involved in tyrosine catabolism, resulting in increased circulating homogentisic acid (HGA). Over time HGA is progressively deposited as a polymer (termed ochronotic pigment) in collagenous tissues, especially the cartilages of weight bearing joints, leading to severe joint disease. Objectives To characterise blood biochemistry and arthropathy in the AKU mouse model (Hgd−/−). To examine the therapeutic effect of long-term treatment with nitisinone, a potent inhibitor of the enzyme that produces HGA. Methods Lifetime levels of plasma HGA from AKU mice were measured by high-performance liquid chromatography (HPLC). Histological sections of the knee joint were examined for pigmentation. The effect of nitisinone treatment in both tissues was examined. Results Mean (±SE) plasma HGA levels were 3- to 4-fold higher (0.148±0.019 mM) than those recorded in human AKU. Chondrocyte pigmentation within the articular cartilage was first observed at 15 weeks, and found to increase steadily with mouse age. Nitisinone treatment reduced plasma HGA in AKU mice throughout their lifetime, and completely prevented pigment deposition. Conclusions The AKU mouse was established as a model of both the plasma biochemistry of AKU and its associated arthropathy. Early-stage treatment of AKU patients with nitisinone could prevent the development of associated joint arthropathies. The cellular pathology of ochronosis in AKU mice is identical to that observed in early human ochronosis and thus is a model in which the early stages of joint pathology can be studied and novel interventions evaluated.

Ochronosis in a murine model of alkaptonuria is synonymous to that in the human condition

OBJECTIVE Alkaptonuria (AKU) is a rare genetic disease which results in severe early onset osteoarthropathy. It has recently been shown that the subchondral interface is of key significance in disease pathogenesis. Human surgical tissues are often beyond this initial stage and there is no published murine model of pathogenesis, to study the natural history of the disease. The murine genotype exists but it has been reported not to demonstrate ochronotic osteoarthropathy consistent with the human disease. Recent anecdotal evidence of macroscopic renal ochronosis in a mouse model of tyrosinaemia led us to perform histological analysis of tissues of these mice that are known to be affected in human AKU. DESIGN The homogentisate 1,2-dioxygenase Hgd(+/)(-)Fah(-)(/)(-) mouse can model either hereditary tyrosinaemia type I (HT1) or AKU depending on selection conditions. Mice having undergone Hgd reversion were sacrificed at various time points, and their tissues taken for histological analysis. Sections were stained with haematoxylin eosin (H&E) and Schmorls reagent. RESULTS Early time point observations at 8 months showed no sign of macroscopic ochronosis of tissues. Macroscopic examination at 13 months revealed ochronosis of the kidneys. Microscopic analysis of the kidneys revealed large pigmented nodules displaying distinct ochre colouration. Close microscopic examination of the distal femur and proximal fibula at the subchondral junctions revealed the presence of numerous pigmented chondrocytes. CONCLUSIONS Here we present the first data showing ochronosis of tissues in a murine model of AKU. These preliminary histological observations provide a stimulus for further studies into the natural history of the disease to provide a greater understanding of this class of arthropathy.

Therapeutic stimulation of denervated muscles: The influence of pattern

Muscular atrophy due to denervation can be substantially reversed by direct electrical stimulation. Some muscle properties are, however, resistant to change. Using a rabbit model of established denervation atrophy, we investigated whether the extent of restoration would vary with the stimulation protocol. Five patterns, delivering 24,000–480,000 impulses/day, were applied for 6 or 10 weeks. The wet weight, cross‐sectional area, tetanic tension, shortening velocity, and power of denervated muscles subjected to stimulation all increased significantly. The fibers were larger and more closely packed and there was no evidence of necrosis. There was a small increase in excitability. Isometric twitch kinetics remained slow and fatigue resistance did not improve. The actual pattern of stimulation had no influence on any of these findings. The results, interpreted in the context of ultrastructural changes and an ongoing clinical study, reaffirm the clinical value of introducing stimulation during the initial non‐degenerative phase. They indicate that there would be little therapeutic benefit in adopting regimes more energetically demanding than those in current use, and that the focus should now shift to protocols that represent the least intrusion into activities of daily living.

Implantable device for long-term electrical stimulation of denervated muscles in rabbits.

Although denervating injuries produce severe atrophic changes in mammalian skeletal muscle, a degree of functional restoration can be achieved through an intensive regime of electrical stimulation. An implantable stimulator was developed so that the long-term effects of different stimulation protocols could be compared in rabbits. The device, which is powered by two lithium thionyl chloride batteries, is small enough to be implanted in the peritoneal cavity. All stimulation parameters can be specified over a wide range, with a high degree of resolution; in addition, up to 16 periods of training (10–180 min) and rest (1–42 h) can be set in advance. The microcontroller-based device is programmed through a bidirectional radiofrequency link. Settings are entered via a user-friendly computer interface and annotated to create an individual study protocol for each animal. The stimulator has been reliable and stable in use. Proven technology and rigorous quality control has enabled 55 units to be implanted to date, for periods of up to 36 weeks, with only two device failures (at 15 and 29 weeks). Changes in the excitability of denervated skeletal muscles could be followed within individual animals. Chronaxie increased from 3.24±0.54 ms to 15.57±0.85 ms (n=55, p<0.0001) per phase in the 2 weeks following denervation.

Pattern Dependence in the Stimulation‐Induced Type Transformation of Rabbit Fast Skeletal Muscle

Little is known of the events that initiate the adaptive response of skeletal muscle to a sustained change in use. This study was designed to distinguish between the role of the electrical activity pattern and that of the resulting contractile force in driving different aspects of the response. A better understanding of these issues would lead to improved clinical protocols for functional electrical stimulation. Rabbit limb muscles were stimulated continuously for 12 weeks either at 2.5 Hz or with an equivalent optimized pattern producing peak forces three‐fold higher. The two patterns induced similar changes in shortening velocity, myosin isoforms, and fatigue resistance. They had markedly different effects on twitch dynamics and summation (“doublet effect”). This pointed to differences in activation that were not, however, attributable to sarcoplasmic reticulum Ca2+ transport ATPase activity. The optimized pattern maintained muscle bulk more effectively. We conclude that changes in myosin isoform composition and fatigue resistance are driven by aggregate impulse activity. Changes in Ca2+ transport and muscle bulk show a distinct pattern dependence.

Adaptive conditioning of skeletal muscle in a large animal model (Sus domesticus)

Recognition of the adaptive capacity of mammalian skeletal muscle has opened the way to a number of clinical applications. For most of these, the fast, fatigue‐susceptible fibres need to be transformed stably to fast, fatigue‐resistant fibres that express the 2A myosin heavy chain isoform. The thresholds for activity‐induced change are size‐dependent, so although the requisite patterns of electrical stimulation are known for the rabbit, in humans these same patterns would produce type 1 fibre characteristics, with an undesirable loss of contractile speed and power. We have used histochemistry, immunohistochemistry and electrophoretic separations to evaluate a possible conditioning regime in a large animal model. Stimulation of the porcine latissimus dorsi muscle with a phasic 30‐Hz pattern for up to 41 days converted all type 2X and 2A/2X fibres to 2A with only a small increase in the type 1 population, from 17% to 22%. Stimulation for longer periods increased the proportion of type 1 fibres to 52%. Based on this model, stimulation regimes designed to achieve a stable 2A phenotype in humans should deliver fewer stimulating impulses, possibly by a factor of 2, than the pattern assessed here. Any such pattern needs to be tested for at least 8 weeks.

Dynamics of stimulation-induced muscle adaptation: insights from varying the duty cycle.

We sought to gain insight into the dynamics of the signalling process that initiates adaptive change in mammalian skeletal muscles in response to chronic neuromuscular stimulation. Programmable miniature stimulators were implanted into rabbits and used to impose one of the following patterns on the dorsiflexors of one ankle: 10 Hz delivered in equal on/off periods of 30 s, 30 min, or 12 h (all equivalent in terms of aggregate impulse activity to continuous 5 Hz). Two further groups received continuous stimulation at 5 Hz or 10 Hz. In every case the stimulation pattern was maintained continuously for 6 weeks. Tibialis anterior muscles stimulated intermittently with equal on/off periods of 30 s, 30 min and 12 h had contractile characteristics that were significantly slower than the contralateral, unstimulated muscles but did not differ from those of muscles stimulated continuously at 5 Hz. Muscles stimulated continuously at 10 Hz were significantly slower than either contralateral muscles or muscles stimulated with any of the other patterns. Corresponding changes were seen in myosin heavy chain isoform composition. The fatigue index, defined as the fraction of tension remaining after 5 min of a standard fatigue test, was 0.4 for muscles in the contralateral group but equal to or greater than 0.85 for muscles of all the stimulated groups. These results were interpreted with the help of a simple model of the growth and decay of a putative signalling substance based on first order kinetics. The model suggests a rate constant for the accumulation of the signalling substance that is greater than 30 h−1, and a rate constant for its removal that is greater than 50 h−1.

Morphological and histological adaptation of muscle and bone to loading induced by repetitive activation of muscle.

Muscular contraction plays a pivotal role in the mechanical environment of bone, but controlled muscular contractions are rarely used to study the response of bone to mechanical stimuli. Here, we use implantable stimulators to elicit programmed contractions of the rat tibialis anterior (TA) muscle. Miniature stimulators were implanted in Wistar rats (n = 9) to induce contraction of the left TA every 30 s for 28 days. The right limb was used as a contralateral control. Hindlimbs were imaged using microCT. Image data were used for bone measurements, and to construct a finite-element (FE) model simulation of TA forces propagating through the bone. This simulation was used to target subsequent bone histology and measurement of micromechanical properties to areas of high strain. FE mapping of simulated strains revealed peak values in the anterodistal region of the tibia (640 µε ± 30.4 µε). This region showed significant increases in cross-sectional area (28.61%, p < 0.05) and bone volume (30.29%, p < 0.05) in the stimulated limb. Histology revealed a large region of new bone, containing clusters of chondrocytes, indicative of endochondral ossification. The new bone region had a lower elastic modulus (8.8 ± 2.2 GPa) when compared with established bone (20 ± 1.4 GPa). Our study provides compelling new evidence of the interplay between muscle and bone.