Terje Lømo

Three myosin heavy chain isoforms in type 2 skeletal muscle fibres

SummaryMammalian skeletal muscles consist of three main fibre types, type 1, 2A and 2B fibres, with different myosin heavy chain (MHC) composition. We have now identified another fibre type, called type 2X fibre, characterized by a specific MHC isoform. Type 2X fibres, which are widely distributed in rat skeletal muscles, can be distinguished from 2A and 2B fibres by histochemical ATPase activity and by their unique staining pattern with seven anti-MHC monoclonal antibodies. The existence of the 2X-MHC isoform was confirmed by immunoblotting analysis using muscles containing 2X fibres as a major component, such as the normal and hyperthyroid diaphragm, and the soleus muscle after high frequency chronic stimulation. 2X-MHC contains one determinant common to 2B-MHC and another common to all type 2-MHCs, but lacks epitopes specific for 2A- and 2B-MHCs, as well as an epitope present on all other MHCs. By SDS-polyacrylamide gel electrophoresis 2X-MHC shows a lower mobility compared to 2B-MHC and appears to comigrate with 2A-MHC. Muscles containing predominantly 2X-MHC display a velocity of shortening intermediate between that of slow muscles and that of fast muscles composed predominantly of 2B fibres.

Calcineurin controls nerve activity-dependent specification of slow skeletal muscle fibers but not muscle growth

Nerve activity can induce long-lasting, transcription-dependent changes in skeletal muscle fibers and thus affect muscle growth and fiber-type specificity. Calcineurin signaling has been implicated in the transcriptional regulation of slow muscle fiber genes in culture, but the functional role of calcineurin in vivo has not been unambiguously demonstrated. Here, we report that the up-regulation of slow myosin heavy chain (MyHC) and a MyHC-slow promoter induced by slow motor neurons in regenerating rat soleus muscle is prevented by the calcineurin inhibitors cyclosporin A (CsA), FK506, and the calcineurin inhibitory protein domain from cain/cabin-1. In contrast, calcineurin inhibitors do not block the increase in fiber size induced by nerve activity in regenerating muscle. The activation of MyHC-slow induced by direct electrostimulation of denervated regenerating muscle with a continuous low frequency impulse pattern is blocked by CsA, showing that calcineurin function in muscle fibers and not in motor neurons is responsible for nerve-dependent specification of slow muscle fibers. Calcineurin is also involved in the maintenance of the slow muscle fiber gene program because in the adult soleus muscle, cain causes a switch from MyHC-slow to fast-type MyHC-2X and MyHC-2B gene expression, and the activity of the MyHC-slow promoter is inhibited by CsA and FK506.

Ras is involved in nerve-activity-dependent regulation of muscle genes

Gene expression in skeletal muscle is regulated by the firing pattern of motor neurons, but the signalling systems involved in excitation–transcription coupling are unknown. Here, using in vivo transfection in regenerating muscle, we show that constitutively active Ras and a Ras mutant that selectively activates the MAPK(ERK) pathway are able to mimic the effects of slow motor neurons on expression of myosin genes. Conversely, the effect of slow motor neurons is inhibited by a dominant-negative Ras mutant. MAPK(ERK) activity is increased by innervation and by low-frequency electrical stimulation. These results indicate that Ras–MAPK signalling is involved in promoting nerve-activity-dependent differentiation of slow muscle fibres in vivo.

Embryonic and neonatal myosin heavy chain in denervated and paralyzed rat skeletal muscle

Using immunofluorescence procedures with specific polyclonal and monoclonal antimyosin antibodies we have found that embryonic and neonatal myosin heavy chains (MHCs), which in rat skeletal muscle disappear during the first weeks after birth, are reexpressed in adult muscle after denervation. Reactivity for embryonic and neonatal MHCs was detected in some fibers as early as 3 days after denervation, became more evident by 7 days, and occurred exclusively in the type 2A fiber population. Paralysis of innervated muscles by tetrodotoxin block of the sciatic nerve also resulted in the reappearance of embryonic and neonatal MHCs in type 2A fibers. Significant variation in the degree of immunoreactivity was observed in different segments of the same muscle fiber, suggesting that coordination of muscle fiber nuclei in the control of myosin heavy chain gene expression is partially lost following denervation.

Slow-to-fast transformation of denervated soleus muscles by chronic high-frequency stimulation in the rat.

1. Adult soleus muscles were denervated and stimulated directly for 2‐130 days with ‘fast’ (short pulse trains at 100 Hz) or slow’ (continuously at 10 Hz, or long pulse trains at 15 Hz) stimulus patterns. 2. At the end of the period of stimulation isometric twitches and tetani and isotonic shortening velocities were measured. Frozen cross‐sections were later examined with antibodies against myosin heavy chains specific for adult fast, adult slow and fetal myosin. 3. Isometric twitch duration (twitch time‐to‐peak and half‐relaxation time) decreased during intermittent 100 Hz stimulation to values that were almost as fast as in the normal extensor digitorum longus (EDL) (95 and 94% transformation). The major part of the decrease occurred between 2 and 21 days after the onset of stimulation, and was accompanied by post‐tetanic potentiation of the twitch, sag’ in tension during an unfused tetanus, lower twitch/tetanus ratio and marked shifts to the right (higher frequencies) of the tension‐frequency curve of the muscle. In contrast, during 10 or 15 Hz stimulation the isometric twitch duration remained slow, the twitch continued to show post‐tetanic depression and absence of ‘sag’, while the twitch/tetanus ratio increased. 4. Denervation per se led to a slight increase and, then, after about a month, to a moderate and gradual decrease in twitch duration. The twitch/tetanus ratio increased markedly and post‐tetanic depression became less pronounced or disappeared. Muscle weight and particularly tetanic tension were markedly reduced and these reductions were to a large extent counteracted by electrical stimulation. 5. Implantation of sham electrodes had no effect on twitch duration of denervated or innervated control muscles, but reduced tetanic tension in the innervated control muscles. 6. Maximum isotonic shortening velocity of the whole muscle (mm/s) increased during intermittent 100 Hz stimulation to a value as fast as in the normal EDL (110% transformation). Since the muscle fibres also increased in length (35%) maximum intrinsic shortening velocity (fibre lengths/s) was only incompletely transformed (55%). The increase in Vmax occurred between 7 and 14 days after the onset of stimulation. 7. All the fibres stimulated intermittently at 100 Hz were strongly labelled with anti‐fast myosin and more than 90% were in addition weakly labelled by anti‐slow myosin. Weak and variable labelling with anti‐fast myosin was first detected 7 days after the onset of stimulation. In contrast, essentially all the fibres stimulated at 10 or 15 Hz showed no binding of anti‐fast but strong binding of anti‐slow myosin.(ABSTRACT TRUNCATED AT 400 WORDS)

Hyperinnervation of Skeletal Muscle Fibers: Dependence on Muscle Activity

After the motor nerve to the rat soleus muscle was blocked reversibly by local anesthesia, individual muscle fibers became innervated by a transplanted motor nerve without losing their original innervation. Such cross-innervation of the denervated soleus muscle by the same foreign nerve was largely reduced by direct electrical stimulation of the muscle. The results demonstrate the importance of muscle activity for synapse formation by a foreign motor nerve.

Control of Hippocampal Output by Afferent Volley Frequency

Publisher Summary This chapter discusses the control of hippocampal output by afferent volley frequency. One of the crucial factors determining the ouput from a neuronal system is the frequency of the input stimulation. The aim of the present investigation has been to study the effect of a relatively shortlasting tetanic stimulation on the efficiency of certain excitatory synapses in the hippocampus. Further, possible correlations between neurophysiological responses and processes of learning have been sought This affects both the electrical and, most likely, the behavioural responses to stimulation of various brain structures. All investigators studying the responses of hippocampal pyramidal neurones are familiar with the great increase, or decrease, down to the level of extinction, of the responses that may occur in different circumstances. In cats and rabbits, anaesthetized with nembutal or urethane and chloralose, the effect of different frequencies of stimulation on synaptic transmission in the hippocampus was studied. Repetitive stimulation led to the development of large negative waves with population spikes superimposed. The evoked potentials could increase by a factor of several hundred.

‘Fast’ and ‘slow’ muscle fibres in hindlimb muscles of adult rats regenerate from intrinsically different satellite cells

Myosin heavy chain (MyHC) expression was examined in regenerating fast extensor digitorum longus (EDL) and slow soleus (SOL) muscles of adult rats. Myotoxic bupivacaine was injected into SOL and EDL and the muscles were either denervated or neuromuscularly blocked by tetrodotoxin (TTX) on the sciatic nerve. Three to 10 or 30 days later, denervated SOL or EDL, or innervated but neuromuscularly blocked EDL received a slow 20 Hz stimulus pattern through electrodes implanted on the muscles or along the fibular nerve to EDL below the TTX block. In addition, denervated SOL and EDL received a fast 100 Hz stimulus pattern. Denervated EDL and SOL stimulated with the same slow stimulus pattern expressed different amounts of type 1 MyHC protein (8%versus 35% at 10 days, 13%versus 87% at 30 days). Stimulated denervated and stimulated innervated (TTX blocked) EDL expressed the same amounts of type 1, 2A, 2X and 2B MyHC proteins. Cross‐sections treated for in situ hybridization and immunocytochemistry showed expression of type 1 MyHC in all SOL fibres but only in some scattered single or smaller groups of fibres in EDL. The results suggest that muscle fibres regenerate from intrinsically different satellite cells in EDL and SOL and within EDL. However, induction by different extrinsic factors arising in extracellular matrix or from muscle position and usage in the limb has not been excluded. No evidence for nerve‐derived trophic influences was obtained.

Regulation of turnover and number of acetylcholine receptors at neuromuscular junctions

The number and metabolic stability of acetylcholine receptors (AChRs) at neuromuscular junctions of rat tibialis anterior (TA) and soleus (SOL) muscles were examined after denervation, paralysis by continuous application of tetrodotoxin to the nerve, or denervation and direct stimulation of the muscle through implanted electrodes. After 18 days of denervation AChR half-life declined from about 10 days to 2.3 days (TA) or 3.6 days (SOL) and after 18 days of nerve conduction block to 3.1 days (TA). In contrast, the total number of AChRs per endplate was unaffected by these treatments. Denervation for 33 days had no further effect on AChR half-life but reduced the total number of AChRs to about 54% (SOL) or 38% (TA) of normal. Direct stimulation of the 33-day denervated SOL from day 18 restored normal AChR stability and counteracted muscle atrophy but had no effect on the decline in AChR number. The results indicate that motoneurons control the stability of junctional AChRs through evoked muscle activity and the number of junctional AChRs through trophic factors.

γ-AChR/ϵ-AChR Switch at Agrin-Induced Postsynaptic-like Apparatus in Skeletal Muscle

Abstract We transfected the extrajunctional region of denervated soleus muscles in adult rats with neural agrin cDNA to induce myofibers to form postsynaptic-like apparatus containing acetylcholine receptor (AChR) aggregates. By 1 week ≈30% of the AChR aggregates contained a mixture of ϵ-AChRs and γ-AChRs while ≈70% had only γ-AChRs. If the transfected muscles were reinnervated in the original junctional region, the postsynaptic-like apparatus, despite the absence of apposed axon terminals, gradually came to have only ϵ-AChRs. We conclude that at the postsynaptic apparatus of ectopic neuromuscular junctions formed by a foreign nerve implanted into the extrajunctional region of denervated muscles, agrin secreted by the axon terminal plays a direct role in the γ-AChR/ϵ-AChR switch that occurs as the apparatus reaches maturity. Our findings, together with results from other studies, indicate further that agrin and acetylcholine are the only nerve-derived factors required for this switch.