Specific labelling of myonuclei by an antibody against pericentriolar material 1 on skeletal muscle tissue sections.

AIM: Skeletal muscle is a heterogeneous tissue containing several different cell types, and only about 40%-50% of the cell nuclei within the tissue belong to myofibres. Existing technology, attempting to distinguish myonuclei from other nuclei at the light microscopy level, has led to controversies in our understanding of the basic cell biology of muscle plasticity. This study aims at demonstrating that an antibody against the protein pericentriolar material 1 (PCM1) can be used to reliably identify myonuclei on histological cross sections from humans, mice and rats. METHODS: Cryosections were labelled with a polyclonal antibody against PCM1. The specificity of the labelling for myonuclei was verified using 3D reconstructions of confocal z-stacks triple-labelled for DNA, dystrophin and PCM1, and by co-localization with nuclear mCherry driven by the muscle-specific Alpha-Actin-1 promoter after viral transduction. RESULTS: The PCM1 antibody specifically labelled all myonuclei, and myonuclei only, in cryosections of muscles from rats, mice and men. Nuclei in other cell types including satellite cells were not labelled. Both normal muscles and hypertrophic muscles after synergist ablation were investigated. CONCLUSION: Pericentriolar material 1 can be used as a specific histological marker for myonuclei in skeletal muscle tissue without relying on counterstaining of other structures or cumbersome and subjective analysis of nuclear positioning.

No change in myonuclear number during muscle unloading and reloading.

Muscle fibers are the cells in the body with the largest volume, and they have multiple nuclei serving different domains of cytoplasm. A large body of previous literature has suggested that atrophy induced by hindlimb suspension leads to a loss of "excessive" myonuclei by apoptosis. We demonstrate here that atrophy induced by hindlimb suspension does not lead to loss of myonuclei despite a strong increase in apoptotic activity of other types of nuclei within the muscle tissue. Thus hindlimb suspension turns out to be similar to other atrophy models such as denervation, nerve impulse block, and antagonist ablation. We discuss how the different outcome of various studies can be attributed to difficulties in separating myonuclei from other nuclei, and to systematic differences in passive properties between normal and unloaded muscles. During reload, after hindlimb suspension, a radial regrowth is observed, which has been believed to be accompanied by recruitment of new myonuclei from satellite cells. The lack of nuclear loss during unloading, however, puts these findings into question. We observed that reload led to an increase in cross sectional area of 59%, and fiber size was completely restored to the presuspension levels. Despite this notable growth there was no increase in the number of myonuclei. Thus radial regrowth seems to differ from de novo hypertrophy in that nuclei are only added during the latter. We speculate that the number of myonuclei might reflect the largest size the muscle fibers have had in its previous history.

Myonuclei acquired by overload exercise precede hypertrophy and are not lost on detraining.

Effects of previous strength training can be long-lived, even after prolonged subsequent inactivity, and retraining is facilitated by a previous training episode. Traditionally, such "muscle memory" has been attributed to neural factors in the absence of any identified local memory mechanism in the muscle tissue. We have used in vivo imaging techniques to study live myonuclei belonging to distinct muscle fibers and observe that new myonuclei are added before any major increase in size during overload. The old and newly acquired nuclei are retained during severe atrophy caused by subsequent denervation lasting for a considerable period of the animal's lifespan. The myonuclei seem to be protected from the high apoptotic activity found in inactive muscle tissue. A hypertrophy episode leading to a lasting elevated number of myonuclei retarded disuse atrophy, and the nuclei could serve as a cell biological substrate for such memory. Because the ability to create myonuclei is impaired in the elderly, individuals may benefit from strength training at an early age, and because anabolic steroids facilitate more myonuclei, nuclear permanency may also have implications for exclusion periods after a doping offense.

Distribution of myonuclei and microtubules in live muscle fibers of young, middle-aged, and old mice.

We have recently published a new technique for visualizing nuclei in living muscle fibers of intact animals, based on microinjection of labeled DNA into single myofibers, excluding satellite cells (Bruusgaard JC, Liestol K, Ekmark M, Kollstad K, and Gundersen K. J Physiol 551: 467-478, 2003). In the present study, we use this technique to study fiber segments of soleus and extensor digitorum longus (EDL) muscles from mice aged 2, 14, and 23 mo. As the animals maturing from 2 to 14 mo, they displayed an increase in size and number of nuclei. Soleus showed little change in nuclear domain size, whereas this increased by 88% in the EDL. For 14-mo-old animals, no significant correlation between fiber size and nuclear number was observed (R2=0.18, P=0.51) despite a fourfold variation in cytoplasmic volume. This suggests that size and nuclear number is uncoupled in middle-aged mice. When animals aged from 14 to 23 mo, EDL IIb, but not soleus, fibers atrophied by 41%. Both EDL and soleus displayed a reduction in number of nuclei: 20 and 16%, respectively. A positive correlation between number of nuclei and size was observed at 2 mo, and this reappeared in old mice. The atrophy in IIb fibers at old age was accompanied by a disturbance in the orderly positioning of nuclei that is so prominent in glycolytic fibers at younger age. In old animals, changes in nuclear shape and in the peri- and internuclear microtubule network were also observed. Thus changes in myonuclear number and distribution, perhaps related to alterations in the microtubular network, may underlie some of the adverse consequences of aging on skeletal muscle size and function.

Muscle hypertrophy induced by the Ski protein: cyto-architecture and ultrastructure.

AIM: Transgenic mice overexpressing the c-ski proto-oncogene driven by the MSV promoter undergo muscle hypertrophy, most notably fast fibres of the lower limb. This hypertrophy is not accompanied by a correspondingly large increase in force, and individual skinned muscle fibres exhibit a 30% reduction in force per cross-sectional area. In this respect, the MSV ski model is different from most other hypertrophy models and we here aim at describing the mechanisms for the reduced specific force. METHODS: Cyoarchitecture and ultrastructure of muscle fibres from the fast extensor digitorum longus muscle of 2-3 months old MSV ski mice was studied. In addition to electron microscopy, we used in vivo intracellular injections of myonuclear dye to investigate nuclear number. RESULTS: The number of nuclei did not increase in proportion to size, and consequently nuclear domains were increased compared with wild type. The fraction of the cytoplasm occupied by contractile material was reduced by 18%. In addition we observed poor intracellular alignment of Z-discs. Such staggering has been reported to reduce force in desmin deficient mice, but the amount and distribution of desmin in the MSV ski mice seemed normal. The mitochondria of MSV ski mice showed irregularly spaced cristae that were frequently disrupted. CONCLUSION: The reduction in specific force observed in MSV ski mice could be explained by a reduced fraction of contractile material and reduced transversal mechanical coupling. The ultrastructural abnormalities could be related to an increase in nuclear domains.

Number and spatial distribution of nuclei in the muscle fibres of normal mice studied in vivo.

We present here a new technique with which to visualize nuclei in living muscle fibres in the intact animal, involving injection of labelled DNA into single cells. This approach allowed us to determine the position of all of nuclei within a sarcolemma without labelling satellite cells. In contrast to what has been reported in tissue culture, we found that the nuclei were immobile, even when observed over several days. Nucleic density was uniform along the fibre except for the endplate and some myotendinous junctions, where the density was higher. The perijunctional region had the same number of nuclei as the rest of the fibre. In the extensor digitorum longus (EDL) muscle, the extrajunctional nuclei were elongated and precisely aligned to the long axis of the fibre. In the soleus, the nuclei were rounder and not well aligned. When comparing small and large fibres in the soleus, the number of nuclei varied approximately in proportion to cytoplasmic volume, while in the EDL the number was proportional to surface area. Statistical analysis revealed that the nuclei were not randomly distributed in either the EDL or the soleus. For each fibre, actual distributions were compared with computer simulations in which nuclei were assumed to repel each other, which optimizes the distribution of nuclei with respect to minimizing transport distances. The simulated patterns were regular, with clear row-like structures when the density of nuclei was low. The non-random and often row-like distribution of nuclei observed in muscle fibres may thus reflect regulatory mechanisms whereby nuclei repel each other in order to minimize transport distances.