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Previous strength training with or without the use of anabolic steroids facilitates subsequent re-acquisition of muscle mass even after long intervening periods of inactivity. Based on in vivo and ex vivo microscopy we here propose a cellular memory mechanism residing in the muscle cells. Female mice were treated with testosterone propionate for 14 days, inducing a 66% increase in the number of myonuclei and a 77% increase in fibre cross-sectional area. Three weeks after removing the drug, fibre size was decreased to the same level as in sham treated animals, but the number of nuclei remained elevated for at least 3 months (>10% of the mouse lifespan). At this time, when the myonuclei-rich muscles were exposed to overload-exercise for 6 days, the fibre cross-sectional area increased by 31% while control muscles did not grow significantly. We suggest that the lasting, elevated number of myonuclei constitutes a cellular memory facilitating subsequent muscle overload hypertrophy. Our findings might have consequences for the exclusion time of doping offenders. Since the ability to generate new myonuclei is impaired in the elderly our data also invites speculation that it might be beneficial to perform strength training when young in order to benefit in senescence.

Non-technical summary Muscle fibres change when they are used differently, such as by exercise. Genetic studies have shown that a hyperactive form of the gene regulatory protein hypoxia inducible factor 1α (HIF-1α) occurs twice as often among strength-trained athletes as in the normal population. HIF-1α is ‘sensing’ the oxygen levels in cells, and the oxygen levels change by an order of magnitude in working muscle. We show that an ‘endurance’ type of activity reduces the level of HIF-1α, while short intense bursts of activity increases it. When HIF-1α was produced in higher quantities by introducing artificial genes in the muscle fibres they became larger and faster, and with a less oxidative metabolism. Thus, oxygen itself could be a trigger for changes in muscle. The composition of muscle fibres is strongly correlated to major lifestyle conditions such as diabetes and chronic obstructive pulmonary disease, and HIF-1α might provide a new molecular link. Abstract Exercise influences muscle phenotype by the specific pattern of action potentials delivered to the muscle, triggering intracellular signalling pathways. can be reduced by an order of magnitude in working muscle. In humans, carriers of a hyperactive polymorphism of the transcription factor hypoxia inducible factor 1α (HIF-1α) have 50% more fast fibres, and this polymorphism is prevalent among strength athletes. We have investigated the putative role of HIF-1α in mediating activity changes in muscle. When rat muscles were stimulated with short high frequency bursts of action potentials known to induce a fast muscle phenotype, HIF-1α increased by about 80%. In contrast, a pattern consisting of long low frequency trains known to make fast muscles slow reduced the HIF-1α level of the fast extensor digitorum longus (EDL) muscle by 44%. Nuclear protein extracts from normal EDL contained 2.3-fold more HIF-1α and 4-fold more HIF-1β than the slow soleus muscle, while von-Hippel-Lindau protein was 4.8-fold higher in slow muscles. mRNA displayed a reciprocal pattern; thus FIH-1 mRNA was almost 2-fold higher in fast muscle, while the HIF-1α level was half, and consequently protein/mRNA ratio for HIF-1α was more than 4-fold higher in the fast muscle, suggesting that HIF-1α is strongly suppressed post-transcriptionally in slow muscles. When HIF-1α was overexpressed for 14 days after somatic gene transfer in adult rats, a slow-to-fast transformation was observed, encompassing an increase in fibre cross sectional area, oxidative enzyme activity and myosin heavy chain. The latter was shown to be regulated at the mRNA level in C2C12 myotubes.

According to the current paradigm, muscle nuclei serve a certain cytoplasmic domain. To preserve the domain size, it is believed that nuclei are injected from satellite cells fusing to fibres undergoing hypertrophy, and lost by apoptosis during atrophy. Based on single fibre observations in and ex vivo we suggest that nuclear domains are not as constant as is often indicated. Moreover, recent time lapse in vivo imaging of single fibres suggests that at least for the first few weeks, atrophy is not accompanied by any loss of nuclei. Apoptosis is abundant in muscle tissue during atrophy conditions, but in our opinion it has not been unequivocally demonstrated that such nuclei are myonuclei. As we see it, the preponderance of current evidence suggests that disuse atrophy is not accompanied by loss of nuclei, at least not for the first 2 months. Moreover, it has not been proven that myonuclear apoptosis does occur in permanent fibres undergoing atrophy; it seems more likely that it is confined to stromal cells and satellite cells. If muscle atrophy is not related to loss of nuclei, design of intervention therapies should focus on protein metabolism rather than regeneration from stem cells.

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.

Previous strength training with or without the use of anabolic steroids facilitates subsequent re-acquisition of muscle mass even after long intervening periods of inactivity. Based on in vivo and ex vivo microscopy we here propose a cellular memory mechanism residing in the muscle cells. Female mice were treated with testosterone propionate for 14 days, inducing a 66% increase in the number of myonuclei and a 77% increase in fibre cross-sectional area. Three weeks after removing the drug, fibre size was decreased to the same level as in sham treated animals, but the number of nuclei remained elevated for at least 3 months (10% of the mouse lifespan). At this time, when the myonuclei-rich muscles were exposed to overload-exercise for 6 days, the fibre cross-sectional area increased by 31% while control muscles did not grow significantly. We suggest that the lasting, elevated number of myonuclei constitutes a cellular memory facilitating subsequent muscle overload hypertrophy. Our findings might have consequences for the exclusion time of doping offenders. Since the ability to generate new myonuclei is impaired in the elderly our data also invites speculation that it might be beneficial to perform strength training when young in order to benefit in senescence.

Exercise influences muscle phenotype by the specific pattern of action potentials delivered to the muscle, triggering intracellular signalling pathways. PO2 can be reduced by an order of magnitude in working muscle. In humans, carriers of a hyperactive polymorphism of the transcription factor hypoxia inducible factor 1α (HIF-1α) have 50% more fast fibres, and this polymorphism is prevalent among strength athletes. We have investigated the putative role of HIF-1α in mediating activity changes in muscle.When rat muscles were stimulated with short high frequency bursts of action potentials known to induce a fast muscle phenotype, HIF-1α increased by about 80%. In contrast, a pattern consisting of long low frequency trains known to make fast muscles slow reduced the HIF-1α level of the fast extensor digitorum longus (EDL) muscle by 44%. Nuclear protein extracts from normal EDL contained 2.3-fold more HIF-1α and 4-fold more HIF-1β than the slow soleus muscle, while von-Hippel-Lindau protein was 4.8-fold higher in slow muscles. mRNA displayed a reciprocal pattern; thus FIH-1 mRNA was almost 2-fold higher in fast muscle, while the HIF-1α level was half, and consequently protein/mRNA ratio for HIF-1α was more than 4-fold higher in the fast muscle, suggesting that HIF-1α is strongly suppressed post-transcriptionally in slow muscles.When HIF-1α was overexpressed for 14 days after somatic gene transfer in adult rats, a slow-to-fast transformation was observed, encompassing an increase in fibre cross sectional area, oxidative enzyme activity and myosin heavy chain. The latter was shown to be regulated at the mRNA level in C2C12 myotubes.

According to the current paradigm, muscle nuclei serve a certain cytoplasmic domain. To preserve the domain size, it is believed that nuclei are injected from satellite cells fusing to fibres undergoing hypertrophy, and lost by apoptosis during atrophy. Based on single fibre observations in and ex vivo we suggest that nuclear domains are not as constant as is often indicated. Moreover, recent time lapse in vivo imaging of single fibres suggests that at least for the first few weeks, atrophy is not accompanied by any loss of nuclei. Apoptosis is abundant in muscle tissue during atrophy conditions, but in our opinion it has not been unequivocally demonstrated that such nuclei are myonuclei. As we see it, the preponderance of current evidence suggests that disuse atrophy is not accompanied by loss of nuclei, at least not for the first 2 months. Moreover, it has not been proven that myonuclear apoptosis does occur in permanent fibres undergoing atrophy; it seems more likely that it is confined to stromal cells and satellite cells. If muscle atrophy is not related to loss of nuclei, design of intervention therapies should focus on protein metabolism rather than regeneration from stem cells.