Kristian Gundersen’s research while affiliated with University of Oslo and other places

One of the ideas stemming from the discovery of a cellular memory in muscle cells has been that an early exercise period could induce a long-term muscle memory, boosting the effects of exercise later in life. In general muscles are more plastic in younger animals, so we devised a 5-week climbing exercise scheme with food reward administered to juvenile rats (post-natal week 4-9). The juvenile exercise increased fiber cross-sectional area (fCSA), and boosted nuclear accretion. Subsequently the animals were subjected to 10 weeks of detraining (week 9-19, standard caging). During this period fCSA became similar in the animals that had been climbing compared to Naive controls, but the elevated number of myonuclei induced by the climbing were maintained. When the Naive rats were subjected to two weeks of adult exercise (week 19-21) there was little effect on fCSA, while the previously trained rats displayed an increase of 19%. Similarly, when the rats were subjected to unilateral surgical overload in lieu of the adult climbing exercise, the increase in fCSA was 20% (juvenile climbing group) and 11% (Naive rats) compared to the contralateral leg. This demonstrated that juvenile exercise can establish a muscle memory. The juvenile climbing exercise with food reward led to leaner animals with lower body weight. These differences were to some extent maintained throughout the adult detraining period in spite of all animals being fed ad libitum, indicating a form of body weight memory.

Muscle cells have different phenotypes adapted to different usage and can be grossly divided into fast/glycolytic and slow/oxidative types. While most muscles contain a mixture of such fiber types, we aimed at providing a genome-wide analysis of chromatin environment by ChIP-Seq in two muscle extremes, the almost completely fast/glycolytic extensor digitorum longus (EDL) and slow/oxidative soleus muscles. Muscle is a heterogeneous tissue where less than 60% of the nuclei are inside muscle fibers. Since cellular homogeneity is critical in epigenome-wide association studies we devised a new method for purifying skeletal muscle nuclei from whole tissue based on the nuclear envelope protein Pericentriolar material 1 (PCM1) being a specific marker for myonuclei. Using antibody labeling and a magnetic-assisted sorting approach we were able to sort out myonuclei with 95% purity. The sorting eliminated influence from other cell types in the tissue and improved the myo-specific signal. A genome-wide comparison of the epigenetic landscape in EDL and soleus reflected the functional properties of the two muscles each with a distinct regulatory program involving distal enhancers, including a glycolytic super-enhancer in the EDL. The two muscles are also regulated by different sets of transcription factors; e.g. in soleus binding sites for MEF2C, NFATC2 and PPARA were enriched, while in EDL MYOD1 and SOX1 binding sites were found to be overrepresented. In addition, novel factors for muscle regulation such as MAF, ZFX and ZBTB14 were identified.

Muscle fibers are the largest cells in the body, and one of its few syncytia. Individual cell sizes are variable and adaptable, but what governs cell size has been unclear. We find that muscle fibers are DNA scarce compared to other cells, and that the nuclear number (N) adheres to the relationship N = aVb where V is the cytoplasmic volume. N invariably scales sublinearly to V (b < 1), making larger cells even more DNA scarce. N scales linearly to cell surface in adult humans, in adult and developing mice, and in mice with genetically reduced N, but in the latter the relationship eventually fails when they reach adulthood with extremely large myonuclear domains. Another exception is denervation-atrophy where nuclei are not eliminated. In conclusion, scaling exponents are remarkably similar across species, developmental stages and experimental conditions, suggesting an underlying scaling law where DNA-content functions as a limiter of muscle cell size.

Mammalian cells exhibit remarkable diversity in cell size, but the factors that regulate establishment and maintenance of these sizes remain poorly understood. This is especially true for skeletal muscle, comprised of syncytial myofibers that each accrue hundreds of nuclei during development. Here, we directly explore the assumed causal relationship between multinucleation and establishment of normal size through titration of myonuclear numbers during mouse neonatal development. Three independent mouse models, where myonuclear numbers were reduced by 75, 55, or 25%, led to the discovery that myonuclei possess a reserve capacity to support larger functional cytoplasmic volumes in developing myofibers. Surprisingly, the results revealed an inverse relationship between nuclei numbers and reserve capacity. We propose that as myonuclear numbers increase, the range of transcriptional return on a per nuclear basis in myofibers diminishes, which accounts for both the absolute reliance developing myofibers have on nuclear accrual to establish size, and the limits of adaptability in adult skeletal muscle.

Transport distances in skeletal muscle fibers are mitigated by these cells having multiple nuclei. We have studied mouse living slow (soleus) and fast (extensor digitorum longus) muscle fibers in situ and determined cellular dimensions and the positions of all the nuclei within fiber segments. We modeled the effect of placing nuclei optimally and randomly using the nuclei as the origin of a transportation network. It appeared that an equidistant positioning of nuclei minimizes transport distances along the surface for both muscles. In the soleus muscle, however, which were richer in nuclei, positioning of nuclei to reduce transport distances to the cytoplasm were of less importance, and these fibers exhibit a pattern not statistically different from a random positioning of nuclei. We also simulated transport times for myoglobin and found that they were remarkably similar between the two muscles despite differences in nuclear patterning and distances. Together, these results highlight the importance of spatially distributed nuclei to minimize transport distances to the surface when nuclear density is low, whereas it appears that the distribution are of less importance at higher nuclear densities.

Transport distances in skeletal muscle fibers are mitigated by these cells having multiple nuclei. We have studied mouse living muscle fibers in situ and determined cellular dimensions and the positions of all the nuclei within fiber segments. We modelled the effect of placing nuclei optimally and randomly using a nucleus as an origin of a transportation network. It appeared that an equidistant positioning of nuclei minimizes transport distances along the surface for both muscles. In the soleus muscle however, which were richer in nuclei, positioning of nuclei to reduce transport distance to the cytoplasm were of less importance, and these fibers exhibit a pattern not statistically different from a random positioning of nuclei. Together, these results highlight the importance of spatially distribute nuclei to minimize transport distances to the surface when nuclear density low, while statistically it appears that the distribution are of less importance at higher nuclear densities.

Previously trained mouse muscles acquire strength and volume faster than naïve muscles; it has been suggested that this is related to increased myonuclear density. The present study aimed to determine whether a previously strength-trained leg (mem-leg) would respond better to a period of strength training than a previously untrained leg (con-leg). Nine men and 10 women performed unilateral strength training (T1) for 10 weeks, followed by 20 weeks of detraining (DT) and a 5-week bilateral retraining period (T2). Muscle biopsies were taken before and after each training period and analyzed for myonuclear number, fiber volume, and cross-sectional area (CSA). Ultrasound and one repetition of maximum leg extension were performed to determine muscle thickness (MT) and strength. CSA (~17%), MT (~10%), and strength (~20%) increased during T1 in the mem-leg. However, the myonuclear number and fiber volume did not change. MT and CSA returned to baseline values during DT, but strength remained elevated (~60%), supporting previous findings of a long-lasting motor learning effect. MT and strength increased similarly in the mem-leg and con-leg during T2, whereas CSA, fiber volume, and myonuclear number remained unaffected. In conclusion, training response during T2 did not differ between the mem-leg and con-leg. However, this does not discount the existence of human muscle memory since no increase in the number of myonuclei was detected during T1 and no clear detraining effect was observed for cell size during DT; thus, the present data did not allow for a rigorous test of the muscle memory hypothesis.

Aim Cachexia is a severe wasting disorder involving loss of body‐ and muscle mass reducing survival and quality of life in cancer patients. We aim at determining if cachexia is a mere perturbation of the protein balance or if the condition also involves a degenerative loss myonuclei within the fiber syncytia or loss of whole muscle fibers. Methods We induced cachexia by xenografting PC3 prostate cancer cells in nu/nu mice. Six weeks later, we counted myonuclei by in vivo microscopic imaging of single live fibers in the extensor digitorum longus muscle (EDL), and the EDL, soleus and tibialis anterior muscles were also harvested for ex vivo histology. Results The mice lost on average 15% of the whole‐body weight. The muscle wet weight of the glycolytic, fast EDL was reduced by 14%, the tibialis anterior by 17%, and the slow, oxidative soleus by 6%. The fiber cross sectional area in the EDL was reduced by 21% with no loss of myonuclei or any significant reduction in the number of muscle fibers. TUNEL‐positive nuclei or fibers with embryonic myosin were rare both in cachectic and control muscles, and hematoxylin‐eosin staining revealed no clear signs of muscle pathology. Conclusion The data suggest that the cachexia induced by xenografted prostate tumors induces a pronounced atrophy not accompanied by a loss of myonuclei or a loss of muscle fibers. Thus, stem‐cell related treatment might not be beneficial, and the quest for treatment options should rather focus on intervening with intracellular pathways regulating muscle fiber‐size. This article is protected by copyright. All rights reserved.

In their Correspondence, Charlotte Peterson's group suggests that methodological weaknesses of our paper ([Egner et al., 2016][1]) preclude the interpretation that satellite cells (SCs) are obligatory for hypertrophy after mechanical overload (OL), a finding conflicting with their own study