Myonuclei Acquired by Overload Exercise Precede Hypertrophy and Are Not Lost on Detraining
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Myonuclei acquired by overload exercise precede hypertrophy and are not lost on detraining J. C. Bruusgaard, I. B. Johansen, I. M. Egner, Z. A. Rana, and K. Gundersen1 Department of Molecular Biosciences, University of Oslo, 0371 Oslo, Norway Edited by Gerald D. Fischbach, The Simons Foundation, New York, NY, and approved July 16, 2010 (received for review December 4, 2009) Effects of previous strength training can be long-lived, even after nuclei newly recruited by overload are kept for at least 3 mo after prolonged subsequent inactivity, and retraining is facilitated by subsequent denervation. This lasting change in the number of a previous training episode. Traditionally, such “muscle memory” myonuclei might provide a substrate for a type of “memory” in has been attributed to neural factors in the absence of any iden- muscle cells. tified local memory mechanism in the muscle tissue. We have used in vivo imaging techniques to study live myonuclei belonging to Results distinct muscle fibers and observe that new myonuclei are added Newly Acquired Myonuclei Precede Hypertrophy. Extensor dig- before any major increase in size during overload. The old and itorum longus (EDL) muscles were overloaded by partially ab- newly acquired nuclei are retained during severe atrophy caused lating their major synergist (day 0), and nuclei of single muscle by subsequent denervation lasting for a considerable period of the fibers were visualized in the intact animal by injection of labeled animal’s lifespan. The myonuclei seem to be protected from the nucleotides 1–21 days after ablation (Fig. 1A). The number of high apoptotic activity found in inactive muscle tissue. A hypertro- nuclei and the cross-sectional area (CSA) were determined from phy episode leading to a lasting elevated number of myonuclei observing the distal half of each fiber in the microscope. During the retarded disuse atrophy, and the nuclei could serve as a cell bi- 21-day period, CSA increased 35% from 1,474 ± 93 μm2 to 1,991 ± ological substrate for such memory. Because the ability to create 150 μm2, whereas the number of myonuclei increased 54% from myonuclei is impaired in the elderly, individuals may benefit from 41 ± 1.5 nuclei per millimeter of fiber length to 63 ± 2.3 nuclei per strength training at an early age, and because anabolic steroids millimeter of fiber length (Fig. 1A). The number of nuclei started CELL BIOLOGY facilitate more myonuclei, nuclear permanency may also have to rise after 6 days and seemed to stabilize after 11 days. This implications for exclusion periods after a doping offense. preceded the rise in CSA, which was increased only after 9 days and stabilized after 14 days (Fig. 1B). This time sequence was muscle memory | muscle nuclei | muscle atrophy | muscle hypertrophy | confirmed in individual fibers by calculating the cytoplasmic vol- apoptosis ume per nucleus for each fiber. This volume decreased 16% from 33,749 ± 1,457 μm3 at 0–3 days after overload to 28,360 ± 956 μm3 ndividuals with a history of previous training acquire force at 6–10 days after overload (P ≤ 0.01). The average volume Iquickly on retraining (1, 2), and this commonly observed phe- (31,235 ± 1,476 μm3) remained lower but was not statistically nomenon has been dubbed “muscle memory.” There is no known different from that of the controls 12–21 days after overload. mechanism for memory in muscle cells, and, to date, the long- lasting effects of previous training have been attributed to motor Elevated Number of Nuclei Persisted During Subsequent Disuse. In learning in the central nervous system (3). However, it has been a separate series of experiments, overload was introduced for 14 reported that muscles can remain hypertrophic after several days. The number of nuclei increased by 37% from 49 ± 1.8 months of detraining (1, 4–8). In one study on elderly individuals nuclei per millimeter to 67 ± 2.4 nuclei per millimeter, and CSA who had strength-trained, force was still 9–14% higher even after was increased by 35% from 1,018 ± 73 μm2 to 1,379 ± 78 μm2 2 y of detraining (7). During 30–32 wk of detraining, a group of (Fig. 2). In a parallel group of animals, the muscle was sub- women lost a considerable part of the extra strength obtained by sequently denervated for 14 days. During this period, CSA de- 20 wk of previous training but regained the strength after only 6 wk creased to 555 ± 48 μm2, which is 40% of the top value. Despite of retraining (1). This suggests the possible presence of a local the atrophy, the number of nuclei was not significantly reduced memory mechanism in the muscle. The present data may provide (Fig. 2). The small nonsignificant reduction in nuclear number such a mechanism in that there is a lasting change of cytoarchi- was similar in muscles that were not denervated but just synergist- tectural features after an episode of overload hypertrophy, even ablated for 35 days (Fig. 2B, ▲). after subsequent prolonged disuse. The small diameter of fibers that had been denervated for the It has been suggested for more than 100 y that a nucleus can long term precluded intracellular injection, and we had to resort only support a certain volume of cytoplasm (9–19). Muscle cells to an ex vivo model in a third series of experiments. Muscles can be five orders of magnitude larger than mononucleated cells, were similarly overloaded for 14 days and then denervated for 3 and muscle fibers are one of the very few multinuclear cell types mo. The muscles were fixed in situ, and nuclei were counted in in vertebrates (18). When the muscle fibers increase in size, it has fibers that had been mechanically and chemically isolated. Sim- been believed that nuclei are added by mitosis and subsequent ilar to the in vivo studies, overload led to an increase in the fusion of muscle stem cells to the muscle fibers and that the number of nuclei (Fig. 3A) and in CSA (Fig. 3B). The number of “excess” myonuclei are removed by selective apoptosis of some nuclei observed after 3 mo of subsequent denervation was not of the nuclei during atrophy (20, 21). Such mechanisms would serve to keep the cytoplasmic nuclear domain size constant. Recent evidence with time-lapse in vivo imaging techniques has Author contributions: J.C.B. and K.G. designed research; J.C.B., I.B.J., I.M.E., Z.A.R., and challenged this simple model, because it has been shown that K.G. performed research; J.C.B., I.B.J., I.M.E., and K.G. analyzed data; and J.C.B., I.B.J., normal levels of myonuclei are preserved during atrophy (22). This I.M.E., and K.G. wrote the paper. might, however, be different in hypertrophic muscles, because The authors declare no conflict of interest. nuclei within the muscle tissue that have undergone mitosis during This article is a PNAS Direct Submission. development of hypertrophy are particularly prone to apoptosis Freely available online through the PNAS open access option. during subsequent disuse (23). The present data show that myo- 1To whom correspondence should be addressed. E-mail: [email protected]. www.pnas.org/cgi/doi/10.1073/pnas.0913935107 PNAS | August 24, 2010 | vol. 107 | no. 34 | 15111–15116 Downloaded by guest on October 1, 2021 Fig. 2. Effect of denervation on overloaded muscles studied in vivo. (A) Micrographs of fibers after overload and subsequent denervation. Nuclei are labeled with fluorescent oligonucleotides. Illustrations represent merged stacks of images from different focal planes. (Scale bar: 50 μm.) (B) Quanti- fication of nuclei per millimeter of fiber length and CSA of single fibers after denervation of hypertrophied muscle. Each data point represents the mean ± SEM (n =23–35 fibers from six to eight animals). Muscles were synergist- ablated and not denervated for 35 days (▲). *Statistical significance difference (P < 0.05). n.s., Nonsignificant difference from 14 days of overload. small increase in the number of nuclei by denervation alone, but this could be attributable to reduced fiber length after the long denervation. Denervation Induces Muscle Apoptosis but Not That of Old or Newly Acquired Myonuclei. It has previously been suggested that newly acquired nuclei in hypertrophic muscles are particularly prone to apoptosis (23). We therefore investigated apoptosis on sections of denervated muscle using stringent criteria for identification of myonuclei. We stained muscle sections for dystrophin, and only nuclei that had their geometrical center inside the dystrophin- stained ring at the fiber cortex were defined as myonuclei. Fig. 1. Effect of overload on fiber size and number of myonuclei studied in On sections, there was an 8-fold increase in the number of fi fl vivo. (A) Micrographs of bers after overload. Nuclei are labeled with uo- TUNEL-positive nuclei by 14 days of denervation (from 1.3 ± 0.4 rescent oligonucleotides. Illustrations represent merged stacks of images ± from different focal planes. (Scale bar: 50 μm.) (B) Number of nuclei per mil- to 11.8 2.7 nuclei per section; Fig. 4). However, although about limeter and CSA. Each data point represents 5–24 fibers from a total of 36 15,000 myonuclei were screened at 7 and 14 days of denervation, animals. Symbols represent the mean ± SEM. A nonlinear fit using a Sigmoid only 1 fulfilled the criterion of being an apoptotic myonucleus. dose–response curve was used for the increase in CSA and nuclei per milli- Thus, although there was a dramatic increase in apoptosis by meter, resulting in R2 = 0.13 and R2 = 0.29, respectively. *Nuclei per millimeter; denervation in these muscles, old and newly acquired myonuclei #CSA significantly different from day 0 (P < 0.05).