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

Abstract

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.

Fig. 1.

Effect of overload on fiber size and number of myonuclei studied in vivo. (A) Micrographs of fibers after overload. Nuclei are labeled with fluorescent oligonucleotides. Illustrations represent merged stacks of images from different focal planes. (Scale bar: 50 μm.) (B) Number of nuclei per millimeter and CSA. Each data point represents 5–24 fibers from a total of 36 animals. Symbols represent the mean ± SEM. A nonlinear fit using a Sigmoid dose–response curve was used for the increase in CSA and nuclei per millimeter, resulting in R² = 0.13 and R² = 0.29, respectively. *Nuclei per millimeter; ^#CSA significantly different from day 0 (P < 0.05). (C) Nuclear domains. Each symbol represents the binned time groups calculated from the dataset in B. **Statistical differences between the indicated time groups (P < 0.01).

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) Quantification 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.

Fig. 3.

The effect of long-term denervation on overloaded muscles studied ex vivo. Number of nuclei counted (A) and CSA measured on isolated EDL muscle fibers (B) taken from muscle before the onset of overload (Control Pre), after 14 days of overload (Overload), after 14 days of overload and the subsequent 2 mo of denervation (Overload + Denervation), after 3 mo of denervation (Denervation), and from normal muscles from the same batch of animals that had not been exposed to experimental treatment (Control Post). Each column represents the mean ± SEM of 134–378 fibers. Columns that were statistically indistinguishable (P > 0.05) are marked with the same letter.

Fig. 4.

Apoptosis in hypertrophied muscle after denervation (Den.). (A) Triple staining with Hoechst 33342 (blue), TUNEL (green), and antibodies against dystrophin (red). Note that all apoptotic nuclei (arrows) are outside the muscle cells; hence, they are either satellite or stroma cells. (Scale bar: 50 μm.) (B) Number of TUNEL-positive nuclei per section. Each column point represents the mean ± SEM (n = 5–8 sections from four muscles). ***Statistical significance (P < 0.0001).

Fig. 5.

Previous hypertrophy episode retards denervation atrophy. (A) Micrograph of a fiber stained for IIb myosin heavy chain (green), dystrophin (red), and nuclei (blue). Arrows indicate myonuclei, defined by having the mass center of the Hoechst stain within the dystrophin ring. (B) Number of nuclei per IIb fiber in normal (Con.) and overloaded (Overl.) muscle. ***Statistical significance (P < 0.0001). (C) Muscle fiber size in muscles overloaded for 14 d (Overl.) relative to nonoverloaded normal muscles (Con.); and muscles overloaded for 14 d and then denervated for 14 d (Overl.+den.) relative to muscles denervated for 14 d (Den.). ^##Statistical significance from denervated muscles (P < 0.001).

Fig. 6.

A model for the connection between muscle size and number of myonuclei. In this model, myonuclei are permanent. Previously untrained muscles acquire newly formed nuclei by fusion of satellite cells preceding the hypertrophy. Subsequent detraining leads to atrophy but no loss of myonuclei. The elevated number of nuclei in muscle fibers that had experienced a hypertrophic episode would provide a mechanism for muscle memory, explaining the long-lasting effects of training and the ease with which previously trained individuals are more easily retrained.