Myonuclear alterations associated with exercise are independent of age in humans

Abstract

Age-related decline in skeletal muscle structure and function can be mitigated by regular exercise. However, the precise mechanisms that govern this are not fully understood. The nucleus plays an active role in translating forces into biochemical signals (mechanotransduction), with the nuclear lamina protein lamin A regulating nuclear shape, nuclear mechanics and ultimately gene expression. Defective lamin A expression causes muscle pathologies and premature ageing syndromes, but the roles of nuclear structure and function in physiological ageing and in exercise adaptations remain obscure. Here, we isolated single muscle fibres and carried out detailed morphological and functional analyses on myonuclei from young and older exercise-trained individuals. Strikingly, myonuclei from trained individuals were more spherical, less deformable, and contained a thicker nuclear lamina than those from untrained individuals. Complementary to this, exercise resulted in increased levels of lamin A and increased myonuclear stiffness in mice. We conclude that exercise is associated with myonuclear remodelling, independently of age, which may contribute to the preservative effects of exercise on muscle function throughout the lifespan. KEY POINTS: The nucleus plays an active role in translating forces into biochemical signals. Myonuclear aberrations in a group of muscular dystrophies called laminopathies suggest that the shape and mechanical properties of myonuclei are important for maintaining muscle function. Here, striking differences are presented in myonuclear shape and mechanics associated with exercise, in both young and old humans. Myonuclei from trained individuals were more spherical, less deformable and contained a thicker nuclear lamina than untrained individuals. It is concluded that exercise is associated with age-independent myonuclear remodelling, which may help to maintain muscle function throughout the lifespan.

Keywords: ageing; exercise; nuclear lamina; nuclear shape; nuclei.

Figure 1. Altered 2D myonuclear shape in trained younger and older individuals

A, representative images of vastus lateralis muscle fibres isolated from younger untrained (YU), older untrained (OU), younger trained (YT) and older trained (OT) individuals, stained with DAPI and myosin heavy chain 7 to visualise myonuclei and slow myosin, respectively. Scale bars: 30 μm and 10 μm main images and zoomed insets of myonuclei, respectively. B–E, above graphs, calculation of aspect ratio (length/width of nucleus) and nuclear area (μm²). B, comparisons of myonuclear aspect ratio in YU, OU, YT and OT individuals; 2053 total nuclei analysed. C, comparisons of myonuclear aspect ratio between groups after normalisation to sarcomere length. D, myonuclear aspect ratio in MYH7⁺ fibres (1385 total nuclei analysed). E, comparisons of nuclear area (μm²) between groups (1453 total nuclei analysed). In B–E, the coloured symbols represent the mean values for each individual, calculated from several pooled muscle fibres, unfilled grey symbols represent myonuclei; mean values for individuals were used for two‐way ANOVA (n = 6, **P < 0.01 ***P < 0.001); error bars represent means ± SD. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2. Lower 3D aspect ratio and greater sphericity in myonuclei from trained younger and older individuals

A, representative three‐dimensional rendering of Z‐stack images of a single human vastus lateralis muscle fibre acquired with a spinning disk confocal microscope equipped with a ×63 oil objective lens. Muscle fibre stained to visualise lamin A, DNA, actin and myosin heavy chain 7 (MYH7). B, representative zoomed images of 3D‐rendered nucleus. C–E, comparisons of nuclear skeletal length/diameter (μm), sphericity and volume in younger untrained (YU), older untrained (OU), younger trained (YT) and older trained (OT) individuals. Coloured symbols represent individual means, grey symbols represent myonuclei; mean values for individuals were used for two‐way ANOVA (n = 4 to 6, **P < 0.01 ***P < 0.001 ****P < 0.0001); error bars represent means ± SD. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3. Organisation of lamin A in trained individuals and untrained counterparts

A, representative images, acquired through confocal microscopy using a ×63 oil objective, of muscle fibres isolated from younger untrained (YU), older untrained (OU) patients, younger trained (YT) and older untrained (OT) individuals. Fibres were stained with DAPI to visualise DNA, actin, lamin A and myosin heavy chain 7 (MYH7). Scale bar: 25 μm in main images, 10 μm in zoomed images. B, representative images of myonuclei from OU and OT muscle fibres acquired through super‐resolution iSIM microscopy. Scale bars: 5 μm. C, quantification of lamin A deposition (μm) in muscle fibres from OT and OU. n = 3–5 per group; unpaired t‐test revealed significant difference between groups (P < 0.05). D, standard deviation projections of lamin A‐stained myonuclei and pixel intensity line scans from YU, OU, YT and OT. E, lamin A total invagination length (μm) in muscle fibres from YU, OU, YT and OT; n = 6. Two‐way ANOVA revealed no significant differences between groups. Coloured symbols represent individual means, grey symbols represent myonuclei; mean values for individuals were used for two‐way ANOVA and t‐test. Error bars represent means ± SD. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4. Lamin A levels are increased in trained mouse tibialis anterior muscle

A, protein levels of lamin A, lamin C, lamin B1 and emerin normalised to histone H3 in tibialis anterior muscle from untrained and high intensity endurance trained mice. B, protein levels of linker of nucleoskeleton and cytoskeleton (LINC) complex proteins SUN1, SUN2, nesprin‐1‐α2 and nesprin‐2‐α1 normalised to histone H3 in tibialis anterior muscle from untrained and trained mice. C and D, images of western blots from which data in A and B were obtained. Note that lamin A levels were significantly increased and SUN2 levels trending to increase. Arrows indicate predicted molecular masses (kDa). Data points represent individual mice; n = 7 per group; *P < 0.05 (t‐test). Error bars represent means ± SD. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 5. Exercise training results in stiffer myonuclei

A, schematic representation of fibre mounting and stretching. B, relationship between sarcomere length and nuclear aspect ratio in muscle fibres from older trained (OT) and untrained (OU) patients. C, variance of aspect ratio/sarcomere length in OT and OU fibres. Note that sarcomere length positively correlates with extent of muscle fibre stretch and that myonuclei in OT fibres were significantly stiffer than myonuclei from OU fibres. *Statistically significant difference between gradients of slopes (linear regression analysis, F‐test to compare variances). D, typical set‐up of individual muscle fibres after isolation and mounting in parallel on an electron microscopy (EM) grid split in half for imaging and nanoindentation. E, nanoindentation of single muscle fibres (brightfield) with myonuclei labelled being probed by nanoindenter (brightfield, left of image). Scale bar, 50 μm. F, comparison of Young's modulus (kPa) in nuclei from untrained and trained mice. G–I, comparisons of E′, E″ and tan δ (E′/E′) at different dynamic mechanical analysis frequencies (Hz) in nuclei from untrained and exercise trained mice. Note that myonuclei were significantly more stiff and more elastic in fibres from trained vs. untrained mice. Each coloured data point represents the average for each mouse, n = 3 per group. Error bars represent means ± SEM. *Statistically significant difference between groups (t‐test and mixed effects analysis). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 6. Summary and proposed effects of myonuclear remodelling with training, and inactivity related defects in nuclear mechanotransduction with age

A, in skeletal muscle fibres from trained individuals, nuclear envelope proteins including the nuclear lamina effectively transduce cytoskeletal forces to the nucleus to regulate signalling pathways. B, in skeletal muscle from trained older individuals, myonuclear shape and mechanotransduction are preserved. C, in skeletal muscle from untrained older individuals, myonuclei are more elongated, nuclear lamina levels are reduced, and myonuclei are more deformable. This may lead to increased susceptibility to myonuclear damage and defective mechanotransduction that results in decline in muscle mass and function. [Colour figure can be viewed at wileyonlinelibrary.com]