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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, we present striking differences 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. We conclude that exercise is associated with age-independent myonuclear remodelling, which may help to maintain muscle function throughout the lifespan.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 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 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. Abstract figure legend Structural and mechanical properties of myonuclei in trained young and aged individuals. In skeletal muscle fibres from trained individuals, myonuclei are more spherical, have greater Lamin A and are stiffer compared to untrained counterparts. This may protect nuclei from damage when subjected to contractile forces during exercise, and permit effective transduction of these forces to regulate gene expression and signalling pathways (mechanotransduction). In skeletal muscle from untrained older individuals, myonuclei are more elongated, nuclear lamina levels are lower, and myonuclei are more deformable. This may increase susceptibility to myonuclear damage and defective mechanotransduction, contributing to declines in muscle mass and function. This article is protected by copyright. All rights reserved.

Background Senescent cells have emerged as bona fide drivers of ageing and age-related cardiovascular disease, with senescent cells accumulating in the aged heart and following damage/injury. We have shown that removal of senescent cells using senolytics can rejuvenate the regenerative capacity of the aged heart. Purpose We investigate the effects of cell senescence and action of the senolytics, Dasatinib (D) and Quercetin (Q), on human cardiomyocyte and endothelial cell viability, survival, proliferation, angiogenesis and senescence in vitro. Methods We developed a transwell insert co-culture stress-induced premature senescence model system in vitro. Doxorubicin was used to induce premature senescence to cardiac stromal progenitor cells or HUVECs, which were seeded on the bottom chamber. In separate well plates, untreated cells (non-senescent) were seeded on the bottom chamber and acted as control. Human iPSC-cardiomyocytes (iPSC-CMs) or HUVECs were seeded on the top chamber insert. Co-cultures were left for 7 days and then iPSC-CMs or HUVECs in the top chamber were analysed for viability, proliferation and markers of senescence, and the conditioned medium analysed for SASP factors. The cultures were then treated with D+Q (0.5μM D with 20μM Q) for 3 days to clear the senescent cells in the bottom chamber. Then 7 days later the iPSC-CMs or HUVECs in the top chamber were analysed for viability, proliferation and the markers of senescence, and conditioned medium analysed for SASP factors (total of 17 days). Results Co-culture of iPSC-CMs with senescent cells led to decreased iPSC-CM viability (64±11% vs. 100±3% control; p Conclusion We show that D+Q senolytics have therapeutic potential in rejuvenating the reparative activity of cardiomyocytes and endothelial cells. These results open the path to further studies on using senolytic therapy in age-related cardiac deterioration and rejuvenation. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): Heart Research UK

Wnt-β-catenin signalling is essential for skeletal muscle myogenesis during development, but its role in adult human skeletal muscle remains unknown. Here we have used human primary CD56Pos satellite cell-derived myogenic progenitors obtained from healthy individuals to study the role of Wnt-β-catenin signalling in myogenic differentiation. We show that dephosphorylated β-catenin (active-β-catenin), the central effector of the canonical Wnt cascade, is strongly upregulated at the onset of differentiation and undergoes nuclear translocation as differentiation progresses. To establish the role of Wnt signalling in regulating the differentiation process we manipulated key nodes of this pathway through a series of β-catenin gain-of-function (GSK3 inhibition and β-catenin overexpression) or loss-of-function experiments (dominant negative TCF4). Our data showed that manipulation of these critical pathway components led to varying degrees of disruption to the normal differentiation phenotype indicating the importance of Wnt signalling in regulating this process. We reveal an independent necessity for active-β-catenin in the fusion and differentiation of human myogenic progenitors and that dominant negative inhibition of TCF4 prevents differentiation completely. Together these data add new mechanistic insights into both Wnt signalling and adult human myogenic progenitor differentiation.

Wnt-β-catenin signalling is essential for skeletal muscle myogenesis during development, but its role in adult human skeletal muscle remains unknown. Here we have used human primary CD56Pos satellite cell-derived myogenic progenitors obtained from healthy individuals to study the role of Wnt-β-catenin signalling in myogenic differentiation. We show that dephosphorylated β-catenin (active-β-catenin), the central effector of the canonical Wnt cascade, is strongly upregulated at the onset of differentiation and undergoes nuclear translocation as differentiation progresses. To establish the role of Wnt signalling in regulating the differentiation process we manipulated key nodes of this pathway through a series of β-catenin gain-of-function (GSK3 inhibition and β-catenin overexpression) or loss-of-function experiments (dominant negative TCF4). Our data showed that manipulation of these critical pathway components led to varying degrees of disruption to the normal differentiation phenotype indicating the importance of Wnt signalling in regulating this process. We reveal an independent necessity for active-β-catenin in the fusion and differentiation of human myogenic progenitors and that dominant negative inhibition of TCF4 prevents differentiation completely. Together these data add new mechanistic insights into both Wnt signalling and adult human myogenic progenitor differentiation.