Constricted Migration of Human Myoblasts Impairs Motility, Nuclear Integrity and Differentiation

Abstract

Introduction: Upon injury, muscle stem cells (MuSCs) undergo activation, proliferation, migration, differentiation and fusion to regenerate muscle. MuSCs sense and respond to the mechanical microenvironment of the tissue while migrating through the interstitial matrix to the cell niche. In fibrosis, a common manifestation of injury, the mechanical microenvironment of the tissue is modified. The extracellular matrix (ECM) has increased stiffness and decreased porosity which may decrease the mobility and differentiation ability of MuSCs. The objective of this study was to determine the capacity of human myoblast to undergo constricted migration through small rigid pores and the deficits in their regenerative capacity following constricted migration. We hypothesized that fewer cells would undergo constricted migration and those that did would incur greater nuclear rupture, increased DNA damage, and impaired differentiation capacity. Methods: Immortalized human myoblasts were allowed to migrate through transwell inserts of pores with 3μm (constricted migration) and 8μm diameter (unconstricted migration) or in 2D conditions (control). Live cell imaging techniques over a period 24 hours were used to quantify relative cell migration. The extent of nuclear rupture and DNA damage were quantified using immunofluorescent imaging of cells on transwell inserts. Differentiation index was calculated by removing the cells from the transwell and placing in differentiation conditions prior to immunofluorescent imaging. Results: Cells migrated more rapidly through large pores than the constrictive pores. For 3μm protrusions through the pores resulted in migration in ~46 % cells compared to 68% for 8μm pores. Following migration through the pores there was no difference in 2D migration, but it was significantly slower than cells that remained in 2D conditions. Human myoblasts undergoing 3μm migration incurred nuclear rupture in ~85% of cells while cells migrating through 8μm or non-migrating cells rarely had ruptured nuclei (<2%). 3μm migration was also related to a significant ~80% increase in DNA damage from cells undergoing 8μm migration. The human myoblasts undergoing 3μm migration maintained impaired differentiation of ~30% compared to 8μm migration or non-migrating cells. Conclusions: The study results support the hypothesis that constricted environments impair migration, cause substantial nuclear rupture, induce DNA damage, and impair differentiation. The use of human myoblasts with different migration speeds than previously used mouse myoblasts had substantially more profound impacts on nuclear rupture and persistence of differentiation defects. Thus, the constrictive environment presented by muscle fibrosis could be a critical aspect of impaired regeneration in fibrotic conditions. Funding sources: This work was supported by grants from NIH NIAMS R01 AR079545 and R00 AR067867 to LRS. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.