The LINC Complex Regulates Tendon Elastic Modulus, Collagen Crimp, and Lateral Expansion During Early Postnatal Development

Abstract

There is limited understanding of how mechanical signals regulate tendon development. The nucleus has emerged as a major regulator of cellular mechanosensation via the linker of nucleoskeleton and cytoskeleton (LINC) protein complex. Specific roles of LINC in tenogenesis have not been explored. In this study, we investigate how LINC regulates tendon development by disabling LINC-mediated mechanosensing via dominant negative (dn) overexpression of the Klarsicht, ANC-1, and Syne Homology (KASH) domain, which is necessary for LINC to function. We hypothesized that LINC regulates mechanotransduction in developing tendons and that disabling LINC would impact tendon's mechanical properties and structure in a mouse model of dnKASH. We used Achilles tendon (AT) and tail tendon (TT) as representative energy-storing and positional tendons, respectively. Mechanical testing at postnatal day 10 showed that disabling the LINC complex via dnKASH significantly impacted tendon mechanical properties and cross-sectional area and that the effects differed between ATs and TTs. Collagen crimp distance was also impacted in dnKASH tendons and was significantly decreased in ATs and increased in TTs. Overall, we show that disruption to the LINC complex specifically impacts tendon mechanics and collagen crimp structure, with unique responses between an energy-storing and limb-positioning tendon. This suggests that nuclear mechanotransduction through LINC plays a role in regulating tendon formation during neonatal development.

Keywords: KASH‐domain; LINC; SUN‐domain; development; mechanobiology; mechanotransduction; nesprin; nuclear mechanosensing; tendon; tenogenesis.

Figure 1.

Schematic of the relationship between the LINC complex, Nesprin, SUN, KASH, and the cell nucleus. Additional proteins not investigated in this study are shown.

Figure 2.

Representative force-displacement and stress-strain curves for (A,B) ATs and (C,D) TTs. Control mice are denoted by +−, and dnKASH mice are denoted by ++, to indicate overexpression of the eGFP-KASH2 fusion protein, which saturates available Sun/KASH binding in a dominant-negative manner. Yellow dashed lines show the linear region used to calculate stiffness and elastic modulus. All yellow lines have a fit with the R² value ≥ 0.95; median R² = 0.998.

Figure 3.

P10 Achilles tendon mechanical properties: (A) Maximum force, (B) displacement at maximum force, (C) cross-sectional area, (D) stiffness, (E) maximum stress, (F) strain, and (G) elastic modulus. dnKASH (++) ATs had a decreased (E) maximum stress and (G) elastic modulus but (C) an increased cross-sectional area, compared to (+−) controls. Bridging lines and asterisks denotes significance (p<0.05). Bars represent mean ± standard deviation.

Figure 4.

P10 tail tendon mechanical properties: (A) Maximum force, (B) displacement at maximum force, (C) cross-sectional area, (D) stiffness, (E) maximum stress, (F) strain, and (G) elastic modulus. dnKASH (++) TTs had a decreased (C) cross-sectional area, compared to (+−) controls. Other mechanical properties did not differ significantly between dnKASH and control TTs. Bridging lines and asterisks denotes significance (p<0.05). Bars represent mean ± standard deviation.

Figure 5.

SEM micrographs of (A) control (+−) and (B) dnKASH (++) ATs, and (C) control (+−) and (D) dnKASH (++) TTs showing crimp pattern and morphology. Though crimp distances differed significantly between control and dnKASH tendons, no obvious ultrastructural or morphological differences are observed. Yellow lines show representative crimp measurements taken between crimp wave “peaks.” Crimp was measured identically in SHG and SEM images. All images captured at 800x. E) dnKASH (++) ATs had significantly decreased crimp distance compared to control (+−) ATs. Conversely, (F) dnKASH (++) TTs had significantly increased crimp distance compared to control (+−) TTs.