Optogenetic-induced muscle loading leads to mechanical adaptation of the Achilles tendon enthesis in mice

Abstract

Skeletal shape depends on the transmission of contractile muscle forces from tendon to bone across the enthesis. Loss of muscle loading impairs enthesis development, yet little is known if and how the postnatal enthesis adapts to increased loading. Here, we studied adaptations in enthesis structure and function in response to increased loading, using optogenetically induced muscle contraction in young (i.e., growth) and adult (i.e., mature) mice. Daily bouts of unilateral optogenetic loading in young mice led to radial calcaneal expansion and warping. This also led to a weaker enthesis with increased collagen damage in young tendon and enthisis, with little change in adult mice. We then used RNA sequencing to identify the pathways associated with increased mechanical loading during growth. In tendon, we found enrichment of glycolysis, focal adhesion, and cell-matrix interactions. In bone, we found enrichment of inflammation and cell cycle. Together, we demonstrate the utility of optogenetic-induced muscle contraction to elicit in vivo adaptation of the enthesis.

Fig. 1.. Study design: Schematic of experimental setup for optogenetic muscle loading (shown here for young mice).

Right hindlimbs of young and adult ActaCre;Ai32 mice were exposed to daily bouts of either 5- or 20-min optogenetic-induced muscle contractions of the triceps surae. Mice were used experimentally after various loading bouts, including for RNA sequencing (RNA-seq) (young mice only; after 5 or 12 days of 20-min loading); for collagen damage assays [collagen hybridizing peptide (CHP); young mice only, after 5 days of 20-min loading]; for microcomputed tomography (young and adult mice; 19 days of 20-min loading); and for mechanical testing (young and adult mice; 19 days of 20-min loading). At each designated end point (i.e., experimental days 5, 12, or 19), experimental and naïve age-matched controls were euthanized to collect Achilles tendon and enthesis for histology, micro–computed topography (microCT), and mechanics and Achilles tendon and calcaneus for RNA isolation (RNA-seq). Created with BioRender.com.

Fig. 2.. Optogenetic activation of triceps surae muscle group is not invasive and does not negatively influence animal weight or generated isometric ankle joint torque in young mice.

(A) Young mice, regardless of exposure to daily bouts of optogenetic muscle stimulation (5- or 20-min duration), gained weight for the duration of the experiment. However, adult mice lost weight (~10%) from onset to end of the experiment. Controls were naïve mice at similar ages as the young group. (B) Normalized torque significantly increased in adult but not young mice with daily bouts of 20-min optogenetic muscle stimulation. Each data point denotes the average weekly change in generated ankle torque normalized to the weight of the animal. (C) Steady state–generated ankle torque (at the end of the 20-min loading bout), measured during isometric ankle plantarflexion, was ~30% of the peak torque in young mice for the first week of daily optogenetic muscle loading. (D) EDL muscle from an adult (3-month-old) ActaCre;Ai32 mouse had a duration-dependent force response to blue light exposure measure contraction when tested in vitro (5 to 70 ms). For (A) and (B), data were compared using two-way analysis of variance (ANOVA) with mixed models (repeated measures between weeks within groups, Sidak correction). Error bars denote means ± 95% confidence interval. *P < 0.05, **P < 0.001, ***P = 0.0004, and ****P < 0.0001.

Fig. 3.. Repeated daily loading of Young mouse Achilles tendons results in expansion and warping deformation and impaired mechanical toughness at the enthesis.

(A) Deformation maps of the posterior, lateral, and medial views of loaded (average shape; right calcaneus) compared to control (average shape, left calcaneus, mirrored for overlay/shape representation) showed reduced height of the calcaneal tuberosity with loading (posterior view; shown in blue) and increased bone expansion and warping (lateral and medial views; shown in red). (B) Total volume of the calcaneal region of interest was significantly reduced with loading (paired t test; P = 0.0338), and BV/TV (%) was significantly increased (paired t test; P = 0.0056) in young loaded compared to young control calcanei. No significant differences in length between young control and loaded calcanei were measured. (C) Representative stress-strain curves for young and adult groups showed structural adaptations in the young enthesis and apophysis coincide with significantly reduced tensile strength and toughness in the loaded compared to nonloaded contralateral limbs. Data were compared using two-way ANOVAs with multiple comparisons (contralateral versus loaded; no correction for sphericity; Sidak correction for multiple comparisons). Error bars denote means ± 95% confidence interval. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 4.. Repeated loading led to denatured collagen fibers and increased prevalence of ECM components at the enthesis and growth plate.

(A) Schematic shows the experimental design for evaluation of the effect of repeated loading on ECM of the young tendon and enthesis. HRP, horseradish peroxidase. DAB, 3,3'-Diaminobenzidine. (B) Repeated loading resulted in the accumulation of collagen denaturation in loaded tendon and enthesis. Data were compared using paired t test (two-tailed; assuming Gaussian distribution, parametric; **P = 0.0062. a.u., arbitrary units. (C and E) Loaded entheses did not exhibit differences in localized aggrecan or type III collagen at the enthesis compared to controls. Type III collagen cell count data were compared using unpaired t tests (nonparametric; Mann-Whitney test; two-tailed; P > 0.9999). (D) However, loading qualitatively influenced the pericellular matrix and cell shape, but not cell number, at the enthesis in young mice. (F) Cell number was not affected by the duration of loading (5 min versus 20 min; unpaired nonparametric t test; Mann-Whitney, two-tailed; P = 0.1342). Scale bars, (B) 10 mm and (C) and (D) 100 μm. Error bars denote mean ± 95% confidence interval. Created with BioRender.com.

Fig. 5.. Optogenetic-induced loading led to transcriptional response in both tendon and bone.

(A) Principal component (PC) analysis plots of differential gene expression for naïve, contralateral, and loaded tendons and bones after 5 and 12 days of loading. (B) Heatmaps of all DEGs for Achilles tendon and calcaneus (bone) in naïve, 5-day loaded, and 12-day loaded mice. (C) In tendon and bone 1193 and 312 genes, respectively, were differentially expressed after 5 and 12 days of loading. DEGs were identified using Wald test in R/Bioconductor/DESeq2 with Benjamini-Hochberg P-adj < 0.05. N = 15 per tissue.

Fig. 6.. Relevant biological processes that were differentially regulated by optogenetically induced loading in young Achilles tendon and bone (calcaneus).

Increased muscle loading on (A) tendon and (B) bone resulted in enrichment of key biological processes, such as angiogenesis, integrin-mediated signaling, and extracellular matrix organization, at one or both time points. Loading enriched processes related to cell adhesion, cytoskeleton and ECM regulation, mechanotransduction, and ossification in both tissues. Biological processes were identified using DAVID’s statistical test of overrepresentation with false discovery rate (FDR) < 0.05 and enrichment > 1.5. TGF-β, transforming growth factor–β; FGF, fibroblast growth factor; GPCR, G protein–coupled receptor; AC, adenylyl cyclase; BMP, bone morphogenetic protein; cAMP, cyclic adenosine monophosphate.

Fig. 7.. After 5 days and 12 days of stimulation, daily bouts of optogenetic loading led to significant down-regulation of genes associated with tendon stem cell activation and ECM synthesis in tendon and up-regulation of proinflammatory markers in bone.

Data shown are log₂-transformed fold change (log2FC, x axis) and −log₁₀ transformed P-adj (−log10(P-adj), y axis) for (A) tendon and (B) bone. Dashed lines denote ±0.5 log2FC.

Fig. 8.. Relevant KEGG pathways that were differentially regulated by optogenetics-induced loading in Young Achilles tendon and bone (calcaneus).

Enriched KEGG pathways, including mechanobiological pathways such as ECM-receptor interaction and focal adhesion, were identified in both (A) tendon and (B) bone at one or both time points. (C) ECM-receptor interaction pathway in tendon is driven by 44 genes of which >90% are down-regulated with loading, while in (D) calcaneus focal adhesion pathway is driven by 36 genes of which >50% are up-regulated with loading. Pathways were identified using DAVID’s statistical test of overrepresentation with an FDR < 0.05 and enrichment > 1.5. VEGF, vascular endothelial growth factor.