Exercise entrainment of musculoskeletal connective tissue clocks.

The musculoskeletal system, crucial for movement and support, relies on the delicate balance of connective tissue homeostasis. Maintaining this equilibrium is essential for tissue health and function. There has been increasing evidence in the last decade that shows the circadian clock as a master regulator of extracellular matrix (ECM) homeostasis in several connective tissue clocks. Very recently, exercise has emerged as a significant entrainment factor for cartilage and intervertebral disc circadian rhythms. Understanding the implications of exercise on connective tissue peripheral clocks holds promise for enhancing tissue health and disease prevention. Exercise-induced factors such as heat, glucocorticoid release, mechanical loading, and inter-tissue crosstalk may play pivotal roles in entraining the circadian rhythm of connective tissues. This mini review underscores the importance of elucidating the mechanisms through which exercise influences circadian rhythms in connective tissues to optimize ECM homeostasis. Leveraging exercise as a modulator of circadian rhythms in connective tissues may offer novel therapeutic approaches to physical training for preventing musculoskeletal disorders and enhancing recovery.

The rat Achilles and patellar tendons have similar increases in mechanical properties but become transcriptionally divergent during postnatal development.

The molecular events that drive post-natal tendon development are poorly characterized. In this study, we profiled morphological, mechanical, and transcriptional changes in the rat Achilles and patellar tendon before walking (P7), shortly after onset of walking (P14), and at motor maturity (P28). The Achilles and patellar tendons increased collagen content and mechanical strength similarly throughout post-natal development. However, at P28 the patellar tendon tended to display a higher maximal tensile load (MTL) (P = 0.0524) than the Achilles tendon, but a similar ultimate tensile strength (UTS; load relative to cross-sectional area) probably due to its increased cross-sectional area during development. The tendons started transcriptionally similar, with overlapping PCA clusters at P7 and P14, before becoming transcriptionally distinct at P28. In both tendons, there was an increase in extracellular matrix (ECM) gene expression and a concomitant decrease in cell cycle and mitochondrial gene expression. The transcriptional divergence at P28 suggested that STAT signalling was lower in the patellar tendon where MTL increased the most. Treating engineered human ligaments with the STAT inhibitor itacitinib increased collagen content and MTL. Our results suggest that during post-natal development, cellular resources are initially allocated towards cell proliferation before shifting towards extracellular matrix development following the onset of mechanical load and provide potential targets for improving tendon function. KEY POINTS: Little is known about mechanisms of post-natal tendon growth. We characterized morphological, mechanical, and transcriptional changes that occur before (P7), and early (P14) and late after (P28) rats begin to walk. From P7 to P28, the Achilles tendon increased in length, whereas the patellar tendon increased in cross-sectional area. Mechanical and material properties of the Achilles and patellar tendon increased from P7 to P28. From P7 to P28, the Achilles and patellar tendons increased expression of ECM genes and decreased mitochondrial and cell cycle gene expression. Ribosomal gene expression also significantly decreased in the Achilles and tended to decrease in the patellar tendon. At P28, STAT1 signalling tended to be lower in the patellar tendon which had grown by increasing cross-sectional area and inhibiting STAT activation in vitro improved mechanical properties in engineered human ligaments.

Saturated fatty acids negatively affect musculoskeletal tissues in vitro and in vivo.

Fish oils rank among the world's most popular nutritional supplements and are purported to have numerous health benefits. Previous work suggested that fish oils increase collagen production; however, the effect of fish oils on musculoskeletal health is poorly understood. Further, the divergent effects of omega-3 (Ω3FA) and saturated fatty acids (SFA) remains poorly understood. We tested the effects of Ω3FA and SFAs on in vitro-engineered human ligament (EHL) function. EHLs were treated with bovine serum albumin (BSA)-conjugated eicosapentaenoic acid (EPA, 20:5(n-3)), palmitic acid (PA, 16:0), or a BSA control for 6 days. EPA did not significantly alter, whereas PA significantly decreased EHL function and collagen content. To determine whether this was an in vitro artifact, mice were fed a control or high-lard diet for 14 weeks and musculoskeletal mass, insulin sensitivity, and the collagen content, and mechanics of tendon and bone were determined. Body weight was 40 % higher on a HFD, but muscle, tendon, and bone mass did not keep up with body weight resulting in relative losses in muscle mass, tendon, and bone collagen, as well as mechanical properties. Importantly, we show that PA acutely decreases collagen synthesis in vitro to a similar extent as the decrease in collagen content with chronic treatment. These data suggest that Ω3FAs have a limited effect on EHLs, whereas SFA exert a negative effect on collagen synthesis resulting in smaller and weaker musculoskeletal tissues both in vitro and in vivo.

Spatial gene expression in the adult rat patellar tendon.

Tendons are dense connective tissues with relatively few cells which makes studying the molecular profile of the tissue challenging. There is not a consensus on the spatial location of various cell types within a tendon, nor the accompanying transcriptional profile. In the present study, we used two male rat patellar tendon samples for sequencing-based spatial transcriptomics to determine the gene expression profile. We integrated our data with a mouse Achilles single cell dataset to predict the cell type composition of the patellar tendon as a function of location within the tissue. The spatial location of the predicated cell types suggested that there were two populations of tendon fibroblasts, one located in the tendon midsubstance, while the other localized with red blood cells, pericytes, and immune cells to the tendon peripheral connective tissue. Of the highest expressed spatially variable genes, there were multiple genes with known function in tendon: Col1a1, Col1a2, Dcn, Fmod, Sparc, and Comp. Further, a novel spatially regulated gene (AABR07000398.1) with no known function was identified. The spatial gene expression of tendon associated genes (Scx, Thbs4, Tnmd, Can, Bgn, Lum, Adamts2, Lox, Ppib, Col2a1, Col3a1, Col6a2) was also visualized. Both patellar tendon samples had similar expression patterns for all these genes. This dataset provides new spatial insights into gene expression in a healthy tendon.

Scleraxis and collagen I expression increase following pilot isometric loading experiments in a rodent model of patellar tendinopathy.

The effect of mechanical load on tendinopathic tissue is usually studied in the context of identifying mechanisms responsible for tendon degradation. However, loading is also one of the most common treatments for tendinopathy. It is therefore possible that different loads result in different cellular responses within a tendon. To test this hypothesis, we first established a rodent model of tendinopathy that has a transcriptional signature similar to human tendinopathy. Tendinopathy was modeled in the rat by producing a lesion in the central core of the patellar tendon using a biopsy punch, followed by two weeks to allow scar formation. We performed 3' Tag RNA-Seq to identify genes that were differentially expressed between the native and scarred rat patellar tendon. Genes involved in extracellular matrix (ECM) structure and turnover were increased, mitochondrial genes were decreased, and there was no inflammatory signature in the tendinopathic tissue. These transcriptional changes phenocopy previously published whole transcriptome analysis in human tendinopathy. After validating the model, the initial response to injury and loading was determined. Two weeks after creation of the patellar tendon lesion, the tendon was loaded using either 4 × 30s isometric or a time-under-tension matched (360 × 0.33s) dynamic protocol. Injured +/- loading and contralateral control tendons were collected eighteen hours after loading, RNA was extracted, and gene expression was quantified using qRT-PCR of the scar with or without loading. The expression of scleraxis and type I collagen increased following isometric loading relative to those loaded dynamically. By contrast, the expression of type II collagen increased in the dynamic samples relative to those loaded isometrically. These data suggest that dynamic loading of a central core tendon injury increases fibrocartilage markers, whereas long isometric loads stimulate markers of tendon regeneration.

2-month ketogenic diet preferentially alters skeletal muscle and augments cognitive function in middle aged female mice.

The effect of a ketogenic diet (KD) on middle aged female mice is poorly understood as most of this work have been conducted in young female mice or diseased models. We have previously shown that an isocaloric KD started at middle age in male mice results in enhanced mitochondrial mass and function after 2 months on diet and improved cognitive behavior after being on diet for 14 months when compared with their control diet (CD) fed counterparts. Here, we aimed to investigate the effect of an isocaloric 2-month KD or CD on healthy 14-month-old female mice. At 16 months of age cognitive behavior tests were performed and then serum, skeletal muscle, cortex, and hippocampal tissues were collected for biochemical analysis. Two months on a KD resulted in enhanced cognitive behavior associated with anxiety, memory, and willingness to explore. The improved neurocognitive function was associated with increased PGC1α protein in the gastrocnemius (GTN) muscle and nuclear fraction. The KD resulted in a tissue specific increase in mitochondrial mass and kynurenine aminotransferase (KAT) levels in the GTN and soleus muscles, with a corresponding decrease in kynurenine and increase in kynurenic acid levels in serum. With KAT proteins being responsible for converting kynurenine into kynurenic acid, which is unable to cross the blood brain barrier and be turned into quinolinic acid-a potent neurotoxin, this study provides a potential mechanism of crosstalk between muscle and brain in mice on a KD that may contribute to improved cognitive function in middle-aged female mice.