Maeda E, Shelton JC, Bader DL, Lee DA. Differential regulation of gene expression in isolated tendon fascicles exposed to cyclic tensile strain in vitro. J Appl Physiol 106: 506 -512, 2009. First published November 26, 2008 doi:10.1152/japplphysiol.90981.2008.-Mechanical stimulus is a regulator of tenocyte metabolism. The present study investigated temporal regulation of the expression of selected genes by tenocytes in isolated fascicles subjected to tensile strain in vitro. Cyclic tensile strain with a 3% amplitude superimposed on a 2% static strain was provided for 10 min, followed by either an unstrained period or continuous cyclic strain until the end of a 24-h incubation period. mRNA expression of selected anabolic and catabolic genes were evaluated with quantitative PCR at 10 min, 1 h, 6 h, and 24 h. The application of 6-h cyclic strain significantly upregulated type III collagen mRNA expression in strained fascicles compared with unstrained controls, but no alterations were observed in mRNA expression of type I collagen and biglycan. Significant downregulation in the expression of the decorin core protein was observed in fascicles subjected to 24-h cyclic strain. MMP3 and MMP13 expression levels were upregulated by the application of 10 min of cyclic strain, followed by a progressive downregulation until the end of the incubation period in both the absence and the presence of the continuing cyclic strain. Accordingly, alterations in the expression of anabolic genes were limited to the upregulation of type III collagen by prolonged exposure to cyclic strain, whereas catabolic genes were upregulated by a small number of strain cycles and downregulated by a prolonged cyclic strain. These findings demonstrate distinctive patterns of mechanoregulation for anabolic and catabolic genes and help our understanding of tenocyte response to mechanical stimulation. cyclic strain; metabolism; mechanobiology MECHANICAL LOADING is one of the regulatory factors influencing tendon metabolism. Tenocytes are responsive to mechanical stimuli through mechanotransduction processes (30) that regulate the expression of both anabolic and catabolic factors, which results in the maintenance of tendon structure and function or may lead to their alteration through remodeling. For example, the mechanical properties of tendon deteriorated when subjected to stress deprivation (33), a process associated with an upregulation of catabolic factors such as matrix metalloproteinases (MMPs) and interleukin-1␤ (10, 29) and an alteration of tissue ultrastructure (19).Details of tenocyte response to mechanical stimuli have been studied in a two-dimensional (2D) model in which the cells are cultured on an elastic membrane and subjected to a variety of mechanical stimulation regimes (30). However, it has been demonstrated that tenocytes form a three-dimensional (3D) communicating network mediated by gap junctions throughout tendon (20) and are subjected not only to tensile strain but also to compressive and shear strains (25). Thus a 3D explant sys...

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Gap junction communication is an essential component in the mechanosensitive response of tenocytes. However, little is known about direct mechanoregulation of gap junction turnover and permeability. The present study tests the hypothesis that mechanical loading alters gap junction communication between tenocyte within tendon fascicles. Viable tenocytes within rat tail tendon fasicles were labelled with calcein-AM and subjected to a fluorescent loss induced by photobleaching (FLIP) protocol. A designated target cell within a row of tenocytes was continuously photobleached at 100% laser power whilst recording the fluorescent intensity of neighbouring cells. A mathematical compartment model was developed to estimate the intercellular communication between tenocytes based upon the experimental FLIP data. This produced a permeability parameter, k, which quantifies the degree of functioning gap functions between cells as confirmed by the complete inhibition of FLIP by the inhibitor 18α-glycyrrhentic acid. The application of 1N static tensile load for 10 min had no effect on gap junction communication. However, when loading was increased to 1 h, there was a statistically significant reduction in gap junction permeability. This coincided with suppression of connexin 43 protein expression in loaded samples as determined by confocal immunofluorescence. However, there was an upregulation of connexin 43 mRNA. These findings demonstrate that tenocytes remodel their gap junctions in response to alterations in mechanical loading with a complex mechanosensitive mechanism of breakdown and remodelling. This is therefore the first study to show that tenocyte gap junctions are not only important in transmitting mechanically activated signals but that mechanical loading directly regulates gap junction permeability.

Tenocytes are known to be mechanoresponsive and the present study tests the hypothesis that distinct mechanical stimulation regimes, associated with the short-term and extended application of cyclic tensile strain, alters the balance between anabolic and catabolic processes. Microarray technology has been used to provide a comprehensive analysis of alterations in gene expression within isolated tendon fascicles in response to cyclic tensile strain using a well-established model system. Isolated rat tail tendon fascicles were subjected to cyclic tensile strain (3% amplitude superimposed on a 2% static strain) for 1 or 24 hr. Messenger RNA expression level was assessed using Illumina microarray. The number of genes significantly altered in strained fascicles from the level of unstrained control fascicles was greater at 24 hr than 1 hr. The expression levels of many extracellular matrix components remained unchanged at both time points; however, a number of members of the matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase with a thrombospondin (ADAMTS) families were significantly downregulated at 24 hr. Functional annotation revealed that upregulated genes were significantly associated with the regulation of transcription at 1 hr and translation at 24 hr. Downregulated genes were associated with inflammatory responses at 1 hr, and genes inhibited at 24 hr were significantly associated with cell apoptosis and a variety of metabolic functions. The present results suggest that the metabolic balance was shifted in favor of catabolism by the application of a small number of tensile strain cycles, whereas an extended number stimulates strong anti-catabolic effects.