Introduction It is known that core temperature in Achilles tendon rises up to around 45°C during intensive exercises in both animal and human models. It may indicate that both overloading and hyperthermia are involved in the onset of tendinopathy. Although a large number of studies have been performed on effects of overloading on tenocyte functions, only a few studies have been performed on effects of hyperthermia on tenocyte activities. In these studies, lethal effects of hyperthermia on tenocytes were mainly reported. However, little has been known about effects of hyperthermia on tenocyte functions, in particular regulation of catabolism.
Gap junctions are known to be involved in mechano-response of tenocytes. It is demonstrated that gap junction and associated intercellular communication (GJIC) are regulated by mechanical loading.1 However, how GJIC in tenocytes is changed under hyperthermia remains unknown. Therefore, the present study was performed to evaluate changes in tenocyte physiological functions and GJIC. It was hypothesised that hyperthermia upregulates tenocyte catabolism and downregulates GJIC.
Methods Tenocytes were isolated from rabbit Achilles tendon and cultured in a microgroove device2 for 24 hours, at cell density of 2000 cells/cm.2 To evaluate cell viability and mRNA expression analysis, tenocytes were maintained at 37, 41 and 43°C for 30 min and then incubated at 37°C for 24 h. Cell viability assay was performed by staining viable cells with calcein-AM and dead cells by ethidium homodimer. qPCR was performed to measure mRNA expressions for COL1 (type I collagen), MMP-1 (Matrix metalloproteinase-1), IL-1β (Interleukin-1β) and CASP3 (Caspase-3).
For determination of GJ diffusivity, tenocytes were stained by calcein-AM and incubated at 37, 43°C for 30 min followed by being subjected to FLIP experiment(1) modified for the present study. Laser photobleaching to a target cell was repeated 100 times with the laser power of 70 μW.
Results Average cell viability at 37, 41, 43°C were 95.5, 76.0 and 60.5% respectively, although the changes were not statistically significant. Fig 1 shows results from mRNA expression analysis. Expressions of MMP-1 and IL-1β were elevated at 43°C compared to that 37°C. Meanwhile, expression of COL1 and CASP3 decreased from 37 to 43°C.
Diffusion coefficient of gap junction at 37 and 43°C was 1.57 and 2.18 µm2/s, respectively. Intracellular diffusion coefficients at 37 and 43°C was 15.68 and 33.55 µm2/s, respectively.
Discussion The observed tend of the decreased cell viability by hyperthermia is in an agreement with a previous report(3). Increases in expressions of MMP-1 and IL-1β and decrease in expressions of COL1 indicate that hyperthermia upregulates tenocyte catabolism and downregulates anabolism. As MMP-1 and IL-1β are markers for inflammation, results suggest that inflammation is triggered by hyperthermia. As expression of CASP3 is a marker of cell apoptosis, decreased expression of CASP3 by hyperthermia indicates that cell death induced by hyperthermia is not due to apoptosis.
Gap junction diffusion coefficient increased at 43ºC, suggests that GJIC is enhanced under hyperthermia, possibly to protect tenocytes from high temperature and/or to recover the damage by heat stress.
References 1 Maeda E, et al. Biomechanics and Modeling in Mechanobiology. 2012;11:439–447
2 Maeda E, et al. Biomedical Microdevices. 2013;15:1067–1075
3 Birch HL, et al. The Journal of Experimental Biology. 1997;200:1703–1708
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