Circadian and permanent pools of extracellular matrix co-exist in tendon tissue, but have distinct rates of turnover and differential responses to ageing

Abstract

Heavy carbon isotopes in the tendons of people who grew up in the age of nuclear bomb testing have shown that the extracellular matrix (ECM), assembled during development, stays with us for life. However, recent work suggests that type-I collagen in ECM-rich mouse tendon exists in two pools: a permanent matrix, and a more soluble, circadian-regulated matrix. Despite this, the underlying regulation of such distinct pools is not understood. Here, we demonstrate using stable isotope labelling coupled with mass spectrometry proteomics that circadian and permanent matrix pools have significantly different half-lives. Furthermore, the properties of the matrix pools are altered during development and ageing. Tail tendon tissue was harvested from mice fed on a heavy-lysine diet; protein was then extracted for analysis using a sequential two-step protocol. The first, soluble fraction ( F1) was found to contain intracellular proteins, and a range of core and associated extracellular matrix proteins, including a pool of type-I collagen shown to be circadian-regulated. The remaining fraction ( F2) contained primarily collagens, including type-I collagen which did not show rhythmicity. In adult mice, matrix proteins extracted in the F1 pool had significantly shorter half-lives than F2, including type-I collagen which had half-lives of 4 {+/-} 2 days in F1, compared to 700 {+/-} 100 days in F2. Circadian-regulated matrix proteins were found to have significantly faster turnover than non-circadian in adult mice, but this distinction was lost in older animals. This work identifies protein turnover as the underlying mechanism for the circadian/permanent model of tendon matrix, and suggests a loss of circadian regulation as a characteristic of ECM ageing.