Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - REBUILD (Mechanisms of sarcomeric protein homeostasis during physical exercise and ageing)

Teaser

During ageing muscle fibers are constantly subjected to high forces resulting in mechanical stress on cells and proteins. As a consequence, they can degenerate, leading to suboptimal force generation, lack of coordinated movements and ultimately to muscle failure in the...

Summary

During ageing muscle fibers are constantly subjected to high forces resulting in mechanical stress on cells and proteins. As a consequence, they can degenerate, leading to suboptimal force generation, lack of coordinated movements and ultimately to muscle failure in the elderly population. This ageing process that leads to a progressive loss of muscle function has a major health impact on the ageing Western society. Physical activity is known to delay this decline.

The REBUILD project aims to determine the temporal dynamics of protein replacement during life of a muscle fiber by SILAC-based mass spectrometry and genetics. It integrates biomechanics with muscle homeostasis and accurately quantify the impact of physical activity on protein turnover and investigate the physiological consequences for the ageing animal. By combining the power of genetics and SILAC-based proteomics with behaviour of free flight under defined exercise conditions, we create a unique position for a discovery orientated large-scale in vivo genetic screen.

We determined the temporal dynamics of protein replacement during the life of flight muscle fibers in Drosophila by SILAC-based mass spectrometry. Protein replacement is assessed by incorporation of heavy Lys8 in newly synthetized proteins, allowing us to obtain turnover rates for the entire proteome in an unbiased manner. By employing a newly designed Flydome, an apparatus to induce Drosophila flight on demand, together with animals incapable of flight-muscle contraction we quantify the impact of muscle contraction on protein turnover rates and investigate the physiological consequences for the ageing subject. Our initial analysis reveals a surprisingly low turnover of sarcomeric and structural proteins in adult animals, that might be a reflection on the high density and stability of muscle fibers. Nevertheless, a subset of these sarcomeric muscle components turns over at higher rates, which is surprising due to their “core sarcomeric” nature. Further validation studies are now due as well as the study of the mechanisms behind this protein turnover.

A precise understanding of mechanisms that control homeostatic replacement of damaged proteins in muscle fibers should foster interventions aimed at maintenance of muscle capacity in older individuals and thus, an important step towards healthy ageing.

Work performed

In order to understand the frequency of exchange of sarcomeric proteins in the muscle, two major state-of-the-art techniques were employed in the REBUILD project:

1) Mass-spectrometry combined with labelled aminoacids. Animals are fed a diet where the sole source of Lysine is a heavier isotope (Lys8) that causes a mass shift of 8Da on a mass spectra, upon digestion of all proteins with an enzyme called lysyl endopeptidase (LysC). In this manner, we can identify newly synthesized proteins after defined time points via incorporation of Lys8 and comparing the ratio of light (before the shift in food source) vs heavy (after introducing the Lys8 food) peptides. This heavy:light ratio informs on the rate of replacement for any given protein in the muscle cell, since mass-spectrometry allows for unbiased detection of the entire proteome.
2) RNA-Sequencing of dissected muscle fibers. To better understand regulators of protein replacement, we performed performed massive next-gen sequencing of RNA from dissected flight muscle of Drosophila after different activity interventions and at several age points, detailed below.
3) A novel “exercise chamber” to stimulate and track flight of free-flying Drosophila. We developed and built a large “Flydome” to stimulate flight-on-demand of populations of hundreds of flies, simultaneously, and combined it with camera-vision and dedicated software to track and quantify flight of all flies. This allowed to obtain muscle from “exercised” flies which we then compared to control flies and “non-flyers” – flies that are unable to contract their flight muscles, due to genetic ablation of their motor neurons. These 3 mechanical/activity interventions (control, exercise, no-flight) we employed to obtain muscle samples with different activation histories, and also, from flies at different stages along their lifespan. These samples were analysed via the techniques mentioned above.
4) Genetic tools for observing directly protein exchange. In order to confirm and study in depth the mechanisms involved in protein turnover, new genetic tools that take advantage of existing regulatory mechanisms were developed. We combined temporal and cell-specific enhancers (both via tissue-specific GAL4 enhancers and flipable fluorescent reporters) with, so-called nanobodies, to control incorporation of green fluorescent proteins attached to newly synthesized sarcomeric proteins. This allowed observation of introduction of new components in sarcomeres after a experimentally defined stimulus.

Final results

From our analysis, which is still on-going, despite considerable gene expression of several sarcomeric components, we find that turnover rates are extremely low for most proteins. This could be a reflection of the crystal-like nature of the sarcomeric apparatus and the challenges for the proteostasis machinery in accessing regions of the sarcomere to both remove and replace components. Nevertheless, we were able to measure incorporation of specific proteins, some of them core members, such as Sallimus, which is surprising due to the aforementioned challenges. This raises the interesting question of how it is possible to exchange such large proteins in an apparatus that is frequently active at high rates (the Drosophila flight muscle cycles through contraction/relaxation at a rate of 200 times per second) and in a muscle type that is hardly subject to changes due to hypertrophy. Highly specialized chaperones must be employed in such a targeted process, for which the fruit fly will continue providing a good starting point for discovery, namely for the easiness in performing gain- and loss-of-function genetic screens. This work could potentially uncover novel regulators associated with loss of muscle function in myopathies and in the elderly.

Website & more info

More info: http://muscledynamics.org/.