Uncovering mechanisms underlying the transdifferentiation of human muscle fibroblasts into adipocytes

Lead Research Organisation: King's College London
Department Name: Ctr of Human & Aerospace Physiolog Sci

Abstract

Skeletal muscle is the largest and one of the most important tissues in the body. Not only does it perform obvious functions in allowing us to move and perform all the necessary physical tasks of daily living, it also has numerous other vital functions which are fundamental to health. These include being the most important source for the uptake of glucose in the body. However, there are numerous conditions where muscle loss occurs and "quality" declines, with the accumulation of intramuscular fat and fibrosis impairing both contractile and metabolic function. This occurs particularly in the muscles of frail elderly people. Given the fact that people are now living longer, this is resulting in a dramatically increasing population of people affected. Furthermore, there are also numerous diseases such as obesity and type 2 diabetes, as well a range of muscular diseases where fibro-fatty accumulation occurs. However, despite the prevalence the basic biological processes that influence these changes remain unclear.

We have recently demonstrated that a population of cells resident within human skeletal muscle, called "fibroblasts" (which give rise to fibrosis), are also the cells that have the capability of giving rise to fat cells and fatty deposits.. The work outlined in this application is targeted at uncovering the molecular mechanisms which drive the process of a fibroblast to become a fat cell, in order to try to prevent or ameliorate fibro-fatty accumulation. With the combined expertise of biomedical researchers at King's College London and GSK, our collaborating industrial partner, we specifically aim to identify the molecules that initiate the events that cause a fibroblast to change into a fat cell; to chart the events that underlie the waning of the fibroblast characteristics and the waxing of those events that produce a fat cell; determine if the fibroblasts are viable and give rise to fat cells in vivo; and show that if you ablate the fibroblasts in skeletal muscle whether this prevents or impairs the fatty accumulation.

To achieve these aims this grant brings together state-of-the-art techniques in human cell culture, cell imaging, RNAdeep sequencing, and in vivo work. It is a multidisciplinary collaboration of expertise in muscle biology at King's College with GlaxoSmithKline who have a parallel research programme targeted at muscle ageing and regeneration.

The results of these experiments promise new insights into the mechanisms driving cell fate in skeletal muscle and have the potential to form the basis of new therapeutic agents directed at preventing fibro-fatty replacement in muscle.

Technical Summary

There are number of conditions such as in sarcopenia, where muscle quality declines, becoming fatty and fibrotic. The mechanisms underlying this process are not known. Recent research in our laboratory using human primary cells cultures has demonstrated clearly that it is the fibroblast cells (identify by a range of markers including TE7, TcF4, vimentin etc.) which not only produce fibrous tissue, but also have the ability to transdifferentiate into fat cells. They accumulate large amounts of lipid, alter their morphology, lose their fibrogenic markers, and increase expression of the adipogenic transcription factors. By contrast, myogenic cells do not possess this ability.

The aim of this project, undertaken in collaboration with our industrial partner, GSK, is to identify and characterise the molecules responsible for the initiation of this transdifferentiation process. We intend to undertake sensitive time course studies where, using the latest transcriptomic technologies (RNA sequencing) we will track the ebb of the fibroblast phenotype with the flow of the adipocyte phenotype as characterised by detailed immunocytochemistry and time lapse cell imaging. We will also undertake experiments using mouse models to demonstrate in vivo viability of the transdifferentiated human cells and to characterize the role of fibroblasts in endogenous fibro-fatty accumulation. The first of these will use immunocompromised mice (Rag2-/- yC-/- C5-/-) to determine the ability of these human cells to form adipocytes in vivo (forming fat pads and in muscle regeneration). The second will use a conditional fibroblast knockout mouse (TCF4CreERT2mice crossed with R26RDTA) to characterise the adipogenic potential of fibroblasts when activated in vivo in a fibro-fatty degeneration model.

This research will provide the mechanistic basis for fibro-fatty transdifferentiation, opening up new avenues for the development of novel and innovative therapeutics.

Planned Impact

Impact summary
Impact from the proposed research will be maximised in five ways:
1. Impact via industry collaboration
For this proposed project we have formed an important collaboration with Dr William Evans (Vice President, Head of Muscle Metabolism Discovery Performance Unit) at GSK. This project aligns closely with the work of Dr Evans and his team in regard to therapies targeted at the treatment of sarcopenia, muscle regeneration and stem cell activation. GSK have identified the synergy of this project with their research programme and strategic plan. Our project will have impact in providing the biological basis for potentially developing new therapeutic target at tackling fat and fibrosis in skeletal muscle in ageing and, in particular, during recovery from muscle damage. In addition a further impact of this project will be the potential for ongoing collaborative studies between our group and the Muscle Metabolism Discovery Performance Unit at GSK in targeting fat and fibrosis specifically, but sarcopenia and muscle regeneration more generally.

2. Impact via the discovery of new molecules
One of the advantages of using mRNA deep sequencing in a process such as transdifferentiation is that new molecules are almost sure to be uncovered. The role of these molecules in determining the phenotype of a cell will be pursued and may lead to new understanding of complex cell metabolic pathways. Understanding the role of these molecules and cell types may lead to discovery of novel therapies to enhance regeneration of skeletal muscle.

3. Impact on the scientific community
There is a wide range of expertise gathered together to bring this work to fruition. The expertise involves cutting edge analyses such as mRNA deep sequencing, cell culture, immunological staining and confocal miscroscopy, state of the art real time imaging (Nikon Imaging Centre) and the use of in vivo animal models to importantly confirm that the in vitro findings are relevant to in vivo biology. It can be readily perceived that data, generated from this range of expertise, will have relevance to many scientific disciplines (cell biology, molecular biology & physiology)and it is anticipated that results will be published in a wide range of high impact scientific journals. Results will also be disseminated at appropriate scientific meetings and at a meeting we ourselves intend to host.

4. Impact via engagement of the public in science
All of the senior members of this application are members of scientific societies and take active roles in trying to engage interested members of the public, particularly younger people, in science, and are STEM ambassadors. The Centre of Human Aerospace and Physiological Sciences has an outstanding track record in endeavours aimed at engaging and enthusing young people about science.

5. Impact by training of a post-doctoral research assistant
As with any reputable scientific centre we are concerned that young scientists who come for training should be given as wide a scope as possible in order to keep their future scientific options open. Our research assistant Chibeza comes with a wide array of cell biology tools that he has developed during his PhD, however, this project will give him excellent opportunities to develop his portfolio of which will include RNA deep sequencings, real time microscopy and train in the use of a variety of in vivo mouse models to confirm our in vitro findings. This will be given added value though our interaction with GSK a global leader in the pharmaceutical industry. We anticipate this will equip him with wide ranging expertise fitted to a wide range of future directions and excellent career development.
 
Description The aim of this project is to gain insight into the mechanisms underpinning the trans-differentiation of human skeletal muscle fibroblasts into adipocytes. We characterised these transdifferentiated fibroblasts using a range of adipocyte markers establishing that they are positive for the adipocyte markers: Acetyl CoA Carboxylase, Fatty Acid Synthase, Perilipin and FABP4. We have performed live cell imaging (time-lapse images were taken every 30 minutes over 72 hours of fatty acid treatment) to shows how highly dynamic the process of transdifferentiation as fibroblasts synthesise lipids and transdifferentiate into adipocytes.
We have also tested the separate effects of different fatty acids (oleic and palmitic) on fibroblasts and observed that if delivered alone, palmitic acid has a clear toxic effect on the cells, while this is rescued by the addition of oleic acid. We have also combined the adipocyte inducing medium (AIM) treatment and oleic acid treatment, which results in the most transdifferentiated phenotype.
The major part of the project has involved a time-course study to analyse the transdifferentiation of fibroblasts into adipocytes. Several time-points have been studied characterise the transcriptome using both microarrays and RNAseq. This will provide further information as the results from both transcriptomic techniques will be compared. These are coupled with a deep immunocytochemical phenotyping and characterisation of the secretome. These data are currently being analysed.
We have also completed a cross-sectional study comparing the phenotype of fibroblasts obtained from different tissues, namely: skin, lung and muscle. We have also compared their respective abilities to transdifferentiate. We have observed similar marker expression between the fibroblasts of different origins, but a different potential for transdifferentiation. These data are currently being analysed and being written up for publication
We have performed in vivo xenotransplantation experiments where GFP labelled human fibroblasts were injected into a mouse model (fat pad assay) to determine if these cells could transdifferentiate in vivo. We are completing the immunohistochemical analysis of these samples.
In work closed allied to this project we have discovered that: i) that Wnt /beta-catenin signalling is a key regulator of muscle progenitor cell differentiation and ii) that DUX4-mediated activation of Ret prevents myogenic differentiation which could contribute to the pathology of Facioscapulohumeral muscular dystrophy by preventing satellite cell-mediated repair. These have been published. Further allied work which is currently under review has shown that Vgll3 proteins in muscle operate via Tead transcription factors to repress gene expression to influence proliferation and differentiation in skeletal muscle.
Exploitation Route This work has formed the basis of a MRC GRCF proposal in collaboration with St John's Research Institute, Bangalore (and Imperial College London and University of Cambridge) in regard to lipid deposition in skeletal muscle of different phenotypes of 2 diabetes present among Indians.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology