Role of TGF beta family members in muscle wasting : towards innovative therapeutical approaches – TGFMyo

Experimental approach: We will generate muscle-specific genetically engineered mouse models, allowing inducible activation of a constitutively active mutant form of type I TGFß receptor (ALK5) either in adult myofibers or in adult muscle stem cells. Multi-omics analyses will provide molecular signatures associated with TGFß-mediated muscle wasting. Further explorations will help to understand the functional impact of these deregulations, and allow the identification of new muscle atrophy biomarkers and new interesting targets to prevent muscle atrophy. Our complementary translational approach will take advantage of the possibility offered by Oncodesign (biopharmaceutical company, France) to test their new inhibitors of TGFß receptors (Nanocyclix®) in our paradigm. We have already generated enough preliminary results to ensure the pertinence of our models and the feasibility of the project.

General characterization of skmRCA mice phenotype (Task1.1) on four different time points 4 days, 1 week, 4 weeks and 12 weeks after tamoxifen treatment: we observed progressive muscle atrophy without evidence for overt myopathy and fibrosis. A shift in muscle fiber type composition towards more oxidative fibers was observed.
skmRCA mice displayed muscle weakness involving altered excitation/contraction coupling.
Gene expression profile at early pre-cachectic stage (4 days after TAM) and in atrophied muscle (4 weeks after TAM): at both stages, RNAseq analyses confirmed upregulation of molecular signatures relative to TGFb pathway activation. Early downregulated signatures were relative to muscle contraction, glycolysis, calcium handling, fatty acid metabolism, whereas the most upregulated signatures were relative to myogenesis, TNF-a signaling, immune system, PI3K/AKT/mTOR signaling, apoptosis signaling and autophagy. At atrophied stage, among the most downregulated biological pathways/signatures, we identified muscle contraction, calcium handling, energy reserve metabolic process, cellular amino acid catabolic process and carbohydrate catabolic process, whereas the most upregulated biological process/signatures were relative to immune response activation, extracellular matrix organization and neuromuscular junction maintenance. These molecular signatures and the most deregulated target genes form a solid and essential basis for our ongoing and future investigations
Interestingly, myofibers showed central fiber areas depleted of mitochondrial activity suggesting mitochondrial metabolism alterations as notably observed in muscle aging. Electron microscopy on late stage (12 weeks after TAM) confirmed presence of mitochondria abnormalities (dilated and giant mitochondrion, onion-like inner membrane structures) suggesting deregulation in mitochondrion fusion/fission dynamics. Mitochondrial content quantified with quantitative PCR of mitochondrial DNA, showed a 25% decrease at 4 weeks after TAM treatment, indicating alteration of mitochondrial biogenesis/maintenance. Interestingly, it was correlated with reduced expression of PGC1a and TFAM, key regulators involved in mitochondrial biogenesis. Thus, activation of TGFb signaling leads to altered mitochondrial biogenesis in the first weeks after TAM treatment.
Overall, we investigated all the topics planned in the WP1, we are currently completing analyses to finish this first part. Data obtained in WP1, especially transcriptomic, metabolomics and lipidomics, provide preliminary and essential results for WP2, WP3, WP4 investigations.

Analysis of RCA muscles allowed us to determine new molecular signatures of TGFß-mediated muscle atrophy. Among them, we identified a strong dysregulation of polyamine (PA) metabolism. The downregulation of PA metabolism is an emerging pathway involved in muscle mass regulation that is notably altered in myopathies and during muscle aging. PA treatments could therefore represent interesting therapeutic options. Thus, we hypothesize that TGFß signaling is a direct/indirect regulator of polyamine synthesis genes expression in skeletal muscle and that deregulation of polyamine content might contribute to muscle atrophy/dysfunction in RCA mice model.
We propose now to specifically target the most interesting candidates/signaling pathways/metabolic pathways identified through transcriptomic/metabolomics approaches as potential atrophy modulators to understand their contribution to muscle mass, muscle metabolism and muscle function regulation (as proposed in WP4). We will use both in vitro and in vivo approaches to modulate levels of these targets and analyze the consequences on muscle cell growth, metabolism and contractile properties. We are first focusing on identified strong downregulation of polyamine (PA) metabolism in RCA muscles (strongly decreased protein levels of key enzymes and 40% decrease in spermidine content). PA are ubiquitous polycations implicated in a large number of cellular process including replication, transcription, translation, post-translational modification, ion channel gating, and membrane stability, protein acetylation. Downregulation of PA metabolism is an emerging metabolic pathway involved in muscle mass regulation, which is notably altered in myopathies and during muscle aging. PA treatments could therefore represent interesting therapeutic options. Investigations are ongoing to (i) to dissect molecular mechanisms involved in TGFb mediated downregulation of polyamine biosynthesis, (ii) investigate on the muscle consequences of reduced polyamine content (iii) investigate if targeting PA metabolism is an interesting therapeutic option to preserve muscle mass in RCA muscles.
RCA mice also provided interesting data regarding the impact of “intrinsic” myofiber atrophy (in contrast to existing models of muscle atrophy that concomitantly affect many cell types) on muscle microenvironment (WP2) and whole body homeostasis (WP3). We observed that reduced muscle mass in RCA mice is associated with a progressive increase in fat mass, impaired muscle glucose metabolism and altered muscle lipid content. In addition, both transcriptomic and metabolomic analyses indicated modifications in the levels of factors secreted by the muscle in RCA mice that might have consequences on the muscle itself, other organs and whole body homeostasis. We will next evaluate the consequences of chronic activation of TGFb signaling in skeletal muscle on the muscle regenerative capacities and whole-body homeostasis as detailed in WP2 and WP3.

12th international SCWD conference on muscle cachexia sarcopenia and muscle wasting (06/12/2019) (poster JSFM Marseille)

Context: Muscle wasting is a life-threatening syndrome involved is a wide range of cachexia-associated chronic diseases such as cardiac failure, myopathies, chronic pulmonary or kidney failure, Cushing syndrome, cancer as well as muscle aging. Despite its clinical importance, there is no approved effective chemical treatment against muscle wasting, which is usually associated with drastic loss of adipose tissue and many organ dysfunctions because of the systemic spread of these diseases. Among the signaling molecules involved in cachexia, members of the Transforming Growth Factor ß (TGFß) family (TGFß, myostatin, GDF11 and activins) are major atrophying factors.

Challenge: Because of the systemic effects of TGFß ligands and the important redundancy existing among the ligands and receptors of this family, data generated so far by ligand or receptor loss or gain of function have been complex to interpret.

Hypothesis: We make the hypothesis that chronic TGFß activation not only involve imbalance muscle catabolism/anabolism (excessive proteolysis leading to muscle fibers shrinkage) but also many, yet poorly explored aspects of muscle biology (energy metabolism, calcium homeostasis, regenerative capacities, muscle endocrine function, neuromuscular junction). Targeting intracellular effectors of TGFß signaling or downstream cellular consequences is then expected to limit muscle weakness associated with chronic diseases.

Objectives: We will provide an unambiguous molecular description of the effects of muscle-specific chronic activation of TGFß signaling pathway on muscle mass function (WP1), muscle regenerative capacity (WP2) and the repercussion of these induced muscle alterations on whole body homeostasis (WP3). In a translational approach (WP4), we will target interesting effectors and cellular processes of muscle atrophy identified in WP 1 ,2 and 3 to initiate innovative therapeutic approaches aiming at decreasing TGFß signaling in skeletal muscle (and/or downstream targets/cellular consequences) for the prevention and the treatment of muscle atrophy.

Experimental approach: We will generate muscle-specific genetically engineered mouse models, allowing inducible activation of a constitutively active TGFß receptor (ALK5) either in adult myofibers or in adult muscle stem cells. Multi-omics analyses will provide molecular signatures of TGFß-mediated muscle wasting. Further explorations will help to understand the functional impact of these deregulations, and allow the identification of new muscle atrophy biomarkers and new interesting targets to prevent muscle atrophy. Our complementary translational approach will take advantage of the possibility offered by Oncodesign (biopharmaceutical company, France) to test their new inhibitors of TGFß receptors Nanocyclix® in our paradigm. We have already generated enough preliminary results to ensure the pertinence of our models and the feasibility of the project.

Consortium: Partner 1 is a specialist of skeletal muscle biology with a wide expertise ranging from molecular biology and gene expression to intracellular signaling, metabolism, and physiology of muscle cells. Partner 2 is a specialist of TGFß signaling. He has developed the molecular tools to generate the mouse models. Thanks to their longstanding expertise in drug screening, the collaboration with Oncodesign will be an asset for the targeted screening part of the project.