BACKGROUND

Muscle atrophy occurs during physiological (i.e., fasting) and pathological conditions (i.e., cancer) and anticipates death. Since not all patients will undergo muscle wasting, it would be highly useful to identify them soon to intervene early.

We aim to generate a reporter system to follow only pathological, but not physiological, muscle wasting through in vivo imaging.

METHODS

Comparing the upstream non-coding regions of a subset of atrophy-related genes or atrogenes, using the MuRF1 promoter as a backbone, we cloned various promoters upstream of Firefly Luciferase. The best hits selected in vitro were further compared in in vivo imaging if able to sense early atrophy induced by MCG101 sarcoma or sciatic nerve resection through plasmid electroporation or AAV9 injections.

The best promoter was used to generate the reporter mouse MyoRep, expressing the cassette in all skeletal and cardiac muscles using the loxP system.

RESULTS

Luciferase assays showed that only the newly generated promoters of MuRF1, one containing glucocorticoid-responsive elements or GRE (TWIST) (p ≤ 0.01, 1.7 FC) and a GRE-less promoter (GREDEL) (p ≤ 0.0001, 1.6 FC), discriminated the supernatants from cachectic tumoural cells (C26) from non-cachectic ones (4T1). Comparing both reporters electroporated in leg muscles, we found that GREDEL, but not TWIST, anticipated atrophy by 6 days in MCG101 carriers (p ≤ 0.05) and by 8 days upon denervation (p ≤ 0.05), recapitulating MuRF1 inductions.

TWIST, but not GREDEL, drove an undesirable bioluminescent signal in vitro to dexamethasone (p ≤ 0.001, 1.5 FC) and in vivo upon fasting (p = 0.0553, 3 FC). GREDEL-carrying AAV9 injected in the legs of Apc Min/+ mice unraveled sex-different cachexia and anticipated body emaciation by 1 week (p ≤ 0.001, 3.7 FC).

GREDEL was then used to generate the MyoRep mouse. Dorsal view of bioluminescent signal of MCG101-carrying MyoRep mice increased already 6 days from tumour injection (p ≤ 0.01, 1.7 FC) when tumour is still unpalpable.

Denervated MyoRep mice emitted a signal already 1 day after surgery (p ≤ 0.05, 1.4 FC), anticipating atrophy. Male Apc Min/+ mice display less musclin in their muscles (p ≤ 0.05, 0.4 FC) and plasma (p ≤ 0.01, 0.6 FC).

Such mice, when expressing MyoRep in their muscle legs, were given the anti-catabolic myokine musclin. The emitted signal was decreased by 30% 3 weeks after musclin-AAV9 administration (p ≤ 0.05), supporting MyoRep useful to test anti-atrophic drugs.

CONCLUSIONS

Since MyoRep detects only pathological atrophy anticipating wasting, it represents an unprecedented tool to predict it early in diseases with local or systemic atrophy.

It could also be useful to identify early biomarkers of atrophy and new drugs at once.