Cellular and molecular dynamics of muscle stem cells during muscle hyperplasia decline in trout – FishMuSC

To understand why muscle hyperplasia decreases during growth, we combined several experimental approaches.

First, we isolated mononuclear cells from the white muscle of juvenile trout of different sizes, ranging from 10 grams to 1.5 kilograms. After removing debris and red blood cells, we counted the living cells and prepared them for single-cell gene expression analysis (snRNA-seq). This approach allowed us to identify and characterize the different cell types present in the muscle, then track changes in their activity as hyperplasia decreased. We also compared the data obtained in trout with data available in humans to highlight mechanisms conserved between species.

To test the ability of cells to form new fibers, we transplanted mononuclear muscle cells from GFP-expressing trout into the muscle of young recipient trout. Three weeks later, we analyzed the formation of new fibers in the transplanted area by quantifying the GFP-expressing fibers derived from the grafted cells.

At the same time, we performed a histological study of the muscle of trout of different sizes to measure fiber size and the number of muscle stem cells. The tissues were stained to distinguish the fiber membranes and identify the muscle stem cells. The images obtained were then analyzed automatically to quantify fiber size and cell distribution.

All of these methods enabled us to draw up a detailed map of muscle cell populations, their location, and their ability to produce new fibers during growth.

We studied the evolution of trout muscle during growth, particularly the formation of new muscle fibers (called hyperplasia). By analyzing trout weighing between 10 g and 2 kg, we observed that the total number of muscle fibers increased sharply up to 500 g, then stabilized. At the same time, the proportion of small fibers (less than 25 µm in diameter), a sign of hyperplastic activity, fell rapidly from 34% in 10 g trout to less than 6% in 500 g trout. The density of muscle stem cells (MuSC), which are essential for the creation of new fibers, also decreased with weight: it was ten times lower in 500 g trout than in 10 g trout.

We transplanted muscle cells from 10 g trout into recipient trout weighing between 10 g and 2 kg. These cells formed new fibers in recipient trout weighing 10 g or 100 g, but much less so in trout weighing more than 500 g. Conversely, cells from trout weighing more than 500 g had a greatly reduced myogenic capacity, even when placed in a favorable environment (in a young recipient). These results suggest that the decrease in hyperplasia is due to both a loss of the myogenic capacity of MuSCs and their niche.

To distinguish the effects of age from those of weight, we compared trout of the same age (12 months) but of very different weights (20 g vs. 1 kg) using dietary restriction. Conclusion: it is not age, but weight, that determines the ability of cells to produce new fibers.

At the same time, we analyzed gene expression in muscle cells to create a comprehensive atlas. We identified 15 different cell types, including myogenic cells (involved in muscle fiber formation) and mesenchymal cells. During growth, the proportion of MuSCs remains stable, while that of myoblasts falls sharply and vascular cells increase. Further analysis reveals two differentiation pathways leading from MuSCs to differentiated cells: the first, composed of highly proliferative cells (marked by mki67), is present only in hyperplastic muscle; the second, present throughout growth, consists of cells that directly engage in differentiation. These changes reflect a gradual transition from a muscle capable of creating new fibers to a muscle that develops only through fiber hypertrophy.

Finally, we observed a reorganization of the extracellular matrix that surrounds muscle cells and plays a key role in their behavior. With growth, this matrix transforms, helping to slow hyperplasia and promote hypertrophy.

This study opens up new perspectives for understanding how muscles develop and regenerate in all vertebrates. We have identified two distinct transcriptomic trajectories in muscle fiber development: one linked to the formation of new fibers (hyperplasia) and the other to the hypertrophy of existing fibers. We also highlighted the existence of a specific population of stem cells expressing both myogenic and fibrogenic marker genes, demonstrating their bipotentiality. These results provide a framework for exploring how the intrinsic programs of stem cells and their environment, known as the “niche,” evolve together during growth.

The question arises as to whether these changes in stem cells are reversible or constitute an irreversible destiny towards hypertrophy.

Our observations also show that the muscle environment undergoes profound changes with growth, as the extracellular matrix reorganizes and influences stem cell behavior. Understanding how signals from vascular and connective tissue cells influence fiber formation will improve our understanding of the mechanisms regulating hyperplasia and hypertrophy.

Beyond developmental biology, these findings could have implications for regenerative medicine. The trout model, which is capable of producing massive amounts of new fibers after hatching, allows for the study of stem cell depletion and niche transformation in a physiological context. This could help to understand the mechanisms of muscle aging or muscle diseases in mammals, and inspire strategies to improve muscle regeneration. Comparing these results with those of other fish with different growth strategies would also provide a better understanding of the evolution of the trade-off between continuous growth and stem cell maintenance.

Skeletal muscle consists mainly of muscle fibers formed during embryonic and fetal development. Except during muscle regeneration following injury, myofiber formation (hyperplasia) ceases around birth in mammals and shortly after hatching in zebrafish. Muscle hyperplasia requires the proliferation and the differentiation of muscle stem cells also called satellite cells (SCs), which are located in a distinct niche, between the basal lamina and the plasma membrane of the myofiber. Once differentiated, these cells either fuse to existing myofibers for generating larger cells (i.e. hypertrophy), or fuse together to form new myofibers (i.e. hyperplasia). Fish of agronomic interest have the extraordinary ability to exhibit a continuous post-larval muscle growth associated with the persistent formation of new myofibres (hyperplasia) that arises from muscle stem cells also called satellite cells. However, the hyperplasic muscle growth declines at the end of the exponential growth phase, and the molecular and cellular mechanisms of muscle hyperplasia arrest are still unexplored. Given that hyperplasia level determines the muscle growth potential, the project objective is to analyse the cellular and molecular dynamic of muscle stem cells during muscle hyperplasia decline in trout. To reach this objective we will study during hyperplasia decline (I) the evolution of the characteristics of satellite cell populations, (ii) the evolution of satellite cell number, (iii) the myogenic capacities of satellite cells. In this project, the use of original biological model (transplantation protocol using mlc2-GFP trout) and innovative technologies allowing in-depth investigation (RNAscope®, 3D imaging and single-cell RNA sequencing) offers a unique opportunity to decipher the cellular and molecular dynamic of muscle stem cells during muscle hyperplasia decline in trout.