Skeletal muscle repair and regeneration
Many devastating degenerative conditions can be traced to a failure of the body's cells to repair or regenerate tissues. This can occur during aging and in greatly accelerated forms in patients who have inherited mutations that make cells prone to injury.
Skeletal muscles allow body movement, e.g. walking but also breathing. There are more than 650 skeletal muscle groups in the human body, making up around 40% of the body weight. The cellular units of the muscle are myofibers, large syncytial cells that can contain hundreds of nuclei and that arise by cell fusion. Each muscle contains many bundles of myofibers, which are surrounded by connective tissue. Myofibers contain the force-generating machinery of the muscle, the sarcomeres.
Skeletal muscle can be repaired by two distinct mechanisms. The first mechanism is fiber-autonomous: it re-seals small damage to the sarcolemma, or removes and replaces damaged sarcomeres. The second, a stem-cell based mechanism, replaces necrotic muscle fibers by new fibers generated from a small pool of muscle-specific stem cells. My laboratory investigates the molecular mechanisms of skeletal muscle repair and regeneration using mouse genetics, innovative life imaging, and modern molecular biology techniques like mRNA sequencing of single nuclei (snRNAseq).
Skeletal muscle regeneration
Skeletal muscle has a remarkable regenerative capacity due to resident stem cells that can form new muscle fibers. Stem cells of the skeletal muscle represent a small cell population in the postnatal muscle that were originally defined as satellite cells by their anatomical location between the basal lamina and plasma membrane of the myofiber. These stem cells derive from myogenic progenitor cells and are marked by Pax7. They proliferate during the postnatal period and generate differentiating cells for muscle growth. In the adult, muscle stem cells are quiescent but become activated and proliferate upon muscle injury. The proliferative muscle stem cells can either self-renew or differentiate to generate new muscle tissue.
The maintenance of the muscle stem cells depends on exogenous Notch signals. Genetic ablation of Notch signaling by mutation of the genes encoding the ligand Dll1 or the transcriptional mediator of Notch signals, RBPj, results in up-regulated MyoD expression, premature myogenic differentiation, and the depletion of the muscle stem cell pool (Vasyutina et al. 2007, doi: 10.1073/pnas.0610647104; Bröhl et al., 2012, doi: 10.1016/j.devcel.2012.07.014). Conversely, forced Notch activation suppresses myogenic differentiation. Genetic evidence demonstrates that suppression of MyoD expression is an important aspect of Notch function, and that uncontrolled MyoD expression is responsible for premature myogenic differentiation and the depletion of the muscle stem cell pool in Notch signaling mutants (Bröhl et al., 2012, doi: 10.1016/j.devcel.2012.07.014).
In recent years, we have found that activated muscle stem cells express a network of genes in an oscillatory manner that regulates the maintenance of muscle stem cells. This Network of oscillating genes encompasses Dll1, Hes1 and MyoD (see below). Moreover, we demonstrated that these oscillations are not only an odd phenomenon, but functionally important.
Oscillations of MyoD and Hes1 proteins regulate the maintenance of activated muscle stem cells
The balance between proliferation and differentiation of muscle stem cells is tightly controlled, ensuring the maintenance of a cellular pool needed for muscle growth and repair. Hes1 is a transcriptional repressor, and Hes1 expression is induced by Notch signaling. Using mouse genetics, we demonstrated that Hes1 controls the balance between proliferation and differentiation of activated muscle stem cells in both developing and regenerating muscle. Interestingly, Hes1 is expressed in an oscillatory manner in activated stem cells where it drives the oscillatory expression of MyoD. MyoD expression oscillates in activated muscle stem cells from postnatal and adult muscle under various conditions: when the stem cells are dispersed in culture (Fig.1A,B), when they remain associated with single muscle fibers, or when they reside in muscle biopsies. Unstable MyoD oscillations and long periods of sustained MyoD expression are observed in differentiating cells (Fig. 1C). Furthermore, ablation of the Hes1 oscillator in stem cells interferes with stable MyoD oscillations and leads to prolonged periods of sustained MyoD expression. This results in an increased differentiation propensity of the muscle stem cells. As a consequence, muscle growth and repair are severely impaired. We conclude that oscillatory MyoD expression allows the cells to remain in an undifferentiated and proliferative state, which is required for amplification of the activated stem cell pool (summarized in Fig. 2; for more information see Lahmann I, Bröhl D, Zyrianova T, Isomura A, Czajkowski MT, Kapoor V, Griger J, Ruffault PL, Mademtzoglou D, Zammit PS, Wunderlich T, Spuler S, Kühn R, Preibisch S, Wolf J, Kageyama R, Birchmeier C., Genes Dev. , 33:524-535. doi: 10.1101/gad.322818.118).
Figure 1: MyoD protein expression oscillates in proliferating muscle stem cells, and the expression dynamics changes when the muscle stem cells differentiate.
A) Schematic display of the MyoD-Luc2 fusion gene construct used to visualize dynamic expression of MyoD. B) Bioluminescence images of a MyoD-Luc2 muscle stem cell kept in culture in proliferation medium. The quantification of the luciferase signal is shown to the right. C) Bioluminescence images of MyoD-Luc2 in a cultured muscle stem cell; differentiation was induced by the addition of differentiation medium (first arrow). The fusion of the cell to a myofiber occurred at the end of the observation period. The quantification of the luciferase signal is shown to the right.
Figure 2: Hes1 and MyoD are dynamically expressed in activated muscle stem cells. Scheme showing oscillatory gene expression of Hes1 (blue) and MyoD (red) in activated muscle stem cells (pale blue). Sustained MyoD expression is observed when muscle stem cells differentiate (dark blue).
Oscillatory expression of Delta-like 1 regulates the balance between differentiation and maintenance of muscle stem cells in development and regeneration
Cell-cell interactions mediated by Notch are critical for the maintenance of skeletal muscle stem cells and suppress their differentiation. However, dynamics, cellular source and identity of functional Notch ligands during expansion of the stem cell pool in muscle growth and regeneration remain poorly characterized. We recently demonstrated that oscillating Delta-like 1 (Dll1) produced by myogenic cells is an indispensable Notch ligand for self-renewal of muscle stem cells in mice (Fig. 3 and Movie 1). Dll1 expression is controlled by the Notch target Hes1 and the muscle regulatory factor MyoD. We generated a mathematical model of the oscillatory network. Consistent with our mathematical model, our experimental analyses showed that Hes1 acts as the oscillatory pacemaker, whereas MyoD regulates robust Dll1 expression. Interestingly, interfering with Dll1 oscillations without changing the overall expression level of Dll1 impairs self-renewal of muscle stem cells during muscle growth and regeneration, and results in premature differentiation. We conclude that the oscillatory Dll1 input into Notch signaling ensures the equilibrium between self-renewal and differentiation in myogenic cell communities. Importantly, sustained Dll1 cannot replace the oscillatory Dll1 input. For more information see Yao Zhang , Ines Lahmann, Katharina Baum, Hiromi Shimojo, Philippos Mourikis, Jana Wolf, Ryoichiro Kageyama and Carmen Birchmeier, Nat Commun . 2021, 12:1318. doi: 10.1038/s41467-021-21631-4.
Figure 3: Dll1 expression oscillates in muscle stem cells in sphere culture
A) Schematic drawing of myogenic stem cells grown in a sphere. A single cell was co-transfected with EpDll1-NanoLuc (a plasmid in which the Dll1 enhancer and promoter drive NanoLuc expression) and nGFP expression plasmids and is surrounded by many untransfected cells. B) Visualization of a transfected cell in a sphere by its GFP expression. C) NanoLuc bioluminescence signal detected over time in the single GFP+ muscle stem cell in a sphere by live imaging. D) Quantification of the NanoLuc bioluminescence signal detected in this cell.
Movie 1: Dynamic Dll1 expression.
Left: Live imaging of the NanoLuc bioluminescence signal detected in a muscle stem cell cultured in a sphere; the corresponding images showing the GFP signal are shown on the right.
Single-nucleus transcriptomics reveals functional compartmentalization in syncytial skeletal muscle cells
Syncytial skeletal muscle cells contain hundreds of nuclei in a shared cytoplasm. We investigated nuclear heterogeneity and transcriptional dynamics in the uninjured and regenerating muscle using single-nucleus RNA-sequencing (snRNAseq) of isolated nuclei from muscle fibers. In addition to the bulk myonuclear population, we detected smaller populations with very distinct transcriptomes that expressed pan-muscle genes like Ttn. Like the bulk nuclei, distinct nuclei in these populations expressed different myosin genes, indicating that the heterogeneity is not driven by fiber type differences. The different populations identified are the nuclei at the neuromuscular junction (NMJ), two types of myonuclei at the myotendinous junction (MTJ-A and -B), Rian+ nuclei that regulate mitochondrial metabolism through the expression of microRNAs, Gssos2+ nuclei that specialize in the expression of genes that control the entry of proteins into the secretory pathway, and Suz12+ and Bcl2+ nuclei (Fig. 4). In addition, a perimysium population was identified after re-clustering the bulk myonuclei; nuclei that express these genes are exclusively found close to the perimysium.
Single-nucleus transcriptomics reveals a muscle fiber repair gene signature in diseased skeletal muscle
We also investigated the muscle of a mouse model of Duchenne muscular dystrophy (Mdx mice). We found two nuclear populations in the Mdx datasets that were not identified in the healthy muscle. Nuclei of the first population expressed various non-coding transcripts, and further experiments demonstrated that these nuclei were located inside dying fibers. Nuclei of the second population expressed a ‘fiber repair’ signature. Genes expressed in ‘fiber repair’ nuclei showed enrichment of ontology terms related to human muscle disease, and many top marker genes were previously reported to be mutated in human myopathies. Combinatorial fluorescence in-situ hybridization on tissue sections confirmed co-expression of such marker genes in a subset of nuclei in the Mdx muscle, but such nuclei were not present in control muscle. Further, nuclei expressing these genes were frequently closely spaced in fibers, like pearls on a string. We also verified that these genes were co-expressed in nuclei from patient biopsies with confirmed DYSTROPHIN mutation, but not in healthy human muscle. Further, co-expression of these marker genes was observed in a dysferlinopathy mouse model and in biopsies from human patients with DYSFERLIN mutations. We propose that the genes that mark this cluster represent a ‘repair’ signature. This indicates that in diseased muscle fibers a repair mechanism is activated. However, the mechanism does not suffice to rescue the damaged fibers over extended periods of time, since myofibers have a high turnover in Mdx mice. Interestingly, a similar nuclear population was identified in the aging muscle by others. For more information see Minchul Kim, Vedran Franke, Bettina Brandt, Elijah D Lowenstein, Verena Schöwel, Simone Spuler, Altuna Akalin, Carmen Birchmeier, Nat Commun. 2020 Dec 11;11(1):6375. doi: 10.1038/s41467-020-20064-9.
Our single nuclei mRNA sequencing data can be freely explored on the MyoExplorer server.
Figure 4: Heterogenous clusters of nuclei detected in nuclei of the healthy muscle using computational tools. (Left) UMAP of nuclei detected in the healthy muscle. The colors identify different nuclear populations in the muscle. The perimysium population was identified after re-clustering the bulk myonuclei and is not shown here. (Right) Heat-map of specific genes enriched in clusters other than the bulk myonuclei. Top representative genes are indicated on the side.