Mitochondrial dynamics maintain muscle stem cell regenerative competence throughout adult life by regulating metabolism and mitophagy.

Skeletal muscle regeneration depends on the correct expansion of resident quiescent stem cells (satellite cells), a process that becomes less efficient with aging. Here, we show that mitochondrial dynamics are essential for the successful regenerative capacity of satellite cells. The loss of mitochondrial fission in satellite cells-due to aging or genetic impairment-deregulates the mitochondrial electron transport chain (ETC), leading to inefficient oxidative phosphorylation (OXPHOS) metabolism and mitophagy and increased oxidative stress. This state results in muscle regenerative failure, which is caused by the reduced proliferation and functional loss of satellite cells. Regenerative functions can be restored in fission-impaired or aged satellite cells by the re-establishment of mitochondrial dynamics (by activating fission or preventing fusion), OXPHOS, or mitophagy. Thus, mitochondrial shape and physical networking controls stem cell regenerative functions by regulating metabolism and proteostasis. As mitochondrial fission occurs less frequently in the satellite cells in older humans, our findings have implications for regeneration therapies in sarcopenia.

Stem cell aging in the skeletal muscle: The importance of communication.

Adult stem cells sustain tissue homeostasis and regeneration; their functional decline is often linked to aging, which is characterized by the progressive loss of physiological functions across multiple tissues and organs. The resident stem cells in skeletal muscle, termed satellite cells, are normally quiescent but activate upon injury to reconstitute the damaged tissue. In this review, we discuss the current understanding of the molecular processes that contribute to the functional failure of satellite cells during aging. This failure is due not only to intrinsic changes but also to extrinsic factors, most of which are still undefined but originate from the muscle tissue microenvironment of the satellite cells (the niche), or from the systemic environment. We also highlight the emerging applications of the powerful single-cell sequencing technologies in the study of skeletal muscle aging, particularly in the heterogeneity of the satellite cell population and the molecular interaction of satellite cells and other cell types in the niche. An improved understanding of how satellite cells communicate with their environment, and how this communication is perturbed with aging, will be helpful for defining countermeasures against loss of muscle regenerative capacity in sarcopenia.

Translational control by DHX36 binding to 5'UTR G-quadruplex is essential for muscle stem-cell regenerative functions.

Skeletal muscle has a remarkable ability to regenerate owing to its resident stem cells (also called satellite cells, SCs). SCs are normally quiescent; when stimulated by damage, they activate and expand to form new fibers. The mechanisms underlying SC proliferative progression remain poorly understood. Here we show that DHX36, a helicase that unwinds RNA G-quadruplex (rG4) structures, is essential for muscle regeneration by regulating SC expansion. DHX36 (initially named RHAU) is barely expressed at quiescence but is highly induced during SC activation and proliferation. Inducible deletion of Dhx36 in adult SCs causes defective proliferation and muscle regeneration after damage. System-wide mapping in proliferating SCs reveals DHX36 binding predominantly to rG4 structures at various regions of mRNAs, while integrated polysome profiling shows that DHX36 promotes mRNA translation via 5'-untranslated region (UTR) rG4 binding. Furthermore, we demonstrate that DHX36 specifically regulates the translation of Gnai2 mRNA by unwinding its 5' UTR rG4 structures and identify GNAI2 as a downstream effector of DHX36 for SC expansion. Altogether, our findings uncover DHX36 as an indispensable post-transcriptional regulator of SC function and muscle regeneration acting through binding and unwinding rG4 structures at 5' UTR of target mRNAs.

Smoothelin-like 2 Inhibits Coronin-1B to Stabilize the Apical Actin Cortex during Epithelial Morphogenesis.

The actin cortex is involved in many biological processes and needs to be significantly remodeled during cell differentiation. Developing epithelial cells construct a dense apical actin cortex to carry out their barrier and exchange functions. The apical cortex assembles in response to three-dimensional (3D) extracellular cues, but the regulation of this process during epithelial morphogenesis remains unknown. Here, we describe the function of Smoothelin-like 2 (SMTNL2), a member of the smooth-muscle-related Smoothelin protein family, in apical cortex maturation. SMTNL2 is induced during development in multiple epithelial tissues and localizes to the apical and junctional actin cortex in intestinal and kidney epithelial cells. SMTNL2 deficiency leads to membrane herniations in the apical domain of epithelial cells, indicative of cortex abnormalities. We find that SMTNL2 binds to actin filaments and is required to slow down the turnover of apical actin. We also characterize the SMTNL2 proximal interactome and find that SMTNL2 executes its functions partly through inhibition of coronin-1B. Although coronin-1B-mediated actin dynamics are required for early morphogenesis, its sustained activity is detrimental for the mature apical shape. SMTNL2 binds to coronin-1B through its N-terminal coiled-coil region and negates its function to stabilize the apical cortex. In sum, our results unveil a mechanism for regulating actin dynamics during epithelial morphogenesis, providing critical insights on the developmental control of the cellular cortex.

FoxO maintains a genuine muscle stem-cell quiescent state until geriatric age.

Tissue regeneration declines with ageing but little is known about whether this arises from changes in stem-cell heterogeneity. Here, in homeostatic skeletal muscle, we identify two quiescent stem-cell states distinguished by relative CD34 expression: CD34High, with stemness properties (genuine state), and CD34Low, committed to myogenic differentiation (primed state). The genuine-quiescent state is unexpectedly preserved into later life, succumbing only in extreme old age due to the acquisition of primed-state traits. Niche-derived IGF1-dependent Akt activation debilitates the genuine stem-cell state by imposing primed-state features via FoxO inhibition. Interventions to neutralize Akt and promote FoxO activity drive a primed-to-genuine state conversion, whereas FoxO inactivation deteriorates the genuine state at a young age, causing regenerative failure of muscle, as occurs in geriatric mice. These findings reveal transcriptional determinants of stem-cell heterogeneity that resist ageing more than previously anticipated and are only lost in extreme old age, with implications for the repair of geriatric muscle.

Assessing Autophagy in Muscle Stem Cells.

The skeletal muscle tissue in the adult is relatively stable under normal conditions but retains a striking ability to regenerate by its resident stem cells (satellite cells). Satellite cells exist in a quiescent (G0) state; however, in response to an injury, they reenter the cell cycle and start proliferating to provide sufficient progeny to form new myofibers or undergo self-renewal and returning to quiescence. Maintenance of satellite cell quiescence and entry of satellite cells into the activation state requires autophagy, a fundamental degradative and recycling process that preserves cellular proteostasis. With aging, satellite cell regenerative capacity declines, correlating with loss of autophagy. Enhancing autophagy in aged satellite cells restores their regenerative functions, underscoring this proteostatic activity's relevance for tissue regeneration. Here we describe two strategies for assessing autophagic activity in satellite cells from GFP-LC3 reporter mice, which allows direct autophagosome labeling, or from non-transgenic (wild-type) mice, where autophagosomes can be immunostained. Treatment of GFP-LC3 or WT satellite cells with compounds that interfere with autophagosome-lysosome fusion enables measurement of autophagic activity by flow cytometry and immunofluorescence. Thus, the methods presented permit a relatively rapid assessment of autophagy in stem cells from skeletal muscle in homeostasis and in different pathological scenarios such as regeneration, aging or disease.