Autophagy is a key factor in maintaining the regenerative capacity of muscle stem cells by promoting quiescence and preventing senescence

ABSTRACT

Autophagy, a highly regulated cellular degradation and recycling process, can occur constitutively at a basal level, and plays an essential role in many aspects of cell physiology. A recently published study (see the related punctum in Autophagy, Vol. 12, No. 4) suggests that basal autophagy is also important for maintaining the regenerative capacity of muscle stem cells, and that the decline of autophagy with aging is the cause of entry into senescence from quiescence in satellite cells.

Satellite cells, long-lived muscle stem cells, are important for the regenerative capacity of skeletal muscle, and these cells are usually in the quiescent state (a G₀ reversible arrested state). When exposed to environmental stimuli, quiescent stem cells can respond and then be activated to re-enter the cell cycle.¹ However, at the geriatric stage of life, skeletal muscle cells may enter a presenescence/senescence state, thereby leading to a loss of mass and functionality of satellite cells and, finally, the impairment of regenerative capacity in skeletal muscle.^2-4 In a recent study by García-Prat et al. the authors identified autophagy as a decisive factor in maintaining the stemness of muscle stem cells by preserving their quiescent state and preventing premature senescence in mice.⁵ The dormant stem cells display continuous basal autophagy, but this activity declines with age, resulting in toxic cellular waste accumulation and entry into senescence.

The authors integrated transcriptome and K-means clustering analysis, revealing that autophagy is prevalent in the quiescent state of mouse muscle stem cells, but autophagy-related (Atg) genes are downregulated as age increases. The quiescent satellite cells were isolated by fluorescence-activated cell sorting from mice at different ages. Next, the authors confirmed that autophagic activity is constitutive in young quiescent satellite cells (˜3-mo old for mice), but that it is impaired in old cells (˜20–24-mo old). However, the autophagy inducer rapamycin is able to restore autophagic activity in aged cells. This finding reveals a difference of autophagic activity in young and old muscle stem cells, providing a basis for answering various questions concerning stem cell regenerative capacity.

The first question the authors examined is whether altered autophagy in aged satellite cells contributes to the entry into a senescent state at a geriatric age. The authors used a tandem mRFP-GFP-LC3 construct to follow the appearance of either autophagosomes (yellow LC3 puncta) or autolysosomes (red LC3 puncta corresponding to the fusion of autophagosomes with lysosomes) in young, old and geriatric satellite cells (˜28-mo old). The results indicated that autolysosomes are only detected at substantial levels in young cells; the greatest defect in autophagy is found in geriatric cells followed by old cells. Moreover, increased accumulation of SQSTM1/p62-ubiquitin aggregates in geriatric cells provided another line of evidence of an autophagic defect in geriatric cells, indicating the presence of damaged/dysfunctional ubiquitinated proteins and/or organelles.

By transplanting an equal amount of lentivirus-GFP-expressing satellite cells from young or geriatric mice (pre-treated with rapamycin or control vehicle) into pre-injured muscles of young mice, the authors found that autophagy reactivation restores the expansion of geriatric cells (indicated by PAX7 expression), prevents senescence (shown by CDKN2A/p16^INK4a and γ-H2AFX reduction), and recovers intrinsic regenerative capacity. A similar result is achieved by overexpressing ATG7, a crucial autophagy-related protein needed for autophagosome formation. Subsequently, the authors intercrossed Atg7-floxed mice with Pax7-Cre and Pax7-Cre^ER mice, blocking autophagy either constitutively or inducibly in PAX7-expressing cells. The analysis of PAX7 immunostaining of muscles and RT-qPCR of senescence markers showed that the deletion of ATG7 leads to an age-associated numerical decline in satellite cells, and regenerative failure. Together, these findings indicate that ATG7 loss causes senescence in satellite cells.

Considering the impairment of autophagy in geriatric cells, the authors turned their attention to 2 additional questions: Whether inhibition of autophagy can induce premature aging in young satellite cells, and the direct cause of senescence in geriatric cells. The quantification of lysosomes and mitochondria showed the atg7Δ young satellite cells display a similar phenotype to old cells, with an accumulation of lysosomes and mitochondria but a lower membrane potential in the latter, indicating the possibility of defective mitophagy. Compared to young cells, more reactive oxygen species (ROS) and colocalization of ROS with mitochondria are found in geriatric satellite cells. Conversely, the inhibition of ROS production by the vitamin E analog Trolox attenuates impairment of mitophagy, prevents senescence, and restores proliferative and regenerative capacity in geriatric satellite cells. Thus, the authors concluded that ROS resulting from impaired autophagy/mitophagy might be the main cause driving senescence in aged satellite cells. Along these lines, CDKN2A concentrations increase dramatically with aging, due to the loss of polycomb repressive complex 1-mediated histone 2A monoubiquitination at the Cdkn2a locus.⁴ The authors confirmed that Trolox treatment restores this modification, thereby reducing senescence and promoting proliferation in both geriatric and ATG7-deficient cells. This result further demonstrates that ROS can epigenetically control CDKN2A expression, connecting impaired autophagy with senescence. Importantly, a similar autophagy defect is seen in aged human cells.

This study has successfully identified autophagy as playing a crucial role in the switch between the quiescent and senescent state of muscle stem cells, and this function is especially important for nondiving stem cells such as those present in muscle cells.⁶ The long-lived satellite cells can maintain a high level of basal autophagy to preserve their regenerative capacity and avoid senescence, but as age increases, the autophagic activity of skeletal muscle stem cells declines and the cells enter a senescent state.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by NIH grant GM053396 to DJK.