Rapamycin improves satellite cells’ autophagy and muscle regeneration during hypercapnia
Joseph Balnis, Emily L. Jackson, Lisa A. Drake, Diane V. Singer, Ramon Bossardi Ramos, Harold A. Singer, Ariel Jaitovich
Joseph Balnis, Emily L. Jackson, Lisa A. Drake, Diane V. Singer, Ramon Bossardi Ramos, Harold A. Singer, Ariel Jaitovich
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Research Article Pulmonology

Rapamycin improves satellite cells’ autophagy and muscle regeneration during hypercapnia

Abstract

Both CO2 retention, or hypercapnia, and skeletal muscle dysfunction predict higher mortality in critically ill patients. Mechanistically, muscle injury and reduced myogenesis contribute to critical illness myopathy, and while hypercapnia causes muscle wasting, no research has been conducted on hypercapnia-driven dysfunctional myogenesis in vivo. Autophagy flux regulates myogenesis by supporting skeletal muscle stem cell — satellite cell — activation, and previous data suggest that hypercapnia inhibits autophagy. We tested whether hypercapnia worsens satellite cell autophagy flux and myogenic potential and if autophagy induction reverses these deficits. Satellite cell transplantation and lineage-tracing experiments showed that hypercapnia undermined satellite cells’ activation, replication, and myogenic capacity. Bulk and single-cell sequencing analyses indicated that hypercapnia disrupts autophagy, senescence, and other satellite cell programs. Autophagy activation was reduced in hypercapnic cultured myoblasts, and autophagy genetic knockdown phenocopied these changes in vitro. Rapamycin stimulation led to AMPK activation and downregulation of the mTOR pathway, which are both associated with accelerated autophagy flux and cell replication. Moreover, hypercapnic mice receiving rapamycin showed improved satellite cell autophagy flux, activation, replication rate, and posttransplantation myogenic capacity. In conclusion, we have shown that hypercapnia interferes with satellite cell activation, autophagy flux, and myogenesis, and systemic rapamycin administration improves these outcomes.

Authors

Joseph Balnis, Emily L. Jackson, Lisa A. Drake, Diane V. Singer, Ramon Bossardi Ramos, Harold A. Singer, Ariel Jaitovich

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Figure 9

Hypercapnia downregulates autophagy, and autophagy knockdown replicates hypercapnia.

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Hypercapnia downregulates autophagy, and autophagy knockdown replicates ...
(A) C2C12 myoblast proliferation is reduced in hypercapnia (n = 5). (B) Cells in hypercapnia express fewer LC3 isoforms (n = 3). (C) Densitometric quantitation of Western blots in B. (D) Autophagy pathway constituents are downregulated in hypercapnia; however, mTOR and p-mTOR (Ser2448) remain unchanged. Despite activation surrogates of AMPK (p-AMPK and p-ACC) increasing in hypercapnia, the canonical AMPK-targeted ULK1S317 is not upregulated (n = 3). (E) Densitometric quantitation of Western blots in D. (F) qPCRs of AMPK and ULK1 demonstrate lack of transcriptional downregulation (n = 16). (G) PLA shows that hypercapnia exposure is associated with reduced AMPK/ULK1-driven puncta (n = 8). (H) When treated with mTOR inhibitor rapamycin and bafilomycin, LC3-II increases in hypercapnia, suggesting retained autophagy flux capacity (n = 5). (I) Densitometric quantitation of Western blots in H. (J) PLA shows that hypercapnia-treated cells significantly improve AMPK/ULK1-driven puncta when treated with rapamycin (n = 8). (K) mTOR and downstream autophagy-inhibitory ULK1S757 are dephosphorylated after rapamycin treatment (n = 5), while AMPK-targeted and autophagy-stimulatory ULK1S317 are upregulated (n = 4). (L) Densitometric quantitation of Western blots in panel K. (M) siRNA Atg7 transfection in C2C12 cells causes a reduction in Atg7 protein products and upregulation of autophagy arrest markers. Scramble (scr) is control, nonspecific siRNA (n = 3). (N) Densitometric quantitation of Western blots in panel M. (O) C2C12 cells transfected with Atg7 siRNA are unable to build normal levels of LC3-II with rapamycin (n = 4). (P) C2C12 cells transfected with Atg7 siRNA demonstrated a normal differentiation pattern (n = 3). (Q) Atg7 silencing significantly reduces C2C12 cell proliferation similarly to hypercapnia (n = 3). Statistical comparisons were performed using Student’s t test in panels A, F, G, J, and Q. Densitometric statistical comparisons in C, E, I, L, and N were performed using a 1-sample t test; *P < 0.05, **P < 0.01, and ***P < 0.001.