Myostatin/HIF2α-Mediated Ferroptosis is Involved in Skeletal Muscle Dysfunction in Chronic Obstructive Pulmonary Disease

Abstract

Objective: Skeletal muscle dysfunction is an important comorbidity in patients with chronic obstructive pulmonary disease (COPD), and is associated with poor quality of life and reduced survival, but the mechanisms involved remain elusive. Ferroptosis is a newly discovered type of cell death resulting from iron-dependent lipid peroxide accumulation. The purpose of this study was to examine whether ferroptosis is involved in COPD-associated skeletal muscle dysfunction.

Methods: A mouse model of COPD was established after 24 weeks of cigarette smoke (CS) exposure, and mRNA sequencing, hematoxylin-eosin (H&E) staining, immunostaining (IF), RT-PCR, and Western blot were utilized to identify the changes in gastrocnemius muscles. In vitro, C2C12 myotubes were treated with CS extract (CSE) and evaluated for ferroptosis-related molecules. The pathways regulating ferroptosis were then explored in CSE-stimulated myotubes.

Results: Compared with controls, COPD mice showed an enriched ferroptosis pathway. Gpx4 was decreased, while hypoxia-inducible factor (Hif) 2α was increased, at gene and protein levels. A reduced level of GSH, but increased cell death, Fe²⁺, lipid ROS, LPO, and 4-HNE were observed in COPD mice or in CSE-stimulated C2C12 myotubes, which could be ameliorated by ferroptosis inhibitors. The expression of myostatin (MSTN) was enhanced in COPD mice and CSE-stimulated myotubes. MSTN up-regulated HIF2α expression and led to ferroptosis in myotubes, whereas inhibition of MSTN binding to its receptor or inhibition/knockdown of HIF2α resulted in decreased cell death, and partially restored GPX4 and GSH.

Conclusion: CS exposure induced ferroptosis in vivo and in vitro. Mechanistically, CS-exposure upregulated MSTN which further induced ferroptosis through HIF2α in skeletal muscles, which may contribute to muscle dysfunction through impairing metabolic capacity and decreasing muscle fiber numbers, revealing a potential novel therapeutic target for COPD-related skeletal muscle dysfunction.

Keywords: chronic obstructive pulmonary disease; ferroptosis; hypoxia-inducible factor 2α; myostatin; skeletal muscle dysfunction.

Figure 1

Long-term CS exposure led to emphysema and skeletal muscle dysfunction. (A–F) Airway resistance (RL), lung compliance, FVC, FEV0.1, FEV0.1/FVC, and FEF25-75% were measured in mice; (G) H&E staining of lung tissues from mice exposed to air and CS; Arrows (left) indicate destruction of the alveolar wall; Arrow (right) indicate Inflammatory cells infiltrates in the lung parenchyma and interstitial spaces; The scale in each image is 100 μm; (H and I) histogram of MLI and DI; (J) FACS showed the neutrophils (CD11B⁺Ly6G⁺) and eosinophils (CD11B⁺SiglecF⁺) in BALF; (K) histogram of grip strength; (L) H&E staining of muscles from mice exposed to air and CS; Arrows indicate changes in muscle fibers and widening of gaps; The scale in each image is 100 μm; (M) WB showed the protein expression of Slow MyHC, Fast MyHC, MuRF1, Atrogin1 and MSTN in muscles of air and CS-exposed mice; (N) the expression of Slow MyHC in muscles from mice. n = 5, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

Chronic CS exposure caused ferroptosis in skeletal muscles from mice. (A) Cell death was enriched in skeletal muscles from COPD patients and healthy control by GSEA. (B and C) Gene expression of Gpx4 and Ncoa4 in skeletal muscles from mice by RNA-sequencing; (D) RT-qPCR examined the expression of Gpx4, Slc7a11, Tfr1, and Ncoa4 in muscles; (E) WB showed the protein expression of GPX4 in muscles; (F) IF showed the expression of GPX4 in muscles from mice exposed to air and CS; The scale in each image is 50 μm; (G) the content of GSH in muscles; (H) the level of LPO in muscles determined by colorimetry; (I) IF showed the expression of 4-HNE in muscles from mice exposed to air and CS; The scale in each image is 50 μm. n = 5, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3

CSE stimulation caused ferroptosis in C2C12 myotubes. (A) Cellular morphology of C2C12 myoblasts and C2C12 myotubes. The scale in each image is 100 μm; (B) the proportion of cell death was detected by flow cytometry with 7AAD staining in Ctrl and 3% CSE-stimulated myotubes; (C) RT-qPCR examined the expression of Gpx4, Slc7a11, Slc3a2, Tfr1, Ncoa4, Acls4 and Lpcat3 in C2C12 myotubes; (D) WB showed the protein expression of GPX4, MuRF1, Atrogin1, and MSTN in C2C12 myotubes; (E) the level of Fe²⁺ was detected by flow cytometry with the fluorescent probe; (F) the content of GSH in C2C12 myotubes; (G) lipid ROS was detected by flow cytometry with C11 BODIPY; (H) the level of LPO in C2C12 myotubes. (I) The proportion of cell death was detected by flow cytometry with 7AAD staining. UAMC-3203, a ferroptosis inhibitor. ***P < 0.001, ****P < 0.0001.

Figure 4

CSE induced ferroptosis by up-regulating the expression of MSTN. (A) WB showed the expression of GPX4 in myotubes; (B) the expression of lipid ROS in myotubes by confocal microscopy with C11 BODIPY; The scale in each image is 100 μm; (C) the proportion of cell death was detected by flow cytometry with 7AAD staining in different concentration of MSTN treated myotubes; (D) WB showed the protein expression of GPX4 and HIF2α; (E) histogram of the content of GSH in myotubes; (F and G) flow cytometry detected the levels of Fe²⁺ and lipid ROS in different concentration of MSTN treated myotubes; (H) histogram of the content of LPO in myotubes. *P < 0.05,**P < 0.01,****P < 0.0001.

Figure 5

Ferroptosis inhibitor intervention alleviated ferroptosis-related indicators caused by MSTN. (A) Cell death was detected by flow cytometry; (B) the protein expression of GPX4; (C) Histogram of the content of GSH; (D and E) flow cytometry detected the levels of Fe²⁺ and lipid ROS; (F) histogram of the content of LPO; (G) the expression of lipid ROS in myotubes using confocal microscopy; The scale in each image is 100 μm. *P < 0.05,**P < 0.01,***P < 0.001, ****P < 0.0001.

Figure 6

HIF2α inhibition alleviated ferroptosis-related indicators caused by CSE. (A) Corplot showed the correlation between Hif1α, Epas1, Hif3α, and Gpx4. (B) Gene expression of Hif1α, Epas1, and Hif3α in skeletal muscles from mice by RNA-sequencing; (C) WB showed the protein expression of HIF2α in muscles (up) and C2C12 myotubes (down); (D) the proportion of cell death was detected by flow cytometry with 7AAD staining in myotubes; (E) RT-qPCR detected the expression of Gpx4 in myotubes; (F) the expression of HIF2α and GPX4 were detected by WB; (G and H) the levels of Fe²⁺ and lipid ROS were detected by flow cytometry; (I and J) histogram of the content of LPO and GSH in C2C12 myotubes. HIF2α IN, an inhibitor of HIF2α. *P < 0.05,**P < 0.01,****P < 0.0001.

Figure 7

HIF2α knocking down alleviated ferroptosis-related indicators caused by CSE. (A) WB showed the protein expression of GPX4 and HIF2α; (B) the proportion of cell death was detected by flow cytometry with 7AAD staining in myotubes; (C) histogram of the content of GSH in myotubes; (D and E) flow cytometry detected the levels of Fe²⁺ and lipid ROS in myotubes; (F) histogram of the content of LPO in myotubes. *P < 0.05,**P < 0.01,***P < 0.001.