Abstract Body: Objective: To test the hypothesis that myostatin signaling limits regenerative capacity in ischemic limbs of obese mice via Galectin-3-mediated skeletal muscle fibro-fatty remodeling and impaired angiogenesis.
Methods: Leptin receptor mutant (db/db) mice were crossed with mice lacking myostatin (MSTN KO), a negative regulator of muscle differentiation and growth, galectin-3 (GAL3 KO), a chimeric lectin that mediates cell crosstalk and inflammation, or NADPH oxidase-1 (NOX1 KO), a primary producer of the reactive oxygen species (ROS) O₂^-, to generate db^+/-MSTN^+/-, db^+/-GAL3^+/-, and db^+/-NOX1^+/- double KO C57BL/6 mice. Femoral artery ligation was used to model peripheral artery disease (PAD). Limb reperfusion was determined via laser speckle contrast imaging. Fibro-fatty remodeling was quantified histologically. MSTN signaling was inhibited post-surgically via AAV-mediated follistatin (FST) (directly binds MSTN) overexpression. Bone marrow-derived macrophages (BMDMs), +/- MSTN treatment (Tx), isolated from control and ischemic hindlimbs were analyzed for gene expression.
Results: MSTN is upregulated in obesity and its KO results in preserved SKM mass and glycemic status in obese db/db mice. RNAseq analysis of db^+/-MSTN^+/- SKM reveals Lgals3 (GAL3) as one of the most significantly upregulated genes in obesity that is consequently restored by MSTN KO. Obesity inhibits and MSTN, GAL3, or NOX1 KO restores limb perfusion and angiogenesis. However, while MSTN or GAL3 KO prevents post-ischemia fibro-fatty degeneration (prognosticator of limb-threatening regeneration failure), restoration of vascular function alone in NOX1 KO mice was insufficient. Additionally, the protective effects of MSTN KO were recapitulated via post-injury FST AAV Tx. Interestingly, relative to lean and MSTN KO mice, the 2-fold elevation in Lgals3 expression at baseline increases to 4-fold 28 days post ischemia (dpi) in obese SKM, mirroring GAL3:F4/80⁺ macrophage colocalization with and expansion during PLIN1⁺ fatty lesion onset (7dpi) and progression (7-28dpi), respectively. Importantly, ischemic limb BMDM Lgals3 expression is potently stimulated by MSTN Tx.
Conclusion: In summary, obesity is sufficient to drive tissue degeneration in historically ischemic injury-resilient C57Bl/6 mice. We identify the novel MSTN-GAL3, myocyte-macrophage, crosstalk axis as a driver of unresolved inflammation and impaired recovery in PAD.