Inhibition of vascular intimal hyperplasia by the myokine Musclin: the role of NPR3/raptor/mTORC1-mediated glycolysis and phenotypic switching of VSMCs

Abstract

Skeletal muscle-derived Musclin exerts multiple effects on the cardiovascular system. However, the role of Musclin in vascular intimal hyperplasia (IH) remains unclear. This study aims to investigate the role and underlying mechanism of Musclin in IH. We overexpress Musclin in skeletal muscle via adeno-associated virus serotype 6 (AAV6)-mediated gene transfer (AAV-Musclin) in an injury-induced mouse vascular IH model. Morphological analyses, including hematoxylin and eosin (H&E) staining and Ki-67 immunohistochemistry, are used to evaluate IH severity. Ki-67 immunofluorescence, transwell assay, wound healing assay, and analysis of vascular smooth muscle cell (VSMC) differentiation markers are conducted to assess VSMC phenotypic switching. The extracellular acidification rate (ECAR) assay is utilized to measure glycolysis in VSMCs. Following AAV-Musclin transfection, Musclin levels are increased in both skeletal muscle and peripheral blood. Muscle-specific Musclin overexpression ameliorates injury-induced vascular IH. In vitro, Musclin represses glycolysis, proliferation, and migration while increasing VSMC differentiation markers in PDGF-BB-stimulated VSMCs. Mechanistically, Musclin inhibits mammalian target of rapamycin complex 1 (mTORC1) activity and induces NPR3-raptor interaction. Restoring mTORC1 activity abolishes the inhibitory effects of Musclin on PDGF-BB-induced VSMC phenotypic switching and its protective role against injury-induced vascular IH. Additionally, NPR3 silencing abrogates Musclin-mediated suppression of mTORC1 activity, glycolysis, and phenotypic switching in PDGF-BB-treated VSMCs. Collectively, external Musclin supplementation may represent a promising therapeutic strategy for preventing vascular IH-related pathologies.

1. Introduction

Vascular intimal hyperplasia (IH) is a key pathological feature of atherosclerosis, coronary in-stent restenosis, and graft failure after coronary artery bypass grafting [1]. Although drug-eluting stents and antiproliferative agents that target IHs have significantly reduced morbidity, restenosis-related complications remain unresolved [2]. IH results from a maladaptive repair response to vascular injury, primarily characterized by abnormal thickening of the intima, phenotypic switching of vascular smooth muscle cells (VSMCs), extracellular matrix deposition, and neointimal formation [3]. As the major component of the vascular wall, VSMC phenotypic switching plays a critical role in IH. The switching process is characterized by excessive proliferation, migration, and dedifferentiation of VSMCs in response to cytokines such as platelet-derived growth factor (PDGF) [4]. Consequently, elucidating novel mechanisms underlying VSMC phenotypic switching is essential for preventing vascular IH.

Physical exercise significantly reduces the morbidity and mortality of cardiovascular diseases (CVDs), such as coronary heart disease [5]. The beneficial effects of exercise on the cardiovascular system are largely mediated by skeletal muscle-derived myokines [6]. Several myokines are known to regulate VSMC function and vascular diseases. For example, irisin inhibits the pyroptosis and osteoblastic transformation of VSMCs [7,8]. Elevated myostatin suppresses VSMC proliferation and inhibits vascular restenosis [9]. Musclin, an exercise-induced myokine, has been implicated in several CVDs, including hypertension, heart failure, and pulmonary hypertension [6,10,11]. Additionally, Musclin exerts anti-proliferative effects on cell types such as fibro-adipogenic progenitors [12]. However, the role of Musclin in VSMC phenotypic switching and vascular IH remains largely unknown.

Musclin has been shown to inhibit glucose metabolism in skeletal muscle, adipose tissue, and pulmonary arteries [11,13,14]. Enhanced glucose metabolism is closely linked to excessive proliferation and migration of VSMCs, as well as vascular IH [15]. Consequently, glycolysis may represent a potential mechanism through which Musclin affects VSMCs. In response to growth factors, energy shifts, and pathological stress, mammalian target of rapamycin (mTOR) functions as a central signal that regulates intracellular metabolism by forming complexes, including mTOR complex 1 (mTORC1) and mTORC2 [16]. mTORC1 promotes glucose uptake and glycolysis, thereby driving VSMC proliferation, migration, and vascular IH [17]. However, whether mTORC1-mediated glucose metabolism acts downstream of Musclin to regulate VSMC phenotypic switching and vascular IH remains unclear.