Benzi Andrea, Baratto Serena, Astigiano Cecilia, Sturla Laura, Panicucci Chiara, Mamchaoui Kamel, Raffaghello Lizzia, Bruzzone Santina, Gazzerro Elisabetta, Bruno Claudio
Department of Experimental Medicine-DIMES, University of Genova, Genova, Italy.
Center of Translational and Experimental Myology, IRCCS Istituto Giannina Gaslini, Genova, Italy.
Lab Invest. 2023 Mar;103(3):100037. doi: 10.1016/j.labinv.2022.100037. Epub 2023 Jan 10.
Sarcoglycanopathies, limb-girdle muscular dystrophies (LGMD) caused by genetic loss-of-function of the membrane proteins sarcoglycans (SGs), are characterized by progressive degeneration of skeletal muscle. In these disorders, muscle necrosis is associated with immune-mediated damage, whose triggering and perpetuating molecular mechanisms are not fully elucidated yet. Extracellular adenosine triphosphate (eATP) seems to represent a crucial factor, with eATP activating purinergic receptors. Indeed, in vivo blockade of the eATP/P2X7 purinergic pathway ameliorated muscle disease progression. P2X7 inhibition improved the dystrophic process by restraining the activity of P2X7 receptors on immune cells. Whether P2X7 blockade can display a direct action on muscle cells is not known yet. In this study, we investigated eATP effects in primary cultures of myoblasts isolated from patients with LGMDR3 (α-sarcoglycanopathy) and in immortalized cells isolated from a patient with LGMDR5 (γ-sarcoglycanopathy). Our results demonstrated that, owing to a reduced ecto-ATPase activity and/or an enhanced release of ATP, patient cells are exposed to increased juxtamembrane concentrations of eATP and display a higher susceptivity to eATP signals. The purinoceptor P2Y2, which proved to be overexpressed in patient cells, was identified as a pivotal receptor responsible for the enhanced ATP-induced or UTP-induced Ca increase in affected myoblasts. Moreover, P2Y2 stimulation in LDMDR3 muscle cells induced chemotaxis of immune cells and release of interleukin-8. In conclusion, a higher eATP concentration and sensitivity in primary human muscle cells carrying different α-SG or γ-SG loss-of-function mutations indicate that eATP/P2Y2 is an enhanced signaling axis in cells from patients with α-/γ-sarcoglycanopathy. Understanding the basis of the innate immune-mediated damage associated with the dystrophic process may be critical in overcoming the immunologic hurdles associated with emerging gene therapies for these disorders.
肌聚糖病是由膜蛋白肌聚糖（SGs）的基因功能丧失引起的肢带型肌营养不良症（LGMD），其特征是骨骼肌进行性退化。在这些疾病中，肌肉坏死与免疫介导的损伤有关，其触发和持续的分子机制尚未完全阐明。细胞外三磷酸腺苷（eATP）似乎是一个关键因素，eATP可激活嘌呤能受体。事实上，体内阻断eATP/P2X7嘌呤能通路可改善肌肉疾病的进展。P2X7抑制通过抑制免疫细胞上P2X7受体的活性改善了营养不良过程。P2X7阻断是否能对肌肉细胞产生直接作用尚不清楚。在本研究中，我们研究了eATP对从LGMDR3（α-肌聚糖病）患者分离的成肌细胞原代培养物以及从LGMDR5（γ-肌聚糖病）患者分离的永生化细胞的影响。我们的结果表明，由于胞外ATP酶活性降低和/或ATP释放增加，患者细胞暴露于更高的膜旁eATP浓度，并对eATP信号表现出更高的敏感性。嘌呤受体P2Y2在患者细胞中被证明过度表达，被确定为导致受影响的成肌细胞中ATP诱导或UTP诱导的钙增加增强的关键受体。此外，LDMDR3肌肉细胞中的P2Y2刺激诱导了免疫细胞的趋化作用和白细胞介素-8的释放。总之，携带不同α-SG或γ-SG功能丧失突变的原代人肌肉细胞中更高的eATP浓度和敏感性表明，eATP/P2Y2是α-/γ-肌聚糖病患者细胞中增强的信号轴。了解与营养不良过程相关的先天性免疫介导损伤的基础可能对于克服与这些疾病新兴基因治疗相关的免疫障碍至关重要。
