Mosqueira Matias, Scheid Lisa-Mareike, Kiemel Dominik, Richardt Talisa, Rheinberger Mona, Ollech Dirk, Lutge Almut, Heißenberg Tim, Pfitzer Lena, Engelskircher Lisa, Yildiz Umut, Porth Isabel
Cardio-Ventilatory Muscle Physiology Laboratory, Institute of Physiology and Pathophysiology, Heidelberg University Hospital, Heidelberg, Germany.
PromoCell GmbH, Heidelberg, Germany.
Front Physiol. 2022 Aug 15;13:946682. doi: 10.3389/fphys.2022.946682. eCollection 2022.
Nitric oxide (NO) is a bioactive gas produced by one of the three NO synthases: neuronal NOS (nNOS), inducible (iNOS), and endothelial NOS (eNOS). NO has a relevant modulatory role in muscle contraction; this takes place through two major signaling pathways: (i) activation of soluble guanylate cyclase and, thus, protein kinase G or (ii) nitrosylation of sulfur groups of cysteine. Although it has been suggested that nNOS-derived NO is the responsible isoform in muscle contraction, the roles of eNOS and iNOS and their signaling pathways have not yet been clarified. To elucidate the action of each pathway, we optimized the generation of myooids, an engineered skeletal muscle tissue based on the C2C12 cell line. In comparison with diaphragm strips from wild-type mice, 180 myooids were analyzed, which expressed all relevant excitation-contraction coupling proteins and both nNOS and iNOS isoforms. Along with the biochemical results, myooids treated with NO donor (SNAP) and unspecific NOS blocker (L-NAME) revealed a comparable NO modulatory effect on force production as was observed in the diaphragm strips. Under the effects of pharmacological tools, we analyzed the myooids in response to electrical stimulation of two possible signaling pathways and NO sources. The nNOS-derived NO exerted its negative effect on force production the sGG-PKG pathway, while iNOS-derived NO increased the excitability in response to sub-threshold electrical stimulation. These results strengthen the hypotheses of previous reports on the mechanism of action of NO during force production, showed a novel function of iNOS-derived NO, and establish the myooid as a novel and robust alternative model for pathophysiological skeletal muscle research.
一氧化氮（NO）是由三种一氧化氮合酶之一产生的生物活性气体：神经元型一氧化氮合酶（nNOS）、诱导型（iNOS）和内皮型一氧化氮合酶（eNOS）。NO在肌肉收缩中具有重要的调节作用；这通过两条主要信号通路发生：（i）可溶性鸟苷酸环化酶的激活，进而激活蛋白激酶G；或（ii）半胱氨酸硫基团的亚硝基化。尽管有人提出nNOS衍生的NO是肌肉收缩中起作用的异构体，但eNOS和iNOS的作用及其信号通路尚未阐明。为了阐明每条通路的作用机制，我们优化了肌样体的生成，肌样体是一种基于C2C12细胞系构建的工程化骨骼肌组织。与野生型小鼠的膈肌条相比，我们分析了180个肌样体，它们表达了所有相关的兴奋-收缩偶联蛋白以及nNOS和iNOS异构体。结合生化结果，用NO供体（SNAP）和非特异性NOS阻断剂（L-NAME）处理的肌样体显示出与膈肌条中观察到的类似的NO对力产生的调节作用。在药理学工具的作用下，我们分析了肌样体对两条可能的信号通路和NO来源的电刺激的反应。nNOS衍生的NO通过sGG-PKG通路对力产生发挥负面影响，而iNOS衍生的NO在亚阈值电刺激下增加了兴奋性。这些结果强化了先前关于NO在力产生过程中作用机制的报道的假设，展示了iNOS衍生的NO的新功能，并将肌样体确立为病理生理骨骼肌研究的一种新型且强大的替代模型。
