Department of Plastic Surgery, BG University Hospital Bergmannsheil, Ruhr University Bochum, Bürkle-de-la-Camp Platz 1, 44789 Bochum, Germany.
Cells. 2021 Jul 3;10(7):1680. doi: 10.3390/cells10071680.
Ischemia reperfusion (IR) injury remains an important topic in clinical medicine. While a multitude of prophylactic and therapeutic strategies have been proposed, recent studies have illuminated protective effects of myostatin inhibition. This study aims to elaborate on the intracellular pathways involved in myostatin signaling and to explore key proteins that convey protective effects in IR injury. We used CRISPR/Cas9 gene editing to introduce a myostatin () deletion into a C2C12 cell line. In subsequent experiments, we evaluated overall cell death, activation of apoptotic pathways, ROS generation, lipid peroxidation, intracellular signaling via mitogen-activated protein kinases (MAPKs), cell migration, and cell proliferation under hypoxic conditions followed by reoxygenation to simulate an IR situation in vitro (hypoxia reoxygenation). It was found that mitogen-activated protein kinase kinase 3/6, also known as MAPK/ERK Kinase 3/6 (MEK3/6), and subsequent p38 MAPK activation were blunted in C2C12- cells in response to hypoxia reoxygenation (HR). Similarly, c-Jun N-terminal kinase (JNK) activation was negated. We also found the intrinsic activation of apoptosis to be more important in comparison with the extrinsic activation. Additionally, intercepting myostatin signaling mitigated apoptosis activation. Ultimately, this research validated protective effects of myostatin inhibition in HR and identified potential mediators worth further investigation. Intercepting myostatin signaling did not inhibit ROS generation overall but mitigated cellular injury. In particular, intrinsic activation of apoptosis origination from mitochondria was alleviated. This was presumably mediated by decreased activation of p38 caused by the diminished kinase activity increase of MEK3/6. Overall, this work provides important insights into HR signaling in C2C12- cells and could serve as basis for further research.
缺血再灌注 (IR) 损伤仍然是临床医学中的一个重要课题。虽然已经提出了许多预防和治疗策略，但最近的研究揭示了抑制肌肉生长抑制素的保护作用。本研究旨在详细阐述肌肉生长抑制素信号转导中的细胞内途径，并探讨在 IR 损伤中传递保护作用的关键蛋白。我们使用 CRISPR/Cas9 基因编辑将肌肉生长抑制素 () 缺失引入 C2C12 细胞系。在随后的实验中，我们评估了总细胞死亡、凋亡途径的激活、ROS 生成、脂质过氧化、缺氧再氧合后通过丝裂原激活的蛋白激酶 (MAPKs) 的细胞内信号传导、细胞迁移和细胞增殖，以模拟体外的 IR 情况（缺氧再氧合）。结果发现，丝裂原激活的蛋白激酶激酶 3/6，也称为 MAPK/ERK 激酶 3/6 (MEK3/6)，以及随后的 p38 MAPK 激活在 C2C12-细胞中对缺氧再氧合 (HR) 的反应减弱。同样，c-Jun N-末端激酶 (JNK) 的激活被否定。我们还发现内在的凋亡激活比外在的激活更为重要。此外，阻断肌肉生长抑制素信号转导减轻了细胞凋亡的激活。最终，这项研究验证了 HR 中抑制肌肉生长抑制素的保护作用，并确定了值得进一步研究的潜在介质。阻断肌肉生长抑制素信号转导并没有总体上抑制 ROS 的产生，而是减轻了细胞损伤。特别是，源自线粒体的凋亡内在激活得到缓解。这可能是由 MEK3/6 的激酶活性增加导致的 p38 激活减少介导的。总的来说，这项工作为 C2C12-细胞中 HR 信号转导提供了重要的见解，并可以为进一步的研究提供基础。
