Abstract
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 (Mstn) 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-Mstn−/− 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-Mstn−/− cells and could serve as basis for further research.
Highlights
Ischemia reperfusion (IR) injury remains a challenging topic in clinical medicine and has led to the proposal of several prophylactic and therapeutic strategies
This study aims to illuminate the role of myostatin signaling in skeletal muscle cells in response to hypoxia reoxygenation (HR)/IR injury and to place it into the pathophysiological cascades of reactive oxygen species (ROS) generation and stress signaling via p38 and Jun N-terminal kinase (JNK) that lead to cell death
We found apoptosis in response to HR to be mainly transduced through the intrinsic branch of the cascade, which is in line with previous studies [29]
Summary
Ischemia reperfusion (IR) injury remains a challenging topic in clinical medicine and has led to the proposal of several prophylactic and therapeutic strategies. Circuits involved in the pathogenesis of IR injury are mainly initiated by adenosine triphosphate (ATP) depletion and reactive oxygen species (ROS) production [4] These cascades culminate in different forms of (programmed) cell death [4]. This study aims to illuminate the role of myostatin signaling in skeletal muscle cells in response to HR/IR injury and to place it into the pathophysiological cascades of ROS generation and stress signaling via p38 and JNK that lead to cell death. For this reason, we used C2C12 cells and a CRISPR/Cas gene-edited C2C12 cell line in an in vitro HR/IR model
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