By promoting atrial structural remodeling, atrial hypoxia contributes to the development of the atrial fibrillation substrate. Our study aimed to investigate the modulatory effect of hypoxia on profibrotic activity in cultured HL-1 cardiomyocytes and explore the possible signaling transduction mechanisms of profibrotic activity in vitro. Hypoxia (1% O-2) significantly and time-dependently increased the expression of hypoxia-inducible factor (HIF)-1 alpha and fibrotic marker proteins collagen I and III (COL1A and COL3A), transforming growth factor (TGF)-beta 1 and alpha-smooth muscle actin (SMA). Western blot or immunohistochemistry analysis showed that hypoxia-induced increase in COL1A and COL3A was significantly attenuated by the addition of SP600125 (a specific c-Jun N-terminal kinase [JNK] inhibitor) or expression of dominant-negative JNK before hypoxia treatment. The inhibition of hypoxia-activated phosphorylation of JNK signal components (JNK, MKK4, nuclear c-Jun and ATF-2) by pre-treatment with SP600125 could suppress hypoxia-stimulated HIF-1 alpha upregulation and fibrotic marker proteins expression. Hypoxia significantly increased reactive oxygen species (ROS) production in cultured HL-1 atrial cells. Pre-treatment with N-acetylcysteine significantly abrogated the expression of nuclear HIF-1 alpha, JNK transduction components and fibrotic marker proteins. Taken together, these findings indicated that the hypoxia-induced atrial profibrotic response occurs mainly via the ROS/JNK pathway, its downstream upregulation of HIF-1 alpha and c-Jun/ATF2 phosphorylation and nuclear translocation to up-regulate the expression of fibrosis-related proteins (COL1A, COL3A, TGF-beta 1 and alpha-SMA). Our result suggests that suppression of ROS/JNK signaling pathway is a critical mechanism for developing a novel therapeutic strategy against atrial fibrillation.
