Mitochondrial Ca2+ Uptake and Superoxide Generation Regulates Angiotensin II-Induced Proliferation in Neonatal Cardiac Fibroblasts

Abstract

Cardiac fibroblasts (CFs) are one o the most abundant cell types in the heart and play key roles in regulating myocardial physiological function and pathophysiological remodeling especially for the cardiac fibrosis. The levels of Angiotensin II (AT-II) are increased in the remodeling heart and Angiotensin signaling participates in pathological CF proliferation. It has been shown that CF proliferation may occur via the increased levels of cellular reactive oxygen species (ROS), but the detailed signal transduction remains unclear. We previously reported that the enhancement of mitochondrial Ca2+ uptake by mitochondrial Ca2+ uniporter (MCU) induces mitochondrial superoxide (mtSO) generation in cardiac myofibroblast cell line H9C2 cells. Therefore, we hypothesize that Ang-II stimulation enhances mitochondrial Ca2+-induced mtSO generation in primary CFs, which can activate ROS-dependent proliferation signaling in primary CFs. First, we confirmed that AT-II (≥1 μM) stimulation induces significant mitochondrial Ca2+ uptake assess by mitochondria-targeted Ca2+ biosensor in response to the Ca2+ release from the endoplasmic reticulum in neonatal rat CFs (NCF). In addition, AT-II stimulation increases the mtSO levels detected by a mtSO indicator MitoSOX Red. We also confirmed that AT-II application activates proliferative pathway, including ERK1/2, p38 and JNK1/2 in time-dependent manner, which was abolished by losartan pretreatment. Lastly, pretreatment of a mitochondria-targeted antioxidant, Mito-tempo significantly inhibited AT-II-mediated activation of the mtSO production as well as proliferative pathway without changing the AT-II-induced the mitochondrial Ca2+ uptake profile. Our results indicate that mtSO generation induced by mitochondrial Ca2+ accumulation via MCU serves as an important regulator for the Ang II-induced proliferation in CFs.