Angiotensin II‐mediated Mitochondrial Ca2+ Uptake and Superoxide Generation Activate Proliferative Pathway in Neonatal Cardiac Fibroblasts

Abstract

Cardiac fibroblasts (CFs) are the most prevalent cell type in the heart in addition to cardiomyocytes and play key roles in regulating myocardial physiological function and pathophysiological remodeling. Clinical observations and basic research data strongly suggest that CFs can respond to various stimuli including, angiotensin II (AT‐II), the levels of which are increased in the remodeling heart and participates in remodeling of the failing heart by AT‐II‐mediated pathological CF proliferation. It has been shown that CF proliferation may occur via enhanced production of 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 cardiomyocytes. Therefore, we hypothesize that Ang‐II stimulation enhances mitochondrial Ca2+‐ induced mtSO generation in CFs, which can activate ROS‐dependent proliferation signaling in CFs. First, using a mitochondria‐targeted Ca2+ biosensor, we found that AT‐II (≥1 μM) stimulation induces significant mitochondrial Ca2+ uptake 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. These effects were significantly blocked by the pretreatment of an AT‐receptor antagonist losartan. Next, we 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 also significantly inhibited AT‐II‐mediated activation of the proliferative pathway without changing the AT‐II‐induced the mitochondrial Ca2+ uptake profile. Our results indicate that mtSO generation induced by mitochondrial Ca2+ accumulation works as a stimulator of the Ang II–induced proliferative pathway in cardiac fibroblasts.Support or Funding InformationThis work was partly supported by American Heart Association (AHA) grant (14BGIA18830032 to J.O.‐U.), Medical Research Grant from W.W. Smith Charitable Trust (H1403 to J.O.‐U.) and NIH grants (2R01HL093671 and 1R01HL122124 to SSS).