Gene expressions underlying mishandled calcium clearance and elevated generation of reactive oxygen species in the coronary artery smooth muscle cells of chronic heart failure rats

Background: The calcium clearance and reactive oxygen species (ROS) generations in the coronary artery smooth muscle cells in chronic heart failure (HF) have not been fully investigated. Therefore, we attempted to understand the gene expressions underlying the mishandling of calcium clearance and the accumulations of ROS. Methods: We initially established an animal model of chronic HF by making the left anterior descending coronary artery ligation (CAL) in rats, and then isolated the coronary artery vascular smooth muscle cells from the ischemic and the nonischemic parts of the coronary artery vessels in 12 weeks after CAL operation. The intracellular calcium concentration and ROS level were measured using flow cytometry, and the gene expressions of sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a), encoding sarcoplasmic reticulum Ca²⁺-ATPase 2a, encoding sodium-calcium exchanger (NCX), and p47phox encoding a subunit of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were examined using real-time quantitative reverse transcription polymerase chain reaction and Western blotting, respectively. Results: We found that the calcium accumulation and ROS generation in the coronary artery smooth muscle cells isolated from either the ischemic or the nonischemic part of the CAL coronary artery vessel were significantly increased irrespective of blood supply (all P < 0.01). Moreover, these were accompanied by the increased expressions of NCX and p47phox, the decreased expression of SERCA2a, and the increased amount of phosphorylated forms of p47phox in NADPH oxidase (all P < 0.05). Conclusions: Our results demonstrated that the disordered calcium clearance and the increased ROS generation occurred in the coronary artery smooth muscle cells in rats with chronic HF produced by ligation of the left anterior descending coronary artery (CAL), and which was found to be disassociated from blood supply, and the increased generation of ROS in the cells was found to make concomitancy to the increased activity of NADPH oxidase in cytoplasm.