Effects of Yunaconitine on Calcium Homeostasis in Arrhythmia Induced by Aconitum vilmorinianum Kom in Cardiomyocytes (H9C2 Cells)

Yunaconitine is a diterpenoid alkaloid, which is the main active component extracted from A. vilmorinianum. Previous studies have shown that although Aconitum vilmorinianum and yunaconitine have obvious analgesic and anti-inflammatory effects, they also have high cardiotoxicity. This study aimed to investigate the effect of yunaconitine on calcium homeostasis in cardiomyocytes (H9C2 cells) to preliminarily explore the molecular mechanism of arrhythmia induced by yunaconitine. The ultrastructural changes of H9C2 cells treated with yunaconitine were observed by TEM. The levels of Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase in H9C2 cells treated with yunaconitine were detected by the kits. The intracellular Ca²⁺ concentration of H9C2 cardiomyocytes treated with yunaconitine was detected by flow cytometry and high-content screening (HCS). Then, the RT-PCR and western blotting were detected to investigate the effects of yunaconitine on the expression of connexins and Ca²⁺ handling proteins. Yunaconitine induced mitochondrial crest expansion, endoplasmic reticulum swelling and apoptotic corpuscle formation in H9C2 cells. Additionally, yunaconitine significantly reduced the activities of Na⁺-K⁺ -ATPase and Ca²⁺-Mg²⁺-ATPase in H9C2 cells. According to flow cytometric and high-content analysis results, yunaconitine markedly increased intracellular Ca²⁺ concentration. The results of the RT-PCR and western blotting tests showed that yunaconitine could downregulate the expression of CX43, CaM, NCX1, Cav1.2 and SERCA2a, while upregulating that of RyR₂. Furthermore, the activation of RyR₂ induced by yunaconitine was notably inhibited by ryanodine. These results demonstrated that yunaconitine disturbed intracellular Ca²⁺ homeostasis in cardiomyocytes by causing dysregulation of calcium regulatory proteins and reducing calcium-related ATPase activity. Furthermore, inhibition of Ca²⁺ release from the RyR2 channel of sarcoplasmic reticulum may be an effective method to prevent yunaconitin-induced arrhythmias.