Calenduloside E suppresses calcium overload by promoting the interaction between L-type calcium channels and Bcl2-associated athanogene 3 to alleviate myocardial ischemia/reperfusion injury

Introduction: Intracellular calcium overload is an important contributor to myocardial ischemia/reperfu- sion (MI/R) injury. Total saponins of the traditional Chinese medicinal plant Aralia elata (Miq.) Seem. (AS) are beneficial for treating MI/R injury, and Calenduloside E (CE) is the main active ingredient of AS. Objectives: This study aimed to investigate the effects of CE on MI/R injury and determine its specific reg- ulatory mechanisms. Methods: To verify whether CE mediated cardiac protection in vivo and in vitro, we performed MI/R sur- gery in SD rats and subjected neonatal rat ventricular myocytes (NRVMs) to hypoxia-reoxygenation (HR). CE’s cardioprotective against MI/R injury was detected by Evans blue/TTC staining, echocardiography, HE staining, myocardial enzyme levels. Impedance and field potential recording, and patch-clamp tech- niques of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were used to detect the function of L-type calcium channels (LTCC). The mechanisms underlying between CE and LTCC was studied through western blot, immunofluorescence, and immunohistochemistry. Drug affinity responsive target stability (DARTS) and co-immunoprecipitation (co-IP) used to further clarify the effect of CE on LTCC and BAG3. Results: We found that CE protected against MI/R injury by inhibiting calcium overload. Furthermore, CE improved contraction and field potential signals of hiPSC-CMs and restored sarcomere contraction and calcium transient of adult rat ventricular myocytes (ARVMs). Moreover, patch-clamp data showed that CE suppressed increased L-type calcium current (ICa,L) caused by LTCC agonist, proving that CE could reg- ulate calcium homeostasis through LTCC. Importantly, we found that CE promoted the interaction between LTCC and Bcl2-associated athanogene 3 (BAG3) by co-IP and DARTS. Conclusion: Our results demonstrate that CE enhanced LTCC-BAG3 interaction to reduce MI/R induced- calcium overload, exerting a cardioprotective effect.