Molecular Mechanism Of Calcium/Calmodulin Involvement In Ferroptosis Mediated Myocardial Infarction

Objectives:Myocardial infarction（MI）is the irreversible death of myocardial cells caused by a decrease in coronary artery blood flow and inadequate oxygen supply to the heart tissue.Because myocardial cells have almost no regenerative potential,inhibiting myocardial cell death is particularly crucial for the prognosis of MI patients.Ferroptosis,a newly discovered iron-dependent cell death pathway that accompanies the excessive accumulation of lipid peroxides,has been found to be closely related to the occurrence and development of various diseases in recent years.The key role of ferroptosis in cardiovascular disease has also attracted attention,but its specific mechanism of involvement has not been elucidated.In the occurrence of MI,calcium overload in myocardial cells has been found to be involved.The occurrence of intracellular calcium overload can activate calmodulin（CaM）,which regulates the function of L-type voltage-dependent calcium channels（LVDCC）.Given that intracellular iron overload is an important mechanism for inducing ferroptosis of myocardial cells,and LVDCC is an important cation channel involved in intracellular iron ion transport,we intend to investigate whether the activation of Ca²⁺/CaM in the process of myocardial infarction can cause iron overload by regulating LVDCC channels,thereby exacerbating the occurrence of ferroptosis.Methods:A hypoxia model of H9c2 rat cardiomyocytes was established.Ferrostatin-1（Fer-1）and Deferoxamine（DFO）were used for intervention.Cell Counting Kit-8（CCK-8）assay and Propidium Iodide（PI）staining were used to detect cell viability and death rate.Spectrophotometry was used to detect the levels of Malondialdehyde（MDA）and Glutathione（GSH）in the myocardium.Western blot was used to detect changes in the expression levels of Solute Carrier Family 7 Member 11（SLC7A11）and Glutathione peroxidase 4（GPX4）.Subsequently,Fluo-4/AM fluorescence probe was used to detect the effect of hypoxia on intracellular Ca²⁺levels,and Western blot and real-time quantitative PCR（RT-qPCR）were used to detect the expression levels of CaM protein and m RNA,as well as the expression of Ca²⁺/calmodulin-dependent protein kinases,including CaMKⅡ and CaMKIV.In addition,myocardial cell activity and ferroptosis-related indicators were detected by inhibiting CaM.Furthermore,the effect of blocking LVDCC on intracellular iron levels in hypoxia-induced myocardial cells was detected using calcein-AM fluorescent probe and total iron ion colorimetric assay kit.Finally,an MI model was established to measure cardiac function and myocardial fibrosis level,as well as changes in CaM expression.At the same time,Calmidazolium chloride（CCL）,a CaM antagonist,was used to treat MI mice after surgery,and the ferroptosis-related indicators were detected in myocardial tissue.Results:At the cellular level,compared with the control group,the Hypoxia group showed decreased cell survival rate,GSH content,and expression of SLC7A11 and GPX4,while cell death rate and MDA content increased.Fer-1 and DFO treatments,compared with the Hypoxia group,both inhibited the hypoxia-induced H9c2 cell damage to varying degrees.After hypoxia induction,H9c2 cells showed increased Ca²⁺concentration,and upregulated expression of CaM,CaMKⅡ,and CaMKIV.Further investigation through cell viability and detection of Ferroptosis-related indicators found that CaM antagonist CCL or CaM knockdown could alleviate hypoxia-induced Ferroptosis in cardiac muscle cells.Results from Calcein-AM and total cellular iron detection showed that LVDCC blocker Verapamil inhibited hypoxia-induced iron accumulation,suggesting that CaM regulates LVDCC to open and cause intracellular iron overload,leading to Ferroptosis.At the animal level,it was further demonstrated that CaM plays an important role in mediating Ferroptosis in myocardial infarction through regulating LVDCC.Conclusions:This study confirms at the cellular level that ferroptosis is an important form of cell death in myocardial cells under hypoxic injury,and reveals that Ca²⁺/CaM and its regulation of LVDCC may serve as a novel target for inhibiting ferroptosis.This has been validated at the animal level,providing a reference for further elucidating its mechanisms and expanding its applications.