β1-AR metoprolol is one of the most commonly used drugs to treat heart failure in clinic[12; 13]. Our study demonstrates that metoprolol can improve cardiac function in HHcy mice. Cardiac ultrasound showed that treatment with metoprolol improved left ventricular systolic function decline in HHcy mice. The biochemical detection of plasma showed that metoprolol decreased the plasma concentration of LDH in HHcy mice, Transmission electron microscope showed that metoprolol reversed myocardial ultrastructural injury in HHcy mice. This is related to metoprolol inhibiting the activation of β1-AR/CaMKII pathway induced by Hcy. Therefore, we first proposed that metoprolol reduces myocardial injury in HHcy mice through inhibiting β1-AR/CaMKII signal pathway.
Hyperhomocysteinemia (HHcy) is one of the independent risk factors of heart diseases, It has been reported that the risk of heart disease will increase by 10% when the concentration of plasma Hcy increases by 10µM. Folic acid is an important determinant of the Hcy metabolic pathway which can reduce the production of Hcy, therefore, exogenous folic acid supplementation is effective for some patients[15; 16]. However, clinical investigation shows that about 40% of HHcy patients do not have a satisfactory effect after taking folic acid treatment[17; 18]. This may be related to the mutation of methylene reductase, therefore it is of great significance to clarify the mechanism involved in HHcy-induced myocardial injury and find new therapeutic targets .
we constructed a HHcy model by feeding C57BL / 6J mice with 2.5% methionine diet. After 28 weeks of feeding, a significant increase in serum Hcy concentration was detected, suggesting that the HHcy model was established successfully and can be used for subsequent experiments. Compared with normal mice, echocardiography showed that cardiac contractility was decreased in HHcy mice, and the serum LDH and CK-MB concentrations were increased, meanwhile the disrupted myocardial ultrastructure, disorganized sarcomeres, and swollen mitochondria, indicating that chronically suffering from HHcy induces cardiac damage.
To further explore the mechanism of HHcy-induced myocardial injury, genome-wide expression analysis was performed on the myocardium of normal mice and HHcy mice, and KEGG analysis showed that the adrenalin signaling pathway and cAMP signaling pathway were significantly enriched in the myocardium of HHcy mice compared with the Control group. Since β-AR is the main AR expressed in the heart, and the β1-AR is the most widely expressed β-AR in the myocardial cells. β1-AR plays an important role in the sympathetic regulation of the heart and is highly conserved during evolution. Meanwhile, the expression of β1-AR changes in myocardial cells of heart failure patients. We wonder whether HHcy can activate β1-AR? The activation form of β1-AR is phosphorylation. There are many phosphorylation sites in β1-AR, at present there is no commercial antibody that can detect all the phosphorylation sites of β1-AR. Therefore, we detected the phosphorylation level of β1-AR by immunoprecipitation. The molecular weight of β1-AR is about 50KD, it is difficult to distinguish the target band from the IgG heavy chain. Therefore, we constructed the β1-AR-GFP fusion plasmid, whose molecular weight is about 77KD. We transfected the plasmid into HEK293 cells and found that Hcy increased the phosphorylation level of β1-AR.
β1-AR activation can be divided into two types: short-term stimulation of β1-AR activates the PKA signaling pathway and induces positive inotropic an chronotropic effects which are the most effective mechanisms for rapidly increasing cardiac output, while chronic activation of β1-AR activates CaMKII which is harmful to the heart[22; 23]. Therefore, in this study different time points were established at 8 weeks, 24 weeks, and 28 weeks to monitor the effects of Hcy on the cardiac function of C57BL/6J mice. The results showed that after 8 weeks of high methionine diet, the myocardial contractility of HHcy mice was significantly stronger than that of the normal mice. However at the 24th week, it’s lower than the Control group, and the same trend remained until 28 weeks, consistent with the trend of cardiac function changes after cardiac β1-AR activation. Then we extracted NRCMs and observed that the beat frequency of NRCMs was significantly increased after the addition of Hcy for 10 minutes, which was effectively reduced by β1-AR blocker metoprolol. To further discuss whether Hcy can activate the β1-AR downstream signaling pathway, we stimulated cardiomyocytes with Hcy for a short term (5–60 minutes), and the results showed that the activation level of PKA was increased in cardiomyocytes. Then, we stimulated cardiomyocytes with Hcy for a long term (48 hours) and found that the phosphorylation level of CaMKII was increased. All of these effects were inhibited by β1-AR blocker metoprolol. Finally, we constructed a mouse model fed with a high methionine diet and treated with metoprolol. After 28 weeks, the serum Hcy concentration showed that the model was successfully established. Compared with HHcy mice, mice treated with metoprolol showed reduced activation levels of CaMKII in myocardial tissue, lower serum LDH concentration, stronger myocardial contractility, and less damage in cardiac ultrastructure. These results suggest that Hcy enhances myocardial contractility through activation of the β1-AR/PKA signaling pathway in the short term, while chronic Hcy stimulation activates the β1-AR/CAMKII signaling pathway and plays a role in myocardial injury.
Our study showed that β1-AR blocker metoprolol is a potential drug for the treatment of myocardial injury induced by HHcy, For HHcy patients with increased myocardial contractility, early administration of metoprolol may have a good effect on alleviating HHcy induced myocardial disease. There's limitation to this study. Due to technical limitations, at present β1-AR protein cannot be purified. Therefore, whether Hcy can directly bind to β1-AR as a ligand to regulate its conformation still remain unclear.