Introduction
Stress is an inevitable response to internal and external stimuli that can impair cardiac function, such as stress cardiomyopathy, arrhythmia, and sudden death [1]. Stress response is significantly regulated by the sympatho-adrenomedullary system, resulting in an increased secretion of catecholamines, which activates of β-adrenoceptors (βAR) [2]. During stress, GRK2, also known as β-adrenoceptor kinase 1 (βARK1), predominately translocates to the cell membrane to catalyze the phosphorylation of an already activated βAR, the main receptor that governs the inotropic and chronotropic effects of the heart. Evidence suggests that the female sex hormone estrogen could improve cardiac function during stress by regulating βAR-Gs/Gi signaling pathway [3]. However, the interplay between estrogen and GRK2 is not well understood.
Aim
Here we explored the effects of estrogen on myocardial GRK2 during stress and its effect on GRK2-mediated internalization of βAR.
Methods and results
In vivo and in vitro experiments were performed using female wild-type (WT) and GPER-KO mice, isolated adult mice cardiomyocytes, and hESC-CM.
WT mice were divided into sham-operated and ovariectomy groups. These mice and isolated adult cardiomyocytes were subcutaneously administered with isoproterenol (ISO) and estrogen (E2). The results showed that (1) Estrogen enhanced cardiac function by measuring ECG and cardiomyocytes' shortening amplitude, and improved APD (action potential duration), INa, Ito, ICa-L of hESC-CMs by patch clam in acute stress. (2) Estrogen reduced total GRK2 and membrane GRK2 content in the myocardium during acute stress in mice and hESC-CM by western blot and immunofluorescence. (3) By inhibiting or overexpressing GRK2, the results showed that estrogen enhanced the contractile function and electrophysiological indexes of cardiomyocytes via inhibiting GRK2 in acute stress. (4) Estrogen reduced GRK2-mediated internalization of β1AR in myocardium during acute stress by Immunoprecipitation and immunofluorescence.
WT and GPER -KO mice were also categorized into groups based on the subcutaneous administration of ISO. hESC-CM were subjected to GPER siRNA transinfection, then cells were pre-treated with ISO and estrogen. The results showed that (1) GPER attenuated GRK2-induced reduction in cardiac function during acute stress by measuring ECG and cardiomyocytes’shortening amplitude. (2) GPER reduced GRK2 content in the myocardium membrane and β1AR internalization in acute stress by immunofluorescence.
Statistical analysis
All data analyses were performed with GraphPad Prism 5.01 and presented as means ± s.e.m. Statistical significance (P ﹤0.05) for each variable was estimated by one-way or two-way ANOVA followed by Bonferroni post hoc tests. For animals,n=6-8, for cells n=3-4.
Conclusion
Exciting new findings from our study demonstrate that estrogen has a powerful protective effect on the heart by reducing the content of GRK2 in the myocardium and preventing the internalization of β1AR during acute stress. These findings not only shed light on the complex mechanisms underlying estrogen's beneficial effect on heart function but also highlight GRK2 as a crucial target for treating stress-induced heart disease. By targeting GRK2, we can potentially avoid the side effects of estrogen and develop more effective therapies for women with estrogen deficiencies or elderly patients.
Ethical standards
All animal procedures complied with the guidelines of the Animal Ethics Committee of Xuzhou Medical University (China) (permit number:L2021701001).