Role Of GRKs In Sustained β AR-stimulated Cardiac Hypertrophy

Objective: 1. To investigate the function of different GRK subtype in the process of myocardial Ca MKII activiction. To screen out GRK subtype with significant actions, and observe the role of it in sustained β AR-stimulated cardiac hypertrophy.2. To examine whether oxidative signal ROS activate cardiac Ca MKII through affect the detected GRK subtype in the sustained β AR-stimulated cardiac hypertrophy.Methods: This project was divided into two part:Part 1 : Male mice(BW 20-28g)WT, GRK2, 3, 5, 6 knockout and GRK(-) mice(transgenic mice with cardiac-specific overexpression of a mutant β1、2-AR that lacks GRK phosphorylation sites) were used in this experiment. Mice were administered with isoproterenol(β-AR agonist, ISO) or saline with the same volume. After sacrificing the mice, heart left ventricle were excised and immediately frozen in liquid nitrogen and stored at-80°C for later biochemical assays. Western Blot was used to detect the expression of Ca MK II, phospho-Ca MK II(Thr286). Ca MK II activity of ventricular homogenates was measured by a Ca MK II assay kit based on ELISA principle. HE staining is to detect the histological change.Part 2 : Healthy male SD rats(body weight from 180-200g) were randomly separated into 4 groups: control(CTRL), ISO treated(ISO), control with NAC supplement(CTRL+NAC) and ISO treated with NAC supplement group(ISO+NAC) with 6 rats in each group. ISO treated method: the rats from ISO and ISO+NAC groups receive intraperitoneal injection of ISO for 3mg/kg/d; rats from CTRL and CTRL+NAC groups receive intraperitoneal injection of same volume of Physiological Saline. NAC supplement method: rats from CTRL+NAC and ISO+NAC groups drink water with 15g/L NAC freely everyday. The animals were sacrificed at the second weekend. Heart weight index(HW / BW) and HE staining were use to detect left ventricular hypertrophy; RT-q PCT, immunohistochemistry and Western blot analysis were applied to examine the expression of left ventricular GRK5.Results: Part1 : After ISO administration, the levels of phosphorylated Ca MK II(p Ca MK II; the active form of Ca MK II) markedly increased in hearts of WT, GRK2, GRK 3, and GRK 6 knockout mice, whereas ISO-mediated Ca MK II phosphorylation was markedly blunted in hearts from GRK5 knockout mice, indicating that GRK5 is necessary for the β-AR stimulated Ca MK II activation(n=5, P<0.05 fold induction by ISO of GRK5 knockout mice vs WT). As expected, in mouse hearts containing mutant β-ARs lacking GRK phosphorylation sites(β-AR GRK(-)), no significant increase in ISO stimulated p Ca MK II expression was observed(n=5, P<0.05 vs WT). Furthermore, Ca MK II activity detected by ELISA method in the heart extracts from WT and GRK2, GRK3, and GRK6 knockout mice were significantly increased followed by ISO stimulation compared with GRK5 knockout mice(n=5, P<0.05).Part2 :(1) ISO treated rats heart weight index(HW / BW) was significantly higher than the CTRL group [(3.99 ± 0.13 vs 3.31 ± 0.10) mg / g, P <0.05], HE staining showed that the cardiomyocyte cross-sectional area of ISO group increased significantly compared with CTRL group [(11117.00 ± 387.57 vs 4572.23 ± 176.39) μ m, P <0.05]; NAC significantly reduced the ISO-induced increases of heart weight index [(3.56 ± 0.12 vs 3.99 ± 0.13) mg/g, P<0.05] and myocyte cross-sectional area [(6160.33 ± 141.44 vs 11117.00 ± 387.57) μm, P <0.05].(2) Heart rate of ISO and ISO + NAC group decreased.(3) There was no significant difference of myocardial GRK5 expression between ISO and CTRL, ISO + NAC group and ISO group(P> 0.05).(4) RT-q PCR detect no significance of myocardial GRK5-m RNA expression between ISO and CTRL, ISO + NAC and ISO group(P> 0.05).(5) Arterial blood pressure showed no significant difference among the four group of rats(P> 0.05).(6) There was no significant difference of the detected index between rats from CTRL+NAC and CTRL group.Conclusion: This study preliminarly demonstrated that GRK5 is necessary for Ca MKII activation induced by the persistent βAR-stimulation. In the mechanisms of sustained βAR-stimulated cardiac hypertrophy, oxidative stress signaling molecules may not activate Ca MKII by affecting the expression of GRK5, and this process needs to be further study in vitro experiments.