Convergence of G protein-coupled receptor and S-nitrosylation signaling determines the outcome to cardiac ischemic injury

Abstract

Heart failure caused by ischemic heart disease is a leading cause of death in the developed world. Treatment is currently centered on regimens involving G protein-coupled receptors (GPCRs) or nitric oxide (NO). These regimens are thought to target distinct molecular pathways. We showed that these pathways were interdependent and converged on the effector GRK2 (GPCR kinase 2) to regulate myocyte survival and function. Ischemic injury coupled to GPCR activation, including GPCR desensitization and myocyte loss, required GRK2 activation, and we found that cardioprotection mediated by inhibition of GRK2 depended on endothelial nitric oxide synthase (eNOS) and was associated with S-nitrosylation of GRK2. Conversely, the cardioprotective effects of NO bioactivity were absent in a knock-in mouse with a form of GRK2 that cannot be S-nitrosylated. Because GRK2 and eNOS inhibit each other, the balance of the activities of these enzymes in the myocardium determined the outcome to ischemic injury. Our findings suggest new insights into the mechanism of action of classic drugs used to treat heart failure and new therapeutic approaches to ischemic heart disease.

Fig. 1

Cardiac eNOS protects against GRK2-mediated injury following ischemia/reperfusion. A, Representative images of Evan’s Blue/triphenyltetrazolium chloride (TTC) staining of hearts after ischemia/reperfusion. Dotted area is the infarct zone. B, Quantification of infarct size as a percentage of left ventricular (LV) ischemic area at risk (AAR) in non-transgenic control (NLC), GRK2 Tg, eNOS Tg and GRK2/eNOS mice. *, P<0.05, #, P<0.01 (ANOVA, n = 8–12 mice/group). C, Cardiac function evaluated by LV ejection fraction (EF%) measured by echocardiography in the mice from (B). *, P<0.05, #, P<0.01 (ANOVA, n = 8–12 mice/group). D, Quantification of infarct size as a percentage of LV AAR in wild-type control (WT), GRK2 Tg, eNOS^null and GRK2/eNOS^null mice. *, P<0.05, #, P<0.01 (ANOVA, n = 10–12 mice/group). E, Quantification of infarct size as a percentage of LV AAR in WT, βARKct Tg, eNOS^null and βARKct/eNOS^null mice. *, P<0.05, #, P<0.001 (ANOVA, n = 6–8 mice/group).

Fig. 2

GRK2 interacts with eNOS in mouse heart, an interaction that is increased after ischemia. A, B, Representative Western blots of GRK2 and eNOS co-immunoprecipitations at baseline (A), and post-ischemia-reperfusion (B). C, Quantification of the amount of cardiac eNOS immunoprecipitated by GRK2 in sham treated mice and mice after ischemia-reperfusion with eNOS/GRK2 (mean±SEM) values shown. *, P<0.05 (Mann-Whitney test, n=5 mice/group). D, Activation of eNOS induced by H₂O₂ treatment is blocked by co-expression of GRK2 in myocytes. eNOS was immunoprecipitated from NRVMs infected with an adenovirus containing eNOS (Ad-eNOS), with or without Ad-GRK2, and treated with H₂O₂. A representative Western blot for eNOS phosphorylated at Ser¹¹⁷⁷ (top panel) and total eNOS (bottom panel) is shown. E, Quantification of 5 separate experiments done as in (D). *, P<0.05, #, P<0.01 (Kruskal Wallis test). F, G, S-nitrosylation of GRK2 in GRK2 immunoprecipitates from hearts of GRK2 Tg and GRFK2/eNOS mice after ischemia-reperfusion, as determined by a Cys-NO antibody. A representative blot for SNO-GRK2 and total GRK2 after immunoprecipitation (F). The fold change (compared to sham-operated GRK2 Tg mice) of SNO-GRK2/GRK2 in GRK2 Tg and GRK2/eNOS mice after ischemia-reperfusion (G). *, P<0.05 (Mann-Whitney test, n=5 mice/group).

Fig. 3

Evaluation of the baseline phenotype of GRK2-C340S knock-in mice. A, Western blot for GRK2 in the heart, liver and brain from WT mice and GRK2-C340S knock-in mice. GAPDH was used as a loading control. A Western blot representative of 4 independent experiments is shown. B, SNO-GRK2 in WT and GRK2-C340S knock-in mouse hearts under basal conditions and 15 min after a bolus injection of isoproterenol (ISO). A representative result from SNO-RAC assay with SNO-GRK2 (top panel) and total GRK2 (bottom panel) under the different conditions is shown. C, S-nitrosylated GRK2 in the various genotypes normalized to S-nitrosylated GRK2 in WT heart under baseline conditions. *, P<0.01, #, P<0.05 (Kruskal Wallis test, n=4–6 mice/group). D, Decline in the in vivo cardiac contractility (indicated by a decline in peak LV dP/dt_max) over 30 min during a maintained ISO infusion in WT and GRK2-C340S mice. *, P<0.05, #, P<0.01, $, P<0.05 C340S curve compared to WT (two way ANOVA, n=4–6 mice/group).

Fig. 4

The susceptibility of GRK2-C340S knock-in mice to ischemia/reperfusion injury. A, Representative images of Evan’s Blue/TTC staining of hearts after ischemia/reperfusion. B, Quantification of infarct size as a percentage of LV AAR in WT and GRK2-C340S knock-in mice with or without NOS inhibition (L-NAME) (*, P<0.05, #, P<0.01, ANOVA, n=6–9 mice/group); with or without GSNO infusion (C). (*, P<0.01, #, P<0.001, ANOVA, n=6–7 mice/group); in WT and GRK2 Tg mice with or without GSNO infusion (D). (*, P<0.01, #, P<0.001, ANOVA, n=6 mice/group). E, Representative Western blot of pAkt (Ser⁴⁷⁶), total Akt and GAPDH in sham and post-ischemia/reperfusion WT and GRK2-C340S hearts. F, pAkt/Akt ratio normalized to that of the WT sham group as done in (E). *, P<0.05, #, P<0.01 (Kruskal Wallis test, n=7 mice/group). G, Representative TUNEL staining (green) of WT and GRK2-C340S heart post-ischemia/reperfusion with or without GSNO infusion. DAPI staining (blue) marked nuclei and Troponin I staining (red) labeled cardiac myocytes. Scale: 50 μM. H, Double labeling of TUNEL and Troponin I staining to show TUNEL positive cardiomyocytes. Scale: 20μM. I, The percentage of TUNEL positive nuclei in WT and GRK2-C340S hearts following ischemia/reperfusion injury. *, P<0.05, #, P<0.01, $, P<0.001 (ANOVA, n=6–8 mice/group). J, Interdependence of GRK2 and eNOS in the heart.