Level of G protein-coupled receptor kinase-2 determines myocardial ischemia/reperfusion injury via pro- and anti-apoptotic mechanisms

Abstract

Rationale: Activation of prosurvival kinases and subsequent nitric oxide (NO) production by certain G protein-coupled receptors (GPCRs) protects myocardium in ischemia/reperfusion injury (I/R) models. GPCR signaling pathways are regulated by GPCR kinases (GRKs), and GRK2 has been shown to be a critical molecule in normal and pathological cardiac function.

Objective: A loss of cardiac GRK2 activity is known to arrest progression of heart failure (HF), at least in part by normalization of cardiac β-adrenergic receptor (βAR) signaling. Chronic HF studies have been performed with GRK2 knockout mice, as well as expression of the βARKct, a peptide inhibitor of GRK2 activity. This study was conducted to examine the role of GRK2 and its activity during acute myocardial ischemic injury using an I/R model.

Methods and results: We demonstrate, using cardiac-specific GRK2 and βARKct-expressing transgenic mice, a deleterious effect of GRK2 on in vivo myocardial I/R injury with βARKct imparting cardioprotection. Post-I/R infarct size was greater in GRK2-overexpressing mice (45.0±2.8% versus 31.3±2.3% in controls) and significantly smaller in βARKct mice (16.8±1.3%, P<0.05). Importantly, in vivo apoptosis was found to be consistent with these reciprocal effects on post-I/R myocardial injury when levels of GRK2 activity were altered. Moreover, these results were reflected by higher Akt activation and induction of NO production via βARKct, and these antiapoptotic/survival effects could be recapitulated in vitro. Interestingly, selective antagonism of β(2)ARs abolished βARKct-mediated cardioprotection, suggesting that enhanced GRK2 activity on this GPCR is deleterious to cardiac myocyte survival.

Conclusion: The novel effect of reducing acute ischemic myocardial injury via increased Akt activity and NO production adds significantly to the therapeutic potential of GRK2 inhibition with the βARKct not only in chronic HF but also potentially in acute ischemic injury conditions.

Figure 1. Post-I/R LV infarct size in hearts overexpressing GRK2 or βARKct

A) Representative photographs of post-I/R TTC-stained heart sections from cardiac βARKct transgenic, GRK2 transgenic or non-transgenic littermate (NLC) control mice. B) LV infarct size expressed as percentage of the area at risk (AR; ischemic) in each group revealing significantly larger injury when GRK2 is elevated and decreased injury in βARKct expressing hearts. C) LV AR expressed as percentage of the total LV. (n=13-25/group; *p<0.05 vs. NLC, ⁺p<0.05 vs. βARKct).

Figure 2. Post-I/R apoptosis in hearts overexpressing GRK2 or βARKct

A) Representative photographs of TUNEL-stained tissue sections from post-I/R NLC, βARKct and GRK2 mice including Sham-negative control sections (scale bar: 50μm). TUNEL (green), apoptotic nuclei, and DAPI (blue), total nuclei. B) TUNEL-positive nuclei quantification represented as number per high power field (HPF). C) Caspase-3 activity represented as pmol of substrate per mg of protein per hour (pmol/mg prot/h). (n=6-9/group; *p<0.05 vs. NLC, ⁺p<0.05 vs. βARKct, ^‡p<0.05 vs. Sham).

Figure 3. Production of nitric oxide (NO) is induced by facilitated adrenergic signaling

A) Reciprocal effects of GRK2 overexpression and its inhibition by βARKct were found in measurements of myocardial NO content in the myocardial area at risk (AR) after 30 min Ischemia and 3 hrs of Reperfusion (n=8-22/group, *p<0.01 vs Control, +<0.01 vs CTR). B) Synthesis of NO under basal conditions and stimulated by TNFα (300ng/ml) and norepinephrine (NE, 1μM) in neonatal rat ventricular myocytes (NRVMs) overexpressing either GRK2 or expressing βARKct. GFP infected cells were used as a control and βARKct expressing cells showed significantly induced NO production in the latter and this effect was abolished by blocking of NO synthases by L-NAME (n=3, experiments done in duplicate, * p<0.001 vs GFP)

Figure 4. Apoptosis in myocytes subjected to in vitro I/R or oxidative stress is linked to activity of survival signaling via Akt

A) NRVMs infected with adenoviruses containing GRK2, βARKct or GFP (control) were subjected to H₂O₂ inducing oxidative stress or an in vitro I/R protocol. Apoptotic index assessed after 24 hrs is depicted as percentage of TUNEL-positive (FITC) apoptotic over total nuclei (DAPI). (n=10/group; *p<0.05 vs. GFP in respective treatment, ⁺p<0.05 vs. Basal). Blocking of up- and downstream signaling of Akt via LY294002 (PI3K) or L-NAME (NOS), respectively, was effective in abolishing the beneficial effect of βARKct (n=3/group, p=n.s. βARKct vs GFP). B) Phosphorylation of Akt in NRVMs expressing control (GFP), βARKct or GRK2 adenoviral construct and treated with H₂O₂. The increase in Akt phosphorylation is significantly higher in Control and βARKct expressing NRVMs whereas in GRK2 overexpressing myocytes Akt phosphorylation is blunted (n=4, independent experiments done in duplicates, *p<0.05 vs CTR, +p<0.05 vs βARKct, ANOVA with Newman-Keuls post hoc test). C) Representative Western blot for phospho-Akt^Serine473 at baseline (lanes 1-6) and stressed conditions (H₂O₂, lanes 7-10).

Figure 5. In vivo post-I/R activation of cardiac Akt and eNOS is enhanced with βARKct expression and blunted with GRK2 overexpression

A) Representative Western blot (left) of pAkt and GAPDH protein levels in LV tissue subjected to I/R for 3 hrs and quantification (right) of the increase in Akt phosphorylation normalized to GAPDH as a percentage of NLC increase (n=5-6 hearts, 3-4 independent experiments, *p<0.05). B) In line with increased pAkt, eNOS phosphorylation at Serine-1177 (left) was also enhanced in βARKct expressing myocardium and shown (right) as fold increase of pENOS/tENOS ratio (n=4, from 2 independent experiments, *p<0.05).

Figure 6. Post-I/R injury in βARKct and GRK2 transgenic mice are reversed by β₂AR modulation

A) Representative photographs of TTC-stained sections from post-I/R NLC and βARKct mice treated with the β₂AR-selective antagonist ICI 118,551. B) LV infarct size (IS) expressed as percentage of the area at risk (IS/AR) in NLC and βARKct groups shows no significant difference (n=11-13/group, p=n.s.). C) AR expressed as percentage of the total LV in NLC and βARKct mice is comparable. D) Representative photographs of TTC-stained sections from post-I/R NLC and cardiac GRK2 overexpressing mice treated with the β₂AR agonist fenoterol. E) IS/AR ratio is the same in NLC and GRK2 groups (n=6-10, p=n.s.). F) AR/LV did not show any difference between NLC and GRK2 mice.

Figure 7. Proposed model of enhanced β₂AR signaling through Akt/NO leading to increased cell survival after acute myocardial injury

This study shows that cardioprotective signaling through GPCRs such as the β₂AR can negatively be affected by GRK2 expression and activity through inhibition of Akt. Moreover, GRK2 inhibition by the βARKct expression eliminates this negative Akt influence increasing downstrewam pro-survival signaling that includes decreased Caspase-3 activity as well as enhanced activation of eNOS and NO production, which reduces apoptosis and cell death.