Research ArticleCardiovascular Physiology

Convergence of G Protein–Coupled Receptor and S-Nitrosylation Signaling Determines the Outcome to Cardiac Ischemic Injury

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Science Signaling  29 Oct 2013:
Vol. 6, Issue 299, pp. ra95
DOI: 10.1126/scisignal.2004225


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.


Nitric oxide (NO) protects the heart against ischemic injury (13), and NO-based therapy is part of the standard of care in patients with heart failure (4). The classic view holds that NO acts primarily as a vasodilator; however, it is not known how NO protects the ischemic heart. In this light, there has been growing appreciation that endogenous nitrosylating compounds called S-nitrosothiols (SNOs) are involved in ischemic cardioprotection (5, 6). The protein targets of SNOs that may ameliorate cardiac injury are largely unknown.

GRK2 [G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor (GPCR) kinase 2] is the primary effector of post-ischemic myocyte death that is downstream of GPCRs (7), particularly β-adrenergic receptors (βARs), which have a central role in the pathogenesis of heart failure (8). Although GPCR-based pathways of injury are viewed as unrelated to NO-based signaling, the inhibition of GRK2 by NO (9) would improve βAR resensitization and coupling to agonists, thus simulating the effect of β-blockade (10) (the lynchpin of the current treatment paradigm for heart failure). GRK2 appears to serve as a critical regulator of myocardial GPCR signaling (11). Here, we consider the possibility that classic GPCR-regulated ischemic injury and SNO-mediated cardioprotection have a shared mechanistic basis that arises through convergence of signaling on GRK2.


Cardiac endothelial nitric oxide synthase protects against GRK2-mediated injury after ischemia

To determine whether classic cardioprotection by NO was mediated through inhibition of GRK2, we asked if endothelial nitric oxide synthase (eNOS) could alleviate the detrimental effect of GRK2 activation after ischemia/reperfusion injury. For this purpose, we bred cardiac-specific GRK2-overexpressing transgenic (Tg) mice (12) with cardiac-specific eNOS Tg mice (1) to generate GRK2 and eNOS double Tg mice (GRK2/eNOS mice). GRK2 and eNOS abundance in the hearts of GRK2/eNOS mice were similar to those in the breeder lines (fig. S1A). We then subjected adult GRK2/eNOS mice and their littermates (including control mice and single-breeder Tg mice) to 30 min of myocardial ischemia followed by 24 hours of reperfusion (13). Consistent with a previous report (7), GRK2 Tg mice had larger infarcts at 24 hours compared to the other lines (Fig. 1, A and B), whereas eNOS Tg mice had reduced infarcts. Infarct size in GRK2/eNOS mice was reduced by 20% compared to that in GRK2 Tg mice and was not different from that in control mice (Fig. 1, A and B). All four groups had similar ischemic areas at risk (fig. S1B), demonstrating the same severity of ischemic stress.

Fig. 1 Cardiac eNOS protects against GRK2-mediated injury after ischemia/reperfusion.

(A) Representative images of Evans blue/triphenyltetrazolium chloride (TTC) staining of hearts after ischemia/reperfusion. Dotted area is the infarct zone. (B) Measurement of infarct size as a percentage of LV ischemic area at risk (AAR) in non-Tg littermate control (NLC), GRK2 Tg, eNOS Tg, and GRK2/eNOS mice. *P < 0.05, #P < 0.01 [analysis of variance (ANOVA); n = 8 to 12 mice per group]. (C) Cardiac function evaluated by LV ejection fraction (LVEF, %) measured by echocardiography in the mice from (B). *P < 0.05, #P < 0.01 (ANOVA; n = 8 to 12 mice per group). (D) Measurement of infarct size as a percentage of LV AAR in wild-type control (WT), GRK2 Tg, eNOSnull, and GRK2/eNOSnull mice. *P < 0.05, #P < 0.01 (ANOVA; n = 10 to 12 mice per group). (E) Measurement of infarct size as a percentage of LV AAR in WT, βARKct Tg, eNOSnull, and βARKct/eNOSnull mice. *P < 0.05, #P < 0.001 (ANOVA; n = 6 to 8 mice per group).

Infarct size reductions in GRK2/eNOS mice translated to marked improvements (~30%) in cardiac function as measured by left ventricular (LV) ejection fraction by echocardiography 24 hours after reperfusion (Fig. 1C and fig. S1C). In addition, LV dilation in GRK2 Tg mice (as assessed by diastolic LV internal diameter) was accounted for, and inversely correlated with, myocardial eNOS expression (fig. S1D). To further explore the functional relationship between eNOS and GRK2 in the ischemic heart, we bred GRK2 Tg mice with eNOSnull mice and subjected these mice to ischemia/reperfusion injury. Deletion of eNOS exacerbated the post-ischemia/reperfusion injury caused by GRK2 as demonstrated by larger infarct size in GRK2/eNOSnull mice compared to GRK2 Tg mice or eNOSnull alone (Fig. 1D) despite similar ischemic areas at risk (fig. S1E). Thus, the injurious effects of GRK2 in the ischemic heart are accentuated by deficiency of eNOS and attenuated by increased eNOS.

Inhibition of GRK2 by the peptide inhibitor βARKct protects against cardiac ischemic injury (7), and βARKct Tg mice showed robust protection against ischemia/reperfusion injury as measured by infarct size at 24 hours (Fig. 1E and fig. S1F). When βARKct mice were bred onto the eNOSnull background, cardioprotection was abolished, and infarct size in βARKct/eNOSnull mice was no different from that in control mice (Fig. 1E and fig. S1F). These data argue that the protection conferred by GRK2 inhibition is transduced through eNOS. Our findings are consistent with a model in which GRK2 inhibits eNOS activity and inhibition of GRK2 allows for increased NO bioavailability.

GRK2 interacts with eNOS in mouse heart

We next explored the mechanism of this apparent interdependence of eNOS and GRK2 activity. eNOS coimmunoprecipitated with GRK2 from cardiac lysates (Fig. 2A), and this interaction was increased substantially after 30 min of ischemia followed by 30 min of reperfusion (Fig. 2, B and C). We overexpressed eNOS in neonatal rat ventricular myocytes (NRVMs) with or without GRK2 coexpression and treated myocytes with H2O2 to simulate oxidative stress in ischemia/reperfusion injury. H2O2 treatment activated eNOS (as measured by phosphorylation of Ser1177), and eNOS activation was blocked by GRK2 coexpression (Fig. 2, D and E). These findings are consistent with reports that GRK2 Tg hearts have lower NO concentrations after ischemia (7) and that GRK2 directly binds Akt (14), leading to lower Akt-mediated phosphorylation of eNOS and thus activity. Akt may be part of a protein complex with GRK2-eNOS.

Fig. 2 GRK2 interacts with eNOS in mouse heart, an interaction that is increased after ischemia.

(A and B) Representative Western blots of GRK2 and eNOS coimmunoprecipitations at baseline (A) and after ischemia/reperfusion (B). (C) Quantification of the amount of cardiac eNOS immunoprecipitated by GRK2 in sham-treated mice and mice after ischemia/reperfusion with mean ± SEM values shown. *P < 0.05 (Mann-Whitney test; n = 5 mice per group). (D) Activation of eNOS induced by H2O2 treatment is blocked by coexpression 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) and total eNOS (bottom) is shown. (E) Quantification of five separate experiments done as in (D). *P < 0.05, #P < 0.01 (Kruskal-Wallis test). (F and G) S-nitrosylation of GRK2 in GRK2 immunoprecipitates from hearts of GRK2 Tg and GRK2/eNOS mice after ischemia/reperfusion, as determined with a Cys-NO antibody. (F) Representative blot for SNO-GRK2 and total GRK2 after immunoprecipitation. (G) Fold change (compared to sham-operated GRK2 Tg mice) of SNO-GRK2/GRK2 in GRK2 Tg and GRK2/eNOS mice after ischemia/reperfusion. *P < 0.05 (Mann-Whitney test; n = 5 mice per group).

We assessed the S-nitrosylation state of GRK2 after ischemia/reperfusion injury. Because S-nitrosylation inhibits GRK2 (9), the amount of nitrosylated GRK2 should reflect the extent to which GRK2 was inhibited by eNOS and thus the tendency for injury (lower amounts of nitrosylated GRK2) compared to protection (higher amounts of nitrosylated GRK2). The amounts of SNO-GRK2 were determined with a Cys-NO–specific antibody (15) after immunoprecipitation of GRK2 from hearts of GRK2 Tg mice and GRK2/eNOS mice. The SNO-GRK2/GRK2 ratio in post-ischemia/reperfusion samples was decreased by 28% in GRK2 Tg mice compared to sham-operated controls, whereas coexpression of eNOS (in GRK2/eNOS mice) normalized the amount of cardiac SNO-GRK2, which was comparable to sham controls (Fig. 2, F and G). Thus, overexpression of eNOS overcomes GRK2-mediated inhibition of eNOS to block GRK2 activity and ultimately portends better outcomes after ischemic injury. Overall, these data demonstrate bidirectional and dynamic regulation of eNOS and GRK2 activities and suggest that the heart’s response to ischemic insult lies in the balance.

GRK2-C340S knock-in mice are resistant to S-nitrosylation–based regulation

To examine the importance of GRK2 in the cardioprotection conferred by eNOS, we generated a knock-in mouse harboring a point mutation in GRK2 (Cys340→Ser; C340S) that prevented the majority of S-nitrosylation by which NO inhibits GRK (9) (fig. S2, A and B). The abundance of GRK2 in multiple organs including the heart was similar to that in wild-type control mice (Fig. 3A). There were also no compensatory changes in the abundance of mRNAs encoding β1AR, β2AR, GRK3, GRK5, GRK6, or adenylyl cyclase (AC) 5 and AC6 in the hearts of GRK2-C340S knock-in mice compared to wild-type mice (fig. S2). The abundance of major limiting factors in βAR signaling, including AC5, AC6, and Gαs, was also not altered (fig. S2), and we observed no cardiac functional differences in C340S mice compared to wild-type mice (fig. S3).

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. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. A Western blot representative of four 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) and total GRK2 (bottom) 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 to 6 mice per group). (D) Decline in in vivo cardiac contractility (indicated by a decline in peak LV dP/dtmax) over 30 min during a maintained isoproterenol 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 to 6 mice per group).

GRK2-C340S knock-in mice were resistant to S-nitrosylation–based regulation. The SNO-RAC (resin-assisted capture) assay (16) showed that the amounts of S-nitrosylated GRK2 increased in the hearts of wild-type mice after isoproterenol infusion but not in GRK2-C340S knock-in hearts (Fig. 3, B and C). Similarly, S-nitrosoglutathione (GSNO) infusion (for 7 days) increased the S-nitrosylation of GRK2 in the hearts of wild-type mice but not in GRK2-C340S hearts (fig. S4, A and B). These data establish that Cys340 in GRK2 is modified by both endogenous and exogenous NO or SNO. S-nitrosylation of Cys340 did not appear to affect the interaction between GRK2 and eNOS because the association between these two proteins was similar in wild-type and GRK2-C340S knock-in hearts (fig. S4, C and D). The GRK2-eNOS interaction in the GRK2-C340S knock-in hearts was increased after ischemia/reperfusion, similar to that in wild-type hearts (fig. S4, E and F). Global amounts of SNOs in the heart were not changed by the C340S mutation under basal conditions (fig. S4, G and H). S-nitrosylated GRK2 was, however, decreased in GRK2-C340S knock-in hearts compared to wild-type hearts after ischemia/reperfusion (fig. S4I).

Ischemic heart failure is characterized by both myocyte injury and βAR desensitization. If the cardioprotective effects of NO are mediated through GRK2 S-nitrosylation, GRK2-C340S mice should be resistant to NO. We first used an in vivo assay for βAR desensitization (9), which analyzes the waning of cardiac pressure over time during a maintained infusion of isoproterenol. GRK2-C340S mice exhibited greater loss of contractility than did wild-type animals (Fig. 3D). Thus, desensitization during maintained catecholamine stimulation was accelerated in GRK2-C340S mice, a finding that is consistent with higher GRK2 activity. Moreover, the NOS inhibitor l-NG-nitroarginine methyl ester (l-NAME), which promoted βAR desensitization in wild-type animals (fig. S5A), had minimal effect in GRK2-C340S mice (fig. S5B). These data establish that agonist stimulation of the βAR is coupled to eNOS-mediated S-nitrosylation of GRK2, which prevents cardiac receptor desensitization. Diminished NO bioactivity causes overt βAR uncoupling from agonist stimulation, a major problem in the failing heart.

GRK2-C340S knock-in mice are more susceptible to ischemia/reperfusion injury

The interdependence of GRK2 and eNOS was further explored using GRK2-C340S mice. GRK2-C340S mice had larger infarcts after ischemia/reperfusion than did wild-type mice despite having similar areas at risk (Fig. 4, A and B, and fig. S6A). These results suggest that GRK2-C340S mutant mice had higher GRK2 activity. Furthermore, the NOS inhibitor l-NAME increased ischemic injury in wild-type but not in mutant mice (Fig. 4B). Moreover, infusion of GSNO reduced infarct size in both wild-type mice and GRK2-overexpressing mice, but not in GRK2-C340S mice (Fig. 4, C and D, and fig. S6, B and C). Functional assessments of cardiac performance correlated well with infarct size: GRK2-C340S mice had significantly worse post-ischemia/reperfusion cardiac function than did wild-type mice; l-NAME treatment worsened cardiac function in wild-type mice but not in GRK2-C340S mice (fig. S6D); GSNO infusion improved post-ischemia/reperfusion cardiac function in wild-type mice and GRK2 Tg mice but not in GRK2-C340S mice (fig. S6, E and F). Similarly, mice that overexpressed eNOS but had GRK2-C340S were not protected after ischemia/reperfusion injury (fig. S7, A and B). Therefore, the cardioprotective effects of both endogenous and exogenous NO are mediated by S-nitrosylation of GRK2. Our data should not be taken to indicate that GRK2 inhibition is the only means by which NO bioactivity may protect the heart (by analogy to the multiple mechanisms by which NO may inhibit apoptosis), and it should be noted that the protective effects of SNOs may vary in different ischemia/reperfusion models (fig. S7, C and D) (17).

Fig. 4 Susceptibility of GRK2-C340S knock-in mice to ischemia/reperfusion injury.

(A) Representative images of Evans blue/TTC staining of hearts after ischemia/reperfusion. (B to D) Measurement of infarct size as a percentage of LV AAR in WT and GRK2-C340S knock-in mice with or without NOS inhibition (l-NAME) (B) (*P < 0.05, #P < 0.01, ANOVA; n = 6 to 9 mice per group) or with or without GSNO infusion (C) (*P < 0.01, #P < 0.001, ANOVA; n = 6 to 7 mice per group) and in WT and GRK2 Tg mice with or without GSNO infusion (D) (*P < 0.01, #P < 0.001, ANOVA; n = 6 mice per 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 per group). (G) Representative terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining (green) of WT and GRK2-C340S hearts after ischemia/reperfusion with or without GSNO infusion. 4′,6-Diamidino-2-phenylindole (DAPI) staining (blue) marked nuclei, and troponin I staining (red) labeled cardiac myocytes. Scale bar, 50 μm. (H) Double labeling with TUNEL and troponin I to show TUNEL-positive cardiomyocytes. Scale bar, 20 μm. (I) Percentage of TUNEL-positive nuclei in WT and GRK2-C340S hearts after ischemia/reperfusion injury. *P < 0.05, #P < 0.01, $P < 0.001 (ANOVA, n = 6 to 8 mice per group). (J) Interdependence of GRK2 and eNOS in the heart.

To test whether the different outcome to ischemia/reperfusion injury in C340S knock-in mice is accompanied by altered survival signaling, we measured the amounts of phosphorylated and activated Akt in the injured hearts (Fig. 4E). Ischemia/reperfusion resulted in robust activation of Akt in both groups, but to a lesser extent in GRK2-C340S knock-in mice, consistent with the worsened phenotype in these mice (Fig. 4F). Also, after ischemia/reperfusion injury, the border zone of C340S hearts showed more apoptotic nuclei in ischemic myocytes than in wild-type hearts (Fig. 4, G to I). GSNO infusion reduced cell death after ischemia/reperfusion injury in wild-type hearts but not in C340S hearts (Fig. 4, G and I). We also found no difference in epidermal growth factor receptor (EGFR) activity in cardiomyocytes overexpressing wild-type GRK2 or GRK2-C340S after stimulation of βARs with isoproterenol (fig. S8, A and B), eliminating the possibility that trans-activation of EGFR by GRK5/6 was involved in cardioprotection (18). Myocyte injury by GRK2 is thus likely mediated through direct prodeath kinase activity. Together, these data provide strong evidence for (i) prodeath effects of GRK2 that involve loss of NO bioactivity and (ii) protective functions of eNOS that involve inhibition of GRK2.


Our results demonstrate that GRK2 and eNOS interact in the heart to form a key nodal point that determines outcomes to ischemic injury. GRK2-eNOS disequilibrium (increased GRK2 activity or decreased eNOS activity) manifests as βAR desensitization and myocyte injury, which are molecular corollaries of heart failure. eNOS inactivates GRK2 by S-nitrosylation to enable signaling through βARs, thereby maintaining cardiac function. Ischemic stress and heart failure are typified by increased abundance of GRK2 (11, 19), which counteracts eNOS cardioprotection (Fig. 4J). The utility of nitrates (a source of NO bioactivity) and β-blockers (a means to inhibit GRK2 activity) (20, 21) in heart failure has preceded detailed molecular understanding. Our data indicate that these drugs may restore the balance between GRK2 and NO activity and suggest new therapeutic approaches.

GRK2 has been previously reported to directly bind to Akt (14), leading to lower Akt-mediated eNOS phosphorylation and activity. The finding that cardioprotection mediated by GRK2 inhibition is dependent on eNOS (because no cardioprotection is seen in βARKct/eNOSnull mice) is consistent with the involvement of Akt. Our data (Fig. 4J) thus raise the possibility that Akt may be part of a signaling complex with GRK2-eNOS. It is interesting to note that activation of the Akt/eNOS pathway is dependent on Gβγ (22) and βARKct inhibits GRK2 by blocking Gβγ.

Current tenets hold that cardioprotection by NO is mediated by various effects on vessel tone, thrombosis, cell death signaling, inflammation, and energy conservation, and a long list of nitrosylated targets (guanylate cyclase, caspases, nuclear factor κB, and mitochondrial proteins) has been investigated (23). More than 1000 S-nitrosylated proteins have been identified in hearts treated with NO donors, and consequently, it is intriguing that a single cysteine in GRK2 (Cys340) is the primary locus through which both endogenous and exogenous NO bioactivities confer protection (Fig. 4J). Inhibition of GRK2 by NO can thus account for a major part of NO’s protective function in the ischemic heart. It is interesting to note in this regard that GRK2 translocates to mitochondria after ischemia/reperfusion (24) and that mitochondrially targeted NO bioactivity is protective in ischemia/reperfusion models (17, 25). Mitochondrial targets of GRK2 could be effectors of death kinase activity.

Our results suggest that GRK2 is a common factor underlying the cardioprotective effects of NO and β blockers as well as newer therapeutic strategies, which yield beneficial effects in heart failure models (2628). Thus, our new results support the idea that GRK2 is a potential therapeutic target in acute ischemia and identify a deficiency of NO bioactivity as a component of the pathophysiology of death kinase signaling. Our studies have implications for GPCR signaling in the ischemic heart, broaden perspectives on βAR blocker therapy, and point to new therapeutic approaches in acute coronary syndromes, chronic myocardial ischemia, and heart failure.

Materials and Methods

Generation of GRK2-C340S knock-in mice

A GRK2-positive clone was screened from a 129/SV mouse genome BAC (bacterial artificial chromosome) library (obtained from Children’s Hospital Oakland Research Institute), and the G was replaced with C in codon 340, leading to Cys340→Ser (C340S) mutation in exon 12. The GRK2-C340S targeting vector consisted of the diphtheria toxin A (DTA) gene, a 3.5-kb 5′ homology sequence, the neomycin resistance gene (Neo), and a 3.4-kb 3′ homology sequence containing the mutation. The linearized targeting vector was electroporated into C57 embryonic stem cells. Positive clones were injected to generate chimeric mice and GRK2-C340S mice (Ingenious Targeting Laboratory). Mutation was confirmed by sequencing of polymerase chain reaction (PCR) products flanking exon 12.

Ischemia/reperfusion injury model

Surgical procedures were carried out according to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals, and all procedures were approved by the Animal Care Committee at Temple University. The ischemia/reperfusion injury protocol was performed as previously described (13). Briefly, 8- to 10-week-old mice were anesthetized with 3% isoflurane inhalation. The heart was externalized through a left thoracotomy at the level of the fourth intercostal space. A slipknot was made around the left anterior descending coronary artery (LAD) at the level of the left auricle with a 6-0 silk suture. After the slipknot was tied, the heart was immediately placed back into the intrathoracic space followed by evacuation of pneumothoraces and closure of the muscle and skin suture through a previously placed purse-string suture. Sham-operated animals were subjected to the same surgical procedures except that the suture was passed under the LAD but was not tied. After 30 min of ischemia, the slipknot was released, and the myocardium was reperfused for either 3 hours to determine myocardial apoptosis or 24 hours to assess myocardial infarct size and cardiac function. For some of the mice, GSNO (10 mg/kg per day) or phosphate-buffered saline (PBS) was infused via implanted micro-osmotic pumps (Alzet) for 24 hours or 7 days before ischemia/reperfusion surgery, and in other experiments, l-NAME (50 nmol/g, bolus) was injected 10 min before ischemia/reperfusion surgery.

Determination of LV infarct size and AAR

LV infarct size and AAR were determined as previously described with slight modifications (13). Briefly, the ligature around the LAD was retied through the previous ligation, and 0.2 ml of 2% Evans blue dye was injected 24 hours after reperfusion. The dye distributed uniformly in the heart to areas perfused by the nonligated coronary arteries. The heart was then excised and sliced into five 1.2-mm-thick sections in the short axis of the heart. The sections were then stained with 2% TTC (Sigma-Aldrich) in PBS at room temperature for 15 min and then digitally photographed. The TTC-negative staining region would be pale, and the TTC-positive staining region would be red. The areas were defined as follows: the infarct area (Inf) consists of the TTC-negative staining region; the AAR consists of the Evans blue–negative staining region, including the TTC-positive staining and TTC-negative staining regions; and the area not at risk (ANAR) or nonischemic region consists of the Evans blue–positive staining regions. These regions were quantified with SigmaScan Pro 5.0 (SPSS Science). Myocardial infarct size was calculated as a percentage of the AAR (Inf/AAR), and the AAR was calculated as a percentage of total LV [AAR/(AAR + ANAR)]).

Transthoracic echocardiographic analysis

Transthoracic two-dimensional echocardiography in mice anesthetized with 2% isoflurane was performed with a 12-MHz probe as described previously (13). M-mode echocardiography was carried out in the parasternal short axis in mice 24 hours after reperfusion to assess heart rate, LV end-diastolic diameter, and LV anterior and posterior wall thickness. LV fractional shortening and ejection fraction were calculated.

Hemodynamic analysis of cardiac function

Hemodynamic analysis was conducted as described previously (9). Briefly, a 1.4-French micromanometer-tipped catheter (Millar Instruments) was inserted into the right carotid artery and then advanced into the LV. A polyethylene-50 catheter was placed in the left external jugular vein for continuous infusion of isoproterenol (20 ng/g per minute) for 30 min. Wild-type or GRK2-C340S knock-in mice were pretreated with either PBS or l-NAME (50 nmol/g, bolus) for 10 min. Steady-state LV maximal (dP/dtmax.) was recorded in closed-chest mode throughout the experiment with a PowerLab DAQ System (Millar Instrument).

Assessment of myocardial apoptosis

Myocardial apoptosis was assessed by TUNEL staining (7). Mice (n = 4 to 6 for sham groups, n = 6 to 8 for I/R groups) were euthanized 3 hours after reperfusion, and hearts were quickly removed and fixed in 4% paraformaldehyde. The hearts were then embedded in paraffin and cut into sections measuring 6 μm in thickness. TUNEL staining of sections was carried out with the in situ cell death detection kit (Roche). Slides were counterstained with DAPI-containing mounting medium. Cardiac myocytes were labeled with troponin I antibody (Cell Signaling Technology). The infarct border zone was visualized under a fluorescence microscope with a DAPI filter (330 to 380 nm) and a fluorescein isothiocyanate (FITC) filter (465 to 495 nm), and digital images were collected for DAPI, FITC, and merged. Images for at least three sections per animal were taken. Apoptotic cells with green fluorescence were counted with NIS-Elements Software (Nikon, Japan) to assess the apoptotic index (number of TUNEL-positive nuclei/number of total nuclei). More than 1000 cells were counted for each heart. For some experiments, sections were imaged with a Carl Zeiss 710 two-photon confocal microscope with a W Plan-Apochromat 63× oil objective, using 1× digital zoom, with excitations at 405, 488, and 561 nm. Images were quantified with Zen 2010 (Zeiss).

Isolation and primary culture of NRVMs

Ventricular cardiomyocytes from 1- to 2-day-old rat hearts (NRVMs) were prepared as described before (7). NRVMs were cultured in Ham’s F-10 supplemented with 5% fetal bovine serum (FBS) and penicillin/streptomycin (100 U/ml) at 37°C in a 95% air/5% CO2 humidified atmosphere for 4 days. NRVMs were infected with the indicated adenoviruses at a multiplicity of infection of 20 on day 2, and experiments were performed 48 hours after infection. NRVMs were starved in 0.5% FBS 8 hours before H2O2 treatment (150 μM, 10 min).

Coimmunoprecipitation and immunoblotting

Cells and tissue were homogenized in ice-cold radioimmunoprecipitation assay buffer [50 mM tris-HCl, 135 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, supplemented with 1 mM phenylmethylsulfonyl fluoride, leupeptin (10 μg/ml), aprotinin (20 μg/ml), and 1% (v/v) phosphatase inhibitor cocktail 1 and 2 (Sigma-Aldrich)]. The lysates were centrifuged at 13,000 rpm for 30 min at 4°C, and protein concentration was determined with BCA Protein Assay Kit (Pierce). Equal amounts of protein were then heated at 95°C for 5 min in 6× protein loading buffer. For coimmunoprecipitation, endogenous GRK2 from equal amounts of heart lysates was immunoprecipitated with rabbit GRK2 antibody conjugated to protein A/G–agarose beads (Santa Cruz Biotechnology). Samples were rotated overnight at 4°C and then centrifuged at 5000 rpm for 5 min. Immunoprecipitated samples were washed three times with lysis buffer and resuspended in 1× gel loading buffer and boiled. Clarified lysates and immunocomplexes were electrophoresed through 4 to 20% polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were blocked in Odyssey Blocking Buffer (Li-COR) and then incubated with primary antibodies detecting total p42/44 extracellular signal–regulated kinase (ERK1/2), p42/44 ERK, total Akt, pAkt, total eNOS, peNOS (Cell Signaling Technology), GRK2, AC5/6, Gαs (Santa Cruz Biotechnology), GAPDH (Millipore), and S-nitroso-cysteine (Sigma-Aldrich) at 4°C overnight. Proteins were then detected with the appropriate Alexa Fluor 680 nm– or IRDye 800 nm–coupled secondary antibodies (Life Technologies) with the Odyssey Infrared Imaging System (Li-COR). Quantitative densitometric analysis was performed with Odyssey infrared imaging software (version 2.1). Phospho-specific antibody signals and S-nitrosylation signals were normalized to total antibody signal.

SNO-RAC assay and global SNO measurement

Protein S-nitrosylation was detected by SNO-RAC as previously described (16). Briefly, hearts were homogenized with a Polytron homogenizer and centrifuged. Supernatants were collected and diluted in HEN buffer (100 mM Hepes, 1 mM EDTA, 0.1 mM neocuproine, pH 7.7) with 1% SDS and 0.1% methyl methanethiosulfonate for blocking thiol for 20 min at 50°C. Samples were then precipitated with acetone and washed with 70% acetone twice. After the addition of 50 μl of thiopropyl Sepharose and 50 mM sodium ascorbate, samples were rotated in the dark for 4 hours. Eluted samples after wash were analyzed by Western blot. Global SNO amounts were measured by photolysis chemiluminescence (29).

Real-time PCR

Total RNA from the hearts of 8-week-old wild-type and GRK2-C340S hearts were extracted with TRIzol (Life Technologies) and reverse-transcribed with random primers (Bio-Rad). The primers used for quantitative real-time PCR were as follows: β1AR, 5′-GTCATGGGATTGCTGGTGGT-3′ (forward) and 5′-GCAAACTCTGGTAGCGAAAGG-3′ (reverse); β2AR, 5′-GGGAACGACAGCGACTTCTT-3′ (forward) and 5′-GCCAGGACGATAACCGACAT-3′ (reverse); GRK3, 5′-GTGTGTGCGCGATACATTGC-3′ (forward) and 5′-GGGCTACATACCCCAGAGATAC-3′ (reverse); GRK5, 5′-CCTCCGAAGGACCATAGACA-3′ (forward) and 5′-GACTGGGGACTTTGGAGTGA-3′; (reverse); GRK6, 5′-TGACCCACGAGTACCTGAG-3′ (forward) and 5′-TCGCTTCTTTATCCGCTTCTTTT-3′ (reverse); AC5, 5′-CTTGGGGAGAAGCCGATTCC-3′ (forward) and 5′-ACCGCTTAGTGGAGGGTCT-3′ (reverse); AC6, 5′-GATGAACGGAAAACAGCTTGGG-3′ (forward) and 5′-GGTGGCTCCGCATTCTTGA-3′ (reverse); GAPDH, 5′-CCACTCTTCCACCTTCGATG-3′ (forward) and 5′-TCCACCACCCTGTTGCTGTA-3′ (reverse). Real-time quantification was performed by SYBR Green (Bio-Rad) with the CFR96 detection system (Bio-Rad). Relative gene expression was normalized to that of GAPDH and compared using the ΔΔCt method between wild type and GRK2-C340S.

Supplementary Materials

Fig. S1. Ischemia/reperfusion injury in GRK2 Tg or βARKct Tg mice in which eNOS abundance was genetically manipulated.

Fig. S2. Generation of GRK2-C340S knock-in mouse and real-time PCR and Western blot measurements of the abundance of βAR signaling pathway components in wild-type and GRK2-C340S knock-in mouse hearts.

Fig. S3. Echocardiography measurements in wild-type and GRK2-C340S mice.

Fig. S4. Biochemical characteristics of GRK2 in wild-type and GRK2-C340S knock-in mouse hearts.

Fig. S5. Decline in the in vivo LV contractility of wild-type and GRK2-C340S mice during isoproterenol infusion.

Fig. S6. Characteristics of ischemia/reperfusion injury in wild-type and GRK2-C340S knock-in mice after GSNO treatment.

Fig. S7. Characteristics of ischemia/reperfusion injury in eNOS/GRK2-C340S knock-in mice and wild-type mice acutely infused with GSNO.

Fig. S8. Activation of the mitogen-activated protein kinase p42/p44 ERK in NRVMs treated with isoproterenol or isoproterenol and EGFR inhibitor.

References and Notes

Acknowledgments: We thank J. Chen, H. Chen, G. Pari, and Y. Gao for assistance with BAC recombineering; J. Rabinowitz for adenoviruses; and Z. Qu, J. Ibetti, S. Baxter, and N. Otis for technical support. Funding: This work was supported in part by NIH grants R37 HL061690 (W.J.K.), P01 HL08806 (Project 3, W.J.K.), P01 HL075443 (Project 2, W.J.K.), and P01 HL075443 (Project 3, J.S.S.) and by a postdoctoral fellowship grant from the Great Rivers Affiliate of the American Heart Association (Z.M.H.). Author contributions: Z.M.H. conducted all of the experiments and wrote the paper. E.G. and X.S. did ischemia/reperfusion, echocardiography, and hemodynamics. H.H. and F.V.F. did the SNO-RAC experiments. J.K.C. and X.T. performed cell studies. N.E.H. did confocal imaging. D.G.T., M.M., and D.J.L. contributed to the writing of the paper. J.S.S. and W.J.K. wrote the paper. W.J.K. conceived and supervised the project. Competing interests: The authors declare that they have no competing interests. Data and materials availability: A material transfer agreement is required by Temple University for the GRK2-C340S knock-in mice.
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