key: cord-0014549-1nip45bv authors: Clemente-Moragón, Agustín; Gómez, Mónica; Villena-Gutiérrez, Rocío; Lalama, Doménica V; García-Prieto, Jaime; Martínez, Fernando; Sánchez-Cabo, Fátima; Fuster, Valentín; Oliver, Eduardo; Ibáñez, Borja title: Metoprolol exerts a non-class effect against ischaemia–reperfusion injury by abrogating exacerbated inflammation date: 2020-10-07 journal: Eur Heart J DOI: 10.1093/eurheartj/ehaa733 sha: 46c607d07c112920e56eb56be5f322c2168c2cb4 doc_id: 14549 cord_uid: 1nip45bv AIMS: Clinical guidelines recommend early intravenous β-blockers during ongoing myocardial infarction; however, it is unknown whether all β-blockers exert a similar cardioprotective effect. We experimentally compared three clinically approved intravenous β-blockers. METHODS AND RESULTS: Mice undergoing 45 min/24 h ischaemia–reperfusion (I/R) received vehicle, metoprolol, atenolol, or propranolol at min 35. The effect on neutrophil infiltration was tested in three models of exacerbated inflammation. Neutrophil migration was evaluated in vitro and in vivo by intravital microscopy. The effect of β-blockers on the conformation of the β1 adrenergic receptor was studied in silico. Of the tested β-blockers, only metoprolol ameliorated I/R injury [infarct size (IS) = 18.0% ± 0.03% for metoprolol vs. 35.9% ± 0.03% for vehicle; P < 0.01]. Atenolol and propranolol had no effect on IS. In the three exacerbated inflammation models, neutrophil infiltration was significantly attenuated only in the presence of metoprolol (60%, 50%, and 70% reductions vs. vehicle in myocardial I/R injury, thioglycolate-induced peritonitis, and lipopolysaccharide-induced acute lung injury, respectively). Migration studies confirmed the particular ability of metoprolol to disrupt neutrophil dynamics. In silico analysis indicated different intracellular β1 adrenergic receptor conformational changes when bound to metoprolol than to the other two β-blockers. CONCLUSIONS: Metoprolol exerts a disruptive action on neutrophil dynamics during exacerbated inflammation, resulting in an infarct-limiting effect not observed with atenolol or propranolol. The differential effect of β-blockers may be related to distinct conformational changes in the β1 adrenergic receptor upon metoprolol binding. If these data are confirmed in a clinical trial, metoprolol should become the intravenous β-blocker of choice for patients with ongoing infarction. Acute myocardial infarction (AMI) is a leading cause of morbidity and mortality worldwide. The advent of reperfusion technologies has dramatically reduced acute mortality associated with AMI. However, the size of the infarct often leaves survivors with severe heart damage, and these patients are at high risk of future heart failure and readmission. 1, 2 Reperfusion, despite being essential for myocardial salvage, triggers an exacerbated sterile inflammatory process that contributes to final infarct size (IS). This inflammation is driven by neutrophils, which infiltrate the damaged myocardium through interactions with platelets contribute to ischaemia-reperfusion (I/R) injury (IRI). [3] [4] [5] Paradoxically, blood flow restoration in the large epicardial coronary artery many times is not accompanied by efficient tissue perfusion due to the obstruction of the microvasculature. Endothelial swelling, external compression of small vessels secondary to oedema formation, and cellular aggregates (neutrophils, platelets, and erythrocytes) generating plugs that restrict tissue perfusion at the capillary level contribute to the phenomenon known as microvascular obstruction (MVO). [6] [7] [8] The latter is a main contributor to IRI and final IS. 1, 2 The b1-selective blocker metoprolol has been demonstrated to reduce myocardial IS in several species, including humans. [9] [10] [11] Metoprolol appears to limit IS largely through its inhibitory effect on neutrophils. 9 Based partly on the cardioprotective effect of metoprolol injection, 10, 12 current clinical practice guidelines recommend early intravenous administration of b-blockers (as a drug class) to patients with an ongoing AMI. 13 However, it is unknown whether different b-blockers exert the same cardioprotective effect, and a trial in patients with ongoing AMI undergoing reperfusion showed no infarct-limiting effect of the b1-selective blocker atenolol. 14 In this study, we explored the cardioprotective effect of three b-blockers approved for clinical i.v. administration (metoprolol, atenolol, and propranolol) in a mouse model of IRI. We further explored the effect of these b-blockers on neutrophil migration and infiltration in three models of exacerbated inflammation: myocardial I/R, thioglycolate-induced peritonitis, and lipopolysaccharide (LPS)-induced acute lung injury (ALI). In silico studies were conducted to evaluate conformational changes in the b1 adrenergic receptor upon binding the different b-blockers. Our results show that metoprolol has a particular action on neutrophils during exacerbated inflammation that affords a cardioprotection not provided by other b-blockers. Full section of material and methods can be found in the Supplementary material online. Metoprolol, but not atenolol or propranolol, limits myocardial infarct size The cardioprotective activity of three clinically approved intravenous b-blocker agents was assessed in an established in vivo model of IRI ( Figure 1A) . 9 In brief, mice were anaesthetized by i.p. administration of ketamine, xylazine, and atropine, and were placed on mechanical ventilation. The left anterior descending coronary artery was accessed by a small thoracotomy and then fully occluded by tying a silk knot around the proximal segment of the artery. After 45 min of coronary artery occlusion, the knot was released to allow reperfusion. Before reperfusion, mice were randomly allocated to i.v. metoprolol, atenolol, propranolol (all 12.5 mg/kg) or vehicle (0.9% NaCl). Operators were blinded to treatment allocation. Drug or vehicle was injected as a single bolus through the retro-orbital sinus 10 min before reperfusion (35 min after ischaemia onset). The metoprolol, atenolol, and propranolol dose was based on a dose-response study, in which 12.5 mg/kg was identified as the highest dose with moderate haemodynamic effect (<20%) for the three b-blockers (Supplementary material online, Figure S1 ). At 24 h post-reperfusion, mice were euthanized, and area at risk (AAR)-normalized IS was calculated. 9 Confirming previous studies, i.v. metoprolol resulted in smaller IS (% AAR) than in vehicle-treated mice (metoprolol, 18 .0% ± 8.11%; vehicle, 35 .9% ± 10.7%; P = 0.0142). In contrast, atenolol and propranolol had no effect on IS (atenolol, 38.0% ± 20.9%; propranolol, 36 .0% ± 10.3%; vehicle, 35 .9% ± 8.11%) ( Figure 1B-D) . In previous studies in pigs and mice, we showed that pre-reperfusion metoprolol injection results in reduced myocardial neutrophil infiltration, 9, 15 accounting for its cardioprotective effect. Here, assessment of myocardial Ly6G protein levels at 24 h post-reperfusion revealed significantly lower neutrophil density in metoprolol-injected mice than in vehicle-treated mice, whereas atenolol and propranolol had no effect ( Figure 1A , E, and F). Ly6G protein levels in metoprololtreated mice were almost 60% lower in left ventricles of metoprololtreated mice than those of controls. Metoprolol, but not atenolol or propranolol, inhibits neutrophil-platelet interactions during myocardial ischaemia-reperfusion Neutrophil-platelet interactions are crucial for neutrophil tissue infiltration during sterile inflammation. [3] [4] [5] Because the cardioprotective effect not shared by the other b-blockers was expected to be driven by altered neutrophil dynamics, we next explored neutrophil interactions with platelets in peripheral blood 24 h after reperfusion. The percentage of circulating neutrophils interacting with platelets was significantly reduced only in the case of metoprolol (metoprolol, 37 .5% ± 20.9%; vehicle, 80.2% ± 10.1%; P = 0.0166), whereas atenolol (67.6% ± 17.5%) and propranolol (67.3% ± 28.1%) had no statistically significant effect ( Figure 1G and H). The percentage and number of circulating neutrophil population were not affected by any of the treatment conditions (Supplementary material online, Figure S2 ). We previously showed that metoprolol exerts its cardioprotective effect during I/R by targeting neutrophils. 9 Here, we wanted to explore whether the action on neutrophils was a drug class effect, and thus shared by other b-blockers, or was particular to metoprolol. The effect of the tested b-blockers on neutrophil migration was assessed in a chemokine-induced transwell migration assay ( Figure 2A) . Mouse neutrophils were exposed across the transwell filter to the chemoattractant CXCL1 in the presence or absence of metoprolol, atenolol, or propranolol (10 mM for every condition), and the number of cells migrating across the transwell membrane was quantified by flow cytometry after 90 min. Metoprolol inhibited baseline neutrophil migration along the CXCL1 gradient (0.73 ± 0.31 vs. vehicle; P = 0.0095), whereas no effect on chemokine-induced migration was seen with either atenolol (1.04 ± 0.27) or propranolol (1.01 ± 0.19) ( Figure 2B and C). To confirm these results, we investigated whether atenolol or propranolol could mimic the ability of metoprolol to inhibit neutrophil tissue infiltration in vivo in a validated mouse model of thioglycolateinduced peritonitis 9 ( Figure 2D ). Thioglycolate induces massive leucocyte migration into the peritoneal cavity within the first 6 h, with most infiltrating cells being neutrophils. The i.v. metoprolol bolus (12.5 mg/kg) steeply inhibited thioglycolate-induced neutrophil infiltration into the mouse peritoneal cavity (4.03 ± 4.70 Â 10 5 vs. 7 .84 ± 5.01 Â 10 5 neutrophils/mL for metoprolol and vehicle, respectively; P = 0.0336) and reduced neutrophils as a percentage of viable cells (55.2% ± 23.3% vs. 78 .5% ± 17.1% for metoprolol and vehicle, respectively; P = 0.0053). In contrast, atenolol and propranolol (12.5 mg/kg each) had no anti-migratory effect on neutrophil infiltration (10.8 ± 5.06 Â 10 5 , 7.26 ± 4.14 Â 10 5 , and 7.84 ± 5.01 Â 10 5 neutrophils/mL for atenolol, propranolol, and vehicle, respectively) or neutrophils as a percentage of viable cells (82.9% ± 5.76%, 77.9% ± 11.23%, and 78.5% ± 17.1% for atenolol, propranolol, and vehicle, respectively) ( Figure 2E -G). To exclude potential dose-dependent effects and differential potency of the three tested b-blockers, we halved and doubled the bblocker dose in the thioglycolate-induced peritonitis model to a single 6.25 or 25 mg/kg i.v. bolus, respectively. At these b-blocker doses, the same pattern was maintained, with neutrophil migration inhibited only by metoprolol, and atenolol and propranolol having no effect (Supplementary material online, Figure S3 ). We next tested the differential effects of i.v. b-blockers on neutrophil migration and infiltration in a mouse model of infection-induced inflammation: LPS-induced ALI ( Figure 3A) . At 24 h after LPS instillation, Broncho-alveolar lavage fluid (BALF) from metoprolol-treated mice contained significantly fewer neutrophils than BALF from vehicletreated mice (1.03 ± 0.81 Â 10 5 vs. 3 .44 ± 2.71 Â 10 5 neutrophils/mL for metoprolol and vehicle, respectively; P = 0.0060). Neither atenolol nor propranolol had any effect on the BALF neutrophil count (3.78 ± 1.36 Â 10 5 , 4.06 ± 1.05 Â 10 5 , and 3.44 ± 2.71 Â 10 5 neutrophils/mL for atenolol, propranolol, and vehicle, respectively) ( Figure 3B and C). Tissue damage in response to an acute inflammatory response is known to involve neutrophil release of nuclear chromatin, known as neutrophil extracellular traps (NETs). 16 Given that the LPS challenge increases citH3, 16 which is strongly implicated in NET formation, 17 we assessed whether b-blocker treatment affects this process. Immunoblot analysis showed that mice receiving i.v. metoprolol exhibited a 65% attenuation of H3 citrullination (on R2 þ R8 þ R17) compared with those receiving vehicle, whereas atenolol and propranolol had no effect ( Figure 3D and E). Confocal microscopy analysis of lung tissue revealed that metoprolol significantly reduced the area of lung tissue covered by citH3 and the area of co-localization between citH3 and neutrophils (Ly6Gþ cells) ( Figure 3F and G). Moreover, reduced H3 citrullination in the lungs of metoprolol-treated mice was accompanied by reductions in neutrophil-elastase and myeloperoxidase (Supplementary material online, Figure S4A and B), neutrophil granule proteins involved in NET generation. 17 These changes were accompanied by a protection against lung tissue damage in metoprolol-treated mice (Supplementary material online, Figure S4C and D). These results confirm attenuation of NET production and the amelioration of ALI in mice receiving metoprolol. Metoprolol has a disruptive effect on neutrophil dynamics in vivo not shared by the other b-blockers tested Myocardial I/R is a paradigm of acute sterile inflammation, in which chemotactic recruitment of inflammatory cells is predominantly mediated by neutrophils. To initiate an acute inflammatory response, neutrophils adhering to the activated endothelium undergo morphological rearrangements that allow them to interact with and recruit other cell types to infiltrate the tissue. 4 Having observed that, unlike metoprolol, atenolol and propranolol showed no effect on neutrophil recruitment, we next explored the effect of these drugs on neutrophil dynamics. For this, we used 2D intravital microscopy (IVM) to image migration in the cremaster muscle vessels of mice injected with tumour necrosis factor a (TNFa), which triggers massive neutrophil recruitment 4 ( Figure 4A ). Of the tested b-blockers, only metoprolol reduced neutrophil migratory velocity (0.16 ± 0.07 mm/s vs. 0.29 ± 0.15 mm/s for metoprolol and vehicle, respectively; P < 0.0001), accumulated distance (9.75 ± 4.33 mm vs. 17 .7 ± 8.82 mm for metoprolol and vehicle, respectively; P < 0.0001), and euclidean crawling distance (5.90 ± 3.66 mm vs. 10 .2 ± 8.18 mm for metoprolol and vehicle, respectively; P < 0.0010). Moreover, metoprolol reduced the percentage of neutrophils interacting with platelets through the uropod (42.5% ± 17.6% vs. 59.4% ± 12.3% for metoprolol and vehicle, respectively; P < 0.0014). Neither atenolol nor propranolol had any effect on any of the in vivo neutrophil dynamics parameters evaluated ( Figure 4B -F and Supplementary material online, Videos S1A-D). 3D IVM studies were performed ( Figure 5A ) to test whether metoprolol specifically altered neutrophil shape or polarization during the acute inflammatory response. Consistent with the disrupted crawling dynamics observed in the 2D analysis, 3D reconstructions revealed that metoprolol impaired neutrophil polarization in TNFa-inflamed cremaster vessels, reducing neutrophil length and preventing the adoption of the typical cigar-like prolate spheroid cell shape Metoprolol exerts a non-class cardioprotective effect ( Figure 5B-D) . These effects were not observed with atenolol and propranolol, indicating that the neutrophil morphological changes needed to initiate intercellular interactions and subsequent tissue infiltration remain intact in mice treated with these drugs. This result might explain the lack of a cardioprotective effect with these drugs during I/R. Conversely, metoprolol blocks neutrophil infiltration and migration through an effect on neutrophil dynamics, and this neutrophil-stunning effect confers a cardioprotective effect during myocardial I/R. The interaction of the three b-blockers with the b1 adrenergic receptor (b1AR) was investigated by in silico approaches. All three selected b-blockers belong to the same pharmaceutical class, signal through G-protein coupled receptors (GPCRs), have high affinity for the b1AR, and are currently authorized for intravenous administration to patients. Simulated ligand binding did not substantially alter the overall topology of the human b1AR, which showed only minor differences upon binding the different b-blockers. As expected for drugs belonging to the same class, the extracellular drug-binding pocket has a small solvent accessible surface, and this pocket was moved slightly and to a similar extent with respect to the unbound protein upon binding of all tested ligands. The model was refined by submitting it to the positioning of proteins in membranes(PPM) server, which positioned the receptordrug complex more precisely in the membrane. The energy and stability of the ligand-b1AR complex was similar for all drugs; however, metoprolol binding induced an affinity-independent increase in the size of the internal cavity significantly greater than seen with the other tested drugs (63 759.34 Å 2 for metoprolol, 37 571.32 Å 2 for epinephrine, 50069.59 Å 2 for atenolol, and 44 371.77Å 2 for propranolol) ( Figure 6A and B and Supplementary material online, Tables S1 and S2). Modelling of the mouse b1AR yielded proportionally similar differences in internal cavity size (Supplementary material online, Figure S6 , Tables S3 and S4). These results strongly suggest that metoprolol binding induces a bigger conformational change in the receptor that opens the intracellular cavity, likely modifying its interactions with intracellular effectors. To elucidate whether the opening of the intracellular cavity of the receptor when bound to metoprolol translates into differences in G s protein signalling, we modelled the binding of the G s a subunit to the complexes established upon docking of the different ligands to the human b1AR. Although large differences in the energy of interface were not documented, the G s a subunit penetrates more in the cavity of metoprolol-b1AR than in the rest of the ligand-b1AR complexes (Figure 7) , possibly making it more difficult for the G s a subunit to interact with other effectors to perpetuate the classical adenylate cyclase-AMPc signalling cascade. We further explored the impact of the b1AR-Gsa interaction (upon binding to different b-blockers) on biased agonism signalling pathways. We focused our computational analyses on the study of two conserved sites (regions) of the intracellular region of the b1AR experimentally described as containing putative phosphoryl-Ser that initiate the receptor signalling and deactivation cascade [Ser461 and Ser462, which are susceptible to being phosphorylated by G-proteincoupled receptor kinases (GRKs); and Ser312 that is susceptible to being phosphorylated by protein kinase A]. Qualitatively, it is noticeable that the Ser 461-462 positions are more exposed when the G s a subunit binds the metoprolol-b1AR complex (Figure 7) , potentially being more prone to be phosphorylated by GRKs, triggering a b-arrestin-mediated signalling cascade. In this study, we have evaluated the cardioprotective effect of different clinically approved i.v. b-blockers to reduce IS in a mouse model of myocardial IRI. We have explored the effect of these drugs on the hyperactive immune response during exacerbated inflammation in models of acute injury in the heart, peritoneum and lung, and neutrophil migration in vitro. Finally, we have studied in silico structural changes occurring in the b1AR when bound to the different bblockers. Our results show that while metoprolol significantly ameliorates myocardial IRI, atenolol and propranolol have no cardioprotective effect. Our in vitro and in vivo studies show that metoprolol is the only studied b-blocker that impairs neutrophil migration and infiltration during exacerbated inflammation, and 2D and 3D IVM studies show that metoprolol exerts a particular disruptive effect on neutrophil dynamics. The in silico analysis reveals that, upon binding to the b1AR, metoprolol provokes a significant conformational change in the intracellular domain that is not observed with atenolol or propranolol. Taken together, these results show that metoprolol has a unique ability among the b-blockers tested to target neutrophils and stun the neutrophil immune response during exacerbated inflammation (Take home figure) . These findings have important clinical implications, given that since clinical practice guidelines on the use of b-blockers during AMI assume that the cardioprotective effect of metoprolol is shared by other drugs of this class. 13 The METOCARD-CNIC clinical trial demonstrated that prereperfusion injection of metoprolol in AMI patients significantly reduces IS and the incidence of long-term heart failure. 10, 12 In another trial in AMI patients undergoing reperfusion, atenolol administration showed no association with reduced IS. 14 While these starkly different outcomes could reflect differences in trial design, they also point to possible differences in the ability of these b-blocker agents to counter injurious mechanisms. The leading mechanism of tissue injury during sterile inflammation is exaggerated neutrophil activation and tissue infiltration, [18] [19] [20] and myocardial I/R serves as a paradigm of this process. A recent study showed that metoprolol ameliorates myocardial IRI through a direct action on neutrophils that prevents intercellular interactions and the cell morphological changes needed to initiate tissue infiltration. 9 This prompted us to explore the potential cardioprotective effect of three clinically approved b-blockers, as well as their effect on neutrophil biology during exacerbated inflammation . We previously showed that metoprolol-induced cardioprotection involves a 'stunning' effect on neutrophils. This effect is b1AR-mediated, since metoprolol did not reduce migration in neutrophils from b1KO mice. 9 In the present study, we show that other b1AR-selective b-blockers do not inhibit neutrophil migration in vitro or in vivo. This result is in line with a previous in vitro study, in which metoprolol but not atenolol reduced neutrophil migration. 21 The lack of an inhibitory effect with another b1AR-selective blocker prompted the authors to conclude that the metoprolol effect was independent of the b1AR. However, the lack of an anti-migratory effect of metoprolol in b1KO neutrophils suggests that the discrepancy between the effects of metoprolol and atenolol might be due to differences in the outcome of b-blocker-b1AR interaction. Our in silico studies confirm that the b1AR undergoes different conformational changes upon binding to these different b-blockers. The lack of an IS-reducing effect with propranolol appears to contradict a classical analysis showing smaller IS upon propranolol injection in a dog model of chronic coronary occlusion. 22, 23 However, there are important differences between that study and ours, the most important being that the canine model did not include reperfusion and thus did not examine IRI. 22, 23 Our work shows that metoprolol achieves its protective effect by targeting neutrophils, which are prominent mediators of reperfusion injury. In the absence of Take home figure Metoprolol exerts a particular protective effect against neutrophil-mediated ischaemia-reperfusion injury. The cardioprotective properties of metoprolol derive from its particular ability to target neutrophils and reduce ischaemia-reperfusion injury, whereas atenolol and propranolol have no effect on this cell population or on IS. Conformational changes induced in the b1AR upon binding to metoprolol differ significantly from those induced by atenolol and propranolol, and this difference may underlie the neutrophil-stunning action of metoprolol. These data have important implications because clinical practice guidelines currently recommend the use of b-blockers during acute myocardial infarction as a drug class, making no distinction among them. reperfusion, the leading mechanism of death is ischaemic damage, in which neutrophils do not play such significant role. The drugs used in this study were selected on the basis of their availability in i.v. formulations and their shared affinity for the b1AR, with no other direct vasodilatory effect and with metoprolol and atenolol being more selective than propranolol. 24 This selectivity was particularly important to avoid interference from non-specific effects. Our results with the mouse IRI model unexpectedly establish that cardioprotection is not a b-blocker class effect and that metoprolol has a differential ability to limit IS by reducing neutrophil migration to cardiac tissue and impeding neutrophil-platelet interactions ( Figure 1) . The inhibitory effect of metoprolol on neutrophil-platelet interactions has been previously shown to be associated with less MVO 9 (a major contributor to IS). The fact that atenolol and propranolol did not show any effect on these cell-to-cell interactions probably resulted in no effect on MVO. Unfortunately, in the present study we have not performed thioflavin-based MVO measurements to definitely demonstrate that only metoprolol breaks the axis neutrophil-platelet interactions-MVO-IS. This non-class effect was confirmed in the other in vitro and in vivo models of exacerbated inflammation examined. The transwell and acute peritonitis results show a characteristically strong blocking effect of metoprolol on neutrophil migration and infiltration that was not observed with atenolol or propranolol even at double the i.v. dose (Figure 2and Supplementary material online, Figure S2 ). The ability of metoprolol to reduce neutrophil counts in BALF from mice with LPS-induced ALI ( Figure 3) confirms that the protective effect is exportable to any inflammation setting. It is also significant that metoprolol attenuated histone three hypercitrullination in the ALI model ( Figure 3 and Supplementary material online, Figure S4 ). Histone 3 hypercitrullination is a hallmark of the generation of NETs, extracellular fibrillary networks primarily composed of neutrophil chromatin. Neutrophil extracellular trap generation is a key feature of the acute inflammatory response in a variety of settings, such as atherothrombosis. 25 The ability to form NETs has recently been implicated in the organ damage and mortality associated with COVID- 19. 26 Impaired NET formation in the ALI model appears to be due to the scarcity of neutrophils in the inflamed lung resulting from the disruptive effect of metoprolol on neutrophil recruitment. The single-cell in vivo 2D and 3D IVM analyses confirm that metoprolol directly targets neutrophils. Metoprolol specifically induced erratic behaviour in neutrophils and altered morphological features required for tissue infiltration. The lack of any effect on these properties in the presence of atenolol or propranolol excludes any effect of atenolol and propranolol on this immune cell type (Figures 4 and 5) . Our previous results showed that the cardioprotective effect of metoprolol is mediated by the b1AR, with no involvement of the b2AR. 9 We therefore focused the in silico analysis exclusively on the b1AR. b-blockers are believed to act by occupying the bAR extracellular domain, thereby blocking ligand-dependent downstream cascade activation. Nevertheless, b-blockers with similar receptor affinities have been suggested to trigger different downstream effects. Our finding that neutrophil migration and infiltration are inhibited only with metoprolol suggests that its protective effect might involve more than simply blocking catecholamine interaction with the receptor. Indeed, the ability of metoprolol to inhibit neutrophil migration in the in vitro transwell assays shows that this metoprolol action is not dependent on the presence of catecholamines. Our in silico analysis clearly shows that metoprolol is the tested bblocker able to induce a more significant change in b1AR conformation, increasing the size of the intracellular cavity ( Figure 6 and Supplementary material online, Tables S1 and S2). Large-scale rearrangement of GPCR residue side-chains can produce different receptor conformations that influence G-protein selectivity and generate differential effects on downstream signalling proteins. 27, 28 Our in silico analysis suggests that when metoprolol-b1AR complex binds to G s a protein induces a specific conformational change in b1AR that affects its intracellular coupling interface, exposing Ser 461 and 462 phosphorylation sites and potentially modifying its interaction with diverse intracellular-binding partners, such as GRKs. A greater exposition of this site could boost the phosphorylation of these Serines by GRK2 (complex GRK2-G bc ) and mediate the recruitment of b-arrestins to the receptor, which uncouples the receptor from its G protein and initiates receptor internalization and desensitization. We speculate that activated b-arrestin through these conformational changes at the receptor level might initiate a biased agonism signalling pathway. This conformational change may deactivate constitutive b1AR function 29 or activate a specific signalling profile that eventually produces cardioprotection by neutrophil stunning. These in silico outcomes suggest recent pharmacological concepts, such as inverse or biased agonism 30-32 as possible mechanisms underlying metoprololinduced neutrophil stunning through b1AR. To date, no intervention aimed at reducing IS has demonstrated a solid clinical benefit in terms of hard endpoints reduction. 33 For the case of i.v. b-blockers in the acute phase of STEMI, the acute benefits in terms of cardioprotection and primary ventricular fibrillation reduction 10 have not been translated into long-term clinical benefits, as shown in a recent meta-analysis including 1150 patients. 34 Several reasons might explain the lack of translation of cardioprotection into improved clinical benefits. 35, 36 The most obvious reason is the small sample size of all trials on the topic performed in the primary angioplasty era. 34 In addition, key aspects, such as type, dose, and timing of b-blocker administration varied significantly between trials included in the meta-analysis. 34 According to experimental data, 11 the trial using the ideal dose and timing of i.v. metoprolol administration was the METOCARD-CNIC study. 10 While in this trial, acute infarctlimiting effect was associated with a reduction in long-term heart failure, the small sample size (N = 270) precludes a definite conclusion. Based on these clinical data, and supported by the results provided in the present study, a definite trial with adequate dose and timing of i.v. metoprolol administration (not other b-blocker) powered to detect clinical benefits is needed to determine the clinical benefits (hard endpoints) of this strategy in haemodynamically stable STEMI patients. In summary, the present study indicates that b-blockers should not be considered a single drug class in the treatment of myocardial IRI. The cardioprotective effect of metoprolol is mediated by a targeting of neutrophils that is not shared by other b-blockers. These findings refine cardiovascular pharmacotherapy and have major implications for clinical cardiology. Evolving therapies for myocardial ischemia/reperfusion injury Cardiovascular remodelling in coronary artery disease and heart failure Involvement of neutrophils in the pathogenesis of lethal myocardial reperfusion injury Neutrophils scan for activated platelets to initiate inflammation Pathophysiology underlying the bimodal edema phenomenon after myocardial ischemia/reperfusion No-reflow phenomenon in the heart and brain The coronary circulation as a target of cardioprotection Coronary microvascular obstruction: the new frontier in cardioprotection Neutrophil stunning by metoprolol reduces infarct size Effect of early metoprolol on infarct size in ST-segment-elevation myocardial infarction patients undergoing primary percutaneous coronary intervention: the Effect of Metoprolol in Cardioprotection During an Impact of the timing of metoprolol administration during STEMI on infarct size and ventricular function Long-term benefit of early pre-reperfusion metoprolol administration in patients with acute myocardial infarction: results from the METOCARD-CNIC Trial (Effect of Metoprolol in Cardioprotection During an Acute Myocardial Infarction) ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: the Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC) Short-term effects of early intravenous treatment with a beta-adrenergic blocking agent or a specific bradycardiac agent in patients with acute myocardial infarction receiving thrombolytic therapy The cardioprotection granted by metoprolol is restricted to its administration prior to coronary reperfusion Neutrophil extracellular traps are indirectly triggered by lipopolysaccharide and contribute to acute lung injury Neutrophil extracellular traps kill bacteria Role of innate and adaptive immune mechanisms in cardiac injury and repair The neutrophil in vascular inflammation Intravascular danger signals guide neutrophils to sites of sterile inflammation Modulation of neutrophil migration and superoxide anion release by metoprolol Infarct size reduction by propranolol before and after coronary ligation in dogs Factors influencing infarct size following experimental coronary artery occlusions Beta-blockers: historical perspective and mechanisms of action Neutrophil extracellular traps: a new source of tissue factor in atherothrombosis Targeting potential drivers of COVID-19: neutrophil extracellular traps Structural studies of G protein-coupled receptors Conformational plasticity of the intracellular cavity of GPCR-G-protein complexes leads to G-protein promiscuity and selectivity Constitutive activity of the human beta(1)-adrenergic receptor in beta(1)-receptor transgenic mice Biased G protein-coupled receptor signaling: changing the paradigm of drug discovery Mechanisms of signalling and biased agonism in G protein-coupled receptors Making sense of pharmacology: inverse agonism and functional selectivity Targeting reperfusion injury in patients with ST-segment elevation myocardial infarction: trials and tribulations Early intravenous beta-blockers in patients undergoing primary percutaneous coronary intervention for ST-segment elevation myocardial infarction: a patientpooled meta-analysis of randomized clinical trials Critical issues for the translation of cardioprotection Myocardial ischaemia-reperfusion injury and cardioprotection in perspective Neutrophils orchestrate post-myocardial infarction healing by polarizing macrophages towards a reparative phenotype Online publish-ahead-of-print 27 Corrigendum to: Exercise-induced improvement of microvascular phenotype and reprogramming of p66 Shc DNA methylation We thank Verónica Labrador and Yeny Rojas-Vega from the microscopy and Advanced Imaging Units. We thank Andrés Hidalgo for sharing his knowledge of neutrophil behaviour in IRI and Carlos Galán for his expertise in graphic design and statistics. Simon Bartlett (CNIC) provided English editing. The individual data will be shared on reasonable request to the corresponding authors. Extrapolation of our data to the clinical scenario is limited by the fact that we have used mouse models only. Validation of these data in a more translational animal model such as the pig would have been desirable, but beyond the scope of the present mechanistic study. The in silico studies performed have intrinsic limitations, such as the lack of modelling of all molecular dynamics occurring in the in vivo setting or the lack of consideration for dose-response effects. In addition, in silico findings were not biochemically validated. Future biological studies (e.g. study of GRK2-mediated Ser 461/462 phosphorylation) should confirm the proposed mechanism responsible for the differential effect of b-blockers on IS and other exacerbated inflammation outcomes observed here. In our study, we focused on the effect of bblockers on neutrophils, but other cell types such as macrophages play a role in final IS. Dynamics of different macrophage subtypes, and the crosstalk between these and neutrophils, 37 impact post-MI healing, and it is plausible that metoprolol can affect these as well. Supplementary material is available at European Heart Journal online.Conflict of interest: none declared.