EBioMedicine 48 (2019) 605–618 Contents lists available at ScienceDirect EBioMedicine journal homepage: www.elsevier.com/locate/ebiom Degradation of GRK2 and AKT is an early and detrimental event in myocardial ischemia/reperfusion Petronila Penelaa,b,c, Javier Insertec,d,e, Paula Ramosa, Antonio Rodriguez-Sinovasc,d,e, David Garcia-Doradoc,d,e, Federico Mayor Jra,b,c,∗ a Departamento de Biología Molecular, Centro de Biología Molecular “Severo Ochoa” (UAM-CSIC), 28049 Madrid, Spain b Instituto de Investigación Sanitaria La Princesa, 28006 Madrid, Spain c CIBER de Enfermedades Cardiovasculares (CIBERCV), 28029 Madrid, Spain d Cardiovascular Diseases Research Group, Vall d’Hebron University Hospital and Research Institute, 08035 Barcelona, Spain e Universitat Autònoma de Barcelona, 08193 Barcelona, Spain a r t i c l e i n f o a b s t r a c t Article history: Background: Identification of signaling pathways altered at early stages after cardiac ischemia/reperfusion Received 10 April 2019 (I/R) is crucial to develop timely therapies aimed at reducing I/R injury. The expression of G protein- Revised 10 September 2019 coupled receptor kinase 2 (GRK2), a key signaling hub, is up-regulated in the long-term in patients and in Accepted 10 September 2019 experimental models of heart failure. However, whether GRK2 levels change at early time points following Available online 5 October 2019 myocardial I/R and its functional impact during this period remain to be established. Keywords: Methods: We have investigated the temporal changes of GRK2 expression and their potential relation- GRK2 ships with the cardioprotective AKT pathway in isolated rat hearts and porcine preclinical models of I/R. AKT Findings: Contrary to the maladaptive up-regulation of GRK2 reported at later times after myocardial Ischemia-reperfusion infarction, successive GRK2 phosphorylation at specific sites during ischemia and early reperfusion elic- Cardioprotection its GRK2 degradation by the proteasome and calpains, respectively, thus keeping GRK2 levels low during Pin1 early I/R in rat hearts. Concurrently, I/R promotes decay of the prolyl-isomerase Pin1, a positive regulator Proteasome Calpains of AKT stability, and a marked loss of total AKT protein, resulting in an overall decreased activity of this pro-survival pathway. A similar pattern of concomitant down-modulation of GRK2/AKT/Pin1 protein levels in early I/R was observed in pig hearts. Calpain and proteasome inhibition prevents GRK2/Pin1/AKT degra- dation, restores bulk AKT pathway activity and attenuates myocardial I/R injury in isolated rat hearts. Interpretation: Preventing transient degradation of GRK2 and AKT during early I/R might improve the potential of endogenous cardioprotection mechanisms and of conditioning strategies. © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license. (http://creativecommons.org/licenses/by-nc-nd/4.0/) Abbreviations: GPCR, G-protein coupled receptor; GRK2, G-protein coupled receptor kinase 2; HF, heart failure; I/R, Ischemia/Reperfusion; MI, myocardial infarction; RISK, reperfusion injury salvage kinase pathway. ∗ Corresponding author at: Centro de Biologia Molecular “Severo Ochoa”, c/Nicolás Cabrera 1, Universidad Autónoma de Madrid, 28049 Madrid, Spain. E-mail address: fmayor@cbm.csic.es (F. Mayor Jr). https://doi.org/10.1016/j.ebiom.2019.09.019 2352-3964/© 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license. (http://creativecommons.org/licenses/by-nc-nd/4.0/) 606 P. Penela, J. Inserte and P. Ramos et al. / EBioMedicine 48 (2019) 605–618 apies adequately targeting reperfusion injury remain elusive and Research in context constitute one of the top unmet needs in cardiology [2,7,8]. Brief cycles of myocardial I/R applied either before (ischemic precon- ditioning) a prolonged coronary artery occlusion plus reperfusion Evidence before this study event or immediately after reflow (ischemic post-conditioning) in experimental models can reduce infarct size by activating different Acute myocardial infarction (MI) is a major cause of death combinations of endogenous cardioprotective pathways [1,2,4,9]. and disability. Prompt restoration of blood flow to the is- Activation of kinases such as AKT and ERK1/2, important com- chemic area is key for reducing infarct size and mortality, ponents of the so-called reperfusion injury salvage kinase path- but can itself trigger additional myocardial damage, termed reperfusion injury. Despite advances in the protocols allowing way (RISK) pathway, plays a key cardioprotective role, and modula- rapid and effective reperfusion in MI patients, therapies ad- tion of this pathway by endogenous mediators or pharmacological equately targeting reperfusion injury remain elusive. There- agents has been postulated as a relevant strategy to prevent I/R fore, it is crucial to gather information about the status of key injury [1,4,10,11]. Given the limited efficacy of these current ther- cardiac signaling pathways at early stages after I/R to identify apeutic strategies, it is crucial to gather information about the sta- potential new targets for intervention. G protein-coupled re- tus of key cardiac signaling pathways at early stages after I/R [6] ceptor kinase 2 (GRK2) is a very relevant signaling hub in car- to identify potential new targets for intervention. diac physiopathology, reported to be upregulated in the long- G protein-coupled receptor kinase 2 (GRK2) is a central regu- term in patients and in experimental models of heart failure. lator of beta–adrenergic and many other G protein-coupled recep- However, whether GRK2 protein levels are altered at early time points following myocardial I/R, the molecular mech- tors (GPCRs) involved in cardiovascular physiopathology. In addi- anisms involved and the impact of such changes in GRK2- tion, data in different cell types put forward GRK2 as a key regu- related signaling networks has not been fully investigated. latory node in non-GPCR pathways via the modulation of insulin and growth factor signaling, the PI3K/AKT route, MAPK cascades, Added value of this study NO bioavailability or mitochondrial function, which are also instru- mental in cardiac function and dysfunction (reviewed in [12–17]. We find that, contrary to the up-regulation taking place In particular, myocardial GRK2 has been reported to participate at later time points after MI, GRK2 levels are transiently in apoptotic pathways after I/R by mechanisms involving its mi- reduced during ischemia and at the onset of reperfusion tochondrial targeting [18,19]. Maladaptive increased GRK2 expres- in rat and porcine preclinical models. GRK2 proteolysis is sion has been described in the failing human heart of patients and achieved by the combined action of proteasome and calpain in experimental models of HF due to chronic hypertensive or is- protein degradation pathways, by mechanisms involving dy- namic changes in the phosphorylation status of GRK2. More- chemic disease, and its genetic ablation or inhibition has been re- over, we suggest that such early GRK2 proteolysis would have ported to be cardioprotective in the long-term in animal models an impact on the levels of the AKT kinase, a very impor- by reducing adverse post-infarction remodeling, purportedly as a tant component of the cardioprotective pathways triggered result of the integrated actions of GRK2 in myocardial contractile in such pathological contexts. GRK2 proteolysis in early I/R function and cardiac metabolism (reviewed in [15,20–23]. How- would favor concurrent degradation of the functionally re- ever, whether GRK2 protein levels are altered at early time points lated Pin1 prolyl-isomerase and AKT proteins, leading to im- following myocardial I/R, the molecular mechanisms involved and paired overall AKT catalytic ability despite hyper-activation of the impact of such changes in GRK2-related signaling networks has remaining protein, resulting in reduced global capacity of this not been fully investigated. pathway to counteract cardiac injury. Importantly, preventing early GRK2/Pin1/AKT degradation by the combined admin- We report that, contrary to the up-regulation reported at later istration of proteasome and calpain inhibitors can attenuate time points after MI, GRK2 levels are transiently reduced during I/R-myocardial injury in isolated rat heart models. ischemia and at the onset of reperfusion in preclinical models, due to the combined action of proteasome and calpain pathways, Implications by mechanisms involving dynamic changes in the phosphoryla- tion status of GRK2. Our data suggest that such early GRK2 pro- The timely use of calpain and proteasome inhibition may teolysis would favor concurrent degradation of the functionally re- help to reinforce therapeutic strategies aimed at reducing I/R lated Pin1 prolyl-isomerase and AKT proteins, thus impairing over- injury. all AKT functionality and cardioprotection, whereas the combined administration of proteasome and calpain inhibitors prevents early GRK2/Pin1/AKT degradation and counteracts I/R myocardial injury. 1. Introduction 2. Materials and methods Acute myocardial infarction (MI) as a result of coronary artery occlusion is a major cause of death and disability [1,2]. Prompt 2.1. Experimental protocol of ischemia/reperfusion in rats restoration of blood flow by means of thrombolytic or primary per- cutaneous coronary intervention (PPCI) is key for reducing infarct The experimental procedures conformed to the Guide for the size and mortality. However, the process of restoring blood flow Care and Use of Laboratory Animals published by the United can itself trigger additional myocardial damage (termed reperfu- States National Institute of Health (NIH Publication Eighth Edi- sion injury), as a result of abrupt changes in pH and calcium home- tion, 2011) and were approved by the Research Commission on ostasis, alterations in metabolic and inflammatory mediators and Ethics of the Hospital Vall d’Hebron. Male Sprague-Dawley rats sudden burst of reactive oxygen species. These changes can pro- (Charles River,Barcelona, Spain) weighing 250 to 300 g were anaes- mote ventricular arrhythmiasmicrovascular obstruction, myocardial thetized with sodium pentobarbital (100 mg/kg). Hearts were re- stunning or myocardial death, leading to increased prevalence of moved, mounted onto a Langendorff apparatus, and perfused with chronic heart failure (HF) in surviving patients [1,3–6]. a modified Krebs-Henseleit bicarbonate buffer as previously de- Whereas the development of systems of care for emergent my- scribed. Hearts were perfused normoxically for 60 min (Nx group) ocardial reperfusion and advances in PPCI technology are increas- or for 20 min and then subjected to 40 min of global ischemia fol- ingly allowing rapid and effective reperfusion in MI patients, ther- lowed by reperfusion of different duration (I/R group). Ischemic P. Penela, J. Inserte and P. Ramos et al. / EBioMedicine 48 (2019) 605–618 607 preconditioning protocol consisted of two cycles of 5 min of is- blue with 100 mM maleimide) or reducing SDS sample buffer (i.e. chemia and 5 min of reperfusion applied before index ischemia supplemented with 5% β -mercaptoethanol). (PreCo group). Ischemic postconditioning was achieved with a previously established protocol commenced immediately after is- 2.4. Western blot and dot blot analysis chemia consisting of 6 cycles of 10-second reperfusion-10-second occlusion (PosCo group) [24–26]. In additional groups of hearts, Proteins were separated by SDS-PAGE for Western blot anal- the membrane-permeable calpain inhibitor SNJ-1945 (Senju Phar- ysis or applied directly on nitrocellulose membranes in a single maceutical Co,Ltd) at 10 μM [27], the proteosome inhibitor borte- spot for immunodetection (dot-blot) as previously described [29]. zomib (Cell Signaling Technology) at 1 μM and 10 μM, the PI3K in- Protein bands were detected by the chemiluminescence method hibitor LY-294002 (10 μM, Sigma-Aldrich), or the indicated combi- (ECL, Amersham Pharmacia Biotech) or with the Odyssey Imag- nations of SNJ-1945; LY-294002 and 10 μM bortezomib or their ve- ing Systems (LI-COR Biosciences). Bands were quantified by laser- hicle (0.01% DMSO) were added to the perfusion media during the scanner densitometry with a Biorad GS-700 scanner or by the soft- 10 min prior to 40 min of ischemia and the first 10 min of reperfu- ware included in the Odyssey Infrared Imaging System. The rab- sion. bit polyclonal antibodies raised against phosho-Ser/Thr-PKA sub- The selective contractile inhibitor blebbistatin (10 μM, Merck) strate, phosho-Ser/Thr-AKT substrate, pSer473-AKT and AKT and was added during the first 5 min of reperfusion in all the experi- the mouse monoclonal anti-Acetylated-Lysine antibody were all ments involving ischemia/reperfusion, except in those experiments from Cell Signaling Technologies. Rabbit primary antibodies raised specifically aimed to assess cell death and infarct size. Blebbistatin against ERK1/2, Lamin B1 and PKA catalytic subunit were from prevents cell death by reducing the mechanical stress caused by Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Mouse anti- the excessive and irreversible contractile activation occurring at the PKA RI-subunit was from BD Transduction Laboratories. Anti-Pin1 onset of reperfusion. It was used to discard the possibility that any rabbit polyclonal and GAPDH mouse monoclonal (6C5) antibodies variation on the measured parameters was a mere consequence of were provided by Upstate Biotechnology and Abcam, respectively. differences in cell death associated with sarcolemmal disruption Anti-pSer670-GRK2 and anti-pSer685-GRK2 polyclonal antibodies [26]. were from Biosource International and from Immunoway. Anti- acetyl-K40-tubulin and anti-tubulin antibodies were purchased from SIGMA. GRK2 protein was immunodetected with a rabbit 2.2. Experimental protocol of ischemia/reperfusion in pigs polyclonal antibody Ab C-15 (sc-562) (Santa Cruz). Vertical dotted lines in western blots indicate juxtaposed lanes that come from the Transient myocardial ischemia in pigs was performed as pre- same gel but were non-adjacent. viously described [28]. Farm pigs (25–30 kg) were pre-medicated with tiletamine–zolazepam (4–6 mg/kg, IM) and xylazine (1– 2.5. Determination of Pin1 acetylation 2 mg/kg, IM), anaesthetised with propofol-lipuro 1% (1.5–2.5 mg/kg, IV, followed by continuous infusion at 11 mg/kg/h) and fentanyl Myocardial tissue was lysed in immunoprecipitation buffer (5 mg/kg, IV, followed by continuous infusion at 3–6 mg/kg/h), and (50 mM HEPES pH 7.5, 150 mM NaCl, 1%Triton, 10% Glycerol, 10 mM mechanically ventilated. A mid-sternotomy was performed, and the NaF,1 mM sodium orthovanadate, plus protease inhibitors). Upon left anterior descending (LAD) coronary artery was dissected free centrifugation (15,000 ×g, 10 min), supernatants were incubated at its midpoint. Lead II of ECG, left ventricular (LV) pressure and with a specific anti-Pin1 polyclonal antibody (Upstate Biotech- LV dP/dt, coronary bloodflow, and regional myocardial function nology or Millipore). Immune complexes were resolved in 15% were continuously recorded. LAD coronary artery was occluded for SDS/PAGE and transferred to nitrocellulose membranes. After in- 40 min followed by 5 min or 120 min of reperfusion. At the end of cubation with antibody anti-Acetyl lysine (Clone 4G12, Millipore), the experiment, animals were sacrificed by a pentobarbital over- blots were stripped and reproved with a polyclonal antibodies di- dose (100 mg/kg, IV) and myocardial tissue samples from both, the rected against the immunoprecipitated Pin1 [30]. The amount of control region and the area at risk were quickly excised and frozen Pin1 acetylation was normalized to the amount of the immuno- in liquid nitrogen. precipitated protein, and data were represented as the fold over control normoxic conditions [29,30]. 2.3. Myocardial tissue processing 2.6. Cell culture and transfection and protein degradation assays Myocardial rat or porcine tissue was minced and homoge- nized in 4 vol (v:w) 20 mm Tris-HCl (pH 7.5), 5 mm EDTA, 5 mm HEK293 were obtained from the American Type Culture Collec- EGTA, and protease inhibitors. The homogenate was centrifuged tion (ATCC) and maintained in DMEM supplemented with 10% (v/v) (2000 ×g, 5 min, 4 °C) to obtain a clarified post-nuclear super- foetal bovine serum (FBS) at 37 °C in a humidified 5% CO2 atmo- natant designated as crude cytoplasmic fraction (SB1). The pellet sphere. Transient transfections with the indicated combinations of was rinsed in the same buffer (1/5 of the initial volume) sup- cDNA were performed using the Lipofectamine/Plus method, fol- plemented with 0.5% NP40, extensively vortexed and centrifuged lowing manufacturer’s instructions. (850 ×g, 10 min, 4 °C) after 15 min ice-cold incubation to obtain a Metabolic labelling and pulse-chase experiments were per- NP40-extracted nuclear fraction (SB2). For determination of GRK2 formed as described [31]. HEK293 cells previously co-transfected levels in some conditions, crude cytoplasmic fractions were pre- with PKA catalytic subunit or empty vector and GRK2wt, GRK2- viously enriched in GRK2 protein by incubating these fractions in K220R or GRK2-S685A constructs were labeled by keeping the the presence of 200–250 mmol/L NaCl, followed by centrifugation cells for 2 h in methionine and cysteine-free Dulbecco’s modi- at 250,000 ×g for 60 min and desalting by using Amicon Ultra- fied Eagle’s medium (DMEM) and then incubated for 15–30 min 0.5 Centrifugal Filter Devices according to the manufacturer’s in- in DMEM supplemented with 250 μCi/ml of [35S]methionine and structions. For detection of redox states of PKA RIα , myocardial [35S]cysteine labeling mixture (NEN Life Science Products).The samples were homogenized using a Polytron grinder with a hypo- plates were washed with phosphate-buffered saline and chased for tonic buffer as above but supplemented with 100 mM maleimide, the indicated times in DMEM plus 10% fetal bovine serum. The and protein lysates electrophoresed with non-reducing SDS sample proteolysis inhibitors lactacystin (Calbiochem, La Jolla, CA), ALLN buffer (100 mM Tris HCl pH 6.8, 4% SDS, 20% glycerol, bromophenol (Sigma), or PD150606 (Calbiochem), the PKA activator Forskolin 608 P. Penela, J. Inserte and P. Ramos et al. / EBioMedicine 48 (2019) 605–618 (Sigma) or the PKA inhibitor PKI (Calbiochem) were added 90 min way ANOVA with Bonferroni’s or Tukey’s post hoc test, respec- before metabolic labeling and maintained during the chase peri- tively, or with unpaired Student’s t-test as indicated in the Figure ods. At different chase times (1−3h), cells were harvested using ra- legends. All results are expressed as mean ± SEM. dioimmune precipitation assay lysis buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% sodium deoxycholate, 0.5% Nonidet P-40, 0.1% SDS, with a mixture of protease inhibitors). GRK2 protein was im- 3. Results munoprecipitated with a specific rabbit polyclonal antibody AbFP1 previously validated [32]. Immunoprecipitates were resolved by 3.1. GRK2 is degraded in ischemia and reperfusion conditions by SDS/PAGE and transferred to PVDF membranes to be treated with different mechanisms the Enhancer Autoradiography Starter Kit (EABiotech Ltd.) accord- ing to the manufacturer’s protocol. Band density of 35S-labeled Potential changes in GRK2 levels were analyzed during my- GRK2 was quantified by laser densitometry analysis and data were ocardial ischemia and the early time points following reperfu- corrected according to total GRK2 protein detected by immunoblot- sion, by using a well-established experimental model of cardiac is- ting [32]. chemia/reperfusion (IR) in isolated rat hearts [24,25,34]. Notably, 40 min of ischemia promoted a marked decay in cardiac GRK2 2.7. Quantification of cell death protein levels compared to control conditions, and GRK2 down- regulation was maintained after 30 min of reperfusion, when a Lactate dehydrogenase (LDH) activity was spectrophotometri- circa 50% decrease compared to normoxic GRK2 expression was cally measured in the coronary effluent throughout the reperfusion noted (Fig. 1A). Neither pre- nor post-conditioning conditions, per- period. After 60 min of reperfusion, heart slices were incubated at formed as described previously [24,25], were able to prevent the 37 °C for 10 min in 1% 2,3,5-triphenyltetrazolium chloride and im- ischemia-promoted GRK2 down-regulation. These treatments did aged under white light to outline the area of necrosis as previously not fully restore GRK2 protein levels during the 30 min of reperfu- described [25]. Previous studies demonstrated that in the Langen- sion, although slightly higher kinase levels at early (precondition- dorff rat model, reperfusion for 60 min is sufficient for acute as- ing) or later (post-conditioning) reperfusion times were observed. sessment of infarct size [26,33]. No overt changes in the subcellular distribution of the kinase were observed in either I or in I/R experimental conditions (Fig. S1), sug- 2.8. Statistical analysis gesting that the rapid down-modulation of GRK2 levels was related to protein degradation. Data analysis was performed using GraphPad Prism for Win- GRK2 can be rapidly degraded by the proteasome, in a pro- dows. Means between groups were compared by 2-way or one- cess dependent on its previous phosphorylation at the S670 a b c (fold of ischemic condition, I-40min) 1,2 Ratio of p-S670-GRK2/GRK2 1,2 ††† (fold of maximun of I/R condition) ††† 1,2 ††† (fold of normoxic condition) 1 *** I/R 1,0 ††† pS685-GRK2 levels †† Levels of total GRK2 1 I/R-PreCo *** ††† * 0,8 †† † ††† ††† †† I/R-PosCo 0,8 † ††† 0,8 ††† ††† † 0,6 I/R ††† ††† ††† † †† 0,6 ††† ††† I/R-PreCo 0,4 †† † † † 0,6 ††† I/R-PosCo † # 0,4 ††† ††† 0,2 † # ††† 0,4 ††† ††† 0 0,2 ††† Nx ### 1 3 5 10 30 0,2 ††† 0 Nx I/R(min) 1 3 5 10 30 0,0 Nx 1 3 5 30 I/R(min) MW I/R I/R- PreCo (kDa) Nx 1 3 5 30 1 3 5 10 30 Time (min) I/R I/R- PreCo pS670-GRK2 MW 75 (kDa) Nx Nx 1 3 5 30 1 3 5 10 30 Time (min) 37 GAPDH MW 75 GRK2 (kDa) 37 GRK2 75 GAPDH 75 37 I/R- PosCo GAPDH I/R MW 75 Nx Nx 1 3 5 30 1 3 5 10 30 Time (min) I/R I/R- PosCo (kDa) MW 37 75 GRK2 Nx 1 3 5 30 1 3 5 10 30 Time (min) (kDa) 37 pS670-GRK2 75 GAPDH 37 GAPDH 75 GRK2 37 GAPDH Fig. 1. Ischemia, reperfusion and conditioning treatments trigger marked and dynamic changes in cardiac GRK2 protein levels and phosphorylation status. Isolated rat hearts were exposed to 40 min of ischemia alone 40or with preconditioning I40PreCo(I) followed by reperfusion for the times indicated (I/R) in the presence or absence of preconditioning (I/R-PreCo) or post-conditioning (I/R-PosCo) interventions as detailed in Methods. Control group (Nx, normoxia condition) was exposed to60 min of continuous perfusion without ischemia. (A) GRK2 protein levels were analyzed by western-blot with a specific total anti-GRK2 antibody as detailed in Methods. GAPDH expression was used as loading control. (B–C) The extent of GRK2 phosphorylation on different residues (Ser670 or Ser685) was monitored in cardiac lysates by western-blot using phosphosite-specific antibodies. Data were normalized to total GRK2 levels GAPDH expression was used as loading control. In all panels, data are mean ± SEM, n = 3–4 rats per condition. In all panels, data were analyzed by comparing the different experimental situations (I/R, I/R-PreCo and I/R-PosCo) to the normoxic condition [2-way ANOVA followed by Bonferroni’s post-hoc test, † p < .05; †† p < .01, ††† p < .001]. In addition, we compared I/R pre-Co and I/R post-Co conditions versus I/R alone (∗ p < .05; ∗∗∗ p < .001) or between conditioning situations (# p < .05; ### p < .001) [1-way ANOVA and Tukey’s post hoc test]. Representative blots are shown. P. Penela, J. Inserte and P. Ramos et al. / EBioMedicine 48 (2019) 605–618 609 Fig. 2. Cardiac ischemia/reperfusion induces rapid and transient oxidation-dependent activation of PKA. (A) Global PKA activity was assessed in lysates from normoxic or reperfused isolated rat hearts (40 min of sustained ischemia followed by the indicated reperfusion times) by dot-blot, using a pan-specific phospho-substrate antibody that broadly detects phosphorylated proteins by PKA as indicated in Methods. GAPDH expression was used as loading control. Data are mean ± SEM, n = 3–4 rats per condition. ∗∗ p < .01; ∗ ∗ ∗ p < .001 compared to I/R 30 min [1-way ANOVA and Tukey’s post hoc test]. A representative dot-blot is shown. (B) I/R promotes oxidation-related dimerization of the regulatory PKA-RIα subunit in parallel to global PKA activation. Lysates as in panel A were probed with a specific PKA-RI antibody that recognizes both monomers and dimers of this regulatory subunit linked by disulfide bond formation. The dimer formation detected in non-reducing SDS-PAGE resolving conditions was fully reversed in the presence of 2-mercaptoethanol (reducing SDS-PAGE). The expression of the PKA catalytic subunit and of GAPDH was used as loading controls. Data are mean ± SEM, n = 3–4 rats per condition. ∗ p < .05 for the indicated comparisons [Student’s t-test]. Representative blots are shown. residue [35,36]. Interestingly, a dramatic increase of GRK2 S670- The pattern of total and phospho-S685 GRK2 levels during phosphorylation status was noted in ischemic conditions (Fig. 1B), reperfusion suggested that this phosphorylation might help to keep a pattern consistent with the involvement of the S670-GRK2 phos- GRK2 downregulated. Pulse-chase experiments in HEK-293 cells, a phorylation/proteasome pathway in kinase downmodulation dur- well-established model for characterizing GRK2 degradation mech- ing this period. However, S670 phosphorylation rapidly returned anisms [20,35,40], indicated that forskolin (FO), a direct stimu- to normoxic levels upon reperfusion in all conditions tested, sug- lator of adenylyl cyclase activity, and thus of the PKA pathway, gesting that additional mechanisms were involved in maintaining increased the basal turnover of GRK2 (Fig. 3A), as well as trig- GRK2 protein levels low during the initial phase of reperfusion. gered phosphorylation of GRK2 on S685 (Fig. S2). Interestingly, FO We searched for other regulatory post-translational modifica- also enhanced the degradation of the catalytically-inactive GRK2- tions during this period. GRK2 is phosphorylated by PKA on S685, K220R mutant (Fig. 3A), previously reported to be impaired in a residue highly conserved across species, leading to increased GPCR-induced proteasome-mediated degradation [41]. This sug- binding to Gβγ subunits and subsequent translocation to the gested that the GRK2-destabilizing effect of FO involved alternative plasma membrane [37]. We uncovered a rapid (1 min) and tran- proteolytic pathways. sient (peak at 5 min) increase of GRK2 phosphorylation at S685 It is worth noting that the fostering effect of FO in GRK2 degra- upon reperfusion (Fig. 1C). A similar pattern was observed un- dation was fully prevented upon addition of a PKA inhibitor (Fig. der pre- or post-conditioning conditions, although the extent of S2B), whereas over-expression of the catalytic subunit of PKA en- S685 phosphorylation was slightly but significantly decreased (pre- hanced the decay of both wild-type GRK2 and the K220R mutant conditioning) or enhanced (post-conditioning) at early reperfusion (Fig. 3B), confirming the involvement of PKA downstream of FO in times. The pattern of phospho-S685 GRK2 during early reperfusion the modulation of GRK2 stability. Moreover, a GRK2-S685A mutant trailed the rapid and transient increase in total PKA activity de- (unable to be phosphorylated by PKA) displayed a stability simi- tected in these cardiac samples (Fig. 2A), consistent with previous lar to WT GRK2 in basal conditions, but lacked the PKA-stimulated reports of PKA activation during I/R as a result of GPCR stimula- component of GRK2 degradation, demonstrating that phosphoryla- tion or oxidative stress [38,39]. Oxidation-dependent formation of tion on S685 was required for PKA activity-induced GRK2 down- a disulfide bond between the RIα subunit of PKA leading to dimer- regulation (Fig. 3C). ization can promote PKA activation independently of cAMP [39]. Consistent with the involvement of alternative PKA-dependent Notably, the transient changes in global PKA activity in reperfused proteolytic pathways, the proteasome inhibitor lactacystin fully in- isolated rat heart were parallel to those observed in the dimeriza- hibited the basal degradation of wt GRK2, whereas only partially tion status of PKA-RIα (Fig. 2B), suggesting that this mechanism of attenuated GRK2 proteolysis in the presence of FO stimulation (Fig. PKA stimulation might play a relevant role in such early reperfu- 3D). On the other hand, PKA-triggered GRK2 degradation was com- sion contexts. pletely abrogated upon addition of broad spectrum (ALLN, Fig. 3D) 610 P. Penela, J. Inserte and P. Ramos et al. / EBioMedicine 48 (2019) 605–618 a b c 120 % of 35S-GRK2 remaining 140 ** % of 35S-GRK2 remaining *** GRK2 wt †† GRK2 wt+PKA 100 *** 120 *** *** GRK2 wt *** ** % of 35S-GRK2 remaining GRK2-K220R 100 100 80 *** GRK2 wt+PKA GRK2 K220R+PKA ### ### ### ## *** ## 80 ### ### 60 GRK2-S685A 80 GRK2 wt 60 ### ### 40 GRK2 S685A+PKA 60 ### GRK2 wt+FO 40 ### 20 GRK2-K220R 20 ### 40 GRK2 K220R+FO 0 0 ### 0 1 * 0 1 20 Duration of chase (hours) Duration of chase (hours) 0 1 2 3 MW Duration Duration of chase (hours) MW Duration of (kDa) 0 1 0 1 0 1 0 1 of chase (hours) (kDa) 0 1 3 0 1 3 chase (hours) 35S-GRK2 35S-GRK2-K220R 75 75 IP: MW Duration of chase (hours) IP: GRK2 0 1 3 (kDa) 75 IB: GRK2 GRK2 35S-GRK2-wt 75 35S-GRK2-K220R 75 - PKA + PKA - PKA + PKA - PKA + PKA - - + - + FO 20μM GRK2 wt GRK2-S685A MW Lysate input (kDa) d MW 0 1 3 Duration of chase (hours) 75 GRK2 % of 35S-GRK2 remaining (kDa) 140 GRK2 wt+Lac 50 120 75 35S-GRK2-wt GRK2 wt+Lac+FO+ALLN Actin 100 - - + - + FO 20μM GRK2 wt+Lac+FO 37 80 * ## 50 GRK2 wt 60 *** # PKA ** 40 ### 37 *** ### 20 - + + - - GRK2-wt 0 1 2 3 - - - + + GRK2-K220R Duration of chase (hours) - - + - + PKA Fig. 3. PKA-mediated phosphorylation stimulates GRK2 degradation in a calpain-dependent manner. (A) Activation of endogenous PKA increases the turnover of both wild- type GRK2 (GRK2-wt) andof the catalytically inactive GRK2-K220R mutant. HEK-293 cells were transiently transfected with GRK2 or GRK2-K220R constructs and the turnover of these proteins in the presence or absence of forskolin (FO, 20 μM), a stimulator of the PKA pathway, was assessed by metabolic labeling with 35 S followed by GRK2 immunoprecipitation with a specific antibody and fluorography detection of 35 S-labeled GRK2 (35 S-GRK2) as described in Methods. (B) GRK2 degradation is enhanced in the presence of the constitutively active PKA catalytic subunit. HEK-293 cells were co-transfected with the indicated constructs and GRK2 turnover monitored as above. (C) GRK2 phosphorylation on S685 is required for PKA-induced downregulation. HEK-293 cells were transiently transfected with GRK2-wt or the PKA phosphorylation-defective site mutant GRK2-S685A and their stability in the presence or absence of the active catalytic subunit of PKA assessed as in previous panels. Immunoprecipitated GRK2 was assessed by fluorography and by western blot as a control (below). (D) PKA-triggered GRK2 degradation is abrogated upon addition of calpain inhibitors. The stability of GRK2 expressed in HEK-293 cells was investigated in the presence of the indicated combinations of the forskolin (FO, 20 μM), the proteasome inhibitor lactacystin Lac, (5 μM) and the broad-spectrum calpain protease inhibitor N-Acetyl-Leu-Leu-norleucinal (ALLN, 5 μM). In all panels data are the mean ± SEM of at least 3 independent experiments performed in duplicate. Panel A, ∗ ∗ ∗ p < .001 compared to untreated GRK2-wt overexpressing cells<, ## p < .01, ### p < .001compared to 0 h, and †† p < .01 comparing GRK2- K220R degradation in the absence or presence of FO. Panels B–C, ∗ ∗ p < .01, ∗ ∗ ∗ p < .001 for the indicated comparisons and ## p < .01, ### p < .001 compared to 0 h. Panel D,∗ p < .05; ∗ ∗ p < .01, ∗ ∗ ∗ p < .001 comparing to GRK2-wt plus lactacystin condition, and # p < .05, ### p < .001 compared to 0 h [One-way ANOVA, Tukey post hoc analysis]. Representative gel fluorographies are shown. or specific (PD150606, Fig. S2C) inhibitors of calpains, a family AKT decrease after ischemia and at very early reperfusion times of Ca2 + -dependent cysteine proteases reportedly over-activated in (Fig. 4A). As for GRK2, no manifest changes in the nu- myocardial reperfusion, leading to myocardial injury through the clear/cytoplasmic distribution of AKT were observed in either I or proteolysis of different proteins [42–44], suggesting that calpain- in I/R experimental conditions (Fig. S3B), suggesting the occurrence mediated proteolyisis of phospho-S685-GRK2 might take place in of protein degradation processes. early I/R conditions. Such pattern of decreased AKT protein levels during I/R was Overall, our data suggested that dynamic and sequential phos- puzzling, since the cardio-protective and pro-survival AKT path- phorylation of GRK2 at S670 and S685 during ischemia and in early way is considered to be robustly enhanced in these conditions reperfusion would trigger GRK2 degradation by the proteasome to counterbalance I/R-promoted injury, as inferred from enhanced and via calpains, respectively, thus maintaining low GRK2 protein AKT phosphorylation at activating residues [1,4,11]. In our exper- levels during such initial period after heart damage. imental setting, we also detected such marked increase of stim- ulated (phosphor-S473) AKT during reperfusion and upon pre- or 3.2. AKT degradation takes place in early I/R resulting in decreased post-conditioning conditions (Fig. 4B). However, the concurrent al- overall activity of this pathway teration in total AKT protein levels appears to have been disre- garded or not addressed in detail in other studies. Our data point We investigated whether such transient GRK2 downmodulation to a more complex scenario in which I/R triggers both AKT pro- was related to changes in other key cardiac signaling pathways. To- tein downmodulation and activation of the remaining kinase. No- tal ERK1/2 protein levels were not significantly altered during I/R tably, when the “global” or bulk AKT activity was tested in car- or upon pre- or post-conditioning (Fig. S3A), indicating that I/R diac extracts using a pan-AKT substrate antibody, we observed a does not promote a general decay in signaling proteins. Interest- marked decrease throughout the I/R period considered (Fig. 4C), ingly however, total AKT protein levels were markedly diminished with a similar pattern upon post- or pre-conditioning, although the by ischemia and maintained low during the first 30 min of reper- later condition allowed slightly higher substrate phosphorylation at fusion (Fig. 4A), with a pattern similar to that detected for GRK2. 5 min after reperfusion. Our data indicated that hyper-activation of Moreover, AKT and GRK2 protein levels displayed a similar trend the remaining AKT during reperfusion might notbe able to fully of changes upon pre- conditioning, which led to a less marked compensate for the decay in protein levels, leading to a clear over- P. Penela, J. Inserte and P. Ramos et al. / EBioMedicine 48 (2019) 605–618 611 a b 3 2,5 c 2 1,2 30 †† 1,5 ### (fold of normoxia, Nx) pS473-AKT/AKT ratio levels 1 *** Levels of total AKT 1 25 0,5 # IR 0 * † ### (fold of nornoxia) 0,8 20 ††† I/R-PreCo ### 0,6 ††† ††† ††† I/R-PosCo 15 †† ††† ††† † ††† 0,4 ††† 10 ††† ††† ## IR ††† ††† †† 0,2 ††† ††† ††† ††† ### I/R-PreCo ††† 5 I/R-PosCo 0 Nx 1 3 5 10 30 0 *** Nx 1 3 5 10 30 I/R(min) I/R(min) I/R I/R- PreCo MW I/R I/R- PreCo MW (kDa) Nx Nx (kDa) 1 3 5 30 1 3 5 30 Time (min) Nx Nx 1 3 5 30 1 3 5 10 30 Time (min) p-AKT AKT 50 50 37 AKT GAPDH 50 37 I/R I/R- PosCo GAPDH MW (kDa) Nx Nx 1 3 5 30 1 3 5 10 30 Time (min) I/R I/R- PosCo MW AKT 50 (kDa) Nx Nx 1 3 5 30 1 3 5 10 30 Time (min) 37 p-AKT GAPDH 50 AKT 50 37 GAPDH Fig. 4. Ischemia/reperfusion promotes a marked loss of total AKT protein and an overall decrease in global AKT-mediated substrate phosphorylation, despite the robust stimulation status of the remaining AKT protein. Rat heart lysates as in Fig. 1 were analyzed with specific antibodies for total AKT (A) or for AKT phosphorylated at Ser473 (B) as detailed in Methods. Data were normalized by GAPDH (panel A) or total AKT (panel B) loading. (C) Global activity of AKT towards its substrates was assessed in cardiac lysates from the indicated conditions by dot-blot, using a pan phospho-AKT substrate-specific antibody, and data normalized by GAPDH loading. In all panels, normalized data were represented as fold-change with respect to normoxic situation and are mean ± SEM, n = 3–4 rats per condition. Data were analyzed by comparing the different experimental situations (I/R, I/R-PreCo and I/R-PosCo) to the normoxic condition (2-way ANOVA followed by Bonferroni’s post-hoc test, † p < .05; †† p < .01, ††† p < .001). In addition, we compared I/R pre-Co and I/R post-Co conditions versus I/R alone (∗ p < .05; ∗ ∗ ∗ p < .001) or between conditioning situations (# p < .05; ## p < .01, ### p < .001) [1-way ANOVA and Tukey’s post hoc test]. Representative blots are shown. all downmodulation of the “catalytic potency” of this key cardio- Importantly, a comparable pattern of concurrent down- protective pathway in early I/R contexts, what would counteract modulation of GRK2, AKT, and Pin1 protein levels in early stages the beneficial effects of treatments specifically aimed at enhancing of myocardial reperfusion was also detected in porcine hearts sub- the AKT activation status. jected to in situ transient ischemia. The levels of these proteins in In search of molecular mechanisms underlying AKT protein de- the area at risk were significantly decreased at 5 min of reperfu- cay and potentially connecting transient GRK2 and AKT downmod- sion with respect to control area of the same animal (Fig. 6A–D). ulation, we focused on the prolyl-isomerase Pin1, a central player After 120 min of reperfusion, total AKT and Pin1 protein expres- in the control of folding, activity, and stability of proteins [45,46]. sion values were still slightly lower than those of the matched con- Pin1 is a critical positive factor in the regulation of AKT stability in trol area, whereas a marked GRK2 down-modulation remained. In cancer cells by binding to activated AKT and preserving the protein line with the mechanistic role of PKA in promoting GRK2 down- from degradation by the proteasome [46,47]. Pin1 has also been modulation observed in the rat model, increased levels of PKA- described as a relevant factor in cardiac hypertrophy, directly bind- phosphorylated substrates were detected at 120 min of reperfu- ing to AKT and MEK and fostering hypertrophic signaling [48]. In- sion (Fig. S4). Of note, although an enhanced AKT phosphoryla- terestingly, we found a clear reduction of Pin1 levels after ischemia tion status was apparent at these I/R stages (Fig. 6C) as previ- and during early reperfusion times (Fig. 5A), with a pattern resem- ously reported in such conditions [50,51], the global or bulk ca- bling that of AKT protein, suggesting a link between Pin1 loss and pacity of active AKT to phosphorylate its targets was markedly re- decreased AKT stability. A similar pattern of Pin1 changes was ev- duced (Fig. 6E), akin to the observed decline in total AKT protein ident in nuclear and cytoplasmic cellular fractions, consistent with levels and in line with our data in the isolated rat heart model Pin1 degradation events (Fig. S3C). (see Fig. 4). Although whether such early and dynamic changes Interestingly, GRK2 is a regulator of Pin1 stability and function- in GRK2/AKT/Pin1 levels take place in patients during cardiac I/R ality in other cell types [30]. GRK2 activates HDAC6, a cytoplas- cannot be directly investigated, the data obtained in this relevant mic lysine deacetylase targeting tubulin and many other proteins, translational large animal model supports the potential clinical rel- thus triggering de-acetylation of Pin1 at a specific residue, there- evance of these alterations. fore favoring Pin1 protein stabilization and stimulation of prolyl- isomerase activity [30,49]. We thus assessed whether Pin1 acety- 3.3. Combined use of proteasome and calpain inhibitors prevents lation might be triggered upon GRK2 downregulation during I/R. GRK2/Pin1/AKT degradation and attenuates I/R myocardial injury This treatment induced a marked increase in Pin1 protein acetyla- tion, which peaked at 5 min of reperfusion (Fig. 5B). A similar pat- Overall, our data suggested a complex interconnection among tern was detected for the acetylation levels of the canonical HDAC6 GRK2, Pin1 and AKT in early cardiac I/R. Changes in the phospho- substrate tubulin (Fig. 5C), strongly suggesting an impaired HDAC6 rylation status of GRK2 would trigger transient reduction of kinase activation by GRK2 in I/R conditions. levels by the combined action of proteasome and calpain path- 612 P. Penela, J. Inserte and P. Ramos et al. / EBioMedicine 48 (2019) 605–618 a b c 1,2 Pin1 levels Acetylated α-Tubulin Acetylated Pin1 Levels of acetylated α-Tubulin 1 16 6,0 * (fold of norrmoxia, Nx) * Levels of acetylated-Pin1 (fold of normoxia, Nx) Levels of total Pin1 14 * I/R 5,0 (fold of normoxia, Nx) 0,8 12 4,0 *** 10 0,6 8 3,0 *** 0,4 *** *** 6 *** *** 2,0 4 0,2 1,0 2 0 0 0,0 Nx I-40min 1 3 5 30 Nx 1 3 5 10 30 Nx 1 3 5 30 I/R(min) I/R(min) I/R(min) I/R MW I/R (kDa) Nx 1 3 5 30 Time (min) MW I/R Nx I40 1 3 5 30 Time (min) MW Acetyl-αTubulin (kDa) (kDa) Nx 50 I40 1 1 3 3 5 5 30 30 Time (min) Pin1 20 15 IB: Acetyl-lysine 50 αTubulin IP:Pin1 20 37 GAPDH IB:Pin1 37 GAPDH Fig. 5. Ischemia/reperfusion alters the protein levels and the acetylation status of the prolyl-isomerase Pin1. (A) Total content of Pin1 protein was analyzed with a specific anti-Pin1 antibody in the indicated conditions. GAPDH expression was used as loading control (B). The acetylation status of Pin1 in the same experimental conditions was determined with a pan anti-Acetyl lysine antibody after immunoprecipitation of total Pin1 from myocardial lysates as detailed in Methods. The amount of Pin1 acetylation was normalized to the amount of the immunoprecipitated protein. (C) Ischemia-reperfusion causes strong hyper-acetylation of myocardial α -tubulin. The levels of acetylated and total α -tubulin in the indicated conditions were analyzed with site-specific anti-acetyl-K40-tubulin and total anti-tubulin antibodies. Total α -tubulin and GAPDH served as loading controls. In all panels, normalized data were represented as fold-change with respect to the normoxic situation, and are the mean ± SEM, n = 3–4 rats per condition. ∗ p < .05, ∗ ∗ ∗ p < .001 compared to normoxia [1-way ANOVA and Tukey’s post hoc test]. Representative blots are shown. a b c d ** * * * * *** * MW MW MW MW (kDa) (kDa) (kDa) (kDa) AKT pAKT 20 75 GRK2 50 50 Pin1 37 37 GAPDH AKT 15 GAPDH 50 #1 #2 #1 #2 #1 #2 #1 #2 #1 #2 #1 #2 #1 #2 37 GAPDH #1 #2 #1 #2 #1 #2 #1 #2 #1 #2 #1 #2 #1 #2 #1 #2 #1 #2 e 5 min 120 min * Ctrl AKT I/R phospho-substrate Ctrl I/R GAPDH Fig. 6. Concurrent down-modulation of GRK2, AKT, and Pin1 protein levels in early stages of reperfusion in porcine hearts. Samples from non-ischemic (controls) or from the ischemic area at risk of lysates obtained from porcine hearts after 40 min of ischemia and 5 or 120 min of reperfusion as detailed in Methods were analyzed with specific antibodies for GRK2 (A), total AKT (B) AKT phosphorylated at Ser473 (C) or total Pin1 levels (D). Data were normalized by GAPDH (panels A, B, D) or total AKT (panel C) loading. (E) Global activity of AKT towards its substrates was assessed in pig cardiac lysates from the indicated conditions by dot-blot, using a pan phospho-AKT substrate-specific antibody, and data normalized by GAPDH loading. In all panels, normalized data were represented as fold-change with respect to control situation (mean value of myocardial control area at 5 min of I/R) and are mean ± SEM, n = 3–5 pigs per condition. ∗ p < .05; ∗ ∗ p < .01, ∗ ∗ ∗ p < .001 compared with the conditions indicated with the lines [1-way ANOVA and Tukey’s post hoc test]. Representative blots are shown. P. Penela, J. Inserte and P. Ramos et al. / EBioMedicine 48 (2019) 605–618 613 a b c d GRK2 levels Levels of phospho-AKT substrates Pin1 levels *** AKT levels ** 2,5 *** 1,4 1,2 1,4 *** * * * 1,2 1,2 Fold of Normoxia Fold of Normoxia 1,0 Fold of Normoxia 2,0 *** Fold of Normoxia 1,0 1,0 0,8 1,5 0,8 0,8 0,6 1,0 0,6 0,6 0,4 0,4 0,4 0,5 0,2 0,2 0,2 0,0 0,0 0,0 0,0 MW MW MW (kDa) (kDa) (kDa) 75 pAKT Phospho-AKT GRK2 Pin1 50 75 substrate 15 75 37 AKT Ponceau GAPDH 50 37 GAPDH staining 37 GAPDH #1 #2 #1 #2 #1 #2 #1 #2 #1 #2 #1 #2 Nx #1 #2 #1 #2 #1 #2 #1 #2 #1 #2 #1 #2 Nx #1 #2 #1 #2 #1 #2 #1 #2 #1 #2 #1 #2 Nx Nx Fig. 7. The combined inhibition of proteasome and calpain activities preserves GRK2, AKT and Pin1 levels and recovers AKT functionality in post-ischemic reperfused hearts. (A–C) Isolated rat hearts were treated with the calpain inhibitor SNJ-1945 (SNJ, 10 μM) or the proteasome inhibitor Bortezomib (BZ) at high (10 μM) or low dose (1 μM), alone or in combination as indicated, during the 10 min prior to ischemia (40 min) and maintained during reperfusion (10 min). Myocardial lysates from the different groups were analyzed for determination of total protein expression levels of GRK2 (A), Pin 1 (B) and AKT (C). GAPDH or non-specific band proteins were used as loading controls for normalization. The AKT activation status assessed in the same samples by using a specific anti-phospho-Ser473 AKT antibody (C). (D) Global activity of AKT towards its substrates is preserved in post-ischemic reperfused hearts upon joint inhibition of proteasome and calpains. Bulk AKT activity was assessed in cardiac lysates from the indicated conditions as in Fig. 4C. Data were normalized with Ponceau protein staining. In all panels, normalized data were represented as fold-change with respect to normoxic situation and are mean ± SEM, n = 4 rats per condition. ∗ p < .05; ∗ ∗ p < .01; ∗ ∗ ∗ p < .001 compared with the conditions indicated with the lines [1-way ANOVA and Tukey’s post hoc test]. Representative blots are shown. ways. GRK2 degradation would favor Pin1 proteolysis by altering 4. Discussion its acetylation status, which in turn would facilitate AKT degra- dation, leading to impaired overall AKT catalytic ability despite Our data reveal several unforeseen alterations taking place in hyper-activation of remaining protein, resulting in reduced global relevant cardiac signaling pathways during the early stages of I/R capacity to counteract cardiac injury. in rat and porcine experimental models that might provide a ra- We therefore tested the effect of the administration of the clin- tionale for therapeutic strategies aimed at fostering cardioprotec- ically used proteasome inhibitor Bortezomib (Bz) and/or the cal- tion mechanisms. First, we uncover that, contrary to the reported pain inhibitor SNJ-1945 (SNJ) on cardiac GRK2, Pin1 and AKT levels up-regulation of cardiac GRK2 taking place at later times after in isolated heartsrats subjected to 40 min of ischemia and reper- MI [21–23], successive GRK2 phosphorylation at specific sites dur- fusion for 10 min. A slight significant prevention of AKT degrada- ing ischemia and in early reperfusion would sequentially elicit tion was observed in the presence of BZ, and a tendency was de- GRK2 degradation by the proteasome and calpains, respectively, tected for GRK2 and AKT protection upon administration of SNJ, thus keeping GRK2 protein levels low during early I/R. Second, but only the combined treatment with BZ and SNJ fully prevented we unveil a marked and previously uncharacterized decrease of the downregulation of GRK2, Pin1 and AKT in such conditions (Fig. AKT protein levels during this period, likely related to concurrent 7A-C), allowing the maintenance of levels close to those detected changes in the Prolyl isomerase Pin1 (a known AKT stabilizing fac- in normoxic conditions. Importantly, the administration of protea- tor), which results in an overall down-modulation of this key car- some or calpain inhibitors or their combined delivery significantly dioprotective pathway in early I/R contexts, despite the high ac- rescued bulk AKT functionality, as assessed by the overall phos- tivation status of the remaining AKT upon reperfusion. Third, we phorylation of AKT substrates during reperfusion (Fig. 7D). This show that hindering GRK2/Pin1/AKT degradation by the combined is consistent with the fact that these treatments tend to protect administration of proteasome and calpain inhibitors can attenu- AKT protein from degradation while preserving kinase activation ate I/R-myocardial injury in isolated ratheart experimental models triggered by reperfusion, as indicated by AKT phospho-S473 sta- (scheme in Fig. 8B). tus (see representative blot in Fig. 7C), thus allowing a normaliza- The prevailing view to date is that GRK2 levels are in- tion of pAKT/AKT ratios. Moreover, the administration of the SNJ creased in the heart of chronic HF patients with dilated or calpain inhibitor or the combined delivery of SNJ and the protea- ischemic cardiomyopathy, as a result of sympathetic nervous some inhibitor BZ prior to a protocol of 40 min of ischemia fol- system hyperactivity, leading to enhanced GRK2 mRNA expres- lowed by 60 min of reperfusion significantly decreased infarct size sion. Such augmented GRK2 levels may initially help the my- and LDH release. Of note, this effect was not observed in the pres- ocardium to counterbalance the beta-adrenergic overdrive and re- ence of a PI3K inhibitor, indicating that calpain and proteasome duce the risk of tachyarrhythmia, but persisting high GRK2 activ- inhibitors exert its protective action by mechanism involving the ity is maladaptive, resulting in GPCR desensitization and down- PI3K/Akt pathway (Fig. 8A). Overall, our results postulate that pre- regulation, defective contractility, insulin resistance, mitochon- venting GRK2/Pin1/AKT degradation at early times in I/R may help drial dysfunction and apoptosis, paving the way to HF [13,21, to counteract myocardial injury. 23]. 614 P. Penela, J. Inserte and P. Ramos et al. / EBioMedicine 48 (2019) 605–618 Fig. 8. Cardiac ischemia-reperfusion injury is attenuated by combined-inhibition of proteasome and calpain activities. (A) Isolated rat hearts were treated during the 10 min prior to ischemia (40 min) and the first 10 min of a total period of 60 min reperfusion with the indicated combinations of the calpain inhibitor SNJ-1945 (SNJ, 10 μM), the proteasome inhibitor Bortezomib (BZ) (10 μM) and the PI3K inhibitor LY-294002 (10 μM). After the reperfusion period, total LDH released during reperfusion (expressed as units of activity released per gram of dry weight during the first 60 min of reperfusion, U/gdw/60 min) and infarct size (expressed as the percentage of ventricular mass in the different experimental groups) were determined as detailed in Methods. Data are the mean ± SEM, n = 6–9 rats per condition. ∗ p < .05 vs untreated I/R control group [1- way ANOVA and Tukey’s post-hoc test]. (B) Scheme of the molecular mechanisms underlying GRK2/Pin1/AKT degradation during myocardial ischemia and early reperfusion. See text for details. Strikingly, we find that following myocardial I/R, GRK2 pro- of ischemia onset [58], further supporting a predominant role for tein levels markedly decrease during the ischemic phase and are GRK2 proteolysis in this context. kept low during the early phase of reperfusion. Our data sug- GRK2 is phosphorylated at S670 by several kinases in a gest that enhanced Ser670-GRK2 phosphorylation during ischemia context-and stimulus-dependent way, including ERK1/2 [49,59,60], would trigger its degradation by the proteasome, whereas phos- p38MAPK [61] or Cdk2 [31]. CDK2 is highly activated during my- phorylation at Ser685 taking place in early reperfusion would favor ocardial ischemia [10,62] and CDK2-mediated GRK2 S670 phospho- calpain-mediated GRK2 proteolysis. Of note, these phosphorylation rylation can trigger proteasome-dependent GRK2 degradation in a sites are highly conserved across species, (murine, dog, pig and hu- GPCR-independent way [31], suggesting that this regulatory axis mans), consistent with preserved modulatory mechanisms. would be active during the ischemic phase. To our knowledge, the pattern of GRK2 protein levels at such The fact that GRK2 protein levels remained low during early initial times of I/R has not been previously described. In murine reperfusion in the absence of enhanced S670 phosphorylation sug- models, cardiac GRK2 protein upregulation has been reported as gested the involvement of an alternative degradation pathway. We early as 3 and 7 days after acute myocardial injury [13,52]. How- find that GRK2 is rapidly and transiently phosphorylated by PKA ever, in line with our results, in a canine model of MI GRK2 levels at S685 upon reperfusion, and that PKA stimulation can trigger were decreased in the subepicardial border and the infarct zone calpain-dependent GRK2 proteolysis in a cellular model. Consistent at 6 and 24 h after ligation of the coronary artery, without ap- with sequential proteasome and calpain GRK2 degradation during parent changes in mRNA levels, and such GRK2 decrease in is- I/R, the combined inhibition of these proteolytic pathways com- chemic cardiac tissue was blocked by treatment with proteasome pletely prevents GRK2 protein decay in vivo. inhibitors, resulting in a significant cardioprotection against ma- Calpains are ubiquitous Ca2 + -dependent cysteine proteases ac- lignant ventricular tachyarrhythmias [53–55]. In the brain, oxygen tivated in pathological conditions associated with Ca2+ overload. deprivation triggers a marked decrease in GRK2 content along en- Calpains are thus strongly activated at the onset of myocardial hanced GRK2 phosphorylation at Ser670 in the absence of changes reperfusion, contributing to myocardial injury through the prote- in mRNA levels, in a process blocked by proteasome inhibitors olysis of key structural membrane, cytoskeletal components and [56,57]. regulatory enzymes [42,44,63]. Notably, PKA-phosphorylated GRK2 Upon phosphorylation at S670 GRK2 can be rapidly degraded would only be targeted by calpains during the reperfusion phase by the proteasome [31,35], and GPCR stimulation facilitates β - upon intracellular pH normalization, since during ischemia ongo- arrestin-dependent ERK1/2 activation and subsequent phosphory- ing acidosis inhibits these proteases [42]. lation of GRK2 at this residue [35]. Thus, the observed burst in However, the functional relationships among PKA stimulation, phospho-Ser670 GRK2 after ischemia points to the occurrence of calpain activation status and GRK2 degradation are not straightfor- a very active proteasome-dependent degradation of the kinase in ward and may involve additional regulatory layers. While phospho- this condition. Although an ischemia-triggered inhibition of GRK2 rylation of GRK2 by PKA switches on degradation by calpain, PKA transcription and/or translation cannot be ruled out, a previous has been reported to inhibit calpains upon β AR-triggered cAMP report showed that during stop-flow and low flow ischemia in stimulation, and such mechanism appears to participate in the the isolated perfused rat heart GRK2 mRNA even increased af- molecular mechanisms of cardioprotection upon ischemic precon- ter 20 min of ischemia and then returned to baseline after 40 min ditioning [64]. This apparent paradox may be reconciled by the P. Penela, J. Inserte and P. Ramos et al. / EBioMedicine 48 (2019) 605–618 615 existence of compartmentalized pools of these proteins and of dis- dation by the proteasome, so decreased Pin1 expression facilitates tinct localization and activation mechanisms of PKA isoforms via AKT degradation [46,47]. Pin1 can also be degraded by the pro- regulatory anchoring proteins termed AKAPs [65]. In the vicinity of teasome [76,77], and decreased expression takes place in liver I/R the plasma membrane, β AR-dependent production of cyclic-AMP by unknown mechanisms [78]). GRK2 controls Pin1 stability and reportedly promotes activation of AKAP79-bound type II PKA II functionality in cancer cells by modulating its acetylation status leading to phosphorylation of GRK2 [37] and potentially also cal- via HDAC6, so decreased GRK2 expression fosters Pin1 acetylation pains, which subsequent inhibition would partially protect sub- and favors its degradation [30]. Consistently, we find that cardiac strates such as fodrin and receptor-bound GRK2 from proteoly- GRK2 downregulation during I/R correlates with a marked reduc- sis in such locations. On the other hand, we find that reperfu- tion in total Pin1 levels, along with its enhanced acetylation. Such sion, in parallel to increased global PKA activity and S685-GRK2 decrease in this AKT stabilizing factor would in turn facilitate the phosphorylation, enhances the oxidation-dependent dimerization observed concurrent AKT degradation. status of PKA-RIα , an event leading to PKA activation indepen- Supporting a potential clinical relevance of our findings, a pat- dently of cAMP and driving its localization at the myofilament tern of concomitant down-modulation of GRK2/AKT/Pin1 protein compartment [39,66], where a significant pool of GRK2 protein levels in early stages of cardiac I/R was also observed in pigs, is present via association with sarcomeric a-actinin [67]. We pos- a very relevant large animal model used in translational studies tulate that PKA-mediated GRK2 phosphorylation at such location [28,51,79]. One limitation of our study is the relatively low number would particularly favor its subsequent degradation by nearby of animals per condition, arising from the logistic complexity of calpains, less likely to be targeted there by the reported β AR- this type of experiments, including comparison of expression levels PKA inhibitory phosphorylation mechanism. Interestingly, at early during myocardial ischemia and different early time points follow- reperfusion times after preconditioning we observed comparatively ing reperfusion, as well as pre- or post-conditioning conditions in higher GRK2 levels and decreased S685 phosphorylation status, the rat model, However, the fact that the key changes reported are consistent with diminished oxidation-dependent PKA activation in observed at different time points, in independent experiments in such conditions due to decreased ROS production [68], despite en- other rat cohorts using calpain and proteasome inhibitors and in a hanced β AR-PKA stimulation also taking place in such conditions large animal pig model support the main conclusions of this work. [64]. Consistent with this notion of related proteolysis events of The cardio-protective and pro-survival AKT pathway is consid- GRK2 (via both proteasome and calpains), Pin1 and AKT (mostly ered to be up-regulated during reperfusion to compensate I/R- via proteasome) during I/R, the combined inhibition of these promoted injury, and enhanced stimulatory AKT phosphorylation degradation pathways prior to I/R fully prevents the degradation is found in such conditions. Activation of the Akt cascade is cen- of GRK2, Pin1 and AKT, rescues global AKT functionality in rat car- tral to several conditioning strategies and plays a relevant cardio- diac extracts, and significantly decreases infarct size and LDH re- protective role within the reperfusion injury salvage kinase path- lease. Both calpain [42–44] and the proteasome [80,81] have been way (RISK) pathway, whereas inhibitors of the upstream kinase reported to target a variety of cardiac substrates, including com- PI3K block conditioning and cardioprotection [1,4,10,11]. However, ponents of several signaling cascades. The susceptibility of specific we uncover that a concurrent and marked reduction in total AKT substrates for degradation may vary depending on their subcel- protein levels is triggered by ischemia and maintained during the lular localization, whereas different proteasome inhibitors inhibit first 30 min of reperfusion, resulting in an overall decrease of cardiac proteasome subtypes to a different extent [82]. Thus, it is global AKT activity in cardiac extracts. The fact that such changes likely that the combined effect of SNJ-1945 and bortezomib may in total Akt levels at early times of I/R has not been investigated alter the levels of other cardiac signaling pathways in addition to in detail before may be explained by the circumstance that in the GRK2/Pin1/AKT axis. However, the fact that the protective ef- this type of studies total AKT levels are usually employed as load- fect of these inhibitors in I/R-induced cardiac damage is lost in the ing controls to normalize the pAkt phosphorylation status, with- presence of an inhibitor of the PI3K/Akt pathway strongly suggests out direct comparison to other loading controls that would allow that preventing GRK2/Pin1/AKT degradation in cardiac cell types at assessing possible changes in Akt protein levels (for instance see early times in I/R and the parallel rescue of global AKT functional- [11,51,69–71] However, a few reports do show an apparent de- ity play a relevant role in helping reduce myocardial injury. crease in total Akt levels compared to normoxia in I/R-related con- Our observation that AKT protein levels fall during early I/R, re- ditions [26,72,73] although this fact or the mechanisms involved sulting in an overall decrease of the functionality of this key car- were not discussed in detail. dioprotective pathway despite the stimulated status of the remain- Our results indicate that hyper-stimulation of the remaining ing AKT upon reperfusion puts forward a strong impediment for AKT enzyme during reperfusion might not be able to fully com- treatment approaches aimed only to enhance AKT activation sta- pensate for the decay in protein levels taking place during early tus, as is the case for a variety of conditioning and pharmacologi- I/R. Such Akt protein down modulation would thus likely coun- cal strategies [1,4,9–11], and suggest that treatments using a com- teract the beneficial effects of therapeutic approaches specifically bination of calpain and proteasome inhibitors at the time of flow aimed at fostering Akt stimulation, suggesting that preventing restoration in order to inhibit GRK2 and AKT degradation would GRK2/Pin1/Akt degradation would safeguard global Akt catalytic foster the efficacy of endogenous cardioprotection, cardioprotective and foster the efficacy of cardioprotective drugs or conditioning drugs or conditioning strategies. strategies (see section below). In addition to helping preserve global AKT functionality, shield- We postulate that GRK2-related changes in the Prolyl isomerase ing GRK2 from degradation during early reperfusion might also Pin1 may provide functionally link altered GRK2 and AKT protein protect against malignant ventricular tachyarrhythmias [53–55] levels during I/R. AKT undergoes proteasomal degradation in dif- and adrenergic-mediated myocardial injury [38]. However, given ferent cell types, and phosphorylation by mTORC2 at S473 can de- the reported increase in GRK2 expression at later time points crease AKT stability in addition to allowing full kinase activation after MI and their overall maladaptive role in cardiac remodel- [47,74]. Of note, Pin1, a known modulator of protein folding, ac- ing and progression to HF [12,13,15,16,23], the therapeutic win- tivity and stability [45] and a relevant player in cardiac hypertro- dow for strategies aimed at decreasing GRK2 degradation should phy [48] and cardiac calcium handling [75], reportedly acts as a be carefully established. In mouse models, genetic GRK2 ablation key positive factor in the regulation of AKT stability in cancer cells 10 days after MI [83] or treatment with the GRK2 catalytic in- by binding to Ser473-activated AKT and protecting it from degra- hibitor paroxetine initiated 2 weeks after MI [22] improved cardiac 616 P. Penela, J. Inserte and P. Ramos et al. / EBioMedicine 48 (2019) 605–618 function and reduced the adverse remodeling of ischemic nature. Spain (grant CB16/11/00479 to DGD and CB16/11/00278 to F.M, co- In this regard, permanent down-regulation of GRK2 in cardiac- funded with European Regional Development Fund-FEDER contri- specific knock-out animals protects [18], whereas increased GRK2 bution), and Programa de Actividades en Biomedicina de la Co- protein levels in cardiac transgenic GRK2 mice at the onset of munidad de Madrid-B2017/BMD-3671-INFLAMUNE to F.M. We also MI aggravates [84] the injury caused by I/R, reportedly related to acknowledge institutional support to the CBMSO from Fundación pro-apoptotic signaling triggered by enhanced targeting of GRK2 Ramón Areces. This work is dedicated to the memory of our col- to the mitochondrial compartment, likely initiated during the is- league and friend Dr. David García-Dorado, who sadly passed away chemic phase of MI injury, as GRK2 mitochondrial shuttling de- during the final revision stage of this manuscript. pends on S670 phosphorylation, which is induced by ischemia. Al- though such data are consistent with a maladaptive role of high Declaration of Competing Interest GRK2 levels in the long term, these models do not recapitulate the dynamic changes in GRK2 levels occurring at different phases The authors have nothing to disclose. of ischemia/reperfusion/remodeling and do not rule out a protec- tive role for timely prevention of GRK2 degradation at early points Appendix A. Supplementary data of reperfusion. Interestingly, adenoviral delivery or cardiac trans- genic expression of the C-terminal region of GRK2 (β ARKct con- Supplementary data to this article can be found online at https: struct, aa 495–689) has a clear cardio-protective effect in both HF //doi.org/10.1016/j.ebiom.2019.09.019. and acute MI experimental settings in murine or porcine preclini- References cal models, by mechanisms involving prevention of the interaction and activation of GRK2 by Gβγ subunits released upon GPCR acti- [1] Hausenloy DJ, Yellon DM. Myocardial ischemia-reperfusion injury: a neglected vation and interference with GRK2 phosphorylation at Ser670 and therapeutic target. J Clin Invest 2013;123(1):92–100. maladaptive targeting to mitochondria (reviewed in [13,21,23]). In [2] Ibanez B, Heusch G, Ovize M, Van de Werf F. Evolving therapies for myocardial other cell types, expression of β ARKct also inhibits phosphoryla- ischemia/reperfusion injury. 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