G Protein-Coupled Receptor Kinase 2 in Patients With Acute Myocardial Infarction Gaetano Santulli, MDa,b,†, Alfonso Campanile, MDc,†, Letizia Spinelli, MDb,c, Emiliano Assante di Panzillo, MDb,c, Michele Ciccarelli, MD, PhDa, Bruno Trimarco, MDa,b, and Guido Iaccarino, MD, PhDa,b,* Lymphocyte G protein– coupled receptor kinase 2 (GRK2) levels are increased in patients with chronic heart failure, and in this condition, they correlate with cardiac function. The aim of this study was to assess the prognostic role of GRK2 during acute cardiac dysfunction in humans. A study was designed to investigate the role of GRK2 levels in patients with acute coronary syndromes. Lymphocyte GRK2 levels were examined at admission and after 24 and 48 hours in 42 patients with acute coronary syndromes, 32 with ST-segment elevation myocardial infarction and 10 with unstable angina as a control group. Echocardiographic parameters of systolic and diastolic function and left ventricular remodeling were evaluated at admission and after 2 years. GRK2 levels increased during ST-segment elevation myocardial infarction and were associated with worse systolic and diastolic function. This association held at 2-year follow-up, when GRK2 was correlated with the ejection fraction and end-systolic volume, indicating a prognostic value for GRK2 levels during acute ST-segment elevation myocardial infarction. In conclusion, lymphocyte GRK2 levels increase during acute myocardial infarction and are associated with worse cardiac function. Taken together, these data indicate that GRK2 could be predictive of ventricular remodeling after myocardial infarction and could facilitate the tailoring of appropriate therapy for high-risk patients. © 2011 Elsevier Inc. All rights reserved. (Am J Cardiol 2011;xx:xxx) In recent years, the overall prevalence of heart failure (HF) has increased.1 Ischemic heart disease represents a leading cause of HF, and a plethora of studies have assessed the clinical evolution of ST-segment elevation myocardial infarction (STEMI) toward HF.2– 6 Given the complexity of HF, interest has intensified in developing biologic markers to provide prognostic information about the disease.7 Indeed, several multivariate prognostic models have been developed to allow risk stratification.6,8 Because biomarkers may reflect various pathophysiologic processes,7 the use of a multimarker approach has been suggested to better identify patients who are at high risk,9 paving the way for more accurate treatment.3–5 A candidate biomarker in cardiovascular scenario is the G protein– coupled receptor kinase 2 (GRK2).10 This protein is involved in the desensitization and downregulation of G protein– coupled receptors, such as the ␤-adrenergic receptor, and is the most important G protein– coupled receptor kinase expressed in the heart. Increased cardiac GRK2 levels have been described in chronic HF and are associated with elevated sympathetic nervous system activity.11–13 We previously showed that increased Divisions of aInternal Medicine and bCardiac Intensive Care Unit and Geriatrics, Department of Clinical Medicine and Cardiovascular and Immunologic Sciences, “Federico II” University, Naples, Italy. Manuscript received October 20, 2010; revised manuscript received and accepted December 15, 2010. *Corresponding author: Tel: 39-081-746-2220; fax: 39-081-746-2256. E-mail address: guiaccar@unina.it (G. Iaccarino). c † Drs. Santulli and Campanile contributed equally to this study. 0002-9149/11/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.amjcard.2010.12.006 cardiac GRK2 levels are associated with the impairment of cardiac function and that cardiac protein levels can be monitored using peripheral lymphocytes, thus circumventing the problem of tissue sampling.14 Nevertheless, the role of GRK2 as a marker for HF progression after acute myocardial infarction (MI) remains to be clarified. Thus, we designed a study to investigate GRK2 levels during the early phases of MI to define its relevance as a biomarker in the evolution toward HF. Methods We enrolled 32 patients consecutively admitted to the Coronary Care Unit of “Federico II” University with a diagnosis of acute (⬍24-hour) STEMI, defined by typical chest pain and persistent ST-segment elevation on electrocardiography.15,16 MI was confirmed by increased serum creatine kinase and assessed by cardiac ultrasound using the wall motion score index. We also recruited 10 age-matched patients with unstable angina pectoris as a control group.3 Angiography was performed in all patients according to standard indications.15 GRK2 levels were evaluated at admission and after 24 hours. All patients provided written informed consent. The study was approved by the ethics committee of “Federico II” University. We used a database reporting characteristics of each patient, including clinical, biochemical, and echocardiographic data and anthropomorphic parameters including age, gender, height, weight, body mass index, and body surface area (BSA). Patients were then reexamined at 2-year follow-up. Outcome variables www.ajconline.org 2 The American Journal of Cardiology (www.ajconline.org) Table 1 Baseline clinical characteristics of patients at admission Variable Men/women Age (years) Weight (kg) Height (cm) Body mass index (kg/m2) BSA (m2) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Heart rate (beats/min) Patients With UAP (n ⫽ 10) Patients With STEMI (n ⫽ 32) High GRK2 (n ⫽ 15) Low GRK2 (n ⫽ 17) ␤-Blocker Therapy (n ⫽ 24) No ␤-Blocker Therapy (n ⫽ 8) 7/3 60.9 ⫾ 3.1 75.7 ⫾ 4.6 164.6 ⫾ 1.6 23.4 ⫾ 3.3 1.8 ⫾ 0.1 133.1 ⫾ 6.0 30/2 59.9 ⫾ 1.9 76.3 ⫾ 1.7 162.3 ⫾ 5.3 27.17 ⫾ 0.59 1.85 ⫾ 0.1 119.8 ⫾ 3.9 13/2 57.7 ⫾ 3.3 75.1 ⫾ 2.8 166.8 ⫾ 1.4 27.1 ⫾ 0.9 1.8 ⫾ 0.3 123.7 ⫾ 4.8 17/0 61.8 ⫾ 2.3 77.4 ⫾ 2.2 158.3 ⫾ 9.9 27.3 ⫾ 0.7 1.86 ⫾ 0.4 116.5 ⫾ 5.9 22/2 58.4 ⫾ 2.7 77.1 ⫾ 1.7 160.8 ⫾ 7 27.4 ⫾ 0.6 1.8 ⫾ 0.03 119.4 ⫾ 4 8/0 63.9 ⫾ 3.2 73.9 ⫾ 4.9 166.9 ⫾ 2.2 26.5 ⫾ 1.6 1.8 ⫾ 0.1 121.2 ⫾ 8.3 76.9 ⫾ 4.0 72.9 ⫾ 2.3 75 ⫾ 2.8 71.2 ⫾ 3.6 73.3 ⫾ 2.7 71.9 ⫾ 4.8 70.5 ⫾ 3.8 72.6 ⫾ 2.1 73.60 ⫾ 2.8 71.65 ⫾ 3.1 75.2 ⫾ 2.2 64.6 ⫾ 3.9* * p ⬍0.05. UAP ⫽ unstable angina pectoris. Table 2 Echocardiographic parameters of patients with ST-segment elevation myocardial infarction Variable Systolic parameters Ejection fraction (%) Shortening fraction (%) Stroke volume (ml) Cardiac output (L/min) Cardiac index (L/min/m2) Wall motion score index Diastolic parameters E (cm/s) A (cm/s) E/A ratio Deceleration time (ms) Isovolumic relaxation time (ms) PVs (cm/s) PVd (cm/s) PVs/PVd ratio E= (cm/s) E/E= ratio Patients With STEMI (n ⫽ 32) High GRK2 (n ⫽ 15) Low GRK2 (n ⫽ 17) 43.2 ⫾ 1.4 27.7 ⫾ 1.1 40.1 ⫾ 1.7 3.0 ⫾ 0.1 1.6 ⫾ 0.1 1.9 ⫾ 0.1 43.9 ⫾ 1.7 29.1 ⫾ 1.9 36.5 ⫾ 1.2 2.8 ⫾ 0.1 1.5 ⫾ 0.1 1.9 ⫾ 0.1 66.6 ⫾ 3.4 68.1 ⫾ 2.8 1.0 ⫾ 0.1 163.9 ⫾ 9.7 92.2 ⫾ 2.8 47.5 ⫾ 1.9 40.5 ⫾ 2.4 1.3 ⫾ 0.1 5.7 ⫾ 0.5 13.9 ⫾ 1.1 68.7 ⫾ 4.45 69.7 ⫾ 4.13 1.0 ⫾ 0.1 143.5 ⫾ 10.4 91.7 ⫾ 2.3 51.7 ⫾ 2.2 38.4 ⫾ 3.1 1.4 ⫾ 0.1 6.2 ⫾ 0.8 13.6 ⫾ 2.01 ␤-Blocker Therapy (n ⫽ 24) No ␤-Blocker Therapy (n ⫽ 8) 41.2 ⫾ 2 26.5 ⫾ 1.2 43.3 ⫾ 2.9* 3.17 ⫾ 0.1* 1.71 ⫾ 0.1 1.91 ⫾ 0.1 41.4 ⫾ 2.7 26.8 ⫾ 2.1 40.9 ⫾ 2.2 3.1 ⫾ 0.2 1.7 ⫾ 0.1 2.0 ⫾ 0.1 45.1 ⫾ 1.9 31.1 ⫾ 2.2 37.6 ⫾ 2.3 2.6 ⫾ 0.2 1.4 ⫾ 0.1 1.8 ⫾ 0.1 64.8 ⫾ 5.1 66.6 ⫾ 4.0 1.0 ⫾ 0.1 183 ⫾ 15.1* 92.8 ⫾ 5.9 44.3 ⫾ 2.9 42.1 ⫾ 3.7 1.2 ⫾ 0.1 5.3 ⫾ 1.3 14.2 ⫾ 1.3 64.62 ⫾ 3.8 64.8 ⫾ 2.9 1.1 ⫾ 0.2 168.2 ⫾ 11.7 90.3 ⫾ 3.9 46.6 ⫾ 2.3 39.9 ⫾ 3.1 1.3 ⫾ 0.1 5.44 ⫾ 0.6 13.96 ⫾ 1.1 72.6 ⫾ 7.5 77.9 ⫾ 6.3* 1.0 ⫾ 0.1 149.1 ⫾ 17 98.2 ⫾ 5.5 50.4 ⫾ 4.1 42.6 ⫾ 3.8 1.3 ⫾ 0.2 6.6 ⫾ 1.05 13.7 ⫾ 3.3 * p ⬍0.05. PVd ⫽ diastolic pulmonary venous flow; PVs ⫽ systolic pulmonary venous flow. analyzed during this examination were echocardiographic (systolic and diastolic function, MI size, and ventricular remodeling) and clinical (compliance with therapy, rehospitalization). Patients’ lymphocytes were extracted from 20-ml blood samples, as previously described, by means of Ficoll purification using Histopaque-1077 (Sigma Aldrich, St. Louis, Missouri) at admission and after 24 and 48 hours.14,17 Samples thus obtained were frozen and stored at ⫺80°C. We performed immunodetection of lymphocyte levels of GRK2 and actin using detergent-solubilized cell extracts or cytosol fractions obtained by centrifugation, using polyclonal anti-GRK2 and antiactin antibodies (Santa Cruz Biotechnology, Santa Cruz, California), as previously described.11,14 The proteins were visualized using standard enhanced chemiluminescence (ECL Kit; Amersham Biosciences, Piscataway, New Jersey).17,18 Quantization was done by scanning the autoradiographic film and using dedicated software (ImageQuant; Molecular Dynamics, Piscataway, New Jersey). GRK2 levels indicate GRK2 expression corrected by actin expression and by lymphocyte levels for each patient (in corrected densitometry units). All laboratory work was performed in blinded fashion with respect to the identity of the samples.17–19 Cardiac ultrasound evaluation was performed using Vivid 7 (GE Healthcare, Milwaukee, Wisconsin) by the same operator (L.S.) in the enrollment phase and at 2-year follow-up. Systolic functional parameters including the ejection fraction, shortening fraction, cardiac output, stroke volume, and cardiac index were analyzed. We also evaluated diastolic functional parameters including E-wave velocity (E), A-wave velocity (A), the E/A ratio, deceleration time, systolic and diastolic pulmonary venous flow, isovolumetric relaxation time, mitral annular motion velocity (E=) with Doppler tissue imaging, and the E/E= ratio. Infarct size was assessed using the wall motion score index. Left ven- Coronary Artery Disease/GRK2 Levels in STEMI 3 Figure 2. Determinants of GRK2 levels on the second day. (A) In patients with STEMI, GRK2 levels were reduced on the second day of observation. (B) This reduction was particularly observed in patients receiving ␤ blockers (␤Bs), whereas those not receiving ␤Bs did not have reduced GRK2 levels. CDU ⫽ corrected densitometry units. *p ⬍0.05 (independentsamples Student’s t test); #p ⬍0.01 (independent-samples Student’s t test). Figure 1. Determinants of GRK2 levels at admission. (A) Western blot analysis of GRK2 on lymphocytes showed that peripheral GRK2 levels were higher in patients with STEMI than in controls with unstable angina pectoris (UAP). (B) Of interest, in patients with STEMI, treatment affected GRK2 levels, as higher GRK2 levels were observed in patients receiving primary percutaneous transluminal coronary angioplasty (PTCA) compared with those receiving late elective PTCA. (C) Furthermore, patients receiving ␤ blockers (␤Bs) had lower levels of GRK2 than patients not receiving ␤Bs. CDU ⫽ corrected densitometry units. *p ⬍0.05 (independent-samples Student’s t test); #p ⬍0.01 (independent-samples Student’s t test). tricular remodeling was assessed by measuring end-systolic volume (ESV) and its long-term variation corrected by BSA (⌬%-ESV/BSA).8,20 Statistical analysis was performed using SPSS version 18.0 (SPSS, Inc., Chicago, Illinois) and GraphPad Prism version 5.01 (GraphPad Software, San Diego, California). Data are expressed as mean ⫾ SE. We performed chi-square tests to compare categorical variables and independent-samples Student’s t tests for continuous variables. All analyses were performed using a 2-sided model. A p value ⬍0.05 was considered significant. Some continuous variables were transformed into categorical variables using the median values as cutoffs. In particular, we divided our population according to GRK2 level at admission into 2 groups using a cut-off value of 0.47 corrected densitometry units: elevated GRK2 and low GRK2. Moreover, we studied the effects of ␤-blocker therapy and revascularization on GRK2 levels. Indeed, we subdivided our STEMI population into patients treated with ␤ blockers and those who did not receive ␤ blockers because of contraindications. Similarly, to investigate whether time of revascularization could affect GRK2 levels, we subdivided our population into patients treated with early percutaneous transluminal coronary angioplasty, defined as angioplasty performed on the infarct-related vessel during the first 24 hours of an acute MI,15 and late percutaneous transluminal coronary angioplasty (patients who received medical therapy and were referred later [⬎24 hours] to coronary intervention).3 We also performed independent-samples Student’s t tests, Pearson’s correlations, and linear regressions between GRK2 levels at admission and echocardiographic variables considered at 2-year follow-up. In this case, we considered as continues variables not only the absolute value of ultrasound parameters but also their variations from admission values. Finally, we performed a linear regression analysis to characterize the most significant variables at admission associated with ⌬%ESV/BSA and a back-step multiple regression analysis to define the best predictive model of ⌬%-ESV/BSA.20 Results Clinical characteristics of patients are listed in Table 1. As expected, patients with STEMI presented with impaired cardiac function, as indicated by the ultrasound parameters listed in Table 2. GRK2 levels at admission were higher in patients with STEMI than in those with unstable angina pectoris (Figure 1). Two pivotal determinants of GRK2 levels were treatment and time. In particular, early revascularization and ␤-blocker therapy clearly affected GRK2 levels. Indeed, GRK2 levels were higher in patients treated with early percutaneous transluminal coronary angioplasty than in those who received late percutaneous transluminal coronary angioplasty (Figure 1). This finding could be related to the reperfusion injury, consistent with previous reports.13,21 Also, patients treated with ␤ blockers had lower GRK2 levels compared with those who did not receive ␤ blockers (Figure 1). Time also affected GRK2 levels. In particular, during the second day of observation, GRK2 levels decreased (Figure 2), 4 The American Journal of Cardiology (www.ajconline.org) Table 3 Admission variables associated with long-term variation of end-systolic volume corrected by body surface area Independent Variable at Admission Shortening fraction Stroke volume Wall motion score index E/E= ratio Creatine kinase GRK2 level Figure 3. GRK2 levels and cardiac function. (A) Patients with high GRK2 levels had worse systolic function as assessed by stroke volume compared to those with low GRK2 levels (independent-samples Student’s t test). (B) GRK2 levels were also associated with worse diastolic function, assessed by deceleration time (DT) (independent-samples Student’s t test). r2 p Value 0.1636 0.1885 0.2283 0.1611 0.4837 0.2547 0.045 0.030 0.016 0.047 0.000 0.012 We found a relation between GRK2 levels at admission and stroke volume, a parameter of systolic function. Patients with high GRK2 levels had worse cardiac function, with lower stroke volumes, than those with low GRK2 levels (Figure 3). Furthermore, we found a relation between peripheral GRK2 levels and diastolic functional parameters such as deceleration time and the E/E= ratio. Indeed, at admission, patients with high GRK2 levels had lower deceleration times than those with low GRK2 levels (Figure 3). Twenty-six patients (81.25%) completed 2-year followup. We found a relation between GRK2 levels on the first day of analysis and stroke volume. Indeed, after 2 years, patients with high GRK2 levels had worse cardiac function, with lower stroke volumes, than those with low GRK2 levels (Figure 4). Furthermore, we observed a correlation and a linear regression between admission GRK2 level and ⌬%-ESV/BSA (Figure 4). The most important admission variables associated with ⌬%-ESV/BSA were shortening fraction, stroke volume, wall motion score index, E/E= ratio, creatine kinase, and GRK2 level (Table 3). Multiple regression analysis conducted using a backward stepwise method including variables showing significant associations on univariate analysis (p ⬍0.05) revealed that admission GRK2 level (p ⫽ 0.011) was the best predictor of ⌬%-ESV/BSA. Discussion Figure 4. GRK2 levels and outcomes. (A) After 2 years, patients with higher GRK2 levels at admission had worse systolic function, with lower stroke volumes, than those with low GRK2 levels (independent-samples Student’s t test). (B) Also, cardiac remodeling, assessed by the change in ESV after 2 years of follow-up (⌬%-ESV/BSA) was correlated with GRK2 level at admission (r2 ⫽ 0.25, p ⬍0.05). and this trend was more evident in patients who received ␤ blockers (Figure 2). Furthermore, when we considered the ␤-blocker group (n ⫽ 24 [75%]), some patients had reductions of GRK2 levels (responders, 62.5% [n ⫽ 15]), whereas others did not have reduced GRK2 levels after treatment (nonresponders, 37.5% [n ⫽ 9]) during the 2 days of observation. Of interest, nonresponders had worse wall motion score indexes (2.27 ⫾ 0.23 vs 1.81 ⫾ 0.1) than responders. The present investigation sheds light on the role of GRK2 in acute MI. The first compelling evidence is that GRK2 increases quickly after STEMI. This increase had already been shown in experimental models of myocardial ischemia in the rat.12 The deleterious effect of this kinase on ␤-adrenergic receptor signaling has been also explored in mice, showing that GRK2 gene deletion could prevent the development of post-MI HF and restore heart function.13 Another major finding of our work concenrs the determinants affecting the increase in GRK2, in particular angioplasty and ␤-blocker therapy. To the best of our knowledge, this is the first study reporting that early reperfusion causes an increase in GRK2 level. Furthermore, the observation that ␤ blockers are able to reduce GRK2 levels is somewhat consistent with previous reports,10,22,23 but we extend it to 2 essential novelties. First, GRK2 level at admission was associated with worse cardiac function acutely and after 2 years. Indeed, we found that patients with high GRK2 levels had worse systolic and diastolic function acutely and after 2 years. At 2-year follow-up, we found that patients with high GRK2 levels had lower stroke volumes and also increases in ⌬%-ESV/BSA (Figure 4) Coronary Artery Disease/GRK2 Levels in STEMI compared to those with low GRK2 levels. These results can be justified by the negative inotropic implications of elevated GRK2 levels.10 –14 Second, in our STEMI population, not all patients showed reduced GRK2 levels in response to ␤-blocker therapy, and interestingly, nonresponders had worse cardiac performance, suggesting the importance of early therapy with ␤ blockers in patients with acute coronary syndromes. Altogether, our results indicate that GRK2 is strictly associated with cardiac remodeling. Indeed, the implications of neurohormonal mechanisms in the progression of left ventricular remodeling have been extensively studied.5,24 –26 It is well known that this process starts rapidly after MI, usually within the first few hours, sustained by activation of the sympathetic activity adrenergic response that, at least initially, provides support for the failing myocardium.5,24 Continuous activation of neurohormonal systems and adrenergic response, leading to excessive vasoconstriction and volume expansion, becomes deleterious to the heart, with progressive deterioration of cardiac function.27 Classic measures to assess left ventricular remodeling include heart size, shape, and mass; ejection fraction; shortening fraction; and ESV. A recent study demonstrated that ESV is the strongest indicator of HF hospitalization in patients with coronary heart disease, being statistically superior to other echocardiographic measures of ventricular enlargement and systolic function such as the ejection fraction and shortening fraction.8 The close statistical relation between GRK2 and ESV supports the prognostic role of peripheral GRK2 levels. Therapeutic agents, such as ␤ blockers, are superior to other classes of drugs to induce reverse remodeling, with a stronger correlation between dose and effect.28,29 It is therefore remarkable to note that ␤-blocker therapy can reduce GRK2 levels and that this phenomenon is associated with better cardiac function.22 Thus, GRK2 levels in the early stages of STEMI could provide valuable information about ventricular remodeling and progression toward HF.1,30 They may also constitute a valid tool to assess and monitor the efficacy of ␤ blockers in patients with acute coronary syndromes, detecting those patients in need of more intensive treatment to improve their outcomes.28,30 The most important limitation of this study was the small sample size, which prevented us from performing a statistical analysis on the basis of a hard end point (death or revascularization). A larger study would also have allowed stratification of the outcome according to ␤-blocker therapy. Moreover, we have no data on GRK2 levels at 2-year follow-up, and we did not perform a crossover analysis between patients treated with ␤ blockers and those not treated. Further studies with larger populations are needed to validate our analysis. This task must be addressed through the development of a rapid bench test to quantify GRK2 levels in humans. Peripheral GRK levels could be used as a biomarker for monitoring prognosis and effectiveness of treatment in patients with STEMI. Indeed, biomarkers linked to mechanisms involved in the development of HF, such as GRK2, seem best suited for serving as early biologic markers to select therapy or assess progression. Further studies ongoing in our laboratory are actively pursuing this issue. Randomized trials are needed to address 5 this translational gap before the use of novel biomarkers becomes common practice to facilitate tailored treatment after an acute coronary event. 1. Ramani GV, Uber PA, Mehra MR. Chronic heart failure: contemporary diagnosis and management. Mayo Clin Proc 2010;85:180 –195. 2. Yeh RW, Sidney S, Chandra M, Sorel M, Selby JV, Go AS. Population trends in the incidence and outcomes of acute myocardial infarction. N Engl J Med 2010;362:2155–2165. 3. Acharjee S, Qin J, Murphy SA, McCabe C, Cannon CP. Distribution of traditional and novel risk factors and their relation to subsequent cardiovascular events in patients with acute coronary syndromes (from the PROVE IT-TIMI 22 trial). Am J Cardiol 2010;105:619 – 623. 4. McCann CJ, Glover BM, Menown IB, Moore MJ, McEneny J, Owens CG, Smith B, Sharpe PC, Young IS, Adgey JA. Prognostic value of a multimarker approach for patients presenting to hospital with acute chest pain. Am J Cardiol 2009;103:22–28. 5. Fertin M, Hennache B, Hamon M, Ennezat PV, Biausque F, Elkohen M, Nugue O, Tricot O, Lamblin N, Pinet F, Bauters C. Usefulness of serial assessment of B-type natriuretic peptide, troponin I, and C-reactive protein to predict left ventricular remodeling after acute myocardial infarction (from the REVE-2 study). Am J Cardiol 2010;106: 1410 –1416. 6. Wang TJ, Gona P, Larson MG, Tofler GH, Levy D, Newton-Cheh C, Jacques PF, Rifai N, Selhub J, Robins SJ, Benjamin EJ, D’Agostino RB, Vasan RS. Multiple biomarkers for the prediction of first major cardiovascular events and death. N Engl J Med 2006;355:2631–2639. 7. Chen WC, Tran KD, Maisel AS. Biomarkers in heart failure. Heart 2010;96:314 –320. 8. McManus DD, Shah SJ, Fabi MR, Rosen A, Whooley MA, Schiller NB. Prognostic value of left ventricular end-systolic volume index as a predictor of heart failure hospitalization in stable coronary artery disease: data from the Heart and Soul Study. J Am Soc Echocardiogr 2009;22:190 –197. 9. Sabatine MS, Morrow DA, de Lemos JA, Gibson CM, Murphy SA, Rifai N, McCabe C, Antman EM, Cannon CP, Braunwald E. Multimarker approach to risk stratification in non-ST elevation acute coronary syndromes: simultaneous assessment of troponin I, C-reactive protein, and B-type natriuretic peptide. Circulation 2002;105:1760 – 1763. 10. Iaccarino G, Tomhave ED, Lefkowitz RJ, Koch WJ. Reciprocal in vivo regulation of myocardial G protein-coupled receptor kinase expression by beta-adrenergic receptor stimulation and blockade. Circulation 1998;98:1783–1789. 11. Hata JA, Williams ML, Schroder JN, Lima B, Keys JR, Blaxall BC, Petrofski JA, Jakoi A, Milano CA, Koch WJ. Lymphocyte levels of GRK2 (betaARK1) mirror changes in the LVAD-supported failing human heart: lower GRK2 associated with improved beta-adrenergic signaling after mechanical unloading. J Card Fail 2006;12:360 –368. 12. Ungerer M, Kessebohm K, Kronsbein K, Lohse MJ, Richardt G. Activation of beta-adrenergic receptor kinase during myocardial ischemia. Circ Res 1996;79:455– 460. 13. Raake PW, Vinge LE, Gao E, Boucher M, Rengo G, Chen X, DeGeorge BR Jr, Matkovich S, Houser SR, Most P, Eckhart AD, Dorn GW II, Koch WJ. G protein-coupled receptor kinase 2 ablation in cardiac myocytes before or after myocardial infarction prevents heart failure. Circ Res 2008;103:413– 422. 14. Iaccarino G, Barbato E, Cipolletta E, De Amicis V, Margulies KB, Leosco D, Trimarco B, Koch WJ. Elevated myocardial and lymphocyte GRK2 expression and activity in human heart failure. Eur Heart J 2005;26:1752–1758. 15. Mangiacapra F, Muller O, Ntalianis A, Trana C, Heyndrickx GR, Bartunek J, Vanderheyden M, Wijns W, De Bruyne B, Barbato E. Comparison of 600 versus 300-mg Clopidogrel loading dose in patients with ST-segment elevation myocardial infarction undergoing primary coronary angioplasty. Am J Cardiol 2010;106:1208 –1211. 16. Galasso G, Santulli G, Piscione F, De Rosa R, Trimarco V, Piccolo R, Cassese S, Iaccarino G, Trimarco B, Chiariello M. The GPIIIA PlA2 polymorphism is associated with an increased risk of cardiovascular adverse events. BMC Cardiovasc Disord 2010;10:41. 17. Izzo R, Cipolletta E, Ciccarelli M, Campanile A, Santulli G, Palumbo G, Vasta A, Formisano S, Trimarco B, Iaccarino G. Enhanced GRK2 6 18. 19. 20. 21. 22. 23. The American Journal of Cardiology (www.ajconline.org) expression and desensitization of betaAR vasodilatation in hypertensive patients. Clin Transl Sci 2008;1:215–220. Lasorella A, Stegmuller J, Guardavaccaro D, Liu G, Carro MS, Rothschild G, de la Torre-Ubieta L, Pagano M, Bonni A, Iavarone A. Degradation of Id2 by the anaphase-promoting complex couples cell cycle exit and axonal growth. Nature 2006;442:471– 474. Santulli G, Ciccarelli M, Palumbo G, Campanile A, Galasso G, Ziaco B, Altobelli GG, Cimini V, Piscione F, D’Andrea LD, Pedone C, Trimarco B, Iaccarino G. In vivo properties of the proangiogenic peptide QK. J Transl Med 2009;7:41. Lanni F, Santulli G, Izzo R, Rubattu S, Zanda B, Volpe M, Iaccarino G, Trimarco B. The Pl(A1/A2) polymorphism of glycoprotein IIIa and cerebrovascular events in hypertension: increased risk of ischemic stroke in high-risk patients. J Hypertens 2007;25:551–556. Brinks H, Boucher M, Gao E, Chuprun JK, Pesant S, Raake PW, Huang ZM, Wang X, Qiu G, Gumpert A, Harris DM, Eckhart AD, Most P, Koch WJ. Level of G protein-coupled receptor kinase-2 determines myocardial ischemia/reperfusion injury via pro- and antiapoptotic mechanisms. Circ Res 2010;107:1140 –1149. Leineweber K, Rohe P, Beilfuss A, Wolf C, Sporkmann H, Bruck H, Jakob HG, Heusch G, Philipp T, Brodde OE. G-protein-coupled receptor kinase activity in human heart failure: effects of beta-adrenoceptor blockade. Cardiovasc Res 2005;66:512–519. Santulli G, Lombardi A, Sorriento D, Anastasio A, Iovino S, Del Giudice C, Miele C, Formisano P, Iaccarino G. Beta 2 adrenergic knock-out mice develop insulin resistance. Eur Heart J 2010;31:936. 24. Lymperopoulos A, Rengo G, Gao E, Ebert SN, Dorn GW II, Koch WJ. Reduction of sympathetic activity via adrenal-targeted GRK2 gene deletion attenuates heart failure progression and improves cardiac function after myocardial infarction. J Biol Chem 2010;285:16378 – 16386. 25. Sorriento D, Santulli G, Fusco A, Anastasio A, Trimarco B, Iaccarino G. Intracardiac injection of AdGRK5-NT reduces left ventricular hypertrophy by inhibiting NF-kappaB-dependent hypertrophic gene expression. Hypertension 2010;56:696 –704. 26. Santulli G, Illario M, Cipolletta E, Sorriento D, Del Giudice C, Anastasio A, Trimarco B, Iaccarino G. Deletion of the CaMK4 gene in mice determines a hypertensive phenotype. Cardiovasc Res 2010; 87:89. 27. Rengo G, Leosco D, Zincarelli C, Marchese M, Corbi G, Liccardo D, Filippelli A, Ferrara N, Lisanti MP, Koch WJ, Lymperopoulos A. Adrenal GRK2 lowering is an underlying mechanism for the beneficial sympathetic effects of exercise training in heart failure. Am J Physiol Heart Circ Physiol 2010;298:H2032–H2038. 28. Frigerio M, Roubina E. Drugs for left ventricular remodeling in heart failure. Am J Cardiol 2005;96(suppl):10L–18L. 29. Udelson JE. Ventricular remodeling in heart failure and the effect of beta-blockade. Am J Cardiol 2004;93(suppl):43B– 48B. 30. Abraham WT, Greenberg BH, Yancy CW. Pharmacologic therapies across the continuum of left ventricular dysfunction. Am J Cardiol 2008;102(suppl):21G–28G.