Chronic pulmonary regurgitation (PR) is a frequent complication in patients who have undergone surgical repair of congenital heart disease. Over time, the regurgitation causes right ventricular (RV) volume overload and progressive dysfunction,1 which are associated with reduced exercise capacity and increased risk of arrhythmias and sudden cardiac death.2

Although pulmonary valve replacement (PVR) is a low-risk intervention,3 multiple reinterventions are required due to subsequent degeneration of the implanted bioprosthesis or homograft.4 Clinical practice guidelines5,6 recommend surgery for severe PR in symptomatic patients or those with moderate-severe RV dilatation/dysfunction.7,8 However, most of the series analyzed showed no evidence of a postoperative increase in exercise capacity or RV systolic function or a prognostic improvement,9 and consequently many authors recommend earlier performance of the PVR.10

Homeostatic regulation of the cardiovascular system is controlled by the autonomic nervous system, with continuous interaction between the 2 systems. Heart failure (HF) patients have increased concentrations of circulating catecholamines, which alter the expression and activity of adrenoceptors and G protein-coupled receptor kinases (GRKs); agonist binding to adrenoceptors triggers their activation and phosphorylation by GRKs. Several isoforms (GRK2, GRK3, and GRK5) are found in the heart and in circulating mononuclear leukocytes (CMLs); once these kinases phosphorylate β-adrenoceptors, they are internalized into the cell and are no longer active in the cell membrane11,12 (Figure 1). The expression of β-adrenoceptors and GRKs (particularly GRK2 and GRK5) is altered by the hemodynamic changes occurring in HF13–16 and can thus be used for disease monitoring. Initially, these changes in the expression of β-adrenoceptors and GRKs were determined in material of cardiac origin (endomyocardial biopsies, cardiac explants), but their expression can also be studied in CMLs,15,17,18 allowing the use of these cells in peripheral blood samples as biomarkers. Indeed, because the CML expression of GRK219 has been used as a molecular marker in the study of HF, a similar approach could be useful in PR.

Figure 1.

Regulation of adrenoceptors and G protein-coupled receptor kinases (GRKs). 1: β-adrenoceptor location; 2: activation; 3: signaling through G proteins with adenylate cyclase (AC) activation; 4: phosphorylation by GRKs; 5: endocytosis; 6: location of endosomes; 7: recycling to the membrane; 8: degradation in lysosomes.

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In the present work, a molecular approach was used to improve the understanding of the pathophysiological process underlying PR and to help establish the optimal moment for PVR. Our main objective was to determine the CML expression of adrenoceptor and GRK genes in patients with severe PR as a sequela of congenital heart disease surgery and to compare their expression levels with those of controls and patients with HF. Secondary objectives were to compare the expression of adrenoceptors and GRKs among patients with severe PR according to the presence of symptoms and RV dysfunction or significant dilatation, because these are the surgical indications.

Patients diagnosed with severe PR followed up in the Adult Congenital Heart Disease Unit were prospectively selected from December 2011 to July 2015. Severe PR was diagnosed according to standard echocardiographic criteria20 and was confirmed by cardiac magnetic resonance imaging. Patients with PR and left ventricular dysfunction or another severe valve disease were excluded.

After participants signed an informed consent form, a peripheral blood sample was taken for analysis of the CML gene expression of adrenoceptors and GRKs. Clinical (New York Heart Association [NYHA] functional class) and electrocardiographic (heart rate and QRS width) data were also collected and RV assessment was performed via cardiac magnetic resonance imaging (ventricular ejection volume and fraction).

The study included 22 healthy volunteers, matched for age and sex with the included patients, and 13 patients with advanced HF of nonischemic origin; these individuals also underwent a single blood extraction for the analysis of adrenoceptor and GRK gene expression. Patients with inflammatory processes that could alter the expression of these molecules were excluded.

The study was carried out in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Hospital Universitario y Politécnico de La Fe.

In this work, we studied patients with PR from a pathophysiological point of view, surmising that analysis of the molecular changes would allow us to better understand the course of the disease and improve its clinical management. Of the 5 genes studied, a significant decrease was found in the expression of GRK2 and β2-adrenoceptor in the CMLs of patients with PR and with HF vs controls; therefore, PR patients exhibit biochemical data of cardiac damage similar to those observed in patients with advanced HF.

PR continues to be a vitally important sequela in patients who undergo surgical repair of tetralogy of Fallot and pulmonary stenosis and is a highly frequent cause of reoperation. The chronic RV volume overload caused by the presence of PR results in pathophysiological changes that lead to its dilatation and dysfunction, which culminate in arrhythmias, exercise intolerance, HF, and death. The indications for PVR in asymptomatic patients are unclear; although surgery is recommended in certain situations such as progressive RV dilatation/dysfunction,5 with there is no consensus on the optimal timing of surgical intervention. Among the tools that could be used in decision-making, the analysis of molecular parameters that indicate ventricular dysfunction could help in the treatment of these patients.

Because there are alterations in the expression of β-adrenoceptors and GRKs in the CMLs of patients with cardiovascular diseases such as hypertension and HF,14–18,21,22 their use has been proposed as biomarkers of the disease course, particularly for HF. For this reason, it was considered appropriate to study the gene expression of the 2 β-adrenoceptor subtypes (β1 and β2) and the 3 GRK subtypes (GRK2, GRK3, and GRK5) in CMLs to determine if their expression patterns also undergo changes in patients with PR.

For HF, changes have been found in the cardiac expression (gene or protein) of β1-adrenoceptor, GRK2, and GRK5,13,16,23 which vary according to the cause24 or stage25,26 of the disease. Evidence suggests that the changes in the heart are reproduced in CMLs,15,27–30 which would allow CMLs to be used as a mirror of cardiac changes. This parallelism between CMLs and the heart can be explained by considering the possible effects of circulating catecholamines or beta-blocker therapy on the expression of adrenoceptors and GRKs. Indeed, a sustained increase in circulating catecholamines decreases the expression of β-adrenoceptor subtypes but augments the expression of GRKs, whereas beta-blockers have the opposite effect.29,31 However, the changes produced in the heart and the CMLs are not always concordant. Although high concentrations of GRK216,21 and β2-adrenoceptor21 have been described in the myocardium of patients with dilated cardiomyopathy, other results indicated reduced expression levels of GRK2 and β2-adrenoceptor in the CMLs of these same patients and that this decrease is reversed after heart transplant.21

Although the mechanisms underlying the altered expression levels of the adrenoceptors and GRKs in CMLs and their pathophysiological significance32 are poorly understood, various animal models and human studies of cardiovascular disease showed a common pattern of changes between β2-adrenoceptor and GRK2 in each territory,21 a pattern also found here in the CMLs of patients with PR, where both genes decrease. Notably, a similar reduction is observed in the CMLs of patients with HF, a decrease that is reversed after heart transplant,21 which supports their correlation with the disease course. Therefore, the analytical value of the CML expression levels of β2-adrenoceptor and GRK2 does not solely lie in identifying a mirror of cardiac changes, but can also involve their use as a biomarker of CMLs themselves, as proposed by other authors for patients with HF.19

Focusing on the group of patients with severe PR, our GRK2 and β2-adrenoceptor expression pattern in CMLs was similar to that of the HF group but significantly different from that of the control group. Regardless of the origin of this change, this pattern indicates that the degree of ventricular dysfunction and the corresponding molecular changes in the CMLs of patients with PR are similar to those of patients with advanced HF, even though patients with PR are largely asymptomatic. If the reduction in β-adrenoceptors in CMLs observed in different cardiac diseases is related to a low-intensity chronic inflammatory response,32 these findings could indicate that there is already highly advanced RV damage in asymptomatic PR that engenders this inflammatory response. These data could also somewhat explain the absence of a significant improvement in RV functional parameters after PVR and indicate that the repair should be performed earlier. Indeed, Hallbergson et al.,33 analyzing long-term RV remodeling data, observed that, although there was a decrease in RV volumes with a slight reduction in RV ejection fraction in the first 2 years after PVR, the RV volume rose again after 10 years to the pre-PVR values, with a lower RV ejection fraction. Although these findings were related to RV pressure overload and volume due to dysfunction of the implanted bioprosthesis, these changes were also observed when the graft dysfunction was not as significant, indicating that the patients’ right ventricle was highly vulnerable to slight hemodynamic changes, possibly because the ventricular remodeling was already established before the PVR. Similarly, a recent study10 showed that the improvement in RV assessment parameters and oxygen consumption data was greater in patients surgically treated at younger ages, probably because there was less RV remodeling. Taken together, the RV volume and function data obtained using cardiac magnetic resonance imaging are probably insufficient in asymptomatic patients to determine the optimal timing of the PVR and biomarkers are needed to support an earlier surgical intervention. In view of the published results, the concentration of GRK2 does show prognostic value in patients with HF19 and, according to our results, could also be useful in patients with PR. First, it is necessary to show that an early intervention improves prognosis and to fine-tune the surgical technique in order to reduce the number of reinterventions.

No studies have analyzed these biomarkers in right-sided heart disease. A recent study identified altered expression of GRK2 and adrenoceptors in the atrial cardiomyocytes of patients with congenital heart disease who underwent surgical repair involving cardiopulmonary bypass.34 These findings were linked to the vulnerability of the myocardium to catecholamine concentrations in this context and the myocardial damage that this can cause. These results, although interesting, are not comparable to those of this work because our patients were stable.

Evaluation of the group of patients with PR according to their symptomatic status and RV assessment parameters revealed a nonsignificant tendency for lower GRK2 gene expression in symptomatic patients vs asymptomatic patients. The absence of a significant difference can be explained by the small sample size. This finding indicates that symptomatic patients have a higher degree of cardiac damage and that there may be an inverse relationship between the GRK2 concentration and the degree of cardiac damage. However, these differences are not present when patients are compared according to RV damage in terms of dilatation or systolic dysfunction. Thus, although the parameters used for the surgical indication are the RV volume and function values on cardiac magnetic resonance imaging, the cutoff points used to indicate surgery may be too late and the myocardial damage may already be established.

This work examines PR from a novel molecular approach and can aid therapeutic decision-making in these patients.

The main finding of the study is that the gene expression levels of GRK2 and β2-adrenoceptor, as molecular markers of cardiac dysfunction, are altered in patients with severe PR vs controls and are similar to those of patients with advanced HF. These findings should encourage further study of these molecular markers in this population and analysis of their prognostic value and usefulness in the clinical treatment of these patients.

Support was received from the Instituto de Salud Carlos III, Health Research Fund (FIS PI070509), Spanish Ministry of Economy and Competitiveness (SAF2013-45362-R), and the Valencian Government (GVACOMP07-182 and GVACOMP2009/261).

E. Oliver is a beneficiary of a research grant from the Marie Sklodowska-Curie Actions FP7 COFUND. The CNIC (National Center for Cardiovascular Research) is supported by the Spanish Ministry of Economy, Industry, and Competitiveness and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505).

None declared.