Ann Thorac Surg 2002;73:610-613
© 2002 The Society of Thoracic Surgeons
Original article: cardiovascular
ß-Receptor downregulation in congenital heart disease: a risk factor for complications after surgical repair?
Reiner Buchhorn, MD*a,
Martin Hulpke-Wette, MDa,
Wolfgang Ruschewski, MDb,
Reinhard Pregla, MDc,
Jens Fielitz, MDc,
Roland Hetzer, MD, PhDc,
Vera Regitz-Zagrosek, MD, PhDc
a Department of Pediatric Cardiology, Georg-August-University Göttingen, Göttingen, Germany
b Department of Cardiothoracic Surgery, Georg-August-University Göttingen, Göttingen, Germany
c Cardiothoracic and Vascular Surgery, Deutsches Herzzentrum Berlin, Berlin, Germany
Accepted for publication October 18, 2001.
* Address reprint requests to Dr Buchhorn, Department of Pediatric Cardiology, Georg-August University of Gottingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
e-mail: rbuchho{at}gwdg.de
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Abstract
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Background. Neurohormonal activation in children with heart failure due to congenital heart disease leads to downregulation of myocardial ß-receptors that may influence the postoperative course after cardiothoracic surgery.
Methods. Myocardial biopsies of 26 children (aged 14 ± 4 months) were obtained from the right atrium during cardiac surgery. Patients were allocated to either of two groups based on the duration of their intensive care unit stay: group 1 comprised those who stayed less than 7 days (n = 17), whereas group 2 comprised those who stayed more than 7 days, plus 3 infants who died during the early postoperative course (n = 9). For ß1- and ß2-mRNA quantitation, real-time polymerase chain reaction with fluorescence-labeled products was used.
Results. Values for myocardial ß-receptor gene expression were twice as high in group 1 children compared with group 2 (ß1-receptor 0.12 ± 0.07 versus 0.06 ± 0.03, p = 0.0016; ß2-receptor 0.12 ± 0.07 versus 0.06 ± 0.03, p = 0.0071). ß-Receptor gene expression in 16 children who received standard treatment for heart failure averaged lower than in the 10 children who received additional propranolol.
Conclusions. ß-Receptor downregulation due to congestive heart failure has an impact on the postoperative course in children with congenital disease and depends on heart failure therapy.
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Introduction
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In congestive heart failure, downregulation of myocardial ß-adrenoceptors due to an elevated sympathetic tone is well known. In infancy and childhood, heart failure is usually related to congenital heart disease. Most infants with left-to-right shunts showed a significant decrease of ß-adrenoceptor density [1], both on mononuclear leukocytes and in the right atrial myocardium [2]. The degree of left-to-right shunt flow and pulmonary systolic pressure correlated directly with plasma norepinephrine levels and inversely with lymphocyte ß-adrenoceptor density [3]. In addition, ß-adrenoceptor density was significantly lower in patients with heart failure than in those without. Moreover, ß-adrenoceptor–stimulated adenyl-cyclase activity was significantly decreased by 65% in patients with severe heart failure when compared with children with no or mild heart failure [4]. In most cases, ß-adrenoceptor downregulation is ß1-subtype selective, but in critically ill infants there is additional significant ß2-subtype downregulation [5]. In 4 tetralogy of Fallot patients treated with the ß-antagonist propranolol, a significantly increased ß-adrenoceptor number was found when compared with untreated patients with this condition [6]. These results are in accordance with the effect of metoprolol treatment for adults with heart failure that was associated with an increase in myocardial ß-adrenoceptor density. In contrast, carvedilol, a third-generation ß-antagonist, did not change myocardial ß-adrenoceptor expression [7]. However the effect of ß-blocker therapy on ß-adrenoceptor downregulation was not measured in children with heart failure due to left-to-right shunts.
We report the interesting observations made during the course of a prospective randomized clinical trial (CHF-PRO-INFANT), which compared the clinical and neurohormonal effects of digoxin and diuretics alone or in addition to propranolol in infants with severe heart failure due congenital heart disease [8]. We analyzed the effect of ß-adrenoceptor downregulation in myocardial biopsies on postoperative recovery after surgical repair for congenital heart disease, along with the influence of preoperative medical treatment.
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Patients and methods
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Patients
We investigated 26 children (aged 14 ± 4 months) with congenital heart disease. Diagnoses and operations are listed in Table 1.
A total of 15 infants were participants of the CHF-PRO-INFANT trial and needed medical treatment due to left-to-right shunts. As previously published [8], patients were treated with either digoxin and diuretics alone or additionally with propranolol (2 mg/kg/d), according to randomization. Another 11 children were patients of a pilot study and were treated with propranolol, or were matched control patients treated with digoxin and diuretics and comparable in age and diagnosis. In summary, 10 of the 26 children received additional propranolol treatment (2 mg/kg/d on average) at the time of their operation.
In accordance with recently published data on risk factors in postoperative recovery after surgical repair for congenital heart disease [9], patients were allocated to either of two groups based on the duration of their stay in the intensive care unit: group 1 (n = 17) comprised patients who stayed less than 7 days, whereas group 2 (n = 9) included those who stayed more than 7 days, as well as 3 infants who died during the early postoperative course. The study protocol was approved by the local ethics committee, and written consent was obtained from the patients’ parents.
For statistical analysis we tested the differences in the data between the two groups by an unpaired nonparametric (Mann-Whitney) test. Spearman correlation coefficients were calculated for the entire patient group.
Myocardial gene expression measurements
Myocardial biopsies were taken from the right atrial atrium during heart surgery, immediately frozen on dry ice, and stored at -80°C.
The amounts of ß-1 and ß2-receptors were estimated based on the expression of the specific mRNAs that encode these genes. For this purpose, total mRNA was extracted from myocardial samples and reverse-transcribed into cDNA. The specific cDNAs sequences encoding for ß1 and ß2 receptors were amplified with specific primers by polymerase chain reaction (PCR; n = 9); PCR amplifies the number of selected mRNA copies and thereby makes it measurable. A newly developed fluorescent dye, SYBR Green, was used for detection of PCR products. Expression of genes of interest should always be related to expression of a nonregulated housekeeping gene; for this purpose,18 S mRNA was used.
The specific primers for ß1, ß2, and 18 S mRNA were developed using PrimerExpress Software (PE Applied Biosystems, Langen, Germany) and by following the manufacturer’s guidelines to obtain a reaction with an almost 100% PCR efficiency. The reaction and measurements were performed with the GeneAmp5700 Sequence Detection system (PE Applied Biosystems) using the SYBR Green fluorescent dye (Core Reagent Kit; Perkin Elmer, Norwalk, CT) with a standard PCR protocol (50°C 2 minutes, 95°C 10 minutes, then 40 cycles 95°C 15 seconds followed by 1 minute 60°C). For each sample, quadruplicates were determined to ensure the reliability of the data. To avoid contaminations between different PCR tubes, the AmpErase UNG reaction was included, while using nucleotides with dUTP. The specificity of the reaction and the uniformity of products was documented by a homogenous melting curve (GenAmp5700). The target gene expression was normalized to the expression of 18S-RNA by using the delta-delta-ct method after proving the 100% efficiency of the reaction in an efficiency experiment according to the guidelines in the GeneAmp5700 manual. The results were then plotted as relative amounts to the first sample, which was designated as 1. The molecular structure of the primer proteins could be referenced by the investigator (R. B.).
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Results
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As shown in Table 2,
there were no differences in age, weight, and preoperative severity of heart failure as measured by the Ross score between the two groups. However, the Ross score showed a significant correlation with ß1-receptor gene expression (r = -0.47, p = 0.015).
There were no differences in hemodynamic risk factors such as ratio of pulmonary to systemic flow, systemic cardiac index, mean pulmonary artery pressure, or pulmonary vascular resistance index. Systemic oxygen saturation was lower in group 2. We found no evidence of an impaired ventricular function in either group, when measured by ejection fraction or mean left atrial pressure. Operative data such as cross-clamp time, cardiopulmonary bypass time, and degree of hypothermia were not significantly different between groups.
Values for myocardial ß1- and ß2-receptor gene expression were twice as high in group 1 children without complications during postoperative follow-up when compared with group 2 (ß1-receptor 0.12 ± 0.07 versus 0.06 ± 0.03, p = 0.0016; ß2-receptor 0.12 ± 0.07 versus 0.06 ± 0.03, p = 0.0071). In addition we found a highly significant correlation between myocardial ß1- and ß2-receptor gene expression (r = 0,59; p < 0.001; Fig 1).
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Fig 1. Values for ß1 and ß2 receptor gene expression in myocardial biopsy specimens of 26 children with congenital heart disease (Spearman r = 0.59; ***p = 0.0008). Target gene expression was normalized to expression of 18S-ribonucleic acid (18S-RNA).
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Myocardial ß1- and ß2-receptor gene expression in 16 children who received standard treatment for heart failure with digoxin and diuretics averaged lower than in the 10 children who received additional propranolol (ß1-receptor 0.08 ± 0.04 versus 0.12 ± 0.09; p = 0.37; ß2-receptor 0.08 ± 0.05 versus 0.12 ± 0.05, p = 0.043).
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Comment
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Age, cardiopulmonary bypass, and aortic cross-clamp durations are known to be significant risk factors compromising postoperative recovery after surgical repair for congenital heart disease [9] that mainly depends on surgical technique. In our high-risk patients who had congestive failure in early infancy, these surgical variables were not significantly different between the two study groups (Table 2). Moreover, our results show myocardial ß-adrenoceptor downregulation as an additional risk factor for postoperative complications after cardiac surgery in children with congenital heart disease. These results are in accordance with previously published data, who showed ß-adrenoceptor downregulation in donor hearts as a predictor of early graft heart failure in the first month after orthotopic cardiac transplantation [10].
A higher degree of cyanosis in the children in group 2 was related to the higher operative risk in these patients, who predominantly received a cavopulmonary anastomosis. No other hemodynamic risk factor was predictive of postoperative complications. Preoperative measurements of ejection fractions or mean left atrial pressures showed that downregulation in children with congenital heart disease is not associated with impaired systolic ventricular function as in adults with heart failure. Moreover downregulation in children with left-to-right shunts seems not to be ß1 selective, indicated by a significant correlation between ß1- and ß2-receptor gene expression (Fig 1). These results are in accordance with previously published data in patients with mitral regurgitation [11], who were also volume loaded.
ß-Adrenoceptor downregulation seems to be related not only to preoperative heart failure but also to preoperative medical therapy, which usually includes digoxin and diuretics. This standard medical treatment, however, is unable to prevent downregulation. Additional propranolol treatment, which was introduced into therapy for heart failure in infants with left-to-right shunts because of its beneficial effect on clinical symptoms and neurohormonal activation [8], may prevent downregulation comparable to the results in tetralogy of Fallot patients [6]. However, the level of significance of different myocardial ß1- and ß2-receptor gene expression between propranolol-treated and untreated patients is low, and some infants develop downregulation despite ß-blocker therapy. Further studies are needed to prove the effect of preoperative medical treatment with ß-adrenoceptor agonists and antagonists on downregulation and postoperative recovery after surgical repair for congenital heart disease.
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Acknowledgments
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The work was supported by grant DFG Re 662/2-3
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References
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Abstract
Introduction
