Radiation-induced changes in gene expression and distribution of atrial natriuretic peptide (ANP) in different anatomical regions of the rat heart.

PURPOSE: To examine the effects of whole-heart irradiation on gene expression and distribution of atrial natriuretic peptide (ANP) in atrial appendages and left ventricles of the rat heart. MATERIAL AND METHODS: Female Sprague-Dawley rats were irradiated with a single dose of 0, 15 or 20 Gy locally to the heart. At intervals up to 16 months post-irradiation, the localization of ANP was examined using immunohistochemical techniques. Absolute mRNA concentrations were determined using the competitive PCR assay. RESULTS: Histological and immunohistochemical studies demonstrated that whole-heart irradiation caused a reduction of atrial ANP, which is due to a substantial loss of ANP-producing atrial myocytes and accumulation of collagen (replacement fibrosis). On the other hand, ANP became detectable in the subendocardium of the irradiated left ventricles. Positive staining of ANP was mainly found in the proximity of injured areas consisting of degeneration of myocytes and fibrosis. At the transcriptional level, reduction of atrial ANP expression at 1 month post-irradiation was followed by recovery at 3 months. Thereafter, ANP mRNA concentrations followed the mRNA pattern of controls and even appeared to increase at 16 months. In the left ventricle, dose-dependent and progressive elevation of ANP gene expression could be observed during the observation period and reached a 20-fold increase as compared with sham-irradiated age-matched controls. CONCLUSIONS: The distribution of ANP after local irradiation of the rat heart depends on the severity of the pathological/structural changes (i.e. myocyte degeneration and fibrosis). In radiation-induced heart disease, elevated ANP expression in the left ventricle is most probably involved in the observed chronic elevation of plasma ANP levels.

Circulating atrial natriuretic peptide plasma levels as a marker for cardiac damage after radiotherapy.

PURPOSE: To investigate whether plasma concentrations of atrial natriuretic peptide (ANP) could be used to identify patients with radiation mediated cardiac dysfunction. MATERIALS AND METHODS: Circulating levels of ANP were measured in patients who have been irradiated on a large part of the heart (50-80%; Hodgkin's disease) or smaller part of the heart (20-30%; primary breast cancer). C-terminal ANP was determined by radioimmunoassay (RIA) using a commercial kit. RESULTS: In this study ANP plasma levels of 121 patients (Hodgkin's disease, 73 patients; breast cancer, 48 patients) and 67 controls were examined. ANP plasma levels of both Hodgkin patients (28.8+/-2.2, P=0.003) and breast cancer patients (20.4+/-2.8 ng/l, P=0.01) were significantly elevated when compared to age-matched controls (13.5+/-1.2 ng/l). Both for the Hodgkin (R=0.42, P=0.05) and breast cancer group (R=0.50, P=0.09) a positive relation between ANP plasma values and age was found. However, no clear relation between ANP plasma levels and time post treatment could be demonstrated. Patients with clinical symptoms of cardiovascular disease (n=25) had significantly higher ANP plasma levels (P<0.001) compared to patients in the same treatment group without evidence of cardiac disease (50.2+/-7.5 vs. 23.3+/-1.3 ng/l, P<0.001, and 38.2+/-12.4 vs. 16.3+/-1.6 ng/l, P<0.001, for Hodgkin's disease and breast cancer, respectively). Eight patients suffered from essential hypertension (n=8), whereas the remaining group of 17 patients showed a variety of cardiac disorders (i.e. myocardial infarction, decreasing ventricular function, and atrial fibrillations). In 11 patients cardiac problems were manifest either before or within a few years after mediastinal therapy. In two patients treated for Hodgkin's disease, and in four patients treated for breast cancer cardiac problems became manifest a long time (>10 years) after radiotherapy. Probably in this group of patients cardiac problems are related to the therapy. CONCLUSIONS: The present study indicates that ANP plasma levels could be used to identify patients with radiation induced cardiac dysfunction.

Structural changes in the auricles of the rat heart after local ionizing irradiation.

BACKGROUND AND PURPOSE: Irradiation of the heart may lead to late cardiovascular complications and depending on the dose to cardiac-related death. There is increasing evidence that left atrial appendages play an important role in left ventricular filling especially in cardiac disease. The aim of the present study was to investigate the radiation response of the atria of the rat heart (auricles in particular) at morphological, histological and transcriptional level. MATERIAL AND METHODS: Sprague-Dawley rats were irradiated with a single dose locally on the heart (0-22.5 Gy). End-diastolic diameters of left auricles were measured during evaluation of cardiac function. Histopathological evaluations were performed at various time points up to 16 months post irradiation. Changes in mRNA expression of procollagen types I and III and pro-fibrogenic cytokines (TGF-beta1 and IL-1beta) were investigated using competitive PCR. RESULTS: Irradiation leads to a dose-dependent decrease in end-diastolic diameter of the left auricles. This decrease was observed at 4 months post-irradiation, where no gross damage of the ventricle has been reported. Histologically, epicardial fibrosis was found already 1 month post irradiation, and the frequency/severity of the structural changes appeared to be dose-dependent and progressive with time post irradiation. At 9 months, fibrosis was observed in all three layers (epicardium, myocardium and endocardium) of both auricles. On the level of gene expression, increases in procollagen types I and III were observed at 12 and 3 months post irradiation, respectively. Increases in IL-1beta and TGF-beta1, cytokines known to influence collagen deposition at different levels, preceded the upregulation of procollagen mRNA. CONCLUSIONS: Auricles of the rat heart show a marked pathological response to ionizing radiation, characterized by generalized accumulation of collagen (fibrosis) and a reduction of end-diastolic diameter. The reduction of auricular volume and loss of elasticity will negatively contribute to the pump function of the irradiated ventricle.

Myocardial enzyme activities in plasma after whole-heart irradiation in rats.

Plasma levels of myocardial enzymes present after local heart irradiation were studied in a rat model. The purpose was to investigate whether, within days after irradiation, these enzyme levels change to such an extent that they may be helpful in assessing the severity of cardiac damage after radiotherapy. Therefore, activities of creatine kinase (CK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alpha-hydroxybutyrate dehydrogenase (alpha-HBDH) were determined in the plasma and left ventricular myocardium of rats following local heart irradiation with a single dose of 20 Gy. A dose of 20 Gy is known to cause irreversible cardiac damage and to reduce survival times of the animals. Cardiac enzyme assays were performed directly after and twice daily for up to 2 weeks after radiation. Plasma CK, LDH, AST and alpha-HBDH levels were increased between 2 h and 24 h after irradiation. Plasma ALT levels remained unchanged. Myocardial enzyme levels, measured between 24 h and 16 days after radiation, did not differ between irradiated and control animals, although acute (first 12 h) reductions were observed in the irradiated group. The elevated enzyme levels in plasma appeared to correlate with the acutely reduced myocardial enzyme levels. Although irradiation with a dose of 20 Gy induced acute rises of cardiac enzyme levels in plasma, it is doubtful that fractionated radiation, as applied clinically for treatment of solid tumors, will induce plasma enzyme elevations that are large enough to indicate the extent of cardiac damage occurring acutely or chronically.

Reirradiation tolerance of the rat heart.

PURPOSE: To investigate the influence of reirradiation on the tolerance of the heart after a previous irradiation treatment. METHODS AND MATERIALS: Female Wistar rats were locally irradiated to the thorax. Development of cardiac function loss was studied with the ex vivo working rat heart preparation (20). To compare the retreatment experiments, initial, and reirradiation doses were expressed as the percentage of the extrapolated tolerance dose (ETD) (1). RESULTS: Local heart irradiation with a single dose led to a dose-dependent and progressive decrease in cardiac function. The progressive nature of irradiation-induced heart disease is shown to affect the outcome of the retreatment, depending on both the time interval between subsequent doses and the size of the initial dose. The present data demonstrate that hearts are capable of repairing a large part of the initial dose of 10 Gy within the first 24 h. However, once biological damage as a result of the first treatment is fixed, the heart does not show any long-term recovery. At intervals up to 6 months between an initial treatment with 10 Gy and subsequent reirradiation, the reirradiation tolerance dose slightly decreased from 74% of the ETDref (at 24-h interval) to 68% of the ETDref (at 6-month interval). Between 6 and 9 months, reirradiation tolerance dose dropped more even to 43% of the ETDref. Treatment of the heart with an initial dose of 17.5 Gy, instead of 10 Gy, 6 months prior to reirradiation, also led to a further decrease of the reirradiation tolerance dose (< 38 vs. 68% of the ETDref). CONCLUSIONS: The outcome of the present study shows a decreased tolerance of the heart to reirradiation at long time intervals (interval > 6 months). This has clinical implications for the estimation of reirradiation tolerance in patients whose mediastinum has to be reirradiated a long time after a first irradiation course.

Changes in cardiac performance and sympathetic stimulation during and after fractionated radiotherapy in a rat model.

The consequences of fractionated irradiation on the number of cardiac alpha- and beta-adrenergic receptors, myocardial norepinephrine concentration and in vitro assessed heart function were studied in Sprague-Dawley rats. Animals were locally irradiated on the thorax with a total dose of 50 Gy, in 5 weeks, using two different fractionation schemes (5 x 2.0 Gy/week and 3 x 3.3 Gy/week). Functional and biochemical assays were performed during treatment and at 6 months after initiation of treatment. During fractionated irradiation, the numbers of alpha- and beta-adrenergic receptors tended to rise. During this period, myocardial norepinephrine concentration remained fairly constant and no decrease in cardiac output was observed. At 6 months, a significant increase of the numbers of alpha- and beta-adrenergic receptors was observed in the 3.3 Gy/fraction group compared to age-matched controls, p = 0.012 and p = 0.02, respectively. At this time point, the myocardial norepinephrine concentration had decreased below control levels (p = 0.008 for the 3.3. Gy/fraction schedule, and p = 0.03 for the 2.0 Gy/fraction schedule). At 6 months, the cardiac output declined to 61% (p = 0.009) and 69% (p = 0.04) of control values for the 3.3 and 2.0 Gy/fraction schedules, respectively. The present data clearly show development of late cardiac sequelae caused by fractionated thorax irradiation with a total dose of 50 Gy. Moreover, this study lends support to the importance of fraction size with regard to the severity of the radiation-induced cardiac damage.

Time dependent changes in myocardial norepinephrine concentration and adrenergic receptor density following X-irradiation of the rat heart.

The hearts of 9 to 12-weeks-old Sprague-Dawley rats were locally irradiated with a single dose of 20 Gy. The effects on myocardial norepinephrine concentrations and on alpha-adrenergic and beta-adrenergic receptor densities was examined up to 16 months post-treatment. Myocardial norepinephrine concentrations were reduced (to 50% of control values between 8 and 16 months) after irradiation. Receptor binding studies using radioactive ligands demonstrated that alpha-adrenergic receptor density was increased to maximally 210% of control values and that beta-adrenergic receptor density was increased to maximally 150% of control values, both measured at 8 months posttreatment. The affinities of both receptor types were not changed after irradiation. An inverse correlation was found between the myocardial norepinephrine concentration and the alpha-adrenergic receptor density. Myocardial norepinephrine concentration was not correlated to the beta-adrenergic receptor density. The changes in myocardial norepinephrine concentration and receptor density observed after irradiation suggest that even 16 months after irradiation overt cardiac failure was not occurring as the radiation-induced alterations differ considerably from those reported for failing hearts.