Accumulating work in experimental animals suggests that bradykinin (BK) exerts cardioprotective effects via bradykinin type-2 receptors (BK-2Rs). In human end-stage heart failure, BK-2Rs are significantly downregulated by mechanisms that have remained elusive. Heart tissues from idiopathic dilated cardiomyopathy (IDC;
Accumulating data indicate that bradykinin (BK) exerts cardioprotective effects, which include both protection of the myocardium from ischemia-reperfusion injuries [
In human end-stage heart failure, due to either idiopathic dilated cardiomyopathy (IDC) or coronary heart disease (CHD), the amount of cardioprotective BK-2Rs is significantly decreased [
Although the molecular mechanisms regulating the expression of BK-2Rs are largely unknown, it is generally thought that the main regulatory mechanism lies at the gene expression level [
In the present study, we have analyzed the age-related regulation of BK-2R expression and the occurrence of two known BK-2R promoter polymorphisms in normal and failing human hearts.
Normal heart samples
BK-2R polymorphism in human hearts. The presence of two BK-2R gene polymorphisms, −58T/C and −9/+9, was analyzed in normal and failing human hearts according to the protocol described in Section
Subject no. | Etiology | Sex | Age | Polymorphism | |
−58 T/C | +9/−9 | ||||
1 | Normal | M | 19 | T/C | +9/−9 |
2 | M | 43 | T/C | +9/+9 | |
3 | M | 49 | C/C | +9/−9 | |
4 | M | 31 | T/T | −9/−9 | |
5 | M | 40 | T/C | +9/+9 | |
6 | F | 50 | C/C | +9/−9 | |
7 | CHD | M | 61 | T/C | −9/−9 |
8 | M | 60 | T/T | −9/−9 | |
9 | M | 55 | C/C | −9/−9 | |
10 | M | 52 | T/C | −9/−9 | |
11 | M | 60 | C/C | −9/−9 | |
12 | M | 54 | T/C | −9/−9 | |
13 | IDC | M | 29 | C/C | −9/−9 |
14 | M | 60 | C/C | −9/−9 | |
15 | M | 46 | C/C | +9/−9 | |
16 | M | 56 | C/C | +9/−9 | |
17 | M | 55 | T/T | +9/−9 | |
18 | M | 56 | T/C | +9/−9 | |
19 | M | 63 | C/C | +9/−9 |
Total RNA was isolated from human heart samples using an ultra-pure TRIzol reagent (GIBCO BRL), and an RNeasy Mini Kit (QIAGEN). One microgram of purified total RNA was transcribed into cDNA, using a Superscript TM pre-amplification system (GIBCO BRL). The primers were as follows: BK-2R: 5′-CACCATCTCCAACAACTTCG (S), 5′-GGTAGCTGATGACACAAGCG (AS); GAPDH: 5′-ACCACAGTCCATGCCATCAC (S), 5′-TCCACCACCCTGTTGCTGTA (AS). The competitor DNA for the BK-2R was obtained by inserting a 129 bp external DNA fragment into the SacI site. The use of equal amounts of mRNA in the RT-PCR assays was confirmed by analyzing the expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (data not shown). The PCR product was verified, by DNA sequencing, to represent the corresponding target and quantitated with a Gel Doc 2000 gel documentation system (Bio-Rad). The logarithm of the target-to-competitor ratio was plotted against the logarithm of the competitor DNA-molecules [
Genomic DNA was isolated from human heart tissue using an RNA/DNA Mini Kit (Qiagen) and stored in TE buffer (pH 8.5) in aliquots at −20°C.
Isolated genomic DNA was subjected to PCR using primers spanning the insertion/deletion (−/+9 bp) site in exon 1 of the BK-2R gene. The primers were as follows: BK-2R: 5′-CAAAGATGAGCTGTTCCCGCC (S), 5′-GGGAACTTTTCCCAACTCCCC (AS). The PCR was run at
Genomic DNA was isolated as described above and subjected to PCR using following primers: BK-2R: 5′-AGGAGTGCAGAGCTCAGCTGGAG (S), 5′-TCGGAGCCCAGAAGCCAGAG (AS). The obtained PCR products were denatured by heating at 94°C for 3 minutes in 95% formamide, 10 mM NaOH, 0.25% bromphenol blue, and 0.25% xylene cyanol, and then were rapidly cooled on ice. The denatured PCR samples were mixed with 9 parts of SSCP Sucrose Dye (10 g sucrose, 62.5 mg BPB.XC, 0.5 ml 0.5 M EDTA (pH 8.0) in 25 ml dH2O), and run on a 14% acrylamide/bis (29 : 1) gel (BIORAD) containing 10% (v/v) 2x concentrated MDE gel solution (BMA) for 5 min at 500 V, and then for 19 hours at 250 V at room temperature. The gel was stained with SYBR Gold nucleic acid gel stain (Molecular Probes) for 30–40 min and analyzed with a Gel Doc 2000 gel documentation system (Bio-Rad).
The data was subjected to linear regression analysis. Statistical significance was accepted at
By simple linear regression analysis, a strong positive correlation
BK-2R mRNA expression correlates with age in normal and failing (IDC) hearts. The level of BK-2R mRNA expression was plotted against age of normal, IDC, and CHD hearts, and the correlation was calculated.
By further plotting the BK-2R protein levels against age of the patients (Figure
(a) BK-2R protein levels in normal and failing hearts show a linear trend (not significant) when plotted against age. (b) BK-2R mRNA expression levels plotted against BK-2R protein levels show a positive linear correlation
To further analyze the possible transcriptional mechanisms involved in the observed downregulation of BK-2R expression in failing hearts, we determined the presence of two polymorphisms, a −58T/C promoter polymorphism and a 9 base pair (bp) exon 1 deletion/insertion polymorphism, both previously known to affect the BK-2R expression levels (Table
Genotypic and allelic frequencies of two BK-2R gene polymorphisms in normal and failing human hearts. The genotypic and allelic frequencies for two common BK-2R gene polymorphisms were calculated in both normal and failing human hearts.
Polymorphism | Genotype | Normal | IDC | CHD |
---|---|---|---|---|
−58T/C | T/T | 17.0% | 14.0% | 17.0% |
T/C | 50.0% | 15.0% | 50.0% | |
C/C | 33.0% | 71.0% | 33.0% | |
−9/+9 | −9/−9 | 17.0% | 29.0% | 100% |
−9/+9 | 50.0% | 71.0% | 0% | |
+9/+9 | 33.0% | 0% | 0% |
Polymorphism | Allele | Frequency | |||
Normal | IDC | CHD | Average | ||
−58T/C | C-allele | 0.58 | 0.81 | 0.58 | 0.66 |
T-allele | 0.42 | 0.19 | 0.42 | 0.34 | |
−9/+9 | −9-allele | 0.42 | 0.64 | 1.00 | 0.69 |
+9-allele | 0.58 | 0.36 | 0.00 | 0.31 |
Here we show that a linear relationship between BK-2R mRNA and protein expression exists in normal and failing human left ventricles, suggesting that the expression of cardioprotective BK-2Rs in human myocardium may be regulated on the transcriptional level, rather than on the translational level. In addition, in normal and IDC hearts, the BK-2R expression was found to correlate positively with age, but the relative increase was clearly lower in the IDC hearts than in the normal hearts. The increased presence of the C-allele in the IDC hearts may explain the age-related lower relative increase in BK-2R expression in IDC hearts, as compared to normal hearts. These results suggest that the human heart adapts to age-related changes in heart function [
In a recent study, a correlation between age and the expression of BK-1Rs and BK-2Rs has also been observed in male Brown Norway rats [
It seems evident that LVH may develop as a compensatory mechanism during the early stages of the disease, whereas the later stages are characterized by a maladaptive loss of myocytes with subsequent fibrosis, LV dysfunction, and heart failure. Thus, if BK-2Rs are an essential part of a local cardioprotective system, a logical adaptive response to an ongoing pathological process would be an increased expression of BK-2Rs. Therefore, it is possible that BK-2R expression is upregulated during LVH, and that the observed receptor downregulation occurs only later, at the stage of heart failure. Similarly, the increased expression of BK-2Rs with increasing age in normal human hearts may occur as an adaptive response to an age-related physiologic process of disease-independent fibrosis [
Recent studies have suggested that patients developing LVH and heart failure may have a genetic background in which their expression levels of cardioprotective BK-2Rs are significantly weaker than in normal hearts, or completely lacking. Indeed, the C allele of the −58T/C promoter polymorphism in the BK-2R gene has been associated with the occurrence of essential hypertension in several ethnic groups, including African Americans [
Military trainees, which were homozygous for the −9 allele and underwent physical training, did not develop exercise-induced myocardial hypertrophy to a similar extent as heterozygous subjects or homozygous for the +9 allele [
The number of human heart samples in this study is too low to allow any conclusions regarding the general distribution of the studied polymorphisms in failing hearts on the population level. Future studies should aim at analyzing the correlation between IDC and CHD patients and specific promoter polymorphisms capable of affecting the expression of BK-2Rs in a large clinical setting. However, due to the advancement of various preventive treatment modes of coronary heart disease, particularly the use of statins, the number of CHD patients subjected to transplantations per year has dramatically dropped. Thus, most of the patients undergoing heart transplantation are diagnosed with primary myocardial disease. Also, acute coronary syndromes hardly lead to massive myocardial necrosis any more. Finally, patients with multiple myocardial infarctions and ensuing end-stage heart failure are also very rare, and if patients have a severe heart failure due to CHD, they are too old for heart transplantation.
The authors thank Mrs. Elina Kaperi and Mrs. Jaana Tuomikangas for excellent technical assistance and Dr. Mikko Mäyränpää for collecting heart specimens. Wihuri Research Institute is maintained by the Jenny and Antti Wihuri Foundation. This work was supported in part by the Finnish Heart Foundation, Helsinki, Finland (I. Liesmaa, N. Shiota, J. O. Kokkonen, K. A. Lindstedt), the Aarne Koskelo Foundation, Helsinki, Finland (I. Liesmaa, N. Shiota, J. O. Kokkonen), the Paavo Nurmi Foundation, Helsinki, Finland (K. A. Lindstedt) and the Academy of Finland, Helsinki, Finland (K. A. Lindstedt).