ARTICLE |
Correspondence to: Heyo K. Kroemer, Institut für Pharmakologie, Ernst-Moritz-Arndt-Universität Greifswald, Friedrich-Loeffler-Str. 23d, D-17489 Greifswald, Germany. E-mail: kroemer@uni-greifswald.de
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Summary |
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ABC-type transport proteins, such as P-glycoprotein (P-gp), modify intracellular concentrations of many substrate compounds. They serve as functional barriers against entry of xenobiotics (e.g., in the gut or the bloodbrain barrier) or contribute to drug excretion. Expression of transport proteins in the heart could be an important factor modifying cardiac concentrations of drugs known to be transported by P-gp (e.g., ß-blockers, cardiac glycosides, doxorubicin). We therefore investigated the expression and localization of P-gp in human heart. Samples from 15 human hearts (left ventricle; five non-failing, five dilated cardiomyopathy, and five ischemic cardiomyopathy) were analyzed for expression of P-gp using real-time RT-PCR, immunohistochemistry, and in situ hybridization. Immunohistochemistry revealed expression of P-gp in endothelium of both arterioles and capillaries of all heart samples. Although P-gp mRNA was detected in all samples, its expression level was significantly reduced in patients with dilated cardiomyopathy. We describe variable expression of P-gp in human heart and its localization in the endothelial wall. Thus, intracardiac concentrations of various compounds may be modified, depending on the individual P-gp level. (J Histochem Cytochem 50:13511356, 2002)
Key Words: P-glycoprotein, heart, drug transport, cardiomyopathy
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Introduction |
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It is increasingly recognized that drug transport across biomembranes is facilitated by membrane proteins. Such drug transporters belonging to the ABC (ATP-binding cassette) family can influence the intracellular concentration and hence the action of many compounds in a variety of cells and tissues (
Expression of P-gp in humans reveals a wide interindividual variability. Both genetic and environmental factors have been identified that contribute to this variability. For example,
Substrates for P-gp exhibit a wide structural diversity and cover a wide range of therapeutic indications. Notably, many cardiovascular active compounds are subject to drug transport by P-gp. Digoxin has been unequivocally identified as a P-gp substrate by experiments in cell lines, in animals, and in humans (
We therefore investigated the expression, localization, and genotype of P-gp in human heart using immunohistochemistry (IHC), in situ hybridization (ISH), real-time RT-PCR, and RFLP. Here we describe variable expression of P-gp in the endothelial wall of cardiac blood vessels. Our data point to reduced expression of P-gp in patients with dilated cardiomyopathy.
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Materials and Methods |
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Tissue Samples
After approval from the local ethics committee, heart tissue samples were taken from excised heart left ventricle during orthotopic heart transplantation due to end-stage heart failure and were immediately frozen in liquid nitrogen or fixed in 4% paraformaldehyde. Of the 15 subjects, five suffered from ischemic cardiomyopathy (ICM) and five from dilated non-ischemic cardiomyopathy (DCM). Medical therapy of patients suffering from DCM and ICM consisted of digitalis, diuretics, nitrates, and angiotensin-converting enzyme inhibitors. Tissue samples from five non-failing hearts (NF), which were not transplanted for surgical reasons or blood group incompatibility, served as controls.
The frozen tissue was homogenized using a vibration grinding mill (Mikro-Dismembrator S; B. Braun Biotech, Melsungen, Germany) for standard RNA and DNA preparation protocols. Fixed tissue was embedded in paraffin for IHC or ISH.
MDR1 Genotype
Genomic DNA was prepared from 30 mg tissue using standard phenol/chloroform extraction. The genotype of each individual at the MDR1 exon 26 C3435T locus was determined using a polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) assay according to
RT-PCR Analysis of MDR1
Total RNA was isolated from 50 mg frozen tissue homogenate using a guanidinium isothiocyanate extraction kit (PeqLab; Erlangen, Germany), and subsequent DNase treatment, followed by spin column purification (Strataprep Total RNA Miniprep Kit; Stratagene, Amsterdam, The Netherlands).
For real-time RT-PCR, 200 ng of total RNA was reverse-transcribed using random hexamers and the TaqMan Reverse Transcription Reagents (Applied Biosystems; Weiterstadt, Germany). RT-PCR of MDR1 and 18S rRNA was performed using the primers MDR1F 5'-TTCGCAACC-CCAAGATCCTC-3', MDR1R 5'-ACAATGGTGGTCCG-ACCTTT-3', and the TaqMan probe 5'-6FAM-ATCCAGAGCCACCTGAACCACTGCT XTp, as well as TaqMan Ribosomal RNA Control Reagents (Applied Biosystems) for 18S rRNA and the TaqMan universal PCR mastermix (Applied Biosystems). PCR products were amplified (50C, 2 min; 95C, 10 min; followed by 40 cycles of 95C, 15 sec and 60C, 1 min) and analyzed on a real-time RT-PCR cycler (ABI Prism 7700; Applied Biosystems).
For relative quantification, fluorescence intensities were plotted against PCR cycle numbers. The amplification cycle displaying the first significant increase of the fluorescence signal was defined as threshold cycle (CT). The CT value of each sample was compared to the CT values of the standardization series, which consisted of cDNA from P-gp-overexpressing L-MDR1 cells (kindly supplied by Dr. A. Schinkel; The Netherlands Cancer Institute, Amsterdam). The ratio of relative copy numbers of MDR1 divided by those of 18S rRNA thus represents the expression level of P-gp coding MDR1 mRNA.
Conventional RT-PCR was performed as described previously (
ISH of MDR1
Non-radioactive ISH was performed in paraffin sections (7 µm) that had been fixed in 4% paraformaldehyde. Sections were rehydrated and permeabilized by pepsin digestion (750 µg/ml pepsin in 0.2 M HCl, 37C, 30 min) and then postfixed (paraformaldehyde 4%, 20 min, 4C) and acetylated using 0.25% acetic anhydride in triethanolamine (0.1 M, pH 8.0, 15 min). After dehydration in ethanol (70, 95, 100%), sections were hybridized for 16 hr (56C) in a solution containing 25% formamide, 0.3 M NaCl, 10% dextran sulfate, 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 8.0, 1 x Denhardt's, 0.1 ng/ml herring sperm DNA, 0.5 mg/ml tRNA, 0.1 mg/ml polyuridylic acid, and 125 ng digoxigenin (DIG)-labeled MDR1 cRNA probe (position 471763 of the human MDR1 cDNA). The corresponding sense cRNA probe served as negative control.
After washing with 50% formamide in 75 mM NaCl, 7.5 mM sodium citrate, pH 7.0, sections were incubated with RNase A followed by additional washing steps and incubation with blocking reagent (Roche; Mannheim, Germany). Bound riboprobe was visualized by incubation with alkaline phosphatase-conjugated anti-DIG antibody (Roche) and subsequent substrate reaction using 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium chloride.
IHC of MDR1
From each heart tissue sample, paraffin sections of 2.0 µm were prepared by standard methods. Staining was performed using the Ventana's NexES IHC Staining System (Ventana Medical Systems; Frankfurt, Germany). For immunostaining, the monoclonal anti-P-gp antibody JSB-1 (mouse, dilution 1:20; Alexis Biochemicals, Grünberg, Germany) was used. The secondary antibody was rabbit anti-mouse diluted 1:100 included in the ABC Detection Kit (Ventana Medical Systems). Renal tissue samples were used as positive controls.
For semi-quantitative evaluation the numbers of specifically stained capillaries in relation to the total numbers of capillaries were determined in DCM, ICM, and NF heart tissue sections.
Statistical Analysis
The amounts of specific mRNA for human P-gp were compared using the MannWhitney U-test; p<0.05 was considered significant. Expression in dependence of MDR1 genotype was compared by the KruskalWallis trend test. All data are presented as mean ± SD.
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Results |
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P-glycoprotein was detected by IHC in all heart tissue samples tested. Immunostained heart tissue sections revealed P-gp localization predominantly in endothelial cells of capillaries and arterioles (Fig 1A). Staining intensity showed wide inter-individual variability. Tissue samples from hearts with dilated cardiomyopathy exhibited less staining compared to ischemic cardiomyopathic or non-failing hearts (Fig 1B; p<0.05). Endothelial localization of P-gp in capillaries and arterioles was confirmed by ISH (Fig 1C and Fig 1D).
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Real-time RT-PCR detected expression of MDR1-specific mRNA in all heart samples. As calculated by comparison with standard curves (MDR1, y = -2.1303x + 38.275; 18S rRNA, y = -2.434x + 28.009) generated from LMDR1 total cellular RNA, ratios of MDR1 to 18S rRNA copy numbers were 0.84 ± 0.22 (NF), 1.15 ± 0.62 (ICM), and 0.41 ± 0.13 (DCM). Patients with DCM had significantly lower MDR1 mRNA expression levels compared to non-failing hearts (p=0.016; Fig 2). Results were confirmed using conventional RT-PCR. Standardized for the housekeeping gene GAPDH, MDR1 mRNA levels of patients with DCM were significantly lower (p=0.032) compared to NF hearts (Fig 3).
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MDR1 C3435T genotype prevalence of the samples did not differ significantly from the known MDR1 frequency in whites (
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Discussion |
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Here we describe expression and localization of the ABC transporter P-gp in human heart. P-gp was detected at the mRNA and protein levels in all 15 left ventricular samples. Both IHC and ISH localize P-gp expression to cardiac arterioles and capillaries. We observed a wide interindividual variability of P-gp. Various factors may be responsible for this phenomenon. Several mutations were described in the P-gp encoding MDR1 gene, some of which had functional consequences (
Moreover, the process of disease may affect individual expression of P-gp. In our study we observed lower expression in patients with DCM compared to ICM and NF. This result was consistent at both the protein and mRNA levels and cannot be attributed to drug therapy because there were no differences in drug therapy between DCM and ICM patients. Two different PCR approaches (conventional vs real-time) employing different housekeeping genes confirmed the observation of reduced P-gp in patients with DCM. Heart failure caused by DCM is believed to be mediated by persistent viral infection or autoimmunity, and a variety of antibodies directed against structural components of human heart have been identified. For example anti-ß1-autoantibody treatment of isolated cardiomyocytes leads to decreased expression of ß-adrenergic receptor protein and mRNA, similar to patients with DCM (
Expression of P-gp in cells of human heart vessels is similar to that of P-gp in brain (
Expression of P-gp may also protect heart tissue against cardiac toxicity of certain drugs. The anthracycline doxorubicin is a P-gp substrate [expression of P-gp in cancer cells results in chemoresistance to anthracyclines (
In summary, we describe expression of the ABC-transporter P-glycoprotein in human heart. Our findings may have implications for drug therapy (alteration of both effects and toxicity). Moreover, our data point to reduced expression in patients with DCM. In view of recent data indicating cardiac expression of various cytochrome P450 enzymes (
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Acknowledgments |
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Supported by research grants from the German Cardiac Society (Düsseldorf, Germany) to KM and from Apogepha GmbH (Dresden, Germany) and the Robert-Bosch-Foundation (Stuttgart, Germany) to IC.
Received for publication December 26, 2001; accepted April 10, 2002.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Agarwala S, Kumar R, Bhatnagar V, Bajpai M, Gupta DK, Mitra DK (2000) High incidence of adriamycin cardiotoxicity in children even at low cumulative doses: role of radionuclide cardiac angiography. J Pediatr Surg 35:1786-1789[Medline]
Cascorbi I, Gerloff T, Johne A, Meisel C, Hoffmeyer S, Schwab M, Schaeffeler E et al. (2001) Frequency of single nucleotide polymorphisms in the P-glycoprotein drug transporter MDR1 gene in white subjects. Clin Pharmacol Ther 69:169-174[Medline]
de Lannoy IA, Silverman M (1992) The MDR1 gene product, P-glycoprotein, mediates the transport of the cardiac glycoside, digoxin. Biochem Biophys Res Commun 189:551-557[Medline]
Dobbs RJ, Royston JP, O'Neill CJ, Deshmukh AA, Nicholson PW, Denham MJ, Dobbs SM (1987) Prescribing digoxin in geriatric units: the unexplained variability in dosage requirements. Eur J Clin Pharmacol 32:611-614[Medline]
Drach J, Gsur A, Hamilton G, Zhao S, Angerler J, Fiegl M, Zojer N et al. (1996) Involvement of P-glycoprotein in the transmembrane transport of interleukin-2 (IL-2), IL-4, and interferon-gamma in normal human T lymphocytes. Blood 88:1747-1754
Fardel O, Lecureur V, Daval S, Corlu A, Guillouzo A (1997) Up-regulation of P-glycoprotein expression in rat liver cells by acute doxorubicin treatment. Eur J Biochem 246:186-192[Abstract]
Fromm MF, Kauffmann HM, Fritz P, Burk O, Kroemer HK, Warzok RW, Eichelbaum M et al. (2000) The effect of rifampin treatment on intestinal expression of human MRP transporters. Am J Pathol 157:1575-1580
Geick A, Eichelbaum M, Burk O (2001) Nuclear receptor response elements mediate induction of intestinal mdr1 by rifampin. J Biol Chem 276:14581-14587
Goorin AM, Chauvenet AR, PerezAtayde AR, Cruz J, McKone R, Lipshultz SE (1990) Initial congestive heart failure, six to ten years after doxorubicin chemotherapy for childhood cancer. J Pediatr 116:144-147[Medline]
Greiner B, Eichelbaum M, Fritz P, Kreichgauer HP, von Richter O, Zundler J, Kroemer HK (1999) The role of intestinal P-glycoprotein in the interaction of digoxin and rifampin. J Clin Invest 104:147-153
Hoffmeyer S, Burk O, von Richter O, Arnold HP, Brockmoller J, Johne A, Cascorbi I et al. (2000) Functional polymorphisms of the human multidrug-resistance gene: multiple sequence variations and correlation of one allele with P- glycoprotein expression and activity in vivo. Proc Natl Acad Sci USA 97:3473-3478
Jette L, Tetu B, Beliveau R (1993) High levels of P-glycoprotein detected in isolated brain capillaries. Biochim Biophys Acta 1150:147-154[Medline]
Karlsson J, Kuo SM, Ziemniak J, Artursson P (1993) Transport of celiprolol across human intestinal epithelial (Caco-2) cells: mediation of secretion by multiple transporters including P-glycoprotein. Br J Pharmacol 110:1009-1016[Abstract]
Kawahara M, Sakata A, Miyashita T, Tamai I, Tsuji A (1999) Physiologically based pharmacokinetics of digoxin in mdr1a knockout mice. J Pharmacol Sci 88:1281-1287[Medline]
Lipshultz SE, Colan SD, Gelber RD, PerezAtayde AR, Sallan SE, Sanders SP (1991) Late cardiac effects of doxorubicin therapy for acute lymphoblastic leukemia in childhood. N Engl J Med 324:808-815[Abstract]
Podlowski S, Luther HP, Morwinski R, Muller J, Wallukat G (1998) Agonistic anti-beta1-adrenergic receptor autoantibodies from cardiomyopathy patients reduce the beta1-adrenergic receptor expression in neonatal rat cardiomyocytes. Circulation 98:2470-2476
Salmon SE, Grogan TM, Miller T, Scheper R, Dalton WS (1989) Prediction of doxorubicin resistance in vitro in myeloma, lymphoma, and breast cancer by P-glycoprotein staining. J Natl Cancer Inst 81:696-701[Abstract]
Schinkel AH (1997) The physiological function of drug-transporting P-glycoproteins. Semin Cancer Biol 8:161-170[Medline]
Schinkel AH, Mayer U, Wagenaar E, Mol CA, van Deemter L, Smit JJ, van der Valk MA et al. (1997) Normal viability and altered pharmacokinetics in mice lacking mdr1-type (drug-transporting) P-glycoproteins. Proc Natl Acad Sci USA 94:4028-4033
Schinkel AH, Wagenaar E, Mol CA, van Deemter L (1996) P-glycoprotein in the blood-brain barrier of mice influences the brain penetration and pharmacological activity of many drugs. J Clin Invest 97:2517-2524
Tanabe M, Ieiri I, Nagata N, Inoue K, Ito S, Kanamori Y, Takahashi M et al. (2001) Expression of P-glycoprotein in human placenta: relation to genetic polymorphism of the multidrug resistance (MDR)-1 gene. J Pharmacol Exp Ther 297:1137-1143
Thum T, Borlak J (2000) Gene expression in distinct regions of the heart. Lancet 355:979-983[Medline]
Tishler DM, Weinberg KI, Hinton DR, Barbaro N, Annett GM, Raffel C (1995) MDR1 gene expression in brain of patients with medically intractable epilepsy. Epilepsia 36:1-6[Medline]
van Asperen J, van Tellingen O, Tijssen F, Schinkel AH, Beijnen JH (1999) Increased accumulation of doxorubicin and doxorubicinol in cardiac tissue of mice lacking mdr1a P-glycoprotein. Br J Cancer 79:108-113[Medline]
Vogelgesang S, Schroeder E, Runge U, Gaab MR, Piek J, Cascorbi I, Siegmund W et al. (2001) Expression of P-glycoprotein in medically intractable epilepsy. Acta Neuropathol (Berl) 102:545
Westphal K, Weinbrenner A, Giessmann T, Stuhr M, Franke G, Zschiesche M, Oertel R et al. (2000) Oral bioavailability of digoxin is enhanced by talinolol: evidence for involvement of intestinal P-glycoprotein. Clin Pharmacol Ther 68:6-12[Medline]
Wijnholds J, Evers R, van Leusden MR, Mol CA, Zaman GJ, Mayer U, Beijnen JH et al. (1997) Increased sensitivity to anticancer drugs and decreased inflammatory response in mice lacking the multidrug resistance-associated protein. Nature Med 3:1275-1279[Medline]