Selective Regulation of Cardiac Organic Cation Transporter Novel Type 2 (OCTN2) in Dilated Cardiomyopathy
2011; Elsevier BV; Volume: 178; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2011.02.020
ISSN1525-2191
AutoresMarkus Grube, Sabine Ameling, Michel Noutsias, Kathleen Köck, Ivonne Triebel, Karina Bonitz, Konrad Meissner, Gabriele Jedlitschky, Lars R. Herda, Markus Reinthaler, Maria Rohde, Wolfgang Hoffmann, Uwe Kühl, Heinz‐Peter Schultheiß, Uwe Völker, Stephan B. Felix, Karin Klingel, Reinhard Kandolf, Heyo K. Kroemer,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoOrganic cation transporters (OCT1-3 and OCTN1/2) facilitate cardiac uptake of endogenous compounds and numerous drugs. Genetic variants of OCTN2, for example, reduce uptake of carnitine, leading to heart failure. Whether expression and function of OCTs and OCTNs are altered by disease has not been explored in detail. We therefore studied cardiac expression, heart failure–dependent regulation, and affinity to cardiovascular drugs of these transporters. Cardiac transporter mRNA levels were OCTN2>OCT3>OCTN1>OCT1 (OCT2 was not detected). Proteins were localized in vascular structures (OCT3/OCTN2/OCTN1) and cardiomyocytes (OCT1/OCTN1). Functional studies revealed a specific drug-interaction profile with pronounced inhibition of OCT1 function, for example, carvedilol [half maximal inhibitory concentration (IC50), 1.4 μmol/L], diltiazem (IC50, 1.7 μmol/L), or propafenone (IC50, 1.0 μmol/L). With use of the cardiomyopathy model of coxsackievirus-infected mice, Octn2mRNA expression was significantly reduced (56% of controls, 8 days after infection). Accordingly, in endomyocardial biopsy specimens OCTN2 expression was significantly reduced in patients with dilated cardiomyopathy, whereas the expression of OCT1-3 and OCTN1 was not affected. For OCTN2 we observed a significant correlation between expression and left ventricular ejection fraction (r = 0.53, P < 0.0001) and the presence of cardiac CD3+ T cells (r = −0.45, P < 0.05), respectively. OCT1, OCT3, OCTN1, and OCTN2 are expressed in the human heart and interact with cardiovascular drugs. OCTN2 expression is selectively reduced in dilated cardiomyopathy patients and predicts the impairment of cardiac function. Organic cation transporters (OCT1-3 and OCTN1/2) facilitate cardiac uptake of endogenous compounds and numerous drugs. Genetic variants of OCTN2, for example, reduce uptake of carnitine, leading to heart failure. Whether expression and function of OCTs and OCTNs are altered by disease has not been explored in detail. We therefore studied cardiac expression, heart failure–dependent regulation, and affinity to cardiovascular drugs of these transporters. Cardiac transporter mRNA levels were OCTN2>OCT3>OCTN1>OCT1 (OCT2 was not detected). Proteins were localized in vascular structures (OCT3/OCTN2/OCTN1) and cardiomyocytes (OCT1/OCTN1). Functional studies revealed a specific drug-interaction profile with pronounced inhibition of OCT1 function, for example, carvedilol [half maximal inhibitory concentration (IC50), 1.4 μmol/L], diltiazem (IC50, 1.7 μmol/L), or propafenone (IC50, 1.0 μmol/L). With use of the cardiomyopathy model of coxsackievirus-infected mice, Octn2mRNA expression was significantly reduced (56% of controls, 8 days after infection). Accordingly, in endomyocardial biopsy specimens OCTN2 expression was significantly reduced in patients with dilated cardiomyopathy, whereas the expression of OCT1-3 and OCTN1 was not affected. For OCTN2 we observed a significant correlation between expression and left ventricular ejection fraction (r = 0.53, P < 0.0001) and the presence of cardiac CD3+ T cells (r = −0.45, P < 0.05), respectively. OCT1, OCT3, OCTN1, and OCTN2 are expressed in the human heart and interact with cardiovascular drugs. OCTN2 expression is selectively reduced in dilated cardiomyopathy patients and predicts the impairment of cardiac function. Cardiovascular diseases are the leading cause of death. Consequently, numerous drugs targeting the cardiovascular system are given to a large number of patients. Little is known about drug concentrations at their target sites, which can be modulated by local factors, among them drug efflux and uptake transporters.1Klaassen C.D. Aleksunes L.M. Xenobiotic, bile acid, and cholesterol transporters: function and regulation.Pharmacol Rev. 2010; 62: 1-96Crossref PubMed Scopus (638) Google Scholar In particular, with regard to tissues such as skeletal or cardiac muscle, knowledge about transporter expression as a prerequisite for intracellular drug concentrations is still limited. Yet, several transport proteins, among them members of the ATP-binding cassette proteins (ABC transporter), have been identified within the cardiovascular system.2Solbach T.F. Konig J. Fromm M.F. Zolk O. ATP-binding cassette transporters in the heart.Trends Cardiovasc Med. 2006; 16: 7-15Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar The potential relevance of transport proteins in these structures is highlighted by a recent study on transporter expression in skeletal muscle cells, indicating an impact of uptake and efflux transporters on local statin concentrations and hence on statin-mediated myotoxicity.3Knauer M.J. Urquhart B.L. Meyer Zu Schwabedissen H.E. Schwarz U.I. Lemke C.J. Leake B.F. Kim R.B. Tirona R.G. Human skeletal muscle drug transporters determine local exposure and toxicity of statins.Circ Res. 2010; 106: 297-306Crossref PubMed Scopus (163) Google Scholar Although several publications address expression of efflux-mediating ABC transport proteins in the human heart, the mode of cardiac drug uptake is unclear. In this context, we attempted to demonstrate that the cardiac expression of two uptake transporters in humans and the expression of one of those, the carnitine transporter OCTN2, were affected by cardiac disease.4Grube M. Kock K. Oswald S. Draber K. Meissner K. Eckel L. Bohm M. Felix S.B. Vogelgesang S. Jedlitschky G. Siegmund W. Warzok R. Kroemer H.K. Organic anion transporting polypeptide 2B1 is a high-affinity transporter for atorvastatin and is expressed in the human heart.Clin Pharmacol Ther. 2006; 80: 607-620Crossref PubMed Scopus (189) Google Scholar, 5Grube M. Meyer Zu Schwabedissen H.E. Prager D. Haney J. Moritz K.U. Meissner K. Rosskopf D. Eckel L. Bohm M. Jedlitschky G. Kroemer H.K. Uptake of cardiovascular drugs into the human heart: expression, regulation, and function of the carnitine transporter OCTN2 (SLC22A5).Circulation. 2006; 113: 1114-1122Crossref PubMed Scopus (104) Google Scholar In the present study, we investigated the cardiac expression of uptake transporters for organic cation transporters [OCT(N)s], namely, the OCT1-3 (SLC22A1-3) and OCTN1 and OCTN2 (SLC22A4 and 5). Recent studies indicate an important pharmacologic role of these transporters.6Koepsell H. Lips K. Volk C. Polyspecific organic cation transporters: structure, function, physiological roles, and biopharmaceutical implications.Pharm Res. 2007; 24: 1227-1251Crossref PubMed Scopus (855) Google Scholar For example, in a functional study of the ubiquitously expressed OCT3 in knockout mice, the distribution of the OCT standard substrate 1-methyl-4-phenylpyridinium (MPP+) was selectively altered in cardiac tissue.7Zwart R. Verhaagh S. Buitelaar M. Popp-Snijders C. Barlow D.P. Impaired activity of the extraneuronal monoamine transporter system known as uptake-2 in Orct3/Slc22a3-deficient mice.Mol Cell Biol. 2001; 21: 4188-4196Crossref PubMed Scopus (175) Google Scholar Furthermore, there is evidence of an interaction of the physiologically important carnitine transporters OCTN1 and OCTN28Yabuuchi H. Tamai I. Nezu J. Sakamoto K. Oku A. Shimane M. Sai Y. Tsuji A. Novel membrane transporter OCTN1 mediates multispecific, bidirectional, and pH-dependent transport of organic cations.J Pharmacol Exp Ther. 1999; 289: 768-773PubMed Google Scholar, 9Nezu J. Tamai I. Oku A. Ohashi R. Yabuuchi H. Hashimoto N. Nikaido H. Sai Y. Koizumi A. Shoji Y. Takada G. Matsuishi T. Yoshino M. Kato H. Ohura T. Tsujimoto G. Hayakawa J. Shimane M. Tsuji A. Primary systemic carnitine deficiency is caused by mutations in a gene encoding sodium ion-dependent carnitine transporter.Nat Genet. 1999; 21: 91-94Crossref PubMed Scopus (496) Google Scholar with certain drugs, thereby acting as an uptake transporter or being inhibited in their physiologic function by these compounds.6Koepsell H. Lips K. Volk C. Polyspecific organic cation transporters: structure, function, physiological roles, and biopharmaceutical implications.Pharm Res. 2007; 24: 1227-1251Crossref PubMed Scopus (855) Google Scholar Such interactions may have consequences for systemic and cardiac carnitine homeostasis as already discussed for the valproic acid–induced carnitine depletion.10Tein I. DiMauro S. Xie Z.W. De Vivo D.C. Valproic acid impairs carnitine uptake in cultured human skin fibroblasts: an in vitro model for the pathogenesis of valproic acid-associated carnitine deficiency.Pediatr Res. 1993; 34: 281-287Crossref PubMed Scopus (63) Google Scholar On the other hand, these transporters may modify drug action itself by controlling local concentrations, which has recently been shown for OCTN1 and the hERG (KCNH2) channel blocker quinidine.11McBride B.F. Yang T. Liu K. Urban T.J. Giacomini K.M. Kim R.B. Roden D.M. The organic cation transporter, OCTN1, expressed in the human heart, potentiates antagonism of the HERG potassium channel.J Cardiovasc Pharmacol. 2009; 54: 63-71Crossref PubMed Scopus (34) Google Scholar Although the tissue-specific mRNA expression of these transporters has been investigated in general studies,12Nishimura M. Naito S. Tissue-specific mRNA expression profiles of human ATP-binding cassette and solute carrier transporter superfamilies.Drug Metab Pharmacokinet. 2005; 20: 452-477Crossref PubMed Scopus (322) Google Scholar knowledge about their cardiac localization and disease-dependent regulation is still limited. In addition to drugs modifying these proteins on a functional level (eg, inhibiting l-carnitine uptake), changes in cardiac expression will result in an altered uptake of both endogenous substrates and drugs. Therefore, this study focused on disease-dependent expression of cardiac OCT(N)s and possible functional interactions with cardiovascular drugs. Taken together, we demonstrate selective regulation of OCTN2 by impaired cardiac function and studied the interaction profile of OCT(N)s and cardiovascular drugs. In the present study, we examined the expression of OCT(N)s in the human heart. The general expression and localization were studied in explanted hearts of potential donors with no indications for cardiac dysfunction, which have been described before.13Meissner K. Sperker B. Karsten C. Zu Schwabedissen H.M. Seeland U. Bohm M. Bien S. Dazert P. Kunert-Keil C. Vogelgesang S. Warzok R. Siegmund W. Cascorbi I. Wendt M. Kroemer H.K. Expression and localization of P-glycoprotein in human heart: effects of cardiomyopathy.J Histochem Cytochem. 2002; 50: 1351-1356Crossref PubMed Scopus (99) Google Scholar, 14Dazert P. Meissner K. Vogelgesang S. Heydrich B. Eckel L. Bohm M. Warzok R. Kerb R. Brinkmann U. Schaeffeler E. Schwab M. Cascorbi I. Jedlitschky G. Kroemer H.K. Expression and localization of the multidrug resistance protein 5 (MRP5/ABCC5), a cellular export pump for cyclic nucleotides, in human heart.Am J Pathol. 2003; 163: 1567-1577Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar We studied the cardiac expression of OCT(N)s in endomyocardial biopsy (EMB) specimens in a cohort of 83 patients. We included patients with a broad range of left ventricular ejection fraction (LVEF) as a measure of left ventricular systolic function and left ventricular end diastolic diameter (LVEDD) as a key measure of left ventricular dilatation. We aimed to encompass patients presenting with acute myocarditis (AMC) and dilated cardiomyopathy (DCM) versus noninflammatory pathogenesis. The latter were composed of non-DCMs (n = 8) and patients with unspecified symptoms [dyspnea on exertion, chest discomfort, palpitations (n = 9)] and normal or mildly reduced LVEF (>45%). Patients' EMB specimens were characterized by immunohistologic proof of inflammatory DCM and PCR proof of cardiac viral infections. For the selection of AMC and DCM patients, we focused on patients without proof of cardiac infections, patients with the most frequently detected parvovirus B19 (PVB19), and patients with enteroviral infection, which have been well characterized in animal models of coxsackievirus-induced myocarditis15Klingel K. Hohenadl C. Canu A. Albrecht M. Seemann M. Mall G. Kandolf R. Ongoing enterovirus-induced myocarditis is associated with persistent heart muscle infection: quantitative analysis of virus replication, tissue damage, and inflammation.Proc Natl Acad Sci U S A. 1992; 89: 314-318Crossref PubMed Scopus (389) Google Scholar and have been associated with adverse prognosis in human disease.16Why H.J. Meany B.T. Richardson P.J. Olsen E.G. Bowles N.E. Cunningham L. Freeke C.A. Archard L.C. Clinical and prognostic significance of detection of enteroviral RNA in the myocardium of patients with myocarditis or dilated cardiomyopathy.Circulation. 1994; 89: 2582-2589Crossref PubMed Scopus (212) Google Scholar, 17Fujioka S. Kitaura Y. Ukimura A. Deguchi H. Kawamura K. Isomura T. Suma H. Shimizu A. Evaluation of viral infection in the myocardium of patients with idiopathic dilated cardiomyopathy.J Am Coll Cardiol. 2000; 36: 1920-1926Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar This selection criterion was met to ascertain possible virus-specific effects on OCTN regulation in virus-associated cardiomyopathy. The patients were randomly selected according to these entry criteria from the biomaterial database of the Sonderforschungsbereich Transregio 19 (SFB TR19). Written consent was obtained from each patient, and the protocol was approved by the Ethics Committee of the Charité – Universitätsmedizin, Berlin, Germany, within the framework of the SFB TR19. Patients' EMB specimens were obtained from the right ventricular septum and were characterized immunohistochemically for the presence of a cardiac inflammation and by PCR techniques to detect cardiotropic viruses as described elsewhere.18Holzmann M. Nicko A. Kuhl U. Noutsias M. Poller W. Hoffmann W. Morguet A. Witzenbichler B. Tschope C. Schultheiss H.P. Pauschinger M. Complication rate of right ventricular endomyocardial biopsy via the femoral approach: a retrospective and prospective study analyzing 3048 diagnostic procedures over an 11-year period.Circulation. 2008; 118: 1722-1728Crossref PubMed Scopus (210) Google Scholar, 19Noutsias M. Seeberg B. Schultheiss H.P. Kuhl U. Expression of cell adhesion molecules in dilated cardiomyopathy: evidence for endothelial activation in inflammatory cardiomyopathy.Circulation. 1999; 99: 2124-2131Crossref PubMed Scopus (176) Google Scholar, 20Noutsias M. Pauschinger M. Ostermann K. Escher F. Blohm J.H. Schultheiss H. Kuhl U. Digital image analysis system for the quantification of infiltrates and cell adhesion molecules in inflammatory cardiomyopathy.Med Sci Monit. 2002; 8: MT59-MT71PubMed Google Scholar, 21Kuhl U. Pauschinger M. Noutsias M. Seeberg B. Bock T. Lassner D. Poller W. Kandolf R. Schultheiss H.P. High prevalence of viral genomes and multiple viral infections in the myocardium of adults with "idiopathic" left ventricular dysfunction.Circulation. 2005; 111: 887-893Crossref PubMed Scopus (559) Google Scholar Significant coronary disease was excluded by coronary angiography in all patients with nonischemic cardiomyopathy. The clinically suspected cardiomyopathy entities of AMC, DCM, and non-DCMs (n = 8 controls with LVEF ≤45%; ischemic cardiomyopathy: n = 5, hypertrophic cardiomyopathy: n = 1, toxic cardiomyopathy: n = 1, and tachycardiomyopathy: n = 1) were determined in accordance with widely accepted classifications,22Richardson P. McKenna W. Bristow M. Maisch B. Mautner B. O'Connell J. Olsen E. Thiene G. Goodwin J. Gyarfas I. Martin I. Nordet P. Report of the 1995 World Health Organization/International Society and Federation of Cardiology Task Force on the Definition and Classification of cardiomyopathies.Circulation. 1996; 93: 841-842Crossref PubMed Scopus (2905) Google Scholar, 23Elliott P. Andersson B. Arbustini E. Bilinska Z. Cecchi F. Charron P. Dubourg O. Kuhl U. Maisch B. McKenna W.J. Monserrat L. Pankuweit S. Rapezzi C. Seferovic P. Tavazzi L. Keren A. Classification of the cardiomyopathies: a position statement from the European Society of Cardiology Working Group on Myocardial and Pericardial Diseases.Eur Heart J. 2008; 29: 270-276Crossref PubMed Scopus (2135) Google Scholar considering the chief clinical presentation, laboratory, echocardiographic, and cardiac catheterization data. AMC was suspected in cases with antecedent viral illness and duration of history up to 4 weeks before first clinical assessment. In AMC cases with preserved or mildly impaired LVEF, a clinical presentation mimicking acute coronary syndrome and arrhythmias was frequent.24Kuhl U. Pauschinger M. Bock T. Klingel K. Schwimmbeck C.P. Seeberg B. Krautwurm L. Poller W. Schultheiss H.P. Kandolf R. Parvovirus B19 infection mimicking acute myocardial infarction.Circulation. 2003; 108: 945-950Crossref PubMed Scopus (215) Google Scholar, 25Angelini A. Calzolari V. Calabrese F. Boffa G.M. Maddalena F. Chioin R. Thiene G. Myocarditis mimicking acute myocardial infarction: role of endomyocardial biopsy in the differential diagnosis.Heart. 2000; 84: 245-250Crossref PubMed Scopus (169) Google Scholar In AMC cases with impaired LVEF (≤45%), cardiac decompensation and progressive heart failure prevailed. Three AMC patients were admitted after cardiopulmonary resuscitation.26Kindermann I. Kindermann M. Kandolf R. Klingel K. Bultmann B. Muller T. Lindinger A. Bohm M. Predictors of outcome in patients with suspected myocarditis.Circulation. 2008; 118: 639-648Crossref PubMed Scopus (514) Google Scholar, 27D'Ambrosio A. Patti G. Manzoli A. Sinagra G. Di Lenarda A. Silvestri F. Di Sciascio G. The fate of acute myocarditis between spontaneous improvement and evolution to dilated cardiomyopathy: a review.Heart. 2001; 85: 499-504Crossref PubMed Scopus (242) Google Scholar The patients' clinical and EMB specimen characteristics are summarized in Table 1.Table 1Patient Characteristics From the First StudyCharacteristicsControls with LVEF ≤45% (n = 8)Controls with LVEF >45% (n = 9)AMC patients with LVEF ≤45% (n = 7)AMC patients with LVEF >45% (n = 32)DCM patients (n = 27)Age, mean ± SD (range), years56.8 ± 15.4 (27–71)43.1 ± 20.3 (16–66)52.0 ± 10.3 (39–63)37.0 ± 12.8 (18–65)51.1 ± 13.7 (30–78)Sex, No. M/F7/14/56/126/623/4LVEF, mean ± SD (range), %30.3 ± 9.5 (17–44)77.1 ± 10.9 (50–84)⁎Significantly (P < 0.05) different from the remaining patient groups (Tukey-Kramer post hoc analysis in comparisons with continuous data).36.6 ± 8.1 (25–45)66.6 ± 9.4 (47–83)⁎Significantly (P < 0.05) different from the remaining patient groups (Tukey-Kramer post hoc analysis in comparisons with continuous data).27.9 ± 11.4 (7–45)LVEDD, mean ± SD (range), mm64.3 ± 8.4 (56–81)49.3 ± 5.2 (39–54) ⁎Significantly (P < 0.05) different from the remaining patient groups (Tukey-Kramer post hoc analysis in comparisons with continuous data).63.3 ± 8.7 (54–78)52.8 ± 5.5 (40–62)⁎Significantly (P < 0.05) different from the remaining patient groups (Tukey-Kramer post hoc analysis in comparisons with continuous data).65.4 ± 9.2 (45–82)LVESD, mean ± SD (range), mm47.4 ± 11.9 (24–62)29.8 ± 6.2 (20–38)51.4 ± 13 (36–72)33.6 ± 6.8 (12–44)51.9 ± 11.1 (26–71)Virus (B19V or EV), No.1⁎Significantly (P < 0.05) different from the remaining patient groups (Tukey-Kramer post hoc analysis in comparisons with continuous data).1⁎Significantly (P < 0.05) different from the remaining patient groups (Tukey-Kramer post hoc analysis in comparisons with continuous data).42115 B19V102134 EV001310 B19V and EV00151Inflammatory DCM (immunohistology)1⁎Significantly (P < 0.05) different from the remaining patient groups (Tukey-Kramer post hoc analysis in comparisons with continuous data).0⁎Significantly (P < 0.05) different from the remaining patient groups (Tukey-Kramer post hoc analysis in comparisons with continuous data).41414M, male; F, female. LVEF, left ventricular ejection fraction; AMC, acute myocarditis; DCM, dilated cardiomyopathy; LVEDD, left ventricular end diastolic diameter; LVESD, left ventricular end systolic diameter. Significantly (P < 0.05) different from the remaining patient groups (Tukey-Kramer post hoc analysis in comparisons with continuous data). Open table in a new tab M, male; F, female. LVEF, left ventricular ejection fraction; AMC, acute myocarditis; DCM, dilated cardiomyopathy; LVEDD, left ventricular end diastolic diameter; LVESD, left ventricular end systolic diameter. A second, independent patient group consisting of 15 DCM and six control patients was used to confirm these findings. Among these patients, coronary heart disease was excluded by angiography and ACM by myocardial biopsy. Patients were treated with angiotensin-converting enzyme inhibitors, angiotensin receptor antagonists, β-blockers, digitalis, and diuretics and received stable oral medication for 3 months before the study. From all patients we obtained six to 10 EMB specimens from the interventricular septum of the right ventricle for clinical reasons. Written consent was obtained from each patient, and the protocol was approved by the Ethics Committee of the University Hospital in Greifswald, Germany (Table 2).Table 2Patient Characteristics From the Second StudyCharacteristicsControl patientsDCM patientsNo.615Age, mean ± SD, years45.7 ± 9.1LVEF, mean ± SD, %58.7 ± 9.235.9 ± 5.0LVEDD, mean ± SD, mm50.5 ± 2.968.6 ± 7.3Sex, No. M/F4/29/6Virus diagnosticNegative8 Negative/7 positiveM, male; F, female; LVEF, left ventricular ejection fraction; LVEDD, left ventricular end diastolic diameter. Open table in a new tab M, male; F, female; LVEF, left ventricular ejection fraction; LVEDD, left ventricular end diastolic diameter. Viral infections are one etiologic factor for the development of DCM. Again, the consequences for expression and function of cardiac uptake transport proteins are unknown. We therefore studied murine Oct(n)transporter expression in a viral myocarditis model. Inbred mice of strains C57BL/6 (H-2b) and ABY/SnJ (H-2b) were infected with coxsackievirus B3 (CVB3) in the animal facilities of the Department of Molecular Pathology, University Hospital Tübingen, as described elsewhere.15Klingel K. Hohenadl C. Canu A. Albrecht M. Seemann M. Mall G. Kandolf R. Ongoing enterovirus-induced myocarditis is associated with persistent heart muscle infection: quantitative analysis of virus replication, tissue damage, and inflammation.Proc Natl Acad Sci U S A. 1992; 89: 314-318Crossref PubMed Scopus (389) Google Scholar Four- to 5-week-old mice were infected i.p. with 5 × 104 plaque-forming units of purified CVB3. Animals were sacrificed at different time points (4, 8, 12, and 28 days) after infection. Noninfected animals of both strains were used as controls. Samples of aseptically removed hearts were snap frozen in liquid nitrogen for real-time RT-PCR analysis. RNA isolation from explanted hearts of patients with no indication for cardiac dysfunction and cDNA synthesis were performed as described before.5Grube M. Meyer Zu Schwabedissen H.E. Prager D. Haney J. Moritz K.U. Meissner K. Rosskopf D. Eckel L. Bohm M. Jedlitschky G. Kroemer H.K. Uptake of cardiovascular drugs into the human heart: expression, regulation, and function of the carnitine transporter OCTN2 (SLC22A5).Circulation. 2006; 113: 1114-1122Crossref PubMed Scopus (104) Google Scholar Three to four EMB specimens from each patient of the second collective were stored in liquid nitrogen, pooled, and homogenized. An aliquot of the cell pellet was transferred to RLT-lysis buffer (RNeasy Micro Kit; Qiagen Inc., Valencia, California). Subsequently, RNA was isolated following the manufacturer's instructions for total RNA isolation from fibrous tissues (Qiagen Inc.). RNA was purified using a Charge Switch Total RNA Cell Kit (Invitrogen, Carlsbad, California), and concentration and quality were assessed using a Nanodrop ND-1000 (NanoDrop Technologies Inc., Wilmington, Delaware) and an RNA 6000 Pico LabChip on a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, California). Before real-time PCR one-round T7-RNA polymerase-mediated linear amplification of 30 ng of total RNA was performed using the first part of the Two Cycle Target labeling protocol (Affymetrix, Santa Clara, California). The amplified RNA was reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, California). For RNA preparation from murine hearts, snap frozen hearts were homogenized in the presence of liquid nitrogen by a mortar. Subsequently, the tissue powder was lysed using 350 μL of RLT-lysis buffer following a proteinase K digestion. RNA isolation was performed according to the manufacturer's protocol (Nucleo Spin Extract II Kit; Macherey-Nagel, Düren, Germany). Finally, the RNA concentration was determined using a Nanodrop ND-1000 system. In explanted hearts, expression of transporter transcripts and the reference gene for 18S rRNA was measured by conventional TaqMan real-time PCR using the following assays on demand (Applied Biosystems): Hs00901881_m1 (OCT1), Hs00161893_m1 (OCT2), Hs00222691_m1 (OCT3), and Hs00268200_m1 (OCTN1). The expression of OCTN2 and 18S rRNA was measured as described before.5Grube M. Meyer Zu Schwabedissen H.E. Prager D. Haney J. Moritz K.U. Meissner K. Rosskopf D. Eckel L. Bohm M. Jedlitschky G. Kroemer H.K. Uptake of cardiovascular drugs into the human heart: expression, regulation, and function of the carnitine transporter OCTN2 (SLC22A5).Circulation. 2006; 113: 1114-1122Crossref PubMed Scopus (104) Google Scholar The transporter expression measurement in the first patient collective was performed by a recently developed preamplification/real-time PCR technique28Noutsias M. Rohde M. Block A. Klippert K. Lettau O. Blunert K. Hummel M. Kuhl U. Lehmkuhl H. Hetzer R. Rauch U. Poller W. Pauschinger M. Schultheiss H.P. Volk H.D. Kotsch K. Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies.BMC Mol Biol. 2008; 9: 3Crossref PubMed Scopus (53) Google Scholar using the mRNA specific assays on demand as already mentioned. Real-time PCR analysis of OCT1, OCTN1, and OCTN2 within the EMB specimens of the second patient collective was performed using a custom-manufactured TaqMan low-density array (TLDA; Applied Biosystems) that, in addition to the target genes, contained the common housekeeping genes B2M, GUSB, TBP, 18S rRNA, HMBS, and GAPDH (due to technical problems OCT3 could not be included in this analysis). The TLDA cards were loaded with reaction mixes from control and patient samples, and PCR amplification was performed using a 7900 HT Sequence Detection System (Applied Biosystems). Thermal cycling was as follows: 2 minutes at 50°C; 10 minutes at 94.5°C, followed by 30 seconds at 97°C; and 1 minute at 59.7°C for 40 cycles. In murine probes, transporter expression and the reference gene for 18S rRNA were measured by conventional TaqMan real-time PCR system (7900HT) using the following assays on demand: Mm00456303_m1 (Oct1), Mm00457295_m1 (Oct2), Mm00488294_m1 (Oct3), Mm00457739_m1 (Octn1), and Mm00441468_m1 (Octn2) (all Applied Biosystems). Real-time PCR data were quantified using the SDS 2.3 software package (Applied Biosystems). Protein localization was investigated by immunofluorescence microscopy. For OCT1, a polyclonal antibody against the human transporter was used (rabbit, dilution 1:50; GenWay Biotech Inc., San Diego, California). OCT3 and OCTN1 were detected using affinity-purified polyclonal rabbit antibodies generated against the rat (OCT3) and murine (OCTN1) transporter (Alpha Diagnostics, San Antonio, Texas). OCTN2 was detected as described before (rabbit, dilution 1:150).5Grube M. Meyer Zu Schwabedissen H.E. Prager D. Haney J. Moritz K.U. Meissner K. Rosskopf D. Eckel L. Bohm M. Jedlitschky G. Kroemer H.K. Uptake of cardiovascular drugs into the human heart: expression, regulation, and function of the carnitine transporter OCTN2 (SLC22A5).Circulation. 2006; 113: 1114-1122Crossref PubMed Scopus (104) Google Scholar Paraffin sections of 2 μm were used (prepared by standard methods). Incubation with primary antibodies was performed at 4°C overnight. After being washed with Tris-buffered saline, the sections were incubated for 2 hours with secondary antibody Alexa Fluor 488–labeled IgG (anti-rabbit IgG, Invitrogen). Nuclei were stained using a 1:1000 dilution of DAPI (Sigma Aldrich, Munich, Germany). Functional studies for OCT1, OCT3, OCTN1, and OCTN2 were performed using transporter overexpressing MDCKII cells. Although the OCTN2 overexpressing cell line has already been described,5Grube M. Meyer Zu Schwabedissen H.E. Prager D. Haney J. Moritz K.U. Meissner K. Rosskopf D. Eckel L. Bohm M. Jedlitschky G. Kroemer H.K. Uptake of cardiovascular drugs into the human heart: expression, regulation, and function of the carnitine transporter OCTN2 (SLC22A5).Circulation. 2006; 113: 1114-1122Crossref PubMed Scopus (104) Google Scholar OCT1, OCT3, and OCTN1 overexpressing cell lines were generated. Therefore, the coding sequence of these transporters was amplified from cDNA preparations (derived from placenta for OCT3 and OCTN1 and from liver for OCT1). Subsequently, transporter fragments were cloned into the mammalian expression vector pcDNA3.1/hygro(-) (Invitrogen) and checked for sequence variations against the reference sequences (OCT1, NM_003057; OCT3, NM_021977; OCTN1, NM_003059). Overexpression of all constructs was performed as described before for OCTN2.5Grube M. Meyer Zu Schwabedissen H.E. Prager D. Haney J. Moritz K.U. Meissner K. Rosskopf D. Eckel L. Bohm M. Jedlitschky G. Kroemer H.K. Uptake of cardiovascular drugs into the human heart: expression, regulation, and function of the carnitine transporter OCTN2 (SLC22A5).Circulation. 2006; 113: 1114-1122Crossref PubMed Scopus (104) Google Scholar Stable overexpressi
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