Cardiomyocyte Hypertrophy in Arrhythmogenic Cardiomyopathy
2017; Elsevier BV; Volume: 187; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2016.12.018
ISSN1525-2191
AutoresMustafa Gerçek, Muhammed Gerçek, Sebastian Kant, Sakine Simsekyilmaz, Astrid Kassner, Hendrik Milting, Elisa A. Liehn, Rudolf E. Leube, Claudia A. Krusche,
Tópico(s)Cardiomyopathy and Myosin Studies
ResumoArrhythmogenic cardiomyopathy (AC) is a hereditary disease leading to sudden cardiac death or heart failure. AC pathology is characterized by cardiomyocyte loss and replacement fibrosis. Our goal was to determine whether cardiomyocytes respond to AC progression by pathological hypertrophy. To this end, we examined tissue samples from AC patients with end-stage heart failure and tissue samples that were collected at different disease stages from desmoglein 2-mutant mice, a well characterized AC model. We find that cardiomyocyte diameters are significantly increased in right ventricles of AC patients. Increased mRNA expression of the cardiac stress marker natriuretic peptide B is also observed in the right ventricle of AC patients. Elevated myosin heavy chain 7 mRNA expression is detected in left ventricles. In desmoglein 2-mutant mice, cardiomyocyte diameters are normal during the concealed disease phase but increase significantly after acute disease onset on cardiomyocyte death and fibrotic myocardial remodeling. Hypertrophy progresses further during the chronic disease stage. In parallel, mRNA expression of myosin heavy chain 7 and natriuretic peptide B is up-regulated in both ventricles with right ventricular preference. Calcineurin/nuclear factor of activated T cells (Nfat) signaling, which is linked to pathological hypertrophy, is observed during AC progression, as evidenced by Nfatc2 and Nfatc3 mRNA in cardiomyocytes and increased mRNA of the Nfat target regulator of calcineurin 1. Taken together, we demonstrate that pathological hypertrophy occurs in AC and is secondary to cardiomyocyte loss and cardiac remodeling. Arrhythmogenic cardiomyopathy (AC) is a hereditary disease leading to sudden cardiac death or heart failure. AC pathology is characterized by cardiomyocyte loss and replacement fibrosis. Our goal was to determine whether cardiomyocytes respond to AC progression by pathological hypertrophy. To this end, we examined tissue samples from AC patients with end-stage heart failure and tissue samples that were collected at different disease stages from desmoglein 2-mutant mice, a well characterized AC model. We find that cardiomyocyte diameters are significantly increased in right ventricles of AC patients. Increased mRNA expression of the cardiac stress marker natriuretic peptide B is also observed in the right ventricle of AC patients. Elevated myosin heavy chain 7 mRNA expression is detected in left ventricles. In desmoglein 2-mutant mice, cardiomyocyte diameters are normal during the concealed disease phase but increase significantly after acute disease onset on cardiomyocyte death and fibrotic myocardial remodeling. Hypertrophy progresses further during the chronic disease stage. In parallel, mRNA expression of myosin heavy chain 7 and natriuretic peptide B is up-regulated in both ventricles with right ventricular preference. Calcineurin/nuclear factor of activated T cells (Nfat) signaling, which is linked to pathological hypertrophy, is observed during AC progression, as evidenced by Nfatc2 and Nfatc3 mRNA in cardiomyocytes and increased mRNA of the Nfat target regulator of calcineurin 1. Taken together, we demonstrate that pathological hypertrophy occurs in AC and is secondary to cardiomyocyte loss and cardiac remodeling. Arrhythmogenic right ventricular cardiomyopathy, which is now referred to as arrhythmogenic cardiomyopathy (AC),1Basso C. Bauce B. Corrado D. Thiene G. Pathophysiology of arrhythmogenic cardiomyopathy.Nat Rev Cardiol. 2012; 9: 223-233Crossref Scopus (174) Google Scholar is an inherited disease with mutations in genes encoding the desmosomal proteins desmoglein 2, desmocollin 2, plakophilin 2, plakoglobin, and desmoplakin,2Awad M.M. 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Arrhythmogenic right ventricular cardiomyopathy: from genetics to diagnostic and therapeutic challenges.World J Cardiol. 2014; 6: 1234-1244Crossref PubMed Google Scholar but a biventricular involvement has also been reported in 52% to 76% of AC patients.5Corrado D. Basso C. Thiene G. McKenna W.J. Davies M.J. Fontaliran F. Nava A. Silvestri F. Blomstrom-Lundqvist C. Wlodarska E.K. Fontaine G. Camerini F. Spectrum of clinicopathologic manifestations of arrhythmogenic right ventricular cardiomyopathy/dysplasia: a multicenter study.J Am Coll Cardiol. 1997; 30: 1512-1520Crossref PubMed Scopus (776) Google Scholar, 6Te Riele A.S. James C.A. Philips B. Rastegar N. Bhonsale A. Groeneweg J.A. Murray B. Tichnell C. Judge D.P. Van Der Heijden J.F. Cramer M.J. Velthuis B.K. Bluemke D.A. Zimmerman S.L. Kamel I.R. Hauer R.N. Calkins H. Tandri H. 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Genetic testing of candidate genes in arrhythmogenic right ventricular cardiomyopathy/dysplasia.Eur J Med Genet. 2012; 55: 225-234Crossref PubMed Scopus (11) Google Scholar AC onset occurs usually in young adults between 20 and 40 years old.8Turrini P. Basso C. Daliento L. Nava A. Thiene G. Is arrhythmogenic right ventricular cardiomyopathy a paediatric problem too?.Images Paediatr Cardiol. 2001; 3: 18-37PubMed Google Scholar In severe cases, the onset is already observed in children at the beginning of puberty (10 to 12 years old). In contrast, disease manifestation in patients with fewer pathogenic mutations may occur only in old age (>60 years9Quarta G. Muir A. Pantazis A. Syrris P. Gehmlich K. Garcia-Pavia P. Ward D. Sen-Chowdhry S. Elliott P.M. McKenna W.J. Familial evaluation in arrhythmogenic right ventricular cardiomyopathy: impact of genetics and revised task force criteria.Circulation. 2011; 123: 2701-2709Crossref PubMed Scopus (182) Google Scholar). Intense physical activity enhances biventricular disease progression, increases the risk for life-threatening arrhythmia, and shortens the time between disease onset and development of heart failure.10Corrado D. Basso C. Rizzoli G. Schiavon M. Thiene G. Does sports activity enhance the risk of sudden death in adolescents and young adults?.J Am Coll Cardiol. 2003; 42: 1959-1963Crossref PubMed Scopus (1023) Google Scholar, 11Saberniak J. Hasselberg N.E. Borgquist R. Platonov P.G. Sarvari S.I. Smith H.J. Ribe M. Holst A.G. Edvardsen T. Haugaa K.H. Vigorous physical activity impairs myocardial function in patients with arrhythmogenic right ventricular cardiomyopathy and in mutation positive family members.Eur J Heart Fail. 2014; 16: 1337-1344Crossref PubMed Scopus (154) Google Scholar The following phases can be distinguished during AC disease progression8Turrini P. Basso C. Daliento L. Nava A. Thiene G. Is arrhythmogenic right ventricular cardiomyopathy a paediatric problem too?.Images Paediatr Cardiol. 2001; 3: 18-37PubMed Google Scholar: i) The concealed preclinical phase with a risk of life-threatening arrhythmia: Minor arrhythmias may or may not occur. Structural cardiac abnormalities are absent. ii) The phase of overt electrical disorders: Ventricular arrhythmias are detectable. Functional and structural abnormalities of the right ventricle are noted. iii) The phase of heart failure: Biventricular pump failure develops. AC disease manifestation is histologically characterized by a replacement of the necrotic right ventricular myocardium with fibrous and adipose tissue.12Corrado D. Basso C. Nava A. Thiene G. Arrhythmogenic right ventricular cardiomyopathy: current diagnostic and management strategies.Cardiol Rev. 2001; 9: 259-265Crossref PubMed Scopus (56) Google Scholar In case of left ventricular involvement, the fibrous tissue replacement predominantly takes place within the lateral and posterior walls.3Pinamonti B. Brun F. Mestroni L. Sinagra G. Arrhythmogenic right ventricular cardiomyopathy: from genetics to diagnostic and therapeutic challenges.World J Cardiol. 2014; 6: 1234-1244Crossref PubMed Google Scholar, 13Bauce B. Basso C. Rampazzo A. Beffagna G. Daliento L. Frigo G. Malacrida S. Settimo L. Danieli G. Thiene G. Nava A. Clinical profile of four families with arrhythmogenic right ventricular cardiomyopathy caused by dominant desmoplakin mutations.Eur Heart J. 2005; 26: 1666-1675Crossref PubMed Scopus (243) Google Scholar In severe cases of childhood AC, extensive infarction-like fibrous scars develop on cardiomyocyte loss.14Bauce B. Rampazzo A. Basso C. Mazzotti E. Rigato I. 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Genetic testing of candidate genes in arrhythmogenic right ventricular cardiomyopathy/dysplasia.Eur J Med Genet. 2012; 55: 225-234Crossref PubMed Scopus (11) Google Scholar A common and ubiquitous hallmark feature of cardiomyopathy is the cardiomyocyte hypertrophy, which has not been investigated in detail during AC disease progression. Cardiomyocyte hypertrophy is a reactive response to intrinsic and/or extrinsic mechanical stress to maintain cardiac function. Physiological hypertrophy occurs during heart development and growth. It is linked to an increase in heart weight. Reactive, reversible hypertrophy is observed during pregnancy and endurance training.16Umar S. Nadadur R. Iorga A. Amjedi M. Matori H. Eghbali M. Cardiac structural and hemodynamic changes associated with physiological heart hypertrophy of pregnancy are reversed postpartum.J Appl Physiol. 2012; 113: 1253-1259Crossref PubMed Scopus (51) Google Scholar, 17George K. Whyte G.P. Green D.J. Oxborough D. Shave R.E. Gaze D. Somauroo J. The endurance athletes heart: acute stress and chronic adaptation.Br J Sports Med. 2012; 46 Suppl 1: i29-i36Crossref PubMed Scopus (60) Google Scholar Pathological cardiac hypertrophy is induced by volume and pressure overload or mutations of genes encoding proteins that are involved in force generation, force distribution, and contraction regulation.18Parvari R. Levitas A. The mutations associated with dilated cardiomyopathy.Biochem Res Int. 2012; 2012: 639250Crossref PubMed Scopus (28) Google Scholar, 19Seidman C.E. Seidman J.G. Identifying sarcomere gene mutations in hypertrophic cardiomyopathy: a personal history.Circ Res. 2011; 108: 743-750Crossref PubMed Scopus (183) Google Scholar, 20Haddad F. Doyle R. Murphy D.J. Hunt S.A. Right ventricular function in cardiovascular disease, part II: pathophysiology, clinical importance, and management of right ventricular failure.Circulation. 2008; 117: 1717-1731Crossref PubMed Scopus (945) Google Scholar Two types of hypertrophy are distinguished: Pressure overload is compensated by concentric cardiac hypertrophy because of increased cardiomyocyte diameters, whereas volume overload is compensated by heart chamber enlargement because of cardiomyocyte elongation and increased cardiomyocyte diameter.21Diwan A. Dorn 2nd, G.W. Decompensation of cardiac hypertrophy: cellular mechanisms and novel therapeutic targets.Physiology. 2007; 22: 56-64Crossref PubMed Scopus (182) Google Scholar, 22Grossman W. Paulus W.J. Myocardial stress and hypertrophy: a complex interface between biophysics and cardiac remodeling.J Clin Invest. 2013; 123: 3701-3703Crossref PubMed Scopus (57) Google Scholar Pathological hypertrophy is accompanied by adverse cardiac remodeling, which is characterized by fibrosis and reexpression of the fetal gene program comprising natriuretic peptide A, natriuretic peptide B (NPPB), α 1 skeletal muscle actin (ACTA1), and, in mice, myosin heavy chain 7 (Myh7).23Luedde M. Katus H.A. Frey N. Novel molecular targets in the treatment of cardiac hypertrophy.Recent Pat Cardiovasc Drug Discov. 2006; 1: 1-20Crossref PubMed Scopus (31) Google Scholar, 24Harvey P.A. Leinwand L.A. The cell biology of disease: cellular mechanisms of cardiomyopathy.J Cell Biol. 2011; 194: 355-365Crossref PubMed Scopus (226) Google Scholar These alterations are believed to contribute to reduction of cardiac preload and to adaptation of the mechanical and contractile properties of cardiomyocytes to increased mechanical stress. Various signaling pathways are involved in the induction of pathological hypertrophy. Among them, calcineurin–nuclear factor of activated T cells (Nfat) signaling resembles a key pathway.25Samak M. Fatullayev J. Sabashnikov A. Zeriouh M. Schmack B. Farag M. Popov A.F. Dohmen P.M. Choi Y.H. Wahlers T. Weymann A. Cardiac hypertrophy: an introduction to molecular and cellular basis.Med Sci Monit Basic Res. 2016; 22: 75-79Crossref PubMed Scopus (99) Google Scholar The hypertrophic response may become insufficient, eventually leading to end-stage heart failure.21Diwan A. Dorn 2nd, G.W. Decompensation of cardiac hypertrophy: cellular mechanisms and novel therapeutic targets.Physiology. 2007; 22: 56-64Crossref PubMed Scopus (182) Google Scholar, 26Ryan J.J. Archer S.L. The right ventricle in pulmonary arterial hypertension: disorders of metabolism, angiogenesis and adrenergic signaling in right ventricular failure.Circ Res. 2014; 115: 176-188Crossref PubMed Scopus (291) Google Scholar The current study was undertaken to analyze the occurrence of cardiomyocyte hypertrophy in AC. Besides cardiac tissue samples obtained from AC patients at end-stage disease, a murine model—desmoglein 2-mutant (Dsg2MT) mice27Krusche C.A. Holthofer B. Hofe V. van de Sandt A.M. Eshkind L. Bockamp E. Merx M.W. Kant S. Windoffer R. Leube R.E. Desmoglein 2 mutant mice develop cardiac fibrosis and dilation.Basic Res Cardiol. 2011; 106: 617-633Crossref PubMed Scopus (62) Google Scholar—was used to study cardiomyocyte hypertrophy in the distinct disease phases. At birth, Dsg2MT mice show no visible pathological changes; however, the animals develop right ventricular dilation and myocardial lesions from 2 to 3 weeks onwards.27Krusche C.A. Holthofer B. Hofe V. van de Sandt A.M. Eshkind L. Bockamp E. Merx M.W. Kant S. Windoffer R. Leube R.E. Desmoglein 2 mutant mice develop cardiac fibrosis and dilation.Basic Res Cardiol. 2011; 106: 617-633Crossref PubMed Scopus (62) Google Scholar Cardiomyocyte death at the onset of the overt disease phase elicits a transient inflammatory response accompanied and followed by replacement and interstitial fibrosis. By the age of 10 to 12 weeks, disease progression slows down and a chronic progression with dilation of all cardiac chambers and heart failure is observed.27Krusche C.A. Holthofer B. Hofe V. van de Sandt A.M. Eshkind L. Bockamp E. Merx M.W. Kant S. Windoffer R. Leube R.E. Desmoglein 2 mutant mice develop cardiac fibrosis and dilation.Basic Res Cardiol. 2011; 106: 617-633Crossref PubMed Scopus (62) Google Scholar, 28Kant S. Krull P. Eisner S. Leube R.E. Krusche C.A. Histological and ultrastructural abnormalities in murine desmoglein 2-mutant hearts.Cell Tissue Res. 2012; 348: 249-259Crossref PubMed Scopus (36) Google Scholar, 29Kant S. Holthofer B. Magin T.M. Krusche C.A. Leube R.E. Desmoglein 2-dependent arrhythmogenic cardiomyopathy is caused by a loss of adhesive function.Circ Cardiovasc Genet. 2015; 8: 553-563Crossref PubMed Scopus (47) Google Scholar In adult animals, a significant prolongation of the QRS complex was noted by electrocardiography.29Kant S. Holthofer B. Magin T.M. Krusche C.A. Leube R.E. Desmoglein 2-dependent arrhythmogenic cardiomyopathy is caused by a loss of adhesive function.Circ Cardiovasc Genet. 2015; 8: 553-563Crossref PubMed Scopus (47) Google Scholar Furthermore, norepinephrine stimulation was shown to induce ventricular extrasystoles, atrial arrhythmias, and repolarization abnormalities.27Krusche C.A. Holthofer B. Hofe V. van de Sandt A.M. Eshkind L. Bockamp E. Merx M.W. Kant S. Windoffer R. Leube R.E. Desmoglein 2 mutant mice develop cardiac fibrosis and dilation.Basic Res Cardiol. 2011; 106: 617-633Crossref PubMed Scopus (62) Google Scholar Herein, cardiomyocyte diameters were analyzed in left and right ventricles of human and mouse hearts. In addition, cardiac hypertrophy was examined by analyzing hypertrophic marker gene expression. Finally, heart function and the spatial and temporal expression of components of the calcineurin-Nfat signaling pathway were assessed in the AC mouse model. Human myocardial tissue samples were obtained from surgical heart transplants at the Heart and Diabetes Center North Rhine-Westphalia. Samples were taken from the explanted hearts of AC patients with end-stage heart failure (n = 5). The patients are identical to Patients 1 to 5 described by Kant et al.30Kant S. Krusche C. Gaertner A. Milting H. Leube R.E. Loss of plakoglobin immunoreactivity in intercalated discs in arrhythmogenic right ventricular cardiomyopathy: protein mislocalization versus epitope masking.Cardiovasc Res. 2016; 109: 260-271Crossref PubMed Scopus (13) Google Scholar The reasons for cardiac transplantation of these patients was right ventricular failure (n = 2), predominantly right ventricular failure (n = 2), and biventricular failure and cardiac arrhythmias (n = 1). Control tissues originate from donor hearts (n = 5) that were not suitable for transplantation. After shock freezing, the tissue samples were cryopreserved at −80°C until use. Patients gave informed consent for the use of their explanted hearts (ethical committee votum 21/2013 by the Medical Faculty of the Ruhr-University Bochum, suboffice Bad Oeynhausen). Mice were housed in the animal facility of the University Hospital Rheinisch-Westfälische Technische Hochschule Aachen. Animals received a standard rodent laboratory diet (Ssniff, Soest, Germany) and had free access to food and water. The experiments were conducted in accordance with the guidelines for the care and use of laboratory animals and approved by the Ministry for Climate Protection, Environment, Agriculture, Conservation and Consumer Protection of the State of North Rhine-Westphalia (reference number 8.87-50.10.37.09.114). Homozygous Dsg2MT mice have been described recently.27Krusche C.A. Holthofer B. Hofe V. van de Sandt A.M. Eshkind L. Bockamp E. Merx M.W. Kant S. Windoffer R. Leube R.E. Desmoglein 2 mutant mice develop cardiac fibrosis and dilation.Basic Res Cardiol. 2011; 106: 617-633Crossref PubMed Scopus (62) Google Scholar Cardiac tissue samples were collected from homozygous Dsg2MT and age-matched wild-type (Dsg2WT) mice of either sex at indicated time points. For the measurement of cardiomyocyte diameter, hearts were cut transversally, fixed in 4% neutrally buffered formaldehyde overnight, dehydrated in a graded isopropanol series, and embedded in paraffin. To study the mRNA expression of markers indicative of cardiac function and/or hypertrophy, hearts of 4-week-old (juvenile), 12-week-old (adult), and 7- to 8-month-old (aged) mice (each genotype and age n = 5 to 7) were collected for RNA isolation (PeqLab Gold RNA Isolation Kit; PEQLAB Biotechnologie GmbH, Erlangen, Germany). After removal of the atria, the right ventricular free wall was separated and the left ventricle (septum and left ventricular free wall) was cleaned from adherent blood. The cardiac samples were separately homogenized in RNA isolation buffer. RNA isolation was performed according to the manufacturer's protocol. Cryostat sections (10 μm thick) were prepared from frozen human heart samples. Sections were fixed for 10 minutes in 4% neutrally buffered formaldehyde. After washing in phosphate-buffered saline (PBS), sections were incubated with tetrarhodamine isothiocyanate–labeled wheat germ agglutinin (100 μg/mL in PBS, 1 hour; catalog number L 5266; Sigma-Aldrich, Seelze, Germany). Sections were washed twice in PBS, and nuclei were counterstained with DAPI (2 μg/mL in PBS). The diameters of murine cardiomyocyte were assessed on paraffin sections (5 μm thick). After paraffin removal with xylene, sections were rehydrated in a graded alcohol series and transferred to PBS. Antigen retrieval was achieved by incubating sections in 10 mmol/L citrate buffer (pH 6) for 30 minutes at 94°C. Thereafter, sections were incubated with tetrarhodamine isothiocyanate–labeled wheat germ agglutinin and counterstained with DAPI. Cardiomyocyte diameters were measured in images taken with 20× and 40× objectives of the ApoTome.2 (Zeiss, Oberkochen, Germany) using Axiovision software version 4.83 (Zeiss). Three to six images were recorded from each right and left ventricle of all studied animals. In mutants and wild-type controls, images were taken from similar myocardial regions. Subsequent analyses were performed blinded to the genotype (Mus.G.). The cell diameter was determined in cardiomyocyte cross sections containing a visible nucleus in the cell center. In each instance, the shortest diameter was determined along a line through the nucleus. For highest accuracy, the inner diameter of the wheat germ agglutinin–labeled cardiomyocytes was measured. The analyses were verified by a second blinded observer (C.A.K.). The interobserver variability was 5.71%. Paraffin sections (5 μm thick) of Dsg2MT and wild-type hearts were used to assess cardiac IgG distribution.31Straub V. Rafael J.A. Chamberlain J.S. Campbell K.P. Animal models for muscular dystrophy show different patterns of sarcolemmal disruption.J Cell Biol. 1997; 139: 375-385Crossref PubMed Scopus (420) Google Scholar Sections were deparaffinized in xylene and rehydrated in a graded alcohol series. The inactivation of the endogenous peroxidase was included in the rehydration process by adding 3% H2O2 to 70% ethanol and incubation for 10 minutes in the dark. After a rinse of 5 minutes with Tris wash buffer (0.05 mol/L Tris/HCl, pH 7.5, 0.3 mol/L NaCl, and 0.045% Tween 20), sections were transferred for 30 minutes to 10 mmol/L citrate buffer (pH 6) heated to 94°C in a water bath. After cooling down to room temperature (20 minutes), murine IgG was detected with the Zytochem Plus HRP-Polymer mouse/rabbit system (catalog number POLHRP-100; Zytomed Systems, Berlin, Germany). At first, the blocking solution was applied for 5 minutes, followed by a 5-minute rinse in Tris wash buffer. Then, sections were incubated with reagent 2 for 30 minutes, washed with Tris wash buffer (2 washes for 5 minutes each), followed by treatment with the horseradish peroxidase (HRP)–polymer solution for 30 minutes. After two washes of 5 minutes each, sections were rinsed with distilled water. The immunoreaction was detected with diaminobenzidine and hydrogen peroxide (diaminobenzidine substrate kit; catalog number TA-060-QHDX; Zytomed Systems). After counterstaining with hematoxylin, sections were mounted with glycerol gelatine (Merck, Darmstadt, Germany). Dsg2MT and wild-type animals, aged 2, 4, 5 to 6, 11 to 13, and 47 to 54 weeks, were assessed (n = 3 to 6 animals per age group and genotype). Total cardiac mouse RNA (1 μg) was reverse transcribed using the Transcriptor First Strand cDNA Synthesis Kit and oligo-(dT)18 primer (Roche, Mannheim, Germany). PCR was performed on cDNA using the LightCycler 96 system and the Fast Start Essential DNA Probes Master Kit (Roche). The PCR volume was 10 μL and contained the equivalent of 30 ng total RNA. Primer and Universal Probe Library (UPL) probes (Roche) were used at a concentration of 0.5 and 0.1 μmol/L, respectively. The PCR program consisted of a preincubation step (95°C, 10 minutes) followed by 45 two-step amplification cycles (denaturation: 10 seconds, 95°C; annealing/elongation: 30 seconds, 60°C) with measurement of PCR product formation at the end of each cycle. Hydroxymethylbilane synthase served as the housekeeping gene. Results are given as means ± SD. To assess the mRNA expression in murine right and left ventricular samples, the following primer pairs and UPL probes were used for quantitative RT-PCR: Nppb: UPL-probe 71, 5′-GTCAGTCGTTTGGGCTGTAAC-3′ (forward) and 5′-AGACCCAGGCAGAGTCAGAA-3′ (reverse); Myh7 (cardiac muscle, β): UPL-probe 85, 5′-TGAAGCTGACGCAGGAGAG-3′ (forward) and 5′-TGAGTGCATTTAACTCAAAGTCC-3′ (reverse); Acta1 (skeletal muscle actin): UPL-probe 9, 5′-TCCTCCCTGGAGAAGAGCTA-3′ (forward) and 5′-ATCCCCGCAGACTCCATAC-3′ (reverse); phospholamban: UPL-probe 27, 5′-TGAGCTTTCCTGCGTAACAG-3′ (forward) and 5′-TGGTCAAGAGAAAGATAAAAAGTTGA-3′ (reverse); sarcoplasmic/endoplasmic reticulum calcium ATPase 2: UPL-probe 15, 5′-TGTTGAGTTCCTTCAGTCCTTTG 3′ (forward) and 5′-GGCAATCCCGATTTCAGAT-3′ (reverse); regulator of calcineurin 1 (Rcan1): UPL-probe 77, 5′-AAAGCCACCAAGTGTCCAGT-3′ (forward) and 5′-GACAATTCCACACGGTTCG-3′ (reverse); hydroxymethylbilane synthase: UPL-probe 42, 5′-AAGTTCCCCCACCTGGAA-3′ (forward) and 5′-GACGATGGCACTGAATTCCT-3′ (reverse). mRNA expression was examined in right and left ventricular samples of five AC patients and five nonfailing donors. Total RNA was isolated with the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's recommendation. Total RNA (250 ng) was reverse transcribed (SuperScript Reverse Transcriptase, random primers; Invitrogen, Life Technologies, Dreieich, Germany). For real-time PCR, 2 μL of cDNA was used in a total volume of 20 μL. Primers and corresponding FAM- and TAMRA-labeled probes were used at concentrations of 0.30 and 0.15 μmol/L, respectively. The PCR program consisted of a preincubation step (95°C, 10 minutes) followed by 40 two-step amplification cycles (denaturation: 15 seconds, 95°C; annealing/elongation: 60 seconds, 60°C) with measurement of the fluorescence signal at the end of each cycle. For the determination of the relative quantity, glyceraldehyde-3-phosphate dehydrogenase was used as reference. The following primer pairs and probes were used to analyze the mRNA expression in human right and left ventricular samples: glyceraldehyde-3-phosphate dehydrogenase, 5′-CTGGGCTACACTGAGCACCA-3′ (forward), 5′-CAGCGTCAAAGGTGGAGGAG-3′ (reverse), 5′-TGGTCTCCTCTGACTTCAACAGCGACAC-3′ (probe); NPPB, 5′-AGGAGCAGCGCAACCATTT-3′ (forward), 5′-TCCAGGGATGTCTGCTCCA-3′ (reverse), 5′-CAGGGCAAACTGTCGGAGCTGCA-3′ (probe); sarcoplasmic reticulum Ca(2+)−ATPase 2 (ATP2A2), 5′-GTCCTTGCTGAGGATGCCC-3′ (forward), 5′-TGACATGGACAGGCAGATGG-3′ (reverse), 5′-CCTGGGAGAACATCTGGCTCGTGG-3′ (probe); ACTA1, 5′-GAGCGTGGCTACTCCTTCGT-3′ (forward), 5′-GTAGCACAGCTTCTCCTTGATGTC-3′ (reverse), 5′-ACCACAGCTGAGCGCGAGATCGT-3′ (probe); and MYH7, 5′-AAGGTCAAGGCCTACAAGCG-3′ (forward), 5′-CTTGCGGAACTTGGACAGGT-3′ (reverse), 5′-AGGAGGCGGAGGAGCAAGCCAAC-3′ (probe). Paraffin sections (5 μm thick) were prepared under RNase free conditions. In situ hybridization was performed with the help of the View RNA ISH Tissue Assay Kit and the View RNA Chromogenic Signal Amplification Kit (both from Affymetrix, Santa Clara, CA; catalog numbers QVT0050 and QVT0200, respectively) using type 1 view RNA probes against Actc1 (Affymetrix; catalog number VB1-13263), and Nfatc1, Nfatc2, Nfatc3, and Nfatc4 (https://www.ncbi.nlm.nih.gov/gene; accession numbers NM_016791.4, NM_010899.3, NM_010901, and NM_023699.3, respectively). The procedure was performed according to the protocol provided by the manufacturer using a programable temperature-controlled slide processing instrument (ThermoBrite; Abbott Laboratories, Abbott Park, IL). The heat and protease pretreatment protocols were optimized in a pilot experiment. As a result, sections were incubated in pretreatment solution for 20 minutes at 95°C to 98°C and digested in protease solution for 20 minutes at 40°C. Specifically, hybridized probes were detected with Fast Red. The reaction was visually controlled using an ApoTome2 microscope setup controlled by AxioVision imaging software version 4.8 (Zeiss, Jena, Germany). To better visualize the fluorescence signals, the contrast was confined between the maximum fluorescence intensity of erythrocytes and the maximum fluorescence intensity of the entire image. The resulting high-contrast fluorescence image was then overlaid with the correspondin
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