Am J Transl Res 2015;7(7):1280-1294 www.ajtr.org /ISSN:1943-8141/AJTR0010909

Original Article Unbalanced upregulation of 2 plays a particular role in early development of daunorubicin cardiomyopathy

Dana Kucerova1,2, Gabriel Doka1, Peter Kruzliak3, Katarina Turcekova1, Jana Kmecova1, Zuzana Brnoliakova4, Jan Kyselovic1, Uwe Kirchhefer2, Frank U Müller2, Peter Krenek1, Peter Boknik2, Jan Klimas1

1Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University, Bratislava, Slovak Re- public; 2Institut für Pharmakologie und Toxikologie, Universitätsklinikum Münster, Münster, Germany; 3Internation- al Clinical Research Center, St. Anne’s University Hospital and Masaryk University, Brno, Czech Republic; 4Institute of Experimental Pharmacology, Slovak Academy of Sciences, Bratislava, Slovak Republic Received May 31, 2015; Accepted July 14, 2015; Epub July 15, 2015; Published July 30, 2015

Abstract: release channel on the of cardiomyocytes ( type 2, RyR2) plays a critical role in the regulation of calcium and was identified as a crucial factor for development of chronic anthracycline cardiomyopathy. Its early stages are less well described although these determine the later development. Hence, we tested the effect of repeated, short-term anthracycline (daunorubicin) administration on cardiac performance, cardiomyocyte function and accompanied changes in calcium regulating expression. Ten-twelve weeks old male Wistar rats were administered with 6 doses of daunorubicin (DAU, 3 mg/kg, i.p., every 48 h), controls (CON) received vehicle. Left ventricular function (left ventricular pressure, LVP; rate of pressure de- velopment, +dP/dt and decline, -dP/dt) was measured using left ventricular catheterization under tribromethanol anaesthesia (15 ml/kg b.w.). shortening was measured in enzymatically isolated cardiomyocytes. The expres- sions of RyR2 and associated intracellular calcium regulating proteins, cytoskeletal proteins (alpha-, alpha- tubul in) as well as oxidative stress regulating enzymes (gp91phox, MnSOD) were detected in ventricular samples using immunoblotting. mRNA expressions of cardiac damage markers (Nppa and Nppb, atrial and natriuretic peptides; Myh6, Myh7 and Myh7b, heavy chain alpha and beta) were detected using RT-PCR. Thiobarbituric acid reactive substances concentration was measured to estimate oxidative stress. DAU rats exhib- ited significantly depressed left ventricular features (LVP by 14%, +dP/dt by 36% and -dP/dt by 30%; for all P<0.05), in line with concomitant increase in Nppa and Nppb expressions (3.23- and 2.18-fold, for both P<0.05), and a 4.34-fold increase in Myh7 (P<0.05). Controversially, we observed increased cell shortening of isolated cardiac cells by 31% (p<0.05). DAU administration was associated with a twofold upregulation of RyR2 (P<0.05), but not of other examined Ca2+ regulating proteins remained. In addition, we observed a significant reduction in alpha- (by 46% when compared to CON P<0.05). Indicators of oxidative injury were unaffected. In conclusion, unbalanced RyR2 overexpression plays a particular role in early development of daunorubicin cardiomyopathy characterized by discrepant in situ versus in vitro cardiac performance.

Keywords: Calcium, (RyR2), oxidative stress, daunorubicin cardiomyopathy

Introduction is of crucial importance to determine the responsible toxic mechanism to find an effec- The therapeutic usage of anthracyclines is lim- tive and targeted prevention of this specific ited because of their well-known serious car- drug-induced cardiac damage. diotoxicity leading to cardiomyopathy and con- sequent cardiac failure. Because cardiac tissue Intriguingly, a huge number of mechanisms pos- has limited capacity to regenerate, it is very sibly responsible for anthracycline cardiotoxici- sensitive to toxic attacks of anthracyclines [1, ty have been introduced [1]. The oxidative 2]. As treatment of consequent dysfunction is stress-based hypothesis involving intramyocar- only transiently or moderately effective [2-4], it dial iron-promoted production of reactive oxy- Upregulation of RyR2 in early daunorubucin cardiomyopathy gen species (ROS) was believed over decades (FKBP12), serving as a stabilizer of as the most plausible [5]. The key point in the RyR2 [24], and cardiac sarco/endoplasmic free radical hypothesis is that anthracyclines reticulum Ca2+-ATPase (SERCA2a), with its and their iron chelates undergo redox cycling, inhibitor (PLB), responsible for resulting in generation of free radicals and Ca2+ uptake into SR [25]. reactive oxygen species. This is supported by successful clinical use of iron-chelator dexra- In experimental chronic anthracycline cardio- zoxane what remains actually the only clinically myopathies, the development of delayed cardi- well documented protective agent against ac failure was linked to downregulation of RyR2 anthracycline cardiotoxicity [6]. Recently, this and SERCA2a [14, 16, 26, 27]. However, early concept has been challenged. Several lines of changes are less well described albeit they may evidence suggest that mechanisms other than substantially determine the later development. the traditionally emphasized “ROS and iron” As anthracyclines and their metabolites modify hypothesis are playing a crucial role in this RyR2 and SERCA2a activity by binding to the pathology [7]. proteins and so influencing Ca2+ cycling [11, 12], we hypothesized that modulation of In general, abnormal calcium cycling is directly expressions of calcium regulating proteins associated with the development of cardiac occurs already at early stages, i.e. in early failure [8-10]. Pharmacologically, the detrimen- anthracycline cardiomyopathy and thereby tal role of calcium overload in anthracycline car- influences its development. Hence, we tested diotoxicity is extremely interesting as anthracy- the effect of repeated, short-term anthracycline clines and their metabolites perform a direct (daunorubicin) administration on cardiac per- pharmacological action on cardiomyocytes as formance, cardiomyocytes function and accom- they interact with sarcoplasmic reticulum (SR) panied changes in calcium regulating proteins proteins and influence intracellular calcium lev- expression. Additionally, we evaluated the pro- els [11, 12]. Indeed, since decades ago, altera- tein or mRNA expression of other potentially tions in calcium cycling have been observed in influential contributors in early anthracycline- the presence of anthracyclines what was linked induced cardiomyopathy such as ROS- to mitochondrial dysfunction, depletion of high generating enzymes, cardiac myosin heavy energy phosphates, increased cardiac stiff- chain isoforms, components of renin-angioten- ness, impaired contractility and cell death [13- sin system and markers of cardiac damage. 16]. Interestingly, a direct bimodal effect of anthracyclines on cardiac Ca2+ release channel Material and methods (ryanodine receptor 2; RyR2) was documented [11, 12, 17]. The influence of anthracyclines on Experimental animals and drug application calcium-regulating proteins expression as well as their action might be the reason also for con- In our study we used male Wistar rats (10-12 troversial effects of anthracyclines on cardiac weeks, Dobra Voda, Slovak Republic), divided preparations exhibiting both depressed or randomly into two experimental groups: control increased performance [18-20]. (CON) and daunorubicin (DAU). In DAU group, six doses of daunorubicin (Daunoblastina inj, SR Ca2+ release is controlled by a tetrameric Pharmacia Italia, Italy) were injected intraperi- protein complex that is localized at the junction- toneally (3 mg/kg every 48 hours) [18]. Controls al SR and consists of the RyR, TRN, junctin received vehicle (0.9% saline solution). Rats (JUN), and CSQ. Functional interactions among were kept under standard conditions and TRN, JUN, and CSQ modulate the function of received food and water ad libitum. All experi- RyR2 [21, 22]. According to this model, the ments were performed 48 h after administra- binding of CSQ to TRN and JUN inhibits the tion of the last dose. RyR2 activity at low luminal Ca2+ concentra- tions. When luminal Ca2+ increases, this inhibi- All animal care and experiments were approved tion is gradually relieved as the Ca2+ binding by the State Veterinary and Food Administration sites on CSQ become occupied by Ca2+, attenu- of the Slovak Republic and by the Ethics ating interactions among CSQ and TRN and/or Committee of the Faculty of Pharmacy, JUN. This results in an increased open probabil- Comenius University in Bratislava and conduct- ity of the RyR2 [23]. Additionally, an important ed according to the EU adopted Directive regulators of calcium cycling are FK506-binding 2010/63/EU of the European Parliament and

1281 Am J Transl Res 2015;7(7):1280-1294 Upregulation of RyR2 in early daunorubucin cardiomyopathy of the Council on the protection of animals striping, without spontaneous contractions and used for experimental and other scientific pur- with good reaction to electrical stimulation poses and to the Slovak law regulating animal were used. experiments. Measurement of relative cell shortening of Left ventricular catheterization isolated cardiac myocytes

A computerized ECG system was used to mea- Measurements of the ventricular myocyte func- sure and analyse standard 12-lead ECG. tion were performed using a videomicroscopy. Electrocardiograms were recorded under tri- The cell suspension was placed in custom- bromoethanol (15 ml/kg, i.p.) anaesthesia. made perfusion chamber mounted on a micro- Duration of QT, as a measure of cardiac repo- scope. This chamber allowed simultaneous larisation, was determined and corrected to rat perfusion with perfusion solution (see Solutions cardiac cycle length as QTc (in milliseconds) = for composition) and electrical field-stimulation 1/2 QT/(RR/150) [28]. (0.5, 1, 2 or 3 Hz, pulse amplitude 10V, pulse Subsequently, left ventricular function (left ven- width 5ms). The cells were then superfused tricular pressure, LVP; rate, HR; rate of with perfusion solution and electrically paced pressure development, +dP/dt; rate of pres- by different frequencies. Myocyte shortening sure decrease, -dP/dt) was measured in situ via was visualized on a PC monitor. The image was catheterization of the left ventricle (Spel processed in software Cell Length Monitor 1.0 Advanced HaemoSys; Experimetria Ltd., (International Laser Centre, Bratislava, Slovak Budapest, Hungary) as described [18]. The Republic), where the longitudinal line for detect- functional response of the left ventricle to beta- ing the changes of the cell edges position dur- adrenergic stimulation was measured upon ing electrical stimulation was used for mea- infusion of increasing doses of isoproterenol (5, surement of the cell shortening. The maximum 15, 30 and 60 ng/min) into the vena jugularis. contraction was normalized to resting cell length and expressed as percentage of short- Isolation of left ventricular cardiac myocytes ening. Cell shortening was measured at stimu- lation frequency 0.5 Hz before and after acute Ventricular myocytes were isolated enzymati- isoproterenol application (10-7 mol/L ISO in per- cally using a slight modification of protocols fusion solution) and at stepwise increasing published before [18, 25]. Shortly, animals stimulation frequency (0.5 Hz, 1 Hz, 2 Hz, and 3 were anesthetized by thiopental 45 mg/kg and Hz). Moreover, the time to 50% decay (T ) and pre-treated with heparin (1.5 IU). After bilateral 50 time to 90% decay (T ) of the cell shortening thoracotomy, the were quickly excised 90 and cannulated through the aorta fixed in the (defined as the time from peak level to 50% or Langendorff apparatus. Isolated hearts were to 90% of the peak amplitude, respectively) at perfused for 5 min at 2.5 ml/min with Ca2+ free stimulation frequency 0.5 Hz was calculated. perfusion buffer (37°C) followed by a perfusion Solutions with the same buffer supplemented by 0.1 mmol/L ethylene glycol tetraacetic acid (EGTA) The basis for solutions used for isolation for 5 min and finally with enzyme solution con- and storage of myocytes was Krebs-Henseleit TM taining 0.9 mg/mL collagenase (type II; GIBCO solution contained (in mmol/L): 130 NaCl, 5.4 Invitrogen Corp., Thermo Scientific, Waltham, KCl, 1.4 MgCl2·6H2O, 1.2 NaH2PO4·H2O, 0.4 MA, USA) in low CaCl2 (50 mmol/L). After enzy- NaH2PO4, 10.0 creatine, 20.0 taurine, 10.0 glu- matic digestion, the left ventricle was separat- cose, 10.0 4-(2-hydroxyethyl)-1-piperazineeth- ed, gently mechanically agitated and filtered enesulfonic acid (HEPES), titrated to pH 7.3 through a nylon mesh (200 µm pore diameter). with NaOH. Filtrate was centrifugated at 42 g for 60 s, supernatant removed and the pellets of myo- Basic external solution perfused on single car- cytes resuspended in enzyme-free isolation diomyocytes during recording contained solution containing 0.75 mmol/L of CaCl (see 2 (mmol/L): 130 NaCl, 5.4 KCl, 1.4 MgCl2·6H2O, Solutions for composition). The isolated left 0.4 NaH2PO4, 10.0 glucose, 10.0 HEPES and ventricular cardiac myocytes were stored at RT 1.8 CaCl adjusted to pH 7.3 with NaOH. and all experiments were performed within the 2 next 6 h. For measurement, only rod-shaped All chemicals were purchased from Sigma- cells with sharp edges and clear Aldrich (Munich, Germany). Thiopental and hep-

1282 Am J Transl Res 2015;7(7):1280-1294 Upregulation of RyR2 in early daunorubucin cardiomyopathy

Table 1. Primer sequences for quantitative RT-PCR GenBank official symbol mRNA PCR product length Primer sequences (5’→3’) accession number (nt) Actb forward: CCGCGAGTACAACCTTCTTG 81 NM_031144.2 reverse: GCAGCGATATCGTCATCCA B2m forward: ATGGAGCTCTGAATCATCTGG 105 NM_012512.1 reverse: AGAAGATGGTGTGCTCATTGC Myh6 forward: GCCCTTTGACATCCGCACAGAGT 152 NM_017239.2 reverse: TCTGCTGCATCACCTGGTCCTCC Myh7 forward: GCGGACATTGCCGAGTCCCAG 133 NM_017240.1 reverse: GCTCCAGGTCTCAGGGCTTCACA Myh7b forward: CCCGATTCTCAACACCAACACCTCT 150 NM_001107794.2 reverse: CATCAGGCACCCAGACCCGT Ryr2 forward: ACTGCTGGGCTACGGCTAC 99 NM_001191043.1 & NM_032078.2 reverse: CTGAAGATGCGGAACCTCTC Cacna1c forward: GTTGCCCTGGGTGTATTTTG 109 NM_012517.2 reverse: GGCTTTCTCCCTCTCTTTGG Atp2a2 forward: CCCGAAACTACCTGGAGCCTGCA 83 NM_001110139.2 reverse: ATGCACGCACCCGAACACCC Trpc3 forward: GCGTCGCTGAGTCGGGTCAAA 130 NM_021771.2 reverse: CGATGGTCTGCTCCCGAAGGC Trpc6 forward: TCGTGGCGCATCCGAACTGC 285 NM_053559.1 reverse: GGAGGAGCTTGGTGCCTTCAAATCT Acta1 forward: TCACTTCCTACCCTCGGCACCC 142 NM_019212.2 reverse: GGGGCATCATCCCCGGCAAAG Nppb forward: GACCGGATCGGCGCAGTCAGT 78 NM_031545.1 reverse: GGAGTCTGCAGCCAGGAGGTCT Nppa forward: GGGGGTAGGATTGACAGGAT 104 NM_012612.2 reverse: GGATCTTTTGCGATCTGCTC Ctf1 forward: GGAAGTCTGGAAGACCACCA 137 NM_017129.1 reverse: TGCTGCACATATTCCTCCAG Kcnq1 forward: CTGGTCTGCCTCATCTTCAG 110 NM_032073.1 reverse: TCTGTCCCAAAGAACACCAC Kcnh2 forward: GACCTGCTTACTGCCCTCTAC 124 NM_053949.1 reverse: GACGTGCATACAGGTTCAGAG Gja1 forward: CCTTTGACTTCAGCCTCCAA 86 NM_012567.2 reverse: CATGTCTGGGCACCTCTCTT Scn5a forward: TGTATGTCCTCAGCCCCTTC 112 NM_013125.2 reverse: ATGAACACGCAGTTGGTCAG Ace forward: tcctgctagacatggagacga 142 NM_012544.1 reverse: cagctcttccacacccaaag Ace2 forward: cgctgtcaccagacaagaa 129 NM_001012006.1 reverse: cgtccaatcctggttcaag Agtr1a forward: cacacaaccctcccagaaag 147 NM_030985.4 reverse: ttggggcagtcatcttgg Mas1 forward: cagagctgggtttacctgga 132 NM_012757.2 reverse: atggctttctcctcagcaaa Thrb forward: TCAGAGCGTCTCAAGCGCCCA 108 NM_012672.3 reverse: TTGTCCCCGCACACTACGCAG Thra1 forward: CGTCCTTGCTCAGCTCGGTCCA 120 NM_001017960.1 reverse: AGAGCTAGGGGCAAGCTGGCT

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Thra2 forward: AACATTCCGCACTTCTGGCCCA 166 NM_031134.2 reverse: GGACCTGCGGACCCTGAACAAC Abbreviations: Actb (, beta); B2m (beta-2 microglobulin); Myh6 (myosin, heavy chain 6, , alpha); Myh7 (myosin, heavy chain 7, cardiac muscle, beta); Myh7b (myosin, heavy chain 7B, cardiac muscle, beta); Ryr2 (ryanodine recep- tor 2, cardiac); Cacna1c (, voltage-dependent, L type, alpha 1C subunit); Atp2a2 (ATPase, Ca++ transporting, cardiac muscle, slow twitch 2); Trpc3 (transient receptor potential cation channel, subfamily C, member 3); Trpc6 (transient re- ceptor potential cation channel, subfamily C, member 6); Acta1 (actin, alpha 1, ); Nppb (natriuretic peptide B); Nppa (natriuretic peptide A); Ctf1 (cardiotrophin 1); Kcnq1 (, voltage-gated KQT-like subfamily Q, member 1); Kcnh2 (potassium channel, voltage gated eag related subfamily H, member 2); Gja1 ( protein, alpha 1); Scn5a (, voltage-gated, type V, alpha subunit); Ace (angiotensin I converting enzyme); Ace2 (angiotensin I converting enzyme 2); Agtr1a (angiotensin II receptor, type 1a); Mas1 (MAS1 proto-oncogene, G protein-coupled receptor); Thrb (thyroid receptor beta); Thra1 (thyroid hormone receptor alpha, transcript variant TRalpha1); Thra2 (thyroid hormone receptor alpha, transcript variant TRalpha2).

Table 2. Basal gravimetric, cardiac and myocyte param- frozen in liquid nitrogen and homoge- eters of control (CON) and daunorubicin (DAU) experi- nized in a buffer containing 10 mM Tris- mental group HCl (pH 7.4), 0.125 M sucrose, 1 mM CON DAU EDTA-Na, 10% sodium dodecylsulphate Gravimetry (n) 13 14 and 1 mM phenylmethylsulphonyl fluo- ride, as described previously [25]. For Body weight (g) 277±9 204±4* immunoblot analysis, 50 µg (for detec- Heart (mg) 857±20 600±14* tion of sarco- Ca2+ Heart/body weight (mg/g) 3.11±0.08 2.95±0.06 ATP-ase, SERCA2a; endothelial NO-syn- Lung (mg) 1202±54 976±36* thase, eNOS; caveolin-1 and -3, cav-1, Liver (g) 10.5±0.4 6.4±0.3* cav-3; heat shock protein, hsp90; glyco- Spleen (mg) 636±19 315±24* sylated subunit of the NADPH oxidase, Kidney (mg) 937±25 650±16* gp91phox; α-tubulin; α-actinin; β-actin; 12-lead electrocardiography 7 8 manganese superoxide dismutase, Mn- RR (ms) 152±4 182±6* SOD), 100µg (for detection of calseques- QT (ms) 76±2 93±2* trin, CSQ; , TRD; phospholamban, PLB; FK506 binding protein, FKBP12) QTc (ms) 75±1 84±2* 150 µg (for detection of inducible Left ventricular catheterization (n) 7 7 NO-synthase, iNOS) or 250 µg (for detec- Heart rate (b/min) 410±9 396±14 tion of RyR2) of homogenate protein Left ventricular pressure (mmHg) 132±5 114±6* were separated on SDS-PAGE. After +dP/dt (mmHg/s) 4976±503 2893±435* transferring of proteins to polyvinylidene -dP/dt (mmHg/s) -4144±304 -2868±297* fluoride (PVDF) transfer membrane Isolated cells (animals/cells) 5/40 5/45 (Immobilon-P, Millipore Corp., Billerica, Cell lenght (μm) 117.1±2.9 120.7±2.5 MA, USA), the blots were incubated with Cell width (μm) 24.8±1.0 27.3±1.2 different . We used various Cell shortening (%) 5.1±0.4 6.7±0.5* antibodies raised against the following proteins: eNOS, iNOS, cav-1, cav-3, hsp- Time to 50% decay (ms) 140±6 117±7* 90, gp91phox, MnSOD (BD Transduction Time to 90% decay (ms) 406±20 333±19* Laboratories, Franklin Lakes, NJ, USA), Data are shown as mean ± SEM. n = number of animals, *P<0.05 vs. CON. α-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), α-actinin (Sigma- Aldrich, Saint Louis, MO, USA). Specific arin were obtained from Lachema (Brno, Czech monoclonal antibodies against RyR2, SERCA2a, Republic). PLB, TRD, FKBP12 and CSQ were used as described [25]. β-actin (Sigma-Aldrich, Saint SDS PAGE and Western blotting Louis, MO, USA) was used as a housekeeping protein for all investigating proteins except pro- For Western blotting analysis, left ventricles of teins of SR (TRD, PLB, FKBP12, RyR2, SERCA2a) rats after catheterization were further used. where the CSQ took over his role. The amount Tissue samples from left ventricles were flash- of bound protein was detected by using chemi-

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Figure 1. Left ventricular catheterisation performed in closed-chest anaesthetized rats from control (CON, n = 7, white circles) and daunorubicin (DAU, n = 7, black circles). Increasing doses of isoproterenol (ISO) were injected into the left jugular vein, determined in ng/min. LVP-left ventricular pressure, HR-heart rate, +dP/dt -rate of pressure development , -dP/dt -rate of pressure decline, data are shown as mean ± SEM, *P<0.05 vs CON. luminescent detection (ECL Plus, Amersham, Grand Island, NY, USA), 1 µg of total RNA were Buckinghamshire, UK) or by 125I-labeled protein reverse-transcribed to cDNA. mRNA levels of A and quantified using Storm 860 (Molecular selected (see Table 1) were evaluated Dynamics, Sunnyvale, CA). Quantification was using real-time PCR (ABI Prism 7300, Applied performed using threedimensional densitome- Biosystems, USA) with gene-specific primers try in Optiquant (Packard Instruments) or with SYBR green detection (Power SYBR Green ImageQuant® (Molecular Dynamics, Sunnyvale, PCR Master Mix, Applied Biosystems, Foster USA), respectively. To allow comparison of the City, California, USA). Relative quantification of protein levels between groups, these arbitrary mRNA expression in RT-PCR experiments was density levels were then normalized to the performed by using the 2-ΔΔCt (Ct-threshold average control level. cycle) method. Results were normalized to the expression of endogenous reference genes Isolation of RNA and real-time PCR (Actb, B2m) and calibrated to the control group. All primers (Table 1) were verified to yield a sin- Separate groups of rats were used. As gle PCR product with correct molecular weight described [25], total RNA was isolated from rat and the absence of signal was verified when cardiac left ventricle using Tri-Reagent (Ambion, reverse transcription was omitted. Grand Island, NY, USA). Isolated RNA was veri- fied to be intact by using agarose gel electro- Estimation of lipid peroxidation phoresis. Reverse transcription to cDNA was performed using High Capacity cRNA RT Kit The concentration of thiobarbituric acid reac- with RNAse inhibitor (Applied Biosystems, tive substances (TBARSs) was used as a bio-

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Figure 2. Cell shortening of single ventricular myocytes. A. Representative time curve of cell shortening of single myocyte from control (CON, dashed line) and daunorubicin (DAU, solid line) group. B. Frequency-dependent effects on myocyte cell shortening. Response to different stimulation frequencies is displayed for CON (white circles) and DAU (black circles) myocytes, displayed as average ± SEM. Frequency was increased stepwise every 5 min from 0.5 to 3 Hz *P<0.05 vs CON, in brackets: no. of animals/myocytes. C. Myocyte cell shortening in response to β-adrenergic receptor stimulation in CON and DAU myocytes. The cell shortening was measured at 0.5-Hz stimula- tion frequency first at basal conditions (superfusion with perfusion solution, white columns, basal) and then in response to isoproterenol (superfusion with perfusion solution supplemented by isoproterenol to final concentration 10-7 mol·l-1, grey columns, ISO). Data are shown as average ± SEM, *P<0.05 vs CON, #P<0.05 vs. basal, in brackets: no. of animals/myocytes. marker to measure the level of oxidative stress Results in left ventricular tissue [29]. Shortly, left ven- tricular samples were homogenized in 10 mM Wasting syndrome and impaired left ventricu- potassium phosphate buffer, pH 7.4. Thereafter, lar function 0.5% solution of thiobarbituric acid in 20% tri- chloroacetic acid was added to homogenates. As expected, cumulative dose of daunorubicine These mixtures were incubated for 30 min at (18 mg/kg) used in our study caused signifi- 90°C. Then, samples were cooled and after cant cachexia manifested as significant loss in centrifugation (at 11000 RPM for 10 min) mea- body weight as well as reduced weights of inter- sured using spectrophotometer Multiscan RC nal organs, including the heart (see Table 2). In (Labsystems, Vantaa, Finland) at 530 nm. The situ, left ventricular catheterisation unveiled a measured TBARs levels were finally expressed depressed cardiac function with significantly as nmol/mg per total protein. reduced LVP (by 13%), +dP/dt (by 42%) and -dP/ dt (by 31%) in DAU group when compared to Data analysis controls (Table 2). This dysfunction persisted also under beta-adrenergic stimulation (Figure All variables are reported as mean and stan- 1). dard error of the mean (SEM). Data were com- pared by Mann-Whitney U test. Values P<0.05 Enhanced cell shortening in single cardiac were considered significant. The data were myocytes handled by GraphPad Prism 4 for Windows (GraphPad Software, Inc., version 4.00, San We examined whether the impaired cardiac Diego, California, USA). function could be due to deterioration in the

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Figure 3. Western blotting analysis of Ca2+ regulating proteins levels. A. Representative immunoblots of total protein level of ryanodine receptor (RyR2), sarco-endoplasmic reticulum ATP-ase 2a (SERCA2a), (CSQ), tria- din (TRD), FK506 binding protein (FKBP12) and phospholamban (PLB) from control (CON) and daunorubicin (DAU) group. B. Quantification of sarcoplasmic reticulum proteins. Data from DAU (grey columns) are normalized to CON (white columns) and are displayed as % of CON ± SEM, *P<0.05 vs CON, n = 7-9 per group.

Figure 4. Western blotting analysis of proteins linked to anthracycline-induced oxidative stress and cytoskeletal proteins. A. Representative immunoblots of total protein level of endothelial NO-synthase (eNOS), inducible NO- synthase (iNOS), heat shock protein 90 (hsp90), caveolin 3 and 1 (cav-3, cav-1), glycosylated subunit of the NADPH oxidase (gp91phox) and manganese superoxide dismutase (MnSOD) from control (CON) and daunorubicin (DAU) group. B. Representative immunoblots of total protein level of cytoskeletal proteins α-tubulin, α-actinin and β-actin from CON and DAU. C. The concentration of thiobarbituric acid reactive substances (TBARSs) in ventricular samples. Data are shown as mean ± SEM, n = 6 per group. function of isolated ventricular cardiomyocytes. nificant augmentation in the relative cell short- Interestingly, these experiments revealed a sig- ening at the stimulation frequency of 0.5 Hz by

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Table 3. Level of protein expression in left (gp91phox, MnSOD) and also proteins of nitric ventricular tissue determined by western blot oxide cascade (eNOS, iNOS, hsp90, cav-3, cav- CON DAU 1). We found no significant alterations in the NO-signalling proteins above mentioned proteins, except for an increased protein levels of cav-1 and cav-3 in eNOS 100±6 95±4 DAU (by 22% and 23%, resp., P<0.05 vs. CON; iNOS 100±16 84±9 Figure 4A, Table 3). Moreover, TBARS determi- hsp90 100±13 102±6 nation, to reveal possible lipid peroxidation, cav-1 100±8 122±5* also showed no significant changes between cav-3 100±10 123±8* DAU and CON group (Figure 4C). ROS regulating enzymes gp91phox 100±5 93±8 Initially, cytoskeletal proteins were used as MnSOD 100±9 100±15 loading controls as they showed stable expres- Cytoskeletal proteins sion (α-actinin and β-myosin). Interestingly, we found a significant decrease in protein expres- α-actinin 100±5 104±10 sion of α-tubulin by 46% in DAU ventricles as α-tubulin 100±9 54±8* compared to CON (P<0.05 vs. CON; Figure 4B). β-actin 100±7 105±4 Mean ± SEM (n = 7-9 per group; *P < 0.05 vs. CON). Higher mRNA expressions of RyR2, Myh7 and heart failure markers

33% in DAU group (Table 2, Figure 2A) with a We performed real-time PCR to determine significant reduction in the T50 by 16% as well mRNA expression of several gene groups as in the T90 by 18%. Similarly, we observed an including genes for calcium signalling, markers enhanced cell shortening in isolated cardiac related to heart failure, genes related to heart myocytes in the DAU group compared to CON at conduction system, components of renin- all other stimulation frequencies (Figure 2B). angiotensin system and thyroid hormone recep- The increased cell shortening in DAU cardio- tors (Table 3). In concert with increased protein myocytes as compared to CON persisted also level, mRNA levels of Ryr2 was also significantly under ISO stimulation (Figure 2C). However, higher in DAU group in compare to CON (by in contrast to CON, DAU cardiomyocytes failed 1.29 fold, P<0.05) with no significant differenc- to respond significantly to beta-adrenergic es in mRNA expression of other genes of Ca2+ stimulation. signalling (Cacna1c, Serca2a, Trpc3, and Trpc6). Gene expression analysis also revealed An upregulation of RyR2 but a stable expres- an increase in mRNAs of all observed heart fail- sion of other SR proteins ure markers (Acta1, Nppb, Nppa, Ctf1) in DAU group as compared to CON (P<0.05). Similarly, As cardiac excitation-contraction cycle is direct- mRNA levels of myosin heavy chain beta (Myh7, ly dependent on a proper cellular calcium han- Myh7b) were in DAU significantly upregulated dling, we investigated the protein levels of the against controls (4.34 fold and 1.57 fold, resp., 2+ 2+ main SR proteins involved in Ca -induced Ca P<0.05). In the DAU group, genes regulating release (Figure 3). Importantly, we found a sig- cardiac electrogenesis were also affected. We nificant, 94% increase of RyR2 expression in found an enhanced expression of Scn5a and DAU. However, this was not accompanied by Kcnq1 (Table 4). alterations in the expressions of RyR2 acces- sory proteins (TRD, CSQ and FKBP12) or of Discussion other tested Ca2+ regulating proteins (SERCA2a and PLB). Anthracyclines have undoubtedly a key role in the treatment of many neoplastic diseases, but Unaffected indicators of oxidative stress cardiotoxic effects limit their chemotherapeutic use. Although the mechanism of anthracycline- Because anthracyclines may activate oxidative induced toxicity is in all likelihood complex and stress, we determined the protein levels of consists of several factors [1], direct modula- several proteins involved in its signalling tion of cellular Ca2+ cycling [11, 12] emphasizes pathways such as ROS regulating enzymes the importance of this component for early

1288 Am J Transl Res 2015;7(7):1280-1294 Upregulation of RyR2 in early daunorubucin cardiomyopathy

Table 4. mRNA levels of selected genes using RT-PCR normalised to alterations in biomark- control group ers of oxidative stress). mRNA product DAU (fold of This indicates that RyR2 Gene group CON of PCR CON) is a key player in the early development of Cardiac myosin heavy chain isoforms Myh6 1.00±0.07 0.81±0.13 anthracycline car dio- Myh7 1.00±0.11 4.34±1.02* myopathy. Myh7b 1.00±0.10 1.57±0.15* Ryr2 1.00±0.09 1.29±0.08* Binding of anthra cycli- Cacna1c 1.00±0.09 1.15±0.07 nes and/or their metab- Serca2a 1.00±0.08 1.14±0.07 olites to SR proteins Trpc3 1.00±0.05 1.12±0.12 (RyR2, SERCA2a and CSQ) is a well known Trpc6 1.00±0.09 1.08±0.11 phenomenon [11, 12, Markers related to cardiac failure Acta1 1.00±0.23 1.46±0.17* 31]. In particular, their Nppb 1.00±0.14 2.18±0.42* binding to RyR2 favours Nppa 1.00±0.16 3.23±0.86* channel opening, lead- Ctf1 1.00±0.11 1.65±0.25* ing to increased cytosol- Conduction system of the heart Kcnq1 1.00±0.07 1.34±0.16* ic Ca2+ [32]. In this study Kcnh2 1.00±0.10 1.17±0.16 we showed that this Gja1 1.00±0.06 1.26±0.13 pharmacological feature causes alteration in Scn5a 1.00±0.03 1.30±0.08* expression of Ca2+ re- Renin-Angiotensin system Ace 1.00±0.07 1.15±0.21 lease channel and, con- Ace2 1.00±0.12 0.84±0.09 sequently, it is directly Agtr1 1.00±0.11 1.31±0.16 involved in early devel- Mas 1.00±0.23 0.97±0.43 opment of cardiomyopa- Thyroid hormone receptors Thrb 1.00±0.07 1.26±0.18 thy. As showed, concom- Thra1 1.00±0.06 1.11±0.12 itantly to alterations in Thra2 1.00±0.06 1.04±0.13 cardiac performance, For abbreviations see table 1. Mean ± SEM (n = 7-9 per group; *P < 0.05 vs. CON). exclusively the level of RyR2 increased follow- ing DAU treatment, cor- development of anthracyclines-induced dam- responding with a slight, but significant increase age. Indeed, several papers describe involve- of its mRNA levels. Presumably, this upregula- ment of reduced RyR2 expression in chronic tion of RyR2 can alter channel stoichiometry cardiac dysfunction) [26, 27, 30]. Some of them dramatically because the expressions of other suggested the influence of iron-dependent oxi- proteins in the complex formed by RyR2 (triadin dative stress in the RyR2 downregulation [27] and calsequenstrin) remained unchanged. that might be a consequence of reciprocal Changes in RyR2 complex stoichiometry could interaction between generation of reactive oxy- attenuate interactions among RyR2 accessory gen species and RyR2 opening [15]. As anthra- proteins, reducing the control of RyR2 from the cyclines directly and indirectly modulate RyR2 luminal site of SR, and consequently increasing function, we hypothesized that the cardiac tis- the open probability of the RyR2 and it thus sue would respond to repeated stimuli by may have detrimental consequences for car- immediately changing the channel expression. diomyocytes function by triggering abnormal Indeed, the main finding of our study is the two- Ca2+ release and/or Ca2+ leak [21, 22]. fold increase in cardiac RyR2 protein expres- sion accompanied by an exaggerated isolated Similarly, cardiac expression of FK506 binding cardiomyocytes function, concomitantly to in protein (FKBP12), a stabilizer of RyR2 [24] situ cardiac dysfunction, in the model of early which prevents aberrant activation of the chan- daunorubicin cardiomyopathy. Interestingly, the nel during the resting phase of the cardiac cycle upregulation of RyR2 was not accompanied by [33], also remained unaltered in our study. alterations in other Ca2+ regulating proteins Importantly, the absent compensatory upregu- (and was also not accompanied by significant lation of FKBP12 can lead to defective RyR2

1289 Am J Transl Res 2015;7(7):1280-1294 Upregulation of RyR2 in early daunorubucin cardiomyopathy function. Additionally, expression of SERCA2a [39]. On the other hand, studies using ²-MHC and its regulator phospholamban remained transgenic mice reported that shift from ±- to unaltered as well, suggesting that the possible ²-MHC may be rather maladaptive and pro- Ca2+ overload remained without compensatory motes cardiac disease progression presumably changes in expression of these proteins of SR. due to the decreased contractile performance Presumably, the unbalanced upregulation of of cardiac myocytes lasting also under beta- RyR2 suggests that several RyR2 units could adrenergic stimulation [40]. Thus, the absent be unbound to FKBP12, and not being correctly response to isoproterenol stimulation in DAU regulated on the surface of the SR. Thus, the cardiomyocytes could be associated with the associated persistent Ca2+ leak and conse- shift from ±- to ²-MHC. quent Ca2+ overload are the most plausible explanation of development of anthracycline With regard to the increased RyR2 expression cardiomyopathy in our study. with an abnormal cell shortening, we next fur- ther analyzed the proteins (sarco- Interestingly, isolated cardiomyocytes from meric skeleton protein α-actinin and microtubu- DAU hearts exhibited abnormal increase of cell lar protein α-tubulin) due to their undoubted shortening in our study. Although anthracy- role in normal myocyte function. Cytoskeletal clines typically lead to depressed cardiac func- transmit mechanical and chemi- tion in situ and in vivo [16, 18, 26], controver- cal stimuli within and between cells, contribute sial findings of increased performance of substantially to cell stability by anchoring sub- cardiac preparations were found also by others. cellular structures, modulate cardiomyocyte In vitro, anthracyclines cause both positive as function [41] and play a role in energy metabo- well as negative inotropic effect, likely depend- lism [42]. In this vein, disruption 2+ ing on concentration of used drug and/or was associated with an altered Ca handling experimental settings [17, 19, 20]. Presumably, [43] and a reduced myocyte stiffness with an the augmented cardiomyocytes shortening increment in sarcomere motion [44]. It has observed in our study might be an effect of been shown that anthracyclines reduce complex modulation of cardiomyocytes by both, β-tubulin [34]. Similarly, we found a reduction in immediate direct and/or indirect action of dau- α-tubulin protein content that could diminish norubicin on RyR2 as well as compensatory viscous intracellular load and reduce cardio- changes in RyR2 expression in the early stage myocytes stiffness [45] and help to enhance of cardiomyopathy. cell shortening and reduce times to myocyte relaxation. Furthermore, in association with In addition to an abnormal Ca2+ release, our increased RyR2 expression and upregulation of finding of an increased cell shortening might be β-MHC, the reduction in α-tubulin could contrib- related also to other observed alterations in our ute to the increased shortening of isolated cells left ventricular samples. In agreement with from DAU hearts. other studies using anthracyclines [34, 35], we In addition to the observed RyR2 upregulation, found an increase of β-myosin heavy chain we also detected elevated mRNA expression of (β-MHC) expression on mRNA level (Myh7, Scn5a (gene encoding Nav1.5, a sodium ion Myh7b genes) concomitantly with only non-sig- channel protein) and Kcnq1 (gene encoding nificantly reduced a-myosin heavy chain level Kv7.1, a potassium channel protein). These (±-MHC; Myh6 gene). These changes were inde- changes could contribute not only to abnormal pendent of changes in mRNA expressions of electrogenesis, manifesting as prolonged QT, thyroid hormone receptors [36] or components but also to an abnormal balance of key ions of RAAS axis [37]. ²-MHC is, in comparison to involved in the excitation-contraction coupling ±-MHC, characterized by a lower ATPase activi- and thus influencing also the process of cardi- ty and lower filament sliding velocity, but it can ac contraction [46, 47]. generate cross-bridge force with a higher econ- omy of energy consumption [38]. Therefore, a Importantly, we found a suspicious discrepancy shift to enhanced ²-MHC expression could lead between an enhanced function of ventricular to better preservation of intracellular energy cardiomyocytes in vitro and a depressed con- and could be viewed as a compensatory altera- tractility of the heart during closed-chest prep- tion tending to improve force-generating ability aration (before and after β-AR stimulation) in

1290 Am J Transl Res 2015;7(7):1280-1294 Upregulation of RyR2 in early daunorubucin cardiomyopathy the DAU group. We expect the involvement of satory changes in other Ca2+ regulatory pro- several possible mechanisms: i) structural teins, was identified as the responsible compo- abnormalities of ventricular tissue like expand- nent resulting into development of cardio- ed extracellular space, e.g. interstitial edema myopathy presumably via increased cytosolic [48] that could impair the Ca2+ overload, transiently stimulating muscle between myocytes, ii) compensatory systemic contraction, manifesting by enhanced cardio- neurohumoral activation/inhibition that is irrel- myocyte shortening in our study. Also, de- evant in isolated ventricular myocytes, and/or creased levels of α-tubulin can contribute to iii) loss of cardiomyocytes due to apoptosis or controversial performance by reducing cell necrosis [16, 49]. Importantly, expression of all stiffness and so facilitating cardiomyocyte observed genes related to the cardiac hypertro- shortening and, particularly, relaxation. Addi- phy and heart failure (Acta1, Nppb, Nppa, Ctf1) tionally, a compensatory upregulation of β-MHC were augmented in DAU group. Altogether, this could theoretically contribute to observed car- points to cardiac dysfunction with dilated car- diomyocytes function. However, whether the diomyopathy rather than hypertrophy, in agree- isoform shift from α- to β-MHC not only main- ment with other studies [50]. tains but could even increase the contraction amplitude must be proven in additional Generally, an increased oxidative (as well as studies. nitrosative) stress is believed to be a trigger mechanism for anthracycline cardiomyopathy Clinical implications [5, 51]. However, we found no alteration in pro- teins involved in activation of oxidative stress Despite tremendous research and testing of or pro/antioxidant factors in DAU group when thousands of potentially protective, predomi- compared to CON. Similarly, we did not nantly oxidative stress reducing agents, leading observed any alterations in lipid peroxidation to disappointing clinical results with classical using TBARS experiment between experimen- antioxidants, only a single drug has been tal groups. Lack of oxidative injury after anthra- approved for use in clinical practice: dexrazox- cyclines, in particular in acute studies, was ane [5]. According to the generally accepted reported also by others [52]. Supportingly, sin- hypothesis, this drug exerts its cardioprotective gle observed difference in NO signalling was effects by prevention of site-specific iron-cata- the slightly but significantly increased protein lyzed ROS production [54]. However, studies level of negative eNOS modulators - cav-1 and with chelators that are stronger and more cav-3 in DAU group. As well accepted, eNOS selective for iron yielded negative or, at best, may cause tissue damage by generating react- mixed outcomes suggesting that other mecha- ing nitrogen species what has been described nism may be better bases for designing also in case of anthracyclines medication [51]. approaches to achieve efficient and safe car- Uncoupling of eNOS by cav-1 deficiency has dioprotection [7]. In parallel, dexrazoxan che- been proposed for anthracycline-induced car- lates also other ions, including calcium [55] diomyopathy [53]. Contrariwise, upregulation of and an experimental study showed that dexra- caveolins will rather decrease eNOS activity. zoxan protection from chronic DAU cardiomy- This finding rather refuses the possibility of opathy was accompanied by prevention of nitrosative stress in hearts of DAU rats. chronic calcium overload of the myocardial tis- Altogether, our findings suggest that oxidative sue, without any significant effect on the myo- as well as nitrosative stress play rather a minor cardial level of iron [7]. In this context, also our role in early daunorubicine cardiomyopathy. findings highlight the prevention of calcium dys- regulation, particularly its overload, as the most Conclusion promising candidate approach for cardiopro- tection against anthracycline injury. Taking together, short-term DAU administration impaired the performance of the heart in situ Acknowledgements but, on the cellular level, single cardiomyocytes displayed an enhanced function. We identified Grant APVV-0887-11 Molecular aspects of drug three potential mechanisms responsible for induced heart failure and ventricular arrhyth- the exaggerated performance. In particular, in- mias from the Slovak Research and creased expression of RyR2, without compen- Development Agency, grants 1/0564/13 and

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1/0294/15 from the Science Grant Agency [9] Wagner R, Piler P, Gabbasov Z, Maruyama J, (VEGA), Slovak Republic and Grant of European Maruyama K, Nicovsky J, Kruzliak P. Adjuvant Regional Development Fund-Project FNUSA- cardioprotection in cardiac surgery: update. ICRC (No.CZ.1.05/1.1.00/02.0123) and the Biomed Res Int 2014; 2014: 808096. doi: grant of Slovak Society of Cardiology 2007. 10.1155/2014/808096. [10] Braunwald E. Heart failure. JACC Heart Fail Disclosure of conflict of interest 2013; 1: 1-20. [11] Hanna AD, Janczura M, Cho E, Dulhunty AF, Authors declare no conflict of interest. Beard NA. Multiple actions of the anthracycline daunorubicin on cardiac ryanodine receptors. Address correspondence to: Dr. Peter Kruzliak, In- Mol Pharmacol 2011; 80: 538-549. [12] Hanna AD, Lam A, Tham S, Dulhunty AF, Beard ternational Clinical Research Center, St. Anne’s Uni- NA. Adverse effects of doxorubicin and its met- versity Hospital and Masaryk University, Pekarska abolic product on cardiac RyR2 and SERCA2A. 53, 656 91 Brno, Czech Republic. Tel: +420 608 Mol Pharmacol 2014; 86: 438-449. 352 569; E-mail: [email protected] [13] Boucek RJ Jr, Dodd DA, Atkinson JB, Oquist N, Olson RD. Contractile failure in chronic doxoru- References bicin-induced cardiomyopathy. J Mol Cell Car- diol 1997; 29: 2631-2640. [1] Chen B, Peng X, Pentassuglia L, Lim CC, Saw- [14] Boucek RJ Jr, Miracle A, Anderson M, Engel- yer DB. Molecular and cellular mechanisms of man R, Atkinson J, Dodd DA. Persistent effects anthracycline cardiotoxicity. Cardiovasc Toxicol of doxorubicin on cardiac gene expression. J 2007; 7: 114-121. Mol Cell Cardiol 1999; 31: 1435-1446. [2] Roziakova L, Mistrik M, Batorova A, Kruzliak P, [15] Kim SY, Kim SJ, Kim BJ, Rah SY, Chung SM, Im Bojtarova E, Dubrava J, Gergel J, Mladosievi- MJ, Kim UH. Doxorubicin-induced reactive oxy- cova B. Can We Predict Clinical Cardiotoxicity gen species generation and intracellular Ca2+ with Cardiac Biomarkers in Patients After Hae- increase are reciprocally modulated in rat car- matopoietic Stem Cell Transplantation? Car- diomyocytes. Exp Mol Med 2006; 38: 535- diovasc Toxicol 2015; 15: 210-6. 545. [3] Georgakopoulos P, Roussou P, Matsakas E, [16] Soga M, Kamal FA, Watanabe K, Ma M, Palani- Karavidas A, Anagnostopoulos N, Marinakis T, yandi S, Prakash P, Veeraveedu P, Mito S, Kuni- Galanopoulos A, Georgiakodis F, Zimeras S, saki M, Tachikawa H, Kodama M, Aizawa Y. Ef- Kyriakidis M, Ahimastos A. Cardioprotective ef- fects of angiotensin II receptor blocker fect of metoprolol and enalapril in doxorubicin- (candesartan) in daunorubicin-induced cardio- treated lymphoma patients: a prospective, myopathic rats. Int J Cardiol 2006; 110: 378- parallel-group, randomized, controlled study 385. with 36-month follow-up. Am J Hematol 2010; [17] Matsushita T, Okamato M, Toyama J, Kodama 85: 894-896. I, Ito S, Fukutomi T, Suzuki S, Itoh M. Adriamy- [4] Lipshultz SE, Lipsitz SR, Sallan SE, Simbre VC cin causes dual inotropic effects through com- 2nd, Shaikh SL, Mone SM, Gelber RD, Colan plex modulation of myocardial Ca2+ handling. SD. Long-term enalapril therapy for left ven- Jpn Circ J 2000; 64: 65-71. tricular dysfunction in doxorubicin-treated sur- [18] Cernecka H, Ochodnicka-Mackovicova K, vivors of childhood cancer. J Clin Oncol 2002; Kucerova D, Kmecova J, Nemcekova V, Doka 20: 4517-4522. G, Kyselovic J, Krenek P, Ochodnicky P, Klimas [5] Berthiaume JM, Wallace KB. Adriamycin-in- J. Enalaprilat increases PPARbeta/delta ex- duced oxidative mitochondrial cardiotoxicity. pression, without influence on PPARalpha and Cell Biol Toxicol 2007; 23: 15-25. PPARgamma, and modulate cardiac function [6] van Dalen EC, Caron HN, Dickinson HO, Kre- in sub-acute model of daunorubicin-induced mer LC. Cardioprotective interventions for can- cardiomyopathy. Eur J Pharmacol 2013; 714: cer patients receiving anthracyclines. Co- 472-477. chrane Database Syst Rev 2005; CD003917. [19] Kelso EJ, Geraghty RF, McDermott BJ, Camer- [7] Simunek T, Sterba M, Popelova O, Adamcova on CH, Nicholls DP, Silke B. Characterisation of M, Hrdina R, Gersl V. Anthracycline-induced a cellular model of cardiomyopathy, in the rab- cardiotoxicity: overview of studies examining bit, produced by chronic administration of the the roles of oxidative stress and free cellular anthracycline, epirubicin. J Mol Cell Cardiol iron. Pharmacol Rep 2009; 61: 154-171. 1997; 29: 3385-3397. [8] Egom EE, Kruzliak P, Rotrekl V, Lei M. The ef- [20] Timolati F, Ott D, Pentassuglia L, Giraud MN, fect of the sphingosine-1-phosphate analogue Perriard JC, Suter TM, Zuppinger C. Neuregu- FTY720 on atrioventricular nodal tissue. J Cell lin-1 beta attenuates doxorubicin-induced al- Mol Med 2015 doi: 10.1111/jcmm.12549. terations of excitation-contraction coupling

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