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Online-Only supplement ONLINE-ONLY MATERIALS AND METHODS All animal procedures and experiments were performed in accordance with the “Guide for the Care and Use of Laboratory Animals” (NIH) and approved by the local “Animal Care and Use Committee” of Baden-Württemberg. Model of post-ischemic heart failure. We employed a model of post-ischemic heart failure (HF) which was previously described and characterized in detail.1,2 HF was induced in german farm pigs (Bräunling; mean body weight: 30±4 kg) by temporary (2 hours) occlusion of the very proximal left circumflex coronary artery (LCX) with a percutaneous transluminal coronary angioplasty balloon (Boston Scientific). AAV vector production and vector titration. pdsAAVCMV-MLC0.26-S100A1 was obtained by replacing the EGFP-cDNA of pdsAAVCMV-MLC0.26-EGFP with a BamHI/BsrGI fragment of the S100A1 cDNA, amplified by linker PCR from pGemex-S100A1.1,3 High titer vectors were produced with a triple transfection approach of 293T cells in cell stacks® (Corning, Munich, Germany) as described before.4-6 For production of AAV6-S100A1, p5E18-VD2-9 providing the AAV-6 cap sequence, and pDGdelVP containing adenoviral helper sequences, were cotransfected with pdsAAVCMV-MLC0.26-S100A1.7,8 Subsequently, vectors were harvested after 48h, purified by filtration cascade (1st 5µm, 2nd 0.8µm, 3rd 0.2µm) and Iodixanol step gradient centrifugation, and quantitated using real-time PCR as reported before.4,9 AAV6-luciferase control vectors were produced analogous.1,3 Efficient myocardial S100A1 overexpression by catheter-based cardiac gene delivery. Myocardial gene transfer in HF pigs (2 weeks post MI) was achieved by selective retroinfusion as described with some modifications.1,10 Briefly, a 7-F balloon wedge pressure catheter (Arrow) was placed in the anterior cardiac vein (ACV). While the left anterior descending coronary artery (LAD) was occluded distal of the first diagonal branch using a 3.5x8mm balloon, 3 times each for 3 minutes, to stop antegrade blood flow, 1.5x1013 tvp of the AAV6 construct in 50 cc saline were retrogradely injected into ACV. Afterwards, the integrity of the LAD was checked by angiography.1 Regional western blot analysis. To investigate the regional S100A1 expression pattern in the pig heart after AAV6-S100A1 and AAV9-S100A1 gene delivery, we analyzed S100A1 protein expression in 36 myocardial segments. At first, the heart was divided into a.) apical, b.) midventricular and c.) basal left ventricular rings. Every ring was further divided into an a.) anterior, b.) septal and c.) posterior area. The lateral area corresponded to the myocardial scar tissue after the LCX occlusion and was not investigated. Because of the large size of the pig heart every area was further divided into 4 segments (final size ~ 3x1.5 cm). Western blots were performed as described previously.11 Samples of myocardium were homogenized at 4°C in 3 w/v PBS with 5 mM EDTA, 5 mM EGTA and protease inhibitor mixture (1836170, complete Mini EDTA free, Roche Diagnostics GmbH, Germany) and centrifuged at 15.000 g for 15 min. Supernatant protein was subjected to electrophoresis, transferred to a PVDF membrane, and probed with anti-S100A1-Ab (SA 5632, 1:1000, custom-made; detecting the human and the pig isoform of S100A1), anti-RyR2 (Thermo Scientific, MA3-916, 1:1000), anti-p-RyR2 ser2808 (Badrilla, A010-30, 1:5000), anti-SERCA2a (Santa Cruz, sc-8095, 1:1500), anti-calsequestrin (Thermo Scientific, PA1-913, 1:1500), anti-PLN (Thermo Scientific, MA3-922, 1:5000), anti-p-PLN Thr 17 (Badrilla, A010-13, 1:5000), anti-p-PLN Ser 16 (Upstate, 07-052, 1:5000). In addition to Bradford analysis probing against mouse anti-GAPDH-AB (Milipore; MAB374, 1:1000) was used to control equal loading. Corresponding secondary antibodies coupled to fluorescent 680 or 800 dyes were used at concentrations 1:2000. For cardic SR isolation, samples of myocardium were homogenized at 4°C in 3 w/v 0,3M sucrose buffer with 10mM imidazole, 30mM histidine, pH7.0, phosphtase inhibitor (P5726, Phosphatase Inhibitor Cocktail 2, P0044, Phosphatase Inhibitor Cocktail 3, Sigma-Aldrich, USA) and protease inhibitor mixture (1836170, complete Mini EDTA free, Roche Diagnostics GmbH, Germany) and centrifuged at 8.000 g for 20 min. Supernatant protein was centrifuged at 45.000g for 90min. The pellet was resuspended in 1 vol of precipitation buffer (same as homogenisation buffer above but also containing 0,6M KCl) and centrifuged at 2000g for 20min. The supernatant was centrifuged at 45.000g for 90min, the pellet resuspended in PBS and used for western blot analysis. Real-time RT-PCR and AAV-S100A1 biodistribution. Genomic DNA and total RNA from myocardial samples as well as from liver, lung, skeletal muscle and brain was isolated as described.11,12 Real-time RT-PCR was performed in duplicates with a 1:100 dilution of the cDNA on a MyIQ real time PCR detection system (BioRad) with the SYBR Green PCR master mix (BioRad). The following GenBank cDNA sequences were used to design oligonucleotide primers to examine expression of porcine BNP (M23596, forward primer 5`-cccgcagtagcatcttcca-3`, reverse primer 5`-ttgctttgaaggggagcag-3`), human S100A1 (NM_006271.1, forward primer 5´-cgatggagaccctcatcaa-3, reverse primer 5´tggaagtccacctccccgtc-3´, porcine sodium/calcium exchanger (NCX) (NM_021097, forward primer 5´gcattggcatcatggaggtgaa-3´, reverse primer 5´-ttgctggtcagtggctgcttgt-3´), porcine ryanodine receptor (RyR2) (XM_001924768.4, forward primer 5´-actggcctttgatgttggct-3´, reverse primer 5´- agatacccttgggctgcttc-3´), porcine SERCA2a (NM_213865.1, forward primer 5´-tgtcactccacttcctaatcc3´, reverse primer 5´-actccagtattgcaggttcc-3´). For normalization, 18S rRNA was used (forward primer 5`-tcaagaacgaaagtcggagg-3`, reverse primer 5`-ggacatctaagggcatcac-3`). Since S100A1 protein was expressed under control of a cardio-selective promoter, AAV-biodistribution was analyzed using isolated genomic DNA in order to investigate infection of non-cardiac organs by the AAV6-S100A1 constructs. PCR conditions were 95ºC, 3 min, and 40 cycles of 95ºC, 10 sec; 60.5ºC, 45 sec. Specificity of PCR products were confirmed by gel electrophoresis. Analyzes of F1-ATPase activity.Adenosine triphosphate (ATP) concentration was determined as described previously.13 In brief, the assays couples hexokinase and glucose 6-phosphate dehydrogenase reaction yealding NADPH. Reduction of NADP was monitored at λ = 320–400 nm and 25 °C. ATP content of samples was calculated using a calibration curve with defined ATP concentrations. Measurement of left ventricular function by cardiac catheterization and echocardiography. At the day of cardiac gene transfer (2 weeks after MI) and at the end of the study (14 weeks after MI) we performed echocardiography and LV catheter-based hemodynamic measurements with a Philips Sonos 5500 imaging system (Philips) and a 5-F pressure catheter (SPC-350; Millar instruments) as previously described.1 The echocardiographer and the examiner in the catheter laboratory were blinded for the individual pigs. Occurrence of monomorphic ventricular tachyarrhythmia (MVT) by right ventricular stimulation in pigs with heart failure. Electrophysiologogical (EP) examination using right ventricular (RV) stimulation (RVS) was performed 14 weeks after myocardial AAV6-S100A1 gene delivery using the Universal Heart Stimulator (UHS) 20 (Biotronic, Berlin) and a bipolar stimulation catheter which was placed into the RV via the jugular vein. In order to investigate the inducibility of monomorphic ventricular tachyarrhythmia (MVT), RVS with a fixed basic cycle length starting at 350 ms, followed by a single premature programmed stimulus, was applied. The coupling interval between the last basic stimulus and the premature stimulus was decreased in 10-ms steps. According to clinical protocols the fixed basic cycle length was further shortened to 300 ms. Afterwards a second and third premature stimulus was coupled until MVT was induced. After 32 steps the RVS protocol ended and the pig was declared “not inducible” in case MVT could not be provoked. A detailed description of the RVS protocol is given in the “online-only” section (online-only Table 3). Six-lead electrocardiogram (ecg) was recorded and heart rate as well as PQ-, QRS- and QTc–intervals were measured according to standard clinical protocols. Isolation and primary culture of neonatal rat cardiomyocytes and calcium transient measurements. Ventricular cardiomyocytes from 1- to 2-day-old neonatal hearts were isolated and intracellular calcium cycling was analyzed as described in detail elsewhere.14 REFERENCES (online-only) 1. Pleger ST, Shan C, Ksienzyk J, Bekeredjian R, Boekstegers P, Hinkel R et al. Cardiac AAV9-S100A1 gene therapy rescues post-ischemic heart failure in a preclinical large animal model. Sci Transl Med 2011; 3(92): 92ra64. 2. Raake PW, Schlegel, P, Ksienzyk, J, Reinkober J, Barthelmes J, Schinkel S et al. AAV6.βARKct cardiac gene therapy ameliorates cardiac function and normalizes the catecholaminergic axis in a clinically relevant large animal heart failure model. Eur Heart J 2013; 34(19): 1437-47. 3. Müller OJ, Schinkel S, Kleinschmidt JA, Katus HA, Bekeredjian R. Augmentation of AAVmediated cardiac gene transfer after systemic administration in adult rats. Gene Therapy 2008; 15: 1558–1565. 4. Hauswirth WW, Lewin AS, Zolotukhin S, Muzyczka N. Production and purification of recombinant adeno-associated virus. Methods Enzymol 2000; 316: 743–761. 5. Müller OJ, Leuchs B, Pleger ST, Grimm D, Franz WM, Katus HA et al. Improved cardiac gene transfer by transcriptional and transductional targeting of adeno-associated viral vectors. Cardiovasc Res 2006; 70: 70-78. 6. Reed SE, Staley EM, Mayginnes JP, Pintel DJ, Tullis GE. Transfection of mammalian cells using linear polyethylenimine is a simple and effective means of producing recombinant adeno-associated virus vectors. J Virol Methods 2006; 138:(1-2), 85-98. 7. Dubielzig R, King JA, Weger S, Kern A, Kleinschmidr JA. Adeno-associated virus type 2 protein interactions: formation of pre-encapsidation complexes. J Virol 1999; 73: 8989–8998. 8. Gao G, Vandenberghe LH, Alvira MR, Lu Y, Calcedo R, Zhou X et al. Clades of adenoassociated viruses are widely disseminated in human tissues. Virol 2004; 78: 6381–6388. 9. Veldwijk MR, Topaly J, Laufs S, Hengge UR, Wenz F, Zeller WJ et al. Development and optimization of a real-time quantitative PCR-based method for the titration of AAV-2 vector stocks. Mol Ther 2002; 6: 272–278. 10. Degenfeld G, Raake P, Kupatt C, Lebherz C, Hinkel R, Gildehaus FJ et al. Selective pressureregulated retroinfusion of fibroblast growth factor-2 into the coronary vein enhances regional myocardial blood flow and function in pigs with chronic myocardial ischemia. J Am Coll Cardiol 2003; 42: 1120-1128. 11. Pleger ST, Remppis A, Heidt B, Völkers M, Chuprun JK, Kuhn M et al. S100A1 gene therapy preserves in vivo cardiac function after myocardial infarction. Mol Ther 2005; 12: 1120-1129. 12. Most P, Pleger ST, Völkers M, Heidt B, Boerries M, Weichenhan D et al. Cardiac adenoviral S100A1 gene transfer rescues failing myocardium. J Clin Invest 2004; 114: 1550-1563. 13. Ruitenbeek W, Sengers RCA, Trijbels JMF, Stadhouders AM, Janssen AJM. Estimation of energy metabolism in human skeletal muscle homogenate as a diagnostic aid. J Inherited Metab Dis 1981; 4: 91–92. 14. Most P, Boerries M, Eicher C, Schweda C, Volkers M, Wedel T et al. Distinct subcellular location of the Ca2_-binding protein S100A1 differentially modulates Ca2+-cycling in ventricular rat cardiomyocytes. Cell Sci 2005; 118: 421–431. LEGENDS (online-only) Figure 6 (online-only). RT-PCR analysis of representative proteins involved in intracellular Ca2+-Cycling. (A and B) High-level myocardial S100A1 overexpression does not alter expression of the ryanodine receptor (RyR2) as well as the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a). (C) Expression of the sodium/calcium exchanger (NCX) was significantly increased in HF controls as compared to the non-failing sham group, while high-level AAV9/S100A1 gene therapy normalized myocardial NCX expression in HF. * P<0.05 vs. HF-Saline or HF-AAV6-Luc. n=5. Data are presented as mean±SEM. Figure 7 (online-only). Dose-dependent effects of S100A1 on intracellular Ca2+-transients in isolated neonatal cardiomyocytes. (A) Infection of isolated neonatal cardiomyocytes (NRCM) with increasing dosages of AAV6-S100A1 resulted in increasing S100A1 protein expression levels. Low(4±1.3-fold) and moderate (10±2.3-fold) S100A1 protein overexpression was achieved by an MOI of 2x104 or MOI of 2x105 while extreme (45±8.2-fold) S100A1 overexpression was achieved by an MOI of 2x106. (B) Low and moderate S100A1 protein overexpression in NRCM resulted in significantly increased intracellular Ca2+-transient amplitudes as compared to adequate AAV6-Luc controls while extreme S100A1 overexpression abrogated this effect. *; P<0.05 vs. sham, AAV6-S100A1 (MOI: 2x106) and AAV6-Luc- controls. #; P<0.05 vs. all groups. n=25, 3 individual experiments. Data are presented as mean±SEM.