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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)
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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.
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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;
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Hauswirth WW, Lewin AS, Zolotukhin S, Muzyczka N. Production and purification of
recombinant adeno-associated virus. Methods Enzymol 2000; 316: 743–761.
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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.
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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.
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Dubielzig R, King JA, Weger S, Kern A, Kleinschmidr JA. Adeno-associated virus type 2
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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.
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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.
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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.
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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.
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Ruitenbeek W, Sengers RCA, Trijbels JMF, Stadhouders AM, Janssen AJM. Estimation of
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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.