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TITLE: S100A1 Genetically Targeted Therapy Reverses Dysfunction of Human Failing
Cardiomyocytes
AUTHORS: Henriette Brinks, MD, David Rohde, MD, Mirko Voelkers, MD, Gang Qiu, MD,
Sven T. Pleger, MD, Nicole Herzog, MS, Joseph Rabinowitz, PhD, Arjang Ruhparwar, MD,
Scott Silvestry, MD, Carolin Lerchenmüller, MD, Paul J. Mather, MD, Andrea D. Eckhart, PhD,
Hugo A. Katus, MD, Thierry Carrel, MD, Walter J. Koch, PhD, Patrick Most, MD
APPENDIX
Materials and Methods:
Human failing cardiomyocyte isolation and culture
Human ventricular myocardium was obtained from 23 patients with severe ischemic heart failure
undergoing orthotopic cardiac transplantation (mean echocardiographic EF 189%). Our
protocols were reviewed and approved by the Thomas Jefferson University Institutional Review
Board and University of Heidelberg ethics committee. Isolation of human failing left ventricular
(LV) cardiomyocytes was performed as previously described (1-2). Briefly, coronary arteries of
explanted hearts were immediately perfused with cold, Ca2+-free Krebs-Henseleit (KH) solution
containing (in mmol/L) glucose 12.5, KCl 5.4, lactic acid 1, MgSO4 1.2, NaCl 130, NaH2PO4
1.2, NaHCO3 25, and sodium pyruvate 2 (pH 7.4, with NaOH). In the laboratory, a small catheter
was placed into the lumen of an artery that supplied a non-infarcted free-wall region of the LV.
The perfused myocardial segment was cut from the heart and rinsed for 30 minutes with a
nonrecirculating KH solution containing 10 mmol/L taurine. The tissue was then perfused for 30
minutes with 200 mL of KH containing 180 U/mL collagenase, 20 mmol/L 2,3-butanedione
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monoxime (BDM), 20 mmol/L taurine, and 0.05 mmol/L CaCl2. This solution was recirculated.
The collagenase-containing solution was washed from the tissue for 10 minutes with 500 mL KH
containing 10 mmol/L taurine, 20 mmol/L BDM, and 0.2 mmol/L CaCl2. The cardiac tissue was
then removed from the cannula, and only mid-myocardial tissue was minced. The resulting cell
suspension was filtered, centrifuged (25g for 1 minute), and resuspended in a KH solution (200
mL KH, 1% weight/volume BSA, 10 mmol/L taurine, and 0.25 mmol/L CaCl 2). All solutions
were equilibrated with 95% O2 and 5% CO2. The temperature was kept at 37°C throughout the
isolation. Initial yields of rod-shaped cells were between 7% and 50%. Cells were subjected to
Ca2+ toleration as described (1-2) and further cultured in BDM-free M199. Cultured cells were
transfected either with S100A1 adenovirus (Ad-S100A1) or control GFP (Ad-control) virus at a
multiplicity of infection of 5 and subjected to 0.2 Hz electrical field-stimulation for 24 hours
employing the C-Pace cell culture stimulator (IonOptix). Generation of the adenovirus has been
described in details elsewhere (3). All experiments were conducted within 30 hours of cell
isolation.
RNA isolation, reverse transcription and semiquantitative real-time PCR
Total RNA isolation from LV tissue samples was performed applying the TRIZOL method,
according to the manufacturer’s protocol (Invitrogen) as previously described (4). Human total
RNA control samples (n=7) from non-failing left ventricular tissue from adult males (age
ranging from 20 to 30 years) were purchased from Biocat (Heidelberg, Germany, Cat.#
R1234138-CDP, Lot. No. A809128, A809126, A809122, A809121, A809125, A809141,
A804068). Quality of RNA was assessed by running an aliquot on a denaturing agarose (1%)
gel. First strand cDNA synthesis from 1µg of total RNA was carried using iScript cDNA
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Synthesis Kit (BioRad). For quantitative PCR, 5 µl of diluted cDNA (1/100) was added to a 25
µl mixture that contained a 1 concentration of iQ SYBR Green Supermix (BioRad) and 100 nM
of gene-specific oligonucleotides. Subsequently, quantitative PCR was carried out on a MyiQ
Single-Color Real-Time PCR detection system (BioRad) for human 18s, brain natriuretic
peptide (BNP), sarcoplasmic reticulum calcium atp-ase 2 (serca 2), sodium-calcium exchanger
(ncx) and s100a1 expression levels as indicated. Sequences of gene-specific oligonucleotide
primers - based on cDNA sequences in the National Center for Biotechnology database - were
generated by the use of PRIMER3 software and available upon request. 18s rRNA that were not
different between groups were used for normalization. After each run, saturation of each
amplification cycle was controlled by the use of MyiQ software (version 1.0) and, subsequently a
melting curve acquired by heating the product to 95°C, cooling to and maintaining at 55°C for 20
seconds, then slowly (0.5°C/s) heating to 95°C was used to determine the specificity of the PCR
products, which were then confirmed by gel electrophoresis.
Immunoblotting
Western blotting was performed as previously reported (5) to assess cardiac protein levels of
S100A1 (SA5632; custom-made by Eurogentec; 1:10.000), calsequestrin (CSQ) (208915;
Calbiochem; 1:10.000), PLB (05-205; Upstate; 1:50.000), phospho-PLB-serine 16 (07-052;
Upstate; 1:10.000), troponin-I (#4002, Cell Signaling, 1:2000), troponin-I-serine 23/24 (#4004,
Cell Signaling, 1:2000). Human total protein extracts (n=4) from non-failing left ventricular
tissue were purchased from Sigma (T5445), Chemicon (CL304-250UG) and Clontech (Cat.No.
635302). Protein content was quantified with the BioRad DC Protein Assay (BioRad
Laboratories, Richmond, CA). After separation of protein samples (50µg per lane) by 4-20%
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SDS-PAGE (Invitrogen Corporation, Carlsbad, CA), proteins were transferred to PVDF
membrane and probed with an appropriate primary antibody at 4°C overnight. After staining
with a corresponding pair of Alexa Fluor 680- (Molecular Probes; 1:20.000) and IRDye 800CWcoupled (Rockland Inc.; 1:20.000) secondary antibodies, respectively, proteins were visualized
with a LI-COR infrared imager (Odyssey), and quantitative densitometric analysis was
performed applying Odyssey version 1.2 infrared imaging software. Signals were normalized to
CSQ densitometric levels that were not different between groups. Comparability between gels
was obtained with data from 1 sample that was run on every gel.
Immunoprecipitation
RyR2 and SERCA2 immunoprecipitation from cardiac SR vesicle preparations was carried out
as previously described (6) to determine S100A1 protein interaction with RyR2 and SERCA2.
All steps were carried out on ice. SR vesicle preparations were diluted in non-denaturing NP40Ca2+ detergent buffer (1% Nonidet-P-40, 10% glycerol, 135mM NaCl, 1mM CaCl2, 20mM TRIS
HCl pH7.4) to 1µg/1µl protein and rotated with bovine serum albumin-treated A/G-PLUSAgarose (20 l/500l; Santa Cruz Biotechnology sc-2003) for 4 hours and centrifuged at 13,000
rcf for 15 min at 4C to remove protein nonspecifically bound to A/G-Sepharose. The
supernatants were then either mixed with precipitating antibodies for RyR2 (ABR #MA3-925,
5l/500g protein) or SERCA2 (Santa Cruz Biotechnology, sc-8094, 10l/500g protein) and
rotated for 8 hours at 4°C. Control experiments were concurrently performed with a mouse IgG1
isotype control antibody (Imgenex # 20109) for the monoclonal IgG1 anti-RyR2 antibody (ABR
#MA3-925) and preincubation of the polyclonal IgG anti-SERCA2 antibody (sc-8094) with the
neutralizing control blocking peptide (sc-8094P) as outlined in the manufacturer protocol. Again,
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A/G-PLUS-Agarose (sc-2003, Santa Cruz Biotechnology) was added and samples were rotated
for additional 4 hours and centrifuged (13,000 rcf, room temperature for 15 min). Pellets were
washed three times with lysis buffer and 50l lysis buffer supplemented with mercaptoethanol
(2% v/v) was added. Samples were heated at 95C for 1 min and centrifuged (800 rcf, room
temperature for 15 min) and 45l of the supernatant were transferred in a new Eppendorf vial
without aspirating the pelleted beads. 45l of loading buffer were added and the sample divided
in two parts that were instantly resolved by SDS-PAGE applying a 4-20% Tris Glycine gel,
transferred to a PVDF, and stained for co-precipitating S100A1 (Acris #SP5355P, 1:1000) and
immunoprecipitated RyR2 (ABR #MA3-916; 1:1000) and SERCA2 (Santa Cruz Biotechnology,
sc-8905; 1:1000). Co-precipitated S100A1 protein levels were normalized to corresponding
RyR2 and SERCA2 signals.
Cardiomyocyte contractility
Contractile properties of isolated ventricular cardiomyocytes were obtained by video-edgedetection as described previously (3). Analysis of in vitro transfected cardiomyocytes was
carried out immediately after isolation and 24 hours following adenoviral gene delivery,
respectively. Analysis of steady state twitches at 2 Hz electrical field stimulation, 37°C and 2
mM extracellular Ca2+-concentration ([Ca2+]e) was performed by custom designed software
written in LabView (version 5.0, National Instruments).
Cardiac SR vesicle preparations and SR flux measurements
SR vesicles from human failing myocardium and microsomal fractions of control and S100A1expressing failing cardiomyocytes were carried out as previously described (4,7). SR Ca2+ uptake
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and SR Ca2+ leak was assessed using fluorescent Ca2+ indicator Fluo-3 salt, as described
previously, by the use of a heated spectrophotometer. Briefly, for Ca2+ uptake measurements, SR
vesicles (0.3 mg/ml) were incubated in 0.2 ml of reaction solution (RS) containing 0.15 mM
potassium gluconate, 1 mM MgCl2, 0.2 mM EGTA-Ca2+ buffer (free [Ca2+] 0.3µM); free [Ca2+]
was calculated by the use of MAX Chelator (www.stanford.edu/~cpatton/maxc.html), 10 mM
NaN3, 20 mM MOPS, pH 7.0, and 10 µM ruthenium red was added to inhibit RyR2 opening.
The solution was heated to 37°C, baseline fluorescence was acquired and Ca2+ uptake was
initiated by the addition of 0.5 mM ATP into the well, and the time course of fluorescence
decline representing SR Ca2+ uptake was monitored spectrophotometrically with Fluo-3 as a Ca2+
indicator. For SR Ca2+ leak measurements, passively Ca2+ preloaded SR vesicles were incubated
in 0.2 ml RS containing 0.15 µM free [Ca2+], 1 µM thapsigargin was added to inhibit SR Ca2+
ATPase activity and the resultant fluorescence increase representing SR Ca2+ leak was
monitored. Human recombinant S100A1 protein was generated as described in detail elsewhere
(3,8) and added at indicated concentration. SR vesicle Ca2+-ATPase activity assays were carried
out according to Hajjar et al. (9) based on pyruvate/NADH coupled reactions. With a photometer
(Beckman DU 640) adjusted at a wavelength of 340 nm, oxidation of NADH in the absence and
presence of thapsigargin (10 µM) was assessed at 37°C in homogenates by the difference of the
total and basal absorbance to specifically determine SERCA2 activity. The reaction was carried
out in a cuvette volume of 1 mL with pCa 6.2 and all experiments were carried out in triplicate.
The rate of ATP hydrolysis (nmol ATP/mg SR protein × min) was estimated from the equation:
ATP hydrolysis = ΔOD340nm / Δt × ε × L × S. ΔOD is the decrease in absorbance at 340 nm
(due to NADH consumption) during the interval Δt (in min), ε is the NADH extinction
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coefficient (6.22 × 106 ml × mol-1 ×·cm-1), L the cuvette length (in cm) and S the amount of SR
protein added to the cuvette (mg/ml).
Cardiomyocyte calcium handling and SR calcium content
Calibration and assessment of intracellular Ca2+-transients and SR Ca2+-load in field-stimulated
ventricular cardiomyocytes was performed as described elsewhere (4,7). Assessment of Ca2+handling properties in isolated, FURA-2AM loaded in vitro transfected cardiomyocytes was
carried out immediately after isolation and 24 hours following adenoviral gene delivery,
respectively. Steady state transients at 2 Hz electrical field stimulation, 37°C and 2 mM [Ca2+]e
were analyzed by T.I.L.L Vision software (version 4.01). Diastolic SR Ca2+-leak in fieldstimulated HFCMs was measured by a previously published protocol by Eisner and co-workers
exposing cells to a combination of isoproterenol (10-7 M) and caffeine (0.5 mM) to provoke
arrhythmogenic SR Ca2+ leak (14). Threshold for detection of diastolic SR Ca2+-waves was set to
an amplitude exceeding 5% of the amplitude of the regular Ca2+ transient.
ATP and phosphocreatine measurement
Enzymatic assays for phosphocreatine (PC) and ATP content in HFCM extracts were performed
as described in detail elsewhere (15).
Isolation of human mitochondria and assessment of mitochondrial function
Intact and vital mitochondria both from cultured human failing control and S100A1 transfected
cardiomyocytes as well as human failing myocardial tissue were isolated using the Miltenyi
Biotec (130-094-532) human mitochondrial isolation kit. Briefly, following the manufacturer’s
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protocol, cells and tissue were lysed on ice employing buffers and reagents provided with the kit
and subsequently labeled with anti-TOM22 magnetic micro beads specifically binding to the
translocase of outer mitochondrial membrane 22 (TOM22). Then, labeled cell and tissue lysates
were loaded onto a MACS column placed in the magnetic field of a MACS separator where
magnetically labeled mitochondria were retained in the column matrix while unlabeled
organelles and cell components ran through. After washing, the columns were removed from the
magnetic field and mitochondria were eluted to immediate experimental use. Purity of isolated
mitochondria was confirmed by Western blotting for nuclear, sarcomeric and SR marker proteins
(Figure 4). Determination of mitochondrial permeability transition (swelling), mitochondrial
electron flow (MTT reduction assay), and membrane potential (rhodamine 123 fluorescence) was
carried out as previously published (10) and described below.
Determination of mitochondrial swelling
Mitochondrial swelling (MPT) was carried out according to Nieminen et al. (2008) with some
modifications (11). Mitochondrial pellet was suspended in swelling buffer (0.15 M KCl, 0.02 M
Tris, pH 7.4). Dilution was also carried out in the same buffer. Increase in mitochondrial volume
(swelling) was estimated by measuring 90° light scattering at 520 nm using Ultrospec 3100pro
(Amersham Biosciences, Sweden) UV/visible spectrophotometer at 25 °C. 2 mg protein/ml was
used in the 2 ml assay system. The swelling was recorded as decrease in the absorbance for a
period of 10 min at 30 s time interval. Percent swelling was calculated with respect to phosphate
(Pi) induced swelling (Pi = 100% swelling) by addition of 2 mM KH2PO4. Calcium overload was
simulated in swelling buffer containing 100 µM CaCl2.
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Determination of mitochondrial electron flow (MTT reduction assay)
Experiments were carried out according to Cohan and Kesler (1999) with some modifications
(12). The assay is based on the amount of water soluble, yellow colored MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) which is reduced to insoluble, purple
colored formazan crystals by mitochondrial succinate dehydrogenase (complex II) and was
measured spectrophotometrically by ELISA plate reader (Synergy HT, Biotek, USA) at 592 nm.
Flow cytometric analysis of mitochondrial membrane potential (ΔΨm)
Flow cytometer (BD-LSR; Becton, Dickinson and Company) was used to measure ΔΨm using
Rhodamine 123 (Rh123), which is a fluorescent dye, and its lipophilic cation accumulates inside
mitochondria in proportion to its membrane potential. The dye binds to the inner and outer sides
of the inner mitochondrial membrane and, as a result, is accumulated by mitochondria in a
greater quantity and released upon membrane depolarization (13). Mitochondrial samples
(control and experimental) were incubated in the dark for 30 min at room temperature with
5 μg/ml of dye. Signals were assessed using FL-1 channel (590 band pass) settings on flow
cytometer and data analyzed on Cell Quest software. Signals were obtained using corresponding
band pass filters. Each determination is based on mean fluorescence intensity of 10,000 events.
Statistical analysis
Results are presented as meanSEM. Results are presented as meanSEM. Comparisons
between treatment groups were made using unpaired 2-tailed Student t test and analysis of
variance (with repeated measures when necessary) followed by the Student-Newman-Keuls
method for post hoc analysis. For all tests, a probability value < 0.05 was considered significant.
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Fisher’s exact test was used to compare incidence of diastolic Ca2+ waves between groups by
comparing paced cardiomyocytes without to cardiomyocytes with diastolic Ca2+ waves. The
authors had full access to the data and take full responsibility for its integrity. All authors have
read and agreed to the manuscript as written.
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