Experimental design

Aim of this study was to explore Cx43 gene therapy in the myocardial scar as means to reduce post-infarct VT incidence. For this purpose we tested first the lentivirus (lentivirus-EGFP or lentivirus-Cx43-IRES-EGFP) constructs in regard to the expression of functional Cx43 gap junctions in vitro and also in vivo using grafting of ex vivo transduced SkM. Next, we established a surgical protocol for direct lentiviral transduction of resident cells in the infarct area 2–3 days after the lesion. Electrical vulnerability was assessed at 2 or 8 weeks after the primary surgery by in vivo electrophysiological testing. For the analysis of conduction velocity ex vivo voltage mapping (2 weeks postoperatively) was performed. Cardiac function was evaluated by left ventricular catheterization or echocardiography, following functional experiments the hearts were harvested and further processed (see also Fig. 2a and Suppl. Fig. 1a).

Virus generation

For production of self-inactivating (SIN) lentiviral vectors (LVs), HEK293T (human embryonal kidney cells) helper cells were co-transfected with the lentiviral plasmid and the packaging plasmids pMDLg/pRRE, RSV-rev43, and pMD2.G44. Recombinant replication deficient LVs of the 3rd generation were purified from cell culture supernatant using ultracentrifugation as described elsewhere45. Briefly, producer cells were seeded on poly-L-Lysine coated cell culture dishes in DMEM (Invitrogen, Darmstadt, Germany), supplemented with 10% fetal calf serum (Biochrom, Berlin, Germany), 100 U/ml penicillin/100 μg/ml streptomycin (Pen/Strep; Biochrom, Berlin, Germany) and incubated at 10% CO 2 and 37 °C. Cells were transfected at ~50% confluency by calcium phosphate transfection and subsequently incubated at 3% CO 2 and 37 °C overnight. The medium was changed the next day and cells were cultured again at 10% CO 2 and 37 °C. For purification of the vesicular stomatitis virus glycoprotein G pseudotyped LVs, cell culture supernatant was collected one day after medium change. Cells were incubated again with fresh medium and virus containing supernatant was collected again on the next day. The supernatants were filtered using a bottle-top filter (SFCA, 0.45 μm, Nalgene, Thermo Fisher Scientific, Waltham, MA, USA) to remove cell debris, transferred to centrifugation tubes and centrifuged in an ultracentrifuge with SW32Ti rotor (Optima L-100 XP, Beckman Coulter Incorporated, Brea, CA, USA) for two hours at 19,400 rpm and 17 °C, respectively. The virus pellets were re-suspended in HBSS (Invitrogen, Darmstadt, Germany) and virus from the first harvest was stored overnight at 4 °C to be combined with the virus from the second harvest. The combined pre-concentrated virus suspension was layered on top of a 20% (w/v) sucrose cushion and ultracentrifuged in a SW55 rotor (Optima L-100 XP, Beckman Coulter Incorporated, Brea, CA, USA) for two hours at 21,000 rpm and 17 °C. The LV pellet was resuspended in HBSS and vortexed for 45 min at 1,400 rpm and 16 °C. After a short spin down of debris for 3 s at 16,000 g the opaque supernatant was aliquoted and LV aliquots were stored at −80 °C.

The original lentiviral plasmids were derived from the lab of Inder Verma (The Salk Institute for Biological Studies, Laboratory of Genetics, La Jolla, CA, USA). The control vector rrl-CMV-EGFP contained a CMV promoter driven EGFP expression cassette. For production of Connexin 43 (Cx43) expressing LVs, the construct rrl-CMV-Cx43-IRES-EGFP was cloned containing a CMV promoter driven murine Cx43 expression cassette combined with an internal ribosome entry site (IRES) for simultaneous EGFP expression. Both constructs contained a central polypurine tract to increase the nuclear transport of the virus pre-integration complex and thus the transduction efficiency46 and the post-transcriptional regulatory element of woodchuck hepatitis virus (WPRE) to increase transgene expression47.

Titration of lentiviral vectors

In order to determine the biological titer of LVs (in infectious particles (IPs) per ml) HEK293T cells were seeded in a 24 well plate. After cell attachment, they were transduced with the vector preparation of serial dilutions in 300 µl supplemented medium and incubated overnight at 10% CO 2 and 37 °C as already described45. Medium was added on the next day. 72 h after transduction, cells were trypsinized and subsequently fixed with 4% (w/v) paraformaldehyde for 15 min on ice. Cells were centrifuged (5 min, 300 g) and re-suspended in phosphate buffered saline. Fixed cells were analyzed using flow cytometry and percentages of EGFP positive cells were determined. Non-transduced cells served as control. The biological titer was determined according to the following formulas: MOI (multiplicity of infection) = −ln (percentage of EGFP-negative cells/100); IP/ml = (number of infected cells) × (MOI) × (dilution factor) × 1,000. The biological titers of LVs were in the range of 1 to 10E + 09 IP/ml and were determined individually for each virus batch. The physical titer of LV preparations was determined by use of a commercially available colorimetric reverse transcriptase assay (Roche Diagnostics, Indianapolis, IN, USA) quantifying active viral reverse transcriptase (RT). The physical titers of LV preparations were in the range of 5 to 20 µl per 200 ng RT and were measured for each virus batch as well.

Lentiviral in vitro transduction of cells

For in vitro tests, 50,000 HEK293T cells were plated in a 24 well dish. 24 h after seeding, cells were transduced with LVs in 300 µl medium overnight. On the next day, medium was filled up. 48 h post infection, cells were analyzed using fluorescence microscopy. The volume of LV preparation was calculated according to the biological titer and the MOI desired according to the following formula: LV volume [µl] = number of cells seeded × MOI/biological titer [IP/µl]. In compliance to the used volume and the physical titer of the virus batch ng RT applied could be determined.

Generation of transgenic myoblasts by lentiviral gene transfer

Skeletal myoblasts (SkM) were harvested from the diaphragm and the hindlimb muscles of E18.5–19.5 CD1 wildtype embryos. Muscles were isolated by blunt dissection and enzymatically digested with collagenase B (1 mg/ml, Roche Diagnostics, Indianapolis, IN, USA) and trypsin (0.05% (w/v), Gibco/Life Technologies, Darmstadt, Germany) at 37 °C for 60 minutes. Then, the cell suspension was filtered through a 100 µm cell strainer and isolated cells were plated for several hours on fibronectin coated 24 well plates (250,000 cells/well) in 300 µl IMDM containing 20% FCS (Gibco/Life Technologies, Darmstadt, Germany) at 37 °C. For lentiviral transduction the virus constructs (CMV-EGFP or CMV-Cx43-IRES-EGFP) were added at a MOI of 25 overnight at 37 °C. The day after cells were washed at least twice with PBS and 1 ml IMDM/FCS was added.

Prior to intramyocardial injection the cells were plated for additional 24 hours, then trypsinized and re-suspended in IMDM/FCS at a concentration of 40,000 cells/µl. SkM, which were not injected, were re-plated and their differentiation in vitro monitored for several days. For in vitro coupling experiments, cells were cultivated for up to 20 days in IMDM/FCS. To avoid fibroblast overgrowth cells were irradiated with 15 Gy at the 3rd day of cell culture48.

Dye dialysis to prove functional gap junctions after lentiviral Cx43 gene transfer

To analyze functional coupling between transduced SkM, single-cell-electroporation was used. Sharp electrodes, filled with 40 mM KCl and two different dyes (Alexa350 and Alexa546-Dextran) were attached to the cell membrane of cultured (10–20 days) myoblasts resulting in a resistance of 140–190 MΩ. Alexa350 (349 Da; 1 ng/nl, Life Technologies, Darmstadt, Germany) emits blue fluorescence and is a small molecule, which is able to pass through Cx43 gap-junctions between adjacent cells. Dextran coupled Alexa546 (10 kDa; 10 ng/nl, Life Technologies, Darmstadt, Germany) emits in the red region of the spectrum and diffuses only to neighbouring cells through cytoplasmic bridges because of its high molecular weight49. Next, the cell membrane was perforated by applying an alternating current of 400 Hz and 10–50 nA in square-pulses and the electroporated myotubes were allowed to load via iontophoresis with the two different dyes. Dye transfer to adjacent EGFP+ cells was observed using fluorescence microscopy (Axiovert 200, Carl Zeiss, Jena, Germany) in 28 EGFP+ cells, dye transfer into an EGFP-SkM was never observed.

Intramyocardial transplantation of Cx43 expressing myoblasts

Cryolesions were generated in 53 adult female CD1 WT mice (age 10–12 weeks). The free left ventricular wall of the heart was exposed by a left lateral thoracotomy under inhalative anesthesia (endotracheal intubation and ventilation, 40% O 2 , 60% N 2 O, 1.5–2.0 Vol% Isoflurane) and a liquid nitrogen precooled copper probe (diameter 3.5 mm) was attached to the surface of the heart (three times, 10–15 seconds). Then, 200.000 myoblasts transduced with CMV-EGFP (n = 25) or CMV-Cx43-IRES-EGFP lentivirus (n = 28) were re-suspended in 5 µl culture medium and injected between the center and the border zone of the acute cardiac lesion using a 10 µl Hamilton syringe equipped with a 29 G insulin needle. To the cell suspension a food dye was added to visualize diffusion of the injected cell suspension from the center to the border zone of the lesion, otherwise a second injection nearby was applied. Afterwards, the chest was closed in layers, the pneumothorax evacuated by a chest drain and the animals allowed to awake, as reported before23.

Direct lentiviral Cx43 transduction of the cardiac lesion in vivo

For direct lentiviral gene transfer of cells in the scar area, 81 CD1 wildtype mice (see also above) were cryoinjured, then 2–3 days after the first operation, the first thoracotomy was surgically re-opened under general anesthesia and the myocardial lesion visualized. Then, via a 10 µl Hamilton syringe equipped with a 33 G insulin needle 5 µl of lentivirus solution (CMV-Cx43-IRES-EGFP, n = 36 or CMV-EGFP, n = 45) was applied by a single injection between the center and the apical margin of the lesion. The infectious particles amounted to 5.8 × 108 to 3.6 × 109 per ml for the CMV-Cx43-IRES-EGFP lentivirus and 1 × 109 to 1.5 × 1010 per ml for the CMV-EGFP lentivirus. Thereafter, the chest was re-closed and the mice allowed to wake up. Peri- and postoperative analgesia (0.1 mg/kg Buprenorphin 2 ×/d and 5 mg/kg Carprofen 1 ×/d s.c.) was administered to all operated animals up to the third postoperative day after each surgery. Sham operations were performed in 9 mice: First, a lateral thoracotomy was performed, the pericardium opened and the chest re-closed. Then, in analogy to virus-injected mice a second re-thoracotomy was performed two days after the first surgical intervention. Mice of the ex vivo imaging and long term groups were treated additionally with Dexamethasone 0.02 mg 2 ×/d s.c up to 7 days.

In vivo electrophysiology, left ventricular catheterization and echocardiography

In vivo electrophysiological testing was performed under inhalative anesthesia (Isoflurane 1.0 to 1.5 Vol %) 12–14 and 56 days after generation of the myocardial lesion by a blinded investigator. As reported before14, a surface 6-lead ECG was recorded (PowerLab 16/30, LabChart 7, ADInstruments, Pty LTD, Australia), then the tip of a 2 French octapolar mouse-electrophysiological catheter (CIBER Mouse Electrophysiology Catheter, NuMED, USA) was inserted to the apex of the right ventricle via the right jugular vein. Bipolar intracardiac electrograms were recorded from neighboring electrode pairs at the atrial, his-bundle and ventricular level. Rectangular stimulus pulses at twice pacing threshold were administered by a multi programmable stimulator (Model 2100, A-M Systems, USA; Stimulus 3.4 Software, Institute for Physiology I, University of Bonn, Germany) via the apical electrode pair. To explore electrical vulnerability both, extrastimulus protocols and the more aggressive burst stimulus were used. The pacing threshold current (1 ms stimulus duration) was between 0.5 and 1.0 mA and rectangular stimulus pulses of 2-fold pacing threshold were applied. Ventricular vulnerability was tested as reported earlier14 by extrastimulus pacing with up to three extra beats (with 10 ms stepwise S1S2 or S2S3 reduction starting 10 ms beneath S1S1) at S1S1 cycle lengths of 120 ms, 100 ms, and 80 ms. Next, ventricular vulnerability was also tested by applying ventricular burst stimulation: S1S1 stimulation at cycle lengths starting at 50 ms with 10 ms stepwise reduction down to 10 ms were performed for 1 second each and repeated 3 times. Between stimulation procedures the hearts were allowed to recover for at least 10 seconds. For the analysis of the in vivo electrophysiology data we have used, as reported in earlier publications50 and also by our groups in Bonn51,52, the clinical definition of VT, namely 4 consecutive ventricular extrabeats with atrioventricular dissociation. Given the high heart rate and the short refractory period in mice, more aggressive stimulation protocols than in humans need to be applied. Electrical noise due to motion and/or other artifacts was reduced by appropriate filtering (10–100 Hz) of the data. Upon VT induction both, mono- and polymorphic VT could be observed.

For in vivo left ventricular catheterization inhalative anesthesia was used as described above. A 1.4 French Millar Aria1 catheter (Millar Instruments Inc., Houston) was inserted retrogradely into the left ventricle via the right carotid artery and pressure-volume loops were recorded with PowerLab 16/30, LabChart 7 (ADInstruments, Pty LTD, Australia) and analyzed by the integrated PVAN-Software.

In long term experiments left ventricular function was measured 1–2 days prior to in vivo electrophysiological testing under inhalative anesthesia with M-mode echocardiography using a HDI-5000 ultrasound system (ATL–Phillips, Oceanside, CA, USA) equipped with a linear array 15 MHz transducer (CL15–7)53. In the parasternal short-axis view, M-mode data were acquired at the level of the papillary muscle and morphological as well as functional parameters measured as described before54.

Histology, immunohistochemistry, and morphometry

After electrophysiological testing mice were heparinized and sacrificed, hearts harvested and imaged with a fluorescence zoom microscope (Axio Zoom V16, Zeiss). Then, hearts were mounted on a Langendorff perfusion apparatus, fixed by perfusion with 4% paraformaldehyde, cryopreserved in 20% sucrose and cut into 10 µm thick slices.

For evaluation of fibrosis and cell engraftment, Sirius Red staining was performed following standard protocols or engrafted cells were identified by their native EGFP fluorescence. Expression of Cx43 in virus-injected hearts was detected by immunohistochemistry after antigen retrieval using a customized polyclonal rabbit Cx43 antibody (PSL GmbH, Heidelberg, Germany). Staining was accomplished using the Vectastain Elite ABC system with AEC substrate (Vector Laboratories, Burlingame, CA, USA) and hematoxylin counterstain. To specify the cell type(s) expressing lentivirus genes we performed fluorescent immunostainings in one heart per virus-construct (20 sections each) for different lineage specific markers. Antibodies against sarcomeric alpha-actinin, alpha-smooth muscle-actin (ASMAC; Sigma-Aldrich, St. Louis, Missouri, USA), Connexin 43 (Cx43; PSL GmbH), CD45 (Lab Vision, Fremont, CA), MyoD (Dako, Hamburg, Germany), and platelet/endothelial cell adhesion molecule (PECAM; BD Pharmingen) were used as previously described23. Visualization was accomplished with appropriate secondary donkey antibodies conjugated to Cy3 or Cy5 (Jackson ImmunoResearch, West Grove, PA). Nuclei were stained with Hoechst dye 33342 (Sigma-Aldrich). Native EGFP fluorescence and immunofluorescence was imaged with a Zeiss Axiovert 200 microscope equipped with an ApoTome and an AxioCam MRm. Images were acquired by use of the AxioVision software (Zeiss).

For morphometric analysis hearts were perfused with cardioplegic solution (HTK Solution, Dr. Franz Köhler Chemie GmbH, Bensheim, Germany), cryopreserved and sectioned at intervals of 300 µm, as described above. Infarct size was determined in clearly relaxed hearts with transmural lesions by measuring the circumference of the damaged area based on autofluorescence; total infarct area was calculated by extrapolation of these measurements.

To quantify cellularity within the infarct area three sections at different levels (lower, mid and upper part of the lesion) were taken from two hearts at 14 days after infarction and stained with Hoechst dye. Tile images were recorded by AxioVision MosaiX and measurements performed using AutMess software (Zeiss). Infarct area was determined based on autofluorescence, and nuclei in this area were counted. Cellularity is given as nuclei per mm2. The number of EGFP+ cells was determined in ten hearts after SkM transplantation (five per construct) and in two hearts (one per construct) after pure lentivirus injection; 6 slides (3 sections each) spanning the complete engrafted area were analyzed by counting of nucleated (Hoechst dye stain) EGFP+ cells and extrapolating numbers to whole hearts.

Analysis of Cx43 expression by western blotting after lvEGFP or lvCx43 injection into the infarct area

Infarct areas (I.A.), into which lvEGFP or lvCx43 was injected, and remote tissue of respective right and left ventricles were excised 10–12 days after lentiviral transduction under a fluorescence stereomicroscope (Leica MZ 16 F, Leica Microsystems) and frozen in liquid nitrogen. Heart tissues were homogenized in RIPA buffer (2 mM EDTA; 25 mM Tris-HCL, pH7.5; 150 mM NaCl; 0.1% Sodiumdoxycholate; 0.1% SDS; 1% Nonidet P40 in H 2 O; 2.5 × cOmplete Proteaseinhibitor, Roche). SDS mini gels (12% separating gel: 12% Acrylamide, 7.5 mM Tris-HCL (pH8.8), 0.1% SDS, 0.05% APS, 0.05% TEMED; 8% stacking gel: 8% Acrylamide, 1.25 mM Tris-HCL, 0.1% SDS, 0.05% APS, 0.1% TEMED) were prepared and protein lysates separated by electrophoresis (ProSieve EX Running Buffer 10x , diluted in H 2 O to 1x , Lonza; Precision Plus Protein WesternC Standards, Biorad), and blotted onto methanol-activated PVDF membrane (Low-fluorescence, 0.2 µm, Biozym) by tankblot (ProSieve EX Western Blot Transfer Buffer 10x , diluted in H 2 O to 1x , Lonza). Membranes were blocked with TBST buffer (20 mM Tris, 150 mM NaCl, 0.1% Tween-20 in H 2 O) containing 5% milk powder (Skim milk powder, VWR) for 1 hour. Cx43 (1:3000, custom-produced rabbit polyclonal antibody, PSL GmbH, Heidelberg, Germany) and horseradish peroxidase (HRP) conjugated-GAPDH (1:5000, Sigma) antibodies were incubated over night at 4 °C, followed by incubation of donkey-anti-rabbit Alexa Fluor 488 conjugated antibody (1:3000, AffiniPure, Jackson Immuno Research) and Precision Protein StrepTactin-HRP Conjugate (1:3000, Biorad) for 1 h at RT. Signals were developed using Pierce ECL Western Blotting Substrate (Thermo Scientific) and detected with ChemiDoc MP Imaging System (Biorad). All antibodies were diluted in 5% milk powder in TBST. Quantification of Cx43 expression was performed using Image Lab Software (Biorad) normalized to GAPDH.

Determination of provirus integration in mouse hearts using a PCR assay after I.A. injection of lvEGFP or lvCx43

Infarct areas with virus injection (I.A.) of either lvEGFP or lvCX43 and areas from the respective right ventricle (RV) were excised 10–12 days after lentiviral transduction. Using the Puregene Core Kit A from QIAGEN (Mat. No. 1042601), genomic DNA was isolated according to the supplier. The forward (fwd) and reverse (rev) primer sequences (Eurofins MWG, Ebersberg, Germany) used for detecting integrated provirus DNA were as follows: 5′-TGTGTGCCCGTCTGTTGTGT-3′ (fwd) and 5′-GAGTCCTGCGTCGAGAGAGC-3′ (rev). PCR reactions were performed on a T-Professional Trio Thermocycler (Biometra). The 139 bp amplification product was stained with ethidium bromide.

Analysis of EGFP expression in mouse hearts by qRT-PCR after I.A. injection of lvEGFP or lvCx43

I.A. of mouse hearts injected with lvCx43 or lvEGFP as well as respective remote areas were excised and stabilized in RNAlater (Qiagen). RNA was extracted following the standard Trizol protocol (Life Technologies) and RNA quality was determined using the Bioanalyzer 2100 (Agilent). RNA was reverse-transcribed and pre-amplified using the Cell Direct kit (Invitrogen) followed by qPCR (CFX96 cycler, Biorad). As controls, H 2 O and non-RT samples were used. TaqMan probes: GAPDH (4352932E), EGFP (custom-designed), TaqMan Gene Expression Master Mix (all from Applied Biosystems).

Ex vivo optical mapping

Hearts were perfused in a Langendorff apparatus, mounted in a custom-designed chamber with a window for optical mapping, side restraints to minimize motion and temperature control to stabilize action potential durations and heart rate. Hearts were loaded with the voltage-sensitive dye di-4-ANEPPS (λ ex = 540 ± 30 nm, λ em > 640 nm) as previously described55. Voltage signals were recorded with a complementary-metal-oxide semiconductor (CMOS) camera (100 pixels × 100 pixels, Ultima-One SciMedia) at 1,000 frames per second. Conduction velocities within the infarct, border- and remote zones of myocardium were evaluated during pacing with a unipolar electrode. Light from a custom-designed 300 W tungsten-halogen lamp (University of Pittsburgh machine and electronic shops) was collimated, passed through an interference filter (540 ± 30 nm) and refocused to illuminate the heart. Fluorescent light from the heart was collected, passed through a high-pass filter (>640 nm) and was refocused on the CMOS camera, which viewed the anterolateral surface of the heart. In this optical alignment, optical maps included the complete lesion, border zone and adjacent native or ‘normal’ myocardium. With this optical configuration, the spatial resolution was close to cellular resolution (70 × 70 µm2 with a depth field of about 130–140 µm). To reduce motion blurring caused by rapid wave propagation the integration time was set to 1 or 2 ms. For stimulation of the hearts, an Ag+/AgCl electrode (200 µm in diameter) was pinned to the native myocardium, proximal to the lesion. Stimulation (impulse duration 2 ms, current 20% above threshold) was performed at a cycle length of 200 ms for at least 20 beats. Activation time-points were determined by dF/dtmax after applying a Savitzky–Golay smoothing filter (11 point width, third order), and isochronal maps of activation were generated as described previously. The local conduction velocity vector was calculated for each pixel by measuring the activation time-point at that pixel and comparing it with the activation time-points of its eight nearest neighbours. The rise time of action potentials was measured from fluorescence signals that showed a signal-to-noise ratio of more than about 20:1, at a sampling rate of 1,000 frames s−1 and a spatial resolution of 100 × 100 µm2 per pixel. The rise time was measured from 10% to 90% of action-potential amplitude from at least five consecutive action potentials and low-pass filtered with an averaging kernel of 1 ms. Border zones were identified visually from autofluorescence images.

Statistics

Electrophysiological data were compared using a multivariant one-way analysis of variance with post hoc subgroup testing, where appropriate (Tukey-Kramer multiple comparisons test). For discrete variables a two-sided Fisher’s exact test was performed. Statistical evaluation of the other data was performed using Student’s t-test. A p value ≤ 0.05 was considered statistically significant, error bars represent SDs or SEMs (functional data).

Study approval

All animal experiments were performed in accordance with National Institutes of Health animal protection guidelines and were approved by the local authorities (Landesamt für Natur, Umwelt und Verbraucherschutz, Nordrhein-Westfalen).

Data availability

All datasets generated during the current study are available from the corresponding author on reasonable request and the results from all data analyzed during this study are included in the published article.