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www.sciencetranslationalmedicine.org/cgi/content/full/11/481/eaau6447/DC1
Supplementary Materials for
An automated hybrid bioelectronic system for autogenous restoration of sinus
rhythm in atrial fibrillation
Emile C.A. Nyns, René H. Poelma, Linda Volkers, Jaap J. Plomp, Cindy I. Bart, Annemarie M. Kip, Thomas J. van Brakel, Katja Zeppenfeld, Martin J. Schalij, Guo Qi Zhang, Antoine A.F. de Vries, Daniël A. Pijnappels*
*Corresponding author. Email: [email protected]
Published 27 February 2019, Sci. Transl. Med. 11, eaau6447 (2019)
DOI: 10.1126/scitranslmed.aau6447
The PDF file includes:
Materials and Methods Fig. S1. Maps of the AAVV genomes. Fig. S2. Transgene expression in different cardiac compartments after gene painting of the RA of adult rat hearts with AAV2/9.45.HsNPPA.ReaChR~citrine.SV40pA. Fig. S3. Transgene expression after gene painting of the RA of adult rat hearts with AAV2/9.45.HsNPPA.citrine.SV40pA. Fig. S4. Masson’s trichrome staining of the RA and LA after gene painting of the RA with AAV2/9.45.HsNPPA.ReaChR~citrine.SV40pA. Fig. S5. Light intensity–duration curve for optical atrial pacing of adult rat hearts after gene painting of the RA with AAV2/9.45.HsNPPA.ReaChR~citrine.SV40pA. Fig. S6. Correlation between surface area and location of right atrial illumination on optogenetic termination of atrial tachyarrhythmias ex vivo. Fig. S7. Efficient autogenous termination of atrial flutter in vivo after gene painting of the RA of adult rat hearts with AAV2/9.45.HsNPPA.ReaChR~citrine.SV40pA. Fig. S8. Implantable LED device. Fig. S9. Schematic overview of the AF detection algorithm and experimental setup. Fig. S10. Prolonged illumination of the RA and ventricle of adult rat hearts after gene painting of the RA with AAV2/9.45.HsNPPA.ReaChR~citrine.SV40pA. Fig. S11. Ex vivo temperature measurements of the RA during LED activation. Fig. S12. Computational simulation of thermal heating after activation of the implantable LED device based on in vivo conditions. Legend for Data file S1 Legends for Movies S1 and S2
Other Supplementary Material for this manuscript includes the following: (available at www.sciencetranslationalmedicine.org/cgi/content/full/11/481/eaau6447/DC1)
Data file S1 (Microsoft Excel format). Raw data. Movie S1 (.avi format). Optical voltage mapping of the ReaChR-expressing RA during AF and subsequent optogenetic restoration of sinus rhythm. Movie S2 (.mp4 format). Summary of all essential experimental steps leading to the development of the automated hybrid bioelectronic system for autogenous restoration of sinus rhythm in AF.
Materials and Methods
Optical cardiac pacing and atrial tachyarrhythmia termination ex vivo
Four to 8 weeks following gene painting of the RA, the animals were prepared for surgery as
described above. Hearts were excised, cannulated and perfused by a Langendorff apparatus as
previously described(23). Action potential propagation was visualized by loading hearts with
12.5 µM of the voltage-sensitive dye di-4-ANBDQBS (AAT Bioquest). Optical images were
captured using a MiCAM ULTIMA-L imaging system (SciMedia). The atria were stimulated
either electrically using a custom-made bipolar electrode or optically with light pulses from a
mounted 470-nm or 730-nm LED with adjustable collimation lens (M470L3-C4 with
additional aspherical lenses LA1131-A-ML and LA1951-A-ML; Thorlabs) powered by a
1200-mA LED driver in trigger mode (T-Cube LED driver, Thorlabs) controlled by a
STG2004 stimulus generator (Multichannel Systems), positioned at approximately 5 cm from
the heart. Irradiance was measured using a PM100D optical power and energy meter
(Thorlabs) equipped with a S130C slim dual range sensor. Atrial tachyarrhythmias were
induced by continuous perfusion of the parasympathomimetic agent carbachol (4 µM final
concentration) and 1-2 s of electrical burst pacing (2000 mV, pulse duration: 10 ms, pulse
shape: square; cycle length: 20-40 ms). Atrial tachyarrhythmias consisted of 62% AF (based
on irregular ventricular activity) and 38% atrial flutter (based on regular ventricular activity).
When atrial tachyarrhythmias lasted for more than 10 s, they were considered sustained and
the RA was subsequently exposed to light pulses of different intensity and duration. A
patterned illumination device (Polygon400; Mightex Systems) that was connected to a 470-
nm, high-power collimator light-emitting diode source (50 W, type-H, also from Mightex
Systems) and controlled by PolyLite software (Mightex Systems) was used to project
different surface areas of illumination on the RA epicardium. The maximum light intensity
output of this setup, projected through a 5× objective lens (Leica Microsystems), was 2.0
mW/mm². Atrial tachyarrhythmias were considered to be optogenetically terminated when
the arrhythmia stopped within 2 s following the start of the light pulse. All control
experiments were performed in an identical fashion. Specialized software was used for data
analysis and construction of activation maps (BrainVision Analyzer 1101; Brainvision).
Immunohistology
Hearts were fixed by overnight incubation at 4°C in 4% paraformaldehyde, dehydrated and
subsequently embedded in paraffin. Transverse and longitudinal 5-µm-thick sections of atria
and ventricles were cut. Sections were deparaffinized and rehydrated in UltraClear
(Klinipath) followed by a graded ethanol series. Antigen retrieval was performed by
incubation with 0.05% trypsin (Sigma-Aldrich), 0.1% CaCl2 in water for 20 minutes at room
temperature (RT). Sections were stained with antibodies directed against cardiac troponin I
(mouse IgG1, clone 6F9; Hytest, Turku, Finland) and GFP (rabbit IgG; Thermo Fisher
Scientific, catalogue number: A-11122). Incubation with primary antibodies (1:200 dilution
in PBS/2% donkey serum (Thermo Fisher Scientific) and corresponding Alexa Fluor 488 or
568-conjugated secondary antibodies (Thermo Fisher Scientific; 1:200 dilution in PBS/2%
donkey serum) was done at RT. Nuclear counterstaining was performed at RT with 1 µg/mL
Hoechst 33342 (Thermo Fisher Scientific). Sections were subsequently mounted in
Vectashield mounting medium (Vector Laboratories). Images were acquired with a Nikon
Eclipse 80i fluorescence microscope (Nikon Instruments). Transduction rate quantification
was performed by threshold pixel analysis of the GFP signal in comparison to the cardiac
troponin I signal. Analysis were performed of the RA, LA and ventricles (n = 6 rats) in both
transversal (n = 3) and longitudinal (n = 3) orientations using Adobe Photoshop CC 2018
(Adobe). Right atrial thickness measurements were performed histologically as the average
distance between the atrial epicardial and endocardial borders in longitudinal sections of the
RA (n = 5). Masson’s trichrome staining was performed to assess the presence of collagen
and cellular infiltrations.
Construction of adeno-associated virus vector (AAVV) shuttle plasmid
Molecular cloning was performed with enzymes from Fermentas (Thermo Fisher Scientific,)
or from New England Biolabs (Bioké) and with oligodeoxyribonucleotides (Sigma-Aldrich)
following the instructions provided with specific reagents or using established procedures.
The AAVV shuttle construct pDD.HsNPPA.ReaChR~citrine.SV40pA was assembled from
DNA fragments of the AAVV shuttle plasmids pDD4(54) (plasmid backbone, AAV serotype
2 [AAV2] inverted terminal repeats and simian virus 40 polyadenylation signal),
pANF.LUC(55) (human NPPA gene promoter) and the lentivirus vector shuttle plasmid
pLenti-ReaChR-Citrine(46) (coding sequence of the ReaChR~citrine fusion protein.
pDD.HsNPPA.ReaChR~citrine.SV40pA was subsequently used to generate
AAV2/9.45.HsNPPA.ReaChR~citrine.SV40pA (see fig. S1 for vector map) particles carrying
the capsid of a liver-detargeted AAV9 variant(56). As a control vector, we used
AAV2/9.45.HsNPPA.citrine.SV40pA (see fig. S1 for vector map). To generate the shuttle
plasmid for making this AAVV, pDD.HsNPPA.ReaChR~citrine.SV40pA was cut with XhoI
and HindIII, the digestion products were treated with Klenow polymerase and the ReaChR-
less vector backbone was religated. The production and titration of AAVV particles were
performed as previously described(23).
Preparation of AAVV/Sealer Protein solution
AAVV particles contained in 0.65-mL Corning Costar low binding microcentrifuge tubes
(Sigma-Aldrich) were transferred to an empty 1.5-mL microcentrifuge tube, diluted with
phosphate-buffered saline (PBS) to a total volume of 50 µL (4×109 genome copies) and
supplemented with 50 µL of TISSEEL Sealer Protein solution containing aprotinin, factor
XIII and fibrinogen (Baxter).
Sharp electrode recordings
Intracellular recordings of dissected right atria (n = 3 rats) were made in Tyrode solution.
Tissue was impaled with a glass microelectrode with a tip resistance of 5-14 MΩ when filled
with 3 M KCl. Measurements were performed using a MultiClamp 700B amplifier and
analogue signals were low-pass filtered (10 kHz) and digitized at a sample rate of 10 kHz via
a Digidata 1440A A/D (both Axon Instruments/Molecular Devices). The optical response of
the optopgenetically modified atria was measured using a collimated LED module (λ = 470
nm, irradiance: 2.55 mW/mm2; Thorlabs) illuminating directly below the recording site.
Experiments were performed with as little ambient light as possible. Data were recorded and
analyzed using pCLAMP 10.7 acquisition software (Axon Instruments).
Experimental RA temperature measurements following LED activation
Hearts were rapidly excised as previously described (23) and placed in an incubator set at
37°C and temperature measurements were performed by a high precision thermometer
(Fisherbrand Traceable Digital Thermometer, Thermo Fisher Scientific) with a temperature
accuracy of 0.05°C and sampling rate of approximately 3 Hz. The thermometer sensor was
placed in between the LED light guide (used in the in vivo closed-chest experiments) and
RA, thereby making physical contact with both structures. The heart and equipment were pre-
equilibrated to reach a temperature of 37°C. The heart was frequently sprayed with PBS of
37°C to prevent tissue drying. Temperature was recorded before, during and after exposure of
the RA to 4 repetitive 500-ms light pulses with 5-s intervals or a single 1000-ms light pulse
of 470 nm at 3.5 mW/mm2 (n = 3).
Computational RA temperature measurements following LED activation
Thermal heating effects of the implantable LED device were also calculated using a finite
element model of the rat heart, body and LED light guide. Fourier’s law was used as the
mathematical model for time-dependent heat transfer using COMSOL Multiphysics 5.3
(Comsol):
p extT
C k T Qt
where k is the thermal conductivity W/ m K , is the density 3kg/m , Cp represents the
specific heat capacity J/ kg K and T is the calculated temperature K . The initial
temperature of the rat body and LED light guide (at t = 0 s) and the temperature boundary
condition on the rat‘s outer skin were set at 37°C. The measured forward voltage of the blue
LUXEON LXZ1-PB1 LED at 180 mA is about 2.81 V. The LED was thus modelled as a heat
source extQ of 0.506 W. The transient simulation is in pulsed mode where 4 heat pulses of
0.506 W and a duration of 0.5 s are provided with 5-s intervals. The maximum temperature of
the heart due to the heat pulses was calculated. fig. S12A shows a cross section of the rat
body which is used for the finite element model. The Cartesian axis gives an indication of the
size of the rat body and its organs. The model is 2D and includes out-of-plane widths of the
rat body (~ 50 mm) and heart (~10 mm). fig. S12B shows the finite element mesh of the
geometry.
Material properties used in the thermal model of the rat heart, body and LED device were
derived from literature or the program used for the thermal simulation(57, 58). The rat body
is modelled as muscle tissue with the following properties: heat capacity 3421 J/ kg K ,
density 1090 3kg/m and thermal conductivity 0.49 W/ m K . The heart heat capacity was
3686 J/ kg K with a density of 1041 3kg/m and thermal conductivity of 0.56 W/ m K .
The PDMS light guide heat capacity was 1460 J/ kg K with a density of 970 3kg/m and
thermal conductivity of 0.16 W/ m K . The polytetrafluoroethylene diffuse reflector heat
capacity was 1050 J/ kg K with a density of 2200 3kg/m and thermal conductivity of 0.24
W/ m K . The LED ceramic substrate Al2O3 heat capacity was 730 J/ kg K with a density
of 3965 3kg/m and the thermal conductivity of 35 W/ m K . The LED chip (In)GaN heat
capacity was 490 J/ kg K , with a density of 6070 3kg/m and thermal conductivity of 130
W/ m K .
RA light penetration measurements
Hearts were rapidly excised as previously described (23) but were not perfused in order to
maintain erythrocytes inside the coronaries. The RA was then carefully cut open in order to
create an unfolded sheet of atrial myocardium. The RA sheet was placed on a transparent
glass slide with the epicardial surface facing upwards. The mounted 470-nm LED with
adjustable collimation lens was used to illuminate approximately 20 mm
2 at 3 different RA
epicardial sites with irradiance of 1.0, 2.0 and 3.5 mW/mm2. Irradiance on the endocardial
side (underneath the glass slide) was measured using the PM100D optical power/ energy
meter (Thorlabs) and averaged based on the 3 different illumination sites. Baseline
measurements were performed on a glass slide without tissue. RA light penetration was
expressed as the average fraction of 470-nm light that passed through the RA in comparison
to the baseline measurement.
Western blotting
The RA, LA and ventricles were separated from each other, cut in small pieces, flash-frozen
in liquid nitrogen and stored in 2-mL round bottom microcentrifuge tubes containing a metal
bead with a diameter of 7 mm at -80°C until processing. Next, ice-cold lysis buffer (150 mM
NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM
Tris-HCl [pH 8.0]) supplemented with protease and phosphatase inhibitors was added. The
tissue was lysed by high-speed shaking for 5 minutes at 50 Hz using a TissueLyser LT
apparatus (QIAGEN). The lysate was then centrifuged at 4°C at 21,130 × g for 5 minutes,
after which the supernatant was collected. Protein concentration was determined using the
BCA protein assay kit (Thermo Fisher Scientific). Proteins were size-fractionated in Bolt
10% Bis-Tris Plus gels (Thermo Fisher Scientific) and transferred to Amersham Hybond P
0.45-µm polyvinylidene difluoride membranes (GE Healthcare) by wet electroblotting using
a Bolt Mini Blot Module (Life Technologies). After blocking for 1 h in 2% ECL Prime
blocking reagent (GE healthcare) dissolved in Tris-based saline/0.1% Tween-20 (TBST),
membranes were incubated for 1 h with primary antibodies directed against GFP (1:12,500;
see above) or GAPDH (1:200,000; mouse IgG1, clone 6C5; Merck) as internal control. After
3 washes with TBST, blots were incubated with 1;25,000 dilutions of corresponding
horseradish peroxidase (HRP)-conjugated secondary antibodies (goat-anti-rabbit IgG-HRP or
goat-anti-mouse IgG-HRP; both from Abcam) for 1 h at RT. All antibodies were diluted in
TBST/2% ECL Prime blocking reagent. After another 3 wash steps with TBST, membranes
were immersed in SuperSignal West Femto Maximum Sensitivity substrate (Thermo Fisher
Scientific) and chemiluminescence was recorded with the ChemiDoc Touch imaging system
(Bio-Rad Laboratories) and analyzed using ImageLab 5.2 software (Bio-Rad Laboratories).
Supplementary Figures
Fig. S1. Maps of the AAVV genomes. Map of the genomes of
AAV2/9.45.HsNPPA.ReaChR~citrine. SV40pA (top image) and
AAV2/9.45.HsNPPA.citrine.SV40pA (bottom image). Black arrows, adeno-associated virus
type 2 inverted terminal repeats. HsNPPA, human NPPA gene promoter. ReaChR, coding
sequence of red-shifted channelrhodopsin variant. Citrine, coding sequence of a slightly red-
shifted green fluorescent protein variant. pA, simian virus 40 polyadenylation signal.
Fig. S2. Transgene expression in different cardiac compartments after gene painting of
the RA of adult rat hearts with AAV2/9.45.HsNPPA.ReaChR~citrine.SV40pA. Typical
immunostaining of transversal sections of the right atrium (left column), left atrium (middle
column) and ventricle (right column) near the right atrioventricular area for green fluorescent
protein (GFP; top row) and cardiac troponin I (cTnI; bottom row), showing minimal
transgene expression in the left atrium and ventricle following the gene paint procedure (2×
magnification). All sections were derived from the same animal and the image acquisition
exposure times for each fluorophore were identical.
Fig. S3. Transgene expression after gene painting of the RA of adult rat hearts with
AAV2/9.45.HsNPPA.citrine.SV40pA. Typical immunostaining of the right atrium of a
ReaChR-expressing heart (left column) and a citrine-expressing control heart (right column)
for green fluorescent protein (GFP; top row) showing similar transduction rates. In the
bottom row the GFP signal is combined with a Hoechst 33342 nuclear staining. Please note
that the GFP signal in AAV2/9.45.HsNPPA.ReaChR~citrine.SV40pA-transduced
cardiomyocytes is largely confined to the sarcolemma, whereas in
AAV2/9.45.HsNPPA.citrine.SV40pA-transduced cardiomyocytes it is located throughout the
cell.
Fig. S4. Masson’s trichrome staining of the RA and LA after gene painting of the RA
with AAV2/9.45.HsNPPA.ReaChR~citrine.SV40pA. Typical Masson’s trichrome staining
of the right atrium (left column) and left atrium (right column) of the same animal, 8 weeks
after the gene paint procedure, showing collagen (blue), myocardium (red) and cell nuclei
(dark brown). The epicardial layer of the gene-painted right atrium is somewhat thickened as
compared to that of the non-treated left atrium. The boxed areas in the upper two panels are
depicted at a higher magnification in the lower two panels and show no signs of
inflammation.
Fig. S5. Light intensity–duration curve for optical atrial pacing of adult rat hearts after
gene painting of the RA with AAV2/9.45.HsNPPA.ReaChR~citrine.SV40pA. The dots
correspond to conditions resulting in capture rates of 100% following 5-Hz (A) ex vivo or (B)
in vivo optical right atrial pacing. The error bar represents one standard error of the mean.
A B
Fig. S6. Correlation between surface area and location of right atrial illumination on
optogenetic termination of atrial tachyarrhythmias ex vivo. (A) Scaled overview of a
ReaChR-expressing heart in the Langendorff setup. Different surface areas of illumination
(20, 10, 5 and 2.5 mm2) are schematically highlighted on the central region of the right atrium
(RA). The dashed line represents the entire RA epicardial surface. (B) Images showing
different surface areas of illumination (470 nm) projected on the anterior region of the RA by
a patterned illumination device. The faint blue area surrounding the projection is the result of
optical scattering. Pictures were taken from a high angle because of the narrow space
between the heart and the objective lens. (C) Quantification of optical termination efficacy
averaged per ReaChR-expressing heart (n = 4) for various surface areas of illumination (λ =
470 nm; irradiance: 2.0 mW/mm2; light pulse duration: 500 ms) projected on the central
(“C”) or anterior (“A”) region of the RA. Illumination was repeated up to 3 times in case of
prior unsuccessful arrhythmia termination. Error bars represent one standard error of the
mean.
Fig. S7. Efficient autogenous termination of atrial flutter in vivo after gene painting of
the RA of adult rat hearts with AAV2/9.45.HsNPPA.ReaChR~citrine.SV40pA.
Quantification of light-induced termination of atrial flutter in vivo expressed as a percentage
of successful attempts averaged for all hearts for the indicated light pulse durations (λ = 470
nm, irradiance: 3.5 mW/mm2). Data are means ± one standard error of the mean. Each dot
represents a minimum of 3 independent measurements in one heart. Kruskal-Wallis test with
Dunn-Bonferroni post-hoc test (n = 5 to 7 hearts). ** P ≤ 0.01, *** P ≤ 0.001.
Fig. S8. Implantable LED device. (A) Exploded schematic view of the LED light guide
assembly. The LED is placed inside the reflector cup at a 90º angle relative to its base for
total internal reflection. The reflector cup is afterwards filled with liquid
polydimethylsiloxane (PDMS) followed by a curing period of 48 h at room temperature. (B)
Photograph of the LED assembly placed next to a ruler. Also shown is the surgical suture
used for fixation. (C) Working principle of the LED assembly. n, refractive index. Θ1, angle
of incidence, Θc, critical angle.
Fig. S9. Schematic overview of the AF detection algorithm and experimental setup. (A)
Sudden changes in heart rate and the inability of the real-time ECG analysis software to
calculate the PR interval ultimately lead to an increasing voltage signal in channel 10. When
this signal surpasses 1.75 V, a programmed delay of 10 s follows, after which a signal is send
to the stimulus generator thereby activating the implanted LED. (B) Schematic overview of
the experimental set-up.
Fig. S10. Prolonged illumination of the RA and ventricle of adult rat hearts after gene
painting of the RA with AAV2/9.45.HsNPPA.ReaChR~citrine.SV40pA. Typical ECG
recordings during 2-s illumination (λ = 470 nm, irradiance: 3.5 mW/mm²) of the right atrium
(top image) and right ventricle (bottom image) showing that prolonged illumination of the
right atrium or right ventricle did not induce ventricular arrhythmias or ectopy.
Fig. S11. Ex vivo temperature measurements of the RA during LED activation. Typical
thermal response of the right atrium following activation of the LED light guide (blue box, λ
= 470 nm, irradiance: 3.5 mW/mm²) with (A) four consecutive 500-ms light pulses with 5-s
intervals or (B) a single 1000-ms light pulse, showing that the right atrial temperature during
LED activation remains well within the physiological range.
Fig. S12. Computational simulation of thermal heating after activation of the
implantable LED device based on in vivo conditions. (A) Geometry of the two-
dimensional rat model with indication of the heart and LED light guide device. (B)
Discretization of the geometry into 42496 mesh elements, a higher mesh density is used in
the region of interest. (C) Maximum cardiac temperature following four consecutive 500-ms
light pulses (λ = 470 nm, irradiance: 3.5 mW/mm²) with 5-s intervals. LED power indicates
the power consumption needed for 3.5 mW/mm² irradiance. D) Section of the heart in closest
contact to the LED light guide and (E) the maximum temperature profile on the heart
periphery and the temperature penetration after the 4th
light pulse. (F) The surface
temperature profile of the LED light guide showing the maximum temperature immediately
after the 4th
consecutive light pulse (solid line) and 1 s later (dashed line).
Data file S1. Raw data. Provided as an Excel file.
Movie S1. Optical voltage mapping of the ReaChR-expressing RA during AF and
subsequent optogenetic restoration of sinus rhythm.
Movie S2. Summary of all essential experimental steps leading to the development of the
automated hybrid bioelectronic system for autogenous restoration of sinus rhythm in
AF.
aau6447_SMaau6447_SupplementalMaterial_v8