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: d.a.pijnappels@lumc.nl

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