Supplementary Materials for

Cell-selective arrhythmia ablation for photomodulation of heart rhythm

Uma Mahesh R. Avula, Hyung Ki Yoon, Chang H. Lee, Kuljeet Kaur, Rafael J. Ramirez,

Yoshio Takemoto, Steven R. Ennis, Fred Morady, Todd Herron, Omer Berenfeld,

Raoul Kopelman,* Jérôme Kalifa*

*Corresponding author. E-mail: kalifaj@med.umich.edu (J.K.); kopelman@umich.edu (R.K.)

Published 28 October 2015, Sci. Transl. Med. 7, 311ra172 (2015)

DOI: 10.1126/scitranslmed.aab3665

This PDF file includes:

Methods

Fig. S1. Characteristics of CTP-Ce6-PEG nanoparticle.

Fig. S2. Nontargeted control experiments in vitro.

Fig. S3. Targeted and nontargeted PDT in a coculture of human cardiac myocytes

and fibroblasts.

Fig. S4. Photoablated lesion depth analysis.

Fig. S5. Thermal imaging during PDT ablation.

References (36–41)

Legends for movies S1 to S3

Other Supplementary Material for this manuscript includes the following:

(available at

www.sciencetranslationalmedicine.org/cgi/content/full/7/311/311ra172/DC1)

Movie S1 (.avi format). Optical mapping of a rat heart 3 days after photoablation.

Movie S2 (.avi format). Optical mapping of the sheep right atrial free wall.

Movie S3 (.avi format). Optical mapping of the sheep right ventricle.

www.sciencetranslationalmedicine.org/cgi/content/full/7/311/311ra172/DC1

METHODS

Preparation of Ce6-conjugated 8-arm PEG (Ce6-PEG)

Unless otherwise noted, reagents were from Sigma Aldrich. The chlorin e6 (Ce6, > 95%, 596.67

Da; Frontier Scientific) was conjugated with amine in 8-arm polyethylene glycol-amine (8-arm

PEG, 40 kDa, Creative PEG Works) through N,N′-dicyclohexylcarbodiimide (DCC) and N-

hydroxysuccinimide (NHS) coupling. The 448 µl of Ce6 solution [20 mg/ml in N,N-

dimethylformamide (DMF)] was activated with 154.8 µl of DCC (20 mg/ml in DMF) and 172.8

µl of NHS, 20 mg/mL in DMF by stirring for 30 to 45 min. Four ml of 8-arm PEG solution (50

mg/ml in DMF) was prepared by stirring and sonicating until the entire PEG was dissolved. The

NHS-activated Ce6 solution was added into the PEG solution and stirred overnight. To remove

unreacted Ce6, the crude product was washed with enough 60% ethanol, PBS (BioReagent, pH

7.4, for molecular biology), and deionized water using Amicon filtration system (10 kDa filter

membrane). After the washing, the Ce6-PEG was filtered by 0.45-µm syringe filter and stored

after freeze-drying.

Preparation of CTP targeted Ce6-PEG (CTP-Ce6-PEG)

For CTP targeting, 1.4 ml of heterobifunctional maleimide-PEG-NHS (MAL-PEG-NHS, 2 kDa,

Creative PEG Works) (100 mg/ml in PBS, pH 7.4) was added into 5 ml of Ce6-PEG solution (20

mg/ml in PBS, pH 7.4) and stirred for 30 min. The unreacted MAL-PEG-NHS was washed out

three times by Amicon centrifugal cell (10 kDa) and the concentration of solution was diluted to

20 mg/ml with PBS. Then, 53.7 mg of cardiac targeting peptide with cysteine attached (CTP-

Cys) APWHLSSQYSRTC, > 95%, 1535.7 Da purchased from RS Synthesis. The mixture

washed with enough D.I. water through Amicon centrifugal cell (10 kDa) and the final CTP-

Ce6-PEG was obtained through freeze drying.

Detection of ROS

The ROS produced from CTP-Ce6-PEG was measured based on previous reported method (36).

In 2 ml of CTP-Ce6-PEG (0.1 mg/ml in PBS, pH 7.4), 80 µl of ADPA (100 µM in pure water,

Life Technologies) was added and measured its fluorescence by FluoroMax-3

Spectrofluorometer (Jobin Yvon Horiba), excited at 370 nm, right after irradiation at 660 nm of

CTP-Ce6-PEG over different time periods (0, 60, 120, 180, 240, 300, 480, and 660 s), under

constant stirring and temperature (25°C). The decay constant of ADPA fluorescence “k value”

was calculated by the previous reported equation (36).

Characterization of CTP-Ce6-PEG

The absorption and fluorescence spectra were taken on a UV-1601 UV-vis spectrometer

(Shimadzu) and a fluorimeter respectively. The number of conjugated CTP obtained based on

amino acid analysis (37) was determined by the Protein Chemistry Laboratory at Texas A&M

University. Hydrodynamic size determination was approximated by weight in comparison to

proteins (38). The ROS production of nanoparticle was determined by ADPA quenching assay

using fluorimeter (9).

Isolation of adult rat myocytes and fibroblasts:

Adult cardiomyocytes from normal adult male rats (200–300 g) were isolated as described in

(32, 33). Briefly, after euthanasia, hearts were retrogradely perfused through the aorta for up to 5

minutes with modified Krebs buffer (KHB) containing (in mM) 118 NaCl, 4.8 KCl, 25 HEPES,

1.25 K2HPO4, 1.25 MgSO4, 11 glucose, 1 CaCl2; the pH was adjusted to 7.40. The perfusate

was then switched to modified Krebs buffer without calcium for 3 minutes. Following calcium-

free KHB perfusion hearts were digested by perfusing calcium-free KHB containing 200 U/ml

collagenase II (Worthington Biochemicals) and blebbistatin (33.3 µM, Cayman Chemical) for 15

min. The collagenase-digested hearts were removed from the apparatus and the tissue was

minced gently to separate cells. A suspension was centrifuged (500×g) for 30 s and the cell pellet

was resuspended in KHB-A containing 2% BSA and blebbistatin. The cell suspension was

centrifuged again and resuspended in culture media (M199) containing glutathione (10 mM),

NaHCO3 (26 mM), 100 U/ml penicillin, 100 µg/ml streptomycin, and 5% fetal bovine serum.

Cells were then plated on laminin-coated (40 µg/ml) tissue culture cover slips. After 2 h, the

medium was changed to serum-free M199. Cell suspension supernatant from both spins was

saved for fibroblast isolation. The suspended fibroblasts were centrifuged at 2000 rpm for 10 min

and the cell pellet was suspended in DMEM (Life Technologies) supplemented with 1%

penicillin/streptomycin, and 10% fetal bovine serum (full medium). Cardiac fibroblasts were

grown in a similar full medium until 70–80% confluent and passaged using 0.05% trypsin

EDTA. After trypsin treatment, the fibroblasts were added to the freshly isolated cardiac

myocytes and cultured overnight or until fibroblasts had grown.

Based on our and others’ publications (32, 33, 38, 39), the co-cultures were prepared in

such a way that other cell types were not accidentally present in the dish. The co-culture

experiments presented in the manuscript used freshly isolated cardiomyocytes and fibroblasts

cultured for several days with fibroblast growth media (Life Technologies). The collagenase

enzyme perfused during the procedure dissolves the connecting tissue and leaves free cells. The

gravity sedimentation for approximately 15 min followed by centrifugation (1000 rpm, 5 s)

results in the elimination of cellular debris, red blood cells and endothelial cells. These processes

yield a pure population of cells. Cardiomyocytes from adult rat are the largest cells in the heart

and were readily identified by their typical rod shape and characteristic sarcomere units (width:

10-35 μm and length: 80-150 μm). As a result of their size, a cardiomyocyte-only sediment was

obtained after gravity sedimentation for approximately 15 min and resuspension. In addition, the

media used to culture cardiomyocytes at 37oC was serum-free (M199) and only allowed for

cardiomyocyte survival, which provided an additional step of purification of the cardiomyocyte

cell population. As expected, the remaining cells were rod-shaped and presented with

morphology similar to that found in the intact tissue.

In comparison, fibroblasts were much smaller, round cells and were isolated as follows.

After gravity sedimentation of cardiomyocytes, fibroblasts were separated by centrifugation of

the supernatant. The purity of fibroblasts was further enhanced by culturing the cells and

passaging them for several days (subculture or splitting) which ensured the removal of any

contaminating cardiomyocytes, as cardiomyocytes do not divide and die rapidly.

In vitro co-culture experiments staining pattern

Several important aspects of Calcein AM and PI staining in cell culture preparations are

summarized as follows. When a cell undergoes necrosis, PI stains the nucleic acids, the great

majority of which is found in the cell’s nuclei. At the same time, the cell loses entirely its

Calcein green stain. As a result, a dead cell shows up as isolated red nucleus/nuclei. When a cell

is “alive”, Calcein stains the entire cell volume. Therefore, the entire cell area shows up green.

To ascertain necrotic cell death, we first localized the live cell area that appeared in green. Then,

cell death confirmation was given by the observation of red PI-stained nuclei dots, within the

previously green area. Cardiomyocytes generally are multinucleated cells whereas fibroblasts are

mononucleuated cells. As a result, a dead fibroblast was characterized by single red nucleus dot,

while dead cardiomyocytes were several confluent red dots. It followed that, to accurately

determine whether a cell did die, we looked for one or several nucleus-sized red dot(s) within the

area that had lost the Calcein green fluorescence.

Human cardiomyocytes and fibroblast co-culture

Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCell cardiac myocytes,

Cellular Dynamics International) were thawed and plated on matrigel-coated glass bottom 35-

mm dish using EB-20 media, at a density of 15,000 cells per dish. Human fibroblasts from a BJ

cell line (Stemgent) were added to the culture (20,000/dish) to obtain a co-culture of myocytes

and fibroblasts. After growing the cells for 3 days, the medium was changed to RPMI with B27

supplement. For targeted PDT, CTP-Ce6-PEG was added to the culture, incubated for 2 hours

and then washed three times to remove any unbound CTP-Ce6-PEG. Fluo-4 AM was also loaded

in to the cells for 30 minutes at 37ºC. Calcium fluorescence line-scanning was performed in the

confocal microscopy setting before the start of PDT therapy so as to differentiate myocytes from

fibroblasts. For non-targeted PDT, the experiment was conducted similarly, but with free Ce6.

Co-localization studies

The goal of using a Pearson correlation coefficient analysis was to obtain a quantitative appraisal

of the co-localization of nanoparticle (Ce6) fluorescence pixels with the myocyte/fibroblast

antibody fluorescence pixels (Fig. 2B). This was done with a Pearson correlation coefficient

analysis using a built-in plugin available in the Zeiss confocal microscope analytical software.

The maximum coefficient is 1 and indicates a 100% correlation.

Histology and immunofluorescence imaging

Immuno-fluorescence analysis was carried out on paraffin embedded tissue samples which were

sectioned into slides. The following antibodies were used for staining: Myocytes: anti-heavy

chain cardiac myosin antibody [3-48] (39) (Abcam, ab15, dilution 1:100) with Alexa Fluor 488

(Abcam)–tagged secondary antibody; Fibroblasts: fluorophore-tagged (Sigma, MX640RS100)

anti-Vimentin antibody (Sigma, V6389, dilution 1:10) (40). Endothelial cells and vascular wall

smooth muscle cells were also identified by anti-vimentin staining (40, 41). DAPI (Life

Technologies) was used to counter stain nuclei. Primary and secondary antibodies were diluted

in PBS plus 0.1% Triton X-100 and 5% donkey serum. Images were acquired using a Nikon

A1R confocal microscope (Nikon Instruments Inc.) with sequential laser firing. In vivo images

were analyzed for co-localization and intensity profile using inbuilt plug-ins. To compare PI

fluorescence in myocytes and in neighboring endocytes’ nuclei, we collected the fluorescence

intensity values (unitless) in multiple myocytes and vascular cells’ nuclei after either

cardiomyocyte-specific or nonspecific ablation. Individual cardiomyocyte nuclei PI fluorescence

was compared with the PI fluorescence of the closest endocyte to obtain delta-cardiomyocyte-

endocyte PI fluorescence values.

Supplemental Figures

Figure S1: Characteristics of CTP-Ce6-PEG nanoparticle. (A) Absorbance (left) and

excitation and fluorescence (right) spectra of Ce6, Ce6-PEG, and CTP-Ce6-PEG.

Concentrations: 0.0022 mg/ml Ce6 in PBS (λex = 655 nm) and 0.1 mg/ml Ce6-PEG or CTP-Ce6-

PEG in PBS (λex = 660 nm). Ex, excitation; em, emission. (B) Efficiency of singlet oxygen

production. (Left) Fluorescence spectra of anthracene-9,10-dipropionic acid (ADPA) with Ce6-

PEG solution (0.1 mg/ml in PBS), λex = 378 nm. (Right) Fluorescence change of ADPA, with

linear fit dependent on irradiation time of Ce6-PEG at λex = 660 nm.

Figure S2: Nontargeted control experiments in vitro. Nontargeted PDT in an in vitro co-

culture of primary adult rat ventricular myocytes and adult rat cardiac fibroblasts. Co-cultures

were incubated with free Ce6 (no CTP) for 2 h. Calcein AM and PI dye was added. (A) Laser

illumination (405 nm, 7-10 mW, 8-10 minutes) was used to ablate cells, which were imaged

before and after PDT. Zoomed in view on right shows the fate of single myocyte and fibroblast.

(B) Quantification of fluorescent changes was shown over 10 min duration of PDT starting at

time 0. Images are representative of three experiments. Graphs are representative of single cells

followed over time (every min) to quantify fluorescence changes.

Figure S3: Targeted and nontargeted PDT in a coculture of human cardiac myocytes and

fibroblasts. Co-cultures of iPSC-derived human cardiomyocytes (iPSC-CMs) and human

fibroblasts (BJ cell line) were incubated with Calcein AM and PI (for live/dead cell staining) and

the calcium oscillation indicator Fluo-4 AM. Myocytes could be identified by the presence of

Fluo-4 AM calcium fluorescence oscillations, which were not seen in fibroblasts. (A) Targeted

PDT. Co-cultures were incubated with CTP-Ce6-PEG for 2 h. After nanoparticles were activated

by laser illumination (405 nm, 7-10 mW, 7-10 minutes), cardiomyocytes, but not fibroblasts,

progressively showed PI uptake (red) and calcein-AM loss (green). Scale bar, 100 µm.

Corresponding Fluo-4 fluorescence oscillations are shown to the right for each cell type over the

course of 6 seconds before PDT. (B) Non-targeted PDT. After incubating the co-culture with free

Ce6 (no CTP) for 2 hours, PDT was performed as in (A). Both cardiomyocytes and fibroblasts -

showed PI uptake. Scale bar, 100 µm. Corresponding Fluo-4 fluorescence oscillations are shown

to the right for each cell type over the course of 6 seconds before PDT.

Figure S4: Photoablated lesion depth analysis. (A) Photographs of Langendorff-perfused rat

hearts showing the area of laser illumination on the anterior left ventricle (yellow dashed circles).

(B) Confocal microscopy of PI fluorescence after CTP-Ce6-PEG or control (laser only)

photoablation shows that lesion depth in this representative targeted PDT heart was 2.47 mm (the

other was 2.28 mm, for n = 2). The same field of views was stained with H&E. Scale bars, 1 mm.

Images are representative of 3 hearts (2 PDT ablation and 1 control laser only illumination).

Figure S5: Thermal imaging during PDT ablation. Thermal imaging of the right atrial

appendage during targeted PDT ablation in a Langendorff-perfused sheep heart. Sequential

snapshots were taken every 1 minute. Graphical presentation of temperature changes in all 3

hearts during PDT is in Fig. 7E.

Supplemental Movie Legends

Movie S1: Optical mapping of a rat heart 3 days after photoablation. The movie is

representative of an ex vivo Langendorff-perfused rat heart during pacing at a cycle length of

150 ms. An area of persistent electrical block was observed at the region of photoablation (see

corresponding Fig. 6C, left).

Movie S2: Optical mapping of the sheep right atrial free wall. The movie is representative of

an ex vivo Langendorff-perfused sheep heart during pacing at a cycle length of 250 ms before

(left half of the movie window) and after CTP-Ce6-PEG photo-ablation (right half of the movie

window). An area of complete block was formed acutely during photoablation (see

corresponding Fig. 7A).

Movie S3: Optical mapping of the sheep right ventricle. The movie is representative of an ex

vivo Langendorff-perfused sheep heart during pacing at a cycle length of 300 ms before (left half

of the movie window) and after CTP-Ce6-PEG photo-ablation (right half of the movie window).

Both 1-min (upper lesion) and 2-min (lower lesion) laser illumination led to the appearance of a

complete electrical block (see corresponding Fig. 7A).