Research ArticleBioengineering

Cell-selective arrhythmia ablation for photomodulation of heart rhythm

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Science Translational Medicine  28 Oct 2015:
Vol. 7, Issue 311, pp. 311ra172
DOI: 10.1126/scitranslmed.aab3665
  • Fig. 1. CTP-Ce6-PEG nanoparticle synthesis and in vitro myocyte-specific uptake and photoablation.

    (A) Schematic of nanoparticle preparation. Primary amine group on the eight-arm PEG is first conjugated to carboxylic acid group on Ce6 by carbodiimide cross-linking chemistry. Then, N-hydroxysuccinimide–PEG-maleimide was used to cross-link CTP- and Ce6-conjugated eight-arm PEG. (B) Coculture of primary adult rat ventricular myocytes (rod-shaped) and adult rat cardiac fibroblasts (irregularly shaped) were incubated with CTP-Ce6-PEG for 2 hours. calcein AM identified all live cells (green), and Ce6 fluorescence is seen in purple. Scale bars, 100 μm. (C and D) Targeted PDT in vitro in a rat myocyte–fibroblast coculture that had been incubated with CTP-Ce6-PEG for 2 hours and then exposed to laser illumination (405 nm, 7 to 10 mW) for 10 to 15 min. Uptake of PI, a dead cell indicator (red), or progressive loss of calcein AM, a live cell indicator (green), was imaged (C) and quantified (D). Images are representative of four experiments. Data are representative single cells followed over time after illumination.

  • Fig. 2. Myocyte-specific targeting of CTP-Ce6-PEG in vivo in rats.

    (A) One hour after CTP-Ce6-PEG injection (1.6 mg of Ce6 per rat), hearts were isolated, fixed, and stained for myocytes [cardiac myosin heavy chain (MHC)] and fibroblasts (vimentin). DAPI (4′,6-diamidino-2-phenylindole) was used to stain nuclei. The Ce6 nanoparticle is the red fluorescence. Scale bar, 50 μm. (B) Colocalization of cardiac or fibroblast fluorescence and Ce6 nanoparticle fluorescence, with corresponding Pearson correlation coefficient analysis (right) presented as a boxplot (n = 3 animals, six images per animal). (C) Heart tissue areas that received PDT, including nonablated surrounding regions, stained for live (calcein AM, green) and dead (PI, red) cells. PI intensity profiles are below the representative images (n = 3 animals, six images per animal).

  • Fig. 3. Targeted photoablation in vivo spares bystander fibroblasts and vascular cells.

    (A) Myocyte-specific (CTP-Ce6-PEG) and nontargeted (Ce6 only) photoablation were performed in n = 3 rats each. The photoablation sites were imaged for PI fluorescence. Cardiac myosin heavy chain identified myocytes, whereas vimentin identified fibroblasts. DAPI was used to stain nuclei. High-magnification images and corresponding fluorescence intensity profiles (right) show nuclear localization of PI. (B) Coronary vessels are not affected by CTP-Ce6-PEG PDT. Myocyte-specific (CTP-Ce6-PEG) and nontargeted (Ce6 only) photoablation were performed in n = 3 rats each. Tissue sections with vessels were stained for myocytes (green), nuclei (DAPI), PI (red), fibroblasts, and vascular endothelial and smooth muscle cells (vimentin, yellow). Fluorescent images from one representative field of view are merged (top), DAPI only (middle), and PI only (bottom). (C) PI fluorescence intensity in cardiomyocyte and vascular cell nuclei after myocyte-targeted and nontargeted PDTs. Data are means ± SD (n = 3 animals, 9 to 13 fields of view, 29 to 48 nuclei). P values were determined by paired Student’s t test. (D) Quantification of the number of nuclei with PI uptake in myocytes, fibroblasts, and vascular endothelial cells after targeted and nontargeted PDTs. Data are means ± SD (n = 3 animals; 11 to 18 fields of view, 257 to 365 nuclei). P values were determined by paired Student’s t test.

  • Fig. 4. In vivo cardiomyocyte-specific photoablation.

    (A) Rat open-chest experimental setup. After anesthesia and lateral thoracotomy, the rat heart was exposed and a bipolar electrode was positioned on the LAA. LV, left ventricle. (B) In vivo PDT. After injection of CTP-Ce6-PEG (n = 5 animals), Ce6 (n = 3 animals), or no injection (sham surgery, n = 5 animals), a laser beam (671 nm, 30 mW, 1-mm diameter) was directed toward the LAA. LAA electrograms were recorded before and during laser illumination. Double, black vertical bars indicate a 6-min break in the electrogram. One representative example in each group is shown.

  • Fig. 5. Electrophysiological effects of myocyte-specific PDT on rat hearts ex vivo.

    (A and B) Inset: Schematic showing the site of laser illumination at the junction PLA-LAA. LAA bipolar electrogram recordings, before and after laser illumination (10 or 60 s), in the presence (A) or absence (B) of CTP-Ce6-PEG (1.6 mg of Ce6 per rat injected before heart extraction). Data are from eight individual animals.

  • Fig. 6. In vivo photoablation and 3-day histology and survival in rats.

    (A) Hematoxylin and eosin (H&E)–stained sections of the photoablation site 3 days after nontargeted (one-half dose of Ce6 only) and cardiomyocyte-targeted (CTP-Ce6-PEG, 1.6 mg of Ce6 per rat) PDTs. Vacuolation and disruption of the tunica media are indicated by arrowheads. Images are representative of n = 2 controls and six targeted animals. Adjacent (1 to 2 mm from the ablated zone) H&E-stained tissue sections from the same animals are shown for comparison. Scale bars, 100 μm. (B) Survival rates 3 days after in vivo PDT. Nontargeted animals received full (1.6 mg of Ce6) or half (0.8 mg of Ce6) dose per rat. Sham animals received only laser illumination and no photosensitizer. P values were determined by log-rank (Mantel-Cox) test. (C) Voltage movie snapshots of Langendorff-perfused rat hearts during pacing removed 3 days after in vivo PDT with CTP-Ce6-PEG or laser only (sham, no Ce6). (The full-dose, Ce6-only animals did not survive up to 3 days postoperatively (B); we therefore did not obtain optical mapping results for these animals.)

  • Fig. 7. Optical mapping of ex vivo sheep hearts after photoablation.

    (A) Sequential voltage movie snapshots obtained before (“laser only,” control) and after PDT in ex vivo sheep hearts (n = 4, same hearts served as both control and PDT group, because nanoparticles were perfused after collecting the data with laser only). Locations of laser illumination site, pacing site, and optically imaged areas are delineated by the red dotted boxes for the right atria (RA) and ventricles (RV). Laser only, no Ce6: 671 nm, 30 mW, 5 min; CTP-Ce6-PEG: the laser illumination was 5 min on right atrial (RA) free wall and 1 and 2 min on right ventricle. (B) Upper panel: Electrogram recordings from the AV node before and after targeted PDT or laser only [n = 3, as one heart from (A) transitioned to ventricular fibrillation before starting AV nodal ablation]. Lower panel: Representative bipolar recordings at a smaller time scale before and after photoablation. (C) Apparent conduction velocity measured in both ablated and nonablated regions (pacing cycle length, 250 ms; laser power, 0.03 W). Thermal imaging of right atrial free wall during PDT shows no significant change in the temperature at the site of ablation. Data are means ± SD (n = 3 to 4). P value was determined by paired Student’s t test.

Supplementary Materials

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

    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.

    Movie S1. Optical mapping of a rat heart 3 days after photoablation.

    Movie S2. Optical mapping of the sheep right atrial free wall.

    Movie S3. Optical mapping of the sheep right ventricle.

    References (3641)

  • Supplementary Material 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{at}med.umich.edu (J.K.); kopelman{at}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 (3641)
    • Legends for movies S1 to S3

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • 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.

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