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We investigated experimentally dependence of light fluence on treated depth with photosensitization reaction shortly after photosensitizer injection in rabbit myocardial tissue in vivo. In this particular photosensitization reaction scheme, the photosensitizer accumulation characteristics for target region are not available. Meanwhile, the photosensitizer dose and hospitalization period under restricted light circumstance might be reduced. Since both photosensitizer and oxygen supply are governed by blood flow, this photosensitization reaction is influenced significantly by blood flow variation in particular blood vessel occlusion. We employed the myocardial tissue to keep tissue blood flow during the photosensitization reaction because vessel blood flow speed in myocardial tissue is fast to resist vascular occlusion. Surgically exposed rabbits myocardial tissues were irradiated with the light fluence ranging 25-100 J/cm2 by a 663 nm diode laser 30 min after the injection of 2 mg/kg water soluble chlorin photosensitizer, Talaporfin sodium. Two weeks after the irradiation, the rabbits were sacrificed and the histological specimens of the irradiated area were made to measure scar layer thickness. The scar layer tissue thickness of 0.2-3.0 mm was observed microscopically by the light fluence ranging 25-100 J/cm2. The scarring threshold in the deposit light fluence was estimated to 15-25 J/cm3 based on the above mentioned relation assuming constant and uniform myocardial effective attenuation coefficient of 0.72 mm-1. The estimated scarring threshold in the deposit light fluence was lower than the threshold of conventional PDT. Large variation of the estimated threshold value might be attributed to unconsidered PDT parameter such as flow rate inhomogeneity in the myocardial tissue. These results suggested that the photosensitization reaction investigated in this study would be available to apply arrhythmia therapy such as atrial fibrillation.

We have proposed non-thermal electrical conduction block for atrial fibrillation treatment by the photosensitization reaction, in which the interval time between the photosensitizer injection and irradiation is less than tenth of that in conventional way. To study the mechanism of photosensitization reaction-induced electrical conduction block, intracellular Ca 2+ concentration change in rat myocardial cells was measured by fluorescent Ca 2+ indicator Fluo-4 AM with confocal laser microscopy. Measured rapid increase in the fluorescence intensity and a change in cell morphology indicated that cell membrane damage; that is Ca 2+ influx and eventually cell death caused by the photosensitization reaction. To demonstrate myocardial electrical conduction block induced by the photosensitization reaction, surgically exposed porcine heart under deep anesthesia was used. The myocardial tissue was paced with a stimulation electrode. The propagated electrical signals were measured by bipolar electrodes at two different positions. Thirty minutes after the injection of 5-10 mg/kg Porfimer sodium or Talaporfin sodium, the red laser light was irradiated to the tissue point by point crossing the measuring positions by the total energy density of less than 200 J/cm 2 . The electrical signal conduction between the measuring electrodes in the myocardial tissue was delayed by each irradiation procedure. The electrical conduction delay corresponded to the block line length was obtained. These results demonstrated the possibility of non-thermal electrical conduction block for atrial fibrillation treatment by the photosensitization reaction.

We demonstrated a possibility of electrical conduction block by ex vivo and in vivo experiments using rat models to establish a non-thermal treatment for atrial fibrillation by photosensitization reaction (PR). One hour after the injection of 2 mg/kg Talaporfin sodium to Wistar rat, the right ventricle (1.4 mmT) was extracted. Paced with a stimulation electrode, this tissue was placed in a tissue bath and immersed in irrigated Tyrode's solution of 37°C with 8 μg/ml Talaporfin sodium and the gas mixture bubbling of 95% CO2 and 5% O2. The propagated electrical signal was measured by two bipolar electrodes. Exciting light of 670 nm in wavelength was irradiated to the tissue between the bipolar electrodes by the power density of 1 W/cm2. After this irradiation, propagation signal blockade was obtained and continued up to three hours. Rat atrioventricular (AV) node was employed as a target region for chronic model. The heart of Wistar rat was surgically exposed. External four-lead electrocardiogram of this rat was measured. Thirty minutes after the injection of 10 mg/kg Talaporfin sodium to the rat, exciting light of 663 nm in wavelength was irradiated to the AV node by the power density of 500 mW/cm2 for ten minutes. Acute AV block was obtained during the irradiation. Two weeks after this procedure, complete AV block was confirmed. The rat was sacrificed to obtain the tissue specimen. We found that the AV node was replaced by scarring tissue under the microscopic observation of the specimen. We verified possibility of permanent electrical conduction block using PR.

We have proposed a new type of atrial fibrillation treatment with the early state photodynamic therapy (PDT), in which the interval time between the photosensitizer injection and irradiation is shorter than that in conventional way. We had demonstrated the acute electrical blockade by the PDT with talaporfin sodium and a red (670 nm) diode laser in ex vivo and in vivo experiment using rat normal myocardial tissue. The previous study of intracellular Ca2+ concentration measurement in rat cardiac myocytes during the PDT indicated that Ca2+ influx induced by the plasma membrane damage might be the main cause of the acute reaction of myocardial tissue. We found that the cell damage of cardiac myocytes triggered by the PDT was mainly influenced by the site where the photosensitizer exists. In this study, we examined the relationship between the sites of talaporfin sodium existing and cell death phenotypes in response to the PDT, in order to clarify the mechanism of the acute electrical blockade induced by the PDT in myocardial tissue. The talaporfin sodium fluorescence was observed after the various incubation times to visualize the time-lapse intracellular photosensitizer localization. The distribution of the photosensitizer was dependent on the incubation time. The change in intracellular Ca2+ concentration during the PDT was examined with a fluorescent Ca2+ indicator by a high-speed Nipkow confocal laser microscope (CSU-X1, Yokogawa Electric Company). We obtained the Ca2+ dynamics during the PDT which can explain the PDT-induced cell death pathways. We concluded that the Ca2+ influx induced by plasma membrane damage is the possible mechanism of the electrical blockade by the early state PDT.