Light Transmittance by the Murine Cardiac Tissue

Before stimulating the heart under different conditions, I aimed to interrogate light trans-mittance through the heart under different conditions. The goal of this experiment was to improve knowledge and understand if a change in the experiment (like adding dye or bleb-bistatin) can potentially affect stimulation thresholds and how do they affect in general on any given mouse heart prior to or during the experiment. Therefore, we decided to measure the mean transmittance of the different mice hearts, ventricles and septum in general without considering the specific thickness for each one of them, especially considering they were all healthy mice and no abnormally enlarged or shrunken hearts were expected. This would give us a head start on where and under which conditions the stimulation thresholds might increase or decrease.

6. Optogenetic Characterization of the ChR2 Mouse Heart

In order to estimate the light transmittance by the heart, more specifically by the ventri-cles and septum, we designed the experimental setup shown in Figure 6.2. The sample tissue was illuminated and the light intensity transmitted through the tissue was measured using a photometer through a 1 mm in diameter pinhole. Detailed methods are explained in section 5.2 Materials & Methods.

Figure 6.2.:Setup for light attenuation measurements. Light is emitted from a LED, com-ing down through the tissue and detected on the other side of a 1 mm in diameter pinhole. Used with permission from [2].

The differences in attenuation by the cardiac tissue were measured directly after one of the following conditions:

• perfusion with tyrode,

• perfusion with tyrode and injected with the potentiometric dye Di-4-ANBDQPQ, with a broad excitation spectra with its peak at 603 nm [54],

• perfusion with tyrode and the electromechanical uncoupler Blebbistatin,

• with blood, right after extraction.

In addition, two different wavelengths were used; 470 nm which is used to stimulate ChR2, and 625 nm, we used to excite the potentiometric dye during optical mapping and can also be used in the future to stimulate other opsins. Table 6.1 summarizes the results obtained shown as percentage of transmitted light (intensity with sample/intensity without sample).

As expected, the RV transmitted more light than the LV, ranging from 5-10 times de-pending on the condition. When comparing the transmittance of light at different wavelengths, 470 nm transmittance was at least 5 times smaller for all the cases than 625 nm, while in the LV and RV for the Tyrode perfused case in the ChR2 heart the difference was about 14-fold

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6.2. Results

Table 6.1.:Light transmittance by the heart under different conditions. The heart and its components are enlisted in the main rows on the left side of the table from most to least light attenuating. On the rows next to them, ChR2-expressing hearts are separated from the wild type hearts and in the third column they are further divided into illuminated with either 470 nm or 625 nm. On the columns the various conditions tested are ordered as tyrode had the highest transmittance of light the blood the least. The numbers represent the % of light detected by the photometer coming through the sample from the amount of light detected by the photometer without sample. ChR2 hearts with blood (n=6), tyrode (n=6), dye (n =3), blebbistatin (n=4). Wild type hearts with blood (n=3), tyrode (n=3).

and about 6-fold higher. This clearly supports the idea of using red-shifted opsins in order to increase the amount tissue excited.

Moreover, all the conditions tested affected light attenuation in a different way. The heart and its elements were most translucent when perfused only with Tyrode, which is important to keep in mind since ex vivoexperiments are an important source of knowledge. When the dye was added, more light was absorbed and this can change the outcome of an experiment performed with or without optical mapping. Blebbistatin attenuated even more light than

6. Optogenetic Characterization of the ChR2 Mouse Heart

the dye, and hearts with blood in their chambers and circulation attenuated light the most.

This behavior was observed for both wavelengths and for the whole heart, LV and septum, where dye, Blebbistatin and blood significantly increased attenuation compared to the Tyrode perfused.

Comparing the attenuation by blood, it was at least 2-fold higher than the tyrode solution for all hearts elements except for the RV. Moreover, illuminating a wild type mouse heart or ChR2 mouse heart with 625 nm did not make a difference in the transmitted light. On the other hand, illumination using 470 nm saw a clear drop in transmittance in the case of ChR2 mice hearts compared to the hearts without the channel, reflecting the absorption by the channels.

Considering measurements of the LV wall thickness (x=1.79 mm) obtained via mag-netic resonance [73] in experiments from a different group, we can use the Lambert-Beer law to calculate attenuation coefficients for the ventricle under the different conditions tested as follows:

µ= ln(1/T(x)) /x

with a wall thickness of x=1.79 mm and T(x) from Table 6.1 Results are shown in Table 6.2. Naturally, the highest attenuation coefficients belong to the conditions with the smallest transmittance. These results provide an indication on what to expect if the ChR2-mouse heart were optogenetically stimulated under these circumstances. Therefore the next step was to measure the response using different illumination parameters.

Table 6.2.:Calculated attenuation coefficientµ of the LV under different conditions. On the rows, ChR2-expressing hearts are separated from the wild type hearts and in the third column they are further divided into illuminated with either 470 nm or 625 nm. On the columns the various conditions tested are ordered as tyrode had the highest transmittance of light the blood the least.

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6.2. Results

6.2.2. The Effect of Di-4-ANBDQPQ and Blebbistatin on the pacing