Optogenetic Control of Cardiomyocytes via Viral Delivery

2.1 Isolating and Preparing Cells for Adenoviral Infection

1. NRVM culture media: M199 media (Invitrogen, Grand Island, NY, USA) supplemented with 10 % fetal bovine serum (FBS), 12 μM L-glutamine, 0.02 μg/mL glucose, 0.05 μg/mL penicillin–streptomycin, and 10 mM HEPES. Store at 4 ºC. Warm to 37 ºC before use.

2. NRVMs prepared according to Jia et al. [20].

2.2 Determining the Optimal Multiplicity of Infection

1. Custom-designed Ad-ChR2(H134R)-eYFP or other virus containing an appropriate light-sensitive opsin. Viral titer is 9×10¹¹ viral particles (VP)/mL (see Note 1).

2. Phosphate-buffered saline (PBS).

3. NRVM culture media: M199 media (Invitrogen, Grand Island, NY, USA) supplemented with 10 % FBS, 12 μM l-glutamine, 0.02 μg/mL glucose, 0.05 μg/mL penicillin–streptomycin, and 10 mM HEPES. Store at 4 ºC. Warm to 37 ºC before use.

4. NRVM infection media: M199 media (Invitrogen, Grand Island, NY, USA) supplemented with 2 % FBS (see Note 2), 12 μM l-glutamine, 0.02 μg/mL glucose, 0.05 μg/mL penicillin–streptomycin, and 10 mM HEPES. Store at 4 ºC. Warm to 37 ºC before use.

5. Fibronectin (BD Biosciences, San Jose, CA, USA): Prepare a stock solution (5 mg/mL) by adding 1 mL distilled water to the vial, aliquot, and store at −20 ºC. For coating cell culture dishes, dilute stock solution in PBS for a final working concentration of 50 μg/mL. Unused fibronectin can be stored at 4 ºC for up to 1 week.

6. Glass-bottomed dishes (14 or 20 mm) (In Vitro Scientific, Sunnyvale, CA, USA).

7. Tyrode’s solution: Prepare a 5× concentrated Tyrode’s solution by dissolving the following amounts of compounds in 1 L of distilled water: 1.02 g MgCl₂, 2.01 g KCl, 39.45 g NaCl, 0.2 g NaH₂PO₄, and 5.96 g HEPES. This concentrated solution can be stored at 4 ºC for several months. To make a working Tyrode’s solution mix 400 mL distilled water with 0.46 g d-glucose, 750 μL CaCl₂ (for 1.5 mM Ca²⁺ in final volume), and 100 mL of 5× Tyrode’s stock. The concentrations (in mM) are as follows: NaCl, 135; MgCl₂, 1, glucose, 5; HEPES, 5; KCl, 5.4; CaCl₂, 1.5; NaH₂PO₄, 0.33. pH should be adjusted to 7.4 with NaOH.

8. Propidium Iodide (Life Technologies, Grand Island, NY, USA): Stain is packaged as a 1 mg/mL solution. For a final working concentration of 2 μg/mL, dilute 2 μL of stock solution in 1 mL of non-phosphate-containing solution, such as Tyrode’s.

2.3 Patterned Transgene Expression

1. Sylgard^® 184 Silicone Elastomer Kit (Fisher Scientific, Pittsburgh, PA, USA).

2.4 Optogenetic Actuation and Feedback

1. Quest Rhod-4, AM (AAT Bioquest, Sunnyvale, CA, USA): Prepare a stock solution of 0.5 mM which can be stored at −20 ºC until experimental use. The solvent contains 20 % Pluronic (F127) and 80 % dimethyl sulfoxide (DMSO). Dilute to 10 μM in room-temperature Tyrode’s solution for cardio-myocyte staining.

3.1 Isolating and Preparing Cells for Adenoviral Infection

Cells specific to this protocol (NRVM) are isolated using a previously published technique [20]. In short, the ventricular apex is excised from 2- to 3-day-old Sprague-Dawley rats and enzymatically digested overnight with trypsin at 4 ºC and then with collage-nase at 37 ºC the following morning. Cardiac fibroblasts are removed from the cell suspension by a two-stage pre-plating procedure. At the end of the isolation procedure, cardiomyocytes are counted and resuspended in NRVM culture media (see Subheading 2 for recipe) to a concentration of 1.125 × 10⁶ cells/mL.

3.2 Determining the Optimal Multiplicity of Infection

This section describes the viral infection of cardiomyocytes with opsin genes using a suspension approach with proper dosing for maximum efficiency while maintaining cell viability. Carry out all procedures under a Biosafety Level 2 (BSL-2) cabinet unless otherwise specified (see Note 3).

1. Prepare a cell suspension of NRVM at a concentration of 1.125×10⁶ cells/mL in NRVM culture media (containing 10 % FBS, see Subheading 2 for recipe).

2. Remove adenovirus from storage at −20 ºC and place on ice (see Note 4).

3. Separate the total volume of cell suspension into three (or more) conicals for multiplicity of infection (MOI) testing (see Note 5). For this protocol, we will describe MOI testing at 0 (control), 25, and 100.

4. Calculate the amount of viral particles needed for infection at the specified MOI (see Note 6):

   $$\text{Viral}\text{particles}(\text{VP})=(\text{total}\text{number}\text{of}\text{cells})\times (\text{MOI})$$

   For example, to infect 2.25 × 10⁶ cells (2 mL cell suspension at 1.125×10⁶ cells/mL) at MOI 25, 56.25 × 10⁶ VP are required.

5. Calculate and measure out the volume of virus needed for infection:

   $$\text{Viral}\text{volume}(\mu \mathrm{L})=\frac{\text{Viral}\text{particles}(\text{VP})}{\text{Viral}\text{titer}(\text{VP}/\mu \mathrm{L})}$$

   For example, to infect a 2 mL suspension of NRVM (2.25×10⁶ total cells), a total viral volume of 62.5 μL is required.

6. Mix the viral volume (μL) calculated in step 5 with infection media (containing 2 % FBS, see Note 2 and Subheading 2 for recipe), so that the volume of virus and infection media maintain the original cell concentration of 1.125 × 10⁶ cells/mL (see Note 7).

7. Spin down the cells at 720 × g for 4 min.

8. Aspirate the culture media from the conical taking care not to disrupt the cell pellet, and resuspend the cells in the infection media and viral particle mixture from step 6.

9. Incubate the conicals at 37 ºC, 5 % CO₂, for 2 h with agitation every 15–20 min during this time (see Note 8).

10. Spin down the cells at 720 × g for 4 min.

11. Aspirate the infection media from the conical, again taking care not to disrupt the cell pellet, and resuspend the cells in fresh NRVM culture media maintaining the original cell concentration of 1.125×10⁶ cells/mL.

12. Plate the cells in a monolayer at a density of 350–470 k cells/ cm² on fibronectin-coated (50 μg/mL, see Note 9) glass- bottomed dishes.

13. Image transgene expression based on fluorescence of the reporter molecule, in this case eYFP (see Fig. 1).

    Since optimization of the MOI requires maximizing expression efficiency and minimizing cytotoxic effects, propidium iodide (PI) staining can be used to quantify cell death. PI is a membrane-impermeant DNA stain, excluded from viable cells and commonly used to detect dead cells in a given population.

14. Prepare a 2 μg/mL solution of PI diluted in a Tyrode’s solution (see Subheading 2 for recipe). This fluorescent stain is light sensitive, and therefore the application must be completed in the dark.

15. Add 0.5 mL of the 2 μg/mL solution to the glass-bottomed dish containing the cells of interest.

16. Incubate the cells with PI for 2 min.

17. Remove the dye solution and wash with fresh Tyrode’s solution.

18. Image cell viability based on the fluorescence of PI using appropriate excitation and emission filter sets (see Fig. 2). When PI is bound to nucleic acids, the maximum excitation and emission are 535 nm and 617 nm, respectively.

19. Based on the overall transgene (ChR2(H134R)-eYFP) expression and cell viability, determine the optimal MOI to be used in future experiments (see Note 10). For NRVM infected with our custom-designed Ad-ChR2(H134R)-eYFP, the optimal MOI was found to be 25 based on maximal expression and minimal cell death.

3.3 Patterned Transgene Expression

The ability to infect NRVM cells in suspension, as opposed to in dish infection after cell plating, allows for patterned optogenetic transgene expression, as described in this section. To date, we have patterned islands of ChR2(H134R)-eYFP-expressing cells using silicone elastomer stencils, in addition to graded mixtures of infected and uninfected cells mimicking potential in vivo transgene expression patterns.

1. Thoroughly mix silicone elastomer in the ratio of 10 parts base to 1 part curing agent (total weight of this mixture is 2.2 g).

2. Pour elastomer mixture into a 60 mm petri dish. Allow the elastomer to spread out and cover the entire bottom of the dish. This volume will yield a 200–250 μm thick layer of silicone.

3. Bake in an oven at 50–60 ºC for 2 h. Alternatively, the elastomer can be cured at room temperature for 24 h.

4. In the meantime, coat glass-bottomed dishes with 50 μg/mL of fibronectin (see Note 9). Cover dishes and leave in the incubator (37 ºC, 5 % CO₂) for 1–2 h.

5. When the elastomer is finished setting, cut out stencils, such as that illustrated in Fig. 3, in the shape of the desired pattern. The stencil illustrated here will create an island of light-sensitive cardiomyocytes surrounded by normal, uninfected cardiomyocytes or vice versa.

6. When the glass-bottomed dishes have been coated with fibronectin, remove the solution and carefully place the stencils on the glass-bottomed dish in the desired location (see Note 11).

7. Plate a droplet of infected cells (350–470 k cells/cm²) in the stencil cutouts and let sit for 30–40 min (see Note 12). This will be sufficient time to allow the infected NRVM to begin adhering to the dish. The droplet volume is determined in such a way that the open stencil area should be covered at a target density of 350–470 k cells/cm².

8. Carefully, remove the stencil and plate uninfected cells at the same plating density in a monolayer on the glass-bottomed dish.

9. To plate uniform, graded mixtures of infected and uninfected cells, mix various concentrations, such as 35 % infected cells and 65 % uninfected cells, that have been maintained in suspension during infection, and plate these mixtures at a density of 350–470 k cells/cm².

10. Image transgene expression based on fluorescence of the reporter molecule (see Fig. 4 for examples of transgene distributions).

3.4 Optogenetic Actuation and Feedback

This section describes means of functional testing of the cardiomyocyte samples transduced with the opsin of interest. It demonstrates a combined optical actuation and optical sensing. The macroscopic (large field of view) optical mapping described in this section is done using a custom-developed ultrahigh-resolution system which has been previously described in detail [20]. Other charge-coupled diode (CCD)-, photodiode array (PDA)-, and complementary metal-oxide-semiconductor (CMOS)-based optical mapping systems can also be adapted to this protocol (for review see ref. 25). Alternatively, the optical sensing can be done with a microscope-integrated photodetector [17]. Here we focus on the choice of optical sensors (i.e., calcium- and voltage-sensitive dyes) and how to trigger optical excitation in NRVM monolayers.

All measurements described here are recorded at room temperature and in the dark so as not to photobleach the fluorescent dyes and/or activate the opsins.

1. Remove a dish containing NRVM monolayer containing cells expressing ChR2(H134R) from the incubator (37 ºC, 5 % CO₂).

2. Remove culture media from the dish, and incubate cells with 10 μM Quest Rhod-4AM (see Subheading 2 for recipe), a robust calcium-sensitive dye (see Note 13), diluted in room- temperature Tyrode’s solution for 20 min.

3. Remove dye solution, and wash the cells with a 20-min incubation in fresh room-temperature Tyrode’s solution.

4. Place a freshly stained glass-bottomed dish on the optical mapping setup (see Note 14).

5. Record electrical activation of the monolayer by pacing with custom-built platinum electrodes connected to a Myopacer Cell Stimulator or equivalent (see Fig. 5b for example of electrical pacing output and Note 15).

6. Record optical activation of the monolayer using light pulses with a wavelength specific to the opsin integrated into the virus (i.e., 470 nm for ChR2(H134R)) (see Fig. 5b for example of optical pacing output and Notes 16–18). According to a strength–duration curve, the pulse duration can be decreased if higher optical power is used. For example, for our samples, 10-ms optical pulses of 0.01 mW/mm² are sufficient to trigger activity in NRVM uniformly expressing ChR2(H134R) (see Note 19).