Pacing of primary murine cardiomyocytes expressing human TRPV1 using infra-red laser

Abstract

The expression of human TRPV1 in cardiomyocytes allowed us to induce action potentials (APs) by pulse irradiation with infra-red (IR) diod laser. Mice cardiomyocytes were transformed by AAV-based vectors bearing construction of TRPV1 with mRuby (for expression detection). We selected cells having their own intrisic AP generation and then set them up for measuring membrane potential in a current-clamp mode. Cells were irradiated using 2 or 7 Hz laser pulses. Laser trigger pulses were recorded along with cell potential. In the dataset 05-12-22.zip, there are some records where electrical stimulation were used alone or together with IR pulses. Experimental conditions are summarized in the file experiment-descriptions.tsv.

Primary cell production and transformation

Experiments were carried out using C57Bl/6J mice (The Jackson Laboratory, #000664, RRID: IMSR_JAX:000664). The mixed mouse primary neonatal cardiomyocyte cell culture was obtained using a neonatal heart dissociation kit (Miltenyi Biotec, 130-098-373) according to the manufacturer’s instructions. The cells were cultured in DMEM/F12, 1:1 mixture (BioloT,  1.3.7.2.) supplemented with 10% FBS, penicillin 100 U/ml /streptomycin 100 mg/ml, and L-glutamine 0.365 g/l. The culture was seeded on 10mm coverslips coated with 10 mg/ml gelatin diluted in PBS and maintained at 37℃ in 5% CO2. For transient expression of the hTRPV1 channel, a reporter protein, and a fluorescent Ca2+ sensor GCaMP6s, we used AAV-based vectors with the encoded genes above. We used AAV-DJ serotype at a MOI of 12,000 VG/cells for cTnT_hTRPV1(sh)_P2A_mRuby based viruses, and a MOI of 2,500 VG/cells for cTnT_GCaMP6s ones. The cells were infected on the next day after plating, and the transgene expression peak was observed on the third day after the infection.

Distant heating system

The system was equipped with a fiber coupled laser diode (LD) 4PN-117 (SemiNex) as a powerful heating laser, providing radiation at a wavelength of 1375 nm with an average power of up to 4.3 W through a multimode fiber with a core diameter of 105 μm and 0.22NA. The LD was mounted onto a TEC-controlled plate “264 TEC HP LaserMount” (A.I.), which was operated by TEC driver TECSource 5305 (A.I.); current stabilization and control for LD were performed with LD driver LaserSource 4320 (A.I.). The laser was controlled via the TTL output from the HEKA EPC-10 amplifier. Different laser intensities, and pulse widths were used. Laser power, P, can be computed from trigger voltage, U, by an equation: P [W] = -0.28 + 1.42 * U [V]

Electrophysiology of single cardiac cells 

Patch electrodes were pulled from hard borosilicate capillary glass (Sutter Instruments flaming/brown micropipette puller) and filled with an intracellular solution consisting of (in mM) K-gluconate, 100; KCl, 40; HEPES, 10; NaCl, 8; MgATP, 4; MgGTP, 0.3; phosphocreatine, 10 (pH 7.3 with KOH). Cells were identified visually using IR-video microscopy using a Hamamatsu ORCA-Flash4.0 V3 Digital sCMOS camera (Hamamatsu Photonics) expression of wild type or mutant TRPV1 was confirmed by the presence of red flourescent protein. Coverslips were placed in a recording chamber continuously perfused with heated Tyrode's solution. Whole-cell recordings were taken at 32°C in current-clamp mode using a HEKA EPC-10 amplifier (List Elektronik) with a sampling rate of 100 μs. Steady state current was injected to achieve a membrane potential of approximately −60 to −90 mV. For experiments using the IR laser the optic fiber was placed near the cell, and light from a green laser diode was shone onto the cell to check correct positioning.