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

<h2>Abstract</h2> <p>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 <em><strong>05-12-22.zip</strong></em>, there are some records where electrical stimulation were used alone or together with IR pulses. Experimental conditions are summarized in the file&nbsp;<em><strong>experiment-descriptions.tsv</strong></em>.</p> <h2>Primary cell production and transformation</h2> <p>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&rsquo;s instructions. The cells were cultured in DMEM/F12, 1:1 mixture (BioloT, &nbsp;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.</p> <h2>Distant heating system</h2> <p>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 &mu;m and 0.22NA. The LD was mounted onto a TEC-controlled plate &ldquo;264 TEC HP LaserMount&rdquo; (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, <em><strong>P</strong></em>, can be computed from trigger voltage, <em><strong>U</strong></em>, by an equation: <em><strong>P [W] = -0.28 + 1.42 * U [V]</strong></em></p> <h2>Electrophysiology of single cardiac cells&nbsp;</h2> <p>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&deg;C in current-clamp mode using a HEKA EPC-10 amplifier (List Elektronik) with a sampling rate of 100 &mu;s. Steady state current was injected to achieve a membrane potential of approximately &minus;60 to &minus;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.&nbsp;</p>