Calcium dynamics upon heating in murine neonatal cardiomyocytes transduced with truncated human TRPV1(−109 a.a.) and the localization of TRPV1 in these cells

<h3>Abstract</h3> <p>The expression of truncated human TRPV1(&minus;109 a.a.) in cardiomyocytes allowed us to induce action potentials (APs) by pulse irradiation with an infrared (IR) diode laser. We studied the calcium dynamics in these cells (Ca_TRPV.zip image set). Murine cardiomyocytes were transducted by AAV-based vectors bearing construction of TRPV1(&minus;109 a.a.) with mRuby (for expression detection) and vectors bearing GCaMP6s calcium sensor. Cardiomyocytes were heated by IR laser with a repetition rate of 2 Hz and 30 ms pulse width in a calcium-free medium.<br>To explain the observed calcium dynamics, we also studied the subcellular localization of human TRPV1 in Murine cardiomyocytes ER_a-flag_100x_param1_Z-stack002.nd2</p> <h3>Primary cell production and transformation</h3> <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 the 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> <h3>Distant heating system</h3> <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, P, can be computed from trigger voltage, U, by an equation: P [W] = -0.28 + 1.42 * U [V].</p> <p>Intracellular calcium recordings of single cardiac cells<br>Neonatal cardiomyocyte cells transduced with GCaMP6s sensor were viewed and acquired under a water immersion Olympus LUMPLFLN40&times;W objective with 40X magnification. Data acquisition was performed at 20 fps using a Scientifica SliceScopePro 2000 microscope (Scientifica, UK) equipped with a Hamamatsu Orca Flash 4.0 CMOS monochrome digital camera (Hamamatsu Photonics) connected to a PC running the free software uManager. A CoolLED pE-300ultra was used as a light source. It was synchronized with the laser heating system via BNC-TTL output from the Heka Elektronik EPC 10 USB Patch Clamp Amplifier. To synchronize the pacing and the GCamp6s signal registration, the light source was switched off for one acquisition cycle at the beginning of the pacing. The GCamp6s signal was analyzed using Fiji software.</p> <h3><br>TRPV1 channel localization</h3> <p>To understand the localization of the expressed TRPV1 channels in neonatal mice cardiomyocytes, we utilized cells infected with AAV-PHP.S serotype viruses with pAAV_cTnT_hTRPV1_P2A_FLAG-tag at a MOI of 2500 VG/cells. To visualize endoplasmic reticulum, we stained the live cells with ER-Tracker&trade; Red (BODIPY&trade; TR Glibenclamide, Thermo Fisher Scientific, E34250) according to the manufacturer&rsquo;s instructions. After the staining with ER-tracker, cardiomyocytes were fixed with 4% paraformaldehyde (Sigma-Aldrich, 158127-100G) for 5 minutes at room temperature and washed trice with 0.3% Tween 20 (Sigma-Aldrich, P1379-250ML) diluted in PBS (5 minutes each). The fixed cells then were blocked with PBS containing 0.12% tween 20, 1% bovine serum albumin (BSA, PanEko, 68100.10г), and 10% goat serum (Thermo Fisher Scientific, 16210072) for 40 minutes at room temperature. After the buffer removal, the cardiomyocytes were labeled with of DYKDDDDK Tag Recombinant Rabbit Monoclonal Antibody (8H8L17, Invitrogen, MA1-142-A488) at 1:500 dilution in 1% BSA, 10% goat serum, and 89% PBS for 2 hours at room temperature. The samples were washed three times with PBS after the incubations. Then, the cells were stained with Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody Alexa Fluor&trade; 488 (Invitrogen, A-11008) at dilution 1:500 for 1 hour at room temperature. The removal of non-conjugated antibodies was performed in parallel with cell nuclei staining. The cells were incubated with PBS supplemented with 2 &mu;g/ml DAPI (Miltenyi Biotec, 130-111-570) for 15 minutes at room temperature. For further experiments, glasses with the labeled cells were placed onto the Superfrost Plus adhesion slides (Epredia, EPBRSF41296SP) in 20 &micro;l VECTASHIELD Vibrance Antifade Mounting Media (Vector Laboratories, H-1700-2) and stored at +4℃ in the dark.<br>The samples were analyzed using an inverted Nikon A1 confocal microscope and visualized using Nikon NIS-Elements software. We pictured the sample in each channel individually exciting DAPI, DYKDDDDK Tag-Alexa Fluor 488, and ER-tracker by laser lines 405 nm, 488 nm and 561 nm, respectively. Colocolization analysis was performed using ImageJ software.</p>