Pacing of primary murine cardiomyocytes expressing human TRPV1 using infra-red laser (optical mapping)

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). The samples (cell culture or mice hearts) were irradiated with laser pulses with a frequency of 4 Hz. Control tests were also performed: electrode stimulation of samples with TRPV1 expression and laser stimulation of samples not expressing TRPV1.

Cardiomyocyte cell cultures and transduction

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 Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (DMEM/F12), 1:1 mixture (BioloT, 1.3.7.2.) supplemented with 10% fetal bovine serum (FBS, Biosera, FB-1001/500), penicillin 100 U/ml /streptomycin 100 mg/ml (PanEko, А065п), and L-glutamine 0.365 g/l (PanEko, Ф032).  As optical mapping requires a dense layer of functional cells, a suspension of isolated cells was seeded at a concentration of 2.5*105 cells/cm2 on 25mm microscope glass coverslips (Heinz Herenz, 1051199). The coverslips were coated with 0.16 mg/mL fibronectin (Imtek, H Fne-С) diluted in phosphate-buffered saline (PBS, PanEko, Р071-1/В-60201) and maintained at 37℃ in 5% CO2 for 12 hours before seeding. For transient expression of the hTRPV1 channel, we used AAV-DJ virus with cTnT_hTRPV1-3×FLAG construct at a MOI of 100,000 VG/cells. The cells were infected on the next day after plating, and the transgene expression peak was observed on the third day after the infection.

Mice heart perfusion

The mouse was anesthetized using an anesthesia machine (R550 Multi-output Animal Anesthesia Machine (RWD, Shenzhen, China)) and decapitated. The heart was isolated and then rinsed with 37°C Tyrode's solution (Sigma-Aldrich, T2145) and heparin (Belmedpreparaty, B01AB01) in concentration 50 ME/ml. Staining with Ca2+ chemical indicator Fluo4-AM (Lumiprobe, 1892-500ug) in concentration 2.8 ug/ml as well as optical mapping were performed in Tyrode's solution at 37°C. Heart fixation to the cannula through the aorta was performed using surgical filament. No time elapsed from the moment of mouse decapitation to the start of perfusion through the cannula was less than 10 minutes. During staining and optical mapping, the heart was perfused using a Langendorff apparatus. The setup consisted of a perfusion circuit and a recording optical system based on a high-speed optical mapping unit (Olympus MVX-10 Macro-View fluorescence microscope (Olympus Co., Tokyo, Japan), Andor iXon-3 Camera 897-U high-speed camera (Andor Technology Ltd., Belfast, UK)). The perfusion circuit was designed to maintain a fixed fluid volume. It consisted of a peristaltic pump (Masterflex L/S Digital Drive, 600 rpm; 115/230 VAC, Masterflex L/S Easy-Load® II Pump Head, SS Rotor; 2-Channel (Cole-Parmer Instrument Company)), a thermostat (Cole-Parmer Polystat Standard 6. 5 L Heated Bath, 150 C, 115 VAC/60 Hz, (Cole-Parmer Instrument Company)), oxygenator (Cole-Parmer Bubble Latcher, Water Shirted Reservoir, Oxygenating Bubbler (Cole-Parmer Instrument Company)). The total volume of fluid circulating in the device was optimized using a compact cardiac chamber made of PDMS polymer (SYLGARD 184, DOWSIL) and Petri dishes (Helicon, N-706201). The minimum volume of perfusion of the heart with oxygenated solution was reduced to 10 mL. For optical mapping, using a syringe pump (Fusion 100 Infusion Pump (Chemix, Stafford, USA)) to perfuse the heart with heated Tyrode's solution, the contour volume was approximately 50 mL.

Optical mapping

Optical mapping with the Ca2+ chemical indicator Fluo4-AM (Lumiprobe, 1892-500ug) in concentration 2.8 ug/ml for mice heart and 4 ug/ml for cell culture was performed in Tyrode's solution (pH 7.25-7.4). The signal was recorded at a resolution of 64×64 pixels and a sampling rate of 239 frames per second (Olympus MVX-10 Macro-View fluorescence microscope (Olympus Co., Tokyo, Japan), Andor iXon-3 EMCCD Camera (Andor Technology Ltd., Belfast, UK) high-speed camera).Data were recorded at 37°C using both thermal and electrode stimulation. The duration and amplitude of the electrode and thermal stimulation ranged from 1 ms to 20 ms duration and from 1 V to 6 V. The stimulation frequency ranged from 2 to 4 Hz unless otherwise stated according to the stimulation protocols. The stimulus was set using a generator (2 MHz USB PC Function Generator, PCGU100 (Velleman, Gavere, Belgium)). Platinum electrodes were used.

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]