Design of a Fluorescence-Based System for Measurement of Transmembrane Potential Variations of Electrically and Mechanically Stimulated Cardiomyocytes

Abstract

Cardiomyocytes are electrically-active heart cells whose electrical properties vary with the local electrical and mechanical environment. Variations in myocytes electrical properties are known to play a role on abnormal rhythms.The purpose of the project is to design a fluorescence-based photodetection system for measurement of transmembrane potential variations by combined electrical and mechanical stimulations in post-culture cardiomyocytes.The isolated cardiomyocytes are seeded on a 10mm x 10mm x 0.127mm silicon sheet held by a pair of pliers, coupled to a stretcher apparatus made of two linear guide systems and two computer controlled linear stepper motors. The cells are kept in bubbled Tyrode solution and are electrically stimulated during the 10 minutes staining by the voltage-sensitive dye di-8-Anepps (Invitrogen) at 5 μmol/L concentration. Field electrical stimulation is done by a pair of parallel carbon electrodes with grounded anode. The cathode voltage is supplied by a bipolar isolation amplifier circuit whose input is a set of pulses from the digital-to-analog converter of a National Instruments card (NI USB-6221). The light source is a green LED array (wavelength = 523nm, NTE Electronics Inc.), with intensity controlled by a Darlington array receiving TTL signals. The emitted fluorescence is filtered (λ > 610nm), converted to voltage with a fast photodiode (S1226-5BK, Hamamatsu), and amplified by an instrumentation amplifier (AD524ADZ, Analog Digital Inc). The voltage is then digitized with a National Instruments card, filtered and saved for post-experiment analysis.Each sub-system has been successfully validated. Testing the whole system with cardiac-derived HL1 cells allowed final improvement on the signal-to-noise ratio and optimization of excitation intensity. This ready to use bioinstrument will play a key role in further studies on cultured cardiomyocytes.