Stimulation of isolated ventricular myocytes within an open architecture microarray

This work is concerned with the physiological responses of single heart cells within microfluidic chambers, in response to stimulation by integrated microelectrodes. To enable these investigations, which included the measurement of action potential duration, intracellular Ca²⁺ and cell shortening, a series of microfluidic chambers (50 μm wide, 180 μm long, 400 μm high, 500 μm pitch) and connecting channels (200 μm wide, 5000 μm long, 50 μm high, 500 μm pitch) were replica-moulded into the silicone elastomer, polydimethylsiloxane (PDMS). The structures were formed against a master of posts and lines, photolithograhically patterned into the high aspect ratio photoresist SU-8. The chambers within the slab of PDMS were aligned against pairs of stimulating gold microelectrodes (50 μm long, 20 μm wide, 0.1-10 μm thick, 180 μm apart) patterned on a microscope coverslip base, thus defining cavities of ∼4 nL volume. The assembly was filled with physiological saline and single isolated rabbit ventricular myocytes were introduced by micropipetting, thus creating limited volumes of saline above individual myocytes that could be varied between 4 nL and ≥4 μL. The application of transient current pulses to the cells via the electrodes caused transient contractions with constant amplitude (recorded as changes in sarcomere length), confirming that excitation contraction coupling (EC coupling) remained functional in these limited volumes. Continuous monitoring of the intracellular Ca²⁺ (using calcium sensitive dyes) showed, that in the absence of bath perfusion, the amplitude of the transients remained constant for ∼3 min in the 4-nL volume and ∼20 min for the 4 μL volume. Beyond this time, the cells became unexcitable until the bath was renewed. The action potential duration (APD) was recorded at stimulation frequencies of 1 Hz and 0.5 Hz using potential sensitive dyes and was prolonged at the higher pacing rate. These studies show the prolonged electrical stimulation of isolated adult cardiac myocytes in microchambers with unimpaired EC coupling as verified on optical records of the action potential, Ca²⁺ transients and cell sho- rtening. The open architecture provided free (pipetting) access for drug dispensation without cross talk between neighboring microwells, and multiplexed optical detection can be realized to study EC coupling on arrays of cells under both control and experimental conditions.