Local regional stimulation of single isolated ventricular myocytes using microfluidics.

The regional manipulation of the microenvironment surrounding single adult cardiac myocytes in a microfluidic structure is described. The flow rates of laminar streams were adjusted such that the fluid interface between an injection flow and a perfusion flow was manipulated laterally to stimulate regions of the cell surface. Using this general principle, we were able to selectively expose defined regions of the cell to test solutions, with predefined pulse durations and frequencies. We demonstrate the transient depolarisation of the cardiomyocyte through the regional chemical stimulation of localized areas of the cell with elevated potassium concentrations (100 mM). The results show that chemical stimulation at frequencies < or = 0.25 Hz evoked Ca(2+) transients and cell shortening, comparable to those induced by electrical (field) stimulation. At higher frequencies the membrane potential failed to recover sufficiently from the depolarisation with the high K(+) solution, possibly because of the slow clearance of the ion from the t-tubular system. To test this hypothesis, the clearance of fluorescently labeled 10 kDa dextran from the t-system was measured and found to be approximately 0.5 s delayed compared to that of the bulk extracellular space, indicating the slow diffusion inside the confined space of the tubular membrane invaginations.