Cathodal stimulation in the recovery phase of a propagating planar wave in the rabbit heart reveals four stimulation mechanisms.

The stimulation of cardiac tissue in the recovery phase has significant importance in relation to reentry induction. In the theoretical experiment proposed by Winfree, termed the 'pinwheel' experiment, a point stimulus (S2) is applied in the wake of a freely propagating planar wave (S1). Reentry induced from this S1-S2 pinwheel protocol has been observed experimentally in heart preparations. However, in these experiments, which focused on activation outcomes, only mapping of extracellular voltages has been conducted. The lack of transmembrane potential (Vm) distribution data makes it impossible to analyse the underlying stimulation mechanisms which precede the reentry induction. In this work we sought to elucidate the stimulation mechanisms throughout the heart cycle using the pinwheel protocol. We examined the cardiac tissue responses during and immediately after cathodal stimulation in the refractory wake of a propagating planar wave. The voltage-sensitive dye di-4-ANEPPS was utilized to measure Vm directly from quasi two-dimensional preparations of cryoablated Langendorff-perfused rabbit hearts. Four stimulation mechanisms were observed that depended on the Vm magnitude during S2 cathodal stimulation. Make stimulation always occurred during diastolic stimulation. When stimulation was at the beginning of the relative refractory period (RRP), transitional make-break stimulation was detected. During the RRP the excitation was due to the break mechanism. While approaching the effective refractory period (ERP), the tissue response is characterized by a damped wave mediated response. These four stimulation mechanisms were observed in all hearts whether the S1 planar wave propagation was parallel or perpendicular to the fibre direction. This study is the first examination of Vm and the stimulation mechanisms throughout the cardiac cycle using the pinwheel protocol, and the results have implications in the development and improvement of pacing protocols for artificial cardiostimulators.