Comparison of Action Potential Characteristics from Intact Rabbit Myocardium Using 2-Photon Excitation, Widefield Epifluorescence and Microelectrode Recordings

Abstract

2-photon excitation of voltage-sensitive dyes enables determination of sub-cellular electrical activity within intact myocardium at a range of depths. The investigation aimed to compare action potential (AP) characteristics derived from sub-cellular 2-photon imaging in an intact cardiac preparation using Di-4-ANEPPS with epifluorescence and microelectrode voltage recordings. Hearts from male New Zealand white rabbits were Langendorff-perfused at 37°C and paced at a cycle length of 350ms. Preparations were loaded with Di-4-ANEPPS and optical APs recorded using widefield epifluorescence and 2-photon (2P) microscopy. BDM (10mM) and Blebbistatin (10µM) were used to eliminate motion artefacts. Membrane potentials were recorded at 26KHz before and after perfusion of mechanical uncouplers. Using surface microelectrodes Vmax values of up to 120 V.s−1 were recorded. Mean 10-90% AP rise times were 2.78±0.29 ms (mean±S.E.M) using microelectrodes (n=3), compared with 3.91±0.30 ms recorded at 50 µm below tissue surface with 2P imaging (at 2.6KHz). Matching sampling frequency between microelectrode and 2P recordings abolished this difference. Perfusion of mechanical uncouplers did not significantly alter rise time (2.44±0.20 ms vs. 2.78±0.29 ms; before vs. after, P=0.4) or APD90 (134.90±4.13 ms vs. 130.25±2.93 ms; before vs. after, P=0.4). Mean AP rise times for 2P recordings lengthened with increasing tissue depth (3.91±0.30 ms vs. 5.35±0.23 ms; 50 vs. 250µm from surface, P<0.05, n=7) while epifluorescence rise times were consistently longer (7.85±0.32 ms, n=7). Composite images from multiple depths using 2P microscopy displayed rise times approaching those recorded from widefield epifluorescence. These data suggest that tissue light-scattering at increasing depths results in lengthening of measured AP rise times without significantly altering APD. Slower rise times combined with more diffuse images obtained at depths greater than 200µm suggests optical aberrations cause loss of signal resolution at these depths.