Properties of new, long-wavelength, voltage-sensitive dyes in the heart

Membrane potential measurements using voltage-sensitive dyes (VSDs) have made important contributions to our understanding of electrophysiological properties of multi-cellular systems. Here, we report the development of long wavelength VSDs designed to record cardiac action potentials (APs) from deeper layers in the heart. The emission spectrum of styryl VSDs was red-shifted by incorporating a thienyl group in the polymethine bridge to lengthen and retain the rigidity of the chromophore. Seven dyes, Pittsburgh I to IV and VI to VIII (PGH I-VIII) were synthesized and characterized with respect to their spectral properties in organic solvents and heart muscles. PGH VSDs exhibited 2 absorption, 2 excitation and 2 voltage-sensitive emission peaks, with large Stokes shifts (> 100 nm). Hearts (rabbit, guinea pig and Rana pipiens) and neurohypophyses (CD-1 mice) were effectively stained by injecting a bolus (10-50 μl) of stock solution of VSD (2-5 mM) dissolved in in dimethylsulfoxide plus low molecular weight Pluronic (16% of L64). Other preparations were better stained with a bolus of VSD (2-5 mM) Tyrode's solution at pH 6.0. Action spectra measured with a fast CCD camera showed that PGH I exhibited an increase in fractional fluorescence, ΔF/F = 17.5 % per AP at 720 nm with 550 nm excitation and AF/ F = - 6% per AP at 830 nm with 670 nm excitation. In frog hearts, PGH1 was stable with ∼30% decrease in fluorescence and AP amplitude during 3 h of intermittent excitation or 1 h of continuous high intensity excitation (300 W Xe-Hg Arc lamp), which was attributed to a combination of dye wash out > photobleaching > dynamic damage > run down of the preparation. The long wavelengths, large Stokes shifts, high ΔF/F and low baseline fluorescence make PGH dyes a valuable tool in optical mapping and for simultaneous mapping of APs and intracellular Ca²⁺.