Development of new voltage-sensitive fluorescent dyes for cardiac optical mapping

Abstract

Cardiac arrhythmias are an important cause of morbidity and mortality. Optical mapping using voltage-sensitive dyes (VSDs) allows to study these disorders in isolated perfused tissues. Until recently, this technique remained limited to the myocardial surface due to the short excitation wavelengths of common VSDs. Using red excitation light allows to by-pass this problem, but availability of such dyes remains limited. To develop new families of red-shifted VSDs and to characterize both their spectral and potentiometric properties. Three families of VSDs were designed. The spectral properties of each VSD was assessed by spectrofluorometry in Langendorff perfused rat hearts. The potentiometric properties were assessed through optical mapping in Langendorff perfused rat hearts. For spectrofluorometry, hearts were freely beating and systolic and diastolic pressures were continuously monitored. In optical mapping experiments hearts were mechanically arrested by supplementation of blebbistatin (10 μM). Out of the three families, one showed a strong shift in excitation and emission wavelengths towards the near-infrared. Within this family, three VSDs, with variations in the hydrophilic tail and charged carrier, were tested. Results showed that these VSDs have peak excitation above 500 nm and peak emission around 730 nm. Although two of these dyes showed a low potentiometric sensitivity (<1%), one VSD had a ΔF/F similar to conventional dyes (>5%) with a stable signal for over 2 hours after a bolus injection in the hearts. In mechanically arrested hearts, the VSDs had no impact on electrophysiological parameters. However, in beating hearts, the VSDs significantly increased diastolic pressure. We have developed new red-shifted VSDs and validate their use in cardiac applications, which might allow optimizing optical mapping of cardiac arrhythmia dynamics in deeper tissue layers and/or in blood-perfused tissues.