One and Two-Photon Calcium Uncaging with Visible Light in Cardiac Myocytes

Abstract

In cardiomyocytes a small amount of Ca2+ enters the cytoplasm upon depolarization and initiates Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR). Spontaneous SR Ca2+ release events can remain highly localized or initiate Ca2+ waves that propagate through the cell. A powerful optical method for spatio-temporal control of [Ca2+]i is one- and two-photon uncaging. We have designed a nitroaromatic photochemical protecting group absorbing visible light very efficiently in the violet to blue range. The chromophore is a dinitro derivative of bisstyrylthiophene bearing two EGTA chelators (BIST-2EGTA). BIST-2EGTA has an absorption maximum at 440 nm, quantum yield of photolysis of 0.23, extinction coefficient at 440 nm of 66,000 M−1cm−1 and binds Ca2+ with high affinity (Kd 84 nM at pH 7.2). Fluorescence imaging with a confocal microscope in point scan mode revealed that rhod-FF showed a rapid change in signal (<200 µs) when BIST-2EGTA was photolyzed using two-photon excitation at 810 nm. Compared to DM-nitrophen the fluorescence signal from BIST-2EGTA was ∼13.7x larger when photolyzed under the same conditions and could be elicited on physiologically relevant time-scales (<1 ms). The efficiency of BIST-2EGTA to create changes in [Ca2+]i that are useful for physiological studies was examined in whole-cell patch-clamped cardiomyocytes loaded with BIST-2EGTA and rhod-2 or X-rhod-5F. Short (1 ms) two-photon excitation at 810 nm produced localized Ca2+ releases that were considerably larger than those produced by BIST-2EGTA photolysis in caffeine-treated cells, suggesting active SR Ca2+ release. Uncaging for longer periods triggered intracellular Ca2+ “mini-waves” or propagating Ca2+ waves. Cells displayed normal excitation-contraction coupling, implying that BIST-2EGTA is nontoxic. One-photon uncaging at 405 nm also produced striking Ca2+ waves. BIST-2EGTA has a set of properties that enables highly efficient and fast control of Ca2+inside living cells using visible light.