New Molecular Scaffolds for Fluorescent Voltage Indicators

Abstract

The ability to non-invasively monitor membrane potential dynamics in excitable cells like neurons and cardiomyocytes promises to revolutionize our understanding of the physiology and pathology of the brain and heart. Here, we report the design, synthesis, and application of a new class of fluorescent voltage indicators that make use of a fluorene-based molecular wire as a voltage-sensing domain to provide fast and sensitive measurements of membrane potential in both mammalian neurons and human-derived cardiomyocytes. We show that the best of the new probes, fluorene VoltageFluor 2 (fVF 2), readily reports on action potentials in mammalian neurons, detects perturbations to the cardiac action potential waveform in human induced pluripotent stem cell-derived cardiomyocytes, shows a substantial decrease in phototoxicity compared to existing molecular wire-based indicators, and can monitor cardiac action potentials for extended periods of time. Together, our results demonstrate the generalizability of a molecular wire approach to voltage sensing and highlight the utility of fVF 2 for interrogating membrane potential dynamics.

Figure 1.

Characterization of fluorene VoltageFluor 2 (fVF 2). a) Live cell fluorescence microscopy image of fVF 2 in HEK cells. Scale bar is 10 μm. b) Normalized absorption and emission spectra of fVF 2. Spectra were acquired in PBS (pH 7.2) +0.1% SDS. For emission scan, excitation was provided at 485 nm. c) Voltage sensitivity of fVF 2 in patch-clamped HEK cells. d) Plot of ΔF/F vs. membrane potential (in mV) for fVF 2. Red line is the line of best fit. Error bars are standard error of the mean for 8 independent determinations. e) Live-cell, wide-field fluorescence images of rat hippocampal neurons stained with 500 nM fVF 2. Scale bar is 20 μm. f) Representative ΔF/F plot of evoked neuronal activity of a single cell recorded optically with fVF 2.

Figure 2.

Fluorene VoltageFluor 2 (fVF 2) reliably reports on cardiac action potential (cAP) dynamics in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). a) Brightfield. b) Membrane localization of fVF 2 (1 μM) in hiPSC-CM monolayers. c) Hoescht 33342 nuclear stain. d) Merge of membrane and nuclear stains. Scale bar is 20 μm. e) Representative image used to acquire functional AP data. Scale bar is 10 μm. f-i) Representative fluorescence traces acquired using 1 μM fVF 2. f) Baseline measurement of spontaneously contracting monolayer. Treatment with cisapride (300 nM) results in g) prolonged APs, h) shorter and more frequent APs, and i) early after depolarizations (EADs). j) Overlay of single APs from f-h to highlight observed waveform changes from cisapride treatment. The baseline trace in black shows a normal ventricular-like shape, the red trace depicts an extended AP after treatment (from panel g), and the blue trace depicts a shortened AP from a tachycardia-like phenotype (from panel h).

Figure 3.. fVF 2 displays low phototoxicity in cardiomyocyte monolayers.

Fluorescence intensity vs time for a) fVF 2 and b) VF2.1.Cl in monolayers of hiSPC-CMs. The raw fluorescence intensity from an entire field of view over an entire 10 s recording session is plotted vs. the total illumination time (in minutes). Individual action potential (AP) traces for c) fVF 2 and d) VF2.1.Cl are indicated by red stars in panels (a) and (b). Plot of mean e) signal-to-noise ratio (SNR ) f) cAPD30, and g) cAPD50 as a function of total illumination time for fVF 2 (black) and VF2.1.Cl (blue). For panels e-g, mean values are determined from n = 3 independent trials, and error bars are ± standard error of the mean. Statistical tests are two-tailed, unpaired t-test for each cAPD at the indicated time vs. t = 0. ** = p<0.005, *** = p<0.001, **** = p<0.0001.

Figure 4.

Fluorene VoltageFluor 2 (fVF 2) reveals changes to cardiac action potentials (AP) upon treatment with cisapride. (a) Action potential duration (APD) values are calculated from the maximum derivative of the depolarization to the repolarization at 70, 50, and 10% of the maximum depolarization value, are corrected for beat-rate, and reported as cAPD30, cAPD50, and cAPD90 values, respectively. Treatment of monolayers cultured for 14 days with doses of cisapride from 0.1 to 300 nM cisapride (red) results in (b) little change in the cAPD30, (c) a moderate increase in the cAPD50, and (d) a clear increase in cAPD90. Black points indicate DMSO-treated control samples. Plots indicate mean values ± standard error of the mean for n = 4 independent experiments.

Scheme 1.

Synthesis of fluorene VoltageFluor dyes (fVF dyes).

Scheme 2.

Synthesis of electron-rich fluorene VoltageFluor dyes