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. 2020 Jan 1;318(1):C163-C173.
doi: 10.1152/ajpcell.00107.2019. Epub 2019 Nov 20.

Real-time visualization of titin dynamics reveals extensive reversible photobleaching in human induced pluripotent stem cell-derived cardiomyocytes

Adrian G Cadar  1   2 , Tromondae K Feaster  3 , Kevin R Bersell  3 , Lili Wang  4 , TingTing Hong  5 , Joseph A Balsamo  3 , Zhentao Zhang  2 , Young Wook Chun  6 , Young-Jae Nam  2 , Michael Gotthardt  7 , Björn C Knollmann  4 , Dan M Roden  4 , Chee C Lim  1   2 , Charles C Hong  6
Affiliations

Real-time visualization of titin dynamics reveals extensive reversible photobleaching in human induced pluripotent stem cell-derived cardiomyocytes

Adrian G Cadar et al. Am J Physiol Cell Physiol. .

Abstract

Fluorescence recovery after photobleaching (FRAP) has been useful in delineating cardiac myofilament biology, and innovations in fluorophore chemistry have expanded the array of microscopic assays used. However, one assumption in FRAP is the irreversible photobleaching of fluorescent proteins after laser excitation. Here we demonstrate reversible photobleaching regarding the photoconvertible fluorescent protein mEos3.2. We used CRISPR/Cas9 genome editing in human induced pluripotent stem cells (hiPSCs) to knock-in mEos3.2 into the COOH terminus of titin to visualize sarcomeric titin incorporation and turnover. Upon cardiac induction, the titin-mEos3.2 fusion protein is expressed and integrated in the sarcomeres of hiPSC-derived cardiomyocytes (CMs). STORM imaging shows M-band clustered regions of bound titin-mEos3.2 with few soluble titin-mEos3.2 molecules. FRAP revealed a baseline titin-mEos3.2 fluorescence recovery of 68% and half-life of ~1.2 h, suggesting a rapid exchange of sarcomeric titin with soluble titin. However, paraformaldehyde-fixed and permeabilized titin-mEos3.2 hiPSC-CMs surprisingly revealed a 55% fluorescence recovery. Whole cell FRAP analysis in paraformaldehyde-fixed, cycloheximide-treated, and untreated titin-mEos3.2 hiPSC-CMs displayed no significant differences in fluorescence recovery. FRAP in fixed HEK 293T expressing cytosolic mEos3.2 demonstrates a 58% fluorescence recovery. These data suggest that titin-mEos3.2 is subject to reversible photobleaching following FRAP. Using a mouse titin-eGFP model, we demonstrate that no reversible photobleaching occurs. Our results reveal that reversible photobleaching accounts for the majority of titin recovery in the titin-mEos3.2 hiPSC-CM model and should warrant as a caution in the extrapolation of reliable FRAP data from specific fluorescent proteins in long-term cell imaging.

Keywords: FRAP; hiPSC-CM; mEos3.2; sarcomere; titin.

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Conflict of interest statement

No conflicts of interest, financial or otherwise, are declared by the authors.

Figures

Fig. 1.
Fig. 1.
Generation of titin-mEos3.2 knock-in model in human induced pluripotent stem cells (hiPSCs). A: titin-mEos3.2 knock-in strategy to replace the stop codon in titin’s terminal exon 363 with an in frame mEos3.2 knock-in into titin’s terminal exon 363. A guide (g)RNA targeting titin exon 363 for cleavage was coelectroporated with a homology directed repair template (mEos3.2, with 600-bp titin flanking homology arms) in hiPSCs. B: gel image of T7 endonuclease assay demonstrates titin-specific gRNA targets exon 363 for cleavage in HEK 293 cells. Briefly, HEK 293 were transiently transfected with 1 μg of PX 459 plasmid encoding a titin-specific gRNA and Cas9 for 36 h. HEK 293 cells were then subjected to antibiotic selection (500 ng/mL, puromycin) for 48 h. HEK 293 cells were then harvested for DNA extraction. A 421-bp loci where the double-stranded breaks occurred was PCR amplified. The PCR product was denatured and reannealed before being subjected to T7 endonuclease. Lane 1: 100-bp ladder. Lane 2: treated with T7 endonuclease. Lane 3: not treated. DNA was visualized by ethidium bromide (EtBr) staining and %cleavage {[sum of cleaved band intensities/(total band intensities)] × 100} was quantified by band densitometry. C: gel image of mEos3.2 sequence integration in hiPSC. hiPSC were electroporated with 10 μg of gRNA and 500 ng of titin-mEos3.2 HDR template. Individual colonies hiPSC were screened for mEos3.2 insertion by PCR using primers flanking outside of the repair template. PCR products were ran on a 1% agarose gel and stained with EtBr. Lane L: 100-bp ladder. Lane C: control. Lanes 1–8: individual hiPSC clones. *mEos3.2 sequence integration. D: chromatogram of genomic sequences from hiPSC with and without mEos3.2 sequence integration. WT, wild type.
Fig. 2.
Fig. 2.
Titin-mEos3.2 human induced pluripotent stem cell-cardiomyocytes (hiPSCs-CMs) express endogenous striated titin-mEos3.2 and are functional. A: live cell bright-field image overlaid with green fluorescence image of day 30 unedited control and titin-mEos3.2 hiPSC-CM. Scale bar = 10 μm. B: day-30 titin-mEos3.2 hiPSC-CMs were fixed and immunostained for Z-disk protein, α-actinin (red, top), or M-band protein, myomesin (red, bottom). Scale bar = 1 μm. Top: alternating bands of titin-mEos3.2 (green) with α-actinin (red). Bottom: colocalziation of myomesin (red) and titin-mEos3.2 (green). Right: corresponding pixel intensity along the white arrow in the merged images. Scale bar = 1 μm. C: representative contraction trace of control hiPSC-CMs and titin-mEos3.2 (unpaced). D: contractility evaluation of cell shortening (left) resting cell length (middle) and cell shortening kinetics in control (n = 6) and titin-mEos3.2 (n = 4) hiPSC-CMs.
Fig. 3.
Fig. 3.
Titin protein dynamics in live and fixed human induced pluripotent stem cell-cardiomyocytes (hiPSCs-CMs). A: representative fluorescence recovery after photobleaching (FRAP) images (prebleach, bleach, recovery) of live and fixed day-30 hiPSC-CMs. Live hiPSC-CMs were treated with 10 μg/mL cycloheximide (CHX), 0.1% DMSO or untreated throughout image acquisition. Fixed hiPSC-CMs were treated with 4% PFA for 15 min and permeabilized with 0.5% Triton X-100 in PBS for 15 min before FRAP. A region of interest (ROI; white outlined box) of 2 sarcomere m-lines were bleached. Scale bar = 5 μm. B: quantification of FRAP images. FRAP images were acquired every 30 min for 9 h. Pixel intensities of the ROI, background, and whole cell were acquired. Background was subtracted from ROI and whole cell measurements. The ROI was normalized to whole cell pixel intensities. Data were fitted into a one-phase association equation to calculate titin-mEos3.2 exchange half-life; n = 4 cells for control and DMSO groups; n = 3 cells for CHX and fixed groups. All data are displayed as means ± SE. AU, arbitrary units.
Fig. 4.
Fig. 4.
Whole cell titin protein dynamics in live and fixed human induced pluripotent stem cell-cardiomyocytes (hiPSCs-CMs). A: representative fluorescence recovery after photobleaching (FRAP) images (prewhole cell postwhole cell bleach and whole cell recovery) of day 30 hiPSC-CMs. Scale bar = 5 μm. B: quantification of FRAP images. FRAP images were acquired every 30 min for 9 h. Pixel intensities of the region of interest (ROI) and background were acquired. Background was subtracted from ROI. Images were normalized to prebleach intensities. Data were fitted into a one-phase association equation to extrapolate a synthesis rate (n = 3 cells for each group). C: titin-mEos3.2 mobile fractions of untreated control, CHX treated, and fixed hiPSC-CMs. AU, arbitrary units.
Fig. 5.
Fig. 5.
Cytosolic mEo3.2 displays reversible photobleaching in HEK 293T cells. A: representative fluorescence recovery after photobleaching (FRAP) images (prebleach, bleach, and recovery) of HEK 293T cells expressing mEos3.2. Scale bar = 10 μm. B: quantification of FRAP images. FRAP images were acquired every 30 min for 9 h. Pixel intensities of the region of interest (ROI), cell, and background were acquired. Background was subtracted from ROI and whole cell. Images were normalized to prebleach intensities. Data were fitted into a one-phase association equation to extrapolate a reversible photoswitching rate (n = 3 cells). C: mobile fractions of fixed mEos3.2-expressing HEK 293T cells. AU, arbitrary units.
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