Sensing Cardiac Electrical Activity With a Cardiac Myocyte–Targeted Optogenetic Voltage IndicatorNovelty and Significance
Rationale: Monitoring and controlling cardiac myocyte activity with optogenetic tools offer exciting possibilities for fundamental and translational cardiovascular research. Genetically encoded voltage indicators may be particularly attractive for minimal invasive and repeated assessments of cardiac excitation from the cellular to the whole heart level.
Objective: To test the hypothesis that cardiac myocyte–targeted voltage-sensitive fluorescence protein 2.3 (VSFP2.3) can be exploited as optogenetic tool for the monitoring of electric activity in isolated cardiac myocytes and the whole heart as well as function and maturity in induced pluripotent stem cell–derived cardiac myocytes.
Methods and Results: We first generated mice with cardiac myocyte–restricted expression of VSFP2.3 and demonstrated distinct localization of VSFP2.3 at the t-tubulus/junctional sarcoplasmic reticulum microdomain without any signs for associated pathologies (assessed by echocardiography, RNA-sequencing, and patch clamping). Optically recorded VSFP2.3 signals correlated well with membrane voltage measured simultaneously by patch clamping. The use of VSFP2.3 for human action potential recordings was confirmed by simulation of immature and mature action potentials in murine VSFP2.3 cardiac myocytes. Optical cardiograms could be monitored in whole hearts ex vivo and minimally invasively in vivo via fiber optics at physiological heart rate (10 Hz) and under pacing-induced arrhythmia. Finally, we reprogrammed tail-tip fibroblasts from transgenic mice and used the VSFP2.3 sensor for benchmarking functional and structural maturation in induced pluripotent stem cell–derived cardiac myocytes.
Conclusions: We introduce a novel transgenic voltage-sensor model as a new method in cardiovascular research and provide proof of concept for its use in optogenetic sensing of physiological and pathological excitation in mature and immature cardiac myocytes in vitro and in vivo.
Optical imaging of transmembrane voltage and intracellular calcium changes during systole and diastole offers a unique possibility to gain insight into pathophysiological processes underlying cardiac development and disease.1–3 Electric recordings by extracellular (eg, surface) or impaling electrodes and patch-clamp techniques are widely used for the characterization of myocardial electrophysiology from the tissue to the cellular level. However, classical intracellular electrophysiology is invasive, time-consuming, resource-intensive, and can typically not be performed repetitively in the same cardiac preparation over extended periods of time. Moreover, limited spatial resolution of extracellular electrophysiological techniques does not allow for precise assessment of conduction disturbances at the micrometer scale. As an alternative methodology, optical imaging of transmembrane voltage by use of voltage-sensitive dyes3–9 offers higher spatial and temporal resolution, but is hampered by cardiac toxicity and the inability to perform repeated measurements in chronic experiments. These limitations can be overcome by innovations in optogenetics, namely by the introduction of genetically encoded voltage indicators (GEVIs). Different GEVIs have been used to date mainly for imaging of electric activity in mouse10,11 and drosophila12 brain, as well as in fish heart.13 Recently, the ArcLight GEVI has been transduced in human induced pluripotent stem cell (iPSC)–derived cardiac myocytes for imaging of action potentials (APs) in drug screening applications.14
Editorial, see p 390
In This Issue, see p 389
The first GEVI was reported in 1997 and consisted of a fusion between GFP and a voltage-gated ion channel.15 Although this and other early GEVIs only functioned well in Xenopus oocytes, more recent efforts by several groups focused on the use of isolated voltage sensing domains and specifically tuned single fluorescent proteins or made use of Förster (fluorescence) resonance energy transfer (FRET).16 In cardiac myocytes and especially in beating heart tissue, FRET-based ratiometric imaging is preferred as it can be applied to minimize motion artifacts.
Because of its reasonably fast kinetics, the apparent biological inertness of the voltage-sensitive domain from Ciona intestinalis, reliable membrane targeting in excitable tissue, and its design for ratiometric imaging, the voltage-sensitive fluorescent protein 2.3 (VSFP2.3) reporter seemed well-suited for recording membrane voltage in cardiac myocytes. To date, sensors of the VSFP family have been mainly used to report membrane voltage changes in cultured neurons as well as in the brains of living mice.10 Whether VSFP2.3 would be of similar use in cardiac myocytes has not been investigated.
The purpose of this study was to establish the first cardiac optogenetic voltage sensor mouse model with robust expression and function of VSFP2.3 under the control of the cardiac myocyte–specific α-myosin heavy chain (αMHC) promoter.17 Analysis at the cellular and whole organ level revealed no transgene toxicity and produced highly reliable optical recordings of cardiac myocyte–specific membrane voltage changes during physiologically and pathologically stimulated contraction cycles. Furthermore, we demonstrate the applicability of VSFP2.3 for the monitoring of cellular function and maturation in iPSC-derived cardiac myocytes.
Experimental animals were maintained in accordance with the guiding principles of the American Physiological Society. Animal experiments were approved by the Niedersächsisches Landesamt fuür Verbraucherschutz und Lebensmittelsicherheit.
Generation of Transgenic Mice
Transgenic mice were generated by pronuclear injection of αMHC-VSFP2.3 cDNA (7783 bp; Figure 1A) released by BamHI/PmeI digestion (Online Figure I). αMHC-GCaMP2 mice were generated as reporter controls with known cardiac toxicity using a similar strategy.
Myocardial structure and function were assessed by echocardiography using a Vevo 2100 system (Visual Sonics Inc, Toronto, Canada).
Isolation of Adult Mouse Cardiac Myocytes
Adult cardiac myocytes were incubated with mouse anti–ryanodine receptor type 2 and anti–calcium channel, voltage-dependent, L-type, α1C subunit antibodies followed by suitable secondary antibodies (Online Table I) for analyses by confocal laser scanning microscopy (LSM710/NLO, Zeiss).
RNA Sequencing and Bioinformatics
RNA sequencing was performed on an Illumina HighSeq-2000 platform (single read 50 bp; >40 Mio reads per sample). Transcripts with an average RPKM (reads per kilobase per million mapped reads)value of >1 were considered expressed and identified as differentially regulated using a minimum absolute log2-fold change of 1 and Benjamin and Hochberg–adjusted P value (false discovery rate) of 0.05. VSFP2.3 transcript abundance was estimated by aligning raw FASTQ RNA reads to the VSFP coding sequence using bowtie2. Gene ontology enrichment analysis of bioprocesses was performed with GOSeq.20 RNA sequencing data were deposited in the National Center for Biotechnology Information’s Gene Expression Omnibus (GEO series accession number GSE69190).
Combined Patch-Clamp and Fluorescence Recordings in Isolated Cardiac Myocytes
Cardiac myocytes were investigated using the patch-clamp technique under whole-cell configuration. Cyan and yellow light was detected with 2 photomultiplier tubes connected to an Optoscan system (Cairn Research; Online Figure II). Voltage-dependent fluorescence changes were assessed in voltage-clamp mode. In current-clamp experiments, APs were triggered by injecting 2 nA/2 ms currents at 1 Hz. Prerecorded APs of human embryonic stem cell–derived cardiac myocytes and human left ventricular cardiac myocytes were imposed on VSFP2.3-expressing mouse cardiac myocytes in voltage-clamp mode to assess VSFP2.3 responses to longer, simulated human APs.
Whole Heart Optical Mapping
Hearts were imaged (MVPLAPO 0.63x, NA 0.15, Olympus) under mercury lamp (HBO103W/2, Olympus) illumination, using a 438±24 nm BP filter (cyan fluorescent protein [CFP] excitation, Semrock). Emitted light was band passed by a 542±27 nm filter (yellow fluorescent protein [YFP] emission, Semrock, Inc) and recorded with a 100×100 pixel CMOS camera (Ultima-L, SciMedia) at a frame rate of 500 Hz.
Fiber Optic Recordings
Three polymethyl methacrylate fibers (1 mm individual fiber diameter; Edmund Optics Inc) were assembled into a triangular array. Illumination provided by a metal halide lamp (Photofluor II, 200 W, AHF analysentechnik AG) passed a filter cube (excitation filter, ET436±20 nm; excitation dichroic, 455 nm LF) and was guided through the optic fiber bundle to directly illuminate the heart. Emission light was collected by the same fiber bundle, passed through a second (emission) filter cube (emission dichroic, 510 nm LPXR; CFP, ET480±40 nm; YFP, ET535±30 nm), and directed to 2 EMCCD cameras (Cascade 128+, Photometrics) for separate recordings of CFP and YFP signals. Proof-of-concept experiments were performed first in Langendorff-perfused hearts and subsequently in anesthetized (2% isoflurane) and mechanically ventilated mice via a left lateral thoracotomy.
Generation of αMHC-VSFP2.3/Neomycin Resistance Gene iPSC Lines
iPSCs were generated from tail tip fibroblasts of a double transgenic mouse–expressing VSFP2.3 and a neomycin resistance gene under the control of the cardiac myocyte–restricted αMHC promoter (Online Figure III) according to standard protocols.21 The genotype was confirmed by polymerase chain reaction and evidence for pluripotency was collected by reverse transcriptase polymerase chain reaction and immunofluorescent staining for stemness markers (Online Table I).
Differentiation and Selection of Cardiac Myocytes From αMHC-VSFP2.3/Neomycin Resistance Gene iPSCs
Cardiac differentiation was performed in spinner flasks for 11 days followed by additional 7 days of G418 (200 μg/mL) for cardiac myocyte selection. Resulting embryoid bodies showed spontaneous beating and were subsequently dissociated enzymatically. Purity of cardiac myocytes was assessed by flow cytometry (BD LSR III) after staining for sarcomeric α-actinin (Sigma). iPSC-derived cardiac myocytes were cultured as monolayers for additional 5 or 12 days. For FRET imaging, iPSC-derived cardiac myocytes were plated on custom-made recording chambers, paced at 1 Hz under field stimulation and recorded with a dual photomultiplier setup (IonOptix) with appropriate CFP and YFP filters (CFP excitation, 436±20 nm; emission, 480±40 nm [CFP] and 535±30 nm [YFP]).
Data are displayed as mean±SEM unless stated otherwise. AP kinetics were calculated by a custom-made algorithm programmed in Matlab. Fluorescence signals were low-pass filtered at 250 Hz before determining AP characteristics. Statistical analyses were performed using Prism5 software (GraphPad) and the respectively indicated tests. The probability of a type I error was set to α=0.05.
Detailed Methods are available in the Online Data Supplement.
Stable Expression of VSFP2.3 With No Evidence for Cardiotoxicity
Forty-three pups were obtained from ≈200 pronuclear injections, and 6 transgenic founder candidates were selected by genotyping. Offspring from 4 of 6 founders (F0-generation) showed unambiguous myocardial YFP fluorescence, indicating VSFP2.3 expression throughout the heart, with differences in expression strength in the individual αMHC-VSFP2.3 founders (Figure 1B). Fluorescence emission spectrum scans demonstrated the anticipated spectral properties of VSFP2.3 with characteristic peaks at 477 nm (Cerulean/CFP) and 527 nm (Citrine/YFP), confirming expression of both fluorescent proteins (Figure 1C).
Based on previous reports and our own experience, overexpression of optogenetic transgenes in the heart (such as GCaMP222) can impair cardiac function or myocardial structure. We tested for potential transgene toxicity by echocardiography in male and female transgenic mice (n=4/sex/mouse line) and did not find differences in myocardial structure and contractility in the αMHC-VSFP2.3 lines and control wild-type mice (Figure 2A–2D; Online Figure IV), confirming that the VSFP2.3 did not cause cardiotoxicity even at high abundance (line no. 123); this was in marked contrast to mice overexpressing the GCaMP2 calcium sensor under αMHC control. We further scrutinized VSFP2.3 transcript abundance and whether transcription of protein encoding genes was altered by VSFP2.3 overexpression. VSFP2.3 was among the 60 most abundant transcripts identified by RNA sequencing, expressed at similar level as myosin-binding protein C (Mybpc3) and ≈4-fold lower as compared with αMHC (Myh6; Figure 2E). Only 170 of the expressed 11 584 protein encoding transcripts were identified as differentially regulated in VSFP2.3 mice (Figure 2F), with 106 of those associated with the gene ontology term immune system process. Nppa (atrial natriuretic peptide) was the only differentially expressed cardiac gene, suggesting a mild molecular, but structurally and functionally unapparent phenotype. This lack of transgene toxicity was further confirmed by patch clamping of cardiac myocytes from WT and VSFP2.3#123 mice (Online Table II).
Targeting of VSFP2.3 to the t-Tubulus/Junctional SR Microdomain
Confocal microscopy of isolated cardiac myocytes suggested that VSFP2.3 was strictly localized to sarcolemmal membranes and the abundant transverse (t)-tubule membrane invaginations typical for mature cardiac myocytes (Figure 3A). Costaining for the sarcolemmal Cav1.2, α1C pore-subunit and the principal intracellular SR calcium release channel in the heart, that is, the ryanodine receptor type 2, confirmed the anticipated localization of VSFP2.3 at the t-tubulus/junctional SR microdomain (Figure 3B and 3C). Interestingly, VSFP2.3 was also highly expressed at the cell surface, both at the intercalated disc and the lateral surface membrane.
VSFP2.3 Reports Membrane Voltage in Cardiac Myocytes
Isolated adult cardiac myocytes subjected to voltage steps under whole-cell patch-clamp configuration showed voltage-dependent fluorescence changes similar to the properties of VSFP2.3 measured in PC12 cells and cortical neurons,23,24 with decreased CFP fluorescence and increased YFP fluorescence on membrane depolarization (Figure 4A). The baseline normalized ratio of YFP over CFP fluorescence (ΔR/Ro; Figure 4B) was measured during the last 10 ms of each 100 ms voltage step and could be well fitted with a Boltzmann function, revealing a half maximal optical response at −48±2 mV (n=10) and a dynamic range of 15% ΔR/Ro (Figure 4C, left).
To assess the kinetics of VSFP2.3 in cardiac myocytes, we fitted the rising and falling fluorescence intensities in response to voltage steps with a single or double exponential function and found that the rising signals were best described by 2 time constants, τON,fast and τON,slow of 3±0.8 and 16±1.6 ms, respectively (Figure 4C, middle; n=10, at +20 mV). Fluorescence signal decay was well fitted by a single exponential function with a time constant τOFF=31±3 ms (Figure 4C, right; n=10, at +20 mV).
The relatively slow on time constant accounted for the lack of the phenotypically characteristic fast phase 0 depolarization of mouse cardiac myocyte APs in VSFP2.3 responses (Figure 4D). Despite the slow off time constant of VSFP2.3, we observed that electric and optical APD (action potential duration) correlated well, with the strongest correlation noted between electric APD90 and optical APD50 (R2=0.88; P<2.2e−16), suggesting that bona fide electric repolarization (APD90) can be approximated robustly from the optical APD50 values in mouse cardiac myocytes (Figure 4E) by linear regression analysis: electric APD90=1.125×optical APD50+19.8 ms. Stimulation with the adrenergic receptor agonist isoprenaline (1 μmol/L) and increasing electric stimulation frequency (0.5–2 Hz) shortened the optical APD as anticipated (Online Figure V). Simulations of the longer lasting human APs from adult cardiac myocytes and the slower APs from human embryonic stem cell–derived cardiac myocytes in VSFP2.3-mouse cardiac myocytes with the AP clamp technique indicated the applicability of the VSFP2.3 sensor for evaluations of upstroke and repolarization in human cardiac myocytes (Figure 4F).
Optical Cardiogram Recordings in VSFP2.3 Transgenic Mouse Hearts
For assessing whether cardiac myocyte-restricted VSFP2.3 could be exploited to record optical cardiograms (OCGs), a fast camera system at a frame rate of 500 Hz was used to visualize the endogenous YFP signal response from the epicardium (8×8 mm field of view) under CFP excitation. Fractional changes of YFP signals (ΔF/F) were derived and denoted as optical APs or OCGs. As the maximum fractional change using this imaging configuration did not exceed 2.5%, spatiotemporal filtering with binning of 3×3 or 5×5 pixels was performed to increase the signal/noise ratio of the OCG. At sinus rhythm, OCGs were synchronous with the simultaneously recorded ECGs (Figure 5A; Online Movie I). Importantly, electric pacing of the heart at the physiological frequency of 10 Hz confirmed 1:1 capture, evidenced by robust OCG signal spread (Figure 5B; Online Movie II). Subsequently, activation times were selected using a 50% threshold and signal averaging from consecutive pacing cycles was used to construct representative activation maps from the cardiac VSFP2.3 signal. These data confirmed that the transgenic VSFP2.3 sensor captures the expected anisotropic activation spread in the heart at maximal conduction velocity of 0.58±0.14 m/s and minor perpendicular conduction velocity of 0.25±0.08 m/s (n=6). Finally, we resolved fast, abnormal cardiac rhythms classified by anatomic origin as ventricular arrhythmia induced by 200-pulse trains administered at 12.5 Hz (Figure 5C; Online Movie III).
Development of a Fiber Optic System for In Vivo OCG Recordings
To advance toward our goal of minimally invasive in vivo recordings of spread of excitation, we assembled a prototype fiber optic imaging system for ratiometric analysis of VSFP2.3 signals, comprising a bundle of 3 optical fibers connected to 2 high-speed cameras (Figure 6A). We first tested this set-up in Langendorff-perfused, spontaneously beating hearts from αMHC-VSFP2.3 mice (line no. 123) without (Figure 6B) and with (Figure 6C) the application of blebbistatin (used for electromechanical uncoupling) and observed the anticipated CFP/YFP signals in both groups. Proof-of-concept in vivo studies confirmed the use of the fiber optic approach in the presence of normal blood perfusion (Figure 6D), with signal kinetics similar to the ones observed in the Langendorff experiments (Table).
VSFP2.3 in iPSC-Derived Cardiac Myocytes for Functional and Morphological Studies
Finally, we tested the use of the VSFP2.3 sensor in documenting structural and functional maturation in iPSC-derived cardiac myocytes. For this, we first generated double transgenic mice by crossing αMHC-neomycin resistance gene (expression of the neomycin resistance in cardiac myocytes25) and αMHC-VSFP2.3 mice (line no. 123; Online Figure IIIA). iPSC lines were then generated from tail tip fibroblast using standard procedures and pluripotency was confirmed (Online Figure IIIB and IIIC). Under G418 selection in spinner flask cultures, cardiac myocyte purities of >85% could be achieved (Figure 7A). In extended cardiac myocyte cultures, that is, 11+7+5 and 11+7+12 days, clear sarcolemma targeting of VSFP2.3 was visible (Figure 7B). However, the lack of t-tubulation at both investigated time points confirmed low structural maturity in iPSC-derived cardiac myocytes (refer to Figure 3 for comparison with the classical signal pattern in an adult cardiac myocyte from a αMHC-VSFP2.3 mouse [line no. 123]). Despite limited structural maturation, we could exploit the VSFP2.3 expression to study membrane voltage changes in iPSC-derived cardiac myocytes (Figure 7C). The fractional change in CFP/YFP-FRET (ΔR/R) in iPSC-derived cardiac myocytes was ≈4% (Figure 7D) and thus comparable with the signals observed in adult cardiac myocytes (Table). Notably, signal increase velocity (max. upstroke, 5.3±0.6 versus 2.8±0.1 ΔR/s) and signal duration (APD90, 209±22 versus 491±46) clearly distinguished adult from fetal-like iPSC-derived cardiac myocytes (Table). Importantly, simultaneous recordings of electric APD90 and optical APD50 correlated similarly well in iPSC-derived cardiac myocytes (Online Figure VI) as in adult cardiac myocytes (Figure 4E) from VSFP2.3 mice; note however that electrophysiological recordings in the small iPSC-derived cardiac myocytes was technically difficult to perform.
Optogenetic tools are evolving rapidly and have been widely introduced to monitor and control neuronal activity.23,26 With the introduction of channelrhodopsin-2 into cardiac myocytes it became possible to control the beating behavior in isolated cardiac myocyte and whole heart preparations.27 For monitoring of electric activity in whole heart, chemical probes remain the gold-standard since their introduction ≈40 years ago.8 Here, we demonstrate the use of an advanced GEVI and classical targeted transgenesis to introduce VSFP2.3 into cardiac myocytes for in vitro and in vivo monitoring of physiological and pathological electric activity in mature and immature cardiac myocytes as well as whole hearts. In addition, we provide proof of concept for the use of VSFP2.3 in benchmarking cardiac myocyte maturation.
Advantages of GEVIs when compared with classical low molecular weight voltage-sensitive dyes include (1) homogeneous signal intensity independent of organ perfusion and cellular uptake, (2) cell-specific targeting of GEVIs, and (3) the applicability in longitudinal studies with repeated measurements in the same tissue sample. For studies in cardiac myocytes, FRET-based GEVIs seem advantageous, because of the possibilities to (1) correct for intensity changes as a consequence of out-of-plane motion during contraction, which typically requires electromechanical uncoupling by pharmacological means (eg, 2,3-butanedione monoxime [BDM]28 or blebbistatin29) and (2) calibrate the ratiometric FRET value for better quantitative comparison of individual experimental results in tissues and cells.
Caveats of transgenic reporters in the heart include unwanted effects on myocardial structure and function. Depending on the mouse model and strain as well as transgene abundance, even the commonly used fluorescent proteins such as GFP can elicit cardiotoxic effects.30 Another example is the inadvertent developmental cardiac toxicity associated with the GCaMP2 calcium reporter, making it necessary to limit its expression to defined postnatal time windows.22 Conversely, VSFP2.3 seemed to be biologically inert and did not cause any notable functional or structural changes, allowing us to study the mouse model with the highest VSFP2.3 expression and thus the strongest signal (αMHC-VSFP-line no. 123). Molecular evidence by RNA sequencing confirmed no dysregulation of cardiac structural and ion channel genes, but suggested an otherwise unapparent immune response, potentially elicited by the transgenic expression of nonmammalian peptides (ie, voltage-sensitive domain of C intestinalis, CFP, and YFP).
FRET-based sensors typically show <10% ΔR/R. Therefore, they require dedicated and low-noise detection systems for proper signal recording and processing. VSFP2.3 showed high maximal ΔR/R (>10% in voltage clamp analyses) with an appropriate dynamic range for monitoring the physiological cardiac myocyte voltage range (−80 to +20 mV; Figure 4C). The on and off kinetics of VSFP2.3 are, however, not fast enough to faithfully report the fastest components of the cardiac myocyte AP and the nonlinear fluorescence–voltage relationship saturates at the peak of the AP. Collectively, this resulted in an apparent plateau and a prolonged duration (APD90, 180–200 ms) of the optical membrane potential recorded in isolated adult mouse cardiac myocytes (Figure 4D). Optical recordings using the voltage-sensitive aminonapthylenylpyridium dye di-4-ANEPPS,31 impaling electrode measurements,32 and monophasic AP recordings33 revealed APD90 values of 30 to 65 ms in mouse ventricular myocardium. Thus, VSFP2.3 cannot replace the state-of-the-art electrophysiological recordings in studies where the shape of the AP is in the center of interest. However, it is important to note that despite these limitations, we found a strong correlation of the VSFP2.3 APD50 and patch-clamp recorded APD90 (Figure 4E), suggesting that using mathematical models,34 the electric AP could be inferred from the optical membrane potential. Moreover, the VSFP2.3 sensor was well suited to study the spread of electric excitation in whole heart preparations ex vivo (Figure 5) and in vivo (Figure 6); the latter is currently not possible with chemical probes. Although not directly demonstrated in this study, performing series of in vivo optical measurements will be possible in αMHC-VSFP2.3 mice or cross-breads with additional genetic modifications. Finally, introducing voltage transients derived from human cardiac myocyte APs in VSFP2.3 mouse cardiac myocytes provided conceptual evidence for the suitability of cardiac myocyte–targeted VSFP2.3 in monitoring cardiac myocyte APs in species with intrinsically slower repolarization (APD90, ≈300 ms in human versus <100 ms in mouse).35–38
Targeting of VSFP2.3 to the sarcolemma is essential for its voltage-dependent activity and may even allow for the analysis of sarcolemma-associated subcompartments such as t-tubules by super-resolution microscopy39 using biophysically optimized fluorochromes (ie, with enhanced stability under high-power laser excitation and reduction in physical size). A particularly exciting application could be the parallel optical assessment of membrane voltage changes and calcium release events at the sarcolemma/SR dyads. Robust labeling of the t-tubular system in adult mouse cardiac myocytes with the VSFP sensor suggests that in principle this would be possible if multi-color super-resolution imaging becomes accessible.
In addition to functional imaging, the use of VSFP to benchmark cardiac myocyte growth and maturation seems to be an attractive methodology. Fetal up to early postnatal cardiac myocytes lack any or have only a residual t-tubular membrane system.40–42 Accordingly, we observed no t-tubulation in iPSC-derived cardiac myocytes even in extended (30 days) monolayer cultures. In line with the resetting of somatic cells to a fetal state, iPSC-derived cardiac myocytes could be also clearly distinguished from adult cardiac myocytes by their slower optical AP upstroke and slower repolarization (Table). Although we cannot provide evidence for enhanced maturation under the conditions studied, our experimental data support the notion that longitudinal studies can be performed minimal invasively in the same cell preparation for screening of to date unknown maturation factors. Similarly and as suggested by others, GEVIs can be used in drug screens for arrhythmogenic potential.14,43 In contrast to studies using voltage indicator dyes, GEVIs would also allow studies on chronic exposure to drugs or other stimuli.
A key objective of our study was to introduce VSFP2.3 for imaging of cardiac electric activity or OCG in vivo. Presently, voltage-sensitive dyes (eg, di-4-ANEPPS) are typically used in ex vivo Langendorff-perfused hearts in combination with excitation–contraction uncouplers (eg, blebbistatin or BDM) to study epicardial spread of electric activation. This strategy cannot be applied in vivo because of the toxicity of the commonly used dyes and the incompatibility of the in vivo use of electromechanical uncouplers as well as their effects on AP morphology.6,44–47 Here, we provide first experimental evidence that a GEVI, expressed stably and homogeneously in cardiac myocyte membranes in vivo, can overcome these limitations. We first scrutinized the VSFP-imaging modality in Langendorff-perfused hearts and validated its use for the visualization of myocardial electric conduction under physiological and pathological heart rate and rhythm (Figure 5). For minimally invasive in vivo recordings, we developed a fiber optic system and collected evidence for its applicability also in the absence of electromechanical uncouplers (Figure 6). In proof-of-concept studies in vivo, we could finally demonstrate its applicability in blood-perfused hearts (Figure 6D). Importantly, AP values recorded ex vivo (Tyrode’s perfused) and in vivo (blood perfused) at physiological heart rates were similar (Table). With further advances of the imaging technology (ie, smaller optical fibers and multiple-fiber arrays; optimized positioning of the fiber array on the epicardial surface), we anticipate that spatial resolution can be further improved. In addition, further advanced GEVIs (eg, with near-infrared reporters) may ultimately also allow for closed chest imaging of heart rate and rhythm with to date unsurpassed spatial and temporal resolution.
Taken together, our study established first proof of concept for the use of a genetically targeted FRET-based GEVI for the visualization of electric activity in isolated cardiac myocytes of different developmental stages and the whole heart and in vivo, with and without electromechanical uncouplers. We anticipate that further optimized GEVIs11 and the application of novel fluorochromes for deep tissue imaging and super-resolution imaging together with advanced engineering of optical detection systems will advance the field to enable reliable membrane voltage recordings (OCGs) in chronic experiments, with applications in studies of fundamental cardiovascular biology and drug screens, in addition to more specific applications such as monitoring of electromechanical coupling of cell grafts for heart regeneration.
We thank Dr S. Verheule (Maastricht) for the introduction into optical imaging and Y. Iwamoto, B. Knocke, U. Leonhardt, R. Blume, and M. Zoremba for excellent technical assistance.
Sources of Funding
We acknowledge financial support from the DZHK (German Center for Cardiovascular Research; W.H. Zimmermann and S.E. Lehnart), the German Research Foundation (DFG ZI 708/7-1, 8-1, 10–1 [W.H. Zimmermann], SFB 1002 TP A03 [L.S. Maier], A04 [K. Guan], A05/B05 [S.E. Lehnart], C03 [S. Luther] and C04/S [W.H. Zimmermann], SFB 937 A18 [S. Luther, W.H. Zimmermann]), the German Federal Ministry for Science and Education (BMBF FKZ 13GW0007A [CIRM] to W.H. Zimmermann), the European Union FP7 CARE-MI (W.H. Zimmermann) and EUTrigTreat (S.E. Lehnart), the National Institutes of Health (U01 HL099997 to W.H. Zimmermann), and RIKEN (H. Mutoh and T. Knöpfel). T.P. de Boer is supported by a Netherlands Heart Foundation fellowship (2010T45).
In May 2015, the average time from submission to first decision for all original research papers submitted to Circulation Research was 15.49 days.
Current address (H.M.): Department of Neurophysiology, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.117.306143/-/DC1.
- Nonstandard Abbreviations and Acronyms
- α-myosin heavy chain
- action potential
- cyan fluorescent protein (Cerulean)
- Förster (fluorescence) resonance energy transfer
- genetically encoded voltage indicators
- induced pluripotent stem cell
- optical cardiograms
- sarcoplasmic reticulum
- voltage-sensitive fluorescent protein
- yellow fluorescent protein (Citrine)
- Received January 30, 2015.
- Revision received June 9, 2015.
- Accepted June 15, 2015.
- © 2015 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Voltage-sensitive dyes are used widely for optical imaging of electric activity ex vivo, but not in vivo.
Optogenetic tools enable optical monitoring and control of cellular activity in vitro and in vivo.
What New Information Does This Article Contribute?
Introduction of the first mouse model with cardiac myocyte–restricted expression of voltage-sensitive fluorescence protein 2.3 (VSFP2.3), a genetically encoded voltage indicator.
Demonstration of robust membrane voltage imaging in cardiac myocytes ex vivo (isolated adult cardiac myocytes), in situ (Langendorff-perfused heart), in vivo (open chest fiber optics), and in vitro (induced pluripotent stem cell–derived cardiac myocytes) without cardiac toxicity.
Transgenic VSFP2.3 enabled benchmarking of structural (t-tubulation) and functional (optical cardiograms) properties in isolated embryonic and adult cardiac myocytes and whole hearts.
Optogenetic tools are evolving rapidly for monitoring and controlling cellular activity. VSFP2.3 is particularly attractive for studying structural and functional properties of cardiac myocytes and whole hearts, because of its targeted expression at the sarcolemma and ratiometric fluorescence energy transfer recordings to minimize motion artifacts in beating hearts. Lack of apparent toxicity is another important prerequisite, especially for longitudinal studies in vivo and in vitro cardiac myocyte cultures. The demonstration that human action potentials can also be visualized by VSFP2.3 documents its use beyond rodent models. Importantly, optical cardiograms could be used to map electric impulse propagation with high temporal and spatial resolution in Langendorff-perfused hearts. In situ fiber optic recordings in spontaneously beating blood-perfused hearts provided proof of concept for the applicability of VSFP2.3 in minimal invasive assessments of impulse propagation in vivo. Collectively, the results of this study provide evidence for the versatile use of VSFP2.3 in assessments of cardiac myocyte structure and function. We anticipate that genetically encoded voltage indicators such as VSFP2.3 will find broad applications in fundamental studies of cardiac myocyte and heart development, assessments of structural and functional alterations in heart disease, and scrutiny of innovative therapeutics, including the assessment of electric integration of cardiac myocyte grafts in attempts to remuscularize failing hearts.