Anatomic localization and autonomic modulation of atrioventricular junctional rhythm in failing human hearts.

BACKGROUND: The structure-function relationship in the atrioventricular junction (AVJ) of various animal species has been investigated in detail; however, less is known about the human AVJ. In this study, we performed high-resolution optical mapping of the human AVJ (n = 6) to define its pacemaker properties and response to autonomic stimulation. METHODS AND RESULTS: Isolated, coronary-perfused AVJ preparations from failing human hearts (n = 6, 53 ± 6 years) were optically mapped using the near-infrared, voltage-sensitive dye, di-4-ANBDQBS, with isoproterenol (1 µmol/L) and acetylcholine (1 µmol/L). An algorithm detecting multiple components of optical action potentials was used to reconstruct multilayered intramural AVJ activation and to identify specialized slow and fast conduction pathways (SP and FP). The anatomic origin and propagation of pacemaker activity was verified by histology. Spontaneous AVJ rhythms of 29 ± 11 bpm (n = 6) originated in the nodal-His region (n = 3) and/or the proximal His bundle (n = 4). Isoproterenol accelerated the AVJ rhythm to 69 ± 12 bpm (n = 5); shifted the leading pacemaker to the transitional cell regions near the FP and SP (n = 4) and/or coronary sinus (n = 2); and triggered reentrant arrhythmias (n = 2). Acetylcholine (n = 4) decreased the AVJ rhythm to 18 ± 4 bpm; slowed FP/SP conduction leading to block between the AVJ and atrium; and shifted the pacemaker to either the transitional cell region or the nodal-His region (bifocal activation). CONCLUSIONS: We have demonstrated that the AVJ pacemaker in failing human hearts is located in the nodal-His region or His bundle regions and can be modified with autonomic stimulation. Moreover, we found that both the FP and SP are involved in anterograde and retrograde conduction.

Single-sensor system for spatially resolved, continuous, and multiparametric optical mapping of cardiac tissue.

BACKGROUND: Simultaneous optical mapping of multiple electrophysiologically relevant parameters in living myocardium is desirable for integrative exploration of mechanisms underlying heart rhythm generation under normal and pathophysiologic conditions. Current multiparametric methods are technically challenging, usually involving multiple sensors and moving parts, which contributes to high logistic and economic thresholds that prevent easy application of the technique. OBJECTIVE: The purpose of this study was to develop a simple, affordable, and effective method for spatially resolved, continuous, simultaneous, and multiparametric optical mapping of the heart, using a single camera. METHODS: We present a new method to simultaneously monitor multiple parameters using inexpensive off-the-shelf electronic components and no moving parts. The system comprises a single camera, commercially available optical filters, and light-emitting diodes (LEDs), integrated via microcontroller-based electronics for frame-accurate illumination of the tissue. For proof of principle, we illustrate measurement of four parameters, suitable for ratiometric mapping of membrane potential (di-4-ANBDQPQ) and intracellular free calcium (fura-2), in an isolated Langendorff-perfused rat heart during sinus rhythm and ectopy, induced by local electrical or mechanical stimulation. RESULTS: The pilot application demonstrates suitability of this imaging approach for heart rhythm research in the isolated heart. In addition, locally induced excitation, whether stimulated electrically or mechanically, gives rise to similar ventricular propagation patterns. CONCLUSION: Combining an affordable camera with suitable optical filters and microprocessor-controlled LEDs, single-sensor multiparametric optical mapping can be practically implemented in a simple yet powerful configuration and applied to heart rhythm research. The moderate system complexity and component cost is destined to lower the threshold to broader application of functional imaging and to ease implementation of more complex optical mapping approaches, such as multiparametric panoramic imaging. A proof-of-principle application confirmed that although electrically and mechanically induced excitation occur by different mechanisms, their electrophysiologic consequences downstream from the point of activation are not dissimilar.

Optical mapping of the isolated coronary-perfused human sinus node.

OBJECTIVES: We sought to confirm our hypothesis that the human sinoatrial node (SAN) is functionally insulated from the surrounding atrial myocardium except for several exit pathways that electrically bridge the nodal tissue and atrial myocardium. BACKGROUND: The site of origin and pattern of excitation within the human SAN has not been directly mapped. METHODS: The SAN was optically mapped in coronary-perfused preparations from nonfailing human hearts (n = 4, age 54 ± 15 years) using the dye Di-4-ANBDQBS and blebbistatin. The SAN 3-dimensional structure was reconstructed using histology. RESULTS: Optical recordings from the SAN had diastolic depolarization and multiple upstroke components, which corresponded to the separate excitations of the SAN and atrial layers. Excitation originated in the middle of the SAN (66 ± 17 beats/min), and then spread slowly (1 to 18 cm/s) and anisotropically. After a 82 ± 17 ms conduction delay within the SAN, the atrial myocardium was excited via superior, middle, and/or inferior sinoatrial conduction pathways. Atrial excitation was initiated 9.4 ± 4.2 mm from the leading pacemaker site. The oval 14.3 ± 1.5 mm × 6.7 ± 1.6 mm × 1.0 ± 0.2 mm SAN structure was functionally insulated from the atrium by connective tissue, fat, and coronary arteries, except for these pathways. CONCLUSIONS: These data demonstrated for the first time, to our knowledge, the location of the leading SAN pacemaker site, the pattern of excitation within the human SAN, and the conduction pathways into the right atrium. The existence of these pathways explains why, even during normal sinus rhythm, atrial breakthroughs could arise from a region parallel to the crista terminalis that is significantly larger (26.1 ± 7.9 mm) than the area of the anatomically defined SAN.

High-precision recording of the action potential in isolated cardiomyocytes using the near-infrared fluorescent dye di-4-ANBDQBS.

The use of voltage-sensitive fluorescent dyes (VSD) for noninvasive measurement of the action potential (AP) in isolated cells has been hindered by low-photon yield of the preparation, dye toxicity, and photodynamic damage. Here we used a new red-shifted VSD, di-4-ANBDQBS, and a fast electron-multiplied charge-coupled device camera for optical AP (OAP) recording in guinea pig cardiac myocytes. Loading di-4-ANBDQBS did not alter APs recorded with micropipette. With short laser exposures (just enough to record one OAP every 1-5 min), di-4-ANBDQBS yielded fluorescent signals with very high signal-to-background ratios (change in fluorescence on depolarization/fluorescence at resting potential: 19.2 ± 4.1%) and signal-to-noise ratios (40 ± 13.2). Quantum chemical calculations comparing the ANBDQ chromophore to the conventional ANEP chromophore showed that the higher wavelength and the greater voltage sensitivity of the former have the same electro-optical origin: a longer path for electron redistribution in the excited state. OAP closely tracked simultaneously recorded electrical APs, permitting measurement of AP duration within 1% error. Prolonged laser exposure caused progressive AP duration prolongation and instability. However, these effects were alleviated or abolished by reducing the dye concentration and by perfusion with antioxidants. Thus the presented technique provides a unique opportunity for noninvasive AP recording in single cardiomyocytes.

Lipid composition affects the rate of photosensitized dissipation of cross-membrane diffusion potential on liposomes.

Hydrophobic or amphiphilic tetrapyrrole sensitizers are taken up by cells and are usually located in cellular lipid membranes. Singlet oxygen is photogenerated by the sensitizer, and it diffuses in the membrane and causes oxidative damage to membrane components. This damage can occur to membrane lipids and to membrane-localized proteins. Depolarization of the Nernst electric potential on cells' membranes has been observed in cellular photosensitization, but it was not established whether lipid oxidation is a relevant factor leading to abolishing the resting potential of cells' membranes and to their death. In this work, we studied the effect of liposomes' lipid composition on the kinetics of hematoporphyrin-photosensitized dissipation of K(+)-diffusion electric potential that was generated across the membranes. We employed an electrochromic voltage-sensitive spectroscopic probe that possesses a high fluorescence signal response to the potential. We found a correlation between the structure and unsaturation of lipids and the leakage of the membrane, following photosensitization. As the extent of nonconjugated unsaturation of the lipids is increased from 1 to 6 double bonds, the kinetics of depolarization become faster. We also found that the kinetics of depolarization is affected by the percentage of the unsaturated lipids in the liposome: as the fraction of the unsaturated lipids increases, the leakage through the membrane is enhanced. When liposomes are composed of a lipid mixture similar to that of natural membranes and photosensitization is being carried out under usual photodynamic therapy (PDT) conditions, photodamage to the lipids is not likely to cause enhanced permeability of ions through the membrane, which would have been a mechanism that leads to cell death.

Dynamics of action potential backpropagation in basal dendrites of prefrontal cortical pyramidal neurons.

Basal dendrites of neocortical pyramidal neurons are relatively short and directly attached to the cell body. This allows electrical signals arising in basal dendrites to strongly influence the neuronal output. Likewise, somatic action potentials (APs) should readily propagate back into the basilar dendritic tree to influence synaptic plasticity. Two recent studies, however, determined that sodium APs are severely attenuated in basal dendrites of cortical pyramidal cells, so that they completely fail in distal dendritic segments. Here we used the latest improvements in the voltage-sensitive dye imaging technique (Zhou et al., 2007) to study AP backpropagation in basal dendrites of layer 5 pyramidal neurons of the rat prefrontal cortex. With a signal-to-noise ratio of > 15 and minimal temporal averaging (only four sweeps) we were able to sample AP waveforms from the very last segments of individual dendritic branches (dendritic tips). We found that in short- (< 150 microm) and medium (150-200 microm in length)-range basal dendrites APs backpropagated with modest changes in AP half-width or AP rise-time. The lack of substantial changes in AP shape and dynamics of rise is inconsistent with the AP-failure model. The lack of substantial amplitude boosting of the third AP in the high-frequency burst also suggests that in short- and medium-range basal dendrites backpropagating APs were not severely attenuated. Our results show that the AP-failure concept does not apply in all basal dendrites of the rat prefrontal cortex. The majority of synaptic contacts in the basilar dendritic tree actually received significant AP-associated electrical and calcium transients.

Imaging activity of neuronal populations with new long-wavelength voltage-sensitive dyes.

We have assessed the utility of five new long-wavelength fluorescent voltage-sensitive dyes (VSD) for imaging the activity of populations of neurons in mouse brain slices. Although all the five were capable of detecting activity resulting from activation of the Schaffer collateral-CA1 pyramidal cell synapse, they differed significantly in their properties, most notably in the signal-to-noise ratio of the changes in dye fluorescence associated with neuronal activity. Two of these dyes, Di-2-ANBDQPQ and Di-1-APEFEQPQ, should prove particularly useful for imaging activity in brain tissue and for combining VSD imaging with the control of neuronal activity via light-activated proteins such as channelrhodopsin-2 and halorhodopsin.

Near-infrared voltage-sensitive fluorescent dyes optimized for optical mapping in blood-perfused myocardium.

BACKGROUND: Styryl voltage-sensitive dyes (e.g., di-4-ANEPPS) have been used successfully for optical mapping in cardiac cells and tissues. However, their utility for probing electrical activity deep inside the myocardial wall and in blood-perfused myocardium has been limited because of light scattering and high absorption by endogenous chromophores and hemoglobin at blue-green excitation wavelengths. OBJECTIVE: The purpose of this study was to characterize two new styryl dyes--di-4-ANBDQPQ (JPW-6003) and di-4-ANBDQBS (JPW-6033)--optimized for blood-perfused tissue and intramural optical mapping. METHODS: Voltage-dependent spectra were recorded in a model lipid bilayer. Optical mapping experiments were conducted in four species (mouse, rat, guinea pig, and pig). Hearts were Langendorff perfused using Tyrode's solution and blood (pig). Dyes were loaded via bolus injection into perfusate. Transillumination experiments were conducted in isolated coronary-perfused pig right ventricular wall preparations. RESULTS: The optimal excitation wavelength in cardiac tissues (650 nm) was >70 nm beyond the absorption maximum of hemoglobin. Voltage sensitivity of both dyes was approximately 10% to 20%. Signal decay half-life due to dye internalization was 80 to 210 minutes, which is 5 to 7 times slower than for di-4-ANEPPS. In transillumination mode, DeltaF/F was as high as 20%. In blood-perfused tissues, DeltaF/F reached 5.5% (1.8 times higher than for di-4-ANEPPS). CONCLUSION: We have synthesized and characterized two new near-infrared dyes with excitation/emission wavelengths shifted >100 nm to the red. They provide both high voltage sensitivity and 5 to 7 times slower internalization rate compared to conventional dyes. The dyes are optimized for deeper tissue probing and optical mapping of blood-perfused tissue, but they also can be used for conventional applications.

Nonlinear optical potentiometric dyes optimized for imaging with 1064-nm light.

Nonlinear optical phenomena, such as two-photon fluorescence (2PF) and second harmonic generation (SHG), in combination with voltage sensitive dyes, can be used to acquire high-resolution spatio temporal maps of electrical activity in excitable cells and tissue. Developments in 1064-nm fiber laser technology have simplified the generation of high-intensity, long-wavelength, femtosecond light pulses, capable of penetrating deep into tissue. To merge these two advances requires the design and synthesis of new dyes that are optimized for longer wavelengths and that produce fast and sensitive responses to membrane potential changes. In this work, we have systematically screened a series of new dyes with varying chromophores and sidechains that anchor them in cell membranes. We discovered several dyes that could potentially be used for in vivo measurements of cellular electrical activity because of their rapid and sensitive responses to membrane potential. Some of these dyes show optimal activity for SHG; others for 2PF. This regulated approach to dye screening also allows significant insight into the molecular mechanisms behind both SHG and 2PF. In particular, the differing patterns of sensitivity and kinetics for these two nonlinear optical modalities indicate that their voltage sensitivity originates from differing mechanisms.

Intracellular long-wavelength voltage-sensitive dyes for studying the dynamics of action potentials in axons and thin dendrites.

In CNS neurons most of synaptic integration takes place in thin dendritic branches that are difficult to study with conventional physiological recording techniques (electrodes). When cellular compartments are too small, or too many, for electrode recordings, optical methods bring considerable advantages. Here we focused our experimental effort on the development and utilization of new kinds of voltage-sensitive dyes (VSD). The new VSDs have bluish appearance in organic solvents, and hence are dubbed "blue dyes". They have preferred excitation windows for voltage recording that are shifted to longer wavelengths (approximately 660nm). Excitation in deep red light and emission in the near-infrared render "blue VSDs" potentially useful in measurements from fluorescent structures below the tissue surface because light scattering is minimized at longer wavelengths. Seven new molecules were systematically tested using intracellular injection. In comparison to the previously used red dye (JPW-3028) the blue dyes have better sensitivity (DeltaF/F) by approximately 40%. Blue dyes take little time to fill the dendritic tree, and in this aspect they are comparable with the fastest red dye JPW-3028. Based on our results, blue VSDs are well suited for experimental exploration of thin neuronal processes in semi intact preparations (brain slice). In some cases only six sweeps of temporal averaging were needed to acquire excellent records of individual action potentials in basal and oblique dendritic branches, or in axons and axon collaterals up to 200microm away from the cell body. Signal-to-noise ratio of these recordings was approximately 10. The combination of blue dyes and laser illumination approach imposed little photodynamic damage and allowed the total number of recording sweeps per cell to exceed 100. Using these dyes and a spot laser illumination technique, we demonstrate the first recording of action potentials in the oblique dendrite and distal axonal segment of the same pyramidal cell.

New near-infrared optical probes of cardiac electrical activity.

Styryl voltage-sensitive dyes (e.g., di-4-ANEPPS) have been widely and successfully used as probes for mapping membrane potential changes in cardiac cells and tissues. However, their utility has been somewhat limited because their excitation wavelengths have been restricted to the 450- to 550-nm range. Longer excitation/emission wavelength probes can minimize interference from endogenous chromophores and, because of decreased light scattering and lower absorption by endogenous chromophores, improve recording from deeper tissue layers. In this article, we report efforts to develop new potentiometric styryl dyes that have excitation wavelengths ranging above 700 nm and emission spectra extending to 900 nm. Three dyes for cardiac optical mapping were investigated in depth from several hundred dyes containing 47 variants of the styryl chromophores. Absorbance and emission spectra in ethanol and multilamellar vesicles, as well as voltage-dependent spectral changes in a model lipid bilayer, have been recorded for these dyes. Optical action potentials were recorded in typical cardiac tissues (rat, guinea pig, pig) and compared with those of di-4-ANEPPS. The voltage sensitivities of the fluorescence of these new potentiometric indicators are as good as those of the widely used ANEP series of probes. In addition, because of molecular engineering of the chromophore, the new dyes provide a wide range of dye loading and washout time constants. These dyes will enable a series of new experiments requiring the optical probing of thick and/or blood-perfused cardiac tissues.

Characterization and application of a new optical probe for membrane lipid domains.

In this article, we characterize the fluorescence of an environmentally sensitive probe for lipid membranes, di-4-ANEPPDHQ. In large unilamellar lipid vesicles (LUVs), its emission spectrum shifts up to 30 nm to the blue with increasing cholesterol concentration. Independently, it displays a comparable blue shift in liquid-ordered relative to liquid-disordered phases. The cumulative effect is a 60-nm difference in emission spectra for cholesterol containing LUVs in the liquid-ordered state versus cholesterol-free LUVs in the liquid-disordered phase. Given these optical properties, we use di-4-ANEPPDHQ to image the phase separation in giant unilamellar vesicles with both linear and nonlinear optical microscopy. The dye shows green and red fluorescence in liquid-ordered and -disordered domains, respectively. We propose that this reflects the relative rigidity of the molecular packing around the dye molecules in the two phases. We also observe a sevenfold stronger second harmonic generation signal in the liquid-disordered domains, consistent with a higher concentration of the dye resulting from preferential partitioning into the disordered phase. The efficacy of the dye for reporting lipid domains in cell membranes is demonstrated in polarized migrating neutrophils.

Synthesis, spectra, delivery and potentiometric responses of new styryl dyes with extended spectral ranges.

Styryl dyes have been among the most widely used probes for mapping membrane potential changes in excitable cells. However, their utility has been somewhat limited because their excitation wavelengths have been restricted to the 450-550 nm range. Longer wavelength probes can minimize interference from endogenous chromophores and, because of decreased light scattering, improve recording from deep within tissue. In this paper we report on our efforts to develop new potentiometric styryl dyes that have excitation wavelengths ranging above 700 nm and emission spectra out to 900 nm. We have prepared and characterized dyes based on 47 variants of the styryl chromophores. Voltage-dependent spectral changes have been recorded for these dyes in a model lipid bilayer and from lobster nerves. The voltage sensitivities of the fluorescence of many of these new potentiometric indicators are as good as those of the widely used ANEP series of probes. In addition, because some of the dyes are often poorly water soluble, we have developed cyclodextrin complexes of the dyes to serve as efficient delivery vehicles. These dyes promise to enable new experimental paradigms for in vivo imaging of membrane potential.

Cholesterol-enriched lipid domains can be visualized by di-4-ANEPPDHQ with linear and nonlinear optics.

We present a membrane-staining dye, di-4-ANEPPDHQ, which differentiates liquid-ordered phases from liquid-disordered phases coexisting in model membranes under both linear and nonlinear microscopies. The dye's fluorescence emission spectrum is blue-shifted 60 nm in liquid-ordered phases compared with liquid-disordered phases, and shows strong second harmonic generation in the liquid-disordered phase compared with the liquid-ordered phase. The ease of staining and the ability of this single dye to detect both phases, should lead to broad applications in biophysical studies of lipid domains in model membranes and cells.

Sensitivity of second harmonic generation from styryl dyes to transmembrane potential.

In this article we present results from the simultaneous nonlinear (second harmonic generation and two-photon excitation fluorescence) imaging and voltage clamping of living cells. Specifically, we determine the sensitivity to transmembrane potential of second harmonic generation by ANEP-chromophore styryl dyes as a function of excitation wavelength and dye structure. We have measured second harmonic sensitivities of up to 43% per 100 mV, more than a factor of four better than the nominal voltage sensitivity of the dyes under "one-photon" fluorescence. We find a dependence of voltage sensitivity on excitation wavelength that is consistent with a two-photon resonance, and there is a significant dependence of voltage sensitivity on the structure of the nonchromophore portion of the dyes.

A fluorometric approach to local electric field measurements in a voltage-gated ion channel.

Site-specific electrostatic measurements have been limited to soluble proteins purified for in vitro spectroscopic characterization or proteins of known structure; however, comparable measurements have not been made for functional membrane bound proteins. Here, using an electrochromic fluorophore, we describe a method to monitor localized electric field changes in a voltage-gated potassium channel. By coupling the novel probe Di-1-ANEPIA to cysteines in Shaker and tracking field-induced optical changes, in vivo electrostatic measurements were recorded with submillisecond resolution. This technique reports dynamic changes in the electric field during the gating process and elucidates the electric field profile within Shaker. The extension of this method to other membrane bound proteins, including transporters, will yield insight into the role of electrical forces on protein function.