Optical Imaging and Functional Characterization of the Transverse Tubular System of Mammalian Muscle Fibers using the Potentiometric Indicator di-8-ANEPPS

Potentiometric dyes are useful tools for studying membrane potential changes from compartments inaccessible to direct electrical recordings. In the past, we have combined electrophysiological and optical techniques to investigate, by using absorbance and fluorescence potentiometric dyes, the electrical properties of the transverse tubular system in amphibian skeletal muscle fibers. In this paper we expand on recent observations using the fluorescent potentiometric indicator di-8-ANEPPS to investigate structural and functional properties of the transverse tubular system in mammalian skeletal muscle fibers. Two-photon laser scanning confocal fluorescence images of live muscle fibers suggest that the distance between consecutive rows of transverse tubules flanking the Z-lines remains relatively constant in muscle fibers stretched to attain sarcomere lengths of up to 3.5 μm. Furthermore, the combined use of two-microelectrode electrophysiological techniques with microscopic fluorescence spectroscopy and imaging allowed us to compare the spectral properties of di-8-ANEPPS fluorescence in fibers at rest, with those of fluorescence transients recorded in stimulated fibers. We found that although the indicator has excitation and emission peaks at 470 and 588 nm, respectively, fluorescence transients display optimal fractional changes (13%/100 mV) when using filters to select excitation wavelengths in the 530–550 nm band and emissions beyond 590 nm. Under these conditions, results from tetanically stimulated fibers and from voltage-clamp experiments suggest strongly that, although the kinetics of di-8-ANEPPS transients in mammalian fibers are very rapid and approximate those of the surface membrane electrical recordings, they arise from the transverse tubular system membranes.     

Electromyographic measures of muscle activation and changes in muscle architecture of human elbow flexors during fatiguing contractions.

The study compared changes in intramuscular and surface recordings of EMG amplitude with ultrasound measures of muscle architecture of the elbow flexors during a submaximal isometric contraction. Ten subjects performed a fatiguing contraction to task failure at 20% of maximal voluntary contraction force. EMG activity was recorded in biceps brachii, brachialis, and brachioradialis muscles using intramuscular and surface electrodes. The rates of increase in the amplitude of the surface EMG for the long and short heads of biceps brachii and brachioradialis were greater than those for the intramuscular recordings measured at different depths. The amplitude of the intramuscular recordings from three muscles increased at a similar rate (P = 0.13), as did the amplitude of the three surface recordings from two muscles (P = 0.83). The increases in brachialis thickness (27.7 +/- 5.7 to 30.9 +/- 3.5 mm; P < 0.05) and pennation angle (10.9 +/- 3.5 to 16.5 +/- 4.8 degrees ; P = 0.003) were not associated with the increase in intramuscular EMG amplitude (P > 0.58). The increase in brachioradialis thickness (22.8 +/- 4.8 to 25.5 +/- 3.4 mm; P = 0.0075) was associated with the increase in the amplitude for one of two intramuscular EMG signals (P = 0.007, r = 0.79). The time to failure was more strongly associated with the rate of increase in the amplitude of the surface EMG than that for the intramuscular EMG, which suggests that the surface measurement provides a more appropriate measure of the change in muscle activation during a fatiguing contraction.     

Fluorescence imaging of cardiac propagation: spectral properties and filtering of optical action potentials

Fluorescence imaging using voltage-sensitive dyes is an important tool for studying electrical propagation in the heart. Yet, the low amplitude of the voltage-sensitive component in the fluorescence signal and high acquisition rates dictated by the rapid propagation of the excitation wave front make it difficult to achieve recordings with high signal-to-noise ratios. Although spatially and temporally filtering the acquired signals has become de facto one of the key elements of optical mapping, there is no consensus regarding their use. Here we characterize the spatiotemporal spectra of optically recorded action potentials and determine the distortion produced by conical filters of different sizes. On the basis of these findings, we formulate the criteria for rational selection of filter characteristics. We studied the evolution of the spatial spectra of the propagating wave front after epicardial point stimulation of the isolated, perfused right ventricular free wall of the pig heart stained with di-4-ANEPPS. We found that short-wavelength (<3 mm) spectral components represent primarily noise and surface features of the preparation (coronary vessels, fat, and connective tissue). The time domain of the optical action potential spectrum also lacks high-frequency components (>100 Hz). Both findings are consistent with the reported effect of intrinsic blurring caused by light scattering inside the myocardial wall. The absence of high-frequency spectral components allows the use of aggressive low-pass spatial and temporal filters without affecting the optical action potential morphology. We show examples where the signal-to-noise ratio increased up to 150 with <3% distortion. A generalization of our approach to the rational filter selection in various applications is discussed.     

Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes.

Nicotinamide adenine dinucleotide (NADH) plays a critical role in oxidative phosphorylation as the primary source of reducing equivalents to the respiratory chain. Using a modified fluorescence microscope, we have obtained spectra and images of the blue autofluorescence from single rat cardiac myocytes. The optical setup permitted rapid acquisition of fluorescence emission spectra (390-595 nm) or intensified digital video images of individual myocytes. The spectra showed a broad fluorescence centered at 447 +/- 0.2 nm, consistent with mitochondrial NADH. Addition of cyanide resulted in a 100 +/- 10% increase in fluorescence, while the uncoupler FCCP resulted in a 82 +/- 4% decrease. These two transitions were consistent with mitochondrial NADH and implied that the myocytes were 44 +/- 6% reduced under the resting control conditions. Intracellular fluorescent structures were observed that correlated with the distribution of a mitochondrial selective fluorescent probe (DASPMI), the mitochondrial distribution seen in published electron micrographs, and a metabolic digital subtraction image of the cyanide fluorescence transition. These data are consistent with the notion that the blue autofluorescence of rat cardiac myocytes originates from mitochondrial NADH.     

Fluorescent styryl dyes applied as fast optical probes of cardiac action potential

Several styryl dyes were tested as fast optical probes of membrane action potentials in mammalian heart muscle tissue. After staining, atrial specimens were superfused in physiological salt solution, and fluorescence was excited by an argon ion laser. Excitation spot size on the surface of the preparation was 60 m in diameter. Dyes RH 160, RH 237, and RH 421 performed excellently as fast fluorescent probes of cardiac membrane potential. Fractional fluorescence changes, F/F, due to the action potential were in the range 2 to 6% at 514.5 nm excitation. Rise times of the action potential onset detected with each of the dyes were less than 0.5 ms, which is as fast or even faster than microelectrode measurements (atria of the rat). Thus membrane potential changes could be monitored with high resolution in both time and space. Emission spectra from heart muscle preparations stained with these dyes were shifted to shorter wavelengths by 70 nm and more as compared to spectra of the dyes in ethanol solution. The fluorescence spectrum of RH 160 at resting potential and the spectrum recorded during the plateau phases of the action potential were measured and showed no difference within the spectral resolution. As can be concluded from measurements of fluorescence changes at different excitation wavelengths, electrochromism cannot be the only mechanism causing the potential response.     

Intramural multisite recording of transmembrane potential in the heart

Heart surface optical mapping of transmembrane potentials has been widely used in studies of normal and pathological heart rhythms and defibrillation. In these studies, three-dimensional spatio-temporal events can only be inferred from two-dimensional surface potential maps. We present a novel optical system that enables high fidelity transmural recording of transmembrane potentials. A probe constructed from optical fibers is used to deliver excitation light and collect fluorescence from seven positions, each 1 mm apart, through the left ventricle wall of the rabbit heart. Excitation is provided by the 488-nm line of a water-cooled argon-ion laser. The fluorescence of the voltage-sensitive dye di-4-ANEPPS from each tissue site is split at 600 nm and imaged onto separate photodiodes for later signal ratioing. The optics and electronics are easily expandable to accommodate multiple optical probes. The system is used to record the first simultaneous measurements of transmembrane potential at a number of sites through the intact heart wall.     

Human diaphragmatic EMG: changes with lung volume and posture during supramaximal phrenic stimulation

If esophageal and chest wall recordings of diaphragmatic electromyographic activity (EMG) accurately reflect neural drive to this muscle, then compound muscle action potentials (CMAPs) produced by supramaximal stimulation of the phrenic nerve should not alter with changes in diaphragmatic position. Maximal CMAPs were therefore recorded 1) during changes in lung volume from near residual volume to near total lung capacity, 2) during isovolume maneuvers at different lung volumes, and 3) while subjects were lying, sitting, and standing. The areas of maximal CMAPs recorded with the gastroesophageal catheter increased 5.1 +/- 3.6 times (mean +/- SD) between these volumes, increased 2.4 +/- 1.3 times as the diaphragm descended during an isovolume maneuver (at functional residual capacity), and increased 4.4 +/- 2.4 times between the lying and standing positions. Because the stimuli were supramaximal, these changes in EMG reflect changes in the relationship between the esophageal electrodes and the diaphragmatic muscle fibers. Artifactual changes were also documented for surface electrodes on the chest wall. Because of these positional changes in maximal CMAPs, previous studies, which used integrated diaphragmatic EMG to document "reflex" changes in neural drive, should be reevaluated.     

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.     

Heart tissue viability monitoring in vivo by using combined fluorescence, thermography and electrical activity measurements.

A prototype system for in vivo monitoring of the heart tissue viability by using combined measurements of fluorescence, thermography and electrical activity has been elaborated for cardiac surgery. The fluorescence imaging of nicotinamide adenine dinucleotide NAD(P)H in the blue light range (lambda=467 nm) by using UV light (lambda=347 nm) excitation was used to detect metabolic disturbances. The method of the principal component analysis was used for the processing of the fluorescence image sequences. Far infrared (lambda=7.5-13 microm) imaging was used to evaluate temperature dynamics of the tissue surface during circulation disturbances. Evaluation of the epicardial electrogram shape by using continuous wavelet transform was used to detect and evaluate ischemia-caused disturbances of the electrical activity of the tissue. The combination of temperature, fluorescence and electrical activity estimates obtained from synchronically registered parameters during the experiments on model systems and experimental animals yielded qualitatively new results for the evaluation of cardiac tissue viability and enabled to achieve a versatile evaluation of the heart tissue viability.     

Fluorescence properties of free and protein bound fluorescein dyes. I. Macrospectrofluorometric measurements.

Excitation and emission properties of fluorescein derivatives were studied macrofluorometrically. Measurements were performed with solutions of various concentrations (0.07-100 microgram/ml) of free sodium fluorescein prepared from fluorescein diacetate (FDA), fluorescein isothiocyanate (FITC) and FITC bound to rabbit gamma-globulin. Both excitation and emission spectra as well as fluorescence intensities at constant excitation/emission wavelengths (496/515 nm) were recorded. The findings indicate that (1) FDA gives about twice the fluorescence intensity compared to equal concentrations of FITC. (2) The fluorescence properties of FITC upon excitation with blue light (lambda = 496 nm) are only slightly altered by the conjugation to rabbit gamma-globulin. (3) Considerable quenching due to conjugation could, however, be shown to occur upon UV excitation (lambda = 340 nm). (4) Fluorescence emission excited by visible blue light (496 nm) increases linearly to dye concentration in a range of 0.07-2.5 microgram/ml. Beginning at 5 microgram/ml (10-(5) M/1) all three compounds show a sharp decrease of fluorescence intensity with further increasing concentration. Practical aspects of these data for the immunofluorescence method are discussed.     

Noninvasive assessment of the effects of glucagon on the gastric slow wave

Hyperglycemic effects on the gastric slow wave are not well understood, and no studies have examined the effects that hyperglycemia has on gastric slow wave magnetic fields. We recorded multichannel magnetogastrograms (MGGs) before and after intravenous administration of glucagon and subsequent modest hyperglycemia in 20 normal volunteers. Normal slow waves were evident in baseline MGG recordings from all 20 subjects, but within 15 min after glucagon had been given, we noted significant effects on MGG signals. In addition to an overall decrease in the slow wave frequency from 2.9 +/- 0.5 cycles per min (cpm) to 2.2 +/- 0.1 cpm (P < 0.05), we observed significant changes in the number and range of spectral peaks recorded. Furthermore, the propagation velocity determined from surface current density maps computed from the multichannel MGG decreased significantly (7.1 +/- 0.8 mm/s to 5.0 +/- 0.3 mm/s, P < 0.05). This is the first study of biomagnetic effects of hyperglycemia in normal subjects. Our results suggest that the analysis of the MGG provides parameter quantification for gastric electrical activity specific to and characteristic of slow wave abnormalities associated with increased serum glucose by injection of glucagon.     

Dual-photon excitation of albumin

Fluorescence emission after two-photon excitation at 580 nm is observed in albumin by means of Nd:YAG laser at room temperature. The two-photon excitation spectral range 550-590 nm was obtained. The experimental results show that albumin fluorescence originates from tryptophan residues.     

Substituted 4-[4-(dimethylamino)styryl]pyridinium salt as a fluorescent probe for cell microviscosity

In aqueous solution, 4-[4-(dimethylamino)styryl]pyridine (DMASP) derivatives displayed dual fluorescence, in which excitation at either 469 or 360 nm produced an emission band near 600 nm. Increasing the viscosity of the environment intensified the fluorescence emission obtained at the longer wavelength of excitation, whereas the emission at the lower wavelength of excitation showed little change in intensity. Thus, using the ratio of the 600 nm emission obtained by exciting at 469 nm to that obtained with 360 nm excitation, it is possible to obtain a value related to the local viscosity that does not depend on the system parameters. The fluorescence emission of the dye in aqueous solution, as well as in living cells, is well suited for use with visible fluorescence spectroscopy. The N-carboxymethyl butyl ester DMASP derivative (1) was found to be irreversibly loaded into living smooth muscle cells, presumably because it is hydrolyzed by cellular esterases, transforming it into a membrane-impermeable fluorescent carboxylate DMASP derivative. (2) After calibrating 2 against glycerol/water and sucrose/water mixtures of known viscosity, the fluorescence ratio generated from cultured smooth muscle cells in dual-excitation mode gave an average intracellular viscosity of 4.5 cP. This value corresponds to those reported in the literature.     

Spectral characteristic of fluorescence induction in a model cyanobacterium, Synechococcus sp. (PCC 7942)

We present here three-dimensional time-wavelength-intensity displays of changes in variable fluorescence, during the O(JI)PSMT transient, observed in cyanobacterium at room temperature. We were able to measure contributions of individual chromophores to fluorescence spectra at various times of fluorescence induction (FI). The method was applied to a freshwater cyanobacterium, Synechococcus sp. (PCC 7942). Analysis of our experimental results provides the following new conclusions: (i) the main chlorophyll (Chl) a emission band at ∼ 685 nm that originates in Photosystem (PS) II exhibits typical fast (OPS) and slow (SMT) FI kinetics with both orange (622 nm) and blue (464 nm) excitation. (ii) Similar kinetics are exhibited for its far-red emission satellite band centered at ∼ 745 nm, where the PS II contribution predominates. (iii) A significant OPS-SMT-type kinetics of C-phycocyanin emission at ∼ 650 nm are observed with the blue light excitation, but not with orange light excitation where the signal rose only slightly to a maximum. The induction of F650 was not caused by an admixture of the F685 fluorescence and thus our data show light-inducible and dark-reversible changes of phycobilin fluorescence in vivo. We discuss possible interpretations of this new observation.     

Electrophysiological interaction through the interstitial space between adjacent unmyelinated parallel fibers.

The influence of interstitial or extracellular potentials on propagation usually has been ignored, often through assuming these potentials to be insignificantly different from zero, presumably because both measurements and calculations become much more complex when interstitial interactions are included. This study arose primarily from an interest in cardiac muscle, where it has been well established that substantial interstitial potentials occur in tightly packed structures, e.g., tens of millivolts within the ventricular wall. We analyzed the electrophysiological interaction between two adjacent unmyelinated fibers within a restricted extracellular space. Numerical evaluations made use of two linked core-conductor models and Hodgkin-Huxley membrane properties. Changes in transmembrane potentials induced in the second fiber ranged from nonexistent with large intervening volumes to large enough to initiate excitation when fibers were coupled by interstitial currents through a small interstitial space. With equal interstitial and intracellular longitudinal conductivities and close coupling, the interaction was large enough (induced Vm approximately 20 mV peak-to-peak) that action potentials from one fiber initiated excitation in the other, for the 40-microns radius evaluated. With close coupling but no change in structure, propagation velocity in the first fiber varied from 1.66 mm/ms (when both fibers were simultaneously stimulated) to 2.84 mm/ms (when the second fiber remained passive). Although normal propagation through interstitial interaction is unlikely, the magnitudes of the electrotonic interactions were large and may have a substantial modulating effect on function.     

Probing field-induced tissue polarization using transillumination fluorescent imaging

Despite major successes of biophysical theories in predicting the effects of electrical shocks within the heart, recent optical mapping studies have revealed two major discrepancies between theory and experiment: 1), the presence of negative bulk polarization recorded during strong shocks; and 2), the unexpectedly small surface polarization under shock electrodes. There is little consensus as to whether these differences result from deficiencies of experimental techniques, artifacts of tissue damage, or deficiencies of existing theories. Here, we take advantage of recently developed near-infrared voltage-sensitive dyes and transillumination optical imaging to perform, for the first time that we know of, noninvasive probing of field effects deep inside the intact ventricular wall. This technique removes some of the limitations encountered in previous experimental studies. We explicitly demonstrate that deep inside intact myocardial tissue preparations, strong electrical shocks do produce considerable negative bulk polarization previously inferred from surface recordings. We also demonstrate that near-threshold diastolic field stimulation produces activation of deep myocardial layers 2-6 mm away from the cathodal surface, contrary to theory. Using bidomain simulations we explore factors that may improve the agreement between theory and experiment. We show that the inclusion of negative asymmetric current can qualitatively explain negative bulk polarization in a discontinuous bidomain model.     

Synthesis of voltage-sensitive fluorescence signals from three-dimensional myocardial activation patterns

Voltage-sensitive fluorescent dyes are commonly used to measure cardiac electrical activity. Recent studies indicate, however, that optical action potentials (OAPs) recorded from the myocardial surface originate from a widely distributed volume beneath the surface and may contain useful information regarding intramural activation. The first step toward obtaining this information is to predict OAPs from known patterns of three-dimensional (3-D) electrical activity. To achieve this goal, we developed a two-stage model in which the output of a 3-D ionic model of electrical excitation serves as the input to an optical model of light scattering and absorption inside heart tissue. The two-stage model permits unique optical signatures to be obtained for given 3-D patterns of electrical activity for direct comparison with experimental data, thus yielding information about intramural electrical activity. To illustrate applications of the model, we simulated surface fluorescence signals produced by 3-D electrical activity during epicardial and endocardial pacing. We discovered that OAP upstroke morphology was highly sensitive to the transmural component of wave front velocity and could be used to predict wave front orientation with respect to the surface. These findings demonstrate the potential of the model for obtaining useful 3-D information about intramural propagation.     

Electrical rhythmicity and spread of action potentials in longitudinal muscle of guinea pig distal colon

Using simultaneous intracellular recordings, we have characterized 1) electrical activity in the longitudinal muscle (LM) of isolated segments of guinea pig distal colon free to contract spontaneously and 2) extent of propagation of spontaneous action potentials around the circumference of the colon. In all animals, rhythmical spontaneous depolarizations (SDs) were recorded that are usually associated with the generation of action potentials. Recordings from pairs of LM cells, separated by 100 microm in the circumferential axis, revealed that each action potential was phase locked at the two electrodes (mean propagation velocity: 3 mm/s). However, at an increased electrode separation distance of 1 mm circumferentially, action potentials and SDs became increasingly uncoordinated at the two recording sites. No SDs or action potentials ever propagated from one circumferential edge to the other (i.e., 13 mm apart). When LM strips were separated from the myenteric plexus and circular muscle, rhythmically firing SDs and action potentials were still recorded. Atropine (1 microM) or tetrodotoxin (1 microM) either reduced the frequency of SDs or temporarily abolished activity, whereas nifedipine (1 microM) always abolished SDs and action potentials. Kit-positive interstitial cells of Cajal were present at the level of the myenteric plexus and circular and longitudinal muscle. In summary, SDs and action potentials in LM propagate over discrete localized zones, usually <1 mm around the circumference of the colon. Furthermore, in contrast to the classic slow wave, rhythmic depolarizations in LM appear to be generated by an intrinsic property of the smooth muscle itself and are critically dependent on opening of L-type Ca(2+) channels.     

培养新生大鼠心肌细胞的电信号传导：多电极记录研究

利用多电极阵列同步记录技术对培养的新生大鼠单层心肌细胞的电活动进行胞外记录,观察心肌细胞在自发搏动和电刺激情况下信号在细胞间的传导模式。通过对记录信号的处理和分析,能获得诸如起搏细胞的数量和位置、动作电位的传导速度和途径以及不同起搏细胞间的相互影响等信息。研究还发现,心肌细胞阈下刺激会影响细胞的搏动和信号传导。