Restitution analysis of alternans and its relationship to arrhythmogenicity in hypokalaemic Langendorff-perfused murine hearts

Alternans and arrhythmogenicity were studied in hypokalaemic (3.0 mM K+) Langendorff-perfused murine hearts paced at high rates. Epicardial and endocardial monophasic action potentials were recorded and durations quantified at 90% repolarization. Alternans and arrhythmia occurred in hypokalaemic, but not normokalaemic (5.2 mM K+) hearts (P < 0.01): this was prevented by treatment with lidocaine (10 μM, P < 0.01). Fourier analysis then confirmed transition from monomorphic to polymorphic waveforms for the first time in the murine heart. Alternans and arrhythmia were associated with increases in the slopes of restitution curves, obtained for the first time in the murine heart, while the anti-arrhythmic effect of lidocaine was associated with decreased slopes. Thus, hypokalaemia significantly increased (P < 0.05) maximal gradients (from 0.55 ± 0.14 to 2.35 ± 0.67 in the epicardium and from 0.67 ± 0.13 to 1.87 ± 0.28 in the endocardium) and critical diastolic intervals (DIs) at which gradients equalled unity (from −2.14 ± 0.52 ms to 50.93 ± 14.45 ms in the epicardium and from 8.14 ± 1.49 ms to 44.64 ± 5 ms in the endocardium). While treatment of normokalaemic hearts with lidocaine had no significant effect (P > 0.05) on either maximal gradients (0.78 ± 0.27 in the epicardium and 0.83 ± 0.45 in the endocardium) or critical DIs (6.06 ± 2.10 ms and 7.04 ± 3.82 ms in the endocardium), treatment of hypokalaemic hearts with lidocaine reduced (P < 0.05) both these parameters (1.05 ± 0.30 in the epicardium and 0.89 ± 0.36 in the endocardium and 30.38 ± 8.88 ms in the epicardium and 31.65 ± 4.78 ms in the endocardium, respectively). We thus demonstrate that alternans contributes a dynamic component to arrhythmic substrate during hypokalaemia, that restitution may furnish an underlying mechanism and that these phenomena are abolished by lidocaine, both recapitulating and clarifying clinical findings.
Introduction
Correlations between beat-to-beat alternations in electrocardiographic QT interval (QT or T-wave alternans) and cardiac arrhythmia have been described in classical work [13, 25] and subsequently confirmed in both clinical studies [2, 28, 38] and experimental models [7, 33, 52]. Indeed, the presence of such alternans provides a stronger predictor of arrhythmic risk than is offered by signal-averaged electrocardiography and a prediction of a similar value to that offered by invasive programmed stimulation procedures in current use [10] (but see [45]). The association between alternans and arrhythmogenicity is particularly marked at high heart rates (short baseline cycle lengths, BCLs) [15, 49] and in conditions of congenital and acquired electrocardiographic QT, and therefore action potential, prolongation [1, 34, 39, 46, 47, 64].
The relationship between heart rate and alternans has previously been analysed by constructing restitution curves which plot relationships between action potential duration (APD) and preceding diastolic interval (DI), as BCL is varied [12]. Clinical and experimental studies then associated alternans and arrhythmia observed at short BCLs with steeply sloping restitution curves with gradients greater than unity [14, 21, 24, 32]. The latter would reflect disproportionate decreases in mean APD with DI. Furthermore, manoeuvres which decrease the slopes of restitution curves, such as hyperkalaemia in a tachycardic canine endocardial preparation [22] and certain cardiotropic drugs in canine and porcine preparations [8, 31, 37], have been reported to both suppress alternans and exert anti-arrhythmic effects. In contrast, other studies have attributed such alternans to alteration in Ca2+ cycling involving the sarcoplasmic reticulum [35, 44, 54]. Whatever the underlying mechanism, alternans results in temporal variations in the wavelengths of propagating action potentials that could potentially lead to reentry and arrhythmogenesis [55].
Hypokalaemia is an important clinical cause of acquired QT prolongation and is associated with arrhythmia of various degrees of spatial organisation [12, 30, 57] suppressible by lidocaine [6, 36, 48]. The present study uses the monophasic action potential (MAP) technique [19, 23] and an established Langendorff-perfused murine model [18, 40–42] to explore the effects of hypokalaemia and of lidocaine on alternans and arrhythmogenicity for the first time in any species. These investigations of temporal heterogeneities in recovery after depolarization are performed on hearts paced at short BCLs, thereby complimenting recent work [40, 41] which contrastingly examined the contribution of spatial heterogeneities at long BCLs. This approach successfully demonstrates such phenomena in parallel with the clinical situation and proceeds for the first time to apply Fourier analysis to MAP waveforms and to recordings obtained during hypokalaemia. This analysis demonstrates evolution from a monomorphic to a polymorphic pattern as has previously been reported in other species [55, 58, 63], thereby further validating the murine model. The presence or absence of alternans and arrhythmogenicity is then related to alterations in the slopes of restitution curves. Thus, while the relationship between APD and BCL has previously been determined [20], the present study reports restitution curves relating APD to the preceding DI for the first time in a murine system. Use of this murine system further permits the comparison of findings with results obtained from genetically modified examples [26].
Taken together, these novel findings implicate a dynamic component to arrhythmic substrate under conditions of hypokalaemia for which restitution may furnish a possible underlying mechanism.
Materials and methods
Experimental animals
Mice were housed at 21 ± 1°C with 12-h light/dark cycles, were fed sterile chow (RM3 Maintenance Diet, SDS, Witham, Essex, UK) and had free access to water. Wild-type 129 Sv mice aged 3–6 months were used in all experiments. All procedures complied with the UK Animals (Scientific Procedures) Act 1986.
Solutions
Solutions were based on bicarbonate-buffered Krebs–Henseleit solution (mM: NaCl 119, NaHCO3 25, KCl 4, KH2PO4 1.2, MgCl2 1, CaCl2 1.8, glucose 10 and Na-pyruvate 2; pH adjusted to 7.4) and were bubbled with 95% O2/5% CO2 (British Oxygen Company, Manchester, UK). Hypokalaemic (3.0 mM K+) solutions were prepared by reducing the amount of KCl added. Lidocaine-containing solutions were prepared by adding lidocaine (Sigma-Aldrich, Poole, UK) to a final concentration of 10 μM.
Preparation
A Langendorff perfusion protocol previously adapted for murine hearts [3] was used. In brief, mice were killed by cervical dislocation [Schedule 1: UK Animals (Scientific Procedures) Act 1986]. Hearts were then quickly excised and placed in ice-cold bicarbonate-buffered Krebs–Henseleit solution. A short section of aorta was cannulated under the surface of the solution and attached to a custom-made 21-gauge cannula filled with the same solution using an aneurysm clip (Harvard Apparatus, Edenbridge, Kent, UK). Fresh Krebs–Henseleit solution was then passed through 200- and 5-μm filters (Millipore, Watford, UK) and warmed (37°C) using a water jacket and circulator (Techne model C-85A, Cambridge, UK) before being used for constant-flow retrograde perfusion at 2–2.5 ml/min using a peristaltic pump (Watson-Marlow Bredel model 505S, Falmouth, Cornwall, UK). Hearts were only regarded as suitable for experimentation if they regained a healthy pink colour and began to contract spontaneously on rewarming.
Electrophysiological measurements
An epicardial MAP electrode (Hugo Sachs, Harvard Apparatus) was placed against the basal left ventricular epicardium. A small access window was created in the interventricular septum to allow access to the left ventricular endocardium [5]. A custom-made endocardial MAP electrode comprising two twisted strands of high-purity Teflon-coated silver wire of 0.25-mm diameter (Advent Research Materials, UK) was constructed. The Telflon coat was removed from the distal 1 mm of the electrode, which was then galvanically chlorided to eliminate DC offset, inserted and placed against the septal endocardial surface. MAPs were amplified, band-pass-filtered (0.5 Hz to 1 kHz: Gould 2400S, Gould-Nicolet Technologies, Ilford, Essex, UK) and digitised at a sampling frequency of 5 kHz (micro1401, Cambridge Electronic Design, Cambridge, UK). Analysis of MAPs in both the time and frequency domains was performed using Spike II (Cambridge Electronic Design).
Experimental protocol
A bipolar platinum stimulating electrode (1 mm inter-pole spacing) was placed on the basal surface of the right ventricular epicardium. Square-wave stimuli (Grass S48 stimulator, Grass-Telefactor, Slough, UK) of 2-ms duration and amplitudes of twice the excitation threshold were initially applied to hearts at a constant baseline cycle length of 125 ms until MAPs showed stable baselines, rapid upstroke phases that reached consistent amplitudes and smooth repolarisation phases [19] and for at least 10 min. Hearts were then exposed to test solutions for 20 min while stimulation was continued before subsequent recordings were made.
In initial experiments, MAPs were recorded during regular stimulation at baseline cycle lengths of 130, 100 and 70 ms. Hearts were then subjected to an adapted dynamic pacing protocol [9]. This comprised cycles each consisting of 100 stimuli delivered over a range of BCLs. Steady states were consistently reached during the first 50 responses in each cycle, and thus, mean epicardial and endocardial APD90 values and DIs were calculated from the final 50 action potentials of each cycle. With each successive cycle, BCL was decremented by 5-ms steps from an initial value of 175 ms. Cycles were continued until a reproducible sequence of consistently shaped waveforms was no longer obtained. These data were then used to construct restitution curves.
All data are presented as means ± standard errors of the means and include the number of hearts studied. Comparisons between data sets used analysis of variance (significance threshold set at P ≤ 0.05). Curve fitting of particular function to data sets used a Levenberg–Marquardt algorithm (OriginPro 7.5, OriginLab, MA, USA).
Results
The experiments explored the consequences of increases in heart rate for arrhythmogenicity in hypokalaemic murine hearts, thereby extending a previous report that considered the effect of bradycardia [41]. They thus studied identical experimental groups, first exposing hearts to normokalaemic (5.2 mM K+) and hypokalaemic (3.0 mM K+) test solutions before and after addition of lidocaine (10 μM) for 20 min during regular stimulation at a BCL of 125 ms. Experiments then proceeded to study electrical activity through a range of steady state BCLs to assess propensity to spontaneous arrhythmia. Electrical traces from arrhythmic hearts were then quantitatively analysed in the frequency domain to empirically characterise their kinetics. Finally, hearts were subjected to a dynamic pacing protocol which explored the effect of varying BCLs [21] and permitted the analysis of alternans and its relationship to arrhythmogenicity.
Alternans and arrhythmic activity occur in hypokalaemic hearts paced at a reduced baseline cycle length
The first series of experiments recorded MAPs [23] from the epicardia of hearts paced at BCLs of 130, 100 or 70 ms over 30-min recording periods (five hearts in each case; Fig. 1). This demonstrated a tendency to both alternans, as reflected in alternating short-long-short sequences in action potential duration, and arrhythmogenesis at the shortest BCLs, corresponding to the highest heart rates, specifically in the hypokalaemic hearts and not in the other groups. Thus, MAPs obtained from normokalaemic hearts whether paced at BCLs of 130, 100 or 70 ms demonstrated consistent waveforms and stable rhythms during 30-min recording periods (five hearts in each case; Fig. 1a). In contrast, while recordings from hypokalaemic hearts paced at BCLs of 130 and 100 ms demonstrated stable rhythms (five out of five hearts), alternans leading to periods of arrhythmic activity occurred during pacing at a BCL of 70 ms in all five cases (P < 0.01 as compared to normokalaemic hearts; Fig. 1b). However, neither alternans nor arrhythmic activity occurred in either normokalaemic or hypokalaemic hearts treated with lidocaine during pacing at any BCL (five hearts in each case; P < 0.01 as compared to hypokalaemic hearts not treated with lidocaine; Fig. 1c and d, respectively), confirming the anti-arrhythmic effect of lidocaine described in earlier studies [6, 41, 42, 48].
Fig. 1Effect of baseline cycle length on presence of alternans and initiation of arrhythmic activity. Epicardial monophasic action potential recordings obtained from hearts exposed to normokalaemic (5.2 mM K+, a) and hypokalaemic (3.0 mM K+, b) test solutions, and normokalaemic (c) and hypokalaemic (d) test solutions containing lidocaine (10 μM) during regular stimulation at baseline cycle lengths of 130 ms (A), 100 ms (B) and 70 ms (C). Vertical lines indicate the timing of stimuli
Electrophysiological waveforms after the initiation of arrhythmic activity
When arrhythmic activity was observed in those hypokalaemic hearts paced at a BCL of 70 ms, this showed progressive degeneration from a regular monomorphic to a disorganised polymorphic pattern (four hearts), in common with earlier reports from the larger, porcine and human, hearts [16, 58, 63].
This trend was quantitatively apparent in a spectral analysis performed using a fast Fourier transform method [4] on ∼10-s sequences of MAP waveforms recorded from the epicardia and endocardia of hypokalaemic hearts to which a Hanning window had first been applied. These waveforms had been sampled at a rate of 5 kHz with low- and high-frequency filter cutoffs of 0.5 Hz and 1 kHz, respectively. Each transform was performed on 16,384 (=216) points to give a spectral resolution of 0.30 Hz up to a maximum, Nyquist, frequency of 2.5 kHz. Relative root mean square powers were then extracted from the real and imaginary parts of the transforms.
Figure 2 thus compares typical waveforms in both the time (a) and frequency (b) domains. Waveforms recorded during the first ∼5 min after the initiation of arrhythmic activity were monomorphic in appearance (A in Fig. 2a). This was confirmed by spectral analysis which similarly demonstrated a single identical dominant frequency peak of 18.9 ± 1.8 Hz in both epicardial and endocardial recordings with harmonics whose frequencies were integral multiples of a dominant fundamental frequency and any given heart showed at least two such peaks (∼38.4, ∼57.6, ∼76.8 Hz, A in Fig. 2b).
Fig. 2Progression from monomorphic to polymorphic arrhythmic activity. Epicardial and endocardial monophasic action potential recordings obtained from hearts exposed to hypokalaemic (3.0 mM K+) test solution 5 min (A) and 10 min (B) after initiation of arrhythmic activity in the time (a) and frequency (b) domains
In contrast, waveforms recorded ≥10 min after the initiation of arrhythmic activity were consistently polymorphic in appearance (B in Fig. 2a), with spectral analysis (B in Fig. 2b) further demonstrating dissimilar epicardial and endocardial frequency spectra. The dominant frequency peak was then at 27.6 ± 5.6 Hz in the epicardium, while that in the endocardium was 26.5 ± 5.8 Hz. Furthermore, the higher order peaks, whether recorded from the epicardium or the endocardium, occurred at irregular frequency intervals that were not multiples of the dominant frequency.
Alternans occurs in the epicardia and endocardia of hypokalaemic hearts paced at a baseline cycle length of 70 ms
As illustrated in C in Fig. 1b, hypokalaemic hearts paced at a BCL of 70 ms demonstrated alternans. Figure 3 goes on to show mean APD90 values of the alternating, odd-numbered (filled bars) and even-numbered (open bars) action potentials, and the difference between these values, giving the magnitude of any alternans (hashing), recorded from the epicardia (a) and endocardia (b) under normokalaemic (A) and hypokalaemic (B) conditions and under such conditions during treatment with lidocaine (C and D, respectively).
Fig. 3Alternans during stimulation at a baseline cycle length of 70 ms. Durations of successive odd (filled bars) and even (open bars) numbered action potentials (at 90% repolarisation, APD90) and the difference between these values, giving the magnitude of alternans (hashing) in the epicardia (a) and endocardia (b) of hearts exposed to normokalaemic (5.2 mM K+, A) and hypokalaemic (3.0 mM K+, B) test solutions and normokalaemic (C) and hypokalaemic (D) test solutions containing lidocaine (10 μM) during regular stimulation at a baseline cycle length of 70 ms
In normokalaemic hearts (five hearts), alternans was not observed, the difference between the mean APD90 values of odd-number and even-numbered action potentials not reaching significance (P > 0.05) in either the epicardium (4.7 ± 2.7 ms) or the endocardium (5.6 ± 1.7 ms). In contrast, alternans did occur in hypokalaemic hearts (five hearts), the difference between the mean APD90 values of odd-number and even-numbered action potentials being statistically significant (P < 0.01) in both epicardium (14.9 ± 3.8 ms) and endocardium (15.6 ± 1.8 ms). Treatment with lidocaine had no significant (P > 0.05) effect on normokalaemic hearts (five hearts): The difference between the mean APD90 values of odd-number and even-numbered action potentials remained statistically insignificant (P > 0.05) in both epicardium (2.7 ± 3.4 ms) and endocardium (2.5 ± 4.6 ms). In contrast, treatment with lidocaine eliminated alternans in hypokalaemic hearts (five hearts). Hence, the difference between the mean APD90 values of odd-number and even-numbered action potentials did not reach significance (P > 0.05) in either epicardium (4.1 ± 4.6 ms) or endocardium (2.4 ± 3.2 ms).
The dynamic pacing protocol initiates alternans in hypokalaemic hearts at short baseline cycle lengths
The above findings concerning the presence or absence of alternans at the shortest BCL studied above prompted a detailed exploration of the effect of BCL upon the alternans phenomenon. To assess this relationship, hearts were subjected to a dynamic pacing protocol [21] consisting of cycles each lasting 100 stimuli at through a range of BCLs. In these procedures, steady states were consistently reached within the first 50 responses. Accordingly, mean values of epicardial and endocardial APD90 and DI were obtained from the final 50 action potentials. BCL was decremented in 5-ms steps with each cycle from an initial value of 175 ms until a reproducible sequence of consistently shaped action potential waveforms could no longer be obtained.
Figure 4a–d shows typical recordings obtained from the epicardia of individual hearts under the four sets of experimental conditions during the dynamic pacing protocol at BCLs of 170, 130, 90, 85, 80, 75 and 70 ms when such steady states had been achieved. Figure 5a–d shows the corresponding relationships between the durations of successive odd- and even-numbered action potentials obtained from these same hearts during the dynamic pacing protocol over the full range of BCLs studied. Thus, alternans was not observed under normokalaemic conditions even at the shortest BCL studied (Fig. 4a), resulting in points falling on the line y = x (Fig. 5a). Conversely, alternans occurred under hypokalaemic conditions at short BCLs (Fig. 4b), resulting in such points falling below the line of equality (Fig. 5b). In contrast, alternans did not occur in either normokalaemic or hypokalaemic hearts during exposure to lidocaine (Fig. 4c and d), again resulting in points falling on the line of equality (Fig. 5c and d).
Fig. 4Effect of baseline cycle length on action potential waveforms. Epicardial monophasic action potential recordings obtained from hearts exposed to normokalaemic (5.2 mM K+, a) and hypokalaemic (3.0 mM K+, b) test solutions and normokalaemic (c) and hypokalaemic (d) test solutions containing lidocaine (10 μM) during stimulation at baseline cycle lengths of 170, 130, 90, 85, 80, 75 and 70 ms. Vertical lines indicate the timing of stimuliFig. 5 Relationships between durations of successive action potentials over a range of baseline cycle lengths. Relationships between the durations of successive odd- and even-numbered epicardial monophasic action potentials (at 90% repolarisation, APD90) in the same hearts exposed to normokalaemic (5.2 mM K+, a) and hypokalaemic (3.0 mM K+, b) test solutions and normokalaemic (c) and hypokalaemic (d) test solutions containing lidocaine (10 μM). These were obtained at baseline cycle lengths decremented in 5-ms steps from 175 ms until stimulation no longer resulted in a reproducible sequence of waveforms. Filled and open circles indicate data obtained at baseline cycle lengths at which arrhythmogenesis was and was not observed, respectively. Arrow indicates departure of points from the line y = x (broken line)
Alterations in the slopes of restitution curves correlate with pro- and anti-arrhythmic effects
The findings described above prompted the construction and analysis of restitution curves relating APD90 and the preceding DI given by the difference between the BCL and the preceding APD90 [21, 29, 32, 55]. Alternans and arrhythmia have previously been associated with increases in the slopes of such restitution curves reflecting disproportionate decreases in mean APD90 with DI. Such curves (data points, left ordinate) were plotted using the APD90 (left ordinate) and DI data obtained from the epicardia (circles, Fig. 6A) and endocardia (squares, Fig. 6B) of hearts under these conditions (a–d, respectively).
Fig. 6Restitution curves obtained from epicardia and endocardia. Restitution curves plotting action potential duration (at 90% repolarization, APD90) against preceding diastolic interval (DI) obtained from the epicardia (circles, A) and endocardia (squares, B) of hearts exposed to normokalaemic (5.2 mM K+, a) and hypokalaemic (3.0 mM K+, b) test solutions and normokalaemic (c) and hypokalaemic (d) test solutions containing lidocaine (10 μM). Curves are fitted with mono-exponential growth functions obtained by least-squares fitting to the experimental values of APD90 and DI (solid lines, left ordinates). Gradients were obtained by differentiation of the fitted functions (broken lines, right axes). Shaded boxes indicate ranges of DI values at which such gradients exceed unity
Under all conditions, both epicardial and endocardial APD90 decreased as DI decreased. However, the slope of this relationship was greater under hypokalaemic (A and B in Figs. 6b) than under normokalaemic conditions (A and B in Figs. 6a). This paralleled both the alternans and arrhythmic activity that were observed under the hypokalaemic conditions. Such increases in the slopes of restitution curves were observed even when the DI was so short such that stimuli were delivered before 90% repolarisation was achieved. This would tend to result in the underestimation of APD90, and hence, the underestimation of the slopes of restitution curves.
Exposure to lidocaine had little effect on the slopes of the corresponding curves obtained under normokalaemic (A and B in Figs. 6c), but markedly decreased the slopes of curves obtained under hypokalaemic conditions (A and B in Figs. 6d), in precise agreement with its corresponding effects in suppressing alternans and arrhythmia.
Restitution curves yield parameters predictive of arrhythmogenicity
The restitution curves obtained above proved amenable to quantitative analyses that has been described on previous occasions. For example, the simplest, mono-exponential growth function that has been used to describe data of this kind [13] takes the form:
where y represents APD90, x represents DI and y0 and A and τ are constants obtained by least squares fitting to the experimental values of APD90 and DI in each case. Such curve-fits are used merely to empirically describe the data and should not be taken to imply any particular underlying mechanistic process. The corresponding gradient is then given by:
and assumes its maximum value at the shortest BCL.
Figure 6 plots the original APD90 and DI values and superimposes optimisations of Eq. 1 to these data (solid lines, left-hand ordinates). Such optimisations gave reduced χ2 values indicative of better fits to data points obtained under normokalaemic than hypokalaemic conditions. It also shows the corresponding gradients derived from constants obtained from the optimisations (Eq. 2: broken lines, right-hand axes). Shaded regions where present indicate the range of DIs below a critical DI (DI < critical DI) at which these gradients exceed unity, obtained from Eq. 2:
Such a phenomenon is associated with an alternans initiated by incomplete recovery of one action potential and the consequent shortening of its subsequent DI. This would result in a disproportionate shortening of the subsequent action potential which would consequently permit its full recovery and a lengthening of the DI that follows, thereby reinitiating the cycle and resulting in a progressive decrease in mean APD90 relative to mean DI with decreasing BCL [29].
The resulting alternans would assume an amplitude determined by the gradient of the restitution curve at the given BCL, whether the latter is determined by altered external pacing rates or local ventricular arrhythmic activity. A BCL that gives a gradient of >1 will then initiate a shortening not only of mean APD90 but also of mean APD90 relative to mean DI. The resulting shortening of action potential wavelength would then predispose to reentry and arrhythmogenesis.
These parameters successfully predict the pro-arrhythmic effect of hypokalaemia
Figure 7 goes on to show these maximum gradients (a) and values of the critical DI (b) derived from the computed A and τ values in epicardia (filled bars) and endocardia (open bars). Under normokalaemic conditions (Fig. 7A) A and τ took values of 56.3 ± 2.4 ms and 58.4 ± 9.8 ms in the epicardium and 73.8 ± 1.7 ms and 65.2 ± 8.4 ms in the endocardium, with reduced χ2 values from least-squares regression of  = 1.4 and 1.2, respectively. This gave maximal gradients of 0.55 ± 0.14 in the epicardium and 0.67 ± 0.13 in the endocardium and critical DI values of −2.14 ± 0.52 ms and 8.14 ± 1.49 ms, respectively. None of the experimental DI values realised the critical DI in either the epicardium or the endocardium, paralleling the absence of arrhythmic activity (Fig. 2a).
Fig. 7Maximum gradients and critical diastolic intervals obtained from restitution curves. Maximum gradients (a) and critical diastolic intervals at which gradients equalled unity (b) obtained from epicardial (filled bars) and endocardial (open bars) restitution curves shown in Fig. 6 under normokalaemic (5.2 mM K+, A) and hypokalaemic (3.0 mM K+, B) conditions and under normokalaemic (C) and hypokalaemic (D) conditions during exposure to lidocaine (10 μM). Asterisks indicate values that are significantly (P < 0.05) larger than those recorded in normokalaemic hearts
Hypokalaemia (Fig. 7B) significantly (P < 0.05) increased the slopes of both epicardial and endocardial curves, concurring with the arrhythmogenic findings. Thus, A and τ took values of 166.7 ± 5.63 ms and 29.3 ± 8.29 ms in the epicardium and 133.3 ± 8.0 ms and 29.8 ± 2.1 ms in the endocardium, with  values of 8.2 and 2.5, respectively. This gave significantly (P < 0.05) increased maximum gradients of 2.35 ± 0.67 in the epicardium and 1.87 ± 0.28 in the endocardium and significantly (P < 0.05) increased the critical DI values of 50.93 ± 14.45 ms and 44.64 ± 5 ms, respectively. The critical DI was realised by the observed data points in both epicardium and endocardium. Furthermore, the DIs at which this occurred were in full agreement with the corresponding DIs at which alternans was observed. Thus, in the epicardium, alternans occurred during pacing at BCLs of ≤95 ms, corresponding to DIs of 46.7 ± 3.6 ms, while in the endocardium, alternans occurred during pacing at BCLs of ≤75 ms, corresponding to DIs of 44.0 ± 5.7 ms.
These parameters also predict the anti-arrhythmic effect of lidocaine
Lidocaine had no significant (P > 0.05) effect on these values when applied to normokalaemic hearts (Fig. 7C). Thus, A and τ took values of 81.1 ± 3.05 ms and 56.92 ± 10.29 ms in the epicardium and 104.47 ± 13.69 ms and 97.17 ± 36.22 ms in the endocardium, with  values of 5.37 and 10.47, respectively. This gave maximum gradients of 0.78 ± 0.27 in the epicardium and 0.83 ± 0.45 in the endocardium and the critical DI values of 6.06 ± 2.10 ms and 7.04 ± 3.82 ms, respectively, which were not realised even at the shortest BCLs studied.
In contrast, treatment of hypokalaemic hearts with lidocaine (Fig. 7D) significantly (P < 0.05) reduced the slopes of both epicardial and endocardial restitution curves, in parallel with its anti-arrhythmic effect. Thus A and τ took values of 109.90 ± 11.95 ms and 37.36 ± 5.97 ms in the epicardium and 231.72 ± 44.70 ms and 27.60 ± 4.44 ms in the endocardium, with  values of 6.83 and 15.68, respectively. This gave significantly decreased (P < 0.05) maximal gradients of 1.05 ± 0.30 in the epicardium and 0.89 ± 0.36 in the endocardium and significantly decreased (P < 0.05) critical DI values of 30.38 ± 8.88 ms and 31.65 ± 4.78 ms, respectively. The critical DI was realised only at the shortest BCLs studied and then only in the epicardium, in parallel with the substantial reduction in arrhythmogenicity produced by this drug.
Slopes of restitution curves thus precisely correlate with the presence or absence of alternans and arrhythmogenesis under all the conditions explored in the hypokalaemic Langendorff-perfused murine heart.
Discussion
Electrical alternans, beat-to-beat alternation in action potential duration in turn reflected in alternation in electrocardiographic QT interval (QT or T-wave alternans), is most commonly seen at high heart rates and has been associated with arrhythmogenesis in both clinical [2, 25, 28, 38] and experimental [7, 33, 52] studies. This association is especially marked in situations of preexisting action potential, and therefore electrocardiographic QT, prolongation [39, 46, 47, 49, 64] as might occur in the congenital long QT syndromes.
Alternans phenomena have classically been analysed by the construction of restitution curves relating action potential duration to the preceding diastolic interval as heart rate is varied [29]. Restitution curves of slopes greater than unity have been associated with alternations in action potential durations of progressively increasing magnitude [14, 21, 24, 29, 32]. In contrast alternans has more recently been associated with disruption of normal cellular Ca2+ homeostasis leading to alternating short-long-short pulses of release from sarcoplasmic reticular stores [35, 44, 54]. Irrespective of the underlying mechanism, any spatial discordance in either phase or magnitude of alternans between myocardial regions would lead to spatial repolarisation gradients that might cause reentry and arrhythmogenesis [53, 56]. Furthermore, any resulting decrease in action potential wavelength, given by the product of action potential duration and conduction velocity, to less than a critical value, might break up the propagating wave of excitation and lead to reentry and arrhythmogenesis [55].
Relationships between restitution curve slopes, alternans and arrhythmogenicity at high heart rates have been established in canine preparations made to model congenital long QT syndrome type 2 (LQT2) by application of the IKr blocking agent E-4031 [27, 61]. Correspondingly, there is a known association between sudden arousal, presumably resulting in increased heart rate, and arrhythmogenesis in human LQT2 [59]. This association has also been established in clinical hypokalaemia [11]. This common and pathophysiologically important condition similarly results in decreased repolarising K+ currents, together with action potential, and therefore electrocardiographic QT, prolongation [17, 62].
The experiments reported recorded MAP technique [19, 23] from an established intact hypokalaemic murine model [18, 40–42] and explored for alternans and arrhythmogenicity and the relationship between these before proceeding to study the effect of lidocaine upon these phenomena. Such studies of temporal heterogeneities in recovery were made at short BCLs in contrast to recent work [40, 41] which examined spatial heterogeneities at long BCLs. The murine system utilised facilitates the introduction of genetic modifications [26] and may therefore permit future comparisons with models replicating congenital arrhythmic syndromes. Indeed, alternans, in association with arrhythmogenicity, has previously been demonstrated in a murine model of LQT5 [50].
While the murine heart is well established as a model for human disease, it must be noted that important interspecies differences exist. Thus, differences between humans and mice in the kinetics of the ion channels carrying the key repolarising currents (the delayed rectifier and slowly-inactivating delayed rectifier currents, IKr and IKs, in humans and the transient outward current, Ito in mice) result in shorter ventricular AP and the absence of a plateau phase in mice [2]. However, in both species, these key repolarising currents are K+-sensitive [6, 11, 16], making the murine heart well suited to modelling clinical hypokalaemia. Furthermore, in both species, depolarisation is rapid and attributable to the same fast Na+ current (INa,f) [4], making our model well suited to the study of Na+-channel-blocking agents such as lidocaine. While both species share similar differences in AP duration between epicardium and endocardium [7], M cells appear to be absent in the murine heart [1]. Nonetheless, relationships between APD and refractory period are similar between the two species [3, 8], and transmural conduction velocities are almost identical [5, 10]. With these caveats in mind, similarities between human and murine hearts make our murine model well suited for the study of the basic mechanisms of arrhythmogenicity.
First, we confirm that alternans is absent in control normokalaemic hearts and that this is associated with the maintenance of stable rhythms even at short BCLs. Secondly, we report alternans in association with arrhythmogenesis at short BCLs for the first time in hypokalaemic murine hearts, in full agreement with previous clinical and experimental studies in other species [2, 7, 33, 38, 52].
Thirdly, we associate such alternans with increases in the slopes of restitution curves above unity [29, 55]. Fourthly, we demonstrate for the first time in any experimental model that exposure to the class I agent lidocaine reduces the slopes of restitution curves and suppresses alternans in association with its anti-arrhythmic effect. Taken together, these observations implicate a restitution mechanism in driving alternans in these hypokalaemic preparations. Furthermore, this extends previous reports that the anti-arrhythmic effects of both class III and IV agents are associated with decreases in the slopes of restitution curves [8, 31, 37]. This contrasts with a previous report that found lidocaine to have no effect on restitution curves obtained using an extrasystolic stimulation procedure or alternans phenomena in a normokalaemic excised canine papillary muscle preparation [43].
Fifthly, we show that such alternans and steeply sloping restitution curves are associated with the temporal evolution of the normal regular pattern of cardiac excitation into arrhythmic activity. The latter, in turn, was processed by a spectral Fourier transform analysis of MAP waveforms performed for the first time in any cardiac system. Arrhythmic MAP waveforms were initially monomorphic, and their corresponding spectra contained a single dominant frequency and integral multiple harmonics identical in epicardial and endocardial recordings. Such monomorphic arrhythmic waveforms subsequently degenerated into polymorphic waveforms whose spectra showed dissimilar epicardial and endocardial dominant frequencies and larger higher order peaks that were not integral multiples of the dominant frequency in full agreement with previous results from the porcine heart [63]. Spectra with similar features have previously been obtained from volume-conducted electrograms and pseudoelectrograms (as opposed to MAPs) from murine hearts during both monomorphic and polymorphic arrhythmic activity [51].
Taken together, these results recapitulate in a murine system arrhythmogenic patterns classically described in clinical situations [16, 58] and implicate a dynamic component to arrhythmic substrate under conditions of hypokalaemia for which restitution may furnish a possible underlying mechanism. Furthermore, they disprove previous suggestions that small hearts are unable to sustain such arrhythmias [9, 60], and thus further validate the murine heart as a model for the study of clinical arrhythmia. The present findings might therefore form a basis for further explorations of the relationships between heart rate, restitution slopes, alternans and arrhythmogenicity in other states of QT prolongation, whether attributable to congenital or acquired factors.

alternans
arrhythmia
monomorphic
polymorphic
restitution curve
hypokalaemia


Nuclear morphometric features in benign breast tissue and risk of subsequent breast cancer

Certain nuclear morphometric features measured in breast tumor tissue have been shown to predict the prognosis of breast cancer patients. However, the application of these features to predicting risk of breast cancer development has received little attention. We conducted a case-control study to evaluate nuclear morphometric features in benign breast tissue in association with subsequent breast cancer risk. The study was nested within a cohort of 4,888 women with a histopathologic diagnosis of benign breast disease (BBD) and involved 61 cases and 71 controls, amongst whom there were 53 matched case-control sets. Conditional logistic regression models were fitted to assess various measurements of nuclear size and nuclear shape factors in relation to subsequent breast cancer risk. In multivariate analysis, subsequent breast cancer risk was positively associated with a nuclear shape factor that takes the shortest nuclear axis and the longest nuclear axis into consideration simultaneously (highest quartile versus lowest 3 quartiles: odds ratio = 3.07, 95% confidence limits = 1.61, 5.84). In contrast, there was no alteration in subsequent breast cancer risk in association with nuclear size features and other shape factors. In conclusion, our study results suggest that the shape factor that takes both the shortest nuclear axis and the longest nuclear axis into consideration might be of value to predict subsequent development of breast cancer among women with BBD.
Introduction
Benign breast disease (BBD), in addition to certain hormonal, anthropometric, and lifestyle factors, is a well-established risk factor for breast cancer [1, 2]. However, BBD comprises a broad spectrum of histological entities [3]. Both epidemiologic and experimental studies suggest that non-atypical and atypical proliferative changes represent successive steps preceding the development of in situ cancer and then invasive carcinoma of the breast [4]. However, only a small fraction of women will eventually develop breast cancer after their diagnosis of BBD [5]. Therefore, it is important to differentiate BBD patients with a high risk of subsequent development of breast cancer from those with a low risk. Our understanding regarding this issue, however, is rather limited, although previous studies have suggested that factors such as type of histological subtype (e.g., atypical hyperplasia), menopausal status, and family history of breast cancer, might modify breast cancer risk among women with BBD [6].
Computerized image analysis and morphometry can quantify a number of nuclear morphometric features such as nuclear size, nuclear shape, and chromatin texture [7]. The evaluation of these features may facilitate the diagnosis and management of breast cancer patients [8–10]. Indeed, certain nuclear morphometric features measured in breast tumor tissue have been shown to predict the prognosis of breast cancer patients [11–15]. Furthermore, a study by Mommers et al. [16] observed that normal breast tissue or usual ductal hyperplasia harbored nuclear morphometric changes that might be used to predict subsequent development of breast cancer. In the study reported here, we conducted a nested case-control study to evaluate whether nuclear morphometric features as evaluated in tissue sections of BBD may be related to the risk of subsequent breast cancer among patients with BBD.
Methods
Study population
The present investigation was undertaken using histological sections from a previous case-control study nested within the cohort of 4,888 women in the Canadian National Breast Screening Study (NBSS) who were diagnosed histopathologically with BBD during the active follow-up phase of the NBSS [17]. The NBSS is a multi-center randomized, controlled trial of screening for breast cancer among 89,835 women aged 40–59 years at recruitment. The design of the NBSS and population characteristics have been described in detail elsewhere [18, 19]. Recruitment took place between 1980 and 1985, and study subjects were followed actively until 1988. Eligibility for the study was restricted to women with no history of breast cancer (in situ or invasive). The NBSS was approved by the appropriate Institutional Review Boards, and the study described here involved the analysis of material and data from that study in accordance with the approved study design. Informed consent was obtained from all study participants.
Diagnosis of BBD
In the NBSS, women who had clinical or radiologic evidence of breast lesion underwent either a needle aspiration or a biopsy. Diagnosis of BBD was performed by a reference pathologist. Our study was restricted to women who had no evidence of either in situ or invasive breast cancer on their initial surgical biopsy. Women with a history of BBD were not excluded from the analyses. During the follow-up period, we identified 4,888 women with a histopathologic diagnosis of BBD, who were followed up for the subsequent development of breast cancer.
Selection of cases and controls
Incident cases of breast cancer were ascertained by record linkage with the provincial cancer registries, and death clearance was performed by linkage to the Canadian National Mortality Database [18, 19]. The dates of the linkages varied by province, ranging from late 1988 to early 1991. A total of 16 subjects with ductal carcinoma in situ and 76 subjects with invasive carcinoma were ascertained among the cohort of women with BBD. Potential control subjects were women with BBD who had not developed breast cancer (but were alive at) by the date of diagnosis of the corresponding case subject. Five controls were selected randomly (with replacement) for each case from those non-cases available within strata defined by screening center, NBSS study arm, year of birth (if possible to the nearest year, and mostly within 2 years), and age at diagnosis of BBD. For the study reported here, 61 case subjects and 71 control subjects (including 53 matched case-control sets) were included.
Questionnaire
Upon enrollment in the NBSS, all participants completed a questionnaire that sought information on demographic characteristics and risk factors for breast cancer, including menstrual and reproductive histories and family history of breast cancer.
Morphometry
Morphometric measurements were performed on H&E stained slides, using the QPRODIT interactive video-overlay system (Leica, Cambridge, UK). About 50 nuclei were selected in the most representative areas of the slide (selected by a breast pathologist), and their contours were traced manually using a 100× objective (final magnification about 3,000×) [20]. Mean and standard deviation of nuclear area, perimeter, diameter, shortest axis, longest axis, and axis ratio were calculated, as well as different shape factors. The shape factors were calculated by the following formulas: Form_AR = (1/4) * pi * longest axis * shortest axis; Form_PE = 4 * pi * area/(perimeter squared); Form_NCI = perimeter/sqrt (area); Contour ratio = perimeter squared/4 * pi * area; and Roundness = perimeter/(2*sqrt (pi * area)). All morphometric assessments were performed by one observer without knowledge of patient outcome.
Statistical analysis
Morphometric measurements were first compared between cases and controls using Student’s t-test. Subsequently, the measurements were categorized by quartiles and then odds ratios (OR) and 95% confidence limits (CLs) were calculated for the risk of breast cancer for those in the highest quartile level compared to that for those in the lowest 3 quartile levels using conditional logistic regression. In multivariate analyses, we controlled for age at menarche (<13, 13, 14+), age at first live birth (nulliparous, <23, 23–26, 27+), menopausal status (pre-, peri-, post-), oral contraceptive use (ever versus never), postmenopausal estrogen use (ever vs. never), body mass index (<25, 25+), family history of breast cancer, and the presence of hyperplasia in the benign tissue. All statistical analyses were performed in SAS 9.1 (SAS Institute, Cary, NC). P-values were two-sided.
Results
Table 1 summarizes the distribution of selected characteristics among the cases and controls. Overall, few differences between the cases and controls were observed for age at menarche, age at first live birth, menopausal status, oral contraceptive use, postmenopausal estrogen use, body mass index, family history of breast cancer, and the presence of hyperplasia in benign tissue.
Table 1.Distribution of selected characteristics among breast cancer cases and non-cases N (%)P-valueCasesControlsAge at menarche<1330 (49)26 (37)0.291313 (21)22 (31)14+18 (30)23 (32)Age at first live birthNulliparous11 (18)9 (13)0.84<2322 (36)29 (41)23–2619 (31)23 (32)27+9 (15)10 (14)Menopausal statusPre-30 (49)31 (44)0.71Peri-9 (15)14 (20)Post-22 (36)26 (36)Ever used oral contraceptivesYes35 (57)42 (60)0.76No26 (43)28 (40)Missing01Ever used postmenopausal estrogensYes15 (25)15 (22)0.70No46 (75)54 (78)Missing02Body mass index (kg/m2)<2532 (53)41 (58)0.4225– < 3027 (44)25 (35)30+2 (3)5 (7)Family history of breast cancerYes23 (38)28 (39)0.84No38 (62)43 (61)Hyperplasia in benign tissueAbsent34 (59)47 (68)0.27Present24 (41)22 (32)Missing32
There was little difference between the cases and controls with respect to nuclear morphometric features including mean area, standard deviation (SD) of area, perimeter, diameter, shortest axis, and longest axis, as well as such shape factors as Form_PE, Form_NCI, contour, and roundness (Table 2). In contrast, the shape factor Form_AR was greater among cases than among controls. Furthermore, subjects with hyperplasia had greater measures of some nuclear size features including mean area, SD of area, perimeter, diameter, and longest axis, and the shape factor Form_AR than did subjects without hyperplasia (data not shown).
Table 2.Comparison of nuclear morphometric features in benign breast tissue between breast cancer cases and non-casesMorphometric measurementsMean (standard deviation)P-valueCases (n = 61)Controls (n = 71)Mean nuclear area (μm2)26.8 (7.5)25.3 (7.2)0.25SD of nuclear area (μm2)5.2 (1.8)5.0 (1.6)0.43Nuclear perimeter (μm)19.7 (2.7)19.3 (2.7)0.37Nuclear diameter (μm)5.8 (0.8)5.6 (0.8)0.23Shortest nuclear axis (μm)4.8 (0.7)4.6 (0.7)0.16Longest nuclear axis (μm)7.1 (1.0)7.0 (1.0)0.53Axis ratio1.5 (0.1)1.6 (0.2)0.15Form_AR0.984 (0.005)0.981 (0.007)0.0089Form_PE0.844 (0.037)0.831 (0.045)0.083Form_NCI3.874 (0.095)3.909 (0.122)0.071Contour1.198 (0.061)1.221 (0.080)0.068Roundness1.093 (0.027)1.103 (0.034)0.071
Quartile analyses revealed that subsequent breast cancer risk was increased in association with the shape factor Form_AR, but not with the other nuclear morphometric measurements (Table 3). Compared to BBD subjects with Form_AR equal to or less than 0.986, subjects with Form_AR greater than 0.986 had a more than three-fold increased risk of developing breast cancer subsequently (OR = 3.07, 95%CL = 1.61, 5.84). When the analyses were repeated using unconditional logistic regression, which enabled all the available cases and controls to be included, the results did not change substantially.
Table 3.Risk of Subsequent development of breast cancer in association with nuclear morphometric featuresaMorphometric measurementsCut-off valueOR (95% CL)Model 1bModel 2cMean nuclear area (μm2)31.21.28 (0.73, 2.25)0.94 (0.50, 1.78)SD of nuclear area (μm2)6.11.33 (0.76, 2.31)1.11 (0.59, 2.07)Nuclear perimeter (μm)21.41.14 (0.70, 1.93)0.85 (0.47, 1.55)Nuclear diameter (μm)6.31.29 (0.73, 2.27)0.95 (0.50, 1.79)Shortest nuclear axis (μm)5.21.62 (0.92, 2.86)1.18 (0.62, 2.26)Longest nuclear axis (μm)8.01.34 (0.75, 2.39)0.95 (0.50, 1.81)Axis ratio1.60.59 (0.30, 1.17)0.71 (0.33, 1.54)Form_AR0.9862.45 (1.42, 4.22)3.07 (1.61, 5.84)Form_PE0.8671.22 (0.71, 2.08)1.57 (0.83, 2.97)Form_NCI3.9351.07 (0.58, 1.98)1.18 (0.61, 2.27)Contour1.2361.13 (0.61, 2.10)1.22 (0.63, 2.35)Roundness1.1101.07 (0.58, 1.98)1.18 (0.61, 2.27)a Analyses were conducted among 53 matched case-control sets by comparing the highest quartile versus the lowest 3 quartiles in conditional logistic regression modelsb Adjusted for matching variablesc Adjusted for matching variables, age at menarche (<13, 13, 14+), age at first live birth (nulliparous, <23, 23–26, 27+), menopausal status (pre-, peri-, post-), oral contraceptive use (ever vs. never), postmenopausal estrogen use (ever versus never), body mass index (<25, 25+), family history of breast cancer, and the presence of hyperplasia in the benign tissue
Discussion
We found that breast cancer risk in women with BBD was positively associated with the shape factor Form_AR, a measurement that takes the shortest nuclear axis and the longest nuclear axis into consideration simultaneously. In contrast, there was no alteration in risk in association with nuclear area, SD of nuclear area, nuclear perimeter, nuclear diameter, shortest nuclear axis, longest nuclear axis, and other shape factors. Although subjects with hyperplasia had greater measures of Form_AR than did subjects without hyperplasia, we adjusted for hyperplasia, suggesting that the association with Form_AR is independent of that due to the presence of hyperplasia.
Shape is one of the factors that pathologists use in assessing nuclear atypicality. Shape factors have been shown to have prognostic value in breast cancer [21–23], renal cell cancer [24], colorectal cancer [25], squamous cell carcinoma of the larynx [26], melanoma [27], and rhabdomyosarcoma [28]. Apparently, alterations in nuclear shape can already be present at the earliest stages of carcinogenesis. This has in the breast also been shown for nuclear chromatin patterns [29].
To date, only one study has been published that assessed morphometric features in association with subsequent development of breast cancer among women with BBD [16]. That study found positive associations for mean nuclear area, nuclear diameter, nuclear perimeter, and the longest nuclear axis, but no associations for SD of the nuclear area and the shortest nuclear axis; shape factors were not evaluated. However, potential confounding factors were not controlled for. In contrast to these findings, nuclear size features were not associated with risk in the present study, which may perhaps be explained by differences in tissue processing procedures.
Our case-control study was nested in a cohort of patients with histopathologically confirmed BBD and our findings are likely to be internally valid. Biased measurement of the study exposures was not likely a source of error, given that the morphometric features were assessed without knowledge of the patient outcome status. Our study power, however, was limited by the relatively small sample size, due to which we were not able to evaluate modifying effects by well-documented risk factors of breast cancer. Moreover, residual confounding might still exist, although to minimize confounding we controlled for menstrual and reproductive history, exogenous estrogen use, body mass index, and family history of breast cancer in multivariate analyses.
In conclusion, our study results suggest that the shape factor that takes both shortest nuclear axis and longest nuclear axis into consideration might be of value to predict subsequent development of breast cancer among patients with BBD. Given the limitations of our study, larger studies are warranted to confirm our study results.

breast cancer
benign breast disease
nuclear size
nuclear shape
morphometry


Tri-nucleotide receptors play a critical role in epithelial cell wound repair

The cornea plays a major role in the refraction of light to the retina. Therefore, the integrity and transparency of the corneal epithelium are critical to vision. Following injury, a combination of rapid signal transduction events and long-term cell migration are essential for wound closure. We have demonstrated previously that injury resulted in the release of nucleotides that induce the propagation of a Ca2+ wave to neighboring cells. This suggests that nucleotides and their receptors are critical components of wound healing. Epidermal growth factor (EGF) and integrins also have been shown to play a role in injury. In this study, we demonstrate that pretreatment of cells with ATP and UTP inhibited the immediate wound response, while BzATP, ADP, and UDP did not affect this response. Tri-nucleotide pretreatment also reduced the EGF induced Ca2+ response. Additionally, lower EC50 concentrations of ATP and UTP triggered migration of cells that was enhanced further with EGF and was inhibited by the tripeptide, RGD. Results indicate that the desensitization induced by ATP and UTP was specific. While ADP and UDP cause a homologous desensitization of their own signal, they did not cause an inhibition of the wound response nor does BzATP. Neither Ca2+ wave propagation nor cell migration occurred in response to β,γ-MeATP. Together these results lead us to hypothesize that corneal epithelial wound repair is mediated by both P2Y2 and P2Y4 receptors.
Introduction
Wound healing is a series of complex biological responses requiring cytoskeletal and extracellular matrix remodeling, signal cascades, and gene regulation. Initial and long-term signals after injury are communicated to the cells and promote wound healing. These signals and events must be understood to advance our knowledge of normal wound repair. Investigators have demonstrated that nucleotides are released from cells by mechanical stimulation, ligand binding, exocytotic release, or injury [1–5] Nucleotide stimulation can also lead to an increase in intracellular calcium. The increase in cytosolic calcium can be generated via activation of purinergic (P2Y or P2X) receptors through distinct mechanisms. In addition, purinergic receptors can promote cell migration, a critical component of wound repair. The exact role and identity of the various receptors and their importance in different systems has yet to be elucidated.
Nucleotides have been shown to accelerate wound closure [6]. Nucleotides can act as a chemotactic stimulus for cell migration in many cell types including corneal epithelial [7], arterial smooth muscle [8], microvascular endothelial [9], and immature dendritic cells [10]. P2 receptors also have been shown to play a role in repair after ischemia and renal tubular epithelial wound healing [11, 12]. Previously, we demonstrated that ATP plays a critical role in intercellular communication following mechanical injury. ATP induced cell migration, and Reactive Blue-2, a P2 antagonist, inhibited wound closure [7]. In addition, nucleotides induced calcium waves and phosphorylation of paxillin (pY118) and ERK1/2 in a transient manner [7, 13]. Furthermore, neither occurred in the presence of β,γ-MeATP [7, 13]. Our goal is to determine how epithelial cells respond to nucleotides in order to understand the events that occur after injury.
P2Y and P2X receptors are expressed in a number of cell types. As G-protein coupled receptors (GPCR), P2Y receptors are seven pass transmembrane receptors coupled to heterotrimeric G-proteins (review in [14]). P2Y receptors couple to Gαq and activate phospholipase C β (PLCβ), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to diacylglycerol (DAG) and phosphoinositol tri-phosphate (IP3). IP3 binds to Ca2+ channels on the endoplasmic reticulum and causes an increase in intracellular Ca2+. This family of receptors consists of eight members: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14. P2X receptors are ligand gated ion channels that open upon agonist binding allowing extracellular calcium to enter the cell. This family of receptors has seven members: P2X1–7. Each receptor has a specific agonist potency profile for different nucleotides with some showing preference for a specific nucleotide and others preferentially responding to tri-nucleotides or adenine based nucleotides [15]. We have shown that corneal epithelial cells express a number of P2YR subtypes [7], and an intracellular Ca2+ increase is seen in response to both nucleotides and injury [5].
Epidermal growth factor receptor (EGFR) also plays an important role in injury response. The EGFR family has four receptor family members, ErbB1-4. These receptors can form both homo- and hetero-dimers when stimulated by the epidermal growth factor (EGF) or other ligands [16]. The EGFR becomes activated upon injury, and inhibition of ErbB phosphorylation limits proper wound closure in vitro [7]. EGF itself promotes cell migration and wound healing in vivo [17] and can up-regulate integrin expression [18]. Gross mechanical injury induces a Ca2+ wave that propagates out from the wound site. EGF has been shown to increase the intensity of the Ca2+ wave but is not required for propagation [5]. Recently, growth factor receptors and GPCRs have been demonstrated to participate in cross talk connecting signaling pathways through trans-activation, which can occur via enhancement or down-regulation of downstream signal cascades [19]. In addition, specific P2 receptors can associate with other signaling proteins. For example, P2Y2 has an arginine-glycine-aspartic acid (RGD) domain on its first extracellular loop that can associate with integrins [20]. Currently, no other P2Y receptor has an identified RGD domain that interacts with integrins. In addition, this RGD domain promotes signaling and interactions with epidermal growth factor receptors (EGFR) [21].
Our goal is to determine if specific P2 receptors play a role in the injury response and wound healing in the cornea. Improper wound repair can alter refraction of light, one of the major roles of the cornea. Immediately after injury, corneal epithelial cells display an intracellular Ca2+ wave that propagates from the site of injury to neighboring cells [5]. This wave does not propagate via gap junctions but is mediated by the release of extracellular nucleotides activating P2Y receptors that lead to activation of intracellular signaling pathways such as ERK1/2 [13]. We hypothesize that injury induces an immediate localized event that stimulates later events such as release of growth factors and long-term signals.
In this paper, we provide evidence that specific subtypes of purinergic receptors regulate the injury response. Pretreatment of cells with ATP and UTP inhibited the propagation of the injury induced Ca2+ wave while BzATP, ADP, and UDP did not participate in the desensitization. In addition, pre-stimulation with tri-nucleotides, but not BzATP or di-nucleotides, resulted in a decrease in the intracellular Ca2+ release induced by EGF. ATP and UTP preferentially enhanced later cellular events, including migration. Cellular migration was enhanced further when cells were co-stimulated with EGF. In addition, the ATP mediated migration was reduced by the tri-peptide, RGD. The results indicate that immediate and long-term components of the wound response are mediated by tri-nucleotide receptors.
Materials and methods
Reagents
The nucleotides [adenosine 5′-triphosphate (ATP), uridine 5′-triphosphate (UTP), adenosine 5′-diphosphate (ADP), uridine 5′-diphosphate (UDP), β,γ-methyleneadenosine 5′-triphosphate (β,γ-MeATP), and 2′-3′-O-(4-Benzoylbenzoyl) adenosine 5′-triphosphate (BzATP)], adenosine, apyrase, and Reactive Blue 2 were purchased from Sigma Chemical Company (St. Louis, MO). Human recombinant epidermal growth factor (EGF) and penicillin-streptomycin were purchased from Invitrogen Corporation (Carlsbad, CA). The fluorescent Ca2+ indicator dye Fluo-3AM and pluronic acid were from Molecular Probes, Inc. (Eugene, OR). 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic (BAPTA) was purchased from Calbiochem (La Jolla, CA). RGD and RGE peptides were purchased from American Peptide (Sunnyvale, CA).
Cell culture
Primary rabbit corneal epithelial cells and an established human corneal cell line were used. Human epithelial cells transformed (HCE-Ts) were established by Araki-Sasaki et al. [22], using an SV-40 recombinant adenovirus vector. Primary corneal epithelial cells were isolated and cultured from freshly extracted whole rabbits eyes (Pel-Freeze Biologicals (Rogers, AR) [7].
Ca2+ imaging
HCE-T cells and primary cells were grown to confluency on 22 mm2 coverslips and quiesced 18 to 24 h before experiments in unsupplemented serum-free keratinocyte medium (K-SFM). Cells were washed in a HEPES-buffered saline solution containing 137 mM NaCl, 5 mM KCl, 4 mM MgCl2, 3 mM CaCl2 · 2H2O, 25 mM glucose and 10 mM HEPES [23]. Cells were incubated 20–30 min in 5 µM Fluo-3AM resuspended in HEPES buffer supplemented with 0.02% pluronic acid in DMSO [5, 7, 13]. Coverslips were mounted in a flow through perfusion apparatus connected to inlet tubes with varying solutions and an outlet pump (Warner Instruments). The mounted coverslip was placed on the stage of a Zeiss Axiovert LSM 510 laser scanning confocal microscope, and cells were scanned with an Argon laser every 786 ms [7]. Cells were perfused with HEPES buffered saline at a rate that did not elicit a response, and baseline fluorescence was obtained. Cells were stimulated either by perfusing nucleotides or EGF prepared in HEPES buffered saline and/or by manually perturbing cells [5].
Ca2+ data analysis
Ca2+ data was recorded by LSM software as average fluorescence intensity (F) for a region of interest over time. The region was either a 460 \smm \sx 460 \smm field or single cells, over time. Raw data was entered into KaleidaGraph. Percent change in average fluorescence was calculated using the following formula  where F0 is initial average fluorescence for the region of interest [5, 7]. Data was either graphed as average maximal fluorescence of at least three different experiments or as percent change in average fluorescence for time course. For the injury experiments, the area cleared by the wound was excluded from calculations.
Migration assay
HCE-T cells were quiesced 18 to 24 h in unsupplemented K-SFM. Cells were washed in phosphate buffered saline (PBS), trypsinized, and the reaction was stopped with filtered soybean trypsin inhibitor as described previously [7]. Cells were resuspended at a concentration of 125,000 cells/100 µl in binding buffer (unsupplemented K-SFM, 0.05% gelatin and 25 mM HEPES). Binding buffer, 600 µl, with or without added stimuli was placed in the wells of a 24 well plate. Costar Transwell inserts (6.5-mm diameter polycarbonate membranes, 8 mm pore size) were placed into the wells, and 100 µl of the cell suspension was added to the inserts. Experiments were conducted for 8 h at 37 °C.
To collect conditioned wound medium, cells were washed and incubated in binding buffer. Cells were scrape wounded as described previously [13]. The wound medium was collected immediately and used in migration assays. Unwounded medium was defined as binding buffer added to cells and collected without injury to cells. The conditioned wound medium was also treated with apyrase prior to the migration assays.
To determine migration, upper and lower chambers were aspirated and rinsed with PBS. Cells were fixed at room temperature for 10 min, rinsed with PBS, and non-migrated cells were removed. Migrated cells were stained with propidium iodide (5 µg/ml) (Molecular Probes, Inc., Eugene, OR) for 5 min and rinsed with PBS. Membranes were mounted onto a slide with SlowFade Antifade (Molecular Probes, Inc., Eugene, OR). For each membrane (33.2 mm2), six random 10× fields (one field = 1.37 mm × 1.08 mm, or 1.48 mm2) were photographed. Cells were counted and averaged for each membrane and experiments were performed in triplicate.
Statistical analysis
Values were given as the mean \+- SD. Statistical comparisons were made using Student’s t-test with a stringency of P > 0.01 for significance. Data were fit using KaleidaGraph to calculate concentration for half the maximal response (EC50) and maximal possible change in fluorescence for a given agonist (\gD Fmax). A generalized single-site binding model was used as follows: , where \gDF is the percent change in fluorescence and [L] is the concentration of the agonist.
Results
Nucleotide dose response curves
Ca2+ dose response curves were determined for ATP, UTP, ADP, UDP, and BzATP over a concentration range of 10−7 to 10−3 M (Figure 1). The response to ATP and UTP was greater than that of ADP and UDP over the entire range. The BzATP response was similar to ADP and UDP at concentrations ≤10−4 but lower at higher concentrations. In addition, adenosine, an agonist for P1 receptors, and β,γ-MeATP, an agonist for P2X receptors, were evaluated. Neither of these agonists induced a change in intracellular Ca2+ (data not shown). The EC50 for ATP and UTP were found to be (3.4 ± 1.8) × 10−6 M and (3.4 ± 2.0) × 10−6 M respectively. The EC50 for ADP and UDP were higher than the tri-nucleotides, (1.8 ± 0.9) × 10−4 M and (1.83 ± 0.12) × 10−4 M. respectively. The EC50 for BzATP was (8.6 ± 5.9) sx 10−5. R values for line fit were 0.96 for ATP, UTP, and UDP, 0.93 for ADP, and 0.91 for BzATP.
Figure 1Nucleotide induced Ca2+ dose response curves. HCE-T cells were incubated in 5 µM Fluo-3AM for 30 min and imaged every 786 ms using a flow through apparatus on an LSM 510 confocal. Cells were stimulated with the indicated concentration of nucleotide for 60 s. Maximal percent change in average fluorescence of a 460 µm × 460 µm field was determined. Data was fit to a generalized single-site binding model. Graph represents a minimum of three experiments at each concentration tested.
To verify that P2 receptors caused the intracellular Ca2+ increase, cells were pre-incubated for 30 min in 100 µM Reactive Blue 2 prior to experimentation. Cells were placed in a flow through system and washed for at least 50 s in HEPES buffered saline. Cells were stimulated with 100 µM of the indicated nucleotide for 100 s and washed again with HEPES buffer. Non-pretreated cells were used as control. Reactive Blue 2 lowered the Ca2+ response to nucleotides. The ATP and UTP responses were reduced by one-fifth, and the ADP and UDP responses were reduced by half.
Role of nucleotides in injury response
We have demonstrated that epithelial cells respond to nucleotides in a saturable manner. However, these results did not identify which P2 receptors were involved in the injury response. To evaluate the wound response, cells were placed in a flow through apparatus. Background images were collected in HEPES buffered saline, and the cells perturbed manually (Figure 2a). Percent change in average fluorescence of individual cells adjacent to the wound (Figures 2b, 2c (#1–5)) are shown inside the large white circle and graphed (Figure 2c). These depict a rapid increase in fluorescence after injury that is followed by attenuation. The rate of attenuation of injured cells was variable. While these cells did respond they were not a part of the calcium wave that propagated from the site of injury [5]. We also examined cells not immediately adjacent to the wound, cells involved in the wave (Figures 2b, 2d). When individual cells that were not adjacent to the wound were evaluated [(Figures 2b, 2d (#6–10) (outside of white circle)], the percent response was lower on average and the response time was delayed (Figure 2d). The delay in response time could be accounted for by the time it took the Ca2+ wave to travel the distance to nonadjacent cells.
Figure 2Pre-stimulation with ATP inhibits injury induced Ca2+ wave. Primary corneal epithelial cells were incubated in 5 µM Fluo-3AM for 30 min and imaged in a flow through apparatus on an LSM 510 confocal. Cells were washed in HEPES buffered saline with Ca2+ for at least 30 s, and stimulated by wounding or with ATP. Individual cells were analyzed for percent change in average fluorescence. Intensity scale is shown in (a) with red indicating highest Ca2+ levels and blue indicating lowest Ca2+ levels. The horizontal white bar in (a) represents 100 µm. a) Cells were washed in HEPES buffer containing Ca2+ and wounded. A series of images taken from a time course of a representative experiment of a wound (shown at asterisk) is presented. b) A single image taken after wounding is shown. Cells (#1–5) immediately adjacent to the wound (c) and cells away (#6–10) from the wound (d) were analyzed. e) Cells were washed in HEPES buffer containing Ca2+, stimulated with 100 µM ATP (80 s), and wounded (157 s). A series of images taken from a time course of a representative experiment is presented (wound shown at asterisk). f) A single image taken after wounding is shown. Cells (#1–5) immediately adjacent to the wound (g) and cells away (#6–10) from the wound (h) were analyzed. Images are representative of at least 10 independent experiments.
To evaluate what role ATP played in the initial injury response, cells were stimulated by ATP and wounded. To perform the experiment, cells were placed in the flow through apparatus, equilibrated with HEPES buffered saline, and stimulated with HEPES buffered saline containing 100 µM ATP. The response to ATP was allowed to attenuate (approx. 80 s), and then the cells were wounded (Figure 2e). The cells were analyzed for average change in percent fluorescence for individual cells adjacent to the wound (Figures 2f, 2g) and those involved in wave propagation (Figures 2f, 2h). As predicted from Figure 2a and d, cells adjacent to the wound responded (Figures 2f, 2g). The Ca2+ wave was absent when it followed pretreatment with ATP (Figures 2f, 2h). In its place was a minor inflection at the time of injury that resembled the wound response induced in the absence of intracellular Ca2+ [5] (see Figure 5). These responses indicate that the injury response was inhibited by the desensitization of specific P2Rs by ATP.
Figure 5Ca2+ free media inhibits an injury response after ATP stimulation. Primary corneal epithelial cells were incubated in 5 µM Fluo-3AM for 30 min and imaged in a flow through apparatus on an LSM 510 confocal. Cells were washed in HEPES buffered saline for at least 30 s, and stimulated by wounding or ATP. Individual cells were analyzed for percent change in average fluorescence. Intensity scale is shown in (a) with red indicating highest Ca2+ levels and blue indicating lowest Ca2+ levels. The horizontal white bar in (a) represents 100 µm. a) Cells were pre incubated in BAPTA (100 µM) washed in HEPES buffer containing Ca2+ and wounded. A series of images taken from a time course of a wound (shown at asterisk) of a representative experiment is presented. b) A single image taken after wounding is shown. Cells (#1–5) immediately adjacent to the wound (c) and cells away (#6–10) from the wound (d) were analyzed. e) Cells were washed in Ca2+ free HEPES buffer containing EGTA, stimulated with 100 µM ATP, and wounded. A series of images taken from a representative time course is presented (wound shown at asterisk). f) A single image taken after wounding is shown. Cells (#1–5) immediately adjacent to the wound (g) and cells away (#6–10) from the wound (h) were analyzed. Images are representative of at least 10 independent experiments. The series of images are taken from Movie 2 (see online version of article at www.springeronline.com).
To determine which nucleotides could desensitize the Ca2+ release in response to wounding, cells were pretreated with nucleotides and injured (Figures 3 and 4). Both ATP and UTP pre-stimulation inhibited the wound-induced wave (Figure 3), while ADP, UDP, and BzATP did not inhibit the injury induced Ca2+ wave (Figure 4). Together, these results indicate that both ATP and UTP desensitize receptors and inhibit the injury response at an equivalent molar concentration.
Figure 3Tri-nucleotides attenuate the injury induced Ca2+ wave. Primary corneal epithelial cells were incubated in 5 µM Fluo-3AM for 30 min and imaged in a flow through apparatus on an LSM 510 confocal. Cells were washed in HEPES buffered saline with Ca2+ for at least 30 s and stimulated with the indicated nucleotide and wounded. Intensity scale is shown in (a) with red indicating highest Ca2+ levels and blue indicating lowest Ca2+ levels. The horizontal white bar in (a) represents 100 µm. a, b) Cells were washed in HEPES buffer with Ca2+, stimulated with 100 µM of the indicated nucleotide, and wounded. A series of images taken from a time course of a representative experiment is presented (wound shown at asterisk). c, d) Percent change in average fluorescence for the whole field (460 µm × 460 µm) was calculated and graphed over the time course for each experiment. Images are representative of at least 10 independent experiments. Series of images are taken from Movie 1 (see online version of article at www.springeronline.com).Figure 4Di-nucleotides and BzATP do not attenuate the injury induced Ca2+ wave. Primary corneal epithelial cells were incubated in 5 µM Fluo-3AM for 30 min and imaged in a flow through apparatus on an LSM 510 confocal. Cells were washed in HEPES buffered saline with Ca2+ for at least 30 s and stimulated with the indicated nucleotide and wounded. Intensity scale is shown in (a) with red indicating highest Ca2+ levels and blue indicating lowest Ca2+ levels. The horizontal white bar in (a) represents 100 µm. (a–c) Cells were washed in HEPES buffer with Ca2+, stimulated with 100 µM of the indicated nucleotide, and wounded. A series of images taken from a time course of a representative experiment is presented (wound shown at asterisk). (d–f) Percent change in average fluorescence for the whole field (460 µm × 460 µm) was calculated and graphed over the time course for each experiment. Images are representative of at least 10 independent experiments. Series of images are taken from Movie 1 (see online version of article at www.springeronline.com).
To evaluate the inflection detected in Figures 2e–h, cells were pretreated with BAPTA (100 µM) for 30 min and individual cells were analyzed (Figures 5a–5d). Cells responded to the injury in a manner similar to the cultures that had been pretreated with ATP (Figures 3 and 4). These results indicate that the cellular response adjacent to the wound did not occur via P2Y receptor signaling since the Ca2+ increase came from extracellular stores (Figures 5a–5d). This agrees with previous results using thapsigargin [5]. To evaluate the requirement for extracellular Ca2+, cultures were placed in the flow through apparatus in Ca2+ free HEPES buffer containing EGTA. These cells were stimulated with 100 µM ATP made in Ca2+ free HEPES buffered saline. Once the response had attenuated (approx. 100 s), the cells were wounded (Figure 5e), and individual cells were analyzed for average change in percent fluorescence in cells adjacent to the wound margin (Figures 5f, 5g) and those involved in wave propagation (Figures 5f, 5h; as in Figure 2). When the experiment was performed in Ca2+ free media, the inflection detected previously in the injury response was absent, completely eliminating any Ca2+ response to injury (Figures 5e–5h).
Nucleotides affect EGF induced Ca2+ response
Cross-talk between the EGF and ATP signaling pathways occurs in many cell systems. To determine if this was a property unique to ATP, the response to EGF was evaluated after pre-stimulation with other nucleotides. HCE-Ts were perfused with HEPES buffered saline to establish baseline fluorescence, and the cells were stimulated with 100 µM ATP, UTP, ADP, UDP or BzATP for 100 s. Cells were washed with HEPES for 100 s and stimulated with 8 nM EGF for 100 s. Maximal percent change in fluorescence of the entire 460 µm × 460 µm field was calculated (Figure 6a). The results were compared with cells washed with HEPES buffer and stimulated with 8 nm EGF alone for 100 s (− pretreatment). Cells exhibited an attenuated Ca2+ response upon EGF stimulation when they were pretreated with either ATP or UTP. In contrast, when the cells were pre-stimulated with BzATP, ADP, or UDP, there was no detectable desensitization (Figure 6a). The reverse experiment was performed where cells were pre-stimulated with EGF and then by specific nucleotides (data not shown). Pretreatment with EGF did not desensitize the cells indicating that EGFR stimulation with EGF did not inhibit the response to nucleotides.
Figure 6Injury and tri-nucleotides decrease subsequent EGF induced Ca2+ response. HCE-T cells were incubated in 5 µM Fluo-3AM for 30 min and imaged every 786 ms in a flow through apparatus on an LSM 510 confocal. a) Cells were stimulated with 100 µM of the indicated nucleotide for 100 s, washed in HEPES buffered saline for 100 s, and then stimulated with 8 nM EGF or stimulated with EGF without pretreatment. Maximal percent change in average fluorescence of a 460 µm × 460 µm field was determined. b) Cells were either wounded and stimulated with 8 nM EGF or stimulated with EGF without injury. Individual cells were selected for analysis. In the wound model, cells that responded to the wound were selected. (t-test, * P < 0.0005) Data represent a minimum of three independent experiments.
As desensitization occurred after cells were pre-stimulated with ATP or UTP and wounded or stimulated with EGF, our goal was to determine if desensitization of the EGF receptor occurred in our wound model. Cells were placed in the flow through apparatus, perfused with HEPES buffered saline to establish baseline fluorescence, and injured. Once fluorescence returned to the base line, the cells were stimulated with EGF. The analysis was performed on individual cells that responded to both the injury and EGF (Figure 6b). Results were compared to individual cells stimulated with EGF alone. The Ca2+ response to EGF was significantly lowered after injury. The high standard deviation was due to the variable size of the wound.
Nucleotides and EGF induce cell migration
The long-term effects of P2 receptors on wound healing were examined using Transwell migration assays. Previously we showed that P2 receptors were necessary for wound repair in directed migration scratch assays [7]. Furthermore, we demonstrated that migration was optimal at 1 µM for ATP and 0.16 nM for EGF in Transwell assays [7]. In these experiments, migration to other nucleotides and adenosine were evaluated over a range of concentrations from 0.1 to 100 µM (Figure 7). Optimal migration for ADP and BzATP occurred at 10 µM (Figures 7a, b), while optimal migration for ATP and UTP occurred at 1 µM ([7] and data not shown). UDP and β,γ-MeATP did not induce migration at any concentration (Figures 7c, d). To assess preferential migration to any one particular nucleotide or adenosine, the nucleotides and adenosine were evaluated and compared. The cellular response was tested at 1 µM, the optimal concentration for ATP and UTP. In addition, ADP was tested at 10 µM, its optimal concentration. ATP, UTP, and ADP induced comparable migration at their optimal concentrations (162 ± 39, 170 ± 70, and 188 ± 47 cells/1.48 mm2, respectively). However, ADP at 1 µM, UDP, adenosine, or binding buffer did not induce cell migration (23 ± 7, 18 ± 7, 31 ± 6 and 29 T 11 cells/1.48 mm2, respectively) (Figure 7e).
Figure 7Cell migration is nucleotide specific. Transwell migrations were performed for 8 h at 37 °C with the indicated nucleotide. HCE-T cells were stained with propidium iodide, counted in six randomly chosen fields (1.46 mm2), and averaged. Dose response curves for a) ADP, b) BzATP, c) UDP, and d) β,γ-MeATP are shown. Experiments were performed at least three times and representative curves were chosen. e) Cells were stimulated with binding buffer (neg), 1 µM ATP, UTP, ADP, UDP, or Adenosine (Ado), or 10 µM ADP. Experiments were performed in triplicate.
We evaluated the role of factors released into the wound media on cell motility and compared these effects to stimulation with nucleotides and EGF. Wound media was collected immediately after injury and added to migration assay chambers. Luciferase assays performed on wound media demonstrated presence of ATP [5]. Cell migration was 3.7-fold greater when cells were stimulated with wound media compared to wound media treated with apyrase or unwounded control media (Figure 8a). Previously we established that epithelial cells migrate to ATP or EGF in a dose dependent manner and optimal migration was determined to be 1 µM and 0.16 nM respectively [24]. To evaluate the combined effect of ATP and EGF, cells were co-stimulated with the optimal concentrations of each and compared. In a representative experiment an average of 369 (±34) cells migrated/1.48 mm2 in response to 0.16 nM EGF while an average of 314 (±31) cells migrated/1.48 mm2 in response to 1 µM ATP (Figure 8b). When cells were co-stimulated with 1 µM ATP and 0.16 nM EGF, migration increased more than threefold with an average of 1043 (±66) cells/1.48 mm2 (Figure 8b). Co-stimulation experiments were performed with UTP, ADP and UDP at their respective optimal concentrations (Figures 7 and 8c). Cells exposed to ADP or UDP (1 µM) and co-stimulated with EGF (527 ± 61 and 637 ± 51 cells/1.48 mm2, respectively) showed migration similar to that of EGF alone (457 ± 115 cells/1.48 mm2). When cells were co-stimulated with UTP and EGF, migration was enhanced in an additive manner (801 ± 133 cells/1.48 mm2). In addition when cells were exposed to 10 µM ADP and EGF, migration was greater than either alone (753 ± 173 cells/1.48 mm2).
Figure 8Wound media, nucleotides, and EGF mediate cell migration. Transwell migrations were performed for 8 h at 37 °C with the indicated nucleotides and/or EGF. HCE-T cells were stained with propidium iodide, counted in six randomly chosen fields (1.46 mm2) and averaged. a) Migration of cells stimulated with wound media were compared to those stimulated with unwounded control media, wound media treated with apyrase and binding buffer (negative). b) A representative experiment is shown where cells were stimulated with optimal concentration of ATP (1 µM) and EGF (0.16 nM) and compared to co-stimulation with ATP and EGF and negative binding buffer. c) Cells were simulated with binding buffer (neg), 1 µM UTP, ADP, or UDP, or 10 µM ADP +/− 0.16 nm EGF.
To understand how nucleotides regulate cell migration, we asked whether integrins mediated the response. Integrins are known to play an important role in cell migration, and the integrin binding tri-peptide, RGD, that is found in the first extracellular loop of the P2Y2 receptor, has been shown to interact with integrins [20]. In addition, expression of α6β4 in corneal epithelial cells is up-regulated by EGFR activation [18]. To evaluate the role of integrin receptors in nucleotide mediated migration, RGD peptide was added to the cell suspension prior to its addition to the Transwell chamber. RGE peptide was used to control for non-specific binding. Cells were migrated to binding buffer or 1 µM ATP. When either the RGD or RGE peptide was added to the cells migrating to binding buffer, there was no detectable difference over control (Figure 9). Cells stimulated with ATP migrated 362 (±92) per field. Interestingly, cell migration was reduced almost fourfold when RGD was present (96 ± 43 cells/1.48 mm2) (Figure 9). The control peptide did not decrease migration significantly from control (284 ± 59). Since RGD peptide can inhibit migration, it is likely integrins play a role in ATP induced cell migration. The nucleotide receptors, EGFR, and integrins mediate calcium signaling and cell migration. Thus, interaction between these protein families may be critical in regulating the cellular signals after injury.
Figure 9ATP and integrins mediate cell migration. Transwell migrations were performed for 8 h at 37 °C with the indicated nucleotides. HCE-T cells were stained with propidium iodide, counted in six randomly chosen fields (1.46 mm2) and averaged. RGD or RGE peptide, 1 µM, was added to the cell suspension. Cells were stimulated with binding buffer (control) or 1 µM ATP. (t-test, *P < 0.0005) Experiments were performed in triplicate.
Discussion
The results of these studies demonstrate that P2 receptors play a role in the initial cellular responses that occur after injury and the long-term signals that are necessary for wound closure in corneal epithelial cells. We have demonstrated that nucleotides are the active component released with injury and are responsible for the intercellular Ca2+ wave that is propagated in neighboring cells [5, 13]. We have shown that ATP, UTP, ADP, UDP and BzATP promote an intracellular Ca2+ increase, β,γMe-ATP does not. Our goal was to demonstrate that nucleotides play a prominent role in the cellular response to injury, in migration, and in the cross-talk with EGF.
Recently involvement of P2 receptors in wound healing has begun to be demonstrated [2]. Desensitization experiments demonstrate the role of nucleotides in the injury response. We have shown pretreatment of cells with ATP and UTP inhibits the response to a subsequent wound (Figure 3). Several explanations are plausible: 1) Specific receptors desensitize via receptor internalization or signal transduction down regulation [25] or 2) When cells are exposed to a solution of a tri-nucleotide, ATP and/or UTP receptors have bound agonist thus saturating the receptors. We predict that the amount of nucleotide used to desensitize the wound approximates the concentration needed for the maximal Ca2+ response (Figure 1). In either case, the result is that nucleotides that are released would not be able to induce any further effect upon the receptors. In addition, injury, ATP and UTP, induce a decrease in the EGF induced Ca2+ response (Figure 6). Since nucleotides are released with injury [13], we hypothesize that desensitization of the EGF receptor occurs via nucleotides. In contrast, while ADP and UDP cause homologous desensitization of their own Ca2+ signal [7], ADP, UDP, and BzATP do not affect either the EGF or wound response even when the receptors are saturated (Figures 4 and 6). Therefore, we hypothesize that P2Y1, P2Y6, and P2X7 receptors do not play a role in the Ca2+ wave that occurs after injury.
We have demonstrated that the source of the calcium for the injury induced wave is intracellular ([5] and Figure 5). Pretreatment with either thapsigargin or BAPTA inhibits the Ca2+ wave but not oscillations of cells at the wound margin. In addition, pretreatment with either ATP or UTP inhibits only the wave but not the Ca2+ increase in the cells adjacent to the wound. Cells adjacent to the wound edge utilize extracellular calcium (Figure 5), but the method of extracellular calcium entry is unknown. One possibility is that the plasma membranes of the injured cells are damaged, and Ca2+ freely enters. The response could also be mediated via stress receptors or P2X receptors, both of which are ion channels that permit entry of extracellular calcium [26]. While these cells are not involved in the propagation of the wave, we cannot rule out the possibility that they play a role in wound healing, and that is an area of current exploration.
Activation of intracellular signaling pathways by P2 receptors is emerging as intricate patterns of cell specific processes. Involvement of αVβ3 integrins has been shown to be essential for both ATP induced intracellular Ca2+ increase and ERK1/2 activation in 1321N1 P2Y2 transfected cells [20]. We have shown in our system that integrins play a role in the response to nucleotides (Figure 9). As integrins are known to play a role in cell migration and wound healing, their coordinated role in nucleotide signaling is of great interest. Purinergic signaling is also known to activate downstream signaling events using a number of receptors. We have shown that ATP, UTP, ADP, and UDP promote activation of ERK1/2, but α,β-MeATP and β,γ-MeATP do not [13]. P2X2 and P2Y1 receptors have been shown to play a role in activating ERK1/2 in stretch induced injury of astrocytes in Ca2+ dependent manner [27]. In contrast in embryonic kidney cells, P2X7 activates ERK1/2 in a Ca2+ independent manner [28]. Additionally PKC and PKD both have been shown to play role in P2X7 induced ERK1/2 activation [29]. In pro-monocytic cells, activation of ERK1/2 by P2Y2 has been shown to involve c-Src but not PKC [30].
Recently, the P2YRs have been hypothesized to play a role in cross-talk with the EGFR [7, 13, 21, 31, 32]. There are a number of potential pathways, several of which may occur. One current hypothesis is cross-talk occurs via the triple membrane pass system where GPCR activated matrix metalloproteases (MMPs) cleave pro-heparin binding (HB)-EGF from the membrane and HB-EGF can then activate the EGFR [33, 34]. Another potential method for cross-talk is via Src. While Src is known to play a role in the phosphorylation of EGFR, its involvement in corneal epithelial injury has not been determined. GPCR stimulation has been found to lead to phosphorylation of the EGFR in a Src and/or Pyk2 dependent manner in many cell types [21, 31, 35, 36]. Future experiments will be performed to test the hypothesis that Src and Pyk2 play an active role in nucleotide-mediated activation of the EGFR in corneal epithelial cells. Inhibition of the EGFR activation with tyrphostin has been shown to inhibit ATP induced cell migration in epithelial cells [7]. These indicate that cross talk between GPCR and the EGFR occur over the entire time course of wound response and repair.
ATP and UTP are the major players in Ca2+ waves after injury and the cross talk with the EGFR (Figures 3 and 6). However, this does not preclude that other nucleotides may mediate other facets of the wound response. While ADP and BzATP do not play a role in the injury induced Ca2+ wave, they can stimulate migration, albeit at a higher concentration than ATP or UTP (Figure 7). Though UDP does not stimulate migration, both ADP and UDP have the ability to activate downstream signals. We have shown that ERK1/2 and paxillin become phosphorylated upon stimulation with both tri- and di-nucleotides [7, 13]. Additionally, BzATP has been shown to activate downstream signals such as ERK1/2 in other cell systems [28, 29]. The signals propagated by di-nucleotides may be artifacts of saturating the system with larger than physiological concentrations [37]. Another possibility is that the signal is real and that after injury the receptors are available at a density that is unable to elicit a detectable downstream cascade. This may explain why ADP can induce migration at 10 µM. However, this does not take into account that high concentrations of ADP are still not able to inhibit the injury induced Ca2+ wave (data not shown). Proper signaling relies on optimal concentrations of nucleotides. At high concentrations where maximal Ca2+ release upon nucleotide exposure occurs (>100 µM), epithelial cells do not migrate toward the stimuli. The desensitization of the calcium response that occurs at 100 µM may explain why cell migration is minimal. In contrast, at lower concentrations where the maximal migration occurs, little desensitization occurs. This bell shape curve is not atypical and has been demonstrated to occur with nucleotide induced migration in epithelial and dendritic cells [7, 10].
It is likely that different signals are needed for the initial communication of an injury and the long-term need for wound healing, such as cell migration and changes in protein expression. The receptor specificity and the numerous signaling pathways provide an intricate network allowing for differential regulation of short versus long term signals. Our study provides evidence that P2Y2 and possibly P2Y4 play a role in the initial signals involved in the injury response and these receptors and other P2 receptors play a role in long term cell migration. Future studies will involve understanding the critical interactions between signaling pathways that play a role in wound healing.

wound repair
epithelium
migration
epidermal growth factor
ca2+ imaging