February 2010
Volume 51, Issue 2
Free
Physiology and Pharmacology  |   February 2010
Effect of HCN Channel Inhibition on Retinal Morphology and Function in Normal and Dystrophic Rodents
Author Affiliations & Notes
  • Luca Della Santina
    From the Dipartimenti di Scienze Fisiologiche, e
  • Muriel Bouly
    the Institute De Recherches Internationales Servier, Courbevoie, France.
  • Antonella Asta
    From the Dipartimenti di Scienze Fisiologiche, e
  • Gian Carlo Demontis
    Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Università di Pisa, Pisa, Italy; and
  • Luigi Cervetto
    From the Dipartimenti di Scienze Fisiologiche, e
  • Claudia Gargini
    Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Università di Pisa, Pisa, Italy; and
  • Corresponding author: Claudia Gargini, Dipartimento di Psichiatria e Neurobiologia, Università di Pisa, Via Bonanno 6, 56126 Pisa, Italy; gargini@farm.unipi.it
Investigative Ophthalmology & Visual Science February 2010, Vol.51, 1016-1023. doi:10.1167/iovs.09-3680
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      Luca Della Santina, Muriel Bouly, Antonella Asta, Gian Carlo Demontis, Luigi Cervetto, Claudia Gargini; Effect of HCN Channel Inhibition on Retinal Morphology and Function in Normal and Dystrophic Rodents. Invest. Ophthalmol. Vis. Sci. 2010;51(2):1016-1023. doi: 10.1167/iovs.09-3680.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To elucidate short- and long-term effects of ivabradine, an inhibitor of the hyperpolarization-activated current (I f) recently approved for treatment of stable angina, on retinal function and integrity. As careful ivabradine administration is recommended for patients with retinitis pigmentosa, an additional objective was to test the consequences of repeated ivabradine delivery on retinal integrity in the rd10 mouse, an animal model of the human degenerative disease.

Methods.: The electroretinogram (ERG) was recorded in intact anesthetized animals in response to flashes or time-varied sinusoidal light stimuli of different frequency. Retinal integrity and hyperpolarization-activated cyclic nucleotide-gated (HCN) channel distribution were assessed by immunocytochemistry, confocal microscopy, and Western blot analysis.

Results.: Neither a- nor b-waves of the flash-ERG were significantly affected by ivabradine administration. Conversely, reversible changes in the response to sinusoidal stimuli were observed during both acute and continued treatment. HCN inhibition enhanced the gain of frequency–response curves (FRCs) at the lowest stimulus frequencies and reduced it in the 1- to 7-Hz range. These effects were dose dependent and reverted to normal 1 week after discontinuation of ivabradine. Retinal morphology and distribution of HCN were preserved and no signs of retinal damage were observed in healthy animals. HCN inhibition in dystrophic mice had no effect on either extent or progression of retinal degeneration.

Conclusions.: The results are consistent with the hypothesis that the visual symptoms reported by patients during prolonged treatment with ivabradine are due only to a reversible pharmacologic effect.

An issue of great relevance encountered in testing new therapeutic agents is the occurrence of adverse reactions associated with the main effect of the drug. Frequently reported secondary effects common to a variety of pharmacologic agents, are visual hallucinations and phosphenes, 1 but the underlying mechanisms are seldom understood. 
A new class of molecules, characterized by linked benzazepinone and benzocyclobutane rings, has been considered for some time as a potential therapeutic approach to prevent and cure coronary artery occlusive disease. These compounds act by blocking the HCN channels responsible for the current (I f) that controls spontaneous diastolic depolarization and regulates heart rate. 2,3 Lowering the heart rate by lengthening the diastole relieves the heart metabolic load and improves coronary perfusion. Recently with ivabradine (Procoralan; IRIS, Courbevoie, France), a practical and effective pharmacologic treatment of stable angina pectoris has been developed, approved, and granted marketing authorization in Europe and in several non-European countries. 4 Molecules from this class, however, may also induce visual symptoms, mainly phosphenes. 5,6  
Therapeutic amounts of ivabradine applied in vitro to isolated rod photoreceptor cells selectively inhibit a delayed rectifying current activated by membrane hyperpolarization (I h), leaving other currents unaffected. 7 Given that HCN1–4 isoforms are widely expressed in most retinal neurons, 811 it is reasonable to suggest that inhibition of retinal HCN is the specific cause of the visual symptoms. We tested this hypothesis by investigating the effects of ivabradine on retinal morphology and function by immunocytochemistry and by in vivo electrophysiology. Single-cell current recordings alone may fail to show adverse reactions that develop slowly in time during prolonged treatment. We therefore resorted to two distinct dose regimens: acute single-dose delivery and continuous 3-week infusion by osmotic pumps. We also tested the consequences of repeated ivabradine delivery on retinal integrity in wild-type (wt) and rd10 mice, a model of human retinitis pigmentosa. 12  
Methods
Animal Models
Adult Long-Evans pigmented rats were used for both acute and 3-week infusion ERG experiments and for immunolabeling of HCN. Other experimental models were wt mice and rd10 mutant mice. All animals were raised in cyclic light (12 hours, 100 lux; 12 hours dark). The experiments were performed in compliance with both the guidelines of the Ethics Committee of the University of Pisa and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
A venous catheter was inserted into the jugular vein with the rats under surgical anesthesia (intraperitoneal injection of urethane 120 mg/100 g) for vehicle and ivabradine acute delivery. Ivabradine (IRIS) dissolved in saline was given at the doses of 3, 6, and 12 mg/kg. The effects of ivabradine were tested on the heart rate (HR), on the ERG in response to flashes and sinusoidal stimuli, and in two cases on the arterial blood pressure (ABP). ERG responses were recurrently tested after the drug injection (sinusoidal response recorded after: 15, 90, 180 minutes, respectively; flash response after 45, 120, 240 minutes). In a separate set of experiments, ivabradine was delivered by means of osmotic pumps (model 2ML4; Alzet Osmotic Pumps, Cupertino, CA); this approach ensured constant delivery rates. The pumps were filled with a solution of ivabradine designed to obtain an average daily amount of 11 mg/kg over 3 weeks. With the rats under surgical anesthesia (2,2,2-tribromethanol 28 mg/100 g; Sigma-Aldrich, Steinheim, Germany). The osmotic pumps were placed subcutaneously between the shoulder blades and inserted into a small pocket, formed by spreading the connective tissue apart. In the first animal group (21-day–treated group), the pumps were maintained for 21 days, in the second (7-day recovery group) on day 21, the pumps were removed, and the animals received no treatment for 7 days. In the third group (control group), the procedure was as for the first group but the pumps were loaded with saline. The amount of drug and vehicle delivered during the infusion time was measured on pump removal by subtraction of the residual from the initial volume. 
The repeated treatment of wt and rd10 mice consisted of daily subcutaneous (SC) ivabradine injections (12 mg/kg/d) for a period of 10 days from the postnatal age of 12 days (P12). 
Before the ERG recording session, the animals were dark adapted overnight. Anesthesia was induced by a peritoneal injection of urethane 120 mg/100 g. Body temperature was continuously monitored and maintained near 37°C with an electric blanket, and any change greater than 0.1°C was readily compensated for by a feedback circuit. The observed temperature changes were 0.2°C with negligible effects on the ERG. 13 Pupils were dilated with drops of tropicamide 1%. A thin layer of methylcellulose solution (Lacrinorm; Farmigea, Pisa, Italy) protected the cornea. The electrocardiogram (ECG) continuously monitored HR (beats/minute) and general conditions. Corneal transparency and pupil size were regularly checked with an ophthalmoscope. 
At the end of the ERG recordings, blood was collected from each anesthetized animal before euthanatization and assayed for ivabradine. After prolonged infusion at a dose of 11 mg/kg, the average ivabradine concentration were 86.3 ± 24 ng/mL (n = 6). These concentrations are sixfold higher than those obtained in patients treated with therapeutic doses of 5 mg. 
Optical Stimulation
The full-field illumination of the eyes was achieved via a Ganzfeld sphere 30 cm in diameter, with the interior surface coated with a highly reflective white paint. Two stimulus patterns were adopted: brief flashes that generated the typical ERG response (a- and b-waves) and sinusoidal time-varied luminance that elicited periodic responses. 
Flash Stimulation.
An electronic flash unit (Sunpak B3600 DX; Tocad Company, Tokyo, Japan) generated a stimulus with energy that decayed over time, with a τ of 1.7 ms. A short-wavelength band-pass filter, 7.5 nm half bandwidth (Spindler and Hoyer, Göttingen, Germany) that gave a scotopic effective λ of 492 nm was used. Because the maximum energy of the band-pass–filtered flashes was not sufficient to elicit saturating a-wave responses, the responses were obtained by delivering flashes of white light with scotopic efficacy evaluated according to Lyubarsky and Pugh. 14 The estimated maximum retinal illuminance was 5.7 × 105 Φ (photoisomerization rod−1) per flash. Calibrated neutral-density filters were used to attenuate the intensity of the flashes. 
Sinusoidal Stimulation.
Sinusoidal changes in luminance at various temporal frequencies and modulation depth were generated by a light-emitting diode (LED) source (peak wavelength: λ = 520). The luminance of sinusoidal stimuli is expressed as   where L is the mean retinal illuminance (38.79 Φ/s) and m is the contrast (85%). A light stimulus unit developed in our laboratory generated sinusoidal temporal patterns. 15  
ERG Recording
ERGs were recorded in complete darkness via coiled gold electrodes making contact with the moist cornea. 16 A small gold plate placed in the mouth served as both reference and ground. Responses were amplified differentially, band-pass filtered at 0.1 to 500 Hz, digitized at 12.8 kHz by a computer interface (LabVIEW 6.1; National Instruments, Austin, TX) and stored on disc for processing. Responses to flashes were averaged with an interstimulus interval ranging from 60 seconds for dim lights to 2 minutes for the brightest flashes. 
Analysis of a-Wave.
The effects of the drug on gain and kinetics of the transduction cascade were tested by analyzing the leading edge of the a-wave, fitted to the model of the activation phase of the rod G-protein transduction cascade according to the relation:   where F(t) is the fraction of circulating current normalized to its dark value, A is a parameter with the units of s −2/Φ expressed in terms of the gain parameters of the cascade (amplification factor), t eff is inclusive of any delay contained in both the response and instrumentation. 17,18 The results of this analysis are illustrated (see Fig. 2). 
Analysis of Responses to Sinusoidal Stimulation.
The recorded signals were averaged in synchrony with the stimulus luminance periodicity and a discrete Fourier analysis was performed to estimate amplitude and phase of the first harmonic. Corrections were made to allow for the amplifier's filter properties. The frequency response curves (FRCs; reported in Figs. 3 and 5B) were obtained by plotting the amplitude of the first harmonic as a function of the temporal frequency. Differences in the FRC shapes were quantified by calculating the ratio of the response amplitude (R) at 3 and 0.3 Hz (R 3Hz/R 0.3Hz). 
ABP Measurements
In two acute experiments in which 12 mg/kg of ivabradine was delivered, the ABP was monitored throughout the entire experimental session. ABP measurements were made by means of a catheter inserted into the common carotid artery. The catheter was connected to a blood pressure transducer (model 800; Ugo Basile Comerio-Varese, Italy), and the measurements were fed into a digital data acquisition system (MP100A; Biopac System, Goleta, CA). 
Immunocytochemistry
This procedure has been described in detail in a previous paper. 10 Briefly, primary antibodies were polyclonal anti-HCN1 and anti-HCN2 (1:200; Alomone, Jerusalem, Israel), anti-protein kinase C (PKC, Sigma-Aldrich; 1:200). Secondary antibodies were goat anti-mouse and goat anti-rabbit IgG conjugated with Alexa Fluor 488 or Alexa Fluor 568 (Molecular Probes, Eugene, OR). In wt and rd10 mice, TUNEL staining for apoptosis determination was performed according to the manufacturer's protocols for frozen sections (Dead End Fluorometric TUNEL System; Promega, Madison, WI). Retinal preparations were examined with a confocal microscope (TCS-NT; Leica, Bannockburn, IL) equipped with a krypton-argon laser, and the images were analyzed (PhotoShop; Adobe Systems Inc. San Jose, CA). 
Western Blot Measurements in wt and rd10 Mice
The Western blot procedure was performed as previously reported. 19 Protein of each sample (15 μg) was electrophoresed on a 12% SDS-polyacrylamide gel. The proteins were transferred to a nitrocellulose membrane in transfer buffer (25 mM Tris-HCl [pH 8.3], 192 mM glycine, 20% methanol). The protein blot was blocked by exposure to 3% nonfat dried milk and 0.05% Tween-20 in 20 mM Tris-HCl, 500 mM NaCl (TBS; pH 8) at room temperature for 45 minutes. The membrane was then incubated overnight at 4°C with the mouse monoclonal anti-opsin diluted 1:10,000 (Sigma-Aldrich) or with the rabbit polyclonal anti-GFAP diluted 1:500 (product G9269; Sigma-Aldrich,) in blocking buffer. The reactions were revealed with HRP-conjugated secondary antibodies (anti-mouse IgG or anti-rabbit IgG; Sigma-Aldrich) for 2 hours at room temperature. The bands were visualized with a chemiluminescence kit (ECL Western blot detection agents; Amersham Biosciences, Buckinghamshire, UK) and quantified by optical density. 
On the same blots, opsin and GFAP content were normalized to the amounts of β-actin. The nitrocellulose membrane was incubated in stripping buffer (glycine 20 mM, 0.1% SDS, 0.1% Tween-20 [pH 2.2]) for 30 minutes. Then, the membrane was washed in TBS and in TBS plus Tween-20 0.05% for 30 minutes at room temperature. The protein blot was exposed to the blocking medium at room temperature for 45 minutes. The membrane was then incubated overnight at 4°C with the mouse monoclonal anti-actin diluted 1:2000 (Sigma-Aldrich) in blocking buffer. The reactions were visualized with HRP-conjugated secondary antibodies (anti-mouse IgG) for 2 hours at room temperature. 
Statistical Analysis
The statistical analysis was applied separately to b-wave amplitude, amplification factor A, and the ratio of first harmonic component of the FRC measured at 3 and 0.3 Hz, respectively (R 3Hz/R 0.3Hz). Two-way ANOVA for the effects of time and treatment was applied to repeated measurements and was followed by one-way ANOVA and, in case of significance (F-test), by the Tukey multiple comparison test (**P < 0.01; *P < 0.05). 
Results
Acute Effects of Ivabradine
Acutely delivered ivabradine induced a dose-dependent reduction of the HR. With 12 mg/kg, 15 to 45 minutes after the drug injection, the HR was on average nearly halved for several hours. Recovery was slow, and after 4 hours, the HR was still approximately 75% of the control level. The systolic ABP remained constant, the diastolic level slightly decreased with a consequent moderate increase in pulse amplitude that lasted several minutes. 
Flash ERG.
An example of the effect of 12 mg/kg ivabradine on the flash ERG, is reported in Figure 1. Traces in the six panels are averaged responses (n = 6) to flashes of increasing brightness in the control and at different times after ivabradine administration. The drug effect on both response amplitude and time to the peak of b-wave was small and well within the range of the normal response variability. A small but reproducible oscillation prolonged the duration of the b-wave decay in response to dim and intermediate intensity flashes. Ivabradine had no significant effect (Fig. 2A) on the a-wave amplitude nor on the amplification factor A, which reflects the kinetics of the phototransduction processes (see the Methods section). Figure 2B illustrates the b-wave amplitude as a function of the flash intensity before and 120 minutes after 12 mg/kg ivabradine. 
Figure 1.
 
Averaged ERG responses of increasing light intensity in the control and after a single dose injection of 12 mg/kg ivabradine in Long-Evans pigmented rats. The intensity of the flash is expressed as the number of photoisomerizations per rod (Φ) per flash. Color coded traces are responses in the control and 45, 120, and 240 minutes after ivabradine injection (Σ = 6 low luminance flashes, Σ = 2 two brightest flashes). The collected control HR and those collected at three successive times after ivabradine injection are calculated from the RR interval of the ECG and reported at the top right of the rightmost panel. Luminances (Φ) are reported at the bottom right of each panel.
Figure 1.
 
Averaged ERG responses of increasing light intensity in the control and after a single dose injection of 12 mg/kg ivabradine in Long-Evans pigmented rats. The intensity of the flash is expressed as the number of photoisomerizations per rod (Φ) per flash. Color coded traces are responses in the control and 45, 120, and 240 minutes after ivabradine injection (Σ = 6 low luminance flashes, Σ = 2 two brightest flashes). The collected control HR and those collected at three successive times after ivabradine injection are calculated from the RR interval of the ECG and reported at the top right of the rightmost panel. Luminances (Φ) are reported at the bottom right of each panel.
Figure 2.
 
(A) Amplification factor A estimated from the analysis of the leading edge of the a-wave, in control conditions and 120 minutes after ivabradine injection (12 mg/kg). To minimize the effect of spontaneous response variability, data were normalized to the control value obtained before injection of the vehicle (saline). Changes in the absolute values of A are not statistically significant. (B) Collected data (n = 6) of the b-wave peak amplitude as a function of the flash intensity in the control and 120 minutes after injection of 12 mg/kg ivabradine. Vertical bars, the SEM. *P < 0.05, statistically significant versus control.
Figure 2.
 
(A) Amplification factor A estimated from the analysis of the leading edge of the a-wave, in control conditions and 120 minutes after ivabradine injection (12 mg/kg). To minimize the effect of spontaneous response variability, data were normalized to the control value obtained before injection of the vehicle (saline). Changes in the absolute values of A are not statistically significant. (B) Collected data (n = 6) of the b-wave peak amplitude as a function of the flash intensity in the control and 120 minutes after injection of 12 mg/kg ivabradine. Vertical bars, the SEM. *P < 0.05, statistically significant versus control.
Sinusoidal Stimuli.
FRCs obtained by plotting the peak-to-peak amplitude of the ERG first harmonic in response to sinusoidal stimuli in the frequency range of 0.3 to 30 Hz were determined before and after vehicle or ivabradine administration. Ivabradine increased dose-dependently the response amplitude to low-frequency stimuli and decreased the response to stimuli of higher frequency. The effects of ivabradine reached maximum in approximately 1 hour and persisted for at least 3 hours. The results in Figure 3 illustrate the FRCs measured before and 90 minutes after vehicle delivery or ivabradine administration. 
Figure 3.
 
FRCs obtained by sinusoidal modulation of a mean luminance equivalent to 38.79 Φ before and 90 minutes after vehicle (saline) and ivabradine injection. Relative amplitudes were normalized at 1 Hz. Ivabradine are given at the top of each panel. Stimulus contrast, 85%. Averaged data (n = 6); vertical bars, SEM.
Figure 3.
 
FRCs obtained by sinusoidal modulation of a mean luminance equivalent to 38.79 Φ before and 90 minutes after vehicle (saline) and ivabradine injection. Relative amplitudes were normalized at 1 Hz. Ivabradine are given at the top of each panel. Stimulus contrast, 85%. Averaged data (n = 6); vertical bars, SEM.
An empiric parameter, useful in quantifying these effects, is the ratio of the response amplitude (R) at 3 Hz to that at 0.3 Hz (R 3Hz/R 0.3Hz). Figure 4 illustrates the dose-dependent effect of ivabradine on this ratio as well as on heart rate. 
Figure 4.
 
Effect of increasing doses of ivabradine on the ratio of the response amplitude to sinusoidal stimuli of 3 and 0.3 Hz (R 3Hz/R 0.3Hz) in the control (before injection) and at different times after drug injection. Gray histograms indicate the corresponding heart rate (in beats/minute). Sample sizes were n = 8, 5, 5, and 6 for the 0-, 3-, 6-, and 12-mg/kg dose groups, respectively. *P < 0.05; **P < 0.01, statistically significant versus control. Vertical bars, SEM.
Figure 4.
 
Effect of increasing doses of ivabradine on the ratio of the response amplitude to sinusoidal stimuli of 3 and 0.3 Hz (R 3Hz/R 0.3Hz) in the control (before injection) and at different times after drug injection. Gray histograms indicate the corresponding heart rate (in beats/minute). Sample sizes were n = 8, 5, 5, and 6 for the 0-, 3-, 6-, and 12-mg/kg dose groups, respectively. *P < 0.05; **P < 0.01, statistically significant versus control. Vertical bars, SEM.
Effects of a 3-Week Infusion of Ivabradine
Continuous ivabradine infusion in rats by means of implanted osmotic pumps (∼11 mg/kg/d, a dose close to the highest efficient dose delivered in acute experiments) for 3 weeks induced a heart rate reduction of 21% (P < 0.01) that returned to control values within 1 week after the treatment was discontinued. 
Flash ERG.
As observed in acute experiments, continued ivabradine delivery to rats for a period of 21 days had small effects on the flash ERG. In Figure 5A, the b-wave amplitude is plotted as a function of the flash intensity in three distinct animal groups: vehicle-treated, ivabradine-treated, 1 week after ivabradine discontinuation. Similarly as in acute experiments, the 3-week treatment had small influence on the amplitude of the b-wave. Prolonged ivabradine treatment had no effect on the amplification factor (data not shown). 
Figure 5.
 
Effect of prolonged (21 days) ivabradine treatment (on average, 11 mg/kg/d) on the ERG response. (A) Peak amplitude of the b-wave as a function of the flash intensity in a control group of animals after 21 days' infusion with vehicle or ivabradine and 1 week after ivabradine treatment cessation. (B) Effect of the 21-day ivabradine regimen on the FRCs in the three distinct animal groups shown in (A). Ratio R 3Hz/R 0.3Hz and corresponding heart rate in the control, ivabradine, and recovery groups (C). Each group consisted of six animals. Both R 3Hz/R 0.3Hz and HR changes are statistically significant (**P < 0.01). Vertical bars, SEM.
Figure 5.
 
Effect of prolonged (21 days) ivabradine treatment (on average, 11 mg/kg/d) on the ERG response. (A) Peak amplitude of the b-wave as a function of the flash intensity in a control group of animals after 21 days' infusion with vehicle or ivabradine and 1 week after ivabradine treatment cessation. (B) Effect of the 21-day ivabradine regimen on the FRCs in the three distinct animal groups shown in (A). Ratio R 3Hz/R 0.3Hz and corresponding heart rate in the control, ivabradine, and recovery groups (C). Each group consisted of six animals. Both R 3Hz/R 0.3Hz and HR changes are statistically significant (**P < 0.01). Vertical bars, SEM.
Sinusoidal Stimuli.
The effect of prolonged treatment with ivabradine on the FRCs was similar to that observed in acutely treated animals (Fig. 5). The ratio R 3Hz/R 0.3Hz is reported for the same three animal groups in Figure 5C. The superimposed diagram plots the heart rate changes. Both ERG and heart rate changes are statistically significant, and completely reverted to the values of the control group 7 days after discontinuation of ivabradine treatment. 
HCN1 and -2 Localization.
The influence of prolonged ivabradine treatment on retinal morphology, density, and distribution of HCN1 and -2 channels was investigated by immunolabeling. Confocal images of rat transretinal sections in Figure 6A illustrate HCN1 distribution in vehicle and ivabradine-treated animals. In both conditions, HCN1 labeling is mainly distributed at the inner segment of photoreceptors and at the outer and inner plexiform layers. Figure 6B shows retinal sections double-labeled with the HCN1 antibody and with the DNA-specific label propidium. The distribution of HCN2 is documented in Figure 6C. In the two panels, propidium red fluorescence labels the nuclei, whereas the green fluorescence labels the HCN2 that is distributed mainly at the outer plexiform layer, with no differences between vehicle- and ivabradine-treated animals. Figure 6D illustrates at larger magnification the HCN2 distribution on the dendrites of PKC stained rod bipolar cells. The same immunolabeling analysis was performed on wt and rd10 mice (data not shown). The genetic defect of the r10 model did not affect HCN distribution, per se, and in both wt and mutant, HCN1 and -2 labeling were similarly distributed but were not affected by ivabradine treatment. Repeated ivabradine treatment did not alter retinal morphology or HCN1 and -2 subunit composition and distribution in rats, wt, and rd10 mice retinas. 
Figure 6.
 
Confocal images of retinal sections immunolabeled with rabbit polyclonal antibodies (green fluorescence) for two distinct HCN isoform proteins in the control and in animals with 21-day ivabradine treatment. (A) Confocal images of retinas labeled with a rabbit polyclonal antibody for HCN1 (green fluorescence). (B) The retinas were also counterstained with the DNA-specific label propidium to highlight the nuclei (red fluorescence). (C) Retinas were stained with a rabbit polyclonal antibody for HCN2 (green fluorescence). (D) In addition to immunolabeling with the antibody for HCN2, the retinas were also stained with the mouse monoclonal antibody against PKC, a specific marker for rod bipolar cells (red). IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GC, ganglion cells. Scale bar, 10 μm.
Figure 6.
 
Confocal images of retinal sections immunolabeled with rabbit polyclonal antibodies (green fluorescence) for two distinct HCN isoform proteins in the control and in animals with 21-day ivabradine treatment. (A) Confocal images of retinas labeled with a rabbit polyclonal antibody for HCN1 (green fluorescence). (B) The retinas were also counterstained with the DNA-specific label propidium to highlight the nuclei (red fluorescence). (C) Retinas were stained with a rabbit polyclonal antibody for HCN2 (green fluorescence). (D) In addition to immunolabeling with the antibody for HCN2, the retinas were also stained with the mouse monoclonal antibody against PKC, a specific marker for rod bipolar cells (red). IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GC, ganglion cells. Scale bar, 10 μm.
Effect of Ivabradine on Retinal Apoptosis in wt and rd10 Mice: TUNEL Analysis.
The presence of apoptotic nuclei in retinal neurons and especially in photoreceptors, was investigated by the TUNEL staining technique in frozen sections of retinas isolated from untreated and ivabradine-treated wt and rd10 mice. Figure 7 shows fluorescent microscope images of retinal sections in which individual retinal layers are highlighted by a specific nuclear marker. Inner and outer nuclear layers appear as a blue fluorescence background on which the apoptotic nuclei stand out as green spots. The frequency of apoptotic nuclei was somewhat higher in rd10 than wt mice, but in no case was there an obvious effect of ivabradine treatment. 
Figure 7.
 
Fluorescence microscope images of vertical sections of wt and rd10 mouse retinas from control and ivabradine-treated animal groups. Two retinal sections for each animal group: central retina (near the optic disc; left); far peripheral retina (right). Apoptotic photoreceptors stained positively with the TUNEL method at P21. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar, 20 μm.
Figure 7.
 
Fluorescence microscope images of vertical sections of wt and rd10 mouse retinas from control and ivabradine-treated animal groups. Two retinal sections for each animal group: central retina (near the optic disc; left); far peripheral retina (right). Apoptotic photoreceptors stained positively with the TUNEL method at P21. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar, 20 μm.
Effect of Ivabradine on Retinal Degeneration in wt and rd10 Mice: WB Analysis.
The contents of opsin and GFAP were measured by WB in various retinal samples. The amount of opsin provides an estimate of the photoreceptor density, whereas the expression of large amounts of GFAP is related to retinal degeneration. In retinas of rd10 mice, the opsin content decreases with the animal's age because of the progressive photoreceptor loss and, correspondingly, GFAP expression increases. 20 Figure 8 shows that repeated treatment with ivabradine has no significant effect on either opsin or GFAP in both wt and rd10 mice. Note, however, that the expression of these proteins is markedly altered in rd10 mice as a consequence of the progressive degeneration to which this strain is highly susceptible. 
Figure 8.
 
Western blot diagram showing the ratios of the optical densities opsin/actin and GFAP/actin on the retinas of wt and rd10 mice in the control and ivabradine-treated animal groups. Both opsin and GFAP were normalized to the actin content of the sample and expressed relative to the values measured in the wt control group. Vertical bars, SEM. n = 4 wt vehicle, n = 5 wt ivabradine, n = 7 rd10 vehicle, and n = 9 rd10 ivabradine.
Figure 8.
 
Western blot diagram showing the ratios of the optical densities opsin/actin and GFAP/actin on the retinas of wt and rd10 mice in the control and ivabradine-treated animal groups. Both opsin and GFAP were normalized to the actin content of the sample and expressed relative to the values measured in the wt control group. Vertical bars, SEM. n = 4 wt vehicle, n = 5 wt ivabradine, n = 7 rd10 vehicle, and n = 9 rd10 ivabradine.
Discussion
The changes observed on the ERG response reflect what at the cellular level occurs in both rods and rod bipolar cells, the sum of whose activities contributes to the main waves of the ERG. The band-pass filtering mode with the 1-Hz resonant peak of the ERG's FRC has also been observed in the voltage response of rods 21 and rod bipolar cells 10 and is associated with the gating of HCN channels. The striking feature of I h inhibition in rods and bipolar cells is therefore reflected in the in vivo recordings of ERG's FRCs as a transient suppression of resonance. We suggest that the band-pass mode enables the visual system to respond selectively to stimuli in a range of preferred, possibly perceptually relevant, frequencies. Under similar circumstances, the low-frequency signals carrying a prominent fraction of noise that limits visual performance may cross through the retinal stages to reach the cortical visual centers unfiltered, thus generating conscious visual experiences in the form of phosphenes. It is therefore reasonable to assume that the probability of perceiving phosphenes increases substantially during HCN inhibition. 
Several lines of evidence support the idea that the ERG changes induced by ivabradine are the consequence of a specific HCN inhibition. Recently, we have shown that ivabradine selectively inhibits I h in rods leaving other currents unaffected. 7 The possibility that the drug affects the ERG by reducing retinal perfusion is not supported by the ABP measurements. The effect of ivabradine on the ERG response is qualitatively similar to that reported for other HCN inhibitors, namely zatebradine and cesium. 22 Consistent with a specific action on the retinal I h is also the observation that ivabradine does not affect other processes such as the kinetics of the transduction cascade, as documented by the analysis of the leading edge of the a-wave showing no effect on the amplification factor A. 18 Retinal neurons express four different isoforms (HCN1–4) variously localized within the retinal layers, with HCN3 mainly expressed on the cone pedicles. 9,11 Differences in functional properties and sensitivity to the inhibitors by the various isoforms are not known. Preliminary data from mice in which the HCN1 gene was deleted (source of HCN1-deficient mice, Jackson Laboratories, Bar Harbor, ME), together with the lack of HCN1 and persistence of HCN2–4 staining, produce ERG responses to flashes and sinusoidal stimuli similar to wt mice treated with ivabradine. 
The possible concern that a prolonged therapeutic use of HCN inhibitors may have adverse irreversible effects on retinal integrity and function or contribute to the acceleration of the progress of an ongoing retinal degeneration is not supported by the present results. Prolonged ivabradine exposure reversibly affected the ERG, as in the acute experiments. Furthermore, immunocytochemical determinations show that repeated exposure to ivabradine had no apparent consequences on retinal morphology, HCN1–2 localization and distribution and on the expression of retina-specific proteins such as opsin and GFAP, an indicator of inflammatory and degenerative processes. This instance is the first in which the effects of acute and long-term delivery of an HCN-inhibitor have been compared, showing that the consequences of the two different dosing approaches are similar and always reversible. Similarly, continuous delivery of ivabradine did not increase the frequency of apoptotic cells in rd10 mice at different ages, indicating that the treatment had no influence on both the extent and progress of the retinal damage. All these results converge pointing to the conclusion that ivabradine only exerts a specific, reversible pharmacologic inhibition of retinal HCNs. 
Footnotes
 Supported by Institut de Recherches Internationales Servier (IRIS).
Footnotes
 Disclosure: L. Della Santina, IRIS (F); M. Bouly, IRIS (E, F); A. Asta, IRIS (F); G.C. Demontis, IRIS (F); L. Cervetto, IRIS (C, F); C. Gargini, IRIS (C, F)
The authors thank IRIS and the nonclinical department of the Cardiovascular Unit for fruitful discussions regarding ivabradine during the development of the project and the manuscript. 
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Figure 1.
 
Averaged ERG responses of increasing light intensity in the control and after a single dose injection of 12 mg/kg ivabradine in Long-Evans pigmented rats. The intensity of the flash is expressed as the number of photoisomerizations per rod (Φ) per flash. Color coded traces are responses in the control and 45, 120, and 240 minutes after ivabradine injection (Σ = 6 low luminance flashes, Σ = 2 two brightest flashes). The collected control HR and those collected at three successive times after ivabradine injection are calculated from the RR interval of the ECG and reported at the top right of the rightmost panel. Luminances (Φ) are reported at the bottom right of each panel.
Figure 1.
 
Averaged ERG responses of increasing light intensity in the control and after a single dose injection of 12 mg/kg ivabradine in Long-Evans pigmented rats. The intensity of the flash is expressed as the number of photoisomerizations per rod (Φ) per flash. Color coded traces are responses in the control and 45, 120, and 240 minutes after ivabradine injection (Σ = 6 low luminance flashes, Σ = 2 two brightest flashes). The collected control HR and those collected at three successive times after ivabradine injection are calculated from the RR interval of the ECG and reported at the top right of the rightmost panel. Luminances (Φ) are reported at the bottom right of each panel.
Figure 2.
 
(A) Amplification factor A estimated from the analysis of the leading edge of the a-wave, in control conditions and 120 minutes after ivabradine injection (12 mg/kg). To minimize the effect of spontaneous response variability, data were normalized to the control value obtained before injection of the vehicle (saline). Changes in the absolute values of A are not statistically significant. (B) Collected data (n = 6) of the b-wave peak amplitude as a function of the flash intensity in the control and 120 minutes after injection of 12 mg/kg ivabradine. Vertical bars, the SEM. *P < 0.05, statistically significant versus control.
Figure 2.
 
(A) Amplification factor A estimated from the analysis of the leading edge of the a-wave, in control conditions and 120 minutes after ivabradine injection (12 mg/kg). To minimize the effect of spontaneous response variability, data were normalized to the control value obtained before injection of the vehicle (saline). Changes in the absolute values of A are not statistically significant. (B) Collected data (n = 6) of the b-wave peak amplitude as a function of the flash intensity in the control and 120 minutes after injection of 12 mg/kg ivabradine. Vertical bars, the SEM. *P < 0.05, statistically significant versus control.
Figure 3.
 
FRCs obtained by sinusoidal modulation of a mean luminance equivalent to 38.79 Φ before and 90 minutes after vehicle (saline) and ivabradine injection. Relative amplitudes were normalized at 1 Hz. Ivabradine are given at the top of each panel. Stimulus contrast, 85%. Averaged data (n = 6); vertical bars, SEM.
Figure 3.
 
FRCs obtained by sinusoidal modulation of a mean luminance equivalent to 38.79 Φ before and 90 minutes after vehicle (saline) and ivabradine injection. Relative amplitudes were normalized at 1 Hz. Ivabradine are given at the top of each panel. Stimulus contrast, 85%. Averaged data (n = 6); vertical bars, SEM.
Figure 4.
 
Effect of increasing doses of ivabradine on the ratio of the response amplitude to sinusoidal stimuli of 3 and 0.3 Hz (R 3Hz/R 0.3Hz) in the control (before injection) and at different times after drug injection. Gray histograms indicate the corresponding heart rate (in beats/minute). Sample sizes were n = 8, 5, 5, and 6 for the 0-, 3-, 6-, and 12-mg/kg dose groups, respectively. *P < 0.05; **P < 0.01, statistically significant versus control. Vertical bars, SEM.
Figure 4.
 
Effect of increasing doses of ivabradine on the ratio of the response amplitude to sinusoidal stimuli of 3 and 0.3 Hz (R 3Hz/R 0.3Hz) in the control (before injection) and at different times after drug injection. Gray histograms indicate the corresponding heart rate (in beats/minute). Sample sizes were n = 8, 5, 5, and 6 for the 0-, 3-, 6-, and 12-mg/kg dose groups, respectively. *P < 0.05; **P < 0.01, statistically significant versus control. Vertical bars, SEM.
Figure 5.
 
Effect of prolonged (21 days) ivabradine treatment (on average, 11 mg/kg/d) on the ERG response. (A) Peak amplitude of the b-wave as a function of the flash intensity in a control group of animals after 21 days' infusion with vehicle or ivabradine and 1 week after ivabradine treatment cessation. (B) Effect of the 21-day ivabradine regimen on the FRCs in the three distinct animal groups shown in (A). Ratio R 3Hz/R 0.3Hz and corresponding heart rate in the control, ivabradine, and recovery groups (C). Each group consisted of six animals. Both R 3Hz/R 0.3Hz and HR changes are statistically significant (**P < 0.01). Vertical bars, SEM.
Figure 5.
 
Effect of prolonged (21 days) ivabradine treatment (on average, 11 mg/kg/d) on the ERG response. (A) Peak amplitude of the b-wave as a function of the flash intensity in a control group of animals after 21 days' infusion with vehicle or ivabradine and 1 week after ivabradine treatment cessation. (B) Effect of the 21-day ivabradine regimen on the FRCs in the three distinct animal groups shown in (A). Ratio R 3Hz/R 0.3Hz and corresponding heart rate in the control, ivabradine, and recovery groups (C). Each group consisted of six animals. Both R 3Hz/R 0.3Hz and HR changes are statistically significant (**P < 0.01). Vertical bars, SEM.
Figure 6.
 
Confocal images of retinal sections immunolabeled with rabbit polyclonal antibodies (green fluorescence) for two distinct HCN isoform proteins in the control and in animals with 21-day ivabradine treatment. (A) Confocal images of retinas labeled with a rabbit polyclonal antibody for HCN1 (green fluorescence). (B) The retinas were also counterstained with the DNA-specific label propidium to highlight the nuclei (red fluorescence). (C) Retinas were stained with a rabbit polyclonal antibody for HCN2 (green fluorescence). (D) In addition to immunolabeling with the antibody for HCN2, the retinas were also stained with the mouse monoclonal antibody against PKC, a specific marker for rod bipolar cells (red). IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GC, ganglion cells. Scale bar, 10 μm.
Figure 6.
 
Confocal images of retinal sections immunolabeled with rabbit polyclonal antibodies (green fluorescence) for two distinct HCN isoform proteins in the control and in animals with 21-day ivabradine treatment. (A) Confocal images of retinas labeled with a rabbit polyclonal antibody for HCN1 (green fluorescence). (B) The retinas were also counterstained with the DNA-specific label propidium to highlight the nuclei (red fluorescence). (C) Retinas were stained with a rabbit polyclonal antibody for HCN2 (green fluorescence). (D) In addition to immunolabeling with the antibody for HCN2, the retinas were also stained with the mouse monoclonal antibody against PKC, a specific marker for rod bipolar cells (red). IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GC, ganglion cells. Scale bar, 10 μm.
Figure 7.
 
Fluorescence microscope images of vertical sections of wt and rd10 mouse retinas from control and ivabradine-treated animal groups. Two retinal sections for each animal group: central retina (near the optic disc; left); far peripheral retina (right). Apoptotic photoreceptors stained positively with the TUNEL method at P21. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar, 20 μm.
Figure 7.
 
Fluorescence microscope images of vertical sections of wt and rd10 mouse retinas from control and ivabradine-treated animal groups. Two retinal sections for each animal group: central retina (near the optic disc; left); far peripheral retina (right). Apoptotic photoreceptors stained positively with the TUNEL method at P21. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar, 20 μm.
Figure 8.
 
Western blot diagram showing the ratios of the optical densities opsin/actin and GFAP/actin on the retinas of wt and rd10 mice in the control and ivabradine-treated animal groups. Both opsin and GFAP were normalized to the actin content of the sample and expressed relative to the values measured in the wt control group. Vertical bars, SEM. n = 4 wt vehicle, n = 5 wt ivabradine, n = 7 rd10 vehicle, and n = 9 rd10 ivabradine.
Figure 8.
 
Western blot diagram showing the ratios of the optical densities opsin/actin and GFAP/actin on the retinas of wt and rd10 mice in the control and ivabradine-treated animal groups. Both opsin and GFAP were normalized to the actin content of the sample and expressed relative to the values measured in the wt control group. Vertical bars, SEM. n = 4 wt vehicle, n = 5 wt ivabradine, n = 7 rd10 vehicle, and n = 9 rd10 ivabradine.
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