Ivabradine

The general procedure for animal preparation, anesthesia, ERG recording, light stimulation and data analysis has been previously described in detail in Della Santina et al. [19] (link). Briefly: ERGs were recorded in complete darkness via coiled gold electrodes making contact with the moist cornea. A small gold plate placed in the mouth served as both reference and ground. HCN inhibition was induced by subcutaneous injections of 12 mg/kg ivabradine. 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 s for dim lights to 120 s for the brightest flashes.
The full field illumination of the eyes was achieved via a Ganzfeld sphere 30 cm in diameter, whose interior surface was 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 varying luminance stimuli eliciting periodic responses.

The cardiomyocytes were loaded with 10 μM Fluo-4, AM (Molecular Probes, Leiden, The Netherlands) dissolved in Transfer Buffer B (in mM: 137 NaCl, 5.4 KCl, 1.8 CaCl₂, 0.5 MgCl₂, 10 HEPES, 5.5 glucose, pH 7.4 at 37 °C) for 30 min. For pharmacological treatments ivabradine (Molekula, Munich, Germany; cat. no. 89982651/155974-00-8) or ORM-10103 (Sigma-Aldrich, Germany; cat. no. SML0972) were dissolved in DMSO for stock solution. For working solution, both ivabradine or ORM-10103 were dissolved in Transfer Buffer B at 3 μM or at 10 µM, respectively, and incubated for 10 min prior to the Ca²⁺ recordings.
[Ca²⁺]_i transients were recorded using an Olympus OSP-3 System fluorescence microscope. Cardiomyocytes were single twitched electrically stimulated at 25 V for 10 ms at constant rate of 0.2 Hz. The fluorescence signal was obtained from cytosolic area and calibrated by a pinhole of 7.5 μm diameter, integrated in the photomultiplier, and A/D converted using PowerLab 4/35 and LabChart V7.0.
Out of 8–10 Ca²⁺ transients recorded, the least five were analyzed, avoiding the non-physiological Ca²⁺ transients due to time with no electrical stimulation between cell recordings. Ca²⁺ transients were eligible for regular analysis when lacking automaticity and showing regular [Ca²⁺]_I clearance under field stimulation at 0.2 Hz. Cells that exhibited pacemaker activity (defined as spontaneous beating rate < 0.2 Hz) or abnormal diastolic [Ca²⁺]_i decay (defined as D₉₀ > 2000ms) were excluded from regular analysis and evaluated in a separate record (please refer to Supplementary Fig. 2).
Calibration was performed at the end of each experiment in freshly loaded cells with Fluo-4, AM to transform voltage values into [Ca²⁺]_i^{55 (link)}. Briefly, cells were treated with high Ca²⁺ solution (in mM: 140 NaCl, 5 KCl, 1.2 KH₂PO₄, 1.2 MgCl₂, 4 CaCl₂, 20 HEPES, 0.005 ionomycin, 0.01 CPA, 5 caffeine, 1 ouabaine) to obtain Fmax values followed by application of zero-Ca²⁺ solution (in mM: 140 LiCl, 5 KCl, 1.2 KH₂PO₄, 1.2 MgCl₂, 20 HEPES, 4 EGTA, 0.005 ionomycin, 0.01 CPA, 5 caffeine, 1 ouabaine) to provide Fmin values. The Ca²⁺ signal (F) was then converted into [Ca²⁺]_i according to Grynkiewicz et al. 1985^{56 (link)}.
[Ca²⁺] = Kd(F-Fmin)/(Fmax-F), with Kd: apparent Ca²⁺ dissociation constant of the Fluo-4 of 345 nM, according to the manufacturer’s information.
Five biophysical parameters were analyzed: baseline (μM), peak area (area under the curve; μM*s), D₅₀ (duration of the transient at 50% of the peak; ms), time to peak (time required to reach the maximum from the baseline; ms), slope Ca²⁺ uptake time (time required to return to the base line from the peak to 5% of the baseline; nM/s).

CCTA and MRI were performed in all patients within the first month after discharge and at the same centre (Hospital de Sant Pau, who acted as core-lab for cardiac imaging). On the previous days of the CCTA, patients were treated with a beta-blocker (atenolol 25–50 mg or ivabradine 5–7.5 mg to achieve a heart rate ≤ 60 beats per minute). A pair of expert evaluators formed by a cardiologist and a radiologist with level 3 training in interpretation of CCTA, read each angiogram using a 17-segment model of the coronary arteries without knowledge of the clinical data. Per-patient anatomical severity was classified according to the Coronary Artery Disease—Reporting and Data System (CAD-RADS) [12] (link). Triple-rule-out CCTA examinations (coronary artery disease, pulmonary embolism, and acute aortic pathology) were also performed. After the CCTA study, an MRI exam was performed to evaluate the global and segmental cardiac contractility and presence of focal fibrosis, using late gadolinium enhancement (LGE) contrast. We classified the LGE pattern as “ischemic” if subendocardial or transmural delayed contrast enhancement in a vascular distribution was observed and “non-ischemic” if enhancement was distributed patchy or diffuse, not following a vascular territory, mainly in mesocardial or epicardial locations [13] (link). Finally, in those with significant CAD in the CCTA (CAD-RADS ≥ 3), a pharmacological stress with adenosine was conducted, to assess functional impact of each coronary stenosis. A more detailed version of the study protocol was previously published [14] (link).