Tissue preparations and electropharmacological experiments in RVOT tissues.
This study received approval from the ethics review board at the National Defense Medical Center (IACUC-20-026), where all animal-related woks were conducted, and adhered to the guidelines outlined in the "Guide for the Care and Use of Laboratory Animals" published by the US National Institutes of Health. Male New Zealand white rabbits weighing between 2.5 and 3.5 kg were employed in this study. Prior to euthanasia, the rabbits were subjected to anesthesia with inhalational isoflurane (administered at a concentration of 2.0–2.5%) via a precision vaporizer for 10 minutes. The effectiveness of anesthesia was validated through the absence of corneal reflexes and motor responses to painful stimuli applied with a scalpel tip. Subsequently, following the intravenous administration of heparin (at a dosage of 1000 units per kilogram), the heart and lungs were promptly excised after performing a midline thoracotomy.
As previously outlined, the heart regions corresponding to the RVOT, measuring approximately 1 cm × 1.5 cm, were isolated from all rabbits after euthanasia for subsequent analysis [29]. The RVOT was surrounded superiorly and inferiorly by the pulmonary valve and supraventricular crest, respectively, and excised within the region ≤ 5 mm below the pulmonary valve. The excised tissues underwent preparation and were immersed in Tyrode’s solution at 37°C, composed of 137 mM NaCl, 4 mM KCl, 15 mM NaHCO3, 0.5 mM NaH2PO4, 0.5 mM MgCl2, 2.7 mM CaCl2, and 11 mM dextrose. A constant superfusion rate of 3 ml/min was applied to the tissues, with the perfusion medium saturated by a gas mixture comprising 97% O2 and 3% CO2 [21, 29]. Tissue preparations were connected to a WPI model FD773 electrometer (Florida, USA) employing a 150 mg load [8]. The endocardial side faced upward, and both electrical and mechanical events were recorded using machine-pulled glass capillary microelectrodes filled with 3 M KCl. The data were simultaneously presented and visualized on a Gould 4072 oscilloscope and Gould TA11 recorder (Gould Electronics Ltd., OH, USA). Signals were digitally recorded with 16-bit precision at a 125 kHz rate. An electrical pulse lasting 1 millisecond was locally administered using a Grass S88 stimulator through a Grass SIU5B stimulus isolation unit [11, 13, 29, 35, 50, 51]. The transmembrane action potentials (APs) of the RVOT tissues were recorded by machine-pulled glass capillary microelectrodes filled with 3 M KCl. The amplitude of the AP (APA) was calculated from the resting membrane potential (RMP) to the peak of the AP depolarization. Furthermore, the AP duration (APD) at 20%, 50%, and 90% repolarization of the amplitude was meticulously measured and documented as APD20, APD50, and APD90, respectively. These measurements of RMP, APA, and APD were conducted under a pacing frequency of 2 Hz applied to the RVOT tissues. The RVOT tissues were subjected to acute treatments with control conditions and varying concentrations of IS (0.1 and 1.0 µM, 30–60 mins, Sigma-Aldrich, Merck, Darmstadt, Germany). These tissues were analyzed both before and after stimulation with isoproterenol (ISO, 1.0 µM, Sigma-Aldrich, Merck, Darmstadt, Germany). Additionally, the RVOT tissues underwent pre-treatment in control conditions and were exposed to different concentrations of SN (3 and 30 nM, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for a duration of 30 minutes. This was done to observe burst firing and the impact of these treatments during high-frequency burst pacing at 20 Hz for a duration of 1 second.
Patch clamp experiments in isolated single RVOT cardiomyocytes
Single cardiomyocytes from the RVOTs were isolated using the methods previously described [29]. In brief, the hearts were excised and placed on a Langendorff apparatus. They underwent superfusion in an antegrade fashion with oxygenated normal Tyrode’s solution at 37°C, comprising (in mM) NaCl 137, KCl 5.4, CaCl2 1.8, MgCl2 0.5, HEPES 10, and glucose 11. The pH was adjusted to 7.4 with NaOH. Subsequently, the regions of the hearts corresponding to the RVOT, located within 5 mm below the pulmonary valve, were removed for further experimentation [21, 29]. The cardiomyocytes were allowed to equilibrate in the bath for a minimum of 30 minutes before commencing the experiments. The whole-cell patch clamp experiment was conducted on isolated RVOT cardiomyocytes, either in their untreated (control) state or after treatment with IS (1.0 µM), both in the presence and absence of SN (30 nM). These experiments were conducted using an Axopatch 1D amplifier (Axon Instruments, Foster City, CA, USA) and were conducted at a controlled temperature of 35°C ± 1°C [10, 42]. Ionic currents were recorded within a similar time period (3–5 minutes) after the rupture or perforation of the cell membrane to ensure that ion channel activity remained stable over time. Series resistance was electronically compensated, typically ranging from 60–80%. The recording of ionic currents was performed in the voltage-clamp mode.
The sodium (Na+) current (INa) was recorded during depolarization from a holding potential of -120 mV to testing potentials spanning from − 90 to 0 mV in 5 mV increments, each lasting for 40 milliseconds. These measurements were conducted at a frequency of 3 Hz and at room temperature (25 ± 1°C). The external solution used in this setup consisted of NaCl (5 mM), CsCl (133 mM), MgCl2 (2 mM), CaCl2 (1.8 mM), nifedipine (0.002 mM), HEPES (5 mM), and glucose (5 mM). Micropipettes were filled with a solution containing CsCl (133 mM), NaCl (5 mM), EGTA (10 mM), Mg2ATP (5 mM), TEACl (20 mM), and HEPES (5 mM), adjusted to a pH of 7.3 using CsOH.
The late Na+ current (INa−Late) was assessed using a step/ramp protocol, involving a transition from − 100 mV to + 20 mV for 100 milliseconds, followed by a return to -100 mV over 100 milliseconds. These measurements were conducted at room temperature. The external solution employed for this purpose consisted of NaCl (130 mM), CsCl (5 mM), MgCl2 (1 mM), CaCl2 (1mM), HEPES (10 mM), and glucose (10 mM), with the pH adjusted to 7.3 using NaOH. Micropipettes were filled with a solution containing CsCl (130 mM), Na2ATP (4 mM), MgCl2 (1 mM), EGTA (10 mM), and HEPES (5 mM); pH was adjusted to 7.3 using NaOH. An equilibration period of approximately 5–10 minutes was allowed for adequate dialysis before clamping the cell currents. INa−Late was quantified as the portion of the current traces that was sensitive to tetrodotoxin (30 µM) when the voltage was ramped back to -100 mV.
The ICa−L was assessed by recording the inward current generated during depolarization from a holding potential of -50 mV to test potentials spanning from − 40 to + 60 mV in 10 mV increments, each lasting for 300 milliseconds. These measurements were conducted at a frequency of 0.1 Hz, utilizing a perforated patch clamp technique facilitated by amphotericin B. The micropipettes were filled with a solution containing CsCl (130 mM), MgCl2 (1 mM), MgATP (5 mM), HEPES (10 mM), NaGTP (0.1 mM), and Na2 phosphocreatine (5 mM). This solution was adjusted to a pH of 7.2 using CsOH. The external solution consisted of the following concentrations (in mM): tetraethylammonium chloride (TEACl) 137, CsCl 5.4, CaCl2 1.8, MgCl2 0.5, HEPES 10, and glucose 10.
The Na+-Ca2+ exchanger (NCX) current was induced by applying depolarizing pulses ranging from − 100 to + 100 mV, originating from a holding potential of -40 mV, and each pulse lasted for 300 milliseconds. These pulses were administered at a frequency of 0.1 Hz. The amplitudes of the NCX current were quantified as 10-millimolar nickel-sensitive currents. The external solution consisted of NaCl (140 mM), CaCl2 (2 mM), MgCl2 (1 mM), HEPES (5 mM), and glucose (10 mM), and its pH was maintained at 7.4. This solution also contained 10 µM strophanthidin, 10 µM nitrendipine, and 100 µM niflumic acid. Micropipettes were filled with a solution consisting of NaCl (20 mM), CsCl (110 mM), MgCl2 (0.4 mM), CaCl2 (1.75 mM), TEACl (20 mM), BAPTA (5 mM), glucose (5 mM), MgATP (5 mM), and HEPES (10 mM). The pH of this solution was adjusted to 7.25 using CsOH.
The transient outward potassium current (Ito) was studied with a double-pulse protocol. A 30-ms pre-pulse from − 80 to -40 mV was used to inactivate the Na+ channels, followed by a 300-ms test pulse to + 60 mV in 10-mV steps at a frequency of 0.1 Hz. The external solution was formulated with the following components: 137 mM NaCl, 4 mM KCl, 15 mM NaHCO3, 0.5 mM NaH2PO4, 0.5 mM MgCl2, 2.7 mM CaCl2, 11 mM dextrose, and 200 µM CdCl2. Ito was measured as the difference between the peak outward current and steady-state current.
The rapid delayed rectifier potassium current (IKr−tail) was assessed by measuring the peak outward tail current density following a 3-second pre-pulse. This pre-pulse was initiated from a holding potential of -40 mV and spanned from − 40 to + 60 mV in 10-millivolt increments. These pre-pulses occurred at a frequency of 0.1 Hz and were conducted in the presence of the external solution comprising 137 mM NaCl, 4 mM KCl, 15 mM NaHCO3, 0.5 mM NaH2PO4, 0.5 mM MgCl2, 2.7 mM CaCl2, 11 mM dextrose, 200 µM CdCl2, and chromanol 293B (30 µM). Micropipettes were filled with a solution consisting of KCl (120 mM), MgCl2 (5 mM), CaCl2 (0.36 mM), EGTA (5 mM), HEPES (5 mM), glucose (5 mM), K2-ATP (5 mM), Na2-CrP (5 mM), and Na-GTP (0.25 mM). The pH of this solution was adjusted to 7.2 using KOH.
Measurement of intracellular Ca2+
After single cardiomyocytes from the RVOT were isolated, they underwent superfusion with oxygenated normal Tyrode’s solution at 37°C, comprising (in mM) NaCl 137, KCl 5.4, CaCl2 1.8, MgCl2 0.5, HEPES 10, and glucose 11. The pH was adjusted to 7.4 with NaOH. Intracellular Ca2+ concentration (Ca2 + i) was recorded using a fluorometric ratio technique, following established protocols [20, 29]. The control or IS-treated (1.0 µM, 30 minutes) RVOT cardiomyocytes combined with and without pretreatment with SN (30 nM, 30 mins) were loaded with fluorescent Ca2+ (10 µM) fluo-3/AM over a 30-minute period at room temperature. To remove excess extracellular dye, the bath solution was changed, and intracellular hydrolysis of fluo-3/AM occurred at a controlled temperature of 35°C ± 1°C after 30 minutes. Fluo-3 fluorescence was excited using the 488-nm line of an argon ion laser, and emission was recorded at wavelengths greater than 515 nm. The cells were repeatedly scanned at 2-ms intervals for a total duration of 6 seconds. Fluorescence imaging was performed using a laser scanning confocal microscope (Zeiss LSM 510, Carl Zeiss, Jena, Germany) and an inverted microscope (Axiovert 100). Fluorescent signals were corrected for variations in dye concentrations by normalizing the fluorescence (represented by F) against baseline fluorescence (F0) to obtain reliable information about transient Ca2 + i changes from baseline values, as (F–F0)/F0, and to exclude variations in the fluorescence intensity by different volumes of injected dye. The Ca2 + i transient, peak systolic Ca2 + i, and diastolic Ca2 + i were measured during 1-Hz field stimulation for 10 milliseconds, utilizing square wave pulses with twice the threshold strength. When adding 20 mM caffeine after electrical stimulation at 1 Hz for at least 30 seconds, the estimated sarcoplasmic reticulum (SR) Ca2+ content as the total SR Ca2+ content was measured from the peak amplitude of the caffeine-induced Ca2 + i transients. During this procedure, the cell was imaged in the linescan mode along the longitudinal line for 15 seconds, which allows caffeine-evoked Ca2+ release to be recorded. The SR Ca2+ leak was measured as the tetracaine (1 mM)-reduced Ca2 + i in Na+ free and Ca2+ free solution after achieving steady-state Ca2+ transients with the repeated pulses (1Hz for 5 seconds).
Statistical analysis
All continuous variables were presented as mean values ± standard error of the mean. Nominal variables were compared using either Chi-square analysis or Fisher's exact test. For comparisons involving rabbit RVOT tissues and cardiomyocytes before and after IS infusion, a paired t-test was employed. An unpaired t-test or one-way analysis of variance with a Duncan’s methods was used to evaluate the dose-dependent effect of IS on RVOT electrophysiological characteristics, tissue contractility, and the differences in variables of intracellular Ca2+ homeostasis. Significance was established at a p-value threshold of < 0.05. All statistical analyses were conducted using SPSS Statistic 18.0 software (Chicago, IL, USA), and SigmaPlot 12 (Systat Software Inc., San Jose, CA, USA).