Hypertonic Solutions

Patch-clamp experiments using mitoplasts were performed as described previously [18] (link), [19] (link). Briefly, mitoplasts were prepared from a sample of human astrocytoma mitochondria placed in a hypotonic solution (5 mM HEPES, 200 µM CaCl₂, pH = 7.2) for approximately 1 min to induce swelling and breakage of the outer membrane. Then, a hypertonic solution (750 mM KCl, 30 mM HEPES, 200 µM CaCl₂, pH = 7.2) was added to restore the isotonicity of the medium. The patch-clamp pipette was filled with an isotonic solution containing 150 mM KCl, 10 mM HEPES, and 200 µM CaCl₂ at pH = 7.2. Mitoplasts are easily recognizable due to their size, round shape, transparency, and presence of a “cap”, characteristics that distinguish these structures from the cellular debris that is also present in the preparation. An isotonic solution containing 200 µM CaCl₂ was used as the control solution for all of the presented data. The low-calcium solution (1 µM CaCl₂) contained the following: 150 mM KCl, 10 mM HEPES, 1 mM EGTA and 0.752 mM CaCl₂ at pH = 7.2. All of the modulators of the channels and the substrates and inhibitors of the respiratory chain were added as dilutions in isotonic solution containing 200 µM CaCl₂. To apply these substances, we used a perfusion system containing a holder with a glass tube (made in our workshop), a peristaltic pump, and Teflon tubing. The mitoplasts at the tip of the measuring pipette were transferred into the openings of a multibarrel “sewer pipe” system in which their outer faces were rinsed with the test solutions (Fig. 1A). The configuration of our patch-clamp mode is presented in Fig. 1A. The experiments were carried out in patch-clamp inside-out mode. This is based on observations with various mitochondrial substrates applied such as NADH or succinate. Reported voltages are those applied to the patch-clamp pipette interior. Hence, positive potentials represent the physiological polarization of the inner mitochondrial membrane (outside positive).
The electrical connection was made using Ag/AgCl electrodes and an agar salt bridge (3 M KCl) as the ground electrode. The current was recorded using a patch-clamp amplifier (Axopatch 200B, Molecular Devices Corporation, USA). The pipettes, made of borosilicate glass, had a resistance of 10–20 MΩ and were pulled using a Flaming/Brown puller.
The currents were low-pass filtered at 1 kHz and sampled at a frequency of 100 kHz. The traces of the experiments were recorded in single-channel mode. The illustrated channel recordings are representative of the most frequently observed conductance for the given condition. The conductance of the channel was calculated from the current-voltage relationship (data not shown). The probability of channel opening (P_o, open probability) was determined using the single-channel search mode of the Clampfit 10.2 software. Calculations were performed using segments of continuous recordings lasting 60 s, with N>1000 events. Data from the experiments are reported as the mean values ± standard deviations (S.D.). Student’s t-test was used for statistical analysis. In figures showing single-channel recordings, “-” indicates the closed state of the channel.

Leukocyte was isolated according to the method of O’Loughlin et al [12 (link)]. Briefly, blood leukocytes were isolated using a hypotonic solution and a hypertonic solution. The leukocyte pellets were suspended in 1 mL of TRI Reagent® solution (TRIzol) (Sigma-Aldrich Ireland Ltd., Dublin, Ireland), and stored in a sterile tube at −70°C until RNA isolation.

The Tergitol-based decellularization protocol was modified from the TRICOL protocol previously reported [19 (link)].
Decellularization protocol was performed in an agitation system: treatment with protease inhibitors cocktail (1% v/v) and DMSO (supplier) (10%) at 4 °C (8 h) was followed by washing with a hypotonic solution (12 h). Subsequently, a second phase with protease inhibitors in combination with 1% Tergitol (12 h) (Sigma Aldrich, Saint Louis, MO, USA) was carried out at room temperature. After further washing, tissues were treated with Tergitol (0.1%) in a hypertonic solution (24 h in two cycles). Thereafter, they were treated for 20 h with sodium cholate (Sigma Aldrich, 4% v/v). Finally, tissue samples were treated with peracetic acid (Sigma Aldrich, 0.1%) and ethanol 4% (Carlo Erba, Cornaredo, MI, Italy) solutions (90′) for bioburden removal and primary decontamination.
Valves were cut into 8 mm patches with a biopsy puncher and treated with endonuclease enzyme (Benzonase 25 k U, Sigma Aldrich, E1014) at 37 °C for 48 h to complete the removal of nucleic acid residues. Several washing cycles with Phosphate Buffered Saline (PBS, Sigma Aldrich) were performed to remove residues of the enzyme. Samples (Native pulmonary wall (PW Native), Leaflets (LL Native) and Myocardia (MYO Native), Decellularized Pulmonary wall (PW DC), Leaflets (LL DC) and Myocardia (MYO DC))were then embedded in OCT (Tissue-Tek, 4583, Sakura Finetek, Torrance, CA, USA) and stored at −80 °C for histological and IF analysis, while the rest of the tissues were lyophilized for DNA and biochemical analysis. A subgroup of the decellularized valves (n = 3) was used for biomechanical tests.

Mature female Xenopus laevis frogs (Xenopus I, Ann Arbor, MI, USA) were kept in dechlorinated tap water at 19–21 °C and fed with beef liver at least twice a week. Clusters of oocytes were removed surgically under tricaine (Sigma, St. Louis, MO, USA) anesthesia (0.15%). Individual oocytes were manually dissected in a solution containing (in mM): NaCl, 88; KCl, 1; NaHCO₃, 2.4; MgSO₄, 0.8; HEPES, 10 (pH 7.5) and stored 2–7 days in modified Barth’s solution containing (in mM): NaCl, 88; KCl, 1; NaHCO₃, 2.4; Ca(NO₃)₂, 0.3; CaCl₂, 0.9; MgSO₄, 0.8; HEPES, 10 (pH 7.5), supplemented with sodium pyruvate 2 mM, penicillin 10,000 IU/L, streptomycin 10 mg/L, and gentamicin 50 mg/L [29 (link),30 (link)]. Oocytes were placed in a 0.2 mL recording chamber and superfused at a constant rate of 5–7 mL/min. The bathing solution consisted of (in mM): NaCl, 95; KCl, 2; CaCl₂, 2; and HEPES 5 (pH 7.5). The cells were impaled at the animal pole with two glass microelectrodes filled with 3 M KCl (1–3 MΩ). The oocytes were routinely voltage clamped at a holding potential of −20 mV using a GeneClamp-500 amplifier (Axon Instruments Inc., Burligame, CA, USA). Current responses were digitized by an A/D converter and analyzed using pClamp 6 (Axon Instruments Inc., Burligame, CA, USA) run on an IBM/PC or directly recorded on a Gould 2400 rectilinear pen recorder (Gould Inc., Cleveland, OH, USA). Current-voltage characteristics were studied using 1 s voltage steps (–120 to 20 mV). All chemicals and drugs including glibenclamide, MB HCl, BAPTA-AM, and 8-Br-cAMP were from Sigma (St. Louis, MO, USA). BAY 58-2667 (dissolved in DMSO) was obtained from Tocris-Bio-Techne (Minneapolis, MN, USA). Drugs were applied externally by addition to the superfusate. Procedures for the injections of BAPTA (50–70 nL, 100 mM) were described earlier in detail [31 (link)].
For inside-out patch experiments, oocytes were chemically defolliculated by collagenase 1A treatment (Sigma, St. Louis, MO, USA; 2 mg/mL, 2 h) and injected with 2 ng cRNA encoding Kir6.2ΔC26 mutant. After 2 days of incubation in modified Barth’s solution, oocytes were placed in hypertonic solution containing 200 mM K⁺-aspartate at pH 7.0, and the vitelline layer was removed with sharpened watchmaker’s forceps. The pipette (external) solution contained (in mM) 140 KCl, 1.2 MgCl₂, 2.6 CaCl₂, and 10 HEPES (pH 7.4). Intracellular (bath) solution contained (in mM) 107 KCl, 10 EGTA, 2 MgCl₂, 1 CaCl₂, and 10 HEPES (pH 7.2 with KOH; final K⁺ ≅140). Patch pipettes had a resistance of 250–400 kΩ when filled with the pipette solution. Currents were recorded at 20–22 °C from giant inside-out patches using Axopatch 200B amplifier (Axon Instruments Inc., Burligame, CA, USA) at a holding potential of 0 mV, sampled at a rate of 2 kHz and filtered at 1 kHz. Currents were evoked by 2 s voltage ramps from −100 mV to +100 mV at a pulse frequency of 0.5 Hz. In each inside-out patch, the efficacy of 1 mM K₂ATP to block the K_ATP current was tested before applying MB. Leak currents recorded at 10 mM K₂ATP at the end of the experiments were subtracted from recordings. The recording chamber had a volume of 250 μL and the perfusion rate was 2 mL/min. Due to light sensitivity of MB, experiments were conducted in the dark and MB containers and perfusion lines kept in the dark by aluminum foils.
Statistical significance at the level of 0.05 was analyzed using the Student’s t-test, paired t-test or ANOVA. Concentration-response curves were obtained by fitting the data to the logistic equation,

where x and y are concentration and response, respectively, E_max is the maximal response, EC₅₀ is the half-maximal concentration, and n is the slope factor. For data analysis, calculations, and fits of the data, the computer software Origin 8.5 (Microcal Software-OriginLab Corp., Northampton, MA, USA) was used.
For radioligand binding experiments, follicle-enclosed oocytes were suspended in 300 mL of buffer containing 50 mM HEPES, 300 mM sucrose and 1 mM EDTA at 4 °C on ice as described earlier [32 (link),33 (link)]. Oocytes were homogenized using a motorized Teflon homogenizer (six strokes, 15 s each at high speed). This was followed by sequential centrifugations at 1000× g (10 min) and 10,000× g (20 min); each time the pellet was discarded and the supernatant was used for the subsequent step. The final centrifugation was at 60,000× g for 25 min. The microsomal pellet, which contains the membranes of follicular cells [34 (link)], was resuspended in 50 mM HEPES buffer and used for the binding studies.
The radioligand binding experiments were carried out at room temperature (20–22 °C) for 1 h. Oocyte membranes were incubated in 1 mL of 50 mM HEPES, pH 7.5, at a protein concentration of 200–500 μg/mL. [³H]glibenclamide was dissolved in ethanol/dimethyl sulphoxide (1:1). For each experiment, freshly made glibenclamide solution was used. For the analysis, calculations, nonlinear curve fitting and regression fits of the radioligand binding data, the computer software Origin 8.5 (Microcal Software-OriginLab Corp., Northampton, MA, USA) was used.