Collagenase a

The hearts of all experimental animals, following being anesthetized by sodium pentobarbital (30 mg/kg, i.p.), were perfused retrogradely and performed standard enzymatic digestion, as described elsewhere [59 (link)]. Briefly, the cannulated hearts through the coronary arteries on a Langendorff apparatus were perfused (3–5 min) with a Ca²⁺-free solution (in mM): 145 NaCl, 5 KCl, 1.2 MgSO₄, 1.4 Na₂HPO₄, 0.4 NaH₂PO₄, 5 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), and 10 glucose at pH 7.4, bubbled with O₂ at 37 °C and then followed the perfusion with a solution containing 1 mg/mL collagenase (Collagenase A, Boehringer) for 30–35 min. Left ventricular cardiomyocytes were isolated following the digestion with and the only the cells with rod-shaped, quiescent, and responses electric-field stimulation were used for all experiments The percentage of viable cells was 70–80% in every heart following increases of Ca²⁺ in the medium to a concentration of 1-mM.
To examine the role of a mitochondrial-targeting antioxidant on aged-cardiomyocytes, half of isolated cardiomyocytes from the 24-month old group was treated with MitoTEMPO (1-μM in the medium that cells were incubated) for 5-h before the experiments.

All work involving the use of animals was performed in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Xenopus laevis frogs (NASCO) were anesthetized with 0.17% (w/v) 3-aminobenzoic acid ethyl ester and had an ovarian lobe removed via an incision in the abdomen. Stage V oocytes were isolated from the lobe via digestion with 2 mg ml⁻¹ collagenase A (Boehringer) at 26 °C for 1 h, and 20 ng of cRNA encoding GlyT2 was injected into each oocyte cytoplasm (Drummond Nanoinject; Drummond Scientific Co). The oocytes were then stored in frog Ringer's solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 5 mM Hepes, pH 7.5), which was supplemented with 2.5 mM sodium pyruvate, 0.5 mM theophylline, 50 μg/ml gentamicin and 100 μM ml⁻¹ tetracycline. The oocytes were stored at 18 °C for 3 to 5 days, until transporter expression was adequate for measurement using the two-electrode voltage clamp technique. Adequate transporter expression was defined as a ≥30 nA current following application of the glycine EC₅₀.

Oocytes were extracted from female Xenopus laevis frogs and detached from follicle cell containing lobes by digestion with 2 mg/mL collagenase A (Boehringer, Mannheim, Germany). Defoliculated stage V-VI oocytes were injected with 4.6 ng of cRNA encoding WT or mutant transporter (Drummond Nanoinject, Drummond Scientific Co., Broomall, PA, USA). Surgical proceedures have been approved by the University of Sydney Animal Ethics Committee (protocol 2016/970). The oocytes were stored at 16–18°C for 2–5 days in ND96 solution (96 mM NaCl, 2 mM KCL, 1 mM MgCl₂, 1.8 mM CaCl₂, 5 mM HEPES, pH 7.55), supplemented with 2.5 mM sodium pyruvate, 0.5 mM theophylline, 50 µg/mL gentamicin and 100 µM/mL tetracycline.
2–5 days following injection, glycine transport currents were measured at −60 mV using Geneclamp 500 amplifier (Axon Instruments, Foster City, CA, USA) with a Powerlab 2/20 chart recorder (ADInstruments, Sydney, Australia) and chart software (ADInstruments). All data were subsequently analysed using GraphPad Prism 7.02 (GraphPad Software, San Diego, CA).