CARDIOVASCULAR JOURNAL OF AFRICA • Volume 34, No 2, May/June 2023 AFRICA 75 Therefore, the current study assessed the role of NFκB and associated respiratory changes in TNF-induced cytoprotection. Methods C2C12 myoblasts (European Collection of Cell Cultures, Centre for Applied Microbiology and Research, UK) were stored in cryovials containing 1 × 106 cells/ml in liquid nitrogen at –196°C. When required, a cryovial was thawed for 30–40 seconds in a 37°C water bath. The contents were sterilely transferred to a 75-cm3 tissue culture flask containing Dulbecco’s modified eagle serum (DMEM) with 4.5 g/l glucose, 0.110 g/l sodium pyruvate and L-glutamine supplemented with 10% foetal calf serum (FCS) and 1% (w/v) penicillin/ streptomycin (PIS) (all purchased from Highveld Biological, RSA). The myoblasts were grown in 5% CO2 trypsinised with 0.25% (w/v) trypsin (Highveld Biological, RSA) supplemented with 0.2% EDTA (w/v) (Sigma, Germany), made up in phosphatebuffered saline (PBS). The trypsinisation process was allowed for three minutes at 37°C before it was neutralised with twice the volume of 10% FCS DMEM. The cells, now in suspension, were transferred to 50 ml polypropylene conical tubes and centrifuged for five minutes at 1 000 rpm. Cells were counted in a Neubauer haemocytometer before centrifugation. Tissue culture flasks (25 cm3) were seeded with enough cells to allow 80% confluency in two to three days. At this point, differentiation was initiated with DMEM supplemented with 1% horse serum (HS) (Sigma, Germany) and 1% PIS. Differentiation was maintained with fresh 1% HS DMEM every two days until day eight. Experiments were performed using differentiated myotubes between days eight and 10. Experimental protocol During physiological ischaemia, the pH becomes mildly acidic, potassium concentration increases and metabolic activity is perturbed. Myocardial ischaemia was simulated in the C2C12 study model under these conditions. The protocol was adapted from Esumi et al.27 The simulated ischaemia (SI) buffer [137 mM NaCl, 12 mM KCI, 0.5 mMMgCI, 0.9 mM CaCl2, 20 mM Hepes and 20 mM 2-deoxy-d-glucose (2-DG)] was adjusted to a pH of 6.4. The inclusion of 2-DG served to inhibit glycolysis and thereby disrupt metabolic activity. To simulate ischaemia, low oxygen consumption was also required. Hence, a multigas incubator (Sanyo Electric Co, Ltd, Japan) supplying 5% CO2, 94% N2 and 1% O2 was employed to house the 25-cm3 tissue culture flasks in a hypoxic environment. The preconditioning protocol involved the following groups, which are schematically represented in Fig. 1. Simulated ischaemia Simulated ischaemia Simulated ischaemia Simulated ischaemia Simulated ischaemia Simulated ischaemia Normoxic control Fig. 1. Schematic representation of the protocol. PDTC = ammonium pyrrolidone derivative dithiocarbamate, TNF = tumour necrosis factor, SI = simulated ischaemia on the graph.
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