A Method Sustaining the Bioelectric, Biophysical, and Bioenergetic Function of Cultured Rabbit Atrial Cells

Abstract

Culturing atrial cells leads to a loss in their ability to be externally paced at physiological rates and to maintain their shape. We aim to develop a culture method that sustains the shape of atrial cells along with their biophysical and bioenergetic properties in response to physiological pacing. We hypothesize that adding 2,3-Butanedione 2-monoxime (BDM), which inhibits contraction during the culture period, will preserve these biophysical and bioenergetic properties. Rabbit atrial cells were maintained in culture for 24 h in a medium enriched with a myofilament contraction inhibitor, BDM. The morphology and volume of the cells, including their ability to contract in response to 1–3 Hz electrical pacing, was maintained at the same level as fresh cells. Importantly, the cells could be successfully infected with a GFP adenovirus. Action potentials, Ca2+ transients, and local Ca2+ spark parameters were similar in the cultured and in fresh cells. Finally, these cultured cells' flavoprotein autofluorescence was maintained at a constant level in response to electrical pacing, a response similar to that of fresh cells. Thus, eliminating contraction during the culture period preserves the bioelectric, biophysical and bioenergetic properties of rabbit atrial myocytes. This method therefore has the potential to further improve our understanding of energetic and biochemical regulation in the atria.

Eliminating contraction during culture maintains global and local Ca2+ dynamics in cultured rabbit pacemaker cells

“…The cells were diluted in one of 4 serum-free culture media (summarized in Table 1) and then centrifuged at 500 RPM for 5 min (Spectrafuge 6C, Labnet): (i) a pure culture medium mixed with BDM: M199 (Sigma Aldrich), 2% PS (penicillin-streptomycin (Gibco)), 1% ITS (insulin-transferrin-selenium, Sigma Aldrich), 0.1% BDM (2, 3 butanedione, Sigma Aldrich), and 0.01% BSA (Sigma Aldrich) similar to [17]; (ii) a mixed culture medium with salts (containing (in mmol/L): 116 NaCl, 5.4 KCl, 0.8 MgCl 2 , 0.9 NaH 2 PO 4 , 5.6 Glucose, 20 HEPES, 1.8 CaCl 2 , 26 NaHCO 3 ), 20% M199 (Sigma, in the presence of (in mmol/L): 5 creatine, 2 L-carnitine, 5 taurine, 0.1% ITS, and 1% penicillin and streptomycin and titrated to pH 7.4 with NaOH at 37 °C); (iii) a mixed culture medium with salts and BDM (containing (in mmol/L): 100 NaCl, 5.4 KCl, 0.8 MgCl 2 , 0.9 NaH 2 PO 4 , 5.6 Glucose, 20 HEPES, 1.8 CaCl 2 , 26 NaHCO 3 ), 20% M199 (Sigma, in the presence of (in mmol/L): 5 creatine, 2 L-carnitine, 5 taurine, 0.1% ITS, and 1% penicillin and streptomycin and titrated to pH 7.4 with NaOH at 37 °C) and 10 mmol/L BDM; (iv) a mixed culture medium with salts and blebbistatin (containing (in mmol/L): 116 NaCl, 5.4 KCl, 0.8 MgCl 2 , 0.9 NaH 2 PO 4 , 5.6 glucose, 20 HEPES, 1.8 CaCl 2 , 26 NaHCO 3 , 20% M199 (Sigma, in the presence of (in mmol/L): 5 creatine, 2 L-carnitine, 5 taurine, 0.1% ITS, and 1% penicillin and streptomycin and titrated to pH 7.4 with NaOH at 37 °C) and 25 μmol/L blebbistatin. The conical tube containing the cells was drained, leaving the cells at the bottom of the tube.…”

“…For full method details, see [17]. In short, to quantify pacemaker cell area, the cells were imaged on an inverted fluorescence microscope (Zeiss Observer Z1, Germany) using a 40×/1.4N.A oil immersion lens.…”

“…Ca 2+ kinetics were measured with Fluo-4 AM (ThermoFisher Scientific) at 37 ± 0.5 °C as described before [16,17]. t 50 and t 90 are defined from the minimal signal to the point of 50% or 90% decay of the signal [18].…”

“…In short, cells were excited with a 488 nm Argon laser line, fluorescence emission was collected with LP 505 nm, and all images were captured in frame mode (4096 × 4096 pixels at 102.1 × 102.1 μm). For analysis see [17,19]. …”

“…Mitochondrial membrane potential was visualized using the potential-sensitive dye TMRM (1:80 (125 nM), tetramethyl rhodamine methyl ester perchlorate, ThermoFisher Scientific) as described before [17]. All images were captured in frame mode (512 × 512 pixels at 105.75 × 105.75 μm).…”