To assess the first possibility, we performed slicing in cold bicarbonate-buffered blebbistatin-containing (instead of HEPES-buffered BDM-containing) solution.
Endoscopically obtained tissue samples are desirable due to their wide availability and the tissues are probably less physiologically altered from its original conditions because of less surgical stress and shorter time at ambient temperature than in surgical specimens, that are often without perfusion for some time before the tissue is placed in cold bicarbonate-Ringer.
The MMA was removed from the dura mater by a dissecting microscope, cut into segments 1 2 mm long that were placed in parallel tissue baths of ice-cold bicarbonate buffer solution aerated with a gas composed of 95% O2 and 5% CO2, with a resulting pH of 7.4 [23].
Biopsies were transported in ice-cold bicarbonate-Ringer solution to the laboratory.
The heart was then quickly excised and submerged in ice-cold bicarbonate-buffered Krebs–Henseleit solution containing in m m: 119 NaCl, 25 NaHCO3, 4 KCl, 1.2 KH2PO4, 1 MgCl2, 1.8 CaCl2, 10 glucose and 2 sodium pyruvate.
To assess whether transient exposure to BDM and/or HEPES buffer (guinea pig, 3 slices, 2 animals) during slice preparation was a major contributor to the AP shortening and triangular shape seen after cutting, the process was repeated in ice-cold bicarbonate-buffered solution containing blebbistatin (guinea pig, 5 slices, 2 animals).
Hearts were excised and placed in ice-cold bicarbonate-buffered Krebs-Henseleit solution (KH) containing (m m) NaCl 119, NaHCO3 25, KCl 4, KH2PO4 1.2, MgCl2 1, CaCl2 1.8, glucose 10 and Na-pyruvate 2; pH 7.4, 95%O2/5%% CO2 (British Oxygen Company, Manchester, UK), then cannulated and perfused with KH as previously described (Zhang et al. 2010, 2011).
The heart was then carefully but rapidly excised and placed in ice-cold bicarbonate-buffered Krebs-Henseleit solution that contained the following (in mM): 119 NaCl, 25 NaHCO3, 4 KCl, 1.2 KH2PO4, 1 MgCl2, 1.8 CaCl2, 10 glucose, and 2 sodium pyruvate (pH 7.4).
90 μL of sugar substrate was incubated with 10 μL of diluted enzyme, incubated for 30 min and quenched with 50 μL of 2% cold sodium bicarbonate.
The thoracic aorta was carefully removed and placed immediately in cold Krebs bicarbonate buffer (pH 7.4, 95% O2; 5% CO2) of the following composition (mmol·L−1): NaCl (119), KCl (4.7), CaCl2 (2.5), KH2PO4 (1.18), MgSO4 (1.19), NaHCO3 (25.1), glucose (11).
Hearts were rapidly excised, placed in cold (4°C) bicarbonate buffer (10), cannulated, and perfused at a constant flow of 12 ml·min−10g tissue−1 (10) as follows: 30 min of aerobic perfusion with bicarbonate buffer followed by 5 min of perfusion with 0 10,000 μM hydrogen peroxide in bicarbonate buffer.
