Spontaneous electrical uncoupling in double-current clamped cardiac myocyte pairs

Whole-cell Double-Voltage Clamp is usually employed rather than Double-Current Clamp (DCC) for measuring intercellular electrical resistance (Rj), which is known to rise spontaneously after double-cell attachment in cardiac myocyte pairs. We tested reliability of DCC estimates of Rj in pairs of rat and guinea pig (gp) ventricular myocytes, artificially coupled with a variable external resistance, showing an accuracy >95% for a coupling range of 4<Rj<80 Mohm. Using a mathematical model and experimental results, we showed that, underestimate of Rj for coupling values above this range, is due to membrane resistance non-linearity for hyperpolarized potentials (Vm) close to resting potential (Vr) (Vr-Vm<3 mV) and can be minimized when such non-linearity is measured in single cells and taken into account in the calculation of Rj. A DCC protocol was therefore applied which allowed, at a frequency of 1Hz, to simultaneously sample Rj and sequentially pace real ventricular myocyte pairs. Within the first 7 min after double-patching, paced gp pairs showed faster spontaneous uncoupling than paced rat pairs ( 11.8 +/- 2.1 Mohm/min, n=6 vs 2.0 +/- 0.8 Mohm/min, n=6) and lower initial Rj (18.0 +/- 3.1 Mohm, n=27 vs 32.6 +/- 4.9 Mohm, n=28). Also, not-pacing or buffering intracellular calcium ([Ca2+]i) with 14 mM BAPTA or 14 mM EGTA, decreased the rate of spontaneous uncoupling in gp pairs (0.9 +/- 0.8 Mohm/min, n=4; 0.3 +/- 0.2 Mohm/min, n=4; 2.7 +/- 1.1 Mohm/min, n=3 respectively). We conclude that DCC approach gives accurate estimates of Rj, which spontaneously increases following cell attachment at different rate in gp and rat myocyte pairs, being the rate of uncoupling pacing- and [Ca2+]i-dependent.