Isolation of human atrial myocytes for simultaneous measurements of Ca2+ transients and membrane currents.

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca(2+) handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).(1) Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca(2+) concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca(2+)-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 μM Ca(2+). Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca(2+) concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca(2+) and Ca(2+) buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca(2+) transient measurements, performed in these isolated myocytes.