Steady-state twitch Ca²⁺ fluxes and cytosolic Ca²⁺ buffering in rabbit ventricular myocytes

Intracellular Ca²⁺ ([Ca²⁺]_i) transients and transsarcolemmal Ca²⁺ currents were measured in indo 1-loaded isolated rabbit ventricular myocytes during whole cell voltage clamp to quantitate the components of cytosolic Ca²⁺ influx and to describe the dynamic aspects of cytosolic Ca²⁺ buffering during steady-state contraction (0.5 Hz, 22°C). Sarcolemmal Ca²⁺ influx was directly measured from the integrated Ca²⁺ current (I_Ca) recorded during the clamp (158 ± 10 attomoles; amol). Sarcoplasmic reticulum (SR) Ca²⁺ content was determined from the integrated electrogenic Na⁺/Ca²⁺ exchange current (I_X) induced during rapid application and sustained exposure of cells to caffeine to elicit the release of the SR Ca²⁺ load (1,208 ± 170 amol). The mean steady-state SR Ca²⁺ load was calculated to be 87 ± 13 μM (μmol/l nonmitochondrial cytosolic volume). Ca²⁺ influx via I_Ca represented ∼ 14% of the stored SR Ca²⁺ and 23% of the total cytosolic Ca²⁺ flux during a twitch (47 ±6 μM). Comparison of electrophysiologically measured Ca²⁺ fluxes with Ca²⁺ transients yields apparent buffering values of 60 for caffeine contractures and 110 for twitches (ΔCa²⁺ total/ΔCa²⁺ free). This is consistent with the occurrence of "active" buffering of cytosolic Ca²⁺ by SR Ca²⁺ uptake during the twitch.