Citrate decreases contraction and Ca current in cardiac muscle independent of its buffering action.

Extracellular Ca (Cao) depletions that occur during cardiac muscle contractions are indicative of net Ca entry. Buffering Cao concentration ([Ca]o) with citrate can limit the magnitude of these Cao depletions [e.g., Shattock and Bers. Am. J. Physiol. 256 (Cell Physiol. 25): C813-C822, 1989] which theoretically would allow more Ca entry and consequently greater force at the same free [Ca]o. However, Shimoni and Ginsburg [Am. J. Physiol. 252 (Cell Physiol. 21): C248-C252, 1987] have shown that citrate inhibits cardiac contractions and suggested that this was due to its Ca-buffering action (i.e., dissipating a local elevation of [Ca] at the outer sarcolemmal surface and thereby decreasing Ca influx). To examine the effects of Ca buffering per se, we compared the effects of four low-affinity Ca buffers [citrate, nitrilotriacetic acid (NTA), dipicolinic acid (DPA), and acetamidoiminodiacetic acid (ADA)] on several cardiac preparations. In Mg-free medium with 2 mM free Ca (measured using murexide), citrate, DPA, and ADA (10 mM) decreased the force of twitch contractions in rabbit ventricle to 76 +/- 2, 60 +/- 2, and 85 +/- 2%, respectively, but 10 mM NTA increased force slightly to 105 +/- 2%. No simple correlation was observed between the Ca affinity of the buffer and its effect on tension. These effects were not due to changes in sarcoplasmic reticulum (SR) Ca loading because rapid cooling contractures were not affected and similar results were observed in the presence of caffeine or ryanodine. The depressant effects of citrate and ADA on tension were greater at pH 5.5-6 and ADA had no effect at pH 8.5. Thus the depressant effect is stronger with more protonated forms of citrate and ADA, which are also poorer Ca buffers. Citrate (but not NTA) decreased Ca current in whole cell voltage clamp and shifted the current-voltage relationship and reversal potential to more negative potentials. Citrate decreased Ca current more effectively at higher citrate and lower Ca concentrations. We conclude that citrate (and some other weak Ca buffers) may directly decrease Ca current and contraction in a manner independent of Ca buffering ability.