Calcium Release from the Sarcoplasmic Reticulum Activated by the Low Affinity Calcium Chelator TPEN in Ventricular Myocytes.

C. Jung 1, A. Zima2, L. A. Blatter2, E. Niggli1.

In cardiac muscle, upon membrane depolarization, Ca entry through L-type Ca channels triggers Ca-induced Ca release from the sarcoplasmic reticulum (SR). Ca is released from the SR through the ryanodine receptors (RyRs) that open and close in response to changes in Ca concentrations within the cytosol but may be regulated by the Ca content of the SR. Experimentally altering the amount of Ca within the SR may help to improve our understanding of the mechanism(s), such as SR Ca depletion, which may terminate SR Ca release. TPEN is a membrane-permeant chelator that binds heavy metal ions with high affinity and Ca ions with low affinity, and thus appeared to be a suitable tool for increasing intra SR Ca buffer capacity and Ca content. Using laser-scanning confocal microscopy and whole-cell patch clamp recordings, we examined SR Ca release in isolated ventricular myocytes loaded with fluo-3-AM. When applying TPEN onto myocytes, we observed that local spontaneous Ca releases from the SR immediately increased in frequency developing into Ca waves spreading across the cell. This unexpected observation could either reflect an unknown direct action of TPEN on the Ca sensitivity of the RyRs or could result from an immediate increase in SR Ca concentration. The latter is unlikely as TPEN rapidly diffuses into the SR where it chelates Ca, and leading to a decrease of SR Ca concentration, at least transiently. If TPEN were indeed to increase Ca release from the SR by changing the open probability of the RyRs, SR Ca content should be reduced after longer applications of TPEN, as a result of Ca leaking out through the more frequently opened channels. We found that the amount of Ca in the SR after TPEN application was only 65% of control and that caffeine-induced Ca transients were reduced by 35%, suggesting an effect of TPEN on the RyRs open probability. This conclusion was confirmed by our findings in purified RyRs reconstituted in lipid bilayers showing a 2-fold increase in the open probability mainly resulting from longer mean open times of the channels. We conclude that TPEN directly activates the RyRs leading to enhanced diastolic Ca leak from the SR and Ca depletion of the SR.