Cytochalasin D reduces Ca2+ currents via cofilin-activated depolymerization of F-actin in guinea-pig cardiomyocytes

1. L-type Ca2+ channel currents (I(Ca)) were measured in guinea-pig ventricular myocytes (22 degrees C, 300 ms steps from -45 to +10 mV). Pulsing at 0.5 Hz reduced I(Ca) within 5 min to 92 +/- 3% (mean +/- S.E.M., n = 14) and within 10 min to 83 +/- 4 % ('run-down' with reference to I(Ca) after a 5 min equilibration period). 2. Bath-applied cytochalasin D (cytD, 10 microM) reduced I(Ca) to 75 +/- 4% within 5 min and to 61 +/- 4% within 10 min ('cytD reduction of I(Ca)') by reduction of maximal Ca2+ conductance (suggested by fits of time course and of current-potential (I-V) curves). 3. Preincubation with phalloidin (bath applied, 100 microM, 5 h) prevented the cytD reduction of I(Ca). Since phalloidin specifically blocks F-actin depolymerization, cytD reduction of I(Ca) is linked to depolymerization of F-actin. 4. CytD did not attenuate the beta-adrenergic stimulation of I(Ca) (30 nM isoproterenol), suggesting that A kinase anchoring proteins are unlikely to mediate the cytD reduction of I(Ca). The cytD reduction of I(Ca) was abolished by extra-/intracellular acidosis (pH(o) 6.9), by cell dialysis of 5 mM BAPTA, or by serine/threonine protein phosphatase inhibitors. 5. Actin-depolymerizing factor (ADF)/cofilin are proteins that bind to actin, mediate a pH-sensitive depolymerization of F-actin, and are activated by dephosphorylation. Western blots from hearts perfused with solutions containing zero or 10 microM cytD indicated that cytD reduces the ratio of phosphorylated to total ADF/cofilin content by 50%. 6. The data support the concept that cytD mediates dephosphorylation and activation of ADF/cofilin, leading to depolymerization of F-actin with a subsequent reduction of I(Ca).