Microtubule disruption by colchicine reversibly enhances calcium signalingin intact rat cardiac myocytes

Using the whole-cell patch-clamp configuration in rat ventricular myocytes, we recently reported that microtubule disruption increases calcium current (I-Ca) and [Ca2+](i) transient and accelerates their kinetics by adenylyl cyclase activation. In the present report, we further analyzed the effects of microtubule disruption by 1 mu mol/L colchicine on Ca2+ signaling in car diac myocytes with intact sarcolemma. In quiescent intact cells, it is poss ible to investigate ryanodine receptor (RyR) activity by analyzing the char acteristics of spontaneous Ca2+ sparks. Colchicine treatment decreased Ca2 spark amplitude (F/F-0: 1.78 +/-0.01, n=983, versus 1.64 +/-0.01, n=1660, recorded in control versus colchicine-treated cells; P <0.0001) without mod ifying the sarcoplasmic reticulum Ca2+ load and enhanced their time to peak (in ms: 6.85 +/-0.09, n= 1185, versus 7.33 +/-0.13, n= 1647; P <0.0001). M icrotubule disruption also induced the appearance of Ca2+ sparks in doublet s. These alterations may reflect RyR phosphorylation. To further investigat e Ca2+ signaling in cardiac myocytes with intact sarcolemma, we analyzed [C a2+](i) transient evoked by field stimulation. Cells were loaded with the f luorescence Ca2+ indicator, Fluo-3 cell permeant, and stimulated at 1 Hz. [ Ca2+](i) transient amplitude was greater and its decay was accelerated in c olchicine-treated, field-stimulated myocytes. This effect is reversible. Wh en colchicine-treated myocytes were placed in a colchicine-free solution fo r 30 minutes, tubulin was repolymerized into microtubules, as shown by immu nofluorescence, and the increase in [Ca2+](i) transient was reversed. In su mmary, we demonstrate that microtubule disruption by colchicine reversibly modulates Ca2+ signaling in cardiac cells with intact sarcolemma. The full text of this article is available at http://www.circresaha.org.