Microtubule Disruption by Colchicine Reversibly Enhances Calcium Signaling in Intact Rat Cardiac Myocytes

Abstract

Abstract —Using the whole-cell patch-clamp configuration in rat ventricular myocytes, we recently reported that microtubule disruption increases calcium current ( I Ca ) and [Ca 2+ ] i transient and accelerates their kinetics by adenylyl cyclase activation. In the present report, we further analyzed the effects of microtubule disruption by 1 μmol/L colchicine on Ca 2+ signaling in cardiac myocytes with intact sarcolemma. In quiescent intact cells, it is possible to investigate ryanodine receptor (RyR) activity by analyzing the characteristics of spontaneous Ca 2+ sparks. Colchicine treatment decreased Ca 2+ 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 modifying the sarcoplasmic reticulum Ca 2+ 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). Microtubule disruption also induced the appearance of Ca 2+ sparks in doublets. These alterations may reflect RyR phosphorylation. To further investigate Ca 2+ signaling in cardiac myocytes with intact sarcolemma, we analyzed [Ca 2+ ] i transient evoked by field stimulation. Cells were loaded with the fluorescence Ca 2+ indicator, Fluo-3 cell permeant, and stimulated at 1 Hz. [Ca 2+ ] i transient amplitude was greater and its decay was accelerated in colchicine-treated, field-stimulated myocytes. This effect is reversible. When colchicine-treated myocytes were placed in a colchicine-free solution for 30 minutes, tubulin was repolymerized into microtubules, as shown by immunofluorescence, and the increase in [Ca 2+ ] i transient was reversed. In summary, we demonstrate that microtubule disruption by colchicine reversibly modulates Ca 2+ signaling in cardiac cells with intact sarcolemma. The full text of this article is available at http://www.circresaha.org.