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(Circulation Research. 2001;88:e59.)
© 2001 American Heart Association, Inc.

Response to Research Commentary

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

B. G. Kerfant, G. Vassort, A. M. Gómez

From the Physiopathologie Cardiovasculaire, INSERM U-390, Montpellier, France.

Correspondence to Ana M. Gómez, INSERM U-390, CHU Arnaud de Villeneuve, 34295 Montpellier, France. E-mail agomez{at}montp.inserm.fr

Abstract—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 µmol/L colchicine on Ca²⁺ 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 Ca²⁺ sparks. Colchicine treatment decreased Ca²⁺ spark amplitude (F/F₀: 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 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). Microtubule disruption also induced the appearance of Ca²⁺ sparks in doublets. These alterations may reflect RyR phosphorylation. To further investigate Ca²⁺ signaling in cardiac myocytes with intact sarcolemma, we analyzed [Ca²⁺]_i transient evoked by field stimulation. Cells were loaded with the fluorescence Ca²⁺ indicator, Fluo-3 cell permeant, and stimulated at 1 Hz. [Ca²⁺]_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 [Ca²⁺]_i transient was reversed. In summary, 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.

Key Words: microtubules • calcium transient • calcium sparks • adenylyl cyclase

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