Modulation of Ca 2+ Signaling by Microtubule Disruption in Rat Ventricular Myocytes and Its Dependence on the Ruptured Patch-Clamp Configuration

Abstract

Abstract —In the absence of hypertrophic proliferation of microtubules, microtubule disruption by colchicine does not modulate contraction of adult cardiac myocytes. However, Gomez et al ( Circ Res. 2000;86:30–36) recently reported that disruption of microtubules by colchicine in ruptured patch-clamped myocytes increased I Ca,L density and [Ca 2+ ] i transient amplitude and depressed the response of these parameters to the β-adrenoceptor agonist isoproterenol. These effects were ascribed to stimulation of adenylyl cyclase by increased intracellular free tubulin. In the present study, we show that in intact rat ventricular myocytes, 2 to 4 hours of exposure to 10 μmol/L colchicine had no effect on shortening or [Ca 2+ ] i transient amplitude or on the amplitude of I Ca,L in perforated patch-clamped cells, under basal conditions and after stimulation with 1 μmol/L isoproterenol. However, in ruptured patch-clamped myocytes, basal I Ca,L was 2-fold higher after treatment with colchicine compared with vehicle and, in contrast to vehicle-treated cells, I Ca,L did not increase in response to isoproterenol. Cell width decreased during ruptured patch-clamp experiments in colchicine-treated but not vehicle-treated myocytes. We conclude that in cells with intact sarcolemma, colchicine does not modulate Ca 2+ signaling or the response to β stimulation. However, the combination of microtubule disruption by colchicine and the ruptured patch configuration activates I Ca,L and attenuates the response to β stimulation. We propose that these effects may be due to loss of free tubulin by intracellular dialysis or to increased sensitivity to mechanical stimulation as a result of microtubule disruption. These findings have important implications for cardiomyopathies associated with decreased free tubulin or a diminished microtubular network. The full text of this article is available at http://www.circresaha.org.