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 2ϩ ] i transient amplitude and depressed the response of these parameters to the ␤-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 mol/L colchicine had no effect on shortening or [Ca 2ϩ ] i transient amplitude or on the amplitude of I Ca,L in perforated patch-clamped cells, under basal conditions and after stimulation with 1 mol/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 colchicinetreated but not vehicle-treated myocytes. We conclude that in cells with intact sarcolemma, colchicine does not modulate Ca 2ϩ signaling or the response to ␤ stimulation. However, the combination of microtubule disruption by colchicine and the ruptured patch configuration activates I Ca,L and attenuates the response to ␤ 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.

Constitutive properties of hypertrophied myocardium: cellular contribution to changes in myocardial stiffness

“…However, for this mechanism, which is dependent on an increase in colchicine induced ␤-adrenergic G protein signaling, to have an effect on myocardial material properties, it must lower resting, quiescent, diastolic calcium concentration and increase the rate of calcium sequestration. In previous studies, we and other investigators (6,52) have been unable to demonstrate that colchicine alters resting, quiescent, diastolic calcium concentration or the rate of calcium resequestration. Whether an increase in ␤-adrenergic G protein signaling itself alters resting, quiescent, diastolic calcium concentration in other experimental conditions is less clear.…”

“…Recent studies have raised but not definitively answered the question of whether free, nonpolymerized ␤-tubulin may act as a ␤-adrenergic agonist increasing calcium, cAMP, and protein kinase A via the ␤-adrenergic G protein signaling cascade (6,23). These data have potentially important implications for the current study.…”

Role of microtubules versus myosin heavy chain isoforms in contractile dysfunction of hypertrophied murine cardiocytes

“…However, other data in that same study show that taxol, which markedly reduces the cardiac ␣␤-tubulin heterodimer concentration (40), is without effect on these same calcium variables, and we found (35) that there is with hypertrophy, along with an increase in microtubules, a significant and persistent increase in free ␣␤-tubulin heterodimer concentration; however, in contrast to what this mechanism would predict if it has functional significance, there is a marked decrement rather than increment in contractile function. Furthermore, it has since been shown by others in intact neonatal (23) and adult (2) cardiocytes that colchicine and tubulin dimers are without effect either on Ca 2ϩ signaling or on con- For the immunoblots, the free ␣␤-tubulin heterodimer fraction (lanes 1) was separated from the insoluble tubulin fraction (lanes 2) (27,35). For vincristine-treated cells, the insoluble fraction presumably consists largely of tubulin paracrystals rather than microtubules (9).…”

Muscle-specific RING finger-2 (MURF-2) is important for microtubule, intermediate filament and sarcomeric M-line maintenance in striated muscle development

“…For example, perturbation of microtubule dynamics with depolymerizing or stabilizing agents inhibits skeletal myoblast fusion, differentiation, and normal myofibrillogenesis (Antin et al, 1981;Toyama et al, 1982;Saitoh et al, 1988). In the heart, disruption of microtubules alters cytoplasmic viscosity, myosin mRNA transport, Ca 2+ signaling, and gene expression (Perhonen et al, 1998;Takahashi et al, 1998;Gomez et al, 2000;Calaghan et al, 2001;Kerfant et al, 2001). Furthermore, microtubules are required for proper contractile function and their levels increase in animal models of pressure-overload cardiac hypertrophy (Klein, 1983;Tsutsui et al, 1993).…”