Microtubule disruption by colchicine reversibly enhances calcium signaling in 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 [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 micromol/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.
Kerfant
B G
BG
Physiopathologie Cardiovasculaire, INSERM U-390, Montpellier, France.
Vassort
G
G
Gómez
A M
AM
D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D002478:Cells, Cultured; D003078:Colchicine / Q000494:pharmacology*; D007424:Intracellular Fluid / Q000378:metabolism; D008297:Male; D008870:Microtubules / Q000187:drug effects* / Q000378:metabolism; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D020127:Recovery of Function / Q000502:physiology; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism
D019837:Ryanodine Receptor Calcium Release Channel; D003078:Colchicine; D002118:Calcium