Taxol, a microtubule stabilizer, prevents ischemic ventricular arrhythmias in rats

Abstract

Microtubule integrity is important in cardio-protection, and microtubule disruption has been implicated in the response to ischemia in cardiac myocytes. However, the effects of Taxol, a common microtubule stabilizer, are still unknown in ischemic ventricular arrhythmias. The arrhythmia model was established in isolated rat hearts by regional ischemia, and myocardial infarction model by ischemia/reperfusion. Microtubule structure was immunohistochemically measured. The potential mechanisms were studied by measuring reactive oxygen species (ROS), activities of oxidative enzymes, intracellular calcium concentration ([Ca(2+) ](i) ) and Ca(2+) transients by using fluorometric determination, spectrophotometric assays and Fura-2-AM and Fluo-3-AM, respectively. The expression and activity of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) was also examined using real-time polymerase chain reaction, Western blot and pyruvate/Nicotinamide adenine dinucleotide-coupled reaction. Our data showed that Taxol (0.1, 0.3 and 1 μM) effectively reduced the number of ventricular premature beats and the incidence and duration of ventricular tachycardia. The infarct size was also significantly reduced by Taxol (1 μM). At the same time, Taxol preserved the microtubule structure, increased the activity of mitochondrial electron transport chain complexes I and III, reduced ROS levels, decreased the rise in [Ca(2+)](i) and preserved the amplitude and decay times of Ca(2+) transients during ischemia. In addition, SERCA2a activity was preserved by Taxol during ischemia. In summary, Taxol prevents ischemic ventricular arrhythmias likely through ameliorating abnormal calcium homeostasis and decreasing the level of ROS. This study presents evidence that Taxol may be a potential novel therapy for ischemic ventricular arrhythmias.

fig 1

Pharmacological intervention protocols and TTC staining pictures. (A) The pharmacological intervention protocol of the ischemic ventricular arrhythmia study. Norm, normoxic; Isch, ischemia. (B) TTC staining pictures. (C) The pharmacological intervention protocol of the myocardial infarction study. Isch, ischemia.

fig 2

Taxol preserves the microtubule structure in ischemia. (A) Representative pictures of microtubule structure in the control group. Double labelling of the myocardium with mouse monoclonal antibodies against β-tubulin (green) and rhodamine-phalloidin (red) (×400). Bar, 40 μm. (B) Representative pictures of microtubule structure in the ischemia group. Double labelling of the myocardium with mouse monoclonal antibodies against β-tubulin (green) and rhodamine-phalloidin (red) (×400). Bar, 40 μm. (C) Representative pictures of the microtubule structure in the Taxol (0.1 μM) group. Double labelling of the myocardium with mouse monoclonal antibodies against β-tubulin (green) and rhodamine-phalloidin (red) (×400). Bar, 40 μm.

fig 3

Taxol decreases infarct size. (A) Representative pictures of the infarct size. Myocardial TTC staining was performed immediately after the ischemia/reperfusion (I/R) protocol. 2.5% Evans blue was perfused to delineate the area of risk. Heart slices were incubated with 1% TTC for 15 min. at 37°C to visualize the unstained infarcted region (white), and then digitally photographed. The infarcted region was weighed, and the infarct size was expressed as a percentage of the risk zone. Arrow, white infracted region. (B) Taxol (1 μM) reduces the infarct size. Values represent the mean ± S.E. (n = 8 per group). *P < 0.05 versus I/R group.

fig 4

Taxol decreases the rise in [Ca²⁺]_i and preserves the amplitude and decay time of Ca²⁺ transients in ischemia. (A) [Ca²⁺]_i changes over time. Values represent the mean ± S.E. (n = 50 per group). *P < 0.05 versus control group, ^□P < 0.05 versus control group and ischemia group. (B) Typical pictures of Ca²⁺ transients at the end of the experiment. (C) The amplitude of the Ca²⁺ transients changes over time. Values represent the mean ± S.E. (n = 50 per group). *P < 0.05 versus control group, ^□P < 0.05 versus control group and ischemia group. (D) The decay time of the Ca²⁺ transients changes over time. Values represent the mean ± S.E. (n = 50 per group). *P < 0.05 versus control group, ^□P < 0.05 versus control group and ischemia group.

fig 5

Taxol does not affect the expression of SERCA2a but preserves its activities in ischemia. (A) SERCA2a mRNA expression. Values represent the mean ± S.E. (n = 5 per group). (B) Representative pictures of SERCA2a protein expression. (C) SERCA2a protein expression. Values represent the mean ± S.E. (n = 5 per group). SERCA2a expression, from left to right: control group, ischemia group, Taxol (1 μM) group, Taxol (0.3 μM) group and Taxol (0.1 μM) group. GAPDH expression, from left to right: control group, ischemia group, Taxol (1 μM) group, Taxol (0.3 μM) group and Taxol (0.1 μM) group. (D) SERCA2a activities. Values represent the mean ± S.E. (n = 5 per group). *P < 0.05 versus control group; ^□P < 0.05 versus control group and ischemia group.

fig 6

Taxol reduces the levels of ROS during ischemia. (A) Representative qualitative assessment pictures of ROS (×400). Bar, 40 μm. (B) The level of ROS. Values represent the mean ± S.E. (n = 15 per group). *P < 0.05 versus control group; ^□P < 0.05 versus control group and ischemia group; ⁺P < 0.05 versus control group, ischemia group and Taxol (0.1 and 0.3 μM) groups.