Molecular And Cellular Mechanism On Aconitine-induced Arrhythmic Toxicity

Introduction:Aconite tuber, roots of aconite is an herbal medicine traditionally used in China and other countries to therapeutically increase the peripheral temperature, relieve rheumatic pain, treat neurological disorders, and improve the cardiovascular function. However, life-threatening toxicity would occur after the consumption of crude aconite roots and has been found to be due to aconitum alkaloids. Aconitine and its structurally related analogs are known to affect heart arrhythmia. Its heart toxicity consists mainly of arrhythmogenic effects including the induction of premature ventricular contractions, ventricular tachycardia, torsades de pointes, ventricular fibrillation, and mortality in a dose-dependent manner. Although several factors have been implicated to explain aconitine-induced toxicity, alterations of the intracellular Ca²⁺ handling in the exciatation-contraction coupling (E-C coupling) system in aconitine-induced cardiomyocytes are presently unknown.To investigate the molecular mechanism of aconitine on the heart arrhythmia, we focused on Ca²⁺ signal handling of the cardiac E-C coupling in primary cultured normal cardiomyocytes and gene knockdown cardiomyocytes with aconitine exposure.Methods and Results:1. Established the primary cultured cardiomyocytes system and the purity of the cardiomyocytes was investigated by immunofluorescence with primary antibodies of ?-actin, ?-MHC and cTnT, and FITC-IgG secondary antibody. Results showed that purity of the cardiomyocytes was over 95%.2. Investigated alterations of the Ca²⁺ handling proteins in cardiac E-C coupling in aconitine-induced cardiomyocytes by Western-Blot and RT-PCR including L-type Ca²⁺ channel (DHPR), sarcoplasmic reticulum (SR) Ca²⁺ release channel type 2 ryanodine receptor (RyR₂), sarcolemmal Na⁺/Ca²⁺ exchanger (NCX), sarco (endo) plasmic reticulum Ca²⁺-ATPase (SERCA), phospholamban (PLB) and calsequestrin (CASQ). We found that aconitine significantly increased mRNA transcription levels and protein expressions of RyR₂ gene and NCX gene.3. Investigated alterations of the intracellular Ca²⁺ signals, including the amplitude and frequence of spontaneous Ca²⁺ oscillations and the relative intracellular Ca²⁺ concentration ([Ca²⁺]i) in aconitine-induced cardiomyocytes; and effects of the Ca²⁺ channel blocking reagents on aconitine-induced alterations of the intracellular Ca²⁺ signals, such as the inhibitor of SR Ca²⁺ release channel-RyR, ruthenium red (RR), the inhibitor of SR Ca²⁺-ATPase, thapsigargin (TG) and the inhibitor of L-type Ca²⁺ channel, verapamil (VP) by Ca²⁺ imaging experiments. Again, results found that RR could reverse aconitine-induced alterations of the intracellular Ca²⁺ signals.4. Established RyR₂ gene knockdown cardiomyocyte model by siRNAs through the reverse transfection of the siPORTTM NeoFXTM lipid-based reagent and evaluated the transfection efficiency by Western-Blot, RT-PCR and immunofluorescence.5. Investigated the effects of reduced RyR₂ levels on Ca²⁺ handling proteins in the E-C coupling. We found there are no significant changes of mRNA transcription levels of NCX, CASQ2, SERCA2, DHPR and PLB in RyR₂ knockdown cardiomyocytes compared to those in normal cardiomyocytes indicating that RyR₂ knockdown has no close relationship with the alterations of Ca²⁺ handling proteins associated with cardiac E-C coupling.6. Investigated aconitine-induced alterations of the spontaneous oscillations, the relative [Ca²⁺]i, SR Ca²⁺ releasable content and L-type Ca²⁺ channel currents in normal and RyR₂ gene knockdown cardiomyocytes by Ca²⁺ imaging and patch clamp. Results found that in normal cardiomyocytes, steady and periodic spontaneous Ca²⁺ oscillations were observed, and the baseline [Ca²⁺]i remained at low levels. Exposure to aconitine increased the frequency and decreased the amplitude of Ca²⁺ oscillations; the baseline [Ca²⁺]i and SR Ca²⁺ releasable content were increased but L-type Ca²⁺ currents were inhibited by aconitine-stimulation. In RyR₂ knockdown cardiomyocytes, the steady and periodic spontaneous Ca²⁺ oscillations almost disappeared, but were re-induced by aconitine without affecting the baseline [Ca²⁺]i level; SR Ca²⁺ releasable content were activated and the increased L-type Ca²⁺ currents were inhibited by aconitine-stimulation.7. It can be concluded that alterations of RyR₂ are important consequences of aconitine-stimulation and malfunctions of RyR₂ appear to have a direct relationship with aconitine-induced arrhythmias. The present study therefore provides an insight into a useful method for preventing aconitine-induced arrhythmias by inhibiting SR Ca²⁺ excess leaks through RyR₂ release channel.Discussion:Our results show that the intracellular Ca²⁺ overloaded in aconitine-induced cardiomyocytes, which can lead to a pro-arrhythmogenic state, and further arrhythmia. These results demonstrate that disruption of the intracellular Ca²⁺ homeostasis in the E-C coupling is a crucial mechanism of arrhythmic cytotoxicity in aconitine-induced cardiomyocytes. And also activations of the RyR₂ channel are important consequences with aconitine stimulation. Moreover, certain inhibitors appear to play an important role in the detoxification of aconitine-induced Ca²⁺-dependent arrhythmias. The present study provides an insight into a useful method for preventing ventricular arrhythmias by inhibiting SR Ca²⁺ excess leaks through the RyR₂ channel.As a whole, our research project is fundamentally the exploration of molecular mechanism in the E-C coupling system of cardiomyocytes with the approach of medication. Many of the heart diseases such as arrhythmia are caused by defective intracellular Ca²⁺ handling, due to abnormalities of various components that mediate and control the cardiac E-C coupling. In this sense, the illumination of the whole regulation mechanism and the patterns of expression of the key genes are sure to provide potential opportunities for novel therapeutic targets for heart diseases that are the leading cause of death worldwide.