Spontaneous Calcium Release Induced By Dieste-aconite Alkaloid On Cardiac Myocytes

Aconiti lateralis radix preparata (Fuzi), a traditional Chinese medicine, is the progressed branch root of Aconitum carmichaeli Debx.Clinically, Fuzi has been widely used for the treatment of heart failure, acute myocardial infarction, shock, bronchial asthma, chronic obstructive pulmonary disease, arthritis and other diseases.Fundamental researches have shown that the main components and metabolites of Fuzi were aconite alkaloids, fatty acid and fatty acid ester and so on. Alkaloids mainly include aconitine (AC), mesaconitine (MAC), hypaconitine (HAC), higenamine, aconine. However, the severe toxic effects of these compounds restrict the clinical application in treating various diseases severely. Besides, modern researches demonstrated that aconite alkaloids, the major effective components of Fuzi, also playedthe chief toxic role.Additionally, the dieste-aconite alkaloids, AC, MAC and HAC for instance, have exhibited remarkable toxicity in the previous studies.Aconitine has been most widely studied among the three dieste-aconite alkaloids in Fuzi during the past decades.Toxic effects related to AC have received much attention recently, especially with the central nervous system toxicity reaction and the cardiovascular system, and the arrhythmogenic effects may be more lethal.Pleomorphic arrhythmias, one-focal ventricular premature beat, monophyletic polymorphic ventricular premature beat, ventricular tachycardia and ventricular fibrillation for instance, along with respiratory center paralysis caused by AC could indeed lead to death.Most studies indicated that aconitine could interact with voltage-dependent Na+ channelsand suppress the conformational change of this Na+ channels from the inactive state to the active state which kept membrane remaining depolarized continuously. But the arrhythmia induced by aconitine cannot be simply explained by the incident that large quantity of Na+ flow into the cytosol bringing about triggered activities (TA) eventually. TA, including early after depolarization (EAD) and delayed after depolarization (DAD), is an important mechanism for arrhythmia which could lead to autonomy properties of spontaneous depolarization. Moreover,DADs can not only result in TA but also lead to cardiac arrhythmia directly.lt is widely accepted that DADs areinduced by the Ca2+ spontaneously released from sarcoplasmic reticulum (SR) at the state of intracellular Ca2+ accumulation.The widely acknowledged that AC induced arrhythmia through the NaC-NCX pathway. Briefly, AC could induce Na+ channels to keep active status to bring large numbers of Na+ flow into the cytoplasm which increases the [Ca2+]i concentration through the reversal transport of Na+/Ca2+ exchanger(NCX) in cardiac myocytes. The increased [Ca2+]i leads to Ca2+ overload,by which arrhythmia is triggered. Duringthe process of calcium releasing from the sarcoplasmic reticulumthe elevated [Ca2+]i through NCX contributes to the intracellular Na+ depolarization which could reach the threshold of DAD to trigger a new depolarization.Aconitine may interact with the ion channels to extend the APD period and then DAD and TA were obviously observed in the action potential.In clinical application,the ventricular tachycardia could only partially be inhibited by simply blocking sodium channels.It has been reported that calcium channel blockers performed more effectively than sodium channel blockers did.During the excitation-contraction coupling, L-type calcium channel could be opened by the membrane depolarization, followed by that Ca2+ flowed into the cytoplasm and activated the ryanodine receptors (RyR) on the sarcoplasmic reticulum to release large amount of calcium ions through the calcium induce calcium release (CICR) mechanism.Excessive increased intracellular calcium ion induced by AC could lead to calcium overload, and then elevate calcium wave frequency which triggered arrhythmia eventually. Previous reports have pointedout that arrhythmia induced by aconitine may directly relate to the activation of RyR2 by disrupting intracellular Ca2+ homeostasis.In addition, recent literatures reported that aconitine could down regulate SERCA protein expression, which may restrain the uptake of cytosol Ca2+ into SR inducing calcium accumulation in the cytoplasm during ECC. Furthermore, this Ca2+ accumulation would elevate RyRs open frequency. Besides, RyRs sensibility to Ca2+ rised when the SR Ca2+ content ([Ca]SRT) was at a high level, and then aggravated the calcium leak from SR, which finally triggered calcium release arrhythmically.We hypothesize that the elevated [Ca2+]i or increased sensitivity of RyRs could contribute to the aconitine arrhythmia toxicity through a variety of pathways. Further experimental researches need to be carried out to demonstrate these above hypotheses.In the present study, we investigated the calcium release toxicology and pharmacology effects including spontaneous calcium release (SCR) rate, calcium transient and positive inotropic effects of the three kinds of dieste-aconite alkaloids (aconitine, mesaconitine, hypaconitine) in cardiac myocytes. Furthermore, the relationships among the spontaneous calcium release, the elevated [Ca2+]i and SR Ca2+, and the calcium leak induced by AC, as well as the connection between SERCA and NCX metergasis during the excitation-contraction coupling were estimated to elucidate the mechanism of calcium homeostasis imbalance and mechanism of AC toxicity. To discover a more efficacious treatment for eliminating aconitine cardiac toxicity, cardiac myocytes SCR was detected under different conditions intervened by sodium channel (NaC), L-type calcium channel (LCC), NCX and adrenergic receptor (AR) blockers.Meanwhile, we also evaluated several monomers or compatibilities suggested to effectively eliminate the AC cardiotoxicity from TCM to discover the effective component that could reduce the SCR induced by AC, and to provide experimental foundation for the synergistic or attenuated use of AC.Those monomers were choosen fromTCM which has been reported to have the potential to block NaC, LCC, NCX or AR.Part One:The effects of the three dieste-aconite alkaloids on calcium release in cardiac myocytes.Objective:To investigate the toxicology and pharmacology effect relationships of the 3 dieste-aconite alkaloids (aconitine, mesaconitine, hypaconitine) on calcium release, including spontaneous calcium release(SCR) rate, end diastolic calcium concentration (F0), calcium transient amplitude (AF) and contractile function, in cardiac myocytes.Methods: The left ventricular cardiac myocytes were isolated from adult SD rat by Langendroff method. Continues pacing cells were perfused by three kinds of dieste-aconite alkaloids at 0.3μmol·L-1,1 μmol·L-1 and 3 μmol·L-1 concentration for 12 minutes. To assess the toxicity effects, SCR rate, FO and △F were detected at 4 min, 8 min and 12 min time points respectively. Additionally, The synchronous change of systolic function kinetics and the calcium transient were detected at 12min time point to illuminate the toxicity-effect relationships of the 3 aconite alkaloids at the concentration of 0.3 μmol·L-1.Results:All of the three kinds of dieste-aconite alkaloids could induce spontaneous calcium release in myocardial cells. Besides, mesconitine induced higher SCR rate than the other two at low concentrations (0.3 μmol·L-1) but aconitine induced the highest SCR rate at high concentration (3 μmol·L-1). All the 3 dieste-aconite alkaloids could dose-dependently elevate FO and AF, and mesaconitine displayed the maximal effect while hypaconitine displayed minimal. At low concentration (0.3 μmol·L-1), the 3 dieste-aconite alkaloids could all increase myocardial contractility and the amplitude and max velocity of calcium transient, especially promote diastolic cardiac function.Conclusion:The three kinds of dieste-aconite alkaloids displayed positive inotropic effect as well as toxic effects inducing SCR, which suggested that the 3 dieste-aconite alkaloids produced toxicity and effect on the cardiac myocytes concurrently, and the elevated [Ca2+]i promoted the positive inotropic effect as well as triggered the spontaneous calcium release.Part Two Toxicity mechanism research and intervention of aconitine on cardiac myocytes excitation contraction couplingObjective:To clarify the effects of AC on calcium transport in cardiac myocytes during excitation contraction coupling process and to investigate the effects of key ion channels and receptors disturbing the triggered activity on the spontaneous calcium release induced by AC.Methods:The left ventricular cardiac myocytes were isolated from adult SD rat by Langendroff method. Continues pacing cells were incubated by aconitine at 0.3μmol·L-1 concentration for 12 minutes, and then intracellular Ca2+, calcium transient, SR Ca2+ content, SR Ca2+ leak, and the effects of SERCA and NCX on diastolic calcium removal were detected. SCR rate, FO and AF were assessed at 12min time point under 1 μmol·L-1 aconitine accompany with different intervention by sodium channel (NaC), L-type calcium channel (LCC), NCX and adrenergic receptor (AR) blocked.Results:Aconitine could accelerate calcium elimination in cardiac myocytes and enhance SERCA and NCX activity accompany with increased sarcoplasmic reticulum calcium content ([Ca]SRT). The raised [Ca]sRT fortified RyR sensitivity to [Ca2+]i causing an increased level of sarcoplasmic reticulum calcium leak. A significant increased calcium release ratio from sarcoplasmic reticulum could be observed during calcium transient process. Aconitine increased calcium ion concentration and induce spontaneous calcium release mainly through the NaC-NCX pathways. SCR rates and FO induced by aconitine were decreased when cardiac myocytes were incubated with lidocaine or KB-R7943 or both of them, and there was no significant difference among the three interferences. Lidocaine could decrease calcium transient amplitude compared with AC but still increase compared with control group. Nifedipine could reduce the SCR incidence, as well as the calcium transients amplitude induced by AC. L-type calcium channel did take part in the SCR, but it was not the dominant factor. The adrenergic receptor mediated signaling pathways were involved in the toxicity of aconitine beside the NaC-NCX pathway. Blocking adrenergic receptors could effectively eliminate SCR rate induced by aconitine. Furthermore, the combination with between AR antagonists and sodium channel blockers could eliminate SCR satisfactorily.Conclusion:Aconitine could disorder calcium transport in cardiac myocytes through increasing sarcoplasmic reticulum calcium content and prompting calcium leak. Blocking sodium channels and adrenergic receptors could effectively eliminate spontaneous calcium release.Part Three Attenuated toxicity and synergy effect of Traditional Chinese Medicine compositions compatibility with aconitineObjective: To provide experimental basis for the clinical application of AC, compositions from TCM were evaluated if anyone of them could reduce the SCR rate induced by aconitine markedly.Methods:The left ventricular cardiac myocytes were isolated from adult SD rat by Langendroff method. Continues pacing cells were perfused by aconitine at 1 μmol·L-1 concentration and co-incubated with compounds from TCM for 12 minutes, then SCR rate, FO and AF were assessed. These compounds, consisting of monomer or ingredient combination from TCM, were reported to inhibit NaC, LCC, NCX or AR potentially.Results:Unfortunately, none of the 15 Traditional Chinese Medicine monomers could effectively eliminate spontaneous calcium release induced by aconitine.Conclusion:FifteenTraditional Chinese Medicine monomers failed to eliminate the spontaneous calcium release induced by aconitine.