High Calcium-induced Cardio-electrical Alternans And The Mechanism Of Its Arrhythmogenesis

BackgroundThe phenomenon of cardio-electrical alternans is referred to as periodical alternative changes of electrocardiogram（ECG）in waveforms and amplitudes at a constant heart rate.Cardiac alternans is an important manifestation of the mechanical dysfunction and the instability of electrical activities in hearts.Cardiac alternans closely associated with the clinical heart diseases can increase the risk for malignant arrhythmias and sudden cardiac death（SCD）.Cardiac alternans is more likely to cause fatal arrhythmias,especially in a wide range of pathophysiological conditions,including ischemic congestive heart disease,heart failure,and myocardial infarction,etc.Cardiac alternans is a well-known pathological factor for malignant ventricular arrhythmias,which are the main cause for the occurrence of SCD.Many prospective clinical studies have demonstrated a direct cause-and-effect relationship between cardiac alternans,ventricular arrhythmias and SCD.However,the specific mechanism of cardiac alternans remains unknown until now.It is now well-recognized that cardiac alternans is a precursor to ventricular arrhythmias.Cardiac repolarization alternans plays a major role in the production of arrhythmic substrates,which promotes reentry and eventually leads to persistent arrhythmias.At the cellular level,cardiac alternans refers to the periodic and intermittent changes of contraction,action potential duration（APD）and intracellular calcium transient（Ca T）under a constant heart rate.Therefore,the understanding of cellular mechanism of cardiac alternans is of great significance in elucidating the mechanism of arrhythmias caused by cardiac alternans.T wave alternans（TWA）on ECG,also called the periodic alternans of ventricular action potential repolarization,increase the susceptibility of ventricular arrhythmia and SCD.TWA is observed in a variety of clinical settings,including congenital or acquired long QT syndrome and Brugada syndrome,ischemic or coronary artery disease,dilated and hypertrophic cardiomyopathy,and advanced heart failure,etc.The fact that TWA promotes the development of malignant ventricular tachycardia under different clinical and experimental conditions suggests that TWA may be widespread in the pathophysiological mechanism of SCD,indicating that cardiac alternans has important clinical significance in antiarrhythmic therapies.The available data show that TWA is an important marker of clinical risk,but the molecular mechanism of TWA remains unclear,and the role of TWA in causing SCD remains unknown.Therefore,a better understanding of arrhythmogenic cardiac alternans is essential for identifying high-risk patients and developing new antiarrhythmic therapies.A lot of simulation studies and experimental studies have proved that cardiac alternans is related to the instability of membrane potential and calcium cycling in cardiomyocytes.Owing to complicated feedback pathways between these two parameters,the cellular mechanism of cardiac alternans remains controversial.In recent years,the instability of calcium cycling has gradually become the research hotspot of arrhythmogenic cardiac alternans.In addition to many kinds of cell membrane ion currents（such as L-type calcium currents,sodium-calcium exchange currents,calcium-dependent ion currents,etc.）,regulatory proteins involved in sarcoplasmic reticulum calcium release and recovery also play a key role in maintaining intracellular calcium homeostasis.The phenomenon suggests that cardiac alternans is not only caused by the abnormal activity of a single ion channel but is the result of the combined action of multiple ion channels and related regulatory proteins.Therefore,there are some limitations that the research and treatment of anti-arrhythmias caused by cardiac alternans mainly focus on a single ion channel.Although a great deal of studies has been done,more studies are needed to find new therapeutic targets and to design better anti-arrhythmias treatments.The activities of cardiac ion channels are regulated at multiple parts,including some key enzymes in the G protein-coupled signal transduction pathway located upstream of ion channels,such as Ca²⁺-dependent protein kinase C（PKC）.PKC is involved in the modulation of many kinds of ion channels in hearts,including intracellular calcium homeostasis,and is also regulated by calcium feedback.More and more evidence show that calcium overload enhances and promotes PKC activation,which is closely related to ventricular arrhythmia.However,the effect of PKC on arrhythmogenic cardiac alternans has not been reported,our study on the relationship among PKC,cardiac alternans and ventricular arrhythmias is helpful to clarify the mechanism of cardiac alternans-caused arrhythmias.This study aimed to establish a pathological model of cardiac alternans induced by high calcium,and to further explore the molecular mechanism of cardiac alternans-induced arrhythmias,thus helping us provides a scientific basis for finding a new way for the treatment of arrhythmias caused by cardiac alternans.Research contents1.To establish the model of cardiac alternans induced by high calcium in ventricular myocytesObjective:To successfully induce cardiac alternans by intracellular high calcium interventionMethods:single middle-layer left ventricular myocytes were obtained by acute enzymatic digestion.Whole-cell patch-clamp technique was used to record the changes of action potential;the changes of intracellular calcium transient were measured by dual-excitation photomultiplier system;the amplitude of sarcomere contraction in ventricular myocytes was recorded by the visual dynamic edge detection system.Results:high intracellular free calcium concentrations successfully induced action potential duration alternans（APD-ALT）.APD-ALT induced by high calcium exerted not only a calcium concentration-dependent manner but also a frequency-dependent manner,and intracellular high calcium lowered the threshold of frequency-induced APD-ALT.Moreover,cardiac alternans,including intracellular calcium transient alternans（Ca T-ALT）and the alternative variations of sarcomeres contraction,were observed in isolated ventricular myocytes.Conclusion:the model of cardiac alternans induced by high calcium was successfully established,which indicated that high calcium-induced cardiac alternans by disturbing the regulation of intracellular calcium homeostasis.2.Relationship between high calcium-induced APD-ALT and changes of L-type calcium currents（ICaL）and intracellular calcium concentrationsObjective:To determine the relationships between APD-ALT and changes of I_CaL and intracellular calcium concentrationsMethods:The changes of action potential and L-type calcium current(I_CaL)before and after drug intervention were recorded by whole-cell patch clamp technique in current-clamp mode and in voltage-clamp mode,respectively.Results:The results showed that APD-ALT induced by high calcium was the most pronounced at the plateau phase of action potential,and L-type calcium channel blocker nifedipine eliminated high calcium-induced APD-ALT.However,no beat-to-beat alternations of I_CaL were observed under high calcium pretreatment,that is to say,the occurrence of APD-ALT induced by high calcium was not caused by the alternations of I_CaL.In addition,the transmembrane calcium chelating agent BAPTA-AM also showed a significant inhibitory effect on APD-ALT induced by high calcium.In addition,the results showed that not only nifedipine but also BAPTA-AM eliminated calcium transient alternation and sarcomere amplitude alternation induced by high calcium content.Conclusion:The alternative variations of I_CaL failed to appear under high calcium conditions,and thecardio-electrical alternans induced by high calcium in cardiomyocytes were caused by calcium cycling dysregulation.Either the inhibition of calcium influx by nifedipine or the direct reduction of intracellular calcium concentration by calcium ion chelator BAPTA-AM eliminated APD-ALT induced by high calcium,which showed that the dynamic changes of I_CaL and intracellular calcium concentrations exerted a strong correlation with the occurrence of APD-ALT.3.Effect of the regulation of PKC activities on arrhythmogenic cardiac alternans induced by high calciumObjective:to verify that PKC signal transduction pathway was involved in the onset and development of arrhythmogenic cardiac alternans induced by high calciumMethods:whole-cell patch clamp technique was used to record the action potential.The dynamic changes of Ca T were measured by Fura-2/AM fluorescence method.ECG monitoring isolated perfused hearts was observed with a ECG signal acquisition and analysis system.In addition,western blotting was used to measure the protein expression of PKCαin left ventricular myocardium after isolated heart perfusion under different treatments.Results:high calcium-induced APD-ALT was completely eliminated not only by PKC inhibitor BIM,but also another selective PKC inhibitor G?6976 showed the similar inhibitory effect on APD-ALT.Moreover,APD-ALT was observed after pretreated with PKC activator PMA under normal calcium condition.In addition,BIM effectively prevented the occurrence of Ca T-ALT in single middle ventricular myocytes perfused with high calcium perfusate,and significantly reduced the incidence of Ca T disorders.What’s more,BIM not only eliminated the T-wave alternans（TWA）in isolated high calcium-perfused hearts,but also significantly reduced the incidence of ventricular arrhythmias,including ventricular fibrillation and ventricular tachycardia.In addition,BIM decreased PKCαprotein levels in cardiac tissues pretreated with high calcium.Conclusion:the over-activation of PKC promoted the onset and development of cardiac alternans.Inhibiting PKC activities reduced the occurrence of ventricular arrhythmias caused by cardiac alternans,which indicated that pharmacological inhibition of PKC activation may be a new target for antiarrhythmic research and therapy.Significance of study:We pioneered a pathological model of cardiac alternans induced by high calcium,and further explored the mechanism of arrhythmogenic cardiac alternans.These results showed that pharmacological regulation of PKC activities can prevent and control arrhythmogenic cardiac alternans,which proved that Ca²⁺-dependent PKC signal pathway plays an important role in regulating calcium homeostasis in the process of arrhythmogenic cardiac alternans.This understanding not only provides new insights into the molecular mechanism of cardiac alternans,it also provides a pharmacological basis for preventing arrhythmogenic cardiac alternans associated with calcium dysregulation.