The presented studies investigate dynamics of action potential duration (APD) tobetter understand the underlying mechanism for repolarization alternans.We recorded trans-membrane potentials (TMP) in canine endocardial muscle tissueusing standard glass microelectrode under the control of an explicit diastolic interval (DI)control pacing protocol, i.e. feedback protocol. During sequential sinusoidal DI activation,the trajectory of APD dynamics has multiple values of APD correspondent to the sameDI, i.e. restitution is a bi-modal relationship. Our results indicate that: 1) there is a delay,similar to hysteresis, of change in APD responding to change in DI, 2) and the timecourse of the delay is asymmetric for fast or slow pacing history. The alternans wasobserved during constant DI pacing, i.e. the DI preceding each APD was invariant orchanged within a limited range. This finding suggests that alternans of APD do not needthe oscillation of preceding DI, i.e. DI dependent restitution is not a necessary conditionfor the alternans. This result implies that DI independent component exists in themechanism of the alternans. Nonetheless, the amplitude of alternans was statisticallylarger during constant pacing cycle length (PCL) pacing than that during constant DIpacing, even though both PCL and DI pacing trials used similar average activation rate.These results also demonstrate the ability of the feedback protocol to analyze the memoryeffects and dissect different components in the mechanism of alternans.Two computational models, Luo-Rudy dynamics (LRD) and cardiac ventricle model(CVM) were used to study the hysteresis in restitution. By perturbing membrane current:L-type calcium current, rapid and slow potassium rectifier, and intracellular calciumtransfer rate in sarcoplasmic reticulum (SR) and using sinusoidal DI pacing sequence, weshowed that the asymmetric calcium current across the membrane and its interaction withcalcium buffer in SR during increasing and decreasing DI phase plays an important rolein the hysteresis. CVM was used to study the alternans during constant DI pacing.However CVM failed to replicate the alternans that occurred in the experiments. Thisresult implies that CVM lacks the electrophysiological kinetics related to alternans thatwas shown in our experiment.

Repolarization alternans, i.e. beat-to-beat variation of repolarization of action potential, is proposed as a predictor of life-threatening arrhythmias. Restitution relates repolarization duration with its previous relaxation time, i.e. diatstolic interval (DI), and is considered a dominant mechanism for alternans. Previously, we observed that different repolarization durations at the same DI during decelerating and accelerating pacing, i.e. restitution displays hysteresis, which is a measure of “cardiac memory”. Objective of the current study was to investigate in the pig 1) the mechanism for a previously observed hysteresis type phenomenon, where alternans, once started at higher heart rate, persists even when heart rate decreases below its initiating rate, 2) regional differences in expression of hysteresis, i.e. memory in restitution in the heart, and 3) changes in restitution and memory during manipulation of an important repolarization current, the slow delayed rectifier, IKs. Action potentials were recorded in pig ventricular tissues using microelectrodes. Regional differences were explored in endocardial and epicardial tissues from both ventricles. DIs were explicitly controlled in real time to separate restitution mechanism from non-restitution related effects. Stepwise protocols were used to explore the existence in hysteresis in alternans threshold, where DIs were held constant for each step and progressively decreased and then increased. Quantification of cardiac memory was achieved by sinusoidally changing DI protocols, which were used to investigate memory changes among myocytes from different regions of the heart and during IKs manipulation. Results show that during stepwise protocol, hysteresis in alternans still existed, which indicates that restitution is not the only mechanism underlying the hysteresis. When comparing hysteresis obtained from sinusoidally oscillatory DIs among different regions, results show memory is expressed differently with endocardium expressing the most and epicardium the least memory. This provides important implications about the location where arrhythmia would initiate. Results also show that measures for hysteresis loops obtained by sinusoidal DI protocols decreased (increased) after enhancement (attenuation) of IKs, suggesting decreased (increased) hysteresis, i.e. memory in restitution. This effect needs to be considered during drug development.

Sudden Cardiac Death (SCD) is the largest cause of natural deaths in the USA, accounting for over 300,000 deaths annually. The major reason for SCD is Ventricular Arrhythmia (VA). Therefore, there is need for exploration of approaches to predict increased risk for VA. Alternans of the T wave in the ECG (TWA) is widely investigated as a potential predictor of VA, however, clinical trials show that TWA has high negative predictive value but poor positive predictive value. A possible reason that TWA has a large number of false positives is that a pattern of alternans known as concordant alternans, may not be as arrhythmogenic as another pattern which is discordant alternans. Currently, it is not possible to discern the pattern of alternans using clinical ECGs. Prior studies from our group have showed that alternans of the maximum rate of depolarization of an action potential also can occur when Action Potential Duration (APD) alternans occurs and the relationship between these two has the potential to create spatial discord. These results suggest that exploration of the co-occurrence of depolarization and repolarization alternans has the potential to stratify the outcome of TWA tests. In order to investigate the link between depolarization alternans and changes in ECGs, we used a mathematical model created previously in our research group which simulated ECGs from the cellular level changes observed in our experimental studies. These results suggest that the changes in ECGs should appear as alternating pattern of the amplitude of the R wave. Because there are a variety of factors which may also cause the R wave amplitude to change, we used signal analysis and statistical modeling to determine the link between the observed changes in R wave amplitude and depolarization alternans. Results from ECGs recorded from patients show that amplitude of the R wave can change as predicted by our experimental results and mathematical model. Using TWA as the marker of repolarization alternans and R Wave Amplitude Alternans (RWAA) as the marker of depolarization alternans, we investigated the phase relation between depolarization and repolarization alternans in clinical grade ECG and observed that this relationship does change spontaneously, consistent with our prior results from animal studies. Results of the present study support further investigation of the use of RWAA as a complementary method to TWA to improve its positive predictive value.