Epicardial Activation Sequence Research Articles

Uncertainty Quantification of Fiber Orientation and Epicardial Activation.

Predictive models and simulations of cardiac function require accurate representations of anatomy, often to the scale of local myocardial fiber structure. However, acquiring this information in a patient-specific manner is challenging. Moreover, the impact of physiological variability in fiber orientation on simulations of cardiac activation is poorly understood. To explore these effects, we implemented bi-ventricular activation simulations using rule-based fiber algorithms and robust uncertainty quantification techniques to generate detailed maps of model variability. Specifically, we utilized polynomial chaos expansion, enabling efficient exploration with reduced computational demand through an emulator function approximating the underlying forward model. Our study focused on examining the epicardial activation sequences of the heart in response to six stimuli locations and five metrics of activation. Our findings revealed that physiological variability in fiber orientation does not significantly affect the location of activation features, but it does impact the overall spread of activation. We observed low variability near the earliest activation sites, but high variability across the rest of the epicardial surface. We conclude that the level of accuracy of myocardial fiber orientation required for simulation depends on the specific goals of the model and the related research or clinical goals.

Dynamic electrophysiological mechanism in patients with long-standing persistent atrial fibrillation.

BackgroundImproved understanding of the mechanisms that sustain persistent and long-standing persistent atrial fibrillation (LSpAF) is essential for providing better ablation solutions. The findings of traditional catheter-based electrophysiological studies can be impacted by the sedation required for these procedures. This is not required in non-invasive body-surface mapping (ECGI). ECGI allows for multiple mappings in the same patient at different times. This would expose potential electrophysiological changes over time, such as the location and stability of extra-pulmonary vein drivers and activation patterns in sustained AF.Materials and methodsIn this electrophysiological study, 10 open-heart surgery candidates with LSpAF, without previous ablation procedures (6 male, median age 73 years), were mapped on two occasions with a median interval of 11 days (IQR: 8–19) between mappings. Bi-atrial epicardial activation sequences were acquired using ECGI (CardioInsight™, Minneapolis, MN, United States).ResultsBi-atrial electrophysiological abnormalities were documented in all 20 mappings. Interestingly, the anatomic location of focal and rotor activities changed between the mappings in all patients [100% showed changes, 95%CI (69.2–100%), p < 0.001]. Neither AF driver type nor their number varied significantly between the mappings in any patient (median total number of focal activities 8 (IQR: 1–16) versus 6 (IQR: 2–12), p = 0.68; median total number of rotor activities 48 (IQR: 44–67) versus 55 (IQR: 44–61), p = 0.30). However, individual zones showed a high number of quantitative changes (increase/decrease) of driver activity. Most changes of focal activity were found in the left atrial appendage, the region of the left lower pulmonary vein and the right atrial appendage. Most changes in rotor activity were found also at the left lower pulmonary vein region, the upper half of the right atrium and the right atrial appendage.ConclusionThis clinical study documented that driver location and activation patterns in patients with LSpAF changes constantly. Furthermore, bi-atrial pathophysiology was demonstrated, which underscores the importance of treating both atria in LSpAF and the significant role that arrhythmogenic drivers outside the pulmonary veins seem to have in maintaining this complex arrhythmia.

Noninvasive diagnostic mapping of supraventricular arrhythmias (Wolf-Parkinson-White syndrome and atrial arrhythmias).

The 12-lead electrocardiogram has limited value in precisely identifying the origin of focal or critical component of reentrant arrhythmias during supraventricular arrhythmias, as well as precisely locating accessory atrioventricular conduction pathways. Because of these limitations, efforts have been made to reconstruct epicardial activation sequences from body surface measurements obtained noninvasively. The last decade has registered significant progress in obtaining clinically useful data from the attempts to noninvasively map the epicardial electrical activity. This article summarizes the recent advances made in this area, specifically addressing the clinical outcomes of such efforts relating to atrial arrhythmias and Wolf-Parkinson-White syndrome.

Layered Activation of Epicardial Scar in Arrhythmogenic Right Ventricular Dysplasia

Background— Ventricular tachycardia ablation in arrhythmogenic right ventricular dysplasia (ARVD) is more successful when including epicardial ablation. Scarring may cause independent, layered epicardial activation and promote epicardially confined ventricular tachycardia circuits. We aimed to characterize transmural right ventricular activation in ARVD patients and to compare this with reference patients without structural heart disease. Methods and Results— Eighteen ARVD patients underwent detailed endocardial and epicardial sinus rhythm electroanatomic mapping. Bipolar activation was annotated at the sharpest intrinsic deflection including late potentials and compared with 6 patients with normal hearts. Total scar area was larger on the epicardium (97±78 cm 2 ) than the endocardium (57±44 cm 2 ; P =0.04), with significantly more isolated potentials. Total epicardial activation time was longer than endocardial (172±54 versus 99±27 ms; P &lt;0.01), and both were longer than in reference patients. Earliest endocardial site was the right ventricular anteroseptum in 17 of 18 ARVD patients versus 5 of 6 controls ( P =0.446), and latest endocardial site was in the outflow tract in 13 of 18 ARVD patients versus 4 of 6 controls and tricuspid annulus in 5 of 18 ARVD patients versus 2 of 6 controls ( P =1.00). In reference patients, epicardial activation directly opposite endocardial sites occurred in 5.2±1.9 ms, suggesting direct transmural activation. In contrast, ARVD patients had major activation delay to the epicardium with laminar central scar activation from the scar border, not by direct transmural spread from the endocardium. Conclusions— Transmural right ventricular activation is modified by ARVD scarring with a delayed epicardial activation sequence suggestive of independent rather than direct transmural activation. This may predispose ventricular tachycardia circuits contained entirely within the epicardium in ARVD and explains observations on the need for direct epicardial ablation to eliminate ventricular tachycardia.

Clinical Summaries

Clinical Summaries

Noninvasive Characterization of Epicardial Activation in Humans With Diverse Atrial Fibrillation Patterns

Various mechanisms of atrial fibrillation (AF) have been demonstrated experimentally. Invasive methods to study these mechanisms in humans have limitations, precluding continuous mapping of both atria with sufficient resolution. In this article, we present continuous biatrial epicardial activation sequences of AF in humans using noninvasive electrocardiographic imaging (ECGI). In the testing phase, ECGI accuracy was evaluated by comparing ECGI with co-registered CARTO images during atrial pacing in 6 patients. Additionally, correlative observations from catheter mapping and ablation were compared with ECGI in 3 patients. In the study phase, ECGI maps during AF in 26 patients were analyzed for mechanisms and complexity. ECGI noninvasively imaged the low-amplitude signals of AF in a wide range of patients (97 procedural success). Spatial accuracy for determining initiation sites from pacing was 6 mm. Locations critical to maintenance of AF identified during catheter ablation were identified by ECGI; ablation near these sites restored sinus rhythm. In the study phase, the most common patterns of AF were multiple wavelets (92), with pulmonary vein (69) and non-pulmonary vein (62) focal sites. Rotor activity was seen rarely (15). AF complexity increased with longer clinical history of AF, although the degree of complexity of nonparoxysmal AF varied widely. ECGI offers a noninvasive way to map epicardial activation patterns of AF in a patient-specific manner. The results highlight the coexistence of a variety of mechanisms and variable complexity among patients. Overall, complexity generally increased with duration of AF.

Challenging cardiac electrophysiology

Challenging cardiac electrophysiology

The relationship of myocardial contraction and electrical excitation—the correlation between scintigraphic phase image analysis and electrophysiologic mapping

BackgroundPhase imaging derived from equilibrium radionuclide angiography presents the ventricular contraction sequence. It has been widely but only indirectly correlated with the sequence of electrical myocardial activation. ObjectivesWe sought to determine the specific relationship between the sequence of phase progression and the sequence of myocardial activation, contraction and conduction, in order to document a noninvasive method that could monitor both. MethodsIn 7 normal and 9 infarcted dogs, the sequence of phase angle was correlated with the epicardial activation map in 126 episodes of sinus rhythm and pacing from three ventricular sites. ResultsIn each episode, the site of earliest phase angle was identical to the focus of initial epicardial activation. Similarly, the serial contraction pattern by phase image analysis matched the electrical epicardial activation sequence completely or demonstrated good agreement in approximately 85% of pacing episodes, without differences between normal or infarct groups. ConclusionsA noninvasive method to accurately determine the sequence of contraction may serve as a surrogate for the associated electrical activation sequence or be applied to identify their differences.

Termination of a tachyarrhythmia by flunarizine is not a specific marker for a triggered mechanism

Prior studies have indicated that tachyarrhythmia termination by flunarizine demonstrates a triggered mechanism. This concept was not confirmed in atrial tachyarrhythmias. The purpose of this study was to test the hypothesis that flunarizine will not terminate reentrant atrial flutter (AFL). We administered flunarizine (2 mg/kg intravenously over 2 minutes) in 11 episodes of reproducibly inducible, sustained AFL in eight canines with sterile pericarditis. If flunarizine terminated AFL, we studied AFL reinducibility. We also studied pacing thresholds, refractoriness, and intra-atrial conduction time during closed-chest studies and pacing at selected cycle lengths (CLs) from selected sites before and after flunarizine administration. Atrial mapping (510 electrodes) assessed the epicardial activation sequence during AFL and its termination in six episodes. Four AFL episodes were studied in the closed-chest state. Flunarizine increased AFL CL in all episodes (mean 21 ms; range 7-49 ms), which is explained by slowing conduction in the AFL reentrant circuit, principally in the area of slow conduction. AFL was terminated in 10/11 episodes after drug initiation (mean 3.7 minutes; range 0.5-6.5 minutes) by block in the area of slow conduction. AFL was then not immediately reinducible until >20 minutes after drug administration. Flunarizine had no meaningful effect on atrial pacing thresholds for capture or refractoriness and only affected conduction time in the area of slow conduction in the reentrant circuit. Flunarizine (1) causes progressive slowing and block in the area of slow conduction of the AFL reentrant circuit in the canine sterile pericarditis model and (2) is effective in terminating reentrant AFL and so is not a specific marker for a triggered mechanism.

Normal Atrial Activation and Voltage During Sinus Rhythm in the Human Heart: An Endocardial and Epicardial Mapping Study in Patients with a History of Atrial Fibrillation

The three-dimensional contributions to human atrial activation in sinus rhythm have not been specifically characterized. We evaluated the sequence of endocardial and epicardial activation and voltage of the atria during normal sinus rhythm. The study group includes 35 patients with history of symptomatic atrial fibrillation. Prior to catheter ablation of atrial fibrillation, we performed multielectrode electroanatomic mapping during sinus rhythm, endocardially of the RA, LA, and coronary sinus (CS) and, in 10 patients, epicardially of the transverse sinus and oblique sinus. Following activation of the atrial region of the sinus node, the epicardial transverse sinus was activated 11 +/- 18 msec later, while the earliest endocardial LA activation occurred in the region of Bachmann's bundle at 31 +/- 13 msec, significantly earlier than the earliest epicardial LA activation of the oblique sinus at 54 +/- 10 msec (P < 0.002). The posterior LA revealed complex types of activation in 66% of patients analyzed, due to the convergence of wavefront propagation from the CS, oblique sinus, and endocardial LA. Bipolar voltage measurements revealed significantly higher values for the epicardium (mean 3.05 +/- 1.31 mv) than for the endocardium (mean 1.65 +/- 0.75 mv), P < 0.0001 between both groups. In sinus rhythm, we have characterized endocardial and epicardial atrial activation and voltage, and provide an analysis and understanding of the genesis of the P wave complex in humans.

Venous Catheter Based Mapping of Ectopic Epicardial Activation: Training Data Set Selection for Statistical Estimation

A source of error in most of the existing catheter cardiac mapping approaches is that they are not capable of acquiring epicardial potentials even though arrhythmic substrates involving epicardial and subepicardial layers account for about 15% of the ventricular tachycardias. In this subgroup of patients, mapping techniques that are limited to the endocardium result in localization errors and failure in subsequent ablation procedures. In addition, catheter-based electrophysiological studies of the epicardium are limited to regions near the coronary vessels or require transthoracic access. We have developed a statistical approach by which to estimate high-resolution maps of epicardial activation from very low-resolution multi-electrode venous catheter measurements. A training set of previously recorded maps is necessary for this technique so that composition of the database becomes an important determinant of accuracy. The specific hypothesis of the study was that estimation accuracy would be best when the training data set matches that of the test beat(s), whereby the matching was according to the site of initiation of the beats. This hypothesis suggests approaches to optimized selection of the training set, three of which we have developed and evaluated. One of these methods, the high-CC refinement method, was able to estimate the earliest activation site of left ventricularly paced maps within an average of 4.67 mm of the true site; in 89% of the cases (a total of 231 cases) the error was smaller than 10 mm. In another method, MHC-Spatial activation, right ventricularly paced maps (239 maps) were estimated with an error of 7.15 mm. The average correlation coefficient between the original and the estimated maps was also very high (0.97), which shows the ability of the training data set refinement methods to estimate the epicardial activation sequence. The results of these tests support the hypothesis and, moreover, suggest that such an approach is feasible for providing accurate reconstruction of complete epicardial activation-time maps in a clinical setting.

Electrocardiographic imaging (ECGI), a novel diagnostic modality used for mapping of focal left ventricular tachycardia in a young athlete

We report the first clinical application of electrocardiographic imaging (ECGI), a new, noninvasive imaging modality for arrhythmias, in an athlete with focal ventricular tachycardia (VT) originating from a left ventricular (LV) diverticulum. A reconstructed map of the epicardial activation sequence during a single premature ventricular complex (PVC) of an identical QRS morphology to the clinical VT, generated from 224-electrode body surface ECGs and a chest CT (ECGI), localized the PVC to the site of the diverticulum. This correlated with subsequent maps obtained using standard techniques. We describe the first case that used ECGI to guide diagnosis and therapy of a clinical tachyarrhythmia.

An experimental model to correlate simultaneous body surface and epicardial electropotential recordings in vivo

Our aim was to simultaneously record dense arrays of electropotential signals from the heart and body surface of a closed-chest anaesthetised pig during an acute period of regional ventricular ischaemia. After fitting a suture snare to the equatorial region of the left anterior descending (LAD) coronary artery, an electrode sock containing 127 stainless steel contact electrodes (spaced approximately 7 mm apart) was positioned over the ventricular epicardium. The chest was re-closed and fitted an elasticated vest containing 256 ECG electrodes (spaced approximately 15 mm apart). Electrode locations were measured using a mechanical digitising arm and projected onto a customised 3D mathematical model of the porcine torso and heart. Finite element fitted body surface potential maps (BSPMs) and epicardial activation sequences were used to interpret the electropotential signals. Data were sampled at 2 kHz and recorded at 20 s intervals during a four minute period of LAD occlusion, followed by a period of reperfusion and recovery. During occlusion, propagation of epicardial activation slowed monotonically across the ischaemic region and this was clearly associated with a zone of ST segment elevation in the BSPM and Lead V 1 ECG, whilst the Lead II ECG remained relatively unchanged. The epicardial activation sequence had largely recovered after 60 s of reperfusion, although there was still evidence of slowed activation across the ischaemic region compared to the control. Electrocardiac activity had fully restored to its control state after six minutes of reperfusion. Simultaneous recordings of this type will provide an experimental model to be used together with our integrated computational framework to assess the accuracy and sensitivity of activation inverse ECG algorithms during normal and patho-physiological states.

Noninvasive electrocardiogram imaging of substrate and intramural ventricular tachycardia in infarcted hearts

ObjectivesThe goal of this study was to experimentally evaluate a novel noninvasive electrocardiographic imaging modality during intramural reentrant ventricular tachycardia (VT). BackgroundMyocardial infarction and subsequent remodeling produce abnormal electrophysiologic substrates capable of initiating and maintaining reentrant arrhythmias. Existing noninvasive electrocardiographic methods cannot characterize abnormal electrophysiologic substrates in the heart or the details of associated arrhythmias. A noninvasive method with such capabilities is needed to identify patients at risk of arrhythmias and to guide and evaluate therapy. MethodsA dog heart with a four-day-old infarction was suspended in a human shaped torso-tank. Measured body surface potentials were used to noninvasively compute epicardial potentials, electrograms and isochrones. Accuracy of reconstruction was evaluated by direct comparison to measured data. Reconstructions were performed during right atrial pacing and nine cycles of VT. ResultsNoninvasively reconstructed potential maps, electrograms and isochrones identified: 1) the location of electrophysiologically abnormal infarct substrate; 2) the epicardial activation sequences during the VTs; 3) the locations of epicardial breakthrough sites; and 4) electrophysiologic evidence for activation of the Purkinje system and septum during the reentrant beats. ConclusionsElectrocardiographic imaging can noninvasively reconstruct electrophysiologic information on the epicardium during VT with intramural reentry, provide information about the location of the intramural components of reentry and image abnormal electrophysiologic substrates associated with infarction.

Ventricular activation during sympathetic imbalance and its computational reconstruction.

We characterized the epicardial activation sequence during a norepinephrine (NE)-induced ventricular arrhythmia in anesthetized pigs and studied factors that modulated it. Subepicardial NE infusion caused the QRS complex to invert within a single beat (n = 35 animals, 101 observations), and the earliest epicardial activation consistently shifted to the randomly located infusion site (n = 14). This preceded right atrial activation, whereas the total ventricular epicardial activation time increased from 20 +/- 4 to 50 +/- 9 ms (P < 0.01). These events were accompanied by a ventricular tachycardia and a drop in left ventricular pressure, which were fully reversed after the infusion was stopped. Epicardial pacing at the infusion site mimicked all electrical and hemodynamic changes induced by NE. The arrhythmia was prevented by propranolol and abolished by cardiac sympathetic or vagal nerve stimulation. Focal automaticity was computationally reconstructed using a two-dimensional sheet of 256 x 256 resistively coupled ventricular cells, where calcium handling was abnormally high in the central region. We conclude that adrenergic stimulation to a small region of the ventricle elicits triggered automaticity and that computational reconstruction implicates calcium overload. Interventions that reduce spatial inhomogeneities of intracellular calcium may prevent this type of arrhythmia.

Prediction of defibrillation outcome by epicardial activation patterns following shocks near the defibrillation threshold.

Ventricular defibrillation is probabilistic and shock strength dependent. We investigated the relationship between defibrillation outcome and postshock activation patterns for shocks of the same strength (approximately 50% probability of success for defibrillation [ED50] to yield an equal number of successful and failed shocks). In five pigs, 10 shocks of approximately ED50 strength (right ventricle-superior vena cava, biphasic, 6/4 msec) were delivered after 10 seconds of ventricular fibrillation (VF). Epicardial activation sequences following shocks were mapped with a 504-electrode shock and analyzed by animating dV/dt of the electrograms. Intercycle interval (ICI, time between the onset of successive postshock cycles), wavefront conduction time (WCT, time between the earliest and latest activation of a cycle), and overlapping index (WCT of cycle[n]/ICI of cycle[n+1]) were determined for the first five postshock cycles. An overlapping index >1 indicates overlap between successive cycles. Of 50 defibrillation attempts, 25 were successes. There was no difference between successful and failed episodes for both ICI (68 +/- 9 msec vs 62 +/- 10 msec) and WCT (97 +/- 24 msec vs 100 +/- 14 msec) of cycle 1. However, starting at cycle 2, the ICI was longer, and the WCT was shorter for successful than failed episodes (P < 0.01). Overlapping cycles (index > 1) were found during the transition from cycles 2 through 5 in all failed (index >1) and in no successful episodes. (1) Defibrillation outcome cannot be determined during the first postshock cycle. (2) At least three rapid successive cycles with overlap of cycles 2 and 3 are present in all failed and in no successful episodes. (3) The overlapping index is a marker to predict defibrillation outcome.

Investigation of the atrial pacemaker site and rate of the dog heart by the multi-electrodes mapping system

The site of impulse origin in the right atrium is generally considered to be a single static locus within the sinoatrial (SA) node. However, it has been recognized that the pacemaker site may shift as a consequence of changes in physiological and pathological conditions. To determine the role of the atrial pacemaker complex including the superior pacemaker site, SA node and inferior pacemaker site quantitatively, we investigated atrial rate and the earliest activation region (EAR) from the isochronal activation sequence map by 48 unipolar electrodes and the mapping system. The epicardial activation sequence of the right atrium including the SA node region was obtained from the 48 electrodes, which were fixed to two flexible templates made of soft plastic plates. Using the mapping system, we found that (1) the EAR was shifted by the sympathetic or parasympathetic neural activity, (2) parasympathetic activity predominates over the sympathetic activity not only on heart rate, but also on the location of the EAR, and (3) the role of the inward Ca2+ current, hyperpolarization inward current and delayed rectifier K+ current, of the pacemaker cells distributed in the atrial pacemaker complex is different in the dog heart in situ.

Characteristics and distribution of M cells in arterially perfused canine left ventricular wedge preparations.

Much of the characterization of the M cell to date has been accomplished using isolated tissues and cells. This study uses an arterially perfused wedge preparation to examine the characteristics and distribution of M cells within the anterior wall of the canine left ventricle under more physiological conditions. Floating microelectrodes were used to record transmembrane action potentials simultaneously from epicardial, M region, and endocardial or subendocardial Purkinje sites in isolated arterially perfused canine left ventricular wedge preparations. A transmural ECG was recorded concurrently. M cells with the longest action potentials were found in the deep subendocardium in wedge preparations isolated from the anterior wall of the left ventricle. Fairly smooth transitions in action potential duration (APD) were observed except in the region between epicardium and deep subepicardium. Tissue resistivity increased 2.8-fold in this region and much more modestly in the deep subendocardium. Dispersion of APD90 across the left ventricular wall averaged 51+/-19 and 64+/-25 ms at basic cycle lengths of 1000 and 2000 ms, respectively, whereas transmural dispersion of repolarization time was smaller (34+/-18 and 45+/-25 ms), owing to the endocardial to epicardial activation sequence. We conclude that the qualitative differences between the 3 ventricular cell types previously described in isolated tissues and cells are maintained in intact canine left ventricular wall preparations in which the myocardial cells are electrically well coupled. As anticipated, differences in APD are quantitatively smaller because of electrotonic interactions among the 3 cell types. Our data indicate that transmural dispersion of repolarization is the result of intrinsic differences in APD of cells spanning the ventricular wall as well as a heterogeneous distribution of tissue resistivity across the wall.

Locally propagated activation immediately after internal defibrillation.

Electrical mapping studies indicate an interval of 40 to 100 ms between a defibrillation shock and the earliest activation that propagates globally over the ventricles (globally propagated activation, GPA). This study determined whether activation occurs during this interval but propagates only locally before being blocked (locally propagated activation, LPA). In five anesthetized pigs, the heart was exposed and a 504-electrode sock with 4-mm interelectrode spacing was pulled over the ventricles. Ten biphasic shocks of a strength near the defibrillation threshold (DFT) were delivered via intracardiac catheter electrodes, and epicardial activation sequences were mapped before and after attempted defibrillation. Local activation was defined as dV/dt < or =-0.5 V/s. Postshock activation times and wave-front interaction patterns were determined with an animated display of dV/dt at each electrode in a computer representation of the ventricular epicardium. LPAs were observed after 40 of the 50 shocks. A total of 173 LPA regions were observed, each of which involved 2+/-2 (mean+/-SD) electrodes. LPAs were observed after both successful and failed shocks but occurred earlier (P<.0001) after failed (35+/-8 ms) than successful (41+/-16 ms) shocks, although the times at which the GPA appeared were not significantly different. On reaching the LPA region, the GPA front either propagated through it (n=135) or was blocked (n=38). The time from the onset of the LPA until the GPA front propagated to reach the LPA region was shorter (P<.01) when the GPA front was blocked (32+/-12 ms) than when it propagated through the LPA region (63+/-20 ms). LPAs exist after successful and failed shocks near the DFT. Thus, the time from the shock to the GPA is not totally electrically silent.

Modulation of quinidine-induced arrhythmias by temperature in perfused rabbit heart.

We used low temperature to slow ion channel kinetics and studied the electrophysiological effects of quinidine at different pacing rates in isolated rabbit hearts. Fifteen epicardial electrograms together with an endocardial monophasic action potential were recorded. Epicardial activation and local recovery times were measured. Arrhythmias together with the characteristics of their mode of induction and rate were analyzed by epicardial activation sequence mapping. In the presence of quinidine, arrhythmias consistent with both triggered activity and reentry were observed. At baseline, triggered activity was not inducible, even though at 25 degrees C the recovery time was greater than that in the presence of quinidine at 36 degrees C. Also, with quinidine, the incidence of triggered activity decreased at 30 and 25 degrees C. Therefore prolongation of the recovery time per se does not cause triggered activity. Quinidine's use-dependent effects on conduction and reverse use-dependent effects on recovery time were amplified by low temperatures. These findings can be understood in terms of the known temperature sensitivities of the kinetics of the membrane ion channels responsible for activation and recovery. The results demonstrate that temperature can be used as a tool to elucidate mechanisms of drug action.