ICH S7A and S7B guidelines recommend the use of conscious animals for assessment of non-clinical cardiovascular safety of new chemical entities prior to testing in humans. There is growing interest in non-invasive ECG recording in rodents to enable an early cardiac assessment thereby possibly reducing later stage development attrition (1 ).
Mice in particular have become essential in pharmacological and toxicological research as the development of genetically modified models of human disorders has rapidly expanded. Surgically-implanted radiotelemetric implants for ECG monitoring are expensive and require not only surgery but also a protracted recovery period before the animals regain their pre-operative weight; up to 3 weeks has been reported for mice (2). The ECGenie and companion EzCG analysis software provide safety pharmacology excellent non-invasive means for recording and analyzing ECG from conscious mice (3 ), rats (4 ), hamsters (5), and guinea pigs (6).
The ECGenie provides significant advantages over implants for essentially the same data, less the opportunities potentially inherent in continuous monitoring. The “when and which data” to curate for rodent ECG analysis via telemetry significantly impacts the results as it is usually challenging and not typical for researchers to correlate the ECG recordings with animal behavior. For example, the heart rate (HR) of a mouse resting quietly is much lower than the HR of the same mouse active in its cage in the dark period. These differences could potentially obfuscate effects of drugs on the QTc. The ECGenie instrumentation is able to noninvasively record ECG without telemetric implants, allowing for quicker and easier ECG monitoring. The animal is simply placed on the patented ECGenie platform, whereby the cardiac signal is detected passively via the ventral surface of the paws. With the ECGenie, all of the animals are studied under essentially the same behavioral conditions, so that all of the data can be interpreted independent of animal activity, time of day, etc. Key advantages of the ECGenie also include significantly lower instrumentation cost, significantly lower operating costs, significantly improved animal welfare, and significantly enhanced lab animal personnel safety and satisfaction. There are no risks of surgical accidents and the allegiances to the mantra of the 3Rs (Replacement, Refinement, and Reduction of Animals in Research, http://www.nc3rs.org.uk) are fairly obvious.
Figure 1: Left panel: Mice resting atop of the ECGenie recording platform; representative signal identified on monitor in background. Right panel: screen shot of raw ECG signals acquired from a mouse resting atop of the ECGenie. Continuous recordings of +20 sec are routinely recorded and stored for analysis.
Long periods of data collection are routinely collected and analyzed via the ECGenie. Continuous recordings of ~20-30 sec in mice provide hundreds of complexes for analysis. While the mean QRS interval duration may not be different in a brief 2-3 sec recording compared to the value from a longer 20-30 sec recording, metrics of heart rate variability (HRV) are more robust with a greater collection of signals for analysis. Beat-to-beat variability of repolarization has been proposed as a new biomarker for proarrhythmia in vivo (7). The ECGenie and EzCG analysis software reports numerous beat-to-beat variability values, including QT interval duration variability. Power spectral analysis of HRV from data derived from ECG recordings has been shown to be applicable in cardiovascular safety pharmacology studies (8) and may provide relevant information about possible drug interaction with the autonomic nervous system. The ECGenie and EzCG analysis software provides the power spectrum and automatically reports total power, the low and high frequency components, and the normalized LF and HF.
Figure 2: HRV depicted via power spectra. On left, normal/baseline. On right, intervention shifted the dominant contribution to the parasympathetic end.
Via the EzCG analysis software, analysis of an ECG recording of ~20 sec with ~200 complexes is fast – usually takes <10 sec – providing a complete tabulation of all of the ECG interval durations, and HRV, both in the time and the frequency domains. A complete electrocardiographic study of 7 mice, at baseline and after drug administration – say the acute effects of isoproterenol – takes ~<60 min, including data acquisition, data analysis, and tabulation of results.
For safety pharmacology, an important objective of animal monitoring is to ensure that the models accurately provide insight into the pharmacological action of a particular dosages intended for human patients. Safety pharmacology must detect the potential threat of adverse effects, safety margin calculations, as well as clinical safety monitoring (9). It is therefore important for the instrumentation used to monitor ECG within the safety pharmacology setting to be accurate and precise. The ECGenie can provide hundreds of ECG complexes of excellent quality from thousands of conscious animals yearly – non-invasively. Rodent ECG data have been criticized for noise obfuscating the morphology such as the P wave. The EzCG analysis software’s robust algorithms are able to rectify and identify the P-wave and identify the return to the isoelectric line beat-to-beat, also generating an ensemble averaged waveform depicting the signal morphology. The advantages of quickly obtaining large amounts of reliable data from large cohorts of conscious mice and translating these findings to humans should significantly mitigate late state attrition of potentially good drugs.
Figure 3. Ensemble averaged ECG waveform derived from ~200 complexes from a conscious B6 mouse at baseline. The PQRST morphology is clearly identifiable; the return of each individual complex to the isoelectric line is determined beat-by-beat to establish QT interval duration.
For example, moxifloxacin is a flouroquinolone antibiotic known to increase the QT interval (10). It is recommended as a positive control for evaluating the propensity of new drugs to have adverse cardiac effects. Studies of the effects of moxifloxacin in rodents, however, are few. Significant decreases in HR were detected via the blood pressure signals in diabetic rats treated with moxifloxacin (11). Yet, the researchers reported no significant alteration in QRS interval in perfused hearts explanted from diabetic rats treated with moxifloxacin, though their data indicated a robust decrease [~30%] in the QRS interval duration; the nuances [coronary perfusion, temperature control, ionic composition of buffer solution] of the Langendorff preparation can make ECG measurements in vitro challenging to interpret. A recent report provides the first unambiguous evidence that preclinical in vivo repolarization assays, when accurately modeled and evaluated, yield results that are consistent with the conservation of moxifloxacin-induced QT prolongation in dog, cynomolgus, minipig, marmoset, and guinea pig (12). It remains to be seen if ECG recordings in conscious mice can also detect moxifloxacin-induced increases in the QT interval, but the observations in moxifloxacin-treated rats (11), taken together, suggest that indeed QT interval is likely prolonged in mice treated with moxifloxacin, making this assay a useful standard control against which other compounds for QT lability can be compared. Considering the notion that the ionic mechanisms of repolarization in mice differ from larger species, including humans, selection of the appropriate strain of mouse for ECG measurements is important; there are, however, thousands of mouse strains. As shown in the Mouse Phenome Database (http://phenome.jax.org) curated by The Jackson Laboratory (http://jax.org), there is a wide spectrum of strain differences HR, HRV, and ECG interval durations, including QT interval among commonly used inbred strains. Accordingly, B6 is a good strain for evaluating the effects of new chemical entities on prolonging the QT interval and other electrocardiographic disturbances in mice via ECGenie.
- Tontodonati M., Fasdelli N., Dorigatti R. An improved method of electrode placement in configuration Lead II for the reliable ECG recording by telemetry in the conscious rat. J Pharmacol Toxicol Methods. 2011 Jan-Feb;63(1):1-6.
- Stiedl O., Spiess J. Effect of tone-dependent fear conditioning on heart rate and behavior of C57BL/6N mice. Behav Neurosci. 1999;111:703–711.
- Chu V., et al. Electrocardiographic findings in mdx mice: a cardiac phenotype of Duchenne muscular dystrophy. Muscle Nerve. 2002 Oct;26(4):513-9.
- Lachance D., et al. Moderate exercise training improves survival and ventricular remodeling in an animal model of left ventricular volume overload. Circ Heart Fail. 2009 Sep;2(5):437-45.
- Hampton T.G., et al. Developmental Changes in the ECG of a Hamster Model of Muscular Dystrophy and Heart Failure. Front Pharmacol. 2012 May 18;3:80.
- Harbert K., et al. Using the Conscious Guinea Pig to Evaluate human Ether-à-go-go Related Gene (hERG) Channel Blockers. 2012 AALAS QUAD Symposium Proceedings.
- Floré V., Willems R. T-wave alternans and beat-to-beat variability of repolarization: pathophysiological backgrounds and clinical relevance. Acta Cardiol. 2012 Dec;67(6):713-8.
- Champeroux P., et al. Power spectral analysis of heart rate variability in cynomolgus monkeys in safety pharmacology studies: Comparative study with beagle dogs. J Pharmacol Toxicol Methods. 2013 Jul-Aug;68(1):166-74.
- Pugsley M.K., Authier S., Curtis M.J. Principles of safety pharmacology. Br J Pharmacol. 2008;154:1382–1399.
- Chen X. , et al. QT prolongation and proarrhythmia by moxifloxacin: concordance of preclinical models in relation to clinical outcome. Br J Pharmacol. 2005 Nov;146(6):792-9.
- Absi M., et al. The effect of levofloxacin and moxifloxacin on cardiovascular functions of rats with streptozotocin-induced diabetes. Diab Vasc Dis Res. 2013 Jan;10(1):65-71.
- Holzgrefe H., et al. Preclinical QT safety assessment: Cross-species comparisons and human translation from an industry consortium. J Pharmacol Toxicol Methods. 2013 May 17.