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Contactless magnetocardiographic study of age- and gender-related variability of ventricular repolarization parameters in guinea pigs
International Congress Series 1300 (2007) 443 – 446
www.ics-elsevier.com
Contactless magnetocardiographic study of age- and gender-related variability of ventricular repolarization parameters in guinea pigs ☆ D. Brisinda, M.E. Caristo, R. Fenici ⁎ Clinical Physiology–Biomagnetism Center, Catholic University of Sacred Heart, Rome, Italy
Abstract. Guinea pigs (GPs) are used for preclinical investigation of the ability of new drugs to induce QT alterations and arrhythmias. The aim of this study was to evaluate the accuracy of multisite magnetocardiographic mapping (MCG) for the assessment of age-related variation of ventricular repolarization (VR) maps in GPs. 18 adult GPs (8 males and 10 females) were investigated with an unshielded 36-channel MCG instrumentation, at the age of 5 months. Only 12 survived until the age of 14 months and were restudied. RR, PR, QRS, QTpeak, QTend, JTpeak, JTend and Tpeak-end intervals were measured from MCG waveforms. Magnetic field (MF) maps and the Equivalent Current Dipole (ECD) inverse solution were automatically computed. At the age of 5 months, average values of VR intervals were slightly longer in males. At the age of 14 months, a statistically significant age-related prolongation of the PR ( p b 0.05) and of the QRS ( p b 0.01) intervals was found. Neither significant age- nor gender-related variations of VR intervals were appreciable. Statistically significant ( p b 0.01) age-related differences were found for the JT α angle, Tpeak α angle and for the distance dynamics. At the same age, the strength of the ECD at the peak of the P, QRS and T waves, was stronger ( p b 0.01) than at 5 months. MCG is reliable for VR assessment in GPs. In contrast with findings in rats, age-related changes and gender-related differences of VR parameters were not statistically significant in GPs. More complex variability of MF patterns was observed, which deserves further investigation. © 2007 Published by Elsevier B.V. Keywords: Magnetocardiography; Cardiac mapping; Guinea pig; Ventricular repolarization; Aging
☆
Partially supported by MIUR grants 2001064829 and by Sigma-Tau Research Contract DS/2004/CR/#22. ⁎ Corresponding author. Largo A. Gemelli, 8, 00168, Rome, Italy. Tel.: +39 06 3051193; fax: +39 06 3051343. E-mail address: [email protected] (R. Fenici).
0531-5131/ © 2007 Published by Elsevier B.V. doi:10.1016/j.ics.2006.12.060
444
D. Brisinda et al. / International Congress Series 1300 (2007) 443–446
1. Introduction The guinea pig (GP) is frequently used for experimental evaluation of ventricular repolarization (VR) electrophysiology, since its myocytes have similar action potentials and ion currents as in the human [1]. For non-invasive assessment of VR inhomogeneity (i.e., QT/QTc interval prolongation and of its dispersion), 12-lead ECG [2] or body surface potential mapping (BSPM) [3] is needed. However BSPM is difficult to be performed [4], whereas magnetocardiographic mapping (MCG) is a feasible alternative for surface cardiac mapping of small animals, even conscious and unrestrained, not only in magnetically shielded rooms, but also with unshielded instrumentation [5,6]. This study was aimed: (1) to evaluate age-related changes of cardiac intervals, taking into account possible gender-related differences; (2) to create a normality database, useful for pharmacological studies and for the interpretation of abnormal MCG patterns in animal models with cardiomyopathy. 2. Methods A 36-channel DC-SQUID MCG system (CardioMag Imaging Inc.) was used for MCG (intrinsic sensitivity of 20 fT/√Hz) (Fig. 1A). Signal acquisition and post processing were carried out as detailed elsewhere [5]. A portable digital fluoroscopy system was used to acquire fluoroscopic images for off-line reconstruction of the animals' three-dimensional (3D) heart model (Fig. 1B and C). Signal analysis was carried out with the Windows NT-based CardioMag and with the UNIX-based Neuromag software packages [5,7]. 18 GPs [8 males (M) and 10 females
Fig. 1. (A) Overview of the laboratory set-up with the 36-channel MCG system (on the right) and the animal placed under the digital fluoroscopy. (B) A frontal view of the guinea pig: the over imposed three-dimensional heart was automatically reconstructed from bidimensional orthogonal fluoroscopic images. (C) X-ray opaque lead markers were matched with 3 MCG-system laser beams to define the position of the heart in respect of the recording sensors. In panel D (from top to bottom) averaged MCG waveforms and their “butterfly” superposition.
D. Brisinda et al. / International Congress Series 1300 (2007) 443–446
445
(F)] were studied under anesthesia (ketamine: 50 mg/kg b.w.; diazepam: 2 mg/kg b.w), in sinus rhythm and spontaneous breathing at the age of 5 months [weight: 279.3 ± 13.2 g (M), 259 ± 14.4 g (F)]. Six animals spontaneously died before the second MCG session; therefore only 12 (6 M and 6 F) were restudied at the age 14 months [weight: 636.6 ± 26.3 g (M), 552.5 ± 114 g (F)]. MCG lasted typically for 300 s and was repeated twice to test for reproducibility. The investigation was approved by the Catholic University Ethical Committee and conforms to the Guide for the Care and Use of laboratory Animals (NIH Publication # 85-23, revised 1996). The RR, PR, QRS length, QTpeak, QTend, JTpeak, JTend, Tpeak-end intervals were measured from the “butterfly” superposition of MCG signals (resolution of 2 cm/pT and 200 mm/s) (Fig. 1D), and corrected for heart rate (HR), according to the Bazett's formula [corrected value = measured value (ms) / v RR (s)]. Magnetic field distribution (MFD) maps were constructed with the time resolution of 1 ms. For the quantitative analysis of VR MFD, JTpeak magnetic field gradient (MFG) orientation, MF α angle (for JT segment and T wave peak) [7] and T wave MFD dynamics were calculated [5]. The orientation (degrees) and strength (μAm) of the Equivalent Current Dipole (ECD) were calculated at the peak of the P, QRS and T waves, after solution of the inverse problem [8]. Data are reported as mean ± standard deviation. Statistical analysis was performed with the paired Student's T-test ( p b 0.05 was considered significant). 3. Results MCG waveform of the GP was characterized by a well-defined JT interval and T wave. Average values of VR intervals (uncorrected and HR-corrected), measured at the age of 5 months, were slightly longer in males. At the age of 14 months, only cumulative age-related prolongation of the PR and of the QRS intervals was statistically significant (Table 1). MFD at the peak of the T wave varied inter-individually, at 5 months defining three main patterns. At 14 months, the MFD at the Tpeak remained unchanged in some animals, but markedly changed in others, with rotation of the dipolar T wave pattern, in the absence of variations of P and QRS MFD. As concerns the quantitative analysis of VR, at the age of 5 months, statistically significant ( p b 0.05) gender-related differences were found for Tpeak α angle and for the T wave distance dynamics. At the age of 14 months, statistically significant ( p b 0.01) age-related differences were found for the JT α angle, Tpeak α angle and for the distance dynamics.
Table 1 Age-related changes of cardiac intervals' duration (in milliseconds) (VR intervals are uncorrected)
5 months 14 months
PR
QRS
QTpeak
QTend
JTpeak
JTend
Tpeak-end
52.7 ± 7.1 58.2 ± 6.4⁎
21.1 ± 1.4 23.6 ± 2.4⁎⁎
114.3 ± 23 113.6 ± 24
135.3 ± 26 134.5 ± 22
93.1 ± 23 94 ± 23.7
113.9 ± 26 115 ± 22.3
20.7 ± 4.5 20.9 ± 3.9
⁎p b 0.05, ∧p b 0.001.
446
D. Brisinda et al. / International Congress Series 1300 (2007) 443–446
The strength of the ECD, measured at the peak of the P, QRS and T waves, was stronger at the age of 14 months as compared to the values measured at 5 months. 4. Discussion In this study MCG has proved feasible for longitudinal non-invasive investigation of cardiac intervals and of VR MFD maps, in GPs. As in rats [5], contactless MCG is easier, faster than ECG recording and might be a reliable method for follow-up. This is relevant for future studies of animal models of cardiomyopathy and for preclinical assessment of drug safety [2]. In contrast with findings in rats, age-related changes and gender-related differences of VR parameters were not statistically significant in the GP. Surely further works is deserved to understand the physiological meaning of unexpected VR MFD variability observed in healthy GPs, and to define on a larger population a normality database useful for the study of animal models with cardiomyopathy or the effect of new drugs. Since MCG can be performed also in the awake GPs [6] and repeated even several times in a day, it can be a non-invasive alternative to the implantation of telemetric devices, which might imply surgical complications and a considerable loss of animals available for longterm longitudinal studies [9]. References [1] J. Zeng, et al., Two components of the delayed rectifier K+ current in ventricular myocytes of the guinea pig type. Theoretical formulation and their role in repolarization, Circ. Res. 77 (1) (1995) 140–152. [2] ICH Draft Consensus Guideline (S7B), Safety Pharmacology Studies for Assessing the Potential for Delayed Ventricular Repolarization (QT Interval Prolongation) by Human Pharmaceuticals, 2002 http://www.ich.org/ MediaServer.jser?@_ID=505&@_MODE=GLB. [3] S. Havranek, et al., QT dispersion estimated from 80 body surface potential map leads and from standard 12leads ECG in psychiatric patients treated with dosulepin, Prague Med. Rep. 105 (1) (2004) 53–63. [4] M. Bernadic, L. Zlatos, Cardioelectrical field in experimental cardiomegaly in rats, Bratisl. Lek. Listy 97 (9) (1996) 543–549. [5] D. Brisinda, M.E. Caristo, R. Fenici, Contactless magnetocardiographic mapping in anaesthetized Wistar Rats: evidence of age-related changes of cardiac electrical activity, Am. J. Physiol, Heart Circ. Physiol. 291 (1) (2005) H368–H378. [6] U. Steinhoff, et al., Magnetocardiography for pharmacology safety studies requiring high patient throughput and reliability, J. Electrocardiol. 37 (2004) 187–192 (Suppl). [7] H. Hänninen, et al., Detection of exercise induced myocardial ischemia by multichannel magnetocardiography in patients with single vessel coronary artery disease, Ann. Noninvasive Electrocardiol. 5 (2000) 147–157. [8] J. Nenonen, Magnetocardiography, in: J. Clarke, A. Braginski (Eds.), SQUID Handbook, Berlin Whiley-VCH, 2005. [9] G. Provan, et al., Development of a surgical approach for telemetering guinea pigs as a model for screening QT interval effects, J. Pharmacol. Toxicol. Methods 52 (2005) 223–228.
www.ics-elsevier.com
Contactless magnetocardiographic study of age- and gender-related variability of ventricular repolarization parameters in guinea pigs ☆ D. Brisinda, M.E. Caristo, R. Fenici ⁎ Clinical Physiology–Biomagnetism Center, Catholic University of Sacred Heart, Rome, Italy
Abstract. Guinea pigs (GPs) are used for preclinical investigation of the ability of new drugs to induce QT alterations and arrhythmias. The aim of this study was to evaluate the accuracy of multisite magnetocardiographic mapping (MCG) for the assessment of age-related variation of ventricular repolarization (VR) maps in GPs. 18 adult GPs (8 males and 10 females) were investigated with an unshielded 36-channel MCG instrumentation, at the age of 5 months. Only 12 survived until the age of 14 months and were restudied. RR, PR, QRS, QTpeak, QTend, JTpeak, JTend and Tpeak-end intervals were measured from MCG waveforms. Magnetic field (MF) maps and the Equivalent Current Dipole (ECD) inverse solution were automatically computed. At the age of 5 months, average values of VR intervals were slightly longer in males. At the age of 14 months, a statistically significant age-related prolongation of the PR ( p b 0.05) and of the QRS ( p b 0.01) intervals was found. Neither significant age- nor gender-related variations of VR intervals were appreciable. Statistically significant ( p b 0.01) age-related differences were found for the JT α angle, Tpeak α angle and for the distance dynamics. At the same age, the strength of the ECD at the peak of the P, QRS and T waves, was stronger ( p b 0.01) than at 5 months. MCG is reliable for VR assessment in GPs. In contrast with findings in rats, age-related changes and gender-related differences of VR parameters were not statistically significant in GPs. More complex variability of MF patterns was observed, which deserves further investigation. © 2007 Published by Elsevier B.V. Keywords: Magnetocardiography; Cardiac mapping; Guinea pig; Ventricular repolarization; Aging
☆
Partially supported by MIUR grants 2001064829 and by Sigma-Tau Research Contract DS/2004/CR/#22. ⁎ Corresponding author. Largo A. Gemelli, 8, 00168, Rome, Italy. Tel.: +39 06 3051193; fax: +39 06 3051343. E-mail address: [email protected] (R. Fenici).
0531-5131/ © 2007 Published by Elsevier B.V. doi:10.1016/j.ics.2006.12.060
444
D. Brisinda et al. / International Congress Series 1300 (2007) 443–446
1. Introduction The guinea pig (GP) is frequently used for experimental evaluation of ventricular repolarization (VR) electrophysiology, since its myocytes have similar action potentials and ion currents as in the human [1]. For non-invasive assessment of VR inhomogeneity (i.e., QT/QTc interval prolongation and of its dispersion), 12-lead ECG [2] or body surface potential mapping (BSPM) [3] is needed. However BSPM is difficult to be performed [4], whereas magnetocardiographic mapping (MCG) is a feasible alternative for surface cardiac mapping of small animals, even conscious and unrestrained, not only in magnetically shielded rooms, but also with unshielded instrumentation [5,6]. This study was aimed: (1) to evaluate age-related changes of cardiac intervals, taking into account possible gender-related differences; (2) to create a normality database, useful for pharmacological studies and for the interpretation of abnormal MCG patterns in animal models with cardiomyopathy. 2. Methods A 36-channel DC-SQUID MCG system (CardioMag Imaging Inc.) was used for MCG (intrinsic sensitivity of 20 fT/√Hz) (Fig. 1A). Signal acquisition and post processing were carried out as detailed elsewhere [5]. A portable digital fluoroscopy system was used to acquire fluoroscopic images for off-line reconstruction of the animals' three-dimensional (3D) heart model (Fig. 1B and C). Signal analysis was carried out with the Windows NT-based CardioMag and with the UNIX-based Neuromag software packages [5,7]. 18 GPs [8 males (M) and 10 females
Fig. 1. (A) Overview of the laboratory set-up with the 36-channel MCG system (on the right) and the animal placed under the digital fluoroscopy. (B) A frontal view of the guinea pig: the over imposed three-dimensional heart was automatically reconstructed from bidimensional orthogonal fluoroscopic images. (C) X-ray opaque lead markers were matched with 3 MCG-system laser beams to define the position of the heart in respect of the recording sensors. In panel D (from top to bottom) averaged MCG waveforms and their “butterfly” superposition.
D. Brisinda et al. / International Congress Series 1300 (2007) 443–446
445
(F)] were studied under anesthesia (ketamine: 50 mg/kg b.w.; diazepam: 2 mg/kg b.w), in sinus rhythm and spontaneous breathing at the age of 5 months [weight: 279.3 ± 13.2 g (M), 259 ± 14.4 g (F)]. Six animals spontaneously died before the second MCG session; therefore only 12 (6 M and 6 F) were restudied at the age 14 months [weight: 636.6 ± 26.3 g (M), 552.5 ± 114 g (F)]. MCG lasted typically for 300 s and was repeated twice to test for reproducibility. The investigation was approved by the Catholic University Ethical Committee and conforms to the Guide for the Care and Use of laboratory Animals (NIH Publication # 85-23, revised 1996). The RR, PR, QRS length, QTpeak, QTend, JTpeak, JTend, Tpeak-end intervals were measured from the “butterfly” superposition of MCG signals (resolution of 2 cm/pT and 200 mm/s) (Fig. 1D), and corrected for heart rate (HR), according to the Bazett's formula [corrected value = measured value (ms) / v RR (s)]. Magnetic field distribution (MFD) maps were constructed with the time resolution of 1 ms. For the quantitative analysis of VR MFD, JTpeak magnetic field gradient (MFG) orientation, MF α angle (for JT segment and T wave peak) [7] and T wave MFD dynamics were calculated [5]. The orientation (degrees) and strength (μAm) of the Equivalent Current Dipole (ECD) were calculated at the peak of the P, QRS and T waves, after solution of the inverse problem [8]. Data are reported as mean ± standard deviation. Statistical analysis was performed with the paired Student's T-test ( p b 0.05 was considered significant). 3. Results MCG waveform of the GP was characterized by a well-defined JT interval and T wave. Average values of VR intervals (uncorrected and HR-corrected), measured at the age of 5 months, were slightly longer in males. At the age of 14 months, only cumulative age-related prolongation of the PR and of the QRS intervals was statistically significant (Table 1). MFD at the peak of the T wave varied inter-individually, at 5 months defining three main patterns. At 14 months, the MFD at the Tpeak remained unchanged in some animals, but markedly changed in others, with rotation of the dipolar T wave pattern, in the absence of variations of P and QRS MFD. As concerns the quantitative analysis of VR, at the age of 5 months, statistically significant ( p b 0.05) gender-related differences were found for Tpeak α angle and for the T wave distance dynamics. At the age of 14 months, statistically significant ( p b 0.01) age-related differences were found for the JT α angle, Tpeak α angle and for the distance dynamics.
Table 1 Age-related changes of cardiac intervals' duration (in milliseconds) (VR intervals are uncorrected)
5 months 14 months
PR
QRS
QTpeak
QTend
JTpeak
JTend
Tpeak-end
52.7 ± 7.1 58.2 ± 6.4⁎
21.1 ± 1.4 23.6 ± 2.4⁎⁎
114.3 ± 23 113.6 ± 24
135.3 ± 26 134.5 ± 22
93.1 ± 23 94 ± 23.7
113.9 ± 26 115 ± 22.3
20.7 ± 4.5 20.9 ± 3.9
⁎p b 0.05, ∧p b 0.001.
446
D. Brisinda et al. / International Congress Series 1300 (2007) 443–446
The strength of the ECD, measured at the peak of the P, QRS and T waves, was stronger at the age of 14 months as compared to the values measured at 5 months. 4. Discussion In this study MCG has proved feasible for longitudinal non-invasive investigation of cardiac intervals and of VR MFD maps, in GPs. As in rats [5], contactless MCG is easier, faster than ECG recording and might be a reliable method for follow-up. This is relevant for future studies of animal models of cardiomyopathy and for preclinical assessment of drug safety [2]. In contrast with findings in rats, age-related changes and gender-related differences of VR parameters were not statistically significant in the GP. Surely further works is deserved to understand the physiological meaning of unexpected VR MFD variability observed in healthy GPs, and to define on a larger population a normality database useful for the study of animal models with cardiomyopathy or the effect of new drugs. Since MCG can be performed also in the awake GPs [6] and repeated even several times in a day, it can be a non-invasive alternative to the implantation of telemetric devices, which might imply surgical complications and a considerable loss of animals available for longterm longitudinal studies [9]. References [1] J. Zeng, et al., Two components of the delayed rectifier K+ current in ventricular myocytes of the guinea pig type. Theoretical formulation and their role in repolarization, Circ. Res. 77 (1) (1995) 140–152. [2] ICH Draft Consensus Guideline (S7B), Safety Pharmacology Studies for Assessing the Potential for Delayed Ventricular Repolarization (QT Interval Prolongation) by Human Pharmaceuticals, 2002 http://www.ich.org/ MediaServer.jser?@_ID=505&@_MODE=GLB. [3] S. Havranek, et al., QT dispersion estimated from 80 body surface potential map leads and from standard 12leads ECG in psychiatric patients treated with dosulepin, Prague Med. Rep. 105 (1) (2004) 53–63. [4] M. Bernadic, L. Zlatos, Cardioelectrical field in experimental cardiomegaly in rats, Bratisl. Lek. Listy 97 (9) (1996) 543–549. [5] D. Brisinda, M.E. Caristo, R. Fenici, Contactless magnetocardiographic mapping in anaesthetized Wistar Rats: evidence of age-related changes of cardiac electrical activity, Am. J. Physiol, Heart Circ. Physiol. 291 (1) (2005) H368–H378. [6] U. Steinhoff, et al., Magnetocardiography for pharmacology safety studies requiring high patient throughput and reliability, J. Electrocardiol. 37 (2004) 187–192 (Suppl). [7] H. Hänninen, et al., Detection of exercise induced myocardial ischemia by multichannel magnetocardiography in patients with single vessel coronary artery disease, Ann. Noninvasive Electrocardiol. 5 (2000) 147–157. [8] J. Nenonen, Magnetocardiography, in: J. Clarke, A. Braginski (Eds.), SQUID Handbook, Berlin Whiley-VCH, 2005. [9] G. Provan, et al., Development of a surgical approach for telemetering guinea pigs as a model for screening QT interval effects, J. Pharmacol. Toxicol. Methods 52 (2005) 223–228.