Author + information
- Received April 19, 2006
- Revision received June 30, 2006
- Accepted July 10, 2006
- Published online February 27, 2007.
- Partho P. Sengupta, MBBS, MD, DM⁎,
- Bijoy K. Khandheria, MBBS, FACC†,
- Josef Korinek, MD⁎,
- Arshad Jahangir, MD⁎,
- Shiro Yoshifuku, MD⁎,
- Ilija Milosevic, PhD‡ and
- Marek Belohlavek, MD, PhD, FACC⁎,⁎ ()
- ↵⁎Reprint requests and correspondence:
Dr. Marek Belohlavek, Division of Cardiovascular Diseases, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905.
Objectives We sought to clarify the role of isovolumic intervals during a cardiac cycle by in vivo visualization of left ventricular (LV) intracavitary flow dynamics.
Background Asynchronous LV deformation during isovolumic contraction (IVC) and isovolumic relaxation (IVR) might represent a transient feature of myocardial wall mechanics that reverses the direction of blood flow.
Methods In 10 beating porcine hearts, the changes in LV intracavitary flow were recorded at baseline and after LV epicardial and right atrial pacing with high-resolution Doppler and contrast echocardiography. Two-dimensional vector flow fields were generated offline from B-mode contrast images with particle imaging velocimetry.
Results During IVC, flow from the LV apex accelerated toward the base, whereas blood from the base was redirected toward the outflow through formation of an anterior vortex. Conversely, during IVR, flow was initially directed toward the apex and then briefly reversed toward the base. Epicardial pacing from the LV base altered the stages of flow redirection during the pre-ejection period and delayed mitral valve closure (28 ± 14 ms vs. 61 ± 13 ms, p < 0.001) and aortic valve opening (77 ± 18 ms vs. 111 ± 18 ms, p = 0.004).
Conclusions Isovolumic intervals are not periods of hemodynamic stasis but, rather, phases with dynamic changes in intracavitary flow. Experimentally induced aberrant epicardial electrical activation alters stages of flow redirection and prolongs the pre-ejection period. Normal electromechanical activation through the His-Purkinje system in mammalian hearts maintains an inherent synchrony with the sequence of intracavitary flow redirection.
The left ventricle (LV) in a mammalian heart cyclically carries out the functions of suction and ejection, transiting functionally through short-lived phases known as isovolumic intervals (1). Myocardial disease or altered myocardial electrical activation primarily prolongs the isovolumic intervals, with either no significant change or a shortening of ejection and filling times (2–4).
At the cellular level, the isovolumic intervals are associated with active fluxes in myoplasmic and sarcolemmal calcium that either initiate or reverse interactions between cardiac myofilaments (5). At the tissue level, isovolumic intervals are associated with asynchronous movements of the subendocardial and subepicardial regions (6). During LV isovolumic contraction (IVC), the subendocardial fibers that form a right-handed helix shorten, whereas the left-handed helically-oriented subepicardial fibers lengthen simultaneously. Conversely, during isovolumic relaxation (IVR), the subepicardial fibers that form the left-handed helix lengthen, whereas the right-handed helically-directed subendocardial fibers shorten briefly. Brief asynchronous deformations of the LV might represent a “flow-directing feature” of the myocardial wall mechanics that reverses the direction of blood flow (7).
Conventionally, the timing of mitral valve closure divides the pre-ejection period into 2 composite intervals (8). The interval from the onset of the Q-wave on surface electrocardiography to mitral valve closure is referred to as “electromechanical delay,” whereas isovolumic contraction is the period that follows mitral valve closure and is characterized by a rapid rise in LV pressure before opening of the aortic valve. Interest has recently grown in the pre-ejection period as a marker of ventricular dyssynchrony in patients with severe heart failure (8). However, fundamental hemodynamic changes and features of intracavitary flow that differentiate the phases of the pre-ejection period from the adjacent phases of cardiac cycle remain poorly characterized. Previous investigations have used echocardiography and magnetic resonance velocity mapping of blood circulation for deciphering the features of LV intracavitary blood flow patterns (9,10). An intriguing aspect of this flow is the occurrence of intracavitary vortices (10). Findings from in vitro experiments suggested that strong vortices would be generated during the pre-ejection period near the LV outflow; without these vortices, the mitral valve would remain open at the onset of ventricular contraction (11). However, the time-resolved 2-dimensional (2D) features of intracavitary flow during isovolumic periods have not been visualized directly in vivo.
The aims and objectives of the present investigation were: 1) to directly visualize intracavitary flow in vivo during isovolumic periods with a high-temporal resolution 2D imaging technique that helps distinguish features of isovolumic flow from the other phases of cardiac cycles; and 2) to learn whether electromechanical interventions that alter the mechanical events during a cardiac cycle also change features of flow during the pre-ejection period and alter the timing of mitral valve and aortic valve opening and closing.
Ten adolescent pigs weighing 50 to 60 kg were anesthetized with an infusion of ketamine, fentanyl, and etomidate. All animals received humane treatment in compliance with the Guide for the Care and Use of Laboratory Animals (12). In addition, the experiments were approved by the Mayo Foundation Institutional Animal Care and Use Committee. First, in 2 pigs, we investigated the optimal dose of contrast infusion and the experimental settings that would yield maximum particle imaging velocimetry (PIV) vectors at the highest temporal resolution. Following standardization of the protocol, flow Doppler, echo contrast, and PIV data from the remaining 8 pigs were included for the final analysis.
After the sternotomy, a pericardial cradle was constructed. Introducer sheaths (SciMed Life Systems, Maple Grove, Minnesota) were placed in both common carotid arteries, in both internal jugular veins, and in the left femoral artery for obtaining blood samples, infusing anesthesia and fluids, monitoring blood pressure, and inserting catheters. Three manometer-tipped catheters (Millar Instruments, Inc., Houston, Texas) were placed: 1 each into the LV, aorta, and left atrium.
Color Doppler echocardiography
Color Doppler echocardiographic images of the LV were recorded at more than 200 frames/s with the apical long-axis view (Vivid 7, GE Healthcare, Milwaukee, Wisconsin). A Doppler sector, 2-cm-deep and 30° wide, was placed over the mitral inflow and outflow regions of the LV. The changes in direction of blood flow along the axial direction of the LV, as indicated by changes in Doppler color flow, were timed with aortic and mitral valve movements with offline anatomical M-mode imaging. Mean Doppler velocity tracings were obtained over a circular region of interest 20 mm in diameter placed within the Doppler sector at the LV base. Magnitude and timing of peak positive and negative velocities were measured during isovolumic, ejection, and early diastolic filling periods.
The contrast agent used in this study was a perfluoropropane gas-filled, lipid-stabilized microbubble (Definity, Bristol-Myers Squibb Medical Imaging, Inc., North Billerica, Massachusetts). After being shaken with an agitator for 45 s, the agent was infused intravenously at about 0.01 to 0.03 ml/min. B-mode images of intraventricular flow were obtained at a mechanical index of 0.1 to 0.4, with the apical long-axis view at more than 200 frames/s. Smaller sector areas (<45°) enabled a higher temporal resolution. Contrast echocardiographic M-mode of the LV was performed with the M-mode cursor placed parallel to the long axis of the LV.
Particle imaging velocimetry uses pairs of sequential digital images for calculating the direction and magnitude of fluid flow (11,13,14). Displacement of the particles in the second image, relative to the position of the particles in the first image, is a measure of the motion of the fluid. In the present study, PIV images were analyzed with a cross-correlation interrogation algorithm combined with fast Fourier transformation (INSIGHT, TSI Inc., Shoreview, Minnesota). The interrogation spot size was 32 × 32 pixels with 50% overlap. Maximal particle displacement was <5 pixels during the first interrogation and adjusted until optimal PIV tracking was achieved. The pixel size was directly available from the Echopac software (GE Healthcare) in Cartesian coordinates and was the same in both horizontal and vertical axes, measuring between 0.20 and 0.28 mm. The deformation grid engine was used because of high-velocity gradients in the flow. This algorithm deforms the grid according to the previous vector field results; hence, all particle displacements within an interrogation spot are at the same location after the deformation is performed. The first processing pass calculates the vector displacement using cross-correlation with or without interrogation spot offset. The second processing pass offsets the spots for the integer pixel value, found during the first pass, in the region of high-velocity gradients. In the third pass, the 4 neighboring vectors in the spot corners were used to deform the spot. This method helps restore elongated and splintered peaks to a Gaussian appearance and creates a more accurate flow field with more good vectors than in a regular undeformed grid. To find subpixel displacement, we used 3 points, 2 directions, and 1-dimensional Gaussian peak fitting. The PIV data were analyzed frame by frame (time-resolved analysis) and also averaged for each cardiac cycle phase (time averaged or ensemble averaged).
Bipolar electrical activation
Two bipolar electrodes were positioned in the anterior wall of the LV midway between the LV apex and base, 1 in the subendocardial, and the other in the subepicardial region. Atrial activity was recorded with a third bipolar electrode placed over the surface of the right atrium. Surface electrocardiography, intracardiac pressures, and bipolar regional myocardial electrical potential were recorded simultaneously at baseline (sinus rhythm) and after right atrial and LV epicardial pacing (Sonometrics Corp., London, Ontario, Canada). Heart rate and pressure data were measured and averaged over 3 continuous cardiac cycles for each sampling period.
Right atrial pacing (n = 6) was performed with a transvenous lead placed inside the right atrium just beyond the opening of the superior vena cava. The LV epicardial pacing (n = 6) was performed through surface electrodes placed at the LV base 0.5 to 1 cm lateral and inferior to the bifurcation of the left main coronary artery. Stimulation parameters (voltage 10% above threshold, duration 8 ms, and frequency) were kept constant in each animal. Pacing was performed by stimulating with a square-wave, constant-voltage electronic stimulator (Medtronic, Inc., Houston, Texas) at 10 to 20 beats above the baseline heart rate to suppress the native sinus rhythm. All measurements were performed after confirming a sustained 1:1 capture for at least 10 beats. The first 3 beats of the paced rhythm and nonejecting beats were excluded, and measurements were averaged for 3 successive paced beats.
The pre-ejection period was defined as the duration between the onset of the QRS complex on surface electrocardiography and the echocardiographic timing of the opening of the aortic valve (8). During normal sinus rhythm, the pre-ejection period included the IVC period, defined as the duration between mitral valve closure and aortic valve opening, and the period from the onset of the QRS complex to mitral valve closure. The ejection period was defined as the duration between aortic valve opening and closing, and IVR was defined as the duration between aortic valve closing and mitral valve opening.
All data are expressed as mean ± SD. Linear regression and Bland and Altman analysis (15) were used to compare the longitudinal velocities obtained by Doppler and PIV. Hemodynamic, anatomical M-mode, 2D echocardiographic, and PIV variables at baseline and after right atrial pacing and epicardial pacing were compared by use of a 2-tailed paired ttest. To ensure that results of the paired ttest were not being influenced by outliers, we also used the Wilcoxon signed rank test for confirming the presence of statistical significance. A p value of <0.05 was considered statistically significant.
Characterization of intracavitary flow with Doppler echocardiography
During sinus rhythm, initial Doppler velocities in the pre-ejection period were directed from the apex toward the LV base (Fig. 1).After the mitral leaflets closed, the direction of flow in the submitral region reversed briefly toward the apex before accelerating again in the apex-to-base direction, which further continued into the period of ejection. During IVR, the flow was bidirectional: an initial postejection flow in the base-to-apex direction was followed by a brief apex-to-base flow. During early and late diastolic filling, ventricular filling continued in the base-to-apex direction, with an intervening period of diastasis that had bidirectional shifts in the flow pattern.
Time-resolved contrast echocardiography
To track complex spatial features of intracavitary flow, we opacified the LV cavity with a low concentration of contrast microbubbles and captured 2D ultrasonographic cine loops at more than 200 frames/s (range 204 to 280 frames/s) (Video 1[see Appendix]). The consistency of this approach with the conventional Doppler flow technique was ensured by obtaining anatomical M-mode images from the 2D scans along the central scanning axis. This acquisition allowed comparison of the axial movement of contrast bubbles with Doppler flow changes (Fig. 1). The flow paths of contrast bubbles appeared as streaks in the dark blood pool. Both speed and direction of blood flow influenced the shape and length of these streaks as the bubbles were temporarily followed within the ultrasonic scan plane.
The apex-to-base redirection of blood flow during a cardiac cycle occurs during intervals when the streak lines become curvilinear. After the QRS wave on surface electrocardiography, contrast bubbles at the LV apex started moving toward the base. The onset of redirection was followed by closure of the mitral valve leaflets. During IVC, the apex-to-base drift of contrast bubbles continued further, merging with the flow from the submitral region, where bubbles moved along a curved trajectory, initially curving downward into the cavity and subsequently streaming toward the outflow. Ejection was characterized by continuous apex-to-base movement of contrast bubbles. When IVR began, the flow reversed, with an initial base-to-apex movement followed by a brief apex-to-base flow. During early and late diastolic phases, a large flow surged from the mitral valve toward the LV apex.
Echo contrast particle imaging velocimetry
High frame rates allowed tracking of bubbles sufficiently to determine the 2D component of local vectors of blood motion before the bubbles moved out of the scan plane (Video 2[see Appendix]). Figure 2shows the time-averaged features of LV blood flow during each phase of the cardiac cycle. The time-resolved features of LV flow during the pre-ejection period and during IVR are shown in Figures 3 and 4.⇓⇓
During the initial pre-ejection period, contrast bubbles from the apical region were directed over the outflow, whereas the contrast bubbles moving from the submitral region formed a single large vortex from the inflow toward the outflow (Video 3[see Appendix]). The continued formation of the vortex after mitral valve closure coincided with bidirectional Doppler flow signals during IVC. Contrast bubble movement during ejection was at a higher velocity and accelerated toward the outflow. The IVR produced a base-to-apex drift in contrast bubbles followed by a brief apex-to-base reversal. Early diastolic flow across the mitral valve resulted in formation of a large anterior vortex and a smaller posterior vortex across the anterior and posterior mitral valve leaflets. The vortical swirl continued in the submitral region during diastasis, resulting in approximation of the mitral leaflets during diastasis. This was followed by a late filling phase of atrial contraction characterized by a base-to-apex drift of contrast bubbles and continued formation of a large anterior vortex that subsequently merged with the apex-to-base flow during IVC (Video 3[see Appendix]).
Effects of aberrant electromechanical activation
Cardiac time intervals and hemodynamic status at baseline and after epicardial and right atrial stimulation are shown in Tables 1 and 2.⇓⇓Changes in intracardiac flow velocities were registered with both Doppler and PIV techniques and showed good correlation (r = 0.90, p < 0.001) (supplementary Fig. 1[see Appendix]). During epicardial pacing, bipolar electrodes in the subendocardial and subepicardial regions confirmed the presence of a subendocardial-to-subepicardial dispersion of electrical activation (supplementary Fig. 2[see Appendix]). There was a delay in the redirection of blood flow and a delay in reaching peak longitudinal velocities directed toward the LV base and outflow (52 ± 17 ms vs. 88 ± 30 ms, p = 0.04) (Figs. 5 and 6).⇓⇓A base-to-apex instead of the normal apex-to-base direction of pre-ejection flow resulted in turbulence and loss of a well-organized vortex formation across the inflow-outflow region of the LV (Videos 4 and 5[see Appendix]). Similar changes were not recorded during right atrial pacing.
In 1921, Carl Wiggers (16) defined the ventricular isometric and expulsive phases with intracardiac pressure tracings and provided understanding regarding hemodynamic events during phases of the cardiac cycle. Knowledge about the transition between cardiac filling and ejection, however, has been revised over the years, initially from “isometric” to “isovolumic,” and has been recently challenged further by intriguing observations that reveal the presence of physiologic asynchrony in cardiac muscle deformation during the isovolumic intervals (6,7).
With high-temporal resolution flow Doppler and time-resolved echo contrast PIV flow dynamics, we demonstrate that the LV pre-ejection period is characterized by rapid preliminary redirection of LV intracavitary flow. During IVC, the LV flow changes such that the intracavitary streams accelerate toward the LV outflow, with formation of a large anterior vortex across the LV inflow-outflow region. During IVR, the flow is directed toward the LV apex and then, after a reversal, toward the LV base. These observations argue against the conventional appreciation of the LV as a chamber that contracts and dilates for ejection and filling, with intervening isovolumic phases of hemodynamic stasis (3). Rather, leading vortices make a dynamic contribution to flow redirection and provide efficiency for momentum transfer. This finding is consistent with other biologic vortex formations that are known to contribute to more efficient energy transfer than the equivalent propulsion of fluid along a straight jet (17). The insights into ventricular flow and its influence on valve movements provide a newer understanding about the fluid continuum of the cardiac phases and uncover the reasoning behind the unusual myocardial behavior during the transiting isovolumic intervals.
Embryologically, the initial blood flow in the primitive tubular heart follows a wave of peristalsis that results from a slow isotropic electromechanical activation sequence spreading from the most caudal portion of the primitive LV heart toward the cranially located outflow (18,19). Subsequent looping of the tubular heart results in the inflow and outflow regions occupying the basal region of a mature LV, accompanied by the emergence of specialized His-Purkinje conduction that progressively alters the immature base-to-apex sequence of electromechanical activation into a mature apex-to-base pattern (18,19). Previous investigations in adult hearts, including our recent observations, have confirmed the presence of an apex-to-base gradient in depolarization and repolarization that is accompanied by apex-to-base differences in regional shortening and lengthening sequences during a cardiac cycle (20–22). The present investigation clarifies that flow during the pre-ejection period also occurs in an apex-to-base direction. The direction of electromechanical activation in adult mammalian hearts, therefore, matches and maintains a synchrony between the timing and direction of activation and blood flow. Conversely, during IVR, lengthening of the LV apex initiates enlargement of the LV cavity near the apex, causing a brisk base-to-apex reversal of blood flow during the IVR period. This implies that although the intrinsic LV and mitral valve geometries are important contributors to asymmetric flow redirection (9), the apex-to-base differences in electromechanical forces at the onset of systole and diastole are critical for further continuation and stabilization of flow reversal.
Color Doppler versus echo PIV: methodological considerations
Although echo PIV and color Doppler show good correlation, limits of agreement between the measured values are rather wide (supplementary Fig. 1[see Appendix]). This is consistent with observations in previous investigations and occurs because color Doppler has limitations in measuring absolute fluid velocities (average uncertainty ± 10 cm/s) (10,23). This random error in color Doppler occurs because a spectral estimation technique is used to determine mean Doppler shift for color imaging rather than a direct calculation via fast Fourier transform as is used for pulsed wave Doppler measurement (24). In addition, color Doppler measures the mean velocities and is angle dependent and therefore underestimates the peak velocities (24). Echo PIV, in contrast, provides robust data of instantaneous flow vectors and shows good agreement with optical PIV for in vitro models of pulsatile flow (10,25). Therefore, echo PIV has several incremental advantages over color Doppler for displaying information related to instantaneous intracavitary flow vector fields during different phases of cardiac cycle.
Knowledge regarding the rheological effects of pacing from the LV base has clinical relevance because: 1) the LV base can be reached clinically by routing the pacemaker leads through the coronary sinus; and 2) the sequence of activation resembles Wolff-Parkinson-White syndrome and tachycardia of LV epicardial origin (26,27). Furthermore, epicardial pacing is commonly performed through leads placed through the coronary sinus during cardiac resynchronization therapy. Results from recent clinical investigations that have evaluated functional parameters of heart failure patients during single site LV epicardial pacing produced equivocal results (28,29). Unfavorable proarrhythmic effects of LV epicardial pacing have also been recently reported (30,31). Therefore, current guidelines have attempted to rationalize the selection of patients for achieving a way of pacing that is as physiologic as possible (32). Mechanical dyssynchrony of the LV might be absent in approximately 30% of patients with congestive heart failure (33,34). In patients who have no detectable dyssynchrony or intraventricular conduction abnormalities, ventricular pacing should be avoided (32). The present study provides mechanistic insights into how LV pacing in such a situation could be detrimental and cause a loss of efficiency in LV performance by altering the synchrony of intracavitary blood flow. This would argue against indiscriminate use of LV pacing, in the absence of a clear electrical activation delay. Furthermore, for patients who have an activation delay, the goals of resynchronization therapy could be optimized by selecting a pacing site that synchronizes the LV mechanical performance with the direction of blood flow. This might have relevance for the 20% to 30% of patients with heart failure who have no response to cardiac resynchronization therapy (35).
An open-chest and open-pericardial model alters the magnitude of regional contraction and relaxation velocities (36). Nevertheless, the sequence of LV intracavitary flow and the vortical features seen in this study are consistent with observations in humans (9). Future investigations for evaluating the feasibility and utility of PIV techniques in clinical settings are warranted. Methods have been outlined that optimize bubble detectability yet minimize the possibility of mechanical rupture of the bubble for PIV analysis during clinical echocardiography (37).
The present study evaluated the effects of epicardial pacing from the LV base, because pacing from this site does not couple into the Purkinje system yet maintains adequate distribution of regional myocardial work with lowest effects on perfusion, metabolism, and pump function and, thus, provides an optimum model for isolating effects of electromechanical asynchrony (38). However, the rheological effects of pacing from other sites or the role of multi-site LV pacing and the effects of an optimum atrioventricular synchrony were not addressed and would require further investigations.
In summary, our investigation demonstrated that LV isovolumic intervals of the cardiac cycle are not periods of hemodynamic stasis. Instead, interactive flow rearrangement and stabilization of redirected streams build a flow momentum that optimizes a smooth transition for subsequent LV suction or ejection. An altered sequence of electromechanical activation prolongs the pre-ejection period and disrupts the organized sequence of blood flow redirection. Tracking the pre-ejection flow in 2 dimensions could be a novel approach for quantifying the effects of electromechanical incoordination on intracardiac rheology.
The authors thank GE Healthcare for the use of the Vivid 7 ultrasound system.
To view the videos and supplemental figures, please see the online version of this article.
Left ventricular (LV) flow sequence on Doppler and contrast echocardiography. Flow Doppler (left) is compared with high-temporal resolution contrast echocardiography (right, 250 frames/s) in apical long-axis view of the LV. Note the vortices and the characteristics of intracavitary flow orthogonal to the direction of the ultrasound beam. Ao = aorta; IVR = isovolumic relaxation; LA = left atrium; LVOT = left ventricular outflow tract.
Quantitative assessment of flow sequence using particle image velocimetry. The movement of contrast bubbles (left) is mapped by particle image velocimetry (right). Intraventricular flow sequence during the transition from diastole to systole and vice versa changes without any phase of hemodynamic stasis. Ao = aorta; LA = left atrium; LV = left ventricular; LVOT = left ventricular outflow tract.
Intracavitary flow during the pre-ejection period. Pre-ejection movement of contrast bubbles (left) has been tracked with particle imaging velocimetry (right). Note that the apex-to-base redirection of blood flow merges with a vortex formed across the edge of a closing anterior mitral leaflet. The apex-to-base movement of intracavitary flow continues further during the period between mitral valve closure and aortic valve opening (isovolumic contraction). Ao = aorta; LA = left atrium; LV = left ventricular.
Comparison of pre-ejection flow during sinus rhythm and epicardial pacing. Left ventricular (LV) pre-ejection flow is shown during sinus rhythm (left; heart rate: 72 beats/min) and epicardial pacing (right; heart rate: 82 beats/min). The redirection of contrast bubbles is well coordinated during normal electrical activation (left). During epicardial pacing, despite a shorter cardiac cycle length, the duration of the pre-ejection period is prolonged. Pacing from the LV epicardial surface near the base produces turbulence and a delay in closure of the mitral valve. Both right and left image sequences were recorded at 250 frames/s and are displayed at the same speed. LA = left atrium.
Sequence of pre-ejection flow during epicardial pacing. Delayed left ventricular (LV) pre-ejection flow sequence by contrast echocardiography during epicardial pacing (left; also shown in Video 4) is mapped by particle image velocimetry (right). The LV epicardial surface has been electrically stimulated near the base. Note the initial base-to-apex direction of flow with turbulence and a delay in apex-to-base redirection. Ao = aorta; LA = left atrium.
Left Ventricular Isovolumic Flow Sequence During Sinus and Paced Rhythms: New Insights From Use of High-Resolution Doppler and Ultrasonic Digital Particle Imaging Velocimetry
This work was supported by grant HL68573 and, in part, by grants HL68555 and HL70363 from the National Institutes of Health. Nothing in this article implies endorsement of any product or manufacturer mentioned in this article.
- Abbreviations and Acronyms
- isovolumic contraction
- isovolumic relaxation
- left ventricle/ventricular
- particle imaging velocimetry
- Received April 19, 2006.
- Revision received June 30, 2006.
- Accepted July 10, 2006.
- American College of Cardiology Foundation
- LeWinter M.M.,
- Osol G.
- Gillebert T.C.,
- Van de Veire N.,
- De Buyzere M.L.,
- De Sutter J.
- Salukhe T.V.,
- Henein M.Y.,
- Sutton R.
- Zhou Q.,
- Henein M.,
- Coats A.,
- Gibson D.
- Sengupta P.P.,
- Khandheria B.K.,
- Korinek J.,
- Wang J.,
- Belohlavek M.
- Goetz W.A.,
- Lansac E.,
- Lim H.S.,
- Weber P.A.,
- Duran C.M.
- ↵(1996) Institute of Laboratory Animal Resources (US) Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, DC), 7th edition.
- Wiggers C.J.
- Dabiri J.O.,
- Gharib M.
- Kirn B.,
- Starc V.
- Shandas R.,
- Gharib M.,
- Sahn D.J.
- Wyman B.T.,
- Hunter W.C.,
- Prinzen F.W.,
- McVeigh E.R.
- Daniels D.V.,
- Lu Y.,
- Morton J.B.,
- et al.
- Vollmann D.,
- Luthje L.,
- Schott P.,
- Hasenfuss G.,
- Unterberg-Buchwald C.
- Hay I.,
- Melenovsky V.,
- Fetics B.J.,
- et al.
- Fish J.M.,
- Brugada J.,
- Antzelvitch C.
- Sweeney M.O.,
- Prinzen F.W.
- Yu C.M.,
- Lin H.,
- Zhang Q.,
- Sanderson J.E.
- Kass D.A.
- Derumeaux G.,
- Ovize M.,
- Loufoua J.,
- et al.
- Zheng H.,
- Mukdadi O.,
- Kim H.,
- Hertzberg J.R.,
- Shandas R.
- Prinzen F.W.,
- Hunter W.C.,
- Wyman B.T.,
- McVeigh E.R.