Left Ventricular Isovolumic Flow Sequence During Sinus and Paced Rhythms

New Insights From Use of High-Resolution Doppler and Ultrasonic Digital Particle Imaging Velocimetry

Animal preparation

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.

Contrast echocardiography

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.

PIV

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.

Pacing protocol

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.

Definitions

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.

Statistical analysis

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.

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Figure 1

M-Mode Characteristics of LV Intracavitary Flow Along the Longitudinal Axis

(A)Doppler velocities are shown in red(flow toward left ventricular [LV] apex) and blue(flow toward LV base). (A, B)Phases of the cardiac cycle have been determined with timing of the movement of the mitral valve (MV) and the aortic valve (AV). (C)The M-mode features of Doppler flow are compared with an anatomical M-mode through the LV cavity during contrast infusion. (C, D)The axial movement of contrast bubbles can be compared with the movement of the MV and the AV. Phases: 1, pre-ejection; 2, ejection; 3, isovolumic relaxation; 4, early diastole; 5, diastasis; and 6, late diastole.

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.⇓⇓

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Figure 2

Digital Particle Image Velocimetry Profiles of LV Flow During Phases of Cardiac Cycle

For each phase, the local ensemble-averaged axis-normal velocity magnitude (Vel Mag) is superimposed on the vector field. (A)Pre-ejection. (B)Ejection. (C)Isovolumic relaxation. (D)Early diastole. (E)Diastasis. (F)Late diastole. LA = left atrium; LV = left ventricle.

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Figure 3

Time-Resolved LV Flow During the Pre-Ejection Phase

(A)Anatomical M-mode through the left ventricle (LV). (B)Two-dimensional snapshots of 4 time frames corresponding to the vertical lines (1 to 4)in panel A. 1, the initial flow during the pre-ejection period is directed from the apex toward the base. 2 and 3, the apex-to-base redirection of blood flow merges with a vortex formed across the edge of a closing anterior mitral leaflet. 4, the apex-to-base movement of intracavitary flow continues further during the period between mitral valve closure and aortic valve opening (isovolumic contraction). LA = left atrium; Vel Mag = velocity magnitude.

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Figure 4

Time-Resolved Left Ventricular Flow During the Isovolumic Relaxation Phase

(A)Anatomical M-mode through the left ventricle (LV). (B)Two-dimensional snapshots of 3 time frames shown by vertical lines (1 to 3)in panel A. 1 and 2, the initial flow after ejection is reversed toward the apex. This is followed by a brief reversal of the flow toward the LV base (3).HR = heart rate; LA = left atrium; Vel Mag = velocity magnitude.

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.

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Figure 5

Anatomical M-Mode Through the LV Cavity

Axial movement of contrast bubbles is shown in relation to the mitral valve (MV) and the aortic valve (AV) during sinus rhythm (A, B)and during epicardial pacing (C, D). Blue arrowsindicate the time of MV closure (A, C)and AV opening (B, D). Yellow arrowsindicate the time of isovolumic flow redirection. The delay in flow redirection (C, D)accompanies a delay in MV closure and AV opening after pacing of the left ventricle (LV) from the LV base. Dashed white linesindicate the onset of the QRS complex on surface electrocardiography. HR = heart rate.

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Figure 6

Time-Resolved Digital Particle Image Velocimetry Profiles Showing the Abnormal Pattern of Pre-Ejection Flow During Epicardial Pacing From the Base of the LV

(A)Anatomical M-mode through the left ventricle (LV). (B)Two-dimensional snapshots of 4 time frames corresponding to the vertical lines (1 to 4)in panel A. The base-to-apex direction of flow persists for a prolonged time and delays blood flow redirection and closure of the mitral valve. AV = aortic valve; MV = mitral valve; Vel Mag = velocity magnitude.

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.

Clinical implications

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).

Study limitations

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.