Abstract

Mitral annular (MA) and leaflet three-dimensional (3-D) dynamics were examined after circumferential phenol ablation of the MA and anterior mitral leaflet (AML) muscle. Radiopaque markers were sutured to the left ventricle, MA, and both mitral leaflets in 18 sheep. In 10 sheep, phenol was applied circumferentially to the atrial surface of the mitral annulus and the hinge region of the AML, whereas 8 sheep served as controls. Animals were studied with biplane video fluoroscopy for computation of 3-D mitral annular area (MAA) and leaflet shape. MAA contraction (MAACont) was determined from maximum to minimum value. Presystolic MAA (PS-MAACont) reduction was calculated as the percentage of total reduction occurring before end diastole. Phenol ablation decreased PS-MAACont (72 ± 6 vs. 47 ± 31%, P = 0.04) and delayed valve closure (31 ± 11 vs. 57 ± 25 ms, P = 0.017). In control, the AML had a compound sigmoid shape; after phenol, this shape was entirely concave to the atrium during valve closure. These data indicate that myocardial fibers on the atrial side of the valve influence the 3-D dynamic geometry and shape of the MA and AML.

  • anterior mitral leaflet
  • three-dimensional
  • end systole
  • end diastole

timely and effective valve closure requires precise temporal and spatial coordination of the mitral leaflets, mitral annulus, and the subvalvular apparatus. The mitral annulus in humans (1) and other mammals (22) is a discontinuous fibrous ring with a variable insertion of atrial and ventricular myocardial fibers. Experimental (17, 21) and clinical (13, 16) studies have shown that the mitral annulus has a “sphincteric” function during valve closure facilitating leaflet coaptation by reducing mitral valve area. Our previous experiments in ovine hearts revealed that 89% of annular reduction occurred before ventricular systole (7) and was abolished by ventricular pacing. Subsequently, mitral annular area change was shown to be closely linked with atrial dynamics during rapid pacing (18) and acute ischemia (19). These and other studies (16, 21) suggest an atrial influence on presystolic mitral annular dynamics. Anatomically, atrial myocardial fibers have been shown to insert on the annular portion of the anterior mitral leaflet in humans (2) and experimental animals (6, 22). Furthermore, these fibers have been demonstrated to have intrinsic contractile properties (6, 15) and dense neural innervation (10), suggesting a neuromuscular role in annular and leaflet dynamics, the significance of which is yet to be defined. Although intrinsic annular and leaflet contractile activity may play an important role in normal valve physiology, the in vivo function of these mitral complex components is not understood.

Using miniature radiopaque marker technology, we studied mitral annular and leaflet dynamics after phenol ablation of mitral annular and anterior leaflet hinge musculature on the atrial aspect of the ovine mitral valve.

METHODS

Surgical Preparation

Eighteen adult sheep underwent miniature radiopaque marker placement as described previously in detail (7). The animals were premedicated with ketamine (25 mg/kg im) for venous and arterial line placement and monitored, and anesthesia was induced with sodium thiopental (6.8 mg/kg iv). The animals were intubated and mechanically ventilated (Servo Anesthesia Ventilator, Siemens-Elema), and anesthesia was maintained with inhalational isofluorane (1–2.2%). A left thoracotomy was performed, and the heart was suspended in a pericardial cradle. Eight tantalum myocardial markers (inner diameter 0.8 mm, outer diameter 1.3 mm, length 1.5 to 3.0 mm) were inserted beneath the left ventricular (LV) epicardial surface along four equally spaced longitudinal meridians for LV volume determinations (see locations in Fig. 1). The right atrium and the descending aorta were cannulated following systemic heparinization (300 IU/kg). After establishment of cardiopulmonary bypass and with the heart arrested, eight tantalum markers were sutured around the circumference of the mitral annulus (Fig. 1). Four miniature gold markers were sutured on the central meridian of the anterior mitral leaflet and two on the posterior leaflet (Fig. 1). One marker was also placed on each papillary muscle tip. After completion of marker placement, the control group animals (n = 8) underwent no further treatment, whereas the phenol group (n = 10) had a 95% phenol solution (Sigma Chemical; St. Louis, MO), a histotoxic chemical, applied (as described previously in Ref. 8) for 2 min on the atrial side of the hinge region of the anterior mitral leaflet and septal annulus and for 5 min on the atrial side of the remaining portions of the annulus. Longer application time was chosen for the lateral annulus because it is known to have a higher myocardial fiber content than the more fibrous septal annulus (2). The valve was thoroughly irrigated with normal saline after phenol application. After atriotomy closure and resuscitation, the animals were weaned from bypass. A micromanometer pressure transducer (PA4.5-X6; Konigsberg Instruments; Pasadena, CA) was placed in the LV chamber through the apex, and a LV apical marker was sutured in place.

Fig. 1.

Left ventricular (dark circles), annular (light circles), and leaflet (dark squares) marker array employed in this experiment. AML, anterior mitral leaflet; PML, posterior mitral leaflet.

Experimental Protocol

After a 7- to 10-day recovery period, each animal was taken to the experimental cardiac catheterization laboratory, sedated with ketamine (1–4 mg · kg1 · h1 iv infusion) and diazepam (5 mg iv bolus as needed), intubated, and mechanically ventilated (veterinary anesthesia ventilator 2000; Halowell EMC). Esmolol (20–50 μg · kg1 · min1) and atropine sulfate (0.01 mg · kg1 · min1) intravenous infusions were utilized to minimize reflex sympathetic and parasympathetic responses. Simultaneous biplane videofluoroscopy and hemodynamic data recordings were obtained during stable steady-state baseline conditions in normal sinus rhythm. Degree of mitral regurgitation was graded by an experienced echocardiographer (D. Liang) based on the width and extent of the regurgitant jet into the left atrium as none (0), mild (+1), moderate (+2), moderate-severe (+3), and severe (+4).

At the time of animal death, the hearts were removed, washed, and fixed with 10% neutral buffered formalin. Sagital sections through the anterior mitral leaflet and posterior annulus were prepared. The sections were routinely processed and embedded in paraffin wax. Five-micrometer sections were prepared and stained with hematoxylin and eosin and Masson's trichrome stains. Slides were evaluated for depth of phenol-induced injury by an experienced cardiac pathologist (G. J. Berry).

All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the Guide for Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (DHEW NIH Publication No. 85-23, Revised 1985). This study was approved by the Stanford Medical Center Laboratory Research Animal Review Committee and conducted according to Stanford University policy

Data Acquisition

Images were acquired with the animal in the right lateral decubitus position with a Philips Optimus 2000 biplane Lateral ARC 2/Poly DIAGNOST C2 system (Phillips Medical Systems, North America; Pleasanton, CA) with the image intensifier in the 9″ fluoroscopic mode. Data from two radio-graphic views were digitized (12) and merged to yield three-dimensional (3-D) coordinates for each of the radiopaque markers every 16.7 s using custom-designed software (5). LV pressure and ECG voltage were digitized and recorded simultaneously during data acquisition.

Data Analysis

Hemodynamic and cardiac cycle timing markers. Two to three consecutive steady-state beats were averaged in each animal and used for analysis. End systole was defined as the frame containing the peak rate of fall of LV pressure (–dP/dt), and end diastole was defined as the videofluoroscopic frame containing the peak of the ECG R-wave. Instantaneous LV volume was computed from the epicardial LV markers using a space-filling multiple tetrahedral volume method (11).

Mitral annular and leaflet dynamics. Mitral annular area was calculated by determining the centroid of the annular markers and dividing the annulus into eight triangular areas, which were summed to yield the total annular area. The septal-lateral diameter of the annulus was calculated as the distance in 3-D space between the markers placed in the middle of the septal and lateral mitral annulus, respectively; the commissure-commissure diameter was calculated as the distance between the two commissural markers. “Presystolic” mitral annular area contraction was defined as the percentage of total area reduction occurring between late diastolic maximum [within 167 ms (10 frames) before end diastole] and end diastole. Valve closure was defined as the time after end diastole at which leaflet separation distance reached its minimum plateau. For 3-D reconstruction of mitral leaflet shape, a right-handed Cartesian coordinate system was used with the origin located at the midseptal annulus marker, the y-axis passing through the LV apex (positive toward the apex), positive x-axis directed toward the midlateral annulus such that the midlateral marker was contained in the x-y plane, and positive z-axis toward the posterior commissure. Maximum angular excursion of the anterior and posterior mitral leaflets was determined by calculating the angle between the leaflet edge markers and the septal-lateral annular diameter throughout the cardiac cycle.

Subvalvular Geometry

To assess any changes in subvalvular geometry associated with annular ablation, the distance from each papillary muscle tip to the center of the fibrous annulus was calculated as a distance in 3-D space between the respective markers.

Statistical Analysis

All data are reported as means ± SD unless otherwise stated. Hemodynamic and marker-derived data from two consecutive steady-state beats were aligned at end diastole (t = 0), and data from the two to three beats were averaged. The data were analyzed over 20 frames before and 20 frames after end diastole, thereby allowing evaluation of the behavior of the studied variables over the entire cardiac cycle. Data were compared using Student's t-test for independent comparisons with the level of significance for statistical comparison set at P < 0.05.

RESULTS

Hemodynamics

The control and phenol groups did not differ in animal weight (75 ± 14 and 77 ± 7 kg, respectively; P = not significant), cardiopulmonary bypass (82 ± 5 and 91 ± 11 min; P = not significant), or aortic cross-clamp time (61 ± 3 and 64 ± 5 min; P = not significant). The depth of tissue ablation in the phenol group as determined from microscopic sections (Fig. 2) of the midseptal and midlateral mitral annulus was 1.68 ± 0.56 and 1.21 ± 0.45 mm for the septal and lateral annulus, respectively (P = 0.053). There was no significant difference in any of the measured hemodynamic variables between the two groups (Table 1). Thus the animals in the control and phenol groups were very comparable in size and physiological state, permitting comparison of the effect of experimental application of phenol on mitral annular and leaflet dynamics.

Fig. 2.

Photomicrographs of mitral annulus and anterior mitral leaflet in sagital section after phenol ablation. A: low-power magnification showing regional orientation of structures around annulus including anterior mitral leaflet (AMVL), left atrium (LA), left ventricle (LV), mitral annulus (MA) (Masson's trichrome ×10). B: base of AMVL showing preservation of structural integrity. Atrial aspect shows phenol ablation (trichrome ×20). C: high-power magnification of ablation site with aggregates of fibrin overlying subannular band of myocytes displaying acute injury characterized by hypereosinophilic wavy fibers. Adjacent atrial musculature is not affected (trichrome ×100).

View this table:
Table 1.

Hemodynamic variables in both groups

Mitral Annular and Leaflet Dynamics

The group mean mitral annular area throughout the cardiac cycle for both control and phenol animals is shown in Fig. 3, and the maximum and minimum mitral annular areas for each group are shown in Table 2. Peak mitral annular area did not differ between the two groups, indicating that annular myocardial ablation did not lead to annular dilatation. There was also no change in either the septal-lateral or commissure-commissure annular diameters with phenol application (Table 2). The extent of presystolic reduction in mitral annular area, however, was substantially less with annular phenol ablation (Table 2). Thus a greater proportion of annular area reduction occurred during early systole in the phenol animals than in the control group, but the total extent of annular contraction did not change between groups, and the annulus was reduced to the same minimal size (Fig. 3 and Table 2).

Fig. 3.

MA area (cm2) throughout the cardiac cycle under control conditions and after phenol ablation. A 650-ms time frame centered at end diastole (ED, verticle dashed line) is illustrated for both conditions. Error bars indicate means ± SE.

View this table:
Table 2.

Annular dynamics in the phenol and control groups

Less presystolic mitral annular area contraction was also associated with delayed valve closure (26 ms delay) in the phenol-treated animals (Fig. 4 and Table 2). This delay led to the valve closing at a higher ventricular pressure in the phenol group (49 ± 16 and 76 ± 33 mmHg for control and phenol, respectively; P = 0.053), but there was no statistical difference in the average severity of mitral regurgitation between the two groups (0.8 ± 0.7 and 1.3 ± 0.6 for control and phenol, respectively; P = not significant). Sagittal plane cross-sectional shape of the anterior and posterior mitral leaflets during valve closure is reconstructed for both groups in Fig. 5. During valve closure subsequent to atrial contraction, the anterior mitral leaflet in the control group assumed a more complex sigmoid shape, whereas the anterior leaflet in the phenol animals was concave to the atrium throughout valve closure. Total angular excursion of the anterior mitral leaflet from its diastolic maximum to systolic minimum did not differ between the groups (48 ± 8° and 43 ± 7° for control and phenol, respectively; P = 0.19). Total angular excursion of the posterior mitral leaflet was 34 ± 12° and 43 ± 11° for control and phenol, respectively (P = 0.09).

Fig. 4.

Leaflet edge separation (cm) throughout the cardiac cycle under control conditions and after phenol ablation. A 650-ms time window centered at ED (verticle dashed line) is illustrated for both conditions. Error bars indicate means ± SE.

Fig. 5.

Sagital plane depiction of cross-sectional leaflet shape, derived from the three-dimensional coordinates of the leaflet markers, during valve closure (from 84 ms before ED to 50 ms after ED) in both the control group (A) and after phenol muscle ablation (B).

Subvalvular Geometry

There were no differences in the distance between the anterior papillary muscle tip and the middle of the fibrous annulus (annular “saddle horn”) between the two groups of animals either at end diastole (4.86 ± 0.57 vs. 4.83 ± 0.36 cm, P = 0.9) or end systole (4.74 ± 0.48 vs. 4.68 ± 0.28 cm, P = 0.7). The distance between the posterior papillary muscle tip and the annular saddle horn also did not change (5.24 ± 0.40 vs. 5.20 ± 0.46 cm at end diastole, P = 0.8; 5.10 ± 0.41 vs. 5.13 ± 0.43 cm at end systole, P = 0.9).

DISCUSSION

The mitral annulus plays an important role in valve closure through its “sphincteric” action (20, 21), which results in a smaller septal-lateral annular diameter before systole (7), thus facilitating leaflet coaptation. The musculature of the annulus seems therefore central to this process, yet little is known about the in vivo function of myocardial fibers, which insert into the mitral annulus and leaflets. In the present study, we used phenol to ablate annular and leaflet musculature on the atrial side of the valve and found decreased presystolic mitral annular area reduction, delayed mitral valve closure, and altered shape of the anterior mitral leaflet during and after closure.

The pioneering canine experiments of Tsakiris and colleagues (20) were the first to demonstrate a considerable presystolic component of annular area reduction suggesting atrial influence on annular contraction. Recent ovine experiments have confirmed these findings (7), and clinical echocardiographic studies have also shown the presence of presystolic annular reduction in human subjects (13, 14). The findings of the present study help establish the functional link between annular musculature and presystolic annular area reduction and lend support to previous findings. Because presystolic annular reduction has been observed to depend on the strength and duration of atrial contraction (18, 21), it may be expected that atrial fiber ablation would lead to a decreased “atriogenic” influence on annular area reduction and mitral valve closure. Although presystolic annular reduction was decreased with phenol application, total annular contraction did not change. Thus “ventriculogenic” annular contraction was capable of reducing the annulus to its minimum size even in the total absence of atrial contraction, as reported previously (7). Such a mode of annular reduction, however, is associated with delayed valve closure and higher regurgitant volume (17). Although we did not observe a change in the degree of mitral regurgitation after annular ablation, it is possible that such might have occurred if we had been able to quantitate the degree of mitral regurgitation echocardiographically using effective regurgitant orifice (ERO) or direct measurements of regurgitant volume because only a small amount of mitral insufficiency is associated with “ventriculogenic” valve closure in sheep (17). It is also noteworthy that the delay in valve closure reported in the current experiment is similar to that described with ventricular pacing (17). A previous study (8) from this laboratory after phenol ablation of only the anterior mitral leaflet musculature also did not reveal any increase in mitral regurgitation. The commissure-commissure annular diameter dilated in that experiment but did not do so in the current study. This discordance in commissure-commissure annular diameter size between these two different sets of animals may be due to differences in the control groups or perhaps due to the balancing effect of additional ablation of the lateral annulus in the current experiment.

Along with annular ablation, phenol was also applied to the musculature of the anterior mitral leaflet in the current experiment. These anterior mitral leaflet muscle fibers have been shown to be of atrial origin in animal species (15), yet their continuity with atrial fibers in human valves has not been firmly established (3). It has been suggested that these leaflet fibers modulate anterior leaflet “stiffness” and aid in timely valve closure (4). It is difficult to differentiate whether delayed valve closure observed in our experiment was due to delayed annular reduction versus altered anterior leaflet motion, but total angular anterior leaflet excursion was not affected by phenol. The baseline shape of the anterior mitral leaflet during valve closure assumed a complex sigmoid curve, as reported previously (9). After phenol ablation, the anterior leaflet became entirely concave to the atrium during valve closure in contrast to the canine experiments of Curtis and Priola (4), who observed greater deflection of the anterior leaflet toward the atrium after phenol application. Curtis and Priola, however, placed their strain gauge on the annular one-third of the anterior mitral leaflet (corresponding to the muscle fibers), which is a relatively adynamic portion of the leaflet (Fig. 5). The change in anterior leaflet shape observed in our study may be attributed to lack of fiber contraction or ablation of neural networks as mitral valve leaflets are densely innervated (23). Indeed, the human anterior mitral leaflet has approximately double the nerve density of the posterior leaflet (10). These nerves arborize from the annular portion of the leaflet and are predominantly found on the atrial surface of the valve. Therefore, phenol ablation of anterior leaflet musculature could affect leaflet shape by altering neural motor input to the leaflet. Whether the change in leaflet shape is due to neural or myocardial perturbations is not discernible from our study.

In the present study there was no difference in the distance between each papillary muscle tip and the annular saddle horn comparing the control and phenoltreated animals, indicating no influence on subvalvular geometry. These findings, however, contrast with observations in our previous study (8) with phenol applied just to the anterior leaflet and annulus, where significant displacement of the anterior papillary muscle anterolaterally and toward the mitral annulus was observed. The control group was different in that study, and the experiment used a reference coordinate system to describe displacements of the papillary muscles relative to a defined origin. The sum of individual displacements in a coordinate system, however, may not necessarily result in change in distance, measured in the current study, between two cardiac structures. The extent of phenol annular ablation may have also contributed to this discrepancy because the more muscular posterior annulus was also painted with phenol in the current experiment. Posterior annular ablation perhaps balanced the effect of phenol application on the anterior annulus and reestablished the normal relationship between the anterior papillary muscle tip and the annular saddle horn. The mechanism of this effect is at this time speculative, and the true reason for the above discrepancy is unknown.

The current study presents the first in vivo evaluation of mitral annular and leaflet dynamics after ablation of muscle on the atrial surface of the valve and annulus. These data add further support to the concept of “atriogenic” mitral annular reduction and underscore the importance of annular integrity to normal valvular function. Furthermore, it appears that anterior leaflet muscle may play a role in modulation of leaflet shape and perhaps normal valve closure. Complete elucidation of these mechanisms, however, will require further investigation.

Study Limitations

Measuring mitral annular dynamics with the use of the myocardial marker method requires suturing miniature tantalum markers to the cardiac structures of interest. Therefore, it is possible that the presence of the markers may alter normal valve mechanics, and, in particular, leaflet motion. The markers utilized in this study, however, had little mass (4–8 mg) and are biologically inert; peak opening velocities of the anterior leaflet edge in sheep with and without markers do not differ as determined by M-mode echocardiography in our previous experimental studies (unpublished data). Our measurements of mitral regurgitation were qualitative; a difference in mitral regurgitation might have been detected if more quantitative measurements of the degree of mitral regurgitation, such as ERO or regurgitant volume, were used. The small jets that were present were frequently eccentric, so quantitation was difficult due to the inherent assumptions about the geometry of the convergence region. Additionally, because of the large distance between the esophagus and the heart in sheep, our transesophageal echocardiographic image quality was not sufficient to allow ERO calculation. It is also possible that the depth of tissue ablation in our experiment was insufficient, and hence did not lead to enough changes in mitral valve closure dynamics to result in mitral regurgitation. Perhaps longer application of phenol or intramuscular injection would have yielded greater valvular perturbations. Other study limitations include the anatomic differences between the ovine and human mitral annulus (22); atrial contribution to the structure and dynamics of the mitral annulus in sheep may not be directly applicable to human physiology. Such interspecies differences may actually not be so important, however, because substantial presystolic annular contraction has been observed in both sheep (7) and humans (13).

DISCLOSURES

This study was supported by National Heart, Lung, and Blood Institute (NHLBI) Grants HL-29589 and HL-67025; T. A. Timek, F. Tibayan, P. Dagum, and D. T. Lai are Carl and Leah McConnell Cardiovascular Surgical Research Fellows; T. A. Timek, F. Tibayan, and P. Dagum were supported by NHLBI Individual Research Service Awards Grants HL-10452, HL-67563, and HL-10000, respectively; T. A. Timek is a recipient of the Thoracic Surgery Foundation Research Fellowship Award; D. T. Lai was supported by a fellowship from the American Heart Association, Western States Affiliate.

Present address of T. A. Timek: Dept. of Surgery, Loma Linda University Medical Center, Loma Linda, CA 92354.

Acknowledgments

We appreciate the technical assistance provided by Mary K. Zasio, Carol W. Mead, and Erin M. Schultz.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

References

View Abstract