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School of Medicine, University of California, San Diego, La Jolla, California 92093
Submitted 23 February 2004 ; accepted in final form 16 August 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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myofiber rearrangement; finite deformation; remodeling strains
Remodeling of the noninfarcted ventricular wall post-MI occurs, in part, because of structural changes in myocytes (14) and in part due to rearrangement of myofibrils and changes in the cardiac interstitium. Myocyte responses to MI may include cellular hypertrophy and reexpression of a fetal phenotype (34). Myocytes in noninfarcted myocardium elongate, which is perhaps structurally more important. Myocyte length increases after MI and is greater in tissue adjacent to the infarct than more remote myocardium (14). Changes in the structure of the "normal" ventricular wall adjacent to the infarcted area involve all three components of the myocardium (myocytes, fibroblasts and the extracellular matrix, and the coronary vasculature) and their three-dimensional structural relationship. Because it is not possible to assess the structure of the tissue before the infarct, assessing changes in these components of the tissue requires tracking material markers in the remodeling tissue over long periods of time with a three-dimensional approach as well as a detailed histological evaluation of the remodeled structure. In the present study, we have defined the condition of the initial end diastolic configuration of the tissue with a transmural array of gold beads before coronary occlusion and calculated the deformation of the tissue occurring at 4 wk (deformed configuration). The resulting deformation represents a remodeling strain. With this approach it is possible to estimate not only tissue loss but also rearrangement of the components of the myocardium (e.g., myofiber orientation or arrangement into radial laminae). Although data exist regarding regional changes in uniaxial dimensions after MI (29), it is not possible to relate these measurements to components of the wall (e.g., myofiber direction). Therefore, the purpose of the present study was to examine the hypotheses that changes in the adjacent tissue are related not only to known myocyte elongation but also to myofiber rearrangement and changes in the extracellular matrix in the adjacent tissue. We tested these hypotheses by determining the structure of the remodeled wall and three-dimensional remodeling deformation during the 3 wk after acute coronary occlusion in the pig.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The surgical protocol and bead implantation technique have been described in detail previously (35). Eleven Hampshire farm-raised pigs weighing 23.3 ± 3.2 kg were sedated with 24 mg/kg ketamine and pretreated with 0.03 mg/kg atropine. Anesthesia was induced with 4–5% isofluorane with the use of a mask. All animals were then intubated, ventilated, and maintained with a mixture of isofluorane (1%), nitrous oxide, and oxygen. The heart was exposed via a fifth interspace thoracotomy. During the procedure, arterial pressure was monitored via a catheter inserted into the right femoral artery via a cutdown. Cardiac support (0.33 µg·kg–1·min–1 dobutamine-HCl) and antiarrhythmics (0.04 ml/kg, single bolus lidocaine-HCl, 2%) were administered as necessary during the procedure to maintain femoral artery pressure at >65 mmHg (dobutamine) and to prevent prolonged episodes of ventricular tachycardia (lidocaine). Inotropic support and lidocaine were discontinued as soon as the chest was closed, and no animals received postoperative support.
A 40-cm-long fluid-filled catheter (model S-54-HL, Tygon) was inserted through the apex into the left ventricular (LV) chamber and tunneled to the back of the neck, allowing for chronic measurement of LV pressure. The larger of the first or the second marginal branch of the left circumflex coronary artery (Fig. 1) was dissected free and temporarily constricted (
5–10 s) to visualize the ischemic border. In the pig with little collateral circulation, the ischemic area appears blanched with a bluish tinge. Three columns of three radio-opaque gold beads (0.8–0.9 mm diameter) were implanted transmurally within the LV wall. The nearest column was placed 1 cm from the ischemic border and in red contracting myocardium. Bead columns were implanted perpendicular to the epicardium by temporarily suturing a platform onto the epicardium above the desired location. A trocar was used to insert each bead to a predetermined depth. This was repeated until three to four beads were in the myocardium in each column and three columns of beads were completed. After the platform was removed, a larger gold bead (1 mm diameter) was sutured on the epicardium to mark each transmural column. Beads were also sutured to the LV apex and the bifurcation of the left coronary artery to define an apex-base cardiac coordinate system for the transmural bead set (Fig. 1). Data are reported on 6 of the 11 animals. Two animals did not survive the initial surgery. Both had arrhythmias after coronary ligation and did not survive the first 24 h. The remaining three animals were excluded because the marker set was not adequate (not transmural or had overlapping markers precluding visualization with the biplane system). No animals developed clinical evidence of heart failure.
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Control studies.
Saline infusion was occasionally used to elevate left ventricular end diastolic pressure >10 mmHg so that data could be captured at the same end-diastolic pressure at control and 3 wk after coronary occlusion. With respiration suspended during expiration, three-dimensional control locations of each bead were assessed by biplane cineradiography for three to four cardiac cycles (3.3 s). The dissected artery was then ligated. Ischemia was verified visually by tissue blanching and akinesis. The infarct region was covered with a patch of pericardium, which was not reapproximated, and the chest was closed. Air was evacuated from the chest, anesthesia was discontinued, and animals were allowed to recover. All pigs received 400 mg Sulfamethoxazole/80 mg Trimethoprim in oral suspension for 5 days after surgery. The catheter in the left ventricle was flushed and filled with heparin every other day.
Conscious animal studies. Before surgery, all animals were habituated to a canvas sling and the laboratory over the course of 1–2 wk. Three weeks after coronary ligation animals, they were placed in the sling and biplane X-ray images of the cardiac beads were taken. The animals were given 0–1.0 mg/kg iv diazepam as necessary for mild sedation. LV pressure and heart rate were monitored throughout the procedure.
Strain calculations. After each biplane X-ray experiment, a calibration phantom with beads embedded in known locations was imaged and used for perspective calibration (30). In particular, digitizing the two views of the phantom allowed calculation of a calibration transformation matrix from the known phantom bead positions and the pixel-based coordinates of the images of the calibration phantom. The resolution of this technique has been calculated at 0.2 mm (30). A least-squares finite-element method was used to calculate nonhomogeneous distributions of three-dimensional finite strain, as described elsewhere (17). Briefly, LV pressure tracings and ECG synchronized with a frame grabber (model DT2651, Data Translation) were used to define end-diastolic frames. At least two frames were averaged to obtain each end-diastolic view and the three-dimensional coordinates of each bead were computed from the calibration transformation matrix. The coordinates were then converted to cardiac coordinates defined by the apex and base beads. Three normal strains (E11, E22, and E33) quantified stretch or shortening along local circumferential (X1), longitudinal (X2), or radial (X3) axes. Three shear strains (E12, E13, and E23) quantified changes in angle between pairs of initially orthogonal axes. Remodeling strains were calculated as the bead set deformed between the control diastolic and the 3 wk postinfarct diastolic configurations (22, 28, 31).
The myofiber and sheet orientations were measured in the remodeled configuration, and to estimate their orientations back to the initial configuration we followed the method outlined in the appendix of Takayama et al. (28). Briefly, it is assumed that circumferential segments in the initial configuration remain circumferential and that planes parallel to the epicardium in the initial configuration remain parallel to the epicardium. These two conditions allow the strain alone to specify the mapping of material segments from the initial configuration to the final configuration. Moreover, this mapping is one-to-one for all of the remodeling strain fields that were measured in this study. Hence, the solution was obtained once we correctly guessed (in an iterative computational manner) an initial myofiber orientation 
0 that (when strained as measured) gave rise to the measured orientation 1. The same approach was used for finding the initial sheet orientation 
0 from the measured strain field and measured orientation 1.
Changes in LV chamber size were estimated from a calculation of the distance from the apex-base axis (linear distance from the apex bead to the base bead) to the epicardial centroid of the bead set.
Tissue processing. At the time of death, animals were again sedated with ketamine (24 mg/kg) and pretreated with atropine (0.03 mg/kg). Anesthesia was induced with isofluorane (4–5%) with the use of a mask. All animals were then intubated, ventilated, and maintained with a mixture of isofluorane (1%), nitrous oxide, and oxygen. The heart was exposed via a medial sternotomy. Once the heart was exposed and any noncardiac tissue dissected away, an overdose of pentobarbital was administered. Hearts were arrested via infusion of a hyperkalemic-cardiopelegic solution, dissected free, trimmed of the great vessels and atria, and weighed. The right and left coronary arteries were cannulated via their respective ostia and hearts were fixed by perfusion at 120 mmHg with 10% buffered formalin (Fisher Scientific) while suspended in saline (zero distending pressure). The entire isolation and fixation process lasted <40 min. The right ventricular wall was removed, and a plastic rod was inserted through the apex, which extended beyond the base along the apex-base axis. The LV chamber was filled with silicone, which was allowed to set for 48 h. Hearts were placed in a cutting apparatus, supported by the rod. The apparatus allowed cuts to be made in the LV wall perpendicular to the apex-base axis. Tissue blocks containing bead sets were removed, dehydrated, embedded in paraffin, and sectioned to 10 µm in the epicardial tangent plane. Sections were mounted and stained for collagen with picrosirius red. Each tissue section was oriented with the cardiac circumferential axis horizontal on the image and acquired at x20 total magnification with the use of a color video camera (Sony CCD/RGB) mounted on a microscope and connected to a Macintosh computer (Quadra 900 with NIH Image version 1.61 software). Images were analyzed for collagen area fraction and muscle fiber orientation relative to the circumferential axis of the heart. Mean angle for each tissue depth was determined by averaging 10 measurements within 2 nonadjacent fields in the tissue section. In this way, we could generate a progression of muscle fiber angles transmurally through the noninfarcted wall.
Methods for identifying sheet orientation. It was possible to determine sheet angle in a block of tissue adjacent to the block of tissue, which contained the bead set in five of the six animals. Because this initial block was used to histologically determine the myocyte angles, an adjacent region of tissue was used. A through-wall block of fixed tissue was excised with dimensions (in mm) 3 x 3 x n, where n is the ventricular wall thickness (rounded down to the mm). Two cuts (3 mm apart) were in the circumferential plane whereas the other two cuts (3 mm apart) were in the apex-base plane. After the apex side was marked, a custom made sledge-type slicer was used to cut the block in cross-section at 1-mm intervals parallel to the epicardial surface to yield n blocks with dimensions (in mm) 3 x 3 x 1. The order of the 1-mm slices from epicardium to endocardium was preserved by gluing them with cyanoacrylic adhesive, to a piece of paper with the epicardial side facing up and with the marked apex side toward the bottom of the paper. The slices were infiltrated for 1 wk and embedded with JB-4 plastic embedding media (Polysciences) according to the product instructions for infiltration and embedding.
The embedded tissue is translucent and the myofiber direction can be seen on transillumination with a dissecting microscope. After the plastic was allowed to soften by exposing the sample to air for 2 h, 2 parallel, cross-fiber cuts (1 mm apart) were made with a utility knife. This cut sample was mounted so it could be sectioned with the microtome blade in a plane transverse to the myofiber. Figure 2 displays such a section. , the sheet orientation angle, is the angle subtended by the sheet gap and the radial direction, and is positive if the sheet gap points toward the positive cross-fiber direction (see Fig. 3 for explanation of fiber and sheet coordinates). For the sample in Fig. 2, = –44° on average.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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End-diastolic remodeling strains referred to cardiac coordinates. Remodeling strains averaged for the inner and outer halves of the wall are shown in Fig. 4. Circumferential strain was positive at all depths and significantly different from zero with the greatest strain magnitude occurring from the midwall to the endocardium. Radial remodeling strains were not significantly different from zero in the outer third of the wall becoming negative and significantly different from zero at depths below the midwall. Shear in the plane of the ventricular wall (in-plane shear, E12) and circumferential-transverse shear (E13) were not different from zero at any depth. Longitudinal-transverse shear strains (E23) were positive and significantly different from zero at the endocardium.
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Changes in fiber and sheet angle. Myocyte fiber angle was –65.4 ± 7.5° at the epicardium and linearly progressed to 50.3 ± 7.2° at the endocardium. Subepicardial, midwall and subendocardial collagen area fractions were similar (9.7 ± 3.6%, 8.8 ± 2.3%, and 8.2 ± 3.4%, respectively).
Because myofiber and sheet orientation were determined only in the remodeled configuration, it was necessary to estimate the original fiber and sheet orientation to use as a reference for calculating strain components. This was done as described earlier from the measured strains. Figure 5 shows the estimated changes in muscle fiber and sheet orientation at all sites across the wall in five animals. There was a small change in muscle fiber angle in the spared myocardium adjacent to an infarction [average 4.1 ± 2.4°: absolute value of (control – 3 wk)], whereas the average change in sheet angle was approximately twofold greater (9.4 ± 8.2°).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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After infarction, global remodeling of the infarcted ventricle occurs rapidly. Initial enlargement of the infarcted ventricle may occur as a direct result of lost contractile tissue and a subsequent decrease in stroke volume that causes increased end diastolic volume (24). Remodeling of the post-MI ventricle is characterized by chamber dilation in mouse (23), rat (4, 19, 32) and large animal models (10). Fifty days post-MI, the transverse LV dimension increased 22% in the rat and 35% in the dog (10). Interestingly, there seems to be some interspecies variability in chamber remodeling. Whereas the two species had similar infarct sizes, the relative increase in ventricular globularity postinfarction was greater in the rat than the dog (10). LV dilation after infarction also occurs in humans. Two weeks after a patient's first MI, end-diastolic volume increased 27.6%, as measured by MRI (18). The hearts in the present study also increased in size, as reflected by an increase in chamber dimension of >2 mm at the same end diastolic pressure 3 wk after infarction.
In the present study, changes in chamber dimensions were also accompanied by changes in the structure of the ventricular wall. Three weeks postinfarction, there was a 10% increase in tissue volume in spared myocardium adjacent to the infarct. This increase in tissue volume was uniform across the wall, and qualitatively agrees with the volume change observed in myocytes isolated from rat hearts 3 to 4 wk postinfarction. Within 72 h of surgically induced infarction, rat myocytes exhibit a 14% increase in length and a 28% increase in volume (1). Eight-weeks post-MI, ovine myocytes in noninfarcted tissue adjacent to the MI exhibited an 18% increase in length and a 36% increase in volume (14). Changes in myocyte diameter are less consistent. In rats, the increased volume and length were accompanied by a 6% increase in diameter (1). However, ovine myocytes showed no change in cell cross-sectional area or myocyte width (14). Our data suggest a 13.3–23.8% strain in the direction of the myocytes (see Fig. 5), which is in close agreement with the increase in length from isolated myocytes (14% to 22%). Thus myocyte length changes may contribute significantly to ventricular remodeling postMI. Although we did not measure myocyte cross-sectional area, the small increase in tissue volume in this study (10%) is substantially less than the 28–36% increase seen in individual myocytes (1, 14) implying either fewer cells across the wall or a change in myocyte density. The latter seems unlikely based on the data of Weisman et al. (33). Apoptosis may be a contributing factor in the decrease in myocytes across the remodeling ventricular wall (21).
Our remodeling data directly show for the fist time substantial cross-fiber remodeling in the "normal" tissue adjacent to an infarct. In our data, both cross-fiber remodeling deformation and radial cross-fiber shear were positive and significantly different from zero in the inner half of the ventricular wall. These data provide strong support for the concept of cell slippage, which has been inferred from measurements of myocyte density (unchanged) and the number of myocytes across the wall (decreased) in several experimental studies after MI (20, 32). However, these and previous results cannot exclude several other potential mechanisms that may explain cross-fiber remodeling. Kramer et al. (14) and Gerdes et al. (7) have mentioned changes in cell shape in the failing myocardium as a possibility and have presented preliminary data supporting this contention. These data provide evidence that the type of hypertrophy is reflected in differential myocyte shape change. In the present study, myocytes with larger in-plane dimension would explain the large positive cross-fiber strains and very large positive sheet normal strains. However, there is no direct evidence for this shape change in our post-infarct spared myocardium. Changes in the interstial space would also explain these changes. However, no changes in myocyte density have been reported. Collagen content in the present study, as reflected by picrosirus red staining, averaged 8.9 ± 3.05% and was not different from values determined with the same technique (12) in pigs. Thus changes in the interstitial volume seem unlikely. However, significant changes in the structure of the wall of the ventricle as observed in this study imply that the organization of the matrix is also altered. Indeed, other recent studies (26), which indicate that collagenase activity is essential for ventricular remodeling, imply that these changes may allow the remodeling to occur. Myocyte dropout due to apoptosis has been shown to be present postinfarction but would not explain the positive cross-fiber strains.
The significant positive cross-fiber radial remodeling strains in the present study could be explained by the following: a rotation of myocytes as proposed by Weisman and others (20, 32); by changes in cell shape; or by alterations in the orientation laminar architecture () (5, 16). However, because there were no significant changes in sheet normal shear, the latter possibility seems unlikely. In the present study, the inner half of the ventricular wall adjacent to a MI thinned substantially. In rats, noninfarcted myocardium may thin (32), remain unchanged (8), or hypertrophy (10). In larger animals and patients, noninfarcted myocardium distal to an infarct either thickens (3) or does not change (10). Fewer data exist for remodeling in tissue adjacent to an infarct. A reexamination of data from Kramer et al. (14) reveals significant thinning of myocardium they referred to as "adjacent" at 8 wk after surgically induced infarction (P = 0.001) and the magnitude of the thinning (8%) was similar to that measured in the current work. In the present study, the average cross-fiber radial shear, calculated in the inner half of the wall, was 0.15. This is equivalent to a 15° angle change (31). Figure 8 shows a schematic diagram of potential mechanisms of wall thinning remodeling that have been proposed by several authors. Cell slippage has been investigated by a change in the number of cells across the wall. Several researchers have shown an 40% reduction in transmural myocyte number (2, 19, 20, 33). A relative rotation of myocytes around the cellular long axis of 20° can explain the 40% wall thinning. This calculated 20° change is in relative agreement with our measured 15° cross-fiber radial sheer strain and the large positive strain normal to sheet remodeling strains. However, it is not possible to eliminate two other likely possibilities. A change in myocyte shape to a cell flatter in the epicardial tangent plane also could explain the decrease in radial strain. There is no information on the absolute loss of myocytes across the wall. A 10% decrease in wall thickness would require a 10% cell loss, which seems unlikely considering the short period of time and the relatively low apoptosis rates seen in the rat infarct model (11).
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8 mm apart in the in epicardial tangent plane direction, and thus we are averaging strains over a relatively large sample of myocardium. Moreover, the resolution of the radiographic system is 0.2 mm and the absolute measurements of cardiac dimensions are of this magnitude, which adds substantial difficulty in resolving small changes. We also assume that the markers (gold beads) remain coupled to the same tissue and do not independently migrate. In support of this assumption, none of our previous studies with this technique, which observed the markers repeated over time, showed rapid discontinuous movement of the markers (9). It was not possible to examine sheet and fiber architecture at the same site. In the present study sample blocks may have been as much as 5–8 mm apart, and this could clearly add to the variability in the fiber-sheet data. Moreover, the hearts were fixed at zero distending pressure and we did not have hemodynamic and marker data at that pressure. This should not affect the fiber data greatly because the fiber direction does not change appreciably with distending pressure (6, 27, 28). However, our present data indicate that sheet angle changes with distending pressure. Thus the sheet angle changes here may well include a component due to the difference between zero distending pressure where the hearts were fixed and the end-diastolic pressure where bead configuration was measured (11 mmHg).
In summary, 3 wk after MI, noninfarcted tissue adjacent to the infarct remodeled by expansion along the direction of the fibers and in the cross-fiber direction. These changes are consistent with myocyte elongation and myofiber rearrangement (slippage) as well as a change in cell shape to a more elliptical cross section with the major axis in the epicardial tangent plane. Although this is the first study to clearly demonstrate cross-fiber remodeling, these data are qualitatively consistent with a wealth of previous data showing increased ventricular dimensions (18, 25), changes in myocyte shape (13, 15), and myocyte rearrangement (33) after MI. Moreover, these data indicate that reorientation of fibers either via cell slippage (32) or changes in orientation of the laminar structure (5, 16) of the ventricular wall are quantitatively important aspects of the remodeling of the adjacent normally perfused myocardium.
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GRANTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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