myocardial structure and function in patients with aortic valve disease and their relation to...

Myocardial Structure and Function in Patients With

Aortic Valve Disease and Their Relation to

Postoperative Results

FRANZ SCHWARZ, MD WILLEM FLAMENG, MD JUTTA SCHAPER, MD FRIEDERIKE LANGEBARTELS, MD MICHAEL SESTO, MD FRITZ HEHRLEIN, MD MARTIN SCHLEPPER, MD

Bad Nauheim and Giessen West Germany

From the Kerckhoff-Klinik, Max-Planck-Gesells- chaft, Bad Nauheim and from the Department of Cardiovascular Surgery, Justus-Liebig University Giissen, West Germany. Manuscript received July 6, 1977; revised manuscript received October 14, 1977, accepted October 19, 1977.

Address for reprints: Martin Schlepper, MD, Kerckhoff-Klinik, D-635 Bad Nauheim. West Germany.

Left ventricular angiographic studies were performed before and 6 months after aortic valve replacement with a Bjiirk-Shiley prosthesis in 21 patients, 5 with aortic stenosis, 8 with mixed aortic valve lesions and 8 with aortic insufficiency. The degree of myocardial fibrosis and myocardial ultrastructural changes were evaluated from transmural needle biopsy specimens obtained from the left ventricular anterior free wall at operation. Twelve patients without heart disease served as control subjects for an- gjographic data. Patients with aortic valve disease had a significantly higher left ventricular mass before operation than control subjects and a lower ejection fraction and mean normalized systolic ejection rate. After operation left ventricular mass decreased considerably but did not reach normal level. Ejection fraction and mean normalized systofk ejection rate became normal in all patients with aortic valve disease. The percent fibrosis determined with morphometry was significantly. higher in the subendocardium than in the subepicardium of pressure-overloaded hearts (predominant stenosis) (19 versus 13 percent) but equal in both layers of volume-overloaded hearts (predominant regurgitation) (19 versus 18 percent). Electron microscopy revealed significant intracell alterations of the nucleus, sarcomeres, mitochondria and cytoplasmic reticulum. When all patients, regardless of type of aortic valve lesion, were considered, there was no significant correlation before operation between percent fibrosis and ejection fraction (f 0.10) or mean normalized systolic ejection rate (r 0.02) but a significant inverse relation between left ventricular mass and ejection fraction (r 0.54) as well as mean normalized systolic, ejection rate (r 0.49).

These data suggest that (1) Depressed left ventricular function in aortic valve disease is associated with ultrastructural degenerative cell changes, but complete recovery of cardiac function after aortic valve replacement is not prevented by these changes. (2) Interstitial myocardial fibrosis is not a primary determinant of depressed cardiac function in aortic valve disease.

The role of histopathologic myocardial changes in the regression of hypertrophy and recovery of cardiac function after removal of pressure or volume overload in man has not been defined.‘T2 It is generally accepted that in advanced left ventricular hypertrophy due to work overload interstitial myocardial fibrosis develops, probably as a result of focal cell destruction.3-5 Recently, degenerative changes of myocardial cells associated with marked interstitial fibrosis have been described in aortic valve disease. These cell changes were considered responsible for impaired cardiac performance in some patients.6T7 In a preliminary report we showed that the decrease in left ventricular function in aortic valve disease is correlated with the increase in left ventricular mass and with

April 1978 The American Journal of CARDIOLOGY Volume 41 881

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the presence of degenerative changes at the ultrastructural leveL8 In this study we quantitated the degree of interstitial myocardial fibrosis in biopsy material from patients who underwent aortic valve replacement. Electron microscopic studies of these tissue samples did not provide exact quantification of myocardial cell degeneration but allowed some qualitative estimation of ultrastructural alterations. The morphologic changes were then related to the pre- and postoperative hemodynamic data of these patients.

Methods

Patients: We studied 21 patients with isolated aortic valve disease between 2 and 6 weeks before aortic valve replacement. It was possible to restudy 13 of these patients 6.2 f 1.7 (mean f standard deviation) months (range 5 to 11 months) after aortic valve replacement by a Bjbrk-Shiley tilting disc valve prosthesis. Patients 5, 11 and 12 (Table I) died in the early postoperative phase; five other patients refused the second investigation. Before operation, predominant aortic stenosis was found in 5 of the 21 patients, a mixed aortic valve lesion in 8 and predominant aortic insufficiency in the remaining 8. Associated mitral valve disease was excluded in all patients by analysis of synchronously registered left atria1 and left ventricular pressure curves and, when necessary, additional angiographic studies of the left ventricle. Obstructive coronary artery disease was not found in any patient during selective coronary arteriography.

Functional class was estimated before and after operation by evaluating symptoms commonly associated with aortic valve disease: angina, syncope or presyncope and dyspnea. The electrocardiogram before and after operation was ana- lyzed with the method of Romhilt and E&es9 and cardiothoracic ratio was measured from the chest X-ray film preoper- atively and at the time of restudy. Age, sex, diagnosis, functional class, Romhilt-Estes score, cardiothoracic ratio, aortic valve pressure gradient, degree of aortic regurgitation,1° time after operation at second study and size of Bjijrk-Shiley valve are listed for each patient in Table 1.

Procedures: Patients with aortic valve disease were studied before and after operation without premeditation in the same catheterization laboratory and with the same equipment. All patients were receiving digoxin before and after operation. A no. 8.5F Brockenbrough catheter was positioned in the left ventricle transseptally with use of a femoral vein puncture and a no. SF pigtail catheter was inserted into the ascending aorta in retrograde fashion through the femoral artery. Before injection df co&rast,material,,left ventricular and aortic pressures were measured with Statham P23Db pressure trans- ducers and recorded on an Oscillomink direct writing system. The catheter manometer systems tested in vitro on a sine wave pressure generator had a uniform amplitude response up to 30 hertz. Single plane 35 mm cineangiograms of the left ventricle were filmed at a rate of 48 frames/set in the 30’ right anterior oblique projection after injection of 50 ml Urogra- fin-76@ into the left ventricle using a Brockenbrough catheter. Simultaneously the aortic pressure was recorded with a paper speed of 100 mm/set. No mitral regurgitation was detected during this procedure during normally conducted sinus beats. In all patients aortic root angiography was performed to es- timate the degree of aortic regurgitation.‘O Thereafter, coronary arteriography was carried out using the Judkins technique.

Calculations: From the cineangiograms silhouettes were drawn of the left ventricular cavity at end-diastole and end-

systole. Where indentations in the ventricular outline were caused by papillary muscles, the outline was drawn to include all opacified areas. The long axis (L) of the left ventricle was taken as the longest measured line from the apex to the aortic valve. The minor axis (M) was calculated as M = 7~. A/4-L, where A = area determined with planimetry. Ventricular volumes (V) were calculated using the single plane area-length method% V = 7r/6.M2.L. Correction factors for magnification and pincushion effect were obtained from a grid filmed in the plane occupied by the left ventricle. The regression equation of Kasser and Kennedyll was used. In 15 patients without heart disease we found no significant difference between stroke volume determined from manually checked cardiac output determinations obtained with the thermodilution technique12 (no. 7F Swan-Ganz thermodilution catheter coupled to a cardiac output computer [Fa. Diefenbach, Ffm] and to a Kipp Bl2 direct writing system) and stroke volume assessed angiographically. Stroke volume assessed with thermodilution was 45.4 f 10.8 ml/m2 (mean f standard deviation) compared with the angiographic value of 44.8 f 10.2 ml/m2. These values are in close agreement with the data reported by others13 in normal persons.

A representative measurement of left ventricular wall thickness was made from the right anterior oblique projection of the ventriculogram in end-diastole at a point approximately midway between the apex and the aortic valve’4.

The ejection fraction was determined as stroke volume divided by end-diastolic volume times 100 percent. The mean normalized systolic ejection rate was calculated by division of ejection fraction by the ejection time as determined from the aortic pulse.

Left ventricular mass was calculated according to the method of Rackley et al.15 as modified by Trenouth et a1.16 as: VM = (Vc+w - V9.1.05, Vc+w = 413 P (M/2 + hJ2.(L/2 + h/2), where VM = left ventricular mass, Vc+w = volume of left ventricular cavity plus wall, V’ = end-diastolic volume, h = left ventricular wall thickness at the minor equator at end- diastole and 1.05 = density of the myocardium.

Collection and preparation of biopsy tissues: Tissues were obtained by biopsy from each patient at the time of open heart surgery. Operation was performed with the patient on cardiopulmonary bypass, utilizing a disposable bubble-type oxygenator. We use a Tru-cut biopsy needle (Travenol Lab- oratories) to take a transmural biopsy specimen from the left ventricular anterior free wall. The specimen was taken before cross-clamping of the aorta from the beating heart. In three patients (Cases 8, 10 and 17) only an intramural biopsy specimen was obtained. Immediately after biopsy the tissues were separated into subepicardial, intramural and subendocardial samples and were fixed in buffered 6 percent glutar- aldehyde. Most patients had more than one biopsy specimen (Table I). After immersion fixation for 24 hours at 4” C the samples were rinsed in 0.1 molar cacodylate buffered with 7.5 percent sucrose during 12 hours at 4O C. After postfixation for 1 hour in 2 percent osmium tetroxide in veronal acetate buffer at pH 7.4 with 4 percent sucrose at 4’ C, the tissues were de- hydrated in graded series of alcohol, treated with propylene oxide and embedded in Epon@. After polymerization semithin sections of 1 to 2 wrn thickness were prepared and stained with alkaline toluidine blue for light microscopy. Each block measured 1 to 4 mm2 in area.

Morphometry was carried out with the light microscope using a special grid with vertical and horizontal lines providing 110 intersections (points). According to the basic principles of morphometry,17 counting of the number of points overlying a structure results in a quantitative determination of the volume of the structure in relation to the volume of the entire

MYOCARDIAL STRUCTURE AND FUNCTION IN AORTIC DISEASE-SCHWARZ ET AL.


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tissue under the square grid. Therefore, the total number of points was regarded as 100 percent and the points counted in the fibrotic area were expressed as the percent of the entire tissue within the limits of the grid. In this investigation all points overlying fibrotic tissue were determined. The axis of the square grid was then rotated about 45”, and determination of the points was repeated. After a random sampling procedure a different part of the same section was chosen and the points overlying the fibrotic area were counted with the grid in control position and after rotation of 45O. In this way, two areas of each section were counted twice. Only longitudinal sections at a magnification of 250X were evaluated, and blood vessels and perivascular interstitial cells were excluded from the fibrotic area. In each patient three to nine tissue samples and two sections from each sample were counted. For electron microscopic examination thin sections were cut with a LKB Ultratome III, mounted on uncoated copper grids, stained with saturated uranyl acetate and lead citrate and examined with a Philips electron microscope 300.

Results

Clinical data: All patients with aortic valve disease showed improvement in functional class and the Romhilt-E&s-score. The cardiothoracic ratio improved in all but Patient 16, who had no reduction of cardiothoracic ratio (Table I). The greatest reduction of cardiothoracic ratio (from 0.64 to 0.42) occurred in Patient 8, who had mixed aortic valve disease.

Hemodynamic data: Before operation, patients with aortic stenosis had increased left ventricular systolic and end-diastolic pressures (P <O.OOl compared with control values), normal values for end-diastolic volume, ejection fraction and mean normalized systolic ejection rate (P >0.05) but an increased left ventricular mass (213 percent of control value, P <O.OOl, Table II). Pa- tients with both aortic stenosis and insufficiency had increased left ventricular systolic and end-diastolic pressures (P <O.Ol and P <O.OOl) and end-diastolic volume (P <O.OOl) with a reduced ejection fraction (P <O.Ol) and mean normalized systolic ejection rate (2’ <O.Ol). Left ventricular mass was increased to 300 percent of control value (P <O.OOl). Patients with aortic insufficiency had increased left ventricular systolic pressure (P <O.Ol) but a normal left ventricular end- diastolic pressure (P >0.05). End-diastolic volume was increased (P <O.OOl), and ejection fraction (P <O.OOl) and mean normalized systolic ejection rate (P <O.Ol) were decreased (Table II). Left ventricular mass was increased (227 percent of control value, P <O.OOl).

The two patients with aortic stenosis who were restudied postoperatively showed a reduction of left ventricular systolic pressure from 180 and 184 to 120 and 126 mm Hg, respectively. Left ventricular end- diastolic pressure and volume decreased in both patients. Whereas left ventricular mass decreased considerably, an increase in ejection fraction and mean normalized systolic ejection rate was noted. Paired data before and after operation in five patients with mixed aortic valve disease showed at comparable heart rates (P >0.05) a significant decrease of left ventricular systolic pressure (P <0.05) and end-diastolic pressure (P CO.01). End-diastolic volume (P <0.05) and left ven-

LV mass - g/in2

t PC.001 p< ,001

n AS

l AS+A I I

0 Al b a b a

FIGURE 1. Individual data on left ventricular (LV) mass and mean normalized systolic ejection rate (MNSER) before and after aortic valve replacement in patients with aortic valve disease. The normal range is indicated by verkal bars (mean value f2 standard deviations). a and b = after surgery and before operation, respectively; Al = aortic insufficiency; AS = aortic stenosis; AS + Al = aortic stenosis plus aortic insufficiency; ed vol = end-diastolic volume.

4OC

MNSER-control ed vol/sec

20C

tricular mass decreased (P <0.05), whereas ejection fraction (P <0.05) and mean normalized systolic ejection rate (P <O.Ol) improved.

The six patients with aortic insufficiency had no further evidence of significant aortic regurgitation after operation (Table I). At comparable heart rates left ventricular systolic pressure was unchanged (P >0.05) as was left ventricular end-diastolic pressure. Left ventricular end-diastolic volume and left ventricular mass decreased significantly whereas ejection fraction and mean normalized systolic ejection rate increased (P <O.Ol).

Figure 1 illustrates the regression of left ventricular mass and the increase of mean normalized systolic ejection rate after operation, indicating improved cardiac function after aortic valve replacement. When we considered all patients regardless of the type of aortic valve lesion, we found a significant inverse relation between left ventricular mass and ejection fraction (r 0.54, P <0.05) and between left ventricular mass and mean normalized systolic ejection rate (r 0.49, P <0.05) before operation but not after operation (r 0.00 and r 0.06, P >0.05).

Myocardial fibrosis: Table I presents the results of evaluation of interstitial fibrosis, and Figures 2 and 3 show representative examples of light micrographs. Table I presents the average percent fibrosis for the subepicardial, intramural and subendocardial muscle layers. The overall mean value was calculated by aver- aging the mean values obtained for the different layers. Between three and nine tissue samples were used to



FIGURE 2. Light micrograph of subendocardial tissue from a patient with aortic stenosis. The cardiac muscle cells (C)have lost their intercon- nections, and targe amounts of interstitial fibrosis (F) are evident (X1,250, reduced by 34 percent).

calculate the percent fibrosis, because fibrosis in hypertrophied hearts is widespread but may be focally accentuated.5 In patients with predominant pressure overload (aortic stenosis and aortic stenosis plus insufficiency) the percent fibrosis was significantly larger in the subendocardium than in the subepicardium (19 versus 13 percent, P <0.05). In the group with predominant volume overload (aortic insufficiency) there was no significant difference between percent fibrosis of both layers (19 versus 18 percent, P >0.05).

We calculated total left ventricular fibrotic mass by multiplying the mean percent fibrosis and left ventricular mass (divided by 100 percent). Left ventricular muscle mass was then determined as left ventricular mass minus left ventricular fibrotic mass. Figure 4 shows the relation between left ventricular fibrotic mass and left ventricular muscle mass for all patients studied. This relation is significant and has a correlation coef- ficient of 0.76 (P I<O.OOl). However, there was no sig-

nificant correlation between percent fibrosis and ejection fraction (r 0.10, P >0.05) or mean normalized systolic ejection rate (r 0.02, P >0.05) when all cases were considered irrespective of type of aortic valve lesion.

Myocardial ultrastructure: Electron microscopy, performed in 15 patients (Table I), revealed features of degeneration of the cardiac muscle cells in 13 patients and their absence in Patients 10 and 20. The morphologic alterations interpreted as degenerative were found in all layers of the myocardium. There ap- peared to be no correlation between the nature of the hemodynamic alterations to play no role and the presence of these changes (Table I). The degenerative alterations consisted of (1) unusually large nuclei exhibiting invagination of the nuclear membrane, irregular distribution of chromatin and the occurrence of tubular nuclear structures; (2) loss of myofibrils and accumulation of Z band material; (3) accumulation of large amounts of mitochondria exhibiting variable shape and

FfGURE 3. Light micrographs. a, a myocardial cell showing areas free of contractile elements (ar- rqwheade). Increased amounts of interstitial fl- brosis (F) are shown close to the cell (X1,250, reduced by 35 percent; Normarski optics). b, myocardial cells showing loss of myofilaments (arrowheads). There are many palely stained areas of cytoplasm in which myofibrils appear to be absent (X1.250, reduced by 35 percent).

666 April 1978 The American Journal of CARDIOLOGY Volume 41


size; (4) occurrence of large cytoplasmic areas that were devoid of contractile material but filled with glycogen, ribosomes, and sometimes increased amounts of sarcoplasmic reticulum; and (5) presence of proliferative fibroplasts, macrophages, mast cells, and increased amounts of collagen fibers in the interstitial space.

Figures 4 to 7 show the typical features of myocardial cells with loss of contractile material and disturbed sarcomeric organization. No attempt was made to correlate the frequency and degree of myocardial cell degeneration with cardiac function. In all 13 patients these ultrastructuralalterations wererelativelyconsistent, and we believed that quantitative estimation or grading of degenerative changes would be a subjective procedure.

Discussion

Critique of methods: Our study was designed to determine the significance of interstitial myocardial fibrosis and degenerative cell changes in postoperative cardiac function in patients with aortic valve disease. The transmural biopsy technique under direct vision when previously used in patients with coronary artery diseasel* was found to be of considerable accuracy when related to electrocardiographic findings and ventricular asynergy in the same region. We tried to minimize the sampling error by taking more than one biopsy specimen in most patients. In addition there is evidence that in left ventricular hypertrophy the fibrotic process and the degenerative cell changes are widespread but not localized.4l5 The presence of increased amounts of interstitial myocardial fibrosis in advanced hypertrophy due to aortic valve disease is well known.3*4~5Jg~20 Reichenbach et a1.5, in a preliminary study of 10 patients with mitral and aortic valve disease but without coronary heart disease, found, a correlation between

801

cu 60 5 Cn _

D a40 E 0 - .- E 5 20-

s

r= .76

p’ .OOl

1 I I 1 100 200 300

LV muscle mass-g/m2-before surgery

n AS l AS+AI 0 Al

FIGURE 4. Linear relation between left ventricular (LV) muscle mass before operation and left ventricular fibrotic mass as determined with morphometry. Al = aortic insufficiency; AS = aortic stenosis: AS -I Al = aortic stenosis plus insufficiency; p = probability.

fibrosis and left ventricular mass. More severe fibrosis was found in aortic than in mitral valve disease and fibrosis tended to be more prominent in the subendocardial area. Sasaki et aL21 found a correlation between the amount of fibrosis and increased heart weight in a morphometric study of hypertrophied hearts. The good correlation between mass of fibrosis and left ventricular muscle mass in our biopsy specimens confirms these



FIGURE 6. Electron micrograph of the contractile elements in a hypertrophied myocardial cell. The myofilaments are irregularly arranged, running in different directions (arrowhead). The 2 band (Z) is thickened and irregufar. Thick filaments are lost near the abnormal Z band leaving thin filaments (arrow) (X56.000, reduced by 34 percent).

reports. Furthermore it demonstrates that our biopsy technique allows the evaluation of the morphologic state of the myocardium within a fair range of accuracy. Al- though objective quantification of ultrastructural changes in myocardial cells is not possible with our technique, degree of qualitative evaluation can be obtained. We accepted degenerative changes of the myocardium as significant when they were found in the majority of tissue samples.

To assess cardiac function in normal and hypertrophied hearts we used the ejection fraction and the mean normalized systolic ejection rate. The ejection phase indexes express function per unit of muscle and are considered useful in detecting changes in level of ino- tropic state.22 Furthermore, others23 have shown that the ejection phase indexes are superior to isovolumetric

FIGURE 7. Electron micrograph showing large vacuoles (V) and tubular and vesicular sarcoplasmic reticulum (R). The cell ten be Identified as a cardiac muscle cell because some remnants of myofilaments (ar- rows) can be detected (X16,800, reduced by 32 percent).

indexes in detecting depressed myocardial function in the basal state.

Regression of hypertrophy and recovery of left ventricular function after operation. After aortic valve replacement there were significant reductions in left ventricular mass and left ventricular end-diastolic volume. However, whereas end-diastolic volume became fully normal, left ventricular mass did not. In addition, the decreased left ventricular function, as evaluated with ejection phase indexes, was reversible and became normal in patients with aortic valve disease. The improvement in cardiothoracic ratio, Romhilt-Estes score and symptoms correlates well with the angiographic data. It is well known that in hypertrophied hearts left ventricular function may be normal at rest but abnormal during stress.24 This fact may limit the significance of our results but it does not alter our finding that resting cardiac function improved considerably after aortic valve replacement. Another recent investigation in this laboratorys5 evaluated a series of patients with aortic valve disease after aortic valve replacement at rest and during isoproterenol-induced stress using two ventricular angiograms. After aortic valve replacement most patients had a normal ejection fraction during isoproterenol stress although a peak transvalve pressure gradient of 30 to 40 mm Hg developed.

We believe that the persisting mild outflow tract obstruction after implantation of a Bjork-Shiley prosthesis prevents complete regression of left ventricular hypertrophy. Reductions in end-diastolic volume and left ventricular mass after homograft aortic valve replacement were reported. 2s Normal cardiac function after aortic valve replacement by a Starr-Edwards prosthesis was observed by Hultgren et a1.,27 who found the greatest decrease of heart size in patients with heart failure before operation. Recently Schuler et a1.2s described an important influence of time on recovery of myocardial function after valve replacement for aortic insufficiency. In our study this time factor may not have

666 April 1976 The American Journal ol CARDIDLDGY Volume 41

played an important role because the data were obtained 5 to 8 months postoperatively in 12 of 13 patients.

Relation between myocardial structure and postoperative result: Before operation, left ventricular function was significantly reduced in patients with aortic valve disease. This reduced function was depen- dent upon the degree of hypertrophy of the myocardium but *did not correlate significantly with percent fibrosis. These findings suggest that the scars thought to have a detrimental effect on contraction, that is, separation of cardiac muscle cells and disruption of the functional myocardial syncytium,3,2g are less important for overall cardiac performance. The ultrastructural myocardial alterations found in most cases are probably more related to the reduced contractile function in these hearts. Our results imply that these alterations were the cause of the reduced contractile function in our patients. Such structural alterations were previously described in detail in a similar group of patients by Maron et a1.,s7 who suggested that degenerative changes in myocardial cells may explain the impaired cardiac function in patients with aortic valve disease. Although we could not

correlate the degree of cell degeneration with cardiac function, we did show that the “myocardial factor” was present in patients with depressed cardiac function and advanced hypertrophy.

Restudy of the same patients 6 months after aortic valve replacement allowed comparison of preoperative morphologic features and functional state of the myocardium with the postoperative clinical and hemodynamic course. Obviously the presence of cardiac muscle cell degeneration was not associated with a lack of improvement after operation. Six months postoperatively left ventricular hemodynamics at rest were restored to normal even in patients with severe myocardial fibrosis and muscle cell degeneration.

Implications: We conclude that the preoperative depressed cardiac function in patients with aortic valve disease is related to ultrastructural myocardial changes and not to degree of myocardial fibrosis. The restoration of depressed myocardial function after aortic valve replacement strongly suggests that the ultrastructural myocardial changes described are fully reversible. The generally accepted term “degenerative” is probably not adequate.


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