morphometric study of human myocardium in acquired valvular diseases

Original Article

Morphometric Study of Human Myocardium in Acquired Valvular Diseases

Esa Jantunen’, Matti 0. Halinen*, Tim0 Romppanen3, Veli-Matti Kosma’ and Yrjo Collan4

To study the effect of various valvular heart diseases on the quantitative histology of myocardium, 38 human hearts with valvular lesions were examined (11 aortic stenoses, nine mitral stenoses, nine mitral incompetences and nine combined aortic and mitral valve lesions). The control group consisted of ten hearts without any valvular lesions. With morphometrical methods the volume fractions of myocardial components (myocardial fibres, interstitial space and diffuse connective tissue), the numerical density of arterioles and the mean fibre diameter were estimated.

Myocardial fibrosis was more severe in hearts with valvular lesions than in the controls (5.4 o/o vs 3.3 %, P<O.Ol), but did not correlate with the anatomical severity of the valvular lesions. The most severe myocardial fibrosis was found in hearts with mitral imcompetence (6.7 %). Fibre hypertrophy was most severe in hearts with aortic stenosis and in hearts with mitral incompetence (22 pm and 23 pm, respectively). In hearts with severe valvular lesions the mean fibre diameter was 23 pm and in hearts with mild to moderate lesions 19 pm (P<O.Ol). Good correlation was observed between the mean fibre diameter and the weight of the left ventricle (r=0.81, P<O.Ol). The volume fractions of connective tissue and interstitial space were significantly higher and the volume fraction of myocardial fibres was correspondingly lower in the subendocardium than in the subepicardium in hearts with either pressure overload (aortic stenosis) or volume overload (mitral incompetence). In conclusion, myocardial fibrosis occurs in patients with various valvular lesions, but the severity of the fibrosis does not correlate with the anatomical severity of valvular lesions. Marked transmural differences were observed in the volume fractions of myocardial components between the subendocardium and the subepicardium in patients with aortic stenosis and in patients with mitral incompetence. These transmural differences should be taken into account when endomyocardial biopsies are used in clinical practice.

Key words: myocardial hypertrophy; morphometry; myocardial fibrosis; valvular disease; transmural differences.

(Annals of Medicine 21: 435-440, 1989)

Introduction

Myocardial hypertrophy is a compensatory response of the heart in valvular disease. Congestive heart failure developes in severe cases when hypertrophy can no longer compensate for the increased work load. Many aspects in the pathogenesis and pathophysiology of heart failure are still incompletely understood. Mor- phological fac!ors that have attracted attention in as-

From the ’ Department of Pathology, University of Kuopio, Kuopio; 2 Department of Medicine, Kuopio University Central Hospital, Kuopio; Department of Pathology, Central Hospi- tal of North Carelia, Joensuu, and Department of Pathology, University of Turku, Turku.

Address: Esa Jantunen, M.D., Department of Pathology, University of Kuopio, P.O. Box 6 SF-70211 Kuopio Finland.

Received: January 9 1989; revision accepted August 3, 1989.

sociation with heart failure are myocardial fibrosis and fibre hypertrophy. Myocardial fibrosis has been shown to be associated with increased diastolic myocardial stiffness in patients with valvular disease (1). On the other hand, it has been proposed that excessive hypertrophy of myocardial fibres decreases the ejection fraction of left ventricle by disturbing the process of con- traction (2).

There are some reports, in which quantitative changes in the myocardium of patients with valvular heart diseases have been studied (1, 3-5). Most of these studies are based on endomyocardial biopsies iaken during cardiac catheterization. Baandrup and others have discussed the problem of how representative the endomyocardial biopsies are (6).

In this study we wanted to characterise the quantitative histological changes in the myocardium in patients

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436 Jantunen Halinen Romppanen Kosma Collan

with various valvular disease. We also wanted to com- pare some histological parameters with the anatomic severity of anatomic valvular lesions. In addition, histoquantitative study was performed separately in the subepicardium and subendocardium in patients with pressure overload (aortic stenosis) and volume overload (mitral incompetence) to find out the possible transmural differences in different histological parameters. To avoid the problem of how representative the samples were the material was selected from autopsy cases.

Material and Methods

We examined the autopsy files of Kuopio University Central Hospital 1981-1983 (about 1100 autopsies) and found 38 cases with valvular heart disease. There was no history of diabetes or treated hypertension in any of these patients, who included 11 cases of pre- dominant aortic stenosis (AS), nine cases of mitral stenosis (MS), nine cases of mitral incompetence (MI) and nine cases of combined aortic and mitral valve disease. As controls we had ten hearts without any valvular ab- normalities, diabetes or treated hypertension. These control cases were matched with the study group by age, sex and the prevalence of previous myocardial infarction, which was thought to be the major confounding factor in this study. The mean age of patients was 70.9 f 8.4 years (range 55-84) in the control group and 69.8 f 10.5 years (range 44-93 years) in the valvular disease group. The prevalence of previous myocardial ii1- farction (as assessed at autopsy) was 44 O/O in the valvular disease group and 40 YO in the control group (NS).

The cause of death was assessed from the death certificates. In the control group all patients died of extracardiac causes, mostly malignancies. In the valvular disease group the basic cause of death was valvular heart disease in 12 patients, coronary heart disease in 14 patients (eight acute myocardial infarctions, five previous infarctions attributable to congestive heart failure, one sudden death) and 12 patients died from extracardiac causes.

Clinical data from every patient were reviewed and special emphasis was laid on the possible aetiology of valvular heart disease and the severity of heart failure. Valvular disease was clinically diagnosed in 23 patients and at autopsy in an additional 15 patients. Four patients with clinically diagnosed valvular disease had had bacterial endocarditis, six had a history of rheu- matic fever, two with mitrat incompetence had suffered previous myocardial infarction that was suspected of being a contributory factor, one patient had clinically diagnosed Barlow’s syndrome with mitral incompetence and in one patient the valvular abnormality was associated with ancylosing spondylitis (Mb. Bech- terew). In the rest of the patients the cause of valvular disease was unknown or a degenerative cause was suspected. Cardiac catheterization had been performed in three patients and valve replacement in two patients (six months and four years before death). These patients who had had operation died from intractable congestive heart failure. Valvular lesions found for the first time at autopsy included seven AS, five MS and three MI.

The autopsy protocol for hearts in our hospital has been previously described (7). Aortic and mitral valves were carefully studied and if an abnormality was found the nature and severity of lesion was recorded in each case. The severity of valvular lesions whenever found was assessed by the pathologist and was based on examination of valvular orifice, valve cusps, chordae and papillary muscles. Patients with valvular lesions were divided into two groups based on the autopsy finding: patients with severe valvular lesions (n = 18) and p a tients with mild to moderate valvular lesions (n = 20). In addition to the dimensions of the valvular orifice possible left atrial enlargement and the degree of left ventricular hypertrophy as assessed at autopsy influenced the pathologist’s judgement on the severity of valvular lesions. The lesion was judged to be severe if the anatomical lesion was sufficiently serious to be capable of causing the patient’s death. The lesion was judged to be mild to moderate i f the anatomical finding was less severe and had caused minor or moderate functional derangement and could not have been considered as the basic cause of death of patient.

After the examination of valves, the left ventricular myocardium was cut from endocardium to epicardium at 5 mm intervals to identify any acute or old myocardial infarctions. Hearts were weighed and after the removal of epicardial adipose tissue (7) the weighing was repeated. After that the right ventricle, the free wall of left ventricle and the interventricular septum were separately weighed (7). Three transmural tissue samples were taken from standard sites (anterior left ventricular free wall, interventricular septum and posteri- or left ventricular free wall). The area of cut surface of these transmural samples ranged from 2 cmz to 4 cmz. The tissue samples were then fixed in 10 O/O neutral buffered formalin, embedded in paraffin, cut at 5 pm and stained with Masson trichrome and van Gieson methods.

The histoquantitative morphometric measurements were performed with WILD M 501 microscope (Heer- brugg, Switzerland) with a projection head and an automatic sampling stage. From sections stained with Masson trichrome method, the volume fractions of myocardial fibres (VVMY), interstitial space (V,,,,) and diffuse connective tissue (VvcT) were estimated with point counting methods (8-11). A square grid of 36 points was placed on the projection head (magnification x 200). Bluestained fibrillar tissue between myocardial fibres was considered as diffusely arranged connective tissue in contrast to non-staining interstitial space. Every fourth microscopic field was measured and the sections were scanned with an automatic sampling stage, which allowed systematic sampling. The volume

fractions were calculated from equation VVi = 3, where Vvi is the volume fraction studied, Pi is the number of point hits on the specific tissue component studied and P, is the total number of point hits on the whole area measured (reference area). The volume fractions were expressed in percentage terms (Vvl x 100). Microscopic fields with acute myocardial infarction, visible scars (more than 2 mm in diameters) as well as areas with staining or processing artifacts were excluded

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Morphometric study of myocardium in valvular diseases 437

from measurements. The number of intramyocardial arterioles (vessels with smooth muscle wall, diameter 15-50 pm) was also counted simultaneously in the same microscopic fields and the numerical density of arterioles was calculated from equation N,,, =

nart , where nart is the number of arterioles counted,

n, is the number of microscopic fields analysed per specimen and A, is the area of a microscopic field. The numerical density of arterioles was expressed as l /mm2.

The mean diameter of the myocardial fibres was measured from slices stained with van Gieson method. A magnification of 400 was used and only slices from left ventricular anterior free wall were studied. Meas- urements were made on the projection screen. Only fields with transsected muscle fibres were studied and the shortest diameter at the level of nucleus was measured. From 100 to 200 muscle fibres were measured per specimen.

To study the possible transmural differences in histological parameters in hearts with pressure overload (AS) and volume overload (MI) the histological slides representing ttie left anterior free wall were divided into three portions with a colour pen: subendocardium, midmyocardium and subepicardium. Measurements of

n, x A,

volume fractions and arteriolar density for the subendocardium and subepicardium were then performed as previously described. For calculation of the mean fibre diameter about 50 fibres were measured from both subendocardium and subepicardium.

For statistical analysis of the mean values between study groups Student's t-test was used. The correlation coefficient was calculated between the mean fibre diameter and the weight of left ventricle. P-values < 0.05 were considered significant.

Results

Age, sex distribution, total heart weight and weight of left and right ventricles grouped by valve pathology are shown in Table 1. There was no significant difference in the mean age of patients in different groups. The mean heart weight was highest in patients with mitral incompetence (525 f 160 9).

The volume fractions of myocardial components and the numerical density of intramyocardial arterioles in different groups are presented in Table 2. The volume fraction of interstitial space (VVINT) was increased in patients with MI compared to the controls (PcO.05). The amount of diffuse myocardial fibrosis (VvcT) was

Table 1. Basic characteristics of different groups.

Com bi ned stenosis stenosis incompetence val vu I ar

lesions

Controls Aortic Mitral Mitral

Number of cases Males Age, mean (SD), years Previous myocardial infarction Heart weight1 mean (SD), g Weight of left ventricle, mean (SD), g Weight of right ventricle, mean (SD), g

~ ~~

10 1 1 9 9 9 6 8 5 6 3 71 (8) 71 (6) 68 (8) 71 (16) 69 (11) 4 6 4 6 2

335 (105) 465 (120)" 370 (100) 525 (160)" 455 (175)

190 (65) 285 (95)' 200 (60) 310 (95)" 265 (105)

65 (30) 80 (40) 75 (35) 90 (25) 70 (20)

* *

t

P<0.05 when compared to controls Pc0.01 when compared to controls After removal of epicardial fat.

Table 2. Volume fractions of myocardial fibres, interstitial space, diffuse myocardial connective tissue and numerical densi. ty of arterioles in control hearts and in hearts with various valvular lesions. Values are presented as mean (SD).

Controls Aortic Mitral stenosis stenosis

Myocardial fibres 74.9 (3.4) 72.1 (4.6) 73.9 (5.9) ( V V M Y ? " 0 )

Interstitial space 25.1 (3.4) 27.9 (4.6) 26.1 (5.9) ( V V I N T ~ 'In) Connective tissue ( V V C T , " 0 )

Arterioles WART, 1lmf-n')

3.3 (0.8) 5.5 (1.3)" 3.4 (0.9)

0.8 (0.3) 0.7 (0.1) 0.8 (0.2)

Mitral Combined incompetence valvular

I e s i o n s

69.5 (7.7)' 73.4 (4.4)

30.5 (7.7)' 26.6 (4.4)

6.7 (1.8)" 5.8 (1.5)"

0.7 (0.2) 0.8 (0.3)

' P<0.05 when compared to controls. P<O.Ol when compared to controls.

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significantly increased in all valvular disease groups except mitral stenosis. Myocardial fibrosis (connective tissue) was most severe in patients with MI. Mean fibre diameter was 16 pm in the control group. In patients with MS the value was only slightly higher(l7 pm). The highest values were found in patients with AS and MI (22 pm and 23 pm, respectively). In the volume fraction of arterioles no significant differences were observed between different groups.

Patients with valvular lesions were divided into two groups according to the anatomical severity of the valvular lesions. This comparison is summarized in Table 3. Patients with severe valvular lesions were significant- l y younger than patients with mild or moderate lesions (P< 0.001). The mean heart weight and the weight of left ventricle were significantly greater in patients with severe valvular lesions. No differences were observed in the different volume fractions of myocardial com-

Table 3. Comparison of data from patients with severe valvular lesions to data from patients with mild or moderate valvular lesions. The values are presented as mean (SD).

Severe Mild or Difference valvular moderate lesions valvular

lesions

Number of cases 18 Age (years) 64 (9) Heart weight * 515 (150) Weight of left ventricle (9) 295 (105) Myocardial fibres VVMY (%) 72.0 (7.2) Interstitial space VVINT (%)28.0 (7.2) Connective tissue VVcr (%) 5.6 (1.6) Fibre diameter D, (pm) 23 (5.0) Cause of death - valvular disease 12 - coronary heart disease 3 - extracardiac 3

20 75 (9) P<O.OOl 400 (110) P<0.05 240 (75) P<0.05 72.4 (4.4) NS 27.6 (4.4) NS 5.4 (2.0) NS 19 (2.0) P<O.O1

0 1 1 9

* Afler removal of epicardial fat

Table 4. Comparison of histoquantitative parameters in subepicardium and subendocardium in patients with pressure overload (aortic stenosis) and in patients with volume overload (mitral incompetence). Values are presented as mean (W.

Aortic stenosis

Subepi- cardium

Myocardial fibres VVMy ("10) 71.8 (8.5) Interstitial space VVINT ( O h ) 28.2 (8.5) Connective tissue V,,, (%) 2.8 (2.3) Arterioles NART (l/mmz) 0.5 (0.2) Fibre diameter (rim) 22 (4.0)

_ _ Subendo- Difference card i u m

60.2 (1 1.4) 39.8 (1 1.4)

P < 0.05 P < 0.01

8.4 (6.1) P < 0.05 0.7 (0.4) NS 23 (3.0) NS

Mitral incompetence

Subepi- Subendo- Difference cardium cardium

Myocardial fibres V,,, (%) 70.1 (6.7) 52.1 (15.6) P ~0.01 Interstitial space VVINT ( O h ) 29.9 (6.7) 47.9 (15.6) P<O.Ol Connective tissue VVCT(%) 4.4 (1.9) 16.4 (12.3) P<0.05 Arterioles NART, (l/mm*) 0.8 (0.4) 1.0 (0.5) NS Fibre diameter (pm) 22 (4.0) 22 (4.0) NS

ponents. The mean fibre diameter was higher in patients with severe valvular lesions than in those with mild or moderate lesions (23 pm vs. 19 pm, P<O.Ol). Valvular heart disease was the principal cause of death in 67 O/O of patients with severe valvular lesions but in none in the group of mild or moderate valvular lesions, and coronary heart disease was the cause of death in 17 YO and 55 YO of patients, respectively.

The results of different histoquantitative parameters in subepicardial and subendocardial regions in patients with pressure overload (AS) and volume overload (MI) are separately listed in Table 4. In the subendocardial region Vvc, was significantly higher and V,,, correspondingly lower than in the subepicardium. In the subendocardium myocardial fibrosis was more severe in patients with MI than in patients with AS (16,4 O/O vs. 8,4 %, PcO.05). The numerical density of arterioles was greater in the subendocardium in both groups, but the difference was insignificant. No transmural difference was observed in the mean diameter of myocardial fibres.

Discussion

The most important confounding factor in this study seems to be coronary heart disease, which may cause diffuse myocardial fibrosis (8, 11). This applies espe- cially to patients with previous myocardial infarction but also to lesser extent to patients with coronary atherosclerosis without myocardial infarction (8). So the control group was matched according to the prevalence of previous myocardial infarction to find out the effect of valvular heart disease on the quantitative structure of left ventricular myocardium. The co-exis- tence of valvular heart disease and coronary heart disease is not uncommon in clinical practice (12), espe- cially in a population with a high prevalence of coronary heart disease.

The division of the study material by the autopsy pathologist into hearts with severe valvular lesions and those with mild to moderate lesions was sometimes difficult. This, however, seemed to be the only way to estimate the effect of the haemodynamic severity of a valvular lesion on the quantitative histology of myocardium, since in most patients echocardiographic andlor catheterization data were not available. On the other hand, it was impossible retrospectively to assess the functional class of every individual patient according to the clinical certificates. Furthermore, the functional class of a patient may not be reliable as a sole indicator of the haemodynamic severity of valvular lesion as it is affected by the age and the general physi- cal fitness of the patient and also by possible co- existing coronary heart disease. That dividing of valvular disease group according to the severity of lesions is avalid method is shown by the fact that in the group of severe valvular lesions the mean age of patient was significantly lower, the prevalence of terminal congestive heart failure was higher and the weight of left ventricle was higher than in patients with mild to moderate valvular lesions.

The heart with a chronic valvular disease responds to overload by myocyte hypertrophy (1,4, 13, 14). This

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Morphometric study of myocardium in valvular diseases 439

holds particularly true in patients with pressure overload, but is also observed in those with volume overload (15). In this study, prominent fibre hypertrophy was found in both aortic stenosis and in mitral incompetence. As expected, the fibre diameter in patients with mitral stenosis was near the control values, since in mitral stenosis the haemodynamic load is mainly placed on the left atrium and right ventricle, which were not investigated in this study with morphometric methods. Whether hyperplasia of individual myocytes occurs in severesoverload is still obscure (16). Linzbach (17) has suggested that above a critical heart weight of 500 g longitudial splitting of myofibres occurs and the fibre diameter is nearly constant thereafter. On the other hand, in adult rats no hyperplasia of myofibres has been observed in experimental hypertrophy (18). We found a close correlation between the fibre diameter and the weight of left ventricle in patients with valvular heart disease. This suggests that the increase in fibre diameter is responsible for the increase in heart weight. With these methods, however, it is impossible to rule out hyperplasia with certainty.

Patients with mitral stenosis showed no increase in the volume fraction of diffuse connective tissue in comparison with the controls. On the other hand, in patients with mitral incompetence and those with aortic stenosis, a significant increase in the severity of diffuse myocardial fibrosis was found. This accords with previous studies ( I , 3, 19). Myocardial fibrosis in patients with valvular heart disease may be the result of ischaemia, which is known to be cabable of stimulating fibroblasts in vitro (20), or myocardial fibrosis may be a response of the myocardium to increased wall tension (16). The importance of myocardial fibrosis to cardiac perfor- mance and the prognosis of patients with valvular heart disease is unclear. Oldershaw et al. (19) suggested that myocardial fibrosis correlated inversely with ejection fraction, symptoms and the prognosis of patients with aortic stenosis. It has been suggested that myocardial fibrosis reduces diastolic chamber compliance, lead- ing to congestive heart failure (21, 22). We found that the amount of connective tissue was increased in patients with valvular heart disease except in those with mitral stenosis, but this did not correlate with the anatomical severity of the valvular lesion or with the severity of hypertrophy. In the group with severe valvular heart disease, where terminal cardiac failure was com- mon, the myocardial fibrosis was not increased compared with hearts with less severe anatomical valvular lesions. So, the diffuse myocardial fibrosis seems not to be the only or even the most important morphological determinant of cardiac failure. On the other hand, the severity of myocardial hypertrophy may be a bet- ter indicator for the severity of a valvular lesion.

The question of possible transmural differences in the morphology of myocardium is of interest not only from pathophysiological point of view, but also be- cause of increasing use of endomyocardial biopsies for diagnostic purposes and in assessing the prog- noses of patients with various heart diseases (5,13, 19, 23,24). In this study the volume fraction of interstitial space and the volume fraction of connective tissue were significantly higher and the volume fraction of myocardial fibres correspondingly lower in the suben-

docardium than in the subepicardium in patients with aortic stenosis and in patients with mitral incompetence. This suggests that the subendocardium is more vulnerable in hearts with pressure or volume overload than the rest of left ventricular myocardium.

In conclusion, myocardial fibrosis occurs in patients with various valvular lesions, but the severity of fibrosis is not correlated with the severity of valvular lesions. Since marked transmural differences were found in the structure of myocardium in patients with pressure or volume overload due to valvular heart disease, caution is necessary in correlating myocardial structure and function and in assessing the prognosis of patients on the basis of the histology of endomyocardial biopsies.

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morphometric study of human myocardium in acquired valvular diseases

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