Back
Issue
 Return to home page
PDF(1.1Mb)
Kuruvilla S,1 Balakrishnan KR,2 Parvathy U.3 Right ventricular myocardium in Fallot’s tetralogy: a light microscopic, morphometric and ultrastructural study. Images Paediatr Cardiol 2004;21:1-30
 
 1 Department of Pathology, Sri Ramachandra Medical College & Research Institute 
2 Department of Cardiothoracic Surgery, Sri Ramachandra Medical College & Research Institute 
 
MeSH
Heart defects, congenital Tetralogy of Fallot, heart, ventricles  Myocardium, mitochondria 
 
Abstract
Aims: To analyze peroperative biopsies of RV myocardium in Tetralogy of Fallot by light microscopy, morphometry and electron microscopy in order to determine the degree of hypertrophy and degenerative  changes and to correlate these changes with clinical and haemodynamic parameters.
Materials and Methods: Right ventricular myocardium obtained peroperatively during surgical correction of Tetralogy of Fallot along with age-matched control samples were processed for routine light and electron microscopy using standard processing techniques. Mean cell diameter was analyzed using manual morphometric methods and ultrastructural study was carried out using a Philips transmission electron microscope.
Results: The most consistent features of hypertrophy were the mitochondrial changes and increased nuclear convolutions. Majority of the patients had hypertrophy with mild to moderate degenerative changes. Severe degeneration was associated with irreversibility and was related to the severity and chronicity of the disease. There was a significant correlation of the morphological changes with clinical and haemodynamic parameters.
Conclusions: Peroperative histomorphometric and ultrastructural evaluation of the RV myocardium in Tetralogy of Fallot   reflects the effect of haemodynamic stresses on the right ventricular muscle and correlates with clinical cardiac dysfunction.  It may be a useful adjunct in determining the time for surgical intervention and in predicting clinical outcome.

Introduction
It is a well known fact that congenital heart disease associated with right ventricular outflow tract obstruction leads on to right ventricular hypertrophy, and Fallot's tetralogy is one such entity. Persistence of hypertrophy leads on to progressive degeneration which may vary in grade, depending on the severity of obstruction and chronicity of the disease process.1 Although there are extensive ultrastructural studies on acquired cardiac disease, particularly the cardiomyopathies, 2,3,4 similar studies on congenital heart disease5, 6 have been very limited in the literature.

In this study, the right ventricular myocardium of patients with tetralogy of Fallot (TOF) was peroperatively biopsied and studied using histomorphometry and electron microscopy. Particular attention was directed to the size, shape and pattern of arrangement of myocytes, intercalated discs, alterations in intracellular organelles, including mitochondria, nuclear abnormalities, degree of atrophy and interstitial changes including fibrosis. These morphological changes were categorized according to the criteria of Maron et al7 as hypertrophic changes with or without degeneration, based on light microscopic and ultrastructural characteristics. The spectrum of morphological changes were correlated with clinical and hemodynamic parameters.

Materials & methods
This study was carried out on materials obtained from 12 patients with Tetralogy of Fallot, ranging in age from 8 months to 22 years (Figure 1), and 3 controls.
 

Figure 1: Age distribution
fig01

The patients underwent surgical correction following evaluation by echocardiography. During surgery, the obstructing right ventricular muscle was excised and a bit sent for sampling. In addition, control myocardial samples were obtained from normal hearts harvested for homograft valves from three brain dead individuals of similar age group with no known cardiac disease.

Sampling
Specimens of the right ventricular myocardium from patients of TOF were taken within 4-8 minutes of aortic cross clamping. Two samples were obtained of which one was fixed in 10% formalin for histomorphometric evaluation. The other sample was immediately minced into 1-2 mm size pieces, fixed for 12 hours in cold 3% glutaraldehyde in sodium cacodylate buffer and set aside for ultra-structural evaluation. Right ventricular myocardial samples obtained from controls were also fixed in a similar manner.

Histomorphometric study
After routine processing, the tissue was embedded in paraffin and 5-micron sections were cut. Haematoxylin and eosin stained sections were used for light microscopic examination and morphometric evaluation. Morphometry was done using an eyepiece micrometer and a slide graticule. In addition to cell size, light microscopic examination included study of alignment and orientation of muscle cells, orientation of myofibrils, nuclear changes, myocyte hypertrophy, atrophy, fibrosis and degenerative changes (table 1).
 

Table 1: Salient light microscopic features
S.
No.
Age in Years
Size in Microns
Edema
Fibrosis
Loss of Myocytes
Defective Myocytes
Sclerotic Myocytes
Necrotic Myocytes
Cellular Atrophy
1.
1
16.26
Nil
Moderate
Nil
Few
Few
Nil
Nil
2.
2.5
19.15
Nil
Mild to moderate
Nil
Nil
Few
Nil
Nil
3.
8/12
16.55
Mild
Moderate
Nil
Nil
Few
Nil
Mild
4.
2.5
21.82
Nil
Mild
Nil
Nil
Few
Few
Mild
5.
2
27.9
Nil
Mild to Moderate
Nil
Few
Nil
Nil
Mild
6.
3
25.35
Focal
Moderate
Nil
Moderate
Nil
Few
Moderate
7.
3
21.7
Nil
Moderate to severe
Nil
Few
Nil
Few
Mild
8.
5
25.15
Nil
Mild to moderate
Nil
Moderate
Few
Few
Nil
9.
12
21.2
Mild
Moderate
Nil
Moderate
Moderate
Few
Moderate
10.
11
24.25
Nil
Moderate to severe
Moderate
Moderate
Nil
Nil
Nil
11.
22
23.7
Moderate
Mild
Moderate
Moderate
Nil
Moderate
Moderate
12.
20
27.95
Moderate
Moderate to severe
Moderate
Moderate
Few
Nil
Moderate
 
Mean myocardial cell diameter was calculated from 100 measurements of cross sections near the nucleus by the method described by Chalkley8 and Arai.9 (figure 2).
 
Figure 2: Histomorphometric evaluation of cell diameter vs. no. of cases
fig02

Electron microscopic study
Post-fixation of the glutaraldehyde fixed samples were carried out with cold 1% osmium tetroxide in Millonig's phosphate buffer for 3 hours. The tissues were then dehydrated in ethanol and propylene oxide and embedded in Maraglas epoxy resin. One-micron thin sections were stained with alkaline Toluidine blue. Ultra-thin sections obtained using a diamond knife were stained with uranylacetate and lead citrate and examined with a Phillips transmission electron microscope.
 
Clinical assessment
The following clinical assessements were carried out: evaluation for the presence of cyanotic spells, hematocrit, peripheral oxygen saturation, RVOT (right ventricular outflow tract) gradient, RVEDP (right ventricular end diastolic pressure), SPO2 (oxygen saturation) and PA/AO (pulmonary artery/aorta) ratio (table 2). All patients were evaluated by echocardiography. Cardiac cathetrisation was done in 11 patients. The severity of the RVOT obstruction was assessed by the gradient. All the patients underwent corrective surgery for Tetralogy of Fallot under cardiopulmonary bypass with moderate hypothermia.
 

Table 2: Haemodynamic/Clinical parameters and relationship to nuclear changes and degree of fibrosis
S.No.
Age in years
Spells
HCT
SPO2
RVEDP
RVOT Gradient
Fibrosis
Nuclear Convolutions
1.
1
Yes
39
79
7
70
Moderate
2+
2.
2.5
Yes
35
78
4
82
Mild to Moderate
1+
3.
8/12
No
32
83
5
51
Moderate
2+
4.
2.5
Yes
38
89
6
87
Mild
1+
5.
2
Yes
42
74
7
76
Mild to Moderate
2+
6.
3
Yes
45
76
4
78
Moderate
1+
7.
3
Yes
48
69
10
83
Mod to severe
3+
8.
5
No
64
55
7
79
Mild to Moderate
3+
9.
12
Yes
73
80
10
80
Moderate
1+
10.
11
Yes
64
80
9
76
Mod to severe
2+
11.
22
No
63
72
9
72
Mild
2+
12.
20
Yes
50
85
10
79
Mod to severe
3+
Hct=Haematocrit, SPO2=Oxygen saturation, RVEDP=Right ventricular end diastolic pressure, RVOT gradient=Right ventricular outflow tract gradient

Results
Control samples of right myocardium were studied by light microscopy using routine H&E, semi thin sections which were stained by Toluidine Blue (figure 3 )and by electron microscopy (figures 4,5) to study size, alignment, cross striations, organelles and nuclear contours (figure 6).
 

Figure 3: Control RV myocardium. Longitudinal section showing cross striations and normal nuclear contours. Semi thin section Toluidine blue LM X400
fig03
 
Figure 4: Electron micrograph control RV muscle. Normal Z bands, smooth nuclear contour. EM magnif 4500X
fig04
 
Figure 5: Electron micrograph control RV muscle. EM magnif 30000X
fig05
 
Figure 6: Electron micrographs control RV muscle. Note the smooth nuclear contours EM magnif 15000X
fig06

Histomorphometric studies
The principal light microscopic changes observed were cellular and interstitial The changes in myocardial cells of the patients were predominantly that of hypertrophy, oedema, myocytolysis (necrosis of myocardial cells), atrophy and sclerosis. The interstitium showed fibrosis, oedema and vascular dilatation.

Cellular changes
Presence of hypertrophy was considered unequivocal when the mean diameter of myocytes exceeded 20 microns as assessed by morphometry. Hypertrophy (figure 7) was observed in 9 out of the 12 cases studied.
 

Figure 7: Cross section TOF RV myocardium showing hypertrophy. Semithin section. Toluidine blue LM X400
fig07

In addition to hypertrophy, degenerative changes were observed characterized by varying degrees of myofibrillar disarray (figure 8).
 

Figure 8: Longitudinal section TOF RV myocardium showing hypertrophy and myofibrillar disarray. Semithin section Toluidine blue LM X200
fig08

In some cases, myocytes of 40 microns diameter were observed. Cellular atrophy of a mild to moderate degree was seen 8 cases (4 adults and 4 children). In severe degeneration atrophy of cardiomyocytes resulted in marked crowding of nuclei (figure 9).
 

Figure 9: CS TOF RV myocardium showing severe degeneration.The cardiomyocytes show marked crowding and atrophy. Semithin section Toluidine blue LM X200
fig09

Other cellular changes (oedema, myocytolysis and sclerosis) were observed in half of the cases. Z band abnormalities were also seen in association with varying grades of myofibrillar disarray (figures 10,11).
 

Figure10: Longitudinal Section TOF RV myocardium showing hypertrophy, myofibrillar disarray and Z band abnormalities. Semithin section Toluidine blue LM X200
fig10
 
Figure 11: Longitudinal Section TOF RV myocardium showing hypertrophy and myofibrillar disarray. Semithin section Toluidine blue LM X400
fig11

Defective myocytes were observed in 50% of the patients studied. Necrotic myocytes were predominantly seen in patients with a hematocrit of more than 60.

Nuclear changes
A prominent feature evident in semi thin sections of TOF RV cardiomyocytes were the nuclear abnormalities seen as elongation and increased irregularities and convolutions of the nuclear membrane, in addition to the above cellular abnormalities (figures 12,13).
 

Figure 12: Longitudinal Section TOF RV myocardium showing hypertrophy, myofibrillar disarray and increased nuclear convolutions. Semithin section Toluidine blue. LM X400
fig12
 
Figure13: Longitudinal section: TOF RV myocardium showing hypertrophy, mild myofibrillar disarray, nuclear elongation and irregularity. Semithin section Toluidine blue LM X400
fig13

Interstitial changes
All the cases in this study had varying degree of fibrosis. The type of fibrosis may be described as focal, perimysial, plexiform or perivascular fibrosis. The types of fibrosis seen in majority of cases were either perimysial or perivascular. Three cases showed mild interstitial oedema, although it tended to be more prominent in the samples obtained from the adults. Moderate to marked increase in interstitial fibrosis (figures 14, 15) was noted in at least 5 cases, the remaining showed only a mild/focal increase.
 

Figure 14: Longitudinal section TOF RV myocardium showing severe degeneration seen as marked interstitial and perivascular fibrosis. The cardiomyocytes appear pale with mild atrophic changes. Semithin section Toluidine blue LM X200
fig14
 
Figure 15: Cross section TOF RV myocardium showing hypertrophy, myofibrillar disarray with interstitial and perivascular fibrosis. Semithin section Toluidine blue LM X200
fig15

Electron microscopic changes
The electron microscopic changes were observed within the cytoplasm, organelles and nucleus (table3).
 

Table 3: Salient electron microscopic changes
S.
No.
Age in years
Myofibrillar Disorganization
Intercellular Widening
Mitochondria
Myelin Figures
BM Thickening
Intracytoplasmic Vacuoles
Nuclear Size
Nuclear Convolutions
1.
1
Mild
Slight
Increased irregular
Few
Slight
Nil
Normal
Moderate
2.
2.5
Mild
Normal
Increased, irregular, ruptured
Nil
Nil
Nil
Increased
Mild
3.
8/12
Moderate
Marked
Increased, dilated
Few
Nil
Several
Increased
Moderate
4.
2.5
Mild
Slight
Increased, dilated
Nil
Nil
Nil
Increased
Mild
5.
2
Mild
Slight
Increase, irregular
Nil
Normal
Few
Increased
Moderate
6.
3
Moderate
Slight
Increased, irregular, ruptured
Nil
Nil
Several
Increased
Mild to Moderate
7.
3
Mild
Slight
Increased, irregular
Nil
Nil
Few
Increased
Severe
8.
5
Mild
Slight
Increased, irregular
Few
Nil
Few
Increased
Severe
9.
12
Moderate
Marked
Increased, dilated
Nil
Nil
Several
Increased
Mild
10.
4
Mild
Slight
Increased irregular
Nil
Nil
Nil
Increased
Moderate
11.
22
Moderate
Marked
Increased irregular
Nil
Nil
Several
Increased
Moderate
12.
20
Moderate
Moderate
Increased irregular
Moderate
Nil
Few
Increased
Severe
 
Highlights of the cytoplasmic changes included myofibrillar disarray, Z band abnormalities in mild and moderate degeneration (fig16) and intracytoplasmic vacuolations.
 
Figure 16: Electron micrograph TOF RV myocardium showing moderate degeneration, increased mitochondria and myofibrillar disarray. EM magnif: 7000X
fig16

The principal organelles affected were the mitochondria. The sarcoplasmic reticulum showed proliferation and dilatation. In addition to these cellular changes, the interstitium and nuclei also showed significant alterations. The ultrastructural observations are as follows.

Cytoplasmic changes
Myofibrillar changes included reduction in the number (in 3 cases), disorganization including non-parallel arrangement in 6 cases, and focal lysis (in all cases). Intracytoplasmic vacuolation was seen to a marked degree in 4 cases (figure 17).
 

Figure 17: Electron micrograph TOF RV muscle. Severe degeneration- intracytoplasmic vacuolation. EM magnif 3000X
fig17

In addition, lipid vacuolation was seen in 2 cases. Myelin figures (figure 18) were seen in 4 cases - 3 children and 1 adult - and in the latter the myelin figures were more prominent.
 

Figure 18: Electron micrograph TOF RV muscle Severe degeneration- myelin figure (arrow) EM magnif 7000X
fig18

Uncommon changes observed include lipofuscin granules and Z band abnormalities (1 case each).

Changes in organelles
Changes in the mitochondria represented one of the important EM findings. There was a uniform increase in their number. In all cases they were irregular in shape and were dilated, and in a few, they appeared ruptured (figure 19). In 7 cases, there were small mitochondria.
 

Figure 19: Electron micrograph TOF RV muscle. Markedly dilated, irregular and ruptured mitochondria. EM Magnif 15000X
fig19

Rough endoplasmic reticulum abnormalities were also seen (figure 20).
 

Figure 20: Electron micrograph TOF RV muscle. Severe degeneration- myelin figures, abnormal Z bands, rough endoplasmic reticulum abnormalities and basement membrane thickening. EM magnif 15000X
fig20

Intercellular junctions and basement membranes
Inter-cellular junction widening was seen in all except one. It was marked in 4 cases. (figures 21-23).
 

Figure 21: Electron micrograph TOF RV muscle. Longitudinal section showing widened intercellular junctions (arrow). EM magnif 7000X
 fig21
 
Figure 22: Electron micrograph TOF RV muscle. Longitudinal section: showing widened intercellular junctions (arrow) EM magnif 15000X
fig22
 
Figure 23: Electron micrograph TOF RV muscle. Longitudinal section showing widened intercellular junctions (arrow). EM magnif 30000X
fig23

Multiple intercalated discs were evident in 2 cases. Intercellular dissociation was seen in majority of cases. Slight thickening of the basement membrane was seen in 2 cases.

Nucleus
The nuclear size was increased in all cases, significantly so in 9 cases. There was elongation of the nucleus with moderate increase in nuclear convolutions (figures 24-26) which was evident in all the patients, but was marked in 3.
 

Figure 24: Electron micrograph TOF RV muscle. Hypertrophied cardiomyocytes,
convoluted nuclei, abundant mitochondria. EM Magnif 7000X
fig24
 
Figure 25: Electron micrograph TOF RV muscle. Convoluted nuclei
with intranuclear tubule formation and abundant mitochondria. EM Magnif 30000X
fig25
 
Figure 26: Electron micrograph TOF RV muscle. Increased nuclear convolutions. EM Magnif 20000X
fig26

Intranuclear cytoplasmic pseudo-inclusions were seen in 3 cases and nuclear irregularities in 5 (figures 27, 28).
 

Figure 27: Electron micrograph TOF RV muscle. Hypertrophied cardiomyocytes, convoluted nuclei with intranuclear tubule formation and abundant mitochondria. EM Magnif 7000X
fig27
 
Figure 28: Electron micrograph TOF RV muscle. Convoluted nuclei with intranuclear tubule formation and abundant mitochondria. EM Magnif 20000X
fig28

In summary, the ultra-structural changes provided evidence for the presence of hypertrophy and degeneration (figure 29).
 

Figure 29: Key electron microscopic findings vs. no. of cases (%)
fig29
 
Degenerative changes were assessed according to the criteria described by Maron et al.,7 as mild/early, moderate and severe. Degenerative changes are tabulated in table 4.
 
Table 4: Morphological changes of myocardial degeneration associated with hypertrophy
No. Age Myofibrillar disorganization Myofibrillar lysis Increase in interstitium Loss of thick myofilaments Intra Cytoplasmic Vacuoles Nonparallel Myofibril Small mito-chondria Interstitial fibrosis Cellular atrophy Myelin figures Intercellular dissociation
1. 1 yr Mild Mild Slight Mild Nil Few Few Moderate Nil Few Mild
2. 2.5 yr Mild Focal Mild Nil Few Nil Mild Mild Nil Nil Nil
3. 8/12 Moderate Mild Slight Mild Several Moderate Nil Mild Mild Few Marked
4. 2.5 yr Mild Mild Slight Mild Nil Few Few Mild Mild Nil Mild
5. 2 yr Mild Mild Moderate Nil Few Few Few Mild Mild Nil Mild
6. 3 yr Moderate Mild Focal Mild Several Moderate Few Mild Moderate Nil Mild
7. 3 yr Mild Mild Marked Mild Few Few Few Moderate Mild Nil Mild
8. 5 yr Mild Mild Focal Mild Focal Few Nil Mild Mild Few Mild
9. 12 yr Moderate Moderate Moderate Mild Several Moderate Few Moderate Moderate Nil Marked
10. 11 yr Mild Nil Nil Mild Nil Nil Few Moderate Mild Nil Mild
11. 22 yr Moderate Moderate Slight Mild Several Few Several Mild Moderate Nil Marked
12. 20 yr Moderate Moderate Moderate Mild Few Few Several Moderate Moderate Moderate Moderate
Data analysis of the findings and the correlative parameters was carried out using SPSS software. The relevant findings have been highlighted.

Correlation of clinical/laboratory parameters with morphological changes in the myocardium
Among the laboratory investigations, hematocrit appeared to influence morphologic changes.. Based on the hematocrit, the patients were divided into 3 groups: (table5). Group 1 included patients with hematocrit less than 45; Group II, those with hematocrit 45-60; and Group III patients had a hematocrit of more than 60.
 

Table 5: Percentage of occurrence of ultra structural changes in relation to haematocrit levels
No.
Histopathologic Changes
Group I
(Hct <40)
Group II
(Hct 45-60)
Group III
Hct>60
1.
Intercellular widening
10%
10%
60%
2.
Mitochondria
Increased in 40%
Increased in 100%
Increased and ruptured in 100%
3.
Lipid Accumulation
20%
35%
  
4.
Myofibrillar disorganization
40%
30-40%
35%
5.
Nuclear convolutions
40%
40%
40%
6.
Nuclear Pseudo inclusions
80%
100%
75%
7.
Intranuclear tubules
Nil
100%
25%
8.
Increase in size & irregularity
100%
80%
50%
9.
Focal fibrosis
20%
100%
100%
10.
Perimysial Fibrosis
40%
33%
100%
11.
Perivascular fibrosis
30%
40%
50%
12.
Defective Myocytes
40%
33%
40%
13.
Sclerotic Myocytes
Nil
50%
30%
14.
Necrotic Myocytes
Nil
Nil
75%
15.
Loss of Myocytes
Nil
Nil
75%
 
The myocardium from group III patients (hematocrit of more than 60) showed reduction in the number of myofibrils, moderate disorganisation of myofibrils, widening of intercellular junctions, increased numbers with rupture of mitochondria and presence of necrotic myocytes. In addition, perivascular fibrosis, nuclear irregularity and increase in size as well as number of intranuclear tubules were more evident in Groups II and III. Incidentally these patients were found to have an RVEDP>6 and peak outflow gradient from 50-82 mm. Hg. Significant association was seen between haematocrit and the light microscopic finding of defective myocytes and of myocyte loss and the correlation is statistically significant (p<0.05). Higher RVEDP was associated with statistically significant loss of myocytes (p value 0.01).

Correlation of increasing age with cellular changes of hypertrophy
As mentioned earlier the mean cardiomyocyte diameter is an unequivocal determinant of hypertrophy. With increase in age, there is an increase in the micron size of the right ventricular cardiomyocytes, and the correlation co-efficient was found to be (r) = 0.5. However the correlation is not statistically significant (P= 0.14).

A careful analysis of the light and electron microscopic changes showed that apart from cardiomyocyte diameter, cellular changes of hypertrophy with increasing age were as follows: myofibrillar disorganization was of moderate degree in older patients and mild in all the young or patients, except in an 8 month old child. Intercellular dissociation was also moderate to marked in the older patients and slight in all the younger patients, except one. There was however no definite correlation of mitochondrial changes and nuclear convolutions with age. It is to be noted that with increased severity and chronicity of the disease, cellular changes of hypertrophy were modified by degenerative changes of a mild, moderate or severe nature.

Discussion
With the advent of modern safe techniques for surgical correction of Fallot's Tetralogy, myocardial function is becoming an important factor in determining the long term prognosis.10 Morphological observations on preoperatively resected right ventricular myocardium could have a predictive value in assessing the surgical outcome in the patients, although there is a paucity of such studies in literature.11, 12,13 Effects due to hypoxia and pressure overload on the right ventricular myocardium have been found to increase with age,1 and it has been found that the hypertrophic changes evolve into various degrees of degeneration depending on the severity and duration of the disease. The ultrastructural alterations that ensue during hypertrophy have been described at length by Maron et al.7 In the first stage there is an increase in the energy production and protein synthesis. The second stage is characterized by a stable state of cardiac hyperfunction, which is followed by the third stage during which there is a gradual exhaustion in the heart's ability to synthesize proteins. This results in a failure to renew myofibrils and mitochondria and leads on to progressive myofibrillar damage and cellular atrophy. The same workers also analyzed the continuum of degenerative changes which occur in the late stage of hypertrophy and graded them as mild, moderate and severe. In Fallot's Tetralogy, a detailed analysis of the hypertrophic changes with or without degeneration becomes mandatory, since the severity of the right ventricular outflow tract obstruction has a significant bearing on the clinical manifestations as well as the timing for surgical intervention.14, 15

Hypertrophy without degeneration is characterized by increase in cell size and number of myofibrils per cell. Other ultrastructural changes include focal thickening and accumulation of Z-band material, normal sarcoplasmic reticulum, focal increase in glycogen, increase in size of golgi complex and number of ribosomes and mitochondria. There is also increase in nuclear size with convolutions, but the basement membranes and interstitium are normal (figures 30, 31).
 

Figure 30: Electron micrograph TOF RV muscle. Cross section of hypertrophic muscle fiber and convoluted nucleus (arrow). EM magnif 3000X
fig30
 
Figure 31: Electron micrograph TOF RV muscle. Hypertrophied cardiomyocytes, convoluted nuclei and abundant mitochondria. EM Magnif 4500X
fig31

Recent studies16 show that during hypertrophy, there is a switch of the contractible proteins to the fetal and neonatal forms. The expression of alpha myosin heavy chain is replaced by that of beta myosin heavy chain, which leads to decreased myosin ATPase activity and a slower, more energetic, economical contraction.

In hypertrophy associated with mild degeneration, (figure 32) the cell size and number of myofibrils per cell are normal or increased, focal decrease in thick myofilaments, Z band abnormalities and sparse proliferation of sarcoplasmic reticulum. Changes in intracellular organelles and nuclear convolutions are the same as in hypertrophy. However there may be basement membrane thickening and increase in interstitial fibrous tissue.
 

Figure 32: Electron micrograph TOF RV muscle. Longitudinal section of myocyte showing hypertrophy, mild degeneration. Note Z bands and nuclear convolutions. EM magnif 3000X
fig32

Moderate degeneration is characterized by additional changes like a marked decrease in thick myofilaments, myofibrillar disarray, abnormalities in Z bands, disorganization and extensive proliferation of the sarcoplasmic reticulum. There is partial dissociation of the intercellular junctions, decrease in size of golgi complex, number of ribosomes and a significant increase in mitochondrial size. Basement membrane is markedly thickened and there is increased interstitial fibrosis (figures 33,34).
 

Figure 33: Longitudinal section TOF RV myocardium showing myofibrillar disarray, nuclear irregularities and interstitial fibrosis. Semithin section Toluidine blue. LM X200
fig33
 
Figure 34: Electron micrograph TOF RV muscle. Moderate degeneration-abnormal Z bands, nuclear convolutions. EM magnif 20000X
fig34

Cardiac cells showing severe, irreversible degenerative changes, by light microscopy appear atrophic and pale in toluidine blue stained semithin sections (figure 35).
 

Figure 35: TOF RV myocardium showing irreversible degeneration seen as marked interstitial and perimysial fibrosis. The cardiomyocytes appear atrophic. Semithin section Toluidine blue LM X200
fig35

Ultrastructurally, there is marked loss of contractile elements with a spectrum of changes affecting every organelle. There is marked atrophy of the cardiomyocytes, vacuolation, disorganization of myofilaments (figures 36,37) and decreased myofibrils with focal proliferation of sarcoplasmic reticulum and extensive loss of T tubules.
 

Figure 36: Electron micrograph TOF RV myocardium showing severe degeneration. The cardiomyocytes are atrophic with vacuolation and mitochondrial abnormalities. EM magnif: 7000X
fig36
 
Figure 37: Electron micrograph TOF RV myocardium showing severe degeneration. The cardiomyocytes are markedly atrophic.EM magnif: 2000x
fig37

Myelin figures are prominently seen and there is marked dissociation of intercellular junctions. Mitochondria of various sizes with or without damaged mitochondria may be present. Basement membrane thickening and a marked increase in interstitial fibrous tissue (figure 38) is also a prominent feature in severe degeneration.
 

Figure 38: Electron micrograph TOF RV muscle. Interstitial fibrosis, fibroblast with nucleus and collagen. EM magnif. 7000X
fig38

In our analysis, the majority of patients had light microscopic and ultrastructural changes of hypertrophy associated with mild to moderate degeneration. Severe degeneration was noted in the adult patients and 3 of the paediatric age group. The clinical and haemodynamic parameters in these patients revealed a very low SPO2, presence of cyanotic spells, elevated RVEDP and haematocrit > 50. The significant morphological changes in these patients were presence of cellular atrophy, myofibrillar disorganization, myelin figures, intercellular dissociation, basement membrane thickening and marked interstitial fibrosis. The surgical outcome in these patients may be less favourable than those who had only mild to moderate degenerative changes associated with hypertrophy.

The earliest study of myocardial changes in congenital heart disease was by Vtiurin et al,17 who noted myofibrillar edema and mitochondrial changes and correlated the severity of these changes with age. Kato et al18 analysed the right ventricular myocardium in tetralogy of Fallot by light microscopy and morphometry. He found a significant correlation between the diameter of the cardiac myocyte with Hb levels and the age of the patients. The histopathological alterations however did not correlate with Hb, Sa O2 and PA/Ao ratio. In our study, although there was no significant increase in diameter with age, the morphological changes did show significant correlation with haemodynamic parameters, particularly Hb and SaO2.

In a detailed ultrastructural evaluation of the crista supraventricularis in young and old patients with congenital heart disease associated with right ventricular outflow tract obstruction, Jones and Ferrans19 observed severe degenerative changes in patients over 30 years. They attributed these changes to the stress of prolonged right ventricular hypertrophy and hypoxia and found a correlation with clinical cardiac dysfunction. In the clinical context, if total corrective surgery is performed in patients above 30 years the perioperative morbidity and late mortality rates are high. Their observations support the time-honoured concept that operative correction of congenital heart disease should be undertaken as early in life as is technically appropriate.

Tanimoto et al, 20 developed a scoring method of evaluating the light microscopic changes in right ventricular myocardial biopsies obtained pre and postoperatively in congenital heart disease. He quantified the various light microscopic parameters, such as hypertrophy, edema, vascular changes, inflammation, fibrosis and other degenerative changes. The scores were found to be significantly increased in patients as compared with controls.

A comparative assessment of pre and postoperative right ventricular wall thickness (W), diameter (D) and the ratio (W/D) in TOF patients before and after 6 months of age, 21 have shown that the right ventricular W and D decreased significantly in patients who underwent surgical repair before 6 months of age. This observation suggests that early repair of TOF has a positive effect on regression of right ventricular hypertrophy during the postoperative period. It also reduces the detrimental long-term effects of persistent hypertrophy on myocardial function and increases the potential for occurrence of arrhythmias.

The light microscopic and ultrastructural findings in the right ventricular myocardial biopsies in TOF in the present series have been described in detail and an attempt has been made to correlate the same with the clinical and hemodynamic data of these patients. Histomorphometric analysis helped in quantitative assessment of the cell diameter, which had a bearing on hypertrophy and showed slight increase with age although the latter did not show statistical significance. This needs to be elaborately studied on a larger series of cases.

The morphological findings that were particularly prominent in this study were the increase in number of mitochondria, associated with irregularity, dilatation and rupture. These mitochondrial changes had a strong negative correlation with sPO2. Lower the sPO2; more were the mitochondrial changes (Chi 2 test 5.6000 p value 0.001, Pearson correlation - 0.68313, p value 0.014). Myocardial biopsies obtained from patients with severe right ventricular outflow tract obstruction showed marked mitochondrial alterations like irregular contours, dilatation and rupture.

Nuclear changes were also found to be fairly consistent in our series. Hypertrophic cardiomyocytes with mild to moderate degeneration showed increased nuclear convolutions. Nuclear enlargement was seen in all the cases, except one. It has been postulated, 22 that during ventricular hypertrophy, the nuclear length increases while the diameter remains normal. Also there is increased synthesis of nuclear membrane in excess of that needed to accommodate the increase in volume. This leads to increased infoldings, convolutions and other irregularities of the nuclear membrane. Presence of intranuclear tubules represent an extreme cellular response to the stimulus of hypertrophy.23 Intranuclear tubules were observed in 5 cases (3 adults and 2 children).

Less frequently occurring ultrastructural changes include foci of myocyte damage, atrophy, intercellular dissociation and interstitial fibrosis. Some authors24 have described cardiomyocyte damage as being defective, sclerotic and necrotic. Necrotic myocytes were seen only in patients with a haematocrit > 60% with a higher RVEDP. Statistically significant correlation existed between myocyte loss versus haematocrit. (Chi 2 test - 5.6, p. 0.05, Pearson 0.68, p value < 0.05), and between myocyte loss versus RVEDP (Chi 2 test 5.6 p value < 0.05 and Pearson 0.681, p. value 0.01).

Interstitial fibrosis which has been found to increase with worsening degeneration was noted to a greater degree in patients with raised haematocrit, RVEDP and decreased sPO2, however it was not statistically significant. Kawai et al25 have classified myocardial fibrosis into 5 types: focal, mild and severe perimysial, perivascular and plexiform. Larger areas of myocardial disarray and severe perimysial fibrosis were seen to a greater extent in patients having hypoxic spells or with chronicity as is also our observation. Other authors26 have found that there was marked interstitial fibrosis in patients with Fallot's tetrad who are > 5 years of age, indicating that if early repair is not done, irreversible fibrosis would reduce ventricular compliance, thus emphasizing the need for early corrective surgery.

Extensive pathohistologic studies in 104 cases of TOF by Kato,18 showed that since irreversible light microscopic changes were first observed in patients above 4 years of age, they proposed the timing for corrective surgery to be in children below 3 years of age. A study by Seliem and coworkers, 21 however have shown that there is significant regression of right ventricular hypertrophy when surgical correction is done before 6 months of age. This is also said to reduce the detrimental effect of longstanding hypertrophy and prevents progressive degeneration. Both these workers however did not study the ultrastructure of the right ventricular myocardium..

Summary and conclusions
In summary, therefore, from a morphological viewpoint, the most consistent and striking findings of hypertrophy with degeneration were the mitochondrial and nuclear alterations. Severe degeneration was also associated with increased amounts of interstitial fibrosis, myofibrillar lysis and disorganization and presence of myelin figures. Peripheral arterial oxygen desaturation and raised haematocrit correlated with greater mitochondrial damage. Increased nuclear convolutions were seen in patients with raised right ventricular end diastolic volume. Patients having cyanotic spells and raised haematocrit > 60% showed advanced changes viz. dilated and ruptured mitochondria, perivascular fibrosis and necrosis of myocytes.

This study was undertaken to assess the preoperative morphological changes in TOF and correlate the clinical and haemodynamic data obtained from these patients, with the degree of hypertrophy and degeneration of the right ventricular myocardium. These morphological data did have a bearing on deteriorating cardiac function particularly when changes due to severe degeneration sets in, which leads to an irreversible situation. The resulting decrease in myocardial compliance can adversely affect preoperative morbidity27 Although these irreversible changes are expected in older patients of TOF,1 as a result of chronicity of the disease process, we have observed these changes in 3 of our younger patients where the severity of the disease was also reflected in the hemodynamic parameters (viz lowered oxygen saturation, cyanotic spells, raised right ventricular end diastolic volume and elevated hematocrit levels).

This work provides preliminary data that the RV myocardial changes in Fallot’s Tetralogy may help clinicians in determining an optimal time for surgical repair and predicting the clinical outcome. A prospective study is under way in this center on a larger number of a variety of congenital cardiac diseases, including Tetralogy of Fallot, in order to study further the spectrum of ultrastructural changes in relation to the clinical cardiac dysfunction.

Acknowledgements
We wish to acknowledge the management of Sri Ramachandra Medical College & Research Institute for partially funding this project and for the generous contribution and encouragement of Dr.K.R.Balakrishnan, Professor and HOD of Cardiothoracic Surgery, SRMC&RI.

We also wish to thank the Wellcome Laboratories, Christian Medical College & Hospital, Vellore, where the Electron Microscopic studies were carried out, with expert advice from Professor Minnie Mathan, Professor of Pathology and Electron Microscopy, and Dr.Anna Devasia, Associate Professor Pathology, CMC Vellore. The technical help from Mr.Jagannadhan, Senior EM Technician is also gratefully acknowledged.

References

  1. Jones M, Ferrans VJ. Myocardial degeneration in congenital heart disease: A comparison of morphological findings in young and old patients with congenital heart disease associated with muscular obstruction to right ventricular outflow. Am J Cardiol.1977; 39:1051.
  2. Hibbs RG, Ferrans VJ, Black WC, Weilbaecher DG, Walsh JJ and Burch GE. Alcoholic cardiomyopathy: An electron microscopic study. Am. Heart J. 1965;766-779.
  3. Van Noorden S, Olsen EG and Pearse AG. Hypertrophic obstructive cardiomyopathy: A histological, histochemical and ultrastructural study of biopsy material. Cardiovasc Res 1971; 5:118-131.
  4. Nishikawa T. Ultrastructural features of the myocardium of children with dilated cardiomyopathy. Heart Vessels 1999; 14:52-56.
  5. Sakashita I, Matsukawa J, Ando T, and Asano K. Morphological comparison of infundibular pulmonary stenosis and Tetralogy of Fallot. Jpn. Heart J 1971; 12:205.
  6. Bojers AJJC, Van der Hearse, A., Vliegen HW, Quaegebear JM, Holland I, Egas JM, Cornelisse CJ. Rohmer. J and Huysmans. HA. Assessment of hypertrophy in myocardial biopsies taken during correction of congenital heart disease. Thorac Cardiovasc Surg 1988; 36: 137-140.
  7. Maron BJ, Ferrans VS, and Roberts WC Ultrastructural features of degenerated cardiac muscle cells in patients with cardiac hypertrophy. Am J.Pathol 1975; 387-434.
  8. Chalkley HW, Cornfield J, Park H. A method of estimating volume - surface ratio. Science 1949; 110:295-297.
  9. Arai S, Machide A and Nakamura T. Myocardial structure and vascularisation of hypertrophied hearts. Tohoku J Exp Med 1968; 95:35-54.
  10. Jarmakani JM, Nakazawa M, Jones I. Right ventricular function in children with Tetralogy of Fallot, before and after aortic pulmonary shunt. Circulation 1976; 53: 555-561.
  11. Mitsuno M, Nakano S, Shimazaki Y, Taniguchi.K, Kawamoto T, Kobayashi J, Matsuda H, Kawashima Y. Fate of right ventricular hypertrophy in Tetralogy of Fallot after corrective surgery. Am.J.Cardiol 1993; 72:694-698.
  12. Kato M., Kawashima Y, Fujita T, Mori T, Manabe H. Right Ventricular hypertrophy in Tetralogy of Fallot. Recent Adv Study Card Struc Metab 1976; 12:149 – 155.
  13. Lee YS, Chen YC. Alteration in ultrastructure and anionic sites in basement membranes of myocardial cells and capillaries in patients with congenital heart disease due to Tetralogy of Fallot. Jpn Heart J 1987; 28:333-347.
  14. Rees GM, Staar A. Total correction of Fallot's Tetralogy in patients aged less than one year. Brit Heart J. 1973; 35:898-901.
  15. Pacifica AD, Bargeroi LM, Kirklin JW. Primary total correction of Tetralogy of Fallot in children less than four years of age. Circulation 1973; 48: 1085-1091.
  16. Hunter JT and Chien KR. Signalling pathways for cardiac hypertrophy and failure. New England Journal Med 1999; 34: 1276 - 1285.
  17. Vtiurin BV, Kharmas SSSH, Klamanova ES. Changes in the ultrastructure of muscle cells of the heart in congenital defects. J Kardiologia 1963; 9:26.
  18. Kato M. Right ventricular hypertrophy in Tetralogy of Fallot; pathohistologic study. Jpn Assoc Thorac Surg 1976; 25:1436-1445.
  19. Jones M, Ferrans VJ, Morrow A, Robert WC. Ultrastructure of crista ventricularis muscle in patients with congenital heart disease associated with right ventricular outflow tract obstruction, Circulation 1975; 51: 39-67.
  20. Tanimoto T. Histologic findings of biopsies right ventricular myocardia in congenital heart disease before and after heart surgery. Med J Hiroshima Univ. 1982; 30:605-609.
  21. Seliem MA, Wu YT, Glenwright K. Relation between age at surgery and regression of right ventricular hypertrophy in Tetralogy of Fallot. Pediatr Cardiol 1995; 16:53-58.
  22. Tounssiant M, Pfister A, Da Lage C. The nucleus of the cardiac myocyte in ventricular hypertrophy. Ultrastructural Study Bull Assoc Anat 1984; 68: 59-68
  23. Ferrans VJ, Jones M, Maron BJ, Roberts WC. The nuclear membranes in hypertrophied human cardiac muscle cells. Am J Pathol 1975; 78:427-60.
  24. Lee YS, Suug YT, Chang CH. The surface morphology of infundibular muscle inpatients with Tetralogy of Fallot. Jpn. Heart J.1981; 22:779-787.
  25. Kawai S, Okada R, Kitamura K, Suzuki A, Saito A. A morphometrical study of myocardial disarray associated with right ventricular outflow tract obstruction. Jpn Circ J 1984; 48: 445-456.
  26. Hegarty A, Anderson RH, Deanfed TI. Myocardial fibrosis in Tetralogy of Fallot; effect of surgery or part of natural history? Br Heart J. 1988;59: 123.
  27. Maron BJ, Ferrans VJ, Jones M. The spectrum of degenerative changes in hypertrophied cardiac muscle cells: an ultrastructural study. Recent Adv. Stud. Card. Metab 1975; 8:447-466.
Contact information 
Dr. Sarah Kuruvilla 
Department of Pathology 
Sri Ramachandra Medical College & Research Institute 
No.1, Ramachandra Nagar, Porur, Chennai - 600 116. 
India 
Tel. 24765512 Ext. 294. 
Fax 24767008 
drsarahkuruvilla@rediffmail.com 
TopHome
BackIssue
Return to home page
 
 
www.impaedcard.com