- Email: [email protected]
Multifacetted morphological alterations are present in the failing human heart
J Mol Cell Cardiol 27. 857-861 (1995)
Comment on the Review by Gerdes and Capasso
Multifacetted Morphological Alterations are Present in the Failing Human Heart Jutta Schaper, Stefan Hein, Dimitri Scholz and Hanke Mollnau Max-Planck-Institute, Department of Experimental Cardiology, Bad Nauheim, Germany (Received and accepted 19th October 1994) either dilated or ischemic cardiomyopathy, we suggest that additional morphological factors are possibly involved in the origin of heart failure. In this short comment we will restrict ourselves to our findings in human myocardinm from patients with dilated cardiomyopathy undergoing cardiac transplantation because of intractable heart failure (ejection fraction lower than 20%). Similar changes, however, have also been observed in longstanding ischemic heart disease. Gerdes and Capasso report that in failing myocardium all myocytes are significantly longer than in normal hearts. Even though the authors deny any influence of the isolation procedure on myocyte size, shape, and function we assume that a certain selection of cells occurs. In our experience with isolated myocytes, there exists even in normal hearts a significant size distribution. Additionally, the size and shape of cardiomyocytes in diseased human myocardium c a n be extremely variable. In tissue sections from myocardinm from patients with dilated cardiomyopathy we found a significant diversity in cell shape ranging from ceils that were elongated but not wider than normal, i.e. 12-15/zm, to cells that were either much wider, up to 40/2m, and rather :short, and other cells were of normal size [fig,1 reprinted from (Scholz et a3., 1994)]. Myocyte width was measured in longitudinal sections at the .level of the intercalated disc assuming that this structure is the least dependent on the contractile state of the cells. From this data it becomes evident that in normal human hearts the percentage of slender cells is higher than in diseased hearts but that there occurs a shift to the right in cell width in hearts with
We have read with great interest the paper by Drs Gerdes and Capasso who studied isolated cardiomyocytes of failing hearts using morphological and functional parameters. The authors postulate that in the failing heart "maladaptive remodeling of cardiomyocyte morphology" occurs and is characterized by the occurrence of slender, very long myocytes with a length/width ratio of 11 instead of seven as in cells from normal hearts. The authors carried out their morphological studies in cardiomyoctyes isolated from animal and human hearts and claim that their findings are compatible with changes noted in human dilated cardiomyopathy as well as ischemic heart disease. The proposed concept is an interesting one and a challenge to the morphologist utilizing a variety of different methods trying to identify the structural correlates of heart failure. The authors propose, on the-basis of their measurements in isolated myocytes, that "differential myocyte growth", i.e., growth in the longitudinal direction by addition of sarcomeres but no growth in cell width, is "the primary event involved in the pathological morphometric changes observed during dilated cardiac failure". Re-expression of fetal genes controling myocyte shape is suggested as a triggering factor. A convincing explanation for the specific mechanism causing sarcomerogenesis in the longitudinal direction only is, however, not presented in the authors' manuscript. According to Gerdes and Capasso, elongated myocytes demonstrate a weaker contractile response than cells of normal size and shape which supports the authors' hypothesis. However, based on our extensive studies on failing human hearts with
Please address all correspondenceto: Professor Dr. lutta Schaper, Max-Planek-Institute, Department of lLxperimentalCardiology; Benekestrasse 2, D-61231 Bad Nauheim, Germany. 0022-28281951030857+05 $08.00•0
857
© !995 Academic Press.:r.!mLte,d,
858
J. Schaper et al. 35 [pm] 30
P
E 15
10
5
0
15
30
45
60
75
90
Mean ± S.E.M.
[pm] Figure 1 Distribution of myocyte width in ([]) human controls (n=8) and (11) patients (n=12) with dilated cardiomyopathy. The percentage of slender cells is higher than controls. In diseased hearts there is a shift of the curve to the right. Cell width is significantly increased in diseased hearts as compared to controls. Reprinted with permission from SchoL~et al. (1994). dilated cardiomyopathy. The number of cells with a diameter larger than 20/2m is significantly higher in diseased hearts which is in contrast with the postulation by Gerdes and Capasso. These quantitative findings are corroborated by immunohistochemical preparations of tissue sections stained for various' cardiac proteins that alSO show a wide distribution of cell size and the occurrence of rather large almost quadratic myocytes (Fig.2). On the other hand, in ultrastructural and immunohistochemical studies, the presence of atrophied cardiomyocytes, i.e. cellular remnants, was especially obvious (Fig. 3). We therefore believe that elongated ceils are present within failing myocardium but that a wide range of cell size can be observed and that the elongated ceils are only one facet of the entire pattern. The report by Gerdes and Capasso is mainly concerned with changes in cell size but fails to take into: account changes observed within the myocytes. In myocardium from patients with dilated cardiomyopathy multiple partially adaptive and also degenerative alterations can be observed. The cardiomyocytes exhibit an altered nucleus/cytoplasm relationship, i,e. the nuclei are increased in size but in relation to the size of the cell their volume is significantly smaller [Fig. 4, repr~ted from (Scholz et a/., 1994)]. Additionally, there were alterations of the contractile material and of the cytoskeleton. The myofilaments were significantly reduced and showed a n irregular arrangement involving all
types of contractile proteins, i.e. myosin and the thin filament complex including the third elastic filament, titin, as well as a-actinin (Hein et al., 1994). The cytoskeletal elements such as tubulin and desmin were increased and/or completely disorganized (Schaper et al., 1991). In addition, vacuoles, myelin figures, an increased amount of lipofuscin and lipid droplets can be observed which are the morphological signs of the occurrence of degeneration. It is interesting to note that these alterations showed an inhomogeneous distribution within the tissue with normal cardiomyocytes neighbouring those of totally abnormal appearance. The fact that according to our immunohistochemical data many different proteins are subject to alterations in expression and arrangement may be interpreted that all of these alterations are secondary events and that the primary event should be sought for at the transcriptional level or even higher. Our own data show that the mRNA for myosin, titin and desmin is reduced, i.e. that the transcription of proteins is disturbed (Hein et al., 1993). One may consider that a concomitant disregulation of transcription factors such as various oncogenes may be present but this is pure speculation at the moment. We believe, however, that the injury to and the degeneration of myocytes is the first event leading to variations in cell size, initial hypertrophy as a compensatory mechanism followed by atrophy, and resulting in the production of an increased amount of fibrotic tissue.
!\luhifacelled Morphological Alteralions
~
8S9
°
¢oN'~ o,
,~ ~ / ~
,~,
Figure 2 hnn3unohistochemical demonstration of different cell width and length in human myocardium with dilated cardiornyopathy. (a) Staining for vinculin reveals the occurrence of slender as well as wide cells, both of different length x 330. Ib) Staining for desmin in another tissue sample shows large ahnost quadratic cardiomyocytes, x 530. In dilated c a r d i o m y o p a t h y the occurrence of cardiac fibrosis is a c o m m o n p h e n o m e n o n . The extracellular space is enlarged and contains the c o m p o n e n t s of the matrix in n o r m a l m y o c a r d i u m , i.e. fibronectin, laminin, the various types of collagen, especially collagen I, III and VI and chondroitin sulfate in increased a m o u n t s . The increase
in collagen VI is especially obvious. Fibronectin. laminin, as well as the collagens I and VI s u r r o u n d the myocytes tightly as can be seen by confocal microscopy (Fig. 5). They all seem to adhere to the basal lamina or to be part of it. The cellular elements, fibroblasts, m a c r o p h a g e s and capillary endothelial cells are n u m e r o u s . At later stages of the
860
1. Schaper et al.
Figure 3 A myocyte (Myo) with loss of contractile material shows sequestration of cellular particles (S). Cellular debris (CD) is evident in the extracellular space as are active macrophages (M). x 4300. 1.2
P<0.05
100
P<0.001
1.0 ~-
80
0.8
60 0.6 40 0.4 20
0.2 0 Vvnu c
Nuclear area M e a n + S.E.M.
Figure 4 Nuclear volume density and nuclear area in diseased human myocardium as compared to human controls. It is evident that despite the increase in the size of individual nuclei (nuclear area) the nuclear volume density is reduced, i.e. the nucleus/cytoplasm relationship is altered. Reprinted with permission from Scholz et al. (1994).
development of fibrosis the various components of the matrix tend to isolate the myocytes from each other and most probably inhibiting not only the transfer of an electrical stimulus from myocyte to myocyte but also of metabolites and oxygen. In summary we suggest that the pathophysiology of heart failure involves a complex series of events for which a variety of different hypotheses have been proposed. Concerning the structural correlate
of the reduction in cardiac function in failure we propose that the injury to cardiomyocytes seems to be the first event leading to cellular hypertrophy and to numerous molecular changes, especially transcriptional disturbances, in the cardiomyocytes. These events result in a lack of contractile material and disarrangement of the cytoskeleton. The consequence of cellular degeneration is the sequestration of cellular particles into the
Multifacetled Morphological Alteralions
861
Figure 5 A double staining procedure lbr collagen VI (green) and 7-actinin (red) shows that the collagen fibers surround the myocytes tightly. Conlbcal microscopy, x 900. extracellular space resulting in final atrophy of the cardiomyocytes. This cellular debris occurring in increased amounts in the interstitium most probably leads to an activation of fibroblasts and macrophages. These in turn produce more collagen, fibronectin and laminin as fibrotic tissue is accumulated which tends to separate and later to isolate myocytes from each other. This leads to further functional and structural degeneration of the cardiomyocytes resulting in a vicious circle leading to heart failure. We therefore believe that the concept proposed by Gerdes and Capasso is an elegant one but it fails to cover all the different aspects of morphological changes occurring in heart failure.
References HI.IN S, FUIITANIN, SCHOLZD, BRANDT, SCHAPERJ, 1993. Transcription and translation of titin and alpha-actinin are disturbed in diseased human myocardium. ] Mol Cell Cardiol 25 (Suppl. I): III P 6. HEINS. SCHOLZD. FuIrL.XNIN. BRANDT, FRIEDLA. SCHAPER J. 1994. Altered expression of titin and contractile proteins in failing human myocardium. ] Mol Cell Cardiol 26. 1291-1306. SCHAPER ], FROEDE R, HEIN S, BUCK A, FRIEDL A, HASHIZUMEH. SPEISERB, BLEESEN, 1991. Impairment of myocardial ultrastructure and changes of the cytoskeleton in dilated cardiomyopathy. Circulation 83: 504-514. SCHOLZD, DIENERW, SCHAPERJ, 1994. Altered nucleus/ cytoplasm relationship and degenerative structural changes in human dilated eardiomyopathy. Cardioscience 5: 127-138.
Comment on the Review by Gerdes and Capasso
Multifacetted Morphological Alterations are Present in the Failing Human Heart Jutta Schaper, Stefan Hein, Dimitri Scholz and Hanke Mollnau Max-Planck-Institute, Department of Experimental Cardiology, Bad Nauheim, Germany (Received and accepted 19th October 1994) either dilated or ischemic cardiomyopathy, we suggest that additional morphological factors are possibly involved in the origin of heart failure. In this short comment we will restrict ourselves to our findings in human myocardinm from patients with dilated cardiomyopathy undergoing cardiac transplantation because of intractable heart failure (ejection fraction lower than 20%). Similar changes, however, have also been observed in longstanding ischemic heart disease. Gerdes and Capasso report that in failing myocardium all myocytes are significantly longer than in normal hearts. Even though the authors deny any influence of the isolation procedure on myocyte size, shape, and function we assume that a certain selection of cells occurs. In our experience with isolated myocytes, there exists even in normal hearts a significant size distribution. Additionally, the size and shape of cardiomyocytes in diseased human myocardium c a n be extremely variable. In tissue sections from myocardinm from patients with dilated cardiomyopathy we found a significant diversity in cell shape ranging from ceils that were elongated but not wider than normal, i.e. 12-15/zm, to cells that were either much wider, up to 40/2m, and rather :short, and other cells were of normal size [fig,1 reprinted from (Scholz et a3., 1994)]. Myocyte width was measured in longitudinal sections at the .level of the intercalated disc assuming that this structure is the least dependent on the contractile state of the cells. From this data it becomes evident that in normal human hearts the percentage of slender cells is higher than in diseased hearts but that there occurs a shift to the right in cell width in hearts with
We have read with great interest the paper by Drs Gerdes and Capasso who studied isolated cardiomyocytes of failing hearts using morphological and functional parameters. The authors postulate that in the failing heart "maladaptive remodeling of cardiomyocyte morphology" occurs and is characterized by the occurrence of slender, very long myocytes with a length/width ratio of 11 instead of seven as in cells from normal hearts. The authors carried out their morphological studies in cardiomyoctyes isolated from animal and human hearts and claim that their findings are compatible with changes noted in human dilated cardiomyopathy as well as ischemic heart disease. The proposed concept is an interesting one and a challenge to the morphologist utilizing a variety of different methods trying to identify the structural correlates of heart failure. The authors propose, on the-basis of their measurements in isolated myocytes, that "differential myocyte growth", i.e., growth in the longitudinal direction by addition of sarcomeres but no growth in cell width, is "the primary event involved in the pathological morphometric changes observed during dilated cardiac failure". Re-expression of fetal genes controling myocyte shape is suggested as a triggering factor. A convincing explanation for the specific mechanism causing sarcomerogenesis in the longitudinal direction only is, however, not presented in the authors' manuscript. According to Gerdes and Capasso, elongated myocytes demonstrate a weaker contractile response than cells of normal size and shape which supports the authors' hypothesis. However, based on our extensive studies on failing human hearts with
Please address all correspondenceto: Professor Dr. lutta Schaper, Max-Planek-Institute, Department of lLxperimentalCardiology; Benekestrasse 2, D-61231 Bad Nauheim, Germany. 0022-28281951030857+05 $08.00•0
857
© !995 Academic Press.:r.!mLte,d,
858
J. Schaper et al. 35 [pm] 30
P
E 15
10
5
0
15
30
45
60
75
90
Mean ± S.E.M.
[pm] Figure 1 Distribution of myocyte width in ([]) human controls (n=8) and (11) patients (n=12) with dilated cardiomyopathy. The percentage of slender cells is higher than controls. In diseased hearts there is a shift of the curve to the right. Cell width is significantly increased in diseased hearts as compared to controls. Reprinted with permission from SchoL~et al. (1994). dilated cardiomyopathy. The number of cells with a diameter larger than 20/2m is significantly higher in diseased hearts which is in contrast with the postulation by Gerdes and Capasso. These quantitative findings are corroborated by immunohistochemical preparations of tissue sections stained for various' cardiac proteins that alSO show a wide distribution of cell size and the occurrence of rather large almost quadratic myocytes (Fig.2). On the other hand, in ultrastructural and immunohistochemical studies, the presence of atrophied cardiomyocytes, i.e. cellular remnants, was especially obvious (Fig. 3). We therefore believe that elongated ceils are present within failing myocardium but that a wide range of cell size can be observed and that the elongated ceils are only one facet of the entire pattern. The report by Gerdes and Capasso is mainly concerned with changes in cell size but fails to take into: account changes observed within the myocytes. In myocardium from patients with dilated cardiomyopathy multiple partially adaptive and also degenerative alterations can be observed. The cardiomyocytes exhibit an altered nucleus/cytoplasm relationship, i,e. the nuclei are increased in size but in relation to the size of the cell their volume is significantly smaller [Fig. 4, repr~ted from (Scholz et a/., 1994)]. Additionally, there were alterations of the contractile material and of the cytoskeleton. The myofilaments were significantly reduced and showed a n irregular arrangement involving all
types of contractile proteins, i.e. myosin and the thin filament complex including the third elastic filament, titin, as well as a-actinin (Hein et al., 1994). The cytoskeletal elements such as tubulin and desmin were increased and/or completely disorganized (Schaper et al., 1991). In addition, vacuoles, myelin figures, an increased amount of lipofuscin and lipid droplets can be observed which are the morphological signs of the occurrence of degeneration. It is interesting to note that these alterations showed an inhomogeneous distribution within the tissue with normal cardiomyocytes neighbouring those of totally abnormal appearance. The fact that according to our immunohistochemical data many different proteins are subject to alterations in expression and arrangement may be interpreted that all of these alterations are secondary events and that the primary event should be sought for at the transcriptional level or even higher. Our own data show that the mRNA for myosin, titin and desmin is reduced, i.e. that the transcription of proteins is disturbed (Hein et al., 1993). One may consider that a concomitant disregulation of transcription factors such as various oncogenes may be present but this is pure speculation at the moment. We believe, however, that the injury to and the degeneration of myocytes is the first event leading to variations in cell size, initial hypertrophy as a compensatory mechanism followed by atrophy, and resulting in the production of an increased amount of fibrotic tissue.
!\luhifacelled Morphological Alteralions
~
8S9
°
¢oN'~ o,
,~ ~ / ~
,~,
Figure 2 hnn3unohistochemical demonstration of different cell width and length in human myocardium with dilated cardiornyopathy. (a) Staining for vinculin reveals the occurrence of slender as well as wide cells, both of different length x 330. Ib) Staining for desmin in another tissue sample shows large ahnost quadratic cardiomyocytes, x 530. In dilated c a r d i o m y o p a t h y the occurrence of cardiac fibrosis is a c o m m o n p h e n o m e n o n . The extracellular space is enlarged and contains the c o m p o n e n t s of the matrix in n o r m a l m y o c a r d i u m , i.e. fibronectin, laminin, the various types of collagen, especially collagen I, III and VI and chondroitin sulfate in increased a m o u n t s . The increase
in collagen VI is especially obvious. Fibronectin. laminin, as well as the collagens I and VI s u r r o u n d the myocytes tightly as can be seen by confocal microscopy (Fig. 5). They all seem to adhere to the basal lamina or to be part of it. The cellular elements, fibroblasts, m a c r o p h a g e s and capillary endothelial cells are n u m e r o u s . At later stages of the
860
1. Schaper et al.
Figure 3 A myocyte (Myo) with loss of contractile material shows sequestration of cellular particles (S). Cellular debris (CD) is evident in the extracellular space as are active macrophages (M). x 4300. 1.2
P<0.05
100
P<0.001
1.0 ~-
80
0.8
60 0.6 40 0.4 20
0.2 0 Vvnu c
Nuclear area M e a n + S.E.M.
Figure 4 Nuclear volume density and nuclear area in diseased human myocardium as compared to human controls. It is evident that despite the increase in the size of individual nuclei (nuclear area) the nuclear volume density is reduced, i.e. the nucleus/cytoplasm relationship is altered. Reprinted with permission from Scholz et al. (1994).
development of fibrosis the various components of the matrix tend to isolate the myocytes from each other and most probably inhibiting not only the transfer of an electrical stimulus from myocyte to myocyte but also of metabolites and oxygen. In summary we suggest that the pathophysiology of heart failure involves a complex series of events for which a variety of different hypotheses have been proposed. Concerning the structural correlate
of the reduction in cardiac function in failure we propose that the injury to cardiomyocytes seems to be the first event leading to cellular hypertrophy and to numerous molecular changes, especially transcriptional disturbances, in the cardiomyocytes. These events result in a lack of contractile material and disarrangement of the cytoskeleton. The consequence of cellular degeneration is the sequestration of cellular particles into the
Multifacetled Morphological Alteralions
861
Figure 5 A double staining procedure lbr collagen VI (green) and 7-actinin (red) shows that the collagen fibers surround the myocytes tightly. Conlbcal microscopy, x 900. extracellular space resulting in final atrophy of the cardiomyocytes. This cellular debris occurring in increased amounts in the interstitium most probably leads to an activation of fibroblasts and macrophages. These in turn produce more collagen, fibronectin and laminin as fibrotic tissue is accumulated which tends to separate and later to isolate myocytes from each other. This leads to further functional and structural degeneration of the cardiomyocytes resulting in a vicious circle leading to heart failure. We therefore believe that the concept proposed by Gerdes and Capasso is an elegant one but it fails to cover all the different aspects of morphological changes occurring in heart failure.
References HI.IN S, FUIITANIN, SCHOLZD, BRANDT, SCHAPERJ, 1993. Transcription and translation of titin and alpha-actinin are disturbed in diseased human myocardium. ] Mol Cell Cardiol 25 (Suppl. I): III P 6. HEINS. SCHOLZD. FuIrL.XNIN. BRANDT, FRIEDLA. SCHAPER J. 1994. Altered expression of titin and contractile proteins in failing human myocardium. ] Mol Cell Cardiol 26. 1291-1306. SCHAPER ], FROEDE R, HEIN S, BUCK A, FRIEDL A, HASHIZUMEH. SPEISERB, BLEESEN, 1991. Impairment of myocardial ultrastructure and changes of the cytoskeleton in dilated cardiomyopathy. Circulation 83: 504-514. SCHOLZD, DIENERW, SCHAPERJ, 1994. Altered nucleus/ cytoplasm relationship and degenerative structural changes in human dilated eardiomyopathy. Cardioscience 5: 127-138.