Data from a study in patients with isolated aortic valve stenosis conduced by Schaper and colleagues showed that correlations of structure to function confirm the hypothesis that transition to heart failure occurs by fibrosis and myocyte degeneration, partially compensated by hypertrophy involving DNA synthesis and transcription. Cell loss, mainly by autophagy and oncosis, contributes significantly to the progression of left ventricular function.  Because of the limited postoperative functional recovery in later stages, early correction of the aortic valvular defect is recommended. This lecture reviewed their findings.

The extracellular matrix comprises a number of different proteins. The major proteins include fibronectin, the major matrical microfilament network all over the extracellular matrix. Laminin and collagen IV are the major components of the basal membrane. Collagen I is microfibrils that stabilize the extracellular matrix. Collagen III is a very fine filament network surrounding the microfibrils of Collagen I. Collagen VI sits atop this complex and is a more global component. These proteins together with cellular structures, such as the microvessel fibroblasts, form fibrotic tissue. Even normal myocardium has about 10% fibrotic tissue.

Their data were obtained in confocal microscopy by using monoclonal or polyclonal antibodies.  In normal human myocardium, fine septa separate the myocytes. The blast vessels are surrounded by fibronectin because it is present in the basement membrane. In the diseased myocardium, more fibronectin is present and the myocytes are more separated from each other. Replacement fibrosis is present where myocytes have disappeared.

Collagen I is the fibular structure in the extracellular matrix. In normal myocardium there is a small amount of this material between the myocytes. In failing hearts, a large amount of collagen is present. A high amount of lipofuscin may be present in the failing heart.
The so-called turtle projection on confocal microscopy, obtained with special image processing, clearly shows that collagen I is increased, in varying amounts in the failing myocardium.

The components of the extracellular matrix are connected with the intracellular milieu.  This occurs via three different systems: the dystrophin complex, the spectrin complex, and the lateral costameric junction. The extracellular matrix proteins, such as laminin or collagen, the basal membrane, and the laminin of the basal membrane bind to the distal glycans, and then bind to the dystroglycans present in the sarcolemma and represent receptors that provide connection between the outer component and the inner part of the dystrophin complex, which is dystrophin itself.  The dystrophin then is connected with the actin cytoskeleton and thereby connects the extracellular matrix to the components of the intracellular matrix, up to the nucleus, which is very important for signal transduction.

In the spectrin complex, via the anchoring receptors, which are ion channels in the sarcolemma connected with the spectrin and the actin cytoskeleton. The lateral costameric junction is a very important complex, involving the integrins as the receptors in the sarcolemma. The extracellular matrix proteins bind to the integrins, which have two different subunits. These bind then to talin, vinculin, alpha-actinin, and actin in the intracellular milieu. Alpha-1 integrin and Beta-1 integrin are greatly increased in the failing heart.

Dystrophin localizes at the inner part of the sarcolemma. In the healthy human heart there is nearly 400 myocytes per square millimeter, but only half that number in the failing myocardium. The cells in the failing heart contain much more dystrophin than in the healthy heart, and there is greater separation between the cells by fibrotic material in the failing heart compared to the healthy heart. The presence of the fibrotic material is the first indication on the structural level in failing myocardium that something is happening.

Some of the functions exerted by the extracellular matrix include acting as 1) supportive scaffolding for myocytes, myofibrils, and vasculature, 2) tethering and proper alignment, 3) transduction of mechanical forces, mechanical signaling, 4) provide tensile strength and is a major determinant of compliance, 5) movement of fluids, 6) storage of growth factors, cytokines, latent proteases, and 7) cellular migration and adhesion.

Schaper and colleagues examined the process of heart failure development in patients with isolated aortic valve stenosis. The patient population was subdivided into 3 groups based on ejection fraction (EF): Control group with a normal EF, Group I with a slightly elevated left ventricular (LV) end diastolic pressure (EDP), and an elevated pressure gradient over the aortic wall; Group II with a significantly downregulated EF (increased LVEDP, elevated pressure gradient); Group III with significantly reduced EF, elevated LVEDP, and somewhat reduced pressure gradient over the aortic wall. The baseline characteristics are shown in Table 1. Changes in the extracellular matrix and in the myocyte were studied in tissue obtained during valve replacement.

Table 1	Control	Group I	Group II	Group III
Age (years)	68	69	69	72
Number	6	12	12	8
EF (%)	61	60	43	24
LVEDP (mmHg)	11.6	15.7	18.0	27.3
(mmHg)	11	63	55	42

Baseline characteristics of study patients with aortic valve stenosis. EF: Ejection Fraction; LVEDP: Left ventricular end diastolic pressure; : mean ventricular-aortic pressure gradient.

Changes in Extracellular Matrix

Fibrosis is significantly increased as one of the primary events in the development of hypertrophy. In Group I, fibrosis was significantly upregulated, with an increase from 10% to 30% (p<0.05). Thus, one-third of the myocardium now consisted of fibrotic material. Remarkably, this occurred while the EF was still normal. The patients usually do not have surgery at this stage. In Group II, about 27% of the myocardium was fibrotic and about 37% in Group III.

Concomitantly with the increase in fibrosis, the number of capillaries decreased in this study. CD31, an endothelial marker, was used to label the capillaries. In the normal human myocardium, there were nearly 2000 capillaries per square millimeter, and this decreased linearly with the increase in fibrosis.

Angiotensin converting enzyme (ACE) localization in myocardium was assessed, to identify cytokines that may be responsible for the development of fibrosis. Most of the capillaries were labeled for ACE. There was a sharp increase from the control group to the study groups in the amount of ACE-positive capillaries, affecting about 28% of all vessels.

TGF-beta was present in fibroblasts and macrophages, and also in a stored form in the extracellular matrix without being bound to any cellular structure. The fluorescence intensity of TGF-beta progressively increased, from about 25% in the control group to about 60% in Group III. So, TGF-beta, which is required for the development of fibrosis and for the action of ACE, is increased during hypertrophy.

Pan-leukocytes, lymphocytes, and macrophages are increased in the hypertrophied myocardium, suggesting a chronic, slow inflammatory process in the myocardium.

A causal relation between the degree of fibrosis and the degree of EF was suggested by their positive correlation; the more fibrosis, the lower the EF. Also, a causal relation was found between fibrosis and LVEDP; the more fibrosis, the higher the LVEDP.

Myocyte Changes

The myocytes are comprised of different proteins, summarized in five different protein families: contractile, cytoskeletal, membrane-associated sarcomeric skeletal, and intercalated disc.

Degenerative changes in a number of the myocytes in these protein families were evaluated in the failing myocardium by electron microscopy and confocal microscopy in the present study. Myocyte degeneration is defined Schaper et al as the involvement of all cellular organelles in a chronic and most probably slow process of degradation that finally results in cellular atrophy, cell death, and finally in replacement fibrosis.
In this study, the degree of myocyte degeneration was 0.5% in the control group, and 5% in Group 1, 11 % in Group II, and 16% in Group III. Importantly, these investigators found that fibrosis occurs earlier than damage to myocytes. Fibrosis is the primary event in the development from hypertrophy to failure. These are positively correlated; the greater the degree of fibrosis, the greater the degree of myocyte degeneration. Hence, myocyte degeneration and fibrosis probably have an additive negative effect on the structural integrity of the myocardium.

Myocyte degeneration also correlates with reduced EF, with the greater the degeneration, the lower the EF.  Both myoctye degeneration and fibrosis seem to play a very important role in causing heart failure.

Replacement fibrosis is present in the entire failing myocardium, indicating that many cells, of different types, must be dying. However, this group did not find a very high number of apoptotic cells. Therefore, they looked for other types of cells occurring in failing human hearts.

Multiple mechanisms for cell death exist in the failing human heart. Three types of cell death have been identified by Schaper and colleagues.

Apoptotic cell death is found in the human heart, but not frequently. These TUNEL-positive cells, contain some lipofuscin granules. The typical symptoms of apoptosis were found in the human heart, such as condensation of the comaten, preservation of the mitochondrial structure, and preservation of cellular membrane.

Oncotic cell death is the second type found in the failing human heart. This is accidental ischemic cell death, in contrast to the programmed cell death in apoptosis. Previously called necrotic cell death, this and other research groups changed the name to oncotic cell death, to identify the killing system, not the necrosis that occurs after the cell dies. A leaky membrane is the main characteristic of oncotic cell death, and is the leading functional symptom. Complement 9 staining identifies the leaky membrane and allows the number of oncotic cells in tissue to be counted. Also seen are lipofuscin granules and myocytes that are still intact. Electron microsopy reveals a swollen nucleus, clustering of the comaten, and defective mitochondria.

Autophagic cell death is the third type of cell death. Schaper and colleagues are the first to describe this type of cell death in the failing human myocardium. It has previously been found in lymphocytes or granulocytes and cells in the thymus. Autophagic cell death involves lysosomal protein degradation via ubiquitin-dependent and independent pathways.  It is caspase-independent but ATP-dependent cell death. Bursch previously described autophagic cell death as a catabolic process involving extensive autophagic degradation of cellular organelles and autophagic vacuoles. Autophagic cell death is a very slow process involving the ubiquitinization of many different proteins. Immunogold labeling of autophagic cells revealed a high amount of ubiquitin. 

Ubiquitin, under normal conditions, is responsible for the degradation of proteins by making a conjugate, a sort of complex between proteins and ubiquitin, and then bringing this complex into the so-called proteosome. Leading investigators of proteosomal protein degradation state that 90% of all proteins under normal circumstances are being degraded in the proteosome. This requires an entire cascade to occur. First, ubiquitin must be bound to an activating enzyme, E1, and then to the conjugating enzyme E2. The protein is ubiquitinated only after formation of this complex, with the help of the ligating enzyme E3. The ubiquitin protein complex is transported to the proteosome. Before it can enter the proteosome, cleavage of the ubiquitin in certain places must occur. The ubiquitin then recycles and begins the whole process again, whereas the protein enters the proteosome, a cylindrical structure, where it is degraded to peptides and finally to amino acids. This is the most important mode of protein degradation in normal and in diseased cells.

The cascade of these enzymes must be intact and the activity of the de-ubiquitinating enzymes must be high for this process to occur. Otherwise, ubiquitin protein complexes cannot enter the proteosome, in which case it will be stored.  This is likely what occurs in the failing myocardium.

A significant increase in the ubiquitin conjugating enzyme was found in dilated cardiomyopathy compared to control, explaining the reason many ubiquitin protein complexes are found in the failing heart. Ligase was unchanged.  Importantly, the deubiquitinating enzyme isopeptidase T and the ubiquitin fusion degradation protein one (UFD-1) were decreased in failing myocardium. This means there is no more cleavage of the protein ubiquitin complexes. This is second reason for the accumulation of these complexes in the cell.

This group found that the proteosome still contains a normal amount of protein and that the proteosomal activity is variable among patients. But the mean values of proteosome in the failing heart and in the controls are similar. So, proteosomes are not involved in autophagic cell death. But, the decrease in the deubiquitinating enzyme can explain autophagic cell death. Further, potepsin, another important proteolytic enzyme, also was reduced. 

The time course between the initiation of a certain type of cell death and the duration of the death cascade must be known to estimate the rate of myocyte death, plus when the removal of the myocyte has been completed.

For oncosis, data from animal experiments may be transferred to the human myocardium.  When assuming that the duration of the death cascade was 45 to 60 minutes and that these cells are usually removed in about 48 hours. in failing myocardium, 0.06% of all cells were positive for oncosis. This translates to 11% of myocytes dying annually from oncotic cell death.

Apoptotic cell death seems not to be an important mechanism of death in heart failure. This group estimated that only 1.1% of myocytes die annually from apoptotic cell death, based on experiments devised by this group. They found that the duration of the death cascade in apoptotic cell death is about 14 hours, after it is initiated by H₂O₂ in myocyte cultures.

Autophagic cell death has not been studied in myocardial tissue. Thus, the annual myocyte death by autophagic cell death is unknown. Autophagic cell death is a very slow process, perhaps taking days or months.  But, because of the large number of degenerating myocytes that are seen, this group believes that the process of autophagic cell death is a very important one.

In the progression from compensated hypertrophy to heart failure, myocyte degeneration contributes significantly to functional deterioration. The final stage of degeneration is cell death. Fibrosis and degeneration have additive effects.

Morphometric data from their present study showed that the cross-sectional area of the cells increase as failure progresses, indicating hypertrophy. The size of the nuclei increase, but not to the degree required to make the ratio between cell volume and nuclear volume similar to that in the control group.

The presence of the splicing factor Sc-35 is marker for ongoing transcription, and can thus indicate whether or not cells are still viable.  A significant increase in the content of Sc-35 and of DNA was found in the hypertrophied heart in the present study. This increase was most significant in Group III. However, this is not counterbalanced by an increase in the concentration of DNA and of SC-35, indicating insufficient SC-35 to ensure transcription.

Fibrosis is a primary event in the development of cardiac hypertrophy. Myocyte degeneration occurs later, when the EF is already reduced. Low-grade inflammation present in both hypertrophied and failing hearts. Upregulation of ACE is an early event, whereas TGF-beta is late. Cell death by 3 different mechanisms is evident. The DNA content is increased in the failing heart, but not the DNA concentration. The Sc-35 content is increased in the failing heart, but not the concentration. Sc-35 elevation indicates cellular viability.

These investigators propose this scheme for the progression from pressure overload to heart failure. Pressure overload is the primary event and causes reactive fibrosis, in which TGF-beta and ACE are involved. Capillary density decreases, which is ensued by myocyte degeneration, hypertrophy, and cellular sequestration. Low-grade inflammation and myocyte hypertrophy further enhances this process. There are some counteracting mechanisms in this scheme. The nuclei try to adapt to the higher requirements of the hypertrophied cells, but when this adaptive process becomes exhausted the consequence is myocyte loss and then replacement fibrosis. This creates a vicious circle, finally leading to heart failure.