Introduction
Apoptosis is a highly programed, energy-dependent mechanism that occurs as a part of cell death and development, tissue turnover, and the immune system without damaging the surrounding tissue (1). Apoptosis is the bridge between pro-death and survival signals, and its outcome is key to cell fate (2). It is characterized by its specific morphology, which includes cell shrinkage, chromatin condensation, DNA fragmentation, membrane blebbing, and formation of apoptotic bodies (1). Data obtained from the examination of human heart tissue demonstrated increased apoptosis in idiopathic dilated cardiomyopathy, ischemic cardiomyopathy, and hypertrophic cardiomyopathy (1). The degree of apoptosis associated with such conditions is usually quite low; however, it is thought that the gradual loss of heart cells over time may contribute to eventual heart failure (1). In addition, the majority of tissues examined were obtained in the last stage of the disease, and it has been suggested that apoptosis may play a role in the transition from mild hypertrophy to end-stage heart failure (2). Apoptosis is regulated by the complex interplay of numerous prosurvival and prodeath signals. In particular, the Bcl-2 protein family consists of both anti-apoptotic (Bcl-2, Bcl-xl) and pro-apoptotic (Bax, Bid) proteins and exerts its effects by altering the integrity of the mitochondrial membrane and by releasing apoptotic intermembrane proteins (2).
Autophagy is a catabolic process involving the degradation and recycling of aged cytoplasmic components, such as long-lived proteins and organelles, with the aid of lysosomes (3). Under normal conditions, it is responsible for maintaining homeostasis. In nutrient-deprived cells, autophagy is a cell-survival mechanism but also mediates cell death under certain conditions (3). It has various physiological and pathophysiological roles such as adaptation to nutrient deprivation, protein intracellular clearance, growth, anti-aging, elimination of microorganisms, cell death, tumor suppression, and antigen presentation (4). Autophagy in the heart appears to be upregulated in conditions such as ischemia -reperfusion and heart failure (3). It is unclear whether autophagy is beneficial or dangerous in the heart because it appears to modulate cell viability and death. The presence of autophagic vacuoles in dying cells can be assessed in one of two ways: cells activate autophagy for survival or as part of cell death (3). In a cell with insufficient autophagic activity, aged proteins and defective organelles accumulate and eventually apoptotic cell death occurs (4). Conversely, if autophagy degrades proteins and organelles beyond a certain threshold, autophagic cell death will occur, especially in cells with an inadequate apoptotic response (4). It can be concluded that the crossover mechanism occurs between the two mechanisms and that the balance between autophagy and apoptosis maintains homeostasis. Autophagy is mainly controlled by autophagy-related genes that regulate autophagosome formation (3). Beclin-1 is required for the vesicle nucleation stage of autophagy (3).
In this study, we evaluated the expression rates of bax (apoptosis), Bcl-2 (anti-apoptosis), and beclin (autophagy) protein in epitadial tissue in patients with coronary artery by-pass graft (CABG) or mitral valve replacement (MVR) or aortic valve replacement (AVR) or Benthall procedure or myectomy.
Materials and Methods
Study Design and Study Population
The study was conducted between 2016 and 2018 at the University of Health Sciences Türkiye Kartal Koşuyolu High Specialization Training and Research Hospital. In the study, patients who underwent CABG because of obstructive coronary artery disease constituted the patient group. The patients were evaluated in the cardiology outpatient clinic because of chest pain, and coronary angiography was performed according to the results of the effort test or myocardial perfusion test. All patients had normal troponin levels. Patients with normal coronary arteries detected on preoperative coronary angiography who underwent MVR for severe mitral regurgitation, or who underwent AVR for severe aortic regurgitation, or who underwent myectomy for hypertrophic obstructive cardiomyopathy (HOCM) or Benthall procedure for ascending aortic aneurysm were included in the study as a control group. The left ventricular systolic functions of the patients were preserved both preoperatively and postoperatively (ejection fraction >50%). One of the patients with severe mitral regurgitation had mitral valve prolapse, whereas the others had rheumatic valve disease. All patients with advanced aortic regurgitation have degenerative valve disease. The study was carried out with the approval of the local ethics committee (2023/06/683). All patients were informed in detail about biopsy procedures, surgical procedures to be performed, all complications that may occur during and after surgical procedures, the follow-up period, and patient consent forms.
Histopathological Examination
Biopsy samples were taken from the epicardial tissues of the patients during surgery. Formalin-fixed paraffin blocks were prepared from the biopsy samples. Sections of 4-5 microns taken from the samples were stained with hematoxylin and eosin (H&E) and examined histopathologically under a Nikon Eclipse E600 light microscope. Inflammation intensities in the patients were assessed by considering the number of inflammatory cells entering the high magnification field in light microscopic examination and were classified as none (score 0), mild (score 1), moderate (score 2) and severe (score 3) (Figure 1). Samples found suitable because of histopathological examination were stained immunohistochemically with BAC, Bcl- and Beclin antibodies.
Immunohistochemical Examination
Samples were stained immunohistochemically using the ABC method for Bax, Bcl- and Beclin antibodies. Sections of 4 µm thickness taken on adhesive slides (Surgipath, X-tra Adhesive Microslides, Illinois, USA) and kept in an oven at 56 °C for 12 h. The sections were dried in xylene for 30 min and kept in 100% (absolute), 96%, 90%, and 80% ethyl alcohol for 15 min for dehydration. The sections were washed with water and distilled water. To block endogenous peroxidase activity, 3% H2O2 prepared in methanol was added for 10 min. Sections were washed with phosphate-buffered saline (PBS) (pH =7.60) using the spray method. To reveal the antigenic structures masked in the tissue due to formalin fixation and paraffin blocking, the sections to which both antibodies will be applied are in antigen retrieval solution (0.01M sodium citrate buffer, pH =6.00), medium for the first 10 min, and high for the last 5 min. It was processed in a microwave oven for 15 min. Subsequently, the sections were incubated at room temperature for 30 min and washed three times with distilled water followed by PBS. The Zymed Histostain Plus Broad Spectrum kit (Lot 1385070, Zymed laboratories Inc., Carlsbad, CA, USA) was used for all antibodies. Sections were kept in non-immune blocking serum for 5-10 min. Then, iNOS (polyclonal antibody RB -1605-P1; Neo Marker, Fremont, CA, USA), HSP27 (monoclonal antibody clone G3.1, MS-101- R7, Neo Marker, Fremont, CA, USA), and HSP 60 (monoclonal antibody clone LK1, 101-R7, Neo Marker, Fremont, CA, USA) at +4 0C for 18-20 hours. After reaching room temperature, the sections were washed with PBS for 5 min by spraying and dried, and then biotin-bound (secondary) antibody was applied for 20-30 min. After the sections were washed again with PBS and dried, alkaline phosphatase conjugated with streptavidin was applied and left for 20-30 min. Subsequently, the sections were washed with PBS and dried. The mixture was incubated with AEC chromogen solution (Zymed Laboratories, San. Francisco, Calif, USA, Lot: 00-0027) for approximately 10-15 min to provide color rendering. Sections were run thrice in distilled water. It was kept in Mayer’s hematoxylin for 30 s for background staining. Sections were washed first in tap water and then in distilled water. The slide was then closed using a water-based sealant (Large volume vision mount, Lot:UG14124, labvision Fremont, CA, USA).
Immunohistochemical Evaluation
Cytoplasmic and nuclear staining of myocyte cells in epicardial fat samples were considered positive for Bax, Bcl-2, and Beclin expression. Each sample considered positive was graded according to the expression intensity. In addition, other cells in the connective tissue that showed positivity were not recorded.
Statistical Analysis
Descriptive statistics were used to summarize the case data by diagnostic group. Continuous variables; were evaluated using mean, standard deviation, minimum and maximum values, and ratio and percentage values for categorical variables. One-way analysis of variance was used to compare the numerical variables with more than two groups that were normally distributed, and the Kruskal-Wallis test was used for the variables that were not normally distributed. Comparisons between the two groups were made using Student’s t-test for normally distributed variables and Mann-Whitney U test for non-normally distributed variables. Categorical variables were analyzed using crosstalk statistics (chi-square test, Fisher test). In crosstalk statistics, when the hypothesis of the test to be used in the analysis was not met, the necessary transformations (group aggregation) were made and the data were reanalyzed. Statistical significance was defined as a p value of 0.05. IBM SPSS Statistics version 12.0 (SPSS Inc, Chicago USA) was used in the analysis.
Results
The study included 28-CABG, 4 MVR, 3 AVR, 1 myectomy, and 1 Benthall procedure patients. There were 5 women and 23 men aged between 44 and 75 years in the patient group, whereas the control group included 3 women and 7 men aged between 24 and 68 years. The comorbid diseases of the patients are shown in Table 1.
In the examination of sections obtained from biopsy samples by H&E staining, it is noteworthy that the inflammation in patients diagnosed with ischemic heart disease and therefore undergoing CABG was considerably higher than that in the control group (Table 2).
Bax, Bcl-2, and Beclin expressions in the patient and control groups are shown in Table 3.
It was observed that Bax, Bcl-2, and Beclin expression was observed in all the patient and control groups, albeit at different levels. In patients with CABG with inflammation, it was observed that Bax expression increased in parallel with inflammation. Bcl-2 expression was significantly increased in patients with MVR, AVR, or myectomy without ischemic heart disease compared with patients with CABG (Figure 2). Bax expression, which was detected at a high rate in patients with CABG, suggested that ischemia also triggers apoptosis. In addition to high Bax expression in CABG patients, Bcl-2 expression was increased in these patients. The highest rate of Beclin expression was observed in HOCM patients (Figure 3). Similar results were obtained in the patient who underwent the Benthall procedure as in the HOCM patient (Figure 4). Beclin expression was also increased in patients who underwent AVR or MVR and showed high Bcl-2 expression.
No myocardial infarction, stroke, or death occurred during the in-hospital follow-up of the patients.
Discussion
In the patient group, there were patients with pre-operative and post-operative LVEF >50% who underwent diagnostic angiography for suspected coronary artery disease and whose coronary artery disease was confirmed; therefore, CABG was performed. The control group included patients with MVR, AVR, Benthall procedure, or myectomy with normal or plaque coronary arteries on preoperative coronary angiography and pre-operative and post-operative LVEF >50%. The expression rates of Bax (apoptosis), Bcl-2 (antiapoptosis), and Beclin (autophagy) proteins were measured in epicardial tissue samples taken from the patients during surgery.
Several proteins can initiate apoptosis in mitochondria. These proteins neutralize cytochrome c and endogenous apoptosis inhibitors. The choice between cell death or survival is determined by mitochondrial permeability, which is controlled by more than 20 proteins, the prototype of which is Bcl-2. Multiple sensors are activated when cells are deprived of growth factors and other life signals or when they encounter DNA-damaging factors or accumulate large amounts of misfolded proteins. These sensors are members of the Bcl-2 family called “BH3 proteins”. BH3 proteins activate two proapoptotic members of the family, Bax and Bak. These dimerize and enter the mitochondrial membrane and form channels through which cytochrome c and other mitochondrial proteins can pass into the cytosol. Again, the same sensors inhibit anti-apoptotic molecules called Bcl-2 and Bcl-xL, making it easier for mitochondrial proteins to pass into the cytosol. Cytochrome c with other cofactors activates caspase-9. Other proteins that pass from mitochondria to the cytosol block caspase antagonists, which act as physiological inhibitors of apoptosis. The net result of all these interactions is the activation of the caspase cascade, which results in fragmentation of the cell nucleus. However, when cells continue to receive growth factors and other life signals, they synthesize anti-apoptotic members of the Bcl-2 family. One of the two main types is Bcl-2 itself, and the other is Bcl-xL. Because these proteins antagonize Bax and Bak proteins, they limit the exit of pro-apoptotic proteins from mitochondria. Cells deprived of growth factors not only activate the Bax and Bak proteins. They also decreased the levels of Bcl-2 and BclxL, causing the balance to shift more toward cell death. The mitochondrial pathway appears to be responsible for apoptosis in most cases, as described later (5-9).
Detection of Bax > Beclin in patients undergoing CABG for myocardial ischemia shows that ischemia causes severe death in myocytes not only by necrosis but also by apoptosis and autophagy (even if LVEF is preserved). In the process of necrosis, which starts with cell membrane damage after the prolongation of the ischemia period and the increase in the severity of the existing ischemia and therefore the decrease in intracellular energy, the calcium entering the cell can directly stimulate the caspases and initiate apoptosis via the intrinsic way through the mitochondria.
Only 30% of the cells in the heart wall are myocytes, but because oxygen consumption is high in these cells, ischemia and apoptotic processes triggered by ischemia should be expected to develop predominantly in these cells. It was thought that the increase in apoptosis determined by the increase in Bax in the heart wall was observed in ischemic myocytes. In this process, it can be thought that the general lack of nutrients in the cells of the heart wall stimulates autophagy through Beclin.
The high expression of Bcl-2 in the control group patients with normal coronary angiography and preserved LVEF indicates that the cells escaped death and that the expression of claims was significantly increased in these patients, the adaptation capacity of the cells to the physical stresses causing the surgery was exceeded, myocyte death started via autophagy, and the heart began to dilate may be an indicator.
In the patient group without ischemic findings in the heart wall, heart wall cells showed Bcl-2 expression and Bax expression was low. This suggested that there was no intense stress that could cause cell death in the heart wall cells. However, as in heart valve diseases, the strain of heart wall cells and high energy needs lead to the depletion of energy sources in the environment, suggesting that autophagy is stimulated through nutrient deficiency. Here, the energy source includes oxygen in the short term, but glucose and other substances can be converted into energy in the long term. It can be thought that the heart wall cells use substances that they can convert into energy in the cell after oxygen deficiency and go to autophagy in the final stage. Similarly, it can be thought that autophagy develops after the use of oxygen and then substances that can be converted into energy in the ischemic heart muscle.
Study Limitations
This study was conducted with a small group of patients. Not all valve diseases were evaluated in this study. Mixed serious valve diseases were not evaluated together. There is no long-term follow-up of patients after discharge.
Conclusion
All of the participants had Bax, Bcl-2, and Beclin expression, albeit at different levels. Inflammation level is determinative for Bax and Beclin expression. In patients with inflammatory CABG, Bax expression increases in parallel with inflammation. In patients with MVR or AVR or myectomy without ischemic heart disease, Bcl-2 expression increased much more than in patients with CABG. The increased rate of inflammation in patients with CABG possibly developed secondary to ischemic necrosis, and the high rate of Bax in the same patient group suggested that ischemia triggered not only coagulation necrosis but also severe apoptosis in coronary artery patients who underwent CABG. Because coronary blood flow will be restored with bypass grafts in the patient group undergoing CABG, free radical damage and apoptosis will continue in myocytes that are in ischemia and encounter oxygenated blood again, which will cause high Bax expression. Considering the increased Bcl-2 expression in patients with CABG in addition to increased Bax expression, Bcl-2 expression represents myocytes that resist death, it was understood that high Bax expression in patients with CABG was directly caused by ischemia, considering the high Bcl-2 levels in the control groups without ischemia. The increase in Beclin expression in patients who had AVR or MVR showed high Bcl-2 expression, indicating that the adaptation capacity of myocytes against increased stress was reached and that myocytes died by autophagy without ischemia. In patients with HOCM, the highest rate of Beclin expression in genetically thickened myocytes leads to the death of myocytes via autophagy when increased protein synthesis in the cell exceeds the storage capacity of the cells, even in the absence of ischemia and physical stress. Patients who underwent ascending aortic grafting for an ascending aortic aneurysm had similar results to those of HOCM patients. In line with these results, it was understood that ischemia is the strongest death herald for myocytes, causing coagulation necrosis by hypoxia, as well as triggering apoptosis at a high rate. We have shown that it is not caused by necrosis or apoptosis, but rather by autophagy. This can be used in the timing of surgery, especially in patients with moderate-to-severe valve disease without coronary artery disease.
Ethics
Ethics Committee Approval: The study was carried out with the approval of the local ethics committee (2023/06/683).
Informed Consent: All patients were informed in detail about biopsy procedures, surgical procedures to be performed, all complications that may occur during and after surgical procedures, the follow-up period, and patient consent forms.
Peer-review: Externally and internally peer reviewed.
Authorship Contributions
Concept: M.K., G.A., Design: M.K., G.A., Data Collection or Processing: M.K., T.Ö., G.A., Analysis or Interpretation: M.K., N.K., T.Ö., G.A., Literature Search: M.K., K.E.P., N.K., Writing: M.K., K.E.P., N.K.
Conflict of Interest: No conflict of interest was declared by the authors.
Financial Disclosure: The authors declared that this study received no financial support.