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© 2004 European Association of Cardio-Thoracic Surgery
Clinical characteristics relevant to myocardial cell apoptosis: analysis of pericardial fluid
a Department of Cardiovascular Surgery, Kyoto University, Kyoto, Japan
* Corresponding author. Tel.: +81-75-751-3932; fax: +81-75-751-3909 Received October 23, 2003; received in revised form January 26, 2004; accepted January 30, 2004
We investigated the pro-apoptotic potential of pericardial fluids from patients with different clinical conditions on cultured neonatal rat cardiomyocytes. Pericardial fluids were obtained during open heart surgery from 88 patients with ischemic heart disease  valvular heart disease  or aortic disease  The terminal deoxynucleotidyl transfer-mediated end labeling fragmented nuclei assay was performed on primary cardiac myocytes from neonatal rats in the presence of 1% pericardial fluid from each patient. We evaluated relations between these patients' clinical characteristics and the extent of myocardial cell apoptosis. Induction of myocardial cell apoptosis by pericardial fluids was observed in 29 of the 88 patients (33.0%). The prevalence of myocardial cell apoptosis was significantly influenced by diabetes mellitus (DM) (53.6% with vs. 23.3% without,  ), acute coronary syndrome (ACS) (64.7% with vs. 25.4% without, ), and poor left ventricular systolic function (60.0% with vs. 25.0% without, ). Multivariate stepwise logistic regression analysis revealed that the presence of DM, ACS, and poor left systolic function were significant predictors of myocardial cell apoptosis. DM, ACS and left ventricular dysfunction may play important roles in the pathogenesis of myocardial cell apoptosis in the clinical setting.
Key Words: Angina; Apoptosis; Diabetes mellitus; Left ventricular function; Pericardium
Apoptosis, or programmed cell death, is an active, gene-directed process in which cells initiate their own deaths in response to internal or external stimuli. This mode of death serves as an orderly means for multicellular organisms to eliminate unwanted cells without adversely affecting surrounding tissues [1,2]. While apoptosis of cardiac myocytes is observed during cardiogenesis, an increasing proportion of cardiac myocytes withdraw from the cell cycle during ontogeny, so that apoptosis of adult cardiac myocytes is no longer detectable under physiological conditions. However, recent studies demonstrate that large numbers of adult cardiac myocytes undergo apoptosis in response to both prolonged ischemia and ischemia followed by reperfusion in rats, rabbits, and humans [3–5]. Apoptosis of adult cardiac myocytes is also observed in various animal models of heart failure, including rapid ventricular pacing [6] and pressure overload due to aortic constriction [7]. However, it remains unclear what pathogenic factors influence myocardial cell apoptosis in the clinical setting. To elucidate this issue we collected pericardial fluid during open heart surgery and cultured the neonatal rat cardiac myocytes with 1% pericardial fluid from each patient with a variety of heart diseases [8]. The presence of apoptosis was determined by the terminal deoxynucleotidyl transfer-mediated end labeling of fragmented nuclei (TUNEL assay) [8]. The purpose of this study, consisting of 88 patients with various heart diseases, was to elucidate the clinical pathogenic factors that independently influence the occurrence of myocardial cell apoptosis.
2.1. Study patients All the 88 patients (43 men and 45 women) in this study underwent open heart surgery due to ischemic heart disease valvular heart disease or aortic disease  (Table 1). The mean age of the patients was 67.0±11.9 years. All patients gave their written informed consent. The study protocol was approved by the ethics committee on human research at Kyoto University.
2.2. Clinical variables Patients were considered to have a history of hypertension if their systolic pressure was  160 mmHg, diastolic pressure was 95 mmHg, or if they were currently undergoing treatment for hypertension. A diagnosis of diabetes mellitus (DM) was established on the basis of any one of the following factors: a history of taking insulin or an oral hypoglycemic agent, and positive results on a 75 g oral glucose tolerance test. We defined obesity as body mass index 26 (kg/m2). Patients were considered to have poor left ventricular systolic function if their left ventricular ejection fraction (LVEF) was  40%. 2.3. Sampling of pericardial fluid Immediately after incision of the pericardium, undiluted pericardial fluid was obtained. The samples were collected in sterile tubes and immediately placed on ice. After clarification of cellular components by centrifugation at for 10 min at 4 °C, these samples were rapidly frozen in liquid nitrogen and stored at –80 °C until use. The variability in the percentage of TUNEL-positive myocytes between the sample stored for 1 week and that stored for 1 year was less than 1%. 2.4. Cell culture Primary ventricular cardiac myocytes were prepared as previously described [8]. Briefly, hearts (30 hearts in one experiment) from 1 to 2 days old Sprague–Dawley rats were removed, the ventricles were pooled, and the ventricular cells were dispersed by digestion with pancreatin (Life Technologies, Gaithersburg, MD). The cells were preplated for 1 h to enrich the culture with myocytes. We confirmed that more than 90% of these cells were myocytes by immunostaining with antimyosin heavy chain antibody. Cells were plated at a high density (1000 cells/mm2) onto 60 mm tissue culture dishes (Primaria, Falcon; Becton Dickinson and Co., Lincoln Park, NJ) and cultured in media consisting of Hanks' salts plus minimal essential medium (MEM) vitamin stock, MEM amino acids, MEM nonessential amino acids, 2 mM L-glutamine, 0.67 mM glycine, 0.92 mM hypoxanthine, 19.6 mM NaHCO3 (pH 7.1–7.2), penicillin, streptomycin, and 10% (vol/vol) fetal bovine serum (all from GIBCO BRL, Gaithersburg, MD) at 37 °C, 5% CO2.2.5. In situ labeling and quantitative analysis of apoptotic cells The TUNEL assay was performed on cardiomyocytes that had been plated on flask-style glass slides (Nalgen Nunc, Naperville, IL) as described previously [13,14]. Briefly, 2 days after the preparation of primary cardiac myocytes from neonatal rats, we added 1% pericardial fluid and further incubated for 48 h. After fixing these cells in 10% neutral buffered formalin for 10 min at room temperature, the in situ TUNEL assay was performed using a commercial kit (Takara Biomedicals, Shiga, Japan). Individual nuclei were visualized at x400 for quantitative analysis. An average of 400–500 nuclei from random fields were analyzed on each slide. The apoptotic index (percentage of apoptotic nuclei) was calculated as (apoptotic nuclei/total nuclei) x100%. Sample indicates were concealed during scoring. In each experiment, we performed all patients' samples in duplicate. The variability in the percentage of TUNEL-positive myocytes between duplicate samples was less than 1%. The data of three independent experiments were averaged per sample. The variability in three experiments was less than 2.5%. In preliminary experiments, we found that 1% of pericardial fluid was the best concentration to analyze the presence of apoptosis. Positively induced myocardial cell apoptosis was defined when the percentage of TUNEL-positive myocytes was 20% [8]. 2.6. Statistical analysis Results are expressed as means±SD. Proportional data were analyzed by the -test. The differences between the groups were analyzed with the Mann–Whitney U-test or analysis of variance with Bonferroni test when appropriate. Multivariate analysis (backward stepwise multiple logistic regression) was performed on all categorical variables. Results were considered significant at the 5% critical level. Analyses were performed with the use of SAS for Windows (SAS Institute Inc., CA).
3.1. Prevalence of myocardial cell apoptosis Myocardial cell apoptosis of cultured rat cardiomyocytes was induced by pericardial fluid from 29 of the 88 patients (33.0%). The prevalence of myocardial cell apoptosis in relation to clinical variables is shown in Fig. 1. On the basis of univariate analysis, there was a positive association between DM and the prevalence of myocardial cell apoptosis, and between acute coronary syndrome (ACS) and the prevalence of myocardial cell apoptosis. There was also a positive association between low LVEF and the prevalence of myocardial cell apoptosis.
3.2. Assessment of predictors of myocardial cell apoptosis by multivariate analysis To identify independent predictors of the induction of myocardial cell apoptosis, various clinical parameters were entered into a multivariate stepwise logistic regression analysis. This analysis revealed that a history of DM, a history of ACS, and low LVEF were significant independent predictors of myocardial cell apoptosis (Table 2).
The present analysis in 88 patients with open heart surgery revealed that myocardial cell apoptosis, as assessed by the TUNEL assay, is pathogenically influenced by myocardial ischemia, DM, and left ventricular dysfunction. In our patients, factors inducing myocardial cell apoptosis are most likely released from the cardiac interstitial tissue into the pericardial space [8]. Thus, it is probable that apoptotic factors contribute to myocardial cell death in an autocrine or paracrine fashion. The present study clearly showed that there are at least three independent pathogenic conditions predisposing for the production of apoptotic factors for cardiac myocytes. These results suggest that there may be different apoptotic factors with individual signal pathways in cardiomyocytes. Further studies are needed to identify apoptosis-inducible factors related to each pathogenic factor, and to elucidate precise intracellular signal pathways. Our data show that pericardial fluid from patients with ACS is more likely to induce apoptosis of cultured rat cardiomyocytes. The presence of myocardial cell apoptosis was also determined by in situ labeling of fragmented nuclei and morphologic features such as chromatin condensation [8]. Myocardial cell apoptosis is the major form of myocardial damage due to coronary occlusion or severe stenosis [8,9]. In an earlier study, we challenged to elucidate the relation between myocardial ischemia and myocardial cell apoptosis [8], and found that an oxidant stress-sensitive p38 mitogen-activated protein kinase pathway is a key element connecting ischemia to apoptosis. Cardiac complications are a major cause of morbidity and mortality in diabetic patients. Diabetic patients develop congestive heart failure more readily and have significantly worse prognosis compared to nondiabetic patients with similar extent of coronary artery involvement [10]. Not only diabetic coronary microangiopathy [11] but also myocardial cell apoptosis may explain the above-mentioned clinical characteristics of diabetic patients. It is tempting to speculate the mechanisms why DM causes myocardial cell apoptosis. First, hyperglycemia-induced upregulation of myocardial endothelin-1 and its receptors may lead to myocardial cell apoptosis [12]. Second, locally increased angiotensin II with diabetes may enhance oxidative stress, resulting in cardiac cell apoptosis [13]. Thus, DM may play an important role in the pathogenesis of systolic and diastolic dysfunction of the left ventricle through myocardial cell apoptosis. In the present study, the lower the LVEF, the higher the myocardial cell apoptosis. This clearly indicates that left ventricular dysfunction is closely associated with the presence of myocardial cell apoptosis. Indeed, in a mouse infarct model, myocardial cell apoptosis increased progressively along with the advancement of left ventricular remodeling [14]. Although it is speculated that mechanical stress as a result of left ventricular dilation may trigger myocardial cell apoptosis [15], underlying mechanisms including signaling pathways should be investigated in future studies. Our method to collect pericardial fluid is quite original, safe, and useful for clinical heart research. However, this approach is not applied to reoperation because of the adhesion of pericardial space. Therefore, it is impossible to evaluate the cardiac status chronologically with a repeat collection of pericardial fluid. In the present study, we examined induction of apoptosis in cardiomyocytes from hearts of 1–2 days old rats in culture. The differences in species (rat vs. human), age (neonatal vs. adult) and environment (in culture vs. in vivo) may modify the results reported here. Nevertheless, the main results of the present study suggest that DM, ACS and left ventricular dysfunction may play important roles in the pathogenesis of myocardial cell apoptosis in the clinical setting. doi:10.1016/j.icvts.2004.01.019
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Abstract
1. Introduction