Ethics declaration
The investigation conforms with the principles outlined in the Declaration of Helsinki. In addition, approval was granted by the Ethics Committee of the Faculty of Medicine of the Eberhard-Karls-University, Tübingen, Germany (approval reference number 40/2007 V).
Patient characteristics
The study protocol was approved by the ethics committee of the Faculty of Medicine of the Eberhard-Karls-University Tübingen. 20 patients undergoing elective CABG surgery were included in this study and gave informed consent for study participation. Mean patient age was 65 years (range 45-70). Mean body mass index 28 kg/m2 (range 25-32). Mean left ventricular ejection fraction 63% (range 55-75). Mean number of diseased coronary vessels 3 (range 2-3). Mean number of infarctions 1 (range 1-3) in patients history. The basic medication of all patients consisted of β-blockers (Beloc Zok™ 47.5 mg twice per die, angiotensin converting enzyme inhibitors, statins and diuretics. All patients had a sinus rhythm.
Material
Human tissue was retrieved from the auricle of the right atrium of patients before cardiopulmonary-bypass (CPB) and was processed immediately. Each biopsy was transmuraly divided in thirteen pieces with [0.5 to 1 cm2 ]size, which were placed separately in microperfusion chambers with continuous perfusion. Cardiac specimens were outside the body before being mounted and tested in the chamber system for a maximum of 30 min, but during this period the oxygen supply was maintained continuously by bubble-oxygenating the Krebs-Henseleit buffer in the petri dish (Greiner Bio-One, Frickenhausen Germany).
Chemicals and buffer solutions
The modified Krebs-Henseleit buffer (KH) consisted of 115 mM NaCl, 4.5 mM KCl, 1.18 mM MgCl2, 1.25 mM CaCl2, 1.23 mM NaH2PO4, 1.19 Na2SO4, 80 mM Glucose, and 10 mM HEPES, pH adjusted to 7.4 at 37°C with NaOH.
Cardioplegic solution
Cardioplegic solution was prepared on the basis of Ca-free KH consisting of 115 mM NaCl, 4.5 mM KCl, 1.18 mM MgCl2, 0.5 mM EGTA, 1.23 mM NaH2PO4, 1.19 mM Na2SO4, 80 mM Glucose, and 10 mM HEPES, pH adjusted to 7.4 at 37°C with NaOH. Furthermore, a solution containing 20 mM Tris hydroxymethyl-aminomethane, 60 mmol K+ and anionic polypeptides to the isoionic point was added in a 1:4 proportion to Ca-free KH buffer. This solution served as cardioplegic solution and was administered at 4°C, in analogy to our clinical regimen. The resulting K+ concentration in this mixture was 16.5 mM.
Ceramide
Sphingolipids are constituents of cellular membranes and of lipoproteins. The common backbone is the long chain amino base sphingosine (trans-4-sphingenine), and the ceramides refer to the N-acyl derivatives of sphingosine. For a decade now, ceramides have been widely studied as regulators of major cellular functions, i.e., apoptosis, proliferation, or senescence [9–11]. Apoptosis induction with short chain ceramide (20-50 μM) supports the view that ceramides are able to trigger apoptosis [12]. The concentration of ceramide employed in this study was 50 μM, similar to previous experimental settings [12].
Amitryptiline
Amitryptiline (systematic taxonomy: 3-(10,11-dihydro-5H-dibenzo[[a, d]]cycloheptene-5-ylidene)-N, N-dimethyl-1-propanamine) is a tricyclic antidepressant. Besides its known clinical use it has been identified as an acid sphingomyelinase inhibitor with lowering ceramide levels and thus carrying out anti-apoptotic properties [13, 14].
Cell viability
The viability of cardiomyocytes in tissue samples was assessed by trypan blue exclusion before each experiment. Only samples consisting of ≥ 99% viable cardiomyocytes were further processed in the experiments of this study.
Microperfusion chamber
Our self developed, previously described [6–8] microperfusion chamber was modified to investigate larger specimens. It consisted of two components (Figure 1). The first component a temperature-controlled plexiglas block contained a rectangular cavity forming the chamber with following dimensions (length × width × height, 5.5 × 1.5 × 1.25 cm). The second component was mounted over the first, and consisted of another plexiglas block forming the ceiling of the chamber. In this chamber nylon net with a pore size of 400 μm was mounted diagonally. To enable perfusion of the chamber, a thin pipe was introduced at one end of the plexiglas component, entered the chamber and exited at the other end. A thin rubber layer between each component sealed the microperfusion chamber. The biopsy was fixed physically at the nylon net by the laminar flow (perfusion velocity of 5 ml/min) of the hydrostatic perfusion system through the chamber.
Experimental groups
The protocol was designed to simulate clinical routine procedures administering cardioplegic solution with the same K+ concentration (16.5 mM) and temperature (4°C). Five different groups (I - V) were arranged as follows: I (untreated control, n = 10), II (treated control cp/rep, n = 10), III (cp/rep + ceramide, n = 10), IV (cp/rep + amitryptiline, n = 10) and V (cp/rep + ceramide + amitryptiline, n = 10). In group III cardiomyocytes were continuously treated with 50 μM ceramid. In In group IV cardiomyocytes were continuously treated with 100 μM amitryptiline. In contrast to that in group V cardiomyocytes were continuously treated with both drugs ceramid [50 μM] and amitryptiline [100 μM]. In general, each assay was carried out with the specimens of one patient, i.e. specimens of patients were analysed separately.
Ischemia/reperfusion assay
The cardiac specimens in the microperfusion chambers were initially equilibrated with KH for 5 min (32°C and continuously bubble-oxygenated with carbogen (95% O2 and 5% CO2) to attain a PO2 of 25-30 kPa and pH 7.4. After that the cardioplegic solution (4°C) was administered for 5 min. To induce ischemic injury during the cardioplegia period the perfusion of the microperfusion chamber was stopped and the oxygen supply was discontinued. The cardiac specimens were subjected to various periods of cardioplegia (30, 60 or 120 min) followed by 1/3 of the chosen cardioplegia time as reperfusion (10, 20 or 40 min), as in our surgical routine. For reperfusion 35°C KH was used. Finally, the cardiac specimens were snap-frozen in liquid nitrogen.
Immunohistochemical apoptosis detection
The slides with the cryosections of the samples (10 μm) were processed prior to the staining according to the manufacturer's recommendation (Epitomics, Inc., Burlingame, CA, USA). The described chemicals were purchased from Biochrom, Berlin Germany. In brief, the cryosections were immersed into the staining dish containing the antigen retrieval solution: 9 ml of stock solution A (0.1 M citric acid solution) and 41 ml of stock solution B (0.1 M sodium citrate solution) were added to 450 ml of destillated H2O and adjusted to pH 6.0. After warming for 30 min in a rice cooker and cooling down the slides were washed with TBST (Tris-Buffered Saline and 0.1% Tween 20) for 5 min on a shaker. For the inactivation of endogenous peroxidases the slides were covered with 3% hydrogen peroxide for 10 min and later washed with TBST. After that the slides were immersed into the blocking solution (PBS (Dulbecco's Phosphate Buffered Salts) and 10% bovine serum albumin) for 1 hour.
Later the cryosections were incubated overnight in a humidified chamber (4°C) with antibodies against PARP-1 (Anti-Poly-(ADP-Ribose)-Polymerase)-cleavage (Epitomics, Inc.). PARP is a zinc-dependent DNA binding protein that recognizes DNA strand breaks and is presumed to play a role in DNA repair. PARP is cleaved in vivo by caspase-3 [15]. The antibody only recognizes p25 cleaved-form of PARP-1.
On the other hand cryosections were stained with antibodies against activated Caspase-3 (Epitomics, Inc.), also. Caspases are a family of cytosolic aspartate-specific cysteine proteases involved in the initiation and execution of apoptosis. Caspase-3 (apopain, SCA-1, Yama and CPP32) is a member of the apoptosis execution functional group of caspases, and is either partially or totally responsible for the proteolytic cleavage of many key proteins during apoptosis. Caspase-3 is a cytosolic protein found in cells as an inactive 35 kDa proenzyme. It is activated by proteolytic cleavage into two active subunits only when cells undergo apoptosis (3).
Later for detection to each section secondary HRP-conjugated anti-rabbit antibody (Epitomics, Inc.) diluted in the blocking solution per manufacturer's recommendation was applied and incubated for 1 hour at room temperature.
Fluorescence microscopy
The number of cells on the cryosections was determined by counting the nuclei of cardiomyocytes after staining with DAPI (4',6-Diamidino-2-phenylindole 2 HCl), a dye known to form fluorescent complexes with natural double-stranded DNA, under a fluorescence microscope (Zeiss, Jena, Germany). In each analysis three different areas of the cryosections were counted using 40-fold magnification. Apoptotic cells were identified by condensation and fragmentation of the nuclei and fluorescent conglomerates in the cytoplasm. They were quantified by counting a total of 200 nuclei from each cryosection and calculating the percentage of apoptotic nuclei. After DAPI counterstaining the greater nuclei of cardiomyocytes allow their distinction from fibroblasts with smaller nuclei. In anti-activated caspase-3 positive, apoptotic cardiomyocytes the cytoplasm reveales an intensive granular fluorescence (Figure 2). In contrast to that PARP-1 cleavage positive, apoptotic cardiomyocytes nuclei feature an intensive granular fluorescence intensity with granular staining of the nucleus.
Fluorescence images (blue) of DAPI loaded cardiac specimens were obtained at an excitation wavelength of 360 nm, with an emission wavelength of 460 nm. DAPI was purchased from Sigma-Aldrich, Germany.
Statistical Analysis
Analysis of calcium recordings and graphics were obtained using Sigma Plot software (version 9.0, SPSS Inc., Chicago, IL). Data are expressed as the mean±standard error of deviation (SD) and statistical analysis was performed using GraphPad Prism (version 5.0, GraphPad Software, Inc., CA, USA). Comparison of groups was performed using repeated measures one-way ANOVA followed by Tukey's HSD post hoc test. A p value of less than 0.05 was considered to indicate a statistically significant difference.