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Ann Thorac Surg 2004;77:1376-1383
© 2004 The Society of Thoracic Surgeons
a Department of Surgery, VA Boston Healthcare System, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA
Accepted for publication July 10, 2003.
* Address reprint requests to Dr Khuri, Department of Surgery, MC 112, VA Boston Healthcare System, 1400 VFW Parkway, West Roxbury, MA, USA 02132
e-mail: shukri.khuri{at}med.va.gov
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Abstract |
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METHODS: Multiphoton microscopy was used to image fluorescence signals generated in myocytes deep within atrial and ventricular biopsies for identification of apoptotic changes. The biopsies, obtained during cardiac surgery, were subjected to ex vivo or in vivo acidosis. Proapoptotic markers such as exposure of phosphatidyl serine, cytochrome c, apoptotic protease-activating factor-1, and caspase-3 were identified using fluorescence-based imaging and biochemical assays.
RESULTS: Within 30 minutes of storage in low pH (<7) buffers, apoptosis was detected in human atrial samples, the severity of which correlated well with low pH. Apoptosis was also detected in atrial and ventricular biopsy samples obtained from three porcine subjects maintained on cardiopulmonary bypass and undergoing 110 minutes of aortic cross-clamp and 10 minutes of reperfusion, in which the cardiac pH was 6.36, 7.14, and 7.48. The apoptosis level detected in postacidotic reperfused cardiac tissue was pH dependent and approximately threefold greater than the precross-clamp levels.
CONCLUSIONS: Using fluorescence multiphoton microscopy and biochemical techniques we have assessed a direct correlation between low pH and induction of apoptosis in cardiac samples obtained both from human patients undergoing cardiac surgery and porcine subjects maintained on cardiopulmonary bypass simulating cardiac surgery.
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Introduction |
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Recent studies have clearly shown that acidosis and not hypoxia or ischemia alone is a major trigger of apoptosis and cell death in cultured cells and cardiac myocytes [4, 10–14].
Congestive heart failure is a major cause of decreased survival and late mortality after cardiac surgery. Considering that apoptotic changes have been observed in human and animal models of heart failure [5, 10, 11] and that acidosis triggers apoptosis, it is possible that late heart failure after cardiac surgery could be the result of myocardial apoptosis induced by the acidosis encountered intraoperatively in the course of cardiac surgery, particularly when the patient is at high risk and the surgery is complex and time-consuming.
Until recently, the intracellular events transpiring within living cardiac myocytes could not be directly visualized because of the inability of conventional fluorescence microscopy to image deep into the tissues. However, it is now possible to overcome these limitations by using fluorescence multiphoton microscopy [15] to visualize and quantitate various biochemical processes in intact tissues in real time [16, 17]. In this study we used multiphoton imaging and biochemical techniques to investigate the relationship between acidosis and apoptosis in biopsies obtained from the human and porcine heart. We evaluated the ability of ex vivo and in vivo acidosis to induce apoptosis in cardiac myocytes using fluorescent apoptotic markers and multiphoton microscopy. The tissues used were atrial biopsies obtained from patients undergoing coronary artery bypass grafting surgery and both atrial and ventricular biopsies obtained from porcine hearts maintained on cardiopulmonary bypass whose tissue pH was continuously monitored with the Khuri Myocardial pH Monitoring System (Terumo cardiovascular Systems Corp, Ann Arbor, MI).
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Material and methods |
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Experimental design
Human samples
Right atrial appendage segments were obtained from male patients, 68 ± 7 years of age (n = 11), who were receiving cardiopulmonary bypass while undergoing elective coronary artery bypass grafting surgery, according to a protocol approved by the Human Studies Subcommittee of our institution. The segments were immediately transferred to the laboratory in Hank's balanced salt solution (HBSS) maintained at 21°C. Excess fat and extraneous tissue was gently excised, and the samples were divided into several sections. Hydrochloric acid (1 mol/L), sodium hydroxide (1 mol/L), and HBSS were used to generate buffers of pH 5.5 to 8.0. The atrial sections were incubated in separate pH buffers for 30 minutes each at 37°C. Control sections were fluorescently labeled and imaged immediately without any temporal incubation.
Porcine study
The study was conducted in three Yorkshire, 12-month-old female pigs, each weighing approximately 68.2 kg (150 pounds), in accordance with a humane protocol approved by our Animal Studies Subcommittee. The pigs were anesthetized with intramuscular ketamine (20 mg/kg), intubated and mechanically ventilated with 100% oxygen, and maintained with isoflurane (1%) inhalation anesthetic. The femoral vein and artery were cannulated for intravenous access and blood pressure monitoring, respectively. The chest was opened through a median sternotomy, and the heart was suspended in a pericardial cradle. After systemic heparinization with 350 U/kg of porcine heparin and confirmation of an activated clotting time of more than 400 seconds, the ascending aorta and right atrium were cannulated with a 17F arterial cannula and a 28F single-stage venous cannula, respectively (DLP-Medtronics, Minneapolis, MN). Before cannulating the right atrium, a right atrial biopsy was obtained from the atrial wall by placing a side-biting clamp, excising a 0.5 x 0.5 cm2 full thickness piece of tissue, and suturing the biopsy site with a 5-0 Prolene (Ethicon, Somerville, NJ) suture. Cardiopulmonary bypass was initiated using a closed heparin-bonded circuit (Duraflo; Baxter, Irving, CA), which included a centrifugal pump and kinetic assisted venous drainage with another centrifugal pump (Terumo Corp, Ann Arbor, MI). Blood gases and pH were measured continuously by the Terumo CDI in-line blood gas analyzer (Terumo Cardiovascular System Inc, Tustin, CA) and maintained within the following ranges during bypass: Po2 250 to 300 mm Hg, Pco2 35 to 45 mm Hg, potassium 3.5 to 4.2 mEq, hematocrit 21% to 24%, and pH 7.35 to 7.45.
Perfusion was maintained at a flow rate that would achieve a venous oxygen saturation of 70%. Arterial blood pressure was maintained at a level of 50 to 70 mm Hg using pharmacologic agents if necessary. A previously described [1] precalibrated combination pH and temperature electrode (Khuri Myocardial pH Monitoring System, Terumo Cardiovascular Systems Corp, Ann Arbor, MI), which was 10 mm in length and 1 mm diameter, was inserted perpendicularly into the anterior left ventricular wall and affixed with a fine epicardial suture. Continuous measurements of myocardial pH and temperature were obtained as previously described [1], and were processed through a monitor that displayed and recorded temperature-corrected pH measurements in real time throughout the experiment. After a 5-minute period of stabilization after the pH probe insertion, a left ventricular biopsy was obtained close to the pH electrode by using an 18-gauge trocar biopsy needle. The right atrial and left ventricular biopsies were immediately transferred to the laboratory in HBSS at 21°C. Aortic cross-clamping was initiated, and 500 mL of 4:1 blood cardioplegic solution containing 28 mEq/L of potassium was administered at the rate of 250 mL/min at 21°C. The cross-clamp was kept on for a period of 110 minutes, during which 500 mL of cardioplegic solution was administered every 30 minutes. Ten minutes after cross-clamp release, right atrial and left ventricular biopsies were obtained from the same sites as before and transferred to the laboratory for analysis. The pigs were sacrificed using a bolus injection of potassium chloride at the end of the operation. The quantitative analyses on the tissues from the 3 pigs were performed in a blinded fashion with the examiner having no prior knowledge of the extent of acidosis encountered in each experiment.
Fluorescence apoptosis assays
Hoechst assay
Hoechst 33258 is a cell-permeant dye that binds to the A-T region of DNA with a 40x increase in fluorescence on binding (excitation/emission maxima at 346/460 nm, blue fluorescence) and therefore is a useful tool in quantitating cell number as well as apoptosis in a microscopic field. Nuclei are considered to have the normal phenotype when glowing bright and homogeneously. Early apoptotic nuclei can be identified by the initial swelling of the nucleus, condensed chromatin gathering at the periphery of the nuclear membrane, and decrease in fluorescence [4]. In this study, Hoechst was used as a general nuclear dye for identification of the nuclei, as well as a tool for identification of apoptotic nuclei. Tissue samples were incubated with 1 µL (1 mg/mL) of Hoechst in 500 µL of HBSS for 30 minutes at 37°C. After the incubation, samples were washed with HBSS and mounted in a chamber for imaging analysis.
Annexin V assay
This fluorescence-based assay identifies the externalization of phosphatidyl serine in apoptotic cells through the binding of fluorescently labeled annexin V [18]. The recombinant annexin V is conjugated to Alexa fluor 488 dye that exhibits excitation/emission maxima of 488/519 nm (green fluorescence). Tissue samples were incubated in 500 µL of HBSS with 1:1 Alexa-annexin solution (of 1 part component A [Alexa fluor 488–annexin V] to 10 parts HBSS) for 30 minutes at 21°C. After incubation, samples were washed with HBSS and mounted on an imaging chamber for microscopy. Presence of green fluorescence indicated myocytes undergoing apoptosis.
EnzChek caspase-3 assay
This kit was used to label and identify the myocytes that were caspase-3-positive by modifying the method provided with the kit. Before labeling, the tissue samples were gently permeabilized in 0.5% Triton X-100 solution in HBSS for 10 minutes at 21°C. Myocytes were labeled with rhodamine 110 (R110)–derived substrate Z-DEVD-R110 for 30 minutes at 21°C [19]. On enzymatic cleavage by caspase-3, the nonfluorescent substrate is converted in a two-step process to a fluorescent product with spectral properties similar to those of fluorescein, with excitation/emission maxima of 496/520 nm (green fluorescence). Myocytes exhibiting green fluorescence were counted as caspase-positive and apoptotic.
Multiphoton fluorescence imaging
Imaging and semiquantitative fluorescence measurements were performed with a BioRad MRC 1024ES multiphoton imaging system (BioRad, Hercules, CA) operating at 82-MHz repetition frequency and 80-fs pulse duration with a wavelength tuned to 790 nm [16, 17]. A Zeiss Axiovert S100 inverted microscope (Carl Zeiss Microimaging, Thornwood, NY) equipped with a high quality water immersion 40x/1.2 NA (numerical aperture), C-apochroma objective was used to image the segments and quantitate fluorescence in transmission and epifluorescence mode. The 512 x 512 pixel images were collected in direct detection configuration at a pixel resolution of 0.484 µm with a Kalman 3 collection filter (BioRad). The apoptotic cardiac myocytes were identified by XYZ scanning and imaged at depths of 50 µm, away from the site of incision or surface of the tissues.
Quantitative analysis
Hoechst-labeled nuclei (blue fluorescence) in a microscopic field were counted either manually or by using image-processing software (MetaMorph Imaging Series; Universal Imaging Corp, West Chester, PA). At least three separate fields were counted for each biopsy sample (120 to 300 nuclei/field), and the values were averaged. Atrial and ventricular myocytes that exhibited green fluorescence (Alexa-annexin and caspase-3 assay, respectively) were counted in three different imaged fields. The images with green fluorescence were superimposed on the blue fluorescence images using MetaMorph. Presence of apoptosis in cardiac myocytes was expressed as a percentage of the total number of green fluorescent cells to the total number of blue fluorescent nuclei in a microscopic field. The data were statistically analyzed using a paired Student's t test, and are expressed as mean ± standard error of the mean for human atrial tissue (n = 11) and porcine experiments (n = 3).
Western blotting
Samples were incubated for 30 minutes in pH 5.5, 6.5, and 7.4 buffers as described above. Samples were then homogenized using a Polytron homogenizer (5-mm tip, setting 5; Daigger, Vernon Hills, IL) for 30 seconds in 200 µL of extraction buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 4 mmol/L ethylenediaminetetraacetic acid, 0.2% Triton X-100, 0.3% Igepal CA-630, pH 7.4) and then boiled for 5 minutes. The boiled samples were centrifuged for 5 minutes at 12,200 g, and the pellet was discarded. Protein concentration was measured using the Bio-Rad protein assay, and samples containing 50 µg of total protein were mixed with 2x loading buffer (BioRad) containing 5% ß-mercaptoethanol and boiled for 4 minutes. The samples were resolved on a 10% sodium dodecylsulfate polyacrylamide gel and electroblotted onto polyvinylidene fluoride membrane (BioRad). The blots were then blocked with 3% nonfat dry milk powder in Tris buffered saline solution (20 mmol/L Tris, 500 mmol/L NaCl, pH 7.5) for 60 minutes at 21°C, and washed twice with Tris buffered saline solution with Tween-20 (TTBS; 20 mmol/L Tris, 500 mmol/L NaCl, 0.05% Tween-20, pH 7.5). Blots were incubated with anti-cytochrome c, anti-APAF-1, and anti-caspase-3 antibodies (1:2000), respectively, in TTBS containing 1% bovine serum albumin for 12 hours at 4°C with gentle shaking. Blots were washed twice with TTBS and subsequently incubated with alkaline phosphatase–conjugated secondary antibodies (1:1000) in TTBS with 1% bovine serum albumin for 2 hours at 21°C. Blots were washed twice with TTBS and then once with Tris buffered saline solution, and the bound antibodies were detected using 5-Bromo-4-chloro-indolyl phosphate/nitroblue tetrozoleum purple liquid substrate system. The blots were imaged and analyzed using MetaMorph.
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Comment |
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During ischemia, glycolysis leads to a rapid decrease in tissue pH as a result of the buildup of lactic acid. It has been established that rather than aggravating injury, such acidosis protects and maintains cell viability during periods of ischemia [3, 4]. However, restoration of normal pH after reperfusion of ischemic tissue can rapidly lead to cell death, a phenomenon that has been described as the "pH paradox" in various liver and heart models of ischemia–reperfusion [3, 4]. The pH-dependent onset of mitochondrial membrane pore transition appears to play a major role in the pH paradox. At pH less than 7 conductance through the permeability transition pore is inhibited; however, mitochondrial depolarization does occur because of anoxia [3, 4]. After establishment of normal pH by reperfusion, reoxygenation leads to mitochondrial repolarization. On repolarization, mitochondria rapidly take up calcium that has accumulated in the cytoplasm during the acidosis stage by the activation of the calcium uniporter, loading the mitochondria with large amounts of calcium. Simultaneously, free radicals are also formed by the sudden influx of oxygen in anoxic cells during reperfusion [3, 4]. Thus, a combination of high levels of calcium, oxidative stress, nitric oxide, and other factors such as adenosine triphosphate depletion leads to the activation of permeability transition pores resulting in the release of apoptotic markers such as cytochrome c, leading to eventual cell death [3–11]. In our study, even though the pH was elevated to near normal after the release of the cross-clamp and reperfusion in pig 2, a substantial amount of apoptosis was observed in both the atrial and ventricular tissue. In contrast, in pig 1 the tissue acidosis was not reversed even after reperfusion. This may have been because of activation of apoptosis-inducing, low pH–dependent endonuclease, transglutaminase, and sphingomyelinase and leakage of apoptotic factors from the mitochondria as a result of acid denaturation of the membranes and activation of permeability transition pores [3, 12–14]. Leakage of apoptotic factors from mitochondria leads to the activation of the caspase cascade, resulting in extensive apoptosis in the cardiac tissue [3–5, 11–14]. In pig 3, the cardiac tissue pH remained at the physiologic levels throughout the course of the experiment, and thus apoptosis of cardiac myocytes remained at a minimum.
Although these studies are preliminary in nature, they suggest that a reversal of acidosis at the end of a cardiac surgical operation does not necessarily indicate complete protection from myocardial damage, which still may have occurred as a result of prolonged regional acidosis encountered during the stages of aortic clamping or reflow. It is therefore of concern that our studies in patients undergoing myocardial pH monitoring during cardiac surgery have indicated that regional myocardial acidosis is frequently encountered during aortic clamping and early reflow, often at pH levels below 6.5, for periods of 30 minutes or more [20]. Unlike the injury from necrosis, damage from induced apoptosis is not expected to present with clinical signs such as electrocardiographic changes or cardiac enzyme elevation in the immediate postoperative period [21]. It may however result in more chronic disease states such as congestive heart failure, which is the most common cause of late mortality after cardiac surgery. The numerous models of heart failure suggest that the rate of occurrence of apoptosis can vary widely from less than 1% to in excess of 35%, depending on the model and types of assays used for apoptosis measurements [5, 22–25]. However, it is clear from these previously published studies that even though the rate of apoptosis in heart failure is relatively low in absolute numbers, it is significantly higher than the minimal baseline apoptosis observed in the normal heart [5]. The degree of baseline apoptosis (approximately 4%) we observed in human atrial samples may reflect already induced apoptosis inherent in diseased hearts, as these samples were obtained from patients undergoing a combination of coronary artery bypass grafting and valve replacement surgeries. It is also conceivable that the higher levels of baseline apoptosis we observed in human and porcine samples may reflect inherent deficiencies of our technique of adapting fluorescence-based apoptosis assays for complex, living cardiac tissues. The preliminary pilot experiments reported here were conducted on a small number of animals to confirm in vivo the findings from the in vitro study of acidosis-induced apoptosis in the human atrial muscle. The data generated from these animals are the basis of studies that are currently under way on a much larger cohort of animals and which, we hope, will provide the definitive observations on the relationship between acidosis and apoptosis in vivo in left ventricular muscle.
However, it is clear from our results that the acidosis-induced apoptosis in both human and porcine cardiac tissues is significantly greater than that observed in the absence of acidosis and low pH. Studies to link intraoperative in vivo tissue acidosis with myocyte apoptosis in patients undergoing cardiac surgery resulting in adverse long-term clinical outcomes are currently under way in our institution. These investigations should complement the findings of the current research, and underscore the prolonged long-term myocardial protection that can be achieved intraoperatively by maintaining a normal acid-base state in the various tissue segments of the heart throughout the totality of the course of the cardiac surgical operation.
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