Acidosis-induced apoptosis in human and porcine heart

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Presentation transcript:

Acidosis-induced apoptosis in human and porcine heart Hemant S Thatte, PhD, Jin-Hwa Rhee, BA, Sofija E Zagarins, BS, Patrick R Treanor, CCP, Vladimir Birjiniuk, MD, Michael D Crittenden, MD, Shukri F Khuri, MD The Annals of Thoracic Surgery Volume 77, Issue 4, Pages 1376-1383 (April 2004) DOI: 10.1016/j.athoracsur.2003.07.047

Fig 1 Biopsies of human atrial tissue were incubated in various pH buffers and labeled with apoptosis-sensitive green Alexa-annexin and nuclei-specific blue Hoechst fluorescence dyes. Control samples were labeled immediately after the biopsy without any other treatments. An increase in green fluorescence as a result of binding of Alexa-annexin to the externalized phosphatidyl serine in apoptotic myocytes can be observed in tissue samples that were incubated in pH 6.5 buffer. Representative image at ×320 magnification, n = 11. The Annals of Thoracic Surgery 2004 77, 1376-1383DOI: (10.1016/j.athoracsur.2003.07.047)

Fig 2 Atrial tissue biopsies were obtained from human patients undergoing coronary artery bypass grafting surgery. Samples were incubated in pH-adjusted buffers for 30 minutes at 37°C before labeling with apoptosis-sensitive Alexa-annexin (green fluorescence) and Hoechst (blue fluorescence) dyes, and imaged using multiphoton microscopy. Apoptosis in cardiac myocytes was quantitated and 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 are the mean ± standard error of the mean of 11 experiments (atrial biopsies from 11 different patients) performed on different days. A significant increase in apoptosis was observed with decreasing pH of the buffers (*p = 0.001, pH 6.5 versus 7.4). Samples incubated in pH 6.5 buffer were compared with the pH 7.4 sample to eliminate the influence of incubation on observed apoptosis. The Annals of Thoracic Surgery 2004 77, 1376-1383DOI: (10.1016/j.athoracsur.2003.07.047)

Fig 3 Atrial biopsies were incubated for 30 minutes in pH 5.5, 6.5, and 7.4 buffers and were resolved by gel electrophoresis and Western blot. Induced apoptotic proteins were identified using anti-cytochrome c and apoptotic protease activating factor-1 (APAF-1) antibodies. The two apoptosis protein markers were visible in cardiac myocytes that were incubated in low pH buffers, but not in pH 7.4 buffer. The Annals of Thoracic Surgery 2004 77, 1376-1383DOI: (10.1016/j.athoracsur.2003.07.047)

Fig 4 A precalibrated combination pH/temperature electrode was inserted perpendicularly into the anterior wall at the mid-myocardium and affixed with a fine epicardial suture. Continuous measurement of temperature-corrected myocardial pH was recorded in real time throughout the experiment. Atrial biopsies were obtained from the right atrium, and the left ventricular biopsies were obtained close to the pH electrode. Aortic cross-clamping was initiated and was kept on for a period of 110 minutes during which 500 mL of cardioplegia was administered every 30 minutes. Ten minutes after the cross-clamp was released tissue samples were collected from the same sites as before and transferred to the laboratory for analysis. All the samples were blinded for laboratory analysis. The Annals of Thoracic Surgery 2004 77, 1376-1383DOI: (10.1016/j.athoracsur.2003.07.047)

Fig 5 Biopsies obtained from porcine hearts were labeled with Hoechst nuclear dye and were assayed for caspase-3 activity indicative of apoptosis by the generation of green fluorescence using the fluorescence caspase-3 assay kit as explained in the Methods. An example of an increase in green fluorescence demonstrating caspase-3 activity as seen in cardiac myocytes obtained from post–cross-clamp ventricular tissue is shown in this figure. From such images, apoptotic cardiac myocytes are quantitated, and the results are expressed as a percentage of the total green fluorescent cells to the total number of Hoechst blue fluorescent nuclei in a microscopic field for both the atrial and ventricular samples. As shown in Table 2, atrial and ventricular tissues obtained after aortic cross-clamp show a substantial increase in caspase-3 activity in pigs 1 and 2, but not in pig 3. Representative image at ×320 magnification, n = 3. The Annals of Thoracic Surgery 2004 77, 1376-1383DOI: (10.1016/j.athoracsur.2003.07.047)