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Human hibernating myocardium is jeopardized by apoptotic and autophagic cell death

Albrecht Elsässer, MD^*,*, Achim M. Vogt, MD, Holger Nef, MD^*, Sawa Kostin, MD, Helge Möllmann, MD^*, Woitek Skwara, MD, Christoph Bode, MD^||, Christian Hamm, MD^* and Jutta Schaper, MD

^* Department of Cardiology, Kerckhoff Clinic, Bad Nauheim, Germany
Department of Cardiology, Ruprecht-Carls-University, Heidelberg, Germany
Department of Experimental Cardiology, Max-Planck-Institute, Bad Nauheim, Germany
Department of Thoracic and Cardiovascular Surgery, Kerckhoff Clinic, Bad Nauheim, Germany
^|| Department of Cardiology, Albert-Ludwigs-University, Freiburg, Germany

View larger version (93K): [in a new window]   Figure 1 Electron microscopic images of human hibernating myocardium (HHM). (A) Degenerative changes with numerous autophagic vacuoles (AV) and a lack of nucleus, indicating autophagic cell death. (B) Widening of the interstitial space that contains fibroblasts (F), macrophages (small arrows), and cellular debris (CB). Note the lack of contractile material in one myocyte (arrow). (C) Apoptotic nuclear disassembly in a myocyte (thick arrows). Note the formation of an apoptotic body (thin arrows). All bars = 5 µm.

View larger version (205K): [in a new window]   Figure 2 Large ubiquitin-protein complexes (green) in myocytes (red) lacking a nucleus in the confocal microscope. Nuclei are stained blue (TOTO). Bar = 10 µm.

View larger version (188K): [in a new window]   Figure 3 One myocyte is clearly labeled for complement-9 (green) in the confocal microscope. All other myocytes are counterstained with phalloidin (red); nuclei are blue (TOTO). Bar = 20 µm.

View larger version (198K): [in a new window]   Figure 4 Proteasome subunit 20S 5 (green). Counterstain for actin is red; nuclei are blue (TOTO). (A, B) Normal myocardium. (A) Numerous nuclei are labeled for 20S 5. (B) Comparison with the nuclear stain TOTO confirms that the labeled structures are nuclei. (C, D) Human hibernating myocardium. (C) Only few structures are labeled for 20S 5. (D) TOTO reveals that a great number of nuclei are not stained for 20S 5. Bar = 20 µm.

View larger version (183K): [in a new window]   Figure 5 Apoptosis by the TUNEL method (green). Myocytes are red (phalloidin), and nuclei are blue (TOTO). Bar = 20 µm.

View larger version (68K): [in a new window]   Figure 6 Polymerase chain reaction analysis. (A) Representative original recordings. (B) Quantitative evaluation of changes in gene expression. *p < 0.05 human hibernating myocardium (HHM) versus control. MRNA = messenger ribonucleic acid.

View larger version (53K): [in a new window]   Figure 7 Western blot analysis. (A) Representative examples of original blots. (B) Quantitative data. *p < 0.05 human hibernating myocardium (HHM) versus control.