Apoptotic pathway activation from mitochondria and death receptors without caspase-3 cleavage in failing human myocardium
Fragile balance of myocyte survival?
Robert J. Scheubel, MD* ,*,
Babett Bartling, MS*,
Andreas Simm, PhD*,
Rolf-Edgar Silber, MD*,
Kostas Drogaris, MS,
Dorothea Darmer, PhD and
Juergen Holtz, MD
* Clinic for Cardiothoracic Surgery, Martin Luther University Halle-Wittenberg, Halle/Saale, Germany
Institute of Pathophysiology, Martin Luther University Halle-Wittenberg, Halle/Saale, Germany
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Figure 1 Comparison of left ventricular procaspase-9, activated caspase-9 and caspase-9S expression in patients with end-stage heart failure with nonfailing donor hearts. The messenger ribonucleic acid (mRNA) expression of caspase-9S was downregulated and the ratio of caspase-9S/procaspase-9 was decreased in patients with heart failure (a). Western blot analyses of procaspase-9, activated caspase-9 and caspase-9S protein in failing left ventricles, as compared with donor ventricles (b), illustrate increases of procaspase-9 by 25% and activated caspase-9 by 65% and an inverse relationship for caspase-9S, with no detection of caspase-9S in the failing myocardium (c). (*)p < 0.1; **p < 0.01; ***p < 0.001.
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Figure 2 Immunoblot analysis of cytochrome c and manganese superoxide dismutase (MnSOD; as a marker of the mitochondrial matrix) in the cytosol (a) and total cell lysate (b) of left ventricular specimens from donor hearts and failing hearts. In the failing myocardium, cytosolic cytochrome c was 53% higher (c), whereas MnSOD was not different between the two groups in terms of the cytosolic fraction and total lysate. In cultured rat cardiomyocytes, cytochrome c release could only be detected after treatment with 10 µmol/l of norepinephrine for 24 h (d). *p < 0.05.
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Figure 3 Analysis of left ventricular protein expression of procaspase-8, activated caspase-8, flice-like inhibitory protein (FLIP)S and FLIPL in the failing human myocardium, as compared with donor organs. The messenger ribonucleic acid (mRNA) expression of FLIPS and FLIPL is significantly downregulated in the failing myocardium (a). For activated caspase-8, there was an increase of 62% in the failing myocardium, as compared with donor hearts (b), whereas procaspase-8 was not different between the two groups. The FLIPS protein decreased significantly in the failing myocardium (b). (*)p < 0.1; *p < 0.05; ***p < 0.001.
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Figure 4 Comparison of left ventricular expression of the inhibitor of apoptosis proteins, hIAP-1, hIAP-2 and XIAP, from patients with end-stage heart failure and donors. The messenger ribonucleic acid (mRNA) expression of hIAP-1, hIAP-2 and XIAP was downregulated in the left ventricles of patients with end-stage cardiomyopathy (a). Immunoblot analysis showed a decrease of hIAP-1 and XIAP in patients with terminal heart failure, as compared with donor organs (b). (*)p < 0.1; *p < 0.05; ***p < 0.001.
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Figure 5 Immunoblot analysis of caspase-3 and its cleavage substrates gelsolin and fodrin in the left ventricles of patients with end-stage heart failure, as compared with donor hearts: 17- and 19-kDa–cleaved caspase-3 was exclusively detectable in cell lysates from cytochrome c–treated Jurkat and NIH 3T3 cell extracts. Neither donor hearts nor terminally failing hearts revealed caspase-3 activation when using an anti-active caspase-3 antibody (Cell Signaling Technology) (a). When performing an immunoblot with an antibody against the 32-kDa inactive and active caspase-3 proteins (Pharmingen), there was no cleavage product detectable, although procaspase-3 was detectable in a similar amount (b). For gelsolin, we found only the uncleaved form in both groups (c). The terminally failing hearts revealed significantly more cleaved (p < 0.001) and uncleaved (p < 0.05) fodrin, as compared with donor hearts (d). Equal protein loading of each patient has been shown by coomassie staining (e).
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Figure 6 Messenger ribonucleic acid (mRNA) expression of pro-atrial natriuretic peptide (ANP) as an overload indicator, several apoptosis inhibitors, apoptosis-inducing caspases and mitochondrially localized proteins in the left ventricles of terminally failing hearts treated with or without beta-blockers. Values are given in optical density units normalized to donor organ values. *p < 0.05. **p < 0.01. ***p < 0.001. AIF = apoptosis-inducing factor; FLIP = flice-like inhibitory protein.
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