Acidosis-Induced Apoptosis in Human and Porcine Heart (original) (raw)
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Ischemic acidosis causes apoptosis in coronary endothelial cells through activation of caspase-12☆
Cardiovascular Research, 2007
Objective: Myocardial ischemia has been shown to induce apoptosis of endothelial cells (EC). However, the mechanism of this endothelial injury is still poorly understood. To analyse the signaling pathway of ischemia-induced EC apoptosis was the aim of the present study. Methods: The primary culture of rat coronary EC was exposed to simulated ischemia (glucose-free anoxia at pH o 6.4). Apoptosis was defined by staining of nuclei with Hoechst-33342 and TUNEL. Cytosolic Ca 2+ and pH were measured with Fura-2 and BCECF, respectively. Results: Apoptosis (29.2 ± 1.7% of cells) induced by exposure to simulated ischemia for 2 h was accompanied by cytosolic Ca 2+ overload (1090 ± 52 nmol/l) and acidosis (pH i = 6.52 ± 0.13). Simulated ischemia had no significant effect on caspase-8 cleavage, but induced cleavage of caspase-3 and caspase-12 and led to a slight release of cytochrome C. Prevention of cytosolic acidosis (anoxia at pH o 7.4) had no effect on cytochrome C release, but significantly reduced apoptosis, attenuated cytosolic Ca 2+ overload, and prevented cleavage of caspase-12. A similar effect was achieved by inhibition of Ca 2+ release channels in the endoplasmic reticulum with ryanodine and xestospongin C. Knockdown of caspase-12 with small interfering RNA suppressed caspase-3 activation and reduced apoptotic cell number by about 70%. Conclusion: Acidosis, rather than anoxia, is an important trigger of apoptosis in EC under simulated ischemia. The main pathway of the simulated ischemia-induced apoptosis consists of the Ca 2+ leak from the ER followed by activation of caspase-12 and caspase-3.
A unique pathway of cardiac myocyte death caused by hypoxia-acidosis
Journal of Experimental Biology, 2004
SUMMARY Chronic hypoxia in the presence of high glucose leads to progressive acidosis of cardiac myocytes in culture. The condition parallels myocardial ischemia in vivo, where ischemic tissue becomes rapidly hypoxic and acidotic. Cardiac myocytes are resistant to chronic hypoxia at neutral pH but undergo extensive death when the extracellular pH (pH[o]) drops below 6.5. A microarray analysis of 20 000 genes (cDNAs and expressed sequence tags)screened with cDNAs from aerobic and hypoxic cardiac myocytes identified>100 genes that were induced by >2-fold and ∼20 genes that were induced by >5-fold. One of the most strongly induced transcripts was identified as the gene encoding the pro-apoptotic Bcl-2 family member BNIP3. Northern and western blot analyses confirmed that BNIP3 was induced by 12-fold(mRNA) and 6-fold (protein) during 24 h of hypoxia. BNIP3 protein, but not the mRNA, accumulated 3.5-fold more rapidly under hypoxia–acidosis. Cell fractionation experiments indicat...
FEBS Letters, 2003
Cardiac myocytes undergo apoptosis under condition of ischemia. Little is known, however, about the molecular pathways that mediate this response. We show that serum deprivation and hypoxia, components of ischemia in vivo, resulted in apoptosis of rat ventricular myoblast cells H9c2. Hypoxia alone did not induce signi¢cant apoptosis for at least 48 h, but largely increased the proapoptotic action of serum deprivation. H9c2 cells apoptosis is evidenced by an increase in terminal (TdT)mediated dUTP nick end-labeling-positive nuclei and by activation of caspases 3, 6, 7 and 9, and loss of mitochondrial functions. In this model of simulated ischemia, represented by serum deprivation plus hypoxia, cardiomyoblasts apoptosis was associated with a p53-independent Bax accumulation and with a down-regulation of Bcl-xL, whereas the levels of cIAP-1, cIAP-2 and X-IAP proteins did not change. Phorbol-12-myristate-13-acetate signi¢cantly reduced the induction of apoptosis, inhibiting caspase 3 cleavage, Bax accumulation, Bcl-xL downregulation as well as restoring cell viability. ß
Cardiomyocyte apoptosis in acute and chronic conditions
Basic Research in Cardiology, 1998
Myocytes can die by necrosis or by apoptosis and the characteristics of both kinds of cell death are so typical that a differentiation can be made by histological and molecularbiological methods using electron microscopy, dUTP labeling with fluorescence or peroxidase staining (TUNEL) and the DNA laddering method. However, the problem of quantification of apoptotic cells has not been completely solved because of lack of standardization as well as uncritical use and interpretation of the TUNEL method. Equally, quantification of apoptotic cells is not optimal until now because of three reasons: methodological (overinterpretation of results, no differentiation between myocytes and non-myocytes), experimental (global or regional acute ischemia, chronic conditions such as heart failure or hibernating myocardium), and interpretation (unknown time period for the completion of apoptosis). This problem is reflected in the large differences in incidence of apoptosis reported. Our own data show that in dog myocardium made globally ischemic for 90min, 8 % of the myocytes showed a positive staining for apoptosis (TUNEL method) after 6 h of reperfusion. Despite these results the question of reperfusion injury and the influence of apoptosis still remains open, because it can not be excluded until now that the apoptotic process is initiated during the ischemic period. Studies in hibernating myocardium and chronic heart failure show a similar situation, because of a wide variation of numbers of apoptotic cells and the limited possibility to investigate human tissue. There is no doubt that apoptosis plays an important role in chronic pathophysiological situations such as heart failure and hibernating myocardium but the importance of apoptosis in the acute situation of ischemia/reperfusion still has to be clarified.
Proceedings of The National Academy of Sciences, 1999
Apoptosis has been shown to contribute to loss of cardiomyocytes in cardiomyopathy, progressive decline in left ventricular function, and congestive heart failure. Because the molecular mechanisms involved in apoptosis of cardiocytes are not completely understood, we studied the biochemical and ultrastructural characteristics of upstream regulators of apoptosis in hearts explanted from patients undergoing transplantation. Sixteen explanted hearts from patients undergoing heart transplantation were studied by electron microscopy or immunoblotting to detect release of mitochondrial cytochrome c and activation of caspase-3. The hearts explanted from five victims of motor vehicle accidents or myocardial ventricular tissues from three donor hearts were used as controls. Evidence of apoptosis was observed only in endstage cardiomyopathy. There was significant accumulation of cytochrome c in the cytosol, over myofibrils, and near intercalated discs of cardiomyocytes in failing hearts. The release of mitochondrial cytochrome c was associated with activation of caspase-3 and cleavage of its substrate protein kinase C ␦ but not poly(ADP-ribose) polymerase. By contrast, there was no apparent accumulation of cytosolic cytochrome c or caspase-3 activation in the hearts used as controls. The present study provides in vivo evidence of cytochrome c-dependent activation of cysteine proteases in human cardiomyopathy. Activation of proteases supports the phenomenon of apoptosis in myopathic process. Because loss of myocytes contributes to myocardial dysfunction and is a predictor of adverse outcomes in the patients with congestive heart failure, the present demonstration of an activated apoptotic cascade in cardiomyopathy could provide the basis for novel interventional strategies.
Apoptosis and Cardiopulmonary Bypass
Journal of Cardiac Surgery, 2007
The aim of this study was to ascertain the percentage of apoptotic myocites in patients who underwent coronary artery bypass surgery. Apoptotic index (AI) obtained with in situ terminal deoxynucleotidyl transferase-labeled dUTP nick end labeling (TUNEL) method and Bak protein expression were compared. Patients and Methods: Twenty consecutive patients who underwent coronary artery bypass surgery, myocardial samples from the right atrium were taken in three stages: before cannulation (the first sample group), after declamping (the second sample group), and 20 minutes after reperfusion (the third sample group). The percentage of apoptotic cells was determined by TUNEL method. Expression of Bak protein was immunohistochemically analyzed. Intermittent ischemia and moderate hypothermia were used as methods of myocardial management during surgery. A statistical analysis was performed by using the Friedman ANOVA analysis of variances, the Kendall coefficient of concordance and the Wilcoxon matched pair test. Results: In the first sample group mean value of Bak expression was 2.61 ± 2.18, compared with AI 5.38 ± 3.58, after declamping (the second sample group) the mean value of Bak expression was 4.31 ± 2.68 while AI was 7.63 ± 4.38 and after 20 minutes of reperfusion in the third sample group mean value of Bak expression was 8.89 ± 4.45, while AI was 15.6 ± 8.45. When compared by using Wilcoxon matched pair test two methods significantly correlated, p > 0.0001. Conclusion: The positive correlation between AI obtained by TUNEL method and expression of Bak protein may suggest that apoptosis is activated mainly through mitochondrial activation pathway in ischemic reperfusion injury. The results suggest that ischemic reperfusion injury increases the AI in the right atrial tissue. If so, immunohistochemical expression of Bak protein could be used as a marker of myocardial ischemia induced injury.
BMC physiology, 2005
The importance of apoptosis in the injury sustained by the human myocardium during ischaemia and reoxygenation and the underlying mechanisms remain unclear. To quantify apoptosis and necrosis induced by simulated ischaemia/reoxygenation in the human atrial myocardium, free-hand sections of right atrial appendage (n = 8/group) were subjected to 90 minutes simulated ischaemia followed by 2, 8 and 24 hours reoxygenation. Apoptosis, as assessed by TUNEL, was greater than necrosis after 90 minutes simulated ischaemia and 2 hours reoxygenation (35.32 +/- 3.22% vs 13.55 +/- 1.3%; p < 0.05) but necrosis was greater than apoptosis by 24 hours reoxygenation (45.20 +/- 2.75% vs 4.82 +/- 0.79%; p < 0.05). Total caspase activation was similar after 90 minutes simulated ischaemia followed by 2 hours and 24 hours reoxygenation (515270 +/- 99570 U vs 542940 +/- 95216 U; p = NS). However, caspase-3 like activation was higher at 2 hours than at 24 hours reoxygenation (135900 +/- 42200 U vs 5497...