Acute myocardial infarction represents the leading cause of morbidity and mortality in the western societies. Importantly, both apoptosis and necrosis of cardiomyocytes have been implicated in the pathomechanism of myocardial infarction. The simplest way to analyze apoptosis in cardiac cells is the application of isolated neonatal primary cardiac myocytes, in which ischemia/reperfusion can be mimicked in vitro by exposing them to hypoxia and serum starvation, followed by restored oxygen and serum conditions, referred to as hypoxia/reoxygenation. In this chapter, we describe protocols routinely applied in our lab for investigating cardiomyocyte apoptosis. In summary, a better understanding of the apoptotic pathways and their regulation in the heart will potentially yield novel therapeutic targets for cardiac infarction.

I. Detection of Apoptosis 1. Analyzing Caspase Activation and Caspase Activity in Apoptotic Cells Sharad Kumar and Loretta Dorstyn 2. Flow Cytometry-Based Apoptosis Detection Donald Wlodkowicz, Joanna Skommer, and Zbigniew Darzynkievicz 3. Live to Dead Cell Imaging Stephen W.G. Tait, Lisa Bouchier-Hayes, Andrew Oberst, Samuel Connell, and Douglas R. Green 4. Detection of Apoptosis in Tissue Sections Eva Csizmadia and Vilmos Csizmadia 5. Detection of Apoptosis in Cell-Free Systems Dhyan Chandra and Dean G. Tang II. Detection of Non-Apoptotic Cell Death 6. Methods to Analyze Cellular Necroptosis Benchun Miao and Alexei Degterev 7. Detection of Cell Death by Autophagy Narasimman Gurusamy and Dipak K. Das III. Modifications of Apoptotic Proteins during Apoptosis 8. Methods to Analyze Transglutamination of Proteins Involved in Apoptosis Zoltan Nemes and Laszlo Fesus 9. Methods to Analyze S-nitrosylation of Proteins Involved in Apoptosis Neelam Azad, Anand Krishnan V. Iyer, and Yon Rojanasakul 10. Application of in vivo EPR for Tissue pO2 and Redox Measurements Nadeem Khan and Dipak K. Das IV. Analysis of the Function of Major Regulators of Apoptosis/Cell Survival 11. Assays to Measure p53-dependent and Independent Apoptosis Darren . Phillips, Sean P. Garrison, John R. Jeffers, and Gerard P. Zambetti 12. Measurement of Changes in Cdk2 and Cyclin O-Associated Kinase Activity in Apoptosis Ramon Roset and Gabriel Gil-Gomez 13. Fluorimetric Methods for Detection of Mitochondrial Membrane Permeabilization in Apoptosis Soumya SinhaRoy and Gyorgy Hajnoczky 14. Regulation of Apoptosis by the Unfolded Protein Response Andrew Fribley, Kezhong Zhang, and Randal Kaufman 15. Detection of Uncoupling Protein-2 (UCP-2) as a Mitochondrial Modulator of Apoptosis Zoltan Derdak, Tamako A. Garcia and Gyorgy Baffy 16. A Multiple Approach to Analyzing the Role of MicroRNAs in Apoptosis Riccardo Spizzo and George Calin 17. Assessment of Apoptotic Cell Phagocytosis by Macrophages Kathleen A. McPhilips and Lars-Peter Erwig V. Analysis of Apoptosis in Different Organs 18. Detection of Apoptosis in Mammalian Development Lin Lin, Carlos Penalosa, Yixia Ye, Richard A. Lockshin, and Zahra Zakeri 19. Detection of Apoptosis in the Central Nervous System Youngsoo Lee and Peter J. McKinnon 20. Genetic Mapping of Anti-apoptosis Pathways in Myeloid Progenitor Cells Dan Liu and Zhou Songyang 21. Analysis of Apoptosis in Isolated Primary Cardiac Myocytes Adel Mandl, Ambrus Toth, and Peter Erhardt 22. Cell Death in Myoblasts and Muscles Lawrence M. Schwartz, Zhengliang Gao, Christine Brown, Sangram S. Parelkar, and Honor Glenn VI. Analysis of Apoptosis in Model Organisms 23. Reliable Method for Detection of Programmed Cell Death in Yeasts Xinchen Teng and J. Marie Hardwick 24. Detection of Cell Death in Drosophila Kimberly McCall, Jeanne S. Peterson, and Tracy L. Pritchett 25. Detecting Apoptotic Cells and Monitoring Their Clearance in the Nematode Caenorhabditis elegans Nan Lu, Xiaomeng Yu, Xiangwei He, and Zheng Zhou 26. Detection of Herpes Simplex Virus Dependent Apoptosis Christopher R. Cotter and John A. Blaho

The endoplasmic reticulum (ER) is a multifunctional organelle responsible for the synthesis and folding of proteins as well as calcium storage and signaling. Perturbations of ER function cause ER stress leading to the unfolded protein response (UPR), which includes inhibition of protein synthesis, protein refolding and clearance of misfolded proteins. The UPR aims at restoring cellular homeostasis, however, prolonged ER stress can trigger apoptosis. ER stress-induced apoptosis has been implicated in the pathogenesis of various diseases such as brain ischemia/reperfusion, neurodegeneration, diabetes and, most recently, myocardial infarction and heart failure. Initial events leading to UPR and apoptosis in the heart include protein oxidation and disturbed calcium handling upon ischemia/reperfusion, and forced protein synthesis during cardiac hypertrophy. While XBP-1 and ATF6-mediated induction of ER chaperones seems to protect the heart from ischemia/reperfusion injury, the PERK/ATF4/CHOP branch of the UPR might transmit proapoptotic signals. The precise mechanism of ER stress-induced cardiomyocyte apoptosis remains elusive, however, recent data suggest that the mitochondrial apoptotic machinery is recruited through the upregulation of Puma, a proapoptotic member of the Bcl-2 family. Importantly, suppression of Puma activity prevented both ER stress and ischemia/reperfusion-induced cardiomyocyte loss, highlighting the ER stress pathways as potential therapeutic targets in cardiovascular diseases.

The inhibition of glycogen synthase kinase-3beta (GSK-3beta) via phosphorylation by Akt or protein kinase C (PKC), or the activation of mitogen-activated protein kinase (MAPK) cascades can play a pivotal role in left ventricular remodeling following myocardial infarction. Our previous data showed that MAPK and phosphatidylinositol-3-kinase/Akt pathways could be modulated by poly(ADP-ribose)polymerase (PARP) inhibition raising the possibility that cardiac hypertrophic signaling responses may be favorably influenced by PARP inhibitors. A novel PARP inhibitor (L-2286) was tested in a rat model of chronic heart failure following isoproterenol-induced myocardial infarction. Subsequently, cardiac hypertrophy and interstitial collagen deposition were assessed; additionally, mitochondrial enzyme activity and the phosphorylation state of GSK-3beta, Akt, PKC and MAPK cascades were monitored. PARP inhibitor (L-2286) treatment significantly reduced the progression of postinfarction heart failure attenuating cardiac hypertrophy and interstitial fibrosis, and preserving the integrity of respiratory complexes. More importantly, L-2286 repressed the hypertrophy-associated increased phosphorylation of panPKC, PKC alpha/betaII, PKC delta and PKC epsilon, which could be responsible for the activation of the antihypertrophic GSK-3beta. This work provides the first evidence that PARP inhibition beneficially modulates the PKC/GSK-3beta intracellular signaling pathway in a rat model of chronic heart failure identifying a novel drug target to treat heart failure.

The p53-upregulated modulator of apoptosis (Puma), a BH3-only member of the Bcl-2 protein family, is required for p53-dependent and -independent forms of apoptosis and has been implicated in the pathomechanism of several diseases, including cancer, acquired immunodeficiency syndrome, and ischemic brain disease. The role of Puma in cardiomyocyte death, however, has not been analyzed. On the basis of the ability of Puma to integrate diverse cell death stimuli, we hypothesized that Puma might be critical for cardiomyocyte death upon ischemia-reperfusion (I/R) of the heart. Here we show that hypoxia-reoxygenation of isolated cardiomyocytes led to an increase in Puma mRNA and protein levels. Moreover, if Puma was delivered by an adenoviral construct, cardiomyocytes died by apoptosis. Under ATP-depleted conditions, however, Puma overexpression primarily induced necrosis, suggesting that Puma is involved in the development of both types of cell death. Consistent with these findings, targeted deletion of Puma in a mouse model attenuated both apoptosis and necrosis. When the Langendorff ex vivo I/R model was used, infarcts were approximately 50% smaller in Puma(-/-) than in wild-type mice. As a result, after I/R, cardiac function was significantly better preserved in Puma(-/-) mice than in their wild-type littermates. Our study thus establishes Puma as an essential mediator of cardiomyocyte death upon I/R injury and offers a novel therapeutic target to limit cell loss in ischemic heart disease.

Objective: Puma (p53-upregulated modulator of apoptosis), a proapoptotic BH3-only member of the Bcl-2 protein family, has been implicated in the pathomechanism of several diseases, including cancer, AIDS, and ischemic brain disease. We have recently shown that Puma is required for cardiac cell death upon ischemia/reperfusion of mouse hearts. Since ischemia/reperfusion is also associated with endoplasmic reticulum (ER) stress, in the present study we investigated whether Puma contributes to the ER stress-dependent component of cardiomyocyte apoptosis.Methods: Primary cultures of rat and mouse neonatal cardiomyocytes were treated with 3 mu M thapsigargin or 100 ng mL(-1) tunicamycin. Puma levels were suppressed by adenoviral delivery of shRNA or targeted deletion of the puma gene. Puma expression was detected by RT-PCR and Western blotting. Apoptosis was assessed by TUNEL assay, caspase-3 cleavage, and cytochrome c release.Results: We have shown that in rat neonatal cardiac myocytes, thapsigargin or tunicamycin treatment led to ER-stress, transcriptional upregulation of Puma, and apoptosis. Most importantly, cardiac myocytes acquired resistance to ER stress-induced apoptosis if Puma expression was downregulated by adenoviral delivery of shRNA or eliminated by targeted deletion in knockout mice.Conclusion: Taken together, our data indicate that Puma is a critical component of ER stress-induced apoptosis in cardiac myocytes, and inhibition of Puma activity may be used to treat cardiac infarcts or prevent heart failure by blocking ER stress-induced apoptosis. (c) 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

MDM2 is an E3 ubiquitin ligase that regulates the proteasomal degradation and activity of proteins involved in cell growth and apoptosis, including the tumor suppressors p53 and retinoblastoma and the transcription factor E2F1. Although the effect of several MDM2 targets on cardiomyocyte survival and hypertrophy has already been investigated, the role of MDM2 in these processes has not yet been established. We have, therefore, analyzed the effect of overexpression as well as inhibition of MDM2 on cardiac ischemia/reperfusion injury and hypertrophy. Here we show that isolated cardiac myocytes overexpressing MDM2 acquired resistance to hypoxia/reoxygenation-induced cell death. Conversely, inactivation of MDM2 by a peptide inhibitor resulted in elevated p53 levels and promoted hypoxia/reoxygenation-induced apoptosis. Consistent with this, decreased expression of MDM2 in a genetic mouse model was accompanied by reduced functional recovery of the left ventricles determined with the Langendorff ex vivo model of ischemia/reperfusion. In contrast to cell survival, cell hypertrophy induced by the alpha-agonists phenylephrine or endothelin-1 was inhibited by MDM2 overexpression. Collectively, our studies indicate that MDM2 promotes survival and attenuates hypertrophy of cardiac myocytes. This differential regulation of cell growth and cell survival is unique, because most other survival factors are prohypertrophic. MDM2, therefore, might be a potential therapeutic target to down-regulate both cell death and pathologic hypertrophy during remodeling upon cardiac infarction. In addition, our data also suggest that cancer treatments with MDM2 inhibitors to reactivate p53 may have adverse cardiac side effects by promoting cardiomyocyte death.

During ischemia-reperfusion, reactive oxygen species are generated along the mitochondrial respiratory chain and induce lipid peroxidation, protein oxidation and DNA damage. Single-strand DNA breaks are the most potent activators of poly(ADP-ribose) polymerase (PARP); prolonged action of PARP culminates in intracellular oxidized nicotinamide adenine dinucleotide (NAD(+)) and ATP depletion. The integrity of cellular components and the myocardial energy metabolism can be preserved by using PARP inhibitors under conditions of ischemia and reperfusion. Oxidative stress is capable of activating the phosphoinositol-3-kinase-Akt/protein kinase B signalling pathway, which is further enhanced if treated with PARP inhibitors. Akt, in turn, promotes the survival of cardiomyocytes by inhibiting apoptotis, and causing metabolic adjustment and vasodilation in the jeopardized myocardium.

Molecular mechanisms of cardioprotection afforded by modified mexiletine compounds were investigated during ischemia-reperfusion (IR) in Langendorff perfused hearts. Rat hearts were subjected to a global 25 min ischemia followed by reperfusion, either untreated or treated with mexiletine, or three substituted mexiletine derivates (5 muM). A modified mexiletine derivative (H-2693) promoted best the recovery of myocardial energy metabolism (assessed by (31)P NMR spectroscopy) compared to untreated and mexiletine-treated hearts. H-2693 also preserved cardiac contractile function and attenuated the IR-induced lipid peroxidation (TBARS formation) and protein oxidation (carbonyl content). Western blot revealed that H-2693 propagated the phosphorylation of Akt (activation) and its downstream substrate glycogen synthase kinase-3beta (GSK-3beta, inactivation) compared to untreated IR. Parallel treatment with the phosphatidylinositol-3-kinase (upstream activator of Akt) inhibitor wortmannin (100 nM) abolished the beneficial effects of H-2693 on energetics and function, and reduced Akt and GSK-3beta phosphorylation. As a result of the antiapoptotic impacts of Akt activation, H-2693 decreased caspase-3 activity, which was neutralized by wortmannin. Here we first demonstrated that a free radical-entrapping compound could activate the prosurvival Akt pathway beyond its proven ability to scavenge reactive oxygen species. In conclusion, the favorable influence of H-2693 on signaling events during IR may have considerably contributed to its cardioprotective effect.

Kutatasaink soran kiserletes szivelegtelenseg modellekben vizsgalatuk a poli(ADP-riboz) polimeraz (PARP) gatlok hatasat. Vizsgalatainkkal igazoltuk, hogy PARP-gatlok merseklik a postinfarctusos remodelinget es vedenek a szivelegtelenseg kialakulasaval szemben. Elsőkent igazoltuk, hogy a PARP-gatlok jelentős hatassal birnak a szivelegtelenseg pathomechanizmusaban alapvető szerepet jatszo jelatviteli utakra is. PARP-gatlas fokozta a tulelest segitő, ugyanakkor csokkentette bizonyos maladaptiv jelatviteli utak aktivitasat. Mindezek kovetkezteben a PARP-gatloval kezelt szivek mind funkcionalisan, mind strukturalisan lenyegesen intaktabbak voltak, mint a kezeletlen szivek. Raadasul az ACE-gatlo enalaprillal osszehasonitva a PARP-gatlo kezeles hatekonyabbnak bizonyult a myocardialis remodeling kivedeseben postinfarktusos szivelegtelenseg modellben. Fiatal spontan hipertenziv patkanyokban a PARP-gatloknak a szivizom hypertrophia kialakulasaval szembeni vedő hatasat igazoltuk. Ezen eredmenyeink publikalasra kerultek mar, illetve egyesek meg publikalas alatt allnak. Emellett idős spontan hipertenziv patkanyokban a pangasos szivelegtelenseg kialakulasaval szemben is kifejezett vedő hatast mutattak a PARP-gatlok, az allatok tuleleset is javitotta a kezeles. Ezen adatain meg reszben feldolgozasra varnak. Doxorubicin kezeles altal kivaltott cardiomyopathia modellben vegzett vizsgalatunkbol szarmazo mintak meg feldolgozas alatt allnak. | The effect of poly(ADP-ribose) polymerase (PARP) inhibitors was studied in various experimental heart failure models. We have demonstrated that PARP-inhibitors can moderate the postinfarction myocardial remodeling and can protect against the development of heart failure. We have firstly proved that PARP-inhibitors have a significant effect on signal transduction pathways which play a central role in the development of heart failure. PARP-inhibition activated the prosurvival signal transduction pathways and blocked the activity of several maladaptive signal transduction pathways. Due to these effects, hearts treated with PARP-inhibitors showed better functional and structural features compared to untreated hearts. In addition, PARP-inhibition was more effective against the development of myocardial remodeling in our postinfarction heart failure model compared to ACE-inhibition. In young spontaneous hypertensive rats the protective effect of PARP-inhibitors against the development of myocardial hypertrophy was demonstrated. These results were already published and several data are under publication. Moreover, in adult spontaneous hypertensive rats PARP-inhibition protects against the transition from hypertrophic cardiomyopathy to decompensated heart failure. These results will be published shortly. Finally, tissue samples derived from a toxic (doxorubicin-induced) cardiomyopathy model are yet under measurements.