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Mechanism of Cell Death in Cardiac Myocytes Exposed to Doxorubicin

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Title:
Mechanism of Cell Death in Cardiac Myocytes Exposed to Doxorubicin
Creator:
FRANCO, IBETH ANDREA MARTINEZ ( Author, Primary )
Copyright Date:
2008

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Subjects / Keywords:
Apoptosis ( jstor )
Cell death ( jstor )
Cells ( jstor )
Death ( jstor )
DNA damage ( jstor )
Dosage ( jstor )
Memory interference ( jstor )
Mitochondria ( jstor )
Necrosis ( jstor )
Viability ( jstor )

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University of Florida
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University of Florida
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Copyright Ibeth Andrea Martinez Franco. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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8/31/2007
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658217928 ( OCLC )

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MECHANISM OF CELL DEATH IN CARDIAC MYOCYTES EXPOSED TO DOXORUBICIN By IBETH ANDREA MARTINEZ FRANCO A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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Copyright 2006 By Ibeth Andrea Martinez Franco

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iii ACKNOWLEDGMENTS I thank my committee members for their s upport. I thank Dr. David Julian for his guidance, support and encouragement through al l these years. I thank Michael McCoy for help with the statistical analysis of my resu lts. I also thank my pare nts for never failing to support and encourage my endless passi on for science and animal health.

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iv TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iii LIST OF TABLES.............................................................................................................vi LIST OF FIGURES..........................................................................................................vii ABSTRACT.....................................................................................................................viii INTRODUCTION...............................................................................................................1 Free Radicals as a Cause of Cardiac Damage..............................................................1 Necrosis and Apoptosis................................................................................................2 Mechanism of Doxorubicin Toxicity............................................................................2 Step 1: Increased ROS production........................................................................3 Step 2: Opening of the Mitochondri al Permeability Transition Pore....................4 Step 3: Mitochondrial DNA Damage....................................................................4 MATERIALS AND METHODS.........................................................................................7 Experimental Design and Pla nned Statistical Analyses...............................................7 Preparation of Doxorubicin and Z-VAD......................................................................8 Cell Culture...................................................................................................................8 Detection of Apoptosis Induced by Doxorubicin.........................................................9 Fluorescence Microscopy......................................................................................9 Membrane Permeability Assay............................................................................10 JC-1 Assay...........................................................................................................10 Bioenergetics Assay............................................................................................10 Measurement of Caspase 3 and 7 Activation......................................................11 RESULTS........................................................................................................................ ..12 Qualitative Observations.....................................................................................12 Quantitative Observations:..................................................................................18 DISCUSSION....................................................................................................................29 Does Doxorubicin Induce H9c2 Cardiac Myocyte Death?........................................29 Are Cells Dying through A poptosis or Necrosis?......................................................29

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v What Is the Mechanism through Which Cells Are Dying?........................................32 Free Radical Generation by Doxorubucin...........................................................32 MPTP Opening....................................................................................................32 Caspase Activation..............................................................................................33 Apoptosis Inhibitors............................................................................................34 PI as a Marker of Early Apoptosis..............................................................................35 Limitations..................................................................................................................36 In vitro vs. in vivo Experiments..........................................................................36 DMSO..................................................................................................................37 Conclusion..................................................................................................................37 LIST OF REFERENCES...................................................................................................39 BIOGRAPHICAL SKETCH.............................................................................................43

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vi TABLE Table page 1 96-well microplate set up. 11 doses of doxorubicin and doxorubicin + Z-VAD to which H9c2 cardiac myocytes were exposed during 6, 12 and 24 hours.................11

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vii LIST OF FIGURES Figure page 1 Effects of doxorubicin on the mito chondrial membrane potential and the mitochondrial permeability transition pore (MPTP)..................................................4 2 Damage produced to nuclear DNA fo llowing doxorubicin administration...............5 3 H9c2 cardiac myocytes exposed to doxorubicin during 24 hours and marked with AV and PI.........................................................................................................13 4 Fraction of cells positive for AV af ter 6 (A), 12 (B) and 24 (C) hours of exposure to doxorubicin or doxorubicin + Z-VAD..................................................20 5 Fraction of cells positive for PI after 6 (A) , 12 (B) and 24 (C) hours of exposure to doxorubicin or doxorubicin + Z-VAD.................................................................22 6 Fraction of cells positive to BPI after 6 (A), 12 (B) and 24 (C) hours of exposure to doxorubicin and doxorubicin + Z-VAD...............................................................23 7 Membrane potential after 6 (A), 12 (B) and 24 (C) hours of exposure to doxorubicin and doxorubicin + Z-VAD...................................................................24 8 Caspase 3 and 7 activities after 6 (A), 12 (B) and 24 (C) hours of exposure to doxorubicin and doxorubicin + Z-VAD...................................................................25 10 ATP availability after 6(A), 12 (B) and 24 (C) hours of exposure to doxorubicin and doxorubicin + Z-VAD.......................................................................................27

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viii Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science MECHANISM OF CELL DEATH IN CARDIAC MYOCYTES EXPOSED TO DOXORUBICIN By Ibeth Andrea Martinez Franco August 2006 Chair: David Julian Major Department: Zoology The long-term cardiac toxicity documente d after doxorubicin treatment limits its effectiveness as a chemotherapeutic agent. Apoptosis and necrosis have both been suggested as causes of cardi ac cellular death following doxor ubicin treatments. In this study, we exposed H9c2 cardiac myocytes to increasing doses of doxorubicin for 6, 12 or 24 hours. A group of H9c2 cardiac myocytes was pre-treated with the pancaspase inhibitor Z-VAD. Cells were assessed for vi ability, type of cell death, cellular energy availability, caspase activity and alterati ons in mitochondrial membrane potential. Apoptosis was mainly observed at low doxor ubicin doses and short exposure times, whereas necrosis was prevalent at highe r doxorubicin doses and prolonged exposure times. Cell counts indicated an increase in cell su rvival in the Z-VAD pretreated group. Cell viability assays and ce ll counts showed a dose depende nt increase in cell death. Activity of caspase 3/7 was increased at low doxorubicin doses, but had no effect at higher concentrations. Cellular ATP availabil ity decreased at prolonged exposure periods

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ix and at higher doxorubicin doses. These results suggest that apoptosis and necrosis together account as cell death mechanisms depending on the doxorubicin concentrations, that caspases are a key mediator of doxorubici n-induced apoptosis, and that the transition from apoptosis to necrosis may be due to ce llular energy availability. We also found that the fluorescent nuclear stain propidium iodide appears to be permeant through the cellular membrane but not the nuclear membrane duri ng early necrosis, whic h may prove to be a useful tool in identifying this stage of cell death.

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1 INTRODUCTION Doxorubicin is an anthracycline antib iotic obtained from the bacteria Streptomyces peucetius (Arcamone et al. 1969). It is used as therapeutic agent in the treatment of malignant lymphomas, acute leukemias, and so lid tumors such as neuroblastomas, bone marrow sarcomas and carcinomas of the breast , bladder and thyroid gland (Blum, Carter 1974, Wallace 2003). The use of cumulative doses of doxorubicin limits its clinical value because it has been implicated in the producti on of irreversible ca rdiac toxicity that consists of a decline of contractile function of the heart muscle, leading to an irreversible and life threatening congestive heart fa ilure (Doroshow 1991, Safra 2003, Wallace 2003). Currently, the best way to control for doxorubi cin side effects in patients undergoing chemotherapy is the frequent assessment of their cardiac function, the irregular administration of doxorubicin to prevent its accumulation within the cardiac myocytes, and using a combination of chemotherapeu tic drugs to reduce the dose of doxorubicin without decreasing the potency of the chemothe rapeutic treatment. Given the toxicity of doxorubicin, it is fundamental to establish how it causes dama ge to cardiac tissue and to determine the doses and times at which doxor ubicin becomes toxic to healthy cells. Free Radicals as a Cause of Cardiac Damage Previous studies have shown that mitochondr ia play a pivotal role in the death of cardiac myocytes and in the production of cardiac toxicity mediated by the excessive production of free radicals. Free radicals can interfere with normal cellular homeostasis, causing alterations in the electron transport chain, mitochondrial permeability transition

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2 pore proteins and DNA stability, triggering apoptotic cell deat h and ultimately weakening the overall performance of the heart muscle (Georgy et al. 2003, Katoch et al. 2003, Korichneva et al. 2003, Wallace 2003, Yamanaka et al. 2003, and Mizutani et al. 2005). Necrosis and Apoptosis In general, severely injured cells will di e by either apoptosis or necrosis. Features of cells dying by necrosis include swelling of cells, disruption of the membranes and lysis of the nuclear chromatin. Typi cally, a large group of cells are involved and their cellular contents are released into the extra-cellular space, i nducing a strong inflammatory reaction. In contrast, apoptosis i nvolves one cell at a time. It re fers specifically to certain morphologic features such as cell shrinkage , membrane blebbing, nuclear condensation and fragmentation to apoptotic bodies that then are digested by local cells, without the induction of an inflammatory response (Kerr et al. 1972). Several pathologies, such as ischemic myocardial infarction (Geng 1997), ir radiation and heat shock, seem to involve the necrosis and apoptosis processes simulta neously (Nakano et al. 1997, Saikumar et al. 1999). While apoptosis has been identified in diseases such as aplastic anemia, lupus erythematosus, multiple sclerosis and ulcerativ e colitis (Saikumar et al. 1999), necrosis has been identified in the cases of tubercul osis and acute tubula r necrosis, plus many others (Wicklow et al. 2006). Mechanism of Doxorubicin Toxicity Although the mechanism by which doxorubicin generates cardiac toxicity is not completely understood, several theories involvi ng different apoptotic death pathways, as well as necrosis, genetic and metabo lic factors, have been proposed.

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3 Step 1: Increased ROS production Once inside the mitochondria, the first step in the metabolism of doxorubicin is its univalent reduction to semiquinone. Semiquinone is a highly unstable free radical that can transfer unpaired electrons to an electron acceptor to ge nerate a more stable free radical. Doxorubicin carries the unpaired electron from th e electron donor to the electron acceptor to generate Reactive oxygen species (ROS), such as superoxide anion (O2 •–) and hydrogen peroxide (H2O2) (Green, Leeuwenburg 2002). This redox reaction is also accompanied by the release of iron and cooper from intracellular st ores. Doxorubicin and iron or copper can form complexes that convert O2 •– and H2O2 into the more potent hydroxyl radical (•OH-) (Myers 1998, Giafrianca 2004, Minotti 2004). NADH dehydrogenase is able to catalyze the reduc tive metabolism of doxor ubicin, and therefore several enzymes, including NADH dehydrogenize, ha ve been implicated in the degree of damage produced by doxorubicin (Wallace 2003). Consequently, mitochondrial metabolism, the overgeneration of ROS and the redox reaction that releases them, are considered the main mediators of the pathol ogies associated with doxorubicin toxicity. Doxorubicin acts as an oxidan t promoter inducing greater mitochondrial generation of oxidants that can interfere with the el ectron transport chai n and mitochondrial permeability transition pore stability, and da mage proteins and DNA (Georgy et al. 2003, Wallace 2003, Yamanaka 2003). The ultimate protein and DNA damage induced by doxorubicin may lead to cellular apoptosis. Therefore, following cu mulative dosages of doxorubicin, the increase in free radicals produc tion in the cellular mitochondria is able to induce an irreversible conge stive cardiac failure that can be life threatening because the heart is a post-mitotic tissue.

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4 MPTP Opening QuickTime and a decompressor are needed to see this picture. membrane potential collapse Doxorubicin Proapoptoti cproteins ROS productio n Cytosol Figure 1: Effects of doxorubicin on the mitochondrial membrane potential and the mitochondrial permeability transition pore (MPTP). Step 2: Opening of the Mitochondrial Permeability Transition Pore The mitochondrial membrane potential is created by proton pumping by the electron transport chain across the inner mitochondrial membrane. The production of ROS during doxorubicin redox cycling within th e mitochondria generates a lower proton pumping activity and a decrease in the inner mitochondria me mbrane potential, resulting in changes in the mitochondrial permeability tr ansition pore (MPTP). As a result, and as illustrated in Fig. 1, the mitochondrial response to the increase in ROS formation is represented by the release of pro-apoptotic proteins into the cytosol, initiating the pathway of apoptosis (Green, Leeuwenbur g 2002, Wallace et al. 2003, Minotti et al. 2004). Step 3: Mitochondrial DNA Damage Following doxorubicin exposure, mitochondrial damage is produced through a cycle in which generation of RO S, mitochondrial alterations and respiratory chain insults

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5 accumulate, eventually compromising the bio-en ergetic capacity of the mitochondria. As illustrated in Fig. 2, such cyclic oxidative damage due to ROS generation can also affect mitochondrial DNA (mtDNA), which, if not promptly repaired, may be transformed into mutations. These would affect the electron tr ansport chain by the overproduction of free radicals, ultimately causing damage to nuc lear DNA due to cellular oxidative stress (Lebrecht 2004, Hagen et al . 2002). Increased oxidan t production would thereby contribute to cardiomyopathy via necrosis or apoptosis depe nding on ATP levels. (Hagen et al. 2002). Damage to nDNA ROS formation mtDNA alterations Damage to respiratory chain Doxorubicin Apoptosis ATP (+) Necrosis ATP (-) Figure 2: Damage produced to nuclear DNA following doxorubicin administration The heart is a post-mitotic tissue and its cells depend on mitochondrial energy production for their proper function. Cardi ac myocytes typically experience oxidative stress due to the production of ROS duri ng normal mitochondrial metabolism (Katoch 2003, Korichneva et al., 2003). Furthermore, the post-mitotic nature of the cardiac myocytes also permits greater accumulati on of mitochondrial mutations and deletions (Fannin et al. 1999, Zarkovic et al. 2003). Cardiac tissue is especially susceptible to doxorubicin administration because the heart is physiologically less tolerant of free radicals as result of its low level of antioxidant activity (Yin et al. 1998).

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6 In this study, we used the H9c2 cardiac myocyte cell line to a ssess the response of mammalian cardiomyocytes to increasing doses of doxorubicin over time. We examined cellular viability through the analysis of cellular membrane inte grity, intracellular ATP availability, necrosis, apoptosis and apoptos is inhibition. We had two main hypotheses: 1) The effect of doxorubicin exposure is dos e dependent, in which more cells die at higher doxorubicin concentrati ons and at longer exposure tim es; and 2) cell death is exclusively via apoptosis at low doxorubicin concentratio ns and at short exposure periods, but that higher concentrations of doxorubicin or longer exposure periods cause cell death via necrosis. Our overall goal was to determin e the doxorubicin dosage (time and concentration) at which there is a transi tion between apoptosis and necrosis in H9c2 cardiac myocytes.

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7 MATERIALS AND METHODS Experimental Design and Pl anned Statistical Analyses H9c2 cells were exposed to 11 different concentrations of doxorubicin alone or doxorubicin with 110 M Z-VAD in 96-well multi-well plates (Table 1). Preliminary data was used to choose the appr opriate doxorubicin concentra tions that allowed us to measure morphological changes. 10 M was our starting dose and was logarithmically scaled to 20 M as our highest and 0.019 M as our lower exposure dose. Z-VAD is a pancaspase inhibitor commonly used to inhi bit cellular apoptosis. Doxorubicin and ZVAD were dissolved in DMSO at the appropr iate dilutions to accurately achieve the concentrations used in the treatments. The control group contained the equivalent DMSO amount as the 20 M doxorubicin group did. Cells were th en incubated at 37 C with 5% CO2 for 6, 12 and 24 hours, as described in ta ble 1. Statistical signi ficances in the cell death of H9c2 cardiac myocytes foll owing doxorubicin and doxorubicin+ Z-VAD exposures could then be determined by us ing a linear mixed effect ANOVA test. This model therefore contains three fixed effects: Treatment : H9c2 cells were exposed to doxor ubicin alone or to doxorubicin+ZVAD. Concentration : H9c2 cells were exposed to 11 clinically relevant doxorubicin concentrations or to 11 different doxorubicin concentrations +Z-VAD. Time : H9c2 cells were exposed to the different doses of doxorubicin or doxorubicin+Z-VAD for periods of either 6, 12 or 24 hours.

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8 Experiments were conducted in triplicate on different days during the course of 1 year. Different sub-clones of the H9c2 cells we re used, so the random effect of this model is assigned to the replicate multi-well plates. This model contains statistical analysis of each individual fixed effect as well as each interaction between the fixed effects on apoptotic outcome. This allowed us to test for effects of all the combinations between time, treatment and concentration, time and treatment and treatment and concentration. Preparation of Doxorubicin and Z-VAD Doxorubicin hydrochloride was purchased fr om MP Biomedicals Inc, OH. It was dissolved in DMSO at 10 mg/ml and caref ully mixed at 37 C for 20 minutes. The caspase inhibitor Z-Val-Ala-DL-Asp-fluor omethylketone (Z-VADFMK) was purchased from Promega and was dissolved at 50mg Z-VAD in 1ml of DMSO. Cell Culture H9c2 cells are a subclone of the origin al cell line derived from embryonic BD1X rat heart myoblast tissue. This cardiac myoc yte cell line was obtained from the American Type Culture Collection (ATCC), Manasses, VA. H9c2 cells were grown and maintained at 37 C and 5% CO2 in 250 ml tissue culture flasks containing 25 ml growth medium (Dulbecco’s modified Eagles medium s upplemented with 4mM glutamine, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, 10% fetal bovine serum (FBS), 1mM sodium pyruvate and 50 IU/ml penici llin combined with 50 M/ml Streptomycin. The cells were subcultured approximately every 3 days or when they reached 75% to 80% confluence. They were then detached from the flask by the addition of 5 ml 0.03% (w/v) trypsinEDTA. After 15 min, 7.2 ml of the cell suspension was gently pipetted out, diluted in

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9 fresh growth medium, and 100 L were placed into each of 72 wells of a black 96-well microplate. This plate was then incubated for 24 hours to allo w re-attachment of the cells. Detection of Apoptosis Induced by Doxorubicin Fluorescence Microscopy To identify apoptotic and necrotic ce lls in H9c2 cells following doxorubicin exposure, Annexin V and propidium iodide (P I) were utilized. Annexin V is a fluorescent FITC-conjugated probe that binds to the protein phosphatidyl se rine (PS), which in viable cells is located in the cyto solic surface of th e cell membrane. In cells undergoing early apoptotic processes, PS is tran slocated from the inner to th e outer part of the membrane, where Annexin V binds to it, marking the apoptotic cells by giving them a fluorescent green coloration (Van Engeland et al., 1998, Balasubramanian et al. 2001 and Gylys et al., 2004). PI is a red fluorescent dye that binds to nucleic acids. PI is membraneimpermeable in viable cells, but it can enter necrotic and late apopt otic cells which have permeable cell membranes that allow PI to enter the nuclei, labeling the cells bright red. When double-labeling with Annexin V and PI, h ealthy cells show no fl uorescence, purely apoptotic cells stain positive for Annexin V, necrotic cells show nuc lear PI uptake, and late apoptotic-necrotic cells show both Annexin V and PI labeling. Annexin V and PI were obtained from Molecular Probes, (Eugene, OR) as the Vybrant Apoptosis Assay Kit #3. After labeling, the cells were then visualized using a 484/15 nm excitation filter for Annexin V and a 555/15 nm excitation filter for PI. Each well was divided into four quadrants, a nd a digital image was obtained from each quadrant. Each of the four images for each we ll were later used to count cells positive for each fluorescent stain using the counter module of Image/J software (NIH).

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10 Membrane Permeability Assay Calcein-AM was used as a cellular marker to provide information about cellular viability. Calcein-AM enters the cytoplasm of healthy cells where the AM group is removed by endogenous esterases. The fluoresce nt product calcein can be identified by measuring the green fluorescence emission intensity. Dead cells with compromised cellular membranes lack sufficient esterase ac tivity to convert th e nonfluorescent caleinAM to calcein, and therefore the fluorescence intensity is proportional to the number of viable cells. Calcein-AM was purchased from Molecular Probes, applied as in the product manual, and the fluorescence was read at 490 nm excitation and 520 nm emission using a multimode microplate reader (Bio-Tek Instruments, Synergy SIAFRT). JC-1 Assay JC-1 is a dye that shows potential-depe ndent accumulation inside the mitochondria. The mitochondrial potential is represented by the production of a fluorescence emission change from green (525nm) to red (590nm). Depolarization of mitochondria is indicated by a reduction in the red:green fluorescence ratio (Mancini et al., 1997, Kulkarni et al.,1998). JC-1 was purchased from Molecular Probes and 7.5 L of a 10 mM stock solution prepared in DMSO was added to th e cells after doxorubici n exposure and the fluorescence was read at 525 nm and 590 nm us ing a multimode microplate reader (BioTek Instruments Synergy SIAFRT). Bioenergetics Assay We quantified ATP to evaluate the energetic status of cells after doxorubicin exposure. We utilized a comm ercial luciferase/luciferin reaction, in which luciferase catalyzes the reaction between oxyluciferin and luciferin. Th e light signal generated in this reaction is proportional to the amount of ATP pres ent and is indicative of

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11 metabolically active viable cells. The CellTiter-Glo reagent (Promega, WI) was added 1:1 with cells in the 96 well plate, shaken for 2 minutes at 500 rpm and incubated in the dark for 10 minutes. Luminescence was measured using a multimode microplate reader (Bio-Tek Instruments, Synergy SIAFRT). Measurement of Caspase 3 and 7 Activation Activities of caspase 3 and 7 were dete rmined with the Apo-One homogeneous Caspase 3/7 assay (Promega, WI). A 1:100 d ilution of the substrat e was prepared in buffer provided by the vendor, and 100 L was added to each well, then incubated for 12 hours. Fluorescence was measured in each well at an excitation wavelength of 485 nm using a using a multimode microplate reader (Bio-Tek Instruments Synergy SIAFRT). The amount of fluorescent generated is proportional to the amount of caspase 3/7 cleavage activity present. Table 1: 96-well microplate set up. 11 dos es of doxorubicin and doxorubicin + Z-VAD to which H9c2 cardiac myocytes were exposed during 6, 12 and 24 hours. 1 2 3 4 5 6 7 8 9 10 11 12 Control 6h 0.019 M Dox 0.039 M Dox 0.07 M Dox 0.156 M Dox 0.312 M Dox 0.625 M Dox 1.25 M Dox 2.5 M Dox 5 M Dox 10 M Dox 20 M Dox Z-VAD 12h 0.019 M Dox + Z-VAD 0.039 M Dox+ Z-VAD 0.078 M Dox+ Z-VAD 0.156 M Dox+ Z-VAD 0.312 M Dox+ Z-VAD 0.62 M Dox+ Z-VAD 1.25 M Dox+ Z-VAD 2.5 M Dox+ Z-VAD 5 M Dox+ Z-VAD 10 M Dox+ Z-VAD 20 M Dox+ Z-VAD Control 24h 0.019 M Dox 0.039 M Dox 0.078 M Dox 0.156 M Dox 0.312 M Dox 0.625 M Dox 1.25 M Dox 2.5 M Dox 5 M Dox 10 M Dox 20 M Dox Z-VAD 6h 0.019 M Dox+ Z-VAD 0.039 M Dox+ Z-VAD 0.078 M Dox+ Z-VAD 0.156 M Dox+ Z-VAD 0.312 M Dox+ Z-VAD 0.62 M Dox+ Z-VAD 1.25 M Dox+ Z-VAD 2.5 M Dox+Z -VAD 5 M Dox+Z -VAD 10 M Dox+ Z-VAD 20 M Dox+Z -VAD Control 12h 0.019 M Dox 0.039 M Dox 0.078 M Dox 0.156 M Dox 0.312 M Dox 0.625 M Dox 1.25 M Dox 2.5 M Dox 5 M Dox 10 M Dox 20 M Dox Z-VAD 24h 0.019 M Dox+ Z-VAD 0.039 M Dox+ Z-VAD 0.078 M Dox+ Z-VAD 0.156 M Dox+ Z-VAD 0.312 M Dox+ Z-VAD 0.62 M Dox+ Z-VAD 1.25 M Dox+ Z-VAD 2.5 M Dox+ Z-VAD 5 M Dox+ Z-VAD 10 M Dox+ Z-VAD 20 M Dox+ Z-VAD

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12 RESULTS Fluorescence Microscopy After 6, 12 and 24 hours of doxorubicin treatme nts, H9c2 cells were exposed to Annexin V (AV) and propidium iodide (PI), observed and photographed under the microscope, and qualitative and quantitative observations were pe rformed. Cell counts were done according to the fluorescence co lor: 1) green fluorescence of AV labeling, which indicates apoptosis, 2) bright red nuclear staining of PI-positive nuclei (bright PI, or BPI), indicating necrosis; 3) light PI labeling of the cytoplasm, which we propose represents early necrosis, and 4) simultaneous green a nd bright red double staining (positive for both AV and PI), indica ting necrosis or late apoptosis. Qualitative Observations Due to a low level of normally occurring “background” apoptosis, which is typical of cultured cells, the cells used as negative control sometimes showed a slight labeling by AV and PI dyes, represented by green staining of the cell membrane and red staining of the nuclei (Fig. 3a and 3b). In cells that had been exposed to 0.019 M doxorubicin for 24 hours, there was little dye labeling although, as seen in Fig. 3c and 3d, some cells were positive for AV and PI, while another cell’s nuclei has been stained exclusively with PI. When cells were exposed to 0.039 M doxorubicin, a few more cells were labeled by AV and some of the cells started to show a mo re defined uptake of PI, characterized by red labeling of the nuclei and of AV in the memb rane (Fig. 3e and 3f). Cells that were

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13 exposed to 0.078 M doxorubicin for 24 hours showed AV labeling along with very bright nucleic PI labeling (Fig. 3g). A: 24 HOUR EXPERIMENTS (0-0.039M Dox) Figure 3 : H9c2 cardiac myocytes exposed to doxorubicin during 24 hours and marked with AV and PI.

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14 B: 24 HOUR EXPERIMENTS (0.078-0.312M Dox g. 0.078 M AV h. 0.078 M PI i. 0.156 M AV j. 0.156 M PI k. 0.312 M AV l. 0.312 M AV Figure 3. Continued

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15 C: 24 HOUR EXPERIMENTS (0.625-2.5M Dox) m. 0.625M AV n. 0.625M PI o. 1. 25M AV p. 1.25M PI q. 2.5M AV r. 2.5M PI Figure 3 Continued

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16 D: 24 HOUR EXPERIMENTS (5-20M Dox) s. 5 M AV t. 5 M PI u. 10 M AV v. 10 M PI w. 20 M AV x. 20 M AV Figure 3 Continued.

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17 At doxorubicin concentrations of 0.156 M and 0.312 M, the number of PI positive cells dramatically increases. However, the vast majority of these PI-positive cells showed cytoplasmic PI labeling with the nuclei remain ing free of dye (Fig. 3j and 3l). At these doses, there is an increase in number of AV-positive cells (Fig. 3l and 3k). When cells were exposed to 0.625 and 1.25 M doxorubicin, they showed a leaf-like appearance of the AV binding, and some of these AV-positive cel ls also showed nuclear labeling by PI (Fig. 3m and 3o). This is characteristic of la te apoptosis. Other cells that are positive for cytoplasmic PI uptake also started to show th e very bright nuclear PI labeling (3Bn and 3p). At 2.5, 5 and 10 M doxorubicin exposure, not only did numerous cells label with either AV or PI dye, but many cells were la beled with both dyes simultaneously (Fig 3q, 3s and 3u). Some cells also showed both AV labeling and dim cytoplasmic PI labeling. At these doxorubicin concentrations some cells morphology changed from elongated to rounded, demonstrating the characteristics of late apoptosis, while others maintain the normal architecture of a cardiac myoc yte (Fig 3t, 3u and 3v). At 20 M doxorubicin, the highest concentration used in our experiments, the cells showed strong cellular double staining; numerous cells had PI labeling of cytoplasm and nuclei, and many others were characterized by very bright PI staining in their nuclei. Significant blebbing and loss of cardiac cell architecture is pr esent as well as some cellula r bursting (Fig. 3w and 3x). H9c2 cardiac myocytes exposed to d oxorubicin + Z-VAD qualit atively showed a similar progression in the patt ern of AV and PI labeling as cells exposed solely to doxorubicin (these data are not shown because the cellular morphol ogical changes were similar between the two treatment groups). At low doxorubicin concentrations (0.019 M

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18 to 0.62 M), few cells labeled positive for AV and none were labeled by PI. As the dose of doxorubicin increased, nuclear PI labeli ng increased, and AV s howed binding to the outer cellular membrane. At th e highest doses of doxorubicin (5 M, 10 M and 20 M), a considerable amount of the cells labeled positively for both AV and PI simultaneously. Cellular morphological changes such as bl ebbing and lose of normal cardiac myocyte morphology were also observed. Quantitative Observations: In the absence of Z-VAD, H9c2 cardiac myocytes counts showed that apoptotic and necrotic cell death con tinuously increases as the dos e of doxorubicin concentration increases. However, apoptotic death is the main mechanism of death at low doxorubicin concentrations and necrosis seems to be mo re prevalent at higher doses. Furthermore, addition of Z-VAD significantly decreases the number of ce lls undergoing apoptosis at all doses and times based on cellular counting. Annexin V : Fig. 4 A and B illustrate that after 6 and 12 hours of doxorubicin treatment, pre-treating cells with Z-VAD cons istently lowered the number of cells that were positive for AV, regardless of doxor ubicin concentration. After 12 hours of treatment, there is a notable increase in number of AV-positive cells at high doxorubicin doses (10 and 20 M), which is substantially redu ced in cells treated with Z-VAD (Fig.4B). Following 24 hours of doxorubicin treatment (Fig. 4C), the number of AVpositive cells rises as the dose of doxorubicin increases. When cells were exposed for 24 hours to both doxorubicin and Z-VAD, the num ber of AV-positive ce lls was generally lower than cells exposed to doxor ubicin alone. This pattern is particularly clear at the highest doxorubicin concentra tion (5, 10 and 20M), at whic h concentrations treatment

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19 and time showed a significant effect on apoptosis outcome (P<0.0001, <0.0001 and 0.0007 respectively). The interaction between co ncentration and time wa s also significant (P <0.0001), as well as the interactions between treatment and time (P=0.044) and concentration and treatment (P<0.0001). The in teraction between all of the three fixed factors all together also s howed a significant effect on apoptosis outcome (P<0.0001). Propidium Iodide : After 6 hours of doxorubicin expos ure, the cells that did not receive a pre-treatment with Z-VAD showed a marked increase in PI labeling at doxorubicin concentrations of 0.039 M and 0.078 M. At higher doxorubicin concentrations, there was no further increase in the number of PI-positive cells (Fig. 5 A). Addition of Z-VAD significantly reduced the number of PI-positive cells, which was especially evident at intermediate doxor ubicin concentrations. Following 12 hours of doxorubicin treatment, the two lowest doxor ubicin concentrations showed few PIpositive cells. At 0.078 M doxorubicin and higher, the cel ls without Z-VAD treatment showed a rapid dose-dependent increase in PI labeling, whereas ce lls that were pretreated with Z-VAD do not st art to show a significant uptake of the dye until the doxorubicin dose has reached 10 M (Fig. 5B). Following 24 hours of doxorubicin treatment and up to 2.5 M the number of Z-VAD pre-treated cells was constantly higher than the number of Z-VAD untreated cells, but at higher doses this relationship becomes inverted (Fig. 5C). The statistical analysis of this findings show ed that the time of exposure, treatment and doxorubicin concentrat ion had a significant effect on apoptosis outcome (P<0.0001), as well as their possi ble interactions (P <0.0001), with the exception of the interaction between concen tration and time, which did not show a significant effect on PI labeling.

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20 A. 0 0.2 0.4 0.6 0.8 1 0.010.1110100 Doxorubicin ConcentrationFraction of cells after 6h Dox Dox+Z-VAD B. 0 0.2 0.4 0.6 0.8 1 0.010.1110100 Doxorubicin concentrationFraction of cells after 12h Dox Dox+Z-VAD C. 0 0.2 0.4 0.6 0.8 1 0.010.1110100 Doxorubicin concentrationFaction of cells after 24hDox Dox+Zvad Figure 4: Fraction of cells positive for AV after 6 (A), 12 (B) and 24 (C) hours of exposure to doxorubicin or doxorubicin + Z-VAD.

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21 We also specifically counted cells that were positive for brig ht PI (BPI) labeling, which indicates nuclear permeability to the dye . As shown in Fig. 6 A, B, there is no clear pattern in BPI labeling. But as seen in Fig 6C, after 24 hours of exposure, BPI labeling appeared to be dependent on the doxorubicin concentration. Time of exposure, treatment, and concentration ha d an effect on BPI counts, si nce all of these factors and the possible interactions were st atistically signifi cant (P<0.0001). JC-1 : Mitochondrial membrane potential wa s measured using the JC1 assay (Figures 7A, B and C). Statistically, th ere was a significant effect of doxorubicin concentration on alteration of the mito chondrial membrane potential (P<0.0017). However, this direction of the effect increa sed red:green ratio) is opposite to that which was expected because it would seem to i ndicate mitochondrial membrane potential was shown to increase at the highest doxorubicin concentrations. Caspase 3/7 : Duration of doxorubicin exposure had a statistically significant effect (P<0.0001) on the activity of caspase 3 and 7 at specific doxorubicin concentrations. However, Z-VAD treatment and overall doxor ubicin concentration were not found to have a significant effect on caspase 3 and 7 activity outcome (Fig. 8 A, B and C). Calcein-AM : When evaluating membrane integrity as a marker of cell viability by the use of the calcein assay (Figures 9 A,B and C), doxorubi cin concentration (P<0.0001), duration of exposure, and Z-VAD treatment (P<0.0001) all have significant effects on cell viability. The dose response effect of doxorubi cin is particularly well demonstrated in Fig 9C, which shows a dose de pendent decrease in cell viability after 24 hours of doxorubicin exposure.

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22 A. 0 0.5 1 1.5 2 2.5 3 3.5 4 0.010.1110100 Doxorubicin concentrationFraction of cells after 6hDox Dox+Zvad B. 0 0.5 1 1.5 2 2.5 3 3.5 4 0.010.1110100 Doxorubicin concentrationFraction of cells after 12hDox Dox+Z-VAD C. 0 0.5 1 1.5 2 2.5 3 3.5 4 0.010.1110100 Doxorubicin concentrationFraction of cells after 24hDox Dox+Z-VAD Figure 5: Fraction of cells positive for PI after 6 (A) , 12 (B) and 24 (C) hours of exposure to doxorubicin or doxorubicin + Z-VAD.

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23 A. -0.1 0 0.1 0.2 0.3 0.4 0.5 0.010.1110100 Doxorubicin concentrationFraction of cells after 6hDox Dox+Z-VAD B. -0.1 0 0.1 0.2 0.3 0.4 0.5 0.010.1110100 Doxorubicin concentrationFraction of cells after 12h Dox Dox+Z-VAD C 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.010.1110100 Doxorubicin concentrationFraction of cells after 24hDox Dox+Z-VAD Figure 6: Fraction of cells positive to BPI after 6 (A), 12 (B) and 24 (C) hours of exposure to doxorubicin a nd doxorubicin + Z-VAD.

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24 A. 0.5 0.7 0.9 1.1 1.3 1.5 1.7 1.9 2.1 2.3 2.5 0.010.1110100 Doxorubicin concentrationMitochondrial potential after 6hDox Dox+Z-VAD B. 0.5 0.7 0.9 1.1 1.3 1.5 1.7 1.9 2.1 2.3 2.5 0.010.1110100 Doxorubicin concentrationMembrane Potential after12h Dox Dox+Z-VAD C 0.5 0.7 0.9 1.1 1.3 1.5 1.7 1.9 2.1 2.3 2.5 0.010.1110100 Doxorubicin concentrationMembrane Potential after 24h Dox Dox+Z-VAD Figure 7: Membrane potential after 6 (A), 12 (B) and 24 (C) hours of exposure to doxorubicin and doxorubicin + Z-VAD .

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25 A. 0 2 4 6 8 10 12 14 16 18 0.010.1110100 Doxorubicin concentrationCaspase 3,7 activity after 6h Dox Dox+Z-VADB. 0 2 4 6 8 10 12 14 16 18 0.010.1110100 Doxorubicin concentrationCaspase 3,7 activity after 12h Dox Dox+Z-VADC. 0 2 4 6 8 10 12 14 16 18 0.010.1110100 Doxorubicin concentrationCaspase 3,7 activities after 24h Dox Dox+Z-VAD Figure 8: Caspase 3 and 7 activ ities after 6 (A), 12 (B) a nd 24 (C) hours of exposure to doxorubicin and doxorubicin + Z-VAD.

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26 A. 0.2 0.4 0.6 0.8 1 1.2 1.4 0.010.1110100 Doxorubicin concentrationCalcein AM cell Viability after 6h Dox Dox+Z-VADB 0.2 0.4 0.6 0.8 1 1.2 1.4 0.010.1110100 Doxorubicin concentrationCalcein cell viability after 12h Dox Dox+Z-VADC. 0.2 0.4 0.6 0.8 1 1.2 1.4 0.010.1110100 Doxorubicin concentrationCalcein cell viability after 24h Dox Dox+Z-VAD Figure 9: Calcein cell viability after 6 (A ), 12 (B) and 24 (C) hours of exposure to doxorubicin and doxorubicin + Z-VAD.

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27 A. 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0.010.1110100 Doxorubicin concentrationATP avaliability after 6h Dox Dox+Z-VAD B. 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 0.010.1110100 Doxorubicin concentrationATP avaliability after 12h Dox Dox+Z-VAD C. 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0.010.1110100 Doxorubicin concentrationATP avaliability after 24hDox Dox+Z-VAD Figure 10: ATP availability after 6(A) , 12 (B) and 24 (C) hours of exposure to doxorubicin and doxorubicin + Z-VAD.

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28 ATP : We estimated cell viability and energetic status with an ATP assay. After 6 and 12 hours of doxorubicin exposure; the am ount of cellular ATP increases as doxorubicin dose increases, opposite to what w ould be expected. ATP availability was consistently less in the cells that were pretre ated with Z-VAD than in the cells that were not (Fig. 10A and B). In contrast, followi ng 24 hours of doxorubicin treatment, cells that were pre treated with Z-VAD had a higher AT P reading than cells that were left untreated; and it is also noticeable how as the doses of doxorubicin increase, ATP availability decrease (Fig. 10C). Doxorubici n concentration, duration of exposure, and ZVAD treatment all had a statis tically significant effect on availability of cellular ATP (P<0.0001, 0.0404 and <0.0001, respectively).

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29 DISCUSSION Does Doxorubicin Induce H9c2 Cardiac Myocyte Death? In our study we used the calcein-AM assa y to show that doxorubicin is able to cause death of the H9c2 cardiac myocytes. This assay measures cellular viability independently of whether the cells die via apop tosis or necrosis. The results of our assay showed that after 24 hours of doxorubicin e xposure, cellular viability dramatically decreases and that the decrease is also dos e dependent. This differed from the 6 and 12 hour time periods, in which the fluorescence si gnal doesn’t change significantly. This may be because at shorter exposure times; ev en at higher doxorubicin concentrations, the majority of the cells are still sufficiently aliv e to be able to cleave calcein-AM in to the fluorescent calcein product. Hong et al. (2003) have demonstrated that H9c2 rat ventricular cells exposed to a transient oxida tive insult show a progressive apoptotic cell death. They estimated cell viability by th e use of MTT, ELISA and dUTP nick end labeling (TUNEL) assays to determine ce ll death through the quantification of DNA fragmentation. Green and Leeuwenburgh (2002) also found a decr ease in cellular viability in H9c2 cardiac myocytes by the use of MTT and calcein-AM assays following low doses of doxorubicin exposures. Are Cells Dying through Apoptosis or Necrosis? Apoptosis has been extensively implicat ed in the induction of cardiotoxicity following cumulative doses of doxorubicin, but its exact mechanism of action remains unclear. In vitro experiments have sh own how ROS production, disruption of the

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30 mitochondrial membrane potential, and caspase activation lead to a poptosis in neonatal rat cardiomyocytes as well as in Jurkat cel ls (Gamen et al. 2000, Wang et al. 2004). In vivo and in vitro experiments using mice have also demonstrated that, after 1 hour of doxorubicin exposure, murine oocytes undert ake the morphological and biochemical changes consistent with apoptosis through free radical generation, DNA damage and lipid peroxidaton (Juriscova et al. 2000). On the other hand, it has also been postulated that necrosis may play a role in cellular death as a consequence of doxorubicin treatment, as in the case of the human pancreatic cell lin e L3.6, where besides apoptosis, necrosis was also found as cause of cellular death (Gervas oni et al. 2004). Sugimoto et al. (2002) also suggested necrosis following long term doxorubicin applications, where necrosis accounted for 80% of th e total cell death. Our experiments showed that H9c2 card iac myocyte cells died by both apoptosis and necrosis. At low doxorubicin concentrations and short exposure times, cell death was mostly caused by apoptosis, whereas at long er exposure durations and higher doxorubicin concentrations, the amount of cells under going necrosis increased. To identify and differentiate apoptotic and necr otic cells, we used Annexin V and PI. In addition, necrosis and apoptosis cells were differentiated by obs ervation of cell shape and cell structural features, which are very di stinct in necrosis and a poptosis. At higher doxorubicin concentrations, necrosis was characterized by cells that appeared swollen and had started to lift from the surface of the culture well, th at had a rounded cytoplas m, and that had PI labeling of the nucleus. At intermediate doxor ubicin concentrations and longer exposure times, a high percentage of the cells were la beled simultaneously with Annexin V and PI. This double staining has also b een reported by, for example, Fabbri et al. (2006) in the

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31 bladder cancer cell line HT 1376 after docetax el treatment, and it is attributed to membrane damage in a formerly apoptotic cel l. Based on the results of our experiments, we believe that at intermediate and hi gh doxorubicin concentrations, the cells were observed undergoing late apoptos is or necrosis. At some poi nt during the cellular insult, the apoptotic cells labeled with AV also tend to become permeable to PI, likely due to progressive loss of membrane integrity. Whether cells go through necrosis or apoptosis may depend on cellular energy status. Yaglom et al. (2003) demonstrat ed that H9c2 cardiac myocytes can undergo necrotic cell death when transiently deprived of energy. This could also be postulated as the cause of the increase in necrotic cell de ath in H9c2 cardiac myocytes following high doxorubicin concentrations and pr olonged duration of exposure, considering that one of the initial and most importan t changes linked to cardiac t oxicity induced by doxorubicin is mitochondrial depolarization and swelling along with a considerable decline in mitochondrial ATP (Friedman et al. 1978; Aversano et al. 1983; Chatham et al. 1990). To gather more information about ATP availabi lity after cellular doxorubicin exposure, we used the enzyme luciferase as indicator of metabolically active cells. We found that ATP availability after doxorubicin exposure dir ectly correlates with cell viability, because there was a marked decreased in ATP 24 hours after doxorubicin exposure, which is proportional to the amount of dead cells c ounted with AV and PI . Doxorubicin-induced myocardial toxicity appears, th erefore, to be at least in part due to a lowering of bioenergetic reserves and to its interferen ce with the regulati on of cellular energy metabolism. However, it could also be po ssible that the lowering in ATP after 24 hours

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32 of doxorubicin exposure is simply explained by a physiological decrease in ATP levels as the metabolism shuts down as the cell dies independent of the mechanism of death. What Is the Mechanism through Which Cells Are Dying? Free Radical Generation by Doxorubucin The mechanism through which apoptosis is produced by doxorubicin is still controversial, but there is general agreement that doxor ubicin induces mitochondrial oxidative stress facilitating the release of pro-apoptotic protei ns in to the cytosol inducing apoptotic activity through th e initiation of the caspase cascade. The metabolism of doxorubicin starts by its mitochondrial reducti on to semiquinone, which is a free radical able to transfer unpaired elec trons to an electron acceptor in such way that generates ROS (Green et al. 2002, Gianfriance et al. 2004; Minotti et al. 2004 and Myers et al. 1998). MPTP Opening The mitochondrial membrane potential is created by proton pumping by the electron transport chain across the inner mitochondrial membrane. The production of ROS during doxorubicin redox cycling with in the mitochondria after doxorubicin treatment previously has been shown to re sult in a lower proton pumping activity and a decrease in the inner mitochondria membrane potential of myocytes (Xu et al. 2002). A decrease in the inner mitochondria membrane potential results in opening of the MPTP with the result of cytochrome c translocation into the cy tosol (Zhou et al. 2002, Green et al. 2002; Wallace et al. 2003 and Minoti et al. 2004). Once in the cytosol, cytochrome c has the ability to facilitate caspase activation through fo rmation of the apoptosome, which is a large molecule formed by the asso ciation of cytochrome c with the apoptosis protease activating factor-1 a nd caspase 3 (Reed et al. 1998 ). The apoptosome activates the downstream caspase cascade, which act as the apoptosis executioner leading to

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33 irreversible cellular deat h (Olson et al. 2001, Wolff et al. 1999 and Philchenkov 2004, Green et al. 2002). One of the objectives of our project was to determine if H9c2 cells exposed to doxorubicin showed alterations in the inner mitochondrial membrane potential, which is lost in the proces s of apoptosis during the opening of the mitochondrial permeability transition pore (Desagher et al. 1999). In our experiments, however, we were not able to detect the expected decrease in mitochondrial membrane potential following do xorubicin exposure, even at the highest doxorubicin concentrations, wher e substantial cell death occurred. We believe that the inability of the JC-1 assay to measure m itochondrial membrane potential changes was due to a technical limitation. In our hands the JC-1 dye tended to form large aggregates at different sites of the individua l wells. While such aggregates may not interfere with flow cytometry readings (the technique used by ma ny other researchers), they would interfere with emission measurements from a plate r eader. Therefore, our findings of the JC-1 assay are not a definitive indi cator of mitochondrial membrane potential in H9c2 cardiac myocytes after doxorubicin exposures. Caspase Activation Caspase are a class of cysteine proteases located in the cellula r cytoplasm that are actively involved in the apoptosis process. There are two types of caspases: initiator caspases and effector caspases. Initiator casp ases (caspase 8 and 9) cleave and activate inactive forms of the effector caspases (cas pase 3 and 7) (Earns haw et al, 1999). Our experiments showed a small but significan t increase in caspase 3/7 activity after doxorubicin exposure. Since caspases 3 and 7 ar e effector caspases, we conclude that there is activation of the caspase cascade l eading to irreversible cellular death. It’s important to point out, however, that alt hough several researchers agree that doxorubicin

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34 is able to induce apoptosis by inducing mitochondrial permeability transition pore opening, release of cytochrome c, apoptosom e formation and caspases activation (Kumar 1999), we can not infer that the preliminary ev ents that induced caspase activation were initiated in the mitochondria. This is because the activation of the initiator caspases may be stimulated by other pathways, such as the death receptor pathway, in which certain receptors can directly activate caspase 8, thereby initiating the caspase cascade and making the apoptosis process imminent (Salvesen et al. 1999). Apoptosis Inhibitors Several researchers have tried to preven t the apoptotic process by the introduction of compounds that are able to inhibit some of the mol ecules or the active processes responsible for the apoptotic process. Game n et al. (2000), showed that the use of ZVAD, a pancaspase inhibitor, produced a to tal blockade of caspase activation and therefore of apoptosis in the hum an T-cell leukemia Jurkat cell line. In addition, Li et al. (2006) developed an in vitro investigation on H9c2 cardiom yocytes where they showed how pre-treating cells with the hematopoiet ic/megakaryocytopoietic growth factor thrombopoietin prevented the cardiotoxicity induced by doxor ubicin, probably due to an antiapoptotic mechanism. Yamanaka et al. ( 2003), used calcium ch annel antagonism and the antioxidant properties of amlodipine to inhibit the apoptosis induced by doxorubicin in neonatal rat cardiac myocytes. Our results show that caspase activity play s an active role in H9c2 cardiac myocyte cell death following doxorubicin exposure. Tw o lines of evidence support this: 1) cell counts showed an increase in cell surviv al when the caspase inhibitor Z-VAD was introduced, and 2) caspase 3/7 activity was increased at sp ecific doses of doxorubicin concentrations after 24 hours of exposure. Th e increase in caspase 3/7 activity is not

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35 continuous as the doxorubicin dose increases ; it reaches a point in the doxorubicin concentration between 0.078 and 0.156M where it starts to decline to then become stable. This may be due to the amount of apoptosis present at low doxorubicin concentration compared to the amount of necr osis at high doxorubicin concentrations. At low concentrations, when apoptosis is more frequently seen, caspase 3/7 seems to be more active than at the highe r doxorubicin doses at which necr osis is more prevalent. Therefore, we conclude that apoptosis is occurring in our experi ments and also that caspase activity plays an active role in the cell death process after low dose of doxorubicin treatment in H9c2 cardiac myocyt es. A similar result was shown in Jurkat cells, in which doxorubicin induces activation of caspase 3, and apoptosis was inhibited by Z-VAD (Gamen et al. 2000). Therefore, the ac tivity of the effector caspase 3 and/or 7 is pivotal to the activatio n of apoptosis following doxorubi cin exposure for 6, 12 and 24 hours in H9c2 cardiac myocytes. However, it’s important to note that this experiment does not determine the molecular events that initiate apoptosis and that precede caspase activation. PI as a Marker of Early Apoptosis In our experiments, after 6, 12 and 24 hours of exposure at 0.078 M doxorubicin, we observed a dramatic increase in the numbe r of H9c2 cardiac myocytes that showed cytoplasmic, non-nuclear PI labeling. Unlike PI nuclear labeling in cells undergoing necrosis, the cytoplasmic PI has not been re ported in the literature. Cytoplasmic PI labeling differs from nuclear PI labeling not only in the loca tion of labeling but also in the intensity of labeling, which is lower in cells undergoing necrosis. We believe that these cells are experiencing very early necrosis and that the cytoplasmic labeling is due to

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36 cellular membrane permeability that allows PI to enter and label cytoplasmic RNA. We postulate that at these lowe r doxorubicin concentrations, the damage created is not sufficient to increase nuclear permeabilit y, thus preventing the binding to DNA and the subsequent classical nuclear labeling. A similar type of cytoplasmic labeling has been described recently by Vitali et al. (2006). Th ese authors concluded that because necrotic cells have extensive damage to their membrane , they readily take up PI even after short incubations periods, and that apoptotic cells are also able to take up PI, but at lower intensity than necrotic cells. According to their observations , it is possible to distinguish apoptotic from necrotic cells so lely by use of PI; apoptotic cells show dim PI coloration and necrotic cells a bright PI coloration. They failed, howev er, to specify whether the apoptotic PI coloration was lim ited to the cytoplasm. Theref ore, our study appears to be the first description of faint cytoplasmic PI labeling as an indicator of early necrosis. Limitations In vitro vs. in vivo Experiments For our experiments we used H9c2 cardiac myocyte cell culture as a model for heart cardiac cells. There are important factor s involved when extrapol ating the results of in vitro experiments to in vivo responses. In cell culture experiments the environment is carefully controlled and is ra rely affected by extraneous f actors. Living animals, on the other hand, are affected by fact ors that potentially affect the outcome of an experiment, such as genetic or congenita l cardiovascular pathologies as well as dysfunction of other tissues that may impact the heart cells a nd their response to doxorubicin. Furthermore, when doxorubicin is given either intravenous ly or intraperitoneally, the drug undergoes metabolism and absorption changes before it ac tually reaches the heart cells. For these

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37 reasons, our results require validation in whole animals before changes in doxorubicin treatments are considered. DMSO Dimethyl sulfoxide (DMSO) is a widely used anti-inflammatory pharmaceutical agent. It is also a free radical scavengers that has also been reported to delay the spread of cancer and prolong survival rates when used in conjunction with chemotherapeutic agents (Salim et al 1992). In our study, doxorubicin and Z-VAD were dissolved in DMSO. It is important to recognize that si nce one of the postulated act ion mechanisms of doxorubicin is through the mitochondrial i nduction of oxidative stress; the antioxidant action of DMSO may have influenced the overall mito chondrial and cellular damage outcome. Furthermore, DMSO additions may also have enhanced Z-VAD anti-apoptotic activity via its antioxidant activity. However, since DMSO was added to the control wells for each experiment, comparisons between cont rol and treatment groups remain valid. Conclusion Our experiments demonstrate that af ter doxorubicin exposure, H9c2 cardiac myocytes die from a dose-dependent combina tion of apoptotic and necrotic mechanisms. At low doxorubicin doses, apoptosis is the predominant type of cell death. This shifts changes to necrosis as the doxorubicin dose incr eases. Further research will be necessary to determine if low chemotherapeutic doxor ubicin doses are less likely to induce heart failure, because the amount of apoptosis seen at low doxorubicin doses is much less that the amount of necrosis seen at high doxorubici n doses, and apoptosis is generally better tolerated by tissues than necrosis. In vivo experiments will be needed to determine whether the amount of apoptosis elicited by doxorubicin exposure can be manipulated by the introduction of caspase inhibitors and if the resulting cardiac failure can therefore be

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38 prevented or avoided. Furthermore, it would be important to evaluate whether multiple small doses of doxorubicin are less likely to induce cell death (or are more likely to shift the cell death from necrosis to apoptosis) , and therefore may be less damaging to the heart than the high single doxorubicin doses; without affecting the chemotherapeutic outcome of doxorubicin

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43 BIOGRAPHICAL SKETCH Andrea Martinez graduated as a Doctor of Veterinary Medicine from La Salle University in Bogota, Colombia, in the year 2000. She came to the University of Florida to work in her master’s degree driven by he r passion for animals and medicine. She will be graduating on August 2006 and then she will continue to promote animal's health by joining a veterinary clinic in Indiana.