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(Circulation Research. 2001;0:hh1501.094115.)
© 2001 American Heart Association, Inc.

Article

Oxidative Stress–Mediated Cardiac Cell Death Is a Major Determinant of Ventricular Dysfunction and Failure in Dog Dilated Cardiomyopathy

Daniela Cesselli, Igor Jakoniuk, Laura Barlucchi, Antonio P. Beltrami, Thomas H. Hintze, Bernardo Nadal-Ginard, Jan Kajstura, Annarosa Leri Piero Anversa

From the Departments of Medicine (D.C., I.J., L.B., A.P.B., B.N.-G., J.K., A.L., P.A.) and Physiology (T.H.H.), New York Medical College, Valhalla, NY.

Correspondence to Piero Anversa, MD, Department of Medicine, Cardiovascular Research Institute, New York Medical College, Vosburgh Pavilion, Room 302A, Valhalla, NY 10595. E-mail piero_anversa{at}nymc.edu

	Abstract

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Abstract— Cell death has been questioned as a mechanism of ventricular failure. In this report, we tested the hypothesis that apoptotic death of myocytes, endothelial cells, and fibroblasts is implicated in the development of the dilated myopathy induced by ventricular pacing. Accumulation of reactive oxygen products such as nitrotyrosine, potentiation of the oxidative stress response by p66^shc expression, formation of p53 fragments, release of cytochrome c, and caspase activation were examined to establish whether these events were coupled with apoptotic cell death in the paced dog heart. Myocyte, endothelial cell, and fibroblast apoptosis was detected before indices of severe impairment of cardiac function became apparent. Cell death increased with the duration of pacing, and myocyte death exceeded endothelial cell and fibroblast death throughout. Nitrotyrosine formation and p66^shc levels progressively increased with pacing and were associated with cell apoptosis. Similarly, p50 (N) fragments augmented paralleling the degree of cell death in the failing heart. Moreover, cytochrome c release and activation of caspase-9 and -3 increased from 1 to 4 weeks of pacing. In conclusion, cardiac cell death precedes ventricular decompensation and correlates with the time-dependent deterioration of function in this model. Oxidative stress may be critical for activation of apoptosis in the overloaded heart.

Key Words: oxidative stress response • p53 fragments • p66^shc • caspases • heart failure

	Introduction

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Ventricular pacing in dogs leads to congestive heart failure and changes in cardiac size and shape, which consist of an increase in cavitary diameter and thinning of the wall that typically are found in the decompensated human heart.¹ Myocyte death in the failing dog heart² results over a period of 4 weeks in a decrease in the aggregate number of ventricular myocytes.¹ A similar condition is present in patients with ischemic and dilated cardiomyopathy,^3,4 strengthening the relevance of ventricular pacing as a model of human disease. Alterations in Ca²⁺ and mitochondrial respiration occur with heart failure,⁵ potentiating the production of superoxide anion, O₂^·-, and hydrogen peroxide, H₂O₂.⁶ Additionally, NO can interact with O₂^·-, forming peroxynitrite, or ONOO^-.⁷ ONOO^- reacts with cellular proteins generating nitrotyrosine, an end product of oxidative damage. Antioxidant enzymes decrease in the decompensated heart,^8,9 depressing its defense mechanisms against these sources of oxidative challenge. Importantly, reactive O₂ may trigger apoptotic cell death.¹⁰ Thus, the dilated myopathy associated with ventricular pacing may be the result of oxidative stress and its ability to promote apoptosis. The effects of oxygen free radicals are regulated at least in part by the expression of a member of the Shc family of proteins, p66^shc, that modulates the oxidative stress response.¹¹ Moreover, reactive O₂ causes DNA strand breaks and the injured DNA is recognized by p53.^12,13 This link stimulates p53 via autoproteolytic activity and cleavage of this protein.¹⁴ p53 fragments lacking the C terminus, p50 (C), may bind to single DNA strand breaks, promoting p21^Cip1 expression and DNA repair.¹⁵ In contrast, p53 fragments with N-terminal cleavage, p50 (N), may interact with double DNA strand breaks, upregulating Bax and leading to apoptosis.¹⁵ Alternatively, reactive O₂ may facilitate the mitochondrial release of cytochrome c, activating the caspase cascade.¹⁶ Therefore, we tested the hypothesis that expression of p66^shc with ventricular pacing may be associated with an enhanced oxidative stress response, DNA damage, formation of p53 fragments, cytochrome c release, caspase activation, and ultimately cell death. Cardiac cell death was expected to precede ventricular dysfunction and subsequently contribute to its evolution to terminal failure.

	Materials and Methods

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Cardiac Function
Dogs were instrumented in the left ventricle with a corkscrew electrode attached to a pacemaker. Hearts were paced at 210 bpm for 1 (n=6) and 3 (n=6) weeks, and at 240 bpm for an additional week (n=8). Control dogs were not paced (n=8). Some control dogs (n=4) and dogs paced for 1 (n=3), 3 (n=3), and 4 (n=4) weeks were used previously.^1,2,17,18 Protocols were approved by New York Medical College. Hemodynamics were measured in conscious dogs.^1,2,17,18 Hearts were arrested in diastole, and a portion of the left ventricle was used for myocyte isolation and biochemistry and the remaining for histochemistry.^1,2,17

Myocytes
Myocytes were dissociated by collagenase^1,2,17 in control hearts (n=8) and hearts paced for 1 (n=6), 3 (n=6), and 4 (n=8) weeks.

Western Blot
Myocyte proteins were separated on SDS-PAGE,^2,19 and filters were exposed to antibodies listed in the online data supplement available at http://www.circresaha.org. The following proteins were measured: caspase-9; caspase-3; DNA fragmentation factor (DFF); caspase-activated DNase (CAD); poly(ADP-ribose)polymerase (PARP); DNA-dependent protein kinase catalytic subunit (DNA-PKcs); lamins A, B, and C; p53; p50 (N); p50 (C); Bax; p21; Shc proteins; and nitrotyrosine. Mitochondrial and postmitochondrial fractions were separated on SDS-PAGE and transferred onto nitrocellulose. Membranes were exposed to cytochrome c antibody. Coimmunoprecipitation of Apaf-1, cytochrome c, and procaspase-9 was done by using 200 µg of myocyte proteins that were incubated with antiapoptotic protease-activating factor (Apaf-1). Immunoprecipitated proteins were separated on SDS-PAGE, transferred onto nitrocellulose filters, and exposed to anti–caspase-9 or anti–cytochrome c.^2,20

Caspase Activity
An in vitro reaction between a synthetic substrate and the enzyme present in the sample was done. Acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin hydrolysis by caspase-3 resulted in the release of the fluorescent 7-amido-4-methylcoumarin moiety. Data were obtained with a spectrofluorometer at 15, 30, 45, 60, and 120 minutes.²⁰

Cell Death
Terminal deoxynucleotidyltransferase (TdT)–mediated dUTP nick end labeling (TUNEL) and ligation assay were performed.^2,4,21 For TdT, numbers of myocyte, endothelial cell, and fibroblast nuclei examined in controls were 96x10⁴, 160x10⁴, and 186x10⁴; at 1 week they were 81x10⁴, 138x10⁴, and 139x10⁴; at 3 weeks they were 98x10⁴, 150x10⁴, and 190x10⁴; and at 4 weeks they were 102x10⁴, 177x10⁴, and 246x10⁴. For the hairpin probe, numbers of nuclei sampled in controls were 97x10⁴, 159x10⁴, and 184x10⁴; at 1 week they were 80x10⁴, 125x10⁴, and 140x10⁴; at 3 weeks they were 89x10⁴, 139x10⁴, and 176x10⁴; and at 4 weeks they were 93x10⁴, 186x10⁴, and 161x10⁴.

Nitrotyrosine
Sections were stained with anti-nitrotyrosine.²¹ In controls, 7374 myocytes, 11 005 endothelial cells, and 12 647 fibroblasts were examined. Values in pacing were 8003, 13 801, and 12 215 at 1 week; 7386, 12 912, and 11 332 at 3 weeks; and 7677, 12 974, and 11 266 at 4 weeks.

Statistics
Results are mean±SD. Significance between two measurements was determined by the Student t test, and among groups by the Bonferroni method.

An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.

	Results

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Hemodynamics and Cell Death
Left ventricular end-diastolic pressure (LVEDP) at rest and with the pacemaker turned off increased 1 week after the imposition of tachycardia, whereas left ventricular +dP/dt decreased. A progressive reduction in +dP/dt and a further elevation in LVEDP occurred at 3 and 4 weeks of pacing. However, systolic function became depressed at 4 weeks (Figure 1). Although the changes in ventricular hemodynamics could not be measured in the same animals during the course of the experiments, pacing leads to an early increase in diastolic wall stress, which continues to rise, reaching high levels when heart failure supervenes.^18,22 In vitro studies mimicking diastolic overloads activate myocyte and nonmyocyte death,²³ suggesting that the chronic increase in diastolic stress with pacing may enhance cardiac cell death. Cell death by apoptosis was evaluated in myocytes (Figures 2A through 2C), endothelial cells (Figures 2D through 2F), and fibroblasts of the left ventricle at 1, 3, and 4 weeks of pacing. Cell death increased consistently with the duration of pacing in all three cell populations. Myocyte death was significantly higher than endothelial cell and fibroblast death. Apoptosis was assessed by two independent histochemical methods that gave essentially identical results (Figures 2G and 2H).

View larger version (38K): [in this window] [in a new window]   Figure 1. Effects of pacing on LVEDP, left ventricular systolic pressure, and +dP/dt. Results are mean±SD. *, **, and ***P<0.05 vs control nonpaced dogs (C, n=8) and vs dogs paced for 1 (P1, n=6) and 3 (P3, n=6) weeks, respectively. P4 (n=8) indicates dogs paced for 4 weeks.

View larger version (42K): [in this window] [in a new window]   Figure 2. A through C, Myocyte apoptosis in a paced heart. Blue fluorescence illustrates nuclei by propidium iodide (A), and green fluorescence shows an apoptotic nucleus (B) by in situ ligation (arrow). C, Red fluorescence reflects myocytes by -sarcomeric actin antibody. Bright fluorescence corresponds to the combination of propidium iodide and in situ ligation of the apoptotic nucleus. Blue fluorescence indicates viable nuclei. D through F, Endothelial cell apoptosis in a paced heart. Blue fluorescence illustrates nuclei by propidium iodide (D), and green fluorescence shows an apoptotic nucleus (E) by in situ ligation (arrow). Red fluorescence depicts endothelial cells by factor VIII antibody (F). Bright fluorescence (F) corresponds to the combination of propidium iodide and in situ ligation of the apoptotic nucleus. Blue fluorescence reflects viable nuclei. A through F, Confocal microscopy; magnification x1500. G through H, Cell apoptosis by TdT (G) and in situ ligation with single base 3` overhang probe (H). Results are mean±SD. *, **, and ***P<0.05 vs C (n=8), P1 (n=6), and P3 (n=6), respectively. P4, n=8. P1, P3, and P4 indicate dogs paced for 1, 3, and 4 weeks, respectively.

Caspases
Cytochrome c release, caspase-9 expression, caspase-3 expression and activity, and cleavage of protease substrates were obtained in myocytes only, because myocytes exhibited the highest cell death. It cannot be excluded that the cell isolation procedure may have eliminated some myocytes prone to cell death, affecting the level of the biochemical measurements. This potential artifact, however, resulted in an underestimation of the multiple parameters analyzed, not in an overestimation. Thus, the relative findings are valid, although the absolute values of the various gene products implicated in the apoptotic process may be higher than those found here.

Cytochrome c was assayed by immunoblotting membrane pellets containing the mitochondrial fraction, and cytosolic supernatants (Figure 3A). In control myocytes, cytochrome c was observed only in the mitochondrial compartment, whereas it was undetectable in the cytosol. Conversely, a significant quantity of cytochrome c was translocated from the mitochondria to the cytosol in myocytes paced for 1, 3, and 4 weeks. Cytochrome c, Apaf-1, and procaspase-9 form ternary complexes from which the two active subunits of caspase-9 are released.²⁴ Apaf-1 increased in myocytes with the duration of pacing (Figure 3B), and its physical interaction with cytochrome c was apparent (Figure 3C). Cytochrome c–Apaf-1 complex, however, decreased at 3 to 4 weeks, possibly reflecting the completion of cytochrome c function in Apaf-1 oligomerization.²⁵ Procaspase-9 is in the intermembrane mitochondrial space, and its translocation to the soluble compartment remains to be identified.²⁶ By immunoprecipitation of myocyte proteins with Apaf-1 antibody and subsequent exposure of the immunoprecipitated proteins to anti–procaspase-9 and Western blot, the fraction of procaspase-9 bound to Apaf-1 was detected in the cytosol (Figure 3D). This complex decreased with pacing. Pacing was also associated with a reduction in the total amount of procaspase-9 in the cytosol (Figure 3E). This may be due to cleavage of the inactive precursor p45 into the active subunits p35 and p10 (Figures 3E and 3F); p35 and p10 increased with pacing.

View larger version (39K): [in this window] [in a new window]   Figure 3. A, Western blot (WB) of cytochrome c in the cytosol (Cy) and mitochondria (M) of control (C), P1, P3, and P4 myocytes. B, Western blot of Apaf-1 in control, P1, P3, and P4 myocytes. (+) indicates K562 cell lysates. C, Immunoprecipitation (IP) of Apaf-1 with Apaf-1 antibody (Ab) and Western blot of fraction of cytochrome c bound to Apaf-1 with cytochrome c antibody using lysates of control, P1, P3, and P4 myocytes. (+) indicates K562 cell lysates. D, Immunoprecipitation of Apaf-1 with Apaf-1 antibody and Western blot of fraction of procaspase-9 bound to Apaf-1 with caspase-9 antibody using lysates of control, P1, P3, and P4 myocytes. (+) indicates A431 cell lysates. E through J, Western blot of procaspase-9, p45, and its cleaved subunit p10 (E); caspase-9 subunit p35 (F); procaspase-3, p32, and its cleaved subunit p17 (G); DFF45 and HeLa cell lysates (+) (H); CAD and K562 nuclear cell lysates (+) (I); and PARP and its fragments (J) in control, P1, P3, and P4 myocytes. A through J, n values were as follows: for control, n=8; for P1, n=6; for P3, n=6; and for P4, n=8. K, Western blot of DNA-PKcs and its cleaved subunit p150. NP indicates nonpaced. n=3 in each group of dogs. L and M, Western blot of lamins A and C (L), and lamin B and its cleaved subunit p45 (M). N, Effects of pacing on caspase-3 activity. Aliquots of myocyte lysates were analyzed for DEVDase activity, expressed as Acetyl-Asp-Glu-Val-Asp-7 amido-4-methylcoumarin (AMC) fluorescence emitted at 460 nm with time. Control (, y=0.008x+66.81; R2=0.87, P<0.001. P1 (), y=0.028x+67.49; R2=0.94, P<0.001. P3 (, y=0.040x+67.37; R2=0.94, P<0.001. P4 (), y=0.071x+67.43; R2=0.99, P<0.001. P1, P3, and P4 indicate dogs paced for 1, 3, and 4 weeks, respectively.

The activated caspase-9 cleaves and stimulates procaspase-3.^24,25 After pacing, procaspase-3, p32, decreased with time, paralleling the increase of its active subunit p17 (Figure 3G). p17, in turn, removed the inhibitory DFF45 from CAD,²⁷ promoting cell death. DFF45 progressively dissociated and decreased with pacing generating the functionally active nuclease CAD (Figures 3H and 3I). After chromatin condensation and DNA fragmentation, specific nuclear proteins undergo caspase-dependent proteolysis, including PARP, DNA-PK, and lamins. During apoptosis, PARP is cleaved between the amino acids Asp214 and Gly215 yielding two fragments of 89 and 24 kDa; these fragments contain the active site of the enzyme and the DNA binding domain, respectively.¹³ The appearance of the 89- and 24-kDa fragments of PARP was consistent with activation of caspase-3 in paced myocytes (Figure 3J). DNA-PKcs is another nuclear substrate of caspase-3.²⁸ The cleavage of this kinase in fragments of 240 and 150 kDa is accompanied by loss of activity. The 240-kDa fragment derives from the N terminus of the original protein, whereas the 150-kDa fragment contains the kinase domain and belongs to the C terminus. The 150-kDa peptide can be further cleaved into a 120-kDa fragment that retains the kinase domain.²⁸ In comparison with control myocytes, the intact molecule of DNA-PKcs is upregulated after 1 week of pacing. At 3 and 4 weeks, the reduced expression of this enzyme is coupled with the appearance of the 150-kDa product of cleavage (Figure 3K). Degradation of the nuclear lamina represents one of the last steps of apoptotic cell death.²⁹ In paced myocytes, the proteolysis of lamin B led to the formation of a 45-kDa subunit; this is consistent with the activation of caspase-3 (Figure 3L). Moreover, the decrease in lamin A level with pacing was probably linked to caspase-3–dependent cleavage of this component of the nuclear envelope (Figure 3M). In contrast, lamin C protein did not change with pacing (Figure 3M). Optical density values of the results shown in Figures 3A through 3M and their statistical comparisons are shown in the online data supplement available at http://www.circresaha.org (Figures 1 through 20 online).

Expression of caspase-3 in myocytes was complemented by fluorometric measurements of caspase-3 activity; minimal levels were detected in control myocytes. Paced myocytes were characterized by a linear increase with time of the release of fluorescent moiety from the peptide substrate (Figure 3N). Moreover, caspase-3 activity increased progressively with pacing. It should be noted that the biochemical changes reflecting apoptotic myocyte death with pacing were very apparent. Myocyte apoptosis, however, comprised nearly 0.5%, suggesting that the molecular results might have reflected changes greater than the extent of cell death measured morphologically. Although it is difficult to compare histochemical data with biochemical findings, the possibility may be advanced that pacing affected the resistance of myocytes to the isolation procedure, exaggerating the level of expression of the various markers of the apoptotic process.

p53 Fragments and Oxidative Response
Oxidative stress can damage DNA promoting the formation of single and double DNA strand breaks.¹² These forms of DNA injury trigger p53 autoproteolytic activity, leading to the generation of p50 fragments lacking either the carboxyl or the amino terminal.¹³ Ventricular pacing was characterized by an increase in total p53 in myocytes, and this change was accompanied by the appearance of p50 (N) (Figure 4A) and p50 (C) (Figure 4B). Both p50 fragments accumulated with the duration of pacing. The upregulation of p53 and the formation of the proapoptotic fragment p50 (N) was accompanied by an increase of the quantity of Bax (Figure 4C). Conversely, the enhanced expression of p21 was consistent with the DNA repair function of p50 (C) (Figure 4D).

View larger version (43K): [in this window] [in a new window]   Figure 4. Western blot of p53, p50 (N), and SVT2 cell lysates (+) (A); p53, p50 (C), and SVT2 cell lysates (+) (B); Bax (C); p21 and HeLa cell lysates (+) (D) in control (C), P1, P3, and P4 myocytes. For control, n=8; for P1, n=6; for P3, n=6; and for P4, n=8. P1, P3, and P4 indicate dogs paced for 1, 3, and 4 weeks, respectively.

p66^shc is a member of the Shc family of proteins, which is implicated in the modulation of oxidative stress at the cellular level.¹¹ p66^shc potentiates the impact of reactive O₂ on various cell populations,¹¹ raising the possibility that modifications favoring the effects of reactive oxygen species (ROS) on the stressed heart may develop with the progression of ventricular pacing. Therefore, the expression of p66^shc was examined and found to increase significantly in paced myocytes (Figure 5A). Additionally, nitrotyrosinylation of proteins ranging from 30 to 70 kDa increased after pacing (Figure 5B), providing supporting evidence of oxidative damage. Optical density values of the biochemical parameters illustrated in Figures 4A through 4D and Figures 5A and 5B and their statistical comparisons are shown in Figures 21 through 28 in the online data supplement available at http://www.circresaha.org.

View larger version (94K): [in this window] [in a new window]   Figure 5. A and B, Western blot of p66shc, p52shc, p46shc, and A431 cell lysates (+) (A), and nitrotyrosine (B) in control (C), P1, P3, and P4 myocytes. Control, n=8; P1, n=6; P3, n=6, and P4, n=8. C through F, Nitrotyrosine distribution in myocytes of control heart (C and D) and paced heart (E and F). Green fluorescence illustrates nuclei by propidium iodide; blue fluorescence, nitrotyrosine in tissue (C and E). Red fluorescence corresponds to -sarcomeric actin in myocyte cytoplasm; pink fluorescence reflects nitrotyrosine in myocytes (D and F). G through J, Nitrotyrosine distribution in nonmyocytes of paced hearts. Green fluorescence illustrates nuclei by propidium iodide; blue fluorescence, nitrotyrosine in tissue (G and I). Red fluorescence corresponds to factor VIII in endothelial cell cytoplasm (H) and vimentin in fibroblasts (J); pink fluorescence, nitrotyrosine in these cells (H and J). Confocal microscopy; magnifications are the following: C through F, x800; G and H, x1500; and I and J, x1000. K, Effects of pacing on nitrotyrosine labeling of cardiac cells. Results are mean±SD. * and **P<0.05 vs C (n=8) and P1 (n=6), respectively. P3 (n=6) and P4 (n=8). P1, P3, and P4 indicate dogs paced for 1, 3, and 4 weeks, respectively.

The immunocytochemical detection of nitrotyrosine in myocytes, endothelial cells, and fibroblasts of control and paced hearts is illustrated in Figures 5C through 5J. This provides a direct visualization of a product of oxidative stress in the paced heart. Quantitative analysis demonstrated that the percentage of nitrotyrosine-positive cardiac cells increased as a function of ventricular pacing (Figure 5K). However, the fraction of myocytes expressing nitrotyrosine was severalfold higher than endothelial cells and fibroblasts. At 4 weeks of pacing, myocytes labeled by nitrotyrosine were 7-fold and 10-fold more numerous than endothelial cells and fibroblasts, respectively. Differences between endothelial cells and fibroblasts were modest.

	Discussion

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p66^shc, Heart Failure, and Oxidative Stress
The Shc family of proteins includes three splicing isoforms, p46^shc, p52^shc, and p66^shc.³⁰ p46^shc and p52^shc are ubiquitously expressed. However, p66^shc varies in abundance from cell to cell, and it is not present in all cells.¹¹ p46^shc and p52^shc share a C-terminal SH2 domain that mediates the transmission of growth signals from tyrosine kinase receptors.³¹ p46^shc and p52^shc activate the Ras oncoprotein,^30,31 and this interaction links Shc proteins to downstream effector responses involving mitogen-activated protein (MAP) kinases and cellular growth.³¹ p66^shc is unable to activate MAP kinases after growth factor stimulation¹¹ and inhibits c-fos,³¹ contrasting the role of the other Shc gene products. Thus, Shc proteins exert opposite functions; p46^shc and p52^shc promote cell proliferation and p66^shc negatively influences cell growth.^30,31 Ablation of p66^shc decreases oxidative stress in vivo, because p66^shc reduces the resistance of cells to ROS, initiating cell death.¹¹ p66^shc was almost undetectable in control dog myocytes. Conversely, ventricular pacing was accompanied by a progressive increase in the quantity of p66^shc in failing myocytes, and this may have potentiated the effects of ROS on these cells. Pacing-induced dilated cardiomyopathy is characterized by an increased production of superoxide anion, O₂^·-, and hydroxyl radicals, · OH.⁶ Mitochondrial dysfunction with uncoupling of the respiratory chain is the major intracellular source of ROS that can damage directly proteins, lipids, and DNA.^6,13,32 Concurrently, oxidative injury may involve the formation of peroxynitrite, ONOO^-, which is the product of NO and O₂^·-.⁷

Cytochrome c release from the intermembrane mitochondrial compartment to the extramitochondrial space, translocation of the inactive procaspase-9 from the mitochondria to the cytosol, and nitrotyrosinylation of proteins in myocytes were identified here at the early and late stages of the dilated myopathy. These biochemical events increased as a function of pacing and the appearance of cardiac failure. Additionally, p53 fragments due to the loss of the C-terminus (p50 [C]), or N-terminus (p50 [N]), were detected in paced myocytes, indicating an interaction between activated p53 and distinct forms of DNA damage. p53 fragments increased with the duration of pacing. These factors support the notion that oxidative damage may have played a role in myocyte death during the onset and evolution of the cardiac disease.

Caspases, Cell Death, and Heart Failure
The activation of mitochondrial permeability pore transition results in the leakage of molecules from the organelle to the cytosol.^15,16 Translocation of cytochrome c to the cytosol of paced myocytes was paralleled by an increased expression of Apaf-1. In the presence of ADP and cytochrome c, Apaf-1 participates in a multistage reaction.^24,25 After hydrolysis of ATP to ADP, cytochrome c binds to Apaf-1, promoting its multimerization from a monomeric form to a large complex. The functional multimeric complex recruits and cleaves procaspase-9; when caspase-9 is activated, cytochrome c and ADP are no longer required.³³ This sequence of events was demonstrated in failing canine myocytes in which the continuous decrease in procaspase-9 with pacing was accompanied by an increase in the active subunits p35 and p10. Moreover, activated caspase-9 resulted in the cleavage of downstream effectors, caspase-3 and DFF45 and, thereby, CAD, which is the only endonuclease induced by the caspase cascade.^27,34 A similar sequence of events has been shown in neonatal myocytes exposed in vitro to serum and glucose deprivation.³⁵

PARP increases in the early phases of apoptosis and is activated by binding to DNA strand breaks. PARP catalyzes the poly(ADP-ribosyl)ation of nuclear peptides and facilitates the recruitment of repair proteins at the site of DNA damage.¹³ The positive effect of PARP on DNA integrity is lost in late apoptosis; caspase-3 induces proteolysis and inactivation of PARP. In paced myocytes, PARP was upregulated, but its cleavage increased. The DNA-PK is essential for the repair of double-strand DNA breaks.²⁸ The catalytic subunit of this kinase is activated by binding to DNA damage; this interaction is stabilized by Ku proteins.^28,36 In pacing dogs, the expression of DNA-PKcs was initially enhanced. With progression of ventricular tachycardia, DNA-PKcs was cleaved, most likely by caspase-3, losing its ability to repair DNA. The structure of the nuclear envelope is preserved until the late stages of cell death.²⁹ Lamin degradation may involve caspase-6 and caspase-3. However, consistent with our observations in which lamin C is not affected by caspase-3, depletion of caspase-3 abrogates the cleavage of lamins A and B, but not lamin C.²⁹ The activation of caspase-3 after cytochrome c release from the mitochondria and its accumulation in the myofibrils has been documented, in combination with apoptosis, in human cardiomyopathy.^37,38 These results strengthen our observations in the pacing dog heart. A pathologic consequence of oxidative stress is linked to its ability to produce DNA damage which, in turn, has the capacity of binding p53 and stimulating p53 autoproteolytic activity.^14,15 This process is initiated by the interaction of the transcription factor with double DNA strand breaks with staggered ends and results in N-terminal cleavage of p53. p50 (N) is formed; this fragment induces p53 target genes, leading to apoptosis.¹⁵ Under this condition, apoptosis proceeds independently from the intervention of caspases and the stimulation of endonucleases; endonucleases contribute to the completion of DNA fragmentation after irreversible double DNA strand cleavage. Conversely, cleavage of the C-terminal of p53 is coupled with single DNA strand breaks and the formation of p50 (C). p50 (C) favors growth arrest in G₁ during DNA repair.¹⁵ Pacing was characterized by an increase in the expression of p50 (N) and p50 (C) in myocytes. The time-dependent increase in p50 fragments with pacing suggests that distinct aspects of DNA damage developed with heart failure. They were accompanied by apoptotic myocyte death, upregulation of Bax, and the expression of the cell cycle inhibitor p21.¹³ The latter may be linked to DNA repair.

Cardiac Cell Death and Heart Failure
Oxidative damage leading to cell death may constitute the most proximal event of heart failure.^4,6 With pacing, apoptosis is implicated in the loss of cells. Because it is unknown whether parenchymal or interstitial cells are more susceptible to oxidative damage in the heart, apoptosis was measured in myocytes, endothelial cells, and fibroblasts. Apoptotic cardiac cell death became apparent at 1 week of pacing, increasing progressively with time. The extent of cell death was higher in myocytes than in endothelial cells and fibroblasts. Endothelial cell death may reflect disappearance of capillaries and regional reduction in blood flow and decreased tissue oxygenation.³⁹ A group of myocytes may die by ischemia leading to collagen accumulation. In this regard, the serum of patients with heart failure has the capacity to enhance endothelial cell death in vitro.⁴⁰ Moreover, the death of fibroblasts may influence the interconnection between myocytes, facilitating wall restructuring, mural thinning, and chamber dilation.³⁹ This leads to an increase in wall stress and oxygen consumption, which influences myocyte size, shape, and mechanical properties. The chronic deterioration in function with pacing appears to be the result of interrelated phenomena initiated by oxidative stress and culminating in cardiac cell death.

It is important to recognize that studies of heart failure in animal models and humans do not permit us to establish a cause-and-effect relationship between specific parameters. This limitation is inherent in all in vivo reports. Although the various biochemical measurements performed here are consistent with the activation and progression of the apoptotic pathway in the paced heart, definitive conclusions cannot be made. This cannot be avoided, because cardiac decompensation has to be addressed in vivo. Difficulties also exist in experimentation in vitro; extrapolation of in vitro findings to in vivo states requires considerable caution. However, the in vitro data briefly summarized above, concerning oxidative stress, upregulation of p53,¹¹ p50 fragment formation,^14,15 and cytochrome c–dependent activation of the caspase cascade,^{35,37,38,41,42} are in agreement with the results of the current study.

	Acknowledgments

 
This work was supported by NIH Grants HL-38132, HL-39902, HL-43023, AG-15756, HL-65577, HL-69923, HL-65573, and AG-17042.

Received February 27, 2001; accepted June 4, 2001.

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