Endogenous endothelium-derived nitric oxide inhibits myocardial caspase activity: implications for treatment of end-stage heart failure

FAILING HEART—BASIC SCIENCE

Endogenous Endothelium-Derived Nitric Oxide Inhibits Myocardial Caspase Activity: Implications for Treatment of End-Stage Heart Failure Seema Mital, MD,a Alessandro Barbone, MD,b Linda J. Addonizio, MD,a Jan M. Quaegebeur, MD,b Ralph J. Mosca, MD,b Mehmet C. Oz, MD,b and Thomas H. Hintze, PhDc Background: Apoptosis contributes to ventricular remodeling in heart failure (HF). Nitric oxide (NO) inhibits caspase 3, a key effector apoptotic enzyme. We hypothesized that reduced endogenous NO in HF disinhibits cardiac caspase 3 to promote apoptosis. Methods: Caspase 3 activity was measured colorimetrically in myocardial cell lysates from endothelial NO synthase (eNOS)-deficient mice (eNOS ⫺/⫺; n ⫽ 18), cardiomyopathic (CMP) hamsters (n ⫽ 8), and explanted failing human hearts (n ⫽ 10). We stimulated myocardial caspase 3 activity by adding upstream caspase 8 or 9. Cell lysates were incubated with 10⫺4 mol/liter NO donor, S-nitroso-N-acetyl penicillamine; NOS inhibitor, nitro-L-arginine-methyl ester (L-NAME); or angiotensinconverting enzyme (ACE) inhibitor, enalaprilat. Hamsters underwent echocardiography so we could study the progression of ventricular dysfunction. Results: Stimulated caspase 3 activity was lower in myocardium of eNOS ⫹/⫹ compared with eNOS ⫺/⫺ mouse hearts (5.1 ⫾ 0.5 vs 7.6 ⫾ 1.0 pmol/10 ␮g/min, p ⬍ 0.05). L-NAME increased enzyme activity only in eNOS ⫹/⫹ mice, indicating that endogenous NO inhibits caspase 3. Stimulated caspase 3 activity was lower in control hamsters, 3.3 ⫾ 0.3 pmol/10 ␮g/min, compared with CMP hamsters, 9.6 ⫾ 0.7 and 6.9 ⫾ 0.4 pmol/10 ␮g/min at 4 and 9 months, respectively. This was associated with progressive ventricular dysfunction, thinning, and dilatation. L-NAME increased enzyme activity in normal but not in CMP hamsters. In failing human myocardium, LNAME failed to alter caspase activity, indicating reduced NO availability. Enalaprilat inhibited caspase 3, which was reversed by L-NAME. S-nitroso-N-acetyl penicillamine reversed caspase 3 activation in all groups. Conclusions: Nitric oxide reversibly inhibits myocardial caspase 3 independent of the apoptotic signaling pathway. Reduced NO in HF increases myocardial caspase 3 From the Divisions of aPediatric Cardiology and bCardiothoracic Surgery, Columbia University, New York, New York, and c Department of Physiology, New York Medical College, Valhalla, New York. Submitted May 22, 2001; revised August 1, 2001; accepted September 20, 2001. This work was supported by HL 50142, HL 61290, and PO-1 HL 43023 from the National Heart, Lung and Blood Institute. Presented in part at the Annual Meeting of the International

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Society for Heart and Lung Transplantation, Vancouver, April 2001. Reprint requests: Seema Mital, MD, Division of Pediatric Cardiology, Babies Hospital, 2 North, 3959 Broadway, New York, NY 10032. Telephone: 212-305-8305. Fax: 212-305-4429. Email: [email protected] Copyright © 2002 by the International Society for Heart and Lung Transplantation. 1053-2498/02/$–see front matter PII S1053-2498(01)00404-1

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activity. Agents that promote NO synthesis, including ACE inhibitors, may prevent caspase activation in HF. J Heart Lung Transplant 2002;21:576 –585.

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rogrammed cell death, or apoptosis, has been implicated in the irreversible process leading to cardiac remodeling and dilatation in heart failure.1 Apoptosis is a process by which cells activate an intracellular death program in a controlled way without eliciting a damaging inflammatory response. The key mediators of apoptosis are a family of cysteine proteases called caspases. They are produced as inactive zymogens and are activated in response to apoptotic stimuli by proteolytic processing at conserved aspartic acid residues.2 The downstream “effector” caspases are largely dependent on upstream “initiator” caspases for their proteolytic processing and activation.3 Two major apoptotic pathways have been identified in most cells: 1) death-receptor–mediated pathway or extrinisic pathway, the best characterized death receptors are CD95/Fas and tumor necrosis factor and are activated by ligands, the majority of which are structurally related molecules of the tumor necrosis factor gene superfamily; and 2) mitochondrial pathway or intrinsic pathway, which involves release of cytochrome c from the mitochondria into the cytosol. Caspase 8 is the apical caspase in the death-receptor pathway, and caspase 9 serves as apical caspase of the mitochondrial pathway. Caspase 3 is activated during apoptotic signaling events by upstream proteases, including caspases 8 and 9, and is an effector of apoptosis.4 – 6 Targets of activated caspase 3 include poly (adenosine diphosphate ribose) polymerase, nuclear lamins, gelsolin, and other cell proteins. Mannick et al7 first reported the ability of nitric oxide (NO) to inhibit caspase 3 activation. NO donors can inhibit several members of the caspase family through S-nitrosylation of the active cysteine site on the enzyme.8,9 However the ability of endogenous NO to inhibit caspases has only recently been recognized.10,11 We, and others, have previously reported that endothelial NO production is markedly reduced in heart failure, partly because of down-regulation of the endothelial NO synthase (eNOS) enzyme.12–14 We hypothesized that decreased endogenous NO in heart failure promotes myocardial caspase activation and that restoring NO availability will decrease caspase activity in failing hearts. The objectives of our study were to evaluate the effect of 1) endogenous NO on caspase 3 using transgenic mice deficient in eNOS, and 2) modula-

tion of local tissue NO availability on baseline and stimulated caspase 3 activity in cardiomyopathic (CMP) hamsters and in explanted failing human hearts. Use of mice with targeted disruption of eNOS gene permitted evaluation of the role of the specific eNOS isoform in regulating apoptotic enzyme activation. The existence of an inbred strain of Syrian hamsters with a genetically transmitted degenerative CMP that terminates in end-stage heart failure offers a unique opportunity to study the sequence of events leading to cardiac decompensation and to analyze the role of intrinsic and extrinsic factors in regulating this process.15 The study of failing human myocardium permitted evaluation of the importance of this regulation in heart failure in humans.

MATERIALS AND METHODS Transgenic 9-month-old female eNOS-deficient (⫺/⫺) mice (n ⫽ 18) and age-matched wild-type (⫹/⫹) controls (n ⫽ 8) that had been back-crossed onto the parental wild-type strain for 5 generations were selected from our breeding colony. Syrian CMP hamsters (BIOTO2 strain) aged 4 months and 9 months and age-matched normal hamsters (BIOF1B strain) were obtained from Bio Breeders Inc. (Fitchburg, MA) (n ⫽ 10 –12 in each group). Mice and hamsters were anesthetized with pentobarbital sodium (65 mg/kg intraperitoneally), the heart was quickly removed, and the left ventricle (LV) was frozen in liquid nitrogen at ⫺70°C. Myocardium was also obtained from 10 explanted failing human hearts at the time of heart transplantation. The protocols in animals were approved by the Institutional Animal Care and Use Committee and conform to the Guiding Principles for the Use and Care of Laboratory Animals of the National Institutes of Health and the American Physiological Society. The Institutional Review Board approved the studies in human hearts, and the investigators were masked to the identity of the patients.

Preparation of Cell Extracts Because caspases are intracellular proteases found in the cytosol, cytosolic extracts were prepared from isolated ventricular myocardium. Approximately 100 to 200 mg of frozen tissue was powdered under liquid nitrogen at ⫺70°C and homogenized (two 10

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second bursts at setting 7 on a Brinkman Kinematica GmBH; Westburg, NY) in 5 times the volume of ice-cold cell lysis buffer containing 50 mmol/liter HEPES, 1 mmol/liter Dithiothreitol (DTT), 0.1 mmol/liter ethylenediaminetetracetic acid (EDTA), 0.1% cholamidopropyldimethylammonio-1-propanesulfonate (CHAPS), pH 7.4. This technique has been successfully used to measure other enzyme activities.16 The extracts were centrifuged at 10,000g for 10 minutes at 4°C and supernatant, i.e., cytosolic fraction was used for assay. Protein content of cell lysates was measured using bicinchoninic acid (BCA)-based colorimetric protein assay (Pierce; Rockford, IL).

Caspase Activity Assay Controls. Caspase 3 enzyme cleaves the p-nitroaniline (pNA)labeled substrate Ac-DEVD-pNA to release pNA, which was assayed colorimetrically at an absorbance wavelength of 405 mmol/liter at 10-minute intervals for an hour. Activity was expressed using slope of absorbance over time as pmol of substrate cleaved/ min. Purified recombinant caspases 3, 8, and 9 served as positive controls. Activity was measured with and without NO modulators: 1) S-nitroso-Nacetyl penicillamine (SNAP, 0.1–1 mmol/liter), a NO donor; 2) nitro-L-arginine methyl ester (LNAME, 0.1 mmol/liter), an inhibitor of endogenous NOS; and 3) enalaprilat, 0.1 mmol/liter, an angiotensin-converting enzyme (ACE) inhibitor. Studies were performed in myocardium from 1) eNOS ⫹/⫹ and eNOS ⫺/⫺ mice, 2) 4- and 9-month-old CMP hamsters and age-matched controls, and 3) explanted failing human hearts. Myocardial caspase activity. Baseline caspase 3 activity was measured by adding substrate to cell lysates. This was confirmed by the ability of caspase 3 inhibitor, Ac-DEVD-CHO, to inhibit this activity. Stimulated caspase 3 activity was measured by adding purified caspase 8 or 9 to cell lysates. The effect of NO agonists and antagonists on myocardial caspase 3 activity was measured by adding L-NAME, SNAP, or enalaprilat to cell lysates. Figure 1 shows the pathway of caspase 3 activation and the proposed role of NO in regulating this pathway. Assessment of ventricular function. Transthoracic echocardiography was performed in CMP and control hamsters at 4 and 9 months (n ⫽ 10 –12 in each group) using an Acuson 256 (Acuson

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Corporation, Siemens Co, CA) equipped with a 15-MHz linear transducer (15L8) with real-time digital acquisition, storage, and review capabilities. Hamsters were sedated for echocardiography using thiobutabarbital sodium 0.2 mg/kg, a short-acting barbiturate that is well-tolerated with no adverse effects. The heart was imaged in 2-dimensional mode in the parasternal short-axis view at the level of papillary muscles to obtain LV wall thickness and fractional shortening, in the apical 4-chamber view to measure aortic flow velocity with pulsed-wave Doppler, and in the parasternal long-axis view to measure aortic root diameter and LV systolic and diastolic volumes and ejection fraction. Three beats were averaged for each measurement. All calculations were derived using standard formulas.17

Statistics Data were analyzed using 2-way analysis of variance between groups and 2-way repeated measures analysis of variance within the same group across different ages for caspase activity. Paired and unpaired t-test were used to compare echocardiographic variables. Student-Newman-Keuls post hoc analysis was used to identify which means were different (Sigma Stat Version 3.0, Jandel Scientific; San Rafael, CA). A p value ⱕ 0.05 was considered significant.

RESULTS Controls We used commercially available purified recombinant caspases 3, 8, and 9 as positive controls in the absence of cell lysates to investigate the ability of NO to directly inhibit caspases: 1 mmol/liter SNAP inhibited all 3 purified caspases— caspase 3 (47%), caspase 8 (13%), and caspase 9 (25%), and 0.1 mmol/liter SNAP selectively inhibited caspase 3 (by nearly 20%) but had no effect on caspase 8 or 9 (Figure 2). This suggests a direct inhibitory effect of NO on caspase 3. Unlike SNAP, L-NAME and enalaprilat had no direct effect on caspase 3 activity in the absence of cell lysates (not shown).

Myocardial Caspase Activity There was low baseline caspase 3 activity (⬍1 pmol/10 ␮g/min) in all cell lysates. Caspases 8 and 9 were added to amplify baseline tissue caspase 3 activity. Transgenic mice. Heart weight/body weight ratio did not significantly differ between 9-month-old eNOS ⫹/⫹ (5.0 ⫾ 0.3 mg/g) and eNOS ⫺/⫺ (5.2 ⫾ 0.2 mg/g) mice.

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FIGURE 1 Pathway of caspase activation and its regulation: Nitric oxide is synthesized in

endothelial cells in part by bradykinin activation of B2 kinin receptor. Nitric oxide synthesized in endothelial cells can diffuse into surrounding myocytes to inhibit caspase activation. Caspase 3 is a common downstream effector caspase that is activated by upstream caspases 8 (by death-receptor pathway) and 9 (by mitochondrial pathway). Caspase 3 promotes apoptosis and ventricular remodeling. The following modulators of NO availability were used: ACE inhibitor that prevents kinin degradation; L-NAME that inhibits NO synthase; and SNAP, a NO donor. Caspase activity was measured. Ventricular morphology and function in hamsters was assessed using echocardiography. ACEI, angiotensin-converting enzyme inhibitor; L-NAME, nitro-L-arginine-methyl ester; NO, nitric oxide; SNAP, S-nitroso-N-acetyl penicillamine.

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FIGURE 3 Caspase 3 activity (pmol/10 ␮g/min) in

FIGURE 2 Effect of SNAP on purified human

recombinant caspase activity (pmol/min). 1 mmol/liter SNAP (hatched bar) inhibited (a) caspase 3 by 47%, (b) caspase 8 by 13%, and (c) caspase 9 by 25%. 0.1 mmol/ liter SNAP (solid bars) inhibited caspase 3 by 20% but had no effect on caspases 8 or 9. Therefore, 0.1 mmol/ liter SNAP selectively and directly inhibited caspase 3. *p ⬍ 0.05 from baseline; ***p ⬍ 0.001 from baseline. SNAP, S-nitroso-N-acetyl penicillamine.

Stimulated caspase 3 activity was lower in cell lysates from eNOS ⫹/⫹ mouse hearts. Addition of LNAME increased caspase 3 activity to levels seen in eNOS ⫺/⫺ mouse hearts (Figure 3A), whereas 0.1 mmol/liter SNAP decreased caspase 3 activity in both groups (Figure 3B).

myocardial cell lysates from 8 eNOS ⫹/⫹ and 18 eNOS ⫺/⫺ mice. (a) Stimulated caspase 3 activity (hatched bars) was lower in cell lysates from eNOS ⫹/⫹ mouse hearts compared with eNOS ⫺/⫺ mouse hearts. NO inhibitor L-NAME increased caspase 3 activity in eNOS ⫹/⫹ mouse hearts to levels seen in eNOS ⫺/⫺ mouse hearts (open bars). L-NAME had no effect in the eNOS ⫺/⫺ group. (b) NO donor SNAP significantly decreased caspase 3 activity in both eNOS ⫹/⫹ (open circles) and eNOS ⫺/⫺ (solid circles) mouse hearts. *p ⬍ 0.05 from baseline. #p ⬍ 0.05 from controls. eNOS, endothelial nitric oxide synthase; L-NAME, nitro-L-arginine-methyl ester; NO, nitric oxide; SNAP, S-nitroso-N-acetyl penicillamine.

CMP hamsters. Heart weight/body weight ratio was significantly higher in 9-month-old CMP hamsters compared with controls and 4-month-old CMP hamsters. Cardiomyopathic hamsters showed progressive LV wall thinning and dilatation and decrease in LV function (Table I). At 4 months, LV mass increased in proportion to increase in volume with preservation of cardiac output. By 9 months, mass did not

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TABLE I

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Echocardiography in normal and cardiomyopathic (CMP) hamsters

n Heart/body weight (mg/g) Heart rate (bpm) LV diastolic wall thickness (mm) LV end-diastolic diameter (mm) Fractional shortening (%) Ejection fraction (%) LV mass (␮g) LV volume (␮l) LV mass/volume (mg/ml) Cardiac output (ml/kg/min)

Control 4 months

CMP 4 months

Control 9 months

CMP 9 months

5 3.4 ⫾ 0.1 384 ⫾ 25 1.32 ⫾ 0.06 4.38 ⫾ 0.2 38.3 ⫾ 3 64 ⫾ 2 256 ⫾ 23 328 ⫾ 26 3.11 ⫾ 0.34 3.8

12 3.5 ⫾ 0.1 379 ⫾ 16 0.86 ⫾ 0.03* 5.75 ⫾ 0.14* 21 ⫾ 2* 35 ⫾ 2* 277 ⫾ 24 457 ⫾ 34* 1.45 ⫾ 0.1 3.7

10 3.2 ⫾ 0.1 367 ⫾ 14 1.17 ⫾ 0.06 4.79 ⫾ 0.11 38 ⫾ 2 65 ⫾ 1 264 ⫾ 20 361 ⫾ 24 2.41 ⫾ 0.18 3.2

10 5.1 ⫾ 0.2*† 216 ⫾ 26*† 0.78 ⫾ 0.05* 6.94 ⫾ 0.2*† 11 ⫾ 2*† 26 ⫾ 2*† 343 ⫾ 34* 667 ⫾ 59*† 1.0 ⫾ 0.05*† 1.4*

*p ⬍ 0.01 from age-matched normal hamster; †p ⬍ 0.01 from 4-month-old CMP hamster. LV, left ventricle.

increase in proportion to the increase in LV volume, and mass/volume ratio, ventricular function, and cardiac output decreased significantly, associated with the development of decompensated heart failure. Hearts from control, nonfailing hamsters showed no significant decrease in mass/volume ratio or change in ventricular function between 4 and 9 months. Stimulated caspase 3 activity was lower in cell lysates from normal hamsters (3.3 ⫾ 0.3 pmol/10 ␮g/min) compared with CMP hamsters (9.6 ⫾ 0.7 and 6.9 ⫾ 0.4 pmol/10 ␮g/min at 4 and 9 months, respectively). L-NAME increased caspase 3 activity in normal hamsters to levels seen in CMP hamsters but had no effect in CMP hamsters (Figure 4A). However, 0.1 mmol/liter SNAP decreased enzyme activity in both groups by ⬎50% (Figure 4B). Explanted failing human hearts. Median age of patients undergoing transplantation from whom myocardium was obtained was 13 years (range, 1 month to 65 years), 5 were male patients. Diagnoses included 3 with ischemic CMP; 2 with idiopathic dilated CMP; 3 with restrictive CMP; and 2 with complex congenital heart disease, including 1 with severe neonatal Ebstein’s anomaly and 1 with failed single ventricle physiology. All patients were in New York Heart Association Class 4 and average hemodynamics were as follows: cardiac index, 2.1 ⫾ 0.6 liter/min/m2; pulmonary capillary wedge pressure, 17 ⫾ 6 mm Hg; and mixed venous hemoglobin saturations, 62% ⫾ 3%. Stimulated caspase 3 activity in cell lysates was 8.5 ⫾ 1.0 pmol/10 ␮g/min. A total of 0.1 mmol/ liter SNAP decreased enzyme activity by 50%. As with CMP hamsters, L-NAME had no effect on

caspase activity, indicating low endogenous NO availability (Figure 5A). S-nitroso-N-acetyl penicillamine prevented the activation of caspase 3 in cell lysates in response to caspase 8 or 9 (Figure 5B). Also, stimulated caspase 3 activity was 28% lower when cell lysates were pre-incubated with 0.1 mmol/liter enalaprilat. Adding L-NAME reversed this effect (Figure 5C), indicating a NOdependent mechanism.

DISCUSSION Reduced endothelial NO availability in heart failure increases myocardial caspase 3 enzyme activity in association with progressive ventricular remodeling and dysfunction. Agents that release NO or increase NO synthesis, including ACE inhibitor enalaprilat inhibit caspase 3 activity independent of the pathway of caspase activation and may prevent or reverse the remodeling process through inhibition of apoptosis. Heart failure is a major health problem, with more than 500,000 new cases diagnosed every year, of which 60,000 progress to end-stage heart failure with only a 50% 1- to 2-year survival. Recent studies suggest that the remodeling process can be reversed to prevent the development or progression of heart failure. A major part of cell loss in heart failure is caused by apoptosis and modulating the factors that control apoptosis can prevent this component of cell death. Two recent reports highlight this: inhibiting caspase 3 prevented the development of dilated cardiomyopathy in pro-caspase 8 overexpressing transgenic mice18 and improved LV function in rabbits with pacing-induced heart failure.19 We in-

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FIGURE 4 Caspase 3 activity (pmol/10 ␮g/min) in

myocardial cell lysates from 8 control and 7 CMP hamsters. (a) Stimulated caspase 3 activity was lower in cell lysates from control hamsters compared with CMP hamsters (open bars). L-NAME increased caspase 3 activity in control but not CMP hamsters (hatched bars). (b) SNAP prevented caspase 3 activation in response to caspase 8 in control (open circles) and CMP hamsters (solid hamsters). *p ⬍ 0.05 from baseline; #p ⬍ 0.05 from control. CMP, cardiomyopathic; L-NAME, nitro-L-arginine-methyl ester; SNAP, S-nitroso-N-acetyl penicillamine.

vestigated the ability of NO to inhibit caspase 3. We found that high-dose NO non-selectively inhibits caspases 3, 8, and 9. This was consistent with the findings of other investigators who have shown that NO can inhibit as many as 7 different caspases by S-nitrosylation.8,9 However, lower dose SNAP selectively inhibited caspase 3, suggesting that different caspases may have different susceptibilities to Snitrosylation by NO.20 This requires confirmation with further experimental work. The ability of NO to selectively inhibit downstream caspase 3 at low

FIGURE 5 Caspase activity (pmol/10 ␮g/min) in explanted failing human myocardium (n ⫽ 10). (a) Stimulated caspase 3 activity in the cell lysates was 8.5 ⫾ 1.0 pmol/10 ␮g/min. NO donor SNAP (open circles) caused a dose-dependent reduction in caspase 3 activity. NO synthase inhibitor L-NAME (solid circles) did not alter caspase-3 activity. (b) SNAP prevented caspase 3 activation in response to caspases 8 (open triangles) and 9 (solid triangles). (c) Angiotensin-converting enzyme inhibitor enalaprilat decreased caspase 3 activity and this effect was reversed by L-NAME, indicating a NOdependent mechanism of action of enalaprilat. *p ⬍ 0.05 from baseline; **p ⬍ 0.01 from baseline. L-NAME, nitro-L-arginine-methyl ester; NO, nitric oxide; SNAP, S-nitroso-N-acetyl penicillamine.

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doses makes it an attractive therapeutic target for preventing apoptosis. A recent study reported that transgenic mice lacking eNOS are more susceptible to ischemia– reperfusion injury.21 In our study, the lower myocardial caspase 3 activity in eNOS wild-type mouse hearts and the ability of L-NAME to increase the activity suggest that eNOS-derived NO tonically inhibits caspase 3 activity in normal hearts. The inability of L-NAME to reverse caspase 3 activity in eNOS-deficient mouse hearts suggests that the higher caspase 3 activity in this group may relate in part to absence of eNOS and that the cardioprotective effect of NO may relate in part to reduced apoptotic enzyme activation. It is important to differentiate this anti-apoptotic effect of endothelial NO production from that of NO derived from inducible NOS (iNOS) isoform.22 Higher rates of NO production as seen after iNOS induction, have been reported to overwhelm cellular protective mechanisms and cause apoptosis partly through formation of peroxynitrite.23,24 In our study, the lack of effect of L-NAME on enzyme activity in eNOSdeficient mice excludes the possibility that iNOSderived NO in these mice up-regulated caspase 3 activity. Several studies have shown that NO availability decreases in heart failure in humans.25–27 We have previously reported that eNOS is down-regulated in dogs with pacing-induced heart failure12 and loss of NO production is associated with transition from compensated to decompensated heart failure.28 The development of end-stage heart failure in hamsters is also associated with reduced NO availability.29 In the current study, serial echocardiographic evaluation of cardiac structure and function in hamsters demonstrated at least 2 distinct phases in the evolution of heart failure. At 4 months, LV mass increased with an increase in LV volume with preservation of cardiac output. By 9 months, the increase in LV mass was unable to compensate for the marked increase in LV volume and was associated with a decrease in cardiac output and decompensated heart failure. Our finding that heart weight/body weight ratio was preserved in 4-monthold hamsters but significantly higher (160%) in 9-month-old hamsters is also consistent with an increase in ventricular mass and volume in end-stage heart failure as seen in explanted failing human hearts.30 Of note, heart rate was significantly lower in end-stage CMP hamsters, which has not been previously reported in published studies using other strains of Syrian CMP hamsters.31,32 However, inhi-

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bition of NO using nitro-L-arginine produces a dose-dependent bradycardia in normal and CMP hamsters.32 Similarly, eNOS-deficient mice have been shown to develop mild hypertension and bradycardia.33 Although we did not evaluate the cause of bradycardia in severe heart failure in hamsters, the lack of NO may be an explanation. To determine whether reduced NO availability in failing hearts is associated with altered regulation of caspase activation, we measured caspase activity in the different stages of CMP. The lower caspase 3 activity in normal hamsters and the ability of LNAME to increase the activity indicate that endogenous NO inhibits caspase 3. The failure of LNAME to increase caspase activity in CMP hamsters indicates lowered endogenous NO availability. Further, caspase 8 caused greater stimulation of tissue caspase 3 in CMP hamsters compared with controls. However, 0.1 mmol/lilter SNAP prevented this activation in both groups. This confirmed our hypothesis that NO can prevent the activation of myocardial caspase 3 by an upstream caspase. Interestingly, in the CMP hamsters, caspase 3 activity was higher at 4 months than at 9 months, when heart failure is end-stage. It is important to recognize that by 9 months, there is gross scarring and fibrosis within the myocardium of CMP hamsters, the hallmark of necrotic cell death. Takeda et al34 reported significantly greater fibrosis (8%–16%) in hearts from hamsters with dilated CMP compared with ⬍2% in control hamster hearts. The presence of fibrosis may result in underestimation of myocardial caspase activity. However, at some point in the evolution of heart failure, probably between 4 to 9 months in hamsters, apoptotic cell death may be replaced by predominantly necrotic cell death. A recent study using explanted human hearts showed evidence for cytochrome c– dependent activation of caspase 3 in human CMP.6 Because the cytochrome c pathway depends on caspase 9 activation, we measured caspase 9 activity in addition to caspase 8 in human hearts. In human tissues, SNAP prevented caspase 3 activation but, as in CMP hamsters, L-NAME failed to increase enzyme activity, indicating reduced NO availability. These data are consistent with our previous studies in coronary microvessels showing that NO production is low in these failing human hearts.30 Use of ACE inhibitors can increase NO production in failing human hearts.35 Incubation of cell lysates with enalaprilat, an ACE inhibitor, decreased caspase 3 activity. This effect was reversed by L-NAME, indicating that

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although baseline NO availability may be reduced in failing hearts, an ACE inhibitor such as enalaprilat can stimulate local NO production to decrease caspase activation. These data are consistent with our previous studies showing that various pharmacologic or surgical interventions can restore NO synthesis in failing human myocardium.36,37 Finally, in human hearts, as in hamster hearts, NO donor SNAP prevented the activation of caspase 3 by upstream caspase 8; SNAP also decreased caspase 3 activation in response to caspase 9 but to a lesser extent (12% reduction in activity). This indicates that NO can inhibit downstream caspase 3 activation independent of the apoptotic signaling pathway. Nonetheless, these data should be interpreted with caution because the small sample size precluded the ability to determine the effect of other potentially confounding variables, including cause and severity of heart failure and pre-transplant use of other medications on myocardial caspase activation. The methodology used in the current study have unique advantages. Most studies of apoptosis in cell culture preparations require the use of growth factors, apoptosis-inducing agents, or transfection of cells with recombinant caspases to amplify caspase activity, which is useful to study signal transduction pathways but less relevant to disease states. Use of tissue from normal and heart failure models in our study permitted evaluation of the effect of endogenous NO on caspase activation in disease states. Because these assays were performed on tissue homogenates, the cell types responsible for enzyme activity cannot be determined, but, given the high proportion of myocytes, at least a portion of this activity likely can be attributed to these cells. Endothelium is the predominant source of NO, and we have previously shown that NO produced in coronary microcirculation can act on surrounding myocytes.38 Use of cell lysates that retain eNOS, which is a membrane-bound protein, provides a means of testing for the effect of endogenous NO on myocardial caspase activity. The major limitation of studying human hearts is the lack of normal, nonfailing tissue. Therefore caspase activity in failing hearts could not be compared with controls. Nonetheless, data obtained from these studies have significant implications because they allow direct clinicopathologic correlation and assessment of the effect of interventions on the pathophysiology of heart failure. In conclusion, endogenous endothelium-derived NO tonically inhibits myocardial caspase activity and prevents caspase 3 activation by upstream

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caspases. The ability of NO to inhibit downstream caspase 3 has the potential to rescue a cell from apoptosis even after the caspase cascade has been activated. Loss of endogenous endothelial NO production up-regulates myocardial caspase 3 activity and is associated with progressive ventricular remodeling and heart failure. Use of agents that increase NO bioavailability can inhibit caspase activation and prevent or reverse progressive cardiomyocyte loss and remodeling in heart failure. REFERENCES 1. Anversa P, Li P, Zhang X, Olivetti G, Capasso JM. Ischaemic myocardial injury and ventricular remodelling. Cardiovasc Res 1993;27:145–57. 2. Reed JC, Tomaselli KJ. Drug discovery opportunities from apoptosis research. Curr Opin Biotechnol 2000;11(6):586 – 92. 3. Nicholson DW, Thornberry NA. Caspases: killer proteases. Trends Biochem Sci 1997;22:299 –306. 4. Ellis RE, Yuan JY, Horvitz HR. Mechanisms and functions of cell death. Annu Rev Cell Biol 1991;7:663–98. 5. Raff M. Cell suicide for beginners. Nature 1998;396:119 –22. 6. Narula J, Pandey P, Arbustini E, et al. Apoptosis in heart failure: release of cytochrome c from mitochondria and activation of caspase-3 in human cardiomyopathy. Proc Natl Acad Sci 1999;96:8144 –9. 7. Mannick JB, Asano K, Izumi K, Kieff E, Stamler JS. Nitric oxide produced by human B lymphocytes inhibits apoptosis and Epstein-Barr virus reactivation. Cell 1994;79:1137–46. 8. Li J, Billiar TR, Talanian RV, Kim YM. Nitric oxide reversibly inhibits seven members of the caspase family via Snitrosylation. Biochem Biophys Res Comm 1997;240:419 –24. 9. Rossig L, Fichtlscherer B, Breitschopf K, et al. Nitric oxide inhibits caspase-3 by S-nitrosation in vivo. J Biol Chem 1999;274(11):6823–6. 10. Weiland U, Haendeler J, Ihling C, et al. Inhibition of endogenous nitric oxide synthase potentiates ischemia-reperfusion induced myocardial apoptosis via a caspase-3 dependent pathway. Cardiovasc Res 2000;45:671–8. 11. Feng Q, Song W, Xiangru L. Endothelial NO synthase deficiency increases apoptosis in embryonic heart and induces congenital septal defects (abstract). Circulation 2000; 102:520. 12. Smith CJ, Sun D, Hoegler C, et al. Reduced gene expression of vascular endothelial nitric oxide synthase and cyclooxygenase-1 in heart failure. Circ Res 1996;78:58 –64. 13. Wang J, Seyedi N, Xu XB, Wolin MS, Hintze TH. Defective endothelium-mediated control of coronary circulation in conscious dogs after heart failure. Am J Physiol 1994;266: H670 –H680. 14. Zhao G, Shen W, Xu X, Ochoa M, Bernstein R, Hintze TH. Selective impairment of vagally mediated, nitric oxide-dependent coronary vasodilation in conscious dogs after pacinginduced heart failure. Circulation 1995;91:2655–63. 15. Sole MJ, Liew CC. Catecholamines, calcium and cardiomyopathy. Am J Cardiol 1988;62:20G–24G. 16. Smith CJ, He J, Ricketts SG, Ding J-Z, Moggio RA, Hintze TH. Downregulation of right ventricular phosphodiesterase

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