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

Presented in part at the Annual Meeting of the International Society for Heart and Lung Transplantation, Vancouver, April 2001.
https://doi.org/10.1016/S1053-2498(01)00404-1Get rights and content

Abstract

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 angiotensin-converting 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 μg/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 μg/min, compared with CMP hamsters, 9.6 ± 0.7 and 6.9 ± 0.4 pmol/10 μg/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, L-NAME 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 activity. Agents that promote NO synthesis, including ACE inhibitors, may prevent caspase activation in HF.

Section snippets

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

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

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

References (38)

  • M Pittis et al.

    Canine coronary microvessel NO production regulates oxygen consumption in ecNOS knockout mouse heart

    J Mol Cell Cardiol

    (2000)
  • P Anversa et al.

    Ischaemic myocardial injury and ventricular remodelling

    Cardiovasc Res

    (1993)
  • R.E Ellis et al.

    Mechanisms and functions of cell death

    Annu Rev Cell Biol

    (1991)
  • M Raff

    Cell suicide for beginners

    Nature

    (1998)
  • J Narula et al.

    Apoptosis in heart failurerelease of cytochrome c from mitochondria and activation of caspase-3 in human cardiomyopathy

    Proc Natl Acad Sci

    (1999)
  • U Weiland et al.

    Inhibition of endogenous nitric oxide synthase potentiates ischemia-reperfusion induced myocardial apoptosis via a caspase-3 dependent pathway

    Cardiovasc Res

    (2000)
  • Q Feng et al.

    Endothelial NO synthase deficiency increases apoptosis in embryonic heart and induces congenital septal defects

    Circulation

    (2000)
  • C.J Smith et al.

    Reduced gene expression of vascular endothelial nitric oxide synthase and cyclooxygenase-1 in heart failure

    Circ Res

    (1996)
  • J Wang et al.

    Defective endothelium-mediated control of coronary circulation in conscious dogs after heart failure

    Am J Physiol

    (1994)
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    This work was supported by HL 50142, HL 61290, and PO-1 HL 43023 from the National Heart, Lung and Blood Institute.

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