Infarct Size and Nitric Oxide Synthase in Murine Myocardium

doi:10.1006/jmcc.1999.1050

Journal of Molecular and Cellular Cardiology

Volume 32, Issue 1, January 2000, Pages 35-42

https://doi.org/10.1006/jmcc.1999.1050 Get rights and content

Abstract

M. S. Sumeray, D. D. Rees and D. M. Yellon. Infarct Size and Nitric Oxide Synthase in Murine Myocardium.Journal of Molecular and Cellular Cardiology (2000) 32, 35–42. Controversy surrounds the involvement of nitric oxide (NO) in myocardial ischaemia-reperfusion injury and the balance between deleterious and beneficial effects. NO synthase (NOS) is expressed constitutively as two isoforms: endothelial (eNOS) and neuronal (nNOS). Knockout mice lacking the gene for either eNOS (eNOS KO) or nNOS (nNOS KO), were compared with wild-types (WT) during a protocol of global ischaemia-reperfusion injury. Thirty-six mouse hearts (12 from each group) were isolated and the aorta cannulated for Langendorff perfusion with modified Krebs solution at constant pressure. An apical suture connected the left ventricle to a force transducer via a light weight coupling rod. Following stabilization hearts were subjected to 30 min of global ischaemia at 37°C. During 30 min reperfusion, the recovery of baseline force-rate product (F%) was recorded. Hearts were then stained with tetrazolium, frozen, sliced, and fixed with formalin. Slices were compressed between plexiglas plates, and a magnified video image digitized to allow planimetry for infarct size (as percentage of ventricular volume I/R). Although recovery of contractile function did not differ between groups, eNOS KOs suffered significantly larger infarcts than WT or nNOS KOs (41 v 33 and 30% respectively,P <0.05 for both comparisons). A protective role for eNOS against global ischaemia-reperfusion injury has been demonstrated for the first time in murine myocardium. This may have important clinical implications for future pharmacotherapy to enhance myocardial protection.

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Cited by (101)

Red blood cell eNOS is cardioprotective in acute myocardial infarction
2022, Redox Biology
Citation Excerpt :
This was somehow unexpected as NO inhalation and NO donors such as nitroglycerine are well known to exert cardioprotective effects via cGMP-dependent signaling in the heart; cardioprotection was also observed with overexpression of eNOS in cardiomyocytes [36] (for a review see Refs. [1,4]). However, previous studies carried out in global eNOS KO strains showed different outcomes after AMI depending on the particular eNOS KO strain under study, and the ischemia time and conditions [5–12]. Specifically, the Huang's global eNOS KO model showed increased infarct size and myocardial necrosis [7,37], which was attributable to a permanent decrease in coronary flow [9].
Red blood cells (RBCs) were shown to transport and release nitric oxide (NO) bioactivity and carry an endothelial NO synthase (eNOS). However, the pathophysiological significance of RBC eNOS for cardioprotection in vivo is unknown. Here we aimed to analyze the role of RBC eNOS in the regulation of coronary blood flow, cardiac performance, and acute myocardial infarction (AMI) in vivo. To specifically distinguish the role of RBC eNOS from the endothelial cell (EC) eNOS, we generated RBC- and EC-specific knock-out (KO) and knock-in (KI) mice by Cre-induced inactivation or reactivation of eNOS. We found that RBC eNOS KO mice had fully preserved coronary dilatory responses and LV function. Instead, EC eNOS KO mice had a decreased coronary flow response in isolated perfused hearts and an increased LV developed pressure in response to elevated arterial pressure, while stroke volume was preserved. Interestingly, RBC eNOS KO showed a significantly increased infarct size and aggravated LV dysfunction with decreased stroke volume and cardiac output. This is consistent with reduced NO bioavailability and oxygen delivery capacity in RBC eNOS KOs. Crucially, RBC eNOS KI mice had decreased infarct size and preserved LV function after AMI. In contrast, EC eNOS KO and EC eNOS KI had no differences in infarct size or LV dysfunction after AMI, as compared to the controls. These data demonstrate that EC eNOS controls coronary vasodilator function, but does not directly affect infarct size, while RBC eNOS limits infarct size in AMI. Therefore, RBC eNOS signaling may represent a novel target for interventions in ischemia/reperfusion after myocardial infarction.
Regular dipyridamole therapy produces sustained protection against cardiac ischemia-reperfusion injury: Is it time to revisit PARIS?
2014, International Journal of Cardiology
Increased activated Akt and eNOS expression coincide with this persistent cardioprotection. Emergent coronary reperfusion therapies are rarely carried out before considerable myocardial injury has occurred. Moreover, reperfusion after prolonged ischemia produces paradoxical ischemia–reperfusion injury, attenuating the efficacy of reperfusion therapies. This has provided impetus for identifying chronic therapies to protect against ischemia–reperfusion injury in those at risk. We previously found that regular dipyridamole therapy produces a chronic preconditioning-like effect mediated through adenosine A1 receptors.
To determine how long this chronic preconditioning effect of dipyridamole remains present after discontinuing therapy, guinea pigs received 4 mg/kg/day in their water for 6 weeks. Ischemia–reperfusion was performed at 0, 2, 3, and 4 days after dipyridamole discontinuation (0 day, 2 days, 3 days and 4 days; n=8 per group). Left ventricular developed pressure (LVDP), end-diastolic pressure (LVEDP), coronary flow (CF), infarct size, and western blot analyses for Akt and endothelial nitric oxide synthase (eNOS) were studied.
After ischemia–reperfusion, 0 day, 2 days and 3 days, but not 4 days, had significantly higher LVDP and lower LVEDP compared to control. Myocardial infarct size was significantly reduced at 0 day, 2 days and 3 days, but not 4 days, compared to control. Western blot analyses demonstrated upregulation of phospho-Akt and phospho-eNOS expression at 0 day, 2 days, and 3 days, but not 4 days.
A chronic preconditioning-like cardioprotection by regular dipyridamole treatment persists for 3 days after discontinuing therapy. Increased activated Akt and eNOS expression may play a role in this persistent cardioprotection.
S-nitrosylation of TRIM72 mends the broken heart: A molecular modifier-mediated cardioprotection
2014, Journal of Molecular and Cellular Cardiology
The effects of iloprost on ischemia-reperfusion injury in skeletal muscles in a rodent model
2014, Journal of Surgical Research
The aim of this study was to investigate the effects of iloprost (IL) on ischemia-reperfusion injury in a rodent model.
Twenty-four Wistar Albino rats were randomized into four groups (n = 6). Laparotomy was performed in all groups under general anesthesia. Only laparotomy was applied in group S (Sham). Ischemia-reperfusion group (group I/R) underwent ischemia and reperfusion performed by clamping and declamping of the infrarenal abdominal aorta for 120 min. The iloprost group (group IL) received intravenous infusion of IL 0.5 ng/kg/min, without I/R. Group I/R + IL received intravenous infusion of IL 0.5 ng/kg/min immediately after 2 h period of ischemia. At the end of the reperfusion period, all rats were killed under anesthesia and skeletal muscle samples of lower extremity were harvested for biochemical and histopathologic analyses.
Tissue levels of endothelial nitric oxide were significantly higher in I/R groups than those in groups S and IL. The heat shock protein 60 levels were higher in group I/R than the other groups. But the heat shock protein 60 levels in group I/R + IL were found to be similar with the groups S and IL. Malondialdehyde levels were significantly higher in group I/R. On the other hand, in group I/R + IL, malondialdehyde levels were higher than those in groups S and IL but lower than those in group I/R. Superoxide dismutase (SOD) enzyme activities were found to be significantly lower in group I/R than the other groups. Also in group I/R/I, the SOD enzyme activities were higher than those in group I/R. But, in group I/R + IL, SOD levels were found to be higher than those in group I/R but lower than those in groups S and IL.
These results indicate that IL has protective effects on I/R injury in skeletal muscle in a rodent model.
Ischemic Heart Disease and its Consequences
2014, Cellular and Molecular Pathobiology of Cardiovascular Disease
Ischemic heart disease remains a leading cause of morbidity and mortality in all industrialized nations, resulting from blockage of one or more coronary arteries. Although the treatment of myocardial ischemia continues to improve with more timely and effective reperfusion, no effective therapy exists to prevent or attenuate the resulting myocardial ischemia–reperfusion injury. Moreover, the major determinant of the long-term outcome is the amount of myocardium that is destroyed as a result of ischemic injury (i.e., the size of infarction). Thus, it is believed that a significant reduction in myocardial infarct size will decrease subsequent morbidity and mortality. As such, interventions designed to effectively reduce myocardial infarction are still needed. This chapter will open with a brief description of the pathophysiology of ischemia–reperfusion injury before transitioning into a discussion on the signaling cascades that have been associated with cardioprotection. Specifically, this chapter will highlight the Reperfusion Injury Salvage Kinase (RISK) pathway, the Survivor Activating Factor Enhancement (SAFE) pathway, and AMP-activated protein kinase (AMPK) signaling, which all confer cardioprotection when specifically activated at the onset of reperfusion following myocardial ischemia by signaling through the mitochondria. This chapter will also provide some insights into several therapeutic strategies, such as gasotransmitters (nitric oxide, hydrogen sulfide, carbon monoxide), micro-RNA, and stem cells, which have the ability to activate multiple cardioprotective signaling cascades. Finally, this chapter will provide a discussion on why therapeutic trials for new drugs designed to reduce ischemia–reperfusion injury have not been successful in the past.
Endothelial nitric oxide synthase mediates the first and inducible nitric oxide synthase mediates the second window of desflurane-induced preconditioning
2013, Journal of Cardiothoracic and Vascular Anesthesia
Nitric oxide synthases (NOSs) mediate the first window of anesthetic-induced preconditioning (APC). The authors tested the hypothesis that endothelial NOS (eNOS) mediates the first window and inducible NOS (iNOS) mediates the second window of APC.
Randomized, prospective, blinded laboratory investigation.
Experimental laboratory.
Mice.
Mice were subjected to a 45-minute coronary artery occlusion (CAO) and a 180-minute reperfusion. C57BL/6 mice received desflurane, 1.0 minimum alveolar concentration, for 30 minutes or 12, 24, 48, or 96 hours before CAO. In eNOS^−/− and iNOS^−/− mice, desflurane was given 30 minutes and 48 hours before CAO. In the control groups, no desflurane was administered. Myocardial infarct size (IS) was determined after staining with Evans blue and triphenyltetrazolium chloride.
The second window of APC was detectable at 48 hours but not at 12, 24, and 96 hours after preconditioning. In the control groups, IS was not different among the wild-type (50 ± 10%), eNOS^−/− (52 ± 14%), and iNOS^−/− (46 ± 10%) mice. The IS decreased significantly (p < 0.05) when desflurane was administered 30 minutes (10 ± 6%) or 48 hours (16 ± 7%) before CAO in wild-type mice, 48 hours (21 ± 13%) before CAO in eNOS^−/− mice, and 30 minutes (13 ± 6%) before CAO in iNOS^−/− mice. Desflurane given 30 minutes before CAO in eNOS^−/− mice (60 ± 10%) and 48 hours before CAO in iNOS^−/− mice (48 ± 21%) did not decrease the IS significantly compared with controls.
Endothelial NOS and iNOS work independently to mediate the first and second windows of APC, respectively. Endothelial NOS is not necessary to trigger the second window of APC.

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^☆: Please address all correspondence to: Professor D. M. Yellon, The Hatter Institute, Department of Academic and Clinical Cardiology, University College Hospital, Grafton Way, London WC1E 6DB, UK.

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Journal of Molecular and Cellular Cardiology

Abstract

References (24)

Cardiovascular actions of inhibitors of endothelium-derived relaxing factor (nitric oxide) formation/release in anesthetized dogs

Eur J Pharmacol

Nitric oxide and its role in the cardiovascular system

Prog Cardiovasc Dis

Targeted disruption of the neuronal nitric oxide synthase gene

Cell

Inhibition of nitric oxide limits infarct size in the in situ rabbit heart

Biochem Biophys Res Commun

Positive inotropic effect of nitric oxide in myocardium

Int J Cardiol

Nitric oxide release accounts for biologic activity of endothelium-derived relaxation factor

Nature

Nitric oxide: physiology, pathophysiology, and pharmacology

Pharmacol Rev

Nitric oxide synthesized from L-arginine regulates vascular tone in the coronary circulation of the rabbit

Br J Pharmacol

Role of basal release of nitric oxide on coronary flow and mechanical performance of the isolated rat heart

J Physiol Lond

Endothelium-mediated regulation of coronary tone

Basic Res Cardiol

Abnormal contractile function due to induction of nitric oxide synthesis in rat cardiac myocytes follows exposure to activated macrophage-conditioned medium

J Clin Invest

Modulation of left ventricular relaxation in isolated ejecting heart by endogenous nitric oxide

Am J Physiol

Cited by (101)

Red blood cell eNOS is cardioprotective in acute myocardial infarction

Regular dipyridamole therapy produces sustained protection against cardiac ischemia-reperfusion injury: Is it time to revisit PARIS?

S-nitrosylation of TRIM72 mends the broken heart: A molecular modifier-mediated cardioprotection

The effects of iloprost on ischemia-reperfusion injury in skeletal muscles in a rodent model

Ischemic Heart Disease and its Consequences

Endothelial nitric oxide synthase mediates the first and inducible nitric oxide synthase mediates the second window of desflurane-induced preconditioning

Regular ArticleInfarct Size and Nitric Oxide Synthase in Murine Myocardium☆

Abstract

Eur J Pharmacol

Prog Cardiovasc Dis

Cell

Biochem Biophys Res Commun

Int J Cardiol

Nitric oxide release accounts for biologic activity of endothelium-derived relaxation factor

Nature

Nitric oxide: physiology, pathophysiology, and pharmacology

Pharmacol Rev

Nitric oxide synthesized from L-arginine regulates vascular tone in the coronary circulation of the rabbit

Br J Pharmacol

Role of basal release of nitric oxide on coronary flow and mechanical performance of the isolated rat heart

J Physiol Lond

Endothelium-mediated regulation of coronary tone

Basic Res Cardiol

Abnormal contractile function due to induction of nitric oxide synthesis in rat cardiac myocytes follows exposure to activated macrophage-conditioned medium

J Clin Invest

Modulation of left ventricular relaxation in isolated ejecting heart by endogenous nitric oxide

Am J Physiol

Regular Article
Infarct Size and Nitric Oxide Synthase in Murine Myocardium☆