| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 4, 2003 as doi:10.1096/fj.03-0032fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Pharmacology and 1st Department of Internal Medicine, Health Science Center, and Department of Physical Chemistry and Research Group of Homogeneous Catalysis of Hungarian Academy of Sciences, University of Debrecen, Debrecen, Hungary;
* University of Connecticut Health Center, School of Medicine, Farmington, Connecticut, USA; and
University of Joseph Fourier, Department of Cardiovascular Pharmacology, Grenoble, France
2Correspondence: Department of Pharmacology, Health and Science Center, University of Debrecen, Nagyerdei krt. 98, 4032-Debrecen, Hungary. E-mail: tosaki{at}king.pharmacol.dote.hu
SPECIFIC AIMS
Recently the role of endogenous carbon monoxide (CO) production has been intensively investigated under physiological and pathological conditions. We studied the role and contribution of heme oxygenase-1 (HO-1) mRNA and its protein expression related to endogenous carbon monoxide (CO) production, and the development of reperfusion-induced ventricular fibrillation (VF) in isolated wild-type (+/+), HO-1 heterozygous (+/-), and HO-1 knockout homozygous (-/-) mouse myocardium.
PRINCIPAL FINDINGS
1. Immunohistochemical detection of HO-1 in nonfibrillated and fibrillated hearts
We assessed the role of HO-1-mediated CO production related to reperfusion-induced VF in isolated mouse heart preparation. Figure 1
shows immunohistochemical detection of HO-1 enzyme (left panel) and HO-1 protein expression (right panel) in isolated hearts subjected to 20 min of ischemia followed by 120 min of reperfusion. Upon reperfusion, HO-1 enzyme localization was detected (blue staining) in nonfibrillated/reperfused (No-VF) myocardium in wild-type (+/+) control mice (Fig. 1A
). However, in reperfused/fibrillated (VF) wild-type (+/+) myocardium, HO-1 enzyme detection by immunohistochemistry was reduced (lighter blue area). The same pattern of HO-1 enzyme detection was obtained in the heterozygous (+/-) nonfibrillated (No-VF) and fibrillated (VF) hearts (Fig. 1B
), indicating there is no substantial difference between the localization of HO-1 enzyme in wild-type (+/+) and heterozygous (+/-) mouse myocardium. Upon reperfusion, because of the absence of HO-1 enzyme in homozygous (-/-) mice, localization of HO-1 enzyme (lack of blue staining) was not possible in nonfibrillated or fibrillated homozygous (-/-) myocardium (Fig. 1C
). In homozygous (-/-) mouse studies, all hearts developed reperfusion-induced VF; therefore, electric defibrillation (Defib.) was required to measure cardiac function.
|
2. Detection of HO-1 protein in nonfibrillated and fibrillated hearts
Figure 1
also shows HO-1 protein expression (right panel) in reperfused/nonfibrillated (No-VF) and fibrillated (VF) wild-type (+/+), HO-1 heterozygous (+/-), and HO-1 knockout homozygous (-/-) mouse myocardium. Thus, corresponding to the immunohistochemical staining, HO-1 protein expression (right panel) was detected in ischemic/reperfused nonfibrillated (No-VF) wild-type (+/+) and nonfibrillated heterozygous (+/-) hearts. However, in HO-1 homozygous hearts (-/-), HO-1 protein was not detected (Fig. 1C
) in either ischemic/reperfused nonfibrillated (electrically defibrillated) or fibrillated myocardium. In reperfused/fibrillated wild-type (+/+) mouse heart (Fig. 1A
), a reduction in HO-1 protein expression was shown in comparison with the nonfibrillated myocardium. HO-1 protein detection was not seen in heterozygous (+/-) fibrillated myocardium (Fig. 1B
).
3. Endogenous CO detection in mouse myocardium
In additional studies, we ascertained whether VF affected endogenous carbon monoxide production in ischemic/reperfused hearts. Figure 2
depicts representative curves of endogenous CO production detected by gas chromatography in wild-type (+/+), HO-1 heterozygous (+/-), and HO-1 homozygous (-/-) mouse heart subjected to 20 min ischemia, followed by 30 min and 120 min of reperfusion. It is shown that in wild-type (+/+) and heterozygous (+/-) hearts subjected to 20 min of ischemia followed by 30 min (Fig. 2, b and c
chromatograms) and 120 min (Fig. 2, d and e
chromatograms) of reperfusion, a substantial increase in CO production was observed compared with the nonischemic wild-type (+/+, Fig. 2, a
chromatogram) myocardium. However, in the homozygous (-/-) mutant mouse myocardium (Fig. 2, f and g
chromatograms), endogenous CO production, because of the lack and functional defect of HO-1 system, was detected at a low level. The results (Fig. 2)
were obtained from wild-type, heterozygous, and homozygous mice showing no VF upon reperfusion, or hearts were immediately electrically defibrillated (in HO-1 homozygous knockout mice) if necessary. However, in the presence of VF, endogenous CO production was not detected (data not shown).
|
CONCLUSIONS AND SIGNIFICANCE
The sudden onset of severe ischemia in previously healthy myocardium sets into motion a series of pathological events (including ventricular arrhythmias) that continue until the cardiocytes die. These changes start seconds after the onset of ischemia and occur because of O2 is insufficient to support oxidative phosphorylation in the tissue. A growing awareness of genetically derived arrhythmias as a clinical encountered event has done much to redress the balance of research interest.
We have known for some time that a gaseous molecule exists besides nitric oxide that can be generated endogenously in various tissues, and this is CO. CO contributes to the regulation of vascular tone and is reported to have anti-ischemic and immunosuppressive properties that may contribute to the cytoprotective action of HO-1 in ischemic/reperfused myocardium.
In the present study, we have shown that HO-1 and HO-1-mediated endogenous CO production play an important role in myocardial homeostasis by protecting the heart from ischemia/reperfusion-induced injury, including reperfusion-induced VF. Indeed, after ischemia/reperfusion, there was extensive HO-1 mRNA and protein down-regulation in fibrillated HO-1 heterozygous and homozygous mouse myocardium. The lack of HO-1 mRNA and its protein was observed in aerobically perfused, without an episode of ischemia/reperfusion, homozygous mouse hearts. Upon reperfusion, all HO-1 knockout homozygous hearts developed reperfusion-induced VF, indicating the importance of the presence of HO-1 mRNA, its protein, and enzyme activity as antiarrhythmic substances in the tissue. Presumably the functional defect in HO-1 system correlates with the HO-1-related endogenous CO production. Thus, endogenous CO production was not detected or was at a very low level in fibrillated HO-1 knockout heterozygous and homozygous myocardium, whereas a substantial presence of endogenous CO was measured in ischemic/nonfibrillated myocardium. Furthermore, we found that infarct size was significantly increased in homozygous mouse hearts; this increase in infarct size related to a significant elevation in myocardial Na+ and Ca2+ accumulation and K+ loss.
In the present study, we endeavored to obtain more circumstantial evidence for the involvement of HO-1-related CO production and its direct measurement in the genesis of reperfusion-induced VF in wild-type, heterozygous, and homozygous mice. We now provide direct evidence by measuring cellular CO production suggesting that this is the case and the protective effect against the development of VF is attributable to the generation of endogenous CO through HO-1 mRNA induction. Thus, the stimulation of HO-1 mRNA, its protein expression, enzyme activity, and endogenous CO production demonstrates that endogenous CO production is essential for the prevention of reperfusion-induced VF.
How can heme oxygenase expression provide protection against ischemia/reperfusion-induced injury? Although the main subject of the present study is the delineation of HO-CO system in the ischemic/reperfused myocardium, some discussion of the putative mechanism of action of this pathway must include some mention of nitric oxide synthase (NOS) system. These two pathway systems show many similarities: both HO and NOS have distinct constitutive and inducible isoforms and both CO and NO stimulate soluble guanylyl cyclase to produce cGMP as the second messenger effector. Because NO an CO mediate their effects through the stimulation of guanylyl cyclase, it is necessary to demonstrate that an increased cardiac cGMP level is at least partly due to CO rather than NO in our model system.
In summary, we have shown using wild-type, HO-1 knockout heterozygous, and homozygous mice that HO-1 and HO-1-related endogenous CO production plays an important role in the adaptation of cardiovascular system to ischemia. We postulate that HO-1 may play a central role in cardiac physiology by protecting the heart from ischemia/reperfusion-induced damage. Thus, our findings suggest that pharmacological stimulation of HO-1 expression could prevent the development of reperfusion-induced VF.
|
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0032fje; doi: 10.1096/fj.03-0032fje 
This article has been cited by other articles:
|
|
 |
|
  I. Bak, I. Lekli, B. Juhasz, N. Nagy, E. Varga, J. Varadi, R. Gesztelyi, G. Szabo, L. Szendrei, I. Bacskay, et al. Cardioprotective mechanisms of Prunus cerasus (sour cherry) seed extract against ischemia-reperfusion-induced damage in isolated rat hearts Am J Physiol Heart Circ Physiol, September 1, 2006; 291(3): H1329 - H1336. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Abuarqoub, R. Foresti, C. J. Green, and R. Motterlini Heme oxygenase-1 mediates the anti-inflammatory actions of 2'-hydroxychalcone in RAW 264.7 murine macrophages Am J Physiol Cell Physiol, April 1, 2006; 290(4): C1092 - C1099. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Wu and R. Wang Carbon Monoxide: Endogenous Production, Physiological Functions, and Pharmacological Applications Pharmacol. Rev., December 1, 2005; 57(4): 585 - 630. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Saito, N. Ishizaka, T. Aizawa, M. Sata, N. Iso-o, E. Noiri, I. Mori, M. Ohno, and R. Nagai Iron chelation and a free radical scavenger suppress angiotensin II-induced upregulation of TGF-{beta}1 in the heart Am J Physiol Heart Circ Physiol, April 1, 2005; 288(4): H1836 - H1843. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
