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HayKoeuü bíchuk ^HYBMET ÍMeni C.3. f^ицbкого
TOM 10 № 3(38) Hacmuna 1, 2008
Liliana Kiczak, Urszula Paslawska*, Jacek Bania**, Izabela Sambor, Agata Kochman****, Anna Chelmonska-Soyta***, Joanna Blach, Maciej Ugorski, Krzysztof Kubiak*, Marcin Jankowski*, Jolanta Spuzak*, Agnieszka Noszczyk-Nowak*, Kamila Glinska-Suchocka* ®
Department of Biochemistry, Pharmacology and Toxicology (L.K., I.S., M.U.)
*Department of Internal and Parasitic Diseases with Clinic for Horses,
Dogs and Cats, Faculty of Veterinary Medicine, Wroclaw University of
Environmental and Life Sciences, Norwida 31, 50-375 Wroclaw (U.P) **Department of Food Hygiene and Consumer Protection (J.B.), ***Department of Immunology (A.C.S, J.B.) Faculty of Veterinary Medicine, and
Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland. ****Department of Pathological Anatomy, Wroclaw Medical University (A.K.),
Wroclaw, Poland L.K. and U.P. contributed equally to this work
CONGESTIVE HEART FAILURE LEADS TO INCREASED EXPRESSION OF INTERLEUKIN-1B AND ITS SPLICE VARIANT IN CANINE HEART
TISSUE
Abstract
Increasing number of experimental and clinical evidences suggest that proinflammatory cytokines, in addition to neurohormonal activation and andrenergic system, are involved in pathogenesis of congestive heart failure. The aim of this study was to examine the myocardial IL-ip gene expression in dogs with severe heart failure due to CDVD. Increase in IL-lfi expression seems to be associated with volume heart overload. Endogenous origin, and certain histopathological findings attributed to IL-lfi indicate its importance in cardiac hypertrophy and failure. Lack of some typical IL-lfi actions i.e. inflammatory, pyrogenic and fibrotic may suggest a different role of this cytokine in myocardium degeneration that known from immune surveillance or high threshold for induction of immune response in heart. It appears that the canine IL-lfi gene can be transcribed in three ways in heart tissue, with IL-ifisvl form present mainly in failing hearts.
Key words: chronic degenerative valvular disease, interleukin-lfi, alternative splicing, canine heart
Introduction
Cardiac insufficiency is one of the most important death reason in dogs and human patient. Its most common type is left sided heart failure being secondary to volume overload result of mitral valvular noninflammatory mucous degeneration (chronic degenerative valvular disease - CDVD). Increasing number of experimental and clinical evidences suggest that proinflammatory cytokines, in addition to
® Liliana Kiczak, Urszula Paslawska, Jacek Bania, Izabela Sambor, Agata Kochman, Anna Chelmonska-Soyta, Joanna Blach, Maciej Ugorski, Krzysztof Kubiak, Marcin Jankowski, Jolanta Spuzak, Agnieszka Noszczyk-Nowak, Kamila Glinska-Suchocka, 2008
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Науковий вгсник ЛНУВМБТ 1мен1 С.З. Гжицького Том 10 № 3(38) Частина 1, 2008
neurohormonal activation and andrenergic system, are involved in pathogenesis of congestive heart failure (CHF) (Yndestadt). Increased interleukin-ip (IL-ip) level in serum of patients with heart insufficiency correlates with the severity of the failure despite of its etiology (8). On the other hand, there are few reports founding IL-ip to be elevated in failing humans and animal hearts (3, 2). The biological effects of IL-ip depends on its amount and localization of synthesis. Low local concentration are related to induction of local inflammatory responses, but in larger quantities can exert potentially lethal effects.
A number of experimental and clinical evidences demonstrate that increased interleukin-ip serum level in patients with heart insufficiency correlates with the severity of the failure despite of its etiology. Very few is known on the IL-ip expression in heart tissue.
The aim of the article
The aim of this study was to examine the myocardial IL-ip gene expression in dogs with severe heart failure due to CDVD in order to better characterize the role of IL-ip in heart insuficiency development and progression. Different heart chambers were studied individually and the results were compared between failing and nonfailing myocardium what allowed for investigating following stages of cardiac failure.
Material and methods
The animals were divided in two groups: dogs with clinical heart failure (n=i7) and control animals (n=9) without the symptoms of cardiac insufficiency. Diagnosis was based on a medical history, physical examination, and echocardiogram. The IL-ip transcript level was determined in real time PCR. Tissue sections from left and right ventricle wall, and septum were taken from dogs presented for euthanasia with informed consent from their owners. The samples were immediately frozen in liquid nitrogen before the preparation of tissue homogenates and RNA extraction. About i00 mg of cardiac tissue was homogenized in liquid nitrogen, and lyzed in RIPA buffer. Protein quantification was performed by the BCA reagent. Thirty-^g protein samples were separated in SDS-PAGE on a i5 % gel and transferred onto PDVF membrane. Blotting efficiency was checked using PonceauS staining. The membrane was blotted for IL-ip using goat anti-human IL-ip, and anti-goat IgG HRP-conjugated antibody, followed by an ECL detection. Total RNA was prepared from 30-mg canine cardiac tissue using the RNeasy Fibrous Tissue Mini Kit for reverse transcription -PCR. The relative amount of canine IL-ip, and IL-ip svi RNA was determined by quantitative real-time PCR using an iQ5 Optical System (BioRad) with the iQ SYBR Green Supermix (BioRad) as appropriate. The primers used were as follows: SPILi/ASILi for IL-ip primary transcript, SPIL34/ASPIL34 for IL-ip splice variant i (IL-ipsvi), SIL-iRa/ASIL-iRa for secreted IL-iRa, and SGAPDH/ASGAPDH for GAPDH. The specificity of the PCR was determined by melt-curve analysis for each reaction. The GAPDH was used as a reference gene. Data are presented as a mean ± SD values. Statistical analysis was performed using
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Науковий вгсник ЛНУВМБТ 1мен1 С.З. Гжицького Том 10 № 3(38) Частина 1, 2008
Man-Whitney U-test. The Sperman's rank correlation coefficient was used to compare the groups. The echocardiography data were corrected in consideration towards body weight, ECG data were corrected for heart rate before statistical analyses. ^-values below 0.05 were considered to be statistically significant.
All the experiments were conducted in compliance with the bioethical committee of Wroclaw University of Environmental and Life Sciences guidelines for experimentation on animals.
Results of resarch
L-ip primary transcript level in failing hearts was found to be higher than in hearts from the control animals. In both groups the highest IL-ip level was detected in left ventricles. Despite IL-ip is one of the most potent proinflammatory cytokine most of the dogs with heart failure displayed no inflammatory infiltrates into the myocardium. Massive fibrosis was observed in group of control animals, unlike the failing hearts in which cardiomiocyte hypertrophy and atrophy dominated. One of the two described here alternative IL-ip transcripts, i.e. IL-ipsvi was significantly elevated in the failing myocardium compared to the control group (correlating with the progression of heart insufficiency). The IL-ipsvi level in different heart chambers reflects the progression of cardiac insufficiency.
Conclusions
The major findings of this study were folowing: i) in the heart tissue of dogs with severe heart insufficiency and chronic degenerative valvular disease the IL-ip has had two forms IL-iPpt and IL-ipsvi 2) IL-ip pt is present in the both groups of dogs and the level of IL-ip pt gene expression in (the both) each groups was similar 3) In contrast gene expression of IL-ipsvi exhibited significantly higher level in the group of dogs with heart insufficiency 4) Expression of the both forms of IL-ip correlates with local concentrations of IL-ip in cardiac tissue.
Reference
1. Cosman D., Wignall J., Anderson D., Tushinski J., Gallis B., Urdal D., Cerretti D.P.: Human macrophage colony stimulating factor (M-CSF) alternative RNA splicing generates three different proteins that are expressed on the cell surface and secreted. Behring Inst. Mitt. i988, 83, i5-26.
2. Ono K., Matsumori A., Shioi T., Furukawa Y., Sasayama S.: Cytokine gene expression after myocardial infarction in rat hearts: possible implicaton in left ventricular remodeling. Circulation, i998, 98, i49-i56.
3. Shioi T., Matsumori A., Kihara Y., Inoko M., Ono K., Iwanaga Y., Yamada T., Iwasaki A., Matsushima K., Sasayama S.: Increased expression of interleukin-ip and monocyte chemotactic and activating factor/monocyte chemoattractant protein-i in the hypertrophied and failing heart with pressure overload. Circ. Res. i997, 8i, 664-67i.
Стаття надшшла до редакцИ 20.09.2008
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