Role Of Kir6.2 Subunit Of ATP-sensitive Potassium Channels In Myocardial Damage Induced By Lipopolysaccharide

Severe sepsis, the leading cause of fatality in critically ill patients, is characterized by an immunologically driven systemic response that underlies host protection against microbial infection.Central to this syndrome is a pronounced cytokine-induced cardiovascular state defined by a hypercontractile heart and altered vascular tone. Despite diverse infectious pathogens, the outcome in severe sepsis is governed by the nature and degree of the host response, with the cardiovascular status an ultimate determinant of survival. The Gram-negative bacterial cell wall endotoxin, LPS (lipopolysaccharide), evokes a surge in tumor necrosis factor-alpha (TNF-α), precipitating an inflammatory mediated cascade of pathophysiologic events that imposes a high demand on the hyperdynamic septic heart.ATP-sensitive potassium (KATP) channels have emerged as protein complexes with the capability to decode signals of metabolic distress. By virtue of an intimate integration with intracellular energetic networks and a capacity for high-fidelity processing of incoming metabolic signals, KATP channels regulate cellular excitability-dependent functions. These channels are situated in high density within metabolically active tissues, where changes in the energetic state are conveyed through the ATP-binding cassette sulfonylurea receptor (SUR) regulatory subunit to the K+ channel pore, Kir6.x of the channel complex. It is the tissue-dependent channel pore that, requiring the regulatory SUR chaperone to function, adjusts membrane potential to match demand and maintain cellular wellbeing. In the vasculature, the pore-forming subunit of the KATP channel has been identified as Kir6.1, and Kir6.1-containing channels have been implicated in the regulation of arterial smooth muscle tone. In the myocardium, where the pore-forming isoform is Kir6.2, defects in KATP channel subunits jeopardize cellular stress tolerance and predispose to injury.Recent studies indicate that the nervous system, through vagus nerve, can modulate circulating TNF-α, IL-1βa?nd IL-6 levels induced by endotoxin. This new mechanism termed"cholinergic anti-inflammatory pathway"is based on the release of acetylcholine (ACh), the principal neurotransmitter of the vagus nerve that inhibits the production of pro-inflammatory cytokines via itsα7 nicotinic acetylcholine receptor (α7nAChR) in resident tissue macrophages. Nicotine receptors belong to the family of ligand-gated ion channels and consist of twelve subunits, nineαand threeβknown to exist so far. Alpha7 nicotinic acetylcholine receptor (α7nAChR) is a type of nicotinic acetylcholine receptor, constructed by five homomericα7 subunits. This receptor, most abundantly distributing in mammalian brain, has been demonstrated to control excitability and neurotransmitter release and to mediate neuroprotective properties. Recent studies indicated thatα7nAChR expressed on macrophages played an important role in the cholinergic anti-inflammatory pathway. During acute condition,α7nAChR attenuated renal failure induced by ischemia/reperfusion through inhibiting pro-inflammatory cytokines expression, and subsequently decreasing cell apoptosis. During chronic inflammation process,α7nAChR was related to the pathogenesis of many diseases such as allergies, Alzheimer's disease, diabetes, hypertension, and hormonal imbalances. Therefore as a potential therapeutic target against inflammation and cognitive disorder, selective agonists forα7nAChR attract a great deal of attention.The first aim of this thesis is to investigate the role of Kir6.2 subunit of ATP-sensitive potassium channels in myocardial damage induced by lipopolysaccharide. Thus, light microscopy was performed on paraffin-embedded myocardial sections stained with hematoxylin-eosin from 4% formalin-fixed left ventricles (LV) taken from WT and Kir6.2-KO mice 90min,180min and 360min after LPS or saline vehicle administration. And TUNEL staining was performed on these sections. Transmitted electron microscopy (EM) was performed on ultramicrotome-cut, lead citrate-stained LV sections with a JEOL 1200 EXII electron microscope. All the results showed that the myocardial damage in LPS-challenged Kir6.2-KO mice were aggravated compared to LPS-challenged WT mice.We postulated that at least there were two major factors involved in myocardial damage in LPS-challenged mice. One might be the direct injury from LPS, and the other was the inflammatory impairment from cytokines, such as TNF-αinduced by LPS. Here we assessed the possible inflammatory impairment from cytokines induced by LPS.To assess whether Kir6.2 was able to modulate cytokines expression induced by LPS, Serum cytokine levels were quantified at baseline, 90,180 min and 360min after LPS administration by ELISA. Expression of TNF-αincreased significantly at 360min after LPS. The results suggested that Kir6.2 decreased the expression of cytokines. Meanwhile, cytokine levels in peritoneal macrophages from WT mice and Kir6.2-KO mice were quantified at baseline and 90min after LPS administration by ELISA. Glibenclamide significantly increased TNF-αexpression in peritoneal macrophages from WT mice. And pinacidil significantly decreased TNF-αexpression in peritoneal macrophages from WT mice. These changes were also found in peritoneal macrophages from Kir6.2-KO mice. These results suggested that both Kir6.2 and Kir6.1 were involved in the regulation of the cytokines expression.The second aim of this study was to investigate interaction between Kir6.2 andα7nAChR. Cytokine levels in peritoneal macrophages from WT mice and Kir6.2-KO mice were quantified at baseline and 90min after LPS administration by ELISA. The selectiveα7nAChR agonist PNU-282987 significantly inhibited TNF-αexpression in peritoneal macrophages from WT mice. PNU- 282987 also significantly decreased TNF-αexpression in peritoneal macrophages from Kir6.2-KO mice. These suggested that Kir6.2 might not involve in the cholinergic anti-inflammatory pathway.