title: Activation of CaMKII as a key regulator of reactive oxygen species production in diabetic rat heart.
abstract: Diabetes mellitus is a risk factor for heart failure. Increased reactive oxygen species (ROS) have been proposed as a possible mechanism of cardiac dysfunction in diabetic patients. However, the mechanisms of ROS increase are still elusive. We hypothesized that activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) induced by impaired intracellular Ca(2+) ([Ca(2+)](i)) metabolism may stimulate ROS production in the diabetic heart. Cultured cardiomyocytes from neonatal rats were exposed to high glucose concentrations (25 mmol/L) and ROS levels were analyzed in 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H(2)DCFDA)-loaded cells by flow cytometry analysis. Exposure to high glucose concentrations for 24h significantly increased CM-H(2)DCFDA fluorescence, which was significantly inhibited by 1,2-bis (o-aminophenoxy) ethane- N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM), a [Ca(2+)](i) chelator, and KB-R7943, an inhibitor of the Na(+)-Ca(2+) exchanger (NCX) in the reverse mode. These results indicate that [Ca(2+)](i) increase by NCX activation may induce ROS increase following exposure to high glucose concentrations. We confirmed that exposure to high glucose concentrations significantly increased [Ca(2+)](i), which was inhibited by KB-R7943. Na(+)-H(+) exchanger (NHE) is a key factor in [Ca(2+)](i) metabolism, and is known to activate NCX by increasing the intracellular Na(+) ([Na(+)](i)) level. We showed that the expression of NHE isoform 1 and NHE activity increased following exposure to high glucose concentrations by evaluating protein expressions and intracellular pH recovery from acid loading. Exposure to high glucose concentrations up-regulated phosphorylated CaMKII expression in cardiomyocytes that was inhibited by KB-R7943. Further, autocamtide 2-related inhibitory peptide (AIP), a CaMKII inhibitor, significantly attenuated the ROS increase following exposure to high glucose concentrations. We confirmed these results obtained in in vitro experiments in an animal model of diabetes. ROS level and components of NADPH oxidase, p47phox and p67phox were up-regulated in streptozotocin-induced diabetic rat heart, which were attenuated by KN-93, a CaMKII inhibitor. Consistently, expression of phosphorylated CaMKII was increased in the diabetic heart. Activation of CaMKII by impaired [Ca(2+)](i) metabolism may be a mechanism of ROS increase in the heart with diabetes mellitus.
authors.affiliation: Department of Laboratory Examination and Diagnostics, Faculty of Medicine, Oita University, 1-1 Idaigaoka, Yufu-city, Oita 879-5593, Japan.
authors.forename: Satoru
authors.initials: S
authors.lastname: Nishio
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Teshima
authors.forename: Naohiko
authors.initials: N
authors.lastname: Takahashi
authors.forename: Luong Cong
authors.initials: LC
authors.lastname: Thuc
authors.forename: Shotaro
authors.initials: S
authors.lastname: Saito
authors.forename: Akira
authors.initials: A
authors.lastname: Fukui
authors.forename: Osamu
authors.initials: O
authors.lastname: Kume
authors.forename: Naoya
authors.initials: N
authors.lastname: Fukunaga
authors.forename: Masahide
authors.initials: M
authors.lastname: Hara
authors.forename: Mikiko
authors.initials: M
authors.lastname: Nakagawa
authors.forename: Tetsunori
authors.initials: T
authors.lastname: Saikawa
mesh_terms: D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D020013:Calcium Signaling; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D016923:Cell Death; D002478:Cells, Cultured; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D003921:Diabetes Mellitus, Experimental / Q000201:enzymology*; D004789:Enzyme Activation; D005947:Glucose / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008929:Mitochondria, Heart / Q000201:enzymology / Q000378:metabolism / Q000502:physiology; D009206:Myocardium / Q000201:enzymology* / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D053140:Organelle Shape; D053141:Organelle Size; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D061251:Primary Cell Culture; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism
chemical_list: D017382:Reactive Oxygen Species; D017923:Sodium-Hydrogen Exchangers; D000080242:8-Hydroxy-2'-Deoxyguanosine; D019255:NADPH Oxidases; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D003849:Deoxyguanosine; D005947:Glucose