Aged TG mice (15-month-old) showed significant reduction in parameters including dP/dtmax, dP/dtmin, dV/dtmin, EF, stroke volume, preload-adjusted maximal power and preload-recruitable stroke work relationship (Mw), together with increased LVEDP, , and LVDd or LVDs (all 0

Aged TG mice (15-month-old) showed significant reduction in parameters including dP/dtmax, dP/dtmin, dV/dtmin, EF, stroke volume, preload-adjusted maximal power and preload-recruitable stroke work relationship (Mw), together with increased LVEDP, , and LVDd or LVDs (all 0.05) in comparison either with 5-month-old TG mice or with age-matched NTG mice. shock protein 27 (HSP27), expression of pro-inflammatory cytokines and collagen, and progressive ventricular dysfunction. 2-adrenoceptor activation similarly increased ROS production and phosphorylation of p38 MAPK and HSP27 in cultured cardiomyocytes. Treatment with apocynin, diphenyliodonium or NAC reduced phosphorylation of p38 MAPK and HSP27 in both cultured cardiomyocytes and the LV of 2-TG mice. NAC treatment (500 mgkg?1day?1) for 2 weeks eliminated the up-regulated expression of pro-inflammatory cytokines and collagen in the LV of 2-TG mice. Chronic NAC treatment to 2-TG mice from 7 to 10 months of age largely prevented progression of ventricular dilatation, preserved contractile function (fractional shortening 37 5% vs. 25 3%, ejection portion 52 5% vs. 32 4%, both 0.05), reduced cardiac fibrosis and suppressed matrix metalloproteinase activity. CONCLUSION AND IMPLICATIONS 2-adrenoceptor activation provoked NADPH oxidase-derived ROS production in the heart. Elevated ROS activated p38 MAPK and contributed significantly to cardiac inflammation, remodelling and failure. LINKED ARTICLE This short article is usually commented on by Di Lisa studies suggested an anti-apoptotic action opposing that of 1-adrenoceptors (Zhu studies, including ours, have revealed several non-classical signalling molecules utilized by 2-adrenoceptors, including -arrestin 1 (Drake systems, not exposing the signalling cascade, or in non-cardiac preparations. To define the adverse and non-classic 2-adrenoceptor signalling mechanisms in the heart, we used a transgenic model with cardiac-restricted 2-adrenoceptor overexpression (2-TG) (Milano and our hypothesis that there exists a 2-adrenoceptor/nicotinamide adenine dinucleotide phosphate (NADPH) oxidase/ROS/p38 MAPK signalling pathway leading to cardiac pathology and HF. Experimental procedures Animals All animal care and experimental procedures were approved by a local Animal Ethics Committee and KRT20 the investigation conformed to the published by the Australian National Health and Medical Research Council (7th edition, 2004). Male 2-adrenoceptor transgenic (2-TG) and non-transgenic (NTG) littermate mice, originally explained by Milano analysis using GraphPad 5 (GraphPad Inc., La Jolla, CA, USA), as appropriate. The least-square method was utilized for linear correlation and regression. 0.05 was considered as statistically significant. Materials The suppliers of the materials used here were as follows: Apocynin, DPI, SB202190, NAC, CGP 20712A, Isoprenaline, PEG-SOD all from Sigma-Aldrich, USA. H2DCF-DA: was from Invitrogen, USA and for the anaesthetic combination, ketamine was from Parnell Laboratories Pty Ltd, Australia; xylazine was from Troy Laboratories Pty Ltd, Australia and atropine was from Pfizer Australia. Receptor and drug nomenclatures follow Alexander 0.05, Figure 1B). Further, NADPH oxidase was shown to be an important source of ROS by results from NADPH-enhanced lucigenin assay showing a 64% increase in NADPH oxidase activity in LVs of 5-month-old 2-TG versus NTG mice ( 0.05, Figure 1C). In keeping with these findings, in 2-TG at 5- and 15-months ( 0.05), and sixfold for NOX4 (both 0.05) relative to NTG mice (Figure 1D). Open in a separate window Physique 1 Increased ROS production in the LV of 2-TG mice. A, representative fluorescent probe DHE staining for oxidative fluorescent transmission of LV sections from NTG and 2-TG mice (5-month-old) and densitometric analysis of DHE fluorescence. (Bar = 50 m). B, ROS production was decided in freshly harvested LV (5-month-old) by electron spin resonance assay using superoxide probe CMH, or C, lucigenin-enhanced chemiluminescence assay for NADPH oxidase activity. D, quantitative real-time PCR for mRNA expression of NOX2 and NOX4 isoforms in the LV of NTG and 2-TG mice at both 5 and 15 months of age. Data are offered as relative changes to age-matched NTG mice ( 0.05 versus NTG mice. 2-adrenoceptor activation induces phosphorylation of p38 4-HQN MAPK and HSP27 via NADPH oxidase and ROS p38 MAPK is usually a critical signalling molecule downstream of 2-adrenoceptor activation (Peter 0.05). These changes seen in the 2-TG were significantly reduced by treatment with the 4-HQN antioxidant, NAC (250 mgkg?1, i.p.), or NADPH oxidase inhibitors, apocynin (2 mgkg?1, i.p.) or DPI (1 mgkg?1, i.p., 0.05). Open in a separate window Physique 2 p38 MAPK activation by NADPH oxidase-derived ROS in the LV of 2-TG. A, 2-TG mice (5- to 7-month-old) were treated with N-acetylcysteine (NAC, 250 mgkg?1, i.p.), or NADPH inhibitors, apocynin (Apo, 2 mgkg?1, i.p.) or diphenyliodonium chloride (DPI, 1 mgkg?1, i.p.). The LV was collected 1 4-HQN h after the treatment. Western blot analyses were performed using antibodies against phospho-p38 MAPK, p38 MAPK, phospho-HSP27, HSP27 and tubulin respectively. B, Levels of phosphorylated and total p38 MAPK or 4-HQN HSP27 and tubulin, quantified by densitometry and offered as changes relative to age-matched NTG mice. * 0.05 versus NTG mice; ? 0.05 versus vehicle-treated 2-TG group, 0.05). The levels of phospho-p38 MAPK and phospho-HSP27, which were increased following 2-adrenoceptor activation, were abolished by pretreatment with 2-adrenoceptor antagonist, ICI 118551. Pretreatment of cultured cardiomyocytes with either the antioxidants, NAC.