The GTP-binding protein Rac regulates diverse cellular functions including activation of NADPH oxidase, a major source of superoxide production (O2?). exposed that improved myocardial ZmRacD gene manifestation is associated with concomitant improved levels of NADPH oxidase subunit gp91phox, O2?, and P21-triggered kinase. Therefore these findings provide direct evidence that improved levels of active myocardial Rac renders the heart susceptible to improved postischemic contractile dysfunction and MI following acute I/R. American Physiological Society (APS) (revision authorized by the APS Council on July 16, 2010). Mice. Details regarding the generation and characterization of ZmRacD transgenic (TG) mice have been explained previously (19). Briefly, the MHC-RacD transgene was constructed using the cDNA of a buy WYE-354 constitutively active mutant of ZmRacD gene (28) and -MHC promoter was used to induce selective overexpression of ZmRacD in myocytes of FVB/N wild-type (WT) mice. Rabbit Polyclonal to TBX2 Each mouse was genotyped when it was weaned and only respective littermates served as control (WT). Experiments were performed in age-matched young adult mice (5C8 mo), and tail-clips were kept to reconfirm the genotypes. Most of the mice used in this study were males except six female mice. Echocardiographic evaluation of remaining ventricular function. In vivo cardiac dimensions and contractile function was evaluated by transthoracic M-mode and two-dimensional (2-D) echocardiography under light isoflurane (0.5C1%) anesthesia while described previously (51). Briefly, having a GE Vivid7 echocardiography system and intraoperative epicardial probe (model i13L; FREQ 14 MHz), the 2-D remaining parasternal short-axis look at was used as a guide and LV M-mode tracings were obtained close to buy WYE-354 the papillary muscle mass. LV internal diameters at diastole and systole (LVIDd and LVIDs, respectively) were measured, and LV fractional shortening (FS) was determined as FS (%) = (LVIDd ? LVIDs)/LVIDd 100. Measurement of superoxide (O2?) in the heart. In situ production of myocardial O2? was assessed by oxidative fluorescent dye, dihydroethidium (DHE), mainly because explained previously (19). The cell-permeable DHE is definitely oxidized to fluorescent hydroxyethidine (HE) by O2? and then intercalated into DNA. Briefly, hearts from WT and TG mice were placed immediately in ice-cold PBS, washed, and then inlayed in OCT for cryosectioning. Frozen sections (6-m) from your hearts were incubated with DHE (10 M; Sigma-Aldrich) for 30 min at 37C inside a dark chamber. After PBS wash, sections were fixed with aqueous mounting medium (Gel Mount, Sigma) and images were obtained using a fluorescence microscope (Nikon TE 300, Tokyo). The reddish fluorescence intensity was identified using the MetaMorph image analysis software 7.1.2.0 (Molecular Products). In vitro myocardial ischemia-reperfusion. Hearts were isolated from age-matched mice of both strains as explained previously (51, 52). Briefly, mice were anesthetized with pentobarbital (50 mg/kg ip), and hearts were excised, aorta cannulated, and perfused inside a Langendorff mode at a constant pressure of 80 mmHg having a revised Krebs-Henseleit buffer (KHB) equilibrated with 95% O2-5% CO2 at 37C. The constituents of KHB were (in mmol/l) 120 NaCl, 5.9 KCl, 25 NaHCO3, 1.2 MgCl2, 2.5 CaCl2, 0.5 EDTA, and 16.7 glucose. A fluid-filled balloon was put into the remaining ventricle (LV) across the mitral valve and connected to a pressure transducer permitting continuous measurement of LV pressure (LVP). Hearts were immersed inside a water-jacketed bath managed at 37C, and the LV balloon was filled with water to yield a remaining ventricular diastolic pressure of 3C6 mmHg. Coronary circulation was continuously monitored via a Doppler circulation probe (T206, Transonic Systems, Ithaca, NY) placed in buy WYE-354 the aortic perfusion collection. Aortic and LV developed pressures were recorded on a PowerLab/400 multichannel data-acquisition system (ADInstruments; Castle Hill, Australia). The LVP transmission was digitally processed (using PowerLab Chart software version 4.2, ADInstruments) to yield diastolic and systolic pressures as well while heart rate. LV developed pressure (LVDP) was determined as the difference between systolic and diastolic pressures. Following 30 min equilibration, hearts underwent 20 min of global ischemia, followed by 45 min of reperfusion. In vivo.