Left ventricular (LV) volume overload (VO) results in cardiomyocyte oxidative stress

Left ventricular (LV) volume overload (VO) results in cardiomyocyte oxidative stress and mitochondrial dysfunction. experienced extensive loss of desmin and 2-tubulin that was paralleled by mitochondrial disorganization, loss of cristae, swelling, and clustering recognized by mitochondria complex IV staining and transmission electron microscopy. MitoQ improved mitochondrial structural damage and attenuated desmin loss/degradation evidenced by immunohistochemistry and protein expression. However, LV dilatation and fractional shortening were unaffected by MitoQ treatment in 8-wk ACF. In conclusion, although MitoQ did not impact LV dilatation or function in ACF, these experiments suggest a connection of cardiomyocyte mitochondria-derived ROS production with cytoskeletal disruption and mitochondrial damage in the VO of ACF. = 6/group). Another set of sham and ACF rats was analyzed for in vivo hemodynamic and echocardiographic measurements before death, and this tissue was utilized for protein analysis and immunohistochemistry (= 5/group). The animal use in these experiments was approved by the University or college of Alabama at Birmingham Animal Resource Program (protocol 130409070). Hemodynamics and echocardiography. Echocardiography and hemodynamics GW 4869 distributor were performed before death using the Visualsonics imaging system (VIVO 2100, Toronto, ON, Canada) combined with simultaneous high-fidelity LV pressure catheter recordings (Millar Devices, Houston, TX). With the rats under isoflurane anesthesia, a high-fidelity LV pressure catheter was advanced into the LV cavity via a right carotid cutdown. LV pressure and echocardiography sizes (wall thickness and chamber diameter) were obtained simultaneously using software included in the Visualsonics system. LV volume was calculated from traced M-mode LV sizes using the following Teicholz formula: volume = [7/(2.4 + LVID)] (LVID)3, where LVID is LV internal dimensions. LV wall stress was calculated from traced M-mode LV sizes and simultaneous LV pressure data using the following equation: LV wall stress = (LV pressure GW 4869 distributor is the LV chamber radius. These LV pressure-volume data were analyzed using the Labscribe2 (iWorx System Dover, NH) software package as previously explained by our laboratory (18, 19). Isolation of LV cardiomyocytes. Cardiomyocytes were isolated from sham and ACF rats as previously explained by our laboratory (18, 19, 42). Briefly, hearts were perfused with Rabbit polyclonal to PRKCH perfusion buffer (120 mmol/l NaCl, 15 mmol/l KCl, 0.5 mmol/l KH2PO4, 5 mmol/l NaHCO3, 10 mmol/l HEPES, and 5 mmol/l glucose at pH GW 4869 distributor 7.0) for 5 min and digested with perfusion buffer containing 2% collagenase type II (Invitrogen, Carlsbad, CA) for 30 min at 37C. The right ventricle, atria, and apex were removed before the perfused heart was minced. The digestion was filtered and washed, and cells were pelleted. Only samples with viability (rod-shaped cells) 80% were used. Application of stretch to isolated adult rat cardiomyocytes. Cells (50,000 cells/well) were allowed to adhere to laminin-coated Flexcell plates (Flexcell, Hillsborough, NC) in DMEM made up of 10% FBS, 2 nM glutamine, 10 U/ml penicillin, and 100 mg/ml streptomycin for 2 h before use. Cells were subjected to cyclical strain GW 4869 distributor (60 cycles/min, 3 h) around the Flexcell strain apparatus (model FX-4000, Flexcell) at a level of distension sufficient to promote an increment of 20% in surface area at the point of maximal distension around the culture surface as previously explained by our laboratory (19, 45). A group of cells stretched for 3 h was also treated with MitoQ (50 nM). Control cells were prepared on identical culture plates but were not exposed to stretch. Live cell imaging. The cationic potentiometric fluorescent dye tetramethylrhodamine methyl ester (TMRM; 50 nM) was used to monitor changes in mitochondrial membrane potential. ROS production was monitored with 5-(and-6)-chloromethyl-2,7-dichlorodihydrofluorescein (CM-DCF; 5 mol/l), an H2O2-sensitive fluorescent indication, as previously explained by our laboratory (42). The dish made up of fluorescent dye-loaded cardiomyocytes was equilibrated at 37C with unrestricted access to atmospheric O2 around the stage of an Olympus microscope. CM-DCF and TMRM images were.