Rterial pressures for control and experimental groups. No statistically significant differences were observed among mean arterial pressures during 5day treatment period. doi:10.1371/journal.pone.0046568.tTNF with or without LOS did not have any effect on blood pressure parameters (Table 2).EchocardiographyWhen JSI-124 cost compared to control animals, TNF treated rats had progressive increases in LV end-diastolic dimension (LVD), LV end-systolic dimension (LVS) and TEI index and decreases in fractional shortening (FS ) measurements (Table 3). Additionally, rats treated with TNF+LOS exhibited significant decreases in LVD, LVS and TIE index and increases in FS when compared to rats given TNF only.EPR Measurements Enzymatic and Respiratory Activities of Mitochondrial Complexes I IIMeasurements of mitochondrial complex I, II and III enzymatic activities were performed as previously described [12]. Briefly, aliquots of mitochondria were 
mixed with oxygenated KHB (20 mm Hg O2) containing 1 mM EGTA. Then, the oxygen spin label NOX-13.1-OS (5 mM), CMH (200 mM), and one of the following substrates were added to the mitochondrial suspension: 20 mM glutamate (complex I), 5 mM succinate (complex II), or 5 mM pyruvate (complex III). After adequate mixing, the samples were taken into capillary tube and mitochondrial complex I, II and III were measured using EPR. Activity of each mitochondrial complex was quantified by EPR under the same settings described previously [12,22]. Total ROS, O2N2 and 25837696 OONO2 production rates in LV tissue, as Octapressin biological activity determined by EPR, were all significantly higher in LV tissues of TNF treated rats than in control and TNF+LOS groups (Figure 1a?c ). Increases in ROS 
generation induced by TNF or ANGII were significantly inhibited by LOS. These results further support the ability of LOS to reduce oxidative stress in LV tissue. Table 3. Echocardiographic data from experimental groups.Parameter IVSD (mm) IVSS mm) LVD (mm) LVS (mm) PWD(mm) PWS (mm) FSControl 1.3760.001 2.3160.08 7.2760.06 5.1960.10 1.2960.02 2.1860.026 36.8660.72 332.2563.2 0.2760.TNF 1.6860.06 2.4860.07 7.9760.06* 5.7460.16* 1.4260.04 2.2460.09 26.8560.45* 329.4064.1 0.3860.04*TNF +LOS 1.3360.06 2.3760.1 7.3460.1 5.2760.1 1.3460.1 2.2760.04 35.8860.7 333.7564.9 0.2960.003Measurement of ATP and ADP/ATP RatioATP production rates and ADP/ATP ratios were quantified in isolated mitochondria using a commercially available kit (BioVision, Mountain View, CA) as described previously [12,22].Statistical AnalysesAll data are expressed as mean 6 SEM. Statistical analyses were performed using GraphPad Prism version 5.00 for Windows. Data were analyzed by ANOVA, followed by Bonferroni’s multiple comparison tests. In all cases, p,0.05 was considered statistically significant.HR TEIResults Blood PressureBlood pressure measurements were obtained for all experimental animals during the 5 day treatment period. Administration ofEchocardiographic analysis revealed that both left ventricular diastolic (LVD) and systolic (LVS) dimensions were significantly greater in TNF treated animals. TNF treatment also decreased fractional shortening (FS) and increased Tei index when compared to controls. Co treatment of TNF treated rats with losartan prevented these changes. IVSD, intraventricular septal thickness at end diastole; IVSS, intraventricular septal thickness at end systole; PWD, posterior wall thickness at end diastole; PWS, posterior wall thickness at end systole; HR, heart rate. Values are expressed.Rterial pressures for control and experimental groups. No statistically significant differences were observed among mean arterial pressures during 5day treatment period. doi:10.1371/journal.pone.0046568.tTNF with or without LOS did not have any effect on blood pressure parameters (Table 2).EchocardiographyWhen compared to control animals, TNF treated rats had progressive increases in LV end-diastolic dimension (LVD), LV end-systolic dimension (LVS) and TEI index and decreases in fractional shortening (FS ) measurements (Table 3). Additionally, rats treated with TNF+LOS exhibited significant decreases in LVD, LVS and TIE index and increases in FS when compared to rats given TNF only.EPR Measurements Enzymatic and Respiratory Activities of Mitochondrial Complexes I IIMeasurements of mitochondrial complex I, II and III enzymatic activities were performed as previously described [12]. Briefly, aliquots of mitochondria were mixed with oxygenated KHB (20 mm Hg O2) containing 1 mM EGTA. Then, the oxygen spin label NOX-13.1-OS (5 mM), CMH (200 mM), and one of the following substrates were added to the mitochondrial suspension: 20 mM glutamate (complex I), 5 mM succinate (complex II), or 5 mM pyruvate (complex III). After adequate mixing, the samples were taken into capillary tube and mitochondrial complex I, II and III were measured using EPR. Activity of each mitochondrial complex was quantified by EPR under the same settings described previously [12,22]. Total ROS, O2N2 and 25837696 OONO2 production rates in LV tissue, as determined by EPR, were all significantly higher in LV tissues of TNF treated rats than in control and TNF+LOS groups (Figure 1a?c ). Increases in ROS generation induced by TNF or ANGII were significantly inhibited by LOS. These results further support the ability of LOS to reduce oxidative stress in LV tissue. Table 3. Echocardiographic data from experimental groups.Parameter IVSD (mm) IVSS mm) LVD (mm) LVS (mm) PWD(mm) PWS (mm) FSControl 1.3760.001 2.3160.08 7.2760.06 5.1960.10 1.2960.02 2.1860.026 36.8660.72 332.2563.2 0.2760.TNF 1.6860.06 2.4860.07 7.9760.06* 5.7460.16* 1.4260.04 2.2460.09 26.8560.45* 329.4064.1 0.3860.04*TNF +LOS 1.3360.06 2.3760.1 7.3460.1 5.2760.1 1.3460.1 2.2760.04 35.8860.7 333.7564.9 0.2960.003Measurement of ATP and ADP/ATP RatioATP production rates and ADP/ATP ratios were quantified in isolated mitochondria using a commercially available kit (BioVision, Mountain View, CA) as described previously [12,22].Statistical AnalysesAll data are expressed as mean 6 SEM. Statistical analyses were performed using GraphPad Prism version 5.00 for Windows. Data were analyzed by ANOVA, followed by Bonferroni’s multiple comparison tests. In all cases, p,0.05 was considered statistically significant.HR TEIResults Blood PressureBlood pressure measurements were obtained for all experimental animals during the 5 day treatment period. Administration ofEchocardiographic analysis revealed that both left ventricular diastolic (LVD) and systolic (LVS) dimensions were significantly greater in TNF treated animals. TNF treatment also decreased fractional shortening (FS) and increased Tei index when compared to controls. Co treatment of TNF treated rats with losartan prevented these changes. IVSD, intraventricular septal thickness at end diastole; IVSS, intraventricular septal thickness at end systole; PWD, posterior wall thickness at end diastole; PWS, posterior wall thickness at end systole; HR, heart rate. Values are expressed.
