Effect of TNF on expression of mitochondrial respiratory complexes I, II and III measured by EPR spectroscopy.

<p>The activities of mitochondrial respiratory a) complex I, b) complex II, and c) complex III were assessed using EPR spectroscopy. TNF decreased the activities of all three complexes in isolated heart mitochondria; these results indicate a derangement in electron transport chain activity. Losartan attenuated these changes. * p<0.05 vs. control; $p<0.05 vs. TNF d) ATP production rates and e) ATP/ADP ratios of heart mitochondria from each experimental group. TNF administration resulted in significant decreases in both ATP production rate and ATP/ADP ratio, which is suggestive of electron transport chain dysfunction. Losartan attenuated the changes seen in ATP production and ATP/ADP ratio. * p<0.05 vs. control;$ p<0.05 vs.TNF.</p

Echocardiographic data from experimental groups.

<p>Echocardiographic 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 as means ± SEM.</p>*<p>p<0.05 vs. control;</p><p>$ p<0.05 vs.TN.</p

EPR spectra and their graphic interpretations are given. TNF administration significantly increased free radical production in LV tissue.

<p>Cytosolic a) total ROS, b) superoxide, and c) peroxynitrite production rates in rat cardiac tissues from each experimental group as measured by electron paramagnetic resonance spectroscopy. Administration of TNF to rats significantly increased production of all reactive species measured; LOS attenuated these increases. These results suggest that in the presence of an AT-1R antagonist, TNF cannot exert some of its detrimental effects.* p<0.05 vs. control; $ p<0.05 vs.TNF.</p

Effect of TNF on superoxide production rates and hydrogen peroxide production rates in isolated heart mitochondria from each experimental group as measured by EPR spectroscopy (a&b).

<p>Administration of TNF to rats resulted in significant increases in superoxide and hydrogen peroxide production rates. Losartan attenuated these changes. * p<0.05 vs. control; $ p<0.05 vs.TNF.</p

Blood pressure data from control and experimental groups.

<p>Mean arterial pressures for control and experimental groups. No statistically significant differences were observed among mean arterial pressures during 5-day treatment period.</p

Effects of losartan treatment on mRNA expression of gene expression levels for a) TNF, b) iNOS, c) eNOS, d) AT-1R, and e) gp91phox as determined by real-time RT-PCR and protein expression levels of TNF, iNOS, and eNOS were measured by western blotting (Fig. 2f).

<p>Administration of TNF to rats induced significant increases in expression of proinflammatory and oxidative stress genes. Losartan attenuated these increases, thereby suggesting that TNF interacts with ANGII to cause some of its effects. * p<0.05 vs. control; $ p<0.05 vs.TNF.</p

Schematic diagram showing TNF-induced mitochondrial dysfunction.

<p>Schematic diagram showing TNF-induced mitochondrial dysfunction.</p

Representative western blot analysis of a left ventricle showing changes in expression of MPTP proteins from isolated mitochondria.

<p>TNF treated rats exhibited a significant decrease in adenine nucleotide translocator (ANT) and cytochrome C content when compared with the control and TNF+LOS groups. In the TNF+LOS treated group, ANT and cytochrome C protein levels were normalized (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046568#pone-0046568-g005" target="_blank">Fig 5a</a>). Mitochondrial gene expression levels for b) PGC1α, c) PGC1β, d) CPT1β, e) CPT2, and f) UCP3 were all significantly decreased in animals given TNF; concomitant treatment with LOS attenuated these changes. * p<0.05 vs. control; $ p<0.05 vs.TNF.</p

Rat primers used for RT-PCR.

<p>Rat primers used for RT-PCR.</p

Effect of BB on renal TLR4 gene and protein expression in MetS animals.

<p>The (A) mRNA (n = 6) and (B) a representative western blot of TLR4 in the renal cortical tissues. (C) Bands were analyzed and quantified by densitometry (n = 6). The gene and protein expression of TLR4 was increased in the MetS rats. The pretreatment with BB was able to inhibit the increased expression of TLR4 in MetS animals and was comparable to the LZR controls. All values are presented as mean ± SEM (*p<0.05, **p<0.01).</p