ng Measurement Left Ventricular Mass Ejection Fraction Fractional Shortening Normal Diet 15267 7561 4361 High-Fat Diet 13267 7462 4262 Data was obtained from M-mode echocardiographic images taken in parasternal short axis mode at the level of the papillary muscles. Data are presented as mean6SEM. There was no statistical significance between the data. doi:10.1371/journal.pone.0100579.t002 Upon reperfusion, after 10 min in rigor, cardiomyocytes regained BGJ 398 beating quicker in the normal diet group compared to high-fat diet, P,0.001 and regained full recovery more often than the cardiomyocytes isolated from mice fed high-fat diet, P, 0.01. The effect of non-obesogenic high-fat diet on oxidative state and antioxidant proteins Cardiac oxidative stress was significantly lower in hearts from mice fed high-fat diet compared to hearts from normal diet mice. There were only two antioxidant proteins which had altered expression level in the high-fat diet group compared to the normal 12695532 diet group determined using proteomics: catalase and mitochondrial superoxide dismutase 2 , P,0.05. The effect of non-obesogenic high-fat diet on ROS levels in isolated cardiomyocytes and mitochondria ROS turnover, as measured using the rate of DCF oxidation, did not differ between the two groups when the cardiomyocytes were incubated in HEPES buffer containing either glucose or palmitate. Nor did hydrogen peroxide production by isolated mitochondria in state 3.5 oxidizing pyruvate plus L-malate or palmitoylcarnitine plus Lmalate differ between the two groups. The effect of non-obesogenic high-fat diet on oxygen consumption by cardiac mitochondria in different respiratory states The rate of oxygen consumption fuelled by NADH from pyruvate plus L-malate oxidation was similar for both groups in all tested respiration states. The oxygen consumption rates were also not different between the normal and high-fat diet groups when comparing the b-oxidation pathway using palmitoylcarnitine plus L-malate as substrates. The respiratory control ratio was also not different between the normal and high-fat diet groups for both sets of substrates. The effect of non-obesogenic high-fat diet on mitochondrial morphology The mitochondria in the high-fat diet group were both smaller in area and shorter in length, P,0.001. The total mitochondrial area, as a percentage 
of total myofilament area, was decreased in the highfat diet group compared to the normal diet, P,0.05. The effect of non-obesogenic high-fat diet on mitochondrial fusion and fission proteins In response to the high-fat diet the expression of the fusionrelated proteins Mfn-2 and OPA1 were significantly increased and decreased respectively, with no change in Mfn-1 expression but an increased expression of the fission-related protein, DRP1. 6 Non-Obesogenic High-Fat Diet and Cardiac Remodeling The effect of non-obesogenic high-fat diet on putative mPTP proteins Isolated cardiac mitochondria were assessed for the relative expression of putative mPTP proteins using western blotting. In the high-fat diet group there was a significant increase in the relative protein expression of PiC and a decrease in VDAC compared to the normal diet group, P,0.05. There was no change in both CypD and ANT in the high-fat diet group compared to the 17628524 normal diet group. Protein Catalase Isoform cytoplasmic & peroxisomal of Peroxiredoxin 5 Peroxiredoxin 1 Peroxiredoxin 2 Peroxiredoxin 6 SOD-1 SOD-2 Thioredoxin-dependent peroxide reductase, mitochondrial N