Markers of injury during I/R.

<p>A) Infarct volume quantified from the heart slices stained with TTC. B) Representative heart slices from the normal diet group and high-fat diet group. C) Flow rate determined at different time points during the reperfusion phase. Pre = pre-ischemia and grey hashed area = 40 min ischemia. Data are presented as mean±SEM (n = 6-7 hearts). Data were analyzed using Student's t-test (A) and two-way repeated measures ANOVA with the Bonferroni post-hoc test (C). ** = P<0.01 and * = P<0.05 vs. normal diet.</p

The diastolic intracellular Ca²⁺ concentration measured using Fura-2 AM fluorescence in isolated cardiomyocytes.

<p>A) An example trace of Ca²⁺ transients using the Fura-2 fluorescent dye. The cell was stimulated at the frequencies indicated. B) Diastolic [Ca²⁺]_i at different frequencies in isolated cardiomyocytes. C) Diastolic [Ca²⁺]_i with the 0.2 Hz diastolic [Ca²⁺]_i subtracted. D) The relative phosphorylated phospholamban (P-PLN Ser16) to phospholamban (PLN) protein expression ratio. Data are presented as mean±SEM (n = 29–32 cardiomyocytes from 4 hearts (B–C) and 6 hearts (D)). Data were analyzed using two-way ANOVA with the Bonferroni post-hoc test (B–C) and Student's t-test (D). *** = P<0.001, ** = P<0.01 and * = P<0.05 vs. normal diet.</p

The relative cardiac protein expression of mitochondrial fusion and fission proteins determined using western blotting.

<p>The fusion proteins were Mfn-1 (A), Mfn-2 (B) and OPA1 (C) and the fission protein was DRP1 (D). Protein bands were normalized to GAPDH. Data are presented as mean±SEM (n = 6 hearts). Data were analyzed using Student's t-test (A, B and D) and Student's t-test with Welch's correction (C). *** = P<0.001 and * = P<0.05 vs. normal diet.</p

Characteristics of mice fed normal or high-fat diet.

<p>IPITT = intra-peritoneal insulin tolerance test. Data are presented as mean±SEM. *** = P<0.001 and * = P<0.05 vs. normal diet. Numbers shown in parenthesis indicate number of mice used except for cholesterol, triglycerides and glucose where n refers to number of measurements each containing a pool of 3 samples from 3 separate mice.</p

Relative cardiac protein expression of putative mitochondrial permeability transition pore components/regulators.

<p>PiC (A), VDAC (B), mitochondrial hexokinase II protein (C) and mitochondrial hexokinase activity (D). Data are presented as mean±SEM (n = 5 mitochondrial isolations). Data were analyzed using Student's t-test with Welch's correction (A) and Student's t-test (B–D). * = P<0.05 vs. normal diet.</p

Oxidative state during I/R.

<p>A) MDA measurements in unperfused heart tissue or hearts collected at the end of an I/R protocol analyzed using HPLC. B) The relative catalase protein expression at basal level and after I/R determined using western blotting with all sample run on the same membrane. Data are presented as mean±SEM (n = 5–6 hearts (A) and 3 hearts (B)). Data were analyzed using two-way ANOVA with the Bonferroni post-hoc test (A). *** = P<0.001, ** = P<0.01, * = P<0.05 vs. basal, and $ = P<0.05 vs. normal diet.</p

Representative electron micrographs of mitochondria and measurements.

<p>Representative electron micrographs from mice fed normal diet (A) and high-fat diet (B). Individual mitochondrion area (C) and length (D) and mitochondrial coverage of myofilament area (E) assessed using transmission electron micrographs. Data are presented as mean±SEM (n = 4 hearts and ≥900 mitochondria per heart from ≥10 electron micrographs per heart). Data were analyzed using Mann Whitney U test (C–D) and Student's t-test (E). *** = P<0.001 and * = P<0.05 vs. normal diet.</p

Cardiomyocyte observations and measurements during metabolic inhibition and reperfusion.

<p>Cardiomyocyte time to stop beating (A) and time to the start of rigor (B) following metabolic inhibition (2 mM NaCN in the absence of substrates). C) Time between the start of reperfusion and the resumption of contractile activity. D) Percentage of cardiomyocytes that had full recovery (beating without arrhythmia) following a period of metabolic inhibition. Data are presented as mean±SEM (n≥25 cardiomyocytes from at least 6 hearts). Data were analyzed using the Mann-Whitney U test (A–C) and Fisher's exact test (D). *** = P<0.001 and ** = P<0.01 vs. normal diet.</p

Echocardiography measurements taken from mice anesthetized with isoflurane.

<p>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 mean±SEM. There was no statistical significance between the data.</p

Antioxidant proteins determined using proteomics.

<p>Data are presented as mean±SEM (n = 4 hearts) and are relative to the pooled sample and normalized to GAPDH. Data were analyzed using Student's t-test. * = P<0.05 vs. normal diet.</p