Mitochondrial stress test of cardiomyocytes at various maturational ages.

<p>XF trace (A) and bar graph (B) illustrates OCR changes (from baseline) in NB1, 3WK, 10WK, and 12–18MNTH CMs during mitochondrial stress testing. For group comparisons OCR was normalized to baseline prior to addition of Oligomycin, FCCP and Rotenone/Antimycin A where indicated. Respiratory control ratio (C) and coupling efficiency (D) as calculated from traces are compared between groups. Data are expressed as means ± SEM of 4–7 independent experiments. *p<0.05 by one-way ANOVA and Tukey’s post-test.</p

XF Analyses of Isolated Cardiomyocytes.

<p>Examples of real-time trace oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) during extracellular flux analyses using (A) mitochondrial stress test, (B) glycolytic stress test and (C) palmitate oxidation test. Data points represent mean of three readings taken at each time-point. Drug injections are shown for each test and boxes visually depict computed data.</p

Proton production rate during palmitate oxidation test.

<p>The positive or notionally negative contribution of lactate from both anaerobic glycolysis (grey) and from CO₂/respiration (white) to the sum (black) proton production rate (PPR) are demonstrated for cardiomyocytes from each maturational time-point. The PPR is demonstrated in response to palmitate (A) and etomoxir (B). Data shown are means ± SEM of 3–5 independent experiments. *p<0.05 by one way ANOVA and Tukey’s post-test.</p

Palmitate oxidation test of cardiomyocytes at various maturational ages.

<p>OCR was measured in NB1, 3WK, 10WK, and 12–18MNTH CMs and normalized to baseline rates to interrogate differences in response among the groups. OCR response to exogenous Palmitate-BSA and Etomoxir (carnitine palmitoyl transport inhibitor) are illustrated in trace (A). Bar graphs illustrate group differences in OCR response from baseline conditions (B). Data are expressed as means ± SEM of 3–4 independent experiments. *p<0.05 by one-way ANOVA and Tukey’s post-test.</p

Proton production rate during glycolytic stress test.

<p>The positive or notionally negative contribution of lactate from both anaerobic glycolysis (grey) and from CO₂/respiration (white) to the sum (black) proton production rate (PPR) are demonstrated for cardiomyocytes from each maturational time-point. The PPR is demonstrated in response to glucose (A) and oligomycin (B). Data shown are means ± SEM of 3–5 independent experiments. *p<0.05 by one way ANOVA and Tukey’s post-test.</p

Glycolysis stress test of cardiomyocytes at various maturational ages.

<p>Extra-cellular acidification rate (ECAR) traces (A) and bar graphs (B) show the glycolytic response of NB1, 3WK, 10WK and 12–18MTH old cardiomyocytes in response to glucose and oligomycin injection where indicated. Data shown are means ± SEM of 3–5 independent experiments. *p<0.05 by one way ANOVA and Tukey’s post-test.</p

Offspring mortality time points.

<p>Data are expressed as sums (percent of total pups) per group that were found alive, dead in the cage, dying or dead in utero on the day of delivery.</p

Maturity assessment in newborn lungs.

<p>(A) Representative glycogen deposition in fixed newborn rat lung stained using PAS stain (60X). (B) SDS-PAGE and immunoblot of newborn rat lung lysates using anti-T1α, anti-SP-B and anti-SP-C with β-actin as a loading control. Each lane represents equal protein contributions of multiple offspring (n = 2–4) from a single litter. Densitometric analysis of (C) T1α (D) SP-B and (E) SP-C protein expression. Data are expressed as mean±SEM. * indicates significant diet and ^± diabetes associated changes by 2-way ANOVA. Significance set at p<0.05. CD-CB, controls; CD-STZ, diabetes exposed; HF-CB, high-fat diet exposed; HF-STZ, combination (high-fat and diabetes) exposed.</p

Offspring echocardiography.

<p>Offspring echocardiography.</p

Experimental paradigm.

<p>Female Sprague Dawley rats were fed either control (CD) or high-fat (HF) diet at least 4 weeks prior to mating and bred with normal males. A positive vaginal swab for spermatozoa was deemed gestational day zero of pregnancy (GD0). On GD14, dams were injected with either citrate buffer (CB) vehicle or streptozotocin (STZ) to induce diabetes, which was controlled with sliding scale insulin twice daily. Offspring from four groups were analyzed on day one of life (NB1) or cross fostered to normal dams and analyzed at 3 weeks of age.</p