Transcript levels of proteasome 19S and 20S subunits, as well as the regulatory subunit PA28α, are not modified in late pregnancy.

<p>Relative transcript expression of the cardiac proteasome measured by Real-Time qPCR in non pregnant (NP, black bars), in late pregnancy (LP, white bars), 1 day post-partum (PP1, grey bars) and 7 days post-partum (PP7, shaded bars) for RPN2 and RPT4, which are subunits of 19 S (A–B), β2 and α7, which are subunits of 20 S (C–D) and the proteasome regulatory subunit PA28α (E). GAPDH was used as the internal reference gene (data not shown). Values are mean ± SEM as normalized to NP (n = 3–5 per group).</p

Protein levels of RPT4, RPN2, α7, PA28α and β5i are unaffected by pregnancy.

<p>Immunoblotting of whole heart lysates (100 µg) from non pregnant (NP, black bars), late pregnant (LP, white bars), 1 day post-partum (PP1, grey bars) and 7 days post-partum (PP7, shaded bars) with anti-RPN2 (A–B), -RPT4 (C–D), -α7 (E–F), -PA28α (G–H) and -β5i (I–J) antibodies. In (I), the upper 30 kDa band is the β5i containing the pro-peptide. The bar graphs represent the quantification of fluorescent signal intensity normalized to Vinculin. For β5i both bands were taken into consideration in the quantification of protein levels. Vinculin was used as the loading control (n = 4 per group). Values are mean ± SEM in arbitrary units.</p

Proteasome activity of 26 S, but not 20 S is reduced in late pregnancy.

<p>Activity of different proteasomal beta subunits of the 26 S (A) and 20 S (B) was measured after initiating the reaction with: Z-LLE-AMC (β1), Boc-LSTR-AMC (β2) and Suc-LLVY-AMC (β5) for non pregnant (NP, black bars), late pregnant (LP, white bars), one day post-partum (PP1, grey bars) and seven days post-partum (PP7, shaded bars). The fluorescence values in arbitrary units are represented as mean ± SEM *p<0.05 vs. NP (n = 4 mice per group) and are normalized to NP levels. The raw proteasome activity values for the NP group are as follows (in nmol/min/mg protein): for the 26 S ATP-dependent activities, β1 was 0.11±0.01, β2 was 0.04±0.01 and β5 was 0.16±0.01, while for the 20 S activity, β1 was 0.21±0.03, β2 was 0.15±0.02 and β5 was 0.12±0.01.</p

The increased in heart weight of LP mice is reversed post-partum.

<p>Heart weights (HW, A), body weights (BW, B) and HW/BW (C) in non-pregnant (NP, black bars, n = 8), late pregnant (LP, white bars, n = 11), one day post-partum (PP1, grey bars, n = 6) and seven days post-partum (PP7, shaded bars, n = 5) mice. Values are mean ± SEM, ^*p<0.05 and ^**p<0.001 vs. NP, ^#p<0.05 and ^##p<0.001 vs. LP, ^∧p<0.05 and ^∧∧p<0.001 vs. PP1.</p

Proteomic Analysis of Hearts from Akita Mice Suggests That Increases in Soluble Epoxide Hydrolase and Antioxidative Programming Are Key Changes in Early Stages of Diabetic Cardiomyopathy

Cardiovascular disease is the leading cause of diabetic morbidity with more than 10% of type 1 diabetes mellitus (T1DM) patients dying before they are 40 years old. This study utilized Akita mice, a murine model with T1DM progression analogous to that of humans. Diabetic cardiomyopathy in Akita mice presents as cardiac atrophy and diastolic impairment at 3 months of age, but we observed cardiac atrophy in hearts from recently diabetic mice (5 weeks old). Hearts from 5 week old mice were analyzed with a rigorous label-free quantitative proteomic approach to identify proteins that may play a critical role in the early pathophysiology of diabetic cardiomyopathy. Eleven proteins were differentially expressed in diabetic hearts: products of <i>GANC</i>, <i>PLEKHN1</i>, <i>COL1A1</i>, <i>GSTK1</i>, <i>ATP1A3</i>, <i>RAP1A</i>, <i>ACADS</i>, <i>EEF1A1</i>, <i>HRC</i>, <i>EPHX2</i>, and <i>PKP2</i> (gene names). These proteins are active in cellular defense, metabolism, insulin signaling, and calcium handling. Further analysis of Akita hearts using biochemical assays showed that the cellular defenses against oxidative stress were increased, including antioxidant capacity (2–3-fold) and glutathione levels (20%). Immunoblots of five and twelve week old Akita heart homogenates showed 30% and 145% increases in expression of soluble epoxide hydrolase (sEH (gene name <i>EPHX2</i>)), respectively, and an approximate 100% increase in sEH was seen in gastrocnemius tissue of 12 week old Akita mice. In contrast, 12 week old Akita livers showed no change in sEH expression. Our results suggest that increases in sEH and antioxidative programming are key factors in the development of type 1 diabetic cardiomyopathy in Akita mice and reveal several other proteins whose expression may be important in this complex pathophysiology

Proteomic Analysis of Hearts from Akita Mice Suggests That Increases in Soluble Epoxide Hydrolase and Antioxidative Programming Are Key Changes in Early Stages of Diabetic Cardiomyopathy

Cardiovascular disease is the leading cause of diabetic morbidity with more than 10% of type 1 diabetes mellitus (T1DM) patients dying before they are 40 years old. This study utilized Akita mice, a murine model with T1DM progression analogous to that of humans. Diabetic cardiomyopathy in Akita mice presents as cardiac atrophy and diastolic impairment at 3 months of age, but we observed cardiac atrophy in hearts from recently diabetic mice (5 weeks old). Hearts from 5 week old mice were analyzed with a rigorous label-free quantitative proteomic approach to identify proteins that may play a critical role in the early pathophysiology of diabetic cardiomyopathy. Eleven proteins were differentially expressed in diabetic hearts: products of <i>GANC</i>, <i>PLEKHN1</i>, <i>COL1A1</i>, <i>GSTK1</i>, <i>ATP1A3</i>, <i>RAP1A</i>, <i>ACADS</i>, <i>EEF1A1</i>, <i>HRC</i>, <i>EPHX2</i>, and <i>PKP2</i> (gene names). These proteins are active in cellular defense, metabolism, insulin signaling, and calcium handling. Further analysis of Akita hearts using biochemical assays showed that the cellular defenses against oxidative stress were increased, including antioxidant capacity (2–3-fold) and glutathione levels (20%). Immunoblots of five and twelve week old Akita heart homogenates showed 30% and 145% increases in expression of soluble epoxide hydrolase (sEH (gene name <i>EPHX2</i>)), respectively, and an approximate 100% increase in sEH was seen in gastrocnemius tissue of 12 week old Akita mice. In contrast, 12 week old Akita livers showed no change in sEH expression. Our results suggest that increases in sEH and antioxidative programming are key factors in the development of type 1 diabetic cardiomyopathy in Akita mice and reveal several other proteins whose expression may be important in this complex pathophysiology

Increased nuclear labeling of core Subunits and RPT4 in late pregnancy was reversed one week postpartum.

<p>A. Representative single confocal sections of cardiomyocytes dissociated from non pregnant (NP), late pregnant (LP), one day post-partum (PP1) and seven days post-partum (PP7) are co-immunostained with anti-core (green) and -RPT4 (red) antibodies. The nuclear overlay of core and RPT4 are also shown at higher resolution. These results are representative of the labeling pattern observed in myocytes from 3 different animals in each group. B. Quantification of nuclear fluorescence labeling in the four groups mentioned above for core (green bars) and RPT4 (red bars) from at least 20–25 cells per group (n = 3 mice/group). Only the nucleus in the confocal plane of focus was taken into account. **denotes <i>p</i><0.001 <i>vs.</i> NP, #<i>p</i><0.05 <i>vs.</i> LP and ^∧<i>p</i><0.05 <i>vs.</i> PP1.</p

Proteomic Analysis of Hearts from Akita Mice Suggests That Increases in Soluble Epoxide Hydrolase and Antioxidative Programming Are Key Changes in Early Stages of Diabetic Cardiomyopathy

Cardiovascular disease is the leading cause of diabetic morbidity with more than 10% of type 1 diabetes mellitus (T1DM) patients dying before they are 40 years old. This study utilized Akita mice, a murine model with T1DM progression analogous to that of humans. Diabetic cardiomyopathy in Akita mice presents as cardiac atrophy and diastolic impairment at 3 months of age, but we observed cardiac atrophy in hearts from recently diabetic mice (5 weeks old). Hearts from 5 week old mice were analyzed with a rigorous label-free quantitative proteomic approach to identify proteins that may play a critical role in the early pathophysiology of diabetic cardiomyopathy. Eleven proteins were differentially expressed in diabetic hearts: products of <i>GANC</i>, <i>PLEKHN1</i>, <i>COL1A1</i>, <i>GSTK1</i>, <i>ATP1A3</i>, <i>RAP1A</i>, <i>ACADS</i>, <i>EEF1A1</i>, <i>HRC</i>, <i>EPHX2</i>, and <i>PKP2</i> (gene names). These proteins are active in cellular defense, metabolism, insulin signaling, and calcium handling. Further analysis of Akita hearts using biochemical assays showed that the cellular defenses against oxidative stress were increased, including antioxidant capacity (2–3-fold) and glutathione levels (20%). Immunoblots of five and twelve week old Akita heart homogenates showed 30% and 145% increases in expression of soluble epoxide hydrolase (sEH (gene name <i>EPHX2</i>)), respectively, and an approximate 100% increase in sEH was seen in gastrocnemius tissue of 12 week old Akita mice. In contrast, 12 week old Akita livers showed no change in sEH expression. Our results suggest that increases in sEH and antioxidative programming are key factors in the development of type 1 diabetic cardiomyopathy in Akita mice and reveal several other proteins whose expression may be important in this complex pathophysiology

Pregnancy is associated with decreased polyubiquitinated protein levels, but not de-ubiqutination levels.

<p>A. Representative Western Blot of polyubiquitinated proteins (using the FK2 antibody) in whole heart lysates (100 µg) from non pregnant (NP), late pregnant (LP), 1 day post-partum (PP1) and 7 days post-partum (PP7). B. PonceauS was used as the loading control (n = 4 per group). C. Quantification of the polyubiquitinated proteins by Western Blot in non-pregnant (NP, black bar), late pregnant (LP, white bar), 1 day post-partum (PP1, grey bar) and 7 days post-partum (PP7, shaded bar). D. Polyubiquitination levels in NP, LP, PP1 and PP7 as determined by ELISA (using the FK1 antibody). E. De-ubiquitination activity levels in NP, LP, PP1 and PP7. Values are mean ± SEM and are normalized to NP, n = 4 per group and ^* denotes <i>p</i><0.05 <i>vs.</i> NP.</p

Proteasome activity of the 26S is unaffected by estrogen treatment.

<p>Activity of the different proteasomal beta subunits of the 26 S were measured after initiating the reaction with: Z-LLE-AMC (β1), Boc-LSTR-AMC (β2) and Suc-LLVY-AMC (β5) in ovariectomized female mice treated with placebo (Placebo, black bars) or with 17 β-estradiol for 10 days (E2, white bars). The fluorescence values in arbitrary units are normalized to Placebo levels and represented as mean ± SEM (n = 4 mice per group).</p