TRB3 function in cardiac endoplasmic reticulum stress

Abstract

Rationale: Tribbles (TRB)3 is an intracellular pseudokinase that modulates the activity of several signal transduction cascades. TRB3 has been reported to inhibit the activity of Akt protein kinases. TRB3 gene expression is highly regulated in many cell types, and amino acid starvation, hypoxia, or endoplasmic reticulum (ER) stress promotes TRB3 expression in noncardiac cells.

Objective: The objective of this work was to examine TRB3 expression and function in cultured cardiac myocytes and in mouse heart.

Methods and results: Agents that induced ER stress increased TRB3 expression in cultured cardiac myocytes while blocking insulin-stimulated Akt activation in these cells. Knockdown of TRB3 in cultured cardiac myocytes reversed the effects of ER stress on insulin signaling. Experimental myocardial infarction led to increased TRB3 expression in murine heart tissue in the infarct border zone suggesting that ER stress may play a role in pathological cardiac remodeling. Transgenic mice with cardiac-specific overexpression of TRB3 were generated and they exhibited normal contractile function but altered cardiac signal transduction and metabolism with reduced cardiac glucose oxidation rates. Transgenic TRB3 mice were also sensitized to infarct expansion and cardiac myocyte apoptosis in the infarct border zone after myocardial infarction.

Conclusions: These results demonstrate that TRB3 induction is a significant aspect of the ER stress response in cardiac myocytes and that TRB3 antagonizes cardiac glucose metabolism and cardiac myocyte survival.

Figure 1

Cardiomyoycte ER stress inhibits Akt activation due to TRB3 induction. A. Treatment of HL-1 atrial myocytes with thapsigargin results in ER stress and reduced insulin-stimulated Akt activation. HL-1 cells were pre-treated with thapsigargin (THAPS; 2 μM) or DMSO for 24 hours. HL-1 cells were treated with insulin (10 nM) or control buffer for 10 minutes. Protein lysates were generated from HL-1 cells and proteins were separated by SDS-PAGE followed by immunoblotting with primary antibodies directed against phospho-Akt1/2 (p-Akt), total-Akt1/2 (t-Akt), GRP78, and CHOP. B. Densitometric analysis of Akt activation in HL-1 cells incubated with thapsigargin or DMSO and then stimulated with insulin (Ins) or buffer as depicted in 1A. Phospho-Akt levels were normalized by total Akt levels for each sample. *, P<0.001 versus HL-1 cells that were not treated with thapsigargin or insulin by Student's t-test; ˆ, P=0.004 versus HL-1 cells treated with DMSO and insulin by Student's t-test. C. TRB3 protein levels increase in HL-1 in response to ER stress. HL-1 cells were treated with DMSO or thapsigargin (THAPS; 2 μM) for 24 hours and protein lysates were obtained. Protein lysates were separated by SDS-PAGE followed by immunoblotting with an anti-TRB3 primary antibody. The anti-TRB3 antibody specifically recognizes a 42 kilodalton band or a doublet (depending on the resolution of the gel). Blots were re-probed with an anti-β-actin antibody to control for protein loading. The TRB3 protein levels were measured by computerized densitometry and were normalized by β-actin protein levels. The TRB3/actin levels are indicated below each lane in arbitrary units. The mean TRB3/actin level was 1.0 ± 0.19 for control DMSO-treated cells, and was 1.88 ± 0.14 for thapsigargin-treated cells (P=0.021 by Student's t-test). D. Agents that promote ER stress cause induction of TRB3 mRNA in HL-1 cardiac myocytes. HL-1 cells were treated with tunicamycin (2 μg/ml), thapsigargin (2 μM) or DMSO (control) for 24 hours. RNA was purified from HL-1 cells and TRB3 and GAPDH mRNA levels were analyzed by quantitative real-time PCR. *, P<0.05 by Kruskal-Wallis one way ANOVA on ranks. ˆ, P<0.05 by Kruskal-Wallis one way ANOVA on ranks. E. ER stress-mediated blockade of Akt activation is dependent on TRB3. HL-1 cells analyzed in this figure were treated with thapsigargin (THAPS; 2 μM) or DMSO for 24 hours. Some cells were also pre-treated with TRB3 siRNA at the indicated doses (20, 50 or 100 nM) or with scrambled siRNA (100 nM) for 24 hours prior to the addition of thapsigargin. In the upper panel, cells were serum starved for 6 hours and then insulin (10 nM) was added to all HL-1 cells for 10 minutes and protein lysates were obtained for SDS-PAGE followed by immunoblotting. Immunoblots depict the levels of activated Akt (p-Akt), total Akt (t-Akt), and GRP78. Under the blots, a computerized densitometry analysis of phospho-Akt levels normalized by total Akt protein levels for each lane is provided in arbitrary units. In the lower panel, HL-1 cells were cultured in parallel with those used for the immunoblot experiment, and were treated with TRB3 siRNA (20, 50 or 100 nM) or with scrambled siRNA (100 nM) for 24 hours. RNA was purified from HL-1 cells and TRB3 and GAPDH mRNA levels were analyzed by quantitative real-time PCR. *, P<0.05 by one way ANOVA (Holm-Sidak method) versus scrambled siRNA.

Figure 1

Cardiomyoycte ER stress inhibits Akt activation due to TRB3 induction. A. Treatment of HL-1 atrial myocytes with thapsigargin results in ER stress and reduced insulin-stimulated Akt activation. HL-1 cells were pre-treated with thapsigargin (THAPS; 2 μM) or DMSO for 24 hours. HL-1 cells were treated with insulin (10 nM) or control buffer for 10 minutes. Protein lysates were generated from HL-1 cells and proteins were separated by SDS-PAGE followed by immunoblotting with primary antibodies directed against phospho-Akt1/2 (p-Akt), total-Akt1/2 (t-Akt), GRP78, and CHOP. B. Densitometric analysis of Akt activation in HL-1 cells incubated with thapsigargin or DMSO and then stimulated with insulin (Ins) or buffer as depicted in 1A. Phospho-Akt levels were normalized by total Akt levels for each sample. *, P<0.001 versus HL-1 cells that were not treated with thapsigargin or insulin by Student's t-test; ˆ, P=0.004 versus HL-1 cells treated with DMSO and insulin by Student's t-test. C. TRB3 protein levels increase in HL-1 in response to ER stress. HL-1 cells were treated with DMSO or thapsigargin (THAPS; 2 μM) for 24 hours and protein lysates were obtained. Protein lysates were separated by SDS-PAGE followed by immunoblotting with an anti-TRB3 primary antibody. The anti-TRB3 antibody specifically recognizes a 42 kilodalton band or a doublet (depending on the resolution of the gel). Blots were re-probed with an anti-β-actin antibody to control for protein loading. The TRB3 protein levels were measured by computerized densitometry and were normalized by β-actin protein levels. The TRB3/actin levels are indicated below each lane in arbitrary units. The mean TRB3/actin level was 1.0 ± 0.19 for control DMSO-treated cells, and was 1.88 ± 0.14 for thapsigargin-treated cells (P=0.021 by Student's t-test). D. Agents that promote ER stress cause induction of TRB3 mRNA in HL-1 cardiac myocytes. HL-1 cells were treated with tunicamycin (2 μg/ml), thapsigargin (2 μM) or DMSO (control) for 24 hours. RNA was purified from HL-1 cells and TRB3 and GAPDH mRNA levels were analyzed by quantitative real-time PCR. *, P<0.05 by Kruskal-Wallis one way ANOVA on ranks. ˆ, P<0.05 by Kruskal-Wallis one way ANOVA on ranks. E. ER stress-mediated blockade of Akt activation is dependent on TRB3. HL-1 cells analyzed in this figure were treated with thapsigargin (THAPS; 2 μM) or DMSO for 24 hours. Some cells were also pre-treated with TRB3 siRNA at the indicated doses (20, 50 or 100 nM) or with scrambled siRNA (100 nM) for 24 hours prior to the addition of thapsigargin. In the upper panel, cells were serum starved for 6 hours and then insulin (10 nM) was added to all HL-1 cells for 10 minutes and protein lysates were obtained for SDS-PAGE followed by immunoblotting. Immunoblots depict the levels of activated Akt (p-Akt), total Akt (t-Akt), and GRP78. Under the blots, a computerized densitometry analysis of phospho-Akt levels normalized by total Akt protein levels for each lane is provided in arbitrary units. In the lower panel, HL-1 cells were cultured in parallel with those used for the immunoblot experiment, and were treated with TRB3 siRNA (20, 50 or 100 nM) or with scrambled siRNA (100 nM) for 24 hours. RNA was purified from HL-1 cells and TRB3 and GAPDH mRNA levels were analyzed by quantitative real-time PCR. *, P<0.05 by one way ANOVA (Holm-Sidak method) versus scrambled siRNA.

Figure 1

Cardiomyoycte ER stress inhibits Akt activation due to TRB3 induction. A. Treatment of HL-1 atrial myocytes with thapsigargin results in ER stress and reduced insulin-stimulated Akt activation. HL-1 cells were pre-treated with thapsigargin (THAPS; 2 μM) or DMSO for 24 hours. HL-1 cells were treated with insulin (10 nM) or control buffer for 10 minutes. Protein lysates were generated from HL-1 cells and proteins were separated by SDS-PAGE followed by immunoblotting with primary antibodies directed against phospho-Akt1/2 (p-Akt), total-Akt1/2 (t-Akt), GRP78, and CHOP. B. Densitometric analysis of Akt activation in HL-1 cells incubated with thapsigargin or DMSO and then stimulated with insulin (Ins) or buffer as depicted in 1A. Phospho-Akt levels were normalized by total Akt levels for each sample. *, P<0.001 versus HL-1 cells that were not treated with thapsigargin or insulin by Student's t-test; ˆ, P=0.004 versus HL-1 cells treated with DMSO and insulin by Student's t-test. C. TRB3 protein levels increase in HL-1 in response to ER stress. HL-1 cells were treated with DMSO or thapsigargin (THAPS; 2 μM) for 24 hours and protein lysates were obtained. Protein lysates were separated by SDS-PAGE followed by immunoblotting with an anti-TRB3 primary antibody. The anti-TRB3 antibody specifically recognizes a 42 kilodalton band or a doublet (depending on the resolution of the gel). Blots were re-probed with an anti-β-actin antibody to control for protein loading. The TRB3 protein levels were measured by computerized densitometry and were normalized by β-actin protein levels. The TRB3/actin levels are indicated below each lane in arbitrary units. The mean TRB3/actin level was 1.0 ± 0.19 for control DMSO-treated cells, and was 1.88 ± 0.14 for thapsigargin-treated cells (P=0.021 by Student's t-test). D. Agents that promote ER stress cause induction of TRB3 mRNA in HL-1 cardiac myocytes. HL-1 cells were treated with tunicamycin (2 μg/ml), thapsigargin (2 μM) or DMSO (control) for 24 hours. RNA was purified from HL-1 cells and TRB3 and GAPDH mRNA levels were analyzed by quantitative real-time PCR. *, P<0.05 by Kruskal-Wallis one way ANOVA on ranks. ˆ, P<0.05 by Kruskal-Wallis one way ANOVA on ranks. E. ER stress-mediated blockade of Akt activation is dependent on TRB3. HL-1 cells analyzed in this figure were treated with thapsigargin (THAPS; 2 μM) or DMSO for 24 hours. Some cells were also pre-treated with TRB3 siRNA at the indicated doses (20, 50 or 100 nM) or with scrambled siRNA (100 nM) for 24 hours prior to the addition of thapsigargin. In the upper panel, cells were serum starved for 6 hours and then insulin (10 nM) was added to all HL-1 cells for 10 minutes and protein lysates were obtained for SDS-PAGE followed by immunoblotting. Immunoblots depict the levels of activated Akt (p-Akt), total Akt (t-Akt), and GRP78. Under the blots, a computerized densitometry analysis of phospho-Akt levels normalized by total Akt protein levels for each lane is provided in arbitrary units. In the lower panel, HL-1 cells were cultured in parallel with those used for the immunoblot experiment, and were treated with TRB3 siRNA (20, 50 or 100 nM) or with scrambled siRNA (100 nM) for 24 hours. RNA was purified from HL-1 cells and TRB3 and GAPDH mRNA levels were analyzed by quantitative real-time PCR. *, P<0.05 by one way ANOVA (Holm-Sidak method) versus scrambled siRNA.

Figure 1

Cardiomyoycte ER stress inhibits Akt activation due to TRB3 induction. A. Treatment of HL-1 atrial myocytes with thapsigargin results in ER stress and reduced insulin-stimulated Akt activation. HL-1 cells were pre-treated with thapsigargin (THAPS; 2 μM) or DMSO for 24 hours. HL-1 cells were treated with insulin (10 nM) or control buffer for 10 minutes. Protein lysates were generated from HL-1 cells and proteins were separated by SDS-PAGE followed by immunoblotting with primary antibodies directed against phospho-Akt1/2 (p-Akt), total-Akt1/2 (t-Akt), GRP78, and CHOP. B. Densitometric analysis of Akt activation in HL-1 cells incubated with thapsigargin or DMSO and then stimulated with insulin (Ins) or buffer as depicted in 1A. Phospho-Akt levels were normalized by total Akt levels for each sample. *, P<0.001 versus HL-1 cells that were not treated with thapsigargin or insulin by Student's t-test; ˆ, P=0.004 versus HL-1 cells treated with DMSO and insulin by Student's t-test. C. TRB3 protein levels increase in HL-1 in response to ER stress. HL-1 cells were treated with DMSO or thapsigargin (THAPS; 2 μM) for 24 hours and protein lysates were obtained. Protein lysates were separated by SDS-PAGE followed by immunoblotting with an anti-TRB3 primary antibody. The anti-TRB3 antibody specifically recognizes a 42 kilodalton band or a doublet (depending on the resolution of the gel). Blots were re-probed with an anti-β-actin antibody to control for protein loading. The TRB3 protein levels were measured by computerized densitometry and were normalized by β-actin protein levels. The TRB3/actin levels are indicated below each lane in arbitrary units. The mean TRB3/actin level was 1.0 ± 0.19 for control DMSO-treated cells, and was 1.88 ± 0.14 for thapsigargin-treated cells (P=0.021 by Student's t-test). D. Agents that promote ER stress cause induction of TRB3 mRNA in HL-1 cardiac myocytes. HL-1 cells were treated with tunicamycin (2 μg/ml), thapsigargin (2 μM) or DMSO (control) for 24 hours. RNA was purified from HL-1 cells and TRB3 and GAPDH mRNA levels were analyzed by quantitative real-time PCR. *, P<0.05 by Kruskal-Wallis one way ANOVA on ranks. ˆ, P<0.05 by Kruskal-Wallis one way ANOVA on ranks. E. ER stress-mediated blockade of Akt activation is dependent on TRB3. HL-1 cells analyzed in this figure were treated with thapsigargin (THAPS; 2 μM) or DMSO for 24 hours. Some cells were also pre-treated with TRB3 siRNA at the indicated doses (20, 50 or 100 nM) or with scrambled siRNA (100 nM) for 24 hours prior to the addition of thapsigargin. In the upper panel, cells were serum starved for 6 hours and then insulin (10 nM) was added to all HL-1 cells for 10 minutes and protein lysates were obtained for SDS-PAGE followed by immunoblotting. Immunoblots depict the levels of activated Akt (p-Akt), total Akt (t-Akt), and GRP78. Under the blots, a computerized densitometry analysis of phospho-Akt levels normalized by total Akt protein levels for each lane is provided in arbitrary units. In the lower panel, HL-1 cells were cultured in parallel with those used for the immunoblot experiment, and were treated with TRB3 siRNA (20, 50 or 100 nM) or with scrambled siRNA (100 nM) for 24 hours. RNA was purified from HL-1 cells and TRB3 and GAPDH mRNA levels were analyzed by quantitative real-time PCR. *, P<0.05 by one way ANOVA (Holm-Sidak method) versus scrambled siRNA.

Figure 1

Cardiomyoycte ER stress inhibits Akt activation due to TRB3 induction. A. Treatment of HL-1 atrial myocytes with thapsigargin results in ER stress and reduced insulin-stimulated Akt activation. HL-1 cells were pre-treated with thapsigargin (THAPS; 2 μM) or DMSO for 24 hours. HL-1 cells were treated with insulin (10 nM) or control buffer for 10 minutes. Protein lysates were generated from HL-1 cells and proteins were separated by SDS-PAGE followed by immunoblotting with primary antibodies directed against phospho-Akt1/2 (p-Akt), total-Akt1/2 (t-Akt), GRP78, and CHOP. B. Densitometric analysis of Akt activation in HL-1 cells incubated with thapsigargin or DMSO and then stimulated with insulin (Ins) or buffer as depicted in 1A. Phospho-Akt levels were normalized by total Akt levels for each sample. *, P<0.001 versus HL-1 cells that were not treated with thapsigargin or insulin by Student's t-test; ˆ, P=0.004 versus HL-1 cells treated with DMSO and insulin by Student's t-test. C. TRB3 protein levels increase in HL-1 in response to ER stress. HL-1 cells were treated with DMSO or thapsigargin (THAPS; 2 μM) for 24 hours and protein lysates were obtained. Protein lysates were separated by SDS-PAGE followed by immunoblotting with an anti-TRB3 primary antibody. The anti-TRB3 antibody specifically recognizes a 42 kilodalton band or a doublet (depending on the resolution of the gel). Blots were re-probed with an anti-β-actin antibody to control for protein loading. The TRB3 protein levels were measured by computerized densitometry and were normalized by β-actin protein levels. The TRB3/actin levels are indicated below each lane in arbitrary units. The mean TRB3/actin level was 1.0 ± 0.19 for control DMSO-treated cells, and was 1.88 ± 0.14 for thapsigargin-treated cells (P=0.021 by Student's t-test). D. Agents that promote ER stress cause induction of TRB3 mRNA in HL-1 cardiac myocytes. HL-1 cells were treated with tunicamycin (2 μg/ml), thapsigargin (2 μM) or DMSO (control) for 24 hours. RNA was purified from HL-1 cells and TRB3 and GAPDH mRNA levels were analyzed by quantitative real-time PCR. *, P<0.05 by Kruskal-Wallis one way ANOVA on ranks. ˆ, P<0.05 by Kruskal-Wallis one way ANOVA on ranks. E. ER stress-mediated blockade of Akt activation is dependent on TRB3. HL-1 cells analyzed in this figure were treated with thapsigargin (THAPS; 2 μM) or DMSO for 24 hours. Some cells were also pre-treated with TRB3 siRNA at the indicated doses (20, 50 or 100 nM) or with scrambled siRNA (100 nM) for 24 hours prior to the addition of thapsigargin. In the upper panel, cells were serum starved for 6 hours and then insulin (10 nM) was added to all HL-1 cells for 10 minutes and protein lysates were obtained for SDS-PAGE followed by immunoblotting. Immunoblots depict the levels of activated Akt (p-Akt), total Akt (t-Akt), and GRP78. Under the blots, a computerized densitometry analysis of phospho-Akt levels normalized by total Akt protein levels for each lane is provided in arbitrary units. In the lower panel, HL-1 cells were cultured in parallel with those used for the immunoblot experiment, and were treated with TRB3 siRNA (20, 50 or 100 nM) or with scrambled siRNA (100 nM) for 24 hours. RNA was purified from HL-1 cells and TRB3 and GAPDH mRNA levels were analyzed by quantitative real-time PCR. *, P<0.05 by one way ANOVA (Holm-Sidak method) versus scrambled siRNA.

Figure 2

ER stress markers and TRB3 expression is induced in cardiac tissue after myocardial infarction. Wild type C57BL/6J mice were subjected to experimental myocardial infarction by ligation of the left anterior descending coronary artery. A. Total left ventricle was isolated 4 and 24 hours after MI or sham operation for RNA isolation. Quantitative real-time PCR analysis of GRP78 was performed. B. Total left ventricle was isolated 4 and 24 hours after MI or sham operation for RNA isolation. Quantitative real-time PCR analysis of CHOP was performed. C. Total left ventricle was isolated 4 and 24 hours after MI or sham operation for RNA isolation. Quantitative real-time PCR analysis of TRB3 was performed. *, P<0.05 by one way ANOVA (Holm-Sidak method) versus sham operation. D. Infarct border zone (defined as one-quarter circumference on either side of the infarct edge) and remote left ventricular tissue was isolated 24 hours after MI surgery for RNA isolation. In addition, the entire left ventricle was isolated 24 hours after a sham operation for RNA isolation. Quantitative real-time PCR analysis of TRB3 was performed. *, P<0.05 by one way ANOVA (Holm-Sidak method) versus remote left ventricle.

Figure 2

ER stress markers and TRB3 expression is induced in cardiac tissue after myocardial infarction. Wild type C57BL/6J mice were subjected to experimental myocardial infarction by ligation of the left anterior descending coronary artery. A. Total left ventricle was isolated 4 and 24 hours after MI or sham operation for RNA isolation. Quantitative real-time PCR analysis of GRP78 was performed. B. Total left ventricle was isolated 4 and 24 hours after MI or sham operation for RNA isolation. Quantitative real-time PCR analysis of CHOP was performed. C. Total left ventricle was isolated 4 and 24 hours after MI or sham operation for RNA isolation. Quantitative real-time PCR analysis of TRB3 was performed. *, P<0.05 by one way ANOVA (Holm-Sidak method) versus sham operation. D. Infarct border zone (defined as one-quarter circumference on either side of the infarct edge) and remote left ventricular tissue was isolated 24 hours after MI surgery for RNA isolation. In addition, the entire left ventricle was isolated 24 hours after a sham operation for RNA isolation. Quantitative real-time PCR analysis of TRB3 was performed. *, P<0.05 by one way ANOVA (Holm-Sidak method) versus remote left ventricle.

Figure 2

ER stress markers and TRB3 expression is induced in cardiac tissue after myocardial infarction. Wild type C57BL/6J mice were subjected to experimental myocardial infarction by ligation of the left anterior descending coronary artery. A. Total left ventricle was isolated 4 and 24 hours after MI or sham operation for RNA isolation. Quantitative real-time PCR analysis of GRP78 was performed. B. Total left ventricle was isolated 4 and 24 hours after MI or sham operation for RNA isolation. Quantitative real-time PCR analysis of CHOP was performed. C. Total left ventricle was isolated 4 and 24 hours after MI or sham operation for RNA isolation. Quantitative real-time PCR analysis of TRB3 was performed. *, P<0.05 by one way ANOVA (Holm-Sidak method) versus sham operation. D. Infarct border zone (defined as one-quarter circumference on either side of the infarct edge) and remote left ventricular tissue was isolated 24 hours after MI surgery for RNA isolation. In addition, the entire left ventricle was isolated 24 hours after a sham operation for RNA isolation. Quantitative real-time PCR analysis of TRB3 was performed. *, P<0.05 by one way ANOVA (Holm-Sidak method) versus remote left ventricle.

Figure 2

ER stress markers and TRB3 expression is induced in cardiac tissue after myocardial infarction. Wild type C57BL/6J mice were subjected to experimental myocardial infarction by ligation of the left anterior descending coronary artery. A. Total left ventricle was isolated 4 and 24 hours after MI or sham operation for RNA isolation. Quantitative real-time PCR analysis of GRP78 was performed. B. Total left ventricle was isolated 4 and 24 hours after MI or sham operation for RNA isolation. Quantitative real-time PCR analysis of CHOP was performed. C. Total left ventricle was isolated 4 and 24 hours after MI or sham operation for RNA isolation. Quantitative real-time PCR analysis of TRB3 was performed. *, P<0.05 by one way ANOVA (Holm-Sidak method) versus sham operation. D. Infarct border zone (defined as one-quarter circumference on either side of the infarct edge) and remote left ventricular tissue was isolated 24 hours after MI surgery for RNA isolation. In addition, the entire left ventricle was isolated 24 hours after a sham operation for RNA isolation. Quantitative real-time PCR analysis of TRB3 was performed. *, P<0.05 by one way ANOVA (Holm-Sidak method) versus remote left ventricle.

Figure 3

Analysis of transgenic mice with cardiac-specific overexpression of TRB3. A. Increased TRB3 protein levels in 1×-TRB3, 2×-TRB3, and 4×-TRB3 transgenic heart tissue. Left ventricular tissue was isolated from12 week old transgenic mice and nontransgenic C57BL/6J littermates. Ventricular protein lysates were analyzed by immunoblotting for TRB3 protein content. Blots were re-probed with anti-14-3-3β primary antibody to control for protein loading. B. Reduced insulin-stimulated intracellular signal transduction in 2×-TRB3 transgenic myocardium. 2×-TRB3 mice and their wild type (WT) littermates were anesthetized and treated with insulin (0.1 units/kg body weight) or vehicle by jugular vein injection. Five minutes later, hearts were isolated and ventricular tissue was used to generate protein lysates. Proteins were separated by SDS-PAGE and immunoblotting was performed to evaluate the phosphorylation status of Akt and GSK-3β. Blots were re-probed with anti-total Akt1/2 primary antibody to control for protein loading. Depicted in the figure are the computerized densitometry results that indicate the relative phospho-Akt or phospho-GSK3β/total-Akt protein level for each sample. *, P<0.001 versus wild type (WT) cardiac tissue not treated with insulin by Student's t-test; ˆ, P=0.014 versus WT cardiac tissue treated with insulin by Student's t-test; **, P<0.001 versus WT cardiac tissue not treated with insulin by Student's t-test;ˆˆ, P=0.004 versus WT cardiac tissue treated with insulin by Student's t-test. C. Increased expression of β-MHC in 2×-TRB3 cardiac tissue. RNA was purified from ventricular tissue isolated from 12-16-week-old 2×-TRB3 mice and their nontransgenic wild type littermates (WT). ANF, β-MHC, SERCA2, GRP78 and CHOP gene expression was analyzed by quantitative real-time PCR. *, P=0.006 by Student's t-test versus WT heart.

Figure 3

Analysis of transgenic mice with cardiac-specific overexpression of TRB3. A. Increased TRB3 protein levels in 1×-TRB3, 2×-TRB3, and 4×-TRB3 transgenic heart tissue. Left ventricular tissue was isolated from12 week old transgenic mice and nontransgenic C57BL/6J littermates. Ventricular protein lysates were analyzed by immunoblotting for TRB3 protein content. Blots were re-probed with anti-14-3-3β primary antibody to control for protein loading. B. Reduced insulin-stimulated intracellular signal transduction in 2×-TRB3 transgenic myocardium. 2×-TRB3 mice and their wild type (WT) littermates were anesthetized and treated with insulin (0.1 units/kg body weight) or vehicle by jugular vein injection. Five minutes later, hearts were isolated and ventricular tissue was used to generate protein lysates. Proteins were separated by SDS-PAGE and immunoblotting was performed to evaluate the phosphorylation status of Akt and GSK-3β. Blots were re-probed with anti-total Akt1/2 primary antibody to control for protein loading. Depicted in the figure are the computerized densitometry results that indicate the relative phospho-Akt or phospho-GSK3β/total-Akt protein level for each sample. *, P<0.001 versus wild type (WT) cardiac tissue not treated with insulin by Student's t-test; ˆ, P=0.014 versus WT cardiac tissue treated with insulin by Student's t-test; **, P<0.001 versus WT cardiac tissue not treated with insulin by Student's t-test;ˆˆ, P=0.004 versus WT cardiac tissue treated with insulin by Student's t-test. C. Increased expression of β-MHC in 2×-TRB3 cardiac tissue. RNA was purified from ventricular tissue isolated from 12-16-week-old 2×-TRB3 mice and their nontransgenic wild type littermates (WT). ANF, β-MHC, SERCA2, GRP78 and CHOP gene expression was analyzed by quantitative real-time PCR. *, P=0.006 by Student's t-test versus WT heart.

Figure 3

Analysis of transgenic mice with cardiac-specific overexpression of TRB3. A. Increased TRB3 protein levels in 1×-TRB3, 2×-TRB3, and 4×-TRB3 transgenic heart tissue. Left ventricular tissue was isolated from12 week old transgenic mice and nontransgenic C57BL/6J littermates. Ventricular protein lysates were analyzed by immunoblotting for TRB3 protein content. Blots were re-probed with anti-14-3-3β primary antibody to control for protein loading. B. Reduced insulin-stimulated intracellular signal transduction in 2×-TRB3 transgenic myocardium. 2×-TRB3 mice and their wild type (WT) littermates were anesthetized and treated with insulin (0.1 units/kg body weight) or vehicle by jugular vein injection. Five minutes later, hearts were isolated and ventricular tissue was used to generate protein lysates. Proteins were separated by SDS-PAGE and immunoblotting was performed to evaluate the phosphorylation status of Akt and GSK-3β. Blots were re-probed with anti-total Akt1/2 primary antibody to control for protein loading. Depicted in the figure are the computerized densitometry results that indicate the relative phospho-Akt or phospho-GSK3β/total-Akt protein level for each sample. *, P<0.001 versus wild type (WT) cardiac tissue not treated with insulin by Student's t-test; ˆ, P=0.014 versus WT cardiac tissue treated with insulin by Student's t-test; **, P<0.001 versus WT cardiac tissue not treated with insulin by Student's t-test;ˆˆ, P=0.004 versus WT cardiac tissue treated with insulin by Student's t-test. C. Increased expression of β-MHC in 2×-TRB3 cardiac tissue. RNA was purified from ventricular tissue isolated from 12-16-week-old 2×-TRB3 mice and their nontransgenic wild type littermates (WT). ANF, β-MHC, SERCA2, GRP78 and CHOP gene expression was analyzed by quantitative real-time PCR. *, P=0.006 by Student's t-test versus WT heart.

Figure 4

Altered cardiac metabolism in 2×- and 4×-TRB3 transgenic mice determined by ex vivo working heart analysis. A. Analysis of cardiac glucose and palmitate oxidation rates in 2×-TRB3 transgenic hearts in the ex vivo working mode. 12-week-old 2×-TRB3 mice (n=5) and their nontransgenic wild type (WT) littermates (n=7) were used for these studies. Hearts were isolated from mice and were analyzed ex vivo in working mode. *, P=0.048 by Student's t-test versus WT heart. B. Analysis of cardiac glucose and palmitate oxidation rates in 4×-TRB3 transgenic hearts in the ex vivo working mode. 12-week-old 4×-TRB3 mice (n=4) and their nontransgenic wild type (WT) littermates (n=6) were used for these studies. Hearts were isolated from mice and were analyzed ex vivo in working mode. *, P=0.040 by Student's t-test versus WT heart.

Figure 4

Altered cardiac metabolism in 2×- and 4×-TRB3 transgenic mice determined by ex vivo working heart analysis. A. Analysis of cardiac glucose and palmitate oxidation rates in 2×-TRB3 transgenic hearts in the ex vivo working mode. 12-week-old 2×-TRB3 mice (n=5) and their nontransgenic wild type (WT) littermates (n=7) were used for these studies. Hearts were isolated from mice and were analyzed ex vivo in working mode. *, P=0.048 by Student's t-test versus WT heart. B. Analysis of cardiac glucose and palmitate oxidation rates in 4×-TRB3 transgenic hearts in the ex vivo working mode. 12-week-old 4×-TRB3 mice (n=4) and their nontransgenic wild type (WT) littermates (n=6) were used for these studies. Hearts were isolated from mice and were analyzed ex vivo in working mode. *, P=0.040 by Student's t-test versus WT heart.

Figure 5

Pathological cardiac remodeling is increased after myocardial infarction in 2×-TRB3 mice. At 12 weeks of age, 2×-TRB3 mice (n=9) and their nontransgenic littermates (n=13) were subjected to experimental myocardial infarction surgery by ligation of the left anterior descending coronary artery. A. Evaluation of the initial infarct size by transthoracic echocardiography one day after surgery. The initial infarct size was evaluated in 2×-TRB3 and nontransgenic mice one day after surgery. The segmental wall motion score index (SWMSI) was employed to evaluate initial infarct size. B. Increased LV scar area in 2×-TRB3 mice 7 days after MI. LV sections from 2×-TRB3 and nontransgenic littermates were stained with Masson's trichrome to determine infarct size. C. Computerized analysis of LV infarct size in 2×-TRB3 (n=9) and nontransgenic littermate (n=13) mice determined 7 days after MI. *, P=0.009 versus nontransgenic littermates by Student's t-test. D. Increased apoptotic cardiac myocytes in infarct border zone of 2×-TRB3 mice. LV sections from 2×-TRB3 and nontransgenic littermates obtained 7 days after MI were analyzed by TUNEL in the infarct border zones (defined as one-quarter circumference and either side of the scar edge). *, P=0.012 versus nontransgenic LV sections by Rank Sum test.

Figure 5

Pathological cardiac remodeling is increased after myocardial infarction in 2×-TRB3 mice. At 12 weeks of age, 2×-TRB3 mice (n=9) and their nontransgenic littermates (n=13) were subjected to experimental myocardial infarction surgery by ligation of the left anterior descending coronary artery. A. Evaluation of the initial infarct size by transthoracic echocardiography one day after surgery. The initial infarct size was evaluated in 2×-TRB3 and nontransgenic mice one day after surgery. The segmental wall motion score index (SWMSI) was employed to evaluate initial infarct size. B. Increased LV scar area in 2×-TRB3 mice 7 days after MI. LV sections from 2×-TRB3 and nontransgenic littermates were stained with Masson's trichrome to determine infarct size. C. Computerized analysis of LV infarct size in 2×-TRB3 (n=9) and nontransgenic littermate (n=13) mice determined 7 days after MI. *, P=0.009 versus nontransgenic littermates by Student's t-test. D. Increased apoptotic cardiac myocytes in infarct border zone of 2×-TRB3 mice. LV sections from 2×-TRB3 and nontransgenic littermates obtained 7 days after MI were analyzed by TUNEL in the infarct border zones (defined as one-quarter circumference and either side of the scar edge). *, P=0.012 versus nontransgenic LV sections by Rank Sum test.

Figure 5

Pathological cardiac remodeling is increased after myocardial infarction in 2×-TRB3 mice. At 12 weeks of age, 2×-TRB3 mice (n=9) and their nontransgenic littermates (n=13) were subjected to experimental myocardial infarction surgery by ligation of the left anterior descending coronary artery. A. Evaluation of the initial infarct size by transthoracic echocardiography one day after surgery. The initial infarct size was evaluated in 2×-TRB3 and nontransgenic mice one day after surgery. The segmental wall motion score index (SWMSI) was employed to evaluate initial infarct size. B. Increased LV scar area in 2×-TRB3 mice 7 days after MI. LV sections from 2×-TRB3 and nontransgenic littermates were stained with Masson's trichrome to determine infarct size. C. Computerized analysis of LV infarct size in 2×-TRB3 (n=9) and nontransgenic littermate (n=13) mice determined 7 days after MI. *, P=0.009 versus nontransgenic littermates by Student's t-test. D. Increased apoptotic cardiac myocytes in infarct border zone of 2×-TRB3 mice. LV sections from 2×-TRB3 and nontransgenic littermates obtained 7 days after MI were analyzed by TUNEL in the infarct border zones (defined as one-quarter circumference and either side of the scar edge). *, P=0.012 versus nontransgenic LV sections by Rank Sum test.

Figure 5

Pathological cardiac remodeling is increased after myocardial infarction in 2×-TRB3 mice. At 12 weeks of age, 2×-TRB3 mice (n=9) and their nontransgenic littermates (n=13) were subjected to experimental myocardial infarction surgery by ligation of the left anterior descending coronary artery. A. Evaluation of the initial infarct size by transthoracic echocardiography one day after surgery. The initial infarct size was evaluated in 2×-TRB3 and nontransgenic mice one day after surgery. The segmental wall motion score index (SWMSI) was employed to evaluate initial infarct size. B. Increased LV scar area in 2×-TRB3 mice 7 days after MI. LV sections from 2×-TRB3 and nontransgenic littermates were stained with Masson's trichrome to determine infarct size. C. Computerized analysis of LV infarct size in 2×-TRB3 (n=9) and nontransgenic littermate (n=13) mice determined 7 days after MI. *, P=0.009 versus nontransgenic littermates by Student's t-test. D. Increased apoptotic cardiac myocytes in infarct border zone of 2×-TRB3 mice. LV sections from 2×-TRB3 and nontransgenic littermates obtained 7 days after MI were analyzed by TUNEL in the infarct border zones (defined as one-quarter circumference and either side of the scar edge). *, P=0.012 versus nontransgenic LV sections by Rank Sum test.