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MuRF2 regulates PPARγ1 activity to protect against diabetic cardiomyopathy and enhance weight gain induced by a high fat diet.

He J, Quintana MT, Sullivan J, L Parry T, J Grevengoed T, Schisler JC, Hill JA, Yates CC, Mapanga RF, Essop MF, Stansfield WE, Bain JR, Newgard CB, Muehlbauer MJ, Han Y, Clarke BA, Willis MS - Cardiovasc Diabetol (2015)

Bottom Line: However, increasing MuRF2:PPARγ1 (>5:1) beyond physiological levels drove poly-ubiquitin-mediated degradation of PPARγ1 in vitro, indicating large MuRF2 increases may lead to PPAR degradation if found in other disease states.Mutations in MuRF2 have been described to contribute to the severity of familial hypertrophic cardiomyopathy.These studies also identify MuRF2 as the first ubiquitin ligase to regulate cardiac PPARα and PPARγ1 activities in vivo via post-translational modification without degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, University of North Carolina, 111 Mason Farm Road, MBRB 2340B, Chapel Hill, NC, USA. heju@email.unc.edu.

ABSTRACT

Background: In diabetes mellitus the morbidity and mortality of cardiovascular disease is increased and represents an important independent mechanism by which heart disease is exacerbated. The pathogenesis of diabetic cardiomyopathy involves the enhanced activation of PPAR transcription factors, including PPARα, and to a lesser degree PPARβ and PPARγ1. How these transcription factors are regulated in the heart is largely unknown. Recent studies have described post-translational ubiquitination of PPARs as ways in which PPAR activity is inhibited in cancer. However, specific mechanisms in the heart have not previously been described. Recent studies have implicated the muscle-specific ubiquitin ligase muscle ring finger-2 (MuRF2) in inhibiting the nuclear transcription factor SRF. Initial studies of MuRF2-/- hearts revealed enhanced PPAR activity, leading to the hypothesis that MuRF2 regulates PPAR activity by post-translational ubiquitination.

Methods: MuRF2-/- mice were challenged with a 26-week 60% fat diet designed to simulate obesity-mediated insulin resistance and diabetic cardiomyopathy. Mice were followed by conscious echocardiography, blood glucose, tissue triglyceride, glycogen levels, immunoblot analysis of intracellular signaling, heart and skeletal muscle morphometrics, and PPARα, PPARβ, and PPARγ1-regulated mRNA expression.

Results: MuRF2 protein levels increase ~20% during the development of diabetic cardiomyopathy induced by high fat diet. Compared to littermate wildtype hearts, MuRF2-/- hearts exhibit an exaggerated diabetic cardiomyopathy, characterized by an early onset systolic dysfunction, larger left ventricular mass, and higher heart weight. MuRF2-/- hearts had significantly increased PPARα- and PPARγ1-regulated gene expression by RT-qPCR, consistent with MuRF2's regulation of these transcription factors in vivo. Mechanistically, MuRF2 mono-ubiquitinated PPARα and PPARγ1 in vitro, consistent with its non-degradatory role in diabetic cardiomyopathy. However, increasing MuRF2:PPARγ1 (>5:1) beyond physiological levels drove poly-ubiquitin-mediated degradation of PPARγ1 in vitro, indicating large MuRF2 increases may lead to PPAR degradation if found in other disease states.

Conclusions: Mutations in MuRF2 have been described to contribute to the severity of familial hypertrophic cardiomyopathy. The present study suggests that the lack of MuRF2, as found in these patients, can result in an exaggerated diabetic cardiomyopathy. These studies also identify MuRF2 as the first ubiquitin ligase to regulate cardiac PPARα and PPARγ1 activities in vivo via post-translational modification without degradation.

No MeSH data available.


Related in: MedlinePlus

High fat diet-induced increases in PPAR-regulated gene (mRNA) levels are exaggerated in cardiac MuRF2−/− hearts. RT-qPCR analysis of cardiac mRNA of genes identified as PPAR isoform “specific” based on cardiac transgenic PPARα, PPARβ, and PPARγ1 studies as described in the text. a Cardiac PPARα target gene expression, b PPARβ-regulated mRNA target genes involved in glucose metabolism, c PPARβ-regulated mRNA target genes involved in fatty acid metabolism. d PPARγ1-regulated mRNA target genes. Values expressed as Mean ± SE. Values expressed as Mean ± SE. The significance of observed differences in grouped mean values was determined using a One Way ANOVA followed by Holm-Sidak pairwise post hoc analysis. N per group indicated above graph. *p ≤ 0.001, **p < 0.01, #p < 0.05.
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Fig4: High fat diet-induced increases in PPAR-regulated gene (mRNA) levels are exaggerated in cardiac MuRF2−/− hearts. RT-qPCR analysis of cardiac mRNA of genes identified as PPAR isoform “specific” based on cardiac transgenic PPARα, PPARβ, and PPARγ1 studies as described in the text. a Cardiac PPARα target gene expression, b PPARβ-regulated mRNA target genes involved in glucose metabolism, c PPARβ-regulated mRNA target genes involved in fatty acid metabolism. d PPARγ1-regulated mRNA target genes. Values expressed as Mean ± SE. Values expressed as Mean ± SE. The significance of observed differences in grouped mean values was determined using a One Way ANOVA followed by Holm-Sidak pairwise post hoc analysis. N per group indicated above graph. *p ≤ 0.001, **p < 0.01, #p < 0.05.

Mentions: Cardiac PPARα, PPARβ, and PPARγ1 have pivotal roles in the pathophysiology of diabetic cardiomyopathy [44]. Therefore, we next investigated the expression of cardiac PPAR isoform regulated genes previously described in vivo [54–56]. Gene expression of the cardiac PPARα target genes (not regulated by cardiac PPARβ i.e. glut1 and cd36) (Fig. 4a), cardiac PPARβ target genes associated with glucose metabolism (not regulated by cardiac PPARα, i.e. glut4, pfk, acc1, mcad, and lcad) (Fig. 4b, c), and cardiac PPARγ1-regulated cardiac genes (i.e. acox1, adrp, cpt1b, and pdk4) (Fig. 4d) were evaluated in MuRF2−/− mouse hearts. Notably, MuRF2−/− hearts challenged with high fat diet exhibited significantly increased levels of PPARα-regulated genes (Fig. 4a), PPARβ-regulated genes associated with fatty acid metabolism (Fig. 4c), and PPARγ1-regulated genes (Fig. 4d). MuRF2−/− hearts did not differ from MuRF2+/+ hearts in PPARβ-regulated target genes associated with glucose metabolism (glut4 and pfk, Fig. 4c). Like the PPAR isoform activities assays of the MuRF2−/− heart nuclei demonstrated, MuRF2−/− hearts exhibited enhanced PPAR activities. At the mRNA level, MuRF2−/− hearts exhibited significant increases in PPARα compared with wildtype mice, but no differences in PPARβ or PPARγ1 (Additional file 3: Figure S3).Fig. 4


MuRF2 regulates PPARγ1 activity to protect against diabetic cardiomyopathy and enhance weight gain induced by a high fat diet.

He J, Quintana MT, Sullivan J, L Parry T, J Grevengoed T, Schisler JC, Hill JA, Yates CC, Mapanga RF, Essop MF, Stansfield WE, Bain JR, Newgard CB, Muehlbauer MJ, Han Y, Clarke BA, Willis MS - Cardiovasc Diabetol (2015)

High fat diet-induced increases in PPAR-regulated gene (mRNA) levels are exaggerated in cardiac MuRF2−/− hearts. RT-qPCR analysis of cardiac mRNA of genes identified as PPAR isoform “specific” based on cardiac transgenic PPARα, PPARβ, and PPARγ1 studies as described in the text. a Cardiac PPARα target gene expression, b PPARβ-regulated mRNA target genes involved in glucose metabolism, c PPARβ-regulated mRNA target genes involved in fatty acid metabolism. d PPARγ1-regulated mRNA target genes. Values expressed as Mean ± SE. Values expressed as Mean ± SE. The significance of observed differences in grouped mean values was determined using a One Way ANOVA followed by Holm-Sidak pairwise post hoc analysis. N per group indicated above graph. *p ≤ 0.001, **p < 0.01, #p < 0.05.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526192&req=5

Fig4: High fat diet-induced increases in PPAR-regulated gene (mRNA) levels are exaggerated in cardiac MuRF2−/− hearts. RT-qPCR analysis of cardiac mRNA of genes identified as PPAR isoform “specific” based on cardiac transgenic PPARα, PPARβ, and PPARγ1 studies as described in the text. a Cardiac PPARα target gene expression, b PPARβ-regulated mRNA target genes involved in glucose metabolism, c PPARβ-regulated mRNA target genes involved in fatty acid metabolism. d PPARγ1-regulated mRNA target genes. Values expressed as Mean ± SE. Values expressed as Mean ± SE. The significance of observed differences in grouped mean values was determined using a One Way ANOVA followed by Holm-Sidak pairwise post hoc analysis. N per group indicated above graph. *p ≤ 0.001, **p < 0.01, #p < 0.05.
Mentions: Cardiac PPARα, PPARβ, and PPARγ1 have pivotal roles in the pathophysiology of diabetic cardiomyopathy [44]. Therefore, we next investigated the expression of cardiac PPAR isoform regulated genes previously described in vivo [54–56]. Gene expression of the cardiac PPARα target genes (not regulated by cardiac PPARβ i.e. glut1 and cd36) (Fig. 4a), cardiac PPARβ target genes associated with glucose metabolism (not regulated by cardiac PPARα, i.e. glut4, pfk, acc1, mcad, and lcad) (Fig. 4b, c), and cardiac PPARγ1-regulated cardiac genes (i.e. acox1, adrp, cpt1b, and pdk4) (Fig. 4d) were evaluated in MuRF2−/− mouse hearts. Notably, MuRF2−/− hearts challenged with high fat diet exhibited significantly increased levels of PPARα-regulated genes (Fig. 4a), PPARβ-regulated genes associated with fatty acid metabolism (Fig. 4c), and PPARγ1-regulated genes (Fig. 4d). MuRF2−/− hearts did not differ from MuRF2+/+ hearts in PPARβ-regulated target genes associated with glucose metabolism (glut4 and pfk, Fig. 4c). Like the PPAR isoform activities assays of the MuRF2−/− heart nuclei demonstrated, MuRF2−/− hearts exhibited enhanced PPAR activities. At the mRNA level, MuRF2−/− hearts exhibited significant increases in PPARα compared with wildtype mice, but no differences in PPARβ or PPARγ1 (Additional file 3: Figure S3).Fig. 4

Bottom Line: However, increasing MuRF2:PPARγ1 (>5:1) beyond physiological levels drove poly-ubiquitin-mediated degradation of PPARγ1 in vitro, indicating large MuRF2 increases may lead to PPAR degradation if found in other disease states.Mutations in MuRF2 have been described to contribute to the severity of familial hypertrophic cardiomyopathy.These studies also identify MuRF2 as the first ubiquitin ligase to regulate cardiac PPARα and PPARγ1 activities in vivo via post-translational modification without degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, University of North Carolina, 111 Mason Farm Road, MBRB 2340B, Chapel Hill, NC, USA. heju@email.unc.edu.

ABSTRACT

Background: In diabetes mellitus the morbidity and mortality of cardiovascular disease is increased and represents an important independent mechanism by which heart disease is exacerbated. The pathogenesis of diabetic cardiomyopathy involves the enhanced activation of PPAR transcription factors, including PPARα, and to a lesser degree PPARβ and PPARγ1. How these transcription factors are regulated in the heart is largely unknown. Recent studies have described post-translational ubiquitination of PPARs as ways in which PPAR activity is inhibited in cancer. However, specific mechanisms in the heart have not previously been described. Recent studies have implicated the muscle-specific ubiquitin ligase muscle ring finger-2 (MuRF2) in inhibiting the nuclear transcription factor SRF. Initial studies of MuRF2-/- hearts revealed enhanced PPAR activity, leading to the hypothesis that MuRF2 regulates PPAR activity by post-translational ubiquitination.

Methods: MuRF2-/- mice were challenged with a 26-week 60% fat diet designed to simulate obesity-mediated insulin resistance and diabetic cardiomyopathy. Mice were followed by conscious echocardiography, blood glucose, tissue triglyceride, glycogen levels, immunoblot analysis of intracellular signaling, heart and skeletal muscle morphometrics, and PPARα, PPARβ, and PPARγ1-regulated mRNA expression.

Results: MuRF2 protein levels increase ~20% during the development of diabetic cardiomyopathy induced by high fat diet. Compared to littermate wildtype hearts, MuRF2-/- hearts exhibit an exaggerated diabetic cardiomyopathy, characterized by an early onset systolic dysfunction, larger left ventricular mass, and higher heart weight. MuRF2-/- hearts had significantly increased PPARα- and PPARγ1-regulated gene expression by RT-qPCR, consistent with MuRF2's regulation of these transcription factors in vivo. Mechanistically, MuRF2 mono-ubiquitinated PPARα and PPARγ1 in vitro, consistent with its non-degradatory role in diabetic cardiomyopathy. However, increasing MuRF2:PPARγ1 (>5:1) beyond physiological levels drove poly-ubiquitin-mediated degradation of PPARγ1 in vitro, indicating large MuRF2 increases may lead to PPAR degradation if found in other disease states.

Conclusions: Mutations in MuRF2 have been described to contribute to the severity of familial hypertrophic cardiomyopathy. The present study suggests that the lack of MuRF2, as found in these patients, can result in an exaggerated diabetic cardiomyopathy. These studies also identify MuRF2 as the first ubiquitin ligase to regulate cardiac PPARα and PPARγ1 activities in vivo via post-translational modification without degradation.

No MeSH data available.


Related in: MedlinePlus