Limits...
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

The ratio of MuRF2 to PPARγ1 determines the ubiquitin post-translational modification and ligand-dependent protein levels. a Immunoblot analysis of cardiac LV PPARα, PPARβ, and PPARγ1 levels normalized to GAPDH. N = 3/group. b Increasing MuRF2 results in a PPARγ1 ligand (Rosiglitazone)-dependent decrease in PPARγ1 in vitro 24 h after transfection. HEK293 cells were co-transfected with MuRF2 and PPARγ1 (as indicated below graph). After 24 h, 1 microM Rosiglitazone was added overnight and cells harvest at 48 h. *p < 0.05 vs. MuRF2:PPARγ1 ratio of 1:1 without Rosiglitazone. c Immunoprecipitation studies identifying MuRF2 interaction with PPARg1. HEK293 cells were transfected p3XFlag-PPARγ1 (or p3XFlag-Empty vector), pcDNA3.1-HA-MuRF2p50a (or HA-MURF2∆Ring) and immunoblotted for MuRF2 (anti-HA). dLeft Proteasome inhibition with MG132 prevents MuRF2’s degradation of PPARγ1 in a Right ligase-dependent (Ring Finger-dependent) manner. HEK293 cells transfected with p3XFlag-PPARγ1, pcDNA3.1-HA-MuRF2p50a and treated with MG132 (50 μM) for 2.5 h before Rosiglitazone added (1 μm). e–g In vitro ubiquitination assays of MuRF2’s ability to ubiquitinate PPARα (e), PPARβ (f), and PPARγ1 (g), with all lanes having Ub, E1, E2, MuRF2, and PPAR (=full reaction), unless otherwise indicated. Immunoblot for MuRF2 illustrates auto-ubiquitination (=MuRF2 activity) present in the same reaction as mono-ubiquitination (PPARα) and poly-ubiquitination (PPARγ1). Values expressed as Mean ± SE of three independent experiments. A one-way ANOVA was performed to determine significance, followed by a Holm-Sidak pairwise comparison to determine significance between groups. #p < 0.05, **p < 0.01.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4526192&req=5

Fig6: The ratio of MuRF2 to PPARγ1 determines the ubiquitin post-translational modification and ligand-dependent protein levels. a Immunoblot analysis of cardiac LV PPARα, PPARβ, and PPARγ1 levels normalized to GAPDH. N = 3/group. b Increasing MuRF2 results in a PPARγ1 ligand (Rosiglitazone)-dependent decrease in PPARγ1 in vitro 24 h after transfection. HEK293 cells were co-transfected with MuRF2 and PPARγ1 (as indicated below graph). After 24 h, 1 microM Rosiglitazone was added overnight and cells harvest at 48 h. *p < 0.05 vs. MuRF2:PPARγ1 ratio of 1:1 without Rosiglitazone. c Immunoprecipitation studies identifying MuRF2 interaction with PPARg1. HEK293 cells were transfected p3XFlag-PPARγ1 (or p3XFlag-Empty vector), pcDNA3.1-HA-MuRF2p50a (or HA-MURF2∆Ring) and immunoblotted for MuRF2 (anti-HA). dLeft Proteasome inhibition with MG132 prevents MuRF2’s degradation of PPARγ1 in a Right ligase-dependent (Ring Finger-dependent) manner. HEK293 cells transfected with p3XFlag-PPARγ1, pcDNA3.1-HA-MuRF2p50a and treated with MG132 (50 μM) for 2.5 h before Rosiglitazone added (1 μm). e–g In vitro ubiquitination assays of MuRF2’s ability to ubiquitinate PPARα (e), PPARβ (f), and PPARγ1 (g), with all lanes having Ub, E1, E2, MuRF2, and PPAR (=full reaction), unless otherwise indicated. Immunoblot for MuRF2 illustrates auto-ubiquitination (=MuRF2 activity) present in the same reaction as mono-ubiquitination (PPARα) and poly-ubiquitination (PPARγ1). Values expressed as Mean ± SE of three independent experiments. A one-way ANOVA was performed to determine significance, followed by a Holm-Sidak pairwise comparison to determine significance between groups. #p < 0.05, **p < 0.01.

Mentions: Like other ubiquitin ligases, MuRF2 interacts with a number of protein substrates. Notably, MuRF2 and MuRF1 redundantly interact with roponin-I (TnI), TnT, myosin light chain 2, and T-cap (telethonin) in yeast two-hybrid studies [74]. Unlike MuRF1, MuRF2 has not been shown to degrade any of these substrates (as recently reviewed [75] ). But critical regulation of microtubule, intermediate filament, and sarcomeric M-line stability during striated muscle development [22] and regulation of E2F activity [40]. Understanding that high fat diet induced MuRF2 expression, we next identified PPARα, PPARβ, and PPARγ1 (as the PPARγ2 isoform is restricted to adipocytes) (Fig. 6a). Interestingly, in steady state conditions, cardiac PPARα and PPARα protein levels in MuRF2−/− mice did not differ compared with wildtype controls. However, PPARγ1 levels were slightly (and significantly) increased at baseline (Fig. 6a, far right). After challenge with PPAR ligands (free fatty acids from high fat diet) for 26 weeks, no differences in MuRF2−/− cardiac PPARα and PPARγ1 were identified by immunoblot analysis, but a significant increase in PPARβ protein expression was identified (Fig. 6a). Taken together, these studies illustrate that the steady state levels of cardiac PPARα and PPARγ1 isoforms are not affected by the presence of MuRF2 or its increase (Fig. 1c) after high fat diet challenge. Moreover, these results suggest that MuRF2’s changes in PPARα and PPARγ1 activities could be due to one of the multiple non-canonical post-translational modifications by ubiquitin (e.g. mono-ubiquitination) that are not associated with proteasome dependent and degradation. How MuRF2 is regulating PPARβ without being able to ubiquitinate it directly (Fig. 6f) is unclear. But the mechanism would be indirect include the possibility that MuRF2 it targeting the inhibition of a yet to be determined ubiquitin ligase(s) that normally degrades PPARβ. For example, PPARβ in cancer cells (HEK293 and NIH3T3) is ubiquitinated and degraded in a ligand (GW501516)-dependent manner [76]. While the identification of the ubiquitin ligase targeting PPARβ is not known at this time, ubiquitin ligases degrading other isoforms (e.g. PPARγ) have been reported in adipocytes (MKRN1) [77]. Conversely, MuRF2 ubiquitination could be enhancing a de-ubiquitinase (DUB) that prevents proteasome-mediated degradation by this unidentified E3(s).Fig. 6


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)

The ratio of MuRF2 to PPARγ1 determines the ubiquitin post-translational modification and ligand-dependent protein levels. a Immunoblot analysis of cardiac LV PPARα, PPARβ, and PPARγ1 levels normalized to GAPDH. N = 3/group. b Increasing MuRF2 results in a PPARγ1 ligand (Rosiglitazone)-dependent decrease in PPARγ1 in vitro 24 h after transfection. HEK293 cells were co-transfected with MuRF2 and PPARγ1 (as indicated below graph). After 24 h, 1 microM Rosiglitazone was added overnight and cells harvest at 48 h. *p < 0.05 vs. MuRF2:PPARγ1 ratio of 1:1 without Rosiglitazone. c Immunoprecipitation studies identifying MuRF2 interaction with PPARg1. HEK293 cells were transfected p3XFlag-PPARγ1 (or p3XFlag-Empty vector), pcDNA3.1-HA-MuRF2p50a (or HA-MURF2∆Ring) and immunoblotted for MuRF2 (anti-HA). dLeft Proteasome inhibition with MG132 prevents MuRF2’s degradation of PPARγ1 in a Right ligase-dependent (Ring Finger-dependent) manner. HEK293 cells transfected with p3XFlag-PPARγ1, pcDNA3.1-HA-MuRF2p50a and treated with MG132 (50 μM) for 2.5 h before Rosiglitazone added (1 μm). e–g In vitro ubiquitination assays of MuRF2’s ability to ubiquitinate PPARα (e), PPARβ (f), and PPARγ1 (g), with all lanes having Ub, E1, E2, MuRF2, and PPAR (=full reaction), unless otherwise indicated. Immunoblot for MuRF2 illustrates auto-ubiquitination (=MuRF2 activity) present in the same reaction as mono-ubiquitination (PPARα) and poly-ubiquitination (PPARγ1). Values expressed as Mean ± SE of three independent experiments. A one-way ANOVA was performed to determine significance, followed by a Holm-Sidak pairwise comparison to determine significance between groups. #p < 0.05, **p < 0.01.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4526192&req=5

Fig6: The ratio of MuRF2 to PPARγ1 determines the ubiquitin post-translational modification and ligand-dependent protein levels. a Immunoblot analysis of cardiac LV PPARα, PPARβ, and PPARγ1 levels normalized to GAPDH. N = 3/group. b Increasing MuRF2 results in a PPARγ1 ligand (Rosiglitazone)-dependent decrease in PPARγ1 in vitro 24 h after transfection. HEK293 cells were co-transfected with MuRF2 and PPARγ1 (as indicated below graph). After 24 h, 1 microM Rosiglitazone was added overnight and cells harvest at 48 h. *p < 0.05 vs. MuRF2:PPARγ1 ratio of 1:1 without Rosiglitazone. c Immunoprecipitation studies identifying MuRF2 interaction with PPARg1. HEK293 cells were transfected p3XFlag-PPARγ1 (or p3XFlag-Empty vector), pcDNA3.1-HA-MuRF2p50a (or HA-MURF2∆Ring) and immunoblotted for MuRF2 (anti-HA). dLeft Proteasome inhibition with MG132 prevents MuRF2’s degradation of PPARγ1 in a Right ligase-dependent (Ring Finger-dependent) manner. HEK293 cells transfected with p3XFlag-PPARγ1, pcDNA3.1-HA-MuRF2p50a and treated with MG132 (50 μM) for 2.5 h before Rosiglitazone added (1 μm). e–g In vitro ubiquitination assays of MuRF2’s ability to ubiquitinate PPARα (e), PPARβ (f), and PPARγ1 (g), with all lanes having Ub, E1, E2, MuRF2, and PPAR (=full reaction), unless otherwise indicated. Immunoblot for MuRF2 illustrates auto-ubiquitination (=MuRF2 activity) present in the same reaction as mono-ubiquitination (PPARα) and poly-ubiquitination (PPARγ1). Values expressed as Mean ± SE of three independent experiments. A one-way ANOVA was performed to determine significance, followed by a Holm-Sidak pairwise comparison to determine significance between groups. #p < 0.05, **p < 0.01.
Mentions: Like other ubiquitin ligases, MuRF2 interacts with a number of protein substrates. Notably, MuRF2 and MuRF1 redundantly interact with roponin-I (TnI), TnT, myosin light chain 2, and T-cap (telethonin) in yeast two-hybrid studies [74]. Unlike MuRF1, MuRF2 has not been shown to degrade any of these substrates (as recently reviewed [75] ). But critical regulation of microtubule, intermediate filament, and sarcomeric M-line stability during striated muscle development [22] and regulation of E2F activity [40]. Understanding that high fat diet induced MuRF2 expression, we next identified PPARα, PPARβ, and PPARγ1 (as the PPARγ2 isoform is restricted to adipocytes) (Fig. 6a). Interestingly, in steady state conditions, cardiac PPARα and PPARα protein levels in MuRF2−/− mice did not differ compared with wildtype controls. However, PPARγ1 levels were slightly (and significantly) increased at baseline (Fig. 6a, far right). After challenge with PPAR ligands (free fatty acids from high fat diet) for 26 weeks, no differences in MuRF2−/− cardiac PPARα and PPARγ1 were identified by immunoblot analysis, but a significant increase in PPARβ protein expression was identified (Fig. 6a). Taken together, these studies illustrate that the steady state levels of cardiac PPARα and PPARγ1 isoforms are not affected by the presence of MuRF2 or its increase (Fig. 1c) after high fat diet challenge. Moreover, these results suggest that MuRF2’s changes in PPARα and PPARγ1 activities could be due to one of the multiple non-canonical post-translational modifications by ubiquitin (e.g. mono-ubiquitination) that are not associated with proteasome dependent and degradation. How MuRF2 is regulating PPARβ without being able to ubiquitinate it directly (Fig. 6f) is unclear. But the mechanism would be indirect include the possibility that MuRF2 it targeting the inhibition of a yet to be determined ubiquitin ligase(s) that normally degrades PPARβ. For example, PPARβ in cancer cells (HEK293 and NIH3T3) is ubiquitinated and degraded in a ligand (GW501516)-dependent manner [76]. While the identification of the ubiquitin ligase targeting PPARβ is not known at this time, ubiquitin ligases degrading other isoforms (e.g. PPARγ) have been reported in adipocytes (MKRN1) [77]. Conversely, MuRF2 ubiquitination could be enhancing a de-ubiquitinase (DUB) that prevents proteasome-mediated degradation by this unidentified E3(s).Fig. 6

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