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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

MuRF2 alters nuclear localization of PPARγ1 in a ubiquitin ligase region (RING)-dependent manner without altering steady state protein levels (as found in vivo). Increased MuRF2 alters PPARγ1 localization from primarily nuclear a to increased cytosolic/perinuclear localization b in HL-1 cardiomyocyte-derived cells. MuRF2 lacking ubiquitin ligase activity (∆RING FingerMuRF2, c does not alter PPARγ1 localization compared controls (top row), indicating MuRF2’s dependence on its ubiquitin ligase activity in mediating these changes. Representative of three biological replicates. Right Percentages based on the number of cells analyzed (N = 58, 68, and 88 in FLAG-PPARγ1, FLAG-PPARγ1 + HA-MuRF2, and FLAG-PPARγ1 + HA-∆RING-MuRF2 groups, respectively).
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Fig7: MuRF2 alters nuclear localization of PPARγ1 in a ubiquitin ligase region (RING)-dependent manner without altering steady state protein levels (as found in vivo). Increased MuRF2 alters PPARγ1 localization from primarily nuclear a to increased cytosolic/perinuclear localization b in HL-1 cardiomyocyte-derived cells. MuRF2 lacking ubiquitin ligase activity (∆RING FingerMuRF2, c does not alter PPARγ1 localization compared controls (top row), indicating MuRF2’s dependence on its ubiquitin ligase activity in mediating these changes. Representative of three biological replicates. Right Percentages based on the number of cells analyzed (N = 58, 68, and 88 in FLAG-PPARγ1, FLAG-PPARγ1 + HA-MuRF2, and FLAG-PPARγ1 + HA-∆RING-MuRF2 groups, respectively).

Mentions: To gain more insight on how MuRF2 may be inhibiting transcriptional activity by ubiquitination, we next performed nuclear localization studies using confocal microscopy (Fig. 7). In control cells, we found that PPARγ1 could be found in both the nucleus and cytosol, with most cells having primarily nuclear localization (81%) (Fig. 7a). Increasing MuRF2 (2:1 ratio of PPARγ1 transfected) interestingly resulted in an increase in the “perinuclear” localization of PPARγ1 (Fig. 7b). Notably, MuRF2 co-localized to these perinuclear regions (Fig. 7b). Parallel studies using the MuRF2 without its ubiquitin ligase activity (∆RING-MuRF2) abrogated the perinuclear targeting of PPARγ and colocalization with MuRF2 (Fig. 7c). Since we demonstrated that MuRF2, but not MuRF2∆Ring, bound to PPARγ1 (Fig. 6c), these studies indicates that MuRF2 regulation of PPARγ1 location may lie in its ubiquitin ligase activity and/or through some structural role required for interaction since MuRF2’s Ring Finger domain is required to bind PPARγ1 (Fig. 6c). Taken together, these studies suggested that MuRF2 targets an ubiquitin-mediated regulation of PPARγ1 activity by altering its localization within the nucleus, paralleling recent studies demonstrating autophagic sequestration of receptors in the endoplasmic reticulum and nucleus [78].Fig. 7


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)

MuRF2 alters nuclear localization of PPARγ1 in a ubiquitin ligase region (RING)-dependent manner without altering steady state protein levels (as found in vivo). Increased MuRF2 alters PPARγ1 localization from primarily nuclear a to increased cytosolic/perinuclear localization b in HL-1 cardiomyocyte-derived cells. MuRF2 lacking ubiquitin ligase activity (∆RING FingerMuRF2, c does not alter PPARγ1 localization compared controls (top row), indicating MuRF2’s dependence on its ubiquitin ligase activity in mediating these changes. Representative of three biological replicates. Right Percentages based on the number of cells analyzed (N = 58, 68, and 88 in FLAG-PPARγ1, FLAG-PPARγ1 + HA-MuRF2, and FLAG-PPARγ1 + HA-∆RING-MuRF2 groups, respectively).
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig7: MuRF2 alters nuclear localization of PPARγ1 in a ubiquitin ligase region (RING)-dependent manner without altering steady state protein levels (as found in vivo). Increased MuRF2 alters PPARγ1 localization from primarily nuclear a to increased cytosolic/perinuclear localization b in HL-1 cardiomyocyte-derived cells. MuRF2 lacking ubiquitin ligase activity (∆RING FingerMuRF2, c does not alter PPARγ1 localization compared controls (top row), indicating MuRF2’s dependence on its ubiquitin ligase activity in mediating these changes. Representative of three biological replicates. Right Percentages based on the number of cells analyzed (N = 58, 68, and 88 in FLAG-PPARγ1, FLAG-PPARγ1 + HA-MuRF2, and FLAG-PPARγ1 + HA-∆RING-MuRF2 groups, respectively).
Mentions: To gain more insight on how MuRF2 may be inhibiting transcriptional activity by ubiquitination, we next performed nuclear localization studies using confocal microscopy (Fig. 7). In control cells, we found that PPARγ1 could be found in both the nucleus and cytosol, with most cells having primarily nuclear localization (81%) (Fig. 7a). Increasing MuRF2 (2:1 ratio of PPARγ1 transfected) interestingly resulted in an increase in the “perinuclear” localization of PPARγ1 (Fig. 7b). Notably, MuRF2 co-localized to these perinuclear regions (Fig. 7b). Parallel studies using the MuRF2 without its ubiquitin ligase activity (∆RING-MuRF2) abrogated the perinuclear targeting of PPARγ and colocalization with MuRF2 (Fig. 7c). Since we demonstrated that MuRF2, but not MuRF2∆Ring, bound to PPARγ1 (Fig. 6c), these studies indicates that MuRF2 regulation of PPARγ1 location may lie in its ubiquitin ligase activity and/or through some structural role required for interaction since MuRF2’s Ring Finger domain is required to bind PPARγ1 (Fig. 6c). Taken together, these studies suggested that MuRF2 targets an ubiquitin-mediated regulation of PPARγ1 activity by altering its localization within the nucleus, paralleling recent studies demonstrating autophagic sequestration of receptors in the endoplasmic reticulum and nucleus [78].Fig. 7

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