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

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 PPARß 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, PPARß, 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.

Muscle ring finger-3 protects against diabetic cardiomyopathy induced by a high fat diet.

BACKGROUND: The pathogenesis of diabetic cardiomyopathy (DCM) involves the enhanced activation of peroxisome proliferator activating receptor (PPAR) transcription factors, including the most prominent isoform in the heart, PPARα. In cancer cells and adipocytes, post-translational modification of PPARs have been identified, including ligand-dependent degradation of PPARs by specific ubiquitin ligases. However, the regulation of PPARs in cardiomyocytes and heart have not previously been identified. We recently identified that muscle ring finger-1 (MuRF1) and MuRF2 differentially inhibit PPAR activities by mono-ubiquitination, leading to the hypothesis that MuRF3 may regulate PPAR activity in vivo to regulate DCM. METHODS: MuRF3-/- mice were challenged with 26 weeks 60% high fat diet to induce insulin resistance and DCM. Conscious echocardiography, blood glucose, tissue triglyceride, glycogen levels, immunoblot analysis of intracellular signaling, heart and skeletal muscle morphometrics, and PPARα, PPARß, and PPARγ1 activities were assayed. RESULTS: MuRF3-/- mice exhibited a premature systolic heart failure by 6 weeks high fat diet (vs. 12 weeks in MuRF3+/+). MuRF3-/- mice weighed significantly less than sibling-matched wildtype mice after 26 weeks HFD. These differences may be largely due to resistance to fat accumulation, as MRI analysis revealed MuRF3-/- mice had significantly less fat mass, but not lean body mass. In vitro ubiquitination assays identified MuRF3 mono-ubiquitinated PPARα and PPARγ1, but not PPARß. CONCLUSIONS: These findings suggest that MuRF3 helps stabilize cardiac PPARα and PPARγ1 in vivo to support resistance to the development of DCM. MuRF3 also plays an unexpected role in regulating fat storage despite being found only in striated muscle.

The protective effects of Ambroxol in Pseudomonas aeruginosa-induced pneumonia in rats.

INTRODUCTION: To evaluate the effect of Ambroxol on the pulmonary surfactant (PS) in rat pneumonia induced by Pseudomonas aeruginosa (PA). MATERIAL AND METHODS: The pneumonic rats were obtained by injecting ATCC27853 intratracheally. One hundred and twenty SD rats were randomized into four groups: normal saline and Ambroxol was injected intraperitoneally following PA challenge in the PA/NS and PA/AM group; the other two groups were NS/AM and NS/NS. The wet/dry weight ratio (W/D), and pathological changes were assayed. Total proteins (TP), total phospholipid (TPL), and dipalmitoylphosphatidylcholine (DPPC) in bronchial alveolar lavage fluid (BALF) were analysed. Some BALF was cultured for colony counts. Ultrastructural change of the lung was observed by electron microscopy. RESULTS: The W/D ratio in the PA/AM group was lower than that in the PA/NS group; both were higher than that in the NS/NS group (p < 0.05). There were more neutrophils in the PA/NS group than in the PA/AM group (p < 0.05), and more in the PA/AM group than in the NS/NS group (p < 0.05). The ratio of DSPC/TPL and DSPC/TP in the BALF in PA/NS group was lower than that in the PA/AM group; DSPC/TPL and DSPC/TP ratios also increased in the NS/AM group. The PA colony numbers in the PA/AM group were lower than in the PA/NS group (p > 0.05). In the PA/NS group, vacuolation occurred in the lamellar body of alveolar type 2 cells (AT2) and the PS layer was rough and broken in some areas. In the PA/AM group, the degree of vacuolation of the lamellar body was less than in the PA/NS group. CONCLUSIONS: Ambroxol could protect rats from pneumonia by improving the level of endogenous PS, especially DPPC.