Abstract 3718: The Cardiac Ubiquitin Ligase Muscle Ring Finger-1 (MuRF1) Ubiquitinates and Degrades PPAR-Alpha to Regulate Fatty Acid and Glucose Metabolism
We recently identified that MuRF1 plays a key role in regulating cardiac muscle mass during the development of cardiac hypertrophy. In cardiomyocytes, we have additionally discovered that increasing MuRF1 results in the dose-dependent inhibition of fatty acid oxidation and parallel increase in glucose oxidation. Since these findings emulate changes that occur during cardiac hypertrophy, we set out to determine the underlying mechanism. During cardiac hypertrophy, the inhibition of fatty acid oxidation and compensatory increase in glucose oxidation results primarily from decreased activity of the transcription factor PPAR (peroxisome proliferator-activated receptor)-alpha. Since cardiac hypertrophy increases MuRF1 levels, we hypothesized that MuRF1 regulates fatty acid and glucose oxidation by decreasing PPAR-alpha activity. To test this, HL-1 cardiomyocytes were co-transfected with PPAR expression constructs, PPAR-response element-activity reporter constructs, and MuRF1. We found that MuRF1 specifically inhibited PPAR-alpha activity (over 5 fold), but did not affect PPAR-beta/delta or PPAR-gamma activity. Immunoblot analysis revealed that decreased PPAR-alpha protein levels paralleled decreases in PPAR-alpha activity. Since MuRF1 has ubiquitin ligase activity, we next determined if MuRF1 interacted directly with PPAR-alpha to ubiquitinate the substrate, targeting it for degradation. By immunoprecipitating MuRF1 or endogenous PPAR-alpha from HL-1 cardiomyocytes, we identified specific binding of the reciprocal protein (PPAR-alpha or MuRF1, respectively) occurred. To determine if MuRF1 was able to ubiquitinate PPAR-alpha, we incubated E1, E2 (UBCH5C), MuRF1, PPAR-alpha, ATP, ubiquitin, and buffer and found that MuRF1 specifically poly-ubiquitinated PPAR-alpha in vitro by immunoblot. Since poly-ubiquitinated proteins are generally targeted for degradation by the 26S proteasome, this explains MuRF1’s ability to decrease PPAR-alpha protein levels in cardiomyocytes. These studies identify a novel mechanism by which cardiac hypertrophy driven increases in MuRF1 might regulate characteristic decreases in fatty acid oxidation and increases in glucose oxidation by its role in the ubiquitin proteasome system.
This research has received full or partial funding support from the American Heart Association, National Center.