Abstract 21194: TRIM63, Encoding MuRF1, is a Novel Gene for Human Hypertrophic Cardiomyopathy
TRIM63 encodes Muscle Ring Finger 1 (MuRF1) protein, which is a sarcomere M line with E3 ubiquitin (Ub) ligase function. MuRF1 regulates muscle trophic homeostasis and is considered a muscle atrophy gene. MuRF1 is implicated in ubiquitination and degradation of sarcomeric proteins by the Ubiquitin-Proteasome System (UPS). Hypertrophic cardiomyopathy (HCM) is primarily a disease of sarcomere proteins. We screened 307 probands with HCM by Sanger sequencing of all exons and intron-exon boundaries of TRIM63 and identified 3 mutations in 5 families. The p.A48V and p.Q247X occurred in 2 independent probands and p.I130M in 1 proband. The mutations were absent in over 1000 normal individuals and affected totally conserved amino acids. To determine the effects of the mutation on E3 Ub ligase activity, we generated specialized HeLa cells in which ubiquitinylated proteins could be labeled with biotin and isolated with streptavidin. Lenti-viral transduction of HeLa-His/Biotin-Ub cells showed that the X247 mutation led to complete loss of E3 Ub ligase activity, which the V48 and M130 had modest effects. The findings were verified by co-immunoprecipitation studies in adult cardiac myocytes. The M130 mutation, located at the muscle creatine kinase (CK-M) binding domain, and X247, which lead to partial loss of the coil-coil domain, impaired co-localization of MuRF1 and CK-M to sarcomere M line. In contrast, the V48 mutation enhanced localization to the M line. MuRF1 is known to regulate expression of DNA-damage-inducible transcript 4 (REDD1), a negative regulator of m-TOR hypertrophic pathway, through interactions with glucocorticoid modulatory element binding protein 1 (GMEB1). Expression level of REDD1 mRNA, detected by qPCR was reduced by ∼40% in adult cardiac myocytes transduced with recombinant adenoviruses expressing the MuRF1-X247. Concordantly, expression level of p-mTOR was increased. Thus, TRIM63 is a novel gene for human HCM. The pathogenesis of HCM caused by TRIM63 mutations involves loss of E3 Ub ligase activity, mislocalization to sarcomere M line and activation of GMEB-1/REDD1/mTOR pathway.
- © 2010 by American Heart Association, Inc.