Abstract 1565: Menage-A-Trois 1 Physically Associates with PGC-1 and is Essential for Cardiac Mitochondrial Function
The menage-a-trois 1 (MAT1)/cyclin H/cyclin-dependent kinase 7 (Cdk7) complex has proposed functions in transcription as the catalytic subunit of the general transcription factor TFIIH (phosphorylating the carboxyl-terminal domain of the largest subunit of RNA polymerase II), and is activated in chronic cardiac hypertrophy and end-stage heart failure. Using cardiac-specific deletion of MAT1 (conditional knockout, CKO) we previously showed that MAT1-deficient hearts grew normally but fatal heart failure ensued after 4 weeks of age: genes for energy metabolism were suppressed selectively, including targets of peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1), and exogenous PGC-1 proteins failed to function in the absence of MAT1. Here we investigated cardiac mitochondrial function in the absence of MAT1, and determined if MAT1 directly binds PGC1, as an explanation for its selective transcriptional effects. By electron microscopy, abnormal mitochondria were observed in MAT1 CKO hearts even at 3 wk. Enzyme activities of TCA cycle and respiratory chain complexes were reduced at 4 wk, as were levels of ATP synthase alpha, the adenine nucleotide translocator, and cytochrome oxidase Valpha. Langendorff perfused-heart studies at 3.5 wk showed that oleate and glucose oxidation were significantly reduced. Respiration studies of isolated cardiac mitochondria showed that state III (ADP-stimulated) respiration was significantly attenuated; no defect was seen in liver mitochondria. By GST pull-down and co-immunoprecipitation assays, in vitro translated PGC-1 alpha bound to MAT1 (and to PPAR gamma, a positive control). Little or no interaction was seen using an alternative nuclear receptor coactivator (Ncoa2/SRC2) or GST alone. PGC-1 alpha also bound Cdk7 but not cyclin H. In vitro translated PGC-1 alpha was incubated with HepG2 nuclear extract, then was recovered by precipitation of endogenous MAT1 or endogenous Cdk7. Thus:
Metabolic defects were substantiated in isolated perfused hearts and cardiac mitochondria from MAT1 CKO mice, consistent with our microarray findings.
Physical interaction of PGC-1 alpha with MAT1 plus Cdk7 suggests a likely basis for the selective impact of MAT1 on gene activation by PGC-1.