Abstract 870: UNC-45B Exhibits Increased Abundance In Heart Failure Patients And Functions As A Novel Dual Regulator Of Myosin Heavy Chain Transcription And Assembly
Myosin Heavy Chain (MHC) is a key contractile protein reduced in Heart Failure (HF) which can be regulated at both the transcriptional and assembly level. Proteins involved in these two processes and the mechanisms for coordination are unclear in cardiac muscle. It has been shown that the assembly of MHC in C2C12 myotubules and lower eukaryotes requires the chaperone UNC-45B which we propose also has a role in transcription based on the presence of an ‘armadillo’ motif that is associated with intracellular signaling and transcriptional regulation. We hypothesized that UNC-45B regulates both MHC filament formation and transcriptional activity with its abundance correlated to myofilament changes in HF patients.
Methods: A specific antibody to UNC45B was generated and human left ventricular tissue from normal (n=5), ischemic cardiomyopathic (ICM ; n=8) or dilated cardiomyopathic (DCM ; n=8) patients were immunoblotted using 1DE and 2DE to determine abundance changes and detect protein modification. The functional role of UNC-45B in cardiomyocytes was determined in neonatal cardiomyocytes by combining immunohistochemistry and siRNA (n=3) to reduce UNC-45B mRNA and protein levels. Transcriptional regulation of MHC was explored by over-expressing UNC-45B in cells containing the luciferase reporter under the control of the α-MHC promoter (n=3).
Results: Compared to normal patients, ICM patients exhibited a 1.42 ± 0.03 fold increase in UNC-45B (P<0.01), whereas DCM patients had a 1.24 ± 0.02 fold increase (P<0.05) in UNC-45B. Administration of siRNA to UNC-45B resulted in a significant reduction in both MHC abundance and contraction frequency within 24 hrs. Over-expression of UNC-45B resulted in a 2.1 ± 0.2 fold activation of the α-MHC promoter. In conclusion, this is the first report of UNC-45B presence in cardiac muscle and an increased abundance in HF. As well, this is the first observation of a protein that has a dual regulation of both the folding and transcriptional regulation of a member of the thick filament. This suggests that MHC is highly regulated with a feedback mechanism in ICM/DCM patients which may reflect an incomplete compensatory mechanism to restore MHC levels.