Pivotal role of Cardiac Lineage Protein-1 (CLP-1) in Positive Transcriptional Elongation Factor b complex activity in cardiac hypertrophy.

Abstract

Emerging evidence illustrates the importance of the Positive Transcription Elongation Factor b (P-TEFb) in control of global RNA synthesis which constitutes a major feature of the compensatory response to diverse hypertrophic stimuli in cardiomyocytes. P-TEFb, a complex composed of cyclin T and Cdk9, is critical for elongation of nascent RNA chains via phosphorylation of the carboxyl terminal domain of RNA polymerase II (CTD RNA Pol II). HEXIM1, the human homolog of mouse cardiac lineage protein 1 (CLP-1), was shown to be associated with P-TEFb and inhibit Cdk9 kinase activity. Therefore, I hypothesize that CLP-1/HEXIM1 dissociation from the P-TEFb complex is required for triggering the increased synthesis of RNA, the hallmark of cardiac hypertrophy. To determine, if the expression pattern of CLP-1 in the heart is consistent with its role in controlling P-TEFb activity, I performed immunoprecipitation and immunofluorescence analysis of CLP-1 and the components of P-TEFb in the fetal mouse heart and in 2 day post-natal mouse cardiomyocytes. I observed that CLP-1 is colocalized with Cdk9 and Cyclin T1 and is associated with the P-TEFb complex. I subjected cardiomyocytes to hypertrophic stimuli, such as mechanical stretch or treatment with hypertrophic agents (endothelin-1 and phenylephrine), which results in dissociation of CLP-1 from P-TEFb. Association and dissociation of CLP-1 with the P-TEFb is regulated by the Jak/STAT signaling pathway as inhibition of Jak2 signaling by AG490 prevented release of CLP-1 from P-TEFb despite the ongoing presence of hypertrophic stimulation by mechanical stretch. To extend these studies to the animal models, I used a transgenic mouse model of hypertrophy obtained by cardiac-specific over-expression of calcineurin. I observed that the level of CLP-1 associated with the P-TEFb complex was reduced in transgenic hypertrophic hearts. I also generated bigenic mice (MHC-cyclin T1/CLP-1+/-) by crossing MHC-cyclin T1 transgenic mice with CLP-1 heterozygote. The bigenic mice exhibit enhanced susceptibility to hypertrophy which is accompanied with an increase in Cdk9 activity via an increase in serine-2 phosphorylation of CTD and an increase in GLUT1/GLUT4 ratio. These mice exhibit compensated systolic function without evidence of fibrosis or reduced life span. Taken together, these data suggest that the reduced level of CLP-1 introduced in the background of elevated level of cyclin T1 de-represses of P-TEFb activity and emphasizes the importance of CLP-1’s role in the mechanism governing compensatory hypertrophy in cardiomyocytes. Therefore, the decrease in CLP-1 binding to the P-TEFb complex is critical to the regulation of the hypertrophic response in cardiomyocytes.