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. 2009 Jun 19;104(12):1347-54.
doi: 10.1161/CIRCRESAHA.108.191726. Epub 2009 May 14.

Positive transcription elongation factor b activity in compensatory myocardial hypertrophy is regulated by cardiac lineage protein-1

Jorge Espinoza-Derout  1 , Michael Wagner, Louis Salciccioli, Jason M Lazar, Sikha Bhaduri, Eduardo Mascareno, Brahim Chaqour, M A Q Siddiqui
Affiliations

Positive transcription elongation factor b activity in compensatory myocardial hypertrophy is regulated by cardiac lineage protein-1

Jorge Espinoza-Derout et al. Circ Res. .

Abstract

Emerging evidence illustrates the importance of the positive transcription elongation factor (P-TEF)b in control of global RNA synthesis, which constitutes a major feature of the compensatory response to diverse hypertrophic stimuli in cardiomyocytes. P-TEFb 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 (Pol) II. We and others have shown that the activity of P-TEFb is inhibited by its association with cardiac lineage protein (CLP)-1, the mouse homolog of human HEXIM1, in various physiological and pathological conditions. To investigate the mechanism of control of P-TEFb activity by CLP-1 in cardiac hypertrophy, we used a transgenic mouse model of hypertrophy caused by overexpression of calcineurin in the heart. We observed that the level of CLP-1 associated with P-TEFb was reduced markedly in hypertrophic hearts. We 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 that is accompanied with an increase in cdk9 activity via an increase in serine 2 phosphorylation of carboxyl-terminal domain and an increase in GLUT1/GLUT4 ratio. These mice have compensated systolic function without evidence of fibrosis and reduced lifespan. These data suggest that the reduced level of CLP-1 introduced in the background of elevated levels of cyclin T1 elevates derepression of P-TEFb activity and emphasizes the importance of the role of CLP-1 in the mechanism governing compensatory hypertrophy in cardiomyocytes.

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Figures

Figure 1
Figure 1
Postnatal changes in the P-TEFb complex in wild type and calcineurin (MHC-CnA) transgenic mice. A, immunoblot with anti-CLP-1 and anti-cyclin T1 antibodies of the heart lysates from wild-type (WT) and MHC-CnA (+) mice. GAPDH was used as loading control (n = 6). B, Association of CLP-1 with P-TEFb in postnatal wild type and MHC-CnA mice as a function of age. Heart lysates from wild type (WT) and MHC-CnA (+) of 1 week, 1 month and 4 month old mice were immunoprecipitated with Cyclin T1 antibody and probed with anti-CLP-1 and anti-Cyclin T1 antibodies. Input is non-immunoprecipitated cell lysates. The data is representative three of independent experiments. C, Phosphorylation of Ser2 RNA Pol II and total RNA Pol II in postnatal wild type and CnA transgenic mice. Immunoblot incubated with anti-phosphorylated Ser2 RNA Pol II and anti-total Pol II of cell lysates of hearts from wild-type (WT) and MHC-CnA (+) of 1 week, 1 month, and 4 months old mice (n =4). Data are expressed as mean ± SE . *P < 0.05 versus control.
Figure 1
Figure 1
Postnatal changes in the P-TEFb complex in wild type and calcineurin (MHC-CnA) transgenic mice. A, immunoblot with anti-CLP-1 and anti-cyclin T1 antibodies of the heart lysates from wild-type (WT) and MHC-CnA (+) mice. GAPDH was used as loading control (n = 6). B, Association of CLP-1 with P-TEFb in postnatal wild type and MHC-CnA mice as a function of age. Heart lysates from wild type (WT) and MHC-CnA (+) of 1 week, 1 month and 4 month old mice were immunoprecipitated with Cyclin T1 antibody and probed with anti-CLP-1 and anti-Cyclin T1 antibodies. Input is non-immunoprecipitated cell lysates. The data is representative three of independent experiments. C, Phosphorylation of Ser2 RNA Pol II and total RNA Pol II in postnatal wild type and CnA transgenic mice. Immunoblot incubated with anti-phosphorylated Ser2 RNA Pol II and anti-total Pol II of cell lysates of hearts from wild-type (WT) and MHC-CnA (+) of 1 week, 1 month, and 4 months old mice (n =4). Data are expressed as mean ± SE . *P < 0.05 versus control.
Figure 2
Figure 2
Exacerbated hypertrophy response in MHC-Cyclin T1/CLP-1+/− mice. A, photograph of adult mice hearts of the indicated genotypes. B, heart/body weight ratios in MHC-Cyclin T1 and MHC-Cyclin T1/CLP-1+/− mice. The data shown are means +/− SE of the Δheart/body ratio. *P < 0.05 versus wild type of three-month old mice (n =13). C, Western blots showing levels of CLP-1, Cyclin T1 and cdk9 hearts of the indicated genotypes of three-month old mice. GAPDH was used as loading control (n = 5). *P < 0.05 versus control; †P < 0.05 versus MHC-Cyclin T1.
Figure 2
Figure 2
Exacerbated hypertrophy response in MHC-Cyclin T1/CLP-1+/− mice. A, photograph of adult mice hearts of the indicated genotypes. B, heart/body weight ratios in MHC-Cyclin T1 and MHC-Cyclin T1/CLP-1+/− mice. The data shown are means +/− SE of the Δheart/body ratio. *P < 0.05 versus wild type of three-month old mice (n =13). C, Western blots showing levels of CLP-1, Cyclin T1 and cdk9 hearts of the indicated genotypes of three-month old mice. GAPDH was used as loading control (n = 5). *P < 0.05 versus control; †P < 0.05 versus MHC-Cyclin T1.
Figure 3
Figure 3
Phosphorylation levels of CTD RNA Pol II in MHC-Cyclin T1 and MHC-Cyclin T1/CLP-1+/− mice. A, anti-Ser2 Pol II immunoblot of heart lysates showing phosphorylation levels of RNA Pol II in MHC-Cyclin T1 and MHC-Cyclin T1/CLP-1+/− mice. B, anti-Ser5 Pol II immunoblot heart lysates showing phosphorylation levels of CTD RNA Pol II. C, anti-RNA Pol II immunoblot heart lysates showing total protein levels of RNA Pol II. Data are expressed as mean ± SE of five independent experiments. *P < 0.05 versus control. *P < 0.05 versus control; †P < 0.05 versus MHC-Cyclin T1.
Figure 4
Figure 4
Glucose transporters GLUT1/GLUT4 ratio in MHC-Cyclin T1/CLP-1+/− mice. A, Western blot shows expression levels of GLUT1, GLUT4, and GAPDH in heart lysates of the indicated genotypes. B, quantification of GLUT1/GLUT4 ratio. Data are expressed as mean ± SE of five independent experiments of three-month old mice. *P < 0.05 versus control; †P < 0.05 versus MHC-Cyclin T1.
Figure 5
Figure 5
Mason trichrome staining of representative ventricular transverse sections of the indicated genotypes (three-month old mice) illustrating the absence of any marked increase in interstitial collagen (which stains blue).
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