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Down-regulation of cardiac lineage protein (CLP-1) expression in CLP-1 +/- mice affords.

Mascareno E, Manukyan I, Das DK, Siddiqui MA - J. Cell. Mol. Med. (2009)

Bottom Line: There was a decrease in Cdk7 and Cdk9 kinase activity and consequently in phosphorylation of serine-5 and serine-2 of Pol II CTD in CLP-1 +/- hearts.However, the levels of mitochondrial proteins, PGC-1alpha and HIF-1alpha, which enhance mitochondrial activity and are implicated in cell survival, were increased in CLP-1 +/- hearts subjected to ischaemic stress compared to that in wild-type CLP-1 +/- hearts treated identically.Taken together, our data suggest that regulation of the CLP-1 levels is critical to cellular adaptation of the survival program that protects cardiomyocytes against stress due collectively to a decrease in RNA Pol II phosphorylation but an increase in expression of target proteins that regulate mitochondrial function and metabolic adaptation to stress.

View Article: PubMed Central - PubMed

Affiliation: Center for Cardiovascular and Muscle Research, Department of Anatomy and Cell Biology, State University of New York Downstate Medical Center, Brooklyn, NY 112031, USA.

ABSTRACT
In order to understand the transcriptional mechanism that underlies cell protection to stress, we evaluated the role of CLP-1, a known inhibitor of the transcription elongation complex (pTEFb), in CLP-1 +/- mice hearts. Using the isolated heart model, we observed that the CLP-1 +/- hearts, when subjected to ischaemic stress and evaluated by haemodynamic measurements, exhibit significant cardioprotection. CLP-1 remains associated with the pTEFb complex in the heterozygous hearts, where as it is released in the wild-type hearts suggesting the involvement of pTEFb regulation in cell protection. There was a decrease in Cdk7 and Cdk9 kinase activity and consequently in phosphorylation of serine-5 and serine-2 of Pol II CTD in CLP-1 +/- hearts. However, the levels of mitochondrial proteins, PGC-1alpha and HIF-1alpha, which enhance mitochondrial activity and are implicated in cell survival, were increased in CLP-1 +/- hearts subjected to ischaemic stress compared to that in wild-type CLP-1 +/- hearts treated identically. There was also an increase in the expression of pyruvate dehydrogenase kinase (PDK-1), which facilitates cell adaptation to hypoxic stress. Taken together, our data suggest that regulation of the CLP-1 levels is critical to cellular adaptation of the survival program that protects cardiomyocytes against stress due collectively to a decrease in RNA Pol II phosphorylation but an increase in expression of target proteins that regulate mitochondrial function and metabolic adaptation to stress.

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Changes in CLP‐1 expression level modulates the pTEFb complex activity. (A) Western blot shows the level of CLP‐1 protein expression in CLP‐1 +/+ and CLP‐1 +/− hearts during each stress. The bar graph indicates the ratio between CLP‐1 and GAPDH expression, and shows a decrease in expression level of CLP‐1 protein in CLP‐1 +/− hearts. This measurement was performed in quadruplicate and the results are shown as mean ± S.E.M. per group. *P < 0.05 versus CLP‐1+/+. (B) Association of CLP‐1 with cyclin T was determined by immunoprecipitation of extracts with antibodies against Cdk9 or CLP‐1 followed by Western blotting with cyclin T1 antibody. (C) Cdk activity was measured as described in Materials and Methods. The Cdk9 activity measurement was performed in triplicate and the results are shown as mean ± SEM per group. *P < 0.05 versus control. (D) Extracts as above were used for Western blotting using antibodies for phosphoserine 2 and 5 and Pol II. Graph bars show the relative increase or decrease in phosphoserine 5 and 2 versus total Pol II expression. At least three experiments were performed and a representative figure is shown. *P < 0.05 versus control CLP‐1+/+, **P < 0.05 versus control CLP‐1 +/−, #P < 0.01 versus control CLP‐1+/+.
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f4: Changes in CLP‐1 expression level modulates the pTEFb complex activity. (A) Western blot shows the level of CLP‐1 protein expression in CLP‐1 +/+ and CLP‐1 +/− hearts during each stress. The bar graph indicates the ratio between CLP‐1 and GAPDH expression, and shows a decrease in expression level of CLP‐1 protein in CLP‐1 +/− hearts. This measurement was performed in quadruplicate and the results are shown as mean ± S.E.M. per group. *P < 0.05 versus CLP‐1+/+. (B) Association of CLP‐1 with cyclin T was determined by immunoprecipitation of extracts with antibodies against Cdk9 or CLP‐1 followed by Western blotting with cyclin T1 antibody. (C) Cdk activity was measured as described in Materials and Methods. The Cdk9 activity measurement was performed in triplicate and the results are shown as mean ± SEM per group. *P < 0.05 versus control. (D) Extracts as above were used for Western blotting using antibodies for phosphoserine 2 and 5 and Pol II. Graph bars show the relative increase or decrease in phosphoserine 5 and 2 versus total Pol II expression. At least three experiments were performed and a representative figure is shown. *P < 0.05 versus control CLP‐1+/+, **P < 0.05 versus control CLP‐1 +/−, #P < 0.01 versus control CLP‐1+/+.

Mentions: (A) Schematic representation of the experimental protocol used for the evaluation of haemodynamic parameters and infarct size. (B) A diagram of the experimental protocols for stress application and analysis of wild‐type and CLP‐1 +/− hearts (see Figs. 4 and 5).


Down-regulation of cardiac lineage protein (CLP-1) expression in CLP-1 +/- mice affords.

Mascareno E, Manukyan I, Das DK, Siddiqui MA - J. Cell. Mol. Med. (2009)

Changes in CLP‐1 expression level modulates the pTEFb complex activity. (A) Western blot shows the level of CLP‐1 protein expression in CLP‐1 +/+ and CLP‐1 +/− hearts during each stress. The bar graph indicates the ratio between CLP‐1 and GAPDH expression, and shows a decrease in expression level of CLP‐1 protein in CLP‐1 +/− hearts. This measurement was performed in quadruplicate and the results are shown as mean ± S.E.M. per group. *P < 0.05 versus CLP‐1+/+. (B) Association of CLP‐1 with cyclin T was determined by immunoprecipitation of extracts with antibodies against Cdk9 or CLP‐1 followed by Western blotting with cyclin T1 antibody. (C) Cdk activity was measured as described in Materials and Methods. The Cdk9 activity measurement was performed in triplicate and the results are shown as mean ± SEM per group. *P < 0.05 versus control. (D) Extracts as above were used for Western blotting using antibodies for phosphoserine 2 and 5 and Pol II. Graph bars show the relative increase or decrease in phosphoserine 5 and 2 versus total Pol II expression. At least three experiments were performed and a representative figure is shown. *P < 0.05 versus control CLP‐1+/+, **P < 0.05 versus control CLP‐1 +/−, #P < 0.01 versus control CLP‐1+/+.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4940775&req=5

f4: Changes in CLP‐1 expression level modulates the pTEFb complex activity. (A) Western blot shows the level of CLP‐1 protein expression in CLP‐1 +/+ and CLP‐1 +/− hearts during each stress. The bar graph indicates the ratio between CLP‐1 and GAPDH expression, and shows a decrease in expression level of CLP‐1 protein in CLP‐1 +/− hearts. This measurement was performed in quadruplicate and the results are shown as mean ± S.E.M. per group. *P < 0.05 versus CLP‐1+/+. (B) Association of CLP‐1 with cyclin T was determined by immunoprecipitation of extracts with antibodies against Cdk9 or CLP‐1 followed by Western blotting with cyclin T1 antibody. (C) Cdk activity was measured as described in Materials and Methods. The Cdk9 activity measurement was performed in triplicate and the results are shown as mean ± SEM per group. *P < 0.05 versus control. (D) Extracts as above were used for Western blotting using antibodies for phosphoserine 2 and 5 and Pol II. Graph bars show the relative increase or decrease in phosphoserine 5 and 2 versus total Pol II expression. At least three experiments were performed and a representative figure is shown. *P < 0.05 versus control CLP‐1+/+, **P < 0.05 versus control CLP‐1 +/−, #P < 0.01 versus control CLP‐1+/+.
Mentions: (A) Schematic representation of the experimental protocol used for the evaluation of haemodynamic parameters and infarct size. (B) A diagram of the experimental protocols for stress application and analysis of wild‐type and CLP‐1 +/− hearts (see Figs. 4 and 5).

Bottom Line: There was a decrease in Cdk7 and Cdk9 kinase activity and consequently in phosphorylation of serine-5 and serine-2 of Pol II CTD in CLP-1 +/- hearts.However, the levels of mitochondrial proteins, PGC-1alpha and HIF-1alpha, which enhance mitochondrial activity and are implicated in cell survival, were increased in CLP-1 +/- hearts subjected to ischaemic stress compared to that in wild-type CLP-1 +/- hearts treated identically.Taken together, our data suggest that regulation of the CLP-1 levels is critical to cellular adaptation of the survival program that protects cardiomyocytes against stress due collectively to a decrease in RNA Pol II phosphorylation but an increase in expression of target proteins that regulate mitochondrial function and metabolic adaptation to stress.

View Article: PubMed Central - PubMed

Affiliation: Center for Cardiovascular and Muscle Research, Department of Anatomy and Cell Biology, State University of New York Downstate Medical Center, Brooklyn, NY 112031, USA.

ABSTRACT
In order to understand the transcriptional mechanism that underlies cell protection to stress, we evaluated the role of CLP-1, a known inhibitor of the transcription elongation complex (pTEFb), in CLP-1 +/- mice hearts. Using the isolated heart model, we observed that the CLP-1 +/- hearts, when subjected to ischaemic stress and evaluated by haemodynamic measurements, exhibit significant cardioprotection. CLP-1 remains associated with the pTEFb complex in the heterozygous hearts, where as it is released in the wild-type hearts suggesting the involvement of pTEFb regulation in cell protection. There was a decrease in Cdk7 and Cdk9 kinase activity and consequently in phosphorylation of serine-5 and serine-2 of Pol II CTD in CLP-1 +/- hearts. However, the levels of mitochondrial proteins, PGC-1alpha and HIF-1alpha, which enhance mitochondrial activity and are implicated in cell survival, were increased in CLP-1 +/- hearts subjected to ischaemic stress compared to that in wild-type CLP-1 +/- hearts treated identically. There was also an increase in the expression of pyruvate dehydrogenase kinase (PDK-1), which facilitates cell adaptation to hypoxic stress. Taken together, our data suggest that regulation of the CLP-1 levels is critical to cellular adaptation of the survival program that protects cardiomyocytes against stress due collectively to a decrease in RNA Pol II phosphorylation but an increase in expression of target proteins that regulate mitochondrial function and metabolic adaptation to stress.

Show MeSH
Related in: MedlinePlus