Limits...
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.

Show MeSH

Related in: MedlinePlus

(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).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4940775&req=5

f1: (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).

Mentions: Isolated mice hearts from wild‐type littermates and CLP‐1 +/− subjected to ischaemic stress as before [22] were divided into two groups (see Fig. 1A): (1) isolated hearts were perfused with Krebs‐Henseleit bicarbonate (KHB) buffer for 15 min and then subjected to 30 min of ischemia followed by 15, 30, 60 or 120 min of reperfusion (control); (2) the hearts were preconditioned to ischaemic stress by four cyclic episodes of 5 min ischemia each followed by another 10 min reperfusion followed by 30 min of ischemia and 15, 30, 60 or 120 min of reperfusion (PC). The isolated hearts from CLP‐1 +/− or wild‐type mice were obtained after injecting the mice with pentobarbital sodium (80 mg/kg body wt ip) and anticoagulant heparin sodium (500 IU/kg body wt ip). The hearts were excised and transferred to ice‐cold modifiedKHB, containing in mM (NaCl 118; KCl 4.7; CaCl2 1.7; NaHCO3 24; KH2PO4 1.2; MgSO4 12; Glucose 10). The aorta and pulmonary vein were cannulated and perfused in retrograde Langendorff mode. Perfusate KHB temperature was maintained at 37°C and saturated with 95% O2 and 5% CO2. The perfusion was then switched to anterograde mode where the buffer enters the cannulated left atrium at pressure equivalent to 10 cm of water (10 kPa). The heart measurements were recorded by Gould p23XL transducer (Gould Instrument System, Inc., Valley View, OH, USA). The signal was amplified by using Gould 6600 6600 series signal conditioner (Gould Instrument System, Inc.) and monitored on a Cordat II real‐time acquisition system (Triton technologies, San Diego, CA, USA). The aortic flow was measured by a flow meter. Once baseline measurements of heart rate, coronary flow, aortic flow, left ventricular developed pressure and its maximum first derivative were performed, the anterograde perfusion line was closed, and the heart was subjected to 30 min of ischemia. Before the initiation of the 2 hrs reperfusion the heart was perfused in a retrograde mode to avoid the development of ventricular fibrillation. Hearts that showed any cardiac disturbance such as ventricle arrhythmia and fibrillation during the experiment, were excluded from the study.


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)

(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).
© Copyright Policy
Related In: Results  -  Collection

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

f1: (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).
Mentions: Isolated mice hearts from wild‐type littermates and CLP‐1 +/− subjected to ischaemic stress as before [22] were divided into two groups (see Fig. 1A): (1) isolated hearts were perfused with Krebs‐Henseleit bicarbonate (KHB) buffer for 15 min and then subjected to 30 min of ischemia followed by 15, 30, 60 or 120 min of reperfusion (control); (2) the hearts were preconditioned to ischaemic stress by four cyclic episodes of 5 min ischemia each followed by another 10 min reperfusion followed by 30 min of ischemia and 15, 30, 60 or 120 min of reperfusion (PC). The isolated hearts from CLP‐1 +/− or wild‐type mice were obtained after injecting the mice with pentobarbital sodium (80 mg/kg body wt ip) and anticoagulant heparin sodium (500 IU/kg body wt ip). The hearts were excised and transferred to ice‐cold modifiedKHB, containing in mM (NaCl 118; KCl 4.7; CaCl2 1.7; NaHCO3 24; KH2PO4 1.2; MgSO4 12; Glucose 10). The aorta and pulmonary vein were cannulated and perfused in retrograde Langendorff mode. Perfusate KHB temperature was maintained at 37°C and saturated with 95% O2 and 5% CO2. The perfusion was then switched to anterograde mode where the buffer enters the cannulated left atrium at pressure equivalent to 10 cm of water (10 kPa). The heart measurements were recorded by Gould p23XL transducer (Gould Instrument System, Inc., Valley View, OH, USA). The signal was amplified by using Gould 6600 6600 series signal conditioner (Gould Instrument System, Inc.) and monitored on a Cordat II real‐time acquisition system (Triton technologies, San Diego, CA, USA). The aortic flow was measured by a flow meter. Once baseline measurements of heart rate, coronary flow, aortic flow, left ventricular developed pressure and its maximum first derivative were performed, the anterograde perfusion line was closed, and the heart was subjected to 30 min of ischemia. Before the initiation of the 2 hrs reperfusion the heart was perfused in a retrograde mode to avoid the development of ventricular fibrillation. Hearts that showed any cardiac disturbance such as ventricle arrhythmia and fibrillation during the experiment, were excluded from the study.

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