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Cardioplegia and Diazoxide Modulate STAT3 Activation and DNA Binding

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Presentation on theme: "Cardioplegia and Diazoxide Modulate STAT3 Activation and DNA Binding"— Presentation transcript:

1 Cardioplegia and Diazoxide Modulate STAT3 Activation and DNA Binding
Yng-Ju Hsieh, PhD, Hidetaka Wakiyama, MD, Sidney Levitsky, MD, James D. McCully, PhD  The Annals of Thoracic Surgery  Volume 84, Issue 4, Pages (October 2007) DOI: /j.athoracsur Copyright © 2007 The Society of Thoracic Surgeons Terms and Conditions

2 Fig 1 Experimental schema of acute myocardial infarct model in the in situ pig heart. Pigs were cannulated placed on cardiopulmonary bypass. After 30 minutes of stabilization, hearts were subjected to regional ischemia by snaring of the distal portion of the left anterior descending artery (LAD). Immediately after 30 minutes of normothermic regional ischemia, the aorta was cross-clamped, and cold blood magnesium supplemented potassium (DSA) cardioplegia or DSA with the addition of 50 μMol/L diazoxide (DSA+DZX) was administered through the aortic root. The hearts were then subjected to hypothermic global ischemia. After 30 minutes of hypothermic global ischemia, the cross clamp and the snare were released and the hearts reperfused for 120 minutes. Tissue samples from the regional ischemia and global ischemia zones were obtained and quick frozen in liquid nitrogen before isolation of nuclear proteins. The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur ) Copyright © 2007 The Society of Thoracic Surgeons Terms and Conditions

3 Fig 2 Representative Western blots of nuclear protein signal transducers and activators of transcription (STATs) STAT1, STAT3, and tyrosine (pTyr)and serine (pSer) phosphorylation levels in control, magnesium supplemented potassium (DSA) and DSA plus diazoxide (DSA+DZX) hearts. Control hearts were subjected to sham regional and global ischemia. Regional ischemia and global ischemia zones are indicated. Molecular mass is shown for each gel. The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur ) Copyright © 2007 The Society of Thoracic Surgeons Terms and Conditions

4 Fig 3 Semiquantitative analysis of Western blot signal transducers and activators of transcription (STATs) STAT1, STAT3, tyrosine (pTyr), and serine (pSer) phosphorylation levels. All results are shown as the mean ± SEM for n = 6 separate blots. Densitometry is shown in arbitrary units. Significant differences between groups are shown in bold type. Significant difference at p < versus regional and global ischemic zones is indicated by an asterisk (*). The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur ) Copyright © 2007 The Society of Thoracic Surgeons Terms and Conditions

5 Fig 4 Representative electrophoretic mobility shift assay (EMSA) of signal transducers and activators of transcription (STATs) STAT1, STAT3 DNA binding. (A) Representative EMSA run for 2 hours at 35 mA. Supershift bands are shown in lanes 9 to 12. (B) Representative EMSA run for 6 hours at 35 mA, as described by Zhong and coworkers [13], to allow for identification of STAT1/STAT1, STAT1/STAT3, and STAT3/STAT3 dimer DNA binding. The STAT1, STAT3 DNA binding in DSA and DSA+DZX in regional and global ischemia zones is shown. Control hearts were subjected to sham regional and global ischemia. The 32P, unincorporated [γ–32P] ATP, 32P SIE, [γ–32P] labeled sis-inducible element consensus oligonucleotides; cold SIE, unlabeled SIE (100-fold molar excess); Ab-STAT3, supershift using 2 μg anti-STAT3 antibody. STAT1/STAT1, STAT1/STAT3, STAT3/STAT3, and supershift STAT1/STAT3 (Ab-STAT1/STAT3) and STAT3/ STAT3 (Ab-STAT3/ STAT3) are indicated. The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur ) Copyright © 2007 The Society of Thoracic Surgeons Terms and Conditions

6 Fig 5 (A) Representative competitive electrophoretic mobility shift assay (EMSA) using admixtures of magnesium supplemented potassium (DSA) and DSA containing diazoxide (DSA+DZX) nuclear proteins. Nuclear protein content for each EMSA is shown. Signal transducers and activators of transcription (STATs) STAT1/STAT1, STAT1/STAT3, and STAT3/STAT3 dimer DNA binding for DSA, DSA+DZX (lanes 1 and 4), and DSA and DSA+DZX admixtures (lanes 2 and 3) are shown. Cold sis-inducible element (SIE), unlabeled SIE (100-fold molar excess) is shown in lane 5. (B) Semiquantitative analysis of STAT3/STAT3, STAT1/STAT3, and STAT1/STAT1 dimer DNA binding. The DSA and DSA+DZX admixtures are indicated (open bars = DSA 10 μg/DSA+DZX 0 μg; hatched bars = DSA 5 μg/DSA+DZX 5 μg; gray bars = DSA 2.5 μg/DSA+DZX 7.5 μg; black bars = DSA 0 μg/DSA+DZX 10 μg). All results are shown as the mean ± SEM for n = 4 separate blots. Densitometry is shown in arbitrary units. Significant differences between groups are shown in bold type. The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur ) Copyright © 2007 The Society of Thoracic Surgeons Terms and Conditions

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