1. Differential Effects of Protein Kinase C Isoform Activation in Endothelin-Mediated Myocyte Contractile Dysfunction With Cardioplegic Arrest and Reperfusion 
Kimberly A. Apple, MD, Julie E. McLean, BS, Christina E. Squires, BS, Brooke Schaeffer, BS, Jeffrey A. Sample, BS, Rebecca L. Murphy, Anne M. Deschamps, BS, Amy H. Leonardi, BS, Claire M. Allen, BS, Jennifer W. Hendrick, BS, Robert E. Stroud, MS, Rupak Mukherjee, PhD, Francis G. Spinale, MD, PhD 
The Annals of Thoracic Surgery 
Volume 82, Issue 2, Pages (August 2006)
DOI: /j.athoracsur
Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

2. Fig 1
Myocyte contractility was measured under normothermic conditions and after simulated cardioplegic arrest (CA), as well as in the presence and absence of endothelin (ET). Additional experiments were performed using a specific peptide that caused inhibition of the classical subfamily of protein kinase-C (PKC) isoforms. Myocyte shortening velocity was reduced after CA and reperfusion and was further reduced in the presence of ET. Incubation with an inhibitor of this entire PKC subfamily increased contractility after CA. Inhibition of the classical PKC subfamily in the presence of ET normalized myocyte contractility under normothermic conditions and was increased significantly after CA. (*p < 0.05 versus normothermic baseline, + vs ET only values, and & versus CA baseline; black bars = normothermic; striated bars = CA.)
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

3. Fig 2
(A) Myocyte contractility was measured under normothermic conditions and after simulated cardioplegic arrest (CA) in which myocytes were exposed to endothelin (ET) and an inhibitory peptide specific for the isoforms of the classical protein kinase-C (PKC) subfamily: beta I, beta II, and gamma. The baseline and ET only histograms have been transposed from Figure 1 for reference purposes. Myocyte contractility in the presence of ET and the beta I isoform yielded a variable response, but contractility appeared higher under normothermic and CA conditions when compared with ET only values. Inhibition of the beta II isoform significantly increased myocyte contractility after CA. Inhibition of the gamma isoform yielded similar results to ET only under normothermic conditions, but significantly higher values after CA. (B) Coincubation with either of the inhibitors for the novel PKC isoforms and ET yielded results similar to that obtained with ET only. However, there was a high degree of variation in the presence of epsilon isoform inhibition under normothermic conditions. Coincubation with ET and the eta inhibitory peptide during and after CA increased contractility from ET and CA-only values. (*p < 0.05 versus normothermic baseline, +versus ET only values, and & versus CA baseline; black bars = normothermic; striated bars = CA).
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

4. Fig 3
Relative levels of the protein kinase-C (PKC) isoforms beta I, beta II, and the phosphorylated form of beta II were examined in isolated myocytes under normothermic (N) conditions and after simulated cardioplegic arrest (CA), as well as in the presence and absence of endothelin (ET). The results are summarized for three independent sets of experiments and normothermic values used for relative comparisons. Robust immunoreactive signals for these PKC isoforms were detected in the myocyte preparations. After CA and ET exposure, total levels of beta I and II were increased from normothermic values. A significant increase in the phosphorylated form of beta II was identified after CA and ET exposure. (*p < 0.05; black bars = normothermic; grey bars = CA.)
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

5. Fig 4
Relative levels of the protein kinase-C (PKC) isoforms gamma, eta, and epsilon were examined in isolated myocytes under normothermic (N) conditions and after simulated cardioplegic arrest (CA), as well as in the presence and absence of endothelin (ET). A clear and specific immunoreactive signal was detected for these PKC isoforms in the cardiac myocyte preparations. Total abundance for these PKC isoforms appeared unchanged with CA or ET when compared with normothermic values.
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

6. Fig 5
A schematic of the endothelin (ET) receptor transduction pathway and subsequent activation of the classical protein kinase-C (PKC) isoforms in a cardiac myocyte. After binding of ET to the ET receptor (primarily the ET-A subtype), which is a G-protein coupled receptor, a cascade of intracellular events occurs which includes activation of phospholipase C (PLC) which in turn forms inositol triphosphate (IP3) and diacylglycerol (DAG), and ultimately activation and mobilization of PKC. The PKC family consists of three broad subfamilies: classical, novel, and atypical. The classical PKC isoforms are activated through the G-protein coupled pathway and require calcium (Ca+2) for full activation and, thus, increased intracellular Ca+2 that occurs with CA and ET exposure would likely cause induction-activation of the classical PKC isoforms. The results from the present study confirm this and identify that the specific classical PKC isoform, beta II, is induced and activated with CA and ET exposure. Beta II PKC activation can, in turn, modify a number of intracellular events critical to myocyte contractile behavior which include changes in myofilament sensitivity to Ca+2, changes in Ca+2 reuptake by the sarcoplasmic reticulum (SR), and modifying the function of the voltage sensitive L-type Ca+2 channel. The present study demonstrated that using a selective inhibitory peptide for the beta II isoform, the negative contractile effects associated with CA and ET exposure were attenuated. (PLB = phospholamban; SERCA = sarcoplasmic reticulum calcium-adenosine triphosphatase, both components of the SR.)
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions