Date of Award

7-1-2008

Degree Type

Restricted Access Dissertation

Degree Name

Doctor of Philosophy

Field of Study

Biomedical Sciences

Department

Graduate School of Biomedical Sciences

First Advisor

Robert T. Mallet

Second Advisor

Albert Olivencia-Yurvati

Third Advisor

Peter B. Raven

Abstract

Ryou, Myoung-Gwi, Novel Cardioprotective and Anti-Inflammatory Mechanisms of Pyruvate-Enhanced Cardioplegia. Doctor of Philosophy (Biomedical Sciences), July 2008, 113 pp, 19 figures, Reference, 176 titles. Open-heart surgery requires a quiescent surgical field, achieved by cardioplegic arrest of the beating heart. Cardioplegia-induced cardiac arrest, however, imposes global ischemia and reperfusion which depletes myocardial energy reserves and generates free radicals that can damage the heart and induce harmful inflammation. In a recent randomized trial in patients undergoing cardiac surgery on CPB, cardioplegia containing the intermediary metabolite pyruvate, an energy-yielding fuel and antioxidant in myocardium, robustly increased post-surgical cardiac performance. The result of this clinical trial raises an important question: How did the temporary administration of pyruvate-enhanced cardioplegia produce such persistent improvements in cardiac function? The first portion of this investigation tested the possibility that pyruvate cardioplegia evoked expression of a cardioprotective and anti-inflammatory gene program directed by hypoxia-inducible factor-1 (HIF-1), a transcription factor known to be stabilized and upregulated by pyruvate. I hypothesized that use of pyruvate-fortified cardioplegia augmented myocardial content of HIF-1α subunit, and activated expression and intracellular signaling by the HIF-1-inducible, cytoprotective hormone erythropoietin (EPO). Also tested were the effects of pyruvate vs. conventional glucose cardioplegia on myocardial contents of EPO and its membrane receptor EPO and its membrane receptor EPO-R activation (i.e., phosphorylation) or Erk and Akt, protein kinases implicated in EPO signaling, and myocardial content and activity of the EPO effector, endothelial nitric oxide synthase (eNOS). In situ swine hearts were arrested for 60 min with cardioplegia containing 188 mM glucose alone (control cardioplegia) or with 24 mM pyruvate (pyruvate-fortified cardioplegia). A sham group was surgically instrumented but not subjected to cardioplegic arrest or CPB. Following 60 min cardioplegic arrest and 30 min reperfusion with cardioplegia free blood, pigs were weaned from the heart-lung machine and recovered for 4 hr. At the end of the recovery, left ventricular myocardium was excised and fixed with formalin for EPO-R immonhistochemistry. Additional myocardium was snap frozen and processed for measurements of HIF-1α, EPO, EPO-R, total and phosphorylated Akt and Erk, and eNOS. Myocardial phosphorylation potential, ATP content and NOS activity were measured by spectrophotometry. There were no differences in myocardial energy state among the groups, indicating that the energy-enhancing effects of pyruvate subsided after pyruvate cardioplegia cleared from the myocardium. HIF-1α content was increased by 60% 4 h after cardiac arrest with pyruvate vs. control cardioplegia, but HIF-1α mRNA abundance was unaltered, indicating pyruvate enhanced HIF-1α content at the post-translational level. Pyruvate cardioplegia effected dramatic increase (c. 1000-fold) in EPO mRNA, and increased EPO and EPO-R contents by 58 and 123% respectively. Pyruvate cardioplegia also increased Akt and Erk phosphorylation, by 38 and 75%, without affecting total contents of these signaling kinases. Pyruvate cardioplegia also increased myocardial NOS activity by 45% and eNOS content by 81%. Thus, administration of pyruvate-fortified cardioplegia to arrest the heart persistently enhanced the cytoprotective EPO-Akt/Erk-eNOS signaling cascade, at least in part by stabilizing HIF-1. Anti-inflammatory effects of pyruvate have been demonstrated in various animal models of inflammation. I hypothesized that pyruvate-fortified cardioplegia mitigates inflammation by increasing anti-inflammatory cytokines, reducing ROS, and minimizing neutrophil infiltration. Arterial and coronary sinus plasma was sampled at predetermined points to measure anti-inflammatory cytokines and GSH/GSSG ratio. Left ventricular myocardial contents of acute inflammatory marker C-reactive peptide (CRP), pro-inflammatory enzyme matrix metalloproteinase-3 (MMP3), and the anti-inflammatory factor, tissue inhibitor of metalloproteinase-2 (TIMP2) were examined by immunoblot. CRP was decreased by 74% in myocardium arrested with pyruvate-fortified vs. control cardioplegia. Circulating IL-10 sharply increased during CPB, then subsided during recovery, while IL-6 plateaued at 2-4 h recovery. Pyruvate cardioplegia intensified and prolonged the increase in IL-10, and caused myocardium to release IL-6 into coronary effluent. Pyruvate suppressed neutrophil infiltration and maintained the structural integrity of the myocardium. This effect may be due to a sharp increase in myocardial TIMP2 content, which would suppress MMP3 degradation of the extracellular matrix. I conclude that pyruvate-fortified cardioplegia 1) activates cytoprotective EPO-signaling pathway by enhancing HIF-1α content, and 2) suppresses inflammation by increasing anti-inflammatory cytokines and glutathione redox state, and by suppressing MMP activity, thereby preventing neutrophil invasion of the myocardial parenchyma. These findings support novel cardioprotective mechanisms afforded by pyruvate-enriched cardioplegia during CPB. The cytoprotective and anti-inflammatory mechanisms may share a common pathway mediated by HIF-1 and EPO.

Comments

Ryou, Myoung-Gwi, Novel Cardioprotective and Anti-Inflammatory Mechanisms of Pyruvate-Enhanced Cardioplegia. Doctor of Philosophy (Biomedical Sciences), July 2008, 113 pp, 19 figures, Reference, 176 titles. W 4 R995N 2008

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