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Transgenic swine: Expression of human CD39 protects against myocardial injury

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Abstract

CD39 (ectonucleoside triphosphate diphosphohydrolase-1; ENTPD-1) rapidly hydrolyzes ATP and ADP to AMP; AMP is hydrolyzed by ecto-5′-nucleotidase (CD73) to adenosine, an anti-thrombotic and cardiovascular protective mediator. While expression of human CD39 in a murine model of myocardial ischemia/reperfusion (I/R) injury confers cardiac protection, the translational therapeutic potential of these findings requires further testing in a large animal model. To determine if transgenic expression of CD39 reduces infarct size in a swine model of myocardial ischemia/reperfusion injury, transgenic pigs expressing human CD39 (hCD39) were generated via somatic cell nuclear transfer and characterized. Expression of hC39 in cardiac tissue was confirmed by immunoblot and immunohistochemistry. Myocardial I/R injury was induced by intracoronary balloon inflation in the left anterior descending (LAD) artery for 60 min followed by 3 hours of reperfusion. The ischemic area was delineated by perfusion with 5% phthalo blue and the myocardial infarct size was determined by triphenyl tetrazolium chloride (TTC) staining. During ischemia, the rate-pressure product was significantly lower in control versus hCD39-Tg swine. Following reperfusion, compared to littermate control swine, hCD39-Tg animals displayed a significant reduction in infarct size (hCD39-Tg: 17.2 ± 4.3% vs. Control: 44.7 ± 5.2%, P = 0.0025). Our findings demonstrate for the first time that the findings in transgenic mouse models translate to large animal transgenic models and validate the potential to translate CD39 into the clinical arena to attenuate human myocardial ischemia/reperfusion injury.

Introduction

Myocardial infarction (MI) affects over 1 million people, resulting in over 300,000 deaths and heart failure in an additional 300,000 patients every year in the United States [1]. While numerous strategies have demonstrated efficacy in rodent models of myocardial injury, most have failed to demonstrate clinical efficacy. Indeed, the mouse cardiovascular physiology differs tremendously from human; the mouse heart beats at 600 bpm with a cardiac output of 20 mL/min while the human heart beats at 60 bpm with a cardiac output of 5 L/min [2]. Therefore how do we begin to translate findings in murine models to human?

Purine nucleotides and nucleosides influence nearly every aspect of cardiac physiology and pathophysiology including myocardial ischemia. Extracellular nucleotides (ATP and ADP) released at the site of cellular damage can induce platelet-mediated thrombosis, inflammation, cell apoptosis and necrosis. These nucleotides are sequentially hydrolyzed by cell surface expressed ectonucleotidases, CD39 (ectonucleoside triphosphate diphosphohydrolase 1; ENTPDase-1) and CD73 (ecto-5′-nucleotidase), to the cellular protective molecule, adenosine [3], [4], [5]. Our laboratory recently demonstrated that human CD39 (hCD39) expression decreases infarct size following myocardial I/R injury in mice [6]. Therefore, to translate our findings to a model more physiologically comparable to humans, we utilized a unique transgenic porcine model of hCD39 expression.

Here we demonstrate for the first time that infarct size is significantly reduced following myocardial ischemia-reperfusion injury in transgenic swine expressing human CD39.

Section snippets

Methods

hCD39-expressing swine were generated using the H-2Kb promoter/hCD39 construct previously used to generate transgenic mice (Fig. 1A) [7] via somatic cell nuclear transfer [8] and examined in an in vivo model of left anterior coronary artery myocardial ischemia-reperfusion injury. A detailed expanded Methods description is available in the Online Data Supplement.

Expression of human CD39 in transgenic swine

Expression of hCD39 in transgenic swine was confirmed on circulating blood cells (Fig. 1B) and in myocardial tissue (Fig. 1C–F).

CD39 expression protects against myocardial ischemia-reperfusion injury in swine

Fourteen swine were entered in the study. Two animals were excluded secondary to poor staining with phthalo blue and an inability to accurately assess the area-at-risk (1 Control and 1 hCD39-Tg) and one animal died secondary to ventricular fibrillation at anesthesia induction (hCD39-Tg). Baseline parameters of the 11 animals completing the protocol including age, sex

Discussion

The current study demonstrates that transgenic hCD39 expression in swine protects against myocardial ischemia/reperfusion injury, providing new rationale for human therapies based on this pathway. While one prior published study examined the effect of intracoronary infusion of apyrase (a soluble CD39-like molecule) on porcine myocardial ischemia-reperfusion injury, no benefit was found [9]. However, in our model CD39 is an intact membrane-expressed molecule in the vasculature and heart where it

Disclosures

None.

Funding

This work is supported by National Institutes of Health (National Heart, Lung, and Blood Institute Grants HL 094703 and HL096038 (RJG), U01 AI066331 and P01 AI045897 (SCR)), the Division of Cardiovascular Medicine, The Dorothy M. Davis Heart and Lung Research Institute and Ross Academic Advisory Committee (RJG). Transgenic pigs were generated using funding from the National Health and Medical Research Council of Australia (PJC, MBN, AJFd'A).

Acknowledgments

None.

References (15)

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This work was presented at the American Heart Association 2011 Scientific Session.

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