Cardiovascular Research Advance Access first published online on July 6, 2009
This version [Corrected Proof] published online on July 28, 2009
Cardiovascular Research, doi:10.1093/cvr/cvp233
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Reducing ischaemia/reperfusion injury through 
-opioid-regulated intrinsic cardiac adrenergic cells: adrenopeptidergic co-signalling
1 Cardiology Division, Department of Internal Medicine, University of Texas Medical Branch, 5.106 John Sealy Annex, 301 University Boulevard, Galveston, TX 77555-0553, USA
2 Research Histopathology Core, University of Texas Medical Branch, Galveston, TX, USA
3 Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, TX, USA
4 Department of Immunology and Microbiology, University of Texas Medical Branch, Galveston, TX, USA
5 Cardiac Department
6 National University Medical Institutes, National University of Singapore, Singapore
* Corresponding author. Tel: +1 409 772 2410; fax: +1 409 772 4982. E-mail address: mihuang{at}utmb.edu
Aims: The purpose of this study was to determine whether intrinsic cardiac adrenergic (ICA) cells release calcitonin gene-related peptide (CGRP), exerting synergistic adrenopeptidergic cardioprotection.
Methods and results: In situ hybridization coupled with immunostaining demonstrated that ICA cells exclusively expressed CGRP mRNA and co-expressed CGRP and 
-opioid receptor in human and rat left ventricular (LV) myocardium. Radioimmunoassay detected constitutive CGRP release from ICA cells in human and rat hearts. The -opioid agonist [D-Pen25]-enkephalin (DPDPE) increased CGRP release from ICA cells in denervated rat heart. In an ischaemia/reperfusion rat model, pre-ischaemic treatment with DPDPE reduced infarct size (IS) by 51 ± 16% (P < 0.01). Co-infusion of β2-adrenergic receptor (β2-AR) and CGRP receptor (CGRP-R) antagonists increased IS by 62 ± 23% (P < 0.01) compared with saline and abolished DPDPE-initiated IS reduction. Pre-treatment of ICA cell–myocyte co-culture with the β2-AR/CGRP-R antagonists increased myocyte death rate by 24 ± 4% (P < 0.01) and abolished DPDPE-initiated myocyte protection against hypoxia/reoxygenation (re-O2). In the ICA cell-depleted myocyte culture, DPDPE did not confer myocyte protection. Supplementing ICA cell-depleted myocyte culture with β2-AR/CGRP-R agonists reduced hypoxia/re-O2-induced myocyte death by 24 ± 5% (P < 0.01), simulating endogenous neurohormonal effects of ICA cells. Western blot analysis showed that DPDPE markedly increased phosphorylated myocardial Akt levels. This effect was abolished in the presence of β2-AR/CGRP-R blockade. Terminal dUTP nick-end labelling staining analysis of the LV infarct zone demonstrated that DPDPE reduced myocyte apoptosis by 58 ± 19% (P < 0.05), an effect that was eliminated in the presence of β2-AR/CGRP-R blockade. Finally, echocardiography showed that DPDPE increased LV contractility in a manner dependent on β-AR/CGRP-R stimulation.
Conclusion: ICA cells constitute a -opioid-regulated adrenopeptidergic paracrine system conferring robust cardioprotection through β2-AR/CGRP-R co-signalling, resulting in the activation of an anti-apoptotic pathway during ischaemia/reperfusion.
KEYWORDS Apoptosis; β2-Adrenergic receptor; CGRP; ICA cell; Ischaemia/reperfusion
Time for primary review: 29 days