Ischaemia-reperfusion (I/R) injury complicates myocardial infarction and stroke by exacerbating tissue damage and mortality. We have recently identified C-type natriuretic peptide (CNP) as an endothelium-derived hyperpolarizing factor in the mesenteric resistance vasculature and described a novel signalling pathway involving activation of natriuretic peptide receptor C (NPR-C) which contributes to the regulation of local blood flow (Chauhan et al., 2003). We tested the hypothesis that CNP/NPR-C signalling is also a novel regulatory pathway governing coronary blood flow and protects against I/R injury.

Hearts were excised from male Wistar rats (260-340g) and perfused retrogradely via the aorta in Langendorff mode. Coronary perfusion pressure (CPP) and left ventricular pressure (LVP) were continuously measured on-line. Vasodilator responses to CNP (0.03-10nmol), the NPR-C selective agonist cANF^4-23 (10nmol) and the endothelium-dependent vasodilator acetylcholine (ACh; 3nmol) were measured in the absence and presence of the NO synthase inhibitor N^G-nitro-L-arginine methyl ester (L-NAME; 300μM), the cyclooxygenase inhibitor indomethacin (5μM), or a combination of barium (30μM) plus ouabain (1mM; blockers of K_IR and Na⁺K⁺ATPase, respectively). Release of CNP into the effluent from endothelium intact and denuded preparations (Triton X-100 1%; 60μl at 30μl min^-1 followed by a 20 min recovery period) was measured using a specific RIA. To investigate the effects of CNP on I/R injury hearts subject to a 25min global ischaemia and 120min reperfusion insult were treated with CNP or cANF^4-23 (30nM for 30min) prior to ischaemia or at reperfusion. Infarct size was determined following estimation of dry weight of the infarcted areas over the total weight of the heart determined by dye exclusion using p-nitro-blue tetrazolium (0.5mg/ml, 20min, 37°C).

CNP elicited dose-dependent decreases in CPP (EC₅₀2.13±0.33nmol; E_max=23.0±4.0mmHg; n4); responses were blocked by Ba²⁺ plus ouabain (E_max=3.0±1.0mmHg; P<0.05, n5). Similarly, cANF^4-23 (3nmol) decreased CPP (20.1±5.1mmHg; n4), a response blocked by Ba 2+ plus ouabain (ΔCPP=-2.2±1.0mmHg; P<0.05; n4). ACh elicited CNP release (183.6±25.7pg/ml; n4), with a concomitant drop in CPP (ΔCPP=-16.4±3.8mmHg; P<0.05; n4); responses abolished in the absence of an intact endothelium (P<0.05, n5). Pre-treatment with CNP and cANF^4-23 (in the presence of L-NAME) reduced infarct size (Control infarct: 49.34±2.05%, + CNP: 25.12±2.67%, + cANF^4-23: 30.52±4.18%; P<0.05; n5), maintaining CPP and LVP at pre-ischaemic values. Addition of CNP (30nM) at reperfusion also reduced infarct size (33.34±3.12%; P<0.05 vs control; n4).

Endothelium-derived CNP regulates the coronary circulation and NPR-C-mediated hyperpolarisation underlies the vasorelaxant activity of this peptide. Moreover, this newly-defined pathway represents a protective mechanism against I/R injury and a novel target for therapeutic intervention in ischaemic cardiovascular disorders.