Activation of TRPV1 by 12(s)‐HpETE and 20‐HETE Releases CGRP and Protects the Heart Against the Cardiac Dysfunction Caused by LPS

Rationale The severity of cardiac dysfunction predicts mortality in patients with sepsis. Activation of transient receptor potential vanilloid receptor type 1 (TRPV1), expressed on sensory neurons innervating the heart, improves outcome in sepsis/endotoxemia. However, the identity of the endogenous activators of TRPV1 and the role of the channel in the cardiac dysfunction caused by sepsis/endotoxemia is unknown. Objectives We investigated the impact of TRPV1 deletion upon the cardiac dysfunction caused by lipopolysaccharide (LPS; endotoxemia). In addition we explored the role of the arachidonic acid metabolites 12‐(S)‐hydroperoxyeicosatetraenoic acid (12(s)‐HpETE) and 20‐hydroxyeicosatetraenoic acid (20‐HETE) (potent ligands of TRPV1) and neuropeptides (downstream mediators of TRPV1 activation) in any effects seen. Methods TRPV1 −/− and wild‐type (WT, C57BL6 background) littermate mice were subjected to low (2 mg/kg, i.p.) or high dose LPS (6 mg/kg + peptidoglycan 0.1 mg/kg, i.p.). After 18 h cardiac function was determined using echocardiography. Neuronal (dorsal root ganglion) TRPV1 phosphorylation state was assessed by western blotting. Plasma levels of the neuropeptides calcitonin gene‐related peptide (CGRP), substance P (SP) and somatostatin were analyzed by ELISA. To investigate mechanisms some mice were treated with the CGRP receptor antagonist CGRP 8–37 (150 mg/kg; 30 min prior to LPS and 1 and 2 h after LPS, i.v.), the 12‐lipoxygenase inhibitor cinnamyl‐3, 4‐dihydroxy‐a‐cyanocinnamate (CDC; 0.2 mg/mouse, i.p. to block 12(s)‐HpETE synthesis) or the cytochrome P450 inhibitor 17‐octadecynoic acid (17ODYA; 0.7 mg/mouse, i.p., 20‐HETE synthesis inhibitor). Data is presented as mean ± SEM and was analyzed by one‐way ANOVA followed by Bonferroni's post hoc test or unpaired Student's t‐test. P <0.05 was considered to be statistically significant. Results Whilst low dose LPS did not alter cardiac function in WT mice [% ejection fraction (EF); vehicle: 72.4 ± 0.8, n=9 vs. LPS: 65.3 ± 4.5, n=8; p> 0.05], in TRPV1 −/− mice a pronounced cardiac dysfunction was evident (%EF; vehicle: 69.9 ± 1.4, n=5 vs. LPS: 27.6 ± 4.5, n=7; p <0.05); an effect similar to that evident in WT mice treated with high dose LPS (%EF; 34.4 ± 1.4; p> 0.05; n=7). Blockade of 12(s)‐HpETE or 20‐HETE synthesis augmented the cardiac dysfunction caused by low dose LPS in WT mice (%EF; vehicle: 72.3 ± 2.4, n=5; CDC: 48.5 ± 4.9, n=8; 17ODYA: 51.8 ± 4.9, n=8; CDC + 17ODYA: 40.5 ± 5.3, n=8; p <0.05 for all groups compared to vehicle). LPS (2 mg/kg) caused increases in plasma CGRP, SP and somatostatin levels in WT mice; however interestingly 17ODYA or CDC/17ODYA co‐administration attenuated only the rise in CGRP; ~69% (P<0.01) and 71% (p<0.001) respectively. Accordingly, CGRP 8–37 treatment increased cardiac dysfunction (%EF; vehicle: 67.4 ± 2.5, n=8 vs. CGRP 8–37 : 47.7 ± 2.5, n=7; p <0.05) induced by low dose LPS, suggesting that the release of endogenous CGRP mediates the cardioprotective action of TRPV1. Western blotting confirmed activation of TRPV1 with low dose LPS treatment in WT mice with increased expression of phosphorylated TRPV1 relative to total TRPV1 (~64%, P<0.05) relative to vehicle control, reflecting increased TRPV1 activation. Conclusions Activation of TRPV1, reflected through increased phosphorylation of the channel, by 12(s)‐HpETE and 20‐HETE likely leads to the release of CGRP, which protects the heart against the cardiac dysfunction caused by LPS. Support or Funding Information J.C. is supported by China Scholarship Council and Queen Mary University of London. A.H. is supported by The Trustees of the Professor Derek Willoughby PhD Fund for Inflammatory Research.