Introduction RAF kinases activate the ERK1/2 cascade, a key pathway involved in cardiac remodelling and cytoprotection. Since activating mutations in BRAF cause cancer, small molecule inhibitors of RAF have been developed. However, a paradoxical effect is observed with some inhibitors which activate rather than inhibit ERK1/2. Therefore, ‘paradox breaker’ inhibitors have been developed as new-generation cancer therapies void of this effect (e.g. PLX8394). Here, we determined the effects of PLX8394 on endothelial cell (EC) ERK1/2 signalling and the heart in vivo. Methods Murine ECs were incubated with PLX8394 and effects on ERK1/2 activity determined by western blotting for the phosphorylated (i.e. activated) kinases. Effects on gene expression were determined by qPCR. The effects of PLX8394 on the heart in vivo were determined by infusing male wildtype C57Bl/6J mice (10-12wks, n= 6/group) with PLX8394 (5mg/kg/d, 7d) using osmotic minipumps. Cardiac function/dimensions were assessed using echocardiography; effects on cardiac morphology were assessed by histological staining. mRNA expression was assessed by qPCR. Statistical tests used 1-way ANOVA with Holm-Sidak’s post-test (in vitro studies) and unpaired t-tests (in vivo studies). Results PLX8394 activated ERK1/2 in ECs in a time (7.4±2.3-fold at 5 min; p=0.0365; n=5) and concentration (>1uM; p=0.0625; n=3) dependent manner. This was associated with significant increases in expression of mRNAs encoding the immediate early gene Fos (6.1±2.6-fold; p<0.0001; n=4) and the vasoconstrictor peptide endothelin-1 (Edn1) (2.7±0.9-fold; p=0.0038; n=4). In vivo, PLX8394 decreased cardiac output (p=0.0092), predominantly through reduced stroke volume (p=0.0103). Structurally, PLX8394 promoted cardiac hypertrophy, with increased diastolic left ventricular (LV) posterior wall thickness (p=0.0425) and decreased LV internal diameter (p=0.0463) at 7 d. Cardiac hypertrophy resulted from increased cardiomyocyte cross-sectional area (p=0.0002) despite no changes in Myh7, Nppa or Nppb mRNAs. Moreover, PLX8394-induced cardiac remodelling was not due to increased fibrosis, with no change in mRNA expression of collagens1-4 and using histological assessment. Conclusion Despite being developed as a ‘paradox breaker’ for cancer, PLX8394 promoted ERK1/2 signalling in murine ECs and cardiac remodelling in vivo. These preliminary findings suggest that such inhibitors, currently in Phase 3 trials for RAF-mutant cancers, have potential to modulate cardiac function in patients. Conflict of Interest N/A

mitochondria was examined in the endothelium in intact blood vessels. In controls, TRPV4 activation with GSK1016790A(GSK) generated repetitive Ca2+ oscillations that required Ca2+ influx. When the Dy m was depolarised, by the uncoupler carbonyl cyanide m-chlorophenyl hydrazine (CCCP) or the complex I inhibitor rotenone, TRPV4 activation generated a much larger Ca2+ rise and propagating multicellular Ca2+ waves. The ATP synthase inhibitor oligo-mycin did not potentiate TRPV4 mediated Ca2+ influx. GSK-evoked Ca2+ waves, that occurred when mitochondria were depolarised, persisted in a Ca2+ free extracellular solu-tion i.e. were independent of Ca2+ influx. These signals were blocked by the TRPV4 channel blocker HC067047 (HC067), the SERCA inhibitor cyclopiazonic acid, the phos-pholipase C (PLC) blocker U73122 and the inositol triphos-phate receptor (IP3R) blocker caffeine. These observations suggest that TRPV4 may directly activate Ca2+ release from the internal store. The large propagating waves were inhib-ited by the pannexin blocker probenecid and the extracellular ATP blockers suramin and apyrase. These results highlight a previously unknown role of mitochondria in shaping TRPV4 mediated Ca2+ signalling and show that TRPV4 may trigger ATP release via a pannexin hemichannel when mitochondria are depolarised.