Recent studies demonstrated a role of sphingosine-1-phosphate (S1P) in the protection against the stress of ischemia/reperfusion (I/R) injury. In experiments reported here, we have investigated the signaling through the S1P cascade by FTY720, a sphingolipid drug candidate displaying structural similarity to S1P, underlying the S1P cardioprotective effect. In ex vivo rat heart and isolated sinoatrial node models, FTY720 significantly prevented arrhythmic events associated with I/R injury including premature ventricular beats, VT, and sinus bradycardia as well as A–V conduction block. Real-time PCR and Western blot analysis demonstrated the expression of the S1P receptor transcript pools and corresponding proteins including S1P1, S1P2, and S1P3 in tissues dissected from sinoatrial node, atrium and ventricle. FTY720 (25 nM) significantly blunted the depression of the levels of phospho-Pak1 and phospho-Akt with ischemia and with reperfusion. There was a significant increase in phospho-Pak1 levels by 35%, 199%, and 205% after 5, 10, and 15 min of treatment with 25 nM FTY720 compared with control nontreated myocytes. However, there was no significant difference in the levels of total Pak1 expression between nontreated and FTY720 treated. Phospho-Akt levels were increased by 44%, 63%, and 61% after 5, 10, and 15 min of treatment with 25 nM FTY720, respectively. Our data provide the first evidence that FTY720 prevents I/R injury-associated arrhythmias and indicate its potential significance as an important and new agent protecting against I/R injury. Our data also indicate, for the first time, that the cardioprotective effect of FTY720 is likely to involve activation of signaling through the Pak1.

Key points Natriuretic peptides (NPs) elicit their effects via multiple NP receptors (including NPR‐A, NPR‐B and NPR‐C, with NPR‐C being relatively poorly understood). We have studied the effects of NPR‐C ablation on cardiac structure, function and arrhythmogenesis using NPR‐C knockout (NPR‐C−/−) mice. NPR‐C−/− mice are characterized by sinoatrial node (SAN) dysfunction and a profound increase in susceptibility to atrial fibrillation. Increased susceptibility to arrhythmias in NPR‐C−/− mice was associated with slowed electrical conduction in the SAN as well as the right and left atria due to enhanced collagen expression and deposition in the atria (structural remodelling), but without changes in action potential morphology (electrical remodelling) in isolated SAN or atrial myocytes. This study demonstrates a critical protective role for NPR‐C in the heart. Abstract Natriuretic peptides (NPs) are critical regulators of the cardiovascular system that are currently viewed as possible therapeutic targets for the treatment of heart disease. Recent work demonstrates potent NP effects on cardiac electrophysiology, including in the sinoatrial node (SAN) and atria. NPs elicit their effects via three NP receptors (NPR‐A, NPR‐B and NPR‐C). Among these receptors, NPR‐C is poorly understood. Accordingly, the goal of this study was to determine the effects of NPR‐C ablation on cardiac structure and arrhythmogenesis. Cardiac structure and function were assessed in wild‐type (NPR‐C+/+) and NPR‐C knockout (NPR‐C−/−) mice using echocardiography, intracardiac programmed stimulation, patch clamping, high‐resolution optical mapping, quantitative polymerase chain reaction and histology. These studies demonstrate that NPR‐C−/− mice display SAN dysfunction, as indicated by a prolongation (30%) of corrected SAN recovery time, as well as an increased susceptibility to atrial fibrillation (6% in NPR‐C+/+ vs. 47% in NPR‐C−/−). There were no differences in SAN or atrial action potential morphology in NPR‐C−/− mice; however, increased atrial arrhythmogenesis in NPR‐C−/− mice was associated with reductions in SAN (20%) and atrial (15%) conduction velocity, as well as increases in expression and deposition of collagen in the atrial myocardium. No differences were seen in ventricular arrhythmogenesis or fibrosis in NPR‐C−/− mice. This study demonstrates that loss of NPR‐C results in SAN dysfunction and increased susceptibility to atrial arrhythmias in association with structural remodelling and fibrosis in the atrial myocardium. These findings indicate a critical protective role for NPR‐C in the heart.

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We investigated whether plasma long-chain sphingoid base (LCSB) concentrations are altered by transient cardiac ischemia during percutaneous coronary intervention (PCI) in humans and examined the signaling through the sphingosine-1-phosphate (S1P) cascade as a mechanism underlying the S1P cardioprotective effect in cardiac myocytes. Venous samples were collected from either the coronary sinus (n = 7) or femoral vein (n = 24) of 31 patients at 1 and 5 min and 12 h, following induction of transient myocardial ischemia during elective PCI. Coronary sinus levels of LCSB were increased by 1,072% at 1 min and 941% at 5 min (n = 7), while peripheral blood levels of LCSB were increased by 579% at 1 min, 617% at 5 min, and 436% at 12 h (n = 24). In cultured cardiac myocytes, S1P, sphingosine (SPH), and FTY720, a sphingolipid drug candidate, showed protective effects against CoCl induced hypoxia/ischemic cell injury by reducing lactate dehydrogenase activity. Twenty-five nanomolars of FTY720 significantly increased phospho-Pak1 and phospho-Akt levels by 56 and 65.6% in cells treated with this drug for 15 min. Further experiments demonstrated that FTY720 triggered nitric oxide release from cardiac myocytes is through pertussis toxin-sensitive phosphatidylinositol 3-kinase/Akt/endothelial nitric oxide synthase signaling. In ex vivo hearts, ischemic preconditioning was cardioprotective in wild-type control mice (Pak1f/f), but this protection appeared to be ineffective in cardiomyocyte-specific Pak1 knockout (Pak1cko) hearts. The present study provides the first direct evidence of the behavior of plasma sphingolipids following transient cardiac ischemia with dramatic and early increases in LCSB in humans. We also demonstrated that S1P, SPH, and FTY720 have protective effects against hypoxic/ischemic cell injury, likely a Pak1/Akt1 signaling cascade and nitric oxide release. Further study on a mouse model of cardiac specific deletion of Pak1 demonstrates a crucial role of Pak1 in cardiac protection against ischemia/reperfusion injury.