cAMP is a powerful second messenger whose known general effector is protein kinase A (PKA). : from 2.4 ± 0.6 to 6.9 ± 1.5, P < 0.01) while reducing their amplitude (F/F 0 : 1.8 ± 0.02 versus 1.6 ± 0.01, P < 0.001) in a Ca 2+ /calmodulin kinase II (CaMKII)-dependent and PKA-independent manner. Accordingly, we found that Epac increased RyR phosphorylation at the CaMKII site. Altogether, our data reveal a new signalling pathway by which cAMP governs Ca 2+ release and signalling in cardiac myocytes.

Abstract-Cardiac] i transients Ⅲ ryanodine receptor Ⅲ excitation-contraction coupling Ⅲ CPVT C atecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease characterized by stress-induced, adrenergically mediated bidirectional or polymorphic ventricular tachycardia occurring in structurally normal hearts. 1 During exercise or acute emotions, CPVT patients develop life-threatening ventricular arrhythmias, leading to syncope or sudden death. The first cardiac ryanodine receptor (RyR2) mutation identified in a CPVT family was R4497C. 2 Today, more than 70 RyR2 mutations have been reported (http://www.fsm.it/cardmoc), and they comprise the most common genetic subtype of CPVT, 3-7 although mutations in the calsequestrin gene can also cause CPVT. 8,9 Diverging results and conclusions have been generated from expression studies of RyR2 R4496C in heterologous systems. Jiang et al showed that RyR2 R4496C (the mouse equivalent of the human RyR2 R4497C mutation), when expressed in human embryonic kidney (HEK) cells, exhibits increased basal activity and increased sensitivity to luminal Ca 2ϩ . 10 However, other authors found no difference in the basal activity of RyR2 R4497C but, instead, showed increased activity and gating frequency after protein kinase A phosphorylation 11 or sarcoplasmic reticulum (SR) Ca 2ϩ overload. 12 The expression studies were carried out in a variety of models, which may explain the inhomogeneous findings. Furthermore, heterologous systems lack cardiac intracellular environment with all the RyR2 accessory proteins 13 and most Ca 2ϩ -handling proteins, so analysis in native cardiac myocytes is now critical to elucidate the mechanisms by which the mutation leads to cardiac arrhythmia.Recently, a knock-in mouse model carrier of the RyR2 R4496C mutation was developed. 14 Their phenotype presents extraordinary similarity with the clinical manifestations of patients carrying the RyR2 R4497C mutation, including the development of bidirectional ventricular tachycardia. When exposed to adrenaline and caffeine, the RyR2 R4496C cardiomyocytes develop delayed afterdepolarizations (DADs), 15 suggesting that triggered arrhythmias are elicited by adrenergic activation. 16 Here we demonstrate that untreated RyR2 R4496C myocytes have increased spontaneous Ca 2ϩ release in diastole during electric pacing, because of the enhanced Ca 2ϩ sensitivity of mutant RyR2; this abnormality is further augmented by exposure to isoproterenol and increasing pacing rates. Materials and Methods Ventricular cardiomyocytes from male and female RyR2R4496Cϩ/Ϫ mice (RyR2 R4496C ) and their wild-type (WT) RyR2 R4496CϪ/Ϫ littermates were isolated using a standard enzymatic digestion. 17 [Ca 2ϩ ] i transients and Ca 2ϩ sparks were viewed in isolated myocytes by confocal microscopy and analyzed using homemade routines. All experiments were carried out according to the ethical principles laid down by the French (Ministry of Agriculture) and European Union

Objective While it is accepted that macrophage glycolysis is up-regulated under hypoxic conditions, it is not known whether this is linked to a similar increase in macrophage pro-inflammatory activation and whether specific energy demands regulate cell viability in the atheromatous plaque. Approach and Results We studied the interplay between macrophage energy metabolism, polarization and viability in the context of atherosclerosis. Cultured human and murine macrophages and an in vivo murine model of atherosclerosis were used to evaluate the mechanisms underlying metabolic and inflammatory activity of macrophages in the different atherosclerotic conditions analyzed. We observed that macrophage energetics and inflammatory activation are closely and linearly related, resulting in dynamic calibration of glycolysis to keep pace with inflammatory activity. Additionally, we show that macrophage glycolysis and proinflammatory activation mainly depend on hypoxia-inducible factor (HIF) and on its impact on glucose uptake, and on the expression of hexokinase II and ubiquitous 6-phosphofructo-2-kinase (PFKFB3). As a consequence, hypoxia potentiates inflammation and glycolysis mainly via these pathways. Moreover, when macrophages’ ability to increase glycolysis through PFKFB3 is experimentally attenuated, cell viability is reduced if subjected to proinflammatory and/or hypoxic conditions, but unaffected under control conditions. In addition to this, GM-CSF enhances anaerobic glycolysis while exerting a mild pro-inflammatory activation. Conclusions These findings, in human and murine cells and in an animal model, show that hypoxia potentiates macrophage glycolytic flux in concert with a proportional up-regulation of pro-inflammatory activity, in a manner that is dependent on both HIF-1α and PFKFB3.

HCN4 is the dominant subunit in canine sinoatrial node and RA; strong sinus node HCN expression likely contributes to its pacemaker function; downregulation of HCN4 and HCN2 expression contribute to CHF-induced sinus node dysfunction; and upregulation of atrial HCN4 may help to promote atrial arrhythmia formation. These findings provide novel information about the molecular basis of normal and disease-related impairments of cardiac impulse formation.

Background-Ca2ϩ release from the sarcoplasmic reticulum via the ryanodine receptor (RyR2) activates cardiac myocyte contraction. An important regulator of RyR2 function is FKBP12.6, which stabilizes RyR2 in the closed state during diastole. ␤-Adrenergic stimulation has been suggested to dissociate FKBP12.6 from RyR2, leading to diastolic sarcoplasmic reticulum Ca 2ϩ leakage and ventricular tachycardia (VT). We tested the hypothesis that FKBP12.6 overexpression in cardiac myocytes can reduce susceptibility to VT in stress conditions. Methods and Results-We developed a mouse model with conditional cardiac-specific overexpression of FKBP12.6.Transgenic mouse hearts showed a marked increase in FKBP12.6 binding to RyR2 compared with controls both at baseline and on isoproterenol stimulation (0.2 mg/kg IP). After pretreatment with isoproterenol, burst pacing induced VT in 10 of 23 control mice but in only 1 of 14 transgenic mice (PϽ0.05). In isolated transgenic myocytes, Ca 2ϩ spark frequency was reduced by 50% (PϽ0.01), a reduction that persisted under isoproterenol stimulation, whereas the sarcoplasmic reticulum Ca 2ϩ load remained unchanged. In parallel, peak I Ca,L density decreased by 15% (PϽ0.01), and the Ca 2ϩ transient peak amplitude decreased by 30% (PϽ0.001). A 33.5% prolongation of the caffeine-evoked Ca 2ϩ transient decay was associated with an 18% reduction in the Na ϩ -Ca 2ϩ exchanger protein level (PϽ0.05). Conclusions-Increased FKBP12.6 binding to RyR2 prevents triggered VT in normal hearts in stress conditions, probably by reducing diastolic sarcoplasmic reticulum Ca 2ϩ leak. This indicates that the FKBP12.6-RyR2 complex is an important candidate target for pharmacological prevention of VT.

Hyperpolarization-activated inward current (If) and changes in the messenger RNA (mRNA) expression levels of hyperpolarization-activated cyclic nucleotide-gated channel (HCN)2 and HCN4 encoding If channels of the rat heart were studied in control and hypertrophied myocytes isolated from three ventricular regions: the septum (S), the left ventricular free wall (LV) and the right ventricular free wall (RV). Electrophysiological experiments were conducted by ruptured and perforated-patch clamp techniques and quantification of mRNA levels was carried out by quantitative reverse transcriptase polymerase chain reaction. The occurrence, density and maximal specific conductance of If were found to be significantly higher in hypertrophied ventricular myocytes isolated from S and LV than in those isolated from RV or sham-operated rats. Half-maximal activation potential, the slope of the activation curve and the threshold for activation were similar in ventricular myocytes from sham and aortic stenosed rats in the three regions studied. Isoproterenol 1 micromol l-1 increased current size by shifting current activation to more positive potentials in both sham and hypertrophied myocytes. When we studied the mRNA levels of If channel isoforms present in the ventricle, we found a significant increase of HCN2 and HCN4 mRNA levels in hypertrophied myocytes from S and LV but not in RV. We conclude that the occurrence, density and conductance of If is higher in hypertrophied than in control ventricular myocytes, S being the region where all these changes were most evident. These findings are associated with a higher expression of HCN2 and HCN4 mRNA levels in the two regions that developed hypertrophy.

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Lipoxin A 4 (LXA 4 ) is an endogenous lipid mediator that requires transcellular metabolic traffic for its synthesis. The targets of LXA 4 on neutrophils are well described, contributing to attenuation of inflammation. However, effects of lipoxins on macrophage are less known, particularly the action of LXA 4 on the regulation of apoptosis of these cells. Our data show that pretreatment of human or murine macrophages with LXA 4 at the concentrations prevailing in the course of resolution of inflammation (nanomolar range) significantly inhibits the apoptosis induced by staurosporine, etoposide and S-nitrosoglutathione or by more pathophysiological stimuli, such as LPS/IFNc challenge. The release of mitochondrial mediators of apoptosis and the activation of caspases was abrogated in the presence of LXA 4 . In addition to this, the synthesis of reactive oxygen species induced by staurosporine was attenuated and antiapoptotic proteins of the Bcl-2 family accumulated in the presence of lipoxin. Analysis of the targets of LXA 4 identified an early activation of the PI3K/Akt and ERK/Nrf-2 pathways, which was required for the observation of the antiapoptotic effects of LXA 4 . These data suggest that the LXA 4 , released after the recruitment of neutrophils to sites of inflammation, exerts a protective effect on macrophage viability that might contribute to a better resolution of inflammation.