María Fernández-Velasco

Increased Ca2+ Sensitivity of the Ryanodine Receptor Mutant RyR2R4496C Underlies Catecholaminergic Polymorphic Ventricular Tachycardia

Cardiac ryanodine receptor (RyR2) mutations are associated with autosomal dominant catecholaminergic polymorphic ventricular tachycardia, suggesting that alterations in Ca2+ handling underlie this disease. Here we analyze the underlying Ca2+ release defect that leads to arrhythmia in cardiomyocytes isolated from heterozygous knock-in mice carrying the RyR2R4496C mutation. RyR2R4496C−/− littermates (wild type) were used as controls. [Ca2+]i transients were obtained by field stimulation in fluo-3–loaded cardiomyocytes and viewed using confocal microscopy. In our basal recording conditions (2-Hz stimulation rate), [Ca2+]i transients and sarcoplasmic reticulum Ca2+ load were similar in wild-type and RyR2R4496C cells. However, paced RyR2R4496C ventricular myocytes presented abnormal Ca2+ release during the diastolic period, viewed as Ca2+ waves, consistent with the occurrence of delayed afterdepolarizations. The occurrence of this abnormal Ca2+ release was enhanced at faster stimulation rates and by &bgr;-adrenergic stimulation, which also induced triggered activity. Spontaneous Ca2+ sparks were more frequent in RyR2R4496C myocytes, indicating increased RyR2R4496C activity. When permeabilized cells were exposed to different cytosolic [Ca2+]i, RyR2R4496C showed a dramatic increase in Ca2+ sensitivity. Isoproterenol increased [Ca2+]i transient amplitude and Ca2+ spark frequency to the same extent in wild-type and RyR2R4496C cells, indicating that the &bgr;-adrenergic sensitivity of RyR2R4496C cells remained unaltered. This effect was independent of protein expression variations because no difference was found in the total or phosphorylated RyR2 expression levels. In conclusion, the arrhythmogenic potential of the RyR2R4496C mutation is attributable to the increased Ca2+ sensitivity of RyR2R4496C, which induces diastolic Ca2+ release and lowers the threshold for triggered activity.

Cardiomyocyte overexpression of neuronal nitric oxide synthase delays transition toward heart failure in response to pressure overload by preserving calcium cycling.

Background— Defects in cardiomyocyte Ca2+ cycling are a signature feature of heart failure (HF) that occurs in response to sustained hemodynamic overload, and they largely account for contractile dysfunction. Neuronal nitric oxide synthase (NOS1) influences myocyte excitation-contraction coupling through modulation of Ca2+ cycling, but the potential relevance of this in HF is unknown. Methods and Results— We generated a transgenic mouse with conditional, cardiomyocyte-specific NOS1 overexpression (double-transgenic [DT]) and studied cardiac remodeling, myocardial Ca2+ handling, and contractility in DT and control mice subjected to transverse aortic constriction (TAC). After TAC, control mice developed eccentric hypertrophy with evolution toward HF as revealed by a significantly reduced fractional shortening. In contrast, DT mice developed a greater increase in wall thickness (P<0.0001 versus control+TAC) and less left ventricular dilatation than control+TAC mice (P<0.0001 for both end-systolic and end-diastolic dimensions). Thus, DT mice displayed concentric hypertrophy with fully preserved fractional shortening (43.7±0.6% versus 30.3±2.6% in control+TAC mice, P<0.05). Isolated cardiomyocytes from DT+TAC mice had greater shortening, intracellular Ca2+ transients, and sarcoplasmic reticulum Ca2+ load (P<0.05 versus control+TAC for all parameters). These effects could be explained, at least in part, through modulation of phospholamban phosphorylation status. Conclusions— Cardiomyocyte NOS1 may be a useful target against cardiac deterioration during chronic pressure-overload–induced HF through modulation of calcium cycling.

HIF-1α and PFKFB3 Mediate a Tight Relationship Between Proinflammatory Activation and Anerobic Metabolism in Atherosclerotic Macrophages

Objective—Although it is accepted that macrophage glycolysis is upregulated under hypoxic conditions, it is not known whether this is linked to a similar increase in macrophage proinflammatory 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. In addition, we show that macrophage glycolysis and proinflammatory activation mainly depend on hypoxia-inducible factor and on its impact on glucose uptake, and on the expression of hexokinase II and ubiquitous 6-phosphofructo-2-kinase. As a consequence, hypoxia potentiates inflammation and glycolysis mainly via these pathways. Moreover, when macrophages’ ability to increase glycolysis through 6-phosphofructo-2-kinase is experimentally attenuated, cell viability is reduced if subjected to proinflammatory or hypoxic conditions, but unaffected under control conditions. In addition to this, granulocyte-macrophage colony-stimulating factor enhances anerobic glycolysis while exerting a mild proinflammatory 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 upregulation of proinflammatory activity, in a manner that is dependent on both hypoxia-inducible factor -1&agr; and 6-phosphofructo-2-kinase.

Conditional FKBP12.6 Overexpression in Mouse Cardiac Myocytes Prevents Triggered Ventricular Tachycardia Through Specific Alterations in Excitation- Contraction Coupling

Background— Ca2+ 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. &bgr;-Adrenergic stimulation has been suggested to dissociate FKBP12.6 from RyR2, leading to diastolic sarcoplasmic reticulum Ca2+ 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 (P<0.05). In isolated transgenic myocytes, Ca2+ spark frequency was reduced by 50% (P<0.01), a reduction that persisted under isoproterenol stimulation, whereas the sarcoplasmic reticulum Ca2+ load remained unchanged. In parallel, peak ICa,L density decreased by 15% (P<0.01), and the Ca2+ transient peak amplitude decreased by 30% (P<0.001). A 33.5% prolongation of the caffeine-evoked Ca2+ transient decay was associated with an 18% reduction in the Na+-Ca2+ exchanger protein level (P<0.05). Conclusions— Increased FKBP12.6 binding to RyR2 prevents triggered VT in normal hearts in stress conditions, probably by reducing diastolic sarcoplasmic reticulum Ca2+ leak. This indicates that the FKBP12.6-RyR2 complex is an important candidate target for pharmacological prevention of VT.

Lipoxin A4 impairment of apoptotic signaling in macrophages: implication of the PI3K/Akt and the ERK/Nrf-2 defense pathways.

Lipoxin A4 (LXA4) is an endogenous lipid mediator that requires transcellular metabolic traffic for its synthesis. The targets of LXA4 on neutrophils are well described, contributing to attenuation of inflammation. However, effects of lipoxins on macrophage are less known, particularly the action of LXA4 on the regulation of apoptosis of these cells. Our data show that pretreatment of human or murine macrophages with LXA4 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/IFNγ challenge. The release of mitochondrial mediators of apoptosis and the activation of caspases was abrogated in the presence of LXA4. 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 LXA4 identified an early activation of the PI3K/Akt and ERK/Nrf-2 pathways, which was required for the observation of the antiapoptotic effects of LXA4. These data suggest that the LXA4, 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.

LA419, a Novel Nitric Oxide Donor, Prevents Pathological Cardiac Remodeling in Pressure-Overloaded Rats Via Endothelial Nitric Oxide Synthase Pathway Regulation

Reduced endogenous NO production has been described in cardiovascular disorders as cardiac hypertrophy and heart failure. The therapy with conventional nitrates is limited by their adverse hemodynamic effects and drug tolerance. The novel NO donor LA419 has demonstrated important antithrombotic and anti-ischemic properties without those adverse effects. The aim of this study was to evaluate the effect of LA419 chronic treatment on cardiac hypertrophy development in a progressive model of left ventricular hypertrophy. Rats were randomly divided into 6 groups: sham and clip (euthanized 7 weeks after aortic stenosis), sham+vehicle, sham+LA419, clip+vehicle, and clip+LA419 (euthanized 14 weeks after the surgery and treated with vehicle or 30 mg/kg of LA419 once left ventricular hypertrophy was established). LA419 treatment for 7 weeks induced a marked reduction in the heart:body weight ratio (4.10±0.28 and 3.38±0.06 mg/g in clip+vehicle versus clip+LA419; P<0.001) and left ventricular diameter (11.96±0.25 and 9.90±0.20 mm in clip+vehicle versus clip+LA419; P<0.001) without modifying the high blood pressure observed in stenosed rats. Histological analysis revealed that LA419 attenuated myocardial and perivascular fibrosis observed in rats with pressure overload for 14 weeks. In addition, LA419 treatment restored endothelial NO synthase and caveolin-3 expression levels, enhanced the interaction between endothelial NO synthase and its positive regulator the heat shock protein 90, and re-established the normal cardiac content of cGMP in stenosed rats. Thus, LA419 prevented the progression to maladaptative cardiac hypertrophy in response to prolonged pressure overload through a mechanism that involved the re-establishment of the endothelial NO synthase signaling pathway.

NOD1 activation induces cardiac dysfunction and modulates cardiac fibrosis and cardiomyocyte apoptosis

The innate immune system is responsible for the initial response of an organism to potentially harmful stressors, pathogens or tissue injury, and accordingly plays an essential role in the pathogenesis of many inflammatory processes, including some cardiovascular diseases. Toll like receptors (TLR) and nucleotide-binding oligomerization domain-like receptors (NLRs) are pattern recognition receptors that play an important role in the induction of innate immune and inflammatory responses. There is a line of evidence supporting that activation of TLRs contributes to the development and progression of cardiovascular diseases but less is known regarding the role of NLRs. Here we demonstrate the presence of the NLR member NOD1 (nucleotide-binding oligomerization domain containing 1) in the murine heart. Activation of NOD1 with the specific agonist C12-iEDAP, but not with the inactive analogue iE-Lys, induces a time- and dose-dependent cardiac dysfunction that occurs concomitantly with cardiac fibrosis and apoptosis. The administration of iEDAP promotes the activation of the NF-κB and TGF-β pathways and induces apoptosis in whole hearts. At the cellular level, both native cardiomyocytes and cardiac fibroblasts expressed NOD1. The NLR activation in cardiomyocytes was associated with NF-κB activation and induction of apoptosis. NOD1 stimulation in fibroblasts was linked to NF-κB activation and to increased expression of pro-fibrotic mediators. The down-regulation of NOD1 by specific siRNAs blunted the effect of iEDAP on the pro-fibrotic TGF-β pathway and cell apoptosis. In conclusion, our report uncovers a new pro-inflammatory target that is expressed in the heart, NOD1. The specific activation of this NLR induces cardiac dysfunction and modulates cardiac fibrosis and cardiomyocyte apoptosis, pathological processes involved in several cardiac diseases such as heart failure.

Involvement of monocytes/macrophages as key factors in the development and progression of cardiovascular diseases

Emerging evidence points to the involvement of specialized cells of the immune system as key drivers in the pathophysiology of cardiovascular diseases. Monocytes are an essential cell component of the innate immune system that rapidly mobilize from the bone marrow to wounded tissues where they differentiate into macrophages or dendritic cells and trigger an immune response. In the healthy heart a limited, but near-constant, number of resident macrophages have been detected; however, this number significantly increases during cardiac damage. Shortly after initial cardiac injury, e.g. myocardial infarction, a large number of macrophages harbouring a pro-inflammatory profile (M1) are rapidly recruited to the cardiac tissue, where they contribute to cardiac remodelling. After this initial period, resolution takes place in the wound, and the infiltrated macrophages display a predominant deactivation/pro-resolution profile (M2), promoting cardiac repair by mediating pro-fibrotic responses. In the present review we focus on the role of the immune cells, particularly in the monocyte/macrophage population, in the progression of the major cardiac pathologies myocardial infarction and atherosclerosis.

Sustained Epac activation induces calmodulin dependent positive inotropic effect in adult cardiomyocytes.

Cardiac actions of Epac (exchange protein directly activated by cAMP) are not completely elucidated. Epac induces cardiomyocytes hypertrophy, Ca(2+)/calmodulin protein kinase II (CaMKII) and excitation-transcription coupling in rat cardiac myocytes. Here we aimed to elucidate the pathway cascade involved in Epac sustained actions, as during the initiation of hypertrophy development, where β-adrenergic signaling is chronically stimulated. Rats were treated with the Epac selective activator 8-pCPT during 4 weeks and Ca(2+) signaling was analyzed in isolated cardiac myocytes by confocal microscopy. We observed a positive inotropic effect manifested by increased [Ca(2+)](i) transient amplitudes. In order to further analyze these actions, we incubated adult cardiomyocytes in the presence of 8-pCPT. The effects were similar to those obtained in-vivo and are blunted by Epac1 knock down. Interestingly, the increase in [Ca(2+)] transients was abolished by protein synthesis blockade or when the downstream effectors of calmodulin (CaMKII or calcineurin) were inhibited, pointing to calmodulin (CaM) as an important downstream protein in Epac sustained actions. In fact, CaM expression was enhanced by 8-pCPT treatment in isolated cells, as found by Western blots. Moreover, the 8-pCPT-induced, PKA-independent, positive inotropic effect was favored by enhanced extracellular Ca(2+) influx via L-type Ca(2+) channels. However, 8-pCPT also induced aberrant Ca(2+) release as Ca(2+) waves and extra [Ca(2+)](i) transients, suggesting proarrhythmic effect. These results provide new insights regarding Epac cardiac actions, suggesting an important role in the initial compensation of the heart to pathological stimuli during the initiation of cardiac hypertrophy, favoring contraction but also arrhythmia risk.

Mitochondrial DAMPs Induce Endotoxin Tolerance in Human Monocytes: An Observation in Patients with Myocardial Infarction

Monocyte exposure to mitochondrial Danger Associated Molecular Patterns (DAMPs), including mitochondrial DNA (mtDNA), induces a transient state in which these cells are refractory to further endotoxin stimulation. In this context, IRAK-M up-regulation and impaired p65 activity were observed. This phenomenon, termed endotoxin tolerance (ET), is characterized by decreased production of cytokines in response to the pro-inflammatory stimulus. We also show that monocytes isolated from patients with myocardial infarction (MI) exhibited high levels of circulating mtDNA, which correlated with ET status. Moreover, a significant incidence of infection was observed in those patients with a strong tolerant phenotype. The present data extend our current understanding of the implications of endotoxin tolerance. Furthermore, our data suggest that the levels of mitochondrial antigens in plasma, such as plasma mtDNA, should be useful as a marker of increased risk of susceptibility to nosocomial infections in MI and in other pathologies involving tissue damage.