Langendorff-perfused hearts of mice harbouring one Cx43 knockout (KO) allele and one K258stop or Cx43 allele (K258stop/KO; Cx43/KO as control) were subjected to 1 h of ischaemia and 4 h of reperfusion by reversibly occluding the left anterior descending (LAD) coronary artery. Inducibility of ventricular tachyarrhythmias (VTs) was tested by applying an endocardial burst-pacing protocol during LAD occlusion. Separately, time course and the extent of acidification-induced closure of gap junction channels were tested by dual-voltage clamp. Infarct volume (as per cent of area at risk) was significantly larger in K258stop/KO hearts compared with Cx43/KO controls (42.2 ± 3 vs. 30.4 ± 1.7%, P = 0.004, n = 8 each). During LAD occlusion, K258stop/KO hearts had a higher incidence of pacing-induced VT and a higher frequency of occurrence of spontaneous premature ventricular beats. The occurrence of ventricular arrhythmias was also significantly larger in the K258stop/KO hearts during reperfusion. In separate experiments, we demonstrated reduced sensitivity to acidification-induced uncoupling in cell pairs obtained from K258stop/KO hearts.

Loss of the regulatory domain of Cx43 leads to an increase in infarct size and increased susceptibility to arrhythmias following acute coronary occlusion.

To determine whether PI3K is required for the maintenance of adrenergic vascular tone, we first constricted rat small mesenteric arteries with phenylephrine (PE) and then perfused with PI3K inhibitors, LY294002 and wortmannin, both of which produced a dose-dependent vasodilatation. Next, to investigate whether MMPs modulate PI3K activity, we cultured rat aortic vascular smooth muscle cells (VSMCs) and stimulated them with GPCR agonists such as PE and angiotensin II. Inhibition of MMPs (by GM6001) or EGFR (by AG1478) or suppressing the expression of MMP-2 or MMP-7 or the EGFR by small interfering RNA blunted the PI3K phosphorylation of Akt induced by PE. Further, in VSMCs, PI3K inhibitors reduced the PE-induced increase in ATP synthesis and glucose transporter-4 translocation, an effect that was also observed with MMP and the EGFR inhibitors. Further, the PE-induced increase in ATP synthesis activated MMP-7 by mechanisms involving purinergic (P2X) receptors and calcium.

These data suggest that the maintenance of adrenergic vascular tone by the MMP–EGFR pathway requires PI3K activation and ATP synthesis. Further, our data support the view that elevated levels of GPCR agonists exaggerate the MMP transactivation of EGFR response and contribute to enhanced vascular tone and development of cardiovascular disease such as hypertension.

Real-time polymerase chain reaction showed the predominant CB2 expression in freshly isolated human monocytes. PMA, a potent inducer of differentiation, upregulated CB1 and increased CB1:CB2 transcript ratio from 1:17.5 to 1:3 in 5 days of culture. Immunohistochemistry showed that CB1 protein was colocalized in CD68- and CD36-positive macrophages in human atheroma. Through selective expression of CB1 or CB2 to thioglycollate-elicited peritoneal macrophages, we proved that CB1 and CB2 mediate opposing influences on the production of reactive oxygen species (ROS). Flow cytometry showed that cannabinoid-induced ROS production by macrophages was CB1-dependent. Immunoblotting assays confirmed that macrophage CB1, not CB2, induced phosphorylation of p38-mitogen-activated protein kinase, which modulated ROS production and the subsequent synthesis of tumour necrosis factor- and monocyte chemoattractant protein-1. Pull-down assays showed that the Ras family small G protein, Rap1 was activated by CB2. Dominant-negative Rap1 profoundly enhanced CB1-dependent ROS production by macrophages, suggesting CB2 Rap1-dependently inhibits CB1-stimulated ROS production.

CB1 promotes pro-inflammatory responses of macrophages through ROS production, which is negatively regulated by CB2 through Rap1 activation. Blocking CB1 together with selective activation of CB2 may suppress pro-inflammatory responses of macrophages.

Post-MI dogs had preserved left ventricular (LV) function and susceptibility to ventricular fibrillation (VF) during exercise. LV wedge preparations from VF dogs were more susceptible to action potential (AP) alternans and the frequency-dependence of Ca²⁺ alternans was shifted towards slower rates in myocytes isolated from VF dogs relative to controls. In both groups of cells, cytosolic Ca²⁺ transients ([Ca²⁺]_c) alternated in phase with changes in diastolic Ca²⁺ in sarcoplasmic reticulum ([Ca²⁺]_SR), but the dependence of [Ca²⁺]_c amplitude on [Ca²⁺]_SR was steeper in VF cells. Abnormal ryanodine receptor (RyR) function in VF cells was indicated by increased fractional Ca²⁺ release for a given amplitude of Ca²⁺ current and elevated diastolic RyR-mediated SR Ca²⁺ leak. SR Ca²⁺ uptake activity did not differ between VF and control cells. VF myocytes had an increased rate of reactive oxygen species production and increased RyR oxidation. Treatment of VF myocytes with reducing agents normalized parameters of Ca²⁺ handling and shifted the threshold of Ca²⁺ alternans to higher frequencies.

Redox modulation of RyRs promotes generation of Ca²⁺ alternans by enhancing the steepness of the Ca²⁺ release–load relationship and thereby providing a substrate for post-MI arrhythmias.

Four weeks induction of cardiac-specific overexpression of rat β_2a-subunits shifted steady-state activation and inactivation of whole-cell currents towards more negative potentials, leading to increased Ca²⁺-current density at more negative test potentials. Activity of single Ca²⁺-channels was increased in myocytes isolated from β_2a-transgenic mice. Ca²⁺-current stimulation by 8-Br-cAMP and okadaic acid was blunted in β_2a-transgenic myocytes. In vivo investigation revealed hypotension and bradycardia upon Ca_v1.2-transgene expression but not in mice only overexpressing β_2a. Double-transgenics showed cardiac arrhythmia. Interstitial fibrosis was aggravated by the β_2a-transgene compared with Ca_v1.2-transgene expression alone. Overt cardiac hypertrophy was not observed in any model.

Cardiac overexpression of a Ca²⁺-channel β_2a-subunit alone is sufficient to induce Ca²⁺-channel properties characteristic of chronic human heart failure. β_2a-overexpression by itself did not induce cardiac hypertrophy or contractile dysfunction, but aggravated the development of arrhythmia and fibrosis in Ca_v1.2-transgenic mice.

We evaluated Ins-induced glucose uptake and signalling responses in vitro in cell overexpressing the β₂AR, the GRK2, or the catalytically dead mutant GRK2-DN. In a model of increased adrenergic activity, IRES and elevated cellular GRK2 levels, the spontaneously hypertensive rats (SHR) we performed the intravenous glucose tolerance test load. To inhibit GRK2, we synthesized a peptide based on the catalytical sequence of GRK2 conjugated with the antennapedia internalization sequence (Ant-124). Ins in human kidney embryonic (HEK-293) cells causes rapid accumulation of GRK2, tyrosine phosphorylation of Ins receptor substrate 1 (IRS1) and induces glucose uptake. In the same cell type, transgenic β₂AR overexpression causes GRK2 accumulation associated with significant deficit of IRS1 activation and glucose uptake by Ins. Similarly, transgenic GRK2 overexpression prevents Ins-induced tyrosine phosphorylation of IRS1 and glucose uptake, whereas GRK2-DN ameliorates glucose extraction. By immunoprecipitation, GRK2 binds IRS1 but not the Ins receptor in an Ins-dependent fashion, which is lost in HEK-GRK2 cells. Ant-124 improves Ins-induced glucose uptake in HEK-293 and HEK-GRK2 cells, but does not prevent GRK2/IRS1 interaction. In SHR, Ant-124 infusion for 30 days ameliorates IRES and IRS1 tyrosine phosphorylation.

Our results suggest that GRK2 mediates adrenergic IRES and that inhibition of GRK2 activity leads to increased Ins sensitivity both in cells and in animal model of IRES.

Cardiac-restricted NF-B inhibition was achieved by expression of a stabilized IB mutant (IBN) in cells with an active -myosin heavy chain (MHC) promoter employing the Cre/lox technique. Upon low-gradient trans-aortic constriction (TAC, gradient 21 ± 3 mmHg), hypertrophy was induced in both male and female control mice after 4 weeks. At this time, LV hypertrophy was blocked in transgenic (TG) male but not female mice with NF-B inhibition. Amelioration of LV hypertrophy was associated with activation of NF-B by dihydrotestosterone in isolated neonatal cardiomyocytes. LV remodelling was not attenuated by NF-B inhibition after 8 weeks TAC, demonstrated by decreased fractional shortening (FS) in both control and TG mice irrespective of gender. Similar results were obtained when TAC was performed with higher gradients (48 ± 4 mmHg). In TG mice, FS dropped to similar low levels over the same time course [FS sham, 29 ± 1% (mean ± SEM); FS control + 14 days TAC, 13 ± 3%; FS TG + 14 days TAC, 9 ± 5%]. Similarly, LV remodelling was accelerated by NF-B inhibition in an AngII-dependent genetic heart failure model (AT1-R^MHC) associated with significantly increased cardiac fibrosis in double AT1-R^MHC/TG mice.

NF-B inhibition attenuates cardiac hypertrophy in a gender-specific manner but does not alter the course of stress-induced LV remodelling, indicating NF-B to be required for adaptive cardiac hypertrophy.

We constructed HVEM-expressing adenovirus (AdHVEM) and fusion protein HVEM-Ig and evaluated their roles in immunoregulation in vitro and in vivo. Immunoregulation of dendritic cells (DCs) infected with recombinant virus or treated with HVEM-Ig was then studied. DCs transfected with AdHVEM (DC-AdHVEM) were protected against EAM, whereas HVEM-Ig had no protective effect. Further study showed that DC-AdHVEMs produced a regulatory cytokine, IL-10, which had further effects on induction of IL-10 producing CD4⁺ T cells. This subset of T cells was then responsible for the protection against EAM.

Myosin-DC-AdHVEM cell gene therapy appears to be a safe and effective way of inhibiting the development of EAM. The signal induced by HVEM seems to play different roles in different cells.

We demonstrated that propranolol reduced the incidence of arrhythmias in a rat model of myocardial infarction by coronary artery occlusion. Overexpression of miR-1 was observed in ischaemic myocardium and strikingly, administration of propranolol reversed the up-regulation of miR-1 nearly back to the control level. In agreement with its miR-1-reducing effect, propranolol relieved myocardial injuries during ischaemia, restored the membrane depolarization and cardiac conduction slowing, by rescuing the expression of inward rectifying K⁺ channel subunit Kir2.1 and gap junction channel connexin 43. Our results further revealed that the β-adrenoceptor–cAMP–Protein Kinase A (PKA) signalling pathway contributed to the expression of miR-1, and serum response factor (SRF), which is known as one of the transcriptional enhancers of miR-1, was up-regulated in ischaemic myocardium. Moreover, propranolol inhibited the β-adrenoceptor–cAMP–PKA signalling pathway and suppressed SRF expression.

We conclude that the β-adrenergic pathway can stimulate expression of arrhythmogenic miR-1, contributing to ischaemic arrhythmogenesis, and β-blockers produce their beneficial effects partially by down-regulating miR-1, which might be a novel strategy for ischaemic cardioprotection.

Male C57BL/6 mice were subjected to myocardial ischaemia (45 min) followed by reperfusion (4 h). In the treatment group, after mice were subjected to ischaemia (45 min), the TIR/BB-loop mimetic (AS-1), which inhibits the interaction of IL-1R with MyD88, was administered immediately before reperfusion. Hearts were harvested and cellular proteins were isolated for immunoprecipitation and immunoblotting. AS-1 administration significantly decreased infarct size by 32.92% compared with the untreated I/R group. Ejection fraction and fractional shortening in AS-1-treated mice were also significantly increased by 18.0 and 25.6%, respectively, compared with the untreated I/R group. AS-1 administration significantly decreased the I/R-increased interaction between IL-1R and MyD88, attenuated the I/R-increased NF-B binding activity, and reduced levels of inflammatory cytokines and adhesion molecules in the myocardium compared with the untreated I/R group. In addition, AS-1 administration significantly decreased myocardial myeloperoxidase activity by 23.6% and neutrophil infiltration in the myocardium compared with the untreated I/R group.

The results demonstrated an important role for the IL-1R-mediated MyD88-dependent signalling pathway in myocardial I/R injury. The data suggest that modulation of the IL-1R/MyD88 interaction could be a strategy for reducing myocardial ischaemic injury.

In situ hybridization coupled with immunostaining demonstrated that ICA cells exclusively expressed CGRP mRNA and co-expressed CGRP and -opioid receptor in human and rat left ventricular (LV) myocardium. Radioimmunoassay detected constitutive CGRP release from ICA cells in human and rat hearts. The -opioid agonist [D-Pen²⁵]-enkephalin (DPDPE) increased CGRP release from ICA cells in denervated rat heart. In an ischaemia/reperfusion rat model, pre-ischaemic treatment with DPDPE reduced infarct size (IS) by 51 ± 16% (P < 0.01). Co-infusion of β₂-adrenergic receptor (β₂-AR) and CGRP receptor (CGRP-R) antagonists increased IS by 62 ± 23% (P < 0.01) compared with saline and abolished DPDPE-initiated IS reduction. Pre-treatment of ICA cell–myocyte co-culture with the β₂-AR/CGRP-R antagonists increased myocyte death rate by 24 ± 4% (P < 0.01) and abolished DPDPE-initiated myocyte protection against hypoxia/reoxygenation (re-O₂). In the ICA cell-depleted myocyte culture, DPDPE did not confer myocyte protection. Supplementing ICA cell-depleted myocyte culture with β₂-AR/CGRP-R agonists reduced hypoxia/re-O₂-induced myocyte death by 24 ± 5% (P < 0.01), simulating endogenous neurohormonal effects of ICA cells. Western blot analysis showed that DPDPE markedly increased phosphorylated myocardial Akt levels. This effect was abolished in the presence of β₂-AR/CGRP-R blockade. Terminal dUTP nick-end labelling staining analysis of the LV infarct zone demonstrated that DPDPE reduced myocyte apoptosis by 58 ± 19% (P < 0.05), an effect that was eliminated in the presence of β₂-AR/CGRP-R blockade. Finally, echocardiography showed that DPDPE increased LV contractility in a manner dependent on β-AR/CGRP-R stimulation.

ICA cells constitute a -opioid-regulated adrenopeptidergic paracrine system conferring robust cardioprotection through β₂-AR/CGRP-R co-signalling, resulting in the activation of an anti-apoptotic pathway during ischaemia/reperfusion.

Infarction was produced in mini-swine by ligating the left anterior descending (LAD) coronary artery. TMDR of 3.0 mm in diameter was made by mechanical drilling in the infarcted area. The animals that had LAD ligation were divided into six groups according to the procedures followed (n = 6 in each): control; T (TMDR); C (cell implantation); TS (TMDR+stent implantation); TC (TMDR+cell implantation); TSC (TMDR+stent implantation+cell implantation). Left ventricular (LV) function, myocardial perfusion, vascular density, and histological and morphological analyses were evaluated pre-operatively and at 30 min and 6 weeks post-operatively. Six weeks after operation, the above indices were significantly better in the TSC group than in other groups (P < 0.001 compared with the control group, and P < 0.05 or 0.01 compared with the TS and TC groups), although TS and TC also showed better results than the control group (P < 0.05).

We have demonstrated in a pig model that an intramyocardial stent implanted with slow release of bFGF, heparin, and BMSC transplantation may significantly increase LV function, cardiac blood flow, and vascular density. Therefore, the present study may provide a new method for the surgical treatment of myocardial infarction.

The triiodinated monomer iohexol caused concentration-dependent reductions in store-operated Ca²⁺ entry (SOCE) in rabbit aortic valve endothelium incubated in Ca²⁺-free buffer with cyclopiazonic acid (CPA, 30 µM) to deplete endoplasmic reticulum Ca²⁺ stores. This action was mimicked by Gd³⁺ ions and 2-aminoethoxydiphenyl borate, two established inhibitors of SOCE, whereas Ca²⁺ entry was unaffected by the osmotic agent mannitol. Immunohistochemistry demonstrated that iohexol did not prevent CPA-evoked membrane clustering of Orai1, the key pore element of the store-operated Ca²⁺ channel (SOC) apparatus. In myograph studies with rabbit iliac artery rings, iohexol, and the hexaiodinated dimer iodixanol (both at 90 mg I/mL) attenuated NO-mediated and EDHF-type arterial relaxations evoked by CPA, but did not affect EDHF-type relaxations to acetylcholine, whose principal mode of action is to mobilize Ca²⁺ via inositol 1,4,5-trisphosphate (InsP₃)-induced Ca²⁺ release. Iohexol also exerted inhibitory effects on NO-mediated relaxation and smooth muscle contraction that were not evident with iodixanol.

The data support the hypothesis that IRCM induce generalized endothelial dysfunction by inhibiting Ca²⁺ influx via SOCs rather than their assembly. The presence of organically bound iodine, rather than osmolar effects, may underpin this previously unrecognized phenomenon. In contrast, direct effects of IRCM on smooth muscle function may correlate with osmolarity rather than iodine concentration.

Human aortic endothelial cells (HAECs) were incubated with C-reactive protein at different concentrations (0, 12.5, 25, and 50 µg/mL) with boiled C-reactive protein as a control. For in vivo experiments, human C-reactive protein was injected into rats and human serum albumin was used as a control. Endothelial glycocalyx thickness was examined by transmission electron microscopy. Hyaluronan (HA) was examined in the supernatant of HAECs and in plasma and surface expression of heparan sulfate (HS) was quantified. C-reactive protein dose-dependently increased HA release in vitro and in vivo (P < 0.01). Also, glycocalyx thickness was significantly decreased (P < 0.05). Western blotting for HS showed significant reduction in expression of HS, one of the main glycosaminoglycans in the glycocalyx, with C-reactive protein treatment. There was a significant positive correlation between HA release and monocyte–endothelial cell adhesion, plasminogen activator inhibitor-1, and intercellular adhesion molecule-1 release and a negative correlation with endothelial nitric oxide synthase activity.

Collectively, these data suggest that C-reactive protein impairs glycocalyx function, resulting in endothelial dysfunction.

Treatment of rats with bindarit (200 mg/kg/day) significantly reduced balloon injury-induced neointima formation by 39% at day 14 without affecting re-endothelialization and reduced the number of medial and neointimal proliferating cells at day 7 by 54 and 30%, respectively. These effects were associated with a significant reduction of MCP-1 levels both in sera and in injured carotid arteries of rats treated with bindarit. In addition, in vitro data showed that bindarit (10–300 µM) reduced rat vascular smooth muscle cell (VSMC) proliferation, migration, and invasion, processes contributing to the injury-induced neointima formation in vivo. Similar results were observed in hypercholesterolaemic apoE^–/– mice in which bindarit administration resulted in a 42% reduction of the number of proliferating cells at day 7 after carotid injury and in a 47% inhibition of neointima formation at day 28. Analysis of the cellular composition in neointimal lesions of apoE^–/– mice treated with bindarit showed that the relative content of macrophages and the number of VSMCs were reduced by 66 and 30%, respectively, compared with the control group.

This study demonstrates that bindarit is effective in reducing neointima formation in both non-hyperlipidaemic and hyperlipidaemic animal models of vascular injury by a direct effect on VSMC proliferation and migration and by reducing neointimal macrophage content. All of these data were associated with the inhibition of MCP-1 production.

During living donor transplantation procedures, arteries from donors (n = 8) and recipients (non-diabetic, n = 8; diabetic, n = 8; matched in age, gender, and dialysis treatments) were harvested. Diabetic arteries had increased MMP-2 and -9 activities by 42 and 116% compared with non-diabetic ones. Diabetic arteries were the stiffest, and the stiffness measurement was highly correlated with the summation of MMP-2 + MMP-9 activities (r = 0.738, P = 0.0002). Pulse wave velocity measurements correlated with MMP activity (r = 0.683, P = 0.005). Elastic fibre degradation and calcification were worst in diabetic vessels. The phosphate level, which was 25% higher in diabetic patients, correlated with MMP activity (r = 0.513, P = 0.04) and in vitro stiffness (r = 0.545, P = 0.03), respectively. Angiostatin expression was doubled, whereas vascular endothelial growth factor was 50% reduced in diabetic compared with non-diabetic vessels. Microvascular density in diabetic vessels was 48% of that in non-diabetic ones, and it was strongly associated with MMP activity (r = –0.792, P < 0.0001) and vasorelaxation (r = 0.685, P = 0.0009).

Using a matched case–control design, we report up-regulation of MMP-2 and -9 in diabetic CKD arteries and correlate those with stiffening, impaired angiogenesis, and endothelial dysfunction. These findings may help to explain the high susceptibility of CVD in diabetic and non-diabetic CKD patients.

Isolated ischaemic/reperfused hearts from TNF knockout, TNF receptor-1 knockout, TNF receptor-2 knockout, cardiomyocyte-specific STAT-3-deficient mice or their respective wild-type, (TNF-WT) or (STAT-3-WT), were postconditioned by ischaemic episodes (IPostC) or with exogenous TNF (0.5 µg/L) (TNF-PostC) at the onset of reperfusion. IPostC reduced infarct size (IS) in TNF-WT and TNFR1^–/– hearts (by 33 and 27%, respectively, P < 0.05), whereas hearts from TNF^–/– or TNFR2^–/– failed to be postconditioned. TNF-PostC reduced IS by 37% (P < 0.05) in STAT-3-WT hearts but failed to protect cardiac-specific STAT-3^–/– hearts. Administration of wortmannin, an inhibitor of PI-3 kinase/Akt, or PD98059, an inhibitor of extracellular regulated kinase 1/2 (Erk1/2), during the postconditioning stimulus did not abolish the infarct-sparing effect of TNF-PostC. AG490, an inhibitor of STAT-3, abrogated the protective effect of TNF. Western blot analysis did not demonstrate the involvement of Akt or Erk1/2 in TNF-PostC, whereas STAT-3 phosphorylation was increased in both IPostC and TNF-PostC.

The protective effect of the SAFE pathway is shown in IPostC, with the activation of TNF, its receptor type 2, and STAT-3. This signalling cascade is activated independently of the well-known Reperfusion Injury Salvage Kinases (RISK) pathway, which involves the kinases Akt and Erk1/2.

We have investigated the mechanism(s) involved in the antiproliferative effect of ANP on cardiac fibroblasts in a cell culture model. We found that cardiac myocytes inhibited DNA synthesis in co-cultured cardiac fibroblasts as did treatment with the ET-1 antagonist BQ610. The effect of co-culture was reversed by antibody directed against ANP or the ANP receptor antagonist HS-142-1. ANP inhibited the expression of the ET-1 gene and ET-1 gene promoter activity in cultured fibroblasts. The site of the inhibition was localized to a GATA-binding site positioned between –132 and –135 upstream from the transcription start site. GATA4 expression was demonstrated in cardiac fibroblasts, GATA4 bound the ET-1 promoter both in vitro and in vivo, and siRNA-mediated knockdown of GATA4 inhibited ET-1 expression. ET-1 treatment resulted in increased levels of phospho-serine¹⁰⁵ GATA4 in cardiac fibroblasts and this induction was partially suppressed by co-treatment with ANP.

Collectively, these findings suggest that locally produced ET-1 serves as an autocrine stimulator of fibroblast proliferation, that ANP produced in neighbouring myocytes serves as a paracrine inhibitor of this proliferation, and that the latter effect operates through a reduction in GATA4 phosphorylation and coincident reduction in GATA4-dependent transcriptional activity.

In studies with rabbit iliac artery (RIA) rings, NO-independent, endothelium-derived hyperpolarizing factor (EDHF)-type relaxations evoked by the sarcoplasmic endoplasmic reticulum Ca²⁺-ATPase inhibitor cyclopiazonic acid and the G protein-coupled agonist acetylcholine (ACh) were enhanced by AA (1 mM) and BH₄ (200 µM), which generated buffer concentrations of H₂O₂ in the range of 40–80 µM. Exogenous H₂O₂ potentiated cyclopiazonic acid (CPA)- and ACh-evoked relaxations with a threshold of 10–30 µM, and potentiation by AA and BH₄ was abolished by catalase, which destroyed H₂O₂ generated by oxidation of these agents in the organ chamber. Adventitial application of H₂O₂ also enhanced EDHF-type dilator responses evoked by CPA and ACh in RIA segments perfused intraluminally with H₂O₂-free buffer, albeit with reduced efficacy. In RIA rings, both control relaxations and their potentiation by H₂O₂ were overcome by blockade of gap junctions by connexin-mimetic peptides (YDKSFPISHVR and SRPTEK) targeted to the first and second extracellular loops of the dominant vascular connexins expressed in the RIA. Superoxide dismutase attenuated the potentiation of EDHF-type relaxations by BH₄, but not AA, consistent with findings demonstrating a differential role for superoxide anions in the generation of H₂O₂ by the two agents.

Pro-oxidant effects of AA and BH₄ can enhance the EDHF phenomenon by generating H₂O₂, which has previously been shown to amplify electrotonic hyperpolarization-mediated relaxation by facilitating Ca²⁺ release from endothelial stores.

Preliminary studies showed that 4 months of HF resulted in irreversible systolic dysfunction (n = 9) and a substrate for sustained inducible AF (>3 months, n = 3). We used a chronic (4-month) canine model of tachypacing-induced HF (n = 10) to assess atrial electrophysiological remodelling, relative to controls (n = 5). Left ventricular fractional shortening was reduced from 37.2 ± 0.83 to 13.44 ± 2.63% (P < 0.05). Left atrial (LA) contractility (fractional area change) was reduced from 34.9 ± 7.9 to 27.9 ± 4.23% (P < 0.05). Action potential durations (APDs) at 50 and 90% repolarization were shortened by ~60 and 40%, respectively, during HF (P < 0.05). HF-induced atrial remodelling included increased fibrosis, increased I_to, and decreased I_K1, I_Kur, and I_Ks (P < 0.05). HF induced increases in LA Kv channel interacting protein 2 (P < 0.05), no change in Kv4.3, Kv1.5, or Kir2.3, and reduced Kir2.1 (P < 0.05). When I_Ca-L was elicited by action potential (AP) clamp, HF APs reduced the integral of I_Ca in control myocytes, with a larger reduction in HF myocytes (P < 0.05). I_CaL measured with standard voltage clamp was unchanged by HF. Incubation of myocytes with N-acetylcysteine (a glutathione precursor) attenuated HF-induced electrophysiological alterations. LA angiotensin-1 receptor expression was increased in HF.

Chronic HF causes alterations in ion channel expression and ion currents, resulting in attenuation of the APD and atrial contractility and a substrate for persistent AF.

Action potentials were recorded from multicellular canine ventricular preparations and isolated cardiomyocytes, at cycle lengths (CLs) varying from 0.3 to 5 s, using conventional sharp microelectrodes. APD was either modified by applying inward and outward current pulses, or by superfusion of agents known to lengthen and shorten APD. Net membrane current (I_m) was calculated from action potential waveforms. The hypothesis that RRD may be implicit in the relationship between I_m and APD was tested by numerical modelling. Both drug-induced lengthening (by veratrine, BAY-K 8644, dofetilide, and BaCl₂) and shortening (by lidocaine and nicorandil) of action potentials displayed RRD, i.e. changes in APD were greater at longer than at shorter CL. A similar dependency of effect on CL was found when repolarization was modified by injection of inward or outward current pulses. I_m measured at various points during repolarization was inversely proportional to APD and to CL. Model simulations showed that RRD is expected as a consequence of the non-linearity of the relationship between I_m and APD.

RRD of APD modulation is shared, although with differences in magnitude, by interventions of very different nature. RRD can be interpreted as a consequence of the relationship between I_m and APD and, as such, is expected in all species having positive APD–CL relationship. This implies that the development of agents prolonging APD with direct rate dependency, or even completely devoid of RRD, may be difficult to achieve.

In 43 mongrel dogs, rapid firing-mediated atrial fibrillation (AF) was induced by local HFS (200 Hz, impulse duration 0.1 ms, train duration 40 ms) to the PVs and atria during myocardial refractoriness. The main GP in the atrial fat pads or the ganglia along the ligament of Marshall (LOM) were then ablated. Ablation of the anterior right GP and inferior right GP significantly increased the AF threshold by HFS at the right atrium and PVs. The AF threshold at left atrium and PVs was significantly increased by ablation of the superior left GP and inferior left GP, and was further increased by ablation of the LOM. Ablation of left- or right-sided GP on the atria had a significant effect on contralateral PVs and atrium. Administration of esmolol (1 mg/kg) or atropine (1 mg) significantly increased AF threshold at all sites.

HFS applied to local atrial and PV sites initiated rapid firing via activation of the interactive autonomic network in the heart. GP in either left side or right side contributes to the rapid firings and AF originating from ipsolateral and contralateral PVs and atrium. Autonomic denervation suppresses or eliminates those rapid firings.

Ca²⁺ sparks and contractility were measured in cardiomyocytes and papillary muscle segments from FKBP12.6 null mice, and western blot analysis was carried out on sarcoplasmic reticulum microsomes prepared from mouse heart. Exposure to ISO resulted in a three- and two-fold increase in Ca²⁺ spark frequency in wild-type (WT) and FKBP12.6 knockout (KO) myocytes, respectively, and Ca²⁺ spark kinetics were also significantly altered in both types of cells. The effects of ISO on Ca²⁺ spark properties in KO cells were inhibited by pre-treatment with thapsigargin or phospholamban inhibitory antibody, 2D12. Moreover, twitch force magnitude and the rate of force development were not significantly different in papillary muscles from WT and KO mice. Unlike β-adrenergic stimulation, cADPR stimulation increased Ca²⁺ spark frequency (2.8-fold) and altered spark kinetics only in WT but not in KO mice. The effect of cADPR on spark properties was not entirely blocked by pre-treatment with thapsigargin or 2D12. In voltage-clamped cells, cADPR increased the peak Ca²⁺ of the spark without altering the decay time. We also noticed that basal Ca²⁺ spark properties in KO mice were markedly altered compared with those in WT mice.

Our data demonstrate that dissociation of FKBP12.6 from the RYR2 complex does not play a significant role in β-adrenergic-stimulated Ca²⁺ release in heart cells, whereas this mechanism does underlie the action of cADPR.

Here we demonstrate that forced expression of Nkx2.5 similar to MesP1 is sufficient to enhance cardiogenesis in murine embryonic stem cells (mES). In comparison to control transfected mES cells, a five-fold increased appearance of beating foci was observed as well as upregulated mRNA and protein expression levels. In contrast to MesP1, no increase of the endothelial lineage within the cardiovasculogenic mesoderm was observed. Likewise, Flk-1, the earliest known cardiovascular surface marker, was not induced via Nkx2.5 as opposed to MesP1. Detailed patch clamping analyses showed electrophysiological characteristics corresponding to all subtypes of cardiac ES cell differentiation in Nkx2.5 as well as MesP1 programmed embryoid bodies, but fractions of cardiomyocytes had distinct characteristics: MesP1 forced the appearance of early/intermediate type cardiomyocytes in comparison to control transfected ES cells whereas Nkx2.5 led to preferentially differentiated ventricular cells.

Our findings show proof of principle for cardiovascular subtype-specific programming of pluripotent stem cells and confirm the molecular hierarchy for cardiovascular specification initiated via MesP1 with differentiation factors such as Nkx2.5 further downstream.

MI was induced in five different mouse strains (BalbC, C57Bl6, FVB, 129S6, and Swiss). At 3, 14, and 28 days after MI, cardiac dimensions were monitored by echocardiography and histology, whereas cardiac function was determined by direct intraventricular pressure measurements (dP/dt). Furthermore, matrix metalloproteinases were measured by zymography, and mRNA expression by quantitative PCR. Infarct rupture, which typically occurred at 3–6 days post-MI, was most frequent in 129S6 mice (62%), followed by C57Bl6 (36%), FVB (29%), Swiss (23%), and BalbC (5%). The high incidence of infarct rupture in 129S6 mice was associated with high systolic blood pressure and increased influx of inflammatory cells. Cardiac dilatation was most marked in Swiss mice and least prominent in 129S6 mice. The degree of dilatation was associated with a reduced ejection fraction and decreased dP/dt values at 14 and 28 days post-MI. At day 14 and 28 post-MI, secondary thinning of the infarct area was marked in BalbC, FVB, and Swiss, but absent in C57Bl6 and 129S6 mice. In the latter two groups, this was paralleled by the highest number of myofibroblasts at day 14 post-MI.

The outcome of infarct healing in mice strongly depends on genetic background. On the basis of our results, we suggest that for studies on infarct rupture, the 129S6 mouse is the background of choice, whereas BalbC and Swiss mice are the preferred models to study infarct thinning post-MI.

In anaesthetized rats, the cardiovascular response to local or systemic injection of moxonidine was observed after treatment with the selective iNOS inhibitor S-methylisothiourea (SMT) in the brain. Using immunohistochemical staining and western blot techniques, the protein expression of iNOS in the RVLM was measured in the moxonidine-infused rats. Intracerebroventricular (ICV) injection of SMT (1–100 nmol) dose-dependently attenuated the moxonidine (20 nmol, ICV)-induced decrease in BP and heart rate. Prior injection of SMT (20 and 200 pmol) into the RVLM also dose-dependently prevented the decrease in BP and renal sympathetic nerve activity evoked by RVLM microinjection of moxonidine (5 nmol) or intravenous injection of moxonidine (50 µg/kg). We further found that expression of iNOS protein following chronic ICV infusion of moxonidine (20 nmol, 2 weeks) is selectively upregulated in the RVLM but not in the nucleus tractus solitarius.

The present data suggest that an NO mechanism generated by iNOS in the RVLM plays an important role in mediating the sympathetic inhibition of the centrally acting drug moxonidine.

IC53 cDNA was cloned from a human aorta cDNA library. Using the endothelium-specific VE-cadherin promoter, we constructed tg mice in which IC53 was specifically overexpressed in vascular endothelia and found that the tg mice exhibit elevated systolic blood pressure (SBP) in contrast to the wild-type (wt) controls. Further studies revealed impaired endothelium-dependent vasodilation, reduced nitric oxide (NO) production and decreased endothelial NO synthase (eNOS) expression, and activity in the tg mice. Inhibition of IC53 in human umbilical vein ECs induces upregulation of eNOS activity.

Our results indicate that IC53 participates in the regulation of vascular homeostasis. Endothelium-specific overexpression of IC53 is associated with elevated SBP, which may be in part attributed to the downregulation of eNOS signalling.

Aortic segments from ovariectomized and control female Sprague-Dawley rats were used. Cyclooxygenase (COX-1 and COX-2) expression was studied. ACh-induced relaxation was analysed in the absence and presence of the COX-2 inhibitor NS-398, the TXA₂ synthesis inhibitor furegrelate, the PGI₂ synthesis inhibitor tranylcypromine (TCP), or the thromboxane-prostanoid receptor antagonist SQ-29548. TXA₂, PGI₂, PGF₂, and PGE₂ release was measured, and the vasomotor effect of exogenous TXA₂, PGI_2, PGF₂, and PGE₂ was assessed. Basal and ACh-induced NO release in the absence and presence of NS-398, furegrelate, TCP, or TCP plus furegrelate was studied. Ovariectomy did not alter or increased COX-1 or COX-2 expression, respectively. NS-398 decreased, and furegrelate did not change, the ACh-induced relaxation in arteries from both groups. SQ29,548 decreased the ACh-induced relaxation only in aortas from ovariectomized rats. TCP decreased the ACh-induced relaxation in both groups, and furegrelate or SQ29,548 totally restored that response only in aortas from control rats. Ovariectomy increased the ACh-induced TXA₂, PGI₂, and PGE₂ release and the contractile responses induced by exogenous TXA₂, PGF₂, or PGE₂, while it decreased the PGI₂-induced vasodilator response. In aortas from control rats, NS-398 did not alter the ACh-induced NO release, and furegrelate, TCP, or TCP plus furegrelate increased that release. In arteries from ovariectomized rats, NS-398, furegrelate, TCP, or TCP plus furegrelate decreased the ACh-induced NO release.

Despite the prevalence of vasoconstrictor prostanoids derived from COX-2 in aortas from ovariectomized rats, the ACh-induced relaxation is maintained, probably as consequence of the positive regulation that prostanoids exert on eNOS activity.

Standard aggregometry, flow cytometry, electron microscopy, and thrombin generation assays were performed. TF-MVs induced platelet aggregation in heparinized platelet-rich plasma (PRP) samples; this aggregation was further accelerated by serotonin. In washed platelets, serotonin enhanced platelet aggregation to TF-MVs with a maximum peak of 55.9 ± 1.8 vs. 48.7 ± 2.1% (P < 0.05). Inhibitory strategies with a selective serotonin re-uptake inhibitor and with lepirudin decreased these aggregations. Ultrastructural analysis revealed that serotonin induced platelet pseudopodia formation, thus facilitating the engulfment of TF-MVs. In general, serotonin significantly enhanced (P < 0.05) thrombin generation and the expression of activation markers and procoagulant activity in platelets measured for TF-MVs alone.

Serotonin enhances the interaction of platelets with TF-MVs, increases platelet activation, and potentiates their overall procoagulant activity. The present results could have significant implications in thrombus formation and in the thrombogenic profile of pathological situations with increased cardiovascular risk.

Radiation or busulfan-induced BM ablation in eNOS^–/– mice on day 6, day 14, or day 28 was followed by a BM transplant consisting of enhanced green fluorescent protein positive (EGFP⁺) cells from C57BL/6J mice. Peripheral blood cell chimerism was always greater than 85% at 4 months after BM transplant. Molecular assays of heart, kidney, and liver revealed low-level chimerism in all treatment groups, consistent with residual circulating EGFP⁺ blood cells. When aorta, coronary, renal, hepatic, and splenic arteries in BM-transplanted eNOS^–/– mice were examined by confocal microscopy, there were no EGFP- or eNOS-positive endothelial cells detected in these vessels in any of the treatment groups. Likewise, telemetry did not detect any reduction in blood pressure. Thus, no differences were observed in our measurements using several different treatment protocols.

We found no evidence for BM-derived EPC renewal of endothelium in this eNOS-deficient mouse model of a chronic vascular disease or in wild-type mice during postnatal growth. Hence, renewal of chronic dysfunctional endothelium and endothelial homeostasis may be dependent on resident vascular progenitor cells.

Using the rat carotid angioplasty model, we assess Smad3 expression following arterial injury. We then test the effect of arterial Smad3 overexpression on the response to injury, and use a conditioned media experimental design to confirm an Smad3-dependent soluble factor that mediates this response. We use small interfering RNA (siRNA) to identify this factor as connective tissue growth factor (CTGF). Finally, we attempt to replicate the effect of medial Smad3 overexpression through adventitial application of recombinant CTGF. Injury induced medial expression of Smad3; overexpression of Smad3 caused neointimal thickening and luminal expansion, suggesting adaptive remodelling. Smad3 overexpression, though exclusively medial, caused adventitial changes: myofibroblast transformation, proliferation, and collagen production, all of which are associated with adaptive remodelling. Supporting the hypothesis that Smad3 initiated remodelling and these adventitial changes via a secreted product of medial smooth muscle cells (SMCs), we found that media conditioned by Smad3-expressing recombinant adenoviral vector (AdSmad3)-infected SMCs stimulated adventitial fibroblast transformation, proliferation, and collagen production in vitro. This effect was attenuated by pre-treatment of SMCs with siRNA specific for CTGF, abundantly produced by AdSmad3-infected SMCs, and significantly up-regulated in Smad3-overexpressing arteries. Moreover, periadventitial administration of CTGF replicated the effect of medial Smad3 overexpression on adaptive remodelling and neointimal hyperplasia.

Medial gene transfer of Smad3 promotes adaptive remodelling by indirectly influencing the behaviour of adventitial fibroblasts. This arterial cell–cell communication is likely to be mediated by Smad3-dependent production of CTGF.

Cardiomyocytes were incubated with DEX, MET, or TNF- for varying durations (0–12 h). TNF--induced cell damage was evaluated by measuring caspase-3 activity and Hoechst staining. Protein and gene estimation techniques were employed to determine the mechanisms mediating the effects of AMPK activators on TNF--induced cardiomyocyte apoptosis. Incubation of myocytes with TNF- for 8 h has increased caspase-3 activation and apoptotic cell death, an effect that was abrogated by DEX and MET. The beneficial effect of DEX and MET was associated with stimulation of AMPK, which led to a rapid and sustained increase in Bad phosphorylation. This event reduced the interaction between Bad and Bcl-xL, limiting cytochrome c release and caspase-3 activation. Addition of Compound C to inhibit AMPK reduced Bad phosphorylation and prevented the beneficial effects of AMPK against TNF--induced cytotoxicity.

Our data demonstrate that although DEX and MET are used as anti-inflammatory agents or insulin sensitizers, respectively, their common property to phosphorylate AMPK promotes cardiomyocyte cell survival through its regulation of Bad and the mitochondrial apoptotic mechanism.

Primary cultures of neonatal rat cardiomyocytes were exposed to phenylephrine (PE) at a final concentration of 100 µM in cultures for 48 h to induce cell hypertrophy. The hypertrophic cardiomyocytes were exposed to copper sulfate at a final concentration of 5 µM in cultures for 24 h with a concomitant presence of PE. Flow cytometry, gene silencing, and ELISA procedures were used to analyse the changes in VEGFRs and their relationship with regression of cardiomyocyte hypertrophy. Cu did not change the concentration of VEGF in culture media, but increased the ratio of VEGFR-1 to VEGFR-2 two-fold. Gene silencing of VEGFR-2, in the absence of Cu addition, reversed PE-induced cardiomyocyte hypertrophy, which was suppressed by an anti-VEGF antibody. Gene silencing of VEGFR-1 blocked Cu-induced regression of cell hypertrophy and decreased the activity of cGMP-dependent protein kinase-1 (PKG-1). A PKG-1 antagonist, Rp-8-pCPT-cGMPS, blocked both Cu- and VEGFR-2 gene silencing-induced regression of cardiomyocyte hypertrophy.

Enhanced VEGFR-1 signalling is involved in Cu regression of cardiomyocyte hypertrophy, and the PKG-1 pathway is likely associated with VEGFR-1.

Three different types of β-blockers, carvedilol, metoprolol, and atenolol, were orally administered to a knock-in mouse model of inherited DCM with a deletion mutation K210 in the cardiac troponin T gene (TNNT2). Therapeutic effects were examined on the basis of survival and myocardial remodelling. The lipophilic β₁-selective β-blocker metoprolol was found to prevent cardiac dysfunction and remodelling and extend the survival of knock-in mice. Conversely, both the non-selective β-blocker carvedilol and the hydrophilic β₁-selective β-blocker atenolol had no beneficial effects on survival and myocardial remodelling in this mouse model of inherited DCM.

The highly lipophilic β₁-selective β-blocker metoprolol, known to prevent ventricular fibrillation via central nervous system-mediated vagal activation, may be especially beneficial to DCM patients showing a family history of frequent sudden cardiac death, such as those with a deletion mutation K210 in the TNNT2 gene.

A Langendorff-perfused rabbit heart model was subjected to low-flow ischaemia and reperfusion. Cardiac metabolism was studied by measuring cardiac high-energy phosphate contents via HPLC, mitochondrial respiration was analysed polarographically, and cardiac redox potentials by HPLC. Cardiac function was determined in terms of the recovery of developed pressure during reperfusion and release of creatine kinase (CK) (spectrophotometrically) and noradrenaline (HPLC) after reperfusion. Four concentrations of ivabradine (0.3, 1, 3, and 6 µM) were tested on aerobically perfused hearts to select the most effective without causing changes in mechanical parameters. This proved to be 3 µM, which was therefore the concentration selected for the ischaemia-reperfusion experiments. Ivabradine concentration-dependently reduced HR with a maximal effect of 41 ± 4% at 3 µM (P < 0.001 vs. vehicle), without a negative inotropic effect. This concentration protected the heart against ischaemia-reperfusion damage by reducing the rise in diastolic pressure (from 66 ± 3 with vehicle to 39 ± 4 mmHg, P < 0.01) and improving developed pressure after 30 min reperfusion (39 ± 3 vs. 18 ± 3 mmHg with vehicle, P < 0.01). Ivabradine reduced both CK and noradrenaline release by 47% (both P < 0.05 vs. vehicle) and improved mitochondrial respiratory control index (from 6.9 ± 0.3 to 11.9 ± 1.3, P < 0.001). It preserved cardiac energy metabolism (ATP, from 3.7 ± 0.3 to 11.0 ± 0.6 µM/g dry weight, P < 0.001) and redox state (NADPH/NADP⁺, from 2.5 ± 0.5 to 4.2 ± 0.5, P < 0.001). There was a significant correlation between HR reduction in the ivabradine-treated hearts and cardiac creatine phosphate (r = 0.574, P = 0.02) and ATP levels (ATP, r = 0.674, P = 0.0042) at the end of ischaemia. These benefits were no longer detectable during pacing.

HR reduction by ivabradine confers a marked anti-ischaemic benefit. It significantly reduces cardiac energy consumption, preserves redox potentials during ischaemia, and enhances recovery at reperfusion.

Real-time quantitative reverse transcriptase–polymerase chain reaction and western blotting were used to assess cardiac expression of PPAR and and two of their downstream target genes—medium-chain acyl-CoA dehydrogenase (MCAD) and phosphoenolpyruvate carboxykinase (PEPCK)—in both RV and left ventricle (LV) from five controls and five ARVC patients. In vitro motility assays were used to analyse functional properties of myosin. In the RV, sliding velocity was nearly two-fold lower in ARVC than in controls, whereas a 10% reduction in velocity values was noted between ARVC and non-failing myocardium in the LV. In controls, PPAR and MCAD mRNA and protein levels were higher in the RV compared with the LV. In ARVC, the expression of PPAR and MCAD mRNA and/or proteins was decreased in both RV and LV. RV from ARVC was also characterized by a dramatic activation of the PPAR pathway, as attested by the increase in PPAR mRNA and protein (500 and 270%, respectively, each P < 0.001) and by the induction of PEPCK gene. In contrast, the LV of ARVC heart exhibited no changes in the expression of the PPAR regulatory pathway compared with control.

ARVC is associated with major disturbances in the PPAR and PPAR signalling pathway in the RV that may contribute to intracellular lipid overload and severe myosin dysfunction.

Expression of optic atrophy 1 (OPA1), a mitochondrial fusion protein, was decreased in both human and rat HF, as observed by western blotting. OPA1 is important for maintaining normal cristae structure and function, for preserving the inner membrane structure and for protecting cells from apoptosis. Confocal and electron microscopy studies demonstrated that the mitochondria in the failing hearts were small and fragmented, consistent with decreased fusion. OPA1 mRNA levels did not differ between failing and normal hearts, suggesting post-transcriptional control. Simulated ischaemia in the cardiac myogenic cell line H9c2 cells reduced OPA protein levels. Reduction of OPA1 expression with shRNA resulted in increased apoptosis and fragmentation of the mitochondria. Overexpression of OPA1 increased mitochondrial tubularity, but did not protect against simulated ischaemia-induced apoptosis. Cytochrome c release from the mitochondria was increased both with reduction in OPA1 and with overexpression of OPA1.

This is the first report, to our knowledge, of changes in mitochondrial fusion/fission proteins in cardiovascular disease. These changes have implications for mitochondrial function and apoptosis, contributing to the cell loss which is part of the downward progression of the failing heart.

In cultured adult rat ventricular cardiomyocytes, high glucose (33 mM) increased calpain activity and induced apoptosis, concomitant with the impairment of Na⁺/K⁺ ATPase activity. These effects of high glucose on cardiomyocytes were abolished by various pharmacological calpain inhibitors, knockdown of calpain-1 but not calpain-2 using siRNA, or over-expression of calpastatin, a specific endogenous calpain inhibitor. The effect of calpain inhibition on cardiomyocyte apoptosis was abrogated by ouabain, a selective inhibitor of Na⁺/K⁺ ATPase. Furthermore, blocking gp91^phox-NADPH oxidase activation, L-type calcium channels, or ryanodine receptors prevented calpain activation and apoptosis in high glucose-stimulated cardiomyocytes. In a mouse model of streptozotocin-induced diabetes, administration of different calpain inhibitors blocked calpain activation, increased the Na⁺/K⁺ ATPase activity, and decreased apoptosis in the heart.

Calpain-1 activation induces apoptosis through down-regulation of the Na⁺/K⁺ ATPase activity in high glucose-stimulated cardiomyocytes and in vivo hyperglycaemic hearts. High glucose-induced calpain-1 activation is mediated through the NADPH oxidase-dependent pathway and associated with activation of L-type calcium channels and ryanodine receptors. Our data suggest that calpain activation may be important in the development of diabetic cardiomyopathy and thus may represent a potential therapeutic target for diabetic heart diseases.

SGLT1 was largely localized to the cardiac myocyte sarcolemma. Changes in SGLT1 expression were observed in disease states in both humans and mouse models. SGLT1 expression was upregulated two- to three-fold in type 2 diabetes mellitus and myocardial ischaemia (P < 0.05). In humans with severe heart failure, functional improvement following implantation of left ventricular assist devices led to a two-fold increase in SGLT1 mRNA (P < 0.05). Acute administration of leptin to wildtype mice increased cardiac SGLT1 expression approximately seven-fold (P < 0.05). Insulin- and leptin-stimulated cardiac glucose uptake was significantly (P < 0.05) inhibited by phlorizin, a specific SGLT1 inhibitor.

Our data suggest that cardiac SGLT1 expression and/or function are regulated by insulin and leptin, and are perturbed in disease. This is the first study to examine the regulation of cardiac SGLT1 expression by insulin and leptin and to determine changes in SGLT1 expression in cardiac disease.

We investigated LXR expression patterns in pressure overload-induced hypertrophic hearts and the hypertrophic growth of the LXR-deficient hearts from mice (C57/B6) in response to pressure overload. The underlying mechanisms were also explored using cultured myocytes. We found that cardiac expression of LXR was upregulated in pressure overload-induced left ventricular hypertrophy in mice. Transverse aorta coarctation-induced left ventricular hypertrophy was exacerbated in LXR-null mice relative to control mice. A synthetic LXR ligand, T1317, suppressed cardiomyocyte hypertrophy in response to angiotensin II and lipopolysaccharide treatments. In addition, LXR activation suppressed NF-B signalling and the expression of associated inflammatory factors. Overexpression of constitutively active LXR and β in cultured myocytes suppressed NF-B activity.

LXRs are negative regulators of cardiac growth and inflammation via suppressing NF-B signalling in cardiomyocytes. This should provide new insights into novel therapeutic targets for treating cardiac hypertrophy and heart failure.

Ox-LDL induced rapid RhoA and Rac1 activation as well as MT1-MMP activity in cultured human aortic ECs. Inhibition of LOX-1 prevented ox-LDL-dependent RhoA and Rac1 activation. Knockdown of MT1-MMP by small interfering RNA prevented ox-LDL-induced RhoA and Rac1 activation, indicating that MT1-MMP is upstream of RhoA and Rac1. Fluorescent immunostaining revealed the colocalization of LOX-1 and MT1-MMP, and the formation of a complex of LOX-1 with MT1-MMP was detected by immunoprecipitation. Blockade of LOX-1 or MT1-MMP prevented RhoA-dependent endothelial NO synthase protein downregulation and cell invasion, Rac1-mediated NADPH oxidase activity, and reactive oxygen species generation.

The present study provides evidence that the LOX-1-MT1-MMP axis plays a crucial role in RhoA and Rac1 activation signalling pathways in ox-LDL stimulation, suggesting that this axis may be a promising target for treating endothelial dysfunction.

Hydrogen peroxide and glucose oxidase were chosen to model oxidative stress in vitro. We demonstrate that oxidative damage activated tumour necrosis factor--converting enzyme (TACE) and induced shedding of its targets, glycoprotein (GP) Ib and GPV, in murine and human platelets. Also, 12-HpETE, a peroxide synthesized in the platelet lipoxygenase pathway, induced TACE-mediated receptor cleavage. The TACE activation was independent of platelet activation, as -granule secretion, activation of IIbβ3, or phosphatidylserine expression was not observed. TACE activation induced by hydrogen peroxide was dependent on p38 mitogen-activated protein kinase signalling, whereas protein kinase C, phosphoinositide 3-kinase, and caspases were not involved. Inhibition of p38 cytoplasmic targets, phospholipase A₂ and heat shock protein 27, did not prevent shedding, whereas blocking 12-lipoxygenase or Src kinase slightly inhibited TACE activation. The loss of the GPIb receptor induced by oxidative stress rendered platelets unable to incorporate into a growing thrombus in vivo.

Oxidative stress can render platelets functionally less active by shedding key adhesion receptors via the activation of p38. This suggests that oxidative injury of platelets may attenuate their function.

The increase in macrophage PON2 mRNA levels in response to uPA was shown to depend on PON2 gene promoter activation and mRNA transcription. LDL abolished these effects, suggesting a possible role for a transcription factor involved in cellular cholesterogenesis. Indeed, uPA upregulated PON2 expression in a sterol regulatory binding protein-2 (SREBP-2)-dependent manner, since blocking SREBP-2 maturation by 4-(2-aminoethyl)-benzenesulfonyl fluoride abolished uPA-stimulation of PON2, whereas inhibition of SREBP-2 catabolism by N-acetyl-leucyl-norleucinal had an opposite effect. The upstream signalling mechanisms include uPA activation of extracellular signal-regulated kinases (ERK1/2), which was dependent on NADPH oxidase and phosphatidylinositol 3-kinase activation, and these latter effects were mediated by the tyrosine kinase activity of the platelet-derived growth factor receptor-β.

These findings provide a framework linking interactions among cellular signalling pathways associated with reactive oxygen species production, macrophage cholesterol biosynthesis, and cellular PON2 expression in vascular pathophysiology.

apoE–/– mice were fed normal mouse chow supplemented with or without a high level of homocysteine (Hcy) (1.8 g/L) in drinking water for 2, 4, and 6 weeks. Atherosclerotic lesion area was slightly increased at 2 weeks and substantially elevated at 4 and 6 weeks in HHcy apoE–/– mice. Cotransfer of normal Tregs significantly attenuated atherosclerotic lesion size and infiltration of T cells and macrophages into plaque. Furthermore, Treg cotransfer reversed HHcy-accelerated proliferation of T cells, -increased pro-inflammatory, and -decreased anti-inflammatory cytokine secretion from activated splenic T cells. With a clinically relevant level of plasma Hcy, the proportion of Tregs and suppressive activity in splenic T cells were reduced in HHcy apoE–/– mice, which was associated with reduced mRNA and protein expression of Foxp3, a factor governing mouse Treg development and function. In addition, Hcy significantly attenuated the proportion and suppressive effects of Tregs in vitro.

HHcy suppresses the function of Tregs, which may be responsible for HHcy-accelerated atherosclerosis in apoE–/– mice.

Obese ob/ob and db/db mice were treated with eplerenone, a MR antagonist, for 3 weeks. 3T3-L1 adipocytes were treated with aldosterone or H₂O₂, with and without eplerenone or MR-siRNA. High levels of MR mRNA were detected in adipose tissue of obese ob/ob and db/db mice. Eplerenone treatment significantly reduced insulin resistance, suppressed macrophage infiltration and ROS production in adipose tissues, and corrected the mRNA levels of obesity-related genes in obese mice. In 3T3-L1 adipocytes, aldosterone and H₂O₂ increased intracellular ROS levels and MR blockade inhibited such increases. H₂O₂ and aldosterone resulted in dysregulation of mRNAs of various genes related to ROS and cytokines, whereas MR blockade corrected such changes.

MR blockade attenuates obesity-related insulin resistance partly through reduction of fat ROS production, inflammatory process, and induction of cytokines.