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.