To determine whether PI3-Kinase is required for the maintenance of adrenergic vascular tone, we first constricted rat small mesenteric arteries with phenylephrine (PE) and then perfused with PI3-kinase inhibitors, LY294002 and wortmannin, both of which produced a dose dependent vasodilatation. Next, to investigate whether MMPs modulate PI3-kinase activity, we cultured rat aortic vascular smooth muscle (VSM) cells and stimulated them with GPCR agonists such as PE and angiotensin II (AT 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 (siRNA) blunted the PI3-kinase phosphorylation of Akt induced by PE. Further, in VSM cells, PI3-kinase inhibitors reduced the PE induced increase in ATP synthesis and glucose transporter-4 (GLUT4) 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 maintenance of adrenergic vascular tone by the MMP-EGFR pathway requires PI3-kinase activation and ATP synthesis. Further, our data supports 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.

Infarction was produced in miniswines by ligating the left anterior descending coronary artery (LAD). TMDR of 3.0 mm in diameter were made by mechanical drilling in the infarcted area. The animals that had LAD ligation and were divided into 6 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 preoperatively and at 30 min and 6 weeks postoperatively.

Six weeks after operation, the above indices were significantly better in the TSC group than in other groups (P<0.001 compared to the control group and p<0.05 or 0.01 compared to 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 left ventricular function, cardiac blood flow, and vascular density. Therefore, the present study may provide a new method for the surgical treatment of myocardial infarction.

Radiation or busulfan-induce 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 C56BL/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.

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 (SR) microsomes prepared from mouse heart.

Exposure to ISO resulted in a 3 and 2 fold increase in Ca²⁺ spark frequency in wildtype (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 pretreatment with thapsigargin or phospholamban (PLB) 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 pretreatment 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.

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 a Smad3-dependent soluble factor that mediates this response. We use small interfering RNA 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 remodeling. Smad3 overexpression, though exclusively medial, caused adventitial changes: myofibroblast transformation, proliferation and collagen production, all of which are associated with adaptive remodeling. Supporting the hypothesis that Smad3 initiated remodeling 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 pretreatment of SMCs with siRNA specific for CTGF, abundantly produced by AdSmad3-infected SMCs, and significantly upregulated in Smad3-overexpressing arteries. Moreover, periadventitial administration of CTGF replicated the effect of medial Smad3 overexpression on adaptive remodeling and neointimal hyperplasia.

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

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, while HVEM-Ig had no protective effect. Further study showed 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.

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.

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.

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.

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.

Left ventricular fractional shortening was reduced from 37.2±0.83% to 13.44±2.63% (p<0.05). Left atrial contractility (fractional area change) was reduced from 34.9±7.9 to 27.9±4.23% (p<0.05). Action potential durations (APD) at 50 and 90% repolarization were shortened by ~60% and 40%, respectively, during heart failure (p<0.05). Heart failure-induced atrial remodeling included increased fibrosis, increased I_to and decreased I_K1, I_Kur and I_Ks (p<0.05). Heart failure induced increases in left atrial Kv channel interacting protein 2 (p<0.05), no change in K_v4.3, K_v1.5, or Kir2.3, and reduced Kir2.1 (p<0.05). When I_CaL was elicited by action potential clamp, heart failure action potentials reduced the integral of I_Ca in control myocytes, with a larger reduction in heart failure myocytes (p<0.05). I_CaL measured with standard voltage clamp was unchanged by heart failure. Incubation of myocytes with N-acetylcysteine (glutathione precursor) attenuated HF-induced electrophysiologic alterations. Left atrial angiotensin-1 receptor expression was increased in heart failure.

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

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 analyzed 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 (TP) 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.

Rodent bone-marrow derived EPCs were isolated, cultured, and transfected to express the marker protein, H-2Kk, on the cell surface. Non-transfected EPCs and EPCs transfected with either null plasmid or firefly luciferase served as controls. Control microbubbles (MB_C) and microbubbles targeted to H-2Kk expressed on EPCs (MB_H2Kk) were constructed. Binding of targeted microbubbles to EPCs was assessed in vitro using a parallel plate flow chamber system. CEU imaging of EPC-targeted microbubbles was assessed in vivo using subcutaneously implanted EPC-supplemented matrigel plugs in rats.

In flow chamber experiments, there was minimal attachment of microbubbles to plated control EPCs. While numbers of adhered MB_C were also low, there was greater and more diffuse attachment of MB_H2Kk to plated H-2Kk transfected EPCs. Targeted CEU demonstrated marked contrast enhancement at the periphery of the H-2Kk-transfected EPC-supplemented matrigel plug for MB_H2Kk whereas contrast enhancement was low for MB_C. Contrast enhancement was also low for both microbubbles within control mock-transfected EPC plugs. The signal intensity within the H-2Kk-transfected EPC plug was significantly greater for MB_H2Kk as compared to MB_C.

Microbubbles targeted to a genetically engineered cell-surface marker on EPCs exhibit specific binding to EPCs in vitro. These targeted microbubbles bind to engrafted EPCs in vivo within matrigel plugs, and can be detected by their enhancement on CEU imaging.

Here we demonstrate that forced expression of Nkx2.5 similar to MesP1 is sufficient to enhance cardiogenesis in murine embryonic stem cells. In comparison to control transfected mES cells a 5-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.

The 20S proteasome from rat heart was dissected into three different subpopulations (groups I-III), each comprising 4-7 different subtypes. The major group (group II) comprises standard proteasome subtypes; the two minor subpopulations (groups I and III) contain intermediate proteasome subtypes. All subtypes exhibit chymotrypsin-, trypsin- and caspase-like activity but to different degrees. We have tested the effect of two common proteasome inhibitors on the chymotrypsin-like activity of all subtypes: 20-30 nmol/L MG132 caused 50% inhibition of all subtypes from groups I and II, whereas 100 nmol/L was necessary to affect group III subtypes to the same extent. However, another inhibitor, bortezomib (VELCADE^TM), already used clinically, inhibited 50% of the activity of group III proteasome subtypes even below 20 nmol/L, a concentration showing almost no effect on group I and II proteasome subtypes. The caspase-like activity of group II proteasome subtypes was not affected by MG132 and was inhibited by bortezomib only at concentrations above 100 nmol/L.

These data show that different inhibitors have differential inhibitory effects on the various cardiac proteasome subtypes. Different cardiac subtypes are inhibited by the same dose of proteasome inhibitor to a different extent.

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.

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.

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.

MMP-2 and GSK-3β were incubated and the cleavage fragments were identified by immunoblotting and silver stain. The intact protein and its primary cleavage fragment were subjected to trypsin digestion and the resultant peptides were analysed by LC–MS/MS. GSK-3β kinase activity was measured using a peptide substrate and [-³²P]-ATP. Oxidative stress in H9c2 cardiomyoblasts was induced by H₂O₂ and the levels and activities of MMP-2 and GSK-3β were measured. Incubation of 47 kDa GSK-3β with MMP-2 resulted in the time- and concentration-dependant cleavage of GSK-3β as seen by appearance of an ~30 kDa fragment. MS analysis and Mascot database search yielded a peptide with an amino acid sequence of GSK-3β lacking the N-terminal region. GSK-3β kinase activity was significantly increased upon incubation with MMP-2 which was abrogated by the MMP inhibitor GM-6001. H₂O₂ challenge of H9c2 cardiomyoblasts significantly increased the activity and level of MMP-2, reduced the level of GSK-3β, and significantly increased GSK-3β kinase activity. Both the loss of intact GSK-3β and increase in its kinase activity were reduced with MMP inhibitors. MMP-2 pull-down assays in H9c2 cell lysates showed the association of MMP-2 with GSK-3β.

GSK-3β may be a target of MMP-2 and its cleavage by MMP-2 enhances its kinase activity. MMP-2 may cleave off the N-terminal of GSK-3β where the inhibitory phosphorylation of serine-9 occurs. MMP-2-mediated augmentation of GSK-3β kinase activity may contribute to cardiac injury resulting from enhanced oxidative stress.

Neonatal rat cardiac monolayers were exposed for 24 h to media conditioned by CFs. Optical mapping, sharp microelectrode recordings, quantitative RT–PCR, and immunostaining were used to assess the changes in the propagation and shape of the action potential and underlying changes in gene and protein expression. The fibroblast paracrine factors produced a 52% reduction in cardiac conduction velocity, a 217% prolongation of action potential duration, a 64% decrease of maximum capture rate, a 21% increase in membrane resting potential, and an 80% decrease of action potential upstroke velocity. These effects were dose dependent and partially reversible with removal of the conditioned media. No fibroblast proliferation, cardiomyocyte apoptosis, or decreased connexin-43 expression, phosphorylation, and function were found in conditioned cardiac cultures. In contrast, the expression of the fast sodium, inward rectifying potassium, and transient outward potassium channels were, respectively, reduced 3.8-, 6.6-fold, and to undetectable levels. The expression of β-myosin heavy chain increased 17.4-fold. No electrophysiological changes were observed from media conditioned by CFs in the presence of cardiomyocytes.

Paracrine factors from neonatal CFs alone produced significant electrophysiological changes in neonatal rat cardiomyocytes resembling those found in several cardiac pathologies.

Action potentials were recorded from multicellular canine ventricular preparations and isolated cardiomyocytes, at cycle lengths (CL) 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 modeling.

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 development of agents prolonging APD with direct rate-dependency, or even completely devoid of RRD, may be difficult to achieve.

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.

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.

MI was induced in immunodeficient mice and was followed by intra-myocardial injection of CMPCs, CMPC-CM, or vehicle. Cardiac function was measured longitudinally up to 3 months post-MI using 9.4 Tesla magnetic resonance imaging. The fate of the human cells was determined by immunohistochemistry. Transplantation of CMPCs or CMPC-CM resulted in a higher ejection fraction and reduced the extent of left ventricular remodelling up to 3 months after MI when compared with vehicle-injected animals. CMPCs and CMPC-CM generated new cardiac tissue consisting of human cardiomyocytes and blood vessels. Fusion of human nuclei with murine nuclei was not observed.

CMPCs differentiated into the same cell types in situ as can be obtained in vitro. This excludes the need for in vitro pre-differentiation, making CMPCs a promising source for (autologous) cell-based therapy.

We screened evolutionarily conserved non-coding sequences (CNSs) that are often involved in the regulation of gene expression. The VISTA Enhancer Browser identified 16 regions, termed CNS 1-16, within the Hcn4 locus. Using the luciferase reporter assay in primary neonatal rat cardiomyocytes, we found that CNS13 conferred a prominent enhancer activity (more than 30-fold) on the Hcn4 promoter. Subsequent mutation analysis revealed that the Hcn4 enhancer function was dependent on myocyte enhancer factor-2 (MEF2) and activator protein-1 (AP1) binding sequences located in CNS13. Electrophoretic mobility shift assay and chromatin immunoprecipitation confirmed that MEF2 and AP1 proteins bound CNS13. Furthermore, overexpression of a dominant negative MEF2 mutant inhibited the enhancer activity of CNS13, decreased Hcn4 mRNA expression and also decreased the amplitude of I_h current in myocytes isolated from the inflow tract of embryonic heart.

These results suggest that the novel enhancer CNS13 and MEF2 may play a critical role in the transcription of Hcn4 in the heart.

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 endothelin 1 (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-Serine ¹⁰⁵GATA4 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 neighboring 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.

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 (MMP) 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. Based on 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.

The culture process optimized to improve in vitro cardiac specification consisted of a primary culture of murine SVF cells in semi-solid methylcellulose medium, a selection of AD-CMG cell clusters, and a secondary culture and expansion in BHK21 medium. AD-CMG cells were CD29⁺, CD31^–, CD34^–, CD44⁺, CD45^–, CD81⁺, CD90^–, CD117^–, and Flk-1^– and expressed several cardiac contractile proteins. After 1, 2, and 4 weeks of their injection in mice having acute myocardial infarction, a strong presence of green fluorescent protein-positive cells was identified by immunohistochemistry as well as quantitative polymerase chain reaction. Echocardiography showed a significant reduction of remodelling and stability of left ventricle ejection fraction in the AD-CMG cell-treated group vs. controls. Vascular density analysis revealed that AD-CMG administration was also associated with stimulation of angiogenesis in peri-infarct areas.

Cardiomyogenic cells can be selected and expanded in large amounts from mouse adipose tissue. They can survive and differentiate in an acute myocardial infarction model, avoiding remodelling and impairment of cardiac function, and can promote neo-vascularization in the ischaemic heart.

Standard aggregometry, flow cytometry, electron microscopy and thrombin generation assays were performed.

TF-MVs induced platelet aggregation in heparinized platelet-rich plasma samples (PRP); 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, as 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 pathologic situations with increased cardiovascular risk.

IC53 cDNA was cloned from a human aorta cDNA library. Using the endothelium-specific VE-cadherin promoter, we constructed transgenic mice in which IC53 was specifically overexpressed in vascular endothelia and found that the transgenic mice exhibit elevated systolic blood pressure in contrast to the wild-type (wt) controls. Further studies revealed impaired endothelium-dependent vasodilation, reduced nitric oxide production and decreased endothelial nitric oxide synthase (eNOS) expression and activity in the tg mice. Inhibition of IC53 in human umbilical vein endothelial cells 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 systolic blood pressure, which may be in part attributed to downregulation of eNOS signaling.

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-infusion 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.

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.

Human cardiac mesoangioblasts were isolated from cardiac muscle biopsies of patients undergoing open heart surgery for correction of mitral regurgitation following an acute myocardial infarction (MR-MI) or correction of mitral and aortic regurgitation with ensuing left ventricular hypertrophy (MAR-LVH). The cells express surface markers and cardiac genes similar to mouse cardiac mesoangioblasts; they have limited self-renewing and clonogenic activity and are committed mainly to cardiogenesis. Although cardiac differentiation can be induced by 5-azacytidine or by co-culture with rat neonatal cardiomyocytes, human cells do not contract spontaneously like their mouse counterparts. When locally injected in the infarcted myocardium of immunodeficient mice, cardiac mesoangioblasts generate a chimeric heart that contains human myocytes and some capillaries; likewise, they colonize chick embryo hearts when transplanted in ovo. At variance with cells from patients with MR-MI, when isolation was performed on biopsies from MAR-LVH, cells could be isolated in much lower numbers, proliferated less extensively and failed to differentiate.

Cardiac mesoangioblasts are present in the human heart but this endogenous progenitor population is progressively exhausted, possibly by continuous and inefficient regeneration attempts.

Contractility was measured in ex vivo Langendorff-perfused hearts isolated from Wistar rats. Isolated ventricular cardiomyocytes were used to analyse intracellular calcium ([Ca²⁺]_i) transients evoked by electrical stimulation and L-type Ca²⁺ current by confocal microscopy and whole-cell patch-clamping, respectively. The application of Ucn to perfused hearts induced progressive, sustained, and potent inotropic and lusitropic effects that were dose-dependent with an EC₅₀ of approximately 8 nM. Ucn effects were independent of protein kinase A (PKA) activation but were significantly reduced by protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) inhibitors and by brefeldin A, an antagonist of guanine nucleotide exchange factor, suggested to be an inhibitor of exchange protein activated by cAMP (Epac). These whole-organ effects were correlated with the inotropic effects observed in isolated cells: Ucn increased I_CaL density, [Ca²⁺]_i transients, cell shortening and Ca²⁺ content of sarcoplasmic reticulum.

Our results show that Ucn evokes potent positive inotropic and lusitropic effects mediated, at least in part, by an increase in I_CaL and [Ca²⁺]_i transient amplitude. These effects may involve the activation of Epac, PKC, and MAPK signalling pathways.

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.

Sprague–Dawley rats with HF or sham-operated control (SHAM) rats were treated for 4 weeks with a continuous intracerebroventricular (ICV) infusion of the cytokine blockers—pentoxifylline (PTX, 10 µg/h and 40 µg/h), etanercept (ETN, 5 µg/h and 10 µg/h), or vehicle. Another set of HF and SHAM rats were treated with intraperitoneal (ip) infusion of a similar dose of PTX or ETN. HF rats had increased neuronal excitation accompanied by higher levels of glutamate, norepinephrine (NE), and tyrosine hydroxylase (TH), and lower levels of -aminobutyric acid (GABA), nNOS, and 67-kDa isoform of glutamate decarboxylase (GAD67) in the PVN when compared with SHAM rats. Plasma cytokines, NE, epinephrine, angiotensin II, and renal sympathetic nerve activity (RSNA) were also increased in HF rats. ICV treatment with low doses of PTX or ETN attenuated, and high doses prevented, increases in levels of glutamate, NE, and TH, and decreases in levels of GABA, nNOS, and GAD67 in the PVN in HF rats. The same ICV treatments also attenuated the increased RSNA seen in HF rats. IP treatment with similar doses of PTX or ETN did not affect glutamate, NE, TH, GABA, nNOS, and GAD67 in the PVN and had no effect on RSNA of HF rats.

This study, for the first time, demonstrates that proinflammatory cytokines modulate neurotransmitters in the PVN and contribute to sympathoexcitation in HF.

In 43 mongrel dogs, rapid firing-mediated atrial fibrillation (AF) was induced by local HFS (200 Hz, impulse duration 0.1 msec, train duration 40 msec) 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 contribute to the rapid firings and AF originating from ipsolateral and contralateral PVs and atrium. Autonomic denervation suppresses or eliminates those rapid firings.

Human saphenous vein VSMC were exposed to phorbol 12-myristate 13-acetate (PMA) to induce endogenous cyclooxygenase-2-dependent prostaglandin generation. This was found to attenuate PAR-1 expression; similar suppression was seen with the EP2-prostaglandin receptor agonist butaprost. Stimulation of the ‘exchange protein directly activated by cyclic AMP’ (EPAC) was without effect. The NFAT inhibitor cyclosporin A (CsA) or NFAT2 siRNA both reduced PAR-1 mRNA and protein expression and prevented the stimulatory effects of thrombin or PAR-1 activating peptide (TFLLRN) on ERK1/2 phosphorylation and interleukin-6 expression. CsA or mutation of the NFAT binding motif in the PAR-1 promoter also blunted PAR-1 promoter activity (luciferase reporter assay). These inhibitory actions of CsA were comparable to those of the prostacyclin-mimetic iloprost, and both CsA and iloprost similarly attenuated nuclear NFAT2 localization and binding to the PAR-1 promoter (chromatin immunoprecipitation assay).

This study provides the first evidence that NFAT2 contributes to the transcriptional control of PAR-1 in human VSMC and that PKA-dependent NFAT2 inhibition represents a mechanism by which vasodilatory prostaglandins regulate the vascular actions of thrombin.

We observed increased expression of both angiotensinogen and TNF in the failing rat myocardium, with a regional gradient matching that of the K_ATP subunit Kir6.1 expression. Both angiotensinogen and TNF expression correlated positively with Kir6.1 and negatively with Kir6.2 expression across the post-infarction myocardium. To further identify a causal relationship, cardiomyocytes isolated from normal rat hearts were exposed in vitro to Ang II or TNF. We observed increased Kir6.1 and SUR subunit and reduced Kir6.2 subunit mRNA expression in cardiomyocytes cultured with Ang II or TNF, similar to what was observed in failing hearts. In patch-clamp experiments, cardiomyocytes cultured with Ang II or TNF exhibited responsiveness to diazoxide, in terms of both K_ATP current and action potential shortening. This was not observed in untreated cardiomyocytes and resembles the diazoxide sensitivity of failing cardiomyocytes that also overexpress Kir6.1. Ang II exerted its effect through induction of TNF expression, because TNF-neutralizing antibody abolished the effect of Ang II, and in failing hearts, regional expression of angiotensinogen matched TNF expression. Finally, Ang II and TNF regulated K_ATP subunit expression, possibly through differential expression of Forkhead box transcription factors.

This study identifies Ang II and TNF as mediators of the remodelling of K_ATP channels in heart failure.

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.

Human umbilical vein ECs were subjected to shear flow for 30 min, and S-nitrosylated proteins were detected by the CyDye switch method. In principle, free thiols in proteins become blocked by alkylation, the S-nitrosylated bond is reduced by ascorbate, and then CyDye labels proteins. Proteins that separately labelled with Cy3 or Cy5 were mixed and subjected to two-dimensional gel electrophoresis for further analysis. More than 100 S-nitrosoproteins were detected in static and shear-treated ECs. Among these, 12 major proteins of heterogeneous function showed a significant increase in S-nitrosylation following shear flow. The S-nitrosylated residues in tropomyosin and vimentin, which were localized in the hydrophobic motif of each protein, were identified as Cys170 and Cys328, respectively.

Post-translational S-nitrosylation of proteins in ECs can be detected by a reliable CyDye switch method. This flow-induced S-nitrosylation of endothelial proteins may be essential for the adaptation and remodelling of ECs under flow conditions.

Whole-cell patch-clamp recordings showed outward-rectifying currents in human umbilical vein endothelial cells (HUVECs), which was potentiated by removing the extracellular Ca²⁺ and Mg²⁺, but inhibited by non-specific TRPM7 blocker Gd³⁺ or 2-aminoethoxydiphenyl borate (2-APB). TRPM7 mRNA was detected in HUVECs by RT–PCR, but TRPM6, its closest homologue, was not. Silencing TRPM7 by small interfering RNA (siRNA) decreased the level of TRPM7 mRNA and the TRPM7-like current. Interestingly, knockdown of TRPM7 with siRNA or inhibition of TRPM7 function with 2-APB increased the phosphorylation of extracellular signal-regulated kinase (ERK) and enhanced growth/proliferation of HUVECs. This enhanced cell growth/proliferation was abolished by an inhibitor of the ERK signalling pathway. In addition to cell growth/proliferation, silencing TRPM7 also increased expression of nitric oxide synthase and nitric oxide production in an ERK pathway-dependent manner.

These observations suggest that TRPM7 channels may play an important role in the function of vascular endothelial cells.

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 2- to 3-fold in type 2 diabetes mellitus and myocardial ischemia (p<0.05). In humans with severe heart failure, functional improvement following implantation of left ventricular assist devices led to a 2-fold increase in SGLT1 mRNA (p<0.05). Acute administration of leptin to wildtype mice increased cardiac SGLT1 expression ~7-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.

In this study we verified the presence of mCx43 by a mass spectrometry-based proteomic approach in purified mitochondrial preparations from mouse myocardium and determined by western blot analysis that the C-terminus of mCx43 is oriented towards the intermembrane space. Cross-linking studies with dimethylsuberimidate indicated the presence of Cx43 hexamers in mitochondrial membranes. The contribution of Cx43 to both mitochondrial dye uptake and K⁺ flux was assessed in wild-type mice using hemichannel blockers and Cx43KI32 mice in which Cx43 had been replaced by Cx32. Uptake of the Cx43 hemichannel-permeant dye Lucifer Yellow was reduced in mitochondria from wild-type mice by two hemichannel blockers (carbenoxolone and heptanol) and in Cx43KI32 compared with wild-type mice. Mitochondrial K⁺ influx (PBFI fluorescence) was decreased in digitonin-permeabilized cardiomyocytes from Cx32 mutants compared with wild-type mice, and addition of the Cx43 hemichannel blocker 18-glycyrrhetinic acid had an inhibitory effect on mitochondrial K⁺ influx in wild-type cardiomyocytes, but not in cardiomyocytes from Cx32 mutants.

These results indicate that mCx43 contributes to mitochondrial K⁺ flux in cardiomyocytes, potentially by forming hemichannel-like structures.

Based on a tetracycline-inducible transactivator, we designed an AAV vector system allowing the pharmacological regulation of VEGF production in vivo and tested its efficacy in inducing functional neoangiogenesis in both normoperfused and ischaemic skeletal muscle in mice by a combination of histological, immunofluorescent, and molecular imaging techniques. We observed that a prolonged expression of VEGF was required to determine the formation of stable vessels, able to persist upon withdrawal of the angiogenic stimulus. However, the vessels formed in the presence of continuous VEGF expression consisted mainly of dilated and leaky capillaries. As determined after pinhole scintigraphy, this abnormal vasculature accounted for a significant drop in functional tissue perfusion. In contrast, transient VEGF expression, followed by a period of VEGF withdrawal, allowed maintenance of functional perfusion under resting conditions and during exercise. This VEGF-inducible system was highly effective in improving vascularization and function in a hind-limb ischaemia model.

Together, these results clearly indicate that the fine tuning of VEGF expression is required to achieve the formation of a stable vasculature able to sustain functional neovascularization.

High-frequency ultrasonography (0.01 mm resolution) was used to quantify the effect of LLLI on aneurysmatic aortic dilatation from baseline to 4 weeks after subcutaneous infusion of angiotensin II by osmotic minipumps in the apolipoprotein E-deficient mouse. At 4 weeks, seven of 15 non-irradiated, but none of the 13 LLLI, mice had aneurysmal dilatation in the suprarenal aneurysm-prone segments that had progressed to ≥50% increase in maximal cross-sectional diameter (CSD) over baseline (P = 0.005 by Fisher's exact test). The mean CSD of the suprarenal segments (normalized individually to inter-renal control segments) was also significantly lower in irradiated animals (LLLI vs. non-irradiated: 1.32 ± 0.14 vs. 1.82 ± 0.39, P = 0.0002 by unpaired, two-tailed t-test) with a 94% reduction in CSD at 4 weeks compared with baseline. M-mode ultrasound data showed that reduced radial wall velocity seen in non-treated was significantly attenuated in the LLLI mice, suggesting a substantial effect on arterial wall elasticity.

These in vivo studies, together with previous in vitro studies from this laboratory, appear to provide strong evidence in support of a role for LLLI in the attenuation of aneurysm progression. Further studies in large animals would appear to be the next step towards testing the applicability of this technology to the human interventional setting.

Cell-penetrating(cp)-G_i1/2 and cp-G_i3 C-terminal peptides were assessed for their ability to attenuate cholinergic-parasympathetic signalling in isolated feline atrial myocytes and in canine left atrium (LA). Confocal fluorescence microscopy indicated that cp-G_i1/2 and/or cp-G_i3 peptides moderated carbachol attenuation of cellular Ca²⁺ transients in isolated atrial myocytes. High-density epicardial mapping of dog PLA, left atrial pulmonary veins (PVs), and left atrial appendage (LAA) indicated that the delivery of cp-G_i1/2 peptide or cp-G_i3 peptide into the PLA prolonged effective refractory periods at baseline and during vagal stimulation in the PLA and to varying extents also in the LAA and PV regions. After delivery of cp-G_i peptides into the PLA, AF inducibility during vagal stimulation was significantly diminished.

These results demonstrate the feasibility of using specific G_i-protein inhibition to achieve selective parasympathetic denervation in the PLA, with a resulting change in vagal responsiveness across the entire LA.

Resveratrol (5–25 µM) completely inhibited platelet aggregation stimulated by collagen. Furthermore, resveratrol time- and concentration-dependently stimulated dissipation of the mitochondrial membrane potential (m), activation of caspases-9, -3, and -8, gelsolin and actin cleavage, Bid cleavage into truncated Bid, Bax translocation, cytochrome c release, and phosphatidylserine exposure but not P-selectin expression in washed human platelets. The presence of z-IETD-fmk, a caspase-8 inhibitor, markedly reversed tBid formation and caspase activation and partially reversed the dissipation of platelet m stimulated by resveratrol. In addition, resveratrol also directly evoked dissipation of m and release of cytochrome c from isolated mitochondria. Furthermore, resveratrol shortened platelet survival or enhanced platelet clearance in an in vivo study.

This study demonstrates for the first time that resveratrol simultaneously inhibits platelet aggregation and stimulates platelet apoptosis. Stimulation of platelet apoptosis by resveratrol may represent the increased therapeutic potential for patients suffering from thrombotic conditions or thrombocytosis to promote platelet destruction and thus prevent pathological clotting. Furthermore, this study also provides a novel conception that rigorous surveillance of platelet numbers may be important during resveratrol treatment in the clinic.

Kv currents recorded using the patch clamp technique with freshly isolated rat mesenteric ASM cells were inhibited by ET-1 or AngII. Inclusion of a PKC inhibitor peptide in the intracellular solution substantially reduced inhibition by AngII, but did not affect that by ET-1. Kv inhibition by ET-1 was reduced by the conventional PKC inhibitor Gö 6976 but not by the PKCβ inhibitor LY333531. Selective peptide inhibitors of PKC and PKC were linked to a Tat carrier peptide to make them membrane permeable and used to show that inhibition of PKC prevented ET-1 inhibition of Kv current, but did not affect that by AngII. In contrast, inhibition of PKC prevented Kv inhibition by AngII but not by ET-1. The Tat-linked inhibitor peptides were also used to investigate the involvement of PKC and PKC in the contractile responses of mesenteric arterial rings, showing that ET-1 contractions were substantially reduced by inhibition of PKC, but unaffected by inhibition of PKC. AngII contractions were unaffected by inhibition of PKC but substantially reduced by inhibition of PKC.

ET-1 inhibits Kv channels of mesenteric ASM through activation of PKC, while AngII does so through PKC. This implies that ET-1 and AngII target Kv channels of ASM through different pathways of PKC-interacting proteins, so each vasoconstrictor enables its distinct PKC isoenzyme to interact functionally with the Kv channel.

Isolated perfused adult rat hearts were subjected to the following treatments: ischaemic preconditioning (PC); diazoxide perfusion; FGF-2 pre-treatment followed by 30 min global ischaemia; 30 min global ischaemia followed by 60 min reperfusion in the presence or absence of FGF-2. Cx43 phosphorylation was assessed by western blotting with phospho-specific antibodies. Neonatal cardiomyocyte cultures were used to examine the effect of expressing Cx43 incapable of being phosphorylated at S262 due to an S to alanine (A) substitution on simulated ischaemia-induced cell death (TUNEL staining) and injury (lactic dehydrogenase release). Ischaemic PC, diazoxide, and FGF-2 pre-ischaemic or post-ischaemic treatments elicited a P*Cx43 state, defined as above-physiological levels of phospho-S262-Cx43 and phospho-S368-Cx43. P*Cx43 was sustained during global ischaemia and was accompanied by attenuation of ischaemia-induced Cx43 dephosphorylation and prevention of Cx43 lateralization. Post-ischaemic FGF-2 treatment also diminished dephosphorylated Cx43. Modest overexpression of S262A-Cx43, but not wild-type Cx43, exacerbated cardiomyocyte death and injury caused by simulated ischaemia in vitro. It also prevented the cytoprotective effects of FGF-2 or overexpressed PKC.

P*Cx43 marks a state of enhanced resistance to ischaemic injury promoted by PKC-activating treatments such as FGF-2 administration or ischaemic PC. Cx43 phosphorylation at S262 likely mediates PKC-dependent cardioprotection.

We subjected mice with genetic deletion of ROCK-1 to I/RC. We found that ROCK-1^–/– mice did not develop the fibrosis and cardiac dysfunction characteristic for I/RC: compared with wild-type, ROCK-1^–/– hearts showed markedly lower numbers of I/RC-induced -smooth muscle actin⁺ fibroblasts and CD34⁺/CD45⁺ fibroblast precursors. Isolated cardiac fibroblasts from ROCK-1^–/– mice undergoing I/RC were large and slowly proliferating, similar to fibroblasts isolated from sham-treated hearts. We also performed in vitro assays in which human peripheral blood mononuclear cells (PBMC) migrated through endothelial cells in response to MCP-1. Prior to migration, PBMC were incubated with ROCK-1-targeting small interfering RNA to silence ROCK-1 expression. We found that an 80% reduction of ROCK-1 protein did not inhibit TEM, but significantly reduced the amount of mononuclear cells that differentiated into fibroblasts by >20-fold.

Our data implicate an important role for ROCK-1 in the differentiation, but not in the TEM of monocytes that mature into cardiac fibroblasts. These cells mediate non-adaptive fibrosis.

When HCAECs were treated with recombinant cHSP60, endothelial nitric oxide synthase (eNOS) mRNA and protein levels, enzyme activities, cellular NO levels, mRNA stability, and promoter activities were significantly decreased. Superoxide anion production was significantly increased due to the inhibition of mitochondrial membrane potential and catalase and superoxide dismutase (SOD) activities as well as activation of NADPH oxidase. Antioxidant seleno-l-methionine (SeMet) or SOD mimetic MnTBAP effectively blocked cHSP60-induced eNOS downregulation. In addition, cHSP60 activated mitogen-activated protein kinases (MAPKs) including p38, c-Jun-N-terminal kinase/stress-activated protein kinase, and extracellular signal-regulated kinases. Specific chemical inhibitors or their dominant-negative mutant forms of these MAPKs effectively blocked cHSP60-induced eNOS downregulation. cHSP60-induced eNOS downregulation and oxidative stress were also demonstrated in porcine coronary artery rings in vitro. Functionally, endothelium-dependent vasorelaxation was significantly reduced in cHSP60-treated vessels.

cHSP60 directly induces eNOS downregulation through oxidative stress and MAPK activation in both HCAECs and porcine coronary arteries, thereby causing endothelial dysfunction.

SHRs were divided into two groups: one received no treatment (SHR-C) and the other (SHR-L) received 5 mg/kg/day L-2286 (PARP-inhibitor) orally for 46 weeks. A third group was a normotensive age-matched control group (CFY) and a fourth was a normotensive age-matched group receiving L-2286 treatment 5 mg/kg/day (CFY+L). At the beginning of the study, systolic function was similar in both CFY and SHR groups. In the SHR-C group at the end of the study, eccentric hypertrophy with poor left ventricular (LV) systolic function was observed, while PARP inhibitor treatment preserved systolic LV function. Due to these favourable changes, the survival rate of SHRs was significantly improved (P < 0.01) by the administration of the PARP inhibitor (L-2286). The PARP inhibitor used did not affect the elevated blood pressure of SHR rats, but moderated the level of plasma-BNP (P < 0.01) and favourably influenced all the measured gravimetric parameters (P < 0.05) and the extent of myocardial fibrosis (P < 0.05). The inhibition of PARP increased the phosporylation of Akt-1/GSK-3β (P < 0.01), ERK 1/2 (P < 0.01), and PKC (P < 0.01), and decreased the phosphorylation of JNK (P < 0.05), p-38 MAPK (P < 0.01), PKC pan βII and PKC / (P < 0.01), and PKC /βII and (P < 0.05).

These data demonstrate that chronic inhibition of PARP induces long-term favourable changes in the most important signalling pathways related to oxidative stress. PARP inhibition also prevents remodelling, preserves systolic function, and delays transition of hypertensive cardiopathy to HF in SHRs.

CD74 expression was analysed in human atherosclerotic plaques (immunohistochemistry), PBMC (real-time PCR), and human aortic VSMC (real-time PCR and western blotting). Nuclear factor-B (NF-B) activation was assessed by southwestern histochemistry and electrophoretic mobility shift assay. Monocyte chemoattractant protein-1 (MCP-1) levels were studied by both real-time PCR and enzyme-linked immunosorbent assay. CD74 immunostaining was increased in the inflammatory vs. the fibrous region of atherosclerotic plaques (n = 70, 18.2 ± 1.3 vs. 7.8 ± 0.6% positive staining/mm², P < 0.001). CD74 colocalized with the transcription factor NF-B in both VSMC and macrophages. In cultured VSMC, CD74 expression was induced by interferon (IFN). Incubation with an agonistic anti-CD74 antibody or with IFN elicited MCP-1 expression, which was prevented by AKT and -secretase inhibitors. Moreover, CD74 small-interfering RNA decreased NF-B activation and MCP-1 production induced by IFN in VSMC. Finally, CD74 mRNA levels in PBMC from patients with carotid stenosis were higher than in healthy subjects (n = 20, 3 ± 0.5 vs. 2 ± 0.5 AU, P < 0.001). Additionally, a linear trend between CD74 mRNA expression tertiles and intima-media thickness (IMT) was observed in PBMC from asymptomatic subjects (n = 185, P < 0.001).

CD74 levels are increased in plaques and PBMC from patients with carotid stenosis and are associated with IMT in subjects free from clinical cardiovascular diseases. CD74 could be a novel therapeutic target to decrease the inflammatory response in atherosclerosis.

We observed that pharmacological concentrations of statins (atorvastatin and simvastatin) modulated LOX transcriptional activity, counteracting the down-regulation of LOX (at the mRNA, protein, and activity level) caused by tumour necrosis factor- (TNF) in porcine, bovine, and human aortic endothelial cells. Geranylgeraniol but not farnesol reversed this effect, suggesting the involvement of geranylgeranylated proteins. In accordance, inhibitors of RhoA/Rho kinase also counteracted LOX down-regulation caused by TNF, and over-expression of a RhoA dominant-negative mutant mimicked statin effects. Statins were also able to counteract the decrease in LOX expression produced by atherogenic concentrations of LDL by a similar mechanism and to partially prevent the increase in endothelial permeability elicited by these lipoproteins. Finally, in the in vivo porcine model of hypercholesterolaemia, we observed that statins abrogated the reduction of vascular LOX expression triggered by high plasma levels of LDL.

These data indicate that statins normalize vascular LOX expression altered by atherogenic risk factors through a RhoA/Rho kinase-dependent mechanism. Thus, modulation of LOX by statins could contribute to vascular protection and to the cardiovascular risk reduction achieved by this therapy.

Despite the fact that TTA treatment decreased plasma concentrations of lipids [FA and triacylglycerols (TG)], hearts from TTA-treated mice showed increased mRNA expression of PPAR target genes. Cardiac substrate utilization, ventricular function, cardiac efficiency, and susceptibility to ischaemia-reperfusion were examined in isolated perfused hearts. In accordance with the mRNA changes, myocardial FA oxidation was increased 2.5-fold with a concomitant reduction in glucose oxidation. This increase in FA oxidation was abolished in PPAR-null mice. Thus, it appears that the metabolic effects of TTA on the heart must be owing to a direct stimulatory effect on cardiac PPAR. Hearts from TTA-treated mice also showed a marked reduction in cardiac efficiency (because of a two-fold increase in unloaded myocardial oxygen consumption) and decreased recovery of ventricular contractile function following low-flow ischaemia.

This study for the first time observed that in vivo administration of a synthetic PPAR ligand elevated FA oxidation, an effect that was also associated with decreased cardiac efficiency and reduced post-ischaemic functional recovery.

Endothelial cell sprouting was investigated in the human umbilical vein endothelial cell spheroid assay. We investigated in vivo angiogenesis in the chick (Gallus gallus) chorioallantoic membrane assay. Expression profiling of proangiogenic factors was done by quantitative PCR. The angiopoietin-2 (Ang2) promoter and deletion fragments thereof were cloned into the pGL3 reporter system for analysis of transcriptional activity. We observed that HoxB5 enhances endothelial cell sprouting and modulates the expression of adhesion molecules in vitro. Accordingly, we observed a modification of vascular growth by HoxB5 in vivo. The HoxB5 effect is reminiscent of the effects of angiopoietins. We demonstrate that Ang2 is upregulated upon HoxB5 overexpression and that the HoxB5 effect is abolished by the angiopoietin antagonist soluble Tie-2.

HoxB5 has an activating effect on Ang2 that is essential for endothelial cell sprouting and coordinated vascular growth.

The canine model of AF was successfully established by nicotine administration and rapid pacing. The atrial fibroblasts isolated from healthy dogs were treated with nicotine. The role of microRNAs (miRNAs) on the expression and regulation of transforming growth factor-β1 (TGF-β1), TGF-β receptor type II (TGF-βRII), and collagen production was evaluated in vivo and in vitro. Administration of nicotine for 30 days increased AF vulnerability by ~eight- to 15-fold in dogs. Nicotine stimulated remarkable collagen production and atrial fibrosis both in vitro in cultured canine atrial fibroblasts and in vivo in atrial tissues. Nicotine produced significant upregulation of expression of TGF-β1 and TGF-βRII at the protein level, and a 60–70% decrease in the levels of miRNAs miR-133 and miR-590. This downregulation of miR-133 and miR-590 partly accounts for the upregulation of TGF-β1 and TGF-βRII, because our data established TGF-β1 and TGF-βRII as targets for miR-133 and miR-590 repression. Transfection of miR-133 or miR-590 into cultured atrial fibroblasts decreased TGF-β1 and TGF-βRII levels and collagen content. These effects were abolished by the antisense oligonucleotides against miR-133 or miR-590. The effects of nicotine were prevented by an 7 nicotinic acetylcholine receptor antagonist.

We conclude that the profibrotic response to nicotine in canine atrium is critically dependent upon downregulation of miR-133 and miR-590.

HEK293 cells were transfected with SCN5A cDNA or its mutant carrying W822X, a nonsense mutation associated with Brugada syndrome and sudden cardiac death. The effects of readthrough-enhancing reagents on Na⁺ channel expression and function were examined in the transfected cells. W822X robustly reduced Na⁺ current, decreasing maximal Na⁺ current to <3% of the wild-type level, and inhibited the expression of full-length Na⁺ channels. When readthrough was enhanced by either reducing translational fidelity with aminoglycosides or decreasing translation termination efficiency with small-interfering RNA against eukaryotic release factor eRF3a, Na⁺ current of the mutant was restored to ~30% of the wild-type level; western blot and immunochemical staining analyses showed the increased expression of full-length channels. When the wild-type and mutant cDNAs were co-transfected, readthrough-enhancing reagents increased Na⁺ current from 56% to 74% of the wild-type level. Analysis of Na⁺ channel kinetics showed that the channels expressed from the mutant cDNA under readthrough-enhancing conditions retained the functions of wild-type channels.

Readthrough-enhancing reagents can effectively suppress SCN5A nonsense mutations and may restore the expression of full-length Na⁺ channels with normal functions, which might prevent sudden cardiac death in mutation carriers.

[³H]-Cholesterol- and [³H]-methylcholine chloride-labelled murine macrophages treated with EP 80317 showed a significant increase in cholesterol and phospholipid efflux to both apolipoprotein A-I and high-density lipoprotein in a CD36-dependent manner. Lipid efflux was associated with enhanced activation of PPAR. The signalling pathway by which this CD36 ligand promoted lipid efflux involved an increase in intracellular 15-deoxy-^12,14-prostaglandin J₂ (15d-PGJ₂) levels induced by extracellular signal-regulated kinase 1/2 (ERK1/2)-dependent cyclooxygenase-2 (COX-2) expression, leading to PPAR activation. In agreement, EP 80317-mediated cholesterol efflux was abrogated by inhibitors of PPAR, ERK1/2, and COX-2 as well as ABC transporter inhibitors, whereas a p38 mitogen-activated protein kinase inhibitor had no effect.

These findings suggest a central role for the prostanoid 15d-PGJ₂ in PPAR activation and the upregulation of the ABC transporter pathway in response to CD36 activation by synthetic GHRPs analogues. The resulting enhanced cholesterol efflux might explain, at least in part, the atheroprotective effect of selective CD36 ligands.

Serum TSP-1 (ELISA), subcutaneous and omental AT TSP-1 mRNA (reverse transcriptase–polymerase chain reaction), and protein (western blotting) were significantly lower in PCOS women (P < 0.05). Corresponding plasminogen activator inhibitor-1 (PAI-1) and PAI-1 activity were significantly higher (P < 0.01). After 6 months of metformin treatment, there was a significant increase in serum TSP-1 (P < 0.05) and a corresponding decrease in PAI-1 and PAI-1 activity (P < 0.01). In vitro migration and angiogenesis were significantly increased in serum from PCOS women (P < 0.01); these effects were significantly attenuated by metformin treatment (P < 0.01) through the regulation of TSP-1 levels via nuclear factor-B (NF-B), extracellular regulated-signal kinase 1/2 (Erk1/2) and Erk5 pathways. Importantly, changes in the intima media thickness were predictive of changes in serum TSP-1 (P = 0.049). In AT explants, glucose significantly decreased TSP-1 protein production and secretion into conditioned media (ELISA) (P < 0.05, P < 0.001).

TSP-1 levels are lower in PCOS women. Metformin treatment increases serum TSP-1 in these women. Our findings provide novel insights into the relationship between obesity, IR, and angiogenesis.

The effects of pressure overload were assessed in male Sprague–Dawley rats 6 weeks after AAB using progression of cardiac hypertrophy to heart failure as the endpoint. The AAB-treated rats had significantly elevated blood pressure, systolic and diastolic cardiac dysfunction, evidence of left ventricular hypertrophy (LVH), and transition to heart failure. LVH was characterized by increases in the ratios of heart and left ventricular weights to body weight, increased myocyte cross-sectional areas, myocardial and perivascular fibrosis, and elevated cardiac hydroxyproline. These could be prevented by treatment with iptakalim at daily oral doses of 1, 3, and 9 mg/kg for 6 weeks. Progression to cardiac failure, demonstrated by increases in relative lung and right ventricular weights, cardiac function disorders and overexpression of atrial and B-type natriuretic peptide mRNA, could also be prevented. The downregulated nitric oxide signalling system was enhanced, whereas the upregulated endothelin signalling system was inhibited, resulting in normalization of the balance between these two systems.

Iptakalim protected the endothelium and prevented progression of cardiac hypertrophy to failure induced by a pressure overload.