Rat carotid balloon injury increased Rac₁ activity. Rho GTPase inhibition with C. difficile Toxin B or specific Rac₁-inhibition with adenovirus-mediated expression of dominant-negative Rac₁ reduced Skp2 levels, and VSMC proliferation in vitro and intima formation in vivo following carotid balloon injury. Inhibition of Skp2 expression and proliferation by dominant-negative Rac₁ was reversed by exogenous Skp2. Elevation of endogenous cAMP with forskolin inhibited Rac₁ activity, reduced Skp2, increased p27^Kip1 and inhibited VSMC proliferation, effects that were reversed by constitutively-active Rac₁. These effects were independent of Rac₁ CRIB-domain effector-proteins but associated with Rac₁-dependent actin polymerisation.

Rac₁ activity regulates VSMC proliferation by controlling Skp2 levels. Activation of Rac₁ induced by balloon injury in vivo increases Skp2 levels, which promotes VSMC proliferation and intima formation. Inhibition of this novel pathway underlies the negative effects of cAMP on VSMC proliferation.

In C57Bl/6j mice exposed to 10% oxygen, signaling molecules were measured in cardiac tissue, sarcoplasmic reticulum (SR)-enriched membrane preparations and serum. Cardiomyocytes isolated from neonatal mice were exposed to interleukin (IL)-18 for 24 hours.

The β-adrenergic pathway in the myocardium was not altered by alveolar hypoxia, as assessed by measurements of β-adrenergic receptor levels, adenylyl cyclase activity and subunits of cyclic AMP-dependent protein kinase. However, alveolar hypoxia led to a significantly higher amount (124%) and activity (234%) of protein phosphatase (PP) 2A in SR-enriched membrane preparations from LV compared to control. Serum levels of an array of cytokines were assayed, and a pronounced increase in IL-18 was observed. In isolated cardiomyocytes, treatment with IL-18 increased the amount and activity of PP2A, and reduced phosphorylation of phospholamban at Ser16 to 54% of control.

Our results indicate that the diastolic dysfunction observed in alveolar hypoxia might be caused by increased circulating IL-18 inducing an increase in PP2A and thereby a reduction in phosphorylation of phospholamban at Ser16.

Force, sarcomere length (SL), membrane potential, and [Ca²⁺]_i dynamics were measured in 31 ventricular trabeculae from rat hearts. Non-uniform muscle contraction was produced by exposing a restricted region of muscle to a jet of solution containing 20 mmol/L 2,3-butanedione monoxime (BDM). DADs were induced by 7.5 s-2 Hz stimulus trains at an SL of 2.0 µm (24°C, [Ca²⁺]_o 2.0 mmol/L). The BDM jet enhanced DADs (n = 6, P < 0.05) and aftercontractions (n = 6, P < 0.05) with or without 100 µmol/L streptomycin and occasionally elicited an action potential. A stretch pulse from an SL of 2.0 µm to 2.1 or 2.2 µm during the last stimulated twitch of the trains accelerated Ca²⁺ waves in proportion to the increment of force by the stretch (P < 0.01) with or without streptomycin. In the presence of 1 mmol/L caffeine, rapid shortening of the muscle after the stretch pulse increased [Ca²⁺]_i within the BZ, whose amplitude correlated with the increment of force by the stretch (n = 15, P < 0.01).

These results suggest that non-uniform muscle contraction can enhance DADs by dissociating Ca²⁺ from myofilaments within the BZ and thereby cause triggered arrhythmias.

SHR were treated with apocynin orally or i.v. Arterial pressure was recorded directly. Rat and mouse arterial function was investigated in vitro by small vessel wire myography. NADPH oxidase activity was measured in human granulocytes and in rat vascular preparations. Rho kinase activity was determined by Western blot analysis.

Apocynin did not reduce arterial pressure acutely in SHR when given at 50, 100, or 150 mg*kg^–1*d^–1 orally over one-week-intervals or when given i.v. Apocynin potently inhibited granulocyte NADPH oxidase but not vascular NADPH oxidase-dependent oxygen radical formation unless exogenous peroxidase was added to vascular preparations. Apocynin dilated rat intrarenal and coronary arteries independently of pharmacological interventions that reduce vascular superoxide radical abundance and actions. Aortic rings from p47phox^–/- mice were more sensitive to apocynin-induced dilation than wild type aortic rings. Rho kinase inhibition reduced or prevented the inhibitory effect of apocynin on agonist-induced vasoconstriction and apocynin inhibited the phosphorylation of Rho kinase substrates.

Apocynin per se does not inhibit vascular NADPH oxidase-dependent superoxide formation. Its in vitro vasodilator actions are not due to NADPH oxidase inhibition but may be explained at least in part by inhibition of Rho kinase activity. The discrepancy between apocynin-induced vasodilation in vitro and the failure of apocynin to lower arterial pressure in SHR suggests opposing effects on arterial pressure regulating systems in vivo. Its use as a pharmacological tool to investigate vascular NADPH oxidase should be discontinued.

Wistar rats underwent ischemia/reperfusion injury by ligation of the coronary artery followed by injection of adenovirus vectors for D1NLS and CDK4 with or without Skp2. Enhanced proliferation of cardiomyocytes in the presence of Skp2 was demonstrated by increased expression of Ki67, a marker of proliferating cells (1.95% vs 4.00%), and mitotic phosphorylated histone H3 (0.24% vs 0.58%). Compared to rats that received only D1NLS and CDK4, expression of Skp2 improved left ventricular function as measured by the maximum and minimum rates of change in left ventricular pressure, the left ventricle end-diastolic pressure, left ventricle end-diastolic volume index, and the lung/body weight ratio.

Expression of Skp2 enhanced the effect of D1NLS and CDK4 on the proliferation of cardiomyocytes and further contributed to improved post-ischemic cardiac function. Skp2 might be a versatile tool to improve the effect of cyclins on post-ischemic regeneration of cardiomyocytes in vivo.

Age-dependent effects on vascular function were determined in aortas of C57/Bl6 wild-type (WT), ALDH-2^–/–, MnSOD^+/+ and MnSOD^+/– mice by isometric tension measurements in organ chambers. Mitochondrial ROS formation was measured by luminol (L-012)-enhanced chemiluminescence and 2-hydroxyethidium formation with an HPLC-based assay in isolated heart mitochondria. ROS-mediated mitochondrial DNA (mtDNA) damage was detected by a novel and modified version of the fluorescent-detection alkaline DNA unwinding (FADU) assay.

Endothelial dysfunction was observed in aged C57/Bl6 WT mice in parallel to increased mitochondrial ROS formation and oxidative mitochondrial DNA damage. In contrast, middle-aged ALDH-2^–/– mice showed a marked vascular dysfunction that was similar in old ALDH-2^–/– mice suggesting that ALDH-2 exerts age-dependent vasoprotective effects. Aged MnSOD^+/– mice showed the most pronounced phenotype such as severely impaired vasorelaxation, highest levels of mitochondrial ROS formation and mtDNA damage.

The correlation between mtROS formation and acetylcholine-dependent relaxation revealed that mitochondrial radical formation significantly contributes to age-dependent endothelial dysfunction.

Using phosphoproteomic approaches, we investigated the profiles of phosphorylated proteins in Wistar rat heart mitochondria protected by pharmacological pre and postconditioning elicited by isoflurane. Sixty-one spots were detected by two-dimensional blue-native gel electrophoresis-coupled Western blotting using a phospho-Ser/Thr/Tyr-specific antibody, and 45 of these spots were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Eleven protein spots related to oxidative phosphorylation, energy metabolism, chaperone, and carrier functions exhibited significant changes in their phosphorylation state when protected mitochondria were compared with unprotected. Using a phosphopeptide enrichment protocol followed by liquid chromatography-MS/MS, 26 potential phosphorylation sites were identified in 19 proteins. Among these, a novel phosphorylation site was detected in adenine nucleotide translocator-1 (ANT1) at residue Tyr¹⁹⁴. Changes in ANT phosphorylation between protected and unprotected mitochondria were confirmed by immunoprecipitation. The biological significance of ANT phosphorylation at Tyr¹⁹⁴ was further tested with site-directed mutagenesis in yeast. Substitution of Tyr¹⁹⁴ with Phe, mimicking the non-phosphorylated state, resulted in the inhibition of yeast growth on non-fermentable carbon sources, implying a critical role of phosphorylation at this residue in regulating ANT function and cellular respiration.

Our analysis emphasizes the regulatory functions of the phosphoproteome in heart mitochondria and reveals a novel, potential link between bioenergetics and cardioprotection.

We used pharmacological, biochemical, and siRNA (small interfering ribonucleic acid) approaches to determine the signalling cascades of statin-induced 78 kDa glucose-regulated protein (GRP78) gene transcription and its role in cytoprotection. Exposure of RAW264.7 macrophages to statins increased the expression of GRP78, activating transcription factor 6, X box protein-1, and phosphorylated eukaryotic translation initiation factor 2, while it had no effect on CCAAT/enhancer binding protein-homologous protein. GRP78 induction was abolished by co-treatment with mevalonate and 1,2-bis(o-aminophenoxy)ethane-N, N, N',N'-tetraacetic acid, indicating the involvement of both 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase-dependent and -independent mechanisms. Studies on promoter activity measurements indicated that phosphoinositide turnover, cellular homologue of v-src (c-Src), protein kinase C (PKC), extracellular signal-regulated kinase (ERK), and p38 are involved in upregulating GRP78 gene transcription. We also observed that elevation of intracellular Ca²⁺ and interruption of small G proteins are two bifurcated but cooperative signalling pathways for c-Src activation, leading to downstream activation of phospholipase C, PKC, ERK, and p38. Functionally we demonstrated that fluvastatin could protect macrophages from hypoxia-induced cell death through GRP78 induction.

We demonstrate a novel action of statins of inducing a cytoprotective UPR, providing new insights into the clinical potential of statins for ameliorating ER stress-related diseases.

Experiments were performed in human umbilical vein endothelial cells (HUVEC) and murine lung endothelial cells (MLEC). A combination of specific inhibitors, RNA interference, and PI3K^–/– mice were used to investigate the role of PI3Kβ and isoforms in endothelial cell migration. Both PI3Kβ and isoforms are required for full migration induced by S1P, with Rac1 being a major mediator downstream of both isoforms. In addition, PI3Kβ but not PI3K mediates migration via Akt but independent of Rac1 and endothelial NO synthase (eNOS). Further, a S1P-mediated activation of extracellular signal-regulated kinases (Erk) 1/2 contributes to the chemotactic response independent of PI3Kβ or PI3K.

Our data demonstrate that both PI3Kβ and PI3K isoforms are required for S1P-induced endothelial cell migration through activation of Rac1. In addition, PI3Kβ initiates an Akt-sensitive chemotactic response which is independent of Rac1 and eNOS. Thus, PI3Kβ and PI3K have both overlapping and distinct roles in regulating endothelial cell migration, which may underlie S1P-triggered angiogenic differentiation.

Using proteomic techniques, two-dimensional electrophoresis, and mass spectrometry, the protein profiles of Sprague–Dawley rat aorta cultured under two levels of shear stress, 5 and 15 dyn/cm², were determined. The results showed a significantly lower expression of protein-Rho-GDP dissociation inhibitor alpha (Rho-GDI) in the LSS vessels. Rho-GDI signalling mechanisms and effects on VSMC migration and apoptosis were then studied to understand the role of Rho-GDI in the LSS-induced vascular remodelling. A decrease in Rho-GDI expression by using target small interfering RNA (siRNA) transfection caused increases in the phosphorylation of Rac1 and Akt and enhancements of VSMC migration and apoptosis. Treatment with the PI3K/Akt-specific inhibitor wortmannin significantly decreased Akt phosphorylation, but had no effect on Rho-GDI expression and Rac1 phosphorylation. Wortmannin was able to reverse the Rho-GDI siRNA-induced enhancement of VSMC migration, but not VSMC apoptosis.

The results indicate that the LSS-induced VSMC migration and apoptosis are mediated by a downregulation of Rho-GDI. The effect of Rho-GDI on VSMC migration is mediated by the PI3K/Akt pathway, but its effect on VSMC apoptosis is not.

Three days after surgery (SAD or sham operation), isolated aortic rings were exposed to phenylephrine and acetylcholine (0.1 nM to 10 µM) in the presence or absence of the gap junction blocker 18β-glycyrrhetinic acid (18β-GA, 100 µM). Vascular reactivity to potassium chloride (KCl, 4.7 to 120 mM) was also examined. The incidence of rats presenting oscillatory contractions was measured. Effects of SAD on the vascular smooth muscle expression of the Cx43 mRNA by RT-PCR and Western blotting for Cx43 protein were examined.

Phenylephrine-induced contraction was higher in SAD rat aortas compared with the control. In the presence of 18β-GA, the response to phenylephrine was similar in both groups. Oscillatory contractions were observed in 10/10 SAD rat aortas vs. 2/10 controls. Relaxing response to acetylcholine was similar in both groups, but in the presence of 18β-GA, the response to acetylcholine decreased significantly in the sham-operated group (82.7±7.6% reduction of relaxation), whereas a half-maximal relaxation (reduction of 46.2±5.3%) took place in SAD rat aortas. KCl-induced contraction was similar in both groups. Following SAD, RT-PCR revealed significantly increased levels of Cx43 mRNA (9.85 fold, P<0.01). Western blot analysis revealed greater levels of Cx43 protein (p<0.05).

Blood pressure variability evoked by SAD leads to increased expression of Cx43, which could contribute to enhanced phenylephrine-induced contraction and oscillatory activity in isolated aortas.

Human HepG2 hepatocarcinoma cells and murine RAW264.7 macrophages were treated with simvastatin, with and without Hcy, to examine the expression of ApoA-I and nuclear factor-B (NF-B) or the NF-B target, inducible nitric-oxide synthase (iNOS), respectively. Mice with methylenetetrahydrofolate reductase (Mthfr) deficiency, an animal model of hyperhomocysteinemia, were administered simvastatin (in diets or by injection) for in vivo assessment of these interactions. In HepG2 cells, Hcy reduced the statin-dependent increases in ApoA-I protein, mRNA, and ApoA-I promoter activity. In RAW264.7 macrophages, simvastatin decreased, whereas Hcy increased, the expression of pro-inflammatory NF-B protein; in the presence of both Hcy and simvastatin, the pro-inflammatory effect of Hcy prevailed. Hcy increased mRNA levels of iNOS in the macrophage line; the combination of Hcy and simvastatin resulted in a trend towards greater induction. In mouse studies, simvastatin decreased cholesterol levels, but levels of ApoA-I in Mthfr-deficient mice remained lower than those in Mthfr^+/+ mice. Simvastatin injection increased iNOS protein and mRNA levels in peripheral blood of hyperhomocysteinemic Mthfr-deficient mice, but not in Mthfr^+/+ mice. The drug also increased MTHFR protein in cells and mouse liver, an effect that was modified by Hcy.

These findings provide a link between statins and folate-dependent Hcy metabolism, and suggest that Hcy interferes with some anti-atherogenic and anti-inflammatory properties of simvastatin. Our work may have clinical relevance for hyperhomocysteinemic individuals on statin therapy.

Exposure of human umbilical vein endothelial cells (HUVECs) to high glucose (HG, 30 mM D-glucose) time-dependently increased both the Ca²⁺_i concentration and calpain activity. Chelation of free Ca²⁺_i with 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) abolished the HG-increased calpain activity. In addition, HG activated NHE in a time-dependent manner, but cariporide, an NHE inhibitor, blocked the HG-induced increase of NHE activity. Furthermore, cariporide or NHE siRNA attenuated the HG-induced increases of both Ca²⁺_i concentration and calpain activity. All of these HG-induced effects in HUVECs, including decreased eNOS activity and NO production and increased dissociation of hsp90 from eNOS, were NHE or calpain reversible. In vivo experiments showed that cariporide treatment via inhibition of NHE activity significantly attenuated the hyperglycemia-induced impairment of acetylcholine-induced, endothelium-dependent relaxation in streptozotocin-injected diabetic rats.

Activation of NHE via calcium-dependent calpain contributes to hyperglycemia-induced endothelial dysfunction through dissociation of eNOS from hsp90.

To study the effect of oral corticosteroids on cardiac function, we treated 8-week-old Sgcd-null mice with prednisolone (1.5 mg/kg body weight/day orally) for 8 weeks. In vivo cardiac function was assessed by pressure–volume loops using a conductance catheter. We found a well-compensated cardiomyopathy at baseline in Sgcd-null mice with decreased myocardial contractility, increased preload, and decreased afterload, maintaining a high cardiac output. Cardiac haemodynamics, surprisingly, did not improve in prednisolone-treated mice, but instead deteriorated with evidence of ventricular stiffening. On histology, after steroid treatment there was increased myocardial cell damage and increased myocardial fibrosis.

Prednisolone led to a decompensation of cardiac haemodynamics in Sgcd-null mice and induced additional cardiac damage. On the basis of these findings, although mouse models may not completely replicate the human situation for LGMD2F, we conclude that careful cardiac monitoring is clearly indicated in patients on long-term corticosteroids.

Analysis of heart rate data collected over 10 days in eight intact and six SCN-lesioned Wistar rats during constant darkness revealed that: (i) as with humans, rats exhibit an endogenous circadian rhythm in the scaling exponent characterizing the hourly fractal structure of heart rate (P= 0.0005) with larger exponents during the biological day (inactive phase for rats; active phase for humans); (ii) SCN lesioning abolished the rhythm in the fractal structure of heart rate and systematically increased the scaling exponent (P= 0.01).

Rats possess a circadian rhythm of fractal structure of heart rate with a similar temporal pattern as previously observed in humans despite opposite rest/activity cycles between the two species. The SCN imparts this endogenous rhythm. Moreover, lesioning the SCN in rats results in a larger scaling exponent, as occurs with cardiovascular disease in humans.

The transgenic zebrafish line, T2EGEZ8, was generated by insertion of a Tol2 transposon gene trap vector, and homozygous mutants (T2EGEZ8^m/m) of this line showed failure of embryonic heartbeat without other detectable phenotypes. Observation by transmission electron microscopy revealed that the ventricular myocytes of mutant fish contained fewer, disorganized myofibrillar filaments. The transposon genome in T2EGEZ8 fish was found by tail-PCR and RT-PCR to have inserted into the 9th intron of the tpm4 locus, which resulted in production of Tpm4-GFP fusion proteins and loss of normal transcripts tpm4-tv1 and tpm4-tv2. Whole-mount in situ hybridization indicated that tpm4-tv1, encoding a peptide of 284 residues, is specifically expressed in the heart of zebrafish embryos while tpm4-tv2, encoding a peptide of 248 residues, is mainly present in the vasculature but absent in the heart. Knockdown of tpm4-tv1 and tpm4-tv2 within wild-type embryos led to the failure of heartbeat, which could be rescued by coinjection with tpm4-tv1 mRNA but not with tpm4-tv2 mRNA.

Tpm4-tv1 is a heart-specific isoform of Tpm4 and is essential for heartbeat in zebrafish embryos.

Experiments were performed in a rabbit model of MI. The infarct size was reduced, and LV remodelling and function were improved 14 days and 2 months after MI but not at 2 days after MI in the EPO-DDS group. The number of cluster of differentiation 31(CD31)-positive microvessels and the expression of erythropoietin receptor (EPO-R), phosphorylated-Akt (p-Akt), phosphorylated glycogen synthase kinase 3β (p-GSK-3β), phosphorylated extracellular signal-regulated protein kinase (p-ERK), phosphorylated signal transducer and activator of transcription 3 (p-Stat3), vascular endothelial growth factor (VEGF), and matrix metalloproteinase-1 (MMP-1) were significantly increased in the myocardium of the EPO-DDS group.

Post-MI treatment with an EPO-DDS improves LV remodelling and function by activating prosurvival signalling, antifibrosis, and angiogenesis without causing any side effect.

The culture medium supernatant of H9c2 cells exposed to sI/R was separated by column chromatography and the fractions examined for survival activity. The protein with survival activity was identified by mass spectrometry, and its physiological role was examined in the models of ischaemia. Cell survival activity was detected in at least three fractions of the cell supernatant collected during sI/R and subjected to a series of column chromatographic steps. Among the proteins measured by mass spectrometry and western blotting, a p36 protein identified as a glycolytic enzyme, lactate dehydrogenase muscle subunit (M-LDH), showed strong survival activity. H₂O₂-induced intracellular calcium overload in H9c2 cells and irregular Ca²⁺ transients in adult rat cardiomyocytes were both found to be inhibited by pretreatment with M-LDH. M-LDH also lowered the frequency and amplitude of early afterdepolarizations induced by H₂O₂ in adult rat cardiomyocytes and suppressed the ischaemia–reperfusion-induced reduction of cardiac output from mouse working heart preparations. M-LDH was found to increase the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), which plays a role in H9c2 cell survival.

M-LDH released from cardiomyocytes after hypoxia and reoxygenation has a role in protecting the heart from oxidative stress-induced injury through an intracellular signal transduction pathway involving ERK1/2.

We used male Wistar rats to examine the effect of hyperglycemia on PPAR expression in streptozotocin-induced diabetic rats, primary neonatal rat cardiomyocytes, and H9c2 embryonic rat cardiomyocytes. PPAR mRNA and protein levels were measured using Northern and Western blotting, respectively. The lipid deposition within the heart section was assessed by oil red O staining. The formation of reactive oxygen species (ROS) and changes in morphology, protein synthesis, and -actinin content in hyperglycemic cells were also examined. Inhibitors of ROS production or mitogen-activated protein kinase (MAPK) activation were employed to investigate the possible mechanisms.

Cardiomyopathy induced in streptozotocin-diabetic rats was associated with a marked decrease in cardiac PPAR expression. Also, ROS production, cell size, and protein synthesis were increased while PPAR expression was reduced in cells exposed to hyperglycemia in vitro. However, these glucose-induced changes were abolished in the presence of tiron or PD98059 (MEK/ERK inhibitor).

Our results suggest that inhibitors of ROS production or MAPK activation are involved in reduction of cardiac PPAR expression in response to hyperglycemia.

This tissue graft (sandwiched patch) was used to replace the infarcted wall in a syngeneic Lewis rat model with an experimentally chronic myocardial infarction (MI). There were four treatment groups (n ≥ 10): sham, MI, empty patch, and sandwiched patch. After a 7 day culture of the sandwiched patch, a tissue graft with relatively uniform cell concentrations was obtained. The cells were viable and tightly adhered to the tissue scaffold, as the endogenous extracellular matrix inherent with multilayered cell sheets can act as an adhesive agent for cell attachment and retention. At retrieval, the area of the empty patch was relatively enlarged, suggesting reduced structural support, while that of the sandwiched patch remained about the same (P = 0.56). In the immunofluorescent staining, host cells together with neo-microvessels were clearly observed in the empty patch; however, there were still a large number of unfilled pores within the patch. In the sandwiched patch, besides host cells, originally seeded cells were populated within the entire patch. No apparent evidence of apoptotic cell death was found in both studied patches. Thus, the sandwiched-patch-treated hearts demonstrated a better heart function to the empty-patch-treated hearts (P < 0.05).

The results demonstrated that this novel bioengineered tissue graft can serve as a useful cardiac patch to restore the dilated left ventricle and stabilize heart functions after MI.

Using the whole-cell patch clamp technique, we found that AB neuron excitability (action potential frequency at 50 pA current stimulation) in the T1D rats was lower than that in the sham rats (0.4 ± 0.5 vs. 4.8 ± 0.6 spikes/s, P < 0.05; AB neurons were identified by DiI staining). In addition, HCN current density in AB neurons from the T1D rats was bigger than that from the sham rats (60.2 ± 6.1 vs. 30.7 ± 4.9 pA/pF at test pulse –140 from holding potential –40 mV, P < 0.05). Furthermore, HCN channel blockers (5 mM cesium chloride and 100 µM ZD7288) significantly reduced HCN currents and increased action potential frequency of the AB neurons in sham and T1D rats. Immunofluorescent and western blot analyses demonstrated that the expression of HCN1 and HCN2 channel protein in the NG from the T1D rats was higher than that from the sham rats.

These results indicate that the HCN channels influence the excitability of AB neurons, and more importantly, contribute to the decreased excitability of AB neurons in T1D rats.

RT-PCR, real-time PCR, immunoblotting, and confocal microscopic image analysis were employed to determine protein expression, small Rho-GTPase Rac1 activation, and ROS production. Inhibition of NADPH oxidase 4 (Nox4) or Rac1 was performed by means of siRNA technology. Inhibition of Rac1 activity was accomplished using dominant-negative form of Rac1 (N17Rac1) plasmid. VSMCs from Sprague-Dawley rat thoracic aortas were used in this work.

IGF-I enhanced ROS production in rat VSMCs. IGF-I increased the protein level of Nox4 but had little effect on its mRNA level. IGF-I induced the activation of Rac1. Either knockdown of Nox4 or inactivation of Rac1 impaired IGF-I-induced ROS. Over-expression of Nox4 increased NADPH oxidase activity, which was not influenced by inactivation of Rac1. Neither over-expression nor knockdown of Rac1 influenced Nox4 expression. Knockdown of Nox4 did not affect IGF-I-induced activation of Rac1. IGF-I increased matrix metalloproteinase (MMP) 2 and 9 activity and promoted VSMC migration, which was inhibited by knockdown of Nox4 and inactivation of Rac1.

Our results suggest that Nox4 and Rac1 mediate IGF-I-induced ROS production and cell migration in VSMCs and that Nox4 is not regulated by Rac1.

Neonatal rat ventricular cardiomyocytes were incubated with DOX (0.5 µM) and/or PGE₂ (1 µM). Apoptosis was assessed by determining caspase3 activation and apoptotic DNA fragmentation. The role of Stat3 was evaluated in vitro and in vivo by transfecting cardiomyocytes with siRNA targeting rat Stat3 and by using cardiomyocyte-restricted Stat3 knockout (Stat3 KO) mice, respectively.

Incubation of ventricular cardiomyocytes with PGE₂ led to a time-dependent decrease in the DOX –induced caspase3 activation, reaching a maximal inhibition of 70±5% after 4 h. Similarly, PGE₂ inhibited DOX-induced DNA fragmentation by 58±5% after 24 h. This antiapoptotic action of PGE₂ was strongly reduced by the ERK1/2 inhibitor, U0126, while the p38 MAP kinase inhibitor, SB203580, had no effect. Depleting Stat3 expression by 50-60% in isolated ventricular cardiomyocytes markedly reduced the protective effect of PGE₂ on DOX-induced caspase3 activation and DNA fragmentation. Likewise, the stable PGE₂ analogue, 16,16-dimethyl-PGE₂, was unable to counteract cardiac apoptosis induced by DOX in Stat3 KO mice.

Our results demonstrate that PGE₂ prevents myocardial apoptosis induced by DOX. This protection requires the activation of the prosurvival pathways of Stat3 and ERK1/2.

First, we analyzed the angiogenic properties of bradykinin and enalapril on wild-type C57Bl/6 and B2 receptor-/- mouse heart under normoxia (21% O2) and hypoxia (1% O2) in vitro and the contribution of B1 and B2 kinin receptors to this effect. Bradykinin induced dose-dependent endothelial sprout formation in vitro in adult mouse heart only under hypoxia (1.7 fold, n = 6, p < 0.05). The B2 receptor mediated sprouting that was induced by bradykinin and vascular endothelial growth factor (VEGF164; n = 6, p < 0.05) but did not mediate sprouting that was induced by growth factors bFGF or PDGF-BB. Enalapril induced sprouting through both the B1 and B2 kinin receptors, but it required the presence of the B2 receptor in both scenarios and was dependent on BK synthesis. B1-receptor agonists induced sprout formation via the B1 receptor (2.5 fold, n = 6, p < 0.05), but it required the presence of the B2 receptor for them to do so. Both B2-receptor and B1-receptor agonist-induced angiogenesis required nitric oxide biosynthesis.

The kinin B2 receptor plays a crucial role in angiogenesis that is induced by different vasoactive molecules, namely bradykinin, ACE inhibitors, B1-stimulating kinin metabolites, and VEGF164 in an in vitro model of angiogenesis of mouse heart under hypoxia. Therapeutic treatment of hypertensive patients by using ACE inhibitors may potentially benefit the ischemic heart through inducing B2-dependent heart neovascularization.

For binding studies after ligation-induced arterial injury, the left common carotid artery of C57BL/6J mice was ligated. For binding studies at spontaneously formed atherosclerotic lesion sites, Apolipoprotein E-deficient (ApoE^–/–) mice were fed a 0.25 % cholesterol diet over 16 weeks. Binding of [¹²⁴I]GPVI-Fc was monitored by autoradiography 48 hours after intravenous injection and by immunostaining. To study the effect of GPVI-Fc on neointima formation versus control-Fc, a wire-induced injury of the left A. carotis communis of ApoE^–/–-mice was performed. Mice were treated intraperitoneally with GPVI-Fc for 8 days and neointima formation was assessed 4 weeks after intervention.

[¹²⁴I]GPVI-Fc preferentially bound to injury sites after carotid ligation in C57BL/6J mice and to lipid-rich atherosclerotic lesions of the carotid artery and aortic arch in uninjured ApoE^–/–-mice. Histological examinations of wire-injured carotid arteries showed that neointima formation was significantly reduced in GPVI-Fc-treated ApoE^–/– mice compared to ApoE^–/– mice receiving control-Fc (p < 0.05).

GPVI-Fc preferentially bound to sites of vascular injury and was able to inhibit neointima formation after wire-induced vascular injury in ApoE^–/– mice. Thus, soluble GPVI-Fc might be also a promising compound to attenuate lesion progression after plaque rupture.

Electrophysiological measurements were performed in cardiomyocytes isolated from Wistar-Kyoto and spontaneously hypertensive rats. Bath application of 100 µM NaHS (a H₂S donor) significantly reduced the time required for the repolarization of the action potential. Inhibition of the peak I_Ca,L by NaHS was determined to be concentration-dependent (25, 50, 100, 200, and 400 µM). NaHS inhibited the recovery from depolarization-induced inactivation. Electric field-induced [Ca²⁺]i transients and contraction of single cardiomyocytes and isolated papillary muscles were reduced by NaHS treatment. In contrast, caffeine induced an increase in [Ca²⁺]i that was not altered by NaHS. NaHS had no effect on the K_ATP current or on the levels of cAMP and cGMP in the current study.

H₂S is a novel inhibitor of L-type calcium channels in cardiomyocytes. Moreover, H₂S-induced inhibition of [Ca²⁺]i appears to be a secondary effect owing to its initial action towards I_Ca,L. The inhibitory effect of H₂S on I_Ca,L requires further investigation, particularly in the exploration of new pathways involved in cardiac calcium homeostasis and disease pathology.

We employed chimeric decoy oligodeoxynucleotides (ODN) to inhibit both NFB and E2F simultaneously, and examined the effects of chimeric decoy ODN on the proliferation and migration of cultured vascular cells and on the formation of neointimal hyperplasia using prosthetic graft placement in a rabbit hypercholesterolemia model. Our in vitro study demonstrated that transfection of chimeric decoy ODN inhibited platelet-derived growth factor (PDGF)-induced proliferation and migration of vascular smooth muscle cells, whereas endothelial cell proliferation was not inhibited. In an in vivo study, treatment with chimeric decoy ODN significantly inhibited proximal and distal anastomotic intimal hyperplasia, and accelerated re-endothelialization. -Smooth muscle actin (-SMA)-positive cell proliferation was inhibited at the anastomotic sites. Expression of PDGF-BB and PDGF receptor-β was also suppressed by chimeric decoy ODN, resulting in a reduction of -SMA-positive cell accumulation. In addition, chimeric decoy ODN treatment inhibited macrophage accumulation, which was accompanied by a reduction of vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1 gene expression.

The present study demonstrates the feasibility of chimeric decoy ODN treatment for preventing neointimal formation. This strategy might be useful to improve the clinical outcome after arterial reconstruction.

The left anterior descending coronary artery was ligated in HMGB1-Tg and wild-type littermate (Wt) mice. After coronary artery ligation, HMGB1 was released into circulation from the necrotic cardiomyocytes of HMGB1-overexpressing hearts. The size of myocardial infarction was smaller in HMGB1-Tg than in Wt mice. Echocardiography and cardiac catheterization demonstrated that cardiac remodeling and dysfunction after myocardial infarction were prevented in HMGB1-Tg mice compared to Wt mice. Furthermore, the survival rate after myocardial infarction of HMGB1-Tg mice was higher than that of Wt mice. Immunohistochemical staining revealed that capillary and arteriole formation after myocardial infarction was enhanced in HMGB1-Tg mice.

We report the first in vivo evidence that HMGB1 enhances angiogenesis, restores cardiac function, and improves survival after myocardial infarction. These results may provide a novel therapeutic approach for left ventricular dysfunction after myocardial infarction.

We have heterologously expressed K_ATP channel subunits in HEK293 and CHO cells and studied their function using ⁸⁶Rb efflux and patch clamping. We show that rodent Kir6.1/SUR2B has direct intrinsic metabolic sensitivity independent of any regulation by protein kinase A. In contrast to Kir6.2 containing complexes, this was not endowed by the ATP sensitivity of the pore forming subunit but was instead a property of the SUR2B subunit. Mutagenesis of key residues within the nucleotide-binding domains (NBD) implicated both domains in governing the metabolic sensitivity.

Kir6.1\SUR2B has intrinsic sensitivity to metabolism endowed by the likely processing of adenine nucleotides at the NBD of SUR2B.

Wistar-Furth rat cardiac allografts were perfused ex vivo with AP-1 decoy ODN, STAT-1 decoy ODN, or buffer solution and transplanted into the abdomen of Lewis rats immunosuppressed with cyclosporine. Treatment with both decoy ODNs but not vehicle significantly attenuated the incidence and severity of CAV. Laser-assisted microdissection/real-time polymerase chain reaction as well as immunohistochemistry analyses revealed a significant increase in CD40 abundance in the coronary endothelial cells and medial smooth muscle cells on day 1 post transplantation which was virtually abolished upon AP-1 or STAT-1 decoy ODN treatment. While the AP-1 decoy ODN primarily attenuated basal CD40 expression, the STAT-1 decoy ODN suppressed tumour necrosis factor--/interferon--stimulated expression of CD40 in rat native endothelial cells.

Treating donor hearts with decoy ODNs neutralizing AP-1 or STAT-1 at the time of transplantation prevents upregulation of CD40 expression in the graft coronary arteries and effectively inhibits CAV.

Rat neonatal cardiomyocytes were used in this study. Proteasome activity was assayed using proteasome peptidase substrates. Cell viability and apoptosis were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide and flow cytometry, respectively. Western blot analysis, real-time polymerase chain reaction (PCR) and reverse transcriptional PCR were used to detect the expression of protein and messenger ribonucleic acid (RNA). The location of overexpressed glucose-regulated protein (GRP) 78 was observed by confocal fluorescence microscopy. Proteasome inhibition induced cardiomyocyte death and activated ER stress-induced transcriptional factor ATF6, but not XBP1 (X-box binding protein 1), without up-regulating ER chaperones. ER-initiated apoptosis signalling, including cytosine-cytosine-adenine-adenine-thymine enhancer-binding protein (C/EBP) homologous protein (CHOP), c-Jun-N-terminal kinase (JNK), and caspase-12, was activated by proteasome inhibition. Short interference RNA targeting CHOP, but not the blockage of caspase-12 or JNK pathway, attenuated cardiomyocyte death. Overexpression of GRP78 suppressed both CHOP expression and cardiomyocyte death by proteasome inhibition.

These findings demonstrate that proteasome inhibition induces ER-initiated cardiomyocyte death via CHOP-dependent pathways without compensatory up-regulation of ER chaperones. Supplement and/or pharmacological induction of GRP78 can attenuate cardiac damage by proteasome inhibition.

Echocardiography and pressure–volume conductance catheters were used to assess left ventricular (LV) structure and function in rats subjected to four ecstasy binges (9 mg/kg i.v. for 4 days, separated by a 10 day drug-free period). Hearts from treated and control rats were used for either biochemical and proteomic analysis or the isolation of adult LV myocytes. After the fourth binge, treated hearts showed eccentric LV dilation and diastolic dysfunction. Systolic function was not altered in vivo; however, the magnitude of the contractile responses to electrical stimulation was significantly smaller in myocytes from rats treated in vivo with ecstasy compared with myocytes from control rats. The magnitude of the peak increase in intracellular calcium (measured by Fura-2) was also significantly smaller in myocytes from ecstasy-treated vs. control rats. The relaxation kinetics of the intracellular calcium transients were significantly longer in myocytes from ecstasy-treated rats. Ecstasy significantly increased nitrotyrosine content in the left ventricle. Proteomic analysis revealed increased nitration of contractile proteins (troponin-T, tropomyosin alpha-1 chain, myosin light polypeptide, and myosin regulatory light chain), mitochondrial proteins (Ub-cytochrome-c reductase and ATP synthase), and sarcoplasmic reticulum calcium ATPase.

The repeated binge administration of ecstasy produces eccentric LV dilation and dysfunction that is accompanied by oxidative stress. These functional responses may result from the redox modification of proteins involved in excitation-contraction coupling and/or mitochondrial energy production. Together, these results indicate that ecstasy has the potential to produce serious cardiac toxicity and ventricular dysfunction.

The hyperpermeability-inducer thrombin caused a Rho kinase-dependent redistribution of β-catenin from the membrane to the cytosol as evidenced by the western blot analysis of membrane and cytosol fractions and by immunohistochemistry. Glycogen synthase kinase 3β, which phosphorylates cytosolic β-catenin and thereby facilitates its proteasomal degradation, was inhibited by thrombin. The analysis of nuclear extracts demonstrated a thrombin-induced nuclear accumulation of β-catenin as well as p120ctn. Thrombin stimulation activated β-catenin-mediated transcriptional activity as evidenced by reporter assays. Finally, real-time-PCR revealed increased mRNA levels of several β-catenin target genes.

Thrombin induced a cytosolic stabilization of membrane-liberated β-catenin, which, together with p120ctn, subsequently translocated to the nucleus where it induces several β-catenin target genes. This supports the suggestion that membrane-liberated β-catenin and p120ctn contribute to angiogenic responses of ECs following episodes of vascular leakage.

Isometric twitch force of human atrial trabeculae (n = 252) was recorded (37°C, 1 Hz stimulation) following stretch from 88 (L88) to 98% (L98) of optimal length. [Na⁺]_i and pH_i were measured using SBFI and BCECF epifluorescence, respectively. Stretch induced a biphasic force increase: an immediate increase [first-phase, Frank–Starling mechanism (FSM)] to ~190% of force at L88 followed by an additional slower increase [5–10 min; slow force response (SFR)] to ~120% of the FSM. FSM and SFR were unaffected by gender, age, ejection fraction, and pre-medication with major cardiovascular drugs. There was a positive correlation between the amplitude of the FSM and the SFR. [Na⁺]_i rose by ~1 mmol/L and pH_i remained unchanged during the SFR. Inhibition of Na⁺/H⁺-exchange (3 µM HOE642), Na⁺/Ca²⁺-exchange (5 µM KB-R7943), or stretch-activated channels (0.5 µM GsMtx-4 and 80 µM streptomycin) did not reduce the SFR. Inhibition of angiotensin-II (AngII) receptors (5 µM saralasin and 0.5 µM PD123319) or pre-application of 0.5 µM AngII, however, reduced the SFR by ~40–60%. Moreover, stretch increased phosphorylation of myosin light chain 2 (MLC2a) and inhibition of MLC kinase (10 µM ML-7 and 5 µM wortmannin) decreased the SFR by ~40–85%.

Stretch elicits a SFR in human atrium. The atrial SFR is mediated by stretch-induced release and autocrine/paracrine actions of AngII and increased myofilament Ca²⁺ responsiveness via phosphorylation of MLC2a by MLC kinase.

Rats were implanted with intracerebroventricular (ICV) cannulae and subjected to coronary artery ligation, or sham surgery (SHAM). Subsequently, animals were ICV treated with the angiotensin type 1 receptor (AT1-R) antagonist losartan (LOS, 20 µg/h), or SN50 (2 µg/h), which inhibits nuclear translocation of NF-B, or tempol (TEMP, 80 µg/h), a membrane-permeable superoxide scavenger, or vehicle for 4 weeks. HF induced a significant increase in the expression of AT1-R, PIC, and NAD(P)H oxidase genes and NF-B p50 in the PVN and in plasma norepinephrine (NE) levels when compared with SHAM rats. In contrast, ICV LOS, SN50, or TEMP attenuated PIC, NF-B p50, AT1-R and NAD(P)H oxidase genes in the PVN compared with vehicle-treated HF rats. Treatment with LOS, SN50, or TEMP also reduced plasma levels of NE, angiotensin II, and PIC, and decreased left ventricular end diastolic pressure.

These findings indicate that NF-B mediates the cross-talk between RAS and PIC in the PVN in HF, and that superoxide stimulates more NF-B in the PVN and contributes to neurohumoral excitation.

S1P inhibition of PDGF-induced cell migration and Rac activation in VSMCs was abolished by the selective S1P₂ receptor antagonist JTE-013. The C-terminal peptides of G subunits (G-CTs) act as specific inhibitors of respective G protein-GPCR coupling. Adenovirus-mediated expression of G₁₂-CT, G₁₃-CT, and G_q-CT, but not that of G_s-CT or LacZ or pertussis toxin treatment, abrogated S1P inhibition of PDGF-induced Rac activation and migration, indicating that both G_12/13 and G_q classes are necessary for the S1P inhibition. The expression of G_q-CT as well as G₁₂-CT and G₁₃-CT also abolished S1P-induced Rho stimulation. C3 toxin, but not a Rho kinase inhibitor or a dominant negative form of Rho kinase, abolished S1P inhibition of PDGF-induced Rac activation and cell migration. The angiotensin II receptor AT₁, which robustly couples to G_q, did not mediate either Rho activation or inhibition of PDGF-induced Rac activation or migration, suggesting that activation of G_q alone was not sufficient for Rho activation and resultant Rac inhibition. However, the AT₁ receptor fused to G₁₂ was able to induce not only Rho stimulation but also inhibition of PDGF-induced Rac activation and migration. Phospholipase C inhibition did not affect S1P-induced Rho activation, and protein kinase C activation by a phorbol ester did not mimic S1P action, suggesting that S1P inhibition of migration or Rac was not dependent on the phospholipase C pathway.

These observations together suggest that S1P₂ mediates inhibition of Rac and migration through the coordinated action of G_12/13 and G_q for Rho activation in VSMCs.

Xenopus laevis embryos were sectioned, and whole-mount antibody staining and immunofluorescence studies were carried out to determine the total number and percentage of differentiated cardiomyocytes in mitosis. Capped RNA and/or translation-blocking Xic1 morpholino antisense oligonucleotides (Xic1Mo) were microinjected into embryos, and their role on cardiac differentiation was assessed by in situ hybridization and/or PCR. We show that cell-cycling post-gastrulation is not essential for cardiac differentiation in Xenopus embryos, and conversely that some cells can express markers of cardiac differentiation even when still in cycle. A targeted knock-down of Xic1 protein by Xic1Mo microinjection decreases the expression of markers of cardiac differentiation, wh