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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.