Hiroshi Asanuma

Human atrial natriuretic peptide and nicorandil as adjuncts to reperfusion treatment for acute myocardial infarction (J-WIND): two randomised trials.

BACKGROUNDnPatients who have acute myocardial infarction remain at major risk of cardiovascular events. We aimed to assess the effects of either human atrial natriuretic peptide or nicorandil on infarct size and cardiovascular outcome.nnnMETHODSnWe enrolled 1216 patients who had acute myocardial infarction and were undergoing reperfusion treatment in two prospective, single-blind trials at 65 hospitals in Japan. We randomly assigned 277 patients to receive intravenous atrial natriuretic peptide (0.025 microg/kg per min for 3 days) and 292 the same dose of placebo. 276 patients were assigned to receive intravenous nicorandil (0.067 mg/kg as a bolus, followed by 1.67 microg/kg per min as a 24-h continuous infusion), and 269 the same dose of placebo. Median follow-up was 2.7 (IQR 1.5-3.6) years for patients in the atrial natriuretic peptide trial and 2.5 (1.5-3.7) years for those in the nicorandil trial. Primary endpoints were infarct size (estimated from creatine kinase) and left ventricular ejection fraction (gauged by angiography of the left ventricle).nnnFINDINGSn43 patients withdrew consent after randomisation, and 59 did not have acute myocardial infarction. We did not assess infarct size in 50 patients for whom we had fewer than six samples of blood. We did not have angiographs of left ventricles in 383 patients. Total creatine kinase was 66,459.9 IU/mL per h in patients given atrial natriuretic peptide, compared with 77,878.9 IU/mL per h in controls, with a ratio of 0.85 between these groups (95% CI 0.75-0.97, p=0.016), which indicated a reduction of 14.7% in infarct size (95% CI 3.0-24.9%). The left ventricular ejection fraction at 6-12 months increased in the atrial natriuretic peptide group (ratio 1.05, 95% CI 1.01-1.10, p=0.024). Total activity of creatine kinase did not differ between patients given nicorandil (70 520.5 IU/mL per h) and controls (70 852.7 IU/mL per h) (ratio 0.995, 95% CI 0.878-1.138, p=0.94). Intravenous nicorandil did not affect the size of the left ventricular ejection fraction, although oral administration of nicorandil during follow-up increased the left ventricular ejection fraction between the chronic and acute phases. 29 patients in the atrial natriuretic peptide group had severe hypotension, compared with one in the corresponding placebo group.nnnINTERPRETATIONnPatients with acute myocardial infarction who were given atrial natriuretic peptide had lower infarct size, fewer reperfusion injuries, and better outcomes than controls. We believe that atrial natriuretic peptide could be a safe and effective adjunctive treatment in patients with acute myocardial infarction who receive percutaneous coronary intervention.

Targeting of both mouse neuropilin-1 and neuropilin-2 genes severely impairs developmental yolk sac and embryonic angiogenesis

Neuropilins (NP1 and NP2) are vascular endothelial growth factor (VEGF) receptors that mediate developmental and tumor angiogenesis. Transgenic mice, in which both NP1 and NP2 were targeted (NP1−/−NP2−/−) died in utero at E8.5. Their yolk sacs were totally avascular. Mice deficient for NP2 but heterozygous for NP1 (NP1+/−NP2−/−) or deficient for NP1 but heterozygous for NP2 (NP1−/−NP2+/−) were also embryonic lethal and survived to E10–E10.5. The E10 yolk sacs and embryos were easier to analyze for vascular phenotype than the fragile poorly formed 8.5 embryos. The vascular phenotypes of these E10 mice were very abnormal. The yolk sacs, although of normal size, lacked the larger collecting vessels and had less dense capillary networks. PECAM staining of yolk sac endothelial cells showed the absence of branching arteries and veins, the absence of a capillary bed, and the presence of large avascular spaces between the blood vessels. The embryos displayed blood vessels heterogeneous in size, large avascular regions in the head and trunk, and blood vessel sprouts that were unconnected. The embryos were about 50% the length of wild-type mice and had multiple hemorrhages. These double NP1/NP2 knockout mice had a more severe abnormal vascular phenotype than either NP1 or NP2 single knockouts. Their abnormal vascular phenotype resembled those of VEGF and VEGFR-2 knockouts. These results suggest that NRPs are early genes in embryonic vessel development and that both NP1 and NP2 are required.

Metformin Prevents Progression of Heart Failure in Dogs Role of AMP-Activated Protein Kinase

Background— Some studies have shown that metformin activates AMP-activated protein kinase (AMPK) and has a potent cardioprotective effect against ischemia/reperfusion injury. Because AMPK also is activated in animal models of heart failure, we investigated whether metformin decreases cardiomyocyte apoptosis and attenuates the progression of heart failure in dogs. Methods and Results— Treatment with metformin (10 &mgr;mol/L) protected cultured cardiomyocytes from cell death during exposure to H2O2 (50 &mgr;mol/L) via AMPK activation, as shown by the MTT assay, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling staining, and flow cytometry. Continuous rapid ventricular pacing (230 bpm for 4 weeks) caused typical heart failure in dogs. Both left ventricular fractional shortening and left ventricular end-diastolic pressure were significantly improved in dogs treated with oral metformin at 100 mg · kg−1 · d−1 (n=8) (18.6±1.8% and 11.8±1.1 mm Hg, respectively) compared with dogs receiving vehicle (n=8) (9.6±0.7% and 22±0.9 mm Hg, respectively). Metformin also promoted phosphorylation of both AMPK and endothelial nitric oxide synthase, increased plasma nitric oxide levels, and improved insulin resistance. As a result of these effects, metformin decreased apoptosis and improved cardiac function in failing canine hearts. Interestingly, another AMPK activator (AICAR) had effects equivalent to those of metformin, suggesting the primary role of AMPK activation in reducing apoptosis and preventing heart failure. Conclusions— Metformin attenuated oxidative stress–induced cardiomyocyte apoptosis and prevented the progression of heart failure in dogs, along with activation of AMPK. Therefore, metformin may be a potential new therapy for heart failure.

Ablation of C/EBP Homologous Protein Attenuates Endoplasmic Reticulum–Mediated Apoptosis and Cardiac Dysfunction Induced by Pressure Overload

Background— Apoptosis may contribute to the development of heart failure, but the role of apoptotic signaling initiated by the endoplasmic reticulum in this condition has not been well clarified. Methods and Results— In myocardial samples from patients with heart failure, quantitative real-time polymerase chain reaction revealed an increase in messenger RNA for C/EBP homologous protein (CHOP), a transcriptional factor that mediates endoplasmic reticulum–initiated apoptotic cell death. We performed transverse aortic constriction or sham operation on wild-type (WT) and CHOP-deficient mice. The CHOP-deficient mice showed less cardiac hypertrophy, fibrosis, and cardiac dysfunction compared with WT mice at 4 weeks after transverse aortic constriction, although the contractility of isolated cardiomyocytes from CHOP-deficient mice was not significantly different from that in the WT mice. In the hearts of CHOP-deficient mice, phosphorylation of eukaryotic translation initiation factor 2&agr;, which may reduce protein translation, was enhanced compared with WT mice. In the hearts of WT mice, CHOP-increased apoptotic cell death with activation of caspase-3 was observed at 4 weeks after transverse aortic constriction. In contrast, CHOP-deficient mice had less apoptotic cell death and lower caspase-3 activation at 4 weeks after transverse aortic constriction. Furthermore, the Bcl2/Bax ratio was decreased in WT mice, whereas this change was significantly blunted in CHOP-deficient mice. Real-time polymerase chain reaction microarray analysis revealed that CHOP could regulate several Bcl2 family members in failing hearts. Conclusions— We propose the novel concept that CHOP, which may modify protein translation and mediate endoplasmic reticulum–initiated apoptotic cell death, contributes to development of cardiac hypertrophy and failure induced by pressure overload.

AMPK controls the speed of microtubule polymerization and directional cell migration through CLIP-170 phosphorylation

AMP-activated protein kinase (AMPK) is an energy-sensing Ser/Thr protein kinase originally shown to be regulated by AMP. AMPK is activated by various cellular stresses that inhibit ATP production or stimulate ATP consumption. In addition to its role in metabolism, AMPK has recently been reported to reshape cells by regulating cell polarity and division. However, the downstream targets of AMPK that participate in these functions have not been fully identified. Here, we show that phosphorylation of the microtubule plus end protein CLIP-170 by AMPK is required for microtubule dynamics and the regulation of directional cell migration. Both inhibition of AMPK and expression of a non-phosphorylatable CLIP-170 mutant resulted in prolonged and enhanced accumulation of CLIP-170 at microtubule tips, and slower tubulin polymerization. Furthermore, inhibition of AMPK impaired microtubule stabilization and perturbed directional cell migration. All of these phenotypes were rescued by expression of a phosphomimetic CLIP-170 mutant. Our results demonstrate, therefore, that AMPK controls basic cellular functions by regulating microtubule dynamics through CLIP-170 phosphorylation.

Role of Phasic Dynamism of p38 Mitogen-Activated Protein Kinase Activation in Ischemic Preconditioning of the Canine Heart

Abstract — Although ischemic stress, including ischemic preconditioning (IP), activates p38 mitogen-activated protein kinase (MAPK), the relationship between p38 MAPK activation and the underlying cellular mechanisms of cardioprotection by IP is not verified in vivo. We examined the effects of the selective p38 MAPK inhibition on the cardioprotective effect of IP in the open-chest dogs. The coronary artery was occluded 4 times for 5 minutes, separated by 5 minutes of reperfusion (IP) followed by 90 minutes of occlusion and 6 hours of reperfusion. We infused SB203580 into the coronary artery during IP and 1 hour of reperfusion, during IP alone, and during sustained ischemia in the IP group. p38 MAPK activity markedly increased during IP but did not additionally increase at the onset of ischemia and was even attenuated at 15 minutes of sustained ischemia, and heat-shock protein (HSP) 27 was phosphorylated and translocated from cytosol to myofibril or nucleus without affecting total protein level at the onset of ischemia compared with the control group. SB203580 treatment (1 &mgr;mol/L) only during IP blunted the infarct size limitation by IP (37.3±6.3% versus 7.4±2.1% in the IP group, P <0.01) and attenuated either phosphorylation or translocation of HSP27 during IP. Although the SB203580 treatment throughout the preischemic and postischemic periods had no significant effect on infarct size (33.3±9.4%) in this model, treatment with SB203580 only during ischemia partially mimicked the infarct size limitation by IP (26.8±3.5%). Thus, transient p38 MAPK activation during ischemic preconditioning mainly mediates the cardioprotection followed by HSP27 phosphorylation and translocation in vivo in the canine heart.

Prolonged targeting of ischemic/reperfused myocardium by liposomal adenosine augments cardioprotection in rats.

OBJECTIVESnThe purpose of this study was to investigate whether liposomal adenosine has stronger cardioprotective effects and fewer side effects than free adenosine.nnnBACKGROUNDnLiposomes are nanoparticles that can deliver various agents to target tissues and delay degradation of these agents. Liposomes coated with polyethylene glycol (PEG) prolong the residence time of drugs in the blood. Although adenosine reduces the myocardial infarct (MI) size in clinical trials, it also causes hypotension and bradycardia.nnnMETHODSnWe prepared PEGylated liposomal adenosine (mean diameter 134 +/- 21 nm) by the hydration method. In rats, we evaluated the myocardial accumulation of liposomes and MI size at 3 h after 30 min of ischemia followed by reperfusion.nnnRESULTSnThe electron microscopy and ex vivo bioluminescence imaging showed the specific accumulation of liposomes in ischemic/reperfused myocardium. Investigation of radioisotope-labeled adenosine encapsulated in PEGylated liposomes revealed a prolonged blood residence time. An intravenous infusion of PEGylated liposomal adenosine (450 microg/kg/min) had a weaker effect on blood pressure and heart rate than the corresponding dose of free adenosine. An intravenous infusion of PEGylated liposomal adenosine (450 microg/kg/min) for 10 min from 5 min before the onset of reperfusion significantly reduced MI size (29.5 +/- 6.5%) compared with an infusion of saline (53.2 +/- 3.5%, p < 0.05). The antagonist of adenosine A(1), A(2a), A(2b), or A(3) subtype receptor blocked cardioprotection observed in the PEGylated liposomal adenosine-treated group.nnnCONCLUSIONSnAn infusion as PEGylated liposomes augmented the cardioprotective effects of adenosine against ischemia/reperfusion injury and reduced its unfavorable hemodynamic effects. Liposomes are promising for developing new treatments for acute MI.

PKA rapidly enhances proteasome assembly and activity in in vivo canine hearts.

Proteasome regulates diverse cellular functions by eliminating ubiquitinated proteins. Protein kinase A (PKA) is a key regulator of proteasome activity. However, it remains unknown how PKA regulates proteasome activity and whether it controls proteasome activity in in vivo hearts. Both the in vitro peptidase assay and the in-gel peptidase assays showed that the treatment with PKA for 30 min dose-dependently activated purified 26S proteasome. Simultaneously, PKA treatment enhanced phosphorylation and assembly of purified 26S proteasome evaluated by non-reducing native polyacrylamide gel electrophoresis, either of which was blunted by the pretreatment with a PKA inhibitor, H-89. In in vivo canine hearts, proteasome assembly and activity were enhanced 30 min after the exogenous or endogenous stimulation of PKA by the intracoronary administration of isoproterenol or forskolin for 30 min or by ischemic preconditioning (IP) with 4 times of repeated 5 min of ischemia. The intracoronary administration of H-89 blunted the enhancement of proteasome assembly and activity by IP. Myocardial proteasome activity at the end of ischemia was decreased compared with the control, however, it did not differ from the control in dogs with IP. IP decreased the accumulation of ubiquitinated proteins in the canine ischemia/reperfusion myocardium, which was blunted by the intracoronary administration of a proteasome inhibitor, epoxomicin. However, proteasome activation by IP was not involved in its infarct size-limiting effects. These findings indicate that PKA rapidly enhances proteasome assembly and activity in in vivo hearts. Further investigation will be needed to clarify pathophysiological roles of PKA-mediated proteasome activation in ischemia/reperfusion hearts.

Long-Acting Ca2+ Blockers Prevent Myocardial Remodeling Induced by Chronic NO Inhibition in Rats

Abstract—Chronic inhibition of nitric oxide (NO) synthesis induces cardiac remodeling independent of systemic hemodynamic changes in rats. We examined whether long-acting dihydropyridine calcium channel blockers block myocardial remodeling and whether the activation of 70-kDa S6 kinase (p70S6K) and extracellular signal-regulated kinase (ERK) are involved. Ten groups of Wistar-Kyoto rats underwent 8 weeks of drug treatment consisting of a combination of NO synthase inhibitor NG-nitro-l-arginine methyl ester (L-NAME), an inactive isomer (D-NAME), amlodipine (1 or 3 mg/kg per day), or benidipine (3 or 10 mg/kg per day). In other groups, L-NAME was also used in combination with a p70S6K inhibitor (rapamycin), a MEK inhibitor (PD98059), and hydralazine. Systolic blood pressure (SBP), heart rate, and left ventricular weight (LVW) were measured, together with histological examinations and kinase assay. L-NAME increased SBP and LVW (1048±22 versus 780±18 mg, P <0.01) compared with the control, showing a significant increase in cross-sectional area of cardiomyocytes after 8 weeks. Amlodipine, benidipine, or hydralazine equally attenuated the increase in SBP induced by L-NAME. However, both amlodipine and benidipine but not hydralazine attenuated the increase in LVW by L-NAME (789±27, 825±20 mg, P <0.01, and 1118±29 mg, NS, respectively), also confirmed by histological analysis. L-NAME caused a 2.2-fold/1.8-fold increase in p70S6K/ERK activity in myocardium compared with the control, both of which were attenuated by both amlodipine and benidipine but not hydralazine. Both rapamycin and PD98059 attenuated cardiac hypertrophy in this model. Thus, long-acting dihydropyridine calcium channel blockers inhibited cardiac hypertrophy induced by chronic inhibition of NO synthesis by inhibiting both p70S6K and ERK in vivo.

Identification of genes related to heart failure using global gene expression profiling of human failing myocardium.

Although various management methods have been developed for heart failure, it is necessary to investigate the diagnostic or therapeutic targets of heart failure. Accordingly, we have developed different approaches for managing heart failure by using conventional microarray analyses. We analyzed gene expression profiles of myocardial samples from 12 patients with heart failure and constructed datasets of heart failure-associated genes using clinical parameters such as pulmonary artery pressure (PAP) and ejection fraction (EF). From these 12 genes, we selected four genes with high expression levels in the heart, and examined their novelty by performing a literature-based search. In addition, we included four G-protein-coupled receptor (GPCR)-encoding genes, three enzyme-encoding genes, and one ion-channel protein-encoding gene to identify a drug target for heart failure using in silico microarray database. After the in vitro functional screening using adenovirus transfections of 12 genes into rat cardiomyocytes, we generated gene-targeting mice of five candidate genes, namely, MYLK3, GPR37L1, GPR35, MMP23, and NBC1. The results revealed that systolic blood pressure differed significantly between GPR35-KO and GPR35-WT mice as well as between GPR37L1-Tg and GPR37L1-KO mice. Further, the heart weight/body weight ratio between MYLK3-Tg and MYLK3-WT mice and between GPR37L1-Tg and GPR37L1-KO mice differed significantly. Hence, microarray analysis combined with clinical parameters can be an effective method to identify novel therapeutic targets for the prevention or management of heart failure.