Search

Heart failure (HF) occurs frequently among older individuals, and dysfunction of cardiac mitochondria is often observed. We here show the cardiac-specific downregulation of a certain mitochondrial component during the chronological aging of mice, which is detrimental to the heart. MitoNEET is a mitochondrial outer membrane protein, encoded by CDGSH iron sulfur domain 1 (CISD1). Expression of mitoNEET was specifically downregulated in the heart and kidney of chronologically aged mice. Mice with a constitutive cardiac-specific deletion of CISD1 on the C57BL/6J background showed cardiac dysfunction only after 12 months of age and developed HF after 16 months; whereas irregular morphology and higher levels of reactive oxygen species in their cardiac mitochondria were observed at earlier time points. Our results suggest a possible mechanism by which cardiac mitochondria may gradually lose their integrity during natural aging, and shed light on an uncharted molecular basis closely related to age-associated HF., Takaaki Furihata et al. report a new mouse model with heart-specific deletion of the mitochondrial protein, mitoNEET. Their results suggest that loss of mitoNEET expression may contribute to age-associated heart failure in older adults.

Aims Exercise intolerance in patients with heart failure (HF) is partly attributed to skeletal muscle abnormalities. We have shown that reactive oxygen species (ROS) play a crucial role in skeletal muscle abnormalities, but the pathogenic mechanism remains unclear. Xanthine oxidase (XO) is reported to be an important mediator of ROS overproduction in ischaemic tissue. Here, we tested the hypothesis that skeletal muscle abnormalities in HF are initially caused by XO-derived ROS and are prevented by the inhibition of their production. Methods and results Myocardial infarction (MI) was induced in male C57BL/6J mice, which eventually led to HF, and a sham operation was performed in control mice. The time course of XO-derived ROS production in mouse skeletal muscle post-MI was first analysed. XO-derived ROS production was significantly increased in MI mice from Days 1 to 3 post-surgery (acute phase), whereas it did not differ between the MI and sham groups from 7 to 28 days (chronic phase). Second, mice were divided into three groups: sham + vehicle (Sham + Veh), MI + vehicle (MI + Veh), and MI + febuxostat (an XO inhibitor, 5 mg/kg body weight/day; MI + Feb). Febuxostat or vehicle was administered at 1 and 24 h before surgery, and once-daily on Days 1–7 post-surgery. On Day 28 post-surgery, exercise capacity and mitochondrial respiration in skeletal muscle fibres were significantly decreased in MI + Veh compared with Sham + Veh mice. An increase in damaged mitochondria in MI + Veh compared with Sham + Veh mice was also observed. The wet weight and cross-sectional area of slow muscle fibres (higher XO-derived ROS) was reduced via the down-regulation of protein synthesis-associated mTOR-p70S6K signalling in MI + Veh compared with Sham + Veh mice. These impairments were ameliorated in MI + Feb mice, in association with a reduction of XO-derived ROS production, without affecting cardiac function. Conclusion XO inhibition during the acute phase post-MI can prevent skeletal muscle abnormalities and exercise intolerance in mice with HF.

BACKGROUND Exercise intolerance is a common clinical feature and is linked to poor prognosis in patients with heart failure (HF). Skeletal muscle dysfunction, including impaired energy metabolism in the skeletal muscle, is suspected to play a central role in this intolerance, but the underlying mechanisms remain elusive. Lysine acetylation, a recently identified post-translational modification, has emerged as a major contributor to the derangement of mitochondrial metabolism. We thus investigated whether mitochondrial protein acetylation is associated with impaired skeletal muscle metabolism and lowered exercise capacity in both basic and clinical settings of HF. METHODS We first conducted a global metabolomic analysis to determine whether plasma acetyl-lysine is a determinant factor for peak oxygen uptake (peak VO2 ) in HF patients. We then created a murine model of HF (n = 11) or sham-operated (n = 11) mice with or without limited exercise capacity by ligating a coronary artery, and we tested the gastrocnemius tissues by using mass spectrometry-based acetylomics. A causative relationship between acetylation and the activity of a metabolic enzyme was confirmed in in vitro studies. RESULTS The metabolomic analysis verified that acetyl-lysine was the most relevant metabolite that was negatively correlated with peak VO2 (r = -0.81, P < 0.01). At 4 weeks post-myocardial infarction HF, a treadmill test showed lowered work (distance × body weight) and peak VO2 in the HF mice compared with the sham-operated mice (11 ± 1 vs. 23 ± 1 J, P < 0.01; 143 ± 5 vs. 159 ± 3 mL/kg/min, P = 0.01; respectively). As noted, the protein acetylation of gastrocnemius mitochondria was 48% greater in the HF mice than the sham-operated mice (P = 0.047). Acetylproteomics identified the mitochondrial enzymes involved in fatty acid β-oxidation (FAO), the tricarboxylic acid cycle, and the electron transport chain as targets of acetylation. In parallel, the FAO enzyme (β-hydroxyacyl CoA dehydrogenase) activity and fatty acid-driven mitochondrial respiration were reduced in the HF mice. This alteration was associated with a decreased expression of mitochondrial deacetylase, Sirtuin 3, because silencing of Sirtuin 3 in cultured skeletal muscle cells resulted in increased mitochondrial acetylation and reduced β-hydroxyacyl CoA dehydrogenase activity. CONCLUSIONS Enhanced mitochondrial protein acetylation is associated with impaired FAO in skeletal muscle and reduced exercise capacity in HF. Our results indicate that lysine acetylation is a crucial mechanism underlying deranged skeletal muscle metabolism, suggesting that its modulation is a potential approach for exercise intolerance in HF.

Loop diuretics are widely used for the management of fluid retention in patients with heart failure (HF). Sarcopenia, defined as decreased skeletal muscle mass, is frequently present in patients with HF and is associated with poor prognosis. The effects of loop diuretics on skeletal muscle in HF patients have not been fully elucidated. Here, we investigated the impact of loop diuretics on the skeletal muscle mass in patients with HF.We conducted a subanalysis of a cross-sectional study from 10 hospitals evaluating 155 patients with HF (age 67 ± 13 yrs, 69% men).We compared the HF patients who were treated with loop diuretics (n = 120) with the patients who were not (n = 35). The thigh and arm circumferences were significantly small in the group treated with loop diuretics compared to those not so treated (39.9 ± 4.8 vs. 43.5 ± 6.9 cm, p 0.001 and 26.7 ± 3.5 vs. 28.9 ± 6.2 cm, p 0.001, respectively). In a univariate analysis, higher age, lower body mass index, lower hemoglobin, and loop diuretic use were significantly associated with smaller thigh circumference. In a multivariable analysis, the use of loop diuretics was independently associated with smaller thigh circumference (β = -0.51, 95% confidence interval -0.98 to -0.046, p = 0.032).Loop diuretics are associated with decreased thigh and arm circumferences in patients with HF, independent of the severity of HF. Our findings revealed for the first time the adverse effects of loop diuretics on skeletal muscle wasting. These findings will have a significant impact in clinical practice regarding the frequent use of loop diuretics in HF patients.

A failing heart shows severe energy insufficiency, and it is presumed that this energy shortage plays a critical role in the development of cardiac dysfunction. However, little is known about the mechanisms that cause energy metabolic alterations in the failing heart. Here, we show that the novel RING-finger protein 207 (RNF207), which is specifically expressed in the heart, plays a role in cardiac energy metabolism. Depletion of RNF207 in neonatal rat cardiomyocytes (NRCs) leads to a reduced cellular concentration of adenosine triphosphate (ATP) and mitochondrial dysfunction. Consistent with this result, we observed here that the expression of RNF207 was significantly reduced in mice with common cardiac diseases including heart failure. Intriguingly, proteomic approaches revealed that RNF207 interacts with the voltage-dependent anion channel (VDAC), which is considered to be a key regulator of mitochondria function, as an RNF207-interacting protein. Our findings indicate that RNF207 is involved in ATP production by cardiomyocytes, suggesting that RNF207 plays an important role in the development of heart failure.

Background Transverse aortic constriction (TAC) operation is used as an experimental model of left ventricular (LV) hypertrophy and LV failure in mice. The severity of LV remodeling or failure may depend on the degree of TAC, but is variable among operated animals. Therefore, we tried to identify the optimal diameter of TAC to create this model with ease and high reproducibility. Methods and results To produce TAC in C57BL/6J mice (7–9 weeks, body weight 19–26 g, n = 109), a 7–0 nylon suture ligature was tightly tied around the transverse aorta against needles with 3 different diameters (mm); 0.40, 0.385 and 0.375. LV wall thickness, end-diastolic dimension, fractional shortening were measured by echocardiography. At 4 weeks after TAC, no mouse with the 0.400 mm gauge progressed in LV failure. The 0.385 mm pin gauge mouse kept a more survival rate compared with the 0.375 mm (59% vs 48%), representing same efficient in LV failure. With the 0.385 mm pin gauge, hearts of mice remained LV hypertrophy at 1 week after TAC, followed by LV failure at 4 weeks. Conclusion TAC with the diameter of 0.385 mm can effectively induce the transition from LV hypertrophy to failure in mice with relatively preserved survival.

Insulin resistance can occur as a consequence of heart failure (HF). Activation of the renin-angiotensin system (RAS) may play a crucial role in this phenomenon. We thus investigated the effect of a direct renin inhibitor, aliskiren, on insulin resistance in HF after myocardial infarction (MI). MI and sham operation were performed in male C57BL/6 J mice. The mice were divided into 4 groups and treated with sham-operation (Sham, n=10), sham-operation and aliskiren (Sham+Aliskiren; 10 mg/kg/day, n=10), MI (n=11), or MI and aliskiren (MI+Aliskiren, n=11). After 4 weeks, MI mice showed left ventricular dilation and dysfunction, which were not affected by aliskiren. The percent decrease of blood glucose after insulin load was significantly smaller in MI than in Sham (14±5% vs. 36±2%), and was ameliorated in MI+Aliskiren (34±5%) mice. Insulin-stimulated serine-phosphorylation of Akt and glucose transporter 4 translocation were decreased in the skeletal muscle of MI compared to Sham by 57% and 69%, and both changes were ameliorated in the MI+Aliskiren group (91% and 94%). Aliskiren administration in MI mice significantly inhibited plasma renin activity and angiotensin II (Ang II) levels. Moreover, (pro)renin receptor expression and local Ang II production were upregulated in skeletal muscle from MI and were attenuated in MI+Aliskiren mice, in tandem with a decrease in superoxide production and NAD(P)H oxidase activities. In conclusion, aliskiren ameliorated insulin resistance in HF by improving insulin signaling in the skeletal muscle, at least partly by inhibiting systemic and (pro)renin receptor-mediated local RAS activation, and subsequent NAD(P)H oxidase-induced oxidative stress.

Iron is an essential trace element for regulation of redox and mitochondrial function, and then mitochondrial iron content is tightly regulated in mammals. We focused on a novel protein localized at the outer mitochondrial membrane. Immunoelectron microscopy revealed transferrin receptor (TfR) displayed an intimate relationship with the mitochondria, and mass spectrometry analysis also revealed mitoNEET interacted with TfR in vitro. Moreover, mitoNEET was endogenously coprecipitated with TfR in the heart, which indicates that mitoNEET also interacts with TfR in vivo. We generated mice with cardiac-specific deletion of mitoNEET (mitoNEET-knockout). Iron contents in isolated mitochondria were significantly increased in mitoNEET-knockout mice compared to control mice. Mitochondrial reactive oxygen species (ROS) were higher, and mitochondrial maximal capacity and reserve capacity were significantly decreased in mitoNEET-knockout mice, which was consistent with cardiac dysfunction evaluated by echocardiography. The complex formation of mitoNEET with TfR may regulate mitochondrial iron contents via an influx of iron. A disruption of mitoNEET could thus be involved in mitochondrial ROS production by iron overload in the heart.

Eccentric distribution of atheroma has been associated with plaques likely to rupture and cause an acute coronary syndrome, but the factors responsible for the development of eccentricity remain unknown. Endothelial shear stress (ESS) drives plaque formation. We aimed to investigate the role of the local ESS characteristics in the de novo development and progressive worsening of plaque eccentricity in humans.Vascular profiling (3-vessel 3D coronary reconstruction by angiography/intravascular ultrasound, and blood flow simulation for ESS computation) was performed in 374 patients at baseline6-10 months follow-up. At baseline, we identified (i) disease-free segments (n=2157), and (ii) diseased regions of luminal obstructions (n=408).In disease-free regions, baseline low ESS magnitude (p0.001), marked ESS circumferential heterogeneity (p=0.001), and their interaction (p=0.026) were associated with an increased probability of de novo eccentric plaque formation at follow-up. In diseased regions, baseline low ESS (odds ratio [OR]: 2.33, p=0.003) and large plaque burden (OR: 2.46, p=0.002) were independent predictors of substantially increasing plaque eccentricity index with worsening lumen encroachment. This combined outcome was more frequent in obstructions with both features vs. all others (33 vs. 12%; p0.001). The incidence of percutaneous coronary intervention in worsening obstructions with increasing plaque eccentricity was higher (13.3 vs. 4.3%, p=0.011).The local hemodynamic environment has a critical effect on the development of eccentric coronary plaques at both an early and advanced stage of atherosclerosis. Local ESS assessment could help in predicting sites prone to plaque disruption and acute coronary syndromes in humans.

A 54-year-old man with polycystic liver disease received a domino liver transplantation (DLT) from a patient of hereditary ATTR amyloidosis with the transthyretin Ser50Arg mutation. Ten years after transplantation, he felt a slight numbness in his toes, and cardiac amyloidosis was simultaneously suspected upon a heart function evaluation. Biopsy specimens from the myocardium revealed transthyretin amyloidosis with the Ser50Arg mutation. Oral tafamidis therapy has inhibited the progression of neurological and cardiovascular symptoms this far. We herein report this first case of amyloid polyneuropathy and myocardial amyloidosis after DLT from hereditary ATTR amyloidosis with a transthyretin Ser50Arg mutation and discuss similar cases of other mutations.

Fatty acids such as oleic and stearic acids having a long hydrocarbon chain are known to exist as dimers in their melt and even in a non-polar solvent. In their melt the dimers arrange longitudinally and alternately to form clusters which resemble a smectic liquid crystal. The clusters determine the liquid properties of the fatty acids such as density, viscosity and fluidity. Then, do the dimers of fatty acid having a moderate-length hydrocarbon chain construct such the clusters? In the present study the dynamic molecular behavior and assembly structure of octanoic acid in its melt and also in CCl4 solution have been investigated by the X-ray diffraction, near infrared spectroscopy, 1H-NMR chemical shift, self-diffusion coefficient and 13C-NMR spin-lattice relaxation time measurements. From these results it has been revealed that the clusters of octanoic acid exist in its melt and also in CCl4 and that the clusters in the melt disintegrate with an increase in temperature. The dissociation profile of dimers of octanoic acid into monomers in CCl4 also has been clarified.

Background: In-stent hyperplasia (ISH) may develop in regions of low endothelial shear stress (ESS), but the relationship between the magnitude of low ESS, the extent of ISH, and subsequent clinical events has not been investigated. Methods and Results: We assessed the association of poststent ESS with neointimal ISH and clinical outcomes in patients treated with percutaneous coronary interventions (PCI). Three-dimensional coronary reconstruction was performed in 374 post-PCI patients at baseline and 6 to 10 months follow-up as part of the PREDICTION Study. Each vessel was divided into 1.5-mm-long segments, and we calculated the local ESS within each stented segment at baseline. At follow-up, we assessed ISH and the occurrence of a clinically indicated repeat PCI for in-stent restenosis. In 246 total stents (54 overlapping), 100 (40.7%) were bare-metal stents (BMS), 104 (42.3%) sirolimus-eluting stents, and 42 (17.1%) paclitaxel-eluting stents. In BMS, low ESS post-PCI at baseline was independently associated with ISH (β=1.47 mm 2 per 1-Pa decrease; 95% CI, 0.38-2.56; P < 0.01). ISH was minimal in drug-eluting stents. During follow-up, repeat PCI in BMS was performed in 21 stents (8.5%). There was no significant association between post-PCI ESS and in-stent restenosis requiring PCI. Conclusions: Low ESS after BMS implantation is associated with subsequent ISH. ISH is strongly inhibited by drug-eluting stents. Post-PCI ESS is not associated with in-stent restenosis requiring repeat PCI. ESS is an important determinant of ISH in BMS, but ISH of large magnitude to require PCI for in-stent restenosis is likely attributed to factors other than ESS within the stent., Hellenic Cardiological Society, Schaubert Family, George D. Behrakis Cardiovascular Research Fellowship

Background: Ring-finger proteins constitute a large protein family in the human genome and play essential roles in various biological processes. However, little is known about heart-specific Ring-finger proteins and those relations with cardiac functions. We performed the comprehensive analysis of the expression profiles of various kinds of Ring-finger proteins and found that Ring-finger protein 207 (RNF207) was largely expressed in the heart. The purpose of our study was to elucidate a role of RNF207 in the heart. Methods and Results: First, we confirmed that RNF207 was predominantly expressed in the heart at the mRNA and protein level. Next, we examined whether the expression of RNF207 changed in transverse aortic constriction (TAC) model mice and coronary ischemic heart failure model mice . 4 weeks after TAC, mRNA level of RNF207 was significantly decreased to approximately 40% of that in sham mice. Moreover, we found that the protein level of RNF207 in the mice with heart failure was significantly reduced to around 70% of that in sham mice. Considering the well-known facts that the heart is a high-energy demanding organ and that the levels of ATP in cardiomyocytes are reduced in those model mice, we hypothesized that RNF207 got involved in cardiac energy metabolism. To investigate the hypothesis, we depleted RNF207 in rat neonatal cardiomyocytes (RNC) and performed metabolomic analysis. Metabolomic analysis revealed that ATP concentration and NADH/NAD + ratio were significantly lower and mitochondrial function was significantly reduced in RNF207 depleted RNC, compared to control NRC. Next, to elucidate the molecular mechanism by which RNF207 had effect on cardiac energy metabolism, we explored RNF207-associated proteins by mass spectrometric analysis. We identified voltage-dependent anion channel 1 (VDAC1) as a RNF207-associated protein. It has been shown that mitochondrial protein VDAC1 plays a crucial role in mitochondrial functions, such as energy metabolism. We confirmed that RNF207 directly interacted with VDAC1 by in vitro binding assay. Our results strongly indicate that RNF207 functions as a regulator of cardiac energy metabolism. Conclusion: RNF207 is a novel heart-specific protein and regulates energy metabolism in cardiomyocytes.

Background and aims The natural history and the role of atherosclerotic plaque located behind the stent (PBS) are still poorly understood. We evaluated the serial changes in PBS following bare-metal (BMS) compared to first-generation drug-eluting stent (DES) implantation and the impact of these changes on in-stent neointimal hyperplasia (NIH). Methods Three-dimensional coronary reconstruction by angiography and intravascular ultrasound was performed after intervention and at 6–10-month follow-up in 157 patients with 188 lesions treated with BMS (n = 89) and DES (n = 99). Results There was a significant decrease in PBS area (−7.2%; p < 0.001) and vessel area (−1.7%; p < 0.001) after BMS and a respective increase in both areas after DES implantation (6.1%; p < 0.001 and 4.1%; p < 0.001, respectively). The decrease in PBS area significantly predicted neointimal area at follow-up after BMS (β: 0.15; 95% confidence interval [CI]: 0.10–0.20, p < 0.001) and DES (β: 0.09; 95% CI: 0.07–0.11; p < 0.001) implantation. The decrease in PBS area was the most powerful predictor of significant NIH after BMS implantation (odds ratio: 1.13; 95% CI: 1.02–1.26; p = 0.02). Conclusions The decrease in PBS area after stent implantation is significantly associated with the magnitude of NIH development at follow-up. This finding raises the possibility of a communication between the lesion within the stent and the underlying native atherosclerotic plaque, and may have important implications regarding the pathobiology of in-stent restenosis and late/very late stent thrombosis.

Skeletal muscle atrophy is induced by an imbalance between protein synthesis and degradation. Our previous studies reported that angiotensin II (AII) directly induced muscle atrophy in mice. This study investigated the role of NAD(P)H oxidase 2 (Nox2) activation by AII in the induction of skeletal muscle atrophy. For 4 weeks, either saline (vehicle: V) or AII (1000 ng kg−1 min−1) was infused into male wild-type (WT) and Nox2 knockout (KO) mice via osmotic minipumps. Experiments were performed in the following 4 groups: WT + V, KO + V, WT + AII, and KO + AII. Body weight, muscle weight, and myocyte cross-sectional area were significantly decreased in WT + AII compared to WT + V mice, and these changes were not observed in KO + AII mice. Akt phosphorylation of Ser473 and p70S6K of Thr389 was decreased, gene expression levels of MuRF-1 and atrogin-1 were increased in WT + AII compared to WT + V, and these changes were significantly attenuated in KO + AII mice. The deletion of Nox2 prevented AII-induced skeletal muscle atrophy via improving the balance between protein synthesis and degradation. Therefore, Nox2 may be a therapeutic target for AII-induced skeletal muscle atrophy.

Atherosclerosis is determined by both systemic risk factors and local vascular mechanisms. The arterial remodeling in response to plaque development plays a key role in atherosclerosis. Compensatory expansive remodeling is an adaptive mechanism that maintains lumen patency as a plaque develops. In contrast, excessive expansive remodeling, signifying an enlargement in vascular and lumen volume as a result of local plaque buildup, is a consistent attribute of high-risk plaques. Local hemodynamic factors, in particular low endothelial shear stress (ESS), is an intensely proinflammatory and proatherogenic stimulus and largely accounts for the spatially diverse distribution of atherosclerotic plaques. However, plaque, remodeling and ESS have hitherto been investigated only in the cross-sectional arterial axis and their distribution in the longitudinal axis of individual plaques has not been characterized.

Background: Brain-derived neurotrophic factor (BDNF), a member of the neurotrophic factor family, has been known to be increased by exercise training (ET) and associated with exercise capacity, which are dependent on mitochondrial function in the skeletal muscle (SKM). We thus hypothesized that an increase in BDNF and an activation of its binding receptor, tropomyosin-related kinase B (TrkB), could increase the exercise capacity by enhancing mitochondrial function in SKM in mice. Therefore, we investigated whether an administration of recombinant human (rh)BDNF into sedentary (SED) mice could mimic the effect of ET on exercise capacity and SKM. Methods and Results: C57BL/6J mice randomly assigned either SED (n=20) or ET (n=10), SED mice was divided into 2 groups of the treatment with vehicle (phosphate-buffered saline; n=10) or rhBDNF (10 mg/kg body weight/day, s.c.; n=10), for 5 weeks. ET was performed by swimming for 90 min/day and 5 times/week. SKM weight did not differ among groups. The work (distance x body weight) and peak oxygen uptake (VO2) evaluated by treadmill test were significantly increased in ET+vehicle compared to SED+vehicle (work 24±1 vs 32±1 J and peak VO2 150±2 vs 166±2 mL/kg/min, p Conclusions: rhBDNF can mimic the effects of ET on exercise capacity. BDNF-TrkB pathway may play an important role in the beneficial effects of ET on the exercise capacity and SKM function.

Introduction: Skeletal muscle atrophy is observed in aging and a variety of diseases including chronic obstructive pulmonary disease, chronic renal failure, and heart failure. It has been considered that it is associated with chronic inflammation in the skeletal muscle. We have previously reported that angiotensin II (Ang II) could directly induce skeletal muscle abnormalities including atrophy in mice. Hypothesis: Here, we hypothesized that the activation of NAD(P)H oxidase by Ang II induced the skeletal muscle atrophy. Methods: Either saline (vehicle) or Ang II (1000 ng/kg/min) was infused into male wild-type (WT) and Nox2 knock out (KO) mice via subcutaneously implanted osmotic minipumps for 4 weeks. Experiments were performed in following 4 groups; WT+vehicle (n=8), KO+vehicle (n=6), WT+Ang II (n=8), and KO+Ang II (n=6). Lower skeletal muscle limbs were removed and used for the analysis. Exercise capacity was measured by using treadmill test. Results: Increased activation of NAD(P)H oxidase and production of superoxide in the skeletal muscle by Ang II were completely inhibited in KO mice. Body and skeletal muscle weight, and myocyte cross sectional area were significantly decreased in WT+Ang II compared to WT+vehicle (24±1 vs. 28±1 g, 356±3 vs. 390±4 mg, and 2351±84 vs. 3221±118 μm2, respectively). These changes were significantly attenuated in KO+Ang II (27±1 g, 392±9 mg, 3012±150 μm2, respectively) Phospho-Akt and phospho-p70S6K, key molecule of protein synthesis, were decreased in WT+Ang II compared to WT+vehicle, which was attenuated in KO+Ang II. On the other hand, protein expressions of Muscle RING Finger-1 and MAFbx/Atrogin-1, key molecule of protein degradation, were significantly increased in WT+Ang II compare to WT+vehicle, which was not affected in KO+Ang II. Mitochondrial electron transfer complex and citrate synthase activities were also impaired in WT+Ang II and were not improved in KO+Ang II. In agreements with these results, exercise, Ang II-induced reduction in exercise capacity was not improved in KO mice. Conclusions: The deletion of Nox2 attenuated the skeletal muscle atrophy and decreased protein synthase induced by Ang II, suggesting that Nox2 plays an essential role in the development of skeletal muscle atrophy.

Signaling adaptor protein Crk regulates cell motility and growth through its targets Dock180 and C3G, those are the guanine-nucleotide exchange factors (GEFs) for small GTPases Rac and Rap, respectively. Recently, overexpression of Crk has been reported in various human cancers. To define the role for Crk in human cancer cells, Crk expression was targeted in the human ovarian cancer cell line MCAS through RNA interference, resulting in the establishment of three Crk knockdown cell lines. These cell lines exhibited disorganized actin fibers, reduced number of focal adhesions, and abolishment of lamellipodia formation. Decreased Rac activity was demonstrated by pull-down assay and FRET-based time-lapse microscopy, in association with suppression of both motility and invasion by phagokinetic track assay and transwell assay in these cells. Furthermore, Crk knockdown cells exhibited slow growth rates in culture and suppressed anchorage-dependent growth in soft agar. Tumor forming potential in nude mice was attenuated, and intraperitoneal dissemination was not observed when Crk knockdown cells were injected into the peritoneal cavity. These results suggest that the Crk is a key component of focal adhesion and involved in cell growth, invasion, and dissemination of human ovarian cancer cell line MCAS.

A flow-through analyzer has been developed for the rapid, potentiometric determination of total alkalinity (TA) in seawater in order to capture large temporal and spatial variations of the CO 2 system in coastal zones, where the carbon cycle is still not well understood despite the potential large contribution of such areas to the global carbon cycle. This analyzer requires small amounts of seawater (3 ml min −1 ), and has realized continuous measurements of TA with a response time of 4–5 min, with long-term precision of about 0.1% (ca. 2 μmol kg −1 ), and accuracy of a similar level over a range of TA (more than 200 μmol kg −1 ) when appropriate standard solutions are used. This analyzer is fully automated and can achieve stable measurements (within 0.15%; 3 μmol kg −1 ) over 1–2 days without regular recalibration for drift, which will enable us to carry out continuous, in situ measurements of TA in coastal waters.

A submersible pCO2 measurement system with a membrane tube equilibrator and a nondispersive infrared gas analyzer was developed. The system was designed to measure the partial pressure of CO2 in seawater and the atmosphere, and physical parameters related to CO2 changes, including light intensity, temperature, salinity, dissolved oxygen, and pH. A CPU controls the operation and logs the data. The analyzer and CPU are contained in a titanium cylinder. The power consumption is 15 W, which is supplied by 1419 dry alkaline manganese dioxide battery cells contained in three PVC cylinders. We tested the system on a barrier reef flat in the Palau Islands, and monitored pCO2 and physical parameters from November 1999 to December 2000. The system worked for 50 hours a week, and we changed the battery unit every 4 months. The accuracy of pCO2 measurement was ±5 μatm and the response time was 3 minutes. The results showed diurnal changes in the pCO2 in reef water on a coral reef flat, which corresponded to daytime photosynthesis and nighttime respiration.

Background: Skeletal muscle abnormalities such as mitochondrial dysfunction, fiber type transition, and atrophy are the main cause of reduced exercise capacity observed in various diseases such as diabetes mellitus and heart failure. Renin-angiotensin system (RAS) was activated in the skeletal muscle in these conditions. We thus hypothesized that angiotensin II (Ang II) could directly induce skeletal muscle abnormalities. Methods and Results: Ang II (1000ng/kg/min, n=8) or vehicle (saline, n=8) was administrated into male C57BL/6J mice (10-12 week of age) via subcutaneously implanted osmotic minipumps for 4 weeks. Ang II significantly decreased body weight (26.6±0.3 vs. 27.6±0.3 g, p Conclusion: Ang II could directly induce the reduction of exercise tolerance in association with the abnormalities in skeletal muscle function and structure.

Background: Exercise intolerance is a major clinical manifestation, and closely related to poor prognosis in heart failure (HF) patients. Metabolic derangements have been shown to play an important role in this phenomenon. Recent metabolomic investigations have highlighted several novel biochemical pathways in HF, however, metabolic profiling associated with exercise capacity has not been elucidated. We thus investigated the metabolites associated with exercise capacity by using global metabolomic analysis of plasma samples in HF patients. Methods: Ten consecutive HF patients with reduced ejection fraction (57 ± 12 years old, New York Heart Association class II - III) and 5 age-matched healthy subjects as controls (56 ± 3 years old) were studied. Charged metabolites in plasma samples were measured by capillary electrophoresis mass spectrometry. Peak oxygen uptake (peak VO 2 ) and VE/VCO 2 slope were measured by cardiopulmonary exercise test. Results: A total of 233 charged metabolites were identified and quantified absolutely using standard chemicals for each metabolite. Significant increases in arginine methylation, phospholipid metabolites, and urea cycle metabolites were observed in HF patients compared to controls (Figure). Peak VO 2 was negatively correlated with methylarginine including asymmetric dimethylarginine (ADMA) (r=-0.77, PN,N -dimethylglycine (r=-0.75, P=0.011) levels in HF patients. VE/VCO 2 slope was positively correlated with N,N -dimethylglycine (r=0.72, P=0.026) and Urea (r=0.82, P=0.006) levels. Conclusions: Increases in metabolites of arginine methylation, phospholipid metabolism, and urea cycle were associated with exercise intolerance in HF. These results provide mechanistic insights into exercise intolerance associated with HF.

Background: The natural history and the role of the atherosclerotic plaque located behind the stent (PBS) are still poorly understood. We evaluated the serial changes in PBS following bare-metal (BMS) compared with first-generation drug-eluting stent (DES) implantation and the impact of these changes on in-stent neointimal hyperplasia (NIH). Methods: 3D coronary reconstruction by angiography and intravascular ultrasound were serially performed after intervention and at 6- to 10-month follow-up in 157 Japanese patients treated with BMS (n=90) and DES (n=98; 68 sirolimus-eluting and 30 paclitaxel-eluting stents) included in the PREDICTION Study. Each reconstructed stented coronary artery was divided into consecutive 1.5-mm segments. External elastic lamina, lumen, stent, and PBS area were measured for each segment at both baseline and follow-up. At follow-up NIH area was assessed. Due to the very low rate of events in our population we used significant NIH (defined as NIH area >50% of stent area) as a binary anatomic outcome. Results: Patient, lesion, and stent characteristics were comparable between BMS and DES. There was a significant decrease in PBS area after BMS (median relative change: -7.2%, IQR -19.3 to 5.2%, p Conclusions: The PBS significantly decreased 6 to 10 months after BMS implantation, whereas after DES it increased. The decrease in PBS area was significantly associated with the development of NIH at follow-up in both stent types. These findings raise the possibility of a communication between the lesion within the stent and the underlying native atherosclerotic plaque, and may have important implications regarding the pathobiology of in-stent restenosis.

Introduction: Plaque inflammation is a critical step in the initiation and progression of atherosclerosis. Such inflammation is thought to originate from the luminal surface of plaque and infiltrate the intima-media in advanced lesions. Low endothelial shear stress (ESS) is known to induce intima-media inflammation and plaque growth. In this study we investigated in-vivo the hypothesis that low ESS induces also adventitial inflammation. Methods: We studied 11 swine at 23 (baseline) and 30 (followup) weeks after the induction of diabetes and hyperlipidemia. Using angiography and intravascular ultrasound data, we performed 3D coronary reconstruction of coronary arteries and calculated the ESS with computational fluid dynamics. In 56 segments, we assessed the adventitial inflammatory (CD45) and antigen-presenting (MHC-II) cell content at followup with immunohistochemistry. Segments were classified as low (≤ 1 Pa) or higher (>1 Pa) ESS. Results: MHC-II content in the adventitia (1.3±0.3%) was higher than in the media (0.3±0.1%, p Conclusion: Although total inflammation is not dependent on ESS, low ESS induces higher adventitial activated inflammatory cell content, as assessed by MHC-II immunostaining. This, in conjunction with the higher MHC-II content in the adventitia than in the media and the presence of an intact IEL suggests an additional source of inflammation in low-ESS plaque regions, originating from the vessel outer wall. The induction of neovascularization possibly accounts for this phenomenon.

The oxidation of vinyl and silyl enol ethers with aqueous hydrogen peroxide was first achieved by the use of peroxotungstophosphate (PCWP) as the catalyst. For example, the oxidation of 1-ethoxy-1-octene with a stoichiometric amount of 35% H2O2 in the presence of PCWP (0.5 mol %) in a mixed solvent of methanol and dichloromethane at room temperature gave 1-ethoxy-1-methoxy-2-hydroxyoctane, a synthetic equivalent of 2-hydroxyoctanal, in 70% yield. The oxidation of acyclic silyl enol ethers such as 1-[(trimethylsilyl)oxy]-1-octene under these conditions gave 1-hydroxy-2-octanone in 72% yield, while the same oxidation in dichloromethane alone resulted in cleavage of the enol double bond to form heptanal in 71% yield. Cyclic silyl enol ethers were converted into the corresponding α-hydroxy ketones in 48−71% yields under similar reaction conditions.

AST-120, an Adsorbent of Uremic Toxins Improved the Cardioprotective Signaling in Pacinginduced Heart Failure Dog HYEMOON CHUNG, HIROSHI ASANUMA, SHIN ITO, KYUNG-DUK MIN, KAZUHIRO SHINDO, MIKI IMAZU, HIROKI FUKUDA, HIROKO TAKAHAMA, MASANORI ASAKURA, MASAFUMI KITAKAZE Clinical Research and Development, National Cerebral and Cardiocascular Center, Suita, Japan; Department of Cardiovascular Medicine, Kyoto Prefectural University School of Medicine, Kyoto, Japan

The oxidation of vinyl and silyl enol ethers with aqueous hydrogen peroxide was first achieved by the use of peroxotungstophosphate (PCWP) as the catalyst. For example, the oxidation of 1-ethoxy-1-octene with a stoichiometric amount of 35% H(2)O(2) in the presence of PCWP (0.5 mol %) in a mixed solvent of methanol and dichloromethane at room temperature gave 1-ethoxy-1-methoxy-2-hydroxyoctane, a synthetic equivalent of 2-hydroxyoctanal, in 70% yield. The oxidation of acyclic silyl enol ethers such as 1-[(trimethylsilyl)oxy]-1-octene under these conditions gave 1-hydroxy-2-octanone in 72% yield, while the same oxidation in dichloromethane alone resulted in cleavage of the enol double bond to form heptanal in 71% yield. Cyclic silyl enol ethers were converted into the corresponding alpha-hydroxy ketones in 48-71% yields under similar reaction conditions.

Three-dimensional (3D) coronary artery reconstruction using intravascular ultrasound (IVUS) and angiographic data has been used in studies highlighting the critical role of endothelial shear stress (ESS) in coronary atherosclerosis. Frequency Domain Optical Coherence Tomography (FD-OCT) was recently