Hideki Katoh

Transient opening of mitochondrial permeability transition pore by reactive oxygen species protects myocardium from ischemia-reperfusion injury

Reactive oxygen species (ROS) production during ischemia-reperfusion (I/R) is thought to be a critical factor for myocardial injury. However, a small amount of ROS during the ischemic preconditioning (IPC) may provide a signal for cardioprotection. We have previously reported that the repetitive pretreatment of a small amount of ROS [hydrogen peroxide (H(2)O(2)), 2 microM] mimicked the IPC-induced cardioprotection in the Langendorff-perfused rat hearts. We further investigated the mechanisms of the ROS-induced cardioprotection against I/R injury and tested the hypothesis whether it could mediate the mitochondrial permeability transition pore (mPTP) opening. The Langendorff-perfused rat hearts were subjected to 35 min ischemia and 40 min reperfusion, and the pretreatment of H(2)O(2) (2 microM) significantly improved the postischemic recoveries in left ventricular developed pressure, intracellular phosphocreatine, and ATP levels. A specific mPTP inhibitor cyclosporin A (CsA; 0.2 microM) canceled these H(2)O(2)-induced effects. In isolated permeabilized myocytes, H(2)O(2) (1 microM) accelerated the calcein leakage from mitochondria in a CsA-sensitive manner, indicating the opening of mPTP by H(2)O(2). However, H(2)O(2) did not depolarize mitochondrial membrane potential (DeltaPsi(m)) even in the presence of oligomycin (F(1)/F(0) ATPase inhibitor; 1 microM) and decreased mitochondrial Ca(2+) concentration ([Ca(2+)](m)) by accelerating the mitochondrial Ca(2+) extrusion via an mPTP. We conclude that the transient mPTP opening could be involved in the H(2)O(2)-induced cardioprotection against reperfusion injury, and the reduction of [Ca(2+)](m) without the change in DeltaPsi(m) might be a possible mechanism for the protection.

Local control of mitochondrial membrane potential, permeability transition pore and reactive oxygen species by calcium and calmodulin in rat ventricular myocytes

Calmodulin (CaM) and Ca(2+)/CaM-dependent protein kinase II (CaMKII) play important roles in the development of heart failure. In this study, we evaluated the effects of CaM on mitochondrial membrane potential (DeltaPsi(m)), permeability transition pore (mPTP) and the production of reactive oxygen species (ROS) in permeabilized myocytes; our findings are as follows. (1) CaM depolarized DeltaPsi(m) dose-dependently, but this was prevented by an inhibitor of CaM (W-7) or CaMKII (autocamtide 2-related inhibitory peptide (AIP)). (2) CaM accelerated calcein leakage from mitochondria, indicating the opening of mPTP, however this was prevented by AIP. (3) Cyclosporin A (an inhibitor of the mPTP) inhibited both CaM-induced DeltaPsi(m) depolarization and calcein leakage. (4) CaM increased mitochondrial ROS, which was related to DeltaPsi(m) depolarization and the opening of mPTP. (5) Chelating of cytosolic Ca(2+) by BAPTA, the depletion of SR Ca(2+) by thapsigargin (an inhibitor of SERCA) and the inhibition of mitochondrial Ca(2+) uniporter by Ru360 attenuated the effects of CaM on mitochondrial function. (6) CaM accelerated Ca(2+) extrusion from mitochondria. We conclude that CaM/CaMKII depolarized DeltaPsi(m) and opened mPTP by increasing ROS production, and these effects were strictly regulated by the local increase in cytosolic Ca(2+) concentration, initiated by Ca(2+) releases from the SR. In addition, CaM was involved in the regulation of mitochondrial Ca(2+) homeostasis.

Effects of nitric oxide on mitochondrial permeability transition pore and thiol-mediated responses in cardiac myocytes.

Nitric oxide (NO) alters the opening of mitochondrial permeability transition pore (mPTP). However, the signaling pathways of NO on mPTP remain elusive. We aimed to clarify the contribution of thiol-mediated responses to the effects of NO on mPTP in permeabilized myocytes. We found that (1) a high concentration of spermine NONOate (an NO donor; 500 μM) opened mPTP and depolarized ΔΨ(m). (2) A low concentration of NONOate (5 μM) prevented atractyloside (an mPTP opener)-induced mPTP opening. (3) Mn(III) tetrakis (4-benzoic acid) porphyrin (Mn-TBAP, ONOO(-) scavenger) attenuated the effect of high-concentration NONOate on mPTP opening, but did not inhibited the preventive effects of low-concentration NONOate. (4) When the interaction of NO with thiol was inhibited by N-ethylmaleimide, the opening (by high-concentration NONOate) and preventive effects (by low-concentration NONOate) of NONOate on mPTP were blocked. (5) Dithiothreitol (an inhibitor of disulfide bonds formation) prevented high-concentration NONOate-induced mPTP opening. (6) Ascorbic acid (an inhibitor of S-nitrosylation) prevented the preventive effects of low-concentration NONOate on mPTP. We conclude that opening of mPTP by high-concentration NO is related to disulfide bonds formation and oxidizing effects of ONOO(-). In contrast, the inhibitory effect of physiological concentrations of NO on mPTP is related to S-nitrosylation.

Extracellular acidosis suppresses endothelial function by inhibiting store-operated Ca2+ entry via non-selective cation channels

AIMSnHypoxia, ischaemia, and exogenous chemicals can induce extracellular and intracellular acidosis, but it is not clear which of these types of acidosis affects endothelial cell function. The synthesis and release of endothelium-derived relaxing factors (EDRFs) are linked to an increase in cytosolic Ca(2+) concentration, and we therefore examined the effects of extracellular and intracellular acidosis on Ca(2+) responses and EDRF production in cultured porcine aortic endothelial cells.nnnMETHODS AND RESULTSnCytosolic pH (pH(i)) and Ca(2+) were measured using fluorescent dyes, BCECM/AM (pH-indicator) and fura-2/AM (Ca(2+)-indicator), respectively. EDRFs, nitric oxide (NO) and prostaglandin I(2) (PGI(2)) were assessed using DAF-FM/DA (NO-indicator dye) fluorometry and 6-keto PGF(1alpha) enzyme immunoassay, respectively. HEPES buffers titrated to pH 6.4, 6.9, and 7.4 were used to alter extracellular pH (pH(o)), and propionate (20 mmol/L) was applied to cause intracellular acidosis. Extracellular acidosis strongly suppressed bradykinin (BK, 10 nmol/L)- and thapsigargin (TG, 1 micromol/L)-induced Ca(2+) responses by 30 and 23% at pH(o) 6.9, and by 80 and 97% at pH(o) 6.4, respectively. During the examinations, there were no significant differences in pH(i) among the three groups at pH(o) 7.4, 6.9, and 6.4. Extracellular acidosis also inhibited BK-stimulated PGI(2) production by 55% at pH(o) 6.9 and by 77% at pH(o) 6.4, and NO production by 38% at pH(o) 6.9 and by 91% at pH(o) 6.4. The suppressive effects of extracellular acidosis on Ca(2+) responses and NO production were reversible. Propionate changed pH(i) from 7.3 to 6.9, without altering pH(o) (7.4). Intracellular acidosis had no effect on BK- and TG-induced Ca(2+) responses or NO production.nnnCONCLUSIONnThese results indicate that extracellular, but not intracellular, acidosis causes endothelial dysfunction by inhibiting store-operated Ca(2+) entry, so helping to clarify the vascular pathophysiology of conditions such as ischaemia, hypoxia, acidosis, and ischaemia-reperfusion.

Distribution of late gadolinium enhancement in end-stage hypertrophic cardiomyopathy and dilated cardiomyopathy: differential diagnosis and prediction of cardiac outcome.

BACKGROUNDnThe prognostic implications of late gadolinium enhancement (LGE) have been evaluated in ischemic and non-ischemic cardiomyopathies. The present study analyzed LGE distribution in patients with end-stage hypertrophic cardiomyopathy (ES-HCM) and with dilated cardiomyopathy (DCM), and tried to identify high risk patients in DCM.nnnMETHODSnEleven patients with ES-HCM and 72 with DCM underwent cine- and LGE-cardiac magnetic resonance and ultrasound cardiography. The patient outcome was analyzed retrospectively for 5years of follow-up.nnnRESULTSnLGE distributed mainly in the inter-ventricular septum, but spread more diffusely into other left ventricular segments in patients with ES-HCM and in a certain part of patients with DCM. Thus, patients with DCM can be divided into three groups according to LGE distribution; no LGE (n=24), localized LGE (localized at septum, n=36), and extensive LGE (spread into other segments, n=12). Reverse remodeling occurred after treatment in patients with no LGE and with localized LGE, but did not in patients with extensive LGE and with ES-HCM. The event-free survival rate for composite outcome (cardiac death, hospitalization for decompensated heart failure or ventricular arrhythmias) was lowest in patients with extensive LGE (92%, 74% and 42% in no LGE, localized LGE, and extensive LGE, p=0.02 vs. no LGE), and was comparable to that in patients with ES-HCM (42%).nnnCONCLUSIONSnIn DCM, patients with extensive LGE showed no functional recovery and the lowest event-free survival rate that were comparable to patients with ES-HCM. The analysis of LGE distribution may be valuable to predict reverse remodeling and to identify high-risk patients.

Delayed Enhancement on Cardiac Magnetic Resonance and Clinical, Morphological, and Electrocardiographical Features in Hypertrophic Cardiomyopathy

BACKGROUNDnThe clinical, morphological, and electrocardiographical relevance of delayed enhancement (DE) in cardiac magnetic resonance (CMR) was studied in patients with hypertrophic cardiomyopathy (HCM).nnnMETHODS AND RESULTSnA total of 56 patients underwent both gadolinium-enhanced CMR and 12-lead electrocardiogram. The CMR demonstrated DE at the left ventricular (LV) wall in 39 patients. The patients with DE included more cases with dilated phase of HCM, higher New York Heart Association (NYHA) classes and incidence of ventricular tachyarrhythmias (VT), lower LV ejection fraction (LVEF) and mean LV wall thickness (WT), and a larger ratio of maximum to minimum LVWT. The QRS duration was prolonged and the QRS axis deviated toward left with increases in the DE volume (r = 0.58 and r = 0.41, P < .01). Abnormal Q waves were present in 5 patients and the location coincided with the DE segments in 4 patients, but the concordance was not significant. The amplitude of T waves correlated with the ratio of the apex to basal LVWT (r = 0.38, P < .01) and was more negative in cases with DE at the apex.nnnCONCLUSIONSnIn HCM, the DE was associated with higher NYHA classes and prevalence of VT, impaired global LV function and asymmetrical hypertrophy, and conduction disturbance, abnormal Q waves, and giant negative T waves.

Mitochondrial dysfunction caused by saturated fatty acid loading induces myocardial insulin-resistance in differentiated H9c2 myocytes: A novel ex vivo myocardial insulin-resistance model

Heart failure (HF) is often accompanied with metabolic disorders and insufficient energy production. Some previous studies have suggested an elevated serum free fatty acid (FA) due to chronic adrenergic stimulation induces myocardial insulin-resistance, which further impairs myocardial energy production. Because little is known about the pathogenesis of FA-induced cardiac insulin-resistance, we established an ex vivo cardiac insulin-resistant model and investigated the relationship between insulin-resistance and mitochondrial dysfunction. The ex vivo insulin-resistant myocytes, which was produced by treating differentiated H9c2 myocytes with palmitate (saturated FA; 0.2mM) for 24h, exhibited insulin-signaling deficiency and attenuated 2-deoxy-d-glucose (2-DG) uptake. When myocytes were pretreated with Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (TMPyP, a ROS scavenger; 200 μM), the insulin-signaling deficiency by palmitate was restored, whereas the attenuated 2-DG uptake was remained. In contrast to TMPyP, the pretreatment with perhexiline (a mitochondrial FA uptake inhibitor; 2 μM) restored the insulin-signaling deficiency and the attenuated 2-DG uptake by palmitate. Perhexiline restored the depolarized mitochondrial membrane potential (ΔΨm) and the reduced intracellular ATP by palmitate, and thereby improved the impaired GLUT4 recruitment to plasma membrane after insulin, whereas TMPyP failed to do so. These results suggested that the mitochondrial dysfunction by saturated FA loading and consequent intracellular energy shortage induced myocardial insulin-resistance in our ex vivo insulin-resistant model.

Intravenous glutathione prevents renal oxidative stress after coronary angiography more effectively than oral N -acetylcysteine

This study proposes the intravenous administration of glutathione (GSH) as a novel strategy to prevent contrast medium-induced renal oxidative stress. Renal oxidative stress is a critical cause of contrast-induced nephropathy (CIN). Recent reports have described that N-acetylcysteine (NAC) may prevent CIN by scavenging reactive oxygen species in the kidney. Twenty-one patients with reduced renal function who underwent coronary angiography (CAG) were equally assigned to the control, NAC and GSH (100xa0mg/min for 30xa0min before CAG) groups. CIN occurred in two patients, one in the control and the other in the NAC group. In the control group, the urinary lipid hydroperoxides (LOOHs) increased to 299.5xa0±xa094.4% of the baseline at 2xa0h after CAG (meanxa0±xa0SE, pxa0<xa00.01). The increase in LOOHs was completely abolished in the GSH group (5.5xa0±xa08.8%, pxa0=xa0ns), but not in the NAC group (196.8xa0±xa081.3%, pxa0<xa00.05). In the control group, the serum GSH level fell by 9.4xa0±xa02.3% at 2xa0h after CAG (pxa0<xa00.01). The decrease was prevented in the GSH group (−1.8xa0±xa08.5%, pxa0=xa0ns), but not in the NAC group (−10.0xa0±xa03.3%, pxa0<xa00.05). The renal damage by contrast medium-induced oxidative stress occurs soon after CAG, and intravenous GSH is more effective in preventing the oxidative stress than oral NAC. This advantage may make GSH a potentially more effective therapeutic strategy against CIN.

Comparison of Global and Regional Abnormalities in 99mTc-sestamibi and Cardiac Magnetic Resonance Imaging in Dilated Cardiomyopathy

OBJECTIVEnThe clinical relevance of perfusion defects and increased washout rate (WOR) in (99m)Tc-sestamibi (Tc MIBI) imaging has not been well characterized in dilated cardiomyopathy (DCM). We analyzed abnormalities in Tc MIBI imaging in relation to those in cardiac magnetic resonance (CMR) imaging.nnnMETHODS AND RESULTSnNineteen DCM patients underwent both Tc MIBI and CMR imaging. The perfusion defects and global and regional MIBI WORs were evaluated with planar and single photon emission computed tomography (SPECT) images. The left ventricular function and the delayed enhancement (DE) were estimated with the cine- and DE-mode CMR. In the DCM patients, the Tc MIBI SPECT showed perfusion defects in 65 segments of 14 patients. The global and regional Tc MIBI WORs were higher than those in 10 normal volunteers (19.4 +/- 9.1% vs. 11.7 +/- 6.8% in global and 13.8 +/- 8.6% vs. 9.6 +/- 8.2% in regional WORs; mean +/- SD, P < .01). The DE-mode CMR demonstrated DE in 103 segments of 14 patients. The severity of perfusion defects was correlated with the extent of DE (r = 0.71, P < .01). The regional Tc MIBI WOR was highest in the segments with perfusion defects or DE, but the Tc MIBI WOR in segments without them was also higher than that in the normal volunteers. There was a weak but significant negative correlation between the regional Tc MIBI WOR and wall thickening (r = -0.23, P < .01).nnnCONCLUSIONSnIn DCM, the severity of perfusion defects and the increase in Tc MIBI WOR reflect the spread of myocardial fibrosis and/or scar. The increase in Tc MIBI WOR is potentially associated with regional dysfunction of LV wall.

Ultrasound analysis of the relationship between right internal jugular vein and common carotid artery in the left head-rotation and head-flexion position

Common carotid artery (CCA) injury is a serious complication of internal jugular vein (IJV) cannulation. To minimize unintentional CCA puncture, the anatomic relationship between the IJV and the CCA and the size of IJV were compared under different head positions. Ultrasound analyses of the IJV and the CCA were performed in 103 consecutive patients. Overlapping angle (OA), real puncture angle (RPA) and diameter of IJV (DIJV) were evaluated with 30° and 60° left rotation and with 30° left flexion. When the head position was changed from 30° left rotation to 60° left rotation, OA increased significantly from 6.5° ± 7.7° to 14.5° ± 7.4° at the cricoid cartilage level (Cricoid-level) and from 14.4° ± 8.4° to 20.6° ± 6.9° at the middle triangle level (Triangle-level), whereas RPA decreased significantly at these levels (from 49.7° ± 11.9° to 43.5° ± 13.1° and from 51.1° ± 14.4° to 44.3° ± 13.9°, respectively; P < 0.01 for both). When the head position was changed from 30° left rotation to 30° left flexion, neither OA nor RPA significantly changed (OA: 6.3° ± 6.1° and 15.0° ± 7.2°, RPA: 48.5° ± 12.4° and 51.8° ± 13.6°, P not significant vs 30° left rotation). There was no difference in DIJV when comparing 30° left rotation and 30° left flexion, although DIJV was largest at 60° left rotation. RPA positively correlated with age, and DIJV positively correlated with body mass index. In conclusion, excessive left rotation should be avoided to minimize the probability of unintentional CCA puncture during IJV cannulation. When 30° left rotation is not feasible, the head-flexion position should be utilized.