Hayato Ohtani

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.

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

BACKGROUND The 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. METHODS Eleven 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. RESULTS LGE 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%). CONCLUSIONS In 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.

Eicosapentaenoic acid ameliorates palmitate-induced lipotoxicity via the AMP kinase/dynamin-related protein-1 signaling pathway in differentiated H9c2 myocytes

Background: Emerging evidence suggested the preferable effects of eicosapentaenoic acid (EPA; n‐3 polyunsaturated fatty acid) against cardiac lipotoxicity, which worsens cardiac function by means of excessive serum free fatty acids due to chronic adrenergic stimulation under heart failure. Nonetheless, the precise molecular mechanisms remain elusive. In this study, we focused on dynamin‐related protein‐1 (Drp1) as a possible modulator of the EPA‐mediated cardiac protection against cardiac lipotoxicity, and investigated the causal relation between AMP‐activated protein kinase (AMPK) and Drp1. Methods and results: When differentiated H9c2 myocytes were exposed to palmitate (PAL; saturated fatty acid, 400 &mgr;M) for 24 h, these myocytes showed activation of caspases 3 and 7, enhanced caspase 3 cleavage, depolarized mitochondrial membrane potential, depleted intracellular ATP, and enhanced production of intracellular reactive oxygen species. These changes suggested lipotoxicity due to excessive PAL. PAL enhanced mitochondrial fragmentation with increased Drp1 expression, as well. EPA (50 &mgr;M) restored the PAL‐induced apoptosis, mitochondrial dysfunction, and mitochondrial fragmentation with increased Drp1 expression by PAL. EPA activated phosphorylation of AMPK, and pharmacological activation of AMPK by 5‐aminoimidazole‐4‐carboxamide ribonucleotide ameliorated the PAL‐induced apoptosis, mitochondrial dysfunction, and downregulated Drp1. An AMPK knockdown via RNA interference enhanced Drp1 expression and attenuated the protective effects of EPA against the PAL‐induced lipotoxicity. Conclusion: EPA ameliorates the PAL‐induced lipotoxicity via AMPK activation, which subsequently suppresses mitochondrial fragmentation and Drp1 expression. Our findings may provide new insights into the molecular mechanisms of EPA‐mediated myocardial protection in heart failure. HIGHLIGHTSEPA reduces mitochondrial fragmentation and protects myocytes from lipotoxicity.EPA activates AMPK and downregulates Drp1.

Functional, morphological and electrocardiographical abnormalities in patients with apical hypertrophic cardiomyopathy and apical aneurysm: correlation with cardiac MR.

Objective The prognosis of apical hypertrophic cardiomyopathy (APH) has been benign, but apical myocardial injury has prognostic importance. We studied functional, morphological and electrocardiographical abnormalities in patients with APH and with apical aneurysm and sought to find parameters that relate to apical myocardial injury. Methods Study design: a multicentre trans-sectional study. Patients: 45 patients with APH and 5 with apical aneurysm diagnosed with transthoracic echocardiography (TTE) in the database of Hamamatsu Circulation Forum. Measure: the apical contraction with cine-cardiac MR (CMR), the myocardial fibrotic scar with late gadolinium enhancement (LGE)-CMR, and QRS fragmentation (fQRS) defined when two ECG-leads exhibited RSR’s patterns. Results Cine-CMR revealed 27 patients with normal, 12 with hypokinetic and 11 with dyskinetic apical contraction. TTE misdiagnosed 11 (48%) patients with hypokinetic and dyskinetic contraction as those with normal contraction. Apical LGE was apparent in 10 (83%) and 11 (100%) patients with hypokinetic and dyskinetic contraction, whereas only in 11 patients (41%) with normal contraction (p<0.01). Patients with dyskinetic apical contraction had the lowest left ventricular ejection fraction, the highest prevalence of ventricular tachycardia, and the smallest ST depression and depth of negative T waves. The presence of fQRS was associated with impaired apical contraction and apical LGE (OR=8.32 and 8.61, p<0.05). Conclusions CMR is superior to TTE for analysing abnormalities of the apex in patients with APH and with apical aneurysm. The presence of fQRS can be a promising parameter for the early detection of apical myocardial injury.

Glycogen synthase kinase-3β opens mitochondrial permeability transition pore through mitochondrial hexokinase II dissociation

Accumulating evidence has revealed pivotal roles of glycogen synthase kinase-3β (GSK3β) inactivation on cardiac protection. Because the precise mechanisms of cardiac protection against ischemia/reperfusion (I/R) injury by GSK3β-inactivation remain elusive, we investigated the relationship between GSK3β-mediated mitochondrial hexokinase II (mitoHK-II; a downstream target of GSK3β) dissociation and mitochondrial permeability transition pore (mPTP) opening. In Langendorff-perfused hearts, GSK3β inactivation by SB216763 improved the left ventricular-developed pressure and retained mitoHK-II binding after I/R. In permeabilized myocytes, GSK3β depolarized mitochondrial membrane potential with accelerated mitochondrial calcein release (suggesting GSK3β-mediated mPTP opening) and decreased mitoHK-II bindings. GSK3β-mediated mPTP opening depended on mitoHK-II binding, i.e., it was accelerated by dissociation of mitoHK-II (dicyclohexylcarbodiimide) and attenuated by enhancement of mitoHK-II binding (dextran). However, inactivation of mitoHK-II by glucose-depletion or glucose-6-phosphate inhibited the GSK3β-mediated mPTP opening. We conclude that GSK3β-mediated mPTP opening may be involved in I/R injury and regulated by mitoHK-II binding and activity.

The Electrical Mechanical Interference of Implantable Cardiac Devices at X-Ray Fluoroscopy

Background: X-ray radiation during computed tomography (CT) scanning has been reported to cause electrical mechanical interference (EMI: oversense and partial electrical reset) in implantable cardiac device. However it remains elusive whether EMI was occurred by X-ray fluoroscopy at clinical scan condition. <PurposeVOur purpose is to investigate the EMI of implantable cardiac devices at X-ray fluoroscopy. Method: Implantable cardiac device (cardiac pacemaker, biventricular pacemaker: CRT-P, biventricular pacemaker with implantable cardioverter defibrillator: CRT-D, Medtronic) with its electrode lead sink in a tank filled with saline. X-ray fluoroscopy (Philips: Allura Xper) scanned Implantable cardiac device with scan conditions in our hospital. Results: (1) On low frequency pulse radiation (2, 3, 3.75, 6 frame/sec), X-ray radiation inhibited the pacing pulses of cardiac pacemakers due to oversense of noise. When X-ray radiation exposed CRT-D, cardioversion was delivered because frequent noise was understood as ventricular fibrillation. (2) On high frequency pulse radiation (15, 30 frame/sec), all implantable cardiac devices EMI was not occurred because remove of high frequency noise by band-path filter. (3) X-ray induced EMI depended on the direction of X-ray radiation. Conclusion: EMI of implantable cardiac devices at X-ray fluoroscopy depended on frequency of pulse radiation and direction of X-ray radiation. The radiological technologists should select the frequency of pulse radiation and remove implantable cardiac device from radiation range.