Masao Saotome

Bidirectional Ca2+-dependent control of mitochondrial dynamics by the Miro GTPase

Calcium oscillations suppress mitochondrial movements along the microtubules to support on-demand distribution of mitochondria. To activate this mechanism, Ca2+ targets a yet unidentified cytoplasmic factor that does not seem to be a microtubular motor or a kinase/phosphatase. Here, we have studied the dependence of mitochondrial dynamics on the Miro GTPases that reside in the mitochondria and contain two EF-hand Ca2+-binding domains, in H9c2 cells and primary neurons. At resting cytoplasmic [Ca2+] ([Ca2+]c), movements of the mitochondria were enhanced by Miro overexpression irrespective of the presence of the EF-hands. The Ca2+-induced arrest of mitochondrial motility was also promoted by Miro overexpression and was suppressed when either the Miro were depleted or their EF-hand was mutated. Miro also enhanced the fusion state of the mitochondria at resting [Ca2+]c but promoted mitochondrial fragmentation at high [Ca2+]c. These effects of Miro on mitochondrial morphology seem to involve Drp1 suppression and activation, respectively. In primary neurons, Miro also caused an increase in dendritic mitochondrial mass and enhanced mitochondrial calcium signaling. Thus, Miro proteins serve as a [Ca2+]c-sensitive switch and bifunctional regulator for both the motility and fusion-fission dynamics of the mitochondria.

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.

Distribution of late gadolinium enhancement in various types of cardiomyopathies: Significance in differential diagnosis, clinical features and prognosis

The recent development of cardiac magnetic resonance (CMR) techniques has allowed detailed analyses of cardiac function and tissue characterization with high spatial resolution. We review characteristic CMR features in ischemic and non-ischemic cardiomyopathies (ICM and NICM), especially in terms of the location and distribution of late gadolinium enhancement (LGE). CMR in ICM shows segmental wall motion abnormalities or wall thinning in a particular coronary arterial territory, and the subendocardial or transmural LGE. LGE in NICM generally does not correspond to any particular coronary artery distribution and is located mostly in the mid-wall to subepicardial layer. The analysis of LGE distribution is valuable to differentiate NICM with diffusely impaired systolic function, including dilated cardiomyopathy, end-stage hypertrophic cardiomyopathy (HCM), cardiac sarcoidosis, and myocarditis, and those with diffuse left ventricular (LV) hypertrophy including HCM, cardiac amyloidosis and Anderson-Fabry disease. A transient low signal intensity LGE in regions of severe LV dysfunction is a particular feature of stress cardiomyopathy. In arrhythmogenic right ventricular cardiomyopathy/dysplasia, an enhancement of right ventricular (RV) wall with functional and morphological changes of RV becomes apparent. Finally, the analyses of LGE distribution have potentials to predict cardiac outcomes and response to treatments.

Roles of mitochondrial fragmentation and reactive oxygen species in mitochondrial dysfunction and myocardial insulin resistance

PURPOSE Evidence suggests an association between aberrant mitochondrial dynamics and cardiac diseases. Because myocardial metabolic deficiency caused by insulin resistance plays a crucial role in heart disease, we investigated the role of dynamin-related protein-1 (DRP1; a mitochondrial fission protein) in the pathogenesis of myocardial insulin resistance. METHODS AND RESULTS DRP1-expressing H9c2 myocytes, which had fragmented mitochondria with mitochondrial membrane potential (ΔΨm) depolarization, exhibited attenuated insulin signaling and 2-deoxy-d-glucose (2-DG) uptake, indicating insulin resistance. Treatment of the DRP1-expressing myocytes with Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (TMPyP) significantly improved insulin resistance and mitochondrial dysfunction. When myocytes were exposed to hydrogen peroxide (H2O2), they increased DRP1 expression and mitochondrial fragmentation, resulting in ΔΨm depolarization and insulin resistance. When DRP1 was suppressed by siRNA, H2O2-induced mitochondrial dysfunction and insulin resistance were restored. Our results suggest that a mutual enhancement between DRP1 and reactive oxygen species could induce mitochondrial dysfunction and myocardial insulin resistance. In palmitate-induced insulin-resistant myocytes, neither DRP1-suppression nor TMPyP restored the ΔΨm depolarization and impaired 2-DG uptake, however they improved insulin signaling. CONCLUSIONS A mutual enhancement between DRP1 and ROS could promote mitochondrial dysfunction and inhibition of insulin signal transduction. However, other mechanisms, including lipid metabolite-induced mitochondrial dysfunction, may be involved in palmitate-induced insulin resistance.

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

AIMS Hypoxia, 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. METHODS AND RESULTS Cytosolic 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. CONCLUSION These 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.

Primary cardiac lymphoma--a case report.

Primary cardiac lymphoma, which is very rare, is generally regarded to have a poor prognosis. A case of a 69-year-old man with primary cardiac lymphoma diagnosed by antemortem exam ination is reported. A computed tomography scan of the chest demonstrated a huge right atrial mass with invasion into the other chambers. No mediastinal lymphadenopathy was detected. Cytologic analysis of pericardial effusion revealed diffuse large B-cell type non- Hodgkin malignant lymphoma. The patient died on the 18th day of chemotherapy (cyclophos phamide, hydroxydaunomycin, oncovin, and prednisone) due to low-output syndrome and multiple organ failure. At autopsy, massive gray-white tumor almost occupied the right atrium and invaded the right inferior lobe of the lung. Although prognosis of primary cardiac lymphoma remains poor, early diagnosis may improve the prognosis.

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.

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

BACKGROUND The clinical, morphological, and electrocardiographical relevance of delayed enhancement (DE) in cardiac magnetic resonance (CMR) was studied in patients with hypertrophic cardiomyopathy (HCM). METHODS AND RESULTS A 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. CONCLUSIONS In 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.