Calcium oscillations suppress mitochondrial movements along the microtubules to support on-demand distribution of mitochondria. To activate this mechanism, Ca 2؉ 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 Ca 2؉ -binding domains, in H9c2

SummaryLocal Ca 2+ transfer between adjoining domains of the sarcoendoplasmic reticulum (ER/SR) and mitochondria allows ER/SR Ca 2+ release to activate mitochondrial Ca 2+ uptake and to evoke a matrix [Ca 2+ ] ([Ca 2+ ] m ) rise. [Ca 2+ ] m exerts control on several steps of energy metabolism to synchronize ATP generation with cell function. However, calcium signal propagation to the mitochondria may also ignite a cell death program through opening of the permeability transition pore (PTP). This occurs when the Ca 2+ release from the ER/SR is enhanced or is coincident with sensitization of the PTP. Recent studies have shown that several pro-apoptotic factors, including members of the Bcl-2 family proteins and reactive oxygen species (ROS) regulate the Ca 2+ sensitivity of both the Ca 2+ release channels in the ER and the PTP in the mitochondria. To test the relevance of the mitochondrial Ca 2+ accumulation in various apoptotic paradigms, methods are available for buffering of [Ca 2+ ], for dissipation of the driving force of the mitochondrial Ca 2+ uptake and for inhibition of the mitochondrial Ca 2+ transport mechanisms. However, in intact cells, the efficacy and the specificity of these approaches have to be established. Here we discuss mechanisms that recruit the mitochondrial calcium signal to a pro-apoptotic cascade and the approaches available for assessment of the relevance of the mitochondrial Ca 2+ handling in apoptosis. We also present a systematic evaluation of the effect of ruthenium red and Ru360, two inhibitors of mitochondrial Ca 2+ uptake on cytosolic [Ca 2+ Mitochondrial Ca 2+ transport mechanismsThe pathways of the mitochondrial Ca 2+ import and export are illustrated in Fig1. Ca 2+ traverses the outer mitochondrial membrane (OMM) primarily through the voltage dependent anion-selective channel (VDAC) [1][2][3]. The molecular nature of the proteins mediating the Ca 2+ transport across the inner mitochondrial membrane (IMM) remains unknown. The protein mediating Ca 2+ uptake is referred as the uniporter (UP) and has been identified as a Ca 2+ selective ion channel [4]. The UP passes Ca 2+ along the electrochemical gradient largely due Correspondence to: Dr. György Hajnóczky, Department of Pathology, Anatomy and Cell Biology, Suite 261 JAH, Thomas Jefferson University, Philadelphia PA 19107, USA, Tel.: (215) 503-1427, Fax.: (215) 923-2218, E-mail: E-mail: Gyorgy.Hajnoczky@jefferson.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. In the sequence from the opening of the IP3Rs to the opening of the PTP, the calcium signal propagation may be affec...

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]m reduction. Cytosolic Na ϩ concentrations that yielded one-half maximal activity levels for mitoNCX were 3.6 mM at normal ⌬⌿ m and 7.6 mM at ⌬⌿m dissipation. We conclude that 1) the mitochondrial Ca 2ϩ uniporter accumulates Ca 2ϩ in a manner that is dependent on ⌬⌿m at the physiological range of [Ca 2ϩ ]c; 2) ⌬⌿m dissipation opens the mPTP and results in Ca 2ϩ influx to mitochondria; and 3) although mitoNCX activity is impaired, mitoNCX extrudes Ca 2ϩ from the matrix even after ⌬⌿m dissipation. permeability transition pore; Na ϩ /Ca 2ϩ exchange; depolarization; ischemia-reperfusion injury ACCUMULATING EVIDENCE REVEALS that mitochondria play primary roles in fatal cell damage during ischemia-reperfusion (33). Key events that occur during ischemia include cytosolic Ca 2ϩ elevation, ATP depletion, high P i concentration, depolarized membrane potential, and acidotic pH. On reperfusion and recovery of normal pH, a burst of reactive oxygen species occurs, mitochondrial Ca 2ϩ overload ensues, and these lead to opening of the mitochondrial permeability transition pore (mPTP; Refs. 8,12,34). Opening of the mPTP allows water and solutes Յ1,500 Da in size to enter the matrix and cause mitochondrial swelling, rupture of the outer mitochondrial membrane, and release of cytochrome c or apoptosis-inducing factor, which initiates apoptotic programmed cell death (12,24,34). Because previous studies (8,12,13) (10,11). Furthermore, recent studies (4,8,10,11,18) also suggest a possible contribution by the mPTP to Ca 2ϩ homeostasis in both the cytosol and mitochondria. Despite the considerable attention given to the pathophysiological significance of mitochondrial Ca 2ϩ , the regulation and/or modulation of mitochondrial Ca 2ϩ during pathophysiological conditions such as ischemia-reperfusion injury are unclear. In previous studies, information about mitochondrial Ca 2ϩ was obtained using isolated mitochondria, whereby the structural and functional properties of organelles were seriously affected, and other cellular architectures were separated from the mitochondria. In this study, we measured [Ca 2ϩ ] m in saponin-permeabilized rat ventricular myocytes and investigated how ⌬⌿ m depolarization affects [Ca 2ϩ ] m and mitochondrial Ca 2ϩ transport systems such as the Ca 2ϩ uniporter, the mPTP, and mitoNCX.

Summary Mitochondrial distribution and motility are recognized as central to many cellular functions but their regulation by signaling mechanisms remains to be elucidated. Here we report that ROS, either derived from an extracellular source or intracellularly generated, controls mitochondrial distribution and function, by dose-dependently, specifically and reversibly decreasing mitochondrial motility in both rat hippocampal primary cultured neurons and cell lines. ROS decrease motility independently of cytoplasmic [Ca2+], mitochondrial membrane potential or permeability transition pore opening, known effectors of oxidative stress. However, multiple lines of genetic and pharmacological evidence support that a ROS-activated MAP kinase, p38α is required for the motility inhibition. Furthermore, anchoring mitochondria directly to kinesins without involvement of the physiological adaptors between the organelles and the motor protein prevents the H2O2–induced decrease in mitochondrial motility. Thus, ROS engage p38α and the motor adaptor complex to exert changes in mitochondrial motility, which likely has both physiological and pathophysiological relevance.

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.

Saotome M, Katoh H, Yaguchi Y, Tanaka T, Urushida T, Satoh H, Hayashi H. Transient opening of mitochondrial permeability transition pore by reactive oxygen species protects myocardium from ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol 296: H1125-H1132, 2009. First published February 6, 2009 doi:10.1152/ajpheart.00436.2008.-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 (H2O2), 2 M] 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 2O2 (2 M) significantly improved the postischemic recoveries in left ventricular developed pressure, intracellular phosphocreatine, and ATP levels. A specific mPTP inhibitor cyclosporin A (CsA; 0.2 M) canceled these H2O2-induced effects. In isolated permeabilized myocytes, H 2O2 (1 M) accelerated the calcein leakage from mitochondria in a CsA-sensitive manner, indicating the opening of mPTP by H 2O2. However, H2O2 did not depolarize mitochondrial membrane potential (⌬⌿ m) even in the presence of oligomycin (F 1/F0 ATPase inhibitor; 1 M) and decreased mitochondrial Ca 2ϩ concentration ([Ca 2ϩ ]m) by accelerating the mitochondrial Ca 2ϩ extrusion via an mPTP. We conclude that the transient mPTP opening could be involved in the H2O2-induced cardioprotection against reperfusion injury, and the reduction of [Ca 2ϩ ]m without the change in ⌬⌿m might be a possible mechanism for the protection. energy metabolism; nuclear magnetic resonance spectroscopy; permeabilized myocytes; mitochondrial calcium ALTHOUGH THE GENERATION OF reactive oxygen species (ROS; e.g., superoxide, hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals) are well-known factors leading to ischemia-reperfusion (I/R) injury, it has also been reported that ROS participated in ischemic preconditioning (IPC) and served as an ameliorating cardioprotective substrate against I/R injury (5,26,29). The generation of ROS during brief and repetitive I/R is suggested to be a possible trigger for the initiation of IPC. However, despite previous intensive efforts, the precise mechanisms of endogenous ROS-mediated cardioprotection and how it mimics the IPC still remained elusive.Mitochondria have been identified as the target organelle responsible for the cell injury during I/R, since mitochondria are closely involved in a process of necrotic and apoptotic cell death through the mitochondrial permeability transition pore (mPTP) opening (14,21,24). The opening and clo...

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.