Wanrui Zhang

Cross-talk between calcium and reactive oxygen species signaling

AbstractCalcium (Ca2+) and reactive oxygen species (ROS) constitute the most important intracellular signaling molecules participating in the regulation and integration of diverse cellular functions. Here we briefly review cross-talk between the two prominent signaling systems that finely tune the homeostasis and integrate functionality of Ca2+ and ROS in different types of cells. Ca2+ modulates ROS homeostasis by regulating ROS generation and annihilation mechanisms in both the mitochondria and the cytosol. Reciprocal redox regulation of Ca2+ homeostasis occurs in different physiological and pathological processes, by modulating components of the Ca2+ signaling toolkit and altering characteristics of local and global Ca2+ signals. Functionally, interactions between Ca2+ and ROS signaling systems can be both stimulatory and inhibitory, depending on the type of target proteins, the ROS species, the dose, duration of exposure, and the cell contexts. Such extensive and complex cross-talk might enhance signaling coordination and integration, whereas abnormalities in either system might propagate into the other system and undermine the stability of both systems.

Mitofusin-2 Is a Major Determinant of Oxidative Stress-mediated Heart Muscle Cell Apoptosis

An inexorable loss of terminally differentiated heart muscle cells is a crucial causal factor for heart failure. Here, we have provided several lines of evidence to demonstrate that mitofusin-2 (Mfn-2; also called hyperplasia suppressor gene), a member of the mitofusin family, is a major determinant of oxidative stress-mediated cardiomyocyte apoptosis. First, oxidative stress with H2O2 led to concurrent increases in Mfn-2 expression and apoptosis in cultured neonatal rat cardiomyocytes. Second, overexpression of Mfn-2 to a level similar to that induced by H2O2 was sufficient to trigger myocyte apoptosis, which is associated with profound inhibition of Akt activation without altering ERK1/2 signaling. Third, Mfn-2 silencing inhibited oxidative stress-induced apoptosis in H9C2 cells, a cardiac muscle cell line. Furthermore, Mfn-2-induced myocyte apoptosis was abrogated by inhibition of caspase-9 (but not caspase-8) and by overexpression of Bcl-xL or enhanced activation of phosphatidylinositol 3-kinase-Akt, suggesting that inhibition of Akt signaling and activation of the mitochondrial death pathway are essentially involved in Mfn-2-induced heart muscle cell apoptosis. These results indicate that increased cardiac Mfn-2 expression is both necessary and sufficient for oxidative stress-induced heart muscle cell apoptosis, suggesting that Mfn-2 deregulation may be a crucial pathogenic element and a potential therapeutic target for heart failure.

Imaging superoxide flash and metabolism-coupled mitochondrial permeability transition in living animals

The mitochondrion is essential for energy metabolism and production of reactive oxygen species (ROS). In intact cells, respiratory mitochondria exhibit spontaneous “superoxide flashes”, the quantal ROS-producing events consequential to transient mitochondrial permeability transition (tMPT). Here we perform the first in vivo imaging of mitochondrial superoxide flashes and tMPT activity in living mice expressing the superoxide biosensor mt-cpYFP, and demonstrate their coupling to whole-body glucose metabolism. Robust tMPT/superoxide flash activity occurred in skeletal muscle and sciatic nerve of anesthetized transgenic mice. In skeletal muscle, imaging tMPT/superoxide flashes revealed labyrinthine three-dimensional networks of mitochondria that operate synchronously. The tMPT/superoxide flash activity surged in response to systemic glucose challenge or insulin stimulation, in an apparently frequency-modulated manner and involving also a shift in the gating mode of tMPT. Thus, in vivo imaging of tMPT-dependent mitochondrial ROS signals and the discovery of the metabolism-tMPT-superoxide flash coupling mark important technological and conceptual advances for the study of mitochondrial function and ROS signaling in health and disease.

Kissing and nanotunneling mediate intermitochondrial communication in the heart

Mitochondria in many types of cells are dynamically interconnected through constant fusion and fission, allowing for exchange of mitochondrial contents and repair of damaged mitochondria. However, constrained by the myofibril lattice, the ∼6,000 mitochondria in the adult mammalian cardiomyocyte display little motility, and it is unclear how, if at all, they communicate with each other. By means of target-expressing photoactivatable green fluorescent protein (PAGFP) in the mitochondrial matrix or on the outer mitochondrial membrane, we demonstrated that the local PAGFP signal propagated over the entire population of mitochondria in cardiomyocytes on a time scale of ∼10 h. Two elemental steps of intermitochondrial communications were manifested as either a sudden PAGFP transfer between a pair of adjacent mitochondria (i.e., “kissing”) or a dynamic nanotubular tunnel (i.e., “nanotunneling”) between nonadjacent mitochondria. The average content transfer index (fractional exchange) was around 0.5; the rate of kissing was 1‰ s−1 per mitochondrial pair, and that of nanotunneling was about 14 times smaller. Electron microscopy revealed extensive intimate contacts between adjacent mitochondria and elongated nanotubular protrusions, providing a structural basis for the kissing and nanotunneling, respectively. We propose that, through kissing and nanotunneling, the otherwise static mitochondria in a cardiomyocyte form one dynamically continuous network to share content and transfer signals.

Superoxide Flashes Reveal Novel Properties of Mitochondrial Reactive Oxygen Species Excitability in Cardiomyocytes

Superoxide flash represents quantal and bursting production of mitochondrial reactive oxygen species (ROS) instigated by transient opening of the mitochondrial permeability transition pore (mPTP). Given their critical role in metabolism, ischemia-reperfusion injury, and apoptosis, characterization of flash properties would be valuable to further mechanistic and physiological studies of this newly discovered mitochondrial phenomenon. Here we developed the flash detector FlashSniper based on segmentation of two-dimensional feature maps extracted from time-lapse confocal image stacks, and on the theory for correcting optical distortion of flash-amplitude histograms. Through large-scale analysis of superoxide flashes in cardiomyocytes, we demonstrated uniform mitochondrial ROS excitability among subsarcolemmal and intermyofibrillar mitochondria, and exponential distribution of intervals between consecutive flash events. Flash ignition displayed three different patterns: an abrupt rise from quiescence (44%), a rise with an exponential foot (27%), or a rise occurring after a pedestal precursor (29%), closely resembling action-potential initiation in excitable cells. However, the optical blurring-corrected amplitudes of superoxide flashes were highly variable, as were their durations, indicating stochastic automaticity of single-mitochondrion ROS excitation. Simultaneous measurement of mitochondrial membrane potential revealed that graded, rather than all-or-none, depolarization mirrored the precursor and the primary peak of the flash. We propose that superoxide flash production is a regenerative process dominated by stochastic, autonomous recruitment of a limited number of units (e.g., mPTPs) in single mitochondria.

Subsarcolemmal mitochondrial flashes induced by hypochlorite stimulation in cardiac myocytes

Abstract Mitochondrial superoxide flash (mitoflash) reflects quantal and bursting superoxide production and concurrent membrane depolarization triggered by transient mitochondrial permeability transition in many types of cells, at the level of single mitochondria. Here we investigate reactive oxygen species (ROS)-mediated modulation of mitoflash activity in cardiac myocytes and report a surprising finding that hypochlorite ions potently and preferentially triggered mitoflashes in the subsarcolemmal mitochondria (SSM), whereas hydrogen peroxide (H2O2) elicited mitoflash activity uniformly among SSM and interfibrillar mitochondria (IFM). The striking SSM mitoflash response to hypochlorite stimulation remained intact in cardiac myocytes from NOX2-deficient mice, excluding local NOX2-mediated ROS as the major player. Furthermore, it occurred concomitantly with SSM Ca2+ accumulation and local Ca2+ and CaMKII signaling played an important modulatory role by altering frequency and unitary properties of SSM mitoflashes. These findings underscore the functional heterogeneity of SSM and IFM and the oxidant-specific responsiveness of mitochondria to ROS, and may bear important ramifications in devising therapeutic strategies for the treatment of oxidative stress-related heart diseases.

P02.99. Acupuncture combined with an antidepressant has a better effect on major depression: a multi-center, randomized, controlled clinical trial

Methods 72 inpatients and outpatients with MD, diagnosed by the ICD-10, were randomly divided into three groups with three different treatments: combination of manualacupuncture and paroxetine (23 cases); combination of electro-acupuncture and paroxetine (32 cases); and paroxetine only (17 cases) for 6 weeks. Two statistical analyzing methods, intention to treat (ITT) and per protocol (PP), were applied to assess the main curative indexes, including Hamilton Depression Scale (HAMD), Self-rating depression scale (SDS), and Rating Scale for Side Effects (SERS) scores.

P02.152. Is electro-acupuncture beneficial for overall quality of life improvement of undergraduates in depressive states? A pragmatic controlled trial

Methods Fifty undergraduates in depressive states (CES-D score ≥16, HAMD score≥7, <17) were assigned to 4 groups based on intervention preference in a pragmatic trial. Electro-acupuncture, cognitive behavior therapy (CBT), and a combined intervention of electro-acupuncture and CBT were implemented as interventions. A rejected intervention group in which no intervention was practiced was considered as a control condition. The electroacupuncture implemented traditional Chinese medicine (TCM)-style acupuncture. The CBT is practiced as 8 sessions of group counseling (1 time/week). Each 8-week course of electro-acupuncture consisted of 16 sessions (2 times/week) in the clinic of Beijing University of Traditional Chinese Medicine. The combined intervention consisted of 16 sessions of electro-acupuncture (2 times/ week) and 8 sessions of CBT (1 time/week) in an 8-week course. WHOQOL-BREF was evaluated at baseline and 8 weeks after interventions.