Wei Shang

Superoxide flashes: early mitochondrial signals for oxidative stress-induced apoptosis.

Irreversible mitochondrial permeability transition and the resultant cytochrome c release signify the commitment of a cell to apoptotic death. However, the role of transient MPT (tMPT) because of flickering opening of the mitochondrial permeability transition pore remains elusive. Here we show that tMPT and the associated superoxide flashes (i.e. tMPT/superoxide flashes) constitute early mitochondrial signals during oxidative stress-induced apoptosis. Selenite (a ROS-dependent insult) but not staurosporine (a ROS-independent insult) stimulated an early and persistent increase in tMPT/superoxide flash activity prior to mitochondrial fragmentation and a global ROS rise, independently of Bax translocation and cytochrome c release. Selectively targeting tMPT/superoxide flash activity by manipulating cyclophilin D expression or scavenging mitochondrial ROS markedly impacted the progression of selenite-induced apoptosis while exerting little effect on the global ROS response. Furthermore, the tMPT/superoxide flash served as a convergence point for pro- and anti-apoptotic regulation mediated by cyclophilin D and Bcl-2 proteins. These results indicate that tMPT/superoxide flashes act as early mitochondrial signals mediating the apoptotic response during oxidative stress, and provide the first demonstration of highly efficacious local mitochondrial ROS signaling in deciding cell fate.

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.

Imaging ROS signaling in cells and animals

Reactive oxygen species (ROS) act as essential cellular messengers, redox regulators, and, when in excess, oxidative stressors that are widely implicated in pathologies of cancer and cardiovascular and neurodegenerative diseases. Understanding such complexity of the ROS signaling is critically hinged on the ability to visualize and quantify local, compartmental, and global ROS dynamics at high selectivity, sensitivity, and spatiotemporal resolution. The past decade has witnessed significant progress in ROS imaging at levels of intact cells, whole organs or tissues, and even live organisms. In particular, major advances include the development of novel synthetic or genetically encoded fluorescent protein-based ROS indicators, the use of protein indicator-expressing animal models, and the advent of in vivo imaging technology. Innovative ROS imaging has led to important discoveries in ROS signaling—for example, mitochondrial superoxide flashes as elemental ROS signaling events and hydrogen peroxide transients for wound healing. This review aims at providing an update of the current status in ROS imaging, while identifying areas of insufficient knowledge and highlighting emerging research directions.

Imaging Ca2+ Nanosparks in Heart with a New Targeted Biosensor

Rationale: In cardiac dyads, junctional Ca2+ directly controls the gating of the ryanodine receptors (RyRs), and is itself dominated by RyR-mediated Ca2+ release from the sarcoplasmic reticulum. Existing probes do not report such local Ca2+ signals because of probe diffusion, so a junction-targeted Ca2+ sensor should reveal new information on cardiac excitation–contraction coupling and its modification in disease states. Objective: To investigate Ca2+ signaling in the nanoscopic space of cardiac dyads by targeting a new sensitive Ca2+ biosensor (GCaMP6f) to the junctional space. Methods and Results: By fusing GCaMP6f to the N terminus of triadin 1 or junctin, GCaMP6f-triadin 1/junctin was targeted to dyadic junctions, where it colocalized with t-tubules and RyRs after adenovirus-mediated gene transfer. This membrane protein-tagged biosensor displayed ≈4× faster kinetics than native GCaMP6f. Confocal imaging revealed junctional Ca2+ transients (Ca2+ nanosparks) that were ≈50× smaller in volume than conventional Ca2+ sparks (measured with diffusible indicators). The presence of the biosensor did not disrupt normal Ca2+ signaling. Because no indicator diffusion occurred, the amplitude and timing of release measurements were improved, despite the small recording volume. We could also visualize coactivation of subclusters of RyRs within a single junctional region, as well as quarky Ca2+ release events. Conclusions: This new, targeted biosensor allows selective visualization and measurement of nanodomain Ca2+ dynamics in intact cells and can be used to give mechanistic insights into dyad RyR operation in health and in disease states such as when RyRs become orphaned.

Superoxide constitutes a major signal of mitochondrial superoxide flash.

AIMS Mitochondrial flashes detected with an N- and C-terminal circularly-permuted yellow fluorescent protein (cpYFP) have been thought to represent transient and quantal bursts of superoxide production under physiological, stressful and pathophysiological conditions. However, the superoxide nature of the cpYFP-flash has been challenged, considering the pH-sensitivity of cpYFP and the distinctive regulation of the flash versus the basal production of mitochondrial reactive oxygen species (ROS). Thus, the aim of the study is to further determine the origin of mitochondrial flashes. MAIN METHODS We investigated the origin of the flashes using the widely-used pH-insensitive ROS indicators, mitoSOX, an indicator for superoxide, and 2, 7-dichlorodihydrofluorescein diacetate (DCF), an indicator for H2O2 and other oxidants. KEY FINDINGS Robust, quantal, and stochastic mitochondrial flashes were detected with either mitoSOX or DCF in several cell-types and in mitochondria isolated from the heart. Both mitoSOX-flashes and DCF-flashes showed similar incidence and kinetics to those of cpYFP-flashes, and were equally sensitive to mitochondria-targeted antioxidants. Furthermore, they were markedly decreased by inhibitors or an uncoupler of the mitochondrial electron transport chain, as is the case with cpYFP-flashes. The involvement of the mitochondrial permeability transition pore in DCF-flashes was evidenced by the coincidental loss of mitochondrial membrane potential and matrix-enriched rhod-2, as well as by their sensitivity to cyclosporine A. SIGNIFICANCE These data indicate that all the three types of mitochondrial flashes stem from the common physiological process of bursting superoxide and ensuing H2O2 production in the matrix of single mitochondrion.

Proinflammatory Cytokines Stimulate Mitochondrial Superoxide Flashes in Articular Chondrocytes In Vitro and In Situ

Objective Mitochondria play important roles in many types of cells. However, little is known about mitochondrial function in chondrocytes. This study was undertaken to explore possible role of mitochondrial oxidative stress in inflammatory response in articular chondrocytes. Methods Chondrocytes and cartilage explants were isolated from wild type or transgenic mice expressing the mitochondrial superoxide biosensor - circularly permuted yellow fluorescent protein (cpYFP). Cultured chondrocytes or cartilage explants were incubated in media containing interleukin-1β (10 ng/ml) or tumor necrosis factor-α (10 ng/ml) to stimulate an inflammatory response. Mitochondrial imaging was carried out by confocal and two-photon microscopy. Mitochondrial oxidative status was evaluated by “superoxide flash” activity recorded with time lapse scanning. Results Cultured chondrocytes contain abundant mitochondria that show active motility and dynamic morphological changes. In intact cartilage, mitochondrial abundance as well as chondrocyte density declines with distance from the surface. Importantly, sudden, bursting superoxide-producing events or “superoxide flashes” occur at single-mitochondrion level, accompanied by transient mitochondrial swelling and membrane depolarization. The superoxide flash incidence in quiescent chondrocytes was ∼4.5 and ∼0.5 events/1000 µm2*100 s in vitro and in situ, respectively. Interleukin-1β or tumor necrosis factor-α stimulated mitochondrial superoxide flash activity by 2-fold in vitro and 5-fold in situ, without altering individual flash properties except for reduction in spatial size due to mitochondrial fragmentation. Conclusions The superoxide flash response to proinflammatory cytokine stimulation in vitro and in situ suggests that chondrocyte mitochondria are a significant source of cellular oxidants and are an important previously under-appreciated mediator in inflammatory cartilage diseases.

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.

β-Adrenergic-stimulated l-type channel Ca2+ entry mediates hypoxic Ca2+ overload in intact heart

Ca(2+) mishandling plays a key role in ischemia- and hypoxia-related cardiac dysfunction and injury. However, the cellular and molecular mechanisms underlying hypoxic intracellular Ca(2+) ([Ca(2+)]i) overload remain incompletely understood. This study aimed to investigate possible mechanisms of [Ca(2+)]i overload during hypoxia in the intact heart. In Langendorff-perfused heart expressing the Ca(2+) indicator GCaMP2, confocal microscopy was used to simultaneously visualize [Ca(2+)]i, mitochondrial membrane potential (ΔΨm, by tetramethylrhodamine methyl ester) and sarcolemmal integrity (by Evans blue). Upon hypoxia (pO2 ~20 mmHg in glucose-free perfusate), [Ca(2+)]i transients were initially enhanced and then became depressed, arrhythmic, and completely abolished within 12 min. At ~20 min, basal [Ca(2+)]i rose to its first peak at a supraphysiological level, coincident with loss of ΔΨm and onset of rigor. A greater [Ca(2+)]i rise occurred at ~2h and was linked to the loss of sarcolemmal integrity. Removal of extracellular Ca(2+) or blockade of the l-type Ca(2+) channel (LTCC) (10 μM diltiazem or nifedipine) prevented [Ca(2+)]i overload and markedly delayed the loss of ΔΨm; by contrast, depletion of the sarcoplasmic reticulum Ca(2+) store by thapsigargin did not have any significant effect. Importantly, β-adrenergic blockade or depletion of the sympathetic catecholamine store by reserpine slowed the Ca(2+) and mitochondrial responses to hypoxia in intact heart. This LTCC-mediated hypoxic [Ca(2+)]i overload was reproduced in isolated cardiomyocytes when β-adrenergic agonist was present. Taken together, we conclude that Ca(2+) entry through β-adrenergic-stimulated LTCC underlies hypoxia-induced [Ca(2+)]i overload and the ensuing loss of mitochondrial function in intact heart.