"Real‐time monitoring of Hyper fluorescence during I/R was achieved through serial 2‐D imaging at a sampling rate of 30 s/frame. Cellular ischemia was achieved by perfusing the cells with oxygen‐deprived solution28 containing (in mmol/L) NaCl 137, KCl 4.9, CaCl2 1, MgSO4 1.2, NaH2PO4 1.2, HEPES 20, and NaS2O4 2, pH 7.4. Reperfusion was achieved with perfusion with the same solution without NaS2O4 and with 15 mmol/L glucose. "
[Show abstract][Hide abstract] ABSTRACT: The NADPH oxidase family (Nox) produces reactive oxygen species by adding the electron donated by NADPH to oxygen. Excessive reactive oxygen species production under a variety of pathological conditions has been attributed to increased Nox activity. Here, we aimed at investigating the role of Nox in cardiac ischemic injury through gain- and loss-of-function approaches.
We modulated Nox activity in the heart by cardiac-specific expression of Nox4 and dominant negative Nox4. Modulation of Nox activity drastically changes the cellular redox status. Increasing Nox activity by cardiac-specific overexpression of Nox4 imposed oxidative stress on the myocardium [increased NAD(P)(+)/NAD(P)H and decreased glutathione/glutathione disulfide ratio] and worsened cardiac energetics and contractile function after ischemia-reperfusion. Overexpression of the dominant negative Nox4 (DN), which abolished the Nox function, led to a markedly reduced state [decreased NAD(P)(+)/NAD(P)H and increased glutathione/glutathione disulfide ratio] at baseline and paradoxically promoted mitochondrial reactive oxygen species production during ischemia resulting in no recovery of heart function after reperfusion. Limiting the generation of reducing equivalent through modulating carbon substrates availability partially restored the NAD(+)/NADH ratio and protected dominant negative Nox4 hearts from ischemic injury.
This study reveals an important role of Nox in cardiac redox regulation and highlights the complexity of developing therapies that affect the intricately connected redox states.
Journal of the American Heart Association 12/2014; 3(1):e000555. DOI:10.1161/JAHA.113.000555 · 4.31 Impact Factor
"The high activity of superoxide flashes, in turn, contributes to activating JNK and p38, essential signals for adaptive cell survival responses . In cultured cardiomyocytes, a flurry of superoxide flash activity occurs in a 5–10 min window after reoxygenation from hypoxia or anoxia [67, 95]. In the pathology of Huntington disease, the elevated flash activity induced by elevated mitochondrial Ca2+ signaling acts to exacerbate mtDNA damage . "
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Molecular Medicine 07/2013; 91(8). DOI:10.1007/s00109-013-1067-4 · 5.11 Impact Factor
"Using Occam's razor, the simplest interpretation of the data is that they arise from the same, rather than different, mitochondrial process. Taken together with previous evidence for the ROS origin of cpYFP-flashes (Huang et al., 2011; Wang et al., 2008) (see also Introduction section), we conclude that all three types of mitochondrial flashes originate from a common, fundamental mitochondrial process featuring bursting ROS production under physiological conditions. By taking RT 90 to approximately reflect the underlying ROS-reacting duration of mitoSOX-flashes and DCF-flashes, we have shown that the kinetic features are also generally comparable among the three types of flashes: mitoSOX-flashes and cpYFP-flashes are ~10 s in duration and DCF-flashes are ~2-fold longer (~20 s). "
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
Life sciences 06/2013; 93:178-186. DOI:10.1016/j.lfs.2013.06.012 · 2.70 Impact Factor
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