Publications by authors named "Guohua Gong"

37 Publications

Mitochondrial Dynamics in Adult Cardiomyocytes and Heart Diseases.

Front Cell Dev Biol 2020 17;8:584800. Epub 2020 Dec 17.

Institute for Regenerative Medicine, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China.

Mitochondria are the powerhouse organelles of cells; they participate in ATP generation, calcium homeostasis, oxidative stress response, and apoptosis. Thus, maintenance of mitochondrial function is critical for cellular functions. As highly dynamic organelles, the function of mitochondria is dynamically regulated by their fusion and fission in many cell types, which regulate mitochondrial morphology, number, distribution, metabolism, and biogenesis in cells. Mature rod-shaped cardiomyocytes contain thousands of end-to-end contacted spheroid mitochondria. The movement of mitochondria in these cells is limited, which hinders the impetus for research into mitochondrial dynamics in adult cardiomyocytes. In this review, we discuss the most recent progress in mitochondrial dynamics in mature (adult) cardiomyocytes and the relationship thereof with heart diseases.
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http://dx.doi.org/10.3389/fcell.2020.584800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773778PMC
December 2020

Modified Protocol for A Mouse Heart Failure Model Using Minimally Invasive Transverse Aortic Constriction.

STAR Protoc 2020 Dec 20;1(3):100186. Epub 2020 Nov 20.

Institute for Regenerative Medicine, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China.

Here, we present a modified protocol for a mouse heart failure (HF) model using minimally invasive transverse aortic constriction (miTAC). miTAC is a more effective method in mice than the standard open-chest transverse aortic constriction (TAC) to generate an HF model. miTAC does not require the cutting of the ribs or tracheal intubation with artificial ventilation; it also has a higher survival rate. The successful outcome of the HF model can be verified using transthoracic echocardiography and histology. For complete details on the use and execution of this protocol, please refer to Hu et al. (2003) and Richards et al. (2019).
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http://dx.doi.org/10.1016/j.xpro.2020.100186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757422PMC
December 2020

Protocol for Imaging of Mitoflashes in Live Cardiomyocytes.

STAR Protoc 2020 Sep 3;1(2):100101. Epub 2020 Sep 3.

Institute for Regenerative Medicine, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China.

We describe a protocol for imaging a mitochondrial fluorescence transient increase event (Mitoflash) in live cardiomyocytes using a confocal microscope. Mitoflash, detected by mitochondria-targeted circularly permuted fluorescent protein (mt-cpYFP), can be used to assess mitochondrial respiration function . The protocol is also suitable for live-cell imaging of other adherent cells, including fibroblasts and hepatocytes. For complete details on the use and execution of this protocol, please refer to Gong et al. (2014) and Gong et al. (2015).
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http://dx.doi.org/10.1016/j.xpro.2020.100101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7580095PMC
September 2020

Protocol for Measurement of Oxygen Consumption Rate in Permeabilized Cardiomyocytes.

STAR Protoc 2020 Sep 25;1(2):100072. Epub 2020 Jul 25.

Institute for Regenerative Medicine, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China.

Analysis of mitochondrial respiration function represented by the oxygen consumption rate is necessary for assessing mitochondrial respiration function. This protocol describes steps to evaluate the respiration function of mitochondria in saponin-permeabilized cardiomyocytes. In permeabilized cells, mitochondria are in a relatively integrated cellular system, and mitochondrial respiration is more physiologically relevant than isolated mitochondria. For complete details on the use and execution of this protocol, please refer to Gong et al. (2015a) and Gong et al. (2015b).
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http://dx.doi.org/10.1016/j.xpro.2020.100072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7580108PMC
September 2020

Protocol for Isolation of Viable Adult Rat Cardiomyocytes with High Yield.

STAR Protoc 2020 Sep 9;1(2):100045. Epub 2020 Jun 9.

Institute for Regenerative Medicine, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China.

Isolation of high-quantity and high-quality ventricular cardiomyocytes from adult rats is critical to study heart physiology and pathology and for drug toxicity screening. It remains challenging to produce a high yield of viable cardiomyocytes from rats. Here, we present our modified enzymatic digestion protocol that relies on the Langendorff device to generate large numbers of viable cardiomyocytes consistently. The most critical parts of this protocol are the selection of rat age and digestion time to obtain viable cardiomyocytes. For complete details on the use and execution of this protocol, please refer to Liu et al. (2019) and Qin et al. (2020).
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http://dx.doi.org/10.1016/j.xpro.2020.100045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7580079PMC
September 2020

Mitoflash generated at the Qo site of mitochondrial Complex III.

J Cell Physiol 2021 Apr 15;236(4):2920-2933. Epub 2020 Sep 15.

Institute for Regenerative Medicine, Research Center for Translational Medicine, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China.

The previous research has shown that mitochondrial flash (mitoflash) genesis are functionally and mechanistically integrated with mitochondrial electron transport chain (ETC) energy metabolism. However, the response of mitoflash to superoxide is not entirely consistent with the response of MitoSOX Red. The generation mechanism of mitoflash is still unclear. Here, we investigated mitoflash activities, using the different combinations of ETC substrates and inhibitors, in permeabilized cardiomyocytes or hearts. We found that blocking the complete electron flow, from Complex I to IV, with any one of ETC inhibitors including rotenone (Rot), antimycin A (AntA), myxothiazol (Myxo), stigmatellin, and sodium cyanide, will lead to the abolishment of mitoflashes triggered by substrates in adult permeabilized cardiomyocytes. However, Myxo boosted mitoflashes triggered by the reverse electron of N,N,N',N'-tetramethyl-p-phenylenediamine/ascorbate. Moreover, Rot and AntA furtherly enhanced mitoflash activity rather than depressed it, suggesting that mitoflashes generated at the Complex III Qo site. Meanwhile, the inhibition of Complex III protein expression resulted in the activity of Complex III decrease, which decreased mitoflash frequency. The function defect (no change of protein level) of the Qo site of Complex III in aging hearts augmented mitoflash generation confirmed the Qo site function was critical to mitoflash genesis. Thus, our results indicate that mitoflash detected by circularly permuted yellow fluorescent protein is generated at the Qo site of Complex III.
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http://dx.doi.org/10.1002/jcp.30059DOI Listing
April 2021

Maternal iron deficiency does not affect the iron status of fetuses with congenital heart defects: Does it affect heart development?

Int J Cardiol 2020 05;306:89

Institute for Regenerative Medicine, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China; Department of Pharmacy, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai 200120, China. Electronic address:

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http://dx.doi.org/10.1016/j.ijcard.2020.01.066DOI Listing
May 2020

Mitochondrial fusion mediated by fusion promotion and fission inhibition directs adult mouse heart function toward a different direction.

FASEB J 2020 01 26;34(1):663-675. Epub 2019 Nov 26.

Institute for Regenerative Medicine, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China.

Mitochondrial fusion and fission are essential for heart function. Abrogating mitochondrial dynamism leads to cardiomyopathy. Excessive mitochondrial fragmentation is involved in most heart diseases, thus enhancing mitochondrial fusion will be a potential therapeutic strategy. To understand the effects of promoting mitochondrial fusion in adult cardiac, we investigated mice hearts, and cultured murine embryonic fibroblasts (MEFs), in which mitofusin 2 (Mfn2) overexpressed or dynamin-related protein 1 (Drp1) was abrogated concomitantly forcing mitochondrial fusion. Parallel studies revealed that fission-defective Drp1 knockout hearts and MEFs evoked stronger mitochondrial enlargement, enhanced mitophagy with mitochondrial volume decrease and increased mitochondrial calcium uptake, superoxide production, and permeability transition pore opening, contributed to cardiomyocyte apoptosis and dilated cardiomyopathy. Mfn2 overexpression in the adult heart is comparable with the control except for slight mitochondrial enlargement and mitochondrial volume increase, but without mitophagy induction. Moreover, Mfn2 overexpression increases mitochondrial biogenesis and fusion could protect against mitochondrial fragmentation and Drp1 deletion evoking mitophagy in MEFs. Our findings indicate that mitochondrial fusion provoked by fusion promotion and fission inhibition direct the different fate of heart, Mfn2 upregulation other than Drp1 downregulation well maintains heart mitochondrial function is a more safe strategy for correcting excessive mitochondrial fragmentation in hearts.
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http://dx.doi.org/10.1096/fj.201901671RDOI Listing
January 2020

Comparative study on isolation and mitochondrial function of adult mouse and rat cardiomyocytes.

J Mol Cell Cardiol 2019 11 12;136:64-71. Epub 2019 Sep 12.

Institute for Regenerative Medicine, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China. Electronic address:

Background: Cultured adult mouse and rat cardiomyocytes are the best and low-cost cell model for cardiac cellular physiology, pathology, drug toxicity screening, and intervention. The functions of mouse cardiomyocytes decline faster than rat cardiomyocytes in culture conditions. However, little is known about the difference of mitochondrial function between cultured mouse and rat myocytes.

Methods And Results: A large number of adult mouse and rat cardiomyocytes were comparative isolated using a simple perfusion system. Cardiomyocytes mitochondrial functions were measured after 2 h, 1 day, 2 days, 3 days, and 4 days culture by monitoring mitoflashes. We found that the mitochondrial function of mouse myocytes was remarkedly declined on the third day. Then, we focused on the third day cultured mouse and rat myocytes, comparatively analyzing the respiration function and superoxide generation stimulated by pyruvate/malate/ADP and the mitochondrial permeability transition pore (mPTP) opening induction. Mouse myocytes showed lower respiration and mitoflash activity, but without the change of maximum uncoupled respiration when compared with rat myocytes. Although the response to superoxide production stimulated by respiration substrates was slower than rat myocytes, the basal superoxide generation is faster than the rat. The faster mitochondrial reactive oxygen species (ROS) generation of mouse myocytes upon laser stimulation triggered the faster mPTP opening compared with the rat. Finally, antioxidant MitoTEMPO pretreatment preserved the mitochondrial function of mouse myocytes on the third day.

Conclusions: The mitochondrial function and stability are different between cultured mouse and rat cardiac myocytes beyond 3 days even though they both belong to Muridae. Mitochondrial ROS impairs the mitochondrial functions of mouse cardiomyocytes on the third day. Suppressing superoxide maintained the mitochondrial function of mouse myocytes on the third day.
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http://dx.doi.org/10.1016/j.yjmcc.2019.09.006DOI Listing
November 2019

Heart specific knockout of Ndufs4 ameliorates ischemia reperfusion injury.

J Mol Cell Cardiol 2018 10 27;123:38-45. Epub 2018 Aug 27.

Mitochondria and Metabolism Center, Department of Anesthesiology and Pain Medicine, University of Washington, Seattle, WA 98109, USA; Department of Pathology, University of Washington, Seattle, WA, 98195, USA. Electronic address:

Rationale: Ischemic heart disease (IHD) is a leading cause of mortality. The most effective intervention for IHD is reperfusion, which ironically causes ischemia reperfusion (I/R) injury mainly due to oxidative stress-induced cardiomyocyte death. The exact mechanism and site of reactive oxygen species (ROS) generation during I/R injury remain elusive.

Objective: We aim to test the hypothesis that Complex I-mediated forward and reverse electron flows are the major source of ROS in I/R injury of the heart.

Methods And Results: We used a genetic model of mitochondrial Complex I deficiency, in which a Complex I assembling subunit, Ndufs4 was knocked out in the heart (Ndufs4H-/-). The Langendorff perfused Ndufs4H-/- hearts exhibited significantly reduced infarct size (45.3 ± 5.5% in wild type vs 20.9 ± 8.1% in Ndufs4H-/-), recovered contractile function, and maintained mitochondrial membrane potential after no flow ischemia and subsequent reperfusion. In cultured adult cardiomyocytes from Ndufs4H-/- mice, I/R mimetic treatments caused minimal cell death. Reintroducing Ndufs4 in Ndufs4H-/- cardiomyocytes abolished the protection. Mitochondrial NADH declined much slower in Ndufs4H-/- cardiomyocytes during reperfusion suggesting decreased forward electron flow. Mitochondrial flashes, a marker for mitochondrial respiration, were inhibited in Ndufs4H-/- cardiomyocytes at baseline and during I/R, which was accompanied by preserved aconitase activity suggesting lack of oxidative damage. Finally, pharmacological blockade of forward and reverse electron flow at Complex I inhibited I/R-induced cell death.

Conclusions: These results provide the first genetic evidence supporting the central role of mitochondrial Complex I in I/R injury of mouse heart. The study also suggests that both forward and reverse electron flows underlie oxidative cardiomyocyte death during reperfusion.
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http://dx.doi.org/10.1016/j.yjmcc.2018.08.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6192835PMC
October 2018

Mitoflash lights single mitochondrial dynamics events in mature cardiomyocytes.

Biochem Biophys Res Commun 2018 09 21;503(2):729-736. Epub 2018 Jun 21.

Institute for Regenerative Medicine, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, 200092, China. Electronic address:

Mitochondria, the powerhouse of eukaryotic cells, are highly dynamic organelle. Mitochondrial fission, fusion, kissing and contraction have been reported over and over again in non-static cells, such as fibroblast, with tubular mitochondrial networks. Even though the fluorescence propagation among mitochondria of mature cardiomyocytes had been captured using mitochondrial matrix targeted photoactivatable GFP (PAGFP) or MitoDendra proteins, there are no direct evidence that single real time mitochondrial dynamics events exist in mature cardiomyocytes with ball-like mitochondria. Here we first time revealed the visualizable single mitochondrial dynamics events in adult mature cardiomyocytes by the mitochondrial flash (mitoflash). We found fission, fusion, contraction and kissing were accompanied by a mitoflash event. Metabolism could increase mitochondrial contraction. Fusion and Kissing mediated inter-mitochondrial communication with higher frequency than fission. These results demonstrate that mitochondria of static mature cardiomyocytes are undergoing the rare, but real dynamics change.
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http://dx.doi.org/10.1016/j.bbrc.2018.06.068DOI Listing
September 2018

Correction: MiR-124 acts as a target for Alzheimer's disease by regulating BACE1.

Oncotarget 2018 05 15;9(37):24871. Epub 2018 May 15.

Medicinal Chemistry and Pharmacology Institute, Inner Mongolia University for The Nationalities, Tongliao, Inner Mongolia, P.R. China.

[This corrects the article DOI: 10.18632/oncotarget.23119.].
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http://dx.doi.org/10.18632/oncotarget.25461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5973865PMC
May 2018

miR-539 acts as a tumor suppressor by targeting epidermal growth factor receptor in breast cancer.

Sci Rep 2018 02 1;8(1):2073. Epub 2018 Feb 1.

Medicinal Chemistry and Pharmacology Institute, Inner Mongolia University for Nationalities, Tongliao, Inner Mongolia, 028000, China.

Breast cancer is the most frequently diagnosed malignancy and the leading cause of cancer-associated death in women worldwide. microRNAs (miRNAs) play critical roles in the cellular processes of breast cancer. However, the crucial roles and underlying mechanisms of miR-539 in breast cancer remain unclear. By RT-qPCR, we found that expression of miR-539 was markedly down-regulated in breast cancer tissues and cell lines compared with that in paired adjacent normal tissues and normal cell lines. The low level of miR-539 expression was positively associated with lymph node metastasis. Furthermore, forced expression of miR-539 inhibited proliferation and migration of breast cancer MDA-MB-231 and MCF7 cells in vitro and suppressed tumor growth in vivo. Moreover, bioinformatics analysis and luciferase reporter assays indicated that epidermal growth factor receptor (EGFR) was a direct target of miR-539. Over-expression of miR-539 decreased the EGFR mRNA and protein levels in MDA-MB-231 and MCF7 cells. In addition, ectopic over-expression of EGFR partly reversed miR-539-inhibited proliferation as well as migration of MDA-MB-231 and MCF7 cells. Taken together, our results demonstrate that miR-539 functions as a tumor suppressor in breast cancer by downregulating EGFR, supporting the targeting of the novel miR-539/EGFR axis as a potentially effective therapeutic approach for breast cancer.
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http://dx.doi.org/10.1038/s41598-018-20431-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5794864PMC
February 2018

MiR-124 acts as a target for Alzheimer's disease by regulating BACE1.

Oncotarget 2017 Dec 9;8(69):114065-114071. Epub 2017 Dec 9.

Medicinal Chemistry and Pharmacology Institute, Inner Mongolia University for The Nationalities, Tongliao, Inner Mongolia, P.R. China.

Although large numbers of microRNAs (miRNAs) expressed in Alzheimer disease (AD) have been detected, their functions and mechanisms of regulation remain to be fully clarified. Beta-site Amyloid precursor protein Cleaving Enzyme 1 (BACE1) has been one of the prime therapeutic targets for AD. Here, we identified that miR-124 levels are gradually decreased in AD. In addition, we demonstrated that miR-124 suppresses BACE1 expression by directly targeting the 3'UTR of Bace1 mRNA . Inhibition of miR-124 significantly increased BACE1 levels in neuronal cells. In contrast, miR-124 overexpression significantly suppressed BACE1 expression in cells. And finally we determined that downregulation of miR-124 alleviated Aβ-induced viability inhibition and decreased apoptosis in SH-SY5Y cells. Our results demonstrated that miR-124 is a potent negative regulator of BACE1 in the cellular AD phenotype and might be involved in the pathogenesis of AD.
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http://dx.doi.org/10.18632/oncotarget.23119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5768386PMC
December 2017

miR-15b represses BACE1 expression in sporadic Alzheimer's disease.

Oncotarget 2017 Oct 21;8(53):91551-91557. Epub 2017 Sep 21.

Medicinal Chemistry and Pharmacology Institute, Inner Mongolia University for The Nationalities, Tongliao, Inner Mongolia, P.R. China.

Beta-site Amyloid precursor protein Cleaving Enzyme 1 (BACE1) is conceived as a potential target for therapies against Alzheimer disease (AD). MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression in a sequence-specific manner. Although miRNAs have been increasingly recognized as important modulators in sporadic AD. In order to confirm whether miR-15b correlates with the BACE1 upregulation in sporadic AD, we firstly evaluated the expression of miR-15b and BACE1 in sporadic AD brain tissues and analyzed the correlation of miR-15b with BACE1. Then we determined the regulation of miR-15b in SH-SY5Y cells on the BACE1 expression. And finally we determined the targeting to 3' UTR of BACE1 by miR-15b by a luciferase reporter. Downregulation of miR-15b alleviated Aβ-induced viability inhibition and decreased apoptosis in SH-SY5Y cells. Our results demonstrated that miR-15b play an important role in the cellular AD phenotype and might be involved in the pathogenesis of AD.
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http://dx.doi.org/10.18632/oncotarget.21177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5710945PMC
October 2017

Screening and identification of potential biomarkers in triple-negative breast cancer by integrated analysis.

Oncol Rep 2017 Oct 21;38(4):2219-2228. Epub 2017 Aug 21.

Medicinal Chemistry and Pharmacology Institute, Inner Mongolia University for Nationalities, Tongliao, Inner Mongolia 028000, P.R. China.

Triple-negative breast cancer (TNBC) has attracted great attention due to its unique biology, poor prognosis, and aggressiveness. TNBC patients are more likely to suffer from metastasis. We screened and identified the TNBC-specific genes as potential biomarkers. A total of 167 breast cancer samples (45 TNBC and 122 non-TNBC) were used in the integrated analysis. Gene expression microarrays were used to screen the differentially expressed genes. We identified 65 core DEGs. According to the GO and KEGG analysis, the gene function enrichment in TNBC was revealed, such as basal cell carcinoma, prostate cancer, oocyte meiosis and choline metabolism in cancer pathways. Moreover, the PPI network reconstruction would benefit the screening of hubs. A RFS analysis of TNBC-specific genes was also conducted. RT-PCR was used to validate the expression pattern of hubs in TNBC. Finally, nine genes were identified and all of them were novel, specific and higher dysregulation expressed genes in TNBC. Such that, these genes will serve as potential biomarkers in TNBC and benefit further research in TNBC.
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http://dx.doi.org/10.3892/or.2017.5911DOI Listing
October 2017

Identification and prognostic value of anterior gradient protein 2 expression in breast cancer based on tissue microarray.

Tumour Biol 2017 Jul;39(7):1010428317713392

1 Medicinal Chemistry and Pharmacology Institute, Inner Mongolia University for Nationalities, Tongliao, Inner Mongolia, China.

Breast cancer has attracted substantial attention as one of the major cancers causing death in women. It is crucial to find potential biomarkers of prognostic value in breast cancer. In this study, the expression pattern of anterior gradient protein 2 in breast cancer was identified based on the main molecular subgroups. Through analysis of 69 samples from the Gene Expression Omnibus database, we found that anterior gradient protein 2 expression was significantly higher in non-triple-negative breast cancer tissues compared with normal tissues and triple-negative breast cancer tissues (p < 0.05). The data from a total of 622 patients from The Cancer Genome Atlas were analysed. The data from The Cancer Genome Atlas and results from quantitative reverse transcription polymerase chain reaction also verified the anterior gradient protein 2 expression pattern. Furthermore, we performed immunohistochemical analysis. The quantification results revealed that anterior gradient protein 2 is highly expressed in non-triple-negative breast cancer (grade 3 excluded) and grade 1 + 2 (triple-negative breast cancer excluded) tumours compared with normal tissues. Anterior gradient protein 2 was significantly highly expressed in non-triple-negative breast cancer (grade 3 excluded) and non-triple-negative breast cancer tissues compared with triple-negative breast cancer tissues (p < 0.01). In addition, anterior gradient protein 2 was significantly highly expressed in grade 1 + 2 (triple-negative breast cancer excluded) and grade 1 + 2 tissues compared with grade 3 tissues (p < 0.05). Analysis by Fisher's exact test revealed that anterior gradient protein 2 expression was significantly associated with histologic type, histological grade, oestrogen status and progesterone status. Univariate analysis of clinicopathological variables showed that anterior gradient protein 2 expression, tumour size and lymph node status were significantly correlated with overall survival in patients with grade 1 and 2 tumours. Cox multivariate analysis revealed anterior gradient protein 2 as a putative independent indicator of unfavourable outcomes (p = 0.031). All these data clearly showed that anterior gradient protein 2 is highly expressed in breast cancer and can be regarded as a putative biomarker for breast cancer prognosis.
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http://dx.doi.org/10.1177/1010428317713392DOI Listing
July 2017

Correcting mitochondrial fusion by manipulating mitofusin conformations.

Nature 2016 12 24;540(7631):74-79. Epub 2016 Oct 24.

Center for Pharmacogenomics, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

Mitochondria are dynamic organelles that exchange contents and undergo remodelling during cyclic fusion and fission. Genetic mutations in MFN2 (the gene encoding mitofusin 2) interrupt mitochondrial fusion and cause the untreatable neurodegenerative condition Charcot-Marie-Tooth disease type 2A (CMT2A). It has not yet been possible to directly modulate mitochondrial fusion, in part because the structural basis of mitofusin function is not completely understood. Here we show that mitofusins adopt either a fusion-constrained or a fusion-permissive molecular conformation, directed by specific intramolecular binding interactions, and demonstrate that mitofusin-dependent mitochondrial fusion can be regulated in mouse cells by targeting these conformational transitions. On the basis of this model, we engineered a cell-permeant minipeptide to destabilize the fusion-constrained conformation of mitofusin and promote the fusion-permissive conformation, reversing mitochondrial abnormalities in cultured fibroblasts and neurons that harbour CMT2A-associated genetic defects. The relationship between the conformational plasticity of mitofusin 2 and mitochondrial dynamism reveals a central mechanism that regulates mitochondrial fusion, the manipulation of which can correct mitochondrial pathology triggered by defective or imbalanced mitochondrial dynamics.
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http://dx.doi.org/10.1038/nature20156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5315023PMC
December 2016

CaMKII induces permeability transition through Drp1 phosphorylation during chronic β-AR stimulation.

Nat Commun 2016 10 14;7:13189. Epub 2016 Oct 14.

Mitochondria and Metabolism Center, Department of Anesthesiology and Pain Medicine, University of Washington, Seattle, Washington 98109, USA.

Mitochondrial permeability transition pore (mPTP) is involved in cardiac dysfunction during chronic β-adrenergic receptor (β-AR) stimulation. The mechanism by which chronic β-AR stimulation leads to mPTP openings is elusive. Here, we show that chronic administration of isoproterenol (ISO) persistently increases the frequency of mPTP openings followed by mitochondrial damage and cardiac dysfunction. Mechanistically, this effect is mediated by phosphorylation of mitochondrial fission protein, dynamin-related protein 1 (Drp1), by Ca/calmodulin-dependent kinase II (CaMKII) at a serine 616 (S616) site. Mutating this phosphorylation site or inhibiting Drp1 activity blocks CaMKII- or ISO-induced mPTP opening and myocyte death in vitro and rescues heart hypertrophy in vivo. In human failing hearts, Drp1 phosphorylation at S616 is increased. These results uncover a pathway downstream of chronic β-AR stimulation that links CaMKII, Drp1 and mPTP to bridge cytosolic stress signal with mitochondrial dysfunction in the heart.
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http://dx.doi.org/10.1038/ncomms13189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5067512PMC
October 2016

Mitochondrial Flash: Integrative Reactive Oxygen Species and pH Signals in Cell and Organelle Biology.

Antioxid Redox Signal 2016 09 14;25(9):534-49. Epub 2016 Jul 14.

4 Department of Medicine, Center for Translational Medicine, Sidney Kimmel Medical College, Thomas Jefferson University , Philadelphia, Pennsylvania.

Significance: Recent breakthroughs in mitochondrial research have advanced, reshaped, and revolutionized our view of the role of mitochondria in health and disease. These discoveries include the development of novel tools to probe mitochondrial biology, the molecular identification of mitochondrial functional proteins, and the emergence of new concepts and mechanisms in mitochondrial function regulation. The discovery of "mitochondrial flash" activity has provided unique insights not only into real-time visualization of individual mitochondrial redox and pH dynamics in live cells but has also advanced understanding of the excitability, autonomy, and integration of mitochondrial function in vivo.

Recent Advances: The mitochondrial flash is a transient and stochastic event confined within an individual mitochondrion and is observed in a wide range of organisms from plants to Caenorhabditis elegans to mammals. As flash events involve multiple transient concurrent changes within the mitochondrion (e.g., superoxide, pH, and membrane potential), a number of different mitochondrial targeted fluorescent indicators can detect flash activity. Accumulating evidence indicates that flash events reflect integrated snapshots of an intermittent mitochondrial process arising from mitochondrial respiration chain activity associated with the transient opening of the mitochondrial permeability transition pore.

Critical Issues: We review the history of flash discovery, summarize current understanding of flash biology, highlight controversies regarding the relative roles of superoxide and pH signals during a flash event, and bring forth the integration of both signals in flash genesis.

Future Directions: Investigations using flash as a biomarker and establishing its role in cell signaling pathway will move the field forward. Antioxid. Redox Signal. 25, 534-549.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5035371PMC
http://dx.doi.org/10.1089/ars.2016.6739DOI Listing
September 2016

Parkin-mediated mitophagy directs perinatal cardiac metabolic maturation in mice.

Science 2015 Dec 3;350(6265):aad2459. Epub 2015 Dec 3.

Center for Pharmacogenomics, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO, USA.

In developing hearts, changes in the cardiac metabolic milieu during the perinatal period redirect mitochondrial substrate preference from carbohydrates to fatty acids. Mechanisms responsible for this mitochondrial plasticity are unknown. Here, we found that PINK1-Mfn2-Parkin-mediated mitophagy directs this metabolic transformation in mouse hearts. A mitofusin (Mfn) 2 mutant lacking PINK1 phosphorylation sites necessary for Parkin binding (Mfn2 AA) inhibited mitochondrial Parkin translocation, suppressing mitophagy without impairing mitochondrial fusion. Cardiac Parkin deletion or expression of Mfn2 AA from birth, but not after weaning, prevented postnatal mitochondrial maturation essential to survival. Five-week-old Mfn2 AA hearts retained a fetal mitochondrial transcriptional signature without normal increases in fatty acid metabolism and mitochondrial biogenesis genes. Myocardial fatty acylcarnitine levels and cardiomyocyte respiration induced by palmitoylcarnitine were concordantly depressed. Thus, instead of transcriptional reprogramming, fetal cardiomyocyte mitochondria undergo perinatal Parkin-mediated mitophagy and replacement by mature adult mitochondria. Mitophagic mitochondrial removal underlies developmental cardiomyocyte mitochondrial plasticity and metabolic transitioning of perinatal hearts.
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http://dx.doi.org/10.1126/science.aad2459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4747105PMC
December 2015

Multiomic analysis of mice epilepsy models suggest that miR-21a expression modulates mRNA and protein levels related to seizure deterioration.

Genet Res (Camb) 2015 Dec 22;97:e22. Epub 2015 Dec 22.

Medicinal Chemistry and Pharmacology Institute,Inner Mongolia University for the Nationalities,Tongliao,Inner Mongolia,P.R. China.

Epilepsy is now recognized as the second most common neurological disease in China. To determine the genetic cause of epileptic encephalopathy, we performed a multiomics study using mouse models of controls, anticonvulsant mice treated with five drugs and epileptic mice. Based on genome-wide profiling analysis, we discovered four genes in the epileptic mouse group with differentially-expressed mRNA. After isobaric tags for relative and absolute quantification (iTRAQ) validation, only one gene, SNCA, remained, which was associated with apoptotic response of neuronal cells, and regulation of dopamine release and transport. We also identified three miRNAs targeting SNCA, out of which mmu-miR-21a-3p demonstrated a seven-fold change in expression between control and epileptic mice.
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http://dx.doi.org/10.1017/S0016672315000245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6863633PMC
December 2015

Mitochondrial flash as a novel biomarker of mitochondrial respiration in the heart.

Am J Physiol Heart Circ Physiol 2015 Oct 14;309(7):H1166-77. Epub 2015 Aug 14.

Mitochondria and Metabolism Center, Department of Anesthesiology and Pain Medicine, University of Washington, Seattle, Washington; and

Mitochondrial respiration through electron transport chain (ETC) activity generates ATP and reactive oxygen species in eukaryotic cells. The modulation of mitochondrial respiration in vivo or under physiological conditions remains elusive largely due to the lack of appropriate approach to monitor ETC activity in a real-time manner. Here, we show that ETC-coupled mitochondrial flash is a novel biomarker for monitoring mitochondrial respiration under pathophysiological conditions in cultured adult cardiac myocyte and perfused beating heart. Through real-time confocal imaging, we follow the frequency of a transient bursting fluorescent signal, named mitochondrial flash, from individual mitochondria within intact cells expressing a mitochondrial matrix-targeted probe, mt-cpYFP (mitochondrial-circularly permuted yellow fluorescent protein). This mt-cpYFP recorded mitochondrial flash has been shown to be composed of a major superoxide signal with a minor alkalization signal within the mitochondrial matrix. Through manipulating physiological substrates for mitochondrial respiration, we find a close coupling between flash frequency and the ETC electron flow, as measured by oxygen consumption rate in cardiac myocyte. Stimulating electron flow under physiological conditions increases flash frequency. On the other hand, partially block or slowdown electron flow by inhibiting the F0F1 ATPase, which represents a pathological condition, transiently increases then decreases flash frequency. Limiting electron entrance at complex I by knocking out Ndufs4, an assembling subunit of complex I, suppresses mitochondrial flash activity. These results suggest that mitochondrial electron flow can be monitored by real-time imaging of mitochondrial flash. The mitochondrial flash frequency could be used as a novel biomarker for mitochondrial respiration under physiological and pathological conditions.
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http://dx.doi.org/10.1152/ajpheart.00462.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4666927PMC
October 2015

Interdependence of Parkin-Mediated Mitophagy and Mitochondrial Fission in Adult Mouse Hearts.

Circ Res 2015 Jul 2;117(4):346-51. Epub 2015 Jun 2.

From the Center for Pharmacogenomics, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO (M.S., G.G., S.J.M., G.W.D.); Department of Biomedical Sciences, University of Montreal, Quebec, Canada (Y.B.); Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA (A.B.G.); and Departments of Medicine (Cardiology) and Cell Biology, and Wilf Family Cardiovascular Research Institute, Albert Einstein College of Medicine, Bronx, NY (R.N.K.).

Rationale: The role of Parkin in hearts is unclear. Germ-line Parkin knockout mice have normal hearts, but Parkin is protective in cardiac ischemia. Parkin-mediated mitophagy is reportedly either irrelevant, or a major factor, in the lethal cardiomyopathy evoked by cardiac myocyte-specific interruption of dynamin-related protein 1 (Drp1)-mediated mitochondrial fission.

Objective: To understand the role of Parkin-mediated mitophagy in normal and mitochondrial fission-defective adult mouse hearts.

Methods And Results: Parkin mRNA and protein were present at low levels in normal mouse hearts, but were upregulated after cardiac myocyte-directed Drp1 gene deletion in adult mice. Alone, forced cardiac myocyte Parkin overexpression activated mitophagy without adverse effects. Likewise, cardiac myocyte-specific Parkin deletion evoked no adult cardiac phenotype, revealing no essential function for, and tolerance of, Parkin-mediated mitophagy in normal hearts. Concomitant conditional Parkin deletion with Drp1 ablation in adult mouse hearts prevented Parkin upregulation in mitochondria of fission-defective hearts, also increasing 6-week survival, improving ventricular ejection performance, mitigating adverse cardiac remodeling, and decreasing cardiac myocyte necrosis and replacement fibrosis. Underlying the Parkin knockout rescue was suppression of Drp1-induced hyper-mitophagy, assessed as ubiquitination of mitochondrial proteins and mitochondrial association of autophagosomal p62/sequestosome 1 (SQSTM1) and processed microtubule-associated protein 1 light chain 3 (LC3-II). Consequently, mitochondrial content of Drp1-deficient hearts was preserved. Parkin deletion did not alter characteristic mitochondrial enlargement of Drp1-deficient cardiac myocytes.

Conclusions: Parkin is rare in normal hearts and dispensable for constitutive mitophagic quality control. Ablating Drp1 in adult mouse cardiac myocytes not only interrupts mitochondrial fission, but also markedly upregulates Parkin, thus provoking mitophagic mitochondrial depletion that contributes to the lethal cardiomyopathy.
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http://dx.doi.org/10.1161/CIRCRESAHA.117.306859DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4522211PMC
July 2015

Regulation of metabolism in individual mitochondria during excitation-contraction coupling.

J Mol Cell Cardiol 2014 Nov 22;76:235-46. Epub 2014 Sep 22.

Mitochondria and Metabolism Center, Department of Anesthesiology and Pain Medicine, University of Washington, Seattle, WA 98109, USA. Electronic address:

The heart is an excitable organ that undergoes spontaneous force generation and relaxation cycles driven by excitation-contraction (EC) coupling. A fraction of the oscillating cytosolic Ca(2+) during each heartbeat is taken up by mitochondria to stimulate mitochondrial metabolism, the major source of energy in the heart. Whether the mitochondrial metabolism is regulated individually during EC coupling and whether this heterogeneous regulation bears any physiological or pathological relevance have not been studied. Here, we developed a novel approach to determine the regulation of individual mitochondrial metabolism during cardiac EC coupling. Through monitoring superoxide flashes, which are stochastic and bursting superoxide production events arising from increased metabolism in individual mitochondria, we found that EC coupling stimulated the metabolism in individual mitochondria as indicated by significantly increased superoxide flash activity during electrical stimulation of the cultured intact myocytes or perfused heart. Mechanistically, cytosolic calcium transients promoted individual mitochondria to take up calcium via mitochondrial calcium uniporter, which subsequently triggered transient opening of the permeability transition pore and stimulated metabolism and bursting superoxide flash in that mitochondrion. The bursting superoxide, in turn, promoted local calcium release. In the early stage of heart failure, EC coupling regulation of superoxide flashes was compromised. This study highlights the heterogeneity in the regulation of cardiac mitochondrial metabolism, which may contribute to local redox signaling.
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http://dx.doi.org/10.1016/j.yjmcc.2014.09.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4250349PMC
November 2014

Super-suppression of mitochondrial reactive oxygen species signaling impairs compensatory autophagy in primary mitophagic cardiomyopathy.

Circ Res 2014 Jul 29;115(3):348-53. Epub 2014 May 29.

From the Department of Internal Medicine, Center for Pharmacogenomics, Washington University School of Medicine, St. Louis, MO (M.S., G.G., G.W.D.); National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD (E.M.); and Department of Pathology, University of Washington, Seattle (P.S.R.).

Rationale: Mitochondrial reactive oxygen species (ROS) are implicated in aging, chronic degenerative neurological syndromes, and myopathies. On the basis of free radical hypothesis, dietary, pharmacological, and genetic ROS suppression has been tested to minimize tissue damage, with remarkable therapeutic efficacy. The effects of mitochondrial-specific ROS suppression in primary mitophagic dysfunction are unknown.

Objective: An in vivo dose-ranging analysis of ROS suppression in an experimental cardiomyopathy provoked by defective mitochondrial clearance.

Methods And Results: Mice lacking mitofusin 2 (Mfn2) in hearts have impaired parkin-mediated mitophagy leading to accumulation of damaged ROS-producing organelles and progressive heart failure. As expected, cardiomyocyte-directed expression of mitochondrial-targeted catalase at modest levels normalized mitochondrial ROS production and prevented mitochondrial depolarization, respiratory impairment, and structural degeneration in Mfn2 null hearts. In contrast, catalase expression at higher levels that supersuppressed mitochondrial ROS failed to improve either mitochondrial fitness or cardiomyopathy, revealing that ROS toxicity is not the primary mechanism for cardiac degeneration. Lack of benefit from supersuppressing ROS was associated with failure to invoke secondary autophagic pathways of mitochondrial quality control, revealing a role for ROS signaling in mitochondrial clearance. Mitochondrial permeability transition pore function was normal, and genetic inhibition of mitochondrial permeability transition pore function did not alter mitochondrial or cardiac degeneration, in Mfn2 null hearts.

Conclusions: Local mitochondrial ROS (1) contribute to mitochondrial degeneration and (2) activate mitochondrial quality control mechanisms. A therapeutic window for mitochondrial ROS suppression should minimize the former while retaining the latter, which we achieved by expressing lower levels of catalase.
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http://dx.doi.org/10.1161/CIRCRESAHA.115.304384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106429PMC
July 2014

Mutation in the γ2-subunit of AMP-activated protein kinase stimulates cardiomyocyte proliferation and hypertrophy independent of glycogen storage.

Circ Res 2014 Mar 6;114(6):966-75. Epub 2014 Feb 6.

From the Department of Anesthesiology and Pain Medicine, Mitochondria and Metabolism Center, University of Washington, Seattle (M.K., L.G.-M., G.G., Y.-Y.Y., S.C.K., J.X., W.W., R.T.); and MRC Protein Phosphorylation unit, College of Life Sciences, University of Dundee, Dundee, United Kingdom (R.W.H., K.S.). R.W.H. and K.S. are currently affiliated with Nestlé Institute of Health Sciences SA, Campus EPFL, Innovation Park, bâtiment G, Lausanne, Switzerland.

Rationale: AMP-activated protein kinase is a master regulator of cell metabolism and an attractive drug target for cancer and metabolic and cardiovascular diseases. Point mutations in the regulatory γ2-subunit of AMP-activated protein kinase (encoded by Prkag2 gene) caused a unique form of human cardiomyopathy characterized by cardiac hypertrophy, ventricular preexcitation, and glycogen storage. Understanding the disease mechanisms of Prkag2 cardiomyopathy is not only beneficial for the patients but also critical to the use of AMP-activated protein kinase as a drug target.

Objective: We sought to identify the pro-growth-signaling pathway(s) triggered by Prkag2 mutation and to distinguish it from the secondary response to glycogen storage.

Methods And Results: In a mouse model of N488I mutation of the Prkag2 gene (R2M), we rescued the glycogen storage phenotype by genetic inhibition of glucose-6-phosphate-stimulated glycogen synthase activity. Ablation of glycogen storage eliminated the ventricular preexcitation but did not affect the excessive cardiac growth in R2M mice. The progrowth effect in R2M hearts was mediated via increased insulin sensitivity and hyperactivity of Akt, resulting in activation of mammalian target of rapamycin and inactivation of forkhead box O transcription factor-signaling pathways. Consequently, cardiac myocyte proliferation during the postnatal period was enhanced in R2M hearts followed by hypertrophic growth in adult hearts. Inhibition of mammalian target of rapamycin activity by rapamycin or restoration of forkhead box O transcription factor activity by overexpressing forkhead box O transcription factor 1 rescued the abnormal cardiac growth.

Conclusions: Our study reveals a novel mechanism for Prkag2 cardiomyopathy, independent of glycogen storage. The role of γ2-AMP-activated protein kinase in cell growth also has broad implications in cardiac development, growth, and regeneration.
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http://dx.doi.org/10.1161/CIRCRESAHA.114.302364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3971100PMC
March 2014

Confocal imaging of single mitochondrial superoxide flashes in intact heart or in vivo.

J Vis Exp 2013 Nov 5(81):e50818. Epub 2013 Nov 5.

Mitochondria and Metabolism Center, Department of Anesthesiology and Pain Medicine, University of Washington.

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 °C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.
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http://dx.doi.org/10.3791/50818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3970564PMC
November 2013

Mitochondrial complex I deficiency increases protein acetylation and accelerates heart failure.

Cell Metab 2013 Aug;18(2):239-50

Mitochondria and Metabolism Center, Department of Anesthesiology and Pain Medicine, University of Washington, Seattle, 98109, USA.

Mitochondrial respiratory dysfunction is linked to the pathogenesis of multiple diseases, including heart failure, but the specific mechanisms for this link remain largely elusive. We modeled the impairment of mitochondrial respiration by the inactivation of the Ndufs4 gene, a protein critical for complex I assembly, in the mouse heart (cKO). Although complex I-supported respiration decreased by >40%, the cKO mice maintained normal cardiac function in vivo and high-energy phosphate content in isolated perfused hearts. However, the cKO mice developed accelerated heart failure after pressure overload or repeated pregnancy. Decreased NAD(+)/NADH ratio by complex I deficiency inhibited Sirt3 activity, leading to an increase in protein acetylation and sensitization of the permeability transition in mitochondria (mPTP). NAD(+) precursor supplementation to cKO mice partially normalized the NAD(+)/NADH ratio, protein acetylation, and mPTP sensitivity. These findings describe a mechanism connecting mitochondrial dysfunction to the susceptibility to diseases and propose a potential therapeutic target.
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http://dx.doi.org/10.1016/j.cmet.2013.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3779647PMC
August 2013

Respective contribution of mitochondrial superoxide and pH to mitochondria-targeted circularly permuted yellow fluorescent protein (mt-cpYFP) flash activity.

J Biol Chem 2013 Apr 1;288(15):10567-77. Epub 2013 Mar 1.

Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York 14642, USA.

Superoxide flashes are transient bursts of superoxide production within the mitochondrial matrix that are detected using the superoxide-sensitive biosensor, mitochondria-targeted circularly permuted YFP (mt-cpYFP). However, due to the pH sensitivity of mt-cpYFP, flashes were suggested to reflect transient events of mitochondrial alkalinization. Here, we simultaneously monitored flashes with mt-cpYFP and mitochondrial pH with carboxy-SNARF-1. In intact cardiac myocytes and purified skeletal muscle mitochondria, robust mt-cpYFP flashes were accompanied by only a modest increase in SNARF-1 ratio (corresponding to a pH increase of <0.1), indicating that matrix alkalinization is minimal during an mt-cpYFP flash. Individual flashes were also accompanied by stepwise increases of MitoSOX signal and decreases of NADH autofluorescence, supporting the superoxide origin of mt-cpYFP flashes. Transient matrix alkalinization induced by NH4Cl only minimally influenced flash frequency and failed to alter flash amplitude. However, matrix acidification modulated superoxide flash frequency in a bimodal manner. Low concentrations of nigericin (< 100 nM) that resulted in a mild dissipation of the mitochondrial pH gradient increased flash frequency, whereas a maximal concentration of nigericin (5 μm) collapsed the pH gradient and abolished flash activity. These results indicate that mt-cpYFP flash events reflect a burst in electron transport chain-dependent superoxide production that is coincident with a modest increase in matrix pH. Furthermore, flash activity depends strongly on a combination of mitochondrial oxidation and pH gradient.
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http://dx.doi.org/10.1074/jbc.M113.455709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3624438PMC
April 2013