Optical redox imaging of ANT1-de-cient muscles

Adenine nucleotide translocator(ANT)is a mitochondrial protein involved in the exchange of ADP and ATP across the mitochondrial inner membrane.It plays a crucial role in cellular energy metabolism by facilitating the transport of ATP synthesized within the mitochondria to the cytoplasm.The isoform ANT1 predominately expresses in cardiac and skeletal muscles.Mutations or dysregulation in ANT1 have been implicated in various mitochondrial disorders and neuromuscular diseases.We aimed to examine whether ANT1 deletion may affect mitochondrial redox state in our established ANT1-de-cient mice.Hearts and quadriceps resected from age-matched wild type(WT)and ANT1-de-cient mice were snap-frozen in liquid nitrogen.The Chance redox scanner was utilized to perform 3D optical redox imaging.Each sample underwent scanning across 3–5 sections.Global averaging analysis showed no signi-cant differences in the redox indices(NADH,flavin adenine dinucleotide containing-flavoproteins Fp,and the redox ratio Fp/(NADH+Fp)between WT and ANT1-de-cient groups.However,quadriceps had higher Fp than hearts in both groups(p¼0:0004 and 0.01,respectively).Furthermore,the quadriceps were also more oxidized(a higher redox ratio)than hearts in WT group(p¼0:004).NADH levels were similar in all cases.Our data suggest that under non-stressful physical condition,the ANT1-de-cient muscle cells were in the same mitochondrial state as WT ones and that the signi-cant difference in the mitochondrial redox state between quadriceps and hearts found in WT might be diminished in ANT1-de-cient ones.Redox imaging of muscles under physical stress can be conducted in future.

MITOCHONDRIAL REDOX IMAGING FOR CANCER DIAGNOSTIC AND THERAPEUTIC STUDIES

Mitochondrial redox states provide important information about energy-linked biological processes and signaling events in tissues for various disease phenotypes including cancer.The redox scanning method developed at the Chance laboratory about 30 years ago has allowed 3D highresolution(∼50×50×10µm^(3))imaging of mitochondrial redox state in tissue on the basis of the fluorescence of NADH(reduced nicotinamide adenine dinucleotide)and Fp(oxidized flavoproteins including flavin adenine dinucleotide,i.e.,FAD).In this review,we illustrate its basic principles,recent technical developments,and biomedical applications to cancer diagnostic and therapeutic studies in small animal models.Recently developed calibration procedures for the redox imaging using reference standards allow quantification of nominal NADH and Fp concentrations,and the concentration-based redox ratios,e.g.,Fp/(Fp+NADH)and NADH/(Fp+NADH)in tissues.This calibration facilitates the comparison of redox imaging results acquired for different metabolic states at different times and/or with different instrumental settings.A redox imager using a CCD detector has been developed to acquire 3D images faster and with a higher in-plane resolution down to 10µm.Ex vivo imaging and in vivo imaging of tissue mitochondrial redox status have been demonstrated with the CCD imager.Applications of tissue redox imaging in small animal cancer models include metabolic imaging of glioma and myc-induced mouse mammary tumors,predicting the metastatic potentials of human melanoma and breast cancer mouse xenografts,differentiating precancerous and normal tissues,and monitoring the tumor treatment response to photodynamic therapy.Possible future directions for the development of redox imaging are also discussed.

Quantitative redox imaging biomarkers for studying tissue metabolic state and its heterogeneity

NAD+/NADH redox state has been implicated in many diseases such as cancer and diabetes aswell as in the regulation of embryonic development and aging,To fluorimetrically sssthemitochondrial redox state,Dr.Chance and co-workers measured the fluorescence of NADH andoxidized flavoproteins(Fp)including favin-adenine-dinucleotide(FAD)and demonstrated theirratio(i.e.the redox ratio)is a sensitive indicator of the mitochondrial redox states.,The Chanceredox sca mer was built to simult aneously measure NADH and Fp in tisue at submilimeter scalein 3D using the freeze-trap protocol.This paper summarizes our recent research experience,development and new applications of the redox scanning technique in collaboration with Dr.Chance beginning in 2005.Dr.Chance initiated or actively involved in many of the projectsduring the last several years of his life.We advanced the redox scanning technigue by measuringthe nominal concentrations(in reference to the frozen solution standards)of the endogenousfuorescent analy tes,i.e.,[NADH]and[rp]to quantify the redox ratios in various biologicaltissues.The advancement has enabled us to identify an array of the redox indices as quantitativeimaging,biomarkers(including[NADH],[Fp],[Fp]/[NADH]+[Fp],[NADH]/[Fp],and theirstandard deviations)for studying some important biological questions on cancer and normaltsue metabolism.We found that the redox indices were asociated or changed with(NADH)tumorigenesis(cancer versus non-cancer of human breast tisue biopsies);(2)tumor metastaticpotential;(3)tumor glucose uptake;(4)tumor p53 stat us;(5)PI3K pathway activation in pre-malignant tissue;(6)therapeutic ffects on tumors;(7)embryonic stem cell diferentiation;(8)the heart under fasting,Together,our work demonstrated that the tisue redox indices obtainedfrom the redox scanning technique may provide usefil information about tisue met abolism and physiology status in normal and diseased tissues.The Chance redox scanner and other redoximaging techniques may have wide-ranging potential applications in many fields,such as cancer,diabetes,developmental process,mitochondrial diseases,neurodegenerative diseases,and aging.

REDOX IMAGING OF THE p53-DEPENDENT MITOCHONDRIAL REDOX STATE IN COLON CANCER EX VIVO

The mitochondrial redox state and its heterogeneity of colon cancer at tissue level have not been previously reported.Nor has how p53 regulates mitochondial respi ration been measured at(deep)tissue level,presumably due to the unavailability of the technology that has sufficient spatial resolution and tissue penetration depth.Our prior work demonstrated that the mito-chondrial redox state and its intnatumor heterogeneity is associated with cancer aggressiveness in human melanoma and breast cancer in mouse models,with the more metastatic tumons exhi-bit ing localized negions of more oxidized redox state.Using the Chance redox scanner with an in-plane spatial resolution of 200 pm,we imaged the mitochondrial redox state of the wild-type p53 colon tumors(HCT116 p53 ut)and the p5-deleted colon tumors(HCT116 p53-/-)by cllcting the fuorescence si gnals of nicotinamide adenine dimucleotide(NA DH)and oxidized flavoproteins [Fp,including favin adenine dinucleotide(FAD)]from the mouse xenogmafts snap frozen at low temperature.Our results show that:(1)both tumor lines have significant degree of intratumor heterogeneity of the redox state,typically exhibiting a distinct bi modal distribution that either correlates with the spatial core-rim pattern or the“hot/oold”oxida tion-roduction patches;.(2)the p531-group is significantly more beterogencous in the mitochondrial redox state and has a more oxidized turmor core compared to the p53 wt group when the tunor sizes of the two groups are matched;(3)the tumor size dependence of the redox indices(such as Fp and Fp redox ratio)is significant in the p531-group with the larger ones being more oxidized and more hetero-geneous in their redox state,particularly more oxidized in the tumor central regions;(4)the H&E staining images of tumor soctions grossly correlate with the redox images.The present work is the first to reveal at the submillimeter scale the intratumor heterogeneity pattem of the mitochon-drial redox state in colon cancer and the first to indicate that at tissue level the mitochondial redox state is p53 dependent.The findings should assist in our understanding on colon cancer pa thology and developing new imaging biomarkers for dlinical applications.

3D imaging of the mitochondrial redox state of rathearts under normal and fasting conditions

The heart requires continuous ATP availability that is generated in the mitochondria.Althoughstudies using the cell culture and perfiused organ models have been carried out to investigate thebiochemistry in the mitochondria in response to a change in substrate supply,mitochondrialbioenergetics of heart under normal feed or fasting conditions has not been studied at the tissuelevel with a sub-millimeter spatial resolution either in vivo or er vivo.Oxidation of many food-derived metabolites to generate ATP in the mitochondria is realized through the NADH/NAD+couple acting as a central electron carrier.We employed the Chance redox scanner thelow-temperat ure fluorescence scanner to image the three-dimensional(3D)spatial distribution of themitochondrial redox states in heart tissues of rats under normai feeding or an overnight star-vation for 14.5 h.Multiple consecutive sections of each heart were imaged to map three redoxindices,i.e,NADH,oxidized favoproteins Fp,including flavin adenine dinucleotide(FAD)andthe redox ratio NADH/Fp.The imaging results revealed the micro-heterogeneity and the spatial distribution of these redox indices.The quantitative analysis showed that in the fasted hearts thestandard deviation of both NADH and Fp,ie.,SD NADH and SDFp,significantly decreasedwith a p value of 0.032 and 0.045,respectively,indicating that the hearts become relatively morehomogeneous after fasting.The fasted hearts contained 28.6%less NADH(p=0.038).No sig.nificant change in Fp was foumnd(p=0.4).The NADH/Fp ratio decreased with a marginalP value(0.076).The decreased NADH im the fasted hearts is consistent with the cardiac celsreliance of fatty acids consumption for energy metabolism when glucose becomes scarce.Theexperimental o bservation of N ADH decrease induced by dietary restriction in the heart at tissuelevel has not been reported to our best knowledge.The Chance redox scanner demonstrated thefeasibility of 3D imaging of the mitochondrial redox st ate in the heart and provides a usefil toolto study heart metabolism and fiunction under normal,dietary-change and pathological con-ditions at tisue level. We would like to thank Dr.Joseph Baur for thehelpful discussion and Dr.Hui Qiao for animalpreparation and organ harvesting.

This research was supported by E.U.FP6 Integrated Project“Molecular Imaging”LSHG-CT-2003-503259 and E.U.FP7 Collaborative Project“FMT-XCT”.R.F.acknowledges support from the Marie Curie Program EST-MolecImag Early Stage Training MEST-CT-2004-007643.

We have imaged mitochondrial oxidation-reduction states by taking a ratio of mitochondrial fluorophores:NADH(reduced nicotinamide adenine dinucleotide)to Fp(oxidized flavoprotein).Although NADH has been investigated for tissue metabolic state in cancer and in oxygen deprived tissues,it alone is not an adequate measure of mitochondrial metabolic state since the NADH signal is altered by dependence on the number of mitochondria and by blood absorption.The redox ratio,NADH/(Fp+NADH),gives a more accurate measure of steady-state tissue metabolism since it is less dependent on mitochondrial number and it compensates effectively for hemodynamic changes.This ratio provides important diagnostic information in living tissues.In this study,the emitted fluorescence of mouse colon in situ is passed through an emission filter wheel and imaged on a CCD camera.Redox ratio images of the healthy and hypoxic mouse intestines clearly showed significant differences.Furthermore,the corrected redox ratio indicated an increase from an average value of 0.51±0.10 in the healthy state to 0.92±0.03 in dead tissue due to severe ischemia(N=5).We show that the CCD imaging system is capable of displaying the metabolic differences in normal and ischemic tissues as well as quantifying the redox ratio in vivo as a marker of these changes.

IMAGING REDOX STATE HETEROGENEITY WITHIN INDIVIDUAL EMBRYONIC STEM CELL COLONIES

Redox state mediates embryonic stem cell(ESC)differentiation and thus offers an important complementary approach to understanding the pluripotency of stem cells.NADH redox ratio(NADH/(Fp t NADH)),where NADH is the reduced form of nicotinamide adenine dinucleotide and Fp is the oxidizedflavoproteins,has been established as a sensitive indicator of mitochondrial redox state.In this paper,we report our redox imaging data on the mitochondrial redox state of mouse ESC(mESC)colonies and the implications thereof.The low-temperature NADH/Fp redox scanner was employed to image mESC colonies grown on a feeder layer of gamma-irradiated mouse embryonicfibroblasts(MEFs)on glass cover slips.The result showed significant heterogeneity in the mitochondrial redox state within individual mESC colonies(size:~200-440μm),exhibiting a core with a more reduced state than the periphery.This more reduced state positively correlates with the expression pattern of Oct4,a well-established marker of pluripotency.Our observation is thefirst to show the heterogeneity in the mitochondrial redox state within a mESC colony,suggesting that mitochondrial redox state should be further investigated as a potential new biomarker for the stemness of embryonic stem cells.

QUANTITATIVE REDOX SCANNING OF TISSUE SAMPLES USING A CALIBRATION PROCEDURE

The fluorescence properties of reduced nicotinamide adenine dinucleotide(NADH)and oxidizedflavoproteins(Fp)including flavin adenine dinucleotide(FAD)in the respiratory chain are sensitive indicators of intracellular metabolic states and have been applied to the studies of mitochondrial function with energy-linked processes.The redox scanner,a three-dimensional(3D)low temperature imager previously developed by Chance et al.,measures the in vivo metabolicproperties of tissue samples by acquiring fluorescence images of NADH and Fp.The redox ratios,i.e.Fp/(Fp+NADH)and NADH/(Fp+NADH),provided a sensitive index of the mitochondrialredox state and were determined based on relative signal intensity ratios.Here we report thefurther development of the redox scanning technique by using a calibration method to quantifythe nominal concentration of the fluorophores in tissues.The redox scanner exhibited very goodlinear response in the range of NADH concentration between 165–1318µM and Fp between90–720µM using snap-frozen solution standards.Tissue samples such as human tumor mousexenografts and various mouse organs were redox-scanned together with adjacent NADH and Fpstandards of known concentration at liquid nitrogen temperature.The nominal NADH and Fpconcentrations as well as the redox ratios in the tissue samples were quantified by normalizing the tissue NADH and Fp fluorescence signal to that of the snap-frozen solution standards.This calibration procedure allows comparing redox images obtained at different time,independent of instrument settings.The quantitative multi-slice redox images revealed heterogeneity inmitochondrial redox state in the tissues.