sdarticle[11]

Brief Communication

Direct intracellular nitric oxide detection in isolated adult

cardiomyocytes:?ow cytometric analysis using the ?uorescent probe,

diamino?uorescein

Hans Strijdom a,*,Christo Muller b ,Amanda Lochner a,c

a

Department of Medical Physiology and Biochemistry,Faculty of Health Sciences,Stellenbosch University,

P .O.Box 19063,Tygerberg 7505,South Africa

b

Department of Anatomy and Histology,Faculty of Health Sciences,Stellenbosch University,Tygerberg 7505,South Africa

c

MRC Diabetes Group,Tygerberg,7505South Africa

Received 3November 2003;received in revised form 11May 2004;accepted 20May 2004

Available online 31July 2004

Abstract

We assessed the possibility to detect intracellular nitric oxide (NO)with the NO-speci?c probe 4,5-diamino?uorescein-2/diacetate (DAF-2/DA),by ?ow cytometry,in fresh adult rat cardiomyocytes,and compared the ?ndings with results obtained from quantitation of cellular nitrate/nitrite (NO x )levels.

Methods.–Cardiomyocytes were isolated by collagenase perfusion,followed by incubation in a Krebs–Henseleit/2%bovine serum albumin buffer in the presence of 10μM DAF-2/DA (~0.5×106cells/ml).Experimental conditions were:(i)baseline control,(ii)NO donor (2-(N,N -diethylamino)-diazenolate 2-oxide,DEA/NO)administration,and (iii)120min simulated ischemia (hypoxia).In addition,control and hypoxic groups were incubated with the NO synthase (NOS)inhibitor,N W -nitro-L -arginine methyl ester (L -NAME).Following incubation and washing,intracellular ?uorescence of DAF-triazol (DAF-2T,oxidized form of DAF-2/DA)was analyzed by ?ow cytometry.NO x levels were determined with an NO x assay.Fluorescence-activated cell sorter (FACS)data were expressed as mean ?uorescence intensity (percentage of control)and NO x levels as pmol/106cells.

Results.–Optimal baseline ?uorescence was obtained when myocytes were incubated with DAF-2/DA for 3h at 37°C.The NO donor DEA/NO (500μM)and hypoxia signi?cantly increased DAF ?uorescence and NO x levels.L -NAME addition signi?cantly reversed these trends in the hypoxia groups.

Conclusions.–We have demonstrated that intracellular NO can be detected in fresh isolated adult cardiomyocytes by ?ow cytometry with 10μM DAF-2/DA.Furthermore,we demonstrated that hypoxia is an activator of adult cardiomyocyte NOS,as demonstrated by both end-points.Reproducibility observed between results obtained by FACS analysis and NO x assays suggests that DAF-2/DA ?uorescence can be regarded as an independent marker for intracellular NO in cardiomyocytes.?2004Elsevier Ltd.All rights reserved.

Keywords:Nitric oxide detection;Adult cardiomyocytes;DAF-2/DA;Flow cytometry;Nitrate/nitrite assay;Hypoxia

1.Introduction

Nitric oxide (NO)is an important biological mediator of cardioprotection during ischemia–reperfusion injury and is-chemic preconditioning (IP),and has become one of the fastest growing ?elds of interest in heart research [1].The exact underlying cellular mechanisms of NO are complex

and remain poorly understood [2].An important way by which more insight into NO’s mechanisms of action could be obtained,is measurement of intracellular NO levels,how-ever,many studies depend on indirect methods to predict changes in intracellular NO levels,such as NO synthase (NOS)activity (citrulline assay and cyclic GMP (cGMP)measurements)[3],nitrate/nitrite (NO x )level determinations [3,4],NOS protein [5,6],and mRNA expression [6].In addition,methods for the direct,single-cell NO detec-tion (e.g.chemiluminescence assays,electron paramagnetic

*Corresponding author.Tel.:+27-21-938-9387;fax:+27-21-938-9476.E-mail address:jgstr@sun.ac.za (H.Strijdom).Journal of Molecular and Cellular Cardiology 37(2004)

897–902

https://www.360docs.net/doc/327047638.html,/locate/yjmcc

?2004Elsevier Ltd.All rights reserved.

doi:10.1016/j.yjmcc.2004.05.018

resonance spectroscopy,and electrochemical electrode methods)are often complicated,insensitive and non-speci?c, and not readily available to the average equipped laboratory [7].This is particularly true for low output NO-generating cells such as cardiomyocytes and endothelial cells[7]. Diamino?uorescein-2/diacetate(DAF-2/DA),a membrane-permeable,?uorescent,real-time indicator for NO[8],has recently been used to detect NO in cells of cardiac origin via ?ow cytometric analysis,viz.endothelial cells[6,7,9]and embryonal rat heart-derived cell lines[10].DAF-2/DA was also used to qualitatively assess NO in cultured embryonic chick ventricular myocytes[11]and isolated cardiomyocytes [12],by means of video?uorescent microscopy and confocal microscopy,respectively.However,there is no evidence of studies using DAF-2/DA?uorescence to detect NO levels in adult cardiomyocytes with?ow cytometry(?uorescence-activated cell sorter,FACS).FACS has major advantages over other?uorescence-detection techniques such as spec-tro?uorimetry and?uorescence microscopy[13].The former requires high labeling intensity and does not distinguish between intracellular and extracellular?uorescence,while the latter is time consuming and analyzes a small number of cells at a time.In contrast,FACS rapidly measures and analyzes thousands of cells,distinguishes between cell sub-populations and analyzes intracellular events on a single-cell level[13].

Therefore,we aimed to design a protocol for the FACS analysis of acutely isolated adult rat cardiomyocytes to as-sess whether baseline intracellular NO release in these cells could be detected with DAF-2/DA.The NO donor,2-(N,N-diethylamino)-diazenolate2-oxide(DEA/NO)was adminis-tered to verify the probe’s reported NO sensitivity.Further-more,since ischemia stimulates cardiac NOS activity[3],we investigated whether hypoxia could enhance DAF-2/DA ?uorescence in cardiomyocytes.N W-nitro-L-arginine methyl ester(L-NAME),a NOS inhibitor,was administered to estab-lish whether the observed effects were due to NOS-induced NO release.Levels of NO x,major oxidative metabolites of NO[13],were determined and compared with DAF-2/DA data in order to validate and quantitate results obtained with FACS analysis.

2.Materials and methods

2.1.Materials

L-NAME,HEPES,and DEA/NO were from Sigma Chemical Co.,(St Louis,MO,USA);bovine serum albumin (BSA fraction V)from Boehringer Mannheim;trypan blue from Merck;collagenase Type2from Worthington(Lake-wood,NJ,USA);and DAF-2/DA from Calbiochem(San Diego,CA,USA).All other chemicals were of Analar grade and obtained from Merck,Cape Town.2.2.Preparation of isolated,adult Ca2+-tolerant cardiomyocytes

This investigation conforms to the Guide for the Care and

Use of Laboratory Animals(US National Institutes of Health;NIH publication no.85-23,revised1985).Adult rat

ventricular myocytes were isolated as described previously

[14,15].Investigations were repeated on myocyte prepara-

tions from different hearts.All experimental groups were

incubated in a2%BSA-containing Krebs–Henseleit buffer at

37°C in a tissue culture incubator(21%O2,5%CO2,and

40–60%humidity).

2.3.Cardiomyocyte viability

Assessment of myocyte viability was performed by trypan

blue exclusion(TBE)and cell morphology(percentage of

rod-shaped myocytes),as described previously[14,16].Time

zero viability varied between70%and80%and all cell

isolates of less than70%viability were discarded.

2.4.DAF-2/DA incubation,experimental groups,

and protocols

Suspensions of~0.5×106myocytes/ml were incubated

with10μM DAF-2/DA(dissolved in2%BSA Krebs–

Henseleit buffer solution)for180min(37°C).Exposure to

light was avoided as far as possible throughout experimenta-

tion.Experimental interventions(Fig.1)were introduced at

different time-points during the incubation period:(i)

DEA/NO at t=60min,(ii)hypoxia at t=60min,and(iii) L-NAME at t=30min.Control groups were incubated in suspension under an O2atmosphere(21%O2,5%CO2,and

40–60%humidity)for the full duration of the experiment

(180min).Simulated ischemia was achieved through the

induction of hypoxia by covering myocyte pellets with a

mineral oil layer,as previously described[14,16].

2.5.Flow cytometry

At t=180min,samples were centrifuged,supernatants

removed and cells resuspended in fresh,DAF-2/DA-free

buffer followed by immediate FACS analysis.A Becton

Dickinson FACSCalibur?analyzer was used to quantify

?uorescence(excitation wavelength:488nm and emission

wavelength:530nm)at the single-cell level,and data were

analyzed using Cellquest?version3.3(Becton Dickinson)

software.In each sample,the mean?uorescence intensity of

the analyzed cells was determined,after gating the cell popu-

lation by forward and side light scatter signals as recorded on

a dot plot(Fig.2A).In total,50,000events were acquired,but

non-cellular particles and debris(located on the bottom left

corner of the dot plot)were excluded by prior gating,thereby

limiting undesired effects on overall?uorescence.Final

gated cell populations usually contained10,000–

15,000cells.Fluorescence in these cells was produced by

898H.Strijdom et al./Journal of Molecular and Cellular Cardiology37(2004)897–902

oxidation of DAF-2/DA to its highly green-?uorescent DAF-triazol (DAF-2T)form,and signals were recorded on a fre-quency histogram (Fig.2B )by logarithmic ampli?ers.Fluo-rescence data are expressed as mean ?uorescence (percentage of control).2.6.NO x measurements

Samples collected at t =180min were stored in liquid nitrogen until a NO x colorimetric assay (Cayman Chemical,Ann Arbor)was performed on homogenized cell suspen-sions.Photometric measurements of absorbance (540nm)determine nitrate +nitrite concentrations,expressed as pmol/106cells.2.7.Statistical analysis

Unless stated otherwise,all data are expressed as percent-ages of control (mean ±S.E.M.).For comparative studies,Student’s t -test (unpaired)or one-way ANOV A tests (with Bonferroni post test if P <0.05)were used for statistical analyses.Differences were considered statistically signi?-cant if a P -value of <0.05was achieved.

3.Results

3.1.DAF-2/DA ?uorescence and F ACS analyses (Fig.2)FACS analysis of DAF-2/DA (10μM)produced detect-able mean baseline ?uorescence in control

cardiomyocytes

Fig.1.Experimental protocols.Isolated myocytes were divided into sample fractions of ~0.5×106cells each on which the respective experimental interventions were performed.Experimental groups consisted of samples from different hearts (n :5–15per group).(A)Control samples were kept in suspension in 1ml of 2%BSA Krebs–Henseleit buffer for 180min at 37°C in a tissue culture incubator.(B)The NO donor,DEA/NO,was administered at different concentrations (100,500,and 1000μM,respectively)for 120min at t =60min to validate the NO-detection properties of DAF-2/DA.(C)Ischemia was simulated by a hypoxic insult (cell pellets covered with a mineral oil layer)starting at t =60min and lasting for 120min.The unshaded portions of the bars represent untreated,oxygenated control condi-tions.L -NAME (50μM)was administered to control and hypoxia groups at t =30min,and incubation lasted until t =180min (represented by black lines under the bars).Asterisks indicate the start of DAF-2/DA incubation for FACS analysis at t =0min.Samples were collected at t =180min for subsequent FACS

analyses.

Fig.2.FACS analysis of DAF-2/DA-treated cardiomyocytes.(A)Typical dot plot of a myocyte suspension showing the spread of the total recorded “events”(cells,particles,and debris)calculated by their forward and side light scatter.The red eclipse-shaped area represents the gated cell popula-tion,which is ultimately analyzed and excludes the black zone in the left bottom corner representing cellular debris and other dissolved particles that may in?uence overall ?uorescence.In total,50,000“events”were acquired per sample,and gated cell populations usually contained 10,000–15,000myocytes.Analysis of intracellular DAF-2/DA ?uorescence was performed on the gated cell populations,and recorded on a frequency histogram.(B)A representative frequency histogram depicting the ?uores-cence intensity (log)on the x -axis (FLH-1:?uorescence channel 1height detecting green ?uorescence)and cell count on the y -axis.The black graph represents control (incubated with 10μM DAF-2/DA),the green graph cells incubated in DAF-2/DA-free buffer and the red graph cells incubated in a Ca 2+-free medium.No signi?cant auto?uorescence was present in cells incubated without DAF-2/DA (green),and,incubation of cells in Ca 2+-free buffer had no effect on ?uorescence compared to control cells containing Ca 2+at physiological concentrations.

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H.Strijdom et al./Journal of Molecular and Cellular Cardiology 37(2004)897–902

after 180min.Fig.2A is a typical dot plot of a myocyte suspension showing the spread of the total recorded events.Cells incubated in DAF-2/DA-free buffer showed a ?vefold attenuation in ?uorescence compared to control cells in the presence of the marker.Incubation of control myocytes in Ca 2+-free buffer had no effect on ?uorescence (Fig.2B ).3.2.NO donors demonstrate NO sensitivity of DAF-2/DA A dose-dependent increase in mean ?uorescence was de-tected in DEA/NO-treated myocytes (100,500,and 1000μM:129.4±11%,282.5±10.5%*,and 343.7±61.6%*of control,respectively,*P <0.05vs.control)(Fig.3A,B ).Sodium nitroprusside (SNP),another NO donor,showed increased mean ?uorescence by 55%(1mM)and 227%(10mM),respectively (P <0.05vs.control,data not shown).The NOS activator cyclosporin A (10μM)[9]also increased baseline ?uorescence by 42%(not shown).

3.3.Hypoxia reduces cell viability and increases mean DAF-2/DA ?uorescence

Myocytes subjected to 120min hypoxia showed a 57%and 25%reduction in the percentage TBE cells and rods,respectively (P <0.05for both parameters)(not shown).A 56.3%increase in mean DAF-2/DA ?uorescence was ob-served in hypoxic cardiomyocytes (P <0.05vs.control;Fig.4A,C ).Hypoxic myocytes incubated in DAF-2/DA-free buffer recorded a mean ?uorescence of 77%of control com-pared to 163%in DAF-2/DA-incubated hypoxic myocytes (P <0.05;not shown).

3.4.L -NAME reverses ?uorescence in control and hypoxic cells

Administration of the NOS inhibitor,L -NAME (50μM),to control myocytes,resulted in a 16%attenuation of mean baseline ?uorescence (P <0.05;not shown)(Fig.4).Addi-tion of 50μM L -NAME to hypoxic cells caused a reduction of 30%in mean ?uorescence compared to untreated hypoxic cells (P <0.05;Fig.4B,C ).3.5.NO x measurements

Hypoxia increased NO x levels by 60%compared to con-trol (control:482.6±42vs.hypoxia:773.4±107pmol/106myocytes;P <0.05;Fig.4D ).Addition of L -NAME to hypoxic cells resulted in an attenuation of NO x to levels observed in control cells (469±42pmol/106myocytes;P <0.05vs.hypoxia;Fig.4D ).DEA/NO (500μM)caused a 140-fold increase in NO x levels compared to control (P <0.05;not shown)(Fig.4).

4.Discussion

To the best of our knowledge,we have shown for the ?rst time that FACS analysis detects NO levels in isolated adult cardiomyocytes incubated with the ?uorescent NO probe,DAF-2/DA.The NO sensitivity of the probe in cardiomyo-cytes was demonstrated by the dose-dependent increase in ?uorescence observed with the NO donor,DEA/NO (Fig.3).The NO sensitivity of DAF-2/DA has previously been ques-tioned due to possible susceptibility to Ca 2+and light [17];however,the authors who developed the probe,subsequently showed that the reaction between DAF-2and NO was inde-pendent of Ca 2+and Mg 2+,and that the role of Ca 2+was rather to release NO from NO donors [18].Our results could not demonstrate a ?uorescence-enhancing role for Ca 2+(Fig.2B ).All possible precautions were taken to avoid light exposure during incubation and experimentation;however,the effect of incidental light cannot be completely excluded.In such an event,all samples would be affected equally.Acutely isolated adult cardiomyocytes,despite their shortcomings,are physiologically superior preparations

to

Fig.3.Dose-dependent enhancement of DAF-2/DA ?uorescence by the NO donor,DEA/NO.Cells were incubated with DEA/NO for 120min starting at t =60min.(A)A representative frequency histogram of the ?uorescence intensity resulting from the administration of 100μM (black graph),500μM (green graph),and 1000μM (red graph)DEA/NO,respectively.(B)Bar chart of control and DEA/NO 100,500,and 1000μM ?uorescence.Results are depicted as mean ?uorescence expressed as percentage of control.DEA/NO 500and 1000μM enhanced mean ?uorescence signi?cantly com-pared to control (283%and 344%of control,respectively,P <0.05vs.control,n =5).

900H.Strijdom et al./Journal of Molecular and Cellular Cardiology 37(2004)897–902

cultured neonatal/embryonic myocytes,since the latter ex-press an immature heart cell genotype and phenotype [19].Furthermore,fresh cardiomyocytes are known to survive in vitro for a suf?cient period of time (8–10h)[19],allowing the investigator to study constitutive NOS (cNOS)–NO me-tabolism.Ischemia has been shown to activate cardiac NOS [3].We investigated whether 120min hypoxia could activate cNOS to produce NO.Our FACS results indicate a signi?cant increase in NO production (Fig.3C ),associated with a pro-nounced reduction in cell viability con?rming the ef?cacy of the

protocol.

Fig.4.The effects of hypoxia.(A)Representative frequency histogram of DAF-2/DA ?uorescence observed in control (black graph)and hypoxia (red graph)myocytes.It is clear that 120min of hypoxia resulted in enhanced ?uorescence intensity compared to control.(B)Frequency histogram of hypoxia myocytes treated with 50μM L -NAME (red graph)compared with untreated hypoxia cells (black graph),showing a clear attenuation in ?uorescence intensity in the former.(C)Bar chart depicting the effects of hypoxia ±L -NAME compared with control.Results are expressed as mean ?uorescence as a percentage of control.LN =50μM L -NAME.Results show that 120min of hypoxia enhanced DAF-2/DA ?uorescence by 56%(control:100%vs.hypoxia:156.3±5.6%;P <0.05,n =15).L -NAME attenuated this effect by 30%(untreated hypoxia:156.3±5.6%vs.L -NAME-treated hypoxia:126.4±7.8%;P <0.05,n =5).(D)Bar chart depicting the effects of hypoxia ±L -NAME on NO x (nitrates +nitrites),as measured at the end of t =180min.Results show increased NO x levels after 120min of hypoxia,reversed by the addition of L -NAME (control:482.6±42*,hypoxia:773.4±107,and hypoxia +L -NAME:468.7±42pmol/106myocytes*;*P <0.05vs.hypoxia,n =6).(E)Bar chart combining the DAF-2/DA FACS analysis and NO x data.Results of both methods are expressed as a percentage of control (control =100%).Hypoxia results for both parameters were signi?cantly greater than control and hypoxia +L -NAME values (DAF hypoxia and NO x hypoxia:156%and 160%,respectively;DAF hypoxia +L -NAME and NO x hypoxia +L -NAME:126%and 97%,respectively).

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H.Strijdom et al./Journal of Molecular and Cellular Cardiology 37(2004)897–902

L-NAME signi?cantly reversed increases in NO levels (Fig.3C),suggesting that the increase was due to activated cNOS.Possible hypoxia-induced auto?uorescence was ex-cluded by incubating hypoxic cells in DAF-2/DA-free buffer. In a previous study,we demonstrated signi?cant increases in cardiomyocyte cGMP(most important second messenger of NO)induced by120min hypoxia[14],supporting the DAF-2/DA?ndings of the present study.

In a further attempt to validate this technique,we assayed cellular NO x levels,an acknowledged end-point for NO pro-duction[4],of all samples.The percentage of changes in NO x between control,hypoxia,and hypoxia+L-NAME groups was similar to observations with DAF-2/DA(Fig.4E).This suggests that the FACS technique is at least as sensitive as NO x measurements,particularly in the case of intracellular control and hypoxia-induced NOS-activated NO generation. In view of these similarities,it can be concluded that the ~60%increase in hypoxia-induced DAF-2/DA?uorescence represents an intracellular release of approximately300pmol NO x/106myocytes.A similar conclusion can be drawn from the respective hypoxia+L-NAME?ndings.Attempts to use known concentrations of NO donors and measurements of the NO x they release,as possible standards to calibrate?uo-rescence readings were unsuccessful.Increases in DEA/NO and SNP-induced(not shown)NO x generation over controls were139-and35.4-fold,respectively,compared to2.4and 1.6in FACS studies.Discrepancies between the results of the two detection methods can be explained by the fact that the NO x assay does not distinguish between intracellular and extracellular NO x,whereas FACS analyzes intracellular NO only.The investigator has limited control over the extent to which donors release NO in the extracellular compartment, particularly in the case of DEA/NO,known to spontaneously release NO on dissolution in aqueous media[20].

In summary,our?ndings suggest that the DAF-2/DA FACS analysis method can be regarded as an independent and validated technique that detects and measures intracellu-lar NOS-induced NO production in isolated cardiomyocytes. The application of this technique in isolated myocyte studies can help to elucidate the complex nature of intracellular NO actions in the physiology and pathology of the myocardium.

Acknowledgements

This study was sponsored in part by the Medical Research Council(MRC)of SA,National Research Fund(NRF)of SA,and the Harry Crossley Foundation.

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