Stretch-induced programmed myocyte cell
death.
W Cheng, … , G Olivetti, P Anversa
J Clin Invest.
1995;
96(5)
:2247-2259.
https://doi.org/10.1172/JCI118280
.
To determine the effects of loading on active and passive tensions, programmed cell death,
superoxide anion formation, the expression of Fas on myocytes, and side-to-side slippage
of myocytes, papillary muscles were exposed to 7-8 and 50 mN/mm2 and these parameters
were measured over a 3-h period. Overstretching produced a 21- and a 2.4-fold increase in
apoptotic myocyte and nonmyocyte cell death, respectively. Concurrently, the generation of
reactive oxygen species increased 2.4-fold and the number of myocytes labeled by Fas
protein 21-fold. Moreover, a 15% decrease in the number of myocytes included in the
thickness of the papillary muscle was found in combination with a 7% decrease in
sarcomere length and the inability of muscles to maintain stable levels of passive and active
tensions. The addition of the NO-releasing drug, C87-3754, prevented superoxide anion
formation, programmed cell death, and the alterations in active and passive tensions with
time of overloaded papillary muscles. In conclusion, overstretching appears to be coupled
with oxidant stress, expression of Fas, programmed cell death, architectural rearrangement
of myocytes, and impairment in force development of the myocardium.
Research Article
Stretch-induced Programmed Myocyte
Cell
Death
Wei Cheng,* BaoshengLi,*Jan Kajstura,* Peng Li,*MichaelS.
Wolin,*
Edmund H.Sonnenblick,*§Thomas H.Hintze,*
Giorgio Olivetti,* and Piero Anversa*§
Departments of *MedicineandtPhysiology,New York MedicalCollege, Valhalla, New York10595; and§Department of Medicine, Albert EinsteinCollege ofMedicine, New York10461
Abstract
To determine the effects of loading on active and passive
tensions, programmed celldeath, superoxideanion
forma-tion, the expression of Fas on myocytes, and side-to-side
slippageof myocytes,papillarymuscleswereexposedto
7-8 and 50 mN/mm2 and these parameters were measured
over a 3-h period. Overstretching produced a 21- and a
2.4-fold increase in apoptoticmyocyteandnonmyocyte cell
death, respectively. Concurrently,thegenerationof reactive
oxygen species increased 2.4-foldand the number of
myo-cytes labeled byFasprotein21-fold.Moreover, a 15%
de-creaseinthe number ofmyocytesincludedinthethickness
of thepapillary musclewasfound in combination witha7%
decreaseinsarcomerelengthand theinabilityofmusclesto
maintain stable levels of passive and active tensions. The addition of theNO-releasing drug, C87-3754, prevented
su-peroxide anionformation, programmedcell death, and the
alterations in active andpassivetensions withtime of
over-loaded papillarymuscles. Inconclusion, overstretching ap-pears tobecoupled with oxidant stress,expression ofFas, programmed cell death, architectural rearrangement of myocytes, andimpairmentinforce development ofthe
myo-cardium. (J. Clin. Invest. 1995.96:2247-2259.) Keywords:
biotinylated dUTP labeling * apoptosis * overstretching
Fasprotein *morphometry * reactiveoxygenspecies
Introduction
Recently, the possibility has been advanced that mechanical
forcesproduced bypathologic hemodynamic loadsmayleadto myocytecell death in themyocardium (1).Thishypothesis has
been made in an attemptto understand the changesin cardiac anatomy aftercoronaryarteryconstriction andglobal
myocar-dial ischemia, and coronary artery occlusion and myocardial
infarction(1, 2). However,nodocumentation ofthis phenome-nonhas beenobtained. This is arelevant issue because acute
reductions inwall thickness andchamber dilation, asa
conse-quenceof sudden increases in ventricular end-diastolicpressure,
may involve mural translocation ofcells, aphenomenon
com-monlydescribedasside-to-side slippageofmyocytes(2). With
this form of wall restructuring, the rings of cells that move
Address correspondence to Piero Anversa, Department of Medicine, Vosburgh Pavilion, Room 302, New York Medical College, Valhalla, NY 10595. Phone:914-993-4168;FAX:914-993-4406.
Receivedfor publication 6 March 1995 and acceptedin revised form17July1995.
radially towards theepicardial region mustexpand toenclose
alargercavitaryvolume. Sincelengtheningof sarcomeres and elongation of myocytes alone are inadequate to explain the increase in dimension of theventricle (2-4), single myocyte
celldeath has been postulated to occur to allow side by side translocation of cells within the wall. In the absence ofmultiple
celldeath, the slidingof myocytebundlesfrom the inner tothe
outer layer of the wall would not take place, limiting mural thinning and chamber dilation. Importantly, single myocytecell
death has been claimedtobe mediated by mechanical forces
within thewall whichmayactivategenesinvolved inapoptotic
celldeath(5). Therefore, experiments were conductedin an in
vitro system in which abnormal resting tension levels were
imposed on musclesobtained from normal adult rat hearts.The
possibility was raised thatthephysical forces may induce
apop-totic cell death in these muscles by enhancing the production of reactive oxygen species (6). Overstretching may also in-creasetheexpression of the cell surface molecule, Fas (7, 8), which has been found to be involved in apoptotic myocyte cell death (9). Since nitric oxide (NO) has been shown to decrease programmed cell death in other cell types (10) and to react with superoxide anion (11) andperhaps reduce oxidant stress (12), thebeneficial effects ofanNO-releasing drug (13)were
examined in this setting.
Methods
Muscle mechanics. Male Sprague-Dawley rats at 3 mo ofage were anesthetized withether.Hearts wererapidly excisedandplaced in oxy-genated Thyrode solution containing potassiumtoinducediastolic arrest. Ineachexperiment,twoleft posterior papillary muscleswereobtained fromtwo animalsand suspendedside-by-side in a muscle bath. The nontendinous endof each papillarymuscle wasmountedto amicrometer assembly usedto adjustexternal musclelength. Thetendinous end of thepapillarymusclewastiedto asteel wire withEthicon 5-0 braided silk (Ethicon, Inc., Somerville, NJ). The wire was attached to a 2-cm stainless lever connected to a servo-controlled galvanometer for
measurementof isometric contraction.Muscles wereperfused continu-ously with normal Thyrode solution maintained at 300C by using a coiled glass heat exchangeratthe inflow line in conjunction with a circulatingwaterbath. The solution was gassed with 95%02/5%CO2 (pH = 7.2). Preparations were stimulated at0.1 Hz by rectangular depolarizing pulses 10msin duration and twicediastolic threshold in intensity(14).
Afteranequilibration period of 60-90min,during which the mus-clescontracted isometricallyat aresting tensionof9.8mN/mm2,the passiveand active stress-strain relations were determined. Inonegroup ofmuscles (n = 12), restingmusclelengthwas increased step-wise untilamaximal active tensionwasdeveloped
(L.,.).'
Resting lengthwasthenchangedthree times betweenL.,,and85%ofL,,. to assess
reproductibilityof thecurve.In the other group of muscles(n = 14),
a similarprocedurewasfollowed but initial musclelength wastaken beyond
Lma,,
untilpassiveand activestress-strain relationswereestab-1.Abbreviation usedinthispaper: L,,., maximal active tension. J.Clin. Invest.
©The American Society for Clinical Investigation, Inc. 0021-9738/95/11/2247/13 $2.00
lished between 106 and 85% of Lmax. Step changes in length were made in a reproducible sequence to minimize the effects of hysteresis. Parameters dependent on muscle length were computed at intervals of 2%Lma, by linear interpolation of the data. At thecompletion of each experiment, muscle lengthwasmeasured withareticlein the eyepiece of adissecting microscope set at amagnification of30. Muscle diameter was computed by averagingfive equally spaced microscopic measure-mentsthroughout the length of thepapillarymuscle. The cross-sectional areawasthen calculatedassumingthe geometry ofacircularcylinder. Thislatter parameter was alsoconfirmed by dividingmuscleweightby itslengthassumingatissuedensity of 1.065 grams/cm3(14).
Histologic detection ofDNAstrand breaks. Forthis portion ofthe study, nine leftposterior papillary muscles whichhad notbeenstretched beyond Lm., were positioned in the muscle bath at a fixed resting tension of nearly 7-8 mN/mm2. A second group of 10leftpapillary muscles, which hadbeenoverstretchedbeyondLm.,were maintained at aresting tension of - 50 mN/mm2. These interventionswerekept
for a 3-h period during which muscles were stimulated as described above. Atthe completion of these mechanical impositions, papillary muscleswerefixed for24h inphosphate buffered4%formalin.Tissue was then embedded inparaffin. Sections, - 4 um in thickness, were
obtained from each papillary muscle and mountedon poly-L-lysine-coatedslides(Sigma Chemical Co.,St.Louis, MO). After
deparaffini-zation andrehydration, tissue sectionswereincubatedinPBS, containing 0.1%saponin and 1 mMEGTAfor 30min(5). Subsequently, sections werecovered with 50/lof staining solutioncontaining5U of terminal deoxynucleotidyltransferase, 2.5mMCoCl2, 0.2Mpotassium
cacodyl-ate, 25 mM Tris-HCl, 0.25% bovineserum albumin and 0.5 nM 2'-deoxyuridine-5'-triphosphate, coupled to biotin via a 16-atom spacer arm(biotin- 16-dUTP).These reagentswereall fromBoehringer Mann-heimBiochemicals(Indianapolis, IN).Sectionswereincubatedinthis solution for 30 min at 370Cinahumidified chamber. Afterrinsing in PBS, sections were incubated for 30 min at room temperature in a
solution containing 4X concentrated SSC buffered and 5% (wt/vol) nonfat dry milk(Sigma Chemical Co.). Finally, the staining solution which contained5jg/mlofHITC-labeledExtravidin(SigmaChemical Co.),4X concentrated SSC buffer,0.1%Triton X-100 and 5%nonfat dry milkwasapplied for 30min.
Positive andnegative controlswereincludedintheprotocolto
con-firm thespecificity of theassay. Asapositivecontrol, myocardial sec-tions weretreated withDNase Itoinduceenzymaticallytheformation ofDNAstrand breaks. The tissuewastreated with thisenzymefor 15 minat37°Cand, afterwashingwithPBS,theterminaldeoxynucleotidyl
transferaseassaywasperformed. Negativecontrols consisted ofstaining
forDNA strand breaks sections similarly treated in which biotin-16-dUTP orterminaldeoxynucleotidyl transferase was notpresent. After terminaldeoxynucleotidyl transferasereaction,the sectionswererinsed inPBS, and stained for a-sarcomeric actin. The tissuewas incubated firstat37°C for 30minwith theprimaryantibody (clone SC5; Sigma
ChemicalCo.) diluted 1:20 inPBS, containing 10% goat serum, and subsequently with anti-mouseIgGtetramethylrhodamineB
isothiocya-nate (TRITC) -labeled antibody (Sigma Chemical Co.), also diluted 1:30 inPBS, containing 10% goat serum. Sections werethen stained withbisbenzimide, 50ng/ml,for 15 mintovisualize nuclei. Afterthis procedure, sectionswererinsed in PBS and embedded in Vectashield (VectorLaboratories, Burlingame,CA) mountingmedium.
Quantitative analysisofDNAstrand breaks in myocytes and non-myocytes. The number of myocyte nuclei in thepapillary muscles la-beledbybiotinylateddUTPwas measuredbycounting the number of stained nuclei per unitareaof tissue sections in eachpapillarymuscle. Thisanalysiswasperformedwithamicroscope workingin
epifluores-cencemode,equippedwithsetsofexcitation emission filters for FITC (excitation blue, emission green), which allowed the identification of incorporateddUTP in nuclei. The numberoflabeled nucleiwasrecorded and the distinction between myocytes and nonmyocytes wasobtained bydetectionof a-sarcomeric actin with theuseofasecondsetof filters specificfor TRITC(excitationgreen, emissionred). Bythisapproach,
thenumber ofmyocytes and nonmyocytes stainedbydUTP permm2
oftissuewasdeterminedin eachmuscle.Sectionswereexaminedat a
magnification of 1,250byviewingsubsequent fields through the entire papillary muscle. A square tissue area equal to 6,084 nm2was delineated inthemicroscopicfieldby an ocular reticle (#105844; Wild Heerbrugg Instruments, Inc., Farmingdale, NY) and an average of 800 fields in eachmuscle were examined.
Morphometric determination of the number of dUTP-labeled myo-cytesandnonmyocytes.Thehistologic preparationdescribed in the pre-ceding section didnotpermitthedetermination of the fraction of myo-cytenuclei labeledby dUTP. To obtainthisparameter the number of myocyte nuclei and nonmyocyte nuclei per unit area of tissue was determinedby countinganaverage of30 fields, 6,084
Atm2
each, at a magnification of 1,250 in each papillary muscle. This analysis was performed with a microscope working in epifluorescence mode, equipped with sets of excitation emission filters for bisbenzimide H33258(excitation UV,emissionblue) andTRITC (excitation green, emissionred). Inrandomly selected fields, nucleiwereidentified first (bisbenzimide staining), then excitation emission filterswerechanged to visualize myocytes labeled with anti- a-sarcomeric actin. In this manner, the numericaldensity ofmyocyte and nonmyocyte nucleiin eachpapillary musclewasobtained. By combining thesedatawiththe preceding estimations of dUTP-labeledmyocyte and nonmyocyte nuclei per unit area ofmyocardium, the numberofapoptotic myocyte and nonmyocyte nuclei per 106 nucleiwasdetermined.DNAgel electrophoresis. Toconfirm that the histochemical detec-tion ofDNAstrand breaks correspondedtointernucleosomal cleavage, thepresence of low molecular weight DNAinpapillary muscleswas determined( 15).Groups of three papillarymuscleseachwere mechani-callyhomogenizedonice and fixed for24hat-20'C in70% ethanol. The tissuewasthencentrifugedat800 gfor5 min and the ethanol was thoroughlyremoved. Thepelletwasresuspendedin40,ulof phosphate-citratebuffer, consisting of 192partsof 0.2MNa2HPO4 and 8parts of 0.1Mcitric acid (pH7.8),atroomtemperature,for1 h.After centrifu-gation at 1,000 gfor 5 min, the supernatant was transferred to new
tubesandconcentratedbyvacuuminaSpeedVacconcentrator(Savant InstrumentsInc.,Farmingdale, NY)for 15 min. A
3-Al
aliquot of 0.25% NP-40(Sigma Chemical Co.) in distilled water wasthenadded, fol-lowedby 3/l ofasolution ofRNase, 1 mg/ml,also inwater. After 30-min incubationat 37°C, 3 IL ofasolution ofproteinase K, 1 mg/ ml (Boehringer Mannheim Biochemicals), wasadded and the extract wasincubated foranadditional 1 hat37°C. After theincubation, 12Al
ofloading buffer,0.25%bromophenol blue,30%glycerol,wasadded andsamplesweresubjectedtoelectrophoresison 1% agarosegelcon-taining5
Ag/ml
ethidium bromide. TheDNA inthegelswasvisualized under UV light. For this portion ofthe study, six nonoverstretched muscles andsix overstretched muscleswereutilized.Immunohistochemicallocalization ofFas.Tissuesections mounted onpoly-L-lysine-coatedglass slidesweredeparaffinizedbyheatingat
90°C followedbythree washes inxylene. After dehydrationinabsolute ethanol for15 s, slideswereincubatedin 2%(vol/vol) H202inmethanol for 30-45sthenwashedwith95% ethanol for 20s,followed sequen-tially by70%ethanol for20s,distilledH20for 1 min,and PBS(120 mmol/literNaCl,11.5mmol/literNaH2PO4, 31.3 mmol/liter KH2PO4, pH7.4-7.6) for5 minand then heated by microwaving for 15 min. Afterwashingtwice in PBS for 5 min, tissue sectionswerepreblocked for 45 min in TNK solution (100 mmol/literTris, pH 7.6-7.8,550 mmol/liter NaCl, 10 mmol/liter
KCl)
containing 2% (wt/vol) BSA (SigmaChemicalCo.) 0.1%TritonX-100,and 1%normal goatserum. Antibodieswereaddedtothe slide in thesame solution and incubated overnight at roomtemperature. Anti-mouse Fas hamster monoclonal antibody (clone Jo2;Pharmingen,SanDiego, CA) wasused at 1:100 (vol/vol)dilution. Insomecases, theprimaryantibodieswereomitted (negativecontrols). Myeloma IgG antibody (JacksonImmunoResearch Laboratories, Inc., WestGrove, PA) was alsoused as asecond form ofnegativecontrol. AfterwashingwithPBS,tissue sectionswere incu-bated for 1 h with 5Ag/ml
ofbiotinylated goat anti-rabbitantibody(Vector Laboratories, Inc.) for thedetection of Fas and then washed and incubated for 30-45 min with anavidin-biotin complexreagent
solutioncontaining 3,3'-diaminobenzidine (DAB) and 0.1% (vol/vol) H202 in TNKbuffer.
Thequantitativeanalysis of the cell localization of Fas proteinwas
performed in4and 5papillary musclesexposedtolow andhighlevels of tension, respectively. Five equally spacedareas,along the longitudi-nalaxisofeachmuscle, weresamples andthenumberof labeled and unlabeled myocytes across the diameterofthe musclewasdetermined in each region. Theseindividual values were then averagedtoyielda meanvalue in each muscle.
Detection of superoxide anion. Papillary musclewere exposedto levels of tension comparable to those described above. Under these conditions, the formation of superoxide anion was examined over a period of 3 hduring whichloading waskeptconstant. At theend of thisprocedure, muscleswerefixedin 4% phosphate buffered formalin and embeddedinparaffin forthesubsequent detection ofprogrammed cell death.Specifically, changes in force and 0- generationwere deter-mined inasingle photoncountingapparatusconstructedin alight tight box. In theseexperiments, stimulated muscleswereincubated inKrebs' bicarbonate buffer containing 0.25 mM lucigenin in a continuously gassed 1 cm2spectrophotometercuvettemounted inathermostatedcell holder on the surface ofa Lucite light guide (with a shutter cover) directed intoacooledThornEMIPhotomultiplier Tube (model9235B; ThornEMIElectron Tubes,Ruslip, UnitedKingdom). AThornEMI amplifier-discriminator (model C604) and photon counter (model C660) were used to quantitate chemiluminescence. The counts were integratedover5-speriods by the photoncounter,andananalog signal of the integratedcounts wascontinuously recordedon a(model7 Poly-graph; Grass Instrument Co., Quincy, MA) recorder together with changes in force (16). This analysis wasconducted infour and five muscles atlow andhigh tension levels, respectively. The effects of superoxide dismutase on the generation of superoxide anion in over-stretchedmuscles were also examined(n =4). Moreover,theability of N-2-mercaptopropionyl glycinetoinhibitsuperoxideanion intracellu-lary was evaluatedin four overstretched papillary muscles. Forthese twoconditions, muscleswere exposedtoeithersuperoxidedismutase, 3
jiM,
orN-2-mercaptopropionyl glycine, 2mM,for 15 min (17, 18) before the determination of chemiluminescence levels (16).Thedose of N-2-mercaptopropionyl glycine used herewasestablishedonthebasis of preliminary experiments showing that the ability of this compoundto affectsuperoxide anion wasmaximal at 2 mMwithoutsignificant in-creasesup to20mM.Effects ofCAS936. Thepotential beneficial influenceofthe NO-releasing drug C87-3754,ametaboliteof CAS 936 (13),onthe genera-tion of superoxide anion and the activagenera-tion of programmed cell death wasexaminedinpapillary muscles. Thiswasperformedby superfusing muscles keptatlowandhigh tension levels(seeabove), withasolution containing 10 ktM C87-3754 during a 3-h period. The formation of reactive oxygenspecieswasmeasured throughout and afterthe histo-logic preparation described above,Fasand dUTP labelingof thetissue was determined. Five muscles of high tension levels were used for chemiluminescencemeasurements,andseven wereused for evaluation ofDNA strand breaks. The effects of C87-3754 on developed and resting tensionswith time wereevaluatedin 10papillarymuscles and comparedwith an identicalnumber ofmuscles notexposedtothedrug. Numberofmyocytes across the muscle. Papillarymusclesexposed tolow andhigh levels of restingtension for a3-hperiodwerefixedin these conditions withasolution containing 5% glutaraldehydeand 4% paraformaldehyde. Papillary muscles were then removed and sliced transversely into four pieces which were embedded in araldite. One fragmentof tissue wasembedded longitudinally for themeasurement ofsarcomerelength. Semithin sections, - 0.5 Hm in thickness, were cutfrom each blockperpendicular tothelong axis of the muscle and showingits whole cross-sectional area. The transversearea was deter-minedby an interacting tracking device and the mean diameter was computed (Jandel Scientific, Corte Madera, CA). Subsequently, thin sections were obtained from these blocks and low power electron
micro-graphs,four from each oftwoblocksfrom eachpapillary muscle,were
collected. These micrographs wereprinted at afinal magnification of 4,500and thevolumecompositionofthemyocardiumand the numerical
density of myocyte profiles per unit areaof tissue were measured as previously described (2). The availability of papillary muscle diameter and myocyte density allowed the calculation of the number of cells across the papillary muscle (2). The mean center-to-center distance (d,-c)between myocytes wascalculated from the number of profiles counted per unitareaoftissue,(N(m)A)intransversemyocardial sections by assumingthetendency for theseroughly cylindrical cellstopack in a closehexagonal pattern (2):
2 -1.0746
VN(m)A IN'.7A
In ahexagonal pattern, the spacing betweenplanes of adjacent cells varies with the orientationofthearray fromamaximumofd.-cto a minimum ofdr-,
6V/2
andhas a meanvalue, d, representativeof random orientation, given by3dc-
63
Ir/6 dOd-C
=~~v~ 6d =0.9085d-ir O cosO
Thus, the number,N(m)t,,,,of myocytesacrossthe thicknessof the papil-lary muscle, W,canbefound from
N(m)tin =W/d =
1.0243.W/ii(.)A
This analysiswasperformed in six musclesatlow and six musclesat high tension levels. The blockoftissueembedded parallelto themajor axis ofthe papillary muscle was sectioned and sarcomere length in myofibrilswasdeterminedineach muscle from100measurements made atamagnification of 30,000. These sections were longitudinally oriented andwere cutperpendiculartotheirlengthtoavoidlongitudinal compres-sion.
Datacollection andanalysis. All tissue sampleswerecoded, and thecodewasbrokenattheendof theexperiment. Results are presented asmeantSD computed from the averagemeasurementsobtainedfrom each muscle. Comparisons betweentwo values wereperformed using atwo-tailed Student'sttest.Statisticalsignificance in multiple compari-sons in which the analysis of variance and the Ftest indicated the presence ofsignificant differenceswasdetermined by the Bonferonni method. Values ofP < 0.05wereconsideredsignificant.
Results
Muscle mechanics. Fig. 1 illustrates the passive and active length tension relationships of papillary muscles between 85 and100% ofL,,,,andbetween 85 and 106% of L,,o. Muscles stretched upto 100% ofL,,, showed that the progressive
in-crease in resting tension was coupledwith aparallel increase in developed tension which peaked at
L.,
(Fig. 1 A). These muscles maintained theirabilitytogenerateexpected levels ofdevelopedtensionupwardanddownward alongthe entire length tension curve. In contrast, papillary muscles stretchedbeyond L,,, exhibited marked increases in resting tension associated withadecrease in isometricdevelopedtension(Fig.1B). From 100to 106% of
L.,,,
restingtension increased fromavalue of 19 mN/mm2 to avalueof 52 mN/mm2. Incontrast, isometricdevelopedtension decreased from avalueof 73 mN/mm2to a
valueof 61 mN/mm2. These differenceswerebothstatistically significant (P < 0.001). Moreover, overstretched papillary muscles were unableto produce thesame developed tensions and when moved downward alongthelength, tension
relation-ships producedless tensionsthroughoutthe range ofphysiologic
loads examined. The resting tension curve was shifted to the
right at all lengths from 104 to 94% of
Lmal
(P < 0.001), whereas the reductions indeveloped tension werestatisticallysignificant
from 102to 85% of L, (P <0.001).
A
80 70 60 50 40 30 286 68 90 92 94 96 96 100
Muscle
Length (%
L..)
B
I/
4
T/
..I*
1
I I
56 66 90 92 04 96 06 100102104106
Muscle
Length (% L.)
Figure 1. Resting and active length tensionrelationships of left posterior papillary muscles. Mus-cles extendedbeyondLmax (n
= 14) were notable to maintain stable levels ofresting and devel-oped tensions (B). This phenome-non was not present in muscles ex-tended up toLmax(n = 12) (A). Results are presented as mean±SD. (A)Corresponds to active tensiondevelopment at in-creasing muscle lengths; (v) cor-responds to active tension devel-opment atdecreasing muscle lengths;(A)corresponds to rest-ing tension development at in-creasing muscle lengths; and (v) correspondstoresting tension de-velopment at decreasing muscle lengths. Seetext for statistics and moredetails.
developedtensions inpapillarymusclesexposedtoresting
ten-sion levels of - 7-8and 50 mN/mm2,respectively. Moderately
stretched papillary muscles maintained comparable levels of
passiveandactive tensionsover a3-hperiod. Conversely,
over-stretched muscles showedaninitial increase in isometric devel-oped tension followedbyaprogressivedecline with time.
Rest-ingtension in these muscles decreasedcontinuouslyduringthe
3-h interval. In summary, overstretching affected the ability
ofpapillary muscles to maintain stable levels ofresting and
developedisometric tensionswith time.
Structural characteristics. Semithin sections of papillary
muscles showed that the normalstructureof muscleswas pre-served in allcases.Thus,consistent withpreviousresults( 19),
the size of the muscles used in thisstudydidnotlimit oxygen-ation of the core of the muscles
producing
cellular damage. Thiswasconfirmedquantitatively, since the volume fraction of myocytes and interstitial compartment in musclesexposed
tomoderate levels of tension was 86±1.0 and 14 1.0, whereas thecorrespondingvalues in muscleskeptathigh tensionwere
87±2.0and 13+±2.0. Measurements ofpapillary muscle cross-sectionalarea wereobtained from thesehistologic preparations
for the subsequent evaluation of papillary muscle diameter. Highrestingtension levelswere associated with an 8%
reduc-tion in muscle diameter which, however, was not statistically significant (Fig.3 A).
The numericaldensityof myocyte profilesper unit area of
papillary muscle wasobtained bylow power electron micros-copy. Myocyte numerical density was
5,405+344
in muscles maintainedfor 3 hat7-8mN/mm2and4,857±444 inmuscles kept for thesametimeat50mN/mm2.Thesevalues, in combi-nation with the volume fraction of myocytes in thetissue,wereutilizedtocompute the averagecross-sectionalareaand
diame-terof myocytes. Incomparisonwithmusclessubjectedto
mod-erate loads, high loads were characterized by a 13 and 6%
A0,
B*
*
*
o
1 2Time
(Hours)
al
a 70 N z 60a
0 50 0 .P.4 400 0 2010
P 10a
-2 3 0Tir
Figure2. Effects ofloading, -
7-8mN/mm2 (n = 9)and 50 mN/ mm2(n= 10)onrestingand de-velopedtension with time. *
Indi-I I cates avaluethat isstatistically
1 2 3 significantlydifferent from the
ne
(Hours)
value obtainedat 30min,
P<0.05. 80 go 70
0
a 60 N.,z
a 50 0 o 40 ml a 30 E--0 20 0 Go 8o 70F
60 _ 50 _ 40 _-a
0 N z 0 a U, 0 .P4 V Ed Tko 0 mPVi
30F
20 _ 10 0A
100111,i
NJ
I
"--I/
I/
,.I
a
S
:t
a
:S ¢
0
0
S U
"4
.0
4.'
U
a
a
0
54 0 0
54 .0
a.
z7-8MN/me' 60mN/mm'
B
D
7-5 mN/mm' 50mN/mu
Figure3.Effects of loading, - 7-8 mN/
mm2(n= 6) and50mN/mm2(n= 6) fora3-hperiod,onpapillarymuscle
di-ameter(A),myocytediameter(B),
sar-comerelength(C), and number of
myo-cyteprofiles within the thickness of the
papillarymuscle (D). Resultsare
pre-sentedasmean±SD. * Indicatesavalue thatisstatistically significantlydifferent, P<0.05.
greater myocyte transverse area and diameter, respectively. However, these changeswerenotstatistically significant (Fig. 3 B). On theotherhand,sarcomerelengthmeasured in
longitu-dinallyoriented sections ofmyocyteswas7% shorter in
over-loaded muscle (Fig. 3 C) and this differencewas statistically significant (P < 0.01). When the number ofmyocytes across
thediameter of the papillary musclewas computed according
totheequation in the Methodssection, muscles maintained at
50 mN/mm2 werefoundtopossess 15% less myocyteprofiles
than muscleskeptat7-8 mN/mm2(Fig.3D).This difference
wasstatistically significant (P < 0.02).Insummary,high ten-sion levels reduced the number ofmyocyteprofiles within the thickness of the papillarymuscle.
Biotinylated dUTP detection in the papillary muscle. The specificityof thetechniqueutilizedtostain DNA strand breaks indifferentcellpopulations wasdocumentedby treatingtissue sections with DNase I before the detection of DNA strand breaksby dUTPlabeling (Fig. 4,A andB). Conversely,DNA strandbreakswerenotdetected whenbiotin-16-dUTP (Fig. 4,
C and D) or terminal deoxynucleotidyl transferase was not
includedintheenzymaticreaction. An identical effectwasalso obtained by staining sections which contained DNA strand breaks in the absence of biotin-16-dUTP or terminal deoxy-nucleotidyltransferase.
Fig. 5 illustrates the localizationof DNA strand breaksin
myocyte nuclei ofleft posterior papillary muscles maintained
for 3 hat50 mN/mm2.Occasional dUTP-stainedmyocyte
nu-cleiwerealsoseenin muscleskeptat7-8mN/mm2. However,
the number ofpositively labeledmyocytenucleiwasgreaterin musclesexposedtohighertension levels. Interstitial cellswere
alsofoundtobe labeledbydUTP and the number ofapoptotic
nonmyocytenuclei also increased in overstretched muscle
prep-arations. Incontrast, aftertreatment withC87-3754, dUTP la-beling ofmyocytesandnonmyocytesin musclesmaintainedat
50 mN/mm2wassignificantly reduced, reachingvalues similar
to those of muscles keptat7-8 mN/mm2.To avoidpotential influences from the damagedendsof themuscle, this analysis
wasrestrictedto the mid-portion of each papillary muscle. Fig. 6 shows quantitatively the effects of overstretching and C87-3754 on the number ofmyocyte nuclei andnonmyocyte
nuclei exhibiting DNAstrandbreaks in papillary muscles. After
a 3-h period of exposure of muscles to high tension levels, 6,400 myocyte nuclei per 106 nuclei exhibited DNA strand breaks. This value was 21-fold higher than that obtained in
muscles keptatmoderate tension, 300myocyte nucleiper 106 nuclei. This differencewasstatistically significant (P<0.001). In addition, overstretching was associated with a 2.4-fold
in-creasein the number of interstitial cells labeled bydUTP.This
difference was also statistically significant (P < 0.02). Con-versely, C87-3754 was capable of maintaining the extent of dUTP labeling of myocytes and nonmyocytes within values comparabletothose foundatmoderatetension, in spiteofthe overstretching. Insummary,overstretchingwasassociatedwith
a 21-fold and a 2.4-fold increase in the magnitude of dUTP
labelingofmyocytesandnonmyocytesinpapillarymuscles and C87-3754preventedthisphenomenon.
Detection of DNA laddering in the myocardium. The
pres-enceof DNAfragmentsof sizeequivalenttothemonoor
oligo-nucleosomeswas determinedtoconfirm the detectionofDNA strandbreaks in myocytenuclei. Becauseonly arelativesmall fraction of myocytes was undergoing apoptosis in the
over-stretched papillary muscles, aprocedurefor extraction oflow
A
1.4
a
a
-4
S 1.0 ob
-4
.--4
m
Pa 0.0
2.2
2.0
as
4 1.5
ID 1.6 w
0
1.6
i
1.4 0di 1.2
Figure4.Detection ofDNAstrand breaks innucleiaftertreatmentof sections of myocardiumwith DNase I. (A) Illustrates by bisbenzimide fluorescence, the nuclei includedin thesection, whereas (B) showsthat mostnuclei werestainedby biotinylated dUTP afterDNaseexposure. Omission of dUTPfrom thereactionresults innegative staining (compareCwithD).Thecontrastin (D) was enhanced todemonstratemore clearly the lack ofdUTPlabeling. (A-D)x400.
molecularweight DNA fromapoptoticcellswasused (15). As illustrated inFig. 7,extractsobtained from the musclesexposed to50mNN/mm2revealedthe presence of DNAfragments
consis-tentwithapoptosis. Stretchesof DNA of sizeequivalentto200 bp were themostabundant. Moreover,400-, 600-, and800-bp
fragments which correspond to two, three, and four
nucleo-somes,respectively,werealso visible. Incontrast,no
degrada-tion of DNAwasfound innonoverstretchedpapillary muscles. In summary,DNAfragmentationcharacteristic ofapoptoticcell deathwasobserved in overstretched papillarymuscles.
Chemiluminescent measurementof superoxideanion gener-ation. To determine whether overstretching of the papillary musclewas associatedwith an enhanced formation of reactive oxygen species, lucigenin-dependent photoemission was
mea-sured in muscles kept for 3 h at 7-8 and 50 mN/mm2. As illustrated in Fig. 8, papillary muscles exposed to the lower levelof tensionexhibited no changesintheformation of super-oxide anion during the 3-h period of stretching. In contrast, overstretching resulted in a marked elevation of superoxide-elicited lucigenin chemiluminescence. When these two values
werecompared,a2.7-fold higher activation of superoxide for-mationwas foundinmuscles at 50
mN/mm2
(P<0.005) thaninmuscles at7-8
mN/mm2.
Toestablish whether NOwas ableto prevent stretch-induced superoxide generation, a separate group of overstretched papillary muscles was exposed to 10 ,tM C87-3754. As shown in Fig. 8, in the presence of the NO-releasing drug, a 42% (P < 0.02) decrease in the formation of
superoxideanionwasnoted. To confirm the intracellular origin ofsuperoxide anion, the effects of superoxide dismutase and N-2-mercaptopropionyl glycine on overstretched muscles were measured. Superoxide dismutase did not affect the level of
chemiluminescence generated by the overstretched muscles. Thisintervention decreased the formation of superoxide anion by 2.65±4.51% (n =4)whichwasnotstatisticallysignificant.
Incontrast, N-2-mercaptopropionyl glycine reduced this
param-eterby 53.51+11.64% (n =4) and this changewasstatistically significant (P < 0.01).
Fig. 9 illustrates the values of active and resting tensions of papillary muscles exposedto aload of - 50
mN/mm2,
in thepresence and absence of C87-3754. This phenomenon was ex-amined over a 3-hperiod. TheNO-releasing drugwas associ-ated with lowerlevels of developed tensionthanthoseobserved
in theabsenceof C87-3754,inspite ofacomparable magnitude ofpreload. Resting tensionwas similarin both groups of
mus-cles. Moreover,developed and resting tension decreased with
statisti-Figure5. Detection of DNA strand breaksin a myocyte (A) nucleus (arrow-head)bybiotinylateddUTP labeling of anoverstretched papillarymuscle. (B) Il-lustrates the samemicroscopic field by a combinationofphase contrast and bis-benzimidefluorescence.Myocytes were identified by labeling with a-sarcomeric actin antibody (C). (A-C) x1,200.
to0
..p4
a)
I-z
a)
P-a)
,0
p-4 lo
10000
*
8000
6000
4000
2000
II -VZZ1D0vQQ I vies/~
Myocytes Non-Myocytes
Figure 6.Effects of loading, - 7-8mN/mm2(n=9)(openbars)and
50mN/mm2(n= 10) (hatchedandcross-hatchedbars) fora3-hr period,inthe presence(n=7)(cross-hatched bars)and absence (open andhatchedbars) ofCAS936on the numberof dUTP-labeled nuclei per 106cellsinpapillarymuscles. Results arepresentedasmean±SD.
*Indicates a valuethatisstatisticallysignificantly different from the value inmusclesexposedtonearly7-8mN/mm2,P <0.05. * * Indi-cates a value thatisstatisticallysignificantly differentfromthevalue in musclesexposedto - 50mN/mm2,P <0.05.
cally significant changes in these parameters occurred during
the3-h interval examined with C87-3754. Insummary, overs-tretchingwascharacterizedbyenhancedformation of superox-ide anion intracellularly and this effect was attenuatedby an
NO donor which also maintained active and passive tension levels stableovertime.
Expression ofFas inpapillarymuscles. Fig. 10,A-C
illus-trate the localization of Fas protein in myocytes of muscles exposedto7-8and 50 mN/mm2,respectively. Although
immu-1 2
3
4
5
6
-1353 _ l - 1 0 7
8~~~-07
-
872
-603
-
310
281
-
234
- 194
Figure 7.Electrophoreticpattern ofDNAfragments extracted from nonoverstretched(lanes2and3) and overstretched (lanes 4 and 5) papillarymuscles. Lanes I and6,molecularweightmarkers. Eachlane correspondstothreepapillarymuscles. Arrowscorrespondto200-, 400-,600-,and800-bpDNAfragments.
P-a)
a) 0
-03
m
0
0
4)
0o
350
200
150
100
50
0
**
Figure8. The relativechange insuperoxide anion-elicited lucigenin chemiluminescence after 3-hexposureofpapillary muscles to 7-8mN/
mm' (n=4)
(open
bar),50mN/mm2 (n=5)(hatchedbar),
and 50 mN/mm2in the presence of10 ,uM CAS 936 (n = 7)(cross-hatchedbar).Data arepresentedasmean±SD *Indicates a value that is significantlydifferentfrommuscles exposed to7-8mN/mm2(P
<0.05). * *Indicatesavalue that issignificantly different from mus-clesexposedto50mN/MM2,P< 0.05.
nostaining for Faswasonly occasionally observed in myocytes of muscles maintainedat7-8mN/mm2(Fig. 10 A), numerous myocytes were labeled in muscles kept at 50 mN/mm2 (Fig.
10, B and C). In contrast, Fas labeling was not detected in interstitial cells of thesamemuscles. Thespecificity of
immuno-staining for Fas was confirmed by the lack oflabeling when sectionswereprocessedin the absenceof the primary antibody
(Fig. 10D)orin the presence ofmyeloma IgG antibody (Fig. 10 E). It should beemphasizedthattissue sections could not be stainedsimultaneously for DNA strand breaks and Fas protein because dUTPlabeling required0.2Mcacodylate buffer which interferes with Fas immunolabeling. Conversely, retrieval of Fas antigen requires microwaving which induces DNA strand breaks(not illustrated). However, attemptsweremadeto
docu-mentFas labeling and dUTP staining in serial sections of the
samemuscle.Bythis approach,itwaspossibletoobserve that,
attimes, Fas overexpression andDNA strand breaks involved the same muscle cell profiles (Fig. 10, F-I). Quantitatively, the number of myocytes labeled by Fas was 67,000±26,000
per
106
cells in overstretched muscles and3,200±2,400per106
cells in nonoverstretched muscles. This differencewas
statisti-cally significant(P<0.003). Importantly,C87-3754treatment
reduced the number of Fas-labeled myocytes in overstretched muscleto7,800±2,300per
106
cells.The 88% decrease in Fas labeling ofmyocyteswas statistically significant (P < 0.01).Theadditionof C87-3754
resulted
inalevel ofFaslabeling inoverstretched
muscles similar to that measured innonover-stretched muscles (P = 0.1). Insummary, overstretching was
associated withanincrease in theexpression ofFas inmyocytes which wasmarkedly attenuated byanNO donor.
Discussion
The results of thecurrentstudy indicatethat theforcegenerating
ability of papillary muscles exposed to high levels ofresting
tension decreased along the entirelength tension relationship.
A
50r
H
FIT
+
[0
30 60 90 120 150
N
40
N1.
s 30 Si 0
0 20
V
E-bA
0 1
0
180
F
B
I
30 60 90 120 150 180
30 60 90 120 150 180
Figure9.Effects ofloading, 50
mN/mm2,inthe absence (n= 10) (openbars) and presence (n
= 10) (hatched bars)ofCAS 936 onactive(A)andpassive(B) ten-sion with time. Resultsare pre-sentedasmean+SD. *Indicates avaluethatisstatistically signifi-cantly different from thevalue ob-tainedat30 min,P <0.05.
in muscle diameter, apoptotic myocyte and nonmyocyte cell
death, and a reduction in the number of muscle cells within
the thickness of the papillary muscle. Moreover, endogenous superoxide production was increased. At the molecular level, anenhancedexpressionof Fasreceptorproteinwasdocumented
inmyocytes.Thus,thepossibilitymaybe raised thatpathologic
alterations ofmyocardialloadmaybecoupledwith the
genera-tion of reactiveoxygenspeciesand theactivation ofgene
prod-uctsimplicatedinprogrammedcelldeath. Inturn,this
phenom-enonmayleadtoanarchitecturalrearrangementof the
myocar-diuminvolving side-to-sideslippageofmyocytes. Importantly, interventionsresultinginthe release ofNOpreventedthe forma-tion of superoxide anion, Fas protein, programmedcell death and the relative reduction in muscle performance associated with theimpositionofabnormal mechanical loads.
Mechanicalperformance ofthemyocardium. Alongthe
as-cending limb of the cardiac length-tension curve, developed
tension is length dependent and thisproperty isrelatedto the calciumactivation ofmyofilamentsmediatedbythe troponin-tropomyosin protein regulatory complex (20, 21).The overlap-ping of thin and thick filaments is unchanged in thepositive portion of the Frank-Starling relation (22), and the changes
inrestinglengthand forcedevelopmentarerelatedtotheextent
of calcium myofilament interaction (22, 23). However, less understood arethe mechanismsimplicatedin thealterations in
restingand active tensionsobserved in musclesexposedto lev-els ofstretchbeyondthepeakof thelength-tensioncurve(22).
Underthis setting, excessivepassive forces are generated and
damagehas been claimedto occurto the ends of the muscle
preparations affectingtensiondevelopment (22). Misalignment
of sarcomeres and/or slippage of myofibrils within the cells
have been considered to represent the in vivo counterpart of these in vitro conditions mimicking pathologic states of the heart(24).
Observations in thecurrentstudydocumentthathighloads altered the contractile behavior of the myocardium and this
phenomenonwascharacterizedbyareductioninmuscle diame-ter.Importantly, sarcomere lengthwaslongerin musclesfixed at moderate than high loads. Damage to the myocyte and nonmyocyte compartments of themuscle wasnotobserved in eithercase. Inaddition, the lack ofregisterbetweenindividual
myofibrils in the cytoplasm was a consistent finding in both
preparations, raising questionsontheroleplayed bythis
struc-tural modification in thedepressionofforcegenerationin
over-stretched papillarymuscles. The decrease ofsarcomerelength
in muscleskept for a3-h periodat 50mN/mm2 suggests that the mechanicalimpositionshifted the muscledownwardonthe
length tension relationship resulting in a decrease in tension
development. This notion is consistent with aslightreduction
in sarcomere lengthat higher loads.
Ventricular dilation after increases in fillingpressureinthe
intactheartinvitro, acuteandchronicventricular dysfunction,
acuteandchronic myocardial infarction, sudden restrictions in
coronaryperfusionwithglobal myocardial ischemia,andduring the alterationsinloading associatedwiththetransitionfromthe
fetal to the adult circulatory system (forreview see reference
25)has all been consideredtobe characterizedby astructural
reorganizationof themyocyte compartmentof themyocardium, resulting inmural thinning and expansion in cavitary volume.
This adaptation, consisting of side-to-side slippage of cells
withinthewall,appearstobetheprincipal determinantofacute
ventricular dilation under these pathologic conditions of the heart. In a few cases, a decrease in the number ofmyocytes included in thethickness of theventricularwall has been
dem-onstrated quantitatively (2, 26) in an attempt to support the
notion ofmural translocation of cells. Findings in the current
study demonstratethatabnormalmechanical forcesresultedin
a reduction in the number of cells within the width of the
papillary muscle, providing the first documentation that load
andmyocyte slippagearecausally related.
Programmed myocyte cell death. Although very little is
known concerning programmedcell death in the myocardium (5, 27),thisphenomenonaffectsvariouscelltypesand is initi-ated by a variety of environmental stimuli (28). During this
process, animportantfactor is theactivation ofanendogenous
endonucleasewhich results inendonucleolysis (28).DNA deg-radation, triggered bythis mechanism,isspecificto the spacer
regions, leaving the DNA associated withthe nucleosomes in-tact(29). The techniqueused here allowedan early detection
of DNA strand breaks inmyocardialcell nucleibytheterminal
deoxynucleotydil transferase assay (28). Moreover, the
mor-phologic documentation ofapoptoticcell death was confirmed
bythe demonstration of intranucleosomalcleavage bytheDNA
ladderingassay.Similarobservations have been made after
isch-emicreperfusion injuryof therabbitheart in vivo(27),and in neonatal cardiacmyocytesincultureexposedtohypoxic condi-tion(9). However, defectsinoxygendiffusiontothepapillary
muscle in our preparation were unlikely, suggesting that
pro-80
N
Z 60
0
-P4
0
IV
0
F
Figure10. Photomicrographs il-lustratingFasmonoclonal anti-bodylabeling ofa nonover-stretched(A)andanoverstretched (Band C)papillary muscle. Omission of the primary antibody (D) orits substitution with my-elomaIgG antibody(E) during thereactionresulted innegative staining ofanoverstretched mus-cle. Serial sectionsof an over-stretched papillary muscle show-ingdUTPlabeling (F and H) and Faslabeling (G and I)ofthe same cells (arrows). (A andB) X400;
(C) Xl,100; (D-I) x600.
grammed cell death occurred via a different mechanism. Isch-emic myocyte cell death is characterized by rupture of the sarco-lemmal lining, contraction bands in myofibrils, and mitochon-drial swelling(28-30). Lossof membranefunction (28) and mitochondrialswellingaredistinct aspectsofnecrotic cell death
in all cellpopulations (28, 30). In contrast, programmed cell deathtypically occurs in the absence of loss of plasma
Figure 10(Continued)
plasma membrane, myofibrils and mitochondria, indicate that the protocols used did not produce necrotic cell death in the papillary muscles. In addition, apoptotic cell death appeared tobe independent from local ischemia. DNA strand breaks in myocyte and nonmyocyte nuclei were detected in the absence of structural abnormalities at the light and electron microscopic levels, suggesting that 3 h of mechanical stretching produced lesionscharacteristic of the early phases of apoptotic cell death. The observation thatoverstretching triggered programmed celldeath, involving nearly 0.5% of myocytes, raises the
possi-bility that loss of contractile function in these apoptotic cells may have influenced the detrimental impact of high loading states on isometric tension development of the myocardium. Although the percentage of cells affected by this phenomenon was less than the reduction in active tension, single cell death mayhaveimpinged upon theforce generating ability of
neigh-boring cells depressing more severely overall muscle
perfor-mance. DNA laddering clearly showed the presence of200-,
thetissue cannot be calculated from these observations. Simi-larly,dUTP labeling of myocytes provides an estimation of the early events of apoptosis only (28). Thus, the percentage of myocytes labeled by dUTP most likely represents an underesti-mation of the real extent of myocyte cell death in the tissue (5). As indicated above, myocyte slippage was accompanied by adecrease in sarcomerelength, modifying the passive and
activelength tension relationships. However, the relative contri-bution of programmed myocyte cell death and myocyte slippage to the impairment in myocardialperformancein overstretched papillary muscles cannot be determinedatpresent. On theother hand, these events characterize a common pathophysiologic
phenomenon in which abnormal loads trigger programmed myocytecell death which leads to side-to-sideslippageof myo-cytesdepressing muscle contractile behavior.
Themagnitude of apoptotic myocyte death in overstretched muscle was lower than the extent of myocytesexpressing Fas protein on the surfaceofthecell and cytoplasm. Fas protein was
barely detectable in myocytes of moderately loaded muscles,
indicating thatitsexpressionwasdependentupon theextentof
load imposedonthemyocardium. Althougha causeandeffect relationship between programmed myocyte cell death and the Fas molecule cannot be claimed, it is noteworthy that Fas mRNAincreases inneonatal cardiac myocytes in culture
under-going programmedcell death(9).The Fas genebelongstothe
tumornecrosis factor and nerve growthfactor receptor family
(7-9) and apoptosis can be triggered by ligand activation of the Fas receptor(8). Whether stressed myocytes cangenerate the Fasligand triggeringtheirownsuicideprogram iscurrently
unknown. Although the lack of Fas labeling in fibroblasts of overstretched muscles was unexpected, this phenomenon ap-pears tobe consistent with the moderate increase in apoptotic
cell death of theinterstitial cellpopulation of themyocardium
underthis setting.
Reactive oxygen species, NO, andprogrammedcell death. Impositions ofhighloadsonthemyocardiumarecharacterized byanincreased oxygenconsumptionandthisphenomenonmay lead to the generationofsuperoxideanion which may activate the suicide program of myocytes and interstitial cells(6).NO may counteract superoxide exerting a protective influence
against apoptotic cell death in vitro (10). The results of the
currentstudyareconsistent with thiscontention, since the
NO-releasingdrug,C87-3754,wascapableofpreventingthe
forma-tionof DNA strand breaks in myocytes and interstitial cells of
papillarymusclesexposedtohighlevelsof tension.Inaddition,
thepreservation ofmyocardial structural integrity was associ-ated with the maintenance ofresting and active tension values of overstretched muscles with time. Thus, NO salvaged the
myocardium from the detrimental impact of overstretching,
functionally andmorphologically.
Althoughit isdifficulttorecognize,atpresent, the effector pathwaythatcouples abnormal mechanical loads, enhanced
ex-pression of Fas, programmed cell deathand muscle restructur-ing,theobservations with NOreleasepoint to the induction of superoxideas arelevantfactorintheinitiation of this cascade of
events. Several cellular mechanisms can form reactive oxygen species, includingtheconversionof xanthine dehydrogenase to xanthine oxidase in reperfusion injury (31), mixed-function oxidase systems (32), and electron transport in the respiratory chain (33). Whether one or multiple cellular processes contrib-utedtomyocardial injury in the overstretchedpapillary muscle
remainstobedetermined. NO may alleviate oxidantstressand
therebyprotect fromapoptosis (34, 35). However,NO
adminis-tration markedly reduced the extent of tension development in the overloaded papillary muscle, and this negative inotropic effect may have contributed to diminish the damaging impact of a load of 50 mN/mm2 on the structure and function of myo-cytes. This reduction in muscle performance is consistent with observation in vivo (36-38) and in vitro (39, 40), indicating that NOreduces oxygen consumption and decreases myocardial contractility.
In conclusion, abnormal levels of resting tension appeared to be associated with the activation of the suicide program of myocytes and reduction in muscle mechanical performance. In addition, an architectural rearrangement of the myocyte com-partment occurred, and this phenomenon of restructuring, in combination with apoptotic cell death, may have contributed to depress the mechanical behavior of the myocardium. The intracellular formation of superoxide anion and the overex-pression of Fas protein in myocytes may have been the critical factors in the initiation of the cascade of events leading to programmed cell death and alteration in myocardial contractil-ity. NO seemed to protect from these detrimental effects of overstretching.
Acknowledgments
The experttechnical assistance of Maria Feliciano and the assistance of Dr. Pawel Kaminski in adapting the superoxide quantitationmethods are greatlyappreciated.
This work was supported by grants 38132, 39902, HL-40561,HL-50142, HL-53053, HL-31069, andPO-1-HL-43023from the NationalHeart, Lung and Blood Institute.
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