288

aB-Crystallin in Cardiac TissueAssociation With Actin and Desmin Filaments

Federico Bennardini, Antoni Wrzosek, and Michele Chiesi

aB-Crystallin is a 20-kd peptide highly homologous to the small heat-shock proteins. This protein formssoluble homomultimeric complexes (M, 300-700 kd) and is very abundant in cardiac muscle cells. In vitroexperiments (affinity column chromatography and binding studies with isolated proteins) have shown thataB-crystallin interacts directly with actin and, in particular, with desmin filaments. The immunocyto-chemical localization of aB-crystallin within the cardiomyocytes showed that the protein is distributedexclusively in the central region of the I bands (Z lines), where desmin is localized. In vitro studies havefurther shown that the binding affinity of aB-crystallin to actin and desmin filaments increasesconsiderably at slightly acidic pH (6.5) or after a heat treatment (45C). Moreover, aB-crystallin wasfound to prevent effectively the tendency of actin filaments to form aggregates (i.e., paracrystals) at acidicpH. These in vitro data suggest a protective role of aB-crystallin during stress conditions such as ischemiaof the heart. Crystallin could prevent the aggregation of filaments, which might occur during theacidification of the cytosol and lead eventually to irreversible structural damage. (Circulation Research1992;71:288-294)KEY WORDs * heart * ischemia * stress * aB-crystallin * desmin * actin

T he a-crystallins are major protein constituents ofthe lenticular tissue.12 Two forms of a-crystal-lin are known, namely, aA and aB, which are

encoded by two distinct genes.3 The two a-crystallinmonomers have a similar Mr (=20 kd) and show a highdegree of homology.4 They occur, in situ, as globularhomopolymers or heteropolymers of up to 1,000 kd. Inspite of their abundance in the cytoplasm of the lensfiber cells, these water-soluble proteins have eluded aclear definition of their function. Indirect evidencesuggests that they serve a structural role and that theirhigh concentration and ordered packing within the cellare required for the transparency of the lenses. Thepossibility of an interaction between eye lens actin,vimentin, and aB-crystallin was suggested as early as1979 by Kibbelaar et al,5 who observed that these threeproteins are copurified by DNAse affinity chromatogra-phy. After the recent discovery that aB-crystallin is alsoexpressed in a variety of nonocular tissues,6-9 the gen-eral interest on the a-crystallins has greatly increased.Also very intriguing was the proof of a striking sequencehomology between the a-crystallins and the mammaliansmall heat-shock proteins.10"11 The latter proteins alsoshare the same general secondary and tertiary struc-tural features of aB-crystallin and associate into bigsoluble spherical particles with a diameter of approxi-mately 15-20 nm. The highest concentration of aB-crystallin in nonocular tissues was found in the heartand slow-twitch muscles.7,9'12 Interestingly, in somepathological states, such as in brain tumors or during

From the Department of Research, Pharmaceuticals Division,CIBA-GEIGY Ltd., Basel, Switzerland.Address for correspondence: Dr. Michele Chiesi, Department

of Research, Pharmaceuticals Division, CIBA-GEIGY Ltd., CH-4002 Basel, Switzerland.

Received March 30, 1991; accepted March 25, 1992.

oncogenic transformation of cells in culture, the proteincan be expressed transiently in very high quantities.6"13In cardiac tissue the concentration was estimated to beapproximately 3-5% of the total soluble protein.9 Im-munocytochemical localization of aB-crystallin hasgiven a very strictly defined localization within thecardiomyocyte: only the central portion of the I bandsinteracts with immunospecific antibodies raised againstcardiac crystallin.'4 These observations suggest a spe-cific interaction of aB-crystallin with some element ofthe myofibrils or the cytoskeleton. In fact, preliminarydata have shown that aB-crystallin interacts weakly withactin and also that it is a major contaminant in cardiacdesmin preparations.14

In this study, the effect of isolated aB-crystallin onsome properties of purified desmin and actin prepara-tions was investigated, and a specific direct interactionwith these filaments could be clearly demonstrated. Ourexperiments have also addressed the question of thepossible role of aB-crystallin during the denaturationprocesses induced by stress conditions such as acidifi-cation or heat treatment.

Materials and MethodsChemicals

a-Crystallin from bovine eye lenses was from SigmaChemical Co., St. Louis, Mo.; cyanogen bromide-acti-vated Sepharose 4B and diethylaminoethyl-Sephacelwere from Pharmacia LKB Biotechnology, Uppsala,Sweden; and Centricon-10 microconcentrators werefrom the Amicon Division of W.R. Grace & Co.,Danvers, Mass. All reagents were of analytical grade.

Immunolocalization of aB-Crystallin in Cardiac CellsPolyclonal antibodies were obtained from rabbits

inoculated with aB-crystallin purified from rat heart as

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Bennardini et al QB-Crystallin in Cardiac Tissue 289

previously described by Longoni et al.14 Immunospecificantibodies were obtained by passing the whole immu-noglobulin G fraction through a column containingisolated aB-crystallin coupled to Sepharose 4B. Cardiacmyocytes were prepared from adult rat hearts as de-scribed by Chiesi et al.15 The cells were adsorbed onmicroscope glass slides immediately after preparation,fixed in 3.7% formaldehyde, and permeabilized with0.1% Triton X-100. Subsequent incubations with theimmunospecific antibodies (at a dilution of 1:10) andwith fluorescein isothiocyanate-labeled goat anti-rabbitantibodies (dilution 1:10) were carried out in 1% goatserum/phosphate-buffered saline medium. Slides werethen dehydrated in xylene, mounted, and analyzedusing a Zeiss Axiophot microscope. Details are given inLongoni et al.14

Actin PurificationSwine heart actin was prepared from acetone powder

as described in Pardee and Spudich.16 After two poly-merization-depolymerization cycles, an actin prepara-tion was obtained with a purity of more than 95% (asjudged by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis [SDS-PAGE]). Actin was stored in so-lution at 0°C as F-actin.

Desmin PurificationDesmin was purified from chicken gizzard as de-

scribed by Lazarides and Granger.17 For the final puri-fication of desmin, the acetone powder was extracted in1 M acetic acid. After neutralization, the precipitatethat formed was collected by centrifugation and redis-solved in 1 M acetic acid. This cycle was repeated threetimes. Two minor contaminations were commonly ob-served. The contamination displaying the highest mo-bility on gels (Mr, =42 kd) was identified as actin byimmunoreaction with anti-actin antibodies on immuno-blots (not shown). The other contamination (Mr, -47kd) was probably a desmin degradation product, since itcross-reacted with anti-desmin antibodies (not shown).Only preparations containing at least 90% desmin werechosen for the binding experiments. After a final solu-bilization step in 1 M acetic acid, desmin was allowed topolymerize into filaments by dialysis against water.

caB-Crystallin PurificationaB-Crystallin was purified from an aA- and aB-

crystallin mixture by ion exchange chromatography,basically as described by Schoenmakers et al.18 Briefly, acolumn (20x 1.6 cm) was packed with diethylamino-ethyl-Sephacel and equilibrated with two bed volumesof 5 mM Tris (pH 9.0) containing 6 M urea. A sample of20 mg a-crystallin was dissolved in 2 ml equilibrationbuffer and applied to the column. Resolution of pro-teins was achieved by a linear gradient elution systemconsisting of 70 ml of 5 mM Tris and 6 M urea (pH 9.0)and 70 ml of the same buffer supplemented with 150mM NaCl. Elution was carried out at 4°C at a flow rateof 15 ml/hr. The effluent was monitored at 280 nm witha Uvicord photometer. This slightly modified elutionprotocol allows the complete separation of the twoisoforms: aB-crystallin eluted from the column between100 and 150 mM NaCI, whereas aA-crystallin remained

concentrated 10-20 times by Centricon-10, and analyzedby SDS-PAGE. After dialysis, the isolated aA- andaB-crystallins occurred as globular soluble homomulti-mers (Mr, 300-700 kd) as judged from their sedimenta-tion properties after sucrose gradient centrifugation.9

Actin-Affinity Column ChromatographyG-Actin (5 mg) was coupled to 1 g cyanogen bro-

mide-activated Sepharose 4B. After blocking the non-reacted residues by 1 M ethanolamine (pH 8.0), twocolumns were prepared and equilibrated with three tofive bed volumes of 20 mM MOPS and 100 mM KCl atpH 7.5 (buffer A) and pH 6.5 (buffer B). aB-Crystallin(200 ,ug) was dissolved in 500 ,nl buffers A and B,respectively, and loaded on the column. Samples wereallowed to incubate for 30 minutes at 4°C beforewashing and elution. Unbound protein was washed outwith 10 ml buffer A or B, and protein bound to thecolumns was removed with 10 ml of 3 M guanidinehydrochloride (pH 8.0) containing 0.5 M sodium ace-tate, 1 mM CaCl2, and 30% glycerol. Pooled sampleswere dialyzed overnight against 5 mM MOPS (pH 7.5),lyophilized, and analyzed by SDS-PAGE on 12% gel.

Binding ExperimentsDesmin filaments, aB-crystallin, and G-actin were

dialyzed for 2 days at 0°C against an actin depolymeri-zation buffer (2 mM Tris-HCl [pH 8.0] at 25°C, 0.2 mMATP, 0.2 mM CaCl2, 0.5 mM 2-mercaptoethanol, and0.005% NaN3) and then used immediately in the bind-ing experiments. Appropriate amounts of the proteinswere mixed in a buffer containing 50 mM HEPES, 1mM ATP, 2 mM MgCl2, and different concentrations ofKCl (from 0 to 200 mM). Samples were incubated for 30minutes at various temperatures and at various pHvalues (from pH 6 to 8). Aggregated desmin and/oractin filaments were then removed by centrifugation at3,000g for 10 minutes. Sedimentation of single actinfilaments was achieved by centrifuging for 30 minutes at150,00g in a tabletop centrifuge (model TL-100, Beck-man Instruments, Inc., Fullerton, Calif.). This differen-tial centrifugation procedure also sedimented the por-tion of crystallin that was bound to either theaggregated filaments and/or the actin filaments. Thesupernatants were collected and the pellets resus-pended in the same initial volume; both fractions wereanalyzed by SDS-PAGE and quantified by densitomet-ric scanning. In the absence of desmin and/or actin, nochange in the solubility characteristics of crystallincould be observed under the experimental conditions ofpH, temperature, and ionic strength that were tested.After high-speed centrifugation (i.e., 30 minutes at150,OOOg), all crystallin remained in the supernatant.

Other MethodsSDS-PAGE electrophoresis was performed according

to Laemmli.19 Protein determination was carried outaccording to Bradford20 using bovine serum albumin asa standard.

ResultsInteraction Between Desmin Filaments andaB-Crystallin

bound to the resin. Appropriate fractions were pooled,dialyzed overnight against 20 mM MOPS (pH 7.5),

In a previous study, preparations of cardiac desminwere found to be contaminated by aB-crystallin. 14 This

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A C

S P S P S P

00C 370C 450CB

S p S P S P

00C 37°C 450C

-- _ _-Actin

S P S P S P

00C 37tC 4 50CD

FIGURE 1. Sodium dodecyl sulfate-polyacrylamidegel electrophoresis showing binding of aB-crystallin to

aoB desmin and actin. Equimolar amounts (12 ,uM) ofdesmin or actin were incubated for 30 minutes atdifferent temperatures with aB-crystallin. Desmin andactin filaments were separated from the soluble frac-tion by centrifugation. Supernatants (s) andpellets (p)were then analyzed by gel electrophoresis. The pH ofthe incubation buffer was 6.0 (panels A and C) or 7.5

sActin (panels B and D). Details are given in "Materials andMethods." Note that the interaction of aB-crystallinwith the actin and desmin filaments was promoted by

ac B higher temperature and acidification.

S p s p s p

00C 37"C 45°C

observation suggested a possible interaction betweenthe two proteins. To prove this hypothesis, the twoproteins were isolated, and their interaction was studiedby means of a centrifugation procedure. Chicken giz-zard preparations of desmin (which are free of endog-enous crystallin) were used for these studies. Themedium that was used to investigate the binding be-tween the isolated aB-erystallin and desmin filamentscontained ATP, MgCl2, and KCI to mimic cytoplasmicconditions. The incubation in the binding buffer in-duced a very rapid aggregation of the desmin filaments,which could then be quantitatively sedimented by low-speed centrifugation. In the absence of desmin, aB-crystallin could not be sedimented even by high-speedcentrifugation (150,000g for 30 minutes) at any condi-tion of pH, temperature, or ionic strength used in thebinding experiments. Thus, the amount of aB-crystallinsedimenting at low-speed centrifugation after incuba-tion with desmin corresponded to the desmin-boundportion. Figure lB shows a typical experiment carriedout at pH 7.5. At 37°C (i.e., at physiological tempera-ture), a consistent amount of aB-crystallin interactedwith desmin. Approximately half of the crystallin was

recovered in the pellet. A slight increase in temperaturefrom 370 to 45°C during the 30-minute incubation of thetwo proteins in the binding buffer (heat treatment)clearly potentiated the interaction, and more crystallincosedimented with the desmin aggregates (Figure IB).A slight acidification of the binding buffer also had avery strong effect on the interaction. Figure 1A showsthat at 37°C aB-crystallin could be pelleted quantita-tively when the pH was lowered to 6.0. A semiquanti-tative analysis of the effect of temperature and pH onthe interaction between desmin and crystallin is given inFigure 2. The affinity of the interaction increasedsharply at pH values below 7.0 (see Figure 2A). Theinteraction between the two proteins also became stron-ger as the temperature increased (Figure 2B). Thetemperature dependence showed a sharp transition atapproximately 40°C. In the absence of desmin, heattreatment or acidification did not affect the solubility ofcrystallin (not shown). In the experiment presented inFigure 3, the stoichiometry of the binding reaction was

investigated. A fixed amount of desmin (3 zM) was

incubated with increasing amounts of crystallin, and theamount of the latter protein coaggregating with desminat pH 6.0 was determined. Figure 3 shows that thedesmin-bound crystallin reached saturation at a levelcorresponding to approximately five to seven crystallinmonomers per desmin monomer.

Interaction Between Actin Filaments andaB-Crystallin

Actin affinity chromatography was carried out usingisolated aB-crystallin. Figure 4 shows that at pH 7.5 theinteraction between crystallin and actin was weak. Lessthan 20%3 of the crystallin applied to the column (total,

100

80

r-

a.. 0

'U 40

c

X 4

E 20

O -

i.

A

--0 8-.5 B.0

B

6.5 7.0 7.5 8.0 6.5

pH

100 -

80 -

JC

,, 60

X0

E 20-10

T-T- I T

0 10 20 30 40 50 60

Temperature ["C]FIGURE 2. Graphs showing the effect oftemperature andpHon the affinity of aB-crystallin to desmin. Desmin andaB-crystallin were mixed at a concentration of 10 gM undervarious conditions ofpH (panel A) or of temperature (panelB). After 30 minutes, the mixtures were centrifuged (3,000gfor 10 minutes), andpellets and supernatants were analyzed bysodium dodecyl sulfate-polyacrylamide gel electrophoresis.Aggregation ofthe two proteins increased sharply atpH valuesbelow 7.0 and at temperatures above 40°C.

Desmi n

aB-

Desmin_

_0 -_m I_

_ _ _

0

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Bennardini et al aB-Crystallin in Cardiac Tissue 291

7

6

.~ 0-RC o..

0 Cu.0 _

c L-

= cdM C)

.E=COCD-o

5

4

3

2

S P S P S P

a -

0 2 4 6 8 10 12

crystallin luMJFIGURE 3. Graph showing stoichiometry of crB-crystallinbinding to desmin at acidic pH. Desmin (3 gM) was incu-

bated at 37°C (pH 6.0) in the presence of various amounts ofaB-crystallin. The amount of aB-crystallin coprecipitatingwith desmin was quantified by laser scanning of the proteinbands after electrophoretic separation, and the molar ratio ofthe two proteins was calculated.

200 gg) was retained by the actin column (2-3 mg

bound actin). A slight decrease in pH induced a largeincrease in the binding affinity between the two proteins(Figure 4): at pH 6.5 crystallin was quantitatively re-

tained by the actin column.Qualitatively similar results were also obtained by

using the differential centrifugation procedure describedabove. F-actin, however, could not be sedimented by thelow-speed centrifugation procedure, which removed onlyaggregated material. Therefore, to study the interactionof aB-crystallin with F-actin, a high-speed centrifugation

Actin-affinity column chromatography

C

-0

100

90

80

70

60

s0

40

30

20

10

0

E1 unboundE3 bound

pH 7.5 pH 6.5 controlpH 6.5

FIGURE 4. Bar graph showing binding of aB-crystallin toactin-Sepharose 4B column at different levels of pH. aB-Crystallin (200 pg) was loaded on an actin column and elutedas described in "Materials and Methods. " As a control, a

Sepharose 4B column with the reactive groups blocked byethanolamine was used. On the ordinate, 100% correspondsto the total amount ofprotein loaded on each column. Emptybars represent unbound crystallin; full bars represent crystallinbound to the column.

e Actin

* aBCA

Control +aB +AFIGURE 5. Sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis showing the effect of cvA- and aB-crystallin onactin aggregation atpH 6.0 at low ionic strength. F-actin (10gM) was incubated at 37°C at pH 6.0 in the absence ofKCI.The relative amount of F-actin remaining in solution (S) oraggregated (P) was determined after low-speed centrifugation(control). The same experiment was carried out in thepresence of equimolar amounts of aB- and aA-crystallin(+caB and +cxA, respectively). Whereas cdB-crystallin pro-moted aggregation, cxA-crystallin kept all F-actin in solution.

step was introduced. Figure 1D shows the results ob-tained after incubation of the two proteins at pH 7.5:about half of the caB-crystallin interacted with F-actinand cosedimented with it. A decrease in pH induced aclear potentiation of the interaction (see Figure 1C), inaccordance with the results obtained with the actinaffinity chromatography column (see Figure 4). In theabsence of actin, aB-crystallin could not be sedimentedby this centrifugation step under any of the experimentalconditions investigated.The binding of aB-crystallin to myofibrils isolated

from cardiac tissue was also investigated using a similarapproach. A strong association between aB-crystallinand myofilaments could be observed, in particular atslightly acidic conditions (not shown).

Effect of aB-Crystallin on F-Actin andDesmin AggregationTo get an insight into the pathological and physiolog-

ical significance of the binding of aB-crystallin to fila-ments, the effect of aB-crystallin on the aggregationcharacteristics of actin was investigated. Under specificexperimental conditions, such as in the presence of highconcentrations of divalent cations and/or at acidic pH,F-actin has the tendency to aggregate and formparacrystals.21 22 The proportion of F-actin aggregatingafter 30 minutes of incubation at 37°C at slightly acidicpH was determined after separation by slow-speedcentrifugation. Figure 5 illustrates the singular effect ofthe crystallins on the acid-induced aggregation of F-ac-tin at low ionic strength (pH 6.0). Whereas aB-crystallinclearly potentiated the formation of aggregates and wasquantitatively recovered in the pellet after low-speedcentrifugation, the highly homologous aA-crystallin iso-form very effectively prevented aggregation.

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100

80

M control+ cc B crystallin

60

40

CD

CD

CD 20CU

0

0 10 30 60 100 130 200

KCI (mM)

FIGURE 6. Bar graphs showing the effect of KCI on thepH-induced formation of actin paracrystals in the absenceandpresence of aB-crystallin. F-actin (10 ,M) was incubatedat 37°C in the presence of various KCl concentrations.Low-speed centrifugation (3,000gfor 10 minutes) was carriedout to sediment the aggregated actin filaments, and the pelletswere quantified. The same experiment was carried out in thepresence of an equimolar concentration (10 ,uM) of aB-crystallin (+aB crystallin). The latter protein promoted ag-

gregation of F-actin at low ionic strength but prevented it at

physiological ionic strength.

Figure 6 shows that the effect of aB-crystallin on

actin aggregation (pH 6.5) depended on the ionicstrength of the medium. At low ionic strength, aB-crystallin potentiated the formation of paracrystals so

that all actin present aggregated and precipitated to-gether with aB-crystallin. On the other hand, at an

almost physiological ionic strength (a condition yieldingmaximal acid-induced aggregation of F-actin; see Figure6), the presence of aB-crystallin in the medium inhib-ited the formation of F-actin aggregates. A quantitationof the protective effect of both aA- and aB-crystallin on

the aggregation of F-actin at physiological ionic strengthis given in Table 1.At physiological ionic strength, desmin filaments

spontaneously and quantitatively aggregated even at pH

TABLE 1. Actin Aggregation (10 ,uM) at pH 6.5 in 50 mMHEPES Buffer Containing 2 mM MgC12 and 200 mM KCI

Crystallin

Control 10l M aB 10 ,uM aA

Actin aggregation 12.5+1.1 4.2+1.6* 2.6+0.9*(AU)

n 7 5 3

AU, arbitrary units; n, number of experiments. Values aremean±+SEM.The proportion of F-actin aggregating after 30 minutes of

incubation in the presence, where indicated, of aA- or aB-crystallin was determined by gel electrophoresis and densitometryscanning. All the values are corrected for the amount of actin thataggregates at pH 7.5 (usually <5% of the total actin).

*p<0.01 vs. control.

7.5 (see Figures 1A and 1B). aB-Crystallin also effec-tively inhibited the aggregation of desmin filaments. Thepresence of an excess of aB-crystallin (a 4:1 molarratio) maintained in solution more than 50% of thedesmin filaments (not shown).

Intracellular Localization of arB-Crystallinin Cardiomyocytes

Immunocytochemical localization at the light micros-copy level revealed that aB-crystallin occurs in verydiscrete regions of the cardiomyocytes. Figure 7A showsthe typical striations of a rat cardiomyocyte as seen in aphase-contrast microscope. Sharp dark lines cross thecell with a periodicity of approximately 1.8 ,um, corre-sponding to the sarcomere length of partially contractedcardiomyocytes. Between the sharp dark striations (Zlines), a more diffuse and discontinuous dark region canalso be recognized. The same cell visualized with epi-fluorescence optics shows the exclusive localization ofcrystallin on continuous sharp striations (see Figure7B). The striations obtained with anti-crystallin anti-bodies are very thin and occupy approximately 24±4%of the sarcomere length. Figure 7C was obtained byexactly superimposing the two images (Figure 7A wasobtained with phase-contrast optics, and Figure 7B wasobtained by epifluorescent optics) of the same cell. Thestriations obtained by immunostaining matched with thesharp dark striations corresponding to the Z lines. Theimmunospecific antibodies used in this study werehighly specific for aB-crystallin. No cross-reactivitycould be observed on Western blots with a- and y-actin,a-actinin, desmin, and myosin (not shown).

DiscussionIntracellular Localization of aB-CrystallinThe intracellular localization of aB-crystallin in car-

diomyocytes has very unusual characteristics. In spite ofits water solubility, the protein is found exclusively inthe central region of the I band, as demonstrated by thethin striations obtained after immunocytochemical dec-oration (see Figure 7). To explain this specific intracel-lular distribution, one has to assume that crystallininteracts with an ordered structure present in thisregion of the cardiac cell. Actin, a-actinin, and desminfilaments are the most likely candidates for the interac-tion. Desmin filaments and a-actinin are localized at thelevel of the Z line, i.e., in the center of the I band, andprevious immunocytochemical studies have shown thatsimilar striations at the level of the I band are obtainedusing anti-a-actinin and anti-desmin antibodies.23 Eventhough actin filaments are present in the I bands as wellas in the A bands, the use of anti-actin antibodies resultsin a selective decoration of the I band. This is due to thefact that only the portion of actin close to the Z line(i.e., in the I region) interacts with the anti-actinantibodies, whereas actin bound to myosin in the Abands is not reactive.23 In spite of their similarity, thestriations obtained with anti-actin, anti-a-actinin, andanti-desmin antibodies can be clearly differentiated by acloser investigation. Whereas the desmin cross-stria-tions of the myocyte are continuous, actin and a-actininstriations are characterized by interruptions at regularintervals corresponding to the bundles of the myofila-ments. In addition, desmin and actin striations in car-

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A t lA

FIGURE 7. Immunocytochemical localization of cardiac cxB-crystallin in rat heart myocytes. Adult rat heart myocytes were fixedand incubated first with anti-cardiac crystallin antibodies and then with fluorescein-labeled secondary antibodies as described in'Materials and Methods." The same cell is observed either by phase-contrast light microscopy (panel A) or by fluorescencemicroscopy (panel B). Note the thin crystallin striations crossing the cell laterally without interruptions. In panel C, the exactsuperposition ofthephotographs presented in panelsA and B is illustrated Note that the crystallin striations correspond to the Z lines.

diac cells have a different thickness. Actin striationsoccupy the whole I-Z-I region (corresponding to ;50%of the whole sarcomere length). On the other hand,staining of desmin, which is localized only on the Z line,gives a finer striation whose thickness is less than onethird that of the sarcomere.23 After immunocytochemi-cal staining, the striations generated by aB-crystallinantibodies were found to be continuous, crossing thecell laterally without interruptions (see Figure 7B).Moreover, the striations obtained with anti-crystallinantibodies are very thin and occupy only approximately24±4% of the sarcomere length. Therefore, the immu-nolocalization data clearly suggest that aB-crystallininteracts with desmin rather than actin filaments ora-actinin in situ.The in vitro binding experiments have shown a very

high stoichiometry of binding between aB-crystallinand desmin: saturation occurs only when approximatelyeight crystallin monomers have bound to one desminmonomer. The interpretation of this stoichiometry isnot straightforward, since desmin and crystallin occuras supramolecular structures. Crystallin moleculesspontaneously associate to form water-soluble globularclusters with an M, of approximately half a million,whereas desmin monomers associate in long filamentswith an M, of several million. Thus, the high stoichiom-etry of binding indicates that crystallin globules do notbehave as capping proteins, which interact only with theends of the filaments. It is more likely that a largenumber of caB-crystallin globules bind to the sides of thefilaments, in analogy to the beaded filaments previouslyobserved in cell cultures from the lenticular tissUe.24

Possible Protective Role of caB-CrystallinaB-Crystallin is not recovered with the insoluble

components of the cell but is released into the water-soluble fraction upon homogenization of the heart.9This indicates that the interaction between desmin andaB-crystallin in situ is fairly weak under normal condi-

tions, thus allowing the release of the protein into thesoluble fraction, while desmin filaments are pelletedwith the nonsoluble matrix of the cell. Interestingly,however, a short period of global normothermic isch-emia of the heart induces a drastic redistribution ofcaB-crystallin in the cell homogenate: the protein aggre-gates with the insoluble elements of the cell.25 Thus,during the stress situation (i.e., ischemia), the affinity ofaB-crystallin for some structural elements of the cellincreases. The in vitro studies presented in this investi-gation clearly show that a slight acidification (a phenom-enon known to occur very early in the cytosol of cardiaccells during an isehemic episode26) is enough to inducean important increase in the binding affinity of aB-crystallin to desmin and actin filaments. Thus, during anisehemic episode, it is possible that caB-crystallin bindsmore strongly to desmin filaments in the Z line. Theprotein might undergo a redistribution in the cardiaccell and bind also to F-actin (either isolated or associ-ated to the myofilaments). In the latter case one couldexpect a reduced capacity of the contractile filaments toslide and contract. This hypothetical phenomenon couldplay a role in the genesis of the stunned myocardium,i.e., the condition of a temporarily impaired contractileperformance of the heart when reperfused at the end ofan ischemic episode.The most interesting questions about cardiac crystal-

lin, of course, concern its possible (patho)physiologicalrole. Unfortunately, the information on its characteris-tics in situ in healthy and pathological states is verylimited. For the time being, one must be content with ahypothesis based on in vitro data and on the strikinghomologies that crystallin shares with the small heat-shock proteins and chaperonins. Recently, a small heat-shock protein (Mr, -.25 kd) -highly homologous toaB-crystallin-was identified in smooth muscle.27 Sincethe protein was found to inhibit actin polymerization, itwas suggested that its overexpression is involved (byfacilitating F-actin depolymerization) in the reorganiza-

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tion of the cytoskeleton observed after stress induction.In our present study, the effect of aB-crystallin on someproperties of actin was investigated to unveil any anal-ogous protective function. In preliminary experiments,we could not detect any gross effect of aB-crystallin onthe polymerization kinetics of F-actin (not shown). Onthe other hand, it was observed that aB-crystallinaffects the tendency of actin filaments to aggregate atacidic pH: aB-crystallin either favors (at low ionicstrength) or prevents (at physiological ionic strength)F-actin aggregation. The behavior observed at low ionicstrength, although interesting, is likely to be irrelevantunder physiological as well as pathological conditions ofthe heart. (Even though a K' loss during the reversiblephase of ischemia occurs, its extent is limited to 10-20mM28 and is partly compensated by an increase in theconcentration of other electrolytes.) On the other hand,the prevention by aB-crystallin of desmin and actinfilament aggregation, which was observed at physiolog-ical ionic strength, could have relevant physiopatholog-ical implications. In fact, an uncontrolled acid-inducedaggregation of filaments, which might occur during anischemic episode, could contribute to the onset of theirreversible morphological collapse of the cell that isobserved after prolonged ischemia. A major effect of aheat shock (and probably also of other stress situations)is an unfolding (or an unproper folding) of cellularproteins29 and, in particular, of the intermediate fila-ment network. This unfolding could also cause theaggregation of the filaments. The interaction with crys-tallin could stabilize the proper conformation of thevarious filaments (in analogy to the function of thehomologous small heat-shock proteins named chaper-onins) and protect them from rupture by mechanicalshear and from degradation by oxidative or proteolyticprocesses and thus prevent aggregation. In conclusion,aB-crystallin could play a role in the defense mecha-nisms of the cardiac cell threatened by stress situations.

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F Bennardini, A Wrzosek and M ChiesiAlpha B-crystallin in cardiac tissue. Association with actin and desmin filaments.

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