166

BRIEF COMMUNICATIONS

Release of Unassembled Rat Cardiac Myosin LightChain 1 Following the Calcium Paradox

Allen M. Samarel, Alan G. Ferguson, Richard S. Vander Heide, Richard Davison, andCharles E. Ganote

From the Departments of Medicine (Cardiology) and Pathology, Northwestern University Medical School, Chicago, Illinois

SUMMARY. To determine the intracellular source and release kinetics of myosin light chain 1immediately following irreversible myocytic injury, we perfused rat hearts in a Langendorffapparatus under control conditions (20 minutes), or during global cellular injury produced byoxygenated, calcium-free perfusion (5 minutes), followed by reperfusion with buffer containing2.5 mM calcium (15 minutes). Light chain 1 concentration (double antibody radioimmunoassay)and creatine kinase activity were measured in both the coronary effluent and the 140,000 gsupernatant extract of perfused ventricular tissue (after homogenization and ultracentrifugation).Calcium reperfusion caused the rapid release of both light chain 1 and creatine kinase activity(peak light chain 1 = 1.09 ± 0.19 Mg/& peak creatine kinase = 74.9 ± 10.7 IU/g at 1 minute,mean ± SD, n = 3); 28.5 ± 13.5% of total light chain 1 and 86.5 ± 0.6% of total creatine kinaseactivity were depleted from the tissue extract during the 15-minute reperfusion. No light chain 1or creatine kinase was detected in the effluents of control-perfused hearts. Dodecyl sulfatepolyacrylamide gel electrophoresis and immunodetection with specific antibody to myosin heavychain and light chain 1 showed that the effluent light chain 1 was of similar molecular weight(mol wt = 27,000) to the subunit bound to myofibrils. In addition, light chain 1 was released inthe absence of myosin heavy chain. Thus, a small soluble pool of unassembled myosin light chain1 subunits exists in the cytoplasm of cardiac myocytes that is released from irreversibly injuredcells. This pool demonstrates initial washout kinetics similar to creatine kinase. (Circ Res 58:166-171, 1986)

CARDIAC myosin light chains are low molecularweight polypeptide subunits of the myosin moleculethat can be detected by radioimmunoassay in theserum of patients and experimental animals follow-ing acute myocardial infarction (Khaw et al., 1978;Gere at al., 1978, Nagai et al., 1979, Katus et al.,1982). The appearance and disappearance of thesestructural proteins in the serum following irreversi-ble ischemic cellular injury differ from that of cyto-solic creatine kinase MB and myoglobin in thatserum levels of both cardiac myosin light chain 1(LCI) and light chain 2 (LC2) rise early after theonset of infarction and remain elevated for severaldays after injury (Trahern et al., 1978; Nagai et al.,1980; Katus et al., 1984). The cellular mechanismsresponsible for both the early and sustained releaseof myosin light chains from irreversibly injuredmyocardium are not known with certainty. Nagai etal. (1979, 1980, 1981), Gere et al. (1978), and Khawet al. (1978) all have suggested that myosin lightchain subunits are initially released after irreversibleischemic injury from an unassembled intracellularlight chain pool. The earlier observations of Horvathand Gaetjens (1972), Morkin et al. (1973), and Zak

et al. (1977) lend support to the existence of asarcoplasmic pool of myosin light chains in bothrabbit skeletal muscle and rat heart myocytes. Pre-sumably, sarcolemmal damage produced early inthe course of ischemic injury leads to the releaseinto the circulation of these unassembled lightchains, along with other cytosolic components ofthe myocyte (CK-MB, LDH, myoglobin, etc.). Sub-sequent continuous release of myosin light chains(noncovalently bound to myosin heavy chain withinmyofibrils) occurs because of acidic dissociation(Smitherman et al., 1980) or because of proteolyticdegradation of myofibrils (Nagai et al., 1980).

With the availability of specific antisera for bothLCI and myosin heavy chain, and experience witha well-characterized model for the rapid productionof global myocytic cellular injury (i.e., the calciumparadox in isolated perfused rat heart), we under-took the present investigation to provide additionalproof for the existence of a soluble pool of unassem-bled LCI within the sarcoplasm. Furthermore, wedemonstrate that this soluble LCI pool is rapidlyreleased from perfused rat heart following sarcolem-mal disruption produced by the calcium paradox.

by guest on May 8, 2018

http://circres.ahajournals.org/D

ownloaded from

Samarel et al./Release of Myosin Light Chain 1

Methods

ReagentsSodium [l2iI]iodide was obtained from Amersham, goat

anti-guinea pig IgG and horseradish peroxidase-conju-gated rabbit anti-guinea pig IgG were obtained from Cap-pel, and nitrocellulose sheets (0.45 fim) were a product ofBiorad. All other reagents were of the highest grade com-mercially available and were obtained from Sigma Chem-ical Co., and Scientific Products.

Experimental AnimalsMale Sprague-Dawley rats (250-300 g) were obtained

from Harlan Industries. Male English short-haired guineapigs (700-800 g) were obtained from Scientific Animals.

Heart PerfusionRats were anesthetized by intraperitoneal injection of

sodium pentabarbital. Hearts were removed and cannu-lated via the aorta to a double-reservoir Langendorff ap-paratus, as previously described (Ganote et al., 1984).Perfusion was at 37°C at a pressure of 8-10 pKa. Coronaryflows were measured by timed collections of effluents,and aliquots were collected for subsequent analysis of CKactivity and LCI content by radioimmunoassay. Controlperfusion medium was a standard Krebs-Henseleit-bicar-bonate buffer consisting of NaCl, 118; NaHCO3, 25; KC1,4.7; KH2PO4, 1.2; CaClj, 2.5; and glucose, 11 mM. Cal-cium-free medium was prepared by exclusion of calciumwithout ionic substitution but with inclusion of 100 ^Methylenediaminetetiaacetic acid (EDTA) to ensure removalof residual calcium contaminants. All solutions were con-tinuously gassed with 95% O2, 5% CO2. All hearts wereperfused with control medium for 15 minutes before theexperiments were initiated.

Tissue Homogenization and ExtractionHearts were immersed in 150 mM NaCl (4°C), and the

ventricular muscle was trimmed of blood vessels, valvularstructures, and atria. The tissue then was homogenized (4bursts of 15 seconds, with a Polytion homogenizer) in 25volumes of 10 mM Tris-HCl buffer 7.4, containing 250 mMsucrose, 10 mM ethyleneglycol-bis-(j9-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), and 1 mM 2-mercaptoeth-anol. The homogenate was subsequently centrifuged at140,000 g for 60 minutes. The supernatant extract thenwas removed for subsequent analysis.

Protein and Creatine Kinase AssaysCreatine kinase activity in aliquots of perfusion ef-

fluents and rat heart extracts was assayed with a Gilford/mode! 3402 automatic analyzer and Worthington re-agents. Protein concentration was determined by themethod of Lowry et al. (1951).

Purification of Myosin Light Chain 1In preparing standards and tracer for LCI radio-

immunoassay, we purified myosin from human ventricu-lar muscle (obtained at autopsy) by the method of Wik-man-Coffelt et al. (1973). Light chains were dissociatedfrom myosin heavy chain by denaturation in 5 M guani-dine-HCl at 25°C, followed by ethanol:water (4:1) precip-itation at 4°C to remove myosin heavy chain (Khaw et al.,1978). The supernatant solution containing light chains aswell as trace protein contaminants was dialyzed against

167

water, concentrated by ultrafiltration, and freeze-dried.LCI then was completely separated from LC2 and othertrace protein contaminants by molecular exclusion high-pressure liquid chromatography (HPLC), using a Sphero-gel-TSK 2000 SW column (7.5 X 30 mm) equilibrated with10 mM sodium phosphate buffer, pH 6.8, containing 150mM NaCl. Flow rate was 0.4 ml/min at a pressure of 800psi.

Radioiodination of Light Chain 1Highly purified LCI (2.5 jig) was radioiodinated by the

chloramine T method of Greenwood et al. (1963) using1 mCi of sodium [125I]iodide. Labeled protein was sepa-rated from unincorporated isotope, as previously de-scribed (Samarel et al., 1981). Specific radioactivities ofthe tracer ranged from 60-120 ^Ci/jig protein.

Preparation of Antisera to Cardiac Myosin LightChain 1 and Myosin Heavy Chain

In preparing antisera to LCI, LCI was completely sep-arated from LC2 (and other minor protein contaminantsin the freeze-dried light chain preparation) by sodiumdodecyl sulfate (SDS) polyacrylamide gel electrophoresis(Laemmli, 1970) using 12.5% vertical slab gels. Proteinswere stained with Coomassie brilliant blue. Gel bandscontaining LCI (mol wt = 27,000) were cut from the slabs,emulsified with complete Freund's adjuvant, and injectedintramuscularly and subcutaneously into guinea pigs. Theanimals received injections on days 0, 7, 28, and 59 of400-500 n% of purified LCI. The antibody response wasmonitored by enzyme-linked immunoadsorbent assay(ELISA) in which purified LCI was "dot-blotted' or elec-trophoretically transferred from SDS-polyacrylamide gelsto nitrocellulose sheets (Towbin et al., 1979) and exposedto diluted antiserum from test bleeds. Primary antibodywas detected following incubation with peroxidase-con-jugated rabbit anti-guinea pig IgG. 4-Chloro-l-naphtholwas used for color development in the sandwich assay.

Antiserum to myosin heavy chain was prepared in asimilar fashion. Myosin was purified from rabbit ventric-ular muscle, and the heavy chain (mol wt = 200,000) wasseparated from the light chains by SDS-polyacrylamidegel electrophoresis on 7% vertical slab gels. After staining,gel bands containing heavy chain were excised and emul-sified in complete Freund's adjuvant. Guinea pigs receivedinjections on days 0, 7, 28, and 38 of 400-500 /ig ofpurified protein. The antibody response was monitored asdescribed above.

Radioimmunoassay of Light Chain 1A double-antibody radioimmunoassay was employed

to measure the concentration of LCI in rat heart extractsand perfusion effluents. Guinea pig antisera to LCI wasfirst titrated by incubating serial dilutions of antiserum[diluted in 50 mM sodium phosphate buffer, pH 7.4,containing 150 mM NaCl and 1% (wt/vol) of bovine serumalbumin] with a constant amount (5000 counts/min) ofl25I-labeled LCI. The dilution capable of approximately50% maximum binding (after precipitation of tracer-anti-body complexes with goat anti-guinea pig IgG) was usedin the radioimmunoassay.

In the final radioimmunoassay mixture, 100 y.\ of di-luted antiserum, 50 fi\ of 12!I-labeled LCI (5000 counts/min), and 500 /il of LCI standard (0-400 ng/ml of thehighly purified LCI preparation obtained followingHPLC) or unknown (perfusion effluent or rat ventricular

by guest on May 8, 2018

http://circres.ahajournals.org/D

ownloaded from

168 Circulation Research/Vo/. 58, No. 1, January 1986

muscle extract) were incubated in duplicate at 4°C for 24hours. Antibody-bound LCI was separated from free an-tigen by adding goat anti-guinea pig IgG (50 1̂) andnonimmune guinea pig serum (5 fi\). After incubation at4°C for an additional 24 hours, the immunoprecipitateswere removed by cenrrifugation (1500 g, 15 minutes) andwere counted for 125I radioactivity in an LKB model 1275Minigamma Counter. Nonspecific binding of labeledtracer (determined by incubating 125I-labeled LCI andstandard or unknown in the absence of primary antibody)was determined, and subtracted from each value of countsbound. LCI concentration of unknowns was read from astandard competitive binding curve of log LCI concentra-tion vs. B:BO (where B:BO is the ratio of precipitatedradioactivity for the standard solutions of LCI to the 0concentration standard). Data were expressed as nano-grams of LCI per milliliter of unknown solution.

Immunoelectrophoretic Detection of Myosin LightChain 1 and Myosin Heavy Chain

Tissue homogenates, resuspended myofibrillar sedi-ments, and 140,000 g supernatant extracts (50-100 jtgprotein) of control hearts and hearts subjected to thecalcium paradox were analyzed by SDS-polyacrylamidegel electrophoresis. In addition, an ulrrafiltration concen-trate of the peak fraction of LCI release following thecalcium paradox (50 ng protein) was applied to 7-17%vertical gradient slab gels. After electrophoresis, the sep-arated proteins were either stained with Coomassie bril-liant blue, or were electrophoretically transferred to nitro-cellulose sheets (Towbin et al., 1979). LCI and myosinheavy chain then were detected by incubating the elec-troblots (1 hour, 25°C) with specific antisera to either LCIor myosin heavy chain. Primary antibody binding thenwas demonstrated by incubation (1 hour, 25°C) withperoxidase-conjugated rabbit anti-guinea pig IgG. 4-Chloro-1-naphthol was used for color development forperoxidase.

Results and DiscussionPurification of Myosin Light Chain 1 Used forRadioiodination and Standards in theRadioimmunoassay

Molecular exclusion HPLC of the freeze-driedhuman myosin light chain preparation yielded mul-tiple peaks, all containing LCI (Fig. 1). Protein elut-ing with a retention time of 15-17 minutes (fractionII) demonstrated a single band (mol wt = 27,000)free of LC2 (mol wt = 20,000) and other higher andlower molecular weight protein contaminants whenanalyzed by SDS-polyacrylamide gel electrophoresis(Fig. 1). The purified LCI from several HPLC sepa-rations was pooled and concentrated by ultrafiltra-tion for subsequent use in radioiodination and asstandards in the radioimmunoassay.

Specificity of Antisera to Myosin Light Chain 1and Myosin Heavy Chain

Immunization of guinea pigs with human cardiacLCI (extracted from SDS-polyacrylamide gels) pro-duced a nonprecipitating IgG antibody highly spe-cific for LCI. This antibody was completely cross-reactive with LCI isolated from human skeletal

.04 r

.03

o00

QO

.01

-V I I I I I I .

0 2 4 6 8 10 12 14 16 18 20 22 24TIME (min)

m'a HPLC FRACTION

I I in IZ STD

't

9 2

69

46

3 0

I I

12,4

FIGURE 1. Purification of myosin light chain 1 (LCI). Myosin waspurified from human left ventricle. Myosin light chains were disso-ciated from myosin heavy chain by denaturation in 5 U guanidineHC1, followed by ethanol precipitation of myosin heavy chain. Afterdialysis, LCI was separated from light chain 2 and other proteincontaminants by molecular exclusion high pressure liquid chromatog-raphy (HPLC, upper panel). In the lower panel, protein peaks fromseveral HPLC separations were pooled and analyzed by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (50 ng of eachfraction applied). Molecular weight standards (STD's) were 30 ng ofphosporylase a (mol wt = 9Z0O0), bovine serum albumin (mol wt =69,000), ovalbumin (mol wt = 46,000), carbonic anhydrase (mol wt =30,000), and cytochrome c (mol wt = 12,400).

muscle, as well as LCI isolated from rat, rabbit, anddog myocardia. In addition, the antibody failed tocross-react with myosin heavy chain, troponin sub-units, or tropomyosin isolated from cardiac and skel-etal muscle of all species examined. Slight cross-reactivity was noted between this antiserum and ratcardiac actin, as well as human and canine cardiacLC2, when analyzed by SDS-polyacrylamide gelelectrophoresis and electroblorting (Towbin et al.,1979).

by guest on May 8, 2018

http://circres.ahajournals.org/D

ownloaded from

Samarel et al./Release of Myosin Light Chain 1

Immunization of guinea pigs with rabbit cardiacmyosin heavy chain (similarly extracted from SDS-polyacrylamide gels) produced a nonprecipitatingIgG antibody highly specific for myosin heavy chainisolated from ventricular muscle of several species,including rabbit, rat, dog, and human myocardia.This antibody did not cross-react with any othermyocardial proteins of all species examined, whenanalyzed by SDS-polyacrylamide gel electrophoresisand electroblotting.

Radioimmunoassay of Myosin Light Chain 1The LCI antiserum displayed typical equilibrium-

binding characteristics with 125I-labeled human car-

H IOO

90

80CD2 70o

I 60§ 501 402 30

* 2 0

10

o l 1 1 1 1 L25 KX) 400 1600 3200 12800

I/ANTISERUM DILUTION

CD

.90

.80

.70

.60

.50

.40

.30

.20

.10

10 20 - 40 60 80100 200 300 400LCI CONCENTRATION (ng/ml)

FIGURE 2. Equilibrium-binding characteristics of guinea pig anti-human myosin light chain 1 (LCI) and 125l-labeled human LCI. PanelA: LCI tracer (5000 counts/min) was incubated with serial dilutionsof guinea pig-anti human LCI antiserum (25 to 51,200-fold dilutions)in a total volume of 200 pi for 24 hours at 4°C Antibody-boundtracer was separated from free LCI by precipitation with goat anti-guinea pig IgG secondary antibody. That dilution capable of 50%maximum binding (1/800) was used in the radioimmunoassay. Eachpoint is the mean of two determinations. Panel B: radioimmunoassaystandard curve of human myosin LCI. Each point was the mean oftwo determinations.

169

diac LCI (Fig. 2A). An 800-fold dilution of the LCIantiserum yielded half-maximal binding of tracer,and this dilution was utilized in the radioimmuno-assay. Assay sensitivity was 3 ng/ml with a workingrange of 12.5-400 ng/ml (Fig. 2B). Within-assayvariation was less than 10% near the middle of thedose-response curve. Perfusion effluents and ratheart extracts were diluted 2-fold (in 50 nw of asodium phosphate buffer, pH 7.4, containing 150mM NaCl and 1% bovine serum albumin) to provideLCI concentrations near the middle of the standardcurve.

We took advantage of the cross-species reactivityof our LCI antiserum to confirm the presence ofLCI in the 140,000 g supernatant extract of nonper-fused rat ventricular myocardium, in a fashion anal-ogous to experiments performed by Horvath andGaetjens (1972) using chicken skeletal muscle. LCIwas readily detected in this cytosolic fraction, at aconcentration of 3.40 ± 0.21 /xg/g wet weight (mean± SD, n = 10). Based upon previous radioimmuno-assay measurements of the myosin content of rabbitventricular muscle (Everett et al., 1983), this cyto-solic pool of LCI accounted for approximately 0.1%of the total amount of LCI present in ventricularmyocardium.

Release of Unassembled Myosin Light Chain 1Following the Calcium Paradox

To determine whether the cytosolic pool of LCIwas the source of rapidly released LCI followingirreversible cellular injury, we employed the radio-immunoassay to measure LCI in the coronary ef-fluent of rat hearts subjected to the calcium paradox.This form of massive cellular injury occurs whenhearts briefly perfused with calcium-free buffer arereexposed to a calcium-containing solution (Zim-merman and Hulsmann, 1966). Calcium reperfusionresults in irreversible contracture, massive cellulardisruption, and rapid loss of cytosolic components(CK, LDH, myoglobin) (Zimmerman and Hulsmann,1966).

Continuous perfusion of rat hearts for 35 minuteswith control medium (2.5 mM Ca++) did not causethe release of either creatine kinase (CK), LCI, orother cellular protein into the coronary effluent.However, sequential perfusion with calcium-freemedium (5 minutes) followed by 2.5 mM Ca++ re-perfusion (15 minutes) led to the rapid release ofboth CK and LCI (Fig. 3). Peak CK activity and LCIrelease at 1 minute after calcium reperfusion were74.9 ± 10.7 IU/g wet weight, and 1.09 ± 0.19 /igLCl/g wet weight, respectively (mean ± SD, threeexperiments). Homogenization and centrifugation ofventricular tissue from calcium parodox-perfusedhearts (with subsequent CK assay and LCI radio-immunoassay of the 140,000 g supernatant fraction)demonstrated that the cytosolic fraction was de-pleted of 86.5 ± 0.6% of total CK activity, and 28.5± 13.5% of immunoreactive LCI (mean ± SD, threeexperiments), compared to extracts prepared fromhearts continuously perfused with medium contain-

by guest on May 8, 2018

http://circres.ahajournals.org/D

ownloaded from

170 Circulation Research/Vo(. 58, No. 1, January 1986

Co2+FREECONTROL 2.5mM Ca 2 +

•+• H -

15 20TIME (min)

25 30

1200

- 1000

- 800 g

- 600 ~LU

- 400 LJLJ

- 200 -n

35

FIGURE 3. Release of creatine kinase (CK) and ratcardiac myosin light chain 1 (LCD following thecalcium paradox. After 15 minutes of retrogradeperfusion with Krebs-Henseleit-bicarbonate buffercontaining 25 mM Ca**, a rat heart was perfusedfor 5 minutes with calcium-free buffer, followed byreperfusion (15 minutes) with medium containing25 mstCa**. Coronary effluents (1-minute collec-tions) were assayed for CK (open circles) and LCI(triangles). Data are expressed as units or nano-grams released per minute per gram of tissue wetweight (duplicate determinations).

ing 2.5 nut Ca++. Thus, LCI was rapidly releasedfrom irreversibly injured myocardium, with initialwashout kinetics similar to that of CK. The LCIreleased immediately after calcium reperfusion ap-peared to originate from the cytosolic pool.

Additional experiments were performed to char-acterize further the cytosolic pool of LCI, and todetermine whether the LCI released after calciumreperfusion was derived from assembled myosin.Protein samples (50-100 jig) from the total homog-

enates, resuspended myofibrillar sediments, and140,000 g supernatant extracts of control and cal-cium paradox-perfused hearts, as well as a concen-trate (50 ng protein) of perfusate effluent collectedduring the first minute following calcium reperfu-sion, were separated by SDS-polyacrylamide gelelectrophoresis on 7-17% vertical gradient slab gels.The presence or absence of myosin heavy chain andLCI was determined by protein staining or electro-blotting and exposure to specific antisera.

MHC-

•2-

69 -

46"

30-LC1-

FIGURE 4. lmmunoelectrophoretic detection of myosin heavy chain (MHO and myosin light chain 1 (LCI) in fractions of perfused rat heart.Protein samples (50-100 fig) were of the total homogenates, resuspended myofibrillar sediments and 140,000 g supernatant fractions of heartsperfused under control conditions (O or after calcium-free perfusion and reperfusion with 25 mM Ca** (P). In addition, 50 fig of perfusion effluentprotein (effluent) from the peak fraction of LCI release after the calcium paradox were separated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis on 7-17% vertical slab gels. Separated proteins were either stained with Coomassie brilliant blue (left panel) or were transferredelectrophoretically to nitrocellulose sheets. MHC (middle panel) and LCI (right panel) were identified by immunodetection with specific antibody.Molecular weight standards (STD's) were as in Figure 1.

by guest on May 8, 2018

http://circres.ahajournals.org/D

ownloaded from

Samarel et a/./Release of Myosin Light Chain 1

As seen in Figure 4, myosin heavy chain wasdetected (by protein staining and immunodetectionwith specific antibody) in the total homogenates andmyofibrillar sediments of both control and calciumparadox-perfused hearts. However, myosin heavychain was not detected in either of the two super-natant fractions, or in the concentrated effluent fol-lowing calcium reperfusion. As expected from theradioimmunoassay results, immunoreactive LCIwas present in all tissue fractions, as well as in thecoronary effluent following the calcium paradox.Furthermore, the immunoreactive LCI released dur-ing the first minute after calcium reperfusion was ofidentical apparent molecular weight (mol wt =27,000) to the LCI present in the total homogenates,140,000 g supernatant fractions, and myofibrillarsediments of both control and calcium-reperfusedhearts (Fig. 4). These results indicate that the LCIpresent in the cytosolic fraction (and released fromthe heart following the calcium paradox) was notderived from assembled myosin.

In summary, calcium-free perfusion followed byreperfusion with buffer containing 2.5 mM Ca++ ledto the rapid release of cardiac LCI from an intracel-lular pool of myosin light chains that was separableby homogenization and centrifugation from the bulkof LCI bound to myofibrils. This LCI pool demon-strated initial wash-out kinetics similar to that ofCK, a sarcoplasmic enzyme, suggesting that the LCIoriginated from a cytosolic pool of unassembledmyosin light chain subunits. Release of LCI afterirreversible myocytic injury appears unrelated to theinitial type of damage produced, because rapid re-lease of LCI occurs after ischemic injury, as well asafter global cellular injury caused by the calciumparadox. This cytoplasmic pool of myosin light chain1 may be important in the assembly and turnoverof myofibrils.

We thank Camellia Moore for assistance in preparing the manu-script.

This study was supported in part by NHLBI Grant HL-19648 anda grant from the Oppenheimer Family Foundation. A.M. Samarel wasa recipient of a Schweppe Foundation Fellowship for the duration ofthese studies.

Address for reprints: Allen M. Samarel, M.D., Department ofMedicine (Cardiology), Northwestern University Medical School, 303E. Chicago Avenue, Chicago, Illinois 60611.

Received April 1, 1985; accepted for publication October 14, 1985.

ReferencesEverett AW, Prior G, Clark WA, Zak R (1983) Quantitation of

myosin in muscle. Anal Biochem 130: 102-107Ganote CE, Grinwald PM, Nayler WG (1984) 2,4-Dinirrophenol

(DNP)-induced injury in calcium-free hearts. J Mol Cell Cardiol16: 547-555

Gere JB, Krauth GH, Trahern CA, Bigham DA (1978) A radio-

171

immunoassay for the measurement of human cardiac myosinlight chains. Am J Clin Pathol 71: 309-318

Greenwood FC, Hunter WM, Glover JS (1963) The preparationof mI-labelled human growth hormone of high specific radio-activity. Biochem J 89: 114-123

Horvath B, Gaetjens E (1972) Immunochemical studies of thelight chains from skeletal muscle myosin. Biochim BiophysActa 263: 779-793.

Katus HA, Hurrell JG, Matsueda GR, Ehrlich P, Zurawski VR Jr,Khaw BA, Haber E (1982) Increased specificity in humancardiac myosin radioimmunoassay utilizing two monoclonalantibodies in a double sandwich assay. Mol Immunol 19: 451-455

Katus HA, Yasuda T, Gold HK, Leinbach RC, Strauss HW,Waksmonski C, Haber E, Khaw BA (1984) Diagnosis of acutemyocardial infarction by detection of circulating cardiac myosinlight chains. Am J Cardiol 54: 965-970

Khaw BA, Gold HK, Fallon JT, Haber E (1978) Detection of serumcardiac myosin light chains in acute experimental myocardialinfarction: Radioimmunoassay of cardiac myosin light chains.Circulation 58: 1130-1136

Laemmli UK (1970) Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (Lond) 277:680-685

Lowry OH, Rosebrough NH, Farr AL, Randall RJ (1951) Proteinmeasurement with the Folin phenol reagent. J Biol Chem 193:265-275

Morkin E, Yazaki Y, Katagiri T, LaRaia PJ (1973) Comparison ofthe synthesis of the light and heavy chains of adult skeletalmuscle myosin. Biochim Biophys Acta 324: 420-429

Nagai R, Yazaki, Y (1981) Assessment of myocardial infarct sizeby serial changes in serum cardiac myosin light chain II indogs. Jpn Circ J 45: 661-666

Nagai R, Ueda S, Yazaki Y (1979) Radioimmunoassay of cardiacmyosin light chain II in the serum following experimentalmyocardial infarction. Biochem Biophys Res Commun 86: 683-688

Nagai R, Ueda S, Yazaki Y (1980) Radioimmunoassay of cardiacmyosin light chain II in the serum following experimentalmyocardial infarction. In Advances in Myocardiology, vol 2,edited by M Tajuddin, B Bhatia, HH Siddiqui, G Rona. Balti-more, University Park Press, pp 415-420

Samarel AM, Ogunro EA, Ferguson AG, Lesch M (1981) A doubleantibody radioimmunoassay for canine cardiac cathepsin D.Cardiovasc Res 15: 68-73

Smitherman TC, Dycus DW, Richards EG (1980) Dissociation ofmyosin light chains and decreased myosin ATPase activity withacidification of synthetic myosin filaments: Possible clues tothe fate of myosin in myocardial ischemia and infarction. J MolCell Cardiol 12: 149-164

Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transferof proteins from polyacrylamide gels to nitrocellulose sheets:Procedure and some applications. Proc Natl Acad Sci USA 76:4350-4354

Trahern CA, Gere JB, Krauth GH, Bigham DA (1978) Clinicalassessment of serum myosin light chains in the diagnosis ofacute myocardial infarction. Am J Cardiol 41: 641-645

Wikman-Coffelt J, Zelis R, Fenner C, Mason DT (1973) Compar-ative purification of myocardial myosin and antigenic specific-ity of the two light chains. Prep Biochem 3: 439-449

Zak R, Martin AF, Prior G, Rabinowitz M (1977) Comparison ofturnover of several myofibrillar proteins and critical evaluationof double isotope method. J Biol Chem 252: 3430-3435

Zimmerman ANE, Hulsmann WC (1966) Paradoxical influenceof calcium ions on the permeability of the cell membranes ofthe isolated rat heart. Nature (Lond) 211: 646-647

INDEX TERMS: Radioimmunoassay • Myocyte injury. Antibod-ies • Myofibrillar protein

by guest on May 8, 2018

http://circres.ahajournals.org/D

ownloaded from

A M Samarel, A G Ferguson, R S Vander Heide, R Davison and C E GanoteRelease of unassembled rat cardiac myosin light chain 1 following the calcium paradox.

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 1986 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

doi: 10.1161/01.RES.58.1.1661986;58:166-171Circ Res. 

http://circres.ahajournals.org/content/58/1/166World Wide Web at:

The online version of this article, along with updated information and services, is located on the

  http://circres.ahajournals.org//subscriptions/

is online at: Circulation Research Information about subscribing to Subscriptions: 

http://www.lww.com/reprints Information about reprints can be found online at: Reprints:

  document. Permissions and Rights Question and Answer about this process is available in the

located, click Request Permissions in the middle column of the Web page under Services. Further informationEditorial Office. Once the online version of the published article for which permission is being requested is

can be obtained via RightsLink, a service of the Copyright Clearance Center, not theCirculation Research Requests for permissions to reproduce figures, tables, or portions of articles originally published inPermissions:

by guest on May 8, 2018

http://circres.ahajournals.org/D

ownloaded from