Simultaneously Measured Isometric Tension and ATP Hydrolysis in Glycerlnated Fibers from Normal and Hypertrophied Rabbit Heart

Transcription

1 Smultaneously Measured Isometrc Tenson and ATP Hydrolyss n Glycerlnated Fbers from Normal and Hypertrophed Rabbt Heart By Phlp D. Henry, Gal G. Ahumada, Wllam F. Fredman, and Burton E. Sobel ABSTRACT Adenosnetrphosphatase (ATPase) actvty, sometrc tenson, and sarcomere length of glycernated fbers from 14 hypertrophed and 18 normal rabbt hearts were measured smultaneously to characterze the altered performance of hypertrophed myocardum. ATPase actvty was assessed radochromatographcally, and contrbutons from mtochondra, sarcolemma, and sarcoplasmc retculum were excluded. The actve length-tenson and the actve tenson-pca relatonshps n fbers from hypertrophed myocardum were normal n sharp contrast to the depressed actve length-tenson curves n correspondng ntact papllary muscles. Total ATPase actvty assocated wth contracton at a gven developed tenson was decreased sgnfcantly n fbers from hypertrophed hearts (P< 0.001). ATPase actvty was dmnshed n glycernated myofbrls, actomyosn, and homogenates from hypertrophed hearts as well. On the other hand, the ncrease n ATPase actvty assocated wth a gven ncrease n developed tenson was vrtually dentcal n fbers from hypertrophed and normal hearts. Thus, depressed total ATPase actvty n glycernated fbers from hypertrophed hearts does not appear to mpar mantenance of sometrc tenson. KEY WORDS myocardal mechancs energy utlzaton actomyosn myofbrls length-tenson relatonshp contractlty myofbrllar adenosnetrphosphatase The decreased performance of the hypertrophed or falng myocardum mght be related to the dmnshed adenosnetrphosphatase (ATPase) actvty of the contractle protens (1-6). However, the functonal mplcatons of depressed myosn and actomyosn ATPase actvty n hypertrophed and falng heart muscle have not yet been elucdated. Accordngly, smultaneous determnatons of sarcomere length, developed tenson, and rate of adenosne trphosphate (ATP) hydrolyss From the Departments of Medcne and Pedatrcs, Unversty of Calforna, San Dego, School of Medcne, La Jolla, Calforna Ths nvestgaton was supported n part by U, S. Publc Health Servce Program Project Grant HL and by Research and Career Development Awards 5-K4-HL-41, (Dr. Fredman) and 1- K4-HL-50, (Dr. Sobel). Receved May 19, Accepted for publcaton September 11, were made n glycernated rabbt myocardal fbers to defne the relatonshp between the mantenance of tenson and the depresson of contractle proten ATPase actvty assocated wth hypertrophy. Methods Reagents. Dsodum ATP, mdazole, neutralzed edyleneglycol bs(y3-amnoethylether)-n, N'-tetraacetate (EGTA), and dthothretol (DTT) were obtaned from Sgma Chemcal Corporaton. Gamma-labeled AT 32 P and "C-ATP were obtaned from New England Nuclear Corporaton. Deonzed, doubly glass-dstlled water was used for reagent preparaton. Anmal Procedures. Male New Zealand rabbts, weghng kg, were anesthetzed wth sodum pentobarbtal (25 mg/kg, v) and mantaned wth postve-pressure ventlaton. Rght ventrcular hypertrophy was produced by an 85$ constrcton of the pulmonary artery wth Teflon clps. (3.4 mm,.d.). Sham-operated rabbts served as controls. Rabbts were klled by 740 Crculaton Rtstsrcb, Vol. XXXI, Normer 1972

2 ISOMETRIC TENSION AND ATP HYDROLYSIS 741 a blow to the head 1 week after surgery, and thenhearts were removed rapdly, mmersed n 0-4 o C sotonc KG, and trmmed. The free rght ventrcular wall and the left ventrcle wth the septum were blotted and weghed. Papllary muscles used for studes of mechancs were transferred mmedately to a myograph contanng oxygenated, buffered Krebs soluton wthout beng mmersed n KC1. Mechancs of Papllary Muscles. Rght ventrcular papllary muscles were suspended vertcally n a myograph; the tendnous end was fxed wth 4-0 slk and the other end was attached wth a clp to a Statham Gl force transducer. The bath contaned modfed Krebs-bcarbonate buffer, ph 7.4 at 30 C, equlbrated wth 95% O 2-5% CO 2. The soluton had the followng mumolar composton: NaCl 121, KC1 2.4, MgSO 4 1.1, KH 2 PO 4 1.1, CaCl 2 2.5, NaHCO a, 25 and glucose 4.5. The muscles were stmulated wth platnum feld electrodes at a frequency of 12/mn wth square waves 4 msec n duraton at a voltage 10% above threshold. Relatvely large muscles from normal hearts and relatvely small muscles from hypertrophed hearts were selected to match the muscles for cross-sectonal area. Isometrc lengthtenson curves were obtaned by ncreasng length n 0.1-mm ncrements untl actvely developed tenson was maxmum (Lmax). Muscle dameters at Lmax were used to calculate cross-sectonal areas T> fl»f! " 15 mm ', assumng a cylndrcal model. When experments were complete, muscles were fxed at Lmax n 10% formaln pror to lght mcroscopy studes. Glycernaton of Fbers and Myofbrls. \Jnstretched rght ventrcular papllary muscles were placed n a soluton contanng 50% glycerol (by volume), 20 mm mdazole, 4 mm EGTA, and 1 mm DTT, ph 7.0 at 0 C. After 24 hours at 0 C, the muscles were transferred to fresh glycernaton medum and stored at 20 C for 1 week. Rght ventrcular walls were cut nto small fragments, mmersed n 30 volumes of glycernaton medum, stored for 24 hours at 0 C, and subsequently stored for 1 week at 20 C. Myofbrls were prepared from glycernated fragments as descrbed below. Mechancs of Glycernated Fbers. Fbers were studed n a mcromyogtaph consstng of a 250-/,lter reacton chamber machned from polycarbonate (Fg. 1). Horzontal stanless steel wres (dameter 0.2 mm) penetrated the chamber wthout touchng ts wall from each end through slts (wdth 0.16 mm) n the chamber walls. Glycernated fbers were suspended horzontally n the chamber by attachment to the wres wth 6-0 slk. The free ends of the fbers were trmmed very closely to the slk tes. One wre was attached to a mcrometer to adjust fber length. The other wre was attached to a Sanbom FT-A-1 stran-gauge transducer. The overall complance 4 -- Nf 1) Mcroscope condenser 2) Modfed mcroscope stage 3) Mcroscope objectve 4) Plexglass muscle bath chamber 5) Muscle suspenson wres 6) Hnge 7) Lever arm 8) Mcrometers used as slops 9) Force transducer FIGURE 1 The mcromyogtaph used to assess mechancs n glycernated fbers s represented dagrammatcally. CrcmUon Rtstsrcb, Vol. XXXI, Novtmbtr 1972

3 742 HENRY, AHUMADA, FRIEDMAN, SOBEL of the force-transducng system was 0.12 /m/mg weght, and the natural frequency was 66 Hz. Transducer sgnals were amplfed wth a Brush carrer amplfer and recorded wth a Brush Mark 200 recorder. The myograph was mounted on a standard Zess mcroscope ftted wth Kpl 12.5 eyepeces, Optovar magnfer, UMK 50 Letz objectve, flar mcrometer, and Polarod camera. The reacton chamber could be moved wthn the vsual feld of the mcroscope wth an x-y manpulator. Relaxaton soluton had the followng mllmolar composton: KC1 100, MgCl 2 5, Na 2 ATP 5, neutralzed ethylenedamnetetraacetate (EDTA) 5, sodum azde 5, and mdazole 20, ph 7.0 at 25 C. Contracton soluton was dentcal except that t contaned calcum (Ca)-EGTA buffer (4 mm EGTA), rather than EDTA. pca was adjusted by changng the rato of Ca 2+ to EGTA (apparent assocaton constant of the Ca-EGTA complex = %-' [7]). Unless otherwse specfed, pca was 5.4. Glycernated papllary muscles were ncubated n relaxaton soluton at 0 C for 1 hour before use. Fber bundles 3-4 mm n length and mm n dameter were dssected from the central core of a papllary muscle, selected mcroscopcally on the bass of regular margns, even wdths, and sharp straton patterns, and mounted on the myograph. Mean sarcomere length was taken as one-tenth the dstance between 11 consecutve stratons. Preloads were vared so that ntal sarcomere length ranged from 1.68 to 2.32 [x,m. Mean fber wdth, from three measurements taken n the mddle thrd of the fber, was used to calculate cross-sectonal area assumng a cylndrcal model. Mcroscopcally derved cross-sectonal area and the rato of fber proten content to fber length correlated closely (r = 0.91). Contractons were elcted by substtutng contracton soluton for relaxaton soluton. The relatonshp between pca and tenson was examned by exposng fbers to contracton solutons of ncreasng Ca 2+ concentraton wthout relaxng the fber between changes of soluton. Proten Determnatons. Proten was measured by a modfed Lowry procedure (8) wth the use of smlarly treated bovne serum albumn as a standard. AT Pose Actvty of Glycernated Fbers. ATPase actvty assocated wth a gven contracton was measured usng a radochromatogtaphc assay. The contracton soluton, 200 //lters, contanng 5 mm ATP was added to the reacton chamber. At a specfc actvty of 3 X 10" counts/mn mmole" 1 the chamber contaned 3 X 10 8 counts/mn (1 yxmole) of gamma-labeled AT 32?. After a 10-mnute sometrc contracton, the soluton was quanttatvely recovered and mxed. Duplcate 5-yulter samples were ppetted onto separate strps of a washed Baker-flex cellulose-polyethylenemne (PEI) thn-layer chromatography plate. Knetc measurements were obtaned by removng 5-/lter samples from the bath at tmed ntervals followng bref strrng. Blanks ncluded both fresh contracton soluton and contracton soluton ncubated n the bath for 10 mnutes wthout a muscle fber. After ascendng chromatography n 0.3M LCl and 0.5N HCOOH, ph 2.0 at 25 C, ATP and norganc phosphate (P ) were vsualzed wth sodum molybdate and SnCl 2 sprays {9). Unform strps encompassng ATP and P, were cut from the plates and placed n vals contanng 15 ml of Bray's soluton (10). Radoactvty was counted n a Packard Tr-Carb scntllaton counter (model 3375). Countng effcency was 972. The ATPase actvty of each fber was calculated usng values for the ntal quantty of ATP n the chamber (1 //.mole) and the rato of 82 P, (counts/mn) to AT 82 P + 82 Pj (counts/mn). In knetc experments, the sequental removal of 5-fjdter samples from the bath was taken nto account n the calculatons. In some experments, phosphate lberaton from ATP was estmated smultaneously by the radoactve method and by a modfcaton of a colormetrc procedure, whch requred larger amounts of tssue (11). The ATPase actvty of a fber was expressed wth respect to the proten content of the fber. ATPase Actvty of Glycernated MyofbrUar Preparatons and of Fresh Myocardum. Glycernated myocardal fragments were mnced, passed through a tssue press (pore sze 1 mm), and homogenzed n a Vrts homogenzer wth three 15-second bursts at a speed settng of 5 n 25 volumes of a medum contanng 0.32M sucrose, 20 mm mdazole, and 1 mm DTT, ph 7.0. The homogenate was centrfuged at 60 g for 2 mnutes. The supernatant fracton was recovered and centrfuged at 600 g for 20 mnutes, and the pellet was resuspended n homogenzng medum, washed fve tmes by the same procedure, and fnally resuspended n 2.5 volumes of a soluton contanng KC1 100 mm, MgCl 2 5 mm, mdazole 20 mm, DTT 1 mm, and sodum azde 5 mm, at ph 7.0. In experments wth soluton contanng low concentratons of Mg 2 +, 1 mm neutralzed EDTA was added to the resuspenson medum and exogenous Mg 2+ was omtted. ATPase actvty was determned at 26 C n a contnuously strred 2-ml reacton medum, ph 7.0, contanng KC1 100 mm, MgCl 2 5 mm (except n experments wth no added Mg 2+ ), Na 2 ATP 5 mm, mdazole 20 mm, sodum azde 5 mm, and Ca-EGTA buffer along wth approxmately 1.0 mg/ml of myofbrllar proten. In some CrcuUtton Retard, Vol. XXXI. Ntwmfr 1972

4 ISOMETRIC TENSION AND ATP HYDROLYSIS 743 I I CO IC O O O d o -H -H o S q o o o HI 4 00 cc o o 2t * d d d d o o -H HI CO t*- do 88 d d HI HI HI HI 5 OS d rn 55 m^ CrcxUton Rttetrcb, Vol. XXXI, Navtmktr 1972 o J3 T3 3 CO g a 3 a 8 s HI -5 experments, azde was omtted. Seral 0.1-ml samples were added to 0.5 ml of 0.5N perchlorc acd. P, was measured colormetrcally (11). The reacton was lnear for at least 10 mnutes. ATPase actvty n whole homogenates of fresh myocardum was measured as descrbed for myofbrls. ATPase Actvty of Actomyosn. Actomyosn content of normal and hypertrophed fresh nonglycernated myocardum was estmated by the extracton procedure of Inchosa (12). Actomyosn ATPase actvty was assayed under the same condtons used for assay of myofbrllar ATPase at a pca of 5.0 wth approxmately 0.8 mg proten /ml reacton mxture. The purpose of the actomyosn studes was prmarly to estmate the yeld from normal and hypertrophed myocardum. Depresson of ATPase actvty n actomyosn wth respect to myofbrllar preparatons s not unexpected, because of loss or depolymerzaton of actn durng extracton procedures. Results Extent of Ventrcular Hypertrophy. The operatve procedure resulted n gross rght ventrcular hypertrophy 1 weelc after surgery (Table 1). The rato of extractable actomyosn to total proten was not sgnfcantly dfferent n normal and hypertrophed hearts. Hstologc study of papllary muscles used n NORMAL (0=51 HYPERTROPHY (l-bi ACTIVE TENSION RESTING TENSION FIGURE 2 Length-tenson curves of normal and hypertrophed rght ventrcular papllary muscles. Muscles were studed n modfed Krebs-bcarbonate soluton, ph 7.4 at 30 C. Cross-sectonal areas n normal and hypertrophed muscles averaged 1.04 ±0.11 mm* (mean ± SB) and 1.09 ±0.12 mm', respectvely. 10

5 744 HENRY, AHUMADA, FRIEDMAN, SOBEL TABLE 2 ATPase Actvty (nmoles P,/mg proten mnr 1 ) of Fresh Homogenate, Glycernated Myofbrls, and Actomyosn pca Normal Hypertrophy P Whole homogeaate of fresh myocardum (4) 9 33 ± ± ± 3.0 ± ± ± 1.5 < 0.01 Glycernated myoflbru (9) Results are means ± BE. Number of prepara(ons tested s trven n parentheses ± ± ± ± 3.0 < 0.01 <( ).01 Actomjoln (4) 5 59 ± ± 3.0 <0.001 experments on mechancs revealed no apprecable ncrease n connectve tssue n those solated from hypertrophed hearts. Mechancs of Papllary Muscles. The length-tenson curves of normal and hypertrophed rght ventrcular papllary muscles matched for cross-sectonal area are shown n Fgure 2. Actve tenson was clearly depressed n hypertrophed muscles at all lengths. Restng tenson dd not dffer sgnfcantly when hypertrophed muscles were compared wth normal muscles. ATPase Actvtes of Whole Homogenates of Nonglycernated Fresh Myocardum, Glycernated Myofbrls, and Actomyosn. ATPase actvtes of these preparatons are summarzed n Table 2. At hgh Mg 2+ concentraton, the ATPase actvty of glycernated myofbrls from hypertrophed hearts was sgnfcantly depressed at all Ca 2 + concentratons examned. However, n the absence of added Mg 2 +, preparatons from hypertrophed hearts exhbted myofbrllar ATPase actvty whch dd not dffer from that n normal preparatons looro ' ' \\ j! /. 1 -h- 1 I ' ' ~ ~ ; =444-!! 1 1 \ ' v \\1 ' I 1 ' ' at any Ca 2+ concentraton examned; ths fndng s n agreement wth that of a prevous report (1). ATPase actvty n the presence of azde was lower than t was n the absence of azde, ndcatng only a modest contrbuton of mtochondral ATPase to total ATPase actvty. ATPase assays were performed under condtons n whch pca was vared through the range used n the studes of mechancs. Mechancs of Glycernated Fbers. Fbers glycernated longer than 2 weeks contracted n contracton soluton but dd not relax completely wth EDTA. Wth fbers glycernated less than 2 weeks, reproducble contractonrelaxaton cycles were obtaned (Fg. 3). The relatonshp between glycernated fber sarcomere length and tenson s shown n Fgure 4. At smlar sarcomere lengths, total tenson was comparable n glycernated fbers from normal and hypertrophed hearts. Ths fndng was n sharp contrast to the depressed length-tenson curves observed n ntact, hypertrophed papllary muscles. I 1 ] 1!.... t- H t t \ I j ' ' k! / / j 1 1! ' - \ L- \ 1 v _ : TIME (mn) FIGURE 3 Sequental contractons of a glycernated fber. Contractons were obtaned by alternatng contracton soluton (pca = 5.4) wth relaxaton soluton (pca' 9/ CnmUton Resurcb, Vol. XXXI, Novnxbar 1972

6 ISOMETRIC TENSION AND ATP HYDROLYSIS o Normal (t'll) Hjptrlrophj ( a '10) 1.6 I fl SARCOMERE LENGTH ( jrn) FIGURE 4 SoTcomere length-tenson relatonshp n glycernated fbers. Ponts on the bottom curve represent the tenson observed at pca 9. Ponts on the top curve represent the dfference bet-ween the tensons at pca 5.4 and those at pca ^ 9. Each pont on the bottom. curve and ts correspondng pont on the top curve belong to the frst contracton cycle of a dfferent fber. Snce tenson at pca *-~> 9 approxmates restng tenson, the top curve corresponds to developed tenson. 24 The tenson-pca response curves of eght normal and eght hypertrophed glycernated fbers studed at the same sarcomere lengths are shown n Fgure 5. It s apparent that the response at optmal sarcomere lengths was smlar n fbers from normal and hypertrophed hearts. The pca at half-maxmum tenson was approxmately 6.43 n both cases. Relatonshp between Glycernated Fber Tenson and Smultaneous ATP Hydrolyss. The ATPase actvty of a representatve glycernated fber durng a 12-mnute contracton s shown n Fgure 6; ATP hydrolyss was lnearly related to tme. ATPase actvty of fbers from normal and hypertrophed hearts ncreased lnearly wth total tenson (Fg. 7). Identty of the count ratos of fresh and ncubated blanks ndcated that nether evaporaton nor spontaneous hydrolyss n the bath nfluenced the results spurously. In addtonal experments, agreement was close between results obtaned wth the radoactve ATPase assay system and the colormetrc 100 K o Normol (n=8) Hypertrophy (n = B) pca50 = pco FIGURE 5 Glycernated fber tenson. The range of ntal sarcomere length was 2.11 to 2.18 (m n the normal fbers and 2.08 to 2.20 pm n the hypertrophed fbers. Tensons were expressed as a percent of the tenson at pca = 5.4, whch were 1.3 ± 2.0 g/mm* (mean 1.8) and 1.5 ±1.8 g/mm' (mean 1.7) n the normal and hypertrophed fbers, respectvely. Crculaton Rtsttrch, Vol. XXXI, Novtmtttr 1972 = 20 - TIME FIGURE 6 (mn) ATPase actvty of a representatve glycernated fber durng a contracton at pca = 5.4. In ths experment, mxng durng ncubaton of the fber was acheved by magnetc strrng wth a 1.5-tnm 25-gauge needle tp. Extrapolaton to zero tme ndcated an apparently elevated ntal rate of hydrolyss. The ntal devaton from, lnearty was less than 5% and was not taken nto account n the calculaton of the steadystate rate of hydrolyss. Condtons: pca 5.4, sarcomere length = 2.12 /m, tenson 1.64 g/mm*. 12

7 746 HENRY, AHUM ADA, FRIEDMAN, SOBEL o Normal (n -18) Hypertrophy (n= 14) TOTAL TENSION (g/mm z ) FIGURE 7 Isometrc tenson and smultaneously measured ATPase actvty n ndvdually studed glycernated fbers. Varatons n tenson were obtaned by ntatng contractons at dfferent sarcomere lengths. Regresson lnes were obtaned by the least-squares method; the slopes are not sgnfcantly dfferent. phosphate procedure (N = 4), excludng sgnfcant nterference due to radosotopc exchange. Glycernated fbers from hypertrophed hearts exhbted lower ATPase actvty at all tensons, but wth any gven ncrement n tenson the ncrease n ATP hydrolyss was nearly dentcal, as reflected by the smlar slopes of the regresson lnes. Thus, although the total ATPase actvty of glycernated fbers from hypertrophed muscle was dmnshed, there was no depresson n the augmentaton of ATP hydrolyss assocated wth ncrements n tenson. Addtonal experments were performed to characterze further the glycernated fber ATPase assay system. To determne whether sgnfcant myoknase actvty was present, glycernated fbers were ncubated n contracton soluton contanng 14 C-ATP. Samples were chromatographed on PEI plates wth a LCl gradent chosen to separate nucleotdes (13). The absence of radoactvty n the regon to whch adenosne monophosphate mgrated excluded the presence of apprecable myoknase actvty under the assay condtons. Sequental contractons of normal and hypertrophed fbers wth and wthout 5 HIM azde ( N = 4) demonstrated that azde dd not nfluence tenson development and that the contrbuton of mtochondral ATPase to total ATPase actvty was less than 10? n both preparatons. Furthermore, polarographc experments wth homogenates of glycernated fbers and wth 8,000-g fractons prepared from the same homogenates demonstrated no oxygen consumpton followng addton of adenosne dphosphate wth 0.01M glutamate or 0.01M pyruvate and 0.01M malate, provdng further evdence that functonally ntact mtochondra were absent from the preparaton. Nether ouaban (10- M) nor detergents ( IO^M neutralzed sodum deoxycholate or Tween 80) affected tenson or ATP hydrolyss n glycernated fber preparatons, excludng a sgnfcant contrbuton of sarcolemmal or sarcoplasmc retculum ATPase to total ATPase actvty. Ths fndng was corroborated by the fact that the ATPase actvty of a mcrosomal fracton prepared from glycernated fbers (14) was less than 2% of the total fber ATPase actvty. Dscusson Glycernated fbers from hypertrophed papllary muscles exhbted normal sometrc tenson wth respect to length and pca and normal augmentaton of ATP hydrolyss assocated wth ncreased tenson. These characterstcs of the glycernated fbers from hypertrophed hearts contrasted sharply wth the depressed actve length-tenson curves of the correspondng ntact papllary muscles n ths nvestgaton and of the muscles from hypertrophed hearts descrbed prevously (3, 15, 16). Cross-sectonal area (17) and cellular composton of papllary muscles (17-19) Crculaton Rtsarcb, Vol. XXXI, Novtmttr 1972

8 ISOMETRIC TENSION AND ATP HYDROLYSIS 747 mght be mportant determnants of results based on tenson normalzed for cross-sectonal area n the ntact muscle. In ths study, normal and hypertrophed muscles were matched for cross-sectonal area {Fg. 2), and there was no hstologc evdence of fbross. Furthermore, actomyosn concentratons and ratos of actomyosn to proten were smlar n hypertrophed and normal tssue. Thus, the decrease n mechancal performance of ntact papllary muscles from hypertrophed hearts observed n ths nvestgaton probably reflects an ntrnsc abnormalty of the myocardal cells. The absolute tenson values n ths study were consstent wth those n prevous reports, ndcatng that normal rabbt papllary muscles develop less tenson n vtro than do cat papllary muscles, possbly because of dffuson lmtatons (20). Decreased tenson has been reported n glycernated trabecular fbers from dogs wth chronc rght ventrcular falure (2), n dsagreement wth the fndngs n the present study. However, trabecular muscle mght become fbrotc under condtons of chronc falure, and the rato of actomyosn to proten mght be decreased (17). The problem of dffuson varaton n studes of mechancs of ntact muscles must be consdered. In the present study, muscles were matched for cross-sectonal area to mnmze dsparate effects of dffuson on muscles from hypertrophed and normal hearts. Our results wth glycernated fbers ndcated that the contractle apparatus n hypertrophed muscles was capable of generatng normal tenson. Accordngly, the decreased actve tenson n ntact muscle preparatons mght be related to abnormal actvaton by Ca 2+ of the contractle protens. Prevous fndngs ndcated that Ca 2+ senstvty was progressvely lost durng storage n glycerol (21-24), a change that was noted also n ths study and that could not be retarded by the addton of a reducng agent (DTT) (25). However, by glycernatng the muscles for only 1 week, preparatons wth largely preserved Ca 2+ senstvty were obtaned. The smlarty of the relatonshp between pca and Crculaton Rtsurcb, Vol. XXXI, Hovrmbtr 1972 sometrc tenson n normal and hypertrophed fbers glycernated for 1 week mpled that there was no major dfference n the factors conferrng Ca 2+ senstvty to mantenance of tenson by the contractle apparatus. Thus, t does not appear that the decreased force development n the ntact muscle s related to a decreased senstvty of the contractle system to actvatng Ca 2 +. ATPase actvty was sgnfcantly decreased n homogenate, glycernated myofbrls, and actomyosn from hypertrophed hearts n the present study. In addton, ATPase actvty was decreased n glycernated fbers from hypertrophed papllary muscles despte the fact that sometrc tenson was normal wth respect to sarcomere length. A fve-fold ncrease n total tenson was assocated wth a doublng of ATPase actvty n glycernated fbers from both normal and hypertrophed hearts. The unformty of ths relatonshp suggests that the effcency of bochemcal processes contrbutng to mantenance of tenson s not mpared n the contractle system from hypertrophed myocardum even when the actve length-tenson curve of the correspondng ntact muscle s depressed. As demonstrated n ths nvestgaton, tenson, sarcomere length, and ATPase actvty can be measured smultaneously n glycernated fbers. The present experments were desgned to characterze sometrc contractons. It has been suggested that muscle shortenng s a functon of myosn ATPase actvty and that force s a functon of regulatory protens conferrng Ca 2+ senstvty to the contractle apparatus (26-28). However, the mechansms responsble for mantanng tenson n the presence of ATP mght depend on cyclng of the cross brdges as well (29-31). Thus, shortenng and force mght not be regulated ndependently. Some data supportng ths vew ndcate that unloaded shortenng velocty, as well as force, s Ca 2+ dependent (20, 32-34). The present results ndcate that the capacty of hypertrophed glycernated fbers to mantan sometrc tenson s not mpared by depressed total ATPase actvty. However, the possblty that

9 748 HENRY, AHUMADA, FRIEDMAN, SOBEL decreased ATPase actvty s related to or reflected by abnormaltes of other mechancal propertes of the glycernated fber preparaton such as shortenng velocty cannot be excluded. Accordngly, t s mportant to explore the relatonshp between unloaded shortenng velocty and ATP hydrolyss n glycernated fbers from heart muscle. Nevertheless, t seems clear that the capacty of hypertrophed glycernated fbers to mantan sometrc tenson s not mpared by depressed total ATPase actvty. Acknowledgment' We apprecate the help of Harley Sybers, M.D., who performed the lght mcroscopc studes, and the techncal assstance of Carol Wagner and Alce K. Robson. References 1. AIJPERT, N.R., AND GORDON, M.S.: Myofbrllar adenosne trphosphatase actvty n congestve heart falure. Am J Physol 202: , BENSON, E.S., HALLAWAY, B.S., AND TDRBAK, C.E.: Contractle propertes of glycerol-extracted muscle bundles from the chroncally falng canne heart. Crc Res 6: , CHANDLER, B.M., SONMENBUCK, E.H., SPANN, J.F., AND POOL, P.E.: Assocaton of depressed adenosne trphosphatase and reduced contractlty n expermental heart falure. Crc Res , GORDON, M.S., AND BROWN, A.L.: Effect of ouaban and calcum on myofbrllar adenosnetrphosphatase actvty of falng human hearts (abstr.). Crculaton 30 (suppl. n): , GORDON, M.S., AND BROWN, A.L.: Myofbrllar adenosne trphosphatase actvty of human heart tssue n congestve heart falure: Effects of ouaban and calcum. Crc Res 18: , Lucm, R.J., KRTTCHER, E.M., AND THYRUM, P.T.: Reduced cardac myosn adenosnetrphosphatase actvty n dogs wth spontaneously occurrng heart falure. Crc Res 24: , PORTZEHL, H., CALDWELL, P.C., AND RUEGG, J.C.: Dependence of contracton and relaxaton of muscle fbers from the crab Maa squlnado on the nternal concentraton of free calcum ons. Bochm Bophys Acta 79: , LAYNE, E.: Spectrophotometrc and tutbdmetrc methods for measurng protens. In Methods n Enzymology, vol. 3, edted by S. P. Colowck and N. O. Kaplan. New York, Academc Press, 1957, pp KREBS, K.G., HEUSSER, D., AND WIMMER, H.: Spray reagents. In Thn Layer Chromatography, edted by E. Stahl. New York, Sprnger- Verlag, 1969, pp BRAY, G.: Smple effcent lqud scntllator for countng aqueous solutons n a lqud scntllaton counter. Anal Bochem 1: , SOBEL, B., JEQUIER, E., SJOEHDSMA, A., AND LOVENBERG, W.: Effect of catecholamnes and adrenergc blockng agents on oxdatve phosphorylaton n rat heart mtochondra. Crc Res 19: , INCHIOSA, M.A., JH.: Actomyosn content of rabbt heart ventrcle. Am J Physol 206: , RANDERATH, E., AND RANDERATH, K.: Resoluton of complex nucleotde mxtures by twodmensonal anon-exchange thn layer chromatography. J Chromatogr 16: , HARICAYA, S., AND SCHWARTZ, A.: Rate of calcum bndng and uptake n normal anmal and falng human cardac muscle. Crc Res 25: , KAUFMAMN, R.L., HOMBURGER, H., AND WrBTH, H.: Dsorder n exctaton-contracton couplng of cardac muscle from cats wth expermentally produced rght ventrcular hypertrophy. Crc Res 28: , SPANN, J.F., JR., BUCCTNO, R.A., SONNENBLICK, E.H., AND BRAUNWALD, E.: Contractle state of cardac muscle obtaned from cats wth expermentally produced ventrcular hypertrophy and heart falure. Crc Res 21: , BINC, O.H.L., MATSUSHITA, S., FANBURC, B.L., AND LEVTNE, H.J.: Mechancal propertes of rat cardac muscle durng expermental hypertrophy. Crc Res 28: , BARTOSOVA, D., CHVAPIL, M., KORECKY, B., POUPA, O., RAKUSAN, K., TUREK, Z., AND VIZEK, M.: Growth of the musculat and collagenous parts of the rat heart n varous forms of cardomegaly. J Physol (Lond) 200: , BUCCTNO, RA, HABHIS, E., SPANN, J.F. JR., AND SONNENBLICK, E.H.: Response of myocardal connectve tssue to development of expermental hypertrophy. Am J Physol 216: , BRUTSAERT, D.L., CLAES, V.A., AND SONNEN- BLICK, E.H.: Velocty of shortenng of unloaded heart muscle and the length-tenson relaton. Crc Res 29:63-75, BOZLER, E.: Mechansm of relaxaton n extracted muscle fbers. Am J Physol 167: , Crculaton Research, Vol. XXXI, Novtmbtr 1972

10 ISOMETRIC TENSION AND ATP HYDROLYSIS EMBRY, R., AND BRIGCS, A.H.: Factors affectng contracton and relaxaton n dog glycernated cardac fbers. Am J Physol 210: , HAY Asm, Y., AND TONOMURA, Y.: Dependence of actvty of myofbrllar ATPase on sarcomere length and calcum on concentraton. J Bochem (Tokyo) 63: , HOTTA, K., AND BOWEN, W.J.: Contracton and ATPase actvty of glycernated muscle fbers and myofbrllar fragments. Am J Physol 218: , HABTSHORNE, D.J., AND DANIEL, J.L.: Importance of sulfhydryl groups for the calcumsenstve response of natural actomyosn. Bochm Bophys Acta 223: , KATZ, A.M., AND BRADY, A.J.: Mechancal and bochemcal correlates of cardac contracton. Mod Concepts Cardovasc Ds 40:39-^8, BARANY, M.: ATPase actvty of myosn correlated wth speed of muscle shortenng. J. Gen Physol 50: , PODOLSKY, R.J., AND TEICHHOLZ, L.E.: Relaton between calcum and contracton knetcs n sknned muscle fbers. J Physol (Lond) 211:19-35, HUXLEY, H.E.: Structural bass of muscular contracton. Proc R Soc Lond [Bol] 178: , MILLER, A., AND TREGEAR, R.T.: Evdence concernng crossbrdge attachment durng muscle contracton. Nature (Lond) 226: , CLEWORTH, D., AND EDMAN, K.A.P.: Laser dffracton studes on sngle skeletal muscle fbers. Scence 163: , JULIAN, F.J.: Effect of calcum on the forcevelocty relaton of brefly glycernated frog muscle fbers. J Physol (Land) 218: , DANKER, P.: Bndng of calcum and magnesum to actomyosn and ts modfcaton by natural tropomyosn. Pfluegers Arch 315: , BREMEL, R., AND WEBER, A.: Role of myosn n relaxaton and actvaton of contracton (abstr.). Bophys J 11:237a, CraUton Rut.rch, Vol. XXXI, Novmbtr 1972