Integrity f Energy Stres in Cat Papillary Muscle EFFECT OF CHANGES IN TEMPERATURE AND FREQUENCY OF NTRACTION ON HIGH ENERGY PHOSPHATE STORES By Peter E. Pl, M.D., Brian M. Chandler, M.D., Edmund H. Snnenblick, M.D., and Eugene Braunwald, M.D. ABSTRACT Althugh islated cat papillary muscles have prved useful in a variety f physilgic and pharmaclgic studies, the energy integrity f this preparatin has been questined. Accrdingly, papillary muscles (avg diam =1.3 mm) were either placed in xygenated Krebs slutin fr 1 hur at 26 C at rest r were made t cntract at varius frequencies and temperatures and fr varius duratins. In additin, samples f right ventricular muscle were btained frm living cats. Specimens were frzen in liquid nitrgen-cled ispentane fr determinatin f ATP and creatine phsphate (CP). In viv CP + ATP f right ventricular muscle was 12.9 ± 0.8 /imles/g. In papillary muscles at rest, CP + ATP was 16.7 ±0.6 /j,mles/g, significantly higher than right ventricular muscle in viv (P <.01). In vitr at 12 cntractins/minute, CP + ATP was 16.0 ± 1.0 /^mles/g, nt different frm resting papillary muscles but higher than in viv stres in right ventricular muscle. At 30 r 60 cntractins/minute, CP + ATP was reduced t 13.7 ±1.1 and 11.1 ±0.7 yamle/g. Neither increasing the in vitr temperature frm 26 t 37 C, nr extending the duratin f stimulatin frm 1 t 3 hurs at 12/minute significantly altered CP + ATP. It is cncluded that the energy stres in papillary muscles, even f mderately large diameter, are intact fr at least 3 hurs while cntracting at frequencies f 12/minute at 26 C. Further, energy stres f papillary muscle are even greater than thse fund in right ventricular muscle in viv. While muscle is cntracting at 30 r 60/minute, its energy stres may be slightly limited. ADDITIONAL KEY WORDS creatine phsphate ndensine triphsphate ventricular muscle energy stres cntractin frequency temperature heart muscle in vitr The islated cat papillary muscle has prvided an extremely useful preparatin fr physilgic and pharmaclgic studies n mammalian mycardium. Hwever, the energy integrity f this preparatin has been questined by sme investigatrs. Since papillary muscles f varius sizes have been used in studies cnducted at a variety f temperatures Frm the Cardilgy Branch, Natinal Heart Institute, Bethesda, Maryland 20014. Dr. Chandler's present address is Victria General Hspital, Halifax, Nva Scria. Dr. Snnenblick's present address is Peter Bent Brigham Hspital, Cardivascular Unit, 721 Huntingtn Avenue, Bstn, Massachusetts. Accepted fr publicatin December 21, 1967. and frequencies f cntractin, and since current understanding f mycardial functin and pharmaclgy is based t a significant extent n investigatins carried ut in this preparatin, we cnsidered it critical t define the adequacy f its energy supply mre clearly. Central t this cntrversy is the questin f whether energy supplies in the papillary muscle, in vitr, are sufficient t meet energy requirements. Adensine triphsphate (ATP), which is in equilibrium with creatine phsphate (CP), is recgnized as the immediate surce f energy fr muscle cntractin (1), and the level f the cmbined stres f CP and ATP (CP + ATP) prvides a sensitive indicatin f the balance between energy sup- CiraOaStn Reiurcb, Vl. XXII, February 1968 213
214 POOL, CHANDLER, SONNENBLICK, BRAUNWALD ply and demand. Hwever, until relatively recently, accurate estimates f high energy phsphates have been difficult t achieve because f the marked lability f these cmpunds. The imprtance f using extremely rapid freezing techniques was first demnstrated by Wllenberger and his assciates in 1958 (2). Subsequent mdificatins f this technique and imprved analytic methds have made pssible accurate determinatin f high energy phsphate stres in cardiac muscle (3-5). It was the aim f this investigatin t determine whether energy stres in islated papillary muscles f cats were adequate under cmmnly used cnditins f study. Accrdingly, the present study was designed t evaluate the high energy phsphate stres in the islated papillary muscle at varius frequencies f cntractin, temperatures, and duratins f islatin, and t cmpare these stres t the high energy phsphate stres in the intact right ventricular muscle. Methds Right ventricular papillary muscles were btained frm adult mngrel cats (2-4 kg), anesthetized by the intraperitneal injectin f pentbarbital (25 mg/kg). The muscles were placed in a mygraph previusly described in detail (6). The bathing medium fr all muscles was a mdified Krebs slutin actively aerated with 95% O 2 and 5% 2. The muscles were stimulated 1 directly via platinum wires n the inner surfaces f tine attachment clip, using squarewave pulses f 3 msec duratin with a vltage apprximately 102 abve threshld. All muscles were initially stimulated t cntract ismetrically at a frequency f 12/minute fr 30 minutes at a resting tensin less than 0.1 g, and at that temperature apprpriate t the grup in which each was included. The length-active tensin curve was determined fr each muscle and the muscle was maintained at the apex f the length-active tensin curve. Muscle diameter was calculated frm the measured length f the muscle at the apex f the length-active tensin curve. The muscles were then stimulated in ne f the six ways shwn in Table 1. There were n significant differences in average muscle diameter r f average ttal creatine cncentratin amng the six grups f muscles. At the cnclusin f each experiment the papillary muscle was rapidly frzen by quickly remving the muscle bath and replacing it with a beaker f 2-methyl-butane (ispentane) previusly cled in liquid nitrgen t 150 t 160 C. This prcedure freezes the muscle withut further cntractin. T cmpare the high energy phsphate stres existing in the papillary muscles studied in vitr with thse in the heart in viv, twelve additinal nrmal cats were anesthetized, and a thractmy was perfrmed under psitive pressure ventilatin with 1002 O 2. Then, while ptimal ventilatin and xygenatin were carefully maintained, transmycardial bipsies f the right ventricle were rapidly perfrmed with a rngeur as previusly described (7), and the samples f muscle were plunged int ispentane ( 160 C). The time between remval f the muscle and freezing was less than 0.5 secnds. The frzen papillary muscles and the right ventricular samples were stred in liquid nitrgen. At the time f analysis, each muscle was reduced t pwder with a stainless steel pestle in a small stainless steel test tube held in an aluminum blck at a temperature belw 50 C (5). The same tube was then transferred t anther aluminum blck at 0 C and 0.4 ml f 0.3 M perchlric acid was added with cntinued grinding. The mixture was allwed t thaw fr 10 minutes at 0 C. In the same tube, the mixture was centrifuged (0 C) at 25,000 X g fr 10 minutes. The supernatant fractin was remved, diluted with 3 vlumes f distilled water, and maintained at 0 C fr determinatin f CP, inrganic phsphate (P,, ATP, and creatine. Ttal creatine was determined by the alphanapthl-diacetyl methd (8, 9) after liberatin frm CP by a 9-minute acid hydrlysis at 65 C in the presence f 0.13 N HCI. CP and Pi cncentratins were determined by the Furchgtt and De Gubareff (3) mdificatin f the Fiske and SubbaRw (10) technique. A 60-minute reading fhwing acid hydrlysis f CP represented the ttal f P, plus CP. ATP was assayed by a mdificatin f the firefly luminescence technique f Strehler and McEIry (11) using firefly lantern extract. 2 Duplicate determinatins f peak luminescence were made n a phtmultiplier micrphtmeter, 3 fllwing the additin f 0.1 ml extract t 0.1 ml distilled water and 1.0 ml dilute (5 mg/ml) firefly lantern extract. T avid pssible errrs in weighing small iamerican Electrnic Labratries, Philadelphia, Pa., Mdel 104A. 2 Sigma Chemical C., St. Luis, M. 8 American Instrument C., Silver Spring, Maryland. Circulatin Research, Vl. XXII, Febnury 1968
ENERGY STORES OF PAPILLARY MUSCLE 215 tissues in the frzen state, sample weights were calculated frm the creatine cntent f the excised sample fllwing the determinatin f creatine cncentratins in tw separately btained samples f the right ventricle. The validity f this metfid has been described previusly (6). Cncentratins are expressed in yuxnles/g wet weight f tissue. Results Effect f Cntractin Frequency. At 26 C the resting papillary muscles and thse cntracting at a frequency f 12/minute had a significantly higher (P<.02) average cncentratin f CP + ATP than the specimens f right ventricle excised in viv (Table 1). The muscles cntracting at 30/minute had a cncentratin similar t that in viv, and the grup cntracting at 60/minute had a slightly but nt significantly lwer cncentratin. There was a significant depressin f the CP and ATP in the muscles stimulated t cntract at frequencies f 30 and 60/minute (P<.01) when cmpared t resting muscles. In the resting muscles and thse cntracting at 12/minute, the average cncentratin f ATP was similar t that f the specimens f right ventricle; the muscles cntracting at 30 and 60/minute had lwer cncentratins (P <.01). Significant depressins (P<.01) f average ATP cncentratin were bserved in the muscles cntracting at frequencies f 30 and 60/minute when cmpared t the resting muscles r thse cntracting at 12/minute. The average cncentratins f CP were significantly higher in the resting muscles (P <.01), and in thse cntracting at 12/minute (P<.02) and 30/minute (P<.02) when cmpared t the specimens f right ventricle, while the muscles cntracting at 60/ minute exhibited a similar value (Table 1). In the muscles cntracting at a frequency f 60/minute there was a significant depressin (P <.01) f the average CP cncentratin when cmpared t the resting grup r thse cntracting at 12/minute. Effect f Temperature.--Ttal energy stres (CP + ATP) in the muscles studied at 37 C were significantly higher (P <.01) than in viv. There were n significant differences in average cncentratins f CP, ATP r m I- fa if 81 1 1 i-h 15 r- 03 12 01 -a i i» i O + 1 t> O i-h» i I 13 16 t^ in r-t i 1 CM c»n in 05 I 16 t-- t~ c O5 O O S H * 00 <D O5 l& <-H CN "^ lo <p ii in in * O3 <N t-; i-j ^ c en t- O c c * 3 00 t"» CM <N CS (M eq M 2 i-h rh SSSSSfe '''' 03 3! 1O c C H c c CircuUlin Rtstarb, Vl. XXII, Febttury 1968
216 POOL, CHANDLER, SONNENBLICK, BRAUNWALD Electrn micrgraph f central prtin f a cat papillary muscle islated in Krebs slutin fr 3 hurs at a length crrespnding t the apex f its length-active tensin curve while being stimulated at a frequency f 12/minute at 26"C. Length 6.0 mm; wet weight = 9.2 mg; diameter = 1.4 mm. CP + ATP, in muscle diameters, r in ttal creatine cncentratins between the muscles studied at 37CC and thse studied at 26 C (Table 1). Effect f Length f Cntractin Perid. The CP + ATP in muscles stimulated fr 180 minutes at a frequency f 12/minute at 26 C was significantly higher (P<.01) (Table 1) than in the samples f right ventricle. There were n significant differences in average cncentratins f CP, ATP, r CP + ATP between muscles stimulated at 12/minute at 26 C fr 60 and 180 minutes (Table 1). Effects n Ultrastructure.--T determine whether the ultrastructure f the papillary muscle was altered fllwing an extended perid f islatin, three muscles were fixed in glutaraldehyde fllwing a 3-hur perid f islatin during which they were stimulated t cntract at a frequency f 12/minute at 26 C. Examinatin f the central cre f these muscles by electrn micrscpy revealed n abnrmalities in either mitchndrial r myfibrillar structure (Fig. 1). Circulatin Restercb, Vl. XXII, Ptbmmf 1968
ENERGY STORES OF PAPILLARY MUSCLE 217 Discussin In mst investigatins emplying cat papillary muscle, prcedures altering mechanical activity are emplyed. The questin naturally arises whether r nt the muscle's respnse t the prcedure is limited by the availability f energy stres and whether the prcedure in questin alters energy stres primarily and the physilgic state secndarily. Bth frm studies f xygen cnsumptin and calculatins f xygen diffusin, Lee (12) and Whalen (13, 14) cncluded that papillary muscles with diameters up t 1.2 mm culd be xygenated adequately. Hwever, these limits were seriusly questined by Cranefield and Greenspan (15), wh cncluded that adequate xygenatin f cat papillary muscle expsed t 95% O 2 at 35 C in vitr culd be accmplished nly with muscles less than 0.64 mm in diameter when resting and less than 0.45 mm in diameter when cntracting. On the basis f these rigid criteria, the papillary muscles used in mst studies wuld be cnsidered inadequately xygenated. This cnclusin was based n the finding that thin papillary muscles cnsumed mre xygen per unit weight than did thicker muscles and that stretching f the papillary muscle increased its xygen cnsumptin. In additin, Fisher et al. (16) fund that the maximum ptentiated frce develped by papillary muscles was prprtinal t crss-sectinal area in muscles up t 0.6 mm nly. On the ther hand, in this labratry we have fund that the maximum ptentiated frce f cntractin in the cat's papillary muscle (at 30 C at 12/minute) is linearly related t crss-sectinal area in muscles up t 1.3 mm in crsssectinal area. Further, in nnptentiated muscles, Kch-Weser (17) reprted that the tensin develped, crrected fr muscle diameter, was independent f muscle diameters less than 1.1 mm when stimulated at a frequency f 19/minute at 38 C and less than 0.86 mm in diameter when stimulated at a frequency f 188/minute. In the present study, the high energy phsphate stres were unchanged in papillary muscles at 26 C cntracting at a frequency f Circulatin Restarcb, Vl. XXII, Ftttruary 1968 12/minute when cmpared t muscles resting under these cnditins (Table 1). In additin, these stres were nt reduced by increasing the temperature f the bath t 37 C. Indeed, the high energy phsphate stres in these papillary muscles studied in vitr were even higher than thse fund in the right ventricle studied in viv under ptimally xygenated cnditins at a heart rate f apprximately 100/minute. The nly previusly reprted cncentratins f high energy phsphate cmpunds in feline mycardium were by Lee et al. (18). In resting papillary muscle, they fund cncentratins f ATP t be 2.45 jumles/g and f CP t be 3.89 ^mles/g, values cnsiderably lwer than thse fund in the present investigatin. It is als interesting t nte that the CP stres fund in viv are nt as high as the muscle is capable f attaining. This finding suggests that the high energy phsphate stres are balanced between energy prductin and energy utilizatin. The transfer f energy frm CP t ATP and the subsequent breakdwn f ATP t adensine diphsphate and inrganic phsphate appears t serve as a feedback f intermediary metablism, and thus this balance als serves as a regulatry functin (19). It wuld be anticipated that in muscles with large diameters a central cre f anxia wuld ccur at sme levels f activity, and that this wuld result in a decline in the ATP cncentratin. Indeed, this is the mst prbable explanatin fr the lwer cncentratins f ATP bserved in the muscles studied at higher frequencies f cntractin (30 and 60/minute) than in resting muscles r muscles cntracting at 12/minute. On the ther hand, in adequately xygenated preparatins, bth in viv and in vitr, variatins in CP may reflect an increase in energy demands but ATP levels are maintained because f the favrable equilibrium tward the latter. In papillary muscles studied under the cnditins f the present study, a central cre f relative hypxia may exist, as has been suggested in sme studies f xygen cnsumptin (15). This relative hypxia, hwever,
218 POOL, CHANDLER, SONNENBLICK, BRAUNWALD des nt seem t be sufficient t cmprmise the prductin f energy t an extent that wuld alter ttal energy stres, at least under cnditins f rest r stimulatin at a frequency f 12/minute. Als, n ultrastructural changes ccurred under these cnditins. On the ther hand, the decreases in ATP stres nted abve when the stimulus frequency was increased t 60/minute may reflect mre severe hypxia r anxia. Althugh the present results wuld suggest that the diffusin f xygen int the papillary muscle was adequate t maintain average energy stres at levels exceeding thse existing in the right ventricular muscle in viv under the cnditins nted abve, the questin f whether diffusin f ther substrates is adequate has nt been answered. It has been shwn previusly that acute hypxic heart failure may be prduced in dgs at a time when high energy phsphate stres are nt affected by this hypxia (4), and these undefined effects may be present in the papillary muscle having intact energy stres. On the ther hand, as nted abve, recent studies indicate that papillary muscles up t a diameter f 1.3 mm are nt limited in their mechanical perfrmance. It is therefre cncluded that the energy in cat papillary muscle f mderately large diameter (1.2 t 1.4 mm) islated in vitr in the presence f 95 O 2 is intact fr at least 3 hurs when stimulated at a frequency f 12/minute at 26 C. At a frequency f 60/ minute, energy stres are clearly limited, cmpared t resting papillary muscles r thse cntracting at a frequency f 12/minute, while at a frequency f 30/minute these stres are essentially identical t thse ccurring in right ventricular muscle in viv, but are lwer than thse bserved in resting papillary muscles. EDITORS' NOTE: The Editrs f Circulatin Research believe that the cmparisns between levels f ATP and CP in papillary muscle at rest and papillary muscle cntracting at varius rates and under different cnditins are valid, but that cmparisns between the energy stres in vitr papillary muscle and in viv right ventricular muscle require further basic knwledge befre they are meaningful. Acknwledgment The expert technical assistance f Shirley C. Seagren and Nancy Dittemre is gratefully acknwledged. References 1. CAIN, D. F., AND DAVLES, R. E.: Breakdwn f adensine triphsphate during a single cntractin f wrking muscle. Bichem. Biphys. Res. Cnunun. 8: 361, 1962. 2. WOLLENBERCER, A., KRAUSE, E. G., AND WAHLER, B. E.: Orthphsphat und Phsphkreatingahalt des Herzmuskels. Natuxwissenschaften 45: 294, 1958. 3. FOHCHGOTT, R. F., AND DE GUBAKEFF, T.: Determinatin f inrganic phsphate and creatinine phsphate in tissue extracts. J. Bil. Chem. 223: 337, 1956. 4. POOL, P. E., VELL, J. W., CHIDSEY, C. A., AND BRAUNWALD, E.: Mycardial high energy phsphate stres in acutely induced hypxic heart failure. Circulatin Res. 19: 221, 1966. 5. POOL, P. E., AND SEAGREN, S. C: LOW temperature extractin f small samples f tissue. Chemist-Analyst 56: 38, 1967. 6. POOL, P. E., AND SONNENDLICK, E. H.: Mechanchemistry f cardiac muscle: I. The ismetric cntractin. J. Gen. Physil. 50: 951, 1967. 7. POOL, P. E., SPANN, J. F., JR., BUCCINO, R. A., SONNENBLICK, E. H., AND BRAUNWALD, E.: Mycardial high energy phsphate stres in cardiac hypertrphy and heart failure. Circulatin Res. 21: 365, 1967. 8. ENNOR, A. H., AND ROSENBERG, H.: Determinatin and distributin f phsphcreatine in animal tissues. Bichem. J. 51: 606, 1952. 9. ENNOR, A. H.: Determinatin and preparatin f N-phsphates f bilgical rigin. In Methds in Enzymlgy, vl. 3, edited by S. P. Clwick and N. O. Kaplan, New Yrk, Academic Press, p. 850, 1957. 10. FISKE, C. H., AND SUBBAROW, Y.: Phsphcreatine. J. Bil. Chem. 81: 629, 1929. 11. STREHLER, B. L., AND MCELROY, W. D.: Assay f adensine triphsphate. In Methds in Enzymlgy, vl. 3, edited by S. P. Clwick and N. O. Kaplan. New Yrk, Academic Press, p. 871, 1957. 12. LEE, K. S.: New technique fr the simultaneus recrding f xygen cnsumptin and cntractin f muscle: Effect f uabain n cat papillary muscle. J. Pharmacl. 109: 304, 1953. 13. WHALEN, J. W.: Relatin f wrk and xygen cnsumptin in islated strips f cat and rat mycardium. J. Physil. 157: 1, 1961. 14. WHALEN, W. J., AND FANGMAN, J.: Respiratin f heart muscle as affected by xygen tensin. Science 141: 274, 1963. 15. CRANEFIELD, P. F., AND GREENSPAN, K.: Rate f Circulatin Riswch, Vl. XXII, Pttmirj 1968
ENERGY STORES OF PAPILLARY MUSCLE 219 xygen uptake f quiescent cardiac muscle. J. Gen. Physil. 44: 235, 1960. 16. FISHER, V. J., LEE, R. J., MAHLOW, A. M., AND KAVALER, F.: Paired electrical stimulatin and the maximal cntractile respnses f the ventricle. Circulatin Res. 20: 520, 1967. 17. KOCH-WESEH, J.: Effect f rate changes n strength and time curse f cntractin f papillary muscle. Am. J. Physil. 204: 451, 1963. 18. LEE, K. S., YU, D. H., AND BUBSTEIN, R.: Effect f uabain n the xygen cnsumptin, the high energy phsphates and the cntractility f the cat papillary muscle. J. Pharmacl. Exptl. Therap. 129: 115, 1960. 19. WILLIAMSON, J. R,: Metablic cntrl in the perfused rat heart In Cntrl f Energy Metablism, edited by B. Chance, R. W. Estabrk, and J. R. Williamsn, New Yrk, Academic Press, 1965, p. 333. CircuUtin R.-uarcb, Vl. XXII, Pebrtury 1968