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42 J AM COLL CARDIOL \983.\ :42- 5 1 Regulation of Myocardial Contractility 1958-1983: An Odyssey ARNOLD M. KATZ , MD , FACC Farmington, Connecticut The past 25 years have seen an unprecedented growth in our understanding of the mechanisms responsible for the regulation of myocardial contractility. Beginning with a demonstration that myocardial contractility represents an important determinant of cardiac function, this quarter century has witnessed a series of attempts to explain length-independent changes in myocardial contractile function. Alterations in the properties of the cardiac contractile proteins and changes in the amount of calcium (Ca 2 +) available for binding to the contractile proteins during excitation-contraction coupling are now recognized as important biochemical bases for physiologic, Sing in me, Muse. and through me tell me the story of that man skilled in all ways of conte nding. the wanderer. harried for years on end, after he plundered the stronghold of the proud heigh t of Troy. He saw the townlands and learned the minds of many men. and weathered many bitter nights and days in his deep heart at sea . . . . Homer. The Odyssey . Bk I As the wanderings of Odysseus revealed many wonders. so have the pursuits of those who , over the past quarter century. searched for an understanding of myocardial contractile function and its regulation . Their intellectual journey to uncover the " true " meaning of myocardia l contractility was begun shortly afte r the collapse of a theory of cardiac regulation that had dom inated scie ntific thought for almost half a century and has been no less fantastic than the legendary wanderings of Od ysseus. This brief and personal history begins in 1954 when. as a junior medical student, I attended a sympos ium organized on behalf of the American Physiological Society by my father. Loui s N. Katz. Thi s meeting toppled the then held view that the pumping action of the heart was controlled From the Cardiology Division . Department of Medicine. University of Connecticut Health Center, Farmington. Connectic ut. Supported by Research Grants HL-21812 and HL-22 l 35 from the National Institutes of Health , Bethesda, Maryland . Address for reprints : Arnold M. Katz, MD , Cardio logy Division . Department of Medicine , University of Connectic ut Health Center. Farmington , Connectic ut 06032. ~ 1983 by the American College of Cardiology pharmacologic and pathologic changes in myocardial contractility. Identification of the central role of Ca 2 + in the initiation of the cardiac contractile process has made possible the analysis of the mechanisms by which several drugs alter myocardial contractile function, including the biochemical processes that allow beta-adrenergic agonists to enhance contractility. The rapid developments in this fieldsince 1958 promisean even greater flow of new knowledge regarding the causes, prevention and treatment of heart failure provided that there is given adequate support for such research activities. excl usively, or even primarily , by changes in the end-diastolic fiber length of the myocardial fibers (I) . As magnificent (and doomed) as the city of the Trojans. the view that the regulation of cardiac work could be explained excl usively or primar ily by the Frank-Starling relation had dominated the thinking of cardiac physiologists since the classic studies of Starling performed during the second decade of this century . Althou gh data challengi ng the primac y of Starling ' s law had been publ ished. it remained generall y accepted that the major determin ant of cardiac work was the end-diastolic volume in the vent ricles. Evidence presented at the 1954 symposium. however. demonstrated conclusively that "end-diastolic volume , while playing an important role in the work performance of the heart. is only one of a number of equally important factors adjusting the heart' s power to changing conditions" (2) . Drawing on earlier co ncepts of Dow and McMichael , Sarnoff (3) bro ught into focus the interrelations between Starling 's law and the new concept of myocardial contractility with the statement, " .. , it has not been genera lly apprecia ted that a single Starling curve cannot always explain the observed phenomena: for any give n heart there is a series or family of curves" (Fig. I). With th is clear statement. the next generation of cardiovascular physiologists-my generation-was drawn to the searc h for an understanding of myocardial contractility, Energy Production I might have made it safely home. that time, but as I came round Malea the current 0735- 1097/83/0 10042-10$03.00 REGULATION OF MYOCARDIAL CONTRACTILITY took me out to sea. and from the north a fresh gale drove me on , past Kythera . Nine days I drifted on the teeming sea before dangerous high winds. Upon the tenth we came to the coastline of the Lotos Eaters. ... who showed no will to do us harm. only offerin g the sweet Lotos to our friendsbut those who ate this honeyed plant . the Lotos. never cared to report , nor to return : they longed to stay forever . browsing on that native bloom forgetting their homeland . Homer , The Odvssey, Bk IX Because cardiac contraction is an energy-consuming process, it was logical that early attempts to understand the mechanism for the regulation of myocardial contractility focused on changes in the availability of chemical energy for the contractile process . As early as 1949, evidence had been obtained that high ener gy phosphate content remained normal during the acute failure of isolated heart preparati ons (4), although a decline in both adenosine tripho sphate (ATP) and phosphocreatine concentrations was noted in the chronically overloaded heart (5) . While a decrease in high energy phosphate compounds remains a plausible explanation for some form s of heart failure. notably ischemi c and hypoxic (6), it now seems a fair generalization that under most physiologic conditions, changes in total high energy phosphate content are the result of alterations in the level of cardiac work (7) rather than a primary determ inant of the work capacity of the myocardium. In the case of the powerful positive inotropic effect of the catecholamines (Fig. I), for Figure 1. Effect of epinephrine (suprarenin) on the work of the heart. illustrating the effect of the beta-adrenergic agonist on the Starling curve. GM . M. = gram-meter; LT. = left; L.V. = left ventricular. (Reprinted from Sarnoff SJ [3], with permission.) 60 .-CONTROL X-SUPRARENIN 1.36 j.Lg/KG .lMIN. 50 L.v. 40 STROKE WORK GM.M. O-CONTROL J AM CaLL CARDIaL 1983;1:42- 51 43 example , it is clear that the enhancement of myocardial contractility is not due to a primary effect to increase energy production (8). A similar conclusion regarding the positive inotropic effect of the cardiac glycosides was report ed by R. Bing (9) in the first volume of the American Journal of Cardiology . He wrote : "[Digitalis] increases the force of myocardial contraction . . . . How this is accompli shed is not clear, j ust as the fundamental reasons for impaired muscular funct ion in clinical heart failure are obscure . . . . It does not affect the (chemical) ener gy forming mechani sm of the cardi ac cell .. . it improves the dimini shed response of the total contractile mass of the failing heart to an undimin ished supply of chemical energy. . . ." Heart Failure Myosin In the next land we found were Kyklopes. giants. louts. without a law to bless them. . . . Kyklopes have no muster and no meeting. no consultation or old tribal ways . but each one dwell s in his own mountain cave dea ling out rough justice to wife and child. indifferent to what the others do. Homer . The Odyssey. Bk IX The demonstration that the work of the heart could be regulated by length-independent change s occurring within the myocardium led a number of investigators to examine the properties of the cardiac contractile protein s under conditions of altered myocardi al contra ctility. During the late 1950s and early 1960s. chan ges in the structure of the cardiac myosin molecule were reported to occur in the failing heart , and one group ( 10) reported that the positive inotropic effect of cardiac glyco sides was accomp anied by a rever sal of the physicochemical abnormalities in "heart failure myosin ." Controversial when first described (11), these obser vations were shown to be incorrect by subsequent work that dem onstrated that the overall molecular characteri stics of the myosin molecule were not altered in a manner that could explain either the effects of heart failure or drugs on myocardial contractility (for review, see reference 12). Role of Myosin Adenosine Triphosphatase in Determining Muscle Shortening Velocity 30 20 When Dawn spread out her finger tips of rose we turned out marvelling , to tour the isle. while Zeus's shy nymph daughters flushed wild goats down from the heights-a breakfast for my men. Homer, The Odyssey. Bk IX 10 o~-"""'_"""_-""---"""'----l"---~------' 10 20 LT. AURICLE MEAN PRESSURE (CM. H20) In the late 1950s , a time of intense controvers y regardin g the exi stence of physicochemical abnormalities in heart failure myosin, several investigators noted that the adeno sine J AM cou, CARDIOl 1983:I:42-51 44 KATZ triphosphatase (ATPase) activity of cardiac myosin was less than that of rabbit fast (white) skeletal muscle, the "standard" source of this contractile protein. In 1966, my colleagues and I (13) confirmed these reports that cardiac myosin had a lower ATPase activity than rabbit skeletal muscle myosin, and obtained evidence that the low enzymatic activity of cardiac myosin might be related to the low shortening velocity of the intact myocardium. A now classic study by Barany (14) related myosin ATPase activity to the mechanical expression of this enzymatic rate by showing a close correlation between the rate of ATP hydrolysis by myosin and the maximal shortening velocity in a variety of different muscles (Fig. 2). At a time when studies of cardiac mechanics had come to dominate our view of the physiologic expression of myocardial contractility (see later), and shortly after A.F. Huxley (15) had provided a theoretical basis for understanding the relation between the rate of cross bridge turnover and muscle shortening velocity, Barany's observations provided a clear biochemical explanation for the early observations of A.V. Hill (16) that "the rate at which chemical transformation would occur, and therefore at which energy would be liberated, would ... [increase) as the force diminished. " The direct relation between myosin ATPase activity and the maximal shortening velocity of unloaded muscle has Figure 2. Relation between myosin adenosine triphosphatase (A'l'Pasc) activity and maximal shortening velocity of a variety of muscles, based on data of Barany (14). (Reprinted with permission from Katz AM. Contractile proteins in normal and failing myocardium. Hosp Pract 1972:7:5769.) 24 M~. ,~...Io gito rum '0..1// 20 / Rat Extensor Digitorum Longus / 2 /-- Cat Fast / 8 i/ Mytilus Posterior Adductor V----. / / / V Mouse Soleus c---- Rat DiaPhralgm / Frog Sartorius v·~""'''' .-Human E1bo1 Flexor _ _ Cat Soleus Pecten IStrlated I Adduct~r Frog Sartorius Dogfish Corac~hyold Tortoise lliofibulans o 10 A1Pase Activrtv In Presence of 15 20 Ca"" (,LLmoles Plfgm/secl 25 been amply confirmed during the past decade, and now represents a firm basis for an understanding of one aspect of myocardial contractility and its tonic regulation (17). In the failing, chronically overloaded heart, for example, a decrease in maximal shortening velocity can be explained by a redirection of protein synthesis that leads to the appearance of a myosin isozyme having a low ATPase activity (18) and subtle alterations in amino acid sequence (19). Similarly, changes in maximal shortening velocity that occur in the hearts of hyperthyroid and hypothyroid animals are paralleled by changes in myosin ATPase activity (20). Cardiac Mechanics . . . inside her quiet house they heard the goddess Kirke. Low she sang in her beguiling voice. while on her loom she wove ambrosial fabric sheer and bright, by that craft known to the goddesses of heaven. ... On thrones she seated them. and lounging chairs. while she prepared a meal of cheese and barley and amber honey mixed with Pramnian wine. adding her own vile punch to make them lose desire or thought of our dear father land. Scarce had they drunk when she flew after them with her long stick and shut them in a pigstybodies, voices. heads and bristles. all swinish now .... Homer. The Odvssev, Bk X Force-velocity relation. Shortly before the role of myosin ATPase activity in determining the shortening velocity of cardiac muscle had become the subject of intensive study, there appeared an approach to the characterization of myocardial contractility that came to dominate the thinking of cardiovascular physiologists for more than a decade. This was the utilization of the classic force-velocity relation, which describes the relatively stable tetanic contractions of frog sartorius muscle (16.21) as characterizing the more dynamic situation during contraction of cardiac muscle. In 1959, Abbott and Mommaerts (22) published the first characterization of the mechanical behavior of cardiac muscle that used the force-velocity relation as a basis for data analysis. This report, however, noted several possible pitfalls in the application of the muscle mechanics observed in tetanized skeletal muscle to the much more complex, twitchlike contraction of the heart. Three years later, Sonnenblick (23) presented a more extensive study of this subject at the annual meeting of the American Physiological Society (Fig. 3). Shortly after the importance of changing myocardial contractility in regulating cardiac performance had been recognized, Sonnenblick's report played a seminal role in bringing together the work then in progress on the biochemistry of the contractile proteins and efforts to define the basis for the changing hemodynamic properties of the J AM cou. CARDIOL 1983:1:42-51 REGULATION OF MYOCARDIAL CONTRACTILITY EFFECTS OF INTERVENTIONS ON THE FORCE VELOCITY RELATIONS OF THE CAT PAPILLARY MUSCLE V mall INCREASING LENGTH INCREASING RATE NOREPINEPHRINE t tt Po tt tar - t Figure 3. Diagrammatic representation of force-velocity relation of cardiac muscle. (--) control: ( - - - - ) increasing Po: (...... ) increasing Vm " . (Reprinted from Sonnenblick EH [23]. with permission.) heart in the intact animal and human beings. Although subsequent developments have generally supported the earlier caution of Abbott and Mommaerts, Sonnenblick's presentation stimulated important efforts to integrate biochemical and physiologic approaches to an understanding of myocardial contractility. Down to the shore and ship at last we went bowed with anguish. cheeks all wet with tears. to find that Kirke had been there before us and tied nearby a black ewe and a ram: for she had gone by like air. For who could see the passage of a goddess unless she wished his mortal eyes aware? Homer. The Odyssey. Bk X Controversies concerning Vmax' A review of the controversies regarding the interpretation of the force and velocity data obtained in cardiac muscle is beyond the scope of this article, but a delightful tongue-in-cheek description 45 of these early years by Harris (24) is highly recommended. These controversies focused on three issues: I) the validity of the extrapolations of velocity measurements made at various muscle loads to the shortening velocity at zero load (V maJ (25). 2) the ability of such extrapolated values for V max to serve as quantitative measurements of myocardial contractility (26), and 3) the relation of these V max determinations to the level of myosin ATPase activity (27). Although none of these issues has been settled to the satisfaction of all workers in these fields. these controversies have gradually drifted to the background as the complexity of cardiac muscle mechanics has become recognized. These complexities, and the ambiguities in the interpretation of velocity data calculated by the use of various equations applied to pressure measurements obtained during isovolumic contractions. have led to a general abandonment of Vmax and related indexes for quantifying the state of the myocardium in patients with heart disease. Ejection fraction, The failure of cardiac mechanics as a clinical tool is eloquently demonstrated by the fact that cardiologists have recently turned to the ejection fraction, an index obtained readily by noninvasive methods, as a means to evaluate the overall pump function of the heart. Although the ejection fraction is of proved value in the classification of patients with heart disease, our continuing inability to quantify myocardial contractility is evidenced by the current (and to this writer, distressing) tendency to use ejection fraction as a measure of the contractile state of the heart muscle despite the obvious and major effects of extramyocardial variables, such as heart rate and peripheral resistance, on the ratio between stroke volume and enddiastolic volume. Role of Potassium in Determining Myocardial Contractility Swiftly she turned and led him to her cave. and they went in, the mortal and immortal. ... where the divine Kalypso placed before him victuals and drink of men: then she sat down facing Odysseus, while her serving maids brought nectar and ambrosia to her side. Then each one' s hands went out on each one' s feast until they had had their pleasure; and she said: 'Son of Laertes, versatile Odysseus, after these years with me, you still desire your old home? Even so, I wish you well. If you could see it all, before you goall the adversity you face at seayou would stay here and guard this house. and be immortal. .. .' Homer. The Odyssey. Bk V Twenty-five years ago, attempts by physiologists to relate changes in potassium (K +) fluxes across the myocardial cell membrane to variations in the contractile state of the myocardium represented an interesting but erroneous effort to 46 J AM COLL CARDIOL 19R3;I ''+2- S1 ex plain bea t to bea t changes in myocardial contractility . Th e idea that changing K + level s wit hin the ce ll could influence myocardial contrac tility had its origi ns iii the classic studie s of Szent-Gyorgyi (28) who obse rved that high K + concentration s reduced the interaction s between actin and myosin in vitro. The often cited study by Hajdu (29), in which the force ge nerated from isol ated ca rdiac muscle prep aration s was found to decrease by interventions that led to K + uptake by the heart and to incre ase when K + left the myocard ial ce lls , coupled with Szent -Gyorgyi' s observations , served as the basis for a widely held hypothesis that changes in cytosol ic K + were directl y responsible for changes in myoca rdial contractility. However, prop onent s of this theory had overloo ked the fact that Hajdu (29) had also found that K + efflux was accompanied by a slight net influx of sodium (Na + ) and, more importantl y . a corresponding loss of water, and that intracellular K + concentration hardl y changed. Furtherm ore, the inhibitory effe cts of K + and Na + on the interaction s between the contrac tile prote ins were alm ost ident ical (30) . Th ese obse rvations , coupled with the rapid grow th of our understanding of the primary role of calcium (Ca~ + ) in the control of the contractile process , led to the gradual aband onment of the conce pt that myocardial contract ility was importantly regul ated by changes in cellular K + content. Calcium and Activation of the Cardiac Contractile Proteins A seco nd course lies betwee n the headlan ds. One is a sharp mountain piercing the sky, with stormcl oud round the peak .. , , No mortal man could scale it. nor so much as land the re . not with twenty hands and feet , so sheer the cliffs are as of pol ished stone. , . , that is the den of Skylla, where she yaps abomi nably. a newborn whelp ' s cry. ' , . , The opposite point seem s more a tongue of land you 'd touch with a good bowshot at the narrows. A great wild fig. a shaggy mass of leaves. grow s on it. and Khary bdis lurks below to swa llow dow n the dark sea tide . Ho mer. The Odvssey , S k XII Our present understanding of the prim ary role that Ca 2 + plays in the regul ation of myocard ial contractilit y arose from a series of observations that began a ce ntury ago when Ringer (3 1) found that this cation was esse ntial for cardiac contraction. Th e finding of He ilbrunn and Wiercinski (32) that injec tion of Ca2+ into sing le muscle fibers caused co ntrac tion pro vided concrete evide nce that this esse ntial role of Ca2 + in myocardial contract ion was due to an action within the cell. Thi s led Sand ow (33) in 1952 to formulate a hypothesis in which Ca 2 + release d from the cell membrane activated the contractile pro cess. KATZ "Soluble relaxing factor." The critical role of Ca 2 + in contrac tile activation found additional ex perimental support in obse rvations made dur ing the 1950s with a "soluble relaxing factor" prepared fro m ske letal muscle homogenates. Th is factor ca used relaxation in a variety of actomyosin preparations. an effec t that co uld be reversed by the additio n of sma ll amounts of Ca 2 + . At that time , however. knowledge of both the nature of the relaxing factor and the actions of Ca 2 + on the co ntractile protein s was in a state of confus ion and controve rsy. Mechanisms. Th e mechanism by which Ca2 + regulates mu scle contraction and the nature of the action of the "soluble relaxing factor" were clarified by two observations. both described in the early 1960s. The first to be defined was the nature of the "soluble relaxing factor," which turned out not to be soluble. but instead to consist of membrane vesicles pre sent in the supernatants obtained after the relatively low speed centrifugations generally used in the 1950s. These membrane ve sicles. derived from an intrace llular membrane structure . the sarcoplasmic reticulum . were found to transport Ca 2 + into their interior . thereby ex plaining both their actio n to relax actomyosi n preparation s and the ability of added Ca 2 + to reverse their relaxing effect. As was the rule in the field of muscle biochemistry, these observation s were first made in skeletal muscle (34.35) and shortly thereafter in ca rdiac muscle (36 -40) . The second observation that defined the role of Ca 2 + in regulating myocardial contractility stemmed from an understand ing of the interaction between Ca 2 + and the co ntracti le proteins. A critica l observation was made by Weber and Winicur (4 1). who clar ified the confusing effects of Ca2 + on skeletal muscle acto myosi n by defining a role for micromo lar Ca 2 + conc ent ration in activating its Mg 2 + -activated ATPase activity. Th eir paper also introduced the use of the Ca 2 + -chelator (EGTA). whose high specificity for Ca 2 + -binding in the pre sence of magnesium (Mg 2 + ) allowed Ca2 + concentration to be regulated experimentall y in the micromolar range. . . . we rowed into the strait. Skylla to port and on our starboa rd beam Kharybd is .. .. Homer. The Odyssey . S k XU Ca 2 + and the troponin-tropomyosin complex. Characterization of the Ca 2 + -sensitivity of actomyosin, which of course ex pla ined the Ca2 + -dependent action of the ' 'soluble relaxing factor," led to intensive inves tigation of the mechani sm by which Ca 2 + activa ted the contractile prote ins (see reference 12 for review) . Within a few years I had found (42) that trop omyosin. a protein described in 1948. was able to regulate the interaction between actin and myosin. More important was the discovery by Ebashi and co-workers (43) that troponin, a newl y discovered muscle protein. along with tropomyosin could sen sitize actomyosin to changes in J AM cou, CARDIOl 1983;I:42- 51 REGULATION OF MYOCARDIAL CONTRACTILITY B A ~ C 0.400 =t E o <0 <0 Figure 4. Effects of regulatory proteins on the EGTA (GEDTAl -sensitivity of superpre cipitation (an in vitro model of contraction) by reconstituted actom yosin. (e l low Ca~ + ; (0) Ca ~ + present. A, Troponin alone added . B, Tropom yosin alone added . C, Troponin plus tropom yosin added . (Reprinted from Ebashi S. Kodama A [45], with permission.) <; 0.300 Control ~ r • )- '::: C/l z 47 Control 0 .200 ...J <l: GEDTA o f= g; 0.100 _--'-_~!_~I- 2 3 I I I 246 I 8 TIM E (minutes) Ca2 + concentration in the micromolar range (Fig. 4) (44,45). An interesting reflection of Ebashi ' s attitude during this phase in the development of our knowledge of the contractile proteins can be seen in a letter that I received in 1965 from Ebashi, who was aware of my effort s to explain why highly purified tropomyosin failed to sensitize the reconstituted actomyosins made from highly purified actin and myosin to Ca 2 + (46). In this letter, Ebashi suggested that a protein mixture I had found to sensitize actomyosin to Ca 2 + contained troponin , and he also sent a draft of his unpublished description of this protein , giving me permission to use his unpubli shed methods. His characterization of the activating effect of Ca2+ on skeletal muscle contractile protein s as a result of an interaction of this cation with the troponintropomyosin complex made it possible for us to show that a similar effect also occurred in the cardiac contractile proteins (Fig . 5) (47). We rowed on. The rocks were now behind ; Kharybdis too . and Skylla dropped astern. Then we were coasting the noble island of the god. where grazed those cattle with wide brows. and bounteous flocks of Helios, lord of noon . who rides high heaven. Horn er, The Odyssey . Bk XII Thi s rapidly unfolding series of discoveries, which occurred during the early 1960s, established that the appearance of CaB in the cytosol represented the biochemical basis for the physiologic activat ion of cardiac contraction. A more far-reaching consequence of this advance in our knowledge of the control of cardiac contraction was to provide a new understanding that variations in the availability of Ca 2 + for binding to troponin could be responsible for .4,---.-- - - - -- - - - - - , a. Skeleta .02'- r-- - - - - - - - - - ....., b Car diac l M yos in M y 0 51 n • Sxeteta I Actin (~ E c E Figure 5. Effects of Ca" + (plotted versus pCa. which is -log[C a! +]) on the adenosine triphosphatase (A'I'Pase) activity of skeletal (a) and cardiac (h) myosins ( - - - - ) and on the corre sponding actornyos ins prepared with actins containing troponin and tropomyosin from skeletal (e) and cardiac (0) muscle. (Reprinted from Katz AM . Repke DI. Cohen BR [47], by permissio n of the American Heart Association . lnc .) 0 """, Actm (~ o a, a:Cl> '" j '0 ~ .2 .01 j e ~ o~ e---e-- - - - - _ / e eo_ _ ~------ i _ o -I-z z-,- -,.--,---,-----i E 8 6 4 Jc--r-----r---------.----l E 8 6 4 48 J AM COLL CARDIOL I<.:xrz 1983:1:42-5 1 impo rtant phasic , beat to beat. changes in myocardial contractil ity ( 17). Control of Calcium Availability for Activation of the Cardiac Contractile Proteins Now six full days my galla nt crew cou ld feast upon the prime beef they had marked for slaughter from Hellos" herd ; and Ze us, the son of Kronos, added one fine morn ing. All the gales had cease d, blown out, and with an offshore breeze we launched again , steppin g the mast and sail. to make for the open sea . . . . Homer. The Odyssey. Bk XII Reco gnition of the mechanism by which Ca 2 + activated the ca rdiac co ntractile proteins provided a clear direction for studies of the phasic regulation of myocardial contractility. The se studies were guided in part by the recognition that two Ca2 + pools could be used for this activation: the extracellul ar space and the sarco plasmic reticulum. Thu s. an incre ase in myocardial contractility could be attributed to an increased Ca 2 + influx into the cell across the sarcolemma. an incre ase in the amount of Ca 2 + released from the sarcoplasmic reticulum , or both. Research into these two mechanisms differed in approach, however. Fluxes of Ca 2 + across the sarcolemma were ge nerally studied by measurements of tracer Ca movements into or out of intact cardiac cells, or by electroph ysiologic studies that characterized the charge movements associated with these Ca2 + fluxes. In contrast. Ca 2 + fluxes into and out of the sarco plasmic reticulum were measured by biochemical techn ique s, using isolated sarcopl asmic reticulum vesicles pre pared from cardiac muscle homogenates . Slow inward Ca 2 + current and level of myocardial contractility. By the earl y 1960s. Hodgkin and Huxle y' s magn ificent characterization of the ionic currents respon sible for the action potential in the squid axo n had been applied to the heart , and a model of the more compl ex cardiac action potential had been constructed assuming that. as in the nerve . Na + ions contributed the only import ant inward curre nt (48) . However , subsequent work showed that although the initial depolarization of the myocard ium could be explained by a fast inward Na 2 + current. a second slow inward current carried largely by Ca 2 + ions was responsible for the plateau phase of the cardiac action potential (49.50). and that the extent of Ca 2 + ent ry by way of this slow inward current was directl y related to the level of myocardial contractility (50) . Thu s, the discovery of the slow inward current provided a major brid ge linkin g the physiolog y of excitation to the biochemistry of contraction. Identification of these sarcolemmal Ca 2 + channels provided the basis for several importan t therapeuti c adva nces in treatment of the patient with heart disease. notably the development of the calcium channel blockers and an understandin g of their interpl ay with the beta-adr energic receptor blocking age nts. Since the discovery that the " soluble relaxing factor " was a sarcoplasmic reticulum membrane fraction having its relaxing ability due to the active transport of Ca 2 + into the vesicle interior. a large bod y of know ledge characterizing the Ca 2 + pump ATPase protein in both skeletal and card iac muscle sarcoplasmic reticulum has been accumulated (5 1,52). Much less is known of the biochem ical mechanisms by which Ca 2 + mo ves down its elec trochemical grad ient out of these membranes to initiate the contractile proce ss, although we have obtained ev idence that the Ca 2 + pump ATP ase protein also can medi ate Ca 2 + release from the sarcoplasmic reticulum (53.54). Molecular Basis for the Positive Inotropic Action of Beta-Adrenergic Agonists Now shrugging off his rags the wiliest fighter of the islands leapt and stood on the broad door still. his ow n bow in his hand. He poured out at his feet a rain of arrows from the quive r and spoke to the crowd : .So much for that. Your clea ncut game is over. Now watch me hit a target that no man has hit before. if I can make this shot. Help me. Apollo' Homer. The Odyssev, Bk XXII Recogniti on of the central role of Ca 2 + in the initiation of the cardiac contractile process and of the existence of two pools from which this activator Ca 2 + could be deri ved has made it possible to anal yze mechanisms by which several drugs modify myocardial cont ractil ity. This is illustrated by the rapid development in our understanding of the mechanism by which beta-adrenergic receptor agonists enh ance myocardi al co ntractility. Cyclic AMP and activator Ca 2 + . As show n in Figure I . the powerful inotropic effec t of the beta-adrene rgic agonists was used by Sarnoff (3) in his demonstration of the variabi lity of myocardial co ntrac tility. That the sympathetic nervous system also has marked effec ts on the duration of systole , first suggested over a century ago by Baxt (cited in reference 55) . was established in 1920 by Wiggers and Katz (the latter was then a sophomore medical student) (55). Based on knowledge of the role of Ca 2 + delivery to and removal from the cardiac co ntract ile proteins. and on use of both electrophys iologic and biochem ical approaches. identification of the mech anism for these actions of the betaadrenergic agonists was finally made possible by the discovery of the role of cyclic adenosine monophosphate (AMP) as an intracellular messenger (56) . Together. these approaches allowed the response of the heart to beta-adrenergic J AM cou, CARDIOL 1983:1 :42-51 REGULATION OF MYOCARDIAL CONTRACTILITY agonists to be defined in terms of effects of cyclic AMP on both Ca 2 + entry by way of the slow inward (Ca 2 +) current and Ca 2 + fluxes across the sarcoplasmic reticulum. Electrophysiologic mechanism (cyclic AMP and the slow inward current). It is now established that through an action of cyclic AMP, beta-adrenergic agonists increase the slow inward current (57); this effect can be described electrophysiologically as due to an increase in the number ofCa 2 + channels that open during the action potential (58). The resulting increase in the amount of activator Ca 2 + within the myocardial cell thus plays a central role in the enhancement of tension development. Biochemical mechanism (cyclic AMP phosphorylation and calcium fluxes across sarcoplasmic reticulum). A biochemical explanation of these effects. especially an acceleration of Ca 2 + fluxes across the sarcoplasmic reticulum, required yet another advance in our understanding of the mechanism by which beta-adrenergic agonists and cyclic AMP, the second messenger, regulate cellular processes. The discovery that cyclic AMP could modify cell function by initiating phosphorylation of various intracellular proteins by cyclic AMP-dependent protein kinases (59,60) allowed my colleagues and me (61) to describe the ability of a cardiac cyclic AMP-dependent protein kinase to stimulate Ca2+ transport by cardiac sarcoplasmic reticulum vesicles. This effect of the cyclic AMP-dependent protein kinase is mediated by the phosphorylation of phospholamban. a small protein in these membranes (62,63) that leads to an increase in the rates of both calcium uptake and calcium release by the sarcoplasmic reticulum (Fig. 6) (8). These effects appear to be of importance to the ability of the beta-adrenergic agonists to accelerate both relaxation and contraction in the intact heart. There is growing, but still inconclusive. evidence that a similar cyclic AMP-induced phosphorylation is also responsible for the ability of these agents to increase Ca 2 + fluxes across the sarcolemma (64). Thus. research in this area has begun to combine the electrophysiologic and biochemical approaches to the regulation of myocardial contractility. Conclusions and Predictions I am a part of all that I have met: Yet all experience is an arch wherethrough Gleams that untravelled world. whose margin fades For ever and ever when I move. How dull it is to pause. to make an end. To rust unburnished. not to shine in use. Tennyson. "Ulysses" Looking back over the past 25 years of research on the mechanisms responsible for the regulation of myocardial contractility, I am struck by the large number of questions that could not even have been asked in 1958, but which Phosphoprotein Phosphatase II 49 Cyclic AMP-Dependent Protein Kinase CH2 I C-H I oI P Figure 6, Schematic representation of the effects of phospholamban phosphorylation on the calcium pump of the cardiac sarcoplasmic reticulum. Top: Phospholamban in the dephospho-form interacts with the calcium pump adenosine triphosphatase (ATf'ase) protein to confer positive cooperativity between its two Ca 2T-binding sites and to slow pump turnover rate. Bottom: Phosphorylation of phospholamban reduces its interaction with the calcium pump ATPase protein, allowing each Ca 2T-binding site to act independently with Ca 2T. thereby increasing both Ca'T -sensitivity and turnover of the calcium pump. MW = molecular weight. (Reprinted from Hicks, Shigekawa and Katz. Circ Res 1979;44:384-91, by permission of the American Heart Association. Inc.) have now been answered. There also emerges a clear sense of an accelerating flow of knowledge, of a steady building on the discoveries and concepts that appeared rapidly during this period. Those of us who had the privilege of pursuing these challenges during this exciting quarter century have been fortunate indeed! In many ways. our experiences since 1958 resemble those of my father 25 years before. In 1933, he had just left Wiggers' laboratory in Cleveland to become Director of 50 J AM COLL CARDIOL KATZ }983:1:42-51 Cardiovascular Research at Michael Reese Hospital in Chicago. At the time. his studies of cardiac hemodynamics in dogs had little impact on the way cardiologists treated their patients. Yet, with the introduction of the clinical use of cardiac catheterization and the many advances that made it possible to operate safely on the human heart (65), the foundations of basic knowledge provided by the great hemodynamic physiologists of my father's generation made possible our modern era of open heart surgery. To what can we look forward, if our present generation of young investigators is allowed to continue the progress in cardiovascular research at its current pace? It is within the realm of possibility that means will be found to promote the regeneration of heart muscle, so as to overcome the disability of patients whose cardiac function has been compromised by myocardial infarction. For others, new approaches to improving myocardial contractile function promise relief of the lost contractility in patients with heart failure. Knowledge of the basis for the cardiomyopathies may allow these disease processes to be arrested or reversed. Research now progressing rapidly in the fields of membrane biochemistry and cellular electrophysiology may allow identification of cellular and membrane abnormalities responsible for clinical cardiac arrhythmias. Coupled with rapidly evolving approaches to antiarrhythmic therapy based on an understanding of the molecular interactions between drugs and membranes, this knowledge can be expected to provide specific. nontoxic treatment for patients suffering from electrophysiologic disorders or at risk for sudden cardiac death. While such predictions may seem fantastic, one need only to look back over the past quarter century to see that they are, in fact, rather modest. From the book THE ODYSSEY by Homer. Translated by Robert Fitzgerald. Copyright © 1961 by Robert Fitzgerald. Published by Doubleday and Co., Inc., New York. I thank Dr. Phyllis B. Katz for help with this text. References 1. Katz LN, ed. Symposium on the regulation of the performance of the heart. Physiol Rev 1955;35:90-168. 2. Katz LN. Analysis of the several factors regulating the performance of the heart. Physiol Rev 1955;35:91-100. 3. Sarnoff S1. Myocardial contractility as described by ventricular function curves; observations on Starling's law of the heart. Physiol Rev 1955:35:107-22. 4. Wollenberger A. The energy metabolism of the failing heart and the metabolic action of the cardiac glycosides. Pharmacol Rev 1949;I :31152. 5. Furchgott RF, Lee KS. High energy phosphates and the force of contraction of cardiac muscle. Circulation 1961;24:416-28. 6. Kubler W, Katz AM. Mechanism of the early "pump" failure of the ischemic heart: possible role of adenosine triphosphate depletion and inorganic phosphate accumulation. Am J Cardiol 1977;40;467-71. 7. Hochrein H, Doring HJ. Die energiereichen Phosphate des Myokards bei Variation der Balastungs Bedingungen. Pflugers Arch 1960;271 :54863. 8. Katz AM. Role of the contractile proteins and sarcoplasmic reticulum in the response of the heart to catecholamines. An historical review. Adv Cycl Nucl Res 1979;11:303-43. 9. Bing R. The treatment of heart failure and the use of digitalis in myocardial infarction. Am 1 Cardiol 1958;1:250-9. 10. Olson RE. Myocardial metabolism in congestive heart failure. 1 Chronic Dis 1959:9:442-64. II. Davis 10. Carroll WR, Trapasso M. Yankopoulos NA. Chemical characteristics of cardiac myosin from normal dogs and from dogs with chronic congestive heart failure. 1 Clin Invest 1960;39:1463-71. 12. Katz AM. Contractile proteins of the heart. Physiol Rev 1970;50:63158. 13. Katz AM, Repke 01, Rubin BB. The adenosine triphosphatase activity of cardiac myosin. Comparison of the enzymatic activities and activation by actin of dog cardiac, rabbit cardiac, rabbit white skeletal and rabbit red skeletal myosins. Circ Res 1966;19:611-21. 14. Barany M. ATPase activity of myosin correlated with speed of muscle shortening. 1 Gen Physiol 1967;50:197-216. 15. Huxley AF. Muscle structure and theories of contraction. Prog Biophys Mol Bioi 1957;7:257-318. 16. Hill AV. The heat of shortening and the dynamic constants of muscle. Proc R Soc Lond [Bioi] 1938;126:136-95. 17. Katz AM. Tonic and phasic mechanisms in the regulation of myocardial contractility. Basic Res Cardiol 1976;71:447-55. 18. Alpert NR. Mulieri LA. Heat. mechanics and myosin ATPase in normal and hypertrophied heart muscle. Fed Proc 1982:41:192-8. 19. Lompre A-M, Schwartz K. D'Albis A. Lacombe G, Van Thiem N. Swynghedauw B. Myosin isozyme redistribution in chronic heart overload. Nature 1979;282:105-7. 20. Bannerjee SK, Kabbas SG, Morkin E. Enzymatic properties of heavy meromyosin subfragment of cardiac myosin from normal and thyrotoxic rabbits. 1 Bioi Chem 1977:252:6925-9. 21. Fenn WO, Marsh BS. Muscular force at different speeds of shortening 1 Physiol (Lond) 1935;85:277-97. 22. Abbott BC, Mommaerts WFHM. A study of inotropic mechanisms in the papillary muscle preparation. 1 Gen Physiol 1959;42:533-51. 23. Sonnenblick EH. Implications of muscle mechanics in the heart. 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The actions of various cations on muscle protoplasm. J Cell Physiol 1947:29:15-32. 33. Sandow A. Excitation-contraction coupling in muscular response. Yale J Bioi Med 1952;25:176-201. 34. Ebashi S, Lippman F. Adenosine triphosphate-linked concentration of calcium ions in a particulate fraction of rabbit muscle. 1 Cell Bioi 1962;14:389-400. REGULATION OF MYOCARDIAL CONTRACTILITY 35. Hassel bach W , Makinose M. Die calcium Pumpe der " Erschlaffungsgrana" des Muskels und ihre Abhangigkeit von der ATP-spaltung. Bioch em Z 1961;333:518- 28. 36. Inesi G, Ebas hi S, Watanabe S. Preparation of vesicular relaxing factor from bov ine heart muscle . Am J Physiol 1964:207:1339-44 . 37 . Carsten ME . The cardiac calcium pump. Proc Natl Acad Sci USA 1964;52:1456-62. 38. Fanbu rg B, Gergely J . Studi es on adenosine triphosphate-supported calci um accumulation by cardiac subcellular particles . J Bioi Chern 1965;240:2721- 8. 39 . Weber A, Herz R, Reiss I. Nature of the cardiac relaxing factor. Biochem Bioph ys Acta 1967 ;131:188- 94 . 40 . Katz AM, Repke DI. Quant itative aspects of dog cardiac microsomal calcium binding and calcium uptake . Circ Res 1967;21:153-62. 41 . Weber A, Wini cur S. The role of calcium in the superprecipitation of actomyosi n. J Bioi Chern 1961;236:3198-202. 42 . Katz AM . Influence of tropomyosin upon the reactions of actomyos in at low ionic strength. J Bioi Chern 1964:239:3304-11. 43 . Ebashi S, Kodama A. A new protei n factor promoting aggregation of tropomyosin . J Biochem (Tokyo) 1965;58:107- 8. 44 . Ebashi S, Ebashi F. A new protein component participat ing in the superpreci pitation of myosin B. J Bioche m (Tokyo) 1964:55:604-1 3. 45 . Ebashi S, Kod ama A. Native tropomyosin-like action of troponin on trypsin-treated myosin B. J Biochem (Tokyo) 1967;60:733-4. 46. Katz AM . Purification and properties of a tropomyosin-containing protein fract ion that sensitizes recon stituted actomyosi n to calci umbinding age nts . J Bioi Chern 1966;241:1522-9. 47 . Katz AM, Repke DJ. Cohen BR . Control of the activ ity of highly purified cardiac actomyos in by Ca ~ +, Na " , and K " . Circ Res 1966;19:1062-70. 48. Noble D . A modification of the Hodgkin-Hu xley equations applicable to Purk inje fiber action and pacemaker potentials. J Physiol (L ond) 1962;160:317- 52. 49 . Deck KA, Trautwein W . Ionic currents in cardiac exci tation. Pfliigers Arch 1964;280:65-80. 50. Reuter H. The dependence of slow inwa rd current in Purkinje fibres on the extracellular calcium concentration. J Physiol (Lond) 1967;192:479-92 . 51. Tada M , Yamamoto T, Tonomura Y . Molecular mechanisms of active calcium transport by sarcoplasmic reticulum. Physiol Rev 1978;58:179. J AMCOLL CARDIOL 1983:I:42- 51 51 52 . Shigekawa M, Finegan J-AM , Katz AM . Calcium transport AT Pase of canine card iac sarcoplasmic reticulum: a co mpariso n with that of rabb it fast skeletal muscle sarcoplasmic reticulum . J Bioi Chern 1976;251:6894-900 . 53. Katz AM , Repke DI, Fudyma G . Shigekawa M. Co ntrol of calc ium efflux fro m sarcoplasmic reticulum vesicles by external calci um. J Bioi Chern 1977;252:42 10-4. 54 . Takenaka H, Adler PN, Katz AM . Calci um fluxes across the membrane of sarco plasmic reticulum vesicles. J Bioi Chern (in press). 55 . Wiggers CJ, Katz LN. The specific influence of the acce lerator nerves on the duratio n of ventricular systo le. Am J Physiol 1920:53:49-61 . 56. Murad F, Chi YM, Rail TM , Sutherland EW . Adenyl cyclase : III. Effect of catecholamines and choline esters on the formation of adenosine 3' ,5 ' -phosphate by preparations from cardiac muscle and liver. J Bioi Chern 1962;237:1233- 8. 57 . Tsien R . Cyclic AMP and contrac tile activity in heart. Adv Cyclic Nucl Res 1977;8:363-420. 58 . Reuter H, Scholz H. The regu lation of the calcium conductances of cardiac muscle by adrenaline . J Physiol (Lond) 1977;264:49-62 . 59. Walsh DA, Perkins JP , Kreb s EG . An adenosine 3' ,5' -monophosphate-dependent protein kinase from rabbit skeletal muscle . J Bioi Chern 1968;243:3763-5 . 60 . Kuo JF , Greengard P. An adenosine 3' ,5 ' -monophosphate-dependent protein kinase from Escherichia coli . J Bioi Chern 1969;244 :3417-9. 6 1. Kirch berger MA, Tada M , Repke DI, Katz AM. Cyclic adenosine 3' ,5 ' -rnonophosphate-dependent protein kinase stimulation of calc ium uptake by canine cardiac microsomes. J Mol Cell CardioI1 972:4:67380 . 62 . LaRaia PJ , Morkin E. Adenosine 3'.5 ' -monophosphate-depe ndent membrane phosphorylation; a possible mec hanism for the control of microsom al calcium transport in heart muscle . Circ Res 1974;35:298306 . 63. Tada M . Kirchberger MA, Katz AM . Phosphorylation of a 22,000 dalton co mponent of the cardiac sarcoplasmic reticulum by adenosine 3' .5' -monophosphate-dependent protein kinase. J Bioi Chern 1975;250:2640-57 . 64. Tada M, Katz AM . Phosphorylation of the sarco plasmic reticulum and sarco lemma. Ann Rev Physiol 1982;44:401-23 . 65. Comroe JH Jr, Dripps RD . Ben Franklin and open heart surgery. Circ Res 1974 ;35:661-9.