LIBRARY hdiflhfiganLStaOc {Juivcnnty This is to certify that the thesis entitled Developmental Changes in Caz+ Concentration in Cultured Embryonic Chicken Skeletal Muscle Cells presented by Christine Jaroslava Nykyforiak has been accepted towards fulfillment of the requirements for M.S. . Human Nutrition degree in q 1/ , - ./ _ ”z/ ; ./ _, I_ *1/14”! -' " 1. ’li 9L7 “.‘4 -I Major professor Date August l0, 1978 0-7 639 Developmental Changes in Ca2+ Concentration in Cultured Embryonic Chicken Skeletal Muscle Cells By Christine Jaroslava Nykyforiak A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1978 flu“ ABSTRACT Developmental Changes in Ca2+ Concentration in Cultured Embryonic Chicken Skeletal Muscle Cells By Christine Jaroslava Nykyforiak 2+ Intracellular Ca levels in muscle cells fluctuate during myogenic differentiation. To determine the magnitude _of developmental alterations in intracellular Ca2+ levels. embryonic chick skeletal muscle cells were examined at dif- ferent stages of muscle development, ranging from replica- ‘ting presumptive myoblasts to fully differentiated multi- nucleated myotubes. The cytoplasmic Ca2+ levels were transiently elevated prior to the onset of fusion followed by an initial decline in Ca2+ concentration that was closely coordinated with the onset of myotube formation. The lowest Ca2+ Concentration coincided with cessation of fusion in maximally differentiated muscle cells. The regulation of the cytoplasmic Ca2+ concentration is due to the sarcoplas- mic reticulum which accumulates during skeletal muscle dif- ferentiation and is extensively developed immediately after fusion. Sarcoplasmic reticulum reaches its maximum accretion and functional capacity upon maximum fusion. Relative quan- 2+ tities of Ca concentrations in developing fibroblasts were Christine J. Nykyforiak also studied because the characteristics of replicating fibroblasts are similar to replicating presumptive myo- blasts, and the two cell populations are virtually insepa- rable in 11359. Cytoplasmic Ca2+ concentrations in the fibroblasts followed a pattern similar to that in the myo- blasts. However, the cytoplasmic Ca2+ decrease in myoblasts was approximately one hundred—fold as compared to the five- fold decrease in fibroblasts. This significant decrease reflects the difference between the two cell populations and indicates a Ca2+ role unique to the myogenic events. ACKNOWLEDGEMENTS I would like to express sincere gratitude to Dr. Ron B. Young for his help and guidance throughout my program; Teresa A. Phillips for her advice on cell culture techniques; Drs. Maurice R. Bennink, Jenny T. Bond and Clarence H. Suelter for serving on my advisory committee. ii TABLE OF CONTENTS LIST OF TABLES. LIST OF FIGURES INTRODUCTION. LITERATURE REVIEW A. Skeletal Muscle Development and Differenti- ation. . . . . . . . . . . . . . . . B. Ca2+ Distribution and Function in Muscle Cells. 2+ C. Effect of Ca on Muscle Cell Fusion MATERIALS AND METHODS A. Preparation of Muscle Cell Cultures. B. Measurement of Ca2+ Uptake by Myogenic Cells. . . . . . . . . . . . . C. Nuclei Counts. D. Preparation of Fibroblast Cell Culutres. E. Measurement of Ca2+ Uptake by Fibroblast and Muscle Cultures. . . . . . . . . . . . . RESULTS AND DISCUSSION. . . . . . . . . . . A. Caz+ Accumulation in Differentiating Muscle Cells. . . . . . . . . . . . . . B. Relative Ca2+ Levels in Fibroblasts. SUMMARY . . . . . . . . . . . . . . . . . BIBLIOGRAPHY. . . . . . . . . . . . . . . iii Page iv l0 16 26 26 28 29 30 3O 3l 3l 59 80 82 LIST OF TABLES Table Page l Changes in number of myotube nuclei and cyto- plasmic Ca2+ relative to the onset of myoblast fusion. . . . . . . . . . . . . . . . . . . . . 52 2 Summary of cytoplasmic Ca2+ content of mononu- cleated myoblasts, fully developed multinucle- ated myotubes. replicating fibroblasts, and stationary fibroblasts. . . . . . . . . . . . . 79 iv Figure 10 ll LIST OF FIGURES Equilibration of exogenous 45Ca2+ with intra- cellular pools in muscle cells during the early stages of differentiation. . Equilibration of exogenous 45Ca2+ with intra- cellular pools in fully differentiated muscle cells. . . . . . . . Relationship between relative cytoplasmic Ca2+ concentration and muscle cell fusion in muscle cell cultures. . . . . . . . Relationship between the quantity of organelle- bound Ca2+ and muscle cell fusion. . . . Relationship between total Ca2+ concentration and muscle cell fusion in muscle cell culture. The relationship betwesn the ratio of cyto- plasmic: organelle4 and the number of Mt nuclei/culture as a function of the age of muscle cells in culture over a seven day period . . . Relationship between initiation of Mb fusion and cytoplasmic Ca2+ content . . . . Relationship between initiation of Mb fusion and organelle- -bound Ca2+ . . . . . . Relationship between initiation of Mb fusion and total Ca2+ concentration (cytoplasmic and organelle- bound) . . . . . . . . . . Kinetics of 45Ca2+ equilibration over a 60 minute period in cell proliferating (or repli- cating) fibroblast culture . . . . . . . Kinetics of 45Ca2+ equilibration over a 60 minute period in stationary (or non-replica- ting) fibroblast cultures. . . . . Page 33 35 39 42 45 47 51 S5 57 63 65 Figure l2 l3 I4 15 Page The relationship between the cytoplasmic 45Ca2+ cpm/nucleus and number of cells/plate in fibroblast cell cultures grown over a 6 day period. . . . . . . . . . . . . . . . . . 68 The relationship between the organelle 45Ca2+ cpm/nucleus and the number of cells/plate in fibroblast cultures grown over a 6 day period. . . . . . . . . . . . . . . . . . . . 71 The relationship between the total 45Ca2+ cpm/nucleus and cells/plate in fibroblast cell cultures grown over a six-day period . . 73 The relationship betwegn the ratio of cyto- plasmiczorganelle 45Ca T cpm and the number fibroblasts/plate over a six-day period . . . 76 vi INTRODUCTION An important myogenic event resulting in muscle matu- ration is myoblast fusion. This process can be regulated by extracellular Ca2+ concentrations which, if less than 50 uM, will prevent spontaneous myotube formation. The exact 2+ is involved in muscle cell fusion 2+ mechanism by which Ca is not known. It is speculated that Ca interacts with Ca2+ binding sites on the plasma membrane and that this interaction facilitates muscle cell contact that is pre- requisite to the onset of fusion. 2+ The Ca concentration in muscle cells is regulated by the sarcoplasmic reticulum. This muscle organelle stores 2+ Ca and releases the divalent ion upon the depolarization of the muscle fiber sarcolemma. After depolarization is 2+ from complete, the sarcoplasmic reticulum reaccumulates Ca muscle cytoplasm and further contraction is inhibited. Although sarcoplasmic reticulum is present in virtually all stages of muscle differentiation, it develops immediately after fusion and operates at its maximum capacity when the fusion process is maximum. Because Ca2+ may be involved in regulation of certain myogenic events associated with differentiation, the relative 2+ amounts of Ca in cytoplasmic and organelle fractions of muscle cells were examined. Particular emphasis was placed on the relationship between Ca2+ concentrations and the onset of myoblast membrane fusion. Since fibroblasts are present in a myogenic cell population and their characteris- tics are comparable to replicating myoblasts, Ca2+ levels in the cytoplasmic and organelle compartments of fibroblasts were also examined. This examination was necessary in order to determine whether the observed changes during myoblast fusion were the result of unique myogenic events, or whether they simply resulted from the fact that the myotube nuclei are no longer capable of mitotic activity. The results are consistent with the former possibility and thereby suggest that myoblast fusion may in some way be 2+ regulated by intracellular Ca levels. LITERATURE REVIEW A. Skeletal Muscle Development and Differentiation Embryonic skeletal muscle development has been shown to involve the transition of replicating presumptive myo- blasts (PMb) intoposmitotic mononucleated myoblasts (Mb) and, subsequently, into multinucleated myotubes or mature muscle fibers (Holtzer £3 al, 1975c). Presumptive myoblasts comprise the penultimate compartment of the myogenic lineage (Chi £5 11., 1975; Dienstman and Holtzer, l975; Holtzer 33 _l., 197st),and they can replicate to produce either more PMb or Mb (Dienstman and Holtzer, l975). The precursor of the PMb is the primitive mesenchyme cell (Ms) which may also differentiate into primitive chondroblasts (PCb) and presumptive fibroblasts (PFb) (Abbott 33 11., 1974; Dienstman and Holtzer, 1975; Holtzer and Bischoff, l970). All cells divide via a cycle that includes four periods: 6,, presynthesis; S, DNA synthesis; 62, postsyn- thesis and M, mitosis (Holtzer and Bischoff, 1970; Stock- dale and O'Neill, 1972). Replicative DNA synthesis is initiated when cells with a 2N complement of DNA enter the 5 stage from the G1 stage. Cells with a 4N DNA complement then enter the G2 phase, which is followed by mitosis (Buckley and Konigsberg, l974). The PMb is a differentiated phenotype (Chi t al., 1975) that can further undergo proliferative mitoses to produce daughter cells of equivalent developmental options to their mother cell. Thus the number of cells within the PMb compartment can be increased. Alternatively, PMb can exhibit quantal mitotic activity whereby the synthetic capacities of the daughter cell (i.e., Mb) differ from those of the mother cell. The quantal cell cycle moves cells from one compartment to another within a given lineage (Bischoff and Holtzer, 1969; Dienstman and Holtzer, 1975; Holtzer, 1970; Ishikawa 33 al., 1968). The ultimate compartment of the myogenic lineage included the Mb. Mb have the potential to fuse with other Mb to form myotubes and then remain in the postmitotic state in which they elaborate and assemble the myofibrillar proteins into contractile myofibrils (Chi 33 31., 1975; Fischman, 1967). That fusion occurs in the G1 stage of the cell cycle has been agreed upon my many investigators (Bischoff and Holtzer, 1969; Buckley and Konigsberg, 1974; Dienstman and Holtzer, 1970; Holtzer, 1970; Okazaki and Holtzer, 1965; O'Neill, 1976). Mononu- cleated Mb in the G.I stage of the cell cycle have also been found to synthesize and organize myosin, actin and tropo- myosin into striated myofibrils, whereas the present PMb in 61 cannot (Dienstman and Holtzer, 1975). Mb can be distinguished morphologically from the PMb only upon fusion and detection of contractile protein ' synthesis (Stromer t al., 1974). For example, myosin synthesis rate has been experimentally observed to acceler- ate several-fold at the time of fusion (Emerson and Beckner, 1975; O'Neill, 1976; Paterson and Strohman, 1972; Yaffe and Dym, 1972; Young gt al., 1975). Muscle cell fusion 1 vivo may require weeks to form multinucleated Mt with several hundred nuclei, yet fusion in vitro is observed within 24 hours of plating with close to a thousand nuclei in the Mt (Okazaki and Holtzer, 1965). ‘ Factors other than mitosis affect Mb fusion (Bischoff and Holtzer, 1968; Bischoff and Holtzer, 1970;.Dienstman and Holtzer, 1977; Holtzer, 1970; Holtzer and Bischoff, 1970; -Holtzer _t._l., 1973; Holtzer £3 11., 1974; Holtzer 33 al., 1975; Keller and Nameroff, 1974; Stockdale 3; al., 1964; Trotter and Nameroff, 1976; Turner gt al., 1976; Zalin, 1977). Fusion can be blocked by Ca2+ dinitrophenol (DNP), cytochalasin B (CB), azide, antimycin-A, colchicine and inappropriate substrates (Bischoff and Holtzer, 1968; Holtzer, 1970; Holtzer gt al., 1973). Several of these fusion inhibitors serve as excellent tools in the study of myogenesis. An example of one such inhibitor is 5-bromo- deoxyuridine (BUDR). Replicating PMb exposed to BUDR incorporate this thymidine analog into their DNA and are reversibly inhibited from Mt formation and myosin synthesis. This inhibition results from the fact that BUDR prevents the transition of PMb into Mb (Bischoff and Holtzer, 1970; Okazaki and Holtzer, 1969; Stockdale gt al., 1964). EGTA, 2+ a specific chelating agent for Ca , also is an excellent reversible inhibitor of fusion (Holtzer et al., 1975; Paterson and Strohman, 1972; Turner 3; al., 1976). Mb lose the potential to fuse when their medium is Ca2+ deficient for over a 48 hour period; however, fusion will occur within 10 hours if Ca2+ is restored to the medium (Holtzer and Bischoff, 1970; Paterson and Strohman, 1972; Turner 33 al., 1976). Although fusion is inhibited by EGTA, myofibril formation and myosin synthesis still occur in Mb blocked in the G1 stage of the cell cycle (Dienstman and Holtzer, 1977). Thus, fusion is not a prerequisite event for coordinated expression of the myosin and actin genes, although fusion is an interesting marker for changes in the cell surfaces (Holtzer 33 11-, 1975a; Holtzer et al., 1975b). A significant regulatory role in the growth and dif- ferentiation of muscle cells has been attributed to cyclic adenosine 3'-5' monophosphate (CAMP). Cell fusion, the primary morphological manifestation of differentiated muscle, has been shown to be sensitive to cyclic nucleotides (Epstein, 1975; Wahrman gt al., 1973; Zalin, 1973; Zalin, 1976; Zalin 1977; Zalin and Leaver, 1975; Zalin and Mon- tague, 1974; Zalin and Montague, 1975). The reported effects of cAMP on muscle cell differentiation, however, have been variable. The onset of cell fusion was inhibited- when dibutyryl cAMP was added to chick Mb cultures 24 hours after plating (Zalin, 1973) and to a rat muscle cell line (Nahrman £3 31., 1973). When intracellular levels of cAMP normally present in differentiating M8 were examined, a ten-fold transient increase in the cyclic nucleotide levels was observed 5 to 6 hours prior to the onset of fusion in the cultures (Zalin and Montague, 1974). This suggests that CAMP may be a signal for stimulating the onset of fusion, or at least a specific intracellular response to some other signal. Other experiments utilizing prostaglan- .din E1 (PGE1) have tested the possibility that the increase in cAMP is the signal for cell fusion (Zalin, 1977; Zalin and Leaver, 1975). PGE1 has the ability to produce both large and transient increases in intracellular cAMP. When 10‘5M Pee1 was added to chick Mb cultures 4 hours earlier than normal, fusion occurred 4 hours sooner (Zalin and Leaver, 1975). PGE1 at a physiological concentration of 10-10M, provoked Mb fusion and when PGE1 inhibitors (aspirin (or indomethacin) were added, no fusion occurred. Thus, this work ilaustrated the importance of prostaglandins and their effect on cell fusion (Zalin, 1977). Since cAMP concentra- tion was found to decrease at the time of fusion of primary rat Mb (Wahrman _3__l,, 1973) and to only undergo a tran- sient increase just prior to fusion of primary chick Mb, it was concluded that elevated levels of the cyclic nucleotide, though necessary for the initiation of fusion, probably are incompatible with fusion itself (Zalin and Montague, 1974). Studies conducted by another group of researchers (Mori- yama 33 11., 1976) reaffirm a correlation between intra- cellular concentration of cyclic nucleotides and myogenesis i vitro. Their findings include an increase in cAMP during early myogenesis and support the regulatory role of intra- cellular concentration of cyclic nucleotides in myogenesis. The results obtained by Moriyama's group (1976) demonstrate that the signal for Mb fusion may be not only the increase in CAMP but also a decrease in cGMP or an increase of the ratio of CAMP to cGMP. Epstein and coworkers (1975) pre- sented a different perspective of CAMP influence on fusion. These investigators claim that membranes may be utilized in the study of molecular phenomena associated with muscle cell fusion, and the remainder of this review will focus pri- marily on the functional and developmental characteristics of SR and the sarcolemma. Emphasis is placed on these two membranous components because the SR regulates intracellu- 2+ lar Ca concentration and because the rate of fusion of the sarcolemma is highly dependent on Ca2+ 1972). concentration (Inesi, The SR is an organized network of tubules, vesicles and cisternae surrounding the myofibril (Inesi, 1972). This membrane system regulates contraction by controlling intracellular Ca2+ concentrations (Ebashi gt gt., 1969; Tillack gt gt., 1974). The function of the SR system has been studied extensively at the molecular 1eVel (reviewed by MacLennan and Holland, 1975). However, much less is known about its assembly during muscle differentiation 2+ (Jorgensen gt gt., 1977) and the Ca pumping mechanism in the SR during muscle maturation lg_vitro (Lough gt gt., 1972). Since extensive regulation of cytoplasmic Ca2+ concentrations may not be essential in mononucleated Mb, SR is virtually absent from these precursors of the multi- nucleated myofibers (Boland 9t 61-, 1974; Ezerman and Ishikawa, 1967). However, SR proteins have been found in mononucleated Mb of rat skeletal muscle through immuno- fluorescent staining techniques (Jorgensen 2+ 2+ t al., 1977). The proteins studied were Ca -Mg dependent ATPase, an intrinsic protein, and calsequestrin, an extrinsic protein, which are synthesized under separate controls (Zubrzycka and MacLennan, 1976). Thus, SR develops early without any structural association with myofibrils (MacLennan and H01- land, 1976). The newly synthesized calsequestrin is transferred to the lumen of the SR membrane system along with the ATPase which is synthesized in a different area of the muscle cell. The ATPase synthesis and the assembly of SR are initiated at multiple foci throughout the cytoplasm. The calsequestrin polypeptide chain is initially synthesized on the rough endoplasmic reticulum and then transferred to the Golgi apparatus where it undergoes synthesis into a glycoprotein. From the Golgi apparatus the calsequestrin is transferred to the SR by an unknown mechanism (Jorgensen t 1., 1977). The SR membrane contains another extrinsic protein in addition to calsequestrin - the high affinity 2+«binding protein. It is postulated that both these 2+ Ca proteins bind Ca transported to the luminal surface of the membrane by the action of the transport ATPase (Zubrzycka and MacLennan, 1976). 10 The development of the mitochondrial membranes has been studied by examining succinate cytochrome C reductase (SCR) activity, which increases subsequent to Mb fusion. When compared with Mb mitochondria, Mt mitochondria appeared larger and contained an increased number of cristae. Elec- tron micrographs showed that the larger mitochondria are present in well-differentiated Mt rather than in Mb (Vertel and Fischman, 1977). Mitochondrial development in myo- genesis may lend important implications to the study of biochemical functions in muscle cell differentiation and maturation; however, this important aspect of intermedi- ary metabolism in cultured muscle cells has been virtually ignored. 8. Ca2+ Distribution and Function in Muscle Cells Ca2+ plays a dynamic role in cell membrane systems. The ion functions as a modulator of membrane phenomena, a cofactor of membrane enzymes, a stabilizer in membrane assembly and a transmembrane charge carrier (Laclette and Montal, 1977). These activities result from the interaction 2+ between Ca and the membrane constituents: lipid, protein and carbohydrate. The basic structural element of mem- 2+ reacts with water soluble branes is the lipid bilayer. Ca lipid - protein complexes and makes them more soluble in the hydrocarbon phase of membranes (Laclette and Montal, 1977). The present concept of the biological membrane as fluid mosaic with a bulk lipid bilayer, in which individual ll functional components are embedded and in which these com- ponents have freedom of translational movement, illustrates the dynamic state of membranes (Fambrough, 1974; Schroeder t al., 1976; Singer and Nicolson, 1972). Skeletal muscle contains two major membranous systems that are directly involved in translation of a nerve impulse into physical movement: the sarcoplasmic reticulum (SR) and the transverse tubular system (T system) (Affolter gt gt., 1976; Ezerman and Ishikawa, 1967; Inesi, 1972). The SR contains four major protein components: (a) the 2+ Ca activated ATPase with a molecular weight of 102,000 2+ that serves as the Ca pump (Sarzala gt gl., 1975); (b) a protein with a molecular weight of 55,000, and with one 2+ high-affinity Ca binding site and a large number of non- specific low—affinity Ca2+ 2+ binding sites; (c) a highly acidic, low-affinity Ca binding protein with a molecular weight of 44,000 that has been termed "calsequestrin" be- 2+ cause of its high Ca binding capacity. Calsequestrin is postulated to function as the primary Ca2+ storage protein of the SR, and (d) a proteolipid of molecular weight 6,000 - 12,000 (MacLennan and Holland, 1975). The exact mechanism by which Ca2+ is transported across the SR membrane is not known; however, this mecha- nism has been the focus of recent investigations (Chyn and Martonosi, 1977; Dupont, 1978; Murphy, 1976). A proposed molecular mechanism of ATP-dependent Ca2+ transport in SR-i is depicted by an enzymatic model, The SR membrane contains 12 2+ - MgZ+- dependent ATPase system which reduces exter- 2+ 2+ a Ca nal Ca to uM concentrations and transports Ca into the SR. The hydrolysis of 1 mole of ATP results in the uptake 2+ (MacLennan and Holland, 1975). The Ca2+ of 2 moles of Ca & M92+-dependent ATPase is the major protein constituent of the SR membrane, constituting approximately 60-70% of the total protein (Sarzala gt gl., 1975). Studies on freeze- etched SR revealed the presence of spherical intramembra- nous particles of 7.5 nm apparent diameter. These 7.5 nm particles increase in number during development and parallel 2+ 2+ the sharp increases observed in the Ca transport and Ca sensitive ATPase activity. This close correlation has been postulated to suggest that the 7.5 nm intramembrane par- ticles represent the Ca2+ transport enzyme. The ATPase protein appears to be closely associated with the phospho- lipid bilayer of the SR membrane (Murphy, 1976), and the SR ATPase polypeptide was reported to be strongly complexed with 20-30 phospholipid molecules. This phospholipid com- ponent is absolutely required for the ATPase activity (Hidalgo gt gt., 1976; MacLennan, 1975; Scales and Inesi, 2+ 1976). The main feature of the SR ATPase is Ca activation. The Ca2+—sensitive event is presumed to be the formation of a phosphoprotein intermediate which involves ATP binding, 2+ followed by Ca binding and ADP release. This event occurs on the exterior of the membrane surface. A conformational change of a Ca2+ Ca2+ «carrying phosphorylated protein transports 2+ to the inside of the membrane. Ca is then released 13 from the inside of the membrane, the ATP is rephosphory- lated, and the initial stage is restored (Van der Kooi and Martonosi, 1971; Van der Kooi and Martonosi, 1971a). A model has been proposed depicting the organization of the proteins in the SR membrane. The relatively non- polar portion of ATPase is buried in the bilayer region of the membrane and may contain the ionophoric site. The otheromore polar portion of the molecule is the site of ATP hydrolysis and is located on the exterior of the SR membrane. 2+ Ca transport across the membrane is controlled through the interaction between the two sites. Calsequestrin and 2+ the high affinity Ca binding protein are loosely attached to the inner side of the SR membrane and mainly function in 2+ (MacLennan gt al., 1972). sequestering Ca Muscle contraction and relaxation are controlled by 2+ 2+ the Ca concentration in the sarcoplasm, and Ca concen- tration is regulated by SR (Ebashi gt al., 1969). Muscular contraction results from the coupled activity of the two muscle proteins, actin and myosin, and requires the pre- sence of M92+, Ca2+ and ATP. Two major ATP-consuming pro- cesses are associated directly with muscle contraction: l) the interaction of myosin with actin, and 2) the ATP splitting associated with the Ca2+ pump of the SR as dis- cussed in the preceding paragraphs (Homsher and Kean, 1978). The important biochemical process responsible for initiating the overall contractile event is the depolari- zation of the muscle fiber membrane during nerve impulse l4 transmission induced by the neurotransmitter, acetylcholine (Ach). Ach is released from nerve terminals and interacts with the acetylcholinesterase. The enzyme hydrolyzes Ach, but the wave of depolarization of the sarcolemma induced by Ach spreads via the T system into the interior of the muscle fiber. Eventually the excitation stimulates the SR (Inesi, 1972; Peachey, 1965) which contains stored Ca2+ (Winegrad, 1965). This Caz+ 2+ is liberated and further stimulates SR to release Ca (Constantin and Podolsky, 1965; Endo, 1977). However, it is not known how the actionpotential of the T tubule is transmitted to the SR, nor how it causes Ca2+ release from that organelle (Ebashi and Endo, 1968). Few methods are available for studying the molecular mechanism of Ca2+ release from SR 1g.gttgg. Several recent attempts have been made to develop experiments lg gtttg which focus on elucidating this process (Inesi and Malan, 1976; Kasai and Miyamoto, 1976; Kasai and Miyamoto, 1976a). ~The SR membrane permeability was increased upon the addition of caffeine or changes in the electrolyte composition in the medium, and this resulted in an increase in Ca2+ release (Inesi and Malan, 1976). Utilizing SR membrane fragments (SRF), Kasai and Miyamoto (1976; 1976a) found that anion 2+ exchange in the medium caused Ca release probably due to SR membrane depolarization. After excitation of the SR, Ca2+ enters the cytoplasm and initiates muscle contraction when it binds to the con- tractile protein, Troponin C (Tn-C). Relaxation results 15 2+ after Ca is removed from the cytoplasm by the SR, which 2+ has a higher affinity for Ca than Tn-C. The concentration 5 4 of the free cation is about 10' to 10' M during the maximal activation of contraction and about 10'7M in relaxed muscle (Weber and Bremel, 1971). The contractile mechanism of skeletal muscle is acti- 2+ vated by the binding of Ca to troponin (Fuchs, 1977). Troponin is a protein complex with a molecular weight of 76,000 and is composed of three subunits which are named according to their function: Tn-T, tropomyosinébinding sub- unit; Tnel, troponin-inhibiting subunit; and Tn-C, Ca2+- binding subunit (Mannherz and Goody, 1976). Tn-C contains 2+ four Ca binding sites which have two classes of differing affinity (Gergely, 1974). One of these classes contains two high affinity Ca2+ binding sites (Kca 107M-1) which 1,2 competitively and thus have been termed 2+ also can bind Mg2+ 2 2+ Ca tMg sites. Ca specific sites comprise the other 2+ class of two sites which do not bind Mg and.have a lower affinity for Ca2+ (KCa 105m“) (Potter gt 11., 1976; . 3’4 2+ 2+ Potter gt al., 1976a). Ca must be bound to the Ca specific sites in order for the myofibrillar ATPase to become activated (Potter and Gergely, 1974); thus the regu- lation of muscle contraction depends on this binding step (Potter t al., 1976). Tropomyosin, a regulatory protein, is a rod-like molecule with dimensions of 1.5 x 40 nm (Dabrowska gt gt., 2+ 1976) which must be present when Ca binds to Tn-C 16 (Ebashi t 1., 1959). The combination of Ca2+ to Tn-C causes a stronger troponin subunit interaction but weakens the Tn-I-F actin link. This causes the tropomyosin mole- cule to move toward the groove of the actin molecule. When the myosin head reacts with the actin,contraction occurs. As depOlarization recedes, SR reaccumulates Ca2+ 2+ and Ca concentration is reduced causing relaxation by a reversal of the sequence of events leading to contraction (Mannherz and Goody, 1976). C. Effect of Ca2+ on Muscle Cell Fusion 2+ Ca has been found to have a significant effect on 2+ muscle cell fusion. At low Ca concentrations, myogenic cells do not fuse but will continue either to proliferate as PMb or remain poised for fusion as mononucleated Mb (Morris t al., 1976; Schudt and Pette, 1976; Shainberg gt 1., 1969; Van der Bosch gt gt., 1972; Van der Bosch I‘D t gl,, 1973; Weidekamm gt gt., 1976). Shainberg £3.21. 2+ (1969) tested the effect of increasing the Ca concentra- tion on cultures of newborn rat skeletal muscle cells. The 2 standard nutritional medium was replaced by Ca +-deficient medium containing 14, 35, 70, 140, 270 or 1400 uM CaClz. At 72 hr after plating, cultures receiving 1400 uM CaCl2 displayed normal fusion and formation of multinucleated 2+ or less consisted fibers. Cultures exposed to 270 uM Ca of only mononucleated cells at 72 hr after plating. How- ever, after 95 hr,cultures in medium containing 270 uM 17 CaCl2 displayed a slight increase in the number of fibers (Shainberg gt 31.. 1969). Ca2+ concentrations below 50 pH quantitatively prevented spontaneous myogenic cell fusion (Schudt and Pette, 1975; :Shainberg gt gt., 1969). This Ca2+ effect on muscle cell fusion is completely reversible (Shainberg gt gt., 1969; Van der Bosch gt gt., 1972), since newly formed fibers have been observed 3-4 hr after the addition of Ca2+ , and a network of Mt is present within 24 hr (Shainberg gt gt., 1969). Fusion occurs when the plasma membranes of mononucle- ated Mb dissolve into a continuous membrane that subse- quently surrounds the Mt (Schudt and Pette, 1976). It has 2+ been suggested that Ca facilitates cell contact and thereby triggers fusion (Morris gt gt., 1976; Peretz _t _t., 1974; Prives and Shinitzky, 1977; Schudt and Pette, 1975; Schudt and Pette, 1976; Van der Bosch gt gt., 1972; Van der Bosch gt gt., 1973) by affecting the charges on the cell membranes Shainberg gt gt., 1969). The fusion process seems to depend on a negatively charged cell surface. These negative charges define the surface potential and may affect the actual concentrations of cations near their binding sites in the membrane (Schudt and Pette, 1976). Experiments by Schudt and Pette (1976) were designed to detect specific surface components essential for fusion. One of these components, neuraminic acid, was found to in- fluence the surface charge density. ‘Artificial variations of the neuraminic acid content of cells changed the surface 18 charge density and influenced the Ca2+ dependency of Mb fusion. However, neuraminic acid has not been reported to 2+ selectively bind Ca , and fusion has been shown to be highly sensitive to changes in phospholipid composition. These results suggest that Ca2+ 2+ specificity, Ca binding and fusion-promoting factors are related to phospholipids or intramembranous proteins of the Mb membrane (Schudt and Pette, 1976). An important probe utilized for studying the physical properties of muscle cell membranes during fusion has been the ionophore A23187 (Schudt gt gt., 1976; Truter, 1976; Weidekamm gt gt., 1976). A 23187 is an ionophorous anti- biotic which specifically associates with biological mem- branes and increases their permeability to divalent cations, 2+ especially Ca Its fluorescence characteristics serve as a sensitive tool in detecting fluidity changes in muscle 2+-modulated fusion membranes undergoing normal or Ca (Schudt and Pette, 1975; Truter, T976; Weidekamm gt gt., 1976). During fusion of lipid vesicles with known phospho- lipid composition, a separation of the different lipid bilayer components occurs along with fluidity changes in different membrane areas (Weidekamm gt gt., 1976). It has been suggested that high lipid fluidity is essential for fusion because there is a marked increase in membrane fluidity prior to Mb fusion (Prives and Shinitzky, 1977). Alternatively, since fusion clearly must involve physical rearrangement of the cell membrane, it would indeed be 19 surprising not to detect alterations in membrane fluidity associated with muscle differentiation. Previous work by Van der Bosch and coworkers (1973) reported inhibitory effects of cholesterol and reduced temperature on Mb fusion which supports Prives and Shinitzky's (1977) findings. It has also been observed that certain fatty acids such as stearic or elaidic which increase membrane microviscosity also retard Mb fusion. In contrast, other fatty acids such as oleic and linoleic which decrease mem- brane microviscosity facilitate fusion (Prives and Shinit- zky, 1977). Muscle membranes contain up to 20% acidic 2+ 2+ phospholipids which might be aggregated by Ca Ca may also induce protein alterations which cause the flu- idity changes (Weidekamm gt gt., 1977). It has been suggested that the ionophohe increases the free intra- cellular concentration of Ca2+ though the extracellular Ca2+ concentration does not change. Thus, in the presence of A23187, processes regulated by intracellular Ca2+ con- centration should be accelerated (Schudt and Pette, l975). 2+ .A23187 was also utilized in determining whether Ca ions act on sites at the outer face of the Mb membrane, the inner face of the Mb membrane or inside the cytoplasm. The results from this study suggested that the Ca2+ concen- tration dependence of the fusion process reflects the concentration dependence of Ca2+ binding sites that are exposed at the outer face of the plasma membrane (Schudt and Pette, 1975). Fusion rate is specifically dependent on 20 Ca2+ concentration at physiological temperature and pH when the Ca2+ binding affinity of the muscle cell membrane is optimum (Van der Bosch gt gt., 1972; Van der Bosch gt gt., 1973). Fusion was observed in protein-free media which sug- gests that this phenomenon occurs independent of all factors which are essential for growth, differentiation and main- tenance of the differentiated state (Schudt and Pette, 1976). Ca2+, though necessary for Mb fusion, has been shown not to affect other vital processes related to growth and differentiation such as thymidine or uridine incorpo- ration into nucleic acids (Shainberg gt gt., 1969). Cul- 2+ tures grown in Ca deficient medium exhibited continued specific gene programs of post-mitotic Mb (Young and Allen, 1978). The transport of glucose into muscle may be the rate limiting step for metabolism (Morgan gt gt., 1959), and studies have been conducted to detect this transport system in developing muscle cell cultures. Since Ca2+ has been reported to be important in the regulation of glucose trans- port in adult muscle, Schudt gt 11- (1976) designed expe— 2+ riments to study the effect that Ca may have on the enzymes necessary for glucose metabolism. In particular, these investigators were interested in the influence of 2+ extracellular Ca concentration on the insulin response, 2+ the influence of intracellular Ca concentration on glu- 2 case transport and the influence of insulin on Ca +transport 21 parameters which define cytoplasmic Ca2+ concentration. It was concluded from these experiments that insulin may increase the free cytoplasmic Ca2+ concentration and that this may, in turn, modulate glucose transport (Gaertner _t gt., 1977; Schudt gt gl., 1976). Other key enzymes in energy metabolism have been studied to determine how their activities respond to altered Ca2+ concentrations in developing muscle cells in culture. Ca2+ dependence of phosphorylase activity, for example, has been found to parallel Mb fusion rate. Both events have been inter- preted to have a similar trigger mechanism at the plasma membrane level (Schudt and Pette, 1975; Schudt gt gt., 1975). Synthesis of specific metabolic enzymes, as well as myofibrillar protein synthesis and fusion, may be modulated by differing levels of Ca2+, but whether these diverse intracellular processes are regulated by a common mechanism is not known. A significant amount of research has been conducted studying the effects of Ca2+ on the enzyme creatine phos- phokinase (CPK) and its differentiation in myogenesis (Keller and Nameroff, 1974; Lough and Bischoff, 1977; Morris gt gt:, 1976; Paterson and Strohman, 1972; Schudt gt gl., 1975; Shainberg gt gt., 1969; Vertel and Fischman, 1977; Zalin, 1976). CPK has received considerable atten- tion because it is present at highest concentrations in skeletal muscle and because CPK activity can be used to assess the degree of differentiation of skeletal muscle 22 tissue (Lough and Bischoff, 1977). Also, CPK increases 50-100 fold during and/or following the period of active cell fusion (Keller and Nameroff, 1974; Zalin, 1973). Shainberg gt _t. (1969) and Paterson and Strohman (1972) 2+ claimed that Ca has a direct effect on cell fusion but not on myosin or CPK synthesis although both processes are inhibited when Ca2+ concentration is lowered. In contrast, 2+ other data showed that Ca concentration has direct affect on specific activity of CPK (Morris gt gl., 1975). Contro- versy and discrepancy still surrounds the issue of whether fusion and enzyme synthesis are independent or related 2+ processes. The mechanism of Ca action also is not known and uncertainty prevails as to its exact role in stimulating enzyme activity and cell fusion (Schudt gt gt., 1975). Cell fusion has been shown to be sensitive, in addi- 2+ tion to Ca , to increased levels of cyclic AMP (CAMP) (Zalin and Leaver, 1975). As we discussed in a previous section, intracellular levels of CAMP in differentiating Chick Mb have been found to undergo a 10-fold transient increase just 5 to 6 hr prior to the onset of Mb fusion in cell cultures (Zalin and Montague, 1974). This transient 2+ increase is still exhibited even when Ca is removed. However, it has been demonstrated that inhibition of cell 2+ fusion is more responsive to withdrawal of Ca than it is to alterations in CAMP levels (Zalin, 1976). Therefore, it is postulated that some connection exists between Ca2+ and CAMP in stimulating the onset of the fusion process 23 (Zalin, 1976). From the literature presented in this review, several independent facts are apparent which collectively form the- rationale for a regulatory model that muscle cell fusion is at least partially regulated by cytoplasmic Ca2+ concentra- tion. In summary, the independent types of information 2+ involve the role of Ca in muscle and are described by the following: (1) contraction in skeletal muscle is initiated by Ca2+ ions interacting with troponin and tropomyosin, and the normal range in free Ca2+ concentration in skeletal muscle is from 10‘7 5 M for the relaxed state to approximately 10- M in the contracting state; (2) the organelle in skele- tal muscle that regulates Ca2+ concentration between 10-5 and 10'7M is called the sarcoplasmic reticulum; (3) muscle cells are involved in membrane fusion which is highly Ca2+ dependent. Muscle cells isolated from embryonic chicken muscle are placed in culture and characteristically dif- ferentiate into multinucleated myotubes. The rate of mem- brane fusion can be regulated by altering the Ca2+ concen- tration in the cell culture medium, and no fusion will occur if Ca2+ concentration is kept below approximately 10'5M; (4) development of muscle cells in culture has been studied extensively and these studies have provided further evidence supporting this project. Lough and coworkers (1972) studied accretion of the sarcoplasmic reticulum in differentiation muscle cell cultures and found that the amount of sarcoplasmic reticulum was maximum after five days 24 in culture. Therefore, fusion reaches its maximum level at exactly the same time that the only organelle in muscle 2+ capable of reducing-the intracellular Ca concentration below 10"5 M reaches its maximum; (5) the events known to occur in all cell types involving membrane fusion on a microscale (including endocytosis, exocytosis, cell Cleavage, membrane assembly and secretion) are inhibited by the absence of Ca2+. The observations discussed above are consistent with the following regulatory model of muscle cell fusion. Com- petent myogenic cells fuse freely with each other during the early stages of muscle differentiation because no sarco- plasmic reticulum is present at this stage to lower the Ca2+ concentration to 10'5 M. Sarcoplasmic reticulum synthesis and assembly are initiated immediately after myotube for- mation, and the quantity of sarcoplasmic reticulum gradually increases during myotube maturation. Once the quantity of the sarcoplasmic reticulum is maximum, the intracellular Ca2+ 7M and both contraction concentration is reduced to 10' and additional fusion are blocked. It is also logical that synthesis of the components of the sarcoplasmic reticulum 2+ feedback mechanism is regulated to some extent by a Ca since adequate amounts of sarcoplasmic reticulum are always present. This model is also consistent with some of the events that are known to occur during exercise-induced muscle hypertrophy and normal cell growth, and it can be extended to encompass all known fusion events in non-muscle 25 cells as well. The purpose of this project, therefore, is to examine the validity of this proposed mechanism in dif- ferentiated muscle cells. Materials and Methods A. Preparation of Muscle Cell Cultures Twelve day chick embryo thigh muscle was utilized for muscle cell cultures (Young gt gt., 1975). The muscle cells were plated in 10 cm Corning tissue culture dishes, pre- viously coated with 0.25 mg of collagen, at an initial cell density of 1x107 cells/dish. Under these conditions, myogenic cell fusion occurred after approximately 25-30 hr and cellular fusion was complete by approximately 75 hr. The cell culture procedure was conducted under the Laminar flow hood which was allowed to run 30 minutes prior to starting the culture procedure to ensure a sterile atmos- phere. A buffered salt solution (855) containing 137 mM NaCl, 2.7 mM KCl, 1.0 mM CaCl 1.0 mM NaH P04, 1.36 mM 2’ 2 NazHP04, 6.0 mM NaHC03, 5.5 mM glucose, pH 7.4, was poured into two small icell dishes, centrifuge tubes were placed in the racks, forceps were unwrapped and eggs and discard beaker were arranged to avoid reaching over the cell dishes when working. One dozen eggs containing lZ-day old embryos were removed from the egg incubator and the exterior of each egg was washed with 70% ethanol. The shell was cracked and an embryo from each egg was removed by grasping the neck with the forceps. The embryo was placed into a 26 27 cell dish cover which contained no BSS. The skin was removed from the legs. The foot was grasped with one forcep and pinched off at the thigh-body junction with the other forceps so that all the thigh muscle was removed. The leg was placed in a dish of 855 and the muscle was checked to ensure that all of the skin has been removed, the residual blood was rinsed from the muscle, and the muscle was placed in the second dish of 855. The hind limb was held with the forceps and as much leg muscle as possible was stripped from the bone. Approximately 1/2 of the bones were discarded and the muscle was placed into a centrifuge tube which already contained 10 m1 of regular complete medium (Eagle's Minimum Essential Medium plus 10% horse serum and 5% chicken embryo extract) per dozen embryos. The above steps were repeated until sufficient tissue was obtained. The muscle tissue was vortexed at maximum speed for 20 seconds and the vortex cell suspension was poured out of the tube into a petri dish. The suspen- sion was immediately poured into a 50 m1:syringe, and a Swinney filter containing nylon cloth (200x200 mesh) was attached to the syringe which was then inverted for 30 seconds. This allowed for the bones and large pieces of tissue in the suspension to settle toward the plunger in the barrel of the syringe and thus prevented the bones and large pieces of tissues from clogging the filter. The suspension was forced through the filter gently and firmly but not applying pressure to avoid forcing large pieces of 28 tissue through the filter. The filter was removed from the syringe and the remaining bones, tissue clumps, etc. back into the centrifuge tube adding 10 ml of regular complete medium. The vortexing procedure was repeated and the two suspensions of cells were combined. The combined suspen- sions were forced through a Swinney filter containing a double layer of lens paper as described above. The cell suspension was centrifuged at 700xg for 5 minutes. Thus cell clumps, debris and intact cells that may have passed through the filter were sedimented. The cells were resuspended in 10 ml of complete medium by carefully aspirating with a pipet until no large clumps were seen. Immediately after aspirating a small aliquot of cells was removed with a sterile Pasteur pipet for counting with a hemocytometer. The cells were than plated in complete medium on collagen cultured dishes (Young gt al., 1975). 2+ 8. Measurement of Ca Uptake by Myogenic Cells 45 2+ The cell cultures were pulse labeled with 1 uCi / ml for the designated time points. At the beginning of a pulse label, cell cultures were rinsed once with approxi- mately 5 ml of warm (37°C) buffered salt solution (355). 1‘45Ca2+ was added to each Complete medium containing 1 uCi/m plate and removed immediately for the zero hour control. Additional plates were labeled and removed at the time indicated in individual experiments. The dishes were rinsed as quickly as possible four times with cold BSS. 29 After the plates were drained, 1.0 ml of 0.15 M KCl, 0.02 M Tris HCl, pH 7.2 was added, and the cells were scraped from the surface of the dish with a plastic spatula and homogeni nized with 25 strokes of a 7 ml Dounce-type glass homoge- nizer (Wheaton Scientific, tightly fitting A pestle). The homogenate was centrifuged in a Ti50 type rotor at 133,000xg for 45 minutes. The supernatant was placed into a liquid scintillation counter vial and 2.2 ml of Aquasol (New England Nuclear, Corp.) was added. The samples in the liquid scintillation counter vials were mixed by vortexing, and the total radioactiVity was counted. The 100,000xg pellet was resuspended in 1.0 ml of 0.15 M KCl, 0.02 M Tris HCl, pH 7.2, by vortexing, and the suspension was added to Aquasol as described above. All measurements were made in duplicate. C. Noclei Counts Muscle cell cultures to be counted were rinsed twice at room temperature (37°C) with nonsterile 855. The cells were fixed for 5 min in absolute methanol and stained for a minimum of 20 min at room temperature with Giemsa stain. Nuclei were counted from ten randomly chosen fields at a final magnification of 320. The number of fused nuclei and the total number of nuclei per dish was calculated 4 by using the constant 3.458x10 which is the number of fields/dish for the 10 cm size cell culture dish. 30 D. Preparation of Fibroblast Cell Cultures Skin was removed from the hind limb, the dorsal and ventral abdominal area of the 12 day old chick embryo and 2+M92+ placed into 5 ml of Ca free 355 in a 6 cm Corning tissue culture dish. The skin sections were collected, drained and placed into a 15 ml centrifuge sterile tube with 4.5 m1 of Ca2+MgZ+ free BSS and 0.5 ml of 250 ug/ml trypsin. The suspension was aspirated for 3 min so that the tissue became uniformly distributed in the solution and this sus- pension was centrifuged at approximately 1500xg for three min. The pellet was resuspended in 10 m1 of regular complete culture medium. The cultures were incubated in a 95% air- 5% co2 atmosphere at 37°C in 4 m1 of complete medium. The fibroblasts were refed every 24 hours. Upon nearing con- fluency, the fibroblasts were subcultured via trypsinization into three additional 10 cm dishes. After approximately five days, the fibroblasts were subcultured the last time. The cells were counted in a hemocytometer and were plated 7 at a constant density of 0.25x10 cells/dish. 2+ E. Measurement of Ca Uptake by Fibroblast and Muscle Cultures The nonmyogenic cultures were pulse labeled with 45 1 uCi Ca2+/m1 for 5, 1o, 15, 30 and so min after 48, 72, and 96 hours in culture. The remainder of the procedure was followed as outlined in the relative measurement of Ca2+ uptake by myogenic cells. RESULTS AND DISCUSSION A. Ca2+ Accumulation in Differentiating Muscle Cells The general model of regulation of muscle cell membrane fusion described at the end of the literature review makes a number of predictions regarding muscle development. One of the major predictions is that the cytoplasmic concentra- 2+ tion of Ca must decrease many-fold during the process of muscle differentiation. This decrease in Ca2+ would result 2+ from increased accumulation of SR, the Ca regulatory organelle in muscle. TherefOre, relative intracellular Ca2+ 45 2+ levels were estimated by measuring the quantity of Ca radioactivity in the cytoplasmic and organelle fractions. As detailed in Materials and Methods, cells at each develop- h 45Ca2+ for a sufficient 45Ca2+ mental stage were incubated wit amount of time to ensure that exogenous had equili- brated with the intracellular pools. Figures 1 and 2 show that complete equilibration occurred within 2 hr regardless of the extent of muscle cell differentiation. Thus, in all subsequent experiments on muscle cells, the quantity of 45Ca2+ radioactivity in each compartment was measured after a standard 2 hr equilibration period. Because the number of cells in each culture Changes drastically during muscle development, all data were normalized by dividing the quan- 45 2+ tity of Ca radioactivity by the number of nuclei in the 31 Figure l. 32 Equilibration of exogenous 45Ca2+ with, intracellular pools in muscle cells during the early stages of differentiation. At each time indicated, cells were cooled to 2°C, rinsed 3 times with a cold isotonic saline solution, homogenized, and centri- fuged at 133,000 x g for 45 min. The quan- tity of radioactivity in the supernatant and pellet was assumed to reflect gelative cytoplasmic and organelle-bound Ca T levels, respectively. The muscle cell cultures used for this experiment were two days old. Each point represents the mean of duplicate determinations. 45Caz‘kcpm x 10”4 33 Total Hours 4 Cyioplasmic A Orgonelle + A j l 2 3 Figure 2. 34 Equilibration of exogenous 45Ca2+ with intracellular pools in fully differentiated muscle cells. At each time indicated, cells were cooled to 2°C, rinsed 3 times with cold isotonic saline solution, homo- genized, and centrifuged at 133,000 x g for 45 min. The quantity of radioactivity in the supernatant and pellet was assumed to reflect Eelative cytoplasmic and organelle- bound Ca T levels, respectively. The muscle cell cultures were five days old. Each point represents the mean of duplicate observations. 35 Toiol Hours ‘0 Orgonelle ‘ Cytoplasmic l l 2 3 36 culture. This normalization permitted direct comparison of data from divergent types of experiments. It should be noted that 45 2+ Ca 'cpm were divided by the number of nuclei in each culture rather than by the number of "cells". This was necessary because of the multinucleated nature of muscle cells, making it virtually impossible to accurately evaluate actual cell number. 1 The results in Figures 1 and 2 indicate that the muscle plasma membrane is not only freely permeable to calcium ions at 37°C, but that a transport-exchange system exists which results in rapid equilibration of Ca2+ among different intra- cellular pools. Taking advantage of this property of rapid equilibration permitted measurement of the relative levels 2+ of Ca in different cellular compartments at several stages of muscle development. Also, an interesting and important preliminary fact is depicted in Figures 1 and 2. The Ca2+ level is highest in the cytoplasmic fraction of Mb during the early stages of differentiation (Figure l), but highest in the organelle fraction of more fully differentiated muscle cells (Figure 2). The focus of the following results and discussion is on this difference and how it relates to ces- sation of myogenic cell proliferation and muscle cell dif- ferentiation 12.21232: The changes in the relative quantity of cytoplasmic and organelle-bound Ca2+ were examined throughout muscle differ- entiation. Muscle cells were cultivated over a seven-day period, and experiments were conducted in duplicate at twelve 37 hour intervals. A seven-day period was chosen because cul- tured myogenic cells undergo a complete transition from replicating PMb to fully-differentiated, contracting muscle fibers during this time. This Characteristic pattern of development (Figure 3) is manifested in culture as a burst Of Mb fusion after approximately 24 hr, followed by cessation of fusion on day 5 when differentiation is complete. This fusion curve is also included in several subsequent figures so that all results can be readily compared with the stage of muscle differentiation. Figure 3 also shows a dramatic reduction in the relative 2+ quantity of cytoplasmic Ca during muscle development. The quantity of cytoplasmic Ca2+ is approximately seventy-fold lower in fully-developed multinucleated Mt than in mono- nucleated myogenic cells, with the most dramatic decline in cytoplasmic Ca2+ associated with the onset of Mb fusion. Prior to the onsetof fusion, however, high levels of cyto- plasmic Ca2+. are present, presumably because the SR is not yet a fully functional organelle and thus does not control intracellular Ca2+ concentration to the extent it can in fully differentiated muscle cells (Boland gt gt., 1974; Lough gt gt., 1972; MacLennan and Holland, 1975; Tillack gt gt., 1974). If the fusion process requires that cyto- 2+ concentration be higher than a minimum level, 2+ plasmic Ca then it follows that the lowest level of intracellular Ca should occur at approximately the same time that fusion is observed to attain its maximum level. Critical examination Figure 3. 38 Regitionship between relative cytoplasmic Ca concentration and musile Eell fusion in muscle cell cultures. 5Ca + was measured as described in Materials and Methods. Muscle cell cultures were grown over a seven day period. The number of Mt nuclei was microscopically evaluated in Giemsa stained cultures. Each value represents the mean of four experiments in which all measurements were conducted in duplicate. Values of SEM were within 10-20% of the mean in all cases. 39 75. f0. x 22.8582 322.2 5 4. 3 2 I. d 1.2 p p P . o. 8. 6. . A 2 I O o o . 3.3 No. x 3282\an 339V 368.830 Culture Age (Days) 40 of the data in Figure 3 illustrates that, indeed, this pre- diction is true on approximately day 5 in culture. Thus, this observation is consistent with the proposed fusion regu- latory model. Since the quantity and functional capacity of the SR is minimal during the early stages of differentiation, competent myogenic cells fuse freely with each other in the presence of high extracellular and cytoplasmic Ca2+ concen- trations (Boland gt gt., 1974; Lough gt gt., 1972). It has been shown previously that Ca2+ is necessary for fusion (Schudt and Pette, 1975; Van der Bosch gt gl., 1972; Van der Bosch gt gl., 1973; Weidekamm gt 1., 1976) and that spon- taneous myogenic cell fusion will not occur in a medium with Ca2+ concentrations below 50 uM (Shainberg gt _l., 1969). f 45Ca2+ During the same period when cytoplasmic level 0 declined seventy-five-fold (Figure 3), the quantity a bound to cellular organelles increased only slightly (Figure 4, day 1-4.5). This organelle fraction consists of the intracellular constituents that sediment at 133,000 xg and contains primarily nuclei, mitochondria, myofibrils, micro- somes and sarcoplasmic reticulum. Between days 4.5 and 7, 45C32+ the quantity of bound increased several-fold, proba- bly as a result of SR accumulation (Figure 4, days 5-7). These results further support the evidence presented by Laugh and coworkers (1972) that a significant increase in Ca2+ uptake by SR occurs between the second and sixth days of maturation 1g liEEQ- SR synthesis and assembly develop rapidly with Mt formation, and the quantity and functional Figure 4. 41 Relationship bgtween the quantity of orga- nglls-bound Ca T and muscle cell fusion. 4 Ca T was measured as described in Materials and Methods. Muscle cell cultures were grown over a seven day period. The number of Mt nuclei was microscopically evaluated in cultures stained with Giemsa. 42 713mb. x 2250:2032 322.2 4 3 2 I. Aolo. 0.0. x m=o_o:z\Eau+~oon¢ 2.8090 Culture Age (Days) 43 capacity of the SR membrane system increases during muscle maturation. Once the SR reaches its maximum it is capable of lowering the intracellular Ca2+ concentration below 10‘5M (Lough gt gt., 1972). These data are also consistent with the proposed model, since the only organelle in skeletal 2+ 7 muscle capable of regulating cytoplasmic Ca at 10' M (i.e. the SR) reaches its maximum activity shortly after cessation of muscle cell fusion (Figure 4). Figure 5 shows the rela- 45 2+ tionship between Mb fusion and the total Ca cpm in nucleus. Total Ca2+ was obtained by adding the contributions of both cytoplasmic and organelle fractions. This pattern 2+ illustrates that the total quantity of cellular Ca de- creases during the initial stages of the fusion process, but eventually increases again after differentiation is complete. 2+ Clearly, there are large changes in both Ca content and intracellular distribution during development. To further 2+ re-distribu- tion, the ratio of cytoplasmic to organelle-bound Ca2+ was illustrate the magnitude of intracellular Ca calculated at each developmental stage (Figure 6). This 2+ ratio of the cytoplasmic to organelle Ca is inversely pro- portional to the number of Mt nuclei/culture and is approxi- mately 200-300 fold lower in fully developed, multinucleated Mt than in mononucleated cells.' In summary, the high level 2+ of Ca in the cytoplasmic fraction of replicating myogenic cells and its rapid decline during the increase in Mb fusion 2+ is consistent with the idea that Ca plays a significant role in the initiation and/or mechanism of muscle cell Figure 5. ,ture. 44 Relationship between total Ca2+ concentration and musige gell fusion in muscle cell cul- Ca T was measured as described in Materials and Methods. Muscle cell cultures were grown over a seven day period, and the number of Mt nuclei was microscopically evaluated in cultures stained with Giemsa. 45 71.5.0. x 22.8332 829.2 5 4 3 2 q. . m . L p p R n p 2 0 B 6 .4. 2 L 1. o Q 0 Q 5013 No. x 8282\an $89.28. Culture Age (Days) Figure 6. 46 The relationship between the ratio of cyto- plasmiczorganelle 45Ca2+ and the number of Mt nuclei/culture as a function of the age of muscle cells in culture over a seven day period. Ratios were calculated by dividing cytoplasmic 45Ca2+ iBm/gucleus (Figure 3) by organelle-bound a + cpm/Nucleus (Figure 4). 47 710.90. x 82386.32 322:). 5 4 3 2 l d - fi - - 0_7 .6 .5 1.4 0 L3 . 12 p_._....h.., H D 9 8 7. 6 5 4 3 2 l O .06. .63 hobo? 9.2690 8.88.8.5 Culture Age (Days) 48 fusion (Figure 6). During the course of the previous set of experiments, it was consistently observed that cytoplasmic Ca2+/nucleus exhibited considerably more variability on day 1 than for any of the other ages. Several factors could have accounted for this variation. First, because the large decline in cytoplasmic Ca2+ level appeared to be closely coordinated with the burst of Mb fusion in the individual experiments in this series, and because the time of onset of Mb fusion in culture is known to be highly dependent on the initial pla- ting density, the variability in cytoplasmic Ca2+ on day 1 could merely have reflected minor differences in initial cell density. Alternatively, there might be brief fluctu- ations in cytoplasmic Ca2+ during this transition period that would not have been detected, since time points were taken every twelve hours. Further evaluation of this second possi- bility seemed in order because of the observations of Zalin and Montague (1974) suggesting that cyclic nucleotide levels undergo transient fluctuations several hours prior to the onset of fusion. The fact that fluctuations in cyclic nu- cleotides often cause similar modifications in divalent cation levels (Rasmussen and Goodman, 1977) strongly sugges- ted the possibility that Ca2+ levels might also undergo a transient increase immediately prior to the onset of fusion. Thus, twelve experiments were conducted as described in Materials and Methods to observe the changes in Ca2+ con- centrations of the cytoplasmic and organelle fractions during 49 the period from 10 hrs prior to the onset of fusion to 26 hrs after the onset of fusion. Cultures were sacrificed at 2 hr intervals, and both the Ca2+ levels (cytoplasmic and organelle-bound) and the number of Mt nuclei was measured. As indicated above, the onset of fusion in individual experi- ments is quite dependent on initial cell density; therefore, all results were standardized relative to a point at which fusion could be first detected based on cell counts in Giemsa-stained cultures. A significant, transient increase in cytoplasmic Ca2+ level was consistently observed at 6 hr prior to the onset of Mb fusion (Figure 7). The actual data utilized in Figure 7 is also shown in Table 1'so that the extent of variability in experiments of this nature can be illustrated. The transient change in cytoplasmic Ca2+ 2+ , along with the high cytoplasmic Ca concentrations in mononu- cleated cells as compared to Mt (Figure 3), may be vital prerequisites for the initiation of fusion. Ca2+ may also be directly involved in the cell-to-Cell contact that pre- cedes fusion. Exactly why the peak occurs is not known. However, Zalin (1976) has demonstrated a brief, elevated CAMP level several hours prior to the onset of fusion, sug- gesting that CAMP may be a specific trigger that stimulates events leading up to and resulting in Mb fusion. To determine whether the transient change in cytoplas— 2+ (Figure 7) was specific for Ca2+ 2+ mic ca in the cytoplasmic pool, Ca levels were also examined in the organelle frac- tion of muscle cells. Results of this eValuation (Figure 8) 50 Figure 7. Relationship betwesn initiation of Mb fusion and cytoplasmic Ca + content. Each point represents the mean of twelve experiments in which measurements were made in duplicate. The number of Mt nuclei was microscopically evaluated in cultures stained with Giemsa. 51 .1310. x 8230:3032 329.5. m M. O - q 100 313%. x mao.o:z\an+ . . m m 70 P b...-_ w w m m m m o we”.g 268.330. 0. . Time Relative to Fusion Onset (Hours) Table l. Change§+i mic Ca the onset of myoblast fusion. n number of myotube nuclei 52 and cytoplas- relative to the onset of myoblast fusion. Twelve experiments were carried out with duplicate measurements, and data were normalized relative to Time Relative to Myotube nuclei/ Cytoplasmic 45Ca2+ Fusion Onset (hr) Culture (x105)* cpm/Nucleus (x10'3)* -10 0.02 i 0.01 42.5 i 1.35 -8 0.05 i 0.03 76.5 i 17.2 -6 0.05 i 0.02 83.9 i 18.5 -4 0.07 t 0.03 58.1 t'12.3 -2 0.05 i 0.01 45.0 i 8.85 O 0.07 i 0.03 57.3 i 12.2 +2 0.16 i 0.05 44.2 i 9.88 +4 0.21 i 0.06 31.1 i 6.59 +5 0.28 i 0.05, 44.8 t'14.4 +8 0.35 t 0.08 31.8 1‘5.42 +10 0.39 i 0.07 37.6 i 8.43 +12 0.47 i 0.10 27.1 i 6.98 +14 0.77 i 0.17 18.1 i 5.52 +16 0.79 i 0.13 14.7 i 5.48 +18 _1.06 i 0.16 17.3 i 7.70 +20 1.14 t 0.28 8.77 42.52 +22 1.07 i 0.28 14.3 i 7.81 +24 1.68 i 1.31 7.13 i 3.98 *Each value represents the mean 1 SEM. 53 show a general pattern of relative Changes similar to that observed for the cytoplasmic p001 (Figure 7). In particular, a transient increase in organelle-bound Ca2+ was observed approximately 8 hr prior to initiation of Mb fusion; how- ever, the quantity of organelle-associated Ca2+ does not decline as markedly during the fusion process as does cyto- plasmic Ca2+ levels (c.f., Figure 7). This latter observa- tion would be anticipated based on similar data already pre- sented (Figure 4) which showed relatively constant levels of organelle-bound Ca2+ during the first four days in cul- ture. It is also noteworthy that only about 4-6% of the total intracellular Ca2+ is associated with the organelle fraction during this initial period of muscle development (i.e. comparing the scales 0f Figure 7 and Figure 8); therefore, the pattern of change in total intracellular Ca2+ throughout Mb fusion (Figure 9) primarily reflects the cytoplasmic fraction. In summary, the transient Change in Ca2+ several hours prior to fusion does not stem from a shift between intracellular pools; rather, it results from a net increase in intracellular Ca2+ (Figure 9). Extensive recent work has been conducted on the effect of CAMP on myogenesis and its possible interaction with Ca2+ to promote muscle cell fusion and maturation (Bornet _e_t__a_]_.,1977; Epstein gtgt.,l975;2alin, 1977;2a1in, ‘ 1976); Zalin, 1973; Zalin and Leaver, 1975; Zalin and Monta- gue, 1975; Zalin and Montague, 1974). Presently, however, 2+ the link between CAMP and Ca in promoting Mb fusion is not Figure 8. 54 Relationship between igitiation of Mb fusion and organelle-bound Ca T Each point represents the mean of twelve experiments in which measurements were made in duplicate. The number of Mt nuclei was microscopically evaluated in cultures stained with Giemsa. 55 2.0 .I..o.o_ x 2238.282 32%: _ 5 m... m 0 d d 1 20 IO O. 4 3.10.0.0. x 3282\an $83 2.835 -10 Time Relative to Fusion Onset (Hours) 56 Figure 9. Relationship between initiation of Mb fusion and total Ca2+ concentration (cytoplasmic and organelle-bound). Each point represents the mean of twelve experiments in which measurements were made in duplicate. The number of Mt nuclei was microscopically evaluated in cultures stained with Giemsa. 57 m w w 7|: 0-0. x 83:30:22.2 3232 5 O ....... 0 O 000 mem4mzi 80" p 0 9 I00 “cling x 3332\an +~oom¢ .20... 20 IO m . Time Relative to Fusion Onset (Hours) 58 readily apparent. It would be instrumental in understanding Mb fusion to determine whether regulation of Ca2+ and cAMP levels prior to fusion occurs by independent processes, or whether their regulation is closely coordinated. The possi- bility that the cyclic nucleotide may be directly associated 2+ stems from the observation that Ca2+ and cAMP with Ca interact in a number of their actions in the cell (Berridge, l975) and from the suggestion that certain effects of raising the cAMP level in a cell are ultimately brought about by CAMP-controlled changes in cytoplasmic Ca2+ concen- tration (Borle, l974). It has been suggested that Ca2+-specific sites are present on the muscle cell plasma membrane and that these sites facilitate the cell-to-cell contact that must precede fusion. Thus, Ca2+ is thought to stimulate or facilitate the fusion mechanism by its interaction with these sites (Schudt and Pette, 1975; Van der Bosch 33 al., l972; Weide- kamm _t _l., 1976; Zalin, l976). Experiments need to be designed to examine the morphological and biochemical changes that occur in the plasma membrane. Close examination of the functional structures involved in fusion may give not only a deeper insight into the fusion mechanism but may unravel 2+ the role of Ca in this process. The present results therefore suggest that, if Ca2+ is not intimately associated with the mechanism of cell fusion, its concentration at least coincidentally undergoes a 100-200 decrease during fusion-associated events. It would seem uncharacteristic 59 of eukaryotic cells that a change of this magnitude was not somehow directly involved in the unique events responsible for muscle differentiation. . B. Relative Ca2+ Levels in Fibroblasts The cell culture system is advantageous because it eli- minates a multitude of factors that influence muscle cells in vivo such as hormonal and neural substances; however, muscle cells cultured in vitro do not represent a pure myo- genic cell population. It is extremely difficult to obtain a pure myogenic cell population in cell culture, no matter what care is taken with the isolation procedures. The major contaminant of the muscle cell cultures is the fibroblast (Fb). Replicating PMb and replicating Fb are difficult to distinguish until fusion commences; however, only the post- mitotic muscle cells committed to further maturation fuse. Thus, the two primary cell types in these cultures during a seven-day experiment (such as that shown in Figure 3) cease proliferation, but for different reasons. Whereas myogenic cells are genetically programmed to withdraw from the cell cycle, the Pb stop proliferatidn because they become com- pletely confluent on the surface of the cell culture dish. This latter property is usually referred to as "contact in- hibition" and results from the fact that normal eukaryotic cells in culture will not grow in multiple layers. Experi- ments were thus conducted in order to determine whether the 2+ large decrease in cytoplasmic Ca in differentiated muscle '7 .1 60 cells was associated with unique myogenic events, or whether it was the simple result of the fact that muscle cells are no longer replicating. Fb microsomal activity has been found to be similar in characteristics to the SR of the skeletal muscle; however, the specific activity of chick 2+ embryo Fb microsomal Ca uptake was observed to be much less than that found in the skeletal muscle system (Moore and Pastan, l977). Moore and Pastan (1977) measured Ca2+ levels in chick embryo Pb and characterized the energy depen- dent Ca2+ uptake activity of the microsomal fraction which may play a role in the regulation of calcium ion concentra- tion in the cytosol Fb. Although the activity of the Fb system is less than one percent of the activity of the mi- crosomal fraction from the skeletal muscle (Moore and Pastan, 1977), it is comparable with microsomal activity isolated from cultured skeletal muscle cells (Lough gt al., 1972). Fb are comparable to skeletal muscle cells in that they con- tain contractile proteins (actin and myosin) whose interac- 2+ 2+ tion, along with Fb movement, are regulated by Ca Ca may be regulated by three membrane systems in the Fb: endo- plasmic reticulum, mitochondria and plasma membrane (Moore and Pastan, 1977) by a mechanism similar to that of skeletal 2+ muscle cells. Thus, Ca is not only important in skeletal muscle but also may have a significant role in Fb growth and 2+ on muscle cells which metabolism. The unique effect of Ca differs from any other cell type is that of its role in Mb fusion which is the main emphasis of this research. 6l Because of the similarity between Fb and skeletal muscle cells and because replicating Fb and replicating PMb differ little, experiments were conducted to closer observe changes 2+ in Ca concentration in Fb during their proliferating and confluent phases. As with the previous experiments on muscle cells, preliminary experiments were performed to determine the minimum amount of time needed for exogenous 45 2+ Ca to equilibrate with the cytoplasmic and organelle pools in both proliferating and stationary phases of these 45 a2+ cells (Figure 10 and ll). Although total C cpm were higher in the confluent cultures because of higher cell density (Figure ll), the relative amounts of cytoplasmic and organelle Ca2+ were not significantly different from the proliferating fibroblasts (Figure l0). These results are 2+ in contrast to the muscle cell Ca equilibration results depicted in Figures 1 and 2 where the cytoplasmic and 2+ organelle Ca concentrations were nearly inversely propor- tional to each other as a function of time. These Fb Ca2+ equilibration results suggest that the large decrease in cytoplasmic Ca2+ in muscle cells is purely characteristic of the myogenic population. Four individual Fb experiments were conducted to assess 2+ content of replicating and sta- the relative changes in Ca tionary Fb; however, the results from only one of the experi- ments are shown and discussed. The pattern of the results obtained from the Fb cultures was the same for the four experiments but the variation between individual experiments 62 Figure l0. Kinetics of 45Ca2+ equilibration over a 60 minute period in cell proliferating (or replicating) fibroblast culture. Ca cpm x IO 45 2+ N 63 IO 20 Total Cytoplasmic Minutes 64 Figure ll. Kinetics of 45Ca2+ equilibration over a 60 minute periOd in stationary (or non-repli- cating) fibroblast cultures. 45Caz"'cpm x IO"4 65 7 .. Total 6 .. 5 - Cytoplasmic 4' - 3 .- 2 ' o rganelle A | .. I o l 1 l L L 0 IO 20 30 40 50 60 Minutes 66 was great because of the differences in initial cell den- sities and in the rates of cell proliferation. Changes in cytoplasmic Ca2+ levels in Fb during the six-day culture period (Figure 12) resembled those of muscle cells in culture (Figure 3). In particular, the initial amount of high cytoplasmic Ca2+ on day l in Fb sharply declines in a half-day period (Figure l2). A similar ob- servation was depicted in muscle cells (Figure 3); however, the initial decrease in the cytoplasmic Ca2+ level in muscle was two-fold (Figure 3), whereas in Fb the decrease was only a quarter-fold (Figure l2). Although the number of repli- cating Fb continued to steadily increase after 3.5 days and until 5.5 days when Fb entered the stationary phase, the 2+ cytoplasmic Ca level remained rather constant (Figure 12). 2+ In muscle cells (Figure 3), the cytoplasmic Ca level con- tinued to decrease until 5.0 days at which time fusion was 45 2+ cpm/nucleus x lo"3 re- maximum, and the cytoplasmic Ca mained within 0.03 and 0.04 x 10'3 (Figure 3). It is impor- tant to note that the organelle responsible for lowering intracellular Ca2+ in muscle cells is the sarcoplasmic reti- culum and, on the basis of the results presented in Figures 3 and 12, it can be seen that a much more extensive sarco- plasmic reticulum is present in muscle. This membrane system also reaches a maximum in functional capacity concomitant with completion 0f muscle fusion. These results illustrate that the dramatic cytoplasmic Ca2+ level decrease occurs 2+ only in muscle cells since the cytoplasmic Ca level changes 67 Figure 12. The relationship between the cytoplasmic 45Ca2+ cpm/nucleus and number of cells/ plate in fibroblast cell cultures grown over a 6 day period. 68 Ailmb. x 3052.8 6 5 4 3 2 I. q u d d u q . v7 ’1 - n n p n n - n n w 9 8 7 6 5 4 3 2 l 3.3 No. x 8282\an 589268.830 Culture Age (Days) 69 observed in the Fb cultures were minimal when compared to changes observed in the Mb cultures. Therefore, this de- crease must be unique to myogenic events. That the extensive development of sarcoplasmic reticulum occurs only in muscle cells, can be seen from the results 45 2+ obtained by measuring relative quantities of Ca bound to the organelle fraction of Fb. The initial decrease in 45 2+ 2+ organelle Ca cpm/nucleus (Figure 13) mimics the cyto- plasmic Ca level decrease in muscle cells in some respects; 2+ however, the relative amount of Ca is relatively constant during the period between 1.5 and 6 days (Figure 13). These results differ considerably from those obtained in Mb cul- tures showing the continued increase in the relative quantity of Cazfi bound to the organelle fraction of muscle cells undergoing fusion (Figure 4). That Fb contain a Ca2+ regu- lating system is not only depicted in these results (Figure 13) but has been presented in the literature (Moore and Pastan, 1977). The microsomal'fraction that is responsible 2+ for regulating Ca in the Fb, may be a modification of the SR but does not exhibit the extensive functional capacity of SR unique to muscle cells. On the basis of these results it can be assumed that the two cell populations (Mb and Pb) 2+ have individual Ca regulating systems and are differently 2+ affected by changes in Ca levels during differentiation. The changes in the total (cytoplasmic and organelle) 2+ Ca levels of replicating and stationary Fb are shown in Figure 14. It is clear from these data that the primary 70 Figure 13. The relationship between the organelle 45Ca2+ cpm/nucleus and the number of cells/plate in fibroblast cultures grown over a 6 day period. 7l forte. x 2o_o\o__oo . . . p p — 6 5 4. 3 2 .I Aalov no. x 920335338? 2.9590 Culture Age (Days) 72 Figure 14. The relationship between the total 45Ca2+ cpm/nucleus and cells/plate in fibroblast cell cultures grown over a six-day period. 73 5 7120. x o.o_a\a__8 4 3 2 I. q . . m . “b 7. . x ma£o=z\an+~oonv_Eo._. Culture Age (Days) 74 decrease in total Ca2+ during the transition from prolifera- tion to confluency is the result of a decrease in cytoplas- 2+ content (Figure 12).rather than from an intra- cellular redistribution of a constant quantity of Ca2+. In mic Ca muscle cells, on the other hand, relative quantities of total Ca2+ increase after day 5 probably due to the fact that the sarcoplasmic reticulum reached maximum functional capacity at maximum fusion which occurs after day 5 (Figure 5). 45 The ratio of cytoplasmiczorganelle Ca2+ cpm/nucleus in relation to the increase in the number of Fb cells/plate as a function of time is also a good indicator of changes in the intracellular distribution of Ca2+ (Figure 15). This 2+ steady decrease in cytoplasmic Ca of Fb (Figure 15) is qualitatively similar to the distribution pattern of Mb (Figure 6); however, a significant quantitative difference exists between the Mb and Fb in the decrease of cytoplasmic 2+ 45 2+ Ca levels. The ratio of cytoplasmiczarganelle Ca cpm/ nucleus decreases lO8-fold in the muscle cells (Figure 6) but only 13-fold in the Fb (Figure 15). This difference suggests that the decrease in cytoplasmic Ca2+ is exclusi- vely associated with myogenesis. Since it has been indicated in the literature that extracellular Ca2+ concentrations regulate fusion, and since these results show that the 2+ decrease in cytoplasmic Ca is concomitant with myogenesis and relatively independent of other cell populations present 2+ in muscle culture, it can be concluded that Ca may be 75 Figure 15. The relationship bgtwegn the ratio of cyto- plasmic:organelle 5Ca + cpm and the number fibroblasts/plate over a six-day period. 76 also? x 22% 2.8 6 5 4 _ 3 - q q 4 . 0 . 5 2 m 09.3 3085\an . m... $89. p m o 0 2.9590 "£58395 Culture Age (Days) 77 involved in the mechanism and/or regulation of the muscle cell fusion process. Future experiments may be designed to more closely examine the rapid fusion rate and the relative quantities 2+ concentrations. Effort should be made to of exogenous Ca quantify measurements and to devise a statistical method applicable to cell culture so that variation in cell pro- liferation of individual experiments can be accounted for. Also, procedures ought to be modified so that cell densi- ties may be better controlled and monitored. In summary, results obtained from the Fb experiments indicate the similar pattern in the changes of Ca2+ levels between replicating Pb and replicating PMb. Altered Ca2+ levels in the cytoplasmic and organelle fractions of Fb cells superficially mimic behavior observed in muscle cell cultures. However, Fb and Mb differ significantly in that the fusion process is unique to Mb. On this basis, the . 2+ large decrease in cytoplasmic Ca levels in Mb are proba- bly due to Ca2+ interaction in cell-to-cell contact which initiates fusion. Also, the fact that the SR develops immediately after fusion is evidenced by the decrease in 2+ the relative quantities of cytoplasmic Ca and increase in the levels of organelle-bound Ca2+. SR regulates Ca2+ and upon reaching its maximum, lowers Ca2+ concentration to 10‘5 M as was discussed in the literature review. Although Fb cultures exhibit similar patterns in 2+ changes of relative Ca levels to Mb cultures, they lack 78 a dramatic decrease in cytoplasmic Ca2+ probably because the microsomal fraction in Fb is not as elaborate as the SR of the skeletal muscle cells. As summarized in Table 2, the large cytoplasmic Ca2+ decrease (68-fold) in muscle com- pared to the relatively smaller decrease (5.5-fold) in fibroblasts emphasizes the possibility that Ca2+ is involved in processes unique to muscle cell fusion. 79 Table 2. Summary of cytoplasmic Ca2+ content of mononu- cleated myoblasts, fully developed multinuclee ated myotubes, replicating fibroblasts, and stationary fibroblasts. 45 2+ Cell Type Ca cpm/nucleus (x10‘3) Mononucleated myoblasts 23.0 i 2.6 Multinucleated myotubes 0.34 i 0.06 Replicating fibroblasts 31.2 i 4.5 Stationary fibroblasts 5.63 i 0.71 Mean i SEM of at least four experiments carried out in duplicate. SUMMARY Muscle cell cultures at different stages of develop- ment, ranging from replicating presumptive myoblasts to fully differentiated multinucleated myotubes, were pulse labeled with l uCi 45Cab/ml for 1 hour in order to deter- mine the magnitude of developmental alterationsof Ca2+ levels in the cytoplasmic and organelle compartments of the muscle cells. Equilibration of intra- and extracellu- 1ar Ca2+ pools was complete during the 1 hour pulse label period. Cell homogenates were centrifuged at 133,000 x g for 45 minutes, and the quantities of supernatant 45Ca2+ and of pellet 45Ca2+ were utilized as measures of relative cytoplasmic Ca2+ and organelle 45Ca2+ 2+ levels, respectively. The cytoplasmic Ca was approximately 68-fold lower in myotubes than in replicating presumptive myoblasts. Prior to the onset of fusion cytoplasmic Ca2+ levels in PMb were high. The initial decline in Ca2+ concentration was coordi- nated with the onset of multinucleated myotube formation, and the occurrence of the lowest cytoplasmic Ca2+ concen- tration coincided with cessation of fusion in maximally differentiated muscle cells. Such a large decrease in cytoplasmic Ca2+ 2+ concentration may be associated with fusion events and Ca may interact with plasma membrane Ca2+ binding sites to facilitate cell-to-cell contact and 80 81 initiate fusion. Immediately after fusion, the sarco- plasmic reticulum develops extensively and functions at maximum capacity. This organelle may also be involved in bringing about such dramatic decreases in cytoplasmic Ca2+ levels. The quantity of sarcoplasmic reticulum was shown to be 3-4 times higher in 6 day cultures as opposed to 1 day cultures. The ratios of cytoplasmic to organelle- bound Ca2+ were 11.9 and 0.11 in presumptive myoblasts and myotube cultures, respectively. Thus, the transition from presumptive myoblast to post-mitotic muscle cells is accom- 2+ levels. panied by a dramatic decline in Ca Because myogenic cell populations contain fibroblasts, cultures of replicating and post-mitotic fibroblasts were examined utilizing the same procedure for the muscle cul- tures. Replicating fibroblasts and replicating myoblasts are virtually indistinguishable; therefore, the changes in Ca2+ concentrations were tested to determine whether they were associated with myogenic events during diffentiation. The cytoplasmic Ca2+ decrease was 68-fold in muscle but only 5.5-fold in fibroblast. (These data are consistant with the idea that the extent of Mb membrane fusion may be 2+ partially regulated by intracellular Ca levels. BIBLIOGRAPHY Abbott, J., Schiltz, J., Dienstman, S. and H. Holtzer. 1974. The phenotypic complexity of myogenic clones. Proc. Natl. Acad. Sci. USA 71 (4):1506-1510. Affolter, H., Chiesi, M., Dabrowska, R. and E. Carafoli. 1976. Calcium regulation in heart cells. Eur. J. Biochem. 67:389-396. Berridge, M.J. 1975. The interaction of cyclic nucleo- tides and calcium in the control of cellular activity. In: Advances in Cyclic Nucleotide Research VI. Ed. by Greengard and A. Robinson. Raven Press, New York. p. 2. Bischoff, R. and H. Holtzer. 1968. The effect of mitotic inhibitors on myogenesis. J. Cell Biol. 36:111-128. Bischoff, R. and H. Holtzer. 1969. Mitosis and the pro- cesses of differentiation of myogenic cells in vitro. J. Cell Biol. 41:188-200. Bischoff, R. and H. Holtzer. 1970. Inhibition of myoblast fusion after one round of DNA synthesis in 5-Bromodeo- xyuridine. J. Cell Biol. 44:134-150. Boland, R., Martonosi, A. and T.w. Tillack. 1974. Develop- mental changes in the composition and function of sarcoplasmic reticulum. J. Biol. Chem. 249(2):612-623. Borle, A,B. 1974. Cyclic AMP stimulation of calcium efflux from kidney, liver and heart mitochondria. J. Memb. Biol. 16:221-236. Bornet, E.P., Entman, M.L., Van Winkle, B.N., Schwartz, A., Lehotay, D.C and 6.5. Levey. 1977. Cyclic AMP modu- lation of Ca2+ accumulation by sarcoplasmic reticulum from fast skeletal muscle. Biochim. Biophys. Act. 468:188-193. Buckley, P.A. and I.R. Konigsberg. 1974. Myogenic fusion and the duration of the post-mitotic gap (61). Devel- op. Biol. 37:193-212. 82 83 Chi, J.C., Fellini, S.A. and H. Holtzer. 1975. Differences among myosins synthesized in non-myogenic cells, pre- sumptive myoblasts, and myoblasts. Proc. Natl. Acad. Sci. USA 72(12):4999-5003. Chyn, T. and A. Martonosi. 1977. Chemical modification of sarcoplasmic reticulum membranes. Biochim. Biophys. Acta. 468:114-126. Constantin, L.L. and R.J. Podolsky. 1965. Calcium locali- zation and the activation of striated muscle fibers. Fed. Proc. 24(3):ll41-ll45. Dabrowska, R., Podlubnaya, Z., Nowak, E. and N. Drabikowski. 1976. Interaction of tropomyosin with troponin com- ponents. J. Biochem. 80(1):89-99. Dienstman, S.R. and H. Holtzer. 1975. Myogenesisza cell lineage interpretation. In: Cell Cycle and Cell Differentiation. Ed. by J. Reinert and H. Holtzer. Berlin: Springer-Verlag. p. 1-25. Dienstman, S.R. and H. Holtzer. 1977. Skeletal myogenesis. Exp. Cell Res. 107:355-364. Dupont, Y. 1 78. TEansient kinetics of sarcoplasmic reti- culum Ca + + Mg ATPase studied by fluorescence. Nature. 273(5661):396-398. Ebashi, S. and M. Endo. 1968. Calcium ion and muscle con- traction. Prog. Bio-Phys. Molec. Biol. 18:123-183. Ebashi, S., Endo, M. and T. Ohtsuki. 1969. Control of muscle contraction. 0. Rev. Biophys. 3:351-384. Emerson, C.P. and S.K. Beckner. 1975. Activation of myosin synthesis in fusing and mononucleated myoblasts. J. Mol. Biol. 93:431-447. Endo, M. 1977. Ca2+ release from sarcoplasmic reticulum. Physiol. Rev. 57(1):7l-108. Epstein, C. J. , Jiminez de Asua, L. and E. Rosengurt. 1975. The role of cyclic AMP in myogenesis. J. Cell Physiol. 86: 83- 90. Ezerman, E.B. and H. Ishikawa. 1967. Differentiation of the sarcoplasmic reticulum anlesystem in developing chick skeletal muscle in_vitro. J. Cell Biol. 35: 405-420. 84 Fambrough, D.M. 1974. Cellular and developmental biology of acetylcholine receptors in skeletal muscle. In: Neurochemistry of Cholinergic Receptors. Ed. by de Robertis and J. Schacht. Raven Press. New York. p. 98. Fischman, D.A. 1967. An electron microscope study of myo- fibril formation in embryonic chick skeletal muscle. J. Cell Biol. 32:557-575. Fuchs, F. 1977. The binding of calcium to glycerated muscle fibers in rigor. The effect of filament over- lap. Biochim. Biophys. Acta. 491:523-531. Gaertner, U., Schudt, C. and D. Pette. 1977. Regulation of glycogen synthase interconversion in cultured muscle cells: actions of insulin calcium, ionophore A 23187 and cytochalasin B. Mol. Cell Endocrin. 8:35-46. Gergely, J. 1974. Some aspects of the role of the sarco- plasmic reticulum and the tropomyosin-troponin system in the control of muscle contraction by calcium ions. Supplement III to Circulation Research. 34 and 35: III-74-III-81. Hidalgo, C., Ikemoto, N. and J. Gergely. 1976. Role of phospholipids in the calcium-dependent ATPase of the sarcoplasmic reticulum. J. Biol. Chem. 251(14): 4224-4232. Holtzer, H. 1970. Myogenesis. In: Cell Differentiation. Ed. by 0. Schjeide and J. de Vellis. Van Nostrand Rein- hold. New York. p. 476-503. . Holtzer, H. and R. Bischoff. 1970. Mitosis and myogenesis. In: The Physiology and Biochemistry of Muscle as a Food, 2. Ed. by E.J. Briskey, R.G. Cassens and 8.8. Marsh. Univ. Wisconsin Press. Madison. p. 29-51. Holtzer, H., Croop, J., Dienstman, S., Ishikawa, H. and A.P. Somlyo. 1975a. Proc. Natl. Acad. Sci. USA 72: 513-517. Holtzer, H., Rubinstein, N., Dienstman, S., Chi, J., Biehl, J. and A.P. Somlyo. 1974. Perspectives in myo- genesis. Biochimie. 56(11-12):1575-1580. Holtzer, H., Rubinstein, N., Fellini, S.,‘Yeoh, 6., Chi, J. Birnbaum, J. and M. Okayama. 1975c. Lineages, quantal cell cycles, and the generation of cell diversity. 0. Rev. Biophys. 8. 4:523-557. 85 Holtzer, H., Strabs, K., Biehl, J., Somlyo, A.P. and H. Ishikawa. 1975b. Science. 188:943-945. Holtzer, H., Weintraub, H., Mayne, R. and B. Mochan. 1973. The cell cycle, cell lineages and cell differentiation. Current Topics in Develop. Biol. 7:229-256. Homsher, E. and C.J. Kean. 1978. Skeletal muscle energe- tics and metabolism. Ann. Rev. Physiol. 40:93-131. Inesi, G. 1972. Active transport of calcium ion in sarco- plasmic membranes. Annu. Rev. Biophys. Bioeng. 1: 191-210. Inesi, G. and N. Malan. 1976. Mechanisms of calcium release in sarcoplasmic reticulum. Life Sciences. 18:773-780. Ishikawa, H., Bischoff, R. and H. Holtzer. 1968. Mitosis and intermediate sized filaments in developing skeletal muscle. J. Cell Biol. 38:538-555. Jorgenflyu A.0., Kalnins, V.I., Zubrzycka, E. and D.H. MacLen- nan. 1977. Assembly of the sarcoplasmic reticulum. J. Cell Biol. 74:287-298. Kasai, M. and H. Miyamoto. 1976. Depolarization-induced calcium release from sarcoplasmic reticulum fragments. 1. Release of calcium taken up upon using ATP. J. Biochem. 79(5):1053-1066. Kasai, M. and H. Miyamoto. l976a. Depolarization-induced calcium release from sarcoplasmic reticulum fragments. II. Release of calcium incorporated without ATP. J. Biochem. 79(5):1067-1076. Keller, J.M. and M. Nameroff. 1974. Induction of creatine phosphokinase in cultures of chick skeletal myoblasts without concomitant cell fusion. Differentiation 2: 19-24. Laclette, J.P. and M. Montal. 1977. Interaction of calcium with negative lipids in planar bilayer membranes. Bio- phys. J. 19:199-202. Lough, J. and R. Bischoff. 1977. Differentiation of areas: tine phosphokinase during myogenesis: quantitative ‘ fractionation of isozymes. Develop. Biol. 57:330-344. Lough, J.W., Entman, M.L., Bossen, E.H. and J.L. Hansen. 1972. Calcium accumulation by isolated sarcoplasmic reticulum of skeletal muscle during development in tissue culture. J. Cell Physiol. 80(3):431-436. 86 MacLennan, D.H. 1975. Resolution of the calcium transport system of sarcoplasmic reticulum. Can. J. Biochem. 53(3):251-261. MacLennan, D.H. and P.C. Holland. 1975. Calcium transport in sarcoplasmic reticulum. Annu. Rev. Biophys. Bioeng. 4:377-404. MacLennan, D.H. and P.C. Holland. 1976. The calcium trans- port ATPase of sarcoplasmic reticulum. In: The Enzymes of Biological Membranes III (Membrane Trans- port). Ed. by A. Martonosi. Plenum Press. New York. p. 239. MacLennan, D.H., Yip, C.C. Iles, G.H. and P. Seeman. 1972. Isolation of sarcoplasmic reticulum proteins. Cold Spring Harbor Symp. Quant. Biol. 37:469-477. Mannhertz, H.C. and R.S. Goody. 1976. Proteins of con- tractile systems. Annu. Rev. Biochem. 45:427-465. Moore, L. and I. Pastan. 1977. Regulation of intracellu- lar calcium in chick embryo fibroblast: calcium uptake by the microsomal fraction. J. Cell Physiol. 91(2): 289-296. Morgan, H.E., Randle, P.J. and D.M. Regen. 1959. Regula- tion of glucose uptake by muscle. Biochem. J. 73:573- 579. Moriyama, Y., Hasegawa, S. and K. Murayama. 1976. cAMP and cGMP changes associated with the differentiation of cultured chick embryo muscle cells. Exp. Cell Res. 101:159-163. Morris, G.E., Piper, M. and R. Cole. 1976. Differential effects of calcium ion concentration on cell fusion, cell division and creatine kinase activity in muscle cell cultures. Exp. Cell Res. 99:106-114. Murphy, A.J. 1976. Sulfhydryl group modification of sarco- plasmic reticulum membranes. Biochemistry. 15(20): 4492-4496. Okazaki, K. and H. Holtzer. 1965. An analysis of myogenesis in vitro using fluorescein labeled antimyosin. J. Histochem. Cytochem. 13(8):726-739. O‘Neill, M.C. 1976. Population modeling in muscle cell culture: comparisons with experiments. Develop. Biol. 53:190-205. 87 Paterson, B. and R.C. Strohman. 1972. Myosin synthesis in cultures of differentiating chicken embryo skeletal muscle. Develop. Biol. 29(2):113-138. Peachy, L.D. 1965. Transverse tubules in excitation-con- traction coupling. Fed. Proc. 24(3):1124-1134. Peretz, H., Toister, Z., Laster, Y. and A. Loyter. 1974. Fusion of intact human erythrocytes and erythrocyte ghosts. J. Cell Biol. 63:1-11. Potter, J. D. and J. Gergely. 1974. roponin, tropomyosin, and actin interactions in the Ca regulation of muscle contraction. Biochemistry. 13(13): 2697-2703. Potter, J.D., Hsu, Fu-J. and H.J. Pownall. 1976. Thermo- dynamics of Ca2+ binding to troponin-C. J. Biol. Chem. 252(7):2452-2454. Potter, J.D., Seidel, J.C. , Leavis, .2 Lehrer, 5.5. and J. Gergely. 1976a. Effect of Ca + binding on tropo- nin-C.J. Biol. Chem. 251(23): 7551- 7556. Prives, J. and M. Shinitzky. 1977. Increased membrane fluidity precedes fusion of muscle cells. Nature. 268(5622): 761-763. Rasmussen, H. and D.B.P. Goodman. 1977. Relationships between calcium and cyclic nucleotides in cell acti- vation. Physiol. Rev. 5(3):421-509. Sarzala, M.G., Pilarska, M., Zubrzycka, E. and M1 Michalak. 1975. Changes in the structure, composition and func- tion of sarcoplasmic reticulum membrane during devel- opment. Eur. J. Biochem. 57:25-34. Scales, D. and G. Inesi. 1976. Assembly of ATPase protein in sarcoplasmic reticulum membranes. Biophysical J. 16:735-751. Schroeder, F., Holland, J.F. and P. Roy Vagelos. 1976. Physical properties of membranes isolated from tissue culture cells with altered phospholipid composition. J. Biol. Chem. 251(21):6747-6756. Schudt, S. and D. Pette. 1975. Influence of the ionophore A23187 on myogenic cell fusion. FEBS Letters. 59(1): 36-38. Schudt, C. and D. Pette. 1976. Influence of monosaccha-. rides, medium factors and enzymatic modification on fusion of myoblasts in vitro. Cytobiologie. 13:74-84. 88 Schudt, C., Gaertner, U. and D. Pette. 1976. Insulin action on glucose transport and calcium fluxes in developing muscle cells in vitro. Eur. J. Biochem. 68:103-111 Schudt, C., Gaertner, U., Dolken, G. and D. Pette. 1975. Calcium-related changes of enzyme activities in energy metabolism of cultured embryonic chick myoblasts and myotubes. Eur. J. Biochem. 60:579-586. Shainberg, A. Yagil, G. and D. Yaffe. 1969. Control of myogenesis jn_vitro by Ca2+ concentration in nutri- tional medium.‘ Exptl. Cell Res. 58:163-167. Singer, S.J. and G.L. Nicolson. 1972. The fluid mosaic model of the structures of membranes. Science, 175: 720-731. Stockdale, F.E. and M.C. O'Neill. 1972. Deoxyribonucleic acid synthesis, mitosis, and skeletal muscle differen- tiation. In Vitro, 8(3):212-225. Stockdale, F., Okazaki, K., Nameroff, M. and H. Holtzer. 1964. 5-Bromodeoxyuridine:effect on myogenesis in vitro. Science, 146:533-535. Stromer, M.H., Gall, D.E., Young, R.B., Robson, R.M. and F.C. Parrish, Jr. 1974. Ultrastructural features of skeletal muscle differentiation and development. J. Anim. Sci. 38:1111-1141. Tillack, T.W., Boland, R. and A. Martonosi. 1974. The ultrastructure of developing sarcoplasmic reticulum. J. Biol. Chem. 249(2):624-633. Trotter, J.A. and M. Nameroff. 1976. Myoblast differentia- tion in vitro: morphological differentiation of mono- nucleated myoblasts. Develop. Biol. 49:548-555. Truter, M.R. '1976. Chemistry of the calcium ionophores. In: Symposia of the Society for Experimental Biology, xxx. Ed. C.J. Duncan. Cambridge University Press. New York. p. 19-40. Turner, D.C., Gmur, R. Siegrist, M. Burckhardt, E. and H.M. Eppenberger. 1976. Differentiation in cultures derived from embryonic chicken muscle. Develop. Biol. 48:258-283. Van der Bosch, J., Schudt, Chr. and D. Pette. 1972. Quan- titative investigation on Ca2+ and pH dependence of muscle cell fusion in vitro. Biochem. Biophys. Res. Comm, 48:326-332. 89 van der Bosch, J., Schudt, Chr. and D. Pette. 1973. Influ- ence of temperature, cholesterol, dipalmitoyllecithin and Ca2+ on the rate of muscle cell fusion. Exptl. Cell Res. 82:433-438. VanderKooi, J.M. and A. Martonosi. 1971. Sarcoplasmic Reticulum XII. The interaction of 8-anilino-l- naphthalene sulfonate with skeletal muscle. Arch. Biochem. Biophys. 144:87-98. VanderKooi, J.M. and A. MartonoSi. 1971a. Sarcoplasmic Reticulum. XIII. Changes in the fluoresgence of 8- anilo-lo-naphthalene sulfonate during Ca + transport. Arch. Biochem. Biophys. 144:99-106. Vertel, B.M. and D.A. Fischman. 1977. Mitochondrial development during myogenesis. Develop. Biol. 58: 356-371. Wahrman, J.P., Winand, R. and D. Luzzati. 1973. Effect of cyclic AMP on growth and morphological differentiation of an established myogenic cell line. Nature New Biology, 245:112-113. Weber, A. and R.D. Bremel. 1971. Regulation of contrac- tion and relaxation. In: Contractility of Muscle Cells and Related Processes. Ed. R.J. Podolsky. Prentice- Hall, Inc. New Jersey. pp. 37-53. Weidekamm, E., Schudt, Chr. and D. Brdiczka. 1976. Physi- cal properties of muscle cell membranes during fusion. A fluorescence polarization study with the ionophore A23187. Biochimica et Biophysica Acta 443:169-180. Winegrad, S. 1965. Role of intracellular calcium movements in excitation-contraction coupling in skeletal muscle. Fed. Proc. 24(3):1146-1152. Yaffe, D. and H. Dym. 1972. Gene expression during dif- ferentiation of contractile muscle fibers. Cold Spring Harbor Symp. Quant. Biol. 37:543-547. Young, R.B. and R.E. Allen. 1978. Gene transitions during development of muscle fibers. J. An. Sci. In press. Young, R.B., Gall, D.E. and M.H. Stromer. 1975. Isolation of myosin synthesizing polysomes from cultures of embryonic chicken myoblasts before fusion. Develop. Biol. 47:123-135. Zalin, R.J. 1973. The relationship of the level of cyclic AMP to differentiation in primary cultures of chick muscle cells. Exp. Cell Res. 78:152-158. 90 Zalin, R.J. 1976, The effect of inhibitors upon intra- cellular cyclic AMP levels and chick myoblast differ- entiation. Develop. Biol. 53:1-9. Zalin, R.J. 1977. Prostaglandins and myoblast fusion. Develop. Biol. 59:241-248. Zalin, R.J. and R. Leaver. 1975. The effect of a transient increase in intracellular cyclic AMP upon muscle cell fusion. FEBS Letters 53(1):33-36. Zalin, R.J. and Wm. Montague. 1974. Changes in adeny- late cyclase, cyclic AMP, and protein kinase levels in thick myoblasts, and their relationship to differ- entiation. Cell 2:103-108. Zalin, R.J. and Wm. Montague. 1975. Changes in cyclic, AMP, adenylate cyclase and protein kinase levels during the development of embryonic chick skeletal muscle. Exp. Cell Res. 93:55-62. Zubrzycka, E. and D.H. MacLennan. 1976. Assembly of sarco- plasmic reticulum. J. Biol. Chem. 251(24):7733-7738.