Anthony J. Rivera

Fibroblast Growth Factor Promotes Recruitment of Skeletal Muscle Satellite Cells in Young and Old Rats

Although the role of satellite cells in muscle growth and repair is well recognized, understanding of the molecular events that accompany their activation and proliferation is limited. In this study, we used the single myofiber culture model for comparing the proliferative dynamics of satellite cells from growing (3-week-old), young adult (8- to 10-week-old), and old (9- to 11-month-old) rats. In these fiber cultures, the satellite cells are maintained in their in situ position underneath the fiber basement membrane. We first demonstrate that the cytoplasm of fiber-associated satellite cells can be monitored with an antibody against the extracellular signal regulated kinases 1 and 2 (ERK1 and ERK2), which belong to the mitogen-activated protein kinase (MAPK) superfamily. With this immunocytological marker, we show that the satellite cells from all three age groups first proliferate and express PCNA and MyoD, and subsequently, about 24 hr later, exit the PCNA+/MyoD+ state and become positive for myogenin. For all three age groups, fibroblast growth factor 2 (FGF2) enhances by about twofold the number of satellite cells that are capable of proliferation, as determined by monitoring the number of cells that transit from the MAPK+ phenotype to the PCNA+/MAPK+ or MyoD+/MAPK+ phenotype. Furthermore, contrary to the commonly accepted convention, we show that in the fiber cultures FGF2 does not suppress the subsequent transition of the proliferating cells into the myogenin+ compartment. Although myogenesis of satellite cells from growing, young adult, and old rats follows a similar program, two distinctive features were identified for satellite cells in fiber cultures from the old rats. First, a large number of MAPK+ cells do not appear to enter the MyoD-myogenin expression program. Second, the maximal number of proliferating satellite cells is attained a day later than in cultures from the young adults. This apparent “lag” in proliferation was not affected by hepatocyte growth factor (HGF), which has been implicated in accelerating the first round of satellite cell proliferation. HGF and FGF2 were equally efficient in promoting proliferation of satellite cells in fibers from old rats. Collectively, the investigation suggests that FGF plays a critical role in the recruitment of satellite cells into proliferation.

Gene Expression Patterns of the Fibroblast Growth Factors and Their Receptors During Myogenesis of Rat Satellite Cells

Satellite cells are the myogenic precursors in postnatal muscle and are situated beneath the myofiber basement membrane. We previously showed that fibroblast growth factor 2 (FGF2, basic FGF) stimulates a greater number of satellite cells to enter the cell cycle but does not modify the overall schedule of a short proliferative phase and a rapid transition to the differentiated state as the satellite cells undergo myogenesis in isolated myofibers. In this study we investigated whether other members of the FGF family can maintain the proliferative state of the satellite cells in rat myofiber cultures. We show that FGF1, FGF4, and FGF6 (as well as hepatocyte growth factor, HGF) enhance satellite cell proliferation to a similar degree as that seen with FGF2, whereas FGF5 and FGF7 are ineffective. None of the growth factors prolongs the proliferative phase or delays the transition of the satellite cells to the differentiating, myogenin+ state. However, FGF6 retards the rapid exit of the cells from the myogenin+ state that routinely occurs in myofiber cultures. To determine which of the above growth factors might be involved in regulating satellite cells in vivo, we examined their mRNA expression patterns in cultured rat myofibers using RT-PCR. The expression of all growth factors, excluding FGF4, was confirmed. Only FGF6 was expressed at a higher level in the isolated myofibers and not in the connective tissue cells surrounding the myofibers or in satellite cells dissociated away from the muscle. By Western blot analysis, we also demonstrated the presence of FGF6 protein in the skeletal musle tissue. Our studies therefore suggest that the myofibers serve as the main source for the muscle FGF6 in vivo. We also used RT-PCR to analyze the expression patterns of the four tyrosine kinase FGF receptors (FGFR1-FGFR4) and of the HGF receptor (c-met) in the myofiber cultures. Depending on the time in culture, expression of all receptors was detected, with FGFR2 and FGFR3 expressed only at a low level. Only FGFR4 was expressed at a higher level in the myofibers but not the connective tissue cell cultures. FGFR4 was also expressed at a higher level in satellite cells compared to the nonmyogenic cells when the two cell populations were released from the muscle tissue and fractionated by Percoll density centrifugation. The unique localization patterns of FGF6 and FGFR4 may reflect specific roles for these members of the FGF signaling complex during myogenesis in adult skeletal muscle. (J Histochem Cytochem 48:1079–1096, 2000)

Thin filament near-neighbour regulatory unit interactions affect rabbit skeletal muscle steady-state force-Ca2+ relations

The role of cooperative interactions between individual structural regulatory units (SUs) of thin filaments (7 actin monomers : 1 tropomyosin : 1 troponin complex) on steady‐state Ca2+‐activated force was studied. Native troponin C (TnC) was extracted from single, de‐membranated rabbit psoas fibres and replaced by mixtures of purified rabbit skeletal TnC (sTnC) and recombinant rabbit sTnC (D27A, D63A), which contains mutations that disrupt Ca2+ coordination at N‐terminal sites I and II (xxsTnC). Control experiments in fibres indicated that, in the absence of Ca2+, both sTnC and xxsTnC bind with similar apparent affinity to sTnC‐extracted thin filaments. Endogenous sTnC‐extracted fibres reconstituted with 100 % xxsTnC did not develop Ca2+‐activated force. In fibres reconstituted with mixtures of sTnC and xxsTnC, maximal Ca2+‐activated force increased in a greater than linear manner with the fraction of sTnC. This suggests that Ca2+ binding to functional Tn can spread activation beyond the seven actins of an SU into neighbouring units, and the data suggest that this functional unit (FU) size is up to 10–12 actins. As the number of FUs was decreased, Ca2+ sensitivity of force (pCa50) decreased proportionally. The slope of the force‐pCa relation (the Hill coefficient, nH) also decreased when the reconstitution mixture contained < 50 % sTnC. With 15 % sTnC in the reconstitution mixture, nH was reduced to 1.7 ± 0.2, compared with 3.8 ± 0.1 in fibres reconstituted with 100 % sTnC, indicating that most of the cooperative thin filament activation was eliminated. The results suggest that cooperative activation of skeletal muscle fibres occurs primarily through spread of activation to near‐neighbour FUs along the thin filament (via head‐to‐tail tropomyosin interactions).

2-Deoxy-ATP Enhances Contractility of Rat Cardiac Muscle

To investigate the kinetic parameters of the crossbridge cycle that regulate force and shortening in cardiac muscle, we compared the mechanical properties of cardiac trabeculae with either ATP or 2-deoxy-ATP (dATP) as the substrate for contraction. Comparisons were made in trabeculae from untreated rats (predominantly V1 myosin) and those treated with propylthiouracil (PTU; V3 myosin). Steady-state hydrolytic activity of cardiac heavy meromyosin (HMM) showed that PTU treatment resulted in >40% reduction of ATPase activity. dATPase activity was >50% elevated above ATPase activity in HMM from both untreated and PTU-treated rats. V(max) of actin-activated hydrolytic activity was also >50% greater with dATP, whereas the K(m) for dATP was similar to that for ATP. This indicates that dATP increased the rate of crossbridge cycling in cardiac muscle. Increases in hydrolytic activity were paralleled by increases of 30% to 80% in isometric force (F(max)), rate of tension redevelopment (k(tr)), and unloaded shortening velocity (V(u)) in trabeculae from both untreated and PTU-treated rats (at maximal Ca(2+) activation), and F-actin sliding speed in an in vitro motility assay (V(f)). These results contrast with the effect of dATP in rabbit psoas and soleus fibers, where F(max) is unchanged even though k(tr), V(u), and V(f) are increased. The substantial enhancement of mechanical performance with dATP in cardiac muscle suggests that it may be a better substrate for contractility than ATP and warrants exploration of ribonucleotide reductase as a target for therapy in heart failure.

Investigation of thin filament near‐neighbour regulatory unit interactions during force development in skinned cardiac and skeletal muscle

Ca2+‐dependent activation of striated muscle involves cooperative interactions of cross‐bridges and thin filament regulatory proteins. We investigated how interactions between individual structural regulatory units (RUs; 1 tropomyosin, 1 troponin, 7 actins) influence the level and rate of demembranated (skinned) cardiac muscle force development by exchanging native cardiac troponin (cTn) with different ratio mixtures of wild‐type (WT) cTn and cTn containing WT cardiac troponin T/I + cardiac troponin C (cTnC) D65A (a site II inactive cTnC mutant). Maximal Ca2+‐activated force (Fmax) increased in less than a linear manner with WT cTn. This contrasts with results we obtained previously in skeletal fibres (using sTnC D28A, D65A) where Fmax increased in a greater than linear manner with WT sTnC, and suggests that Ca2+ binding to each functional Tn activates < 7 actins of a structural regulatory unit in cardiac muscle and > 7 actins in skeletal muscle. The Ca2+ sensitivity of force and rate of force redevelopment (ktr) was leftward shifted by 0.1–0.2 −log [Ca2+] (pCa) units as WT cTn content was increased, but the slope of the force–pCa relation and maximal ktr were unaffected by loss of near‐neighbour RU interactions. Cross‐bridge inhibition (with butanedione monoxime) or augmentation (with 2 deoxy‐ATP) had no greater effect in cardiac muscle with disruption of near‐neighbour RU interactions, in contrast to skeletal muscle fibres where the effect was enhanced. The rate of Ca2+ dissociation was found to be > 2‐fold faster from whole cardiac Tn compared with skeletal Tn. Together the data suggest that in cardiac (as opposed to skeletal) muscle, Ca2+ binding to individual Tn complexes is insufficient to completely activate their corresponding RUs, making thin filament activation level more dependent on concomitant Ca2+ binding at neighbouring Tn sites and/or crossbridge feedback effects on Ca2+ binding affinity.

Influence of PDGF-BB on Proliferation and Transition Through the MyoD-myogenin-MEF2A Expression Program During Myogenesis in Mouse C2 Myoblasts

We have previously demonstrated that PDGF-BB enhances proliferation of C2 myoblasts. This has led us to examine whether the mitogenic influence of PDGF-BB in the C2 model correlates with modulation of specific steps associated with myogenic differentiation. C2 myoblasts transiting through these differentiation specific steps were monitored via immunocytochemistry. We show that the influence of PDGF on enhancing cell proliferation correlates with a delay in the emergence of cells positive for sarcomeric myosin. We further monitored the influence of PDGF-BB on differentiation steps preceding the emergence of myosin+ cells. We demonstrate that mononucleated C2 cells first express MyoD (MyoD+/myogenin- cells) and subsequently, myogenin. Cells negative for both MyoD and myogenin (the phenotype preceding the MyoD+ state) were present at all times in culture and comprised the majority, if not all, of the cells which responded mitogenically to PDGF. Additionally, the frequency of the MyoD+/myogenin+ cell phenotype was reduced in cultures receiving PDGF, suggesting that PDGF can modulate the transition of the cells into the myogenin+ state. We determined that many of the myogenin+ cells subsequently become MEF2A+ and this phenomenon is not influenced by PDGF-BB. FGF-2 also enhanced the proliferation of C2 myoblasts and suppressed the appearance of the myogenin+ cells, but did not influence the subsequent transition into the MEF2A+ state. The study raises the possibility that PDGF-BB and FGF-2 might delay the transition of the C2 cells into the MyoD+/myogenin+ state by depressing a paracrine signal that enhances differentiation.

REGULATION OF SKELETAL MUSCLE TENSION REDEVELOPMENT BY TROPONIN C CONSTRUCTS WITH DIFFERENT CA2+ AFFINITIES

In maximally activated skinned fibers, the rate of tension redevelopment (ktr) following a rapid release and restretch is determined by the maximal rate of cross-bridge cycling. During submaximal Ca2+ activations, however, ktr regulation varies with thin filament dynamics. Thus, decreasing the rate of Ca2+ dissociation from TnC produces a higher ktr value at a given tension level (P), especially in the [Ca2+] range that yields less than 50% of maximal tension (Po). In this study, native rabbit TnC was replaced with chicken recombinant TnC, either wild-type (rTnC) or mutant (NHdel), with decreased Ca2+ affinity and an increased Ca2+ dissociation rate (koff). Despite marked differences in Ca2+ sensitivity (>0.5 DeltapCa50), fibers reconstituted with either of the recombinant proteins exhibited similar ktr versus tension profiles, with ktr low (1-2 s-1) and constant up to approximately 50% Po, then rising sharply to a maximum (16 +/- 0.8 s-1) in fully activated fibers. This behavior is predicted by a four-state model based on coupling between cross-bridge cycling and thin filament regulation, where Ca2+ directly affects only individual thin filament regulatory units. These data and model simulations confirm that the range of ktr values obtained with varying Ca2+ can be regulated by a rate-limiting thin filament process.

Thin‐filament regulation of force redevelopment kinetics in rabbit skeletal muscle fibres

Thin‐filament regulation of isometric force redevelopment (ktr) was examined in rabbit psoas fibres by substituting native TnC with either cardiac TnC (cTnC), a site I‐inactive skeletal TnC mutant (xsTnC), or mixtures of native purified skeletal TnC (sTnC) and a site I‐ and II‐inactive skeletal TnC mutant (xxsTnC). Reconstituted maximal Ca2+‐activated force (rFmax) decreased as the fraction of sTnC in sTnC: xxsTnC mixtures was reduced, but maximal ktr was unaffected until rFmax was <0.2 of pre‐extracted Fmax. In contrast, reconstitution with cTnC or xsTnC reduced maximal ktr to 0.48 and 0.44 of control (P < 0.01), respectively, with corresponding rFmax of 0.68 ± 0.03 and 0.25 ± 0.02 Fmax. The ktr–pCa relation of fibres containing sTnC: xxsTnC mixtures (rFmax > 0.2 Fmax) was little effected, though ktr was slightly elevated at low Ca2+ activation. The magnitude of the Ca2+‐dependent increase in ktr was greatly reduced following cTnC or xsTnC reconstitution because ktr at low levels of Ca2+ was elevated and maximal ktr was reduced. Solution Ca2+ dissociation rates (koff) from whole Tn complexes containing sTnC (26 ± 0.1 s−1), cTnC (38 ± 0.9 s−1) and xsTnC (50 ± 1.2 s−1) correlated with ktr at low Ca2+ levels and were inversely related to rFmax. At low Ca2+ activation, ktr was similarly elevated in cTnC‐reconstituted fibres with ATP or when cross‐bridge cycling rate was increased with 2‐deoxy‐ATP. Our results and model simulations indicate little or no requirement for cooperative interactions between thin‐filament regulatory units in modulating ktr at any [Ca2+] and suggest Ca2+ activation properties of individual troponin complexes may influence the apparent rate constant of cross‐bridge detachment.

Cooperative Activation of Skeletal and Cardiac Muscle

Both skeletal and cardiac muscles show a steep force-pCa relationship indicative of cooperative activation, but there are differences in some of the underlying mechanisms of this cooperativity. As we have discussed previously (Gordon et al, 2000), these give rise to significant differences in the properties of skeletal and cardiac muscle that are important for their various physiological roles and methods of control. Cardiac contractions occur spontaneously and rhythmically, driven by the cardiac pacemaker cells in the SA node, with spread of electrical activity from cell to cell. This activates cardiac cells in sequence to eject blood allowing the heart to function as a periodic pump. Since each cell contracts on each beat, variations in cardiac output occur with variations in heart rate and the strength of contraction on each beat. Through intrinsic and extrinsic regulation via the autonomic nervous system, the rate and strength of each contraction can be regulated to meet the circulatory needs. In contrast, skeletal muscle contraction is controlled through the central nervous system as motor units, defined as a motoneuron and the muscle fibers it innervates. Although force varies with frequency of stimulation of each motor unit, the major means of regulation is by recruitment of motor units, a mechanism unavailable to the heart cells.