Kinetic Analysis of the Slow Skeletal Myosin MHC-1 Isoform from Bovine Masseter Muscle

Several heavy chain isoforms of class II myosins are found in muscle fibres and show a large variety of different mechanical activities. Fast myosins (myosin heavy chain (MHC)-II-2) contract at higher velocities than slow myosins (MHC-II-1, also known as β-myosin) and it has been well established that ADP binding to actomyosin is much tighter for MHC-II-1 than for MHC-II-2. Recently, we reported several other differences between MHC-II isoforms 1 and 2 of the rabbit. Isoform II-1 unlike II-2 gave biphasic dissociation of actomyosin by ATP, the ATP-cleavage step was significantly slower for MHC-II-1 and the slow isoforms showed the presence of multiple actomyosin–ADP complexes. These results are in contrast to published data on MHC-II-1 from bovine left ventricle muscle, which was more similar to the fast skeletal isoform. Bovine MHC-II-1 is the predominant isoform expressed in both the ventricular myocardium and slow skeletal muscle fibres such as the masseter and is an important source of reference work for cardiac muscle physiology. This work examines and extends the kinetics of bovine MHC-II-1. We confirm the primary findings from the work on rabbit soleus MHC-II-1. Of significance is that we show that the affinity of ADP for bovine masseter myosin in the absence of actin (represented by the dissociation constant KD) is weaker than originally described for bovine cardiac myosin and thus the thermodynamic coupling between ADP and actin binding to myosin is much smaller (KAD/KD ∼ 5 instead of KAD/KD ∼ 50). This may indicate a distinct type of mechanochemical coupling for this group of myosin motors. We also find that the ATP-hydrolysis rate is much slower for bovine MHC-II-1 (19 s−1) than reported previously (138 s−1). We discuss how this work fits into a broader characterisation of myosin motors from across the myosin family.
Introduction
The myosin superfamily consists of at least 18 classes of ATP-dependent motor proteins that interact with actin filaments and are involved in a large variety of physiological processes, such as muscle contraction, phagocytosis, cell motility and vesicle transport.1,2 Class II myosins (found in muscle) consist of two heavy chains (MHC) and two pairs of light chains: the regulatory light chains (RLC) and essential light chains (ELC). The C termini of the myosin heavy chains dimerize and form a coiled-coil (“myosin tail”) whereas the N-termini form the two myosin “heads” or “motor-domains”. The most familiar activity of class II myosins is muscle contraction. All myosins appear to undergo the same ATP driven cycle of interaction of myosin with actin, known as the cross-bridge cycle, yet myosins show a wide variety of different mechanical activities. We are interested in the variety of different types of mechanochemical activities shown both within and between the members of the myosin family.
Myosins from fast contracting, anaerobic, muscle fibres are amongst the best characterized myosins with events of the biochemical cross-bridge cycle having been correlated with mechanical and structural data from contracting muscle fibres. These fast myosins comprise a subgroup of the myosins family known as MHC-II-2 with three primary isoforms found in the mammalian adult skeletal muscle, 2a, 2b and 2x, each expressed from a different gene.3 The isoform MHC-II-1 is found in slow, aerobic skeletal muscle fibres and in ventricular cardiac muscle tissue where it is known as MHC-β. The myosin light chains (MLC) also differ between myosins with muscle-specific isoforms of both light chains.4 Whereas a single regulatory light chain isoform (MLC2s) is found in both the slow skeletal muscle and in the ventricle of mammals that have been examined, there are two isoforms of slow muscle essential light chains. One is found predominantly in the adult slow skeletal and the ventricle muscle (called MLC-1s/v or MLC-1s/b). The other (MLC-1sa) predominates in smooth and non-muscle tissue and in some skeletal muscle during development.5 The MLC-1s/b is larger and includes and an N-terminal extension, rich in proline and lysine. Unlike molluscan and smooth muscle myosins, where light chain phosphorylation and calcium binding are the primary regulators of myosin's activity, the light chains of striated muscles seem to have relatively little effect on enzymatic activity of the isolated myosin head.6 However, the presence of specific light chain isoforms and their removal does have substantial effects on the generation of force and motility by striated myosin.6 The MLC stabilise the lever-arm or neck of the myosin head and they are in a position to modulate the translation of the isomerisation of the head into mechanical force or movement.7
Fast muscle fibres containing the MHC-II-2, as their name suggests, contract at higher velocities and are more susceptible to fatigue than slow muscle fibres containing MHC-II-1 and both properties have been related to the properties of the myosin heavy chain isoform expressed in the muscle. It is well established that ADP binding to actomyosin is much tighter for MHC-II-1 (slow isoform) than for MHC-II-2 (fast isoform).8 We recently examined the solution biochemical kinetic properties of the slow isoform MHC-II-1 from rabbit soleus muscle and compared it to the well characterised fast isoform MHC-II-2 from rabbit psoas muscle.9 This showed several significant differences between the two isoforms including:(1)Unlike MHC-II-2, the MHC-II-1 gave biphasic dissociation of actomyosin by ATP.(2)The tight binding of ADP to actomyosin was confirmed for MHC-II-I with at least three conformations of actomyosin–ADP present in equilibrium.(3)The rate constant for ADP release from the predominant actomyosin–ADP complex was the correct size to limit the shortening velocity of MHC-II-1 containing soleus muscle fibres.(4)MHC-II-1 has a significantly reduced rate constant for the ATP hydrolysis step.
Together these results indicate that the kinetic differences between the fast and slow MHC-II isoforms are greater than at first expected and that MHC-II-1 has much in common with some of the much slower smooth and non-muscle myosins (see Discussion).
Three of the above novel observations (numbers (1), (2) and (4)) were in contrast to published data on the same isoform isolated from bovine ventricle muscle. Bovine MHC-II-1 has been widely studied from cardiac ventricle tissue and is an important source of reference work for cardiac muscle physiology.8,10 It is therefore essential to test if the differences observed between the rabbit and bovine isoforms are due to the experimental differences or species differences. The previous studies on bovine cardiac S1 (BCS1) reported that the ATP-hydrolysis rate is much higher (k+3 + k-3 = 138 s−1)11,12 than the hydrolysis rate reported for rabbit soleus S1 under similar conditions (RSS1, k+3 + k-3 = 22 s−1).9 Also, unlike RSS1, the ATP-induced dissociation of AM, in the presence or absence of ADP, could not be fitted unambiguously to two components.10 In order to address these differences we used bovine MHC-II-1 isolated from the slow, bovine masseter muscle. Bovine masseter muscle is large, easily accessible and contains >95% MHC-II-1, which makes it convenient for large scale biochemical preparations.13 This tissue is readily available and the isolated myosin and S1 are stable when stored at −20 °C. In order to further examine potential species differences we also isolated myosin S1 from pig soleus fibres as another source of MHC-II-1 myosin. This tissue is not as pure in myosin isoform content14 but does provide a second reference material. Masseter muscle fibres have a maximum shortening velocity (V0 = 0.27 μm s−1) of 40% of the value for rabbit soleus fibres (V0 = 0.67 μm s−1) at 12 °C13 whereas the pig soleus fibres are even slower (V0 = 0.17 μm s−1).13,15 Since V0 has been shown to be a property of the MHC isoform, the species differences in the isoforms used here are expected to be significant. The work presented here confirms the primary finding from the work of rabbit soleus, and extends these studies to slow bovine and porcine myosins. Of significance is that we show that the affinity of ADP for bovine masseter myosin is weaker than originally described for bovine cardiac myosin and thus the thermodynamic coupling between ADP and actin binding to myosin is much smaller10,11 (KAD/KD ∼5 instead of KAD/KD ∼ 50). This may indicate a distinct type of mechanochemical coupling for this group of myosin motors. We also find that the ATP-hydrolysis rate is much slower for BMS1 (19 s−1) than reported previously for BCS1 (138 s−1).
We discuss how this work fits into a broader characterization of myosin motors from across the myosin family. Indeed the relevance of our observations on the significant differences between fast and slow muscle myosin isoforms in the release of ADP has been underlined by a recent study by Iwamoto et al.,16 published as we submitted this work. They have shown, using a novel X-ray scattering approach, that slow muscle acto––S1 has a distinct movement of the lever arm on binding ADP, whereas little movement was detected with a fast muscle isoform. Larger movements were observed with the S1 from smooth and non-muscle myosin II isoforms. These observations are consistent with earlier electron micrograph images of ADP induced movements of the lever arm of smooth muscle17 and cytoplasmic myosin from the wider myosin family, such as myosin I18 and myosin VI.19 Thus, biochemical and structural studies are coming together to define how ADP release is a key event in defining the mechano-chemical coupling of different types of myosin.
Results
Nucleotide binding to BMS1 in the absence of actin
MHC-II-1 myosin was purified from bovine masseter muscle fibres (see Materials and Methods). Figure 1 shows the isolated myosin with two light chains and the purified BMS1 with only one light chain bound. The binding of ATP or ADP to myosin S1 is characterised by an increase in intrinsic protein fluorescence associated with nucleotide binding. Figure 2(a) shows the fluorescence change observed on rapidly mixing 25 μM or 400 μM ATP with 0.5 μM BMS1 at 20 °C (concentrations are post mixing). At 25 μM the transient increase in fluorescence (ΔF) shown can be satisfactorily described by a two exponential function with kobs = 19.6 s−1 (ΔF +11.8%) and 2.5 s−1 (ΔF +4%). The fast phase is easily separated from the slower component and at low ATP concentrations (<50 μM) appears linearly dependent on ATP concentration with an apparent second-order rate constant of 0.97 × 106 M−1s−1. At higher ATP concentrations the kobs value saturates and a hyperbolic fit to the data gives a K0.5 value of 56 μM with a kmax of 117 s−1 (Figure 2(b)). The slow phase only appears as a single process at low ATP concentrations (≤50 μM); at higher ATP there appear to be two components. Separating out these two phases across the full range of ATP concentrations is problematic but at ATP concentrations above 50 μM, the data can all be described by a small slow phase of amplitude 1–1.5% with a kobs of 2 s−1 and a medium rate phase with kobs of 12-15 s−1 and an amplitude of 4%. This is illustrated in Figure 2(a) for [ATP] = 400 μM where the transient increase in fluorescence (ΔF) shown can be satisfactorily described by a triple exponential function (with kobs = 111 s−1 (ΔF +10%), kobs = 13.5 s−1 (ΔF +3.7%) and kobs = 2 s−1 (ΔF +1%) for the fast, medium and slow phase, respectively). The analysis of the data shown in Figure 2(b) has been fitted assuming a constant slow phase of 2 s−1. The medium phase process at 12–15 s−1 is then only apparent when the fast component is faster than ∼30 s−1 and then appears to be constant. In order to rule out contamination with ADP as the cause of the slower phases, BMS1 was extensively treated with apyrase but this had little effect on the observed transients.
The K0.5 and kmax of the fast phase can be assigned to K1 and k+2 of Scheme 1, respectively, the steps thatcontrol ATP binding as observed for many other myosin isoforms. One of the two slower phases could be due to the ATP hydrolysis step of ATP (k+3 + k-3 in Scheme 1) that is known to generate changes in fluorescence for other myosins as described.20,21 In order to determine what is being observed here, quench flow measurements were done in order to establish the rate of ATP hydrolysis.
BMS1 (5 μM) was mixed with excess ATP (50 μM) and incubated at 20 °C for desired time intervals (up to 350 ms). Figure 3 shows a rapid initial burst of ADP production, which can be described by a single exponential function with kobs of 19 s−1 and an amplitude of 0.61 ADP/S1. The amount of irreversibly bound ATP to BMS1 at the end of the burst was determined by quenching the reaction into a hexokinase/glucose buffer after 1 s and then adding 6.5% trichloroacetic acid (TCA) after a further 1 s. In controls this treatment hydrolysed any free ATP to ADP. After HPLC analysis of the reaction mixture 0.14 ATP/S1 remained and therefore at the end of the burst phase 0.14 ATP/S1 remains as tightly bound M*.ATP and 0.61 ADP/S1 as M**.ADP.Pi (Scheme 1). Thus the value of K3 = [M.ADP.Pi]/ [M*.ATP] = 4.3. This is similar to the values reported for fast muscle S1.22,23 The value of the kobs can be assigned to k+3 + k-3 and at 19 s−1 is similar to the medium phase of the stopped-flow fluorescence transient (kobs = 15 s−1). Thus, as for other myosins the hydrolysis step is accompanied by a fluorescence change. It is worth noting that this is quite a slow process, compared to the hydrolysis rate for fast skS1 from rabbit, which is tenfold faster, but similar to the hydrolysis rate for the slow myosin S1 from rabbit or pig soleus muscle9 (see Table 1). The origin of the slowest component of the fluorescence transient remains to be defined. This is small (1–1.5%) and has a constant kobs value of 2 s−1. This could arise from a small fraction of damaged myosin, from a contaminant myosin or from a fraction of the myosin that must isomerise before ATP can bind to it (see Discussion of ATP binding to actomyosin). The signals are very weak and represent a small fraction of the total making it very difficult to investigate and we have not pursued this further. Arrhenius plots of the maximal observed rates of the fast phase (k+2) and the medium phase (k+3 + k-3) and van 't Hoff plot of K1 (not shown) show that between 8 °C–20 °C K1 does not depend on temperature. k+2 and k+3 + k-3 have a marked temperature dependence with similar activation energies of 71 kJ/mol and 75 kJ/mol, respectively. The ratio of the amplitudes for the fast and medium phase remains fairly constant between 8 °C–20 °C (ratio ∼ 4:1).
The rate of ADP-association with BMS1 was also measured. In this case the fluorescence increase was smaller (ΔF 3%) than seen for ATP. There was no evidence of a slow component of the reaction and the transient increase in fluorescence could be fitted to a single exponential. At high ADP concentrations kobs reaches a maximum value of 26 s−1 when fitted to a hyperbola (Figure 2(c)). The ADP-release rate k+6 (=k-D) can be determined from the intercept, k+6 = 1.4(±0.5) s−1. The maximal rate of ADP-binding represents k+6 + k-6 (kmax,ADP) = 26 s−1 and K7 = K0.5,ADP = 21 μM. The second-order constant of ADP binding k-6/K7 (or k+D) can be defined by kmax,ADP/K0.5,ADP = 1.24 × 106 M-1s−1 (see also Table 1) from which a value for the equilibrium dissociation constant can be calculated: KD (1/K6K7) = 1.2 μM. Repeating the ADP-binding experiment using the slow myosin S1 isolated from pig soleus muscle, PSS1, showed that this myosin S1 had a slightly tighter affinity for ADP (KD = 0.9 μM; see Table 1).
An alternative method of determining k+6 (or k-D) is the displacement of ADP from BMS1 by addition of an excess ATP. Figure 4(a) and (b) shows the reaction observed on adding 100 μM ATP to 0.5 μM BMS1 in the presence of 0.2 μM ADP. The best fit of the fluorescence changes is the sum of three exponentials with kobs of 68 s−1, 9.6 s−1 and 1.1 s−1. Two of the observed three phases are due to the binding of ATP to ADP-free BMS1 (68 s−1) and the hydrolysis step (9.6 s−1), as described above, and the third phase, the binding of ATP, is limited by ADP release from BMS1-ADP (see Figure 4(a) and (b)), with an ADP-off rate of 1.1 s−1. Using a triphasic fit the observed rate constants are fairly independent of ADP concentration (average rate constants for the fast, medium and slow phase are 75 s−1, 8 s−1 and 0.9 s−1, respectively) but the measured amplitudes depend on the ADP concentrations. The amplitude of the fast phase (ATP-binding to BMS1) and medium phase (ATP-hydrolysis) decrease and of the slow phase (ADP dissociation) increases as the ADP concentration is increased (Figure 4(c)). The relative ratio of the amplitudes for the fast and medium phase remain constant up to 2 μM ADP (Afast: Amedium ∼ 4: 1). At higher ADP concentrations the amplitude of the ATP hydrolysis process, Amedium, becomes very small but the observed rate constant, kobs, is not affected by [ADP]. In order to be able to separate Afast and Amedium at high ADP concentrations, the relative ratio Afast/Amedium was fixed at 4. The ADP dependence of the amplitudes could be described by hyperbolas with independent best-fit dissociation constants of 2.6 μM (fast phase), 3.0 μM (medium phase) and 3.0 μM (slow phase), resulting in an average KD = 2.8 μM. This is in reasonable agreement with the value found previously using the ADP binding experiment in Figure 2 (KD = 1.2 μM) and we will use the median value of 2.0. A tighter affinity of ADP for the S1 product of bovine cardiac MHC-1 myosin was reported (KD = 0.33 μM) when the interaction of ATP and ADP with bovine cardiac S1 was investigated.11
Nucleotide binding to MHC1 myosin S1 in the presence of actin
The ATP-induced dissociation of acto–MHC-1 complexes can be followed by monitoring the fluorescence of a pyrene label covalently attached to Cys374 of actin. At ATP concentrations above 100 μM, the ATP-induced dissociation of bovine masseter myosin S1 (BMS1) is biphasic, as is illustrated in Figure 5(a) and (b), whereas below 100 μM ATP the fluorescence can be fitted to a single exponential. Figure 5(a) shows the resulting change in fluorescence upon mixing 0.5 μM pyrene-labelled acto–BMS1 complex with 200 μM ATP. At this ATP concentration the reaction can be best described by a double exponential with kobs = 278 s−1 (ΔF 77%) and 58 s−1 (ΔF 10%) for the fast and the slow phase, respectively. The fast phase shows a hyperbolic dependence on [ATP] and saturates at kobs = 900–1100 s−1 whereas the slow phase remains constant at 56(±13) s−1 at [ATP] > 100 μM (Figure 5(b)). The rate constant of the slow phase (kslow = 56 s−1) is similar to the rate constant of ADP-release (94 s−1) and one could argue it is possible that the slow phase is due to contamination with ADP. However, prolonged incubation of BMS1 with apyrase did not remove the slow phase. The presence of another myosin isoform contamination might also cause the biphasic behavior. Bovine masseter fibres are not 100% MHC-1β but contain a small amount (5%) of MHC-1α isoform but this is a fast isoform and is therefore unlikely to cause an additional slow phase.13 The presence of non-muscle myosins (either class 2 or others) could produce a slow phase but the amplitude of the slow phase (15%) is far too large to come from such a source. For BMS1, according to scheme II, the kobs for the slow phase observed here can be assigned to k+α = 56 s−1. From the ratio of the amplitudes of the fast and slow phase of ATP-induced acto–BMS1 dissociation (77:10) the value of Kα = 7.7, can be assigned. Since Kα = k+α/k-α and k+α = 56 s−1 then k-α = 7.3 s−1. The temperature-dependence of the ATP-induced dissociation of acto–BMS1 was measured between 8 °C −32 °C at a low ATP-concentration (50 μM). From the dependence of kobs on temperature the activation energy can be calculated as Ea = 22 kJmol−1 (not shown).
The effect of ADP on the dissociation rate constant was measured at two different ATP concentrations: [ATP] = 50 μM and [ATP] = 200 μM. At [ATP] = 50 μM, the ATP-induced dissociation of 0.5 μM pyrene-labelled acto–BMS1, pre-incubated with 0–250 μM ADP, yielded a fluorescence change of 55% that could be fitted to a single exponential. The resulting kobs was plotted against ADP concentration (see Figure 6(a)) and fitting against equation (4) resulted in a KAD of 9.6(±1.4) μM. Presuming the on-rate is diffusion- limited (∼107 M−1s−1) one can calculate the ADP-off-rate (k-AD = 96 s−1). Reducing the temperature to 12 °C reduced the measured KAD to 5 μM and thus also the calculated ADP off-rate (50 s−1). At higher ATP concentration (200 μM) the fluorescence change of the ATP-induced dissociation of BMS1-actin, in the absence of ADP, can be fitted to a double exponential and both kobs values decreased with increasing ADP concentrations. Plotting the kobs values versus the ADP-concentration (Figure 6(b)) yielded KAD values of 6.8(±1.7) μM (fast phase) and 6.6 μM(±1.9) μM (slow phase), indicating that the kobs of both phases are inhibited by ADP.
The temperature dependences of these two phases are measured at very high ATP concentrations ([MgATP] = 16 mM) in order to define the rate of ADP release. The ATP-induced dissociation of the acto–BMS1 complex in the presence of saturating ADP concentrations (150 μM) is biphasic with a fast kobs = 94 s−1 (ΔF 55%) and a slow kobs = 9.6 s−1 (ΔF 10%) at 20 °C (see Figure 7(a)). According to the model depicted in Scheme 2, these two processes are due to the presence of two actomyosin complexes with ADP bound in solution, A·M'·D and A·M·D. The fast phase measured here (94 s−1) is due to the A·M'·D complex and corresponds well with the calculated ADP off-rate (96 s−1) and, according to Scheme 2 represents k-ADP. The other complex, A·M·D, is responsible for the slow phase observed here and, according to Scheme 2, represents the rate-limiting step of ADP-release (k+αD = 9.6 s−1). Between 12 °C–25 °C the temperature dependences of these observed fast and slow rates are almost identical (see Figure 6(b)) with similar activation energy values Ea = 106 kJ (fast phase) and 98 kJ (slow phase). The ratio of amplitudes of the two phases, Afast:Aslow had a small dependence on the temperature with Af:As = 6.3 at 12 °C and 5.1 at 25 °C.
The small thermodynamic coupling (KAD/KD) between actin and ADP binding to BMS1 (KAD/KD = 4.8) can be validated by measuring the affinity of S1 for actin in the presence and absence of ADP (KDA/KA). Phalloidin-stabilised pyr-actin (15 nM) incubated with various concentrations of BMS1 were mixed with ATP (10 μM), and the amplitude of the dissociation reaction was used to estimate the fraction of actin bound to BMS1. Without ADP present, kobs remained constant at all S1 concentrations (kobs = 19 s−1) and the amplitude increased hyperbolically with [BMS1]. In the presence of 250 μM ADP, the value for kobs was reduced (15 s−1) and the amplitudes increased hyperbolically with [S1] (see Figure 8). In the absence of ADP the best fit to the titration gave a KA ∼ 4–7 nM, whereas in the presence of ADP the best fit yielded a KDA of 37 nM. Thus, ADP reduced the affinity of S1 for actin by about fivefold (KDA/KA = 37/7 = 5.2), which is similar to the reduction in ADP affinity for S1 caused by actin (KAD/KD = 4.8).
Using MHC-II-1 myosin S1 from pig soleus muscle (PSS1) very similar results were obtained and these are summarised in Tables 1 and 2. The ATP-induced dissociation of acto–PSS1 was biphasic at high ATP with a fast phase (kmax = 800–900 s−1) and a slow phase (kmax = 67 s−1), and a relative ratio Afast:Aslow = 4.8 (data not shown). This yields a value for Kα = 4.8, and together with the measured value for k+α = 67 s−1 gives k-α = 14 s−1 (Table 2). In the presence of ADP (0–200 μM) the ATP-induced dissociation of acto–PSS1 also gave fluorescence transients that fitted a single exponential at low ATP concentrations (<100 μM) and a double exponential at high ATP concentrations (data not shown) and a KAD of 15 μM. At very high ATP concentrations (4 mM MgATP) the ATP-induced dissociation of the acto–PSS1 complex in the presence of saturating ADP concentrations (50 μM) is also biphasic with a fast rate kobs,f = 48 s−1 and a slow phase kobs,s = 6.5 s−1 at 20 °C. The ratio of the two amplitudes Afast:Aslow is 6.5 (20 °C) and represents KαD (see Table 2).
Discussion
This paper set out to reexamine and extend the kinetic analysis of the bovine MHC-II-1 isoform of myosin found in slow skeletal muscle and the ventricle of cardiac muscle. Bovine masseter muscle fibres were successfully used to isolate the S1 construct (BMS1) of bovine MHC-II-1 myosin. Comparison of the kinetic parameters of BMS1 and the S1 construct isolated from the left ventricle of bovine cardiac tissue (BCS1),10,11 which is 100% MHC-II-1 and has the same light chain content, shows that theresults agree reasonably well with each other (Tables 1 and 2). The two parameters that deviate significantly between BMS1 and BCS1 are discussed below.
The ATP hydrolysis rate constant reported here is almost tenfold slower than that reported.12 The previously reported rate constant was based on the assumption that when ATP binds to S1, the maximum rate of the fluorescence change represents k+3 + k-3 (Scheme 1), in analogy with measurements for fast myosin S1.20,21 However, the quench flow data reported here show that k+3 + k-3 defines the medium phase observed in the stopped flow measurements and the maximum observed rate constant of the medium phase is k+3 + k-3 = 19 s−1, whereas the fast phase represents k+2. The ATP hydrolysis step of BMS1 is much slower than that of fast skeletal S1 from rabbit (k+3 + k-3 = 131 s−1) but it is similar to the slow skeletal myosin II, MHC-II-1 from rabbit soleus (22 s−1)9 and pig MHC-II-1 (24.5 s−1).24 It may therefore be a general feature of slow myosin II isoforms. The role of this reduced ATP hydrolysis rate for MHC-II-1 could be related tothe slow ADP-release rates also observed for MHC-II-1. A slow ADP-release rate increases the time myosin is bound to actin (i.e. enhances the duty ratio). MHC-II-1 can compensate for this by a slow hydrolysis step thereby maintaining the low duty ratio typical of striated muscle myosins.
The affinity of ADP for BMS1 in the absence of actin (KD = 2 μM) is weaker than the value reported for BCS1 (KD = 0.33 μM) when the interaction of ATP and ADP with BCS1 was investigated.11 Due to aggregation problems in the BCS1 work, the interaction with ATP was reinvestigated a year later12 but as far as we are aware, the interaction with ADP has never been re-examined and this may therefore account for the discrepancy between the KD values. Table 1 shows that the k+6 values measured for BMS1 and BCS1 differ two- to threefold (k+6 = 0.9–1.4 s−1 and 0.5 s−1, respectively) while the values for k-6/K7 are fairly similar (1.24 × 106 M−1s−1 and 1.4 × 106 M−1s−1). The revised KD value for bovine MHC-II-1 S1 has implications for the thermodynamic coupling constant between the affinity of myosin for ADP and actin, KAD/KD (see below).
Comparison of fast and slow muscle myosin II isoforms shows that in the absence of actin, most kinetic parameters are quite similar (Table 1). The major exception is the ATP-hydrolysis rate (k+3 + k-3), which is about tenfold slower for the slow MHC-II-1 S1 isoforms compared to rabbit psoas S1. The ADP dissociation rate constant (k+6) is very similar for all slow isoforms (k+6 0.5–0.9 s−1) and about twofold slower than the ADP dissociation rate constant of fast skeletal S1 (k+6 = 1.4 s−1). The ADP association rate constant (k-6/K7) is similar for all slow isoforms. The different estimates of ADP binding and release result in a surprisingly wide range of values for the ADP-affinity for S1 isoforms listed from 0.4 μM (rabbit soleus) to 2 μM (bovine masseter). However, these KD values are well defined by both the ratio of rate constants (Figure 2) and the amplitude titration data (Figure 4), suggesting that this parameter does vary across the series of isoforms.
The data in the presence of actin (Table 2) indicate that the affinity of S1 for actin in the presence (KDA) and absence (KA) of ADP shows a sursprisingly large variation within the group of slow isoforms with a tenfold difference in KA value of bovine masseter andrabbit soleus and an even larger difference in KDA value. However, despite these differences in individual equilibrium constants, the slow isoforms show a similar range of values for their thermodynamical coupling constants (KAD/KD and KDA/KA). The slow isoforms show ratios of 3–20, all lower than the value for fast rabbit isoforms (30–60) and at the lower end similar to smooth muscle myosin (4–7)25,26 and non-muscle isoforms (∼1–5).
Table 2 shows the parameters that control the ATP induced dissociation of acto–S1 vary relatively little for the fast and slow muscle isoforms listed (K1k+2' and k+2' vary less than twofold). The major difference is that all of the slow isoforms show a two-phase ATP-induced dissociation of acto–S1 whereas the fast muscle protein shows a single phase. Siemankowski & White10 stated that the ATP-induced dissociation of actoBCS1 (in the presence or absence of ADP) could not be fitted unambiguously to a single exponential, suggesting that it too has two components. Additional indications of two phases comes from data measured for other slow MHC-II-1 isoforms of the chicken, from slow skeletal, cardiac, and smooth muscle27 which all show the presence of an additional small slow phase. Thus, a biphasic reaction for ATP-induced dissociation of acto–S1 appears to be common for slow muscle myosin isoforms. ATP-induced biphasic dissociation of acto–S1 has also been reported for a some class I mammalian myosins and non-muscle myosin II.26 The biphasic transient has been interpreted in terms of scheme II in which a fraction of the acto–S1 must isomerise before nucleotide can bind.
The work presented here also confirms the complex nature of the ADP release from acto–S1 reported for soleus S1. It is not simply that ADP release is much slower for this group of myosins than for the fast muscle myosin but there is clear evidence for several A.M–ADP complexes. We show here evidence for two complexes with ADP with an equilibrium constant between them (KαD) of 5.3 and rate constants of ADP release of 94 (k-ADP) and 9.6 (k+αD) s−1. Similar behavior is seen with the pig soleus (Table 2) and rabbit soleus (Table 3) but with variation across the three slow isoforms particularly in the value of KαD. The data with rabbit soleus provided evidence for a third AMD complex and by analogy we might expect a rapid collision complex between ADP and acto–S1 not characterised here.
The value of the ADP release rate constant (k-ADP) has been assigned to the event during the actin-attached part of the ATPase cycle, which limits the maximum shortening velocity (V0) of a muscle fibre. The velocities of contraction are best characterised at 12 °C (0.17 M ionic strength) and the published values of V0 for the three slow muscle isoforms are listed in Table 3 along with the values of the ADP release constant at 12 °C. The maximum unloaded shortening velocity of muscle fibres (V0) of bovine masseter is 0.27 μm s−1 at 12 °C.13 From this, one can estimate the minimum value of the rate constant that limits the detachment of the cross-bridge (kmin) using the relationship:with V0 = maximum sliding velocity (nm s−1) and d = working stroke (nm).8 Depending on the size of the working stroke, kmin is 54 s−1 (d = 5 nm) or 27 s−1 (d = 10 nm) for bovine masseter MHC-II-1. From Table 3 one can see that at 12 °C k-ADP is 27 s−1, which is close to the calculated kmin value. The data from the pig shows a similar correlation between kmin and k-ADP, as does the previously published data from rabbit.9 For all three MHC-II-1 isoforms the measured values of k+αD are much slower than the calculated kmin, indicating that this isomerisation step cannot be part of the main pathway of the unloaded cross-bridge cycle but represents a branched pathway that could become significant if the motor is contracting under loaded conditions. The velocity measurements are, by definition, made under zero load and the isomerisations controlling the ADP release steps are events which are potentially sensitive to the load on the actomyosin cross-bridge.
The evidence that ADP release is load-dependent has been discussed in general terms by Nyitrai & Geeves28 and specifically for the rabbit MHC-II-1 isoform by Iorga et al.9 The data for bovine and porcine isoforms are shown here to be similar to the rabbit isoform and a similar interpretation is possible. Briefly, there is substantial evidence from single-molecule mechanical measurements29–32 and electron micrographs of S1 decorated actin filaments17–19 that, for a range of non-muscle myosins and the smooth-muscle myosin there is a load-dependent structural change after the major force generating event. The force-generating event is associated with phosphate release and the second structural change is probably associated with ADP release. More recent studies with improved laser traps33 and X-ray scattering from S1-decorated, orientated actin filaments16 have extended these observations to striated muscle myosins. The laser trap observed a ∼1 nm movement of the lever arm in the second step for the rat MHC-II-1 isoform and the rate constant we expect for the event limiting ADP release is compatible with the lifetimes measured in the laser trap. The X-ray data report a similar sized movement of the lever arm on binding/release of ADP for the rabbit slow muscle S1. Together, the structural mechanical and biochemical kinetic studies support the hypothesis of a load dependent isomerisation of the actomyosin complex required to allow release of ADP.
The results presented here on the protein isomerisations that limit ADP release from the actomyosin of slow muscle may provide some insight into why, despite a tight affinity for ADP, slow muscles are more resistant to fatigue than fast muscles, or at least the part of fatigue associated with ADP build up. Fatigue is classically defined from studies of the effect of prolonged stimulation on the isometric tension of intact muscle fibres. The complete explanation of the loss of tension remains to be defined but any build up of ADP is likely to result in the enhancement of the lifetime of strongly bound A.M.–ADP states as ADP competes with ATP induced detachment of the cross-bridge. The increased lifetime will increase steady-state tension as is seen on addition of ADP to skinned muscle fibres.34–36 Thus, ADP build up may compensate for other effects of fatigue which tend to reduce tension and the compensatory effect will be greater in slow than in fast muscle fibres.
The effect of ADP on velocity is different; the evidence is for a slowing of maximal shortening velocity in skinned muscle fibres.34,37 In a fast muscle the velocity is limited by the rate of ATP-induced dissociation of actin from A.M., and ADP competes with ATP for binding to the nucleotide pocket.38 The net rate constant for actin detachment is defined by:Thus, at mM ATP concentrations, ADP of the order of the KAD (∼100 μM) can produce a significant inhibition of the detachment rate constant. For slow muscle the detachment of myosin from actin is limited not by the ATP induced reaction but by the slower isomerisation preceding ADP release, controlled by k-ADP (Table 3 gives this as 27 s−1 for masseter at 12 °C). The following ATP-induced dissociation of AM remains much faster than this (∼1000 s−1 at mM ATP concentrations, in fact the K1, and k+2 values are similar for fast and slow myosin isoforms; see Table 2) and the velocity will only be slowed once the ADP competition effect reduces kdetach to <100 s−1 (equation (7)). This requires the ADP concentration to be many times the KAD value or for the ATP concentration to fall to ∼100 μM.
In summary we have shown that the MHC-II-1 isoforms isolated from slow skeletal muscles from the cow and the pig are similar in general properties to MHC-II-1 isolated from bovine cardiac tissue and to the MHC-II-1 of the rabbit and these are quite distinct from the MHC-II-2 isoforms. Of significance is the slow ATP-hydrolysis rate, the low thermodynamic coupling between ADP and actin binding, the slow rates of ADP release (which are of the correct size to limit the maximum shortening velocity) and multiple actomyosin ADP complexes. Nucleotide access to and release from the binding site requires relatively slow isomerisations and these may be load-dependent. These properties are distinct from the fast muscle MHC-II-2 isoforms and similar to smooth and some non-muscle myosin isoforms, e.g. mammalian myosins 1b and 1c, and non-muscle myosin II. However, unlike the non-muscle isoforms, the MHC-II-1 isoforms have a low duty ratio and can produce relatively rapid movement in motility assays or in muscle fibres. These parameters suggest that slow muscle myosin isoforms may form a distinct functional group from the fast muscle isoforms and the slower group which we have called the strain-sensors; non-muscle myosins particularly a sub-set of class I myosins (myo1b and 1c) that appear to operate less as transport motors than as load-bearing and strain-sensing molecules. Slow muscle myosin, like fast muscle myosin, is required for movement but also has an additional role in efficient load bearing in postural muscles. Thus, the molecular properties of the MHC-II-1 motor are consistent with its physiological role.
Materials and Methods
Proteins
MHC-II-1 myosin was purified from bovine masseter muscle fibres that had been frozen in liquid nitrogen prior to storage at −80 °C. Purification was based on the method described.39 Briefly, the tissue (40 g) was homogenized in ice-cold Guba-Straub buffer (pH 6.6) and the suspension was stirred for 30 min. After centrifugation, the supernatant was diluted tenfold with water. The precipitated protein was collected by centrifugation. The pellet was taken up in 3M KCl and ice-cold water was then added to precipitate the myosin and the protein was collected by centrifugation. This procedure was repeated three times and the pellet was then taken up in a small amount of 3M KCl and then adjusted to 125 mM KCl, 10 mM KPi, 2 mM EDTA, 4 mM dithiothreitol (DTT, final concentrations) for digestion. Subfragment 1 (BMS1) was prepared by digesting myosin using chymotrypsin using the method described.40 The soluble BMS1 protein was separated from the non-digested myosin and the insoluble myosin tails by centrifugation and purified over a Q-Sepharose column in 50 mM imidazole (pH 7) with a linear gradient from 0–500 mM KCl. The fractions containing BMS1 were pooled and dialysed into 10 mM KPi buffer (pH 7.5) and typical yields were around 140 mg of protein. Figure 1 shows the isolated myosin with two light chains and the purified BMS1 with only one light chain bound. The gel suggests only one isoform of each light chain and the molecular mass of the essential light chain is consistent with this being the MLC-1b/v isoform expected in slow skeletal and ventricle muscle. For myosin S1, isolated from pig soleus muscle, a similar procedure was followed to isolate the protein and from 58 g of tissue about 70 mg of pig soleus S1 (PSS1) was obtained. After dialysis, BMS1 and PSS1 were lyophilized in the presence of 1% (w/v) sucrose and stored at −20 °C. Prior to use, BMS1 or PSS1 was dialyzed into 20 mM Mops, 30 mM KCl, 5 mM MgCl2, 1 mM sodium azide and remained stable in solution for several days. Rabbit skeletal muscle actin was prepared according to methods described41 and, when necessary, labelled with pyrene at Cys374.42
ATPase assay
ATP-ase activity was measured using an NADH-coupled assay at 20 °C in buffer containing 20 mM Mops (pH 7), 5 mM MgCl2, 30 mM KCl, 1 mM ATP, 40 units/ml of lactate dehydrogenase, 200 units/ml of pyruvate kinase, 1 mM phosphoenolpyruvate, and 200 μM NADH. Changes in A340 (ε = 6220 M−1cm−1) were followed using a UV-VIS spectrophotometer. The basal Mg2+-ATPase activity of bovine masseter myosin S1 (0.012 s−1) agrees well with the value reported (0.017 s−1) for bovine cardiac myosin S1 isolated from bovine cardiac left ventricle.12 In the presence of 50 μM actin the Mg2+-ATPase activity of BMS1 increased more than tenfold (0.182 s−1) and the ATP-ase rate was linearly related to the actin concentration over the range of 0–50 μM. (Not shown).
Transient kinetics
All kinetic experiments were performed at 20 °C in 20 mM Mops, 100 mM KCl, 5 mM MgCl2 and 1 mM azide (pH 7), unless indicated otherwise. All measurements were done with a High-Tech Scientific SF-61 DX2 stopped-flow system. Pyrene actin fluorescence was excited at 365 nm and emission was detected after passing through a KV389 nm cut-off filter (Schott, Mainz, Germany). Tryptophan fluorescence was excited at 295 nm and observed through a WG320 filter. The stated concentrations of reactants are those after mixing in the stopped-flow observation cell (“post”) unless indicated otherwise. Stopped-flow data were analysed using the software provided by Hi-Tech (KinetAsyst) and the Origin software (Microcal).
Without actin present, the kinetics of S1 (either BMS1 or PSS1; M) with nucleotide (T,ATP; D,ADP) were interpreted based on the seven-step model described by Bagshaw etal.,43 where k+i and k-i are the forward and reverse rate constants and Ki (k+i/k-i) represents the equilibrium constant of the reaction (Scheme 1).
In the presence of actin the kinetics of the interaction ofS1 with nucleotide were based on the model proposed for myo1b26,44 and is depicted in Scheme 2. In this model, AM exists in two conformations in the absence of nucleotide (A.M and A.M', equilibrium defined by Kα = k+α/k-α) and in the presence of ADP (A.M.D and A.M'.D, equilibrium defined by KαD = k+αD/ k-αD). In the AM' state, the nucleotide can exchange with the solvent (“open” conformation) but not in the AM state (“closed” conformation). Conversion of M to M' requires opening of the nucleotide binding pocket. The data presented here and summarised in Tables 1 and 2 allow assignment of all the rate and equilibrium constants of Scheme 2. The assignment of the constants to the data is summarized here.
In the absence of ADP, high ATP concentrations induce the dissociation of actin from S1 in two phases. The fast phase represents dissociation from A.M' with:
The slow phase is limited by the isomerisation from A.M to A.M' with the maximum value of kobs,slow = k+α. The ratio of the amplitudes of the fast and the slow phase of the ATP-induced dissociation defines the equilibrium constant between the two conformations in solution and since Kα = k+α/k-α, k-α can be determined:
For fast myosin S1, such as skS1 and psoS1, Kα >> 1 and only a single phase is observed.
For slow myosin S1, in the presence of ADP, the overall dissociation constant of ADP for the actin-S1 complex (KAD) is defined by equation (3):where KADP is the dissociation constant for ADP from A.M' and KαD the equilibrium constant between AM'D and AMD. If the binding of ADP is a rapid equilibrium event, controlled by KADP, ADP and ATP compete effectively for binding to AM and the observed rate constant kobs is defined by equation (4):where k0 is the observed rate constant of ATP-induced actin–S1 dissociation in the absence of ADP and defined by equation (1). To titrate both A.M complexes to A.M.D, actin–S1 was pre-equilibrated with a range of ADP concentrations before adding excess ATP. ATP-induced dissociation of actin from A.M.D occurs via the intermediate A.M'.D and A.M' and is limited by k+αD. If k+α and k+αD differ by more than a factor of 3, then both fast and slow amplitudes seen at zero ADP are reduced as ADP is increased and replaced by a new phase with kobs = k+αD (this is the case for BMS1). The effect of ADP on the amplitude of the fast phase of ATP-induced dissociation of actin-S1 was analysed according to:where Ampfast represents the observed amplitude of the fast phase of the reaction, Amp0 is the amplitude at zero ADP concentration, [ADP] is the concentration of ADP before mixing, KAD is the dissociation equilibrium constant of ADP for actin–S1, and C is the end point. The end point of the reaction defined by C was defined by the fluorescence at infinite high ADP concentrations. Amplitude is calculated as a percentage of the end point of the reaction, according to:
Where amplitude, expressed as %, equals the change in fluorescence observed during the reaction (ΔF) divided by the fluorescence at the end point of the reaction (Ft∞), multiplied by 100.
Quenched-flow experiments
The quenched-flow experiments were carried out with a Hi-Tech RQF-63 using a buffer containing 100 mM KCl, 5 mM MgCl2, 20 mM Mops (pH 7.0). BMS1 (5 μM) was mixed with excess ATP (50 μM) and incubated at 20 °C for desired time intervals (up to 350 ms). The reaction was quenched by the addition of an equal volume of 6.25% (w/v) trichloroacetic acid (TCA). After neutralisation with NaOH and a clarification spin at 3000g for 5 min, the samples were diluted 1:10 with the fast protein liquid chromatography running buffer containing 125 mM KPi (pH 5.5). The separation of ADP and ATP was carried out on a fast protein liquid chromatography (FPLC) system (Amersham Biosciences) using a Hypersil ODS (3 μm) column and an isocratic flow. Integration of the peak areas provided the ratio of ADP and ATP at each time point. The ratio of the ADP to the total nucleotide concentration at each given time point was then used to calculate the hydrolysis rate of the myosin. The amount of irreversibly bound ATP to BMS1 at the end of the burst was determined by quenching the reaction into a 100 mM KCl, 20 mM Mops, 5 mM MgATP buffer containing hexokinase (2 μg/μl) and glucose (1 mM) after 1 s and then adding 6.5% TCA after a further 1 s. In controls this treatment hydrolysed any free ATP to ADP. HPLC analysis of the reaction mixture gives information about the amount of ATP bound to S1 at the end of the burst phase.

adp
actin
atpase
subfragment 1
mhc, myosin heavy chain
mlc, myosin light chain
rlc, regulatory light chain
elc, essential light chain
s1, myosin subfragment s1
bms1, bovine masseter s1
pss1, pig soleus s1
bcs1, bovine cardiac s1
rss1, rabbit soleus s1
tca, trichloroacetic acid
strain-sensor


Public Finance Policy Strategies to Increase Access to Preconception Care

Policy and finance barriers reduce access to preconception care and, reportedly, limit professional practice changes that would improve the availability of needed services. Millions of women of childbearing age (15–44) lack adequate health coverage (i.e., uninsured or underinsured), and others live in medically underserved areas. Service delivery fragmentation and lack of professional guidelines are additional barriers. This paper reviews barriers and opportunities for financing preconception care, based on a review and analysis of state and federal policies. We describe states’ experiences with and opportunities to improve health coverage, through public programs such as Medicaid, Medicaid waivers, and the State Children's Health Insurance Program (SCHIP). The potential role of Title V and of community health centers in providing primary and preventive care to women also is discussed. In these and other public health and health coverage programs, opportunities exist to finance preconception care for low-income women. Three major policy directions are discussed. To increase access to preconception care among women of childbearing age, the federal and state governments have opportunities to: (1) improve health care coverage, (2) increase the supply of publicly subsidized health clinics, and (3) direct delivery of preconception screening and interventions in the context of public health programs.
Introduction
The overarching goal of preconception care is to provide health promotion, screening, and interventions for the more than 62 million women of childbearing age [1] to reduce risk factors that might affect future pregnancies [2–4]. As elsewhere in this supplement, preconception care is defined as a set of interventions that aim to identify and modify biomedical, behavioral, and social risks to a woman's health or pregnancy outcome through prevention and management, emphasizing those factors which must be acted on before conception or early in pregnancy to have maximal impact. It includes care before a first pregnancy or between pregnancies (commonly known as interconception or internatal care) [5], and care that would typically be delivered in primary care settings [4].
Health professional leaders and professional organizations’ guidelines have called for improvements in preconception care for more than 20 years [4, 6–9]. and several reviews of the literature have assessed the evidence for specific interventions and documented their effectiveness [10, 11]. Without changes in financing, however, it appears unlikely that the nation will improve its preconception health and, thereby, improve the health of women, their children, and their families. Since federal and state policies are important drivers of health care financing, this paper provides suggestions on how policymakers can improve financing for preconception care [12].
Although many observers have suggested that providers would deliver more preconception care if they could bill payers for the service [13], a closer look at the situation suggests that more complex changes in women's health care financing may be necessary to support widespread use of preconception care. Affordability of care is a major concern for many women [14]. Many women lack basic health coverage, particularly non-pregnant, low-income women who do not meet of Medicaid's low income eligibility levels [15]. Securing private insurance coverage for additional preventive services may require more and better evidence about the effectiveness of interventions [4]. Other barriers to financing are more mechanical. Financing is required for an ongoing care process over a period of years and across the lifespan, not a single visit. Billing mechanisms and codes need refinement; in addition, cost-benefit analyses, and ongoing alignment with newly emerging approaches to care will also be required [16].
Improving health coverage for women of childbearing age
Financial barriers to health care access are widespread in the United States, with many low-income individuals having no or inadequate health coverage. The Kaiser Family Foundation’s, Women's Health Survey [17] does not focus on preconception care, but some of its results are relevant to understanding barriers to women's health care. Among women of childbearing age (18 to 44 years) 30 percent reported that they delayed or went without care because of cost. Moreover, 21% of women of all non-elderly women (18 to 64 years) in the survey reported that they did not fill a prescription for medication because of cost, including 40% of uninsured women [17].
Uninsured women
Millions of Americans live without health coverage. Nearly 17 million American women are uninsured [17]. One-third of low income women (with income less than 200 percent of the federal poverty level), half of women with disabilities, and 18 percent of all non-elderly women were uninsured in 2003 [18]. Younger women were more likely than older women to be uninsured. Hispanic, Asian, and Black women were more likely than non-Hispanic white women to be uninsured, reflecting their income and employment status patterns (i.e., more likely to have incomes below 200 percent of poverty and less likely to be employed in jobs that offer health insurance) [17]. Moreover, recent data indicate that women are more likely to lose coverage than men. Between 2002 and 2003, nearly 900,000 women ages 18 to 64 became uninsured, compared to 600,000 men (53 and 47 percent, respectively). Such losses are attributed primarily to welfare reform policies and employer-based coverage trends [19].
Data for 2002–2003 reveal that the percentage of women ages 18–64 who were uninsured varies substantially, ranging from 30 percent in Texas to 8 percent in Minnesota. The proportion of uninsured low-income adult women shows similar variation, ranging from more than 50 percent in Texas to less than 25 percent in Massachusetts, Minnesota, Tennessee, and Wisconsin. Although to some extent these differences reflect Medicaid eligibility policy, the state-to-state variations also reflect employment patterns. For example, Minnesota had the highest percentage of adult women with private coverage and a fairly low proportion of women covered by Medicaid (83 percent and 8 percent, respectively). In contrast in Tennessee (under policies in effect at that time), 17 percent of adult women were Medicaid beneficiaries and only 70 percent had private coverage [20].
Women who are young, single, working part-time, or unemployed are most likely to uninsured. These factors are reflected in an age gradient, the percent of uninsured women ranging from 22 percent among 18 to 24 year olds, 21 percent among 25 to 34 year olds, 19 percent among 35 to 44 year olds, and 13 percent among 45 to 54 year olds (Fig. 1). Moreover, women ages 25–44 accounted for more than half (53 percent) of all uninsured women under age 65 [17].
Fig. 1Percent of Non-Elderly U.S. Women who are Unisured, By Age, 2004
Medicaid as a source of coverage for women's health and PCC
Medicaid is effective in improving access to health care for low income women. Medicaid coverage affords women access to essential preventive, primary, and reproductive health care services, thereby allowing them to reduce unintended pregnancy, choose the number and spacing of their pregnancies and, when desired, to plan a healthy birth [21]. Medicaid plays a particularly important role in financing ambulatory care for women of childbearing age [22]. While Medicaid coverage and enrollment for women declined during the mid-1990s as a result of changes in health and welfare policies, Medicaid demonstration waiver projects are now being used by some states expand to access to health care for women of childbearing age.
Under current Federal and State policies, Medicaid is the primary mechanism for extending health coverage for low-income, uninsured women. In 2003, 12 percent of all women of childbearing age (15–44 years) and 37 percent of poor women in that age group relied on Medicaid for health care coverage. Because nearly two-thirds (63 percent) of the women covered by Medicaid are of childbearing age (19–44 years), the program's performance is related to preconception care access and to the outcomes of pregnancy [17].
Federal and state policies together define eligibility rules for Medicaid. Data from the Centers for Medicare and Medicaid Services (CMS) for female beneficiaries in the year 2000 indicate that 26 percent became eligible because they were pregnant and had low income, while 42 percent had welfare/poverty-linked eligibility [23]. Federal law requires that states extend Medicaid eligibility to pregnant women with income below 133 percent of the federal poverty level (FPL) and many states exceed this level. No similar protections exist for non-pregnant women. As of July 2004, income eligibility thresholds, mainly parents with dependent children, ranged from 19 percent of FPL in Alabama to 275 percent FPL in Connecticut. Other women were enrolled because they are over age 65 and had below poverty income; this is a national policy that does not vary by state. Many other low income women do not, however, qualify for Medicaid because they do not have children under age 18, are not over age 65, or are undocumented. In addition, Medicaid managed care arrangements have had an impact on the source of services for women in their childbearing years [24].
Some States have used waiver authority to expand Medicaid coverage to low income, uninsured adults – particularly the parents of children with publicly subsidized coverage. Parents in low-income families may be covered under so-called Section 1931 expansions, which do not require a waiver. More than 40 states and the District of Columbia provide Medicaid coverage to low-income parents through this option. The majority extend short-term coverage to unemployed parents, while other states extend coverage to parents with incomes to 50, 100, or 199 percent FPL, without regard to employment status. Washington State is one state that has used this option to large effect, extending eligibility to 200 percent FPL.
In nearly half of states, special waivers include a focus on adults and may have positive impact on coverage for low-income women of childbearing age. Three types of waivers are of particular importance here: (1) comprehensive waivers broadly designed to cover uninsured adults and make changes to benefits and cost sharing; (2) Health Insurance Flexibility and Accountability (HIFA) waivers, and (3) so-called “family planning” waivers [25]. All three types are authorized under Section 1115 of Medicaid and must be budget neutral. Each type of 1115 requires that states apply for and receive approval by the federal Centers for Medicare and Medicaid Services (CMS).
Comprehensive and HIFA waivers designed to increase health coverage among low-income adults
Nine states offer Medicaid coverage to low-income parents through Section 1115 waivers. All of these nine states extend coverage to families with incomes at or about 100 percent FPL. Again, some states extend coverage only to unemployed parents, whereas others extend coverage to parents with incomes to 50, 100, or 199 percent FPL, without regard to employment status. Minnesota's waiver extends coverage up to 275 percent FPL [26].
At the beginning of 2006, 11 states had active HIFA waivers, with the primary goal being to encourage new state approaches that increase the number of low-income individuals with health insurance coverage within current-level Medicaid and SCHIP resources [26]. Some of these (e.g., Maine and Michigan) extend coverage only to childless adults. More target the expansions to low-income parents with children enrolled in publicly subsidized coverage (e.g., Arizona, California, Illinois). Still other states sought a waiver for more general expansion of adult coverage.
With enactment of the Deficit Reduction Act of 2005 (DRA), states have increased flexibility to extend coverage, structure alternative benefit packages, and adopt premium and cost sharing requirements without a federal waiver. The DRA creates the potential for states to expand coverage to additional low-income adults, and some states (e.g., Kentucky, West Virginia) are considering expansions to parents of children with Medicaid or other publicly subsidized coverage under the State Children's Health Insurance Program (SCHIP). Such expansions may use limited benefit packages.
Family planning and interconception care waivers
States have received federal approval for Medicaid waivers programs that offer coverage to low-income women for family planning and reproductive health services. In 2001, nine state programs served 1.7 million women, and recent studies documented cost savings, reductions of unintended pregnancies, and improved use of family planning services in states with these programs [27]. By February 2005, 21 states had created such family planning waivers.
In recent years, 21 states have developed special programs to expand family planning services to women who do not otherwise qualify for Medicaid. Some states offer coverage to women who lose coverage after the birth of a baby or starting a job, while others offer family planning coverage based on income status to men and/or women [28].
Of review of 15 state family planning waivers prepared for the National Association of State Medicaid Directors (NASMD) found that core services covered were comprehensive health history and physical examination including appropriate laboratory testing, health education and counseling, approved birth control methods and supplies, and infertility services. Some states provide coverage for services that may be considered preconception care, such as screening for nutrition, folic acid intake, alcohol and tobacco use, genetic conditions, rubella serostatus, domestic violence, and other health risks [29].
An evaluation prepared for CMS of family planning waiver projects in six states found that they resulted in significant savings to both the Federal and State governments, as well as some evidence for expanded geographic availability of services and measurable reductions in unintended pregnancy [30]. This is true, despite the fact that the federal government matches state family planning expenditures at 90/10 for each dollar spent. Greater potential savings and prevention, however, could result if States offered coverage for more comprehensive risk screening, health promotion, and interventions leading toward higher levels of preconception wellness. This would require an extension of states waiver authority aimed at coverage of preconception and interconception care.
Public financing to improve access to primary care
Preconception care is part of a continuum of women's health services, typically provided in the context of well-woman visits or integral to chronic care management [31–33]. Most components of preconception care can be embedded in the process of primary and preventive care, rather than an isolated visit [34, 35]. Integration of preconception components into primary care can better serve women across the lifespan and at varying levels of risk [36, 37]. Primary care integrates a variety of health promotion, prevention, and acute care services to address a majority of personal health care needs and common health problems in a community setting [38]. It also may include screening for and ongoing management of chronic conditions in a primary care setting.
National survey data for 2004 indicate that 75 percent of U.S. women ages 18 to 44 had a health care visit in the past year, and most women of reproductive age obtain preventive health services in any given year [17], offering opportunities for clinicians to deliver preconception care. More than half (55 percent) of women had obstetrician/gynecologist visits in the past year. Notably, however, one-third to one-half of women have more than one primary care providers (generally a family physician or internal medicine physician, and an obstetrician/gynecologist) [14]. Some research indicates that having both a reproductive health provider and a primary care provider may increase utilization of clinical preventive services [39].
Having a usual or routine source of care (i.e., a place or provider they usually go when they need health care) is one indicator of health care access and a predictor of certain health behaviors [40]. Women who have a usual source of care are more likely to receive preventive care, to receive continuous care, and to have lower health care costs [14]. As with health coverage, the percentage of women reporting a usual source of care increases with age. Based on detailed survey data from 2002–03, the proportion of women with a usual source of care was 82 percent for ages 18–24, 86 percent for ages 25–34, and 91 percent for ages 35–44 [17]. Proportions also varied by race and ethnicity, with Hispanic women most likely to report no usual source of care. Non-Hispanic Black women were more likely to use a clinic and Non-Hispanic White women more likely to use a physician office as their routine source of care.
These data suggest that publicly subsidized clinics such as community health centers and other federally qualified health centers (FQHCs) might be used to increase access to preconception care among low-income and uninsured women. Community health centers—local, non-profit, community-owned health clinics—serve more than 15 million people in 3,600 low-income, medically underserved, urban and rural communities. Health centers are an important source of primary care for millions of low-income and uninsured women. Nearly 30 percent of all patients are women of childbearing age, and health centers provided prenatal care to over 330,000 women in 2003 [41]. Their patients account for one out of every 10 U.S. births [42]. Research on health centers shows that patients have less infant mortality and low birthweight [43], fewer health disparities [44], improved care for chronic conditions [42], and improved access to primary and preventive care [45]. A study of four states clinics suggests that FQHCs may be an important source of reproductive health services to reduce unintended pregnancies and teen births [46].
Among FQHCs, the Health Disparities Collaboratives Initiative is underway to improve the quality of primary care delivered, and over 600 FQHCs have participated [47]. This quality improvement model is tailored to fit with the staffing and patient patterns of FQHC and is supported by a partnership among community clinics, federal agencies, and national professional organizations. The Health Disparities Collaboratives started with a Chronic Diseases Care Model for quality improvement [48], but now are developing a Primary Health Care model and integrating with the Perinatal Care Collaboratives and other efforts [49].
Health centers receive about one-quarter of their total revenue from direct federal grants. Their single largest source of support is Medicaid, representing more than one-third of total revenues. Patients self-payments, other insurance, and large amounts of non-federal grants and contracts make up most of the remainder of health center funding [41]. Thus, federal and state fiscal decisions, in Medicaid and other health program appropriations, affect the fiscal well-being of FQHCs.
Financing public health programs and projects
Existing public health programs serve millions of women each year. Preconception interventions can be incorporated into these existing programs targeted to reach women at highest risk [50]. Two program examples illustrate the potential, as well as the funding gaps.
The federal Family Planning program is authorized under Title X of the Public Health Service Act. Created in 1970, the Title X program is the only Federal program solely dedicated to family planning and reproductive health with a mandate to provide a broad range of voluntary, affordable, and effective family planning methods and services. The program is particularly designed to provide family planning services to low-income women, many of whom are uninsured and who would have no other source of care. The program provides funds to approximately 4,500 of the estimated 7,000 family planning clinics in the United States and provides reproductive health services to approximately 6.5 million persons each year [27]. Title X-funded clinics provide family planning education, contraceptives, and pregnancy tests; however, many do not offer more comprehensive risk screening and reproductive health promotion and reproductive life planning [51]. If Title X funding had been increased at the rate of inflation from its FY 1980 funding level, it would have been funded at over $590 million in FY 2002 [52, 53]. Federal funding was set at only $288 million for FY 2005 [54]. Without additional funding, it seems unlikely that Title X clinics will obtain the financial and staff resources to add components of preconception care [55].
Federal and State public health programs funded by the Title V Maternal and Child Health Services Block Grant have the flexibility to give priority to preconception health and offer support for demonstration projects and evaluations of prevention programs. Authorized under Title V of the 1935 Social Security Act, this program provides block grants to states to improve the health of all women, children, youth and families. Title V funding is an important source of funding for infrastructure building, population-based services, enabling services, and direct health care services. Preconception care is a priority area for some state Title V programs and is being monitored with performance measures in additional states [51]. Many state Title V agencies have preconception or interconception projects underway or in the design phase, but most of these operate on a small scale or are targeted only to a special population such as teens. Opportunities exist to use Title V block grant and special project funds more broadly to fund related needs assessment, community-based prevention projects, health education, or preconception care for women. Without additional funding, however, it seems unlikely that new priorities will be adopted and new programs implemented by federal or state Title V-funded entities. As part of an overall funding cut for domestic discretionary programs, Congress reduced FY 2006 funding for the Title V program from $724 million to $700 million [56].
Although the federally funded Healthy Start projects have interconception health activities, these projects located in communities with high infant mortality have opportunities to offer more systematic pre/interconception screening, health promotion, and interventions. Healthy Start grantees are required to include interconception care activities as part of their overall project. Such activities might include linkage to local providers, tracking postpartum visits, providing case management services, and/or directly provide interconception services [57]. Because Healthy Start funding is linked to Title V funding overall, resources for more extensive or systematic efforts are limited.
Public health programs funded through the Centers for Disease Control and Prevention (CDC), particularly those offering screening and related services for sexually transmitted diseases and HIV/AIDS also could do more to provide preconception risk assessment and health promotion. Such programs targeted to high risk women are not, however, substitutes for access to primary care and health care coverage.
Conclusions
Public health policy can be correlated to women's health outcomes [58]. Three major policy and finance directions are proposed by this review paper. To increase access to care among women of childbearing age, the federal and state governments have opportunities to improve health care coverage, increase the supply of publicly subsidized health clinics, and encourage delivery of preconception screening and interventions in the context of public health programs. First and foremost, state and federal policymakers might increase health coverage for low-income women of childbearing age through Medicaid policy changes and waivers. Although health coverage in and of itself may not change health behaviors and health care utilization [59], having access–financial and geographic–to a provider can be seen as threshold requirements for improved health care utilization and delivery of preconception care. A second opportunity is to continue to increase support for community health centers and other federally qualified health centers in medically underserved communities. Last, but not least, policymakers might give greater attention to financing for health promotion and prevention programs, particularly programs such as Title X and Title V which focus on services to women of childbearing age [60–62].

finance
policy
preconception
medicaid
title v
family planning
maternal health
infant health


Preconception and the Young Cancer Survivor

Introduction
While the diagnosis of cancer is not common in young men and women, cancer survivors younger than 40 are likely to be very concerned about the impact of their cancer on future fertility, their risk of cancer recurrence after treatment and risks to future offspring. It is estimated that 1 in 900 persons between 16 and 44 are cancer survivors [1], and that there are at least 250,000 women age 40 and younger who are breast cancer survivors in the United States. As cancer survival increases, the focus on quality of life issues increases. When questioned, at least 73% of young breast cancer survivors reported concerns about infertility due to cancer treatment, and 29% reported that this concern impacted their choice of cancer treatment [2].
Premature ovarian failure
In women, the options for preserving fertility and risk of ovarian failure depends on the age at the time the cancer is treated, the type and location of cancer, and the specific treatment chosen. Some treatments, such as bone marrow transplantation (BMT) are accompanied by very high rates (>99%) of ovarian failure [3]. Even for those women who resume normal menstruation after chemotherapy, premature ovarian failure (POF) is not uncommon [4], and the risk of premature ovarian failure is 8-fold higher in cancer survivors [5]. The risk of POF increases with the woman's age, with less than 20% of women under 30 years experiencing POF, whereas most women older than 40 will become menopausal after chemotherapy [6].
POF and menopause are dependent on the type of therapy used. Rates of ovarian failure range from 15% for acute myelocytic leukemia (AML) to 50% for breast cancer, with the overall rate of ovarian failure of 34% [4]. The chemotherapy agents with the highest risk for ovarian failure were the alkylating agents. The higher the dose of these drugs, the higher the likelihood of ovarian failure.
Ovarian tissue damage is well documented with the use of radiation therapy. Radiotherapy is used for treatment of several cancer types affecting pre-menopausal women. The degree of damage to ovarian tissue is related to a woman's age, the total dose of radiation used, and the number of exposures. In one study, when the same total dose of radiation was delivered, exposure to multiple fractionated doses versus a single dose resulted in less damage to the ovaries [4]. When used in conjunction with chemotherapy, the risk of POF increases. Byrne et al. assessed the risk of premature ovarian failure in childhood cancer survivors and found the relative risk of early menopause was 9.2 for those treated with chemotherapy alone, and 27 for those treated with radiation plus chemotherapy [5].
Fertility preservation techniques
Although an increasing number of techniques have become available for the preservation of fertility, many are experimental and have not been tested in randomized controlled trials. Table 1 reviews the types of options available and their benefits and limitations.
Table 1Fertility preservation options for women with malignanciesMethodBenefitsLimitationsIn vitro Fertilization (IVF)Most successful, widespreadDelay of therapy, use of fertility medicationsFrozen oocytesNo partner is neededDifficult to preserveOvarian tissue preservationLess tissue damage from freezing, can be used in pre-menarchal and young womenExperimentalTransposition of ovarian tissueLess radiation exposureLimited to women having radiation therapyPharmacological protectionLimit damage to ovarian tissueAdequate prospective studies needed
In vitro fertilization (IVF)
The best-studied and most successful option for preserving fertility in female cancer patients is ovarian stimulation followed by IVF and embryo cryopreservation. Ovarian stimulation needs to occur prior to cancer therapy because treatment can adversely affect ovarian reserve and response. The pregnancy rate with frozen embryos is lower compared to fresh IVF embryo transfer. A meta-analysis by the American Society for Reproductive Medicine reviewed over 88000 cycles of assisted reproductive technology (ART) treatment. When fresh oocytes were used, a delivery rate of 41.8% was found in more than 6500 cycles analyzed, compared to a delivery rate of 18.6% for frozen embryos [7].
One concern about ovarian stimulation with IVF is that it requires a delay of therapy, which could impact growth rate of tumors and survival rates. In addition, concerns have been voiced that the medications used to stimulate ovarian production could impact on estrogen-receptor positive tumors such as breast carcinoma. Anti-estrogens, such as tamoxifen, or aromatase inhibitors, (e.g. letrozole), have both been used in ovarian stimulation in breast cancer patients to maintain low estradiol levels during IVF cycles.
Frozen oocytes
Frozen oocytes are more difficult to preserve, resulting in a lower survival, fertilization and pregnancy rates. Damage is in part due to ice formation during the freezing process. Unfertilized frozen oocytes have a low success rate for live birth (2% per oocyte) [8], although the rates are now increasing. Rapid freezing of eggs by vitrification limits damage, which improves oocyte survival. Using intracytoplasmic sperm injection (ICSI) increases the fertilization rate even further.
Ovarian tissue cryopreservation
For pre-menarchal girls, young women with POF, women that cannot delay therapy or those in whom high doses of fertility medications are contraindicated, ovarian tissue cryopreservation is offered in some centers. Unlike frozen oocytes, primordial follicles in ovarian tissue are smaller and have less follicular fluid, and therefore may be at less risk from damage from ice crystals that form during freezing. It is hoped that transplanting thawed ovarian tissue will result in the recovery of fertility. Transplantation should be used with caution in women with ovarian or metastatic cancer, to prevent transferring malignant cells back after treatment. This treatment is still considered experimental. Although success in animals has been demonstrated, transplantation of cryopreserved ovarian tissue has resulted in only two livebirths [9, 10].
Ovarian transposition
For women having pelvic radiation, transposition of ovarian tissue outside of the field of radiation successfully preserves ovarian function. The radiation dose to the transposed ovary is approximately 5–10% the level of the non-transposed ovary [11].
Pharmacological protection
To limit or prevent chemotherapy-induced POF, some centers use gonadatropin releasing hormone (GnRH) agonists to reduce gonadatropins. This results in mimicking a pre-pubertal state, because the ovary appears to be less sensitive to gonadotoxic drugs prior to menarche. Because dividing cells are more susceptible to damage from cytotoxic agents, ovarian follicular cells are vulnerable. The mechanism of protection by GnRH agonists is unknown. One prospective clinical case series found that 70 of 75 women (93%) co-treated with GnRH agonists and chemotherapy resumed cyclic ovarian function verses 38 of 82 women (46%) treated with chemotherapy alone [12]. Adequately controlled prospective studies still are required, although data from animal studies are promising. Anti-apoptotic agents, such as sphingosine-1-phosphate (SIP), have prevented radiation-induced oocyte loss in mice [13]. However, since apoptosis is needed for the treatment of cancer, this method faces some obstacles for implementation.
Male fertility
In men, sperm banking is routine and should be offered to all men prior to undergoing cancer treatment. When combined with intracytoplamic sperm injection (ICSI), sperm banking should preserve fertility for almost all males. There is no increased risk for birth defects when sperm is banked prior to therapy [14, 15]. Even in azoospermic males, testicular sperm extraction (TESE) with ICSI has resulted in successful pregnancies. By 2005, 11 of 29 men with azoospermia that had used TESE with ICSI had achieved a pregnancy which resulted in a livebirth [16]. For pre-pubertal males, sperm banking is not available because there is no spermatogenesis. The possibility of cryobanking of testicular tissue is being explored [16, 17]. An increase in sperm aneuploidy is seen up to 18 months post-chemotherapy, so it has been suggested that a delay of conception for 12–18 months after therapy is completed is reasonable [18].
Pregnancy after cancer
Once a woman has recovered from cancer treatment, no clear guidelines exist about the length of time to wait before attempting to become pregnant.
Effect of cancer treatment on pregnancy
Once a cancer survivor achieves pregnancy, her health and the fetal health may be adversely affected by her prior treatment for cancer. Clinically significant heart disease occurs in 0.5–1% of women treated with anthracycline-based chemotherapy drugs, and may not develop until months or years after therapy is completed [19]. Also, women who have radiation to the left side of the abdomen or chest are at risk for heart damage, which could worsen during pregnancy or labor. Women with radiation to the abdomen or pelvic area or total body radiation are at increased risk for miscarriage, preterm birth and delivery of low birthweight infants [20]. A report from the Childhood Cancer Survivor Study (CCSS) did not show any increase in adverse pregnancy outcomes in women treated with most chemotherapy agents. However, an increased risk of stillbirth in women treated with abdominal radiation for Wilm's tumor has been noted [21].
Women who have completed treatment for breast cancer are often placed on selective estrogen receptor modulators (SERMs) such as tamoxifen to reduce the incidence of a second cancer. However, tamoxifen use during pregnancy is to be avoided because it is potentially teratogenic.
Effect of pregnancy on cancer
Some women are concerned that the hormonal changes from pregnancy may increase the risk of cancer recurrence, but pregnancy after breast cancer management does not increase the risk of cancer recurrence or cancer-related death [22, 23]. However, women who are carriers of BRCA1 or BRCA2 germline mutations and who have had a full-term pregnancy are at significantly higher risk of developing breast cancer by age 40 than mutation carriers who do not have children [24]. The risk for breast cancer is increased by 70% in the first 2 years after pregnancy for BRCA2 carriers, but not BRCA1 carriers [25].
Risk to offspring
Since cancer treatment by radiation and many chemotherapy agents are known to cause DNA mutations, cancer survivors are often concerned that the treatment may increase the risk for birth defects, genetic conditions or cancer in their offspring. Several studies have found no increased risk of congenital abnormalities in the offspring of cancer survivors. Epidemiologic data from Japanese survivors of the atomic bomb and their children did not show any evidence for new mutations. The CCSS also did not find any link between radiation therapy or chemotherapy for childhood cancer and genetic diseases or birth defects [26, 27].
Risk of cancer in offspring
No increased risk for childhood cancer was identified in the offspring of childhood cancer survivors, but additional follow-up is needed [26]. In rare instances, the childhood cancer may be hereditary, with a risk of passing on the mutated gene to the offspring. Some cancers, such as breast, colon or ovarian cancer, are more likely to be hereditary when the cancer occurs at an earlier age. For women with breast cancer diagnosed prior to age 40 and no other family history of early breast cancer, the risk of a mutation in one of the hereditary breast cancer genes, BRCA1 or BRCA2 is estimated to be at least 13% [28].
Summary
All women should be counseled about the impact of cancer treatment on fertility prior to undergoing chemotherapy, ovarian surgery or BMT, and offered fertility preservation options, of which ovarian stimulation with IVF is the most successful. Once women achieve pregnancy, the risk for birth defects or genetic conditions is not increased because of either radiation or chemotherapy. Because treatment options may increase the risk for adverse pregnancy outcome, review of therapy records is essential for determining potential risk to the pregnancy. For most young cancer survivors, fertility can be preserved and a successful pregnancy achieved, but discussing these issues prior to treatment is essential.

preconception
ovarian failure
fertility preservation
pregnancy after cancer