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Expression of cardiac troponin T with COOH-terminal truncation accelerates cross-bridge interaction kinetics in mouse myocardium

¹Department of Physiology, University of Wisconsin Medical School, Madison, Wisconsin 53706; and ²Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309

Submitted 23 February 2004 ; accepted in final form 25 May 2004

	ABSTRACT

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Transgenic mice expressing an allele of cardiac troponin T (cTnT) with a COOH-terminal truncation (cTnT_trunc) exhibit severe diastolic and mild systolic dysfunction. We tested the hypothesis that contractile dysfunction in myocardium expressing low levels of cTnT_trunc (i.e., <5%) is due to slowed cross-bridge kinetics and reduced thin filament activation as a consequence of reduced cross-bridge binding. We measured the Ca²⁺ sensitivity of force development [pCa for half-maximal tension generation (pCa₅₀)] and the rate constant of force redevelopment (k_tr) in cTnT_trunc and wild-type (WT) skinned myocardium both in the absence and in the presence of a strong-binding, non-force-generating derivative of myosin subfragment-1 (NEM-S1). Compared with WT mice, cTnT_trunc mice exhibited greater pCa₅₀, reduced steepness of the force-pCa relationship [Hill coefficient (n_H)], and faster k_tr at submaximal Ca²⁺ concentration ([Ca²⁺]), i.e., reduced activation dependence of k_tr. Treatment with NEM-S1 elicited similar increases in pCa₅₀ and similar reductions in n_H in WT and cTnT_trunc myocardium but elicited greater increases in k_tr at submaximal activation in cTnT_trunc myocardium. Contrary to our initial hypothesis, cTnT_trunc appears to enhance thin filament activation in myocardium, which is manifested as significant increases in Ca²⁺-activated force and the rate of cross-bridge attachment at submaximal [Ca²⁺]. Although these mechanisms would not be expected to depress systolic function per se in cTnT_trunc hearts, they would account for slowed rates of myocardial relaxation during early diastole.

calcium ion sensitivity of force; kinetics of actin-myosin interaction; skinned myocardium

In the present study we examined the effects of the cTnT_trunc mutation on the kinetics of cross-bridge binding to the thin filament and thin filament responsiveness to the activating effects of strong-binding cross bridges. We show that cTnT_trunc directly affects thin filament activation by accelerating the rate of cross-bridge binding under basal conditions and enhances the sensitivity of the thin filament to the activating effects of Ca²⁺ and strong-binding cross bridges.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Transgenic mice expressing truncated cTnT. Transgenic mice expressing truncated cTnT (cTnT_trunc) were generated as described previously (19). Expression of the transgene was driven by the murine -myosin heavy chain promoter, which restricts expression of cTnT_trunc to cardiac muscle. Previous studies documented that transgenic mice expressing >10% of total cTnT as cTnT_trunc die within hours of birth (19). To ensure survival for mechanical measurements, transgenic mice that expressed relatively low levels of cTnT_trunc, i.e., <5% total TnT, were studied (19). Analysis of global ventricular function has documented that mice expressing cTnT_trunc at levels comparable to those in the current study exhibit significant diastolic dysfunction (19). Compared with wild-type (WT) littermate controls, transgenic mice expressing low levels of cTnT_trunc exhibited no gross phenotypic abnormalities, no change in mortality, and no apparent ventricular hypertrophy. Transgenic animal production and animal usage were conducted under strict guidelines established by animal use committees at the University of Colorado and the University of Wisconsin.

Skinned myocardial preparations. The methods used for preparing skinned myocardium for mechanical measurements under isometric conditions were described previously (13). After intraperitoneal injection of 5,000 U heparin/kg body wt, WT and cTnT_trunc mice (8–12 mo old) were anesthetized with inhaled isoflurane. Their hearts were excised, and right and left ventricles were dissected in Ringer solution (in mM: 118 NaCl, 4.8 KCl, 2 NaH₂PO₄, 1.2 MgCl₂, 25 HEPES, 11 glucose, and 0.5 CaCl₂; pH 7.4, 22°C). Both ventricles were then rapidly frozen in liquid nitrogen, a step that was essential for obtaining high-quality preparations (13). To prepare skinned myocardium, pieces of frozen ventricle were thawed and homogenized for 4 s in ice-cold relaxing solution (in mM: 100 KCl, 20 imidazole, 7 MgCl₂, 2 EGTA, and 4 ATP; pH 7.0) with a Polytron homogenizer, which yielded multicellular preparations of 100–250 µm x 600–900 µm. The homogenate was centrifuged at 120 g for 1 min, and the resulting pellet was washed with fresh relaxing solution and resuspended in relaxing solution containing 250 µg/ml saponin and 1% Triton X-100. After 30 min, the skinned preparations were washed three times with fresh relaxing solution and were dispersed in 50 ml of relaxing solution in a glass petri dish. The dish was kept on ice at all times except during the selection of individual preparations for mechanical experiments.

Experimental solutions, apparatus, and protocol. Solution compositions were calculated with the computer program of Fabiato (4) and stability constants (corrected to pH 7.0 and 15°C) listed by Godt and Lindley (7). All solutions contained (in mM) 100 N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 14.5 creatine phosphate, and 5 DTT. In addition, 1) pCa 9.0 solution contained (in mM) 7 EGTA, 0.02 CaCl₂, 5.42 MgCl₂, and 4.74 ATP; 2) pCa 4.5 solution contained (in mM) 7 EGTA, 7.01 CaCl₂, 5.26 MgCl₂, and 4.81 ATP; and 3) preactivating solution contained (in mM) 0.07 EGTA, 5.42 MgCl₂, and 4.74 ATP. The ionic strength of all solutions was adjusted to 180 mM with potassium propionate. A range of solutions containing different free Ca²⁺ concentrations ([Ca²⁺]_free) (i.e., pCa 6.5–5.5) were prepared by mixing appropriate volumes of solutions of pCa 9.0 and pCa 4.5.

Skinned preparations with well-defined edges and no free ends evident in the middle region were transferred from the petri dish to a stainless steel experimental chamber (11) containing relaxing solution. The ends of a preparations were attached to the arms of a motor (model 312B, Aurora Scientific) and a force transducer (model 403; Aurora Scientific) as described previously (11). The chamber assembly was then placed on the stage of an inverted microscope (Olympus) fitted with a x40 objective and a CCTV camera (model WV-BL600; Panasonic). Light from a halogen lamp was passed through a cut-off filter (transmission >620 nm) and was used to illuminate the skinned preparations. Bitmap images of the preparations were acquired with an AGP 4X/2X graphics card and associated software (ATI Technologies) and were used to assess mean sarcomere length (SL) during the course of each experiment. Changes in force and motor position were sampled (16-bit resolution, DAP5216a; Microstar Laboratories, Bellevue, WA) at 2.0 kHz with SLControl software developed in our laboratory (http://www.slcontrol.com) and saved to computer files for later analysis. Changes in force were also recorded on a chart recorder with a slow time base.

At the start of each experiment, the preparation was stretched to a mean SL of 2.20 µm for measurements of steady-state Ca²⁺-activated force and the rate constant of force redevelopment (k_tr) after a release and restretch maneuver (2). The force recorded in a solution of pCa 9.0 was taken as resting force and then subtracted from the total force in activating solutions to yield the Ca²⁺-activated force (P). The values of k_tr reported here were determined as the rate constant of force redevelopment after the release and restretch of Ca²⁺-activated preparations (Fig. 1). Mechanical measurements of steady-state Ca²⁺-activated force and the rate of force redevelopment were repeated after 15-min incubations of the preparations in pCa 9.0 solution containing either 3 µM or 6 µM non-force-generating derivative of myosin subfragment-1 (NEM-S1) (5). At the end of each experiment, the preparation was cut free, placed in SDS sample buffer, and stored at –80°C until subsequent analysis of contractile protein content analysis by 12% SDS-PAGE (5). Gels were stained with silver and scanned with a densitometer (Molecular Analyst; Bio-Rad).

View larger version (14K): [in this window] [in a new window]   Fig. 1. Experimental protocol for determination of Ca2+-activated force and rate constant of force redevelopment (ktr) in skinned myocardium. Bottom: changes in force recorded before, during, and after a step change in length (top) of the skinned myocardium. Once Ca2+-activated force reached steady state in a solution of pCa 4.5, muscle length was rapidly reduced by 20%. The amplitude of Ca2+-activated force was determined by subtracting the resting force measured at pCa 9.0 from the total force generated at pCa 4.5. After 20 ms of unloaded shortening, the preparation was restretched to its original length. ktr was calculated as the apparent rate constant of force redevelopment after restretch of the muscle to its original length.

All data are reported as means ± SE. Comparisons between WT and cTnT_trunc data in the absence of NEM-S1 were made with an unpaired t-test and, when appropriate (nonparametric data), with the Wilcoxon Mann-Whitney test. To evaluate the effects of NEM-S1 on WT or cTnT_trunc myocardium, ANOVA with a Tukey post hoc test was used or, when appropriate, the Wilcoxon Mann-Whitney test. Significance level was set at P < 0.05.

	RESULTS

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Mechanical properties of skinned WT and TnT_trunc myocardium. TnT_trunc and WT myocardium generated comparable resting forces and maximum Ca²⁺-activated forces (Table 1). In both cases, the force-pCa relationship was sigmoidal and was well fit with the Hill equation (Fig. 2). In the absence of NEM-S1, mean pCa₅₀ was 5.81 ± 0.01 in WT myocardium and 5.92 ± 0.02 in TnT_trunc myocardium, indicating that the Ca²⁺ sensitivity of force was greater in TnT_trunc myocardium (pCa₅₀ = 0.11 ± 0.01; P < 0.01). Furthermore, the steepness of the force-pCa relationship was markedly reduced in TnT_trunc compared with WT myocardium (n_H was 3.6 ± 0.1 in TnT_trunc vs. 4.3 ± 0.2 in WT; P < 0.001), indicating that low levels of TnT_trunc expression reduce the apparent cooperativity of force development.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Effects of non-force-generating derivative of myosin subfragment-1 (NEM-S1; 3 µM) on force-pCa relationships in skinned wild-type and transgenic myocardium. Data points are means and the error bars are SE. Smooth lines were generated by fitting the mean data with the Hill equation described in MATERIALS AND METHODS. Values for Ca2+ sensitivity of force (i.e., pCa50) in the absence (open symbols) and the presence (closed symbols) of 3 µM NEM-S1 were as follows: wild type: , 5.81 ± 0.01 (n = 10) and , 5.90 ± 0.03 (n = 7); cardiac troponin T with COOH-terminal truncation (cTnTtrunc): , 5.92 ± 0.02 (n = 10) and , 6.03 ± 0.01 (n = 5). P/P0, force normalized to maximum Ca2+-activated force.

View larger version (20K): [in this window] [in a new window]   Fig. 3. Force records obtained during measurements of ktr in skinned wild-type and transgenic myocardium. The force records have been scaled to the peak steady-state force attained after the step change in muscle length to facilitate direct visualization of differences in rates of force recovery in skinned myocardium from wild-type (A) and TnTtrunc (B) mice.

View larger version (13K): [in this window] [in a new window]   Fig. 4. Effects of 3 µM NEM-S1 on the activation dependence of ktr in wild-type and transgenic myocardium. Force redevelopment after rapid release and restretch was measured in the absence (open symbols) and the presence (closed symbols) of 3 µM NEM-S1 in wild-type (circles) and TnTtrunc (squares) myocardium. Data points are means, and the error bars are SE. A: Ca2+ dependence of ktr. B: ktr as a function of force (expressed as a fraction of maximal Ca2+-activated force) measured at each pCa.

Effects of NEM-S1 on mechanical properties of skinned WT and TnT_trunc myocardium. The effects of 3 and 6 µM NEM-S1 on resting force, maximum Ca²⁺-activated force, Ca²⁺ sensitivity of force (pCa₅₀), and the steepness of the force-pCa relationship are summarized in Table 1. In both WT and TnT_trunc myocardium, NEM-S1 increased both resting force and submaximal Ca²⁺-activated forces in a concentration-dependent manner. Mean pCa₅₀ increased significantly in both WT and TnT_trunc myocardium after treatment with 3 µM NEM-S1, indicating an increase in the Ca²⁺ sensitivity of force. Qualitatively similar increases in mean pCa₅₀ were observed after treatment with 6 µM NEM-S1. In addition, treatment with 3 or 6 µM NEM-S1 significantly reduced the steepness of the force-pCa relationships in both WT and TnT_trunc myocardium, an observation consistent with results reported previously in skinned ventricular myocardium (6).

Treatment with either 3 or 6 µM NEM-S1 had no appreciable effect on the kinetics of force redevelopment in maximally activated preparations. However, at low levels of Ca²⁺ activation, NEM-S1 had concentration-dependent effects on k_tr. In both WT and TnT_trunc myocardium, treatment with 3 or 6 µM NEM-S1 increased k_tr at low levels of activation to values identical to k_tr obtained in maximally activated preparations. At intermediate levels of activation, NEM-S1 increased k_tr to greater than control values, but k_tr was still less than maximal. Also, the k_tr values obtained at intermediate levels of activation in the presence of NEM-S1 were significantly greater in TnT_trunc myocardium than in WT myocardium.

	DISCUSSION

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Summary. Earlier functional studies on whole hearts isolated from cTnT_trunc transgenic mice indicated that the mutation induced severe diastolic dysfunction and mild hypocontractile systolic dysfunction (19), whereas studies in reduced systems found that the cTnT_trunc mutation increased the Ca²⁺ sensitivity of force (10), reduced the inhibition of actin-activated myosin ATPase activity in regulated thin filaments, and reduced inhibition of sliding in an in vitro motility assay comprised of regulated thin filaments (14). Although the mechanisms underlying this range of phenotypes are likely to be complex, an hypothesis that is consistent with these findings is that cTnT_trunc directly affects cross-bridge transitional rate constants such that the rate of force development is slowed by reductions in the apparent rate constants of both attachment and detachment of cross bridges from actin. In fact, our results showed that the opposite was the case: at submaximal activations cTnT_trunc myocardium exhibited faster rates of force development (Fig. 4A), implying that the rates of cross-bridge attachment or detachment or both were accelerated compared with WT myocardium. Our results further show that at least some of these effects of the cTnT_trunc mutation were a consequence of greater cross-bridge recruitment and increased thin filament responsiveness to the activating effects of Ca²⁺ and strong-binding cross bridges.

Altered Ca²⁺ sensitivity of force and apparent cooperativity in cTnT_trunc myocardium. Previous studies reported that expression of cTnT_trunc reduced maximal Ca²⁺-activated force compared with controls (12, 24). Our results did not reproduce this effect, and because the maximal rates of force redevelopment (k_tr) did not differ between cTnT_trunc and WT myocardium, a reduction in maximal Ca²⁺-activated force is not likely to contribute to the changes in cross-bridge kinetics observed in cTnT_trunc myocardium at submaximal activations.

Our data also show that transgenic expression of small amounts (<5%) of cTnT_trunc produced dramatic effects on Ca²⁺ sensitivity of force and the apparent cooperativity of thin filament activation, as indicated by changes in n_H. The pCa required at half-maximal activation (pCa₅₀) was shifted to lower [Ca²⁺] by 0.11 units (Fig. 2) in transgenic myocardium compared with WT myocardium, indicating that less Ca²⁺ is required to produce the same amount of force as controls. This finding agrees well with results of Montgomery et al. (10), who reported a 0.14-pCa unit shift in the tension-pCa relationship in the same cTnT_trunc mice compared with WT myocardium.

Another notable finding is that n_H, an index of apparent cooperativity in the activation of force, was reduced in cTnT_trunc myocardium as evidenced by the shallower slope of the force-pCa relationship (Fig. 2). This phenomenon was observed earlier by other investigators (3) and may manifest a diminished capacity of the Tm-Tn complex to inhibit cross-bridge binding. Impaired suppression of myosin binding at low levels of Ca²⁺ is consistent with slowed and incomplete diastolic relaxation and may indirectly give rise to diminished systolic function in cTnT_trunc mice (19). It has been suggested that the TnT truncation destabilizes the blocked conformation of the thin filament regulatory strand, which enhances the transition from the blocked to the active state. Thus, in cTnT_trunc myocardium, there is greater likelihood that the thin filament will assume the "switched-on" state even at low [Ca²⁺] and thereby reduce cooperativity in myosin binding to actin (3, 23).

Accelerated cross-bridge kinetics in cTnT_trunc myocardium. Although cTnT_trunc had no effect on the maximal rate of force redevelopment compared with control, the mutation increased k_tr at submaximal [Ca²⁺] (Fig. 4). Such increases could be due to a direct effect of the mutation on cross-bridge cycling kinetics or an indirect effect of increased cross-bridge binding to the thin filament. Both mechanisms appear to be operational in cTnT_trunc myocardium, because some of the difference in rates between cTnT_trunc and WT disappeared when results were plotted against relative force as a measure of Ca²⁺ activation; however, at P/P_o less than 0.4, k_tr was still faster in cTnT_trunc myocardium. The reduction in difference between cTnT_trunc and WT myocardium when k_tr was plotted against force implies that some of the difference (Fig. 4A) is due to acceleration of k_tr in cTnT_trunc myocardium secondary to the activating effects of greater numbers of cross bridges bound to the thin filament. The observation that some of the difference persists at low levels of activation suggests that cTnT_trunc also has direct effects to accelerate cycling kinetics. In this regard, it is important to reiterate that the acceleration of k_tr at low levels of Ca²⁺ activation was observed despite low levels of cTnT_trunc expression (19). Incorporation of <5% cTnT_trunc into the thin filament was sufficient to elicit a significant increase in the rate of force redevelopment in the absence of NEM-S1 (Fig. 4B).

Earlier measurement of ATPase activity is consistent with these results. Montgomery et al. (10) reported that maximal ATPase cycling rates did not differ between WT and cTnT_trunc; however, the ATPase activity-pCa relationship was shifted to lower [Ca²⁺] in cTnT_trunc, indicating that cross-bridge cycling rates were increased at submaximal [Ca²⁺]. Reports of increased sliding speed of actin filaments in in vitro motility assays (14, 22), decreased inhibition of ATPase activity in the absence of Ca²⁺ (18), and the accelerated k_tr at low Ca²⁺ observed in the present study are consistent with the idea that cTnT_trunc attenuates inhibition of thin filaments at low Ca²⁺.

This phenomenon can also be interpreted in terms of a three-state model of thin filament activation (9) in which the thin filament exists in a blocked state (unable to bind myosin) in the absence of Ca²⁺ but rapidly transitions to a closed state on Ca²⁺ binding to TnC, with subsequent transition to an open state when cross bridges bind to actin. When Ca²⁺ binds to TnC, alterations in interactions among thin filament proteins are thought to release the thin filament from the blocked state and to promote the transition of weakly bound cross bridges to a strongly bound, force-generating state. The impairment in function caused by a low level of expression of TnT_trunc can be explained as an inability of those regions of the thin filament containing cTnT_trunc to completely assume the blocked state in the absence of Ca²⁺, which could result in incomplete relaxation. An increased number of strongly bound cross bridges at low levels of Ca²⁺ activation would also be expected to accelerate the rate of force redevelopment because of the greater activating affects of cross bridges already bound to the thin filament. This model implies that incomplete inactivation of cTnT_trunc thin filaments is most likely due to an incomplete return of Tm to the fully blocked position in the absence of Ca²⁺.

Effects of NEM-S1 on Ca²⁺ sensitivity of force and cooperativity in activation of force. In this study, NEM-S1 was used to assess the effects of strong-binding cross bridges on thin filament activation in WT and cTnT_trunc myocardium. As previously shown in rat myocardium (6), NEM-S1 slightly increases Ca²⁺ independent tension at pCa 9.0, a phenomenon that was similar in WT and cTnT_trunc myocardium. Furthermore, NEM-S1 markedly increased the Ca²⁺ sensitivity of force in both WT and cTnT_trunc myocardium, shifting the force-pCa relationships to lower [Ca²⁺] by similar amounts and having similar effects on WT and transgenic myocardium. Thus, although the cTnT_trunc mutation increased the Ca²⁺ sensitivity of force, it had no obvious effects on the cooperative activation of steady-state isometric force by strong-binding cross bridges.

Effects of NEM-S1 on rate constant of force redevelopment. Earlier studies from this laboratory (6) showed that NEM-S1 accelerates the rate of force development in rat skinned myocardium, presumably due to increased activation of the thin filament as a consequence of greater numbers of cross bridges bound to actin. In the present work, NEM-S1 increased k_tr in both cTnT_trunc and WT myocardium but had greater effects in cTnT_trunc myocardium. In both WT and transgenic myocardium, the NEM-S1-induced acceleration of force development at submaximal [Ca²⁺] reduced the activation dependence of k_tr. NEM-S1 had no effect on maximal k_tr (measured at pCa 4.5) and increased k_tr at very low [Ca²⁺] to maximal values (Fig. 4). The difference in NEM-S1-induced effects on k_tr was observed only at intermediate levels of activation, where k_tr was increased to values less than maximal with greater increases in k_tr in cTnT_trunc myocardium.

In vitro studies of thin filament proteins in the presence of TnT_trunc suggest that Tm is held less tightly by Tn in its inhibitory position (3), thus reducing steric hindrance of myosin binding to actin. Results from the present study showing reduced cooperativity of activation and accelerated cross-bridge kinetics during submaximal activation of TnT_trunc myocardium support this view. Likewise, the greater activating effects of NEM-S1 on cross-bridge kinetics in TnT_trunc myocardium can be explained in terms of increased likelihood of cross-bridge binding at low levels of activation.

Functional significance of cTnT_trunc mutation. The primary function of the thin filament regulatory strand is to inhibit myosin binding in the absence of Ca²⁺ and to relieve this inhibition in the presence of Ca²⁺. Several published reports show that incorporation of cTnT_trunc into the thin filament impairs the ability of the regulatory strand to completely inhibit myosin cross-bridge cycling at very low Ca²⁺ or in its absence (3, 14, 18). Results from the present study support this view. Furthermore, incorporation of cTnT_trunc into the thin filament appears to accelerate cross-bridge cycling at submaximal Ca²⁺ both by increasing the responsiveness of thin filaments to the activating effects of Ca²⁺ and strong-binding cross bridges and by increasing the rate of cross-bridge interaction with actin. Together, our results could account for deficits in global ventricular function, including incomplete diastolic relaxation, which leads to decreased ventricular filling and a lower ejection fraction due to impaired systolic function (19).

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. E. Stelzer, Dept. of Physiology, Univ. of Wisconsin School of Medicine, 1300 Univ. Ave., Madison, WI 53706 (E-mail: stelzer{at}physiology.wisc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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				J. E. Stelzer and R. L. Moss Contributions of Stretch Activation to Length-dependent Contraction in Murine Myocardium J. Gen. Physiol., October 1, 2006; 128(4): 461 - 471. [Abstract] [Full Text] [PDF]

				J. E. Stelzer, J. R. Patel, and R. L. Moss Acceleration of Stretch Activation in Murine Myocardium due to Phosphorylation of Myosin Regulatory Light Chain J. Gen. Physiol., August 28, 2006; 128(3): 261 - 272. [Abstract] [Full Text] [PDF]

				J. E. Stelzer, S. B. Dunning, and R. L. Moss Ablation of Cardiac Myosin-Binding Protein-C Accelerates Stretch Activation in Murine Skinned Myocardium Circ. Res., May 12, 2006; 98(9): 1212 - 1218. [Abstract] [Full Text] [PDF]

				J. E. Stelzer, L. Larsson, D. P. Fitzsimons, and R. L. Moss Activation Dependence of Stretch Activation in Mouse Skinned Myocardium: Implications for Ventricular Function J. Gen. Physiol., January 30, 2006; 127(2): 95 - 107. [Abstract] [Full Text] [PDF]

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