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Am J Physiol Heart Circ Physiol 286: H1785-H1792, 2004. First published December 23, 2003; doi:10.1152/ajpheart.00170.2003
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Augmented systolic response to the calcium sensitizer EMD-57033 in a transgenic model with troponin I truncation

David G. Soergel,1,* Dimitrios Georgakopoulos,2,* Linda B. Stull,1 David A. Kass,2 and Anne M. Murphy1

Departments of 1Pediatrics and 2Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Submitted 21 February 2003 ; accepted in final form 18 December 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Myocardial stunning is a form of acute reversible cardiac dysfunction that occurs after brief periods of ischemia and reperfusion. In several animal models, stunning is associated with proteolytic truncation of troponin I (TnI). Mice expressing the same proteolytic TnI fragment [TnI-(1–193)] demonstrate cardiac depression with a decreased maximal calcium-activated tension. We therefore hypothesized preferential improvement in mice expressing TnI-(1–193) treated with the calcium-sensitizing drug EMD-57033. TnI-(1–193) and nontransgenic myofibrils exhibited significant sensitization to calcium in Mg-ATPase assays after EMD-57033 exposure. However, only transgenic myofibrils exhibited an increase in maximal activity (P = 0.023). EMD-57033 also increased maximal calcium-activated force in TnI-(1–193) muscle, such that it was comparable to nontransgenic cardiac muscle. EMD-57033 enhanced in vivo systolic function modestly in controls but had a marked effect in transgenic mice, with an almost threefold greater leftward shift of the end-systolic pressure-volume relation (P = 0.0005). These data indicate a targeted efficacy of EMD-57033 in offsetting the contractile defect in TnI-(1–193) mice, and this may have therapeutic implications in models displaying this myofilament defect.

myocardial stunning; inotropic agents; contractile function; heart failure

MYOCARDIAL STUNNING IS A FORM of acute reversible cardiac dysfunction that occurs after brief periods of ischemia and reperfusion (2, 3). Although the pathophysiology of stunning is multifactorial, evidence has accumulated that partial proteolysis of troponin I (TnI) can play a role (10, 28, 46, 48). A similar TnI truncation has been observed in the myocardium of humans undergoing cardiac surgery for ischemic disease and in humans with end-stage ischemic cardiomyopathy (29, 31, 33). TnI proteolysis is not observed in all models of myocardial stunning, however, particularly in regional models of stunning in dogs and pigs (8, 27, 43). To further define the pathophysiological consequences of this truncation, we generated transgenic mice expressing a mutated TnI that mimicked the major proteolytic fragment demonstrated in animal models of cardiac stunning (31). Hearts from these animals displayed depressed systolic function and decreased maximal calcium-activated tension, suggesting possible targeted responsiveness to a calcium-sensitizing agent. Regardless of its generalizability to the pathophysiology of myocardial stunning, the model is valuable, inasmuch as it reflects contractile dysfunction associated with altered regulatory thin-filament proteins.

The present study therefore addressed whether the molecular defect in transgenic TnI-(1–193) mice is preferentially amenable to a calcium sensitizer. We employed the thiadiazinone compound EMD-57033, which influences troponin C (TnC), to alter myofilament protein interaction and augment force generation (40). EMD-57033 has been shown to improve systolic function in in vivo and in vitro models of heart failure (6, 22, 36) and enhances diastolic function in vivo (5, 37). We provide novel evidence that the TnI-(1–193) mutation is preferentially sensitive to this agent.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Transgenic mouse model. Transgenic mice in which cardiac expression of the major proteolytic product of TnI [TnI-(1–193)] was observed in stunned myocardium were generated as described elsewhere (31). Transgene status was assessed by polymerase chain reaction from genomic DNA prepared from tail clips using the Gentra Puregene kit (Minneapolis, MN) and transgene-specific oligonucleotides. Nontransgenic (NTG) control mice were littermates of transgenic mice or were obtained from the same parental strain (C57BL/6) and matched for age.

Myofibrillar Mg-ATPase activity measurement. Myofibrils were prepared from cardiac ventricle as described previously (32) with careful use of protease inhibitors. Assays were performed using incubation conditions established by varying the total concentration of metals, salts, and ligands and maintaining ionic strength using stability constants compiled by Fabiato (7); assays were performed at pH 7.0 with 50 mM imidazole, 50 mM KCl, and 2 mM MgATP. Inorganic phosphate liberation was measured using a microtiter plate version of the standard assay as described by Rarick et al. (35). Protein concentration was determined by a variation of the Lowry method (Bio-Rad Laboratories, Hercules, CA). In the final assay conditions, myofibrillar protein concentration was diluted in buffer to be 0.2 mg/ml in the assay. The protein concentration of the myofibrillar stock used in the assay was then determined again for protein concentration for final calculations. Mg-ATPase activity was then calculated in nanomoles of inorganic phosphate liberated per milligram of myofibrillar protein per minute. EMD-57033 (Merck, Darmstadt, Germany) was initially solubilized in DMSO and then diluted 1:100. Control and EMD-57033 samples were preincubated with 1% DMSO alone or with EMD-57033 + 1% DMSO for 10 min on ice.

Force-calcium dependence in skinned trabeculae. Steady-state force development and calcium sensitivity were measured on Triton-skinned trabeculae. Harvesting and mounting of trabeculae were performed as described elsewhere (41). Average dimension was ~200 x 100 µm. Contractile properties were recorded to ensure muscle viability. Complete skinning was verified by visual inspection. Force-calcium relations were studied by exposing the muscle to increasing calcium concentrations. After each activation, muscles were bathed in relaxing solution for 5 min before the next activation at increased calcium concentration. After the initial curve was constructed, muscles were exposed to EMD-57033 in 1% DMSO as described above. The drug was then washed off, and curves were reassessed after 30 min. Control studies were also performed in both muscle types exposed to 1% DMSO and confirmed no effect on force or calcium sensitivity of the vehicle alone (data not shown).

In vivo ventricular function studies. Cardiac function was determined using a miniaturized conductance-micromanometer catheter to derive pressure-volume relations, as previously described (12). Mice were induced with methoxyflurane anesthesia and then injected intraperitoneally with urethane (800–1,000 mg/kg), morphine (1–2 mg/kg), and etomidate (5–10 mg/kg). Supplemental doses (20% total volume) were provided if animals became responsive to tail pinch as assessed by a blood pressure increase. Vascular access was obtained via the jugular vein, and animals were ventilated via a tracheotomy using a custom ventilator. A limited thoracotomy was performed, and the catheter was advanced through the left ventricular (LV) apex so that the electrodes lay just within the LV cavity and its distal tip was in the aortic root. Measures of chamber systolic and diastolic function were obtained from resting pressure-volume loops or families of loops generated by brief obstructions of inflow due to inferior vena caval compression. Data were assessed under baseline conditions and after intravenous administration EMD-57033 at 0.4 mg·kg–1·min–1 dissolved in 1,2-propanediol on the basis of a prior canine protocol (35). The drug was administered for 20 min, with the fluid volume was restricted to ~2 µl/min, during which time a steady-state change in ventricular function was noted. Data are provided after this steady state was achieved. Absolute LV volume was calibrated with an ultrasonic flow probe placed around the descending aorta to independently assess cardiac output and with the saline calibration method (1, 11) to derive the parallel conductance. Parameters included heart rate, LV end-systolic and end-diastolic pressures and volumes, rates of maximum pressure rise (dP/dtmax) and decline (dP/dtmin), time constant of relaxation ({tau}) (50), maximal ventricular power normalized to chamber preload (Pmax/EDV, where EDV is end-diastolic volume), ejection fraction, and peak diastolic filling rate (PFR) normalized to preload (PFR/EDV).

Positive inotropic agents typically shift the end-systolic pressure-volume relation (ESPVR) leftward with an increase in slope. However, the ESPVR values in the present study were often curvilinear, limiting the interpretation of a single linear slope. To better quantify the leftward ESPVR shift, we therefore measured the net increase in pressure-volume area within a physiological pressure range due to the leftward ESPVR displacement. Baseline and post-EMD-57033 ESPVR were fit to logarithmic nonlinear relations (18), and the area between relations spanning end-systolic pressures from 70 to 110 mmHg was numerically integrated (areaEes, where Ees is end-systolic elastance).

Statistical analysis. To characterize the relation of Mg-ATPase activity and force to calcium concentration, the data from each individual preparation were fitted to the Hill equation as follows: Vx – Vmin = (Vmax – Vmin)[Cax]nH/([Ca50]nH + [Cax]nH), where Vx is measured activity, Vmin is basal activity, Vmax is maximal activity, [Cax] is calcium concentration at individual data points in the assay, [Ca50] is calcium concentration required for half-maximal activity, and nH is the Hill coefficient. To fit curves, the measurement at the highest calcium concentration for the Mg-ATPase, 10–4.875 M, was extrapolated to 10–3 M; however, data reported in Table 1 are actual measurements at 10–4.875 M. Paired and unpaired t-tests were used to compare calculated [Ca50], maximal and minimal force, and nH for the skinned trabeculae studies. Two-way repeated-measures analysis of variance was used to determine significant drug effect within and between transgenic and NTG groups for minimal and maximal activity, [Ca50], and nH. Hemodynamic parameters were analyzed by comparing baseline values between the genotypes and by comparing the changes in response to the drug administration between the genotypes using an unpaired Student's t-test. P < 0.05 was considered significant.


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  		Table 1. Effect of EMD-57033 on transgenic TnI-(1-193) and NTG myofibrillar Mg-ATPase activities

 

Animal care. The investigation conforms with the Guide for the Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 85-23, Revised 1996], and the use of animals in this study was approved by the institutional committee.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Biochemical studies. Mg-ATPase activities were determined at baseline and after exposure to EMD-57033 at two doses (Table 1 and Fig. 1). The NTG and TnI-(1–193) mice displayed similar minimum Mg-ATPase, and in both groups there was a significant increase with the drug. However, the overall drug effect did not differ between genotypes. Maximal Mg-ATPase activities were slightly lower in the TnI-(1–193) than in the NTG mice (Table 1), although this difference was not statistically significant. Significantly lower maximal Mg-ATPase activity in the TnI-(1–193) mice was noted in a previous study (21). The maximal Mg-ATPase activity only increased in TnI-(1–193) myofibrils, with a significant drug effect by repeated-measures analysis (P = 0.023); there was no significant increase in the NTG group. This suggests that EMD-57033 might have a specific effect on maximal activation in the transgenic mice.



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  		Fig. 1. Mg-ATPase activity vs. calcium concentration in nontransgenic (NTG) mice (A) and mice expressing troponin I-(1–193) [TnI-(1–193), B] at baseline and with 10 and 30 µM EMD-57033. Values are means ± SE.

 

In controls, EMD-57033 resulted in a significant sensitization of Mg-ATPase activity to calcium in a dose-dependent manner, with a shift in [Ca50] from 2.34 ± 0.20 µM at baseline to 0.51 ± 0.07 µM at 10 µM EMD-57033 and 0.16 ± 0.02 µM at 30 µM EMD-57033 (P = 0.0001). Likewise, TnI-(1–193) myofibrils exhibited an increase in calcium sensitivity in response to EMD-57033, resulting in end points similar to those in NTG mice. However, because the TnI-(1–193) myofibrils were slightly sensitized at baseline, the overall magnitude of shift was less in the transgenic group. Thus there was a significant overall difference in drug effect between the genotype groups as determined by multivariate repeated-measures analysis (P = 0.014). Myo-fibrillar sensitization of Mg-ATPase to [Ca50] at baseline in TnI-(1–193) mice compared with NTG mice has also been recapitulated with recombinant truncated TnI compared with wild-type recombinant TnI (9). This contrasts with the desensitization of tension to calcium in the TnI-(1–193) hearts and implies a decreased economy of tension development at submaximal calcium.

Cooperativity as measured by nH was not significantly changed with EMD-57033, and there was no significant difference between genotypes for drug effect.

In vitro skinned muscle studies. Baseline maximal force (Fig. 2) was lower in the muscle from TnI-(1–193) than from NTG hearts: 37.9 ± 5.6 vs. 83.5 ± 13.1 mN/mm2 (P = 0.04). [Ca50] values were comparable in the muscle from TnI-(1–193) and NTG mice at baseline (0.61 ± 0.30 vs. 0.53 ± 0.05 µM) in these steady-state measurements. nH was very similar between the groups [2.1 ± 0.3 and 3.2 ± 0.4 for TNI-(1–193) and NTG, respectively] and did not change significantly with EMD-57033. With addition of EMD-57033, the responses differed between hearts from mice expressing TnI-(1–193) and NTG controls. The NTG muscle primarily demonstrated a sensitization of the force-calcium curve (to 0.27 ± 0.12 µM, P = 0.03) and a borderline decrease in maximal force (P = 0.04). The maximal force in the TnI-(1–193) transgenic muscle rose such that it was comparable to that in the NTG muscle: 67.5 ± 22.6 and 65.5 ± 13.4, respectively. [Ca50] after EMD-57033 did not shift significantly in the TnI-(1–193) transgenic muscle (to 0.33 ± 0.3, not significant).



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  		Fig. 2. Force-calcium relations in skinned fibers from NTG (A) and TnI-(1–193) (B) mice before () and after ({blacksquare}) exposure to 30 µM EMD-57033. Values are means ± SE. Calculated forces, Hill coefficients, and calcium concentration required for half-maximal activity are described in MATERIALS AND METHODS.

 

In vivo studies: baseline hemodynamics. Baseline cardiac physiology of the TnI-(1–193) hearts has been previously described (21, 31), and we observed similar features in the present experiments. The one exception is that the present cohort did not display significant chamber enlargement, likely because of their younger age. This allowed examination of the effects of TnI-(1–193) and EMD-57033 without the confounding factor of chronic LV remodeling. Importantly, the TnI-(1–193) and NTG mice had similar baseline heart rates (575 ± 43 and 534 ± 63 beats/min, respectively), and neither mean heart rate nor end-diastolic LV changed significantly in either group with drug treatment.

Pertinent hemodynamic findings from this study are provided in Figs. 3 and 4. Basal systolic function was reduced in TnI-(1–193), as reflected in a lower dP/dtmax and Pmax/EDV and a trend to lower ejection fraction. Diastolic function was also abnormal, with dP/dtmin, {tau}, and normalized peak filling rate significantly depressed in the transgenic animals. These findings support earlier data (31).



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  		Fig. 3. Systolic function of NTG (solid bars, n = 8) and TnI-(1–193) (open bars, n = 7) mice before (A, C, and E) and the amount of change after (B, D, and F) treatment with EMD-57033. A and B: rate of maximum pressure rise (dP/dtmax); C and D: maximum ventricular power normalized to end-diastolic volume (Pmax/EDV); E and F: ejection fraction. Values are means ± SE. TnI-(1–193) mice have significantly reduced systolic function, as determined by dP/dtmax and the relatively load-independent index Pmax/EDV, and significantly greater improvements in systolic function after treatment.

 


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  		Fig. 4. Diastolic function of NTG (solid bars, n = 8) and TnI-(1–193) (open bars, n = 7) mice before (A, C, and E) and the amount of change after (B, D, and F) treatment with EMD-57033. A and B: rate of maximum pressure decline (dP/dtmin); C and D: time constant of relaxation ({tau}); E and F: peak filling rate normalized for EDV. TnI-(1–193) mice have significantly reduced diastolic function, as determined by dP/dtmin, {tau}, and peak filling rate/EDV, which is relatively load independent. In vivo diastolic parameters are improved after EMD-57033, and improvement is not significantly different in TnI-(1–193) mice.

 

In vivo response to EMD-57033. EMD-57033 improved systolic function in NTG and TnI-(1–193) hearts, but the effect was markedly amplified in the transgenic animals. This is illustrated in Fig. 3, B, D, and F, which shows changes in each parameter after EMD-57033 infusion. Increases in systolic function were near or more than twofold higher in TnI-(1–193) mutants than in controls. Figure 5A displays representative pressure-volume relations at baseline and after EMD-57033 administration in NTG and TnI-(1–193) mice. In NTG mice, there was a small but significant leftward shift of the ESPVR (increase in areaEes, P < 0.02), accompanied by an increase in loop width (stroke volume) and area (stroke work). However, these changes were markedly augmented in the TnI-(1–193) heart, which demonstrated a substantial leftward shift of the ESPVR (P < 0.00001) and an accompanying increase in cardiac output. The threefold disparity in the areaEes increase was highly significant (P = 0.0005; Fig. 5B).



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  		Fig. 5. A: representative pressure-volume relations of the left ventricle in TnI-(1–193) (TG) and NTG mice before and after administration of EMD-57033 (EMD). A family of pressure-volume curves was generated by brief obstructions of inflow, and end-systolic pressure-volume relation was determined for curves. B: area of end-systolic elastance (areaEes) between pre- and posttreatment curves in NTG (solid bar) and TnI-(1–193) mice as described in MATERIALS AND METHODS. Shift in areaEes curve after EMD-57033 is ~3-fold greater in TnI-(1–193) mice.

 

Baseline diastolic function was abnormal in the TnI-(1–193) heart, raising concerns about potential further deterioration of diastole by EMD-57033 due to increased cross-bridge formation (Mg-ATPase activity of myofibrils) at diastolic calcium levels (14). However, diastolic function as indexed by relaxation rate and peak filling rates improved in NTG and TnI-(1–193) hearts (Fig. 4, B, D, and F). There was a tendency for this effect to be even augmented in TnI-(1–193) hearts; however, these changes fell short of significance.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The major novel finding of this study is that the calcium sensitizer EMD-57033 is potently effective in acutely restoring systolic function in a mouse model harboring a proteolytic truncation of TnI, a model of a myofilament protein defect causing contractile dysfunction. This augmentation occurred to a substantially greater extent in transgenic hearts than in controls, suggesting a specific capacity of this agent to affect the underlying defect. Despite basal diastolic dysfunction in TnI-(1–193) hearts, diastolic function was enhanced by EMD-57033, consistent with prior in vivo studies (5, 37). These data support a particular utility of EMD-57033 (and potentially similar calcium sensitizers) to treat myocardial dysfunction associated with TnI-proteolytic fragmentation.

Unlike some calcium sensitizers, EMD-57033 does not appear to directly influence calcium binding to TnC. Li et al. (25) and Wang et al. (49) demonstrated in elegant NMR studies that EMD-57033 binds to the COOH-terminal domain of TnC, in a region in which an NH2-terminal region of TnI binds in competitive fashion [cardiac TnI-(34–71)]. Wang et al. proposed that, by altering binding of the NH2-terminal region of TnI, EMD-57033 may shift the balance to favor TnC interactions with the so-called inhibitory peptide of TnI [TnI-(128–147)], resulting in decreased affinity of TnI for actin. The removal of this inhibitory effect would facilitate actin-myosin interaction and tension development, as initially proposed by Solaro et al. (40). There is some evidence to support a role for EMD-57033 in alterations in the cross-bridge kinetics or numbers of cross bridges formed (38, 47); however, this may depend on the species and whether the data were obtained in cardiac or skeletal muscle (26). Others have suggested that EMD-57033 may directly influence actin-myosin interactions, leading to increased unitary force per cross bridge (4, 23, 34).

The TnI-(1–193) mice were initially created to mimic loss of 17 amino acid residues from the COOH terminus of TnI, a mutation that recapitulates a proteolytic modification observed with myocardial stunning in a rat model of global ischemia-reperfusion (10, 28, 46) and in human myocardium with ischemic disease (29, 31). The most striking cellular characteristic of the TnI-(1–193) transgenic model is a decreased maximal calcium-activated force, associated at the chamber level with depressed systolic function. This defect in tension development can be directly attributed to the truncated TnI molecule, inasmuch as recent studies using an in vitro motility sliding assay have shown the recombinant truncated TnI to be sufficient for reducing maximal activation of tension development (9).

Precisely how the EMD-57033 interacts with the defect in the TnI-(1–193) mice is not clear. The function of the extreme COOH-terminal region of TnI is not well understood, although binding assays indicate that the truncated TnI does not have altered interactions with actin-tropomyosin, TnC, or troponin T (9). Myofibrils from TnI-(1–193) mice have a lower absolute shift of [Ca50] than those from control mice when exposed to EMD-57033. Therefore, it does not appear that the effect on [Ca50] can explain the more potent inotropic effect of EMD-57033 on in vivo ventricular function. Thus the underlying defect in maximal activation is likely what is alleviated by EMD-57033, resulting in the greater inotropic effect in the TnI-(1–193) mice. Given the ability of EMD-57033 to directly influence actin-myosin interactions, it is speculated that a direct effect of EMD-57033 in stabilizing the force-generating cross-bridge state may occur in this model. It is notable that this effect may occur independently of the effect on calcium sensitization, as has been discussed by Palmer et al. (34). Whether EMD-57033 acts on the kinetics of the cross-bridge formation or on force developed per cross bridge or both, the present data demonstrate that EMD-57033 is capable of over-coming, at least in part, the defect in maximal activation associated with the TnI-(1–193) mutation augmenting systolic function to a greater extent in the mutant hearts.

EMD-57033 has been demonstrated to be effective in treating cardiac dysfunction in a number of animal models, including regional and global models of cardiac stunning, tachycardia-induced cardiac failure, volume overload-induced failure, and myocardial infarction-induced cardiac dysfunction (5, 6, 16, 36, 42, 45). In these models, and unlike the present data, EMD-57033 was similarly effective in augmenting systolic function in the pathological model, as it was in parallel studies of control muscles, regional ventricular segments, or whole hearts. Some studies of regional myocardial stunning in the pig have also found similar responses in control and depressed myocardium (5). However, the pathophysiology of stunning in the pig appears to principally relate to altered calcium transients, rather than myofilament defects (20). On the other hand, one prior study using an alternative sensitizer reported enhanced responsiveness in stunned myocardium (39). However, substantial differences in heart rate with this agent may have contributed to this difference. The present data support the concept that drugs such as EMD-57033 are particularly effective in hearts with an intrinsic TnI myofilament defect.

The present studies recapitulate disparities that have been previously noted between in vitro assays using EMD-57033 (14, 40), in which the sensitizer substantially elevates resting muscle tone, diastolic pressures, or Mg-ATPase activity at low (diastolic) calcium levels, and in vivo data, in which net diastolic function is improved (5, 37). There are some likely mechanisms for this disparity. First, although the rate of pressure relaxation in vivo is influenced by calcium handing, it is also affected by the extent of cardiac ejection (EDV). The latter increases the elastic restoring force most likely linked to titin compression (13), and this recoil improves pressure decay. Thus models in which muscle length or heart volume is constant have generally showed reduced diastolic function (15, 44), whereas those with ejection revealed improved function (5, 37). Also there are differences between steady-state and dynamic calcium-myofilament interactions that might produce effects in vitro that are not observed in vivo. For example, transgenic mice that express slow skeletal TnI have cardiac muscle with higher steady-state calcium sensitivity than NTG controls; however, cardiac muscle from these transgenic mice has faster relaxation rates (19). These disparities stress the importance of assessing inotropic drug effects in a variety of in vivo and in vitro models.

The precise cellular physiology of stunning in human hearts after global or regional ischemia-reperfusion is not fully understood. However, previous work on human tissue samples and human sera suggests that the TnI molecule is capable of being proteolyzed in a fashion similar to that noted in rodent models of stunning (24, 29, 31). In addition, there are humans with genetically mediated dilated cardiomyopathies due to inherited thin filament mutations (17, 30), and it is at least plausible that systolic function in these myopathies might improve with a calcium sensitizer.

In summary, EMD-57033 has been demonstrated to alleviate the molecular defect in activation as well as the in vivo systolic dysfunction in a transgenic model that mimics aspects of myocardial stunning. This effect has been achieved without a negative impact on diastolic function. This class of calcium sensitizer may offer specific benefit in some forms of human myocardial dysfunction.


   ACKNOWLEDGMENTS
 
We thank John Robinson for technical assistance.

GRANTS

This work was funded by National Heart, Lung, and Blood Institute Grants RO1 HL-63038 (A. M. Murphy), F32 HL-10401 (D. G. Soergel), and PO1 HL-59408 (D. A. Kass) and a grant from the Mid-Atlantic Affiliate of the American Heart Association (A. M. Murphy).


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. M. Murphy, Ross 1144, Johns Hopkins Univ. School of Medicine, 720 Rutland Ave., Baltimore, MD 21205 (E-mail: murphy{at}jhmi.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.

* D. G. Soergel and D. Georgakopoulos contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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