Intracellular Manganese Ions Provide Strong T 1 Relaxation in Rat Myocardium

Transcription

1 Magnetic Resonance in Medicine 52: (2004) Intracellular Manganese Ions Provide Strong T 1 Relaxation in Rat Myocardium Wibeke Nordhøy, 1 Henrik W. Anthonsen, 2 Morten Bruvold, 1 Heidi Brurok, 1 Sissel Skarra, 1 Jostein Krane, 2 and Per Jynge 1 * The efficacy of manganese ions (Mn 2 ) as intracellular (ic) contrast agents was assessed in rat myocardium. T 1 and T 2 and Mn content were measured in ventricular tissue excised from isolated perfused hearts in which a 5-min wash-in with 0, 30, 100, 300, or 1000 M of Mn dipyridoxyl diphosphate (MnDPDP) was followed by a 15-min wash-out to remove extracellular (ec) Mn 2. An inversion recovery (IR) analysis at 20 MHz revealed two T 1 components: an ic and short T 1-1 ( ms), and an ec and longer T 1-2 ( ms). Intensities were about 68% and 32%, respectively. Tissue Mn content correlated particularly well with ic R 1-1. A two-site water-exchange analysis of T 1 data documented slow water exchange with ic and ec lifetimes of 11.3 s and 7.5 s, respectively, and no differences between apparent and intrinsic relaxation parameters. Ic relaxivity induced by Mn 2 ions in ic water was as high as 56 (s mm) 1, about 8 times and 36 times higher than with Mn 2 aqua ions and MnDPDP, respectively, in vitro. This value is as high as any reported to date for any synthetic protein-bound metal chelate. The increased rotational correlation time ( R ) between proton and electron (Mn 2 ) spins, and maintained inner-sphere water access, might make ic Mn 2 ions and Mn 2 -ion-releasing contrast media surprisingly effective for T 1 -weighted imaging. Magn Reson Med 52: , Wiley-Liss, Inc. Key words: manganese; MnDPDP; heart; T 1 relaxation; R 1 relaxivity Recent studies (1 3) have shown that divalent manganese ions (Mn 2 ) are promising intracellular (ic) contrast agents, and that Mn 2 -releasing contrast media may be used for cardiac MRI in ischemic heart disease (4). The key factors in the potential success of these agents are that Mn-based MRI (MnMRI) utilizes physiological pathways, and Mn 2 ions are tightly bound to both extracellular (ec) and ic proteins. Thus, cardiac cell uptake of Mn 2 occurs in competition with the calcium ion (Ca 2 ) (5), and Mn- MRI may mirror slow Ca 2 channel function (2). Competition with Ca 2 for cell efflux is less effective, since it may lead to cell Mn 2 retention for hours, as well as to the possibility of delayed MRI. After uptake, Mn 2 ions exert paramagnetic properties inside cardiac cells, and, as we recently demonstrated (3), proton relaxation is greatly enhanced compared to Mn 2 ions in vitro most probably due to extensive binding to slowly tumbling macromolecules (6). These unique endogenous properties of Mn 2 ions may be exploited in both basic research and future clinical diagnostic techniques. The development of MnMRI has been hampered by the notion that Mn 2 ions are cardiotoxic (7,8), since they may inhibit Ca 2 channels in the cardiac cell membrane. However, recent studies have shown that the fear of cardiotoxicity in this case is largely unfounded (9 11). Thus, while Mn 2 entry in cardiomyocytes undoubtedly occurs via Ca 2 channels (2,5,9,12), an inhibition of Ca 2 influx that initiates cardiac contraction will not occur before ec [Mn 2 ] exceeds 25 M (1,3). However, extensive plasma protein binding ensures that only a very few M ofmn 2 may exist in the free form in the blood pool or the interstitial space (13,14). We recently studied ic proton relaxation in rat myocardium with MnCl 2 present in the perfusate of isolated rat hearts (3). Relaxography performed after wash-in plus wash-out experiments revealed slow water exchange in the excised nonperfused cardiac tissue. Biexponential T 1 behavior dominated, and revealed a short T 1-1 and a longer T 1-2. However, both components responded to Mn 2 enrichment. More importantly, we found that Mn 2 -induced relaxivity in the ic water compartment was close to one order of magnitude higher than that measured in vitro with free Mn 2 ions in deionized water. In the present isolated rat heart study, we repeated our relaxography analyses of excised cardiac tissue after Mn 2 enrichment, but this time with the aim of examining cellular T 1 relaxation properties after wash-in and wash-out experiments using Mn-dipyridoxyl-diphosphate (MnD- PDP). This agent is present in a single commercial Mnbased contrast medium (Teslascan ; Amersham Health), which is approved for liver MRI. After MnDPDP is infused intravenously, it undergoes two-way metabolism (15): transmetallation with plasma zinc releases Mn 2 ions, and dephosphorylation produces monophosphate (MnDPMP and ZnDPMP)- and phosphate (MnPLED and ZnPLED)- free metabolites. 1 Department of Circulation and Medical Imaging, Faculty of Medicine, Norwegian University of Science and Technology, Trondheim, Norway. 2 Department of Chemistry, Faculty of Natural Sciences, Norwegian University of Science and Technology, Trondheim, Norway. Grant sponsors: Norwegian University of Science and Technology; Research Council of Norway; Amersham Health. *Correspondence to: Professor Per Jynge, Department of Circulation and Medical Imaging, Faculty of Medicine, Norwegian University of Science and Technology, Medisinsk Teknisk Forskningssenter, N-7489 Trondheim, Norway. per.jynge@medisin.ntnu.no Received 15 August 2003; revised 6 April 2004; accepted 22 April DOI /mrm Published online in Wiley InterScience ( Wiley-Liss, Inc. 506 MATERIALS AND METHODS In Vitro Relaxography Experiments Baseline information about proton relaxation was obtained with MnDPDP and MnCl 2 dissolved in deionized and N 2 - equilibrated water at 37 C (ph 7.0), and with relaxography (T 1 and T 2 ) as described below. The relaxation rate constants R 1 1/T 1 and R 2 1/T 2 were calculated by M z t M e 1/T1 [1]

2 T 1 Relaxation by Intracellular Manganese Ions 507 M xy t M 0 e 1/T2 [2] where M z (t) is the instantaneous longitudinal magnetization, M xy (t) is the instantaneous transversal magnetization, M 0 is its Boltzmann equilibrium value, is a correction factor for deviation from a perfect 180 pulse, T 1 is the longitudinal relaxation time, T 2 is the transversal relaxation time, and t is the time constant between the 180 and 90 pulses for T 1 and the echo time (TE) for T 2. Experimental Heart Model The current experiments followed guidelines set forth by the local ethics committee for animal research. Male Wistar rats ( g body wt.; N 24) were anesthetized by diethyl ether and heparinized (0.25 ml Heparin 1000 IU/ml i.v.). The hearts were rapidly excised and placed in cold (4 C) perfusion medium, and within 1 min they were mounted onto the aortic cannula of a modified Langendorff perfusion system (16). The system consisted of thermostated (37 C) reservoirs, perfusion lines, and heart chambers. Cardiac temperature (thermistor right ventricle) was maintained at 37.0 C 0.4 C. Throughout the experiments all of the hearts were perfused at a constant flow rate of 10 ml/min by means of flow-adjustable pumps (Alitea AB, Stockholm, Sweden). The hearts were subjected to an initial control perfusion period, perfusion with MnDPDP present (wash-in), and perfusion without MnDPDP (wash-out). The perfusate was Krebs-Henseleits bicarbonate buffer (17) containing (in mm) NaCl , NaHCO , KCl 4.7, KH 2 PO 4 1.2, MgSO 4 1.2, CaCl , and glucose During exposure to MnDPDP, we replaced phosphate and sulfate in the perfusate with chloride in order to prevent precipitation of Mn salts. At the end of the experiments, the left and right ventricular tissues were excised and blotted dry from extraneous water. Immediately thereafter, a proton relaxation analysis was performed and the tissues were weighed. Finally, the ventricular specimens were frozen in liquid nitrogen and freeze-dried. The dried specimens were also weighed to obtain dry-to-wet-weight ratios. Ex Vivo Relaxography Fresh ventricular specimens were placed in 10-mm NMR tubes. Measurements of T 2 followed by T 1 were started min after the end of the experiment and completed within 60 min by means of a 0.47 Tesla Bruker Minispec spectrometer (Bruker AG, Rheinstetten, Germany) at 37 C. We measured T 1 using the inversion recovery (IR) method, and collected at most 22 data point, four scans, and two duplicates. We measured T 2 using a Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence, and collected data points, four scans, and two duplicates. The data were fit to a two-component, nonlinear, least-square regression curve, with one short and one long relaxation component (T 1-1 and T 1-2,orT 2-1 and T 2-2 ) containing approximately 60 70% and 30 40% of the data points, respectively. The apparent relaxation rate constants (R 1-1, R 1-2, R 2-1, and R 2-2 ) and population fractions (p 01 and p 02 ) were calculated. Analysis of Relaxography Data T 1 and T 2 The regression equation for T 1 (below) is M z t M 0 p e 1/T1 1 p e 1/T1 2 [3] Equation [3] contains four variables (M 0, p 01, T 1-1, and T 1-2 ), is set to 1.0, and the sum of p 01 and p 02 equals one (or 100%). Similarly, the regression equation for T 2 (Eq. [4]) contains four variables (M 0, p 01, T 2-1 and T 2-2 ): M xy t M 0 p 01 e 1/T2 1 p 02 e 1/T2 2 [4] Relaxivity Relaxivity (r) is another parameter that describes the effectiveness of the contrast agent in reducing T 1 or T 2 as a function of the concentration (19,20). Physiological Parameters and Analysis A water-filled latex balloon (0.15 ml) placed in the left ventricle (LV) was connected to a pressure transducer to record LV pressures (LVP), LV developed (systolic-diastolic) pressure (LVDP), and heart rate (HR). Analog signals (including temperature) were amplified, digitally converted, and processed by a computer (Quadbridge and MacLab units and software; AD Instruments, London, UK). Mn Content Measurements The freeze-dried ventricular specimens were homogenized, and extracts were made in nitric acid. Measurements of Mn in tissue extracts (18) were performed with the use of a flame atomic absorption spectrophotometer (FAAS) (Spectr-AA-400, Varian), and tissue Mn contents were expressed in nmol/g dry weight (wt). R i 1 T i 1 T obs i 1 T i, i 1,2 [5] p where (1/T i ) obs is the observed relaxation rate constant (R i ) in the presence of a paramagnetic species, and (1/T i )isthe (diamagnetic) relaxation rate constant in the absence of a paramagnetic species. (1/T i ) p represents the additional paramagnetic contribution. In the absence of solute solute interactions, the solvent relaxation rate constants are linearly dependent on the concentration of the paramagnetic contrast agent ([CA]), which is the case for the standards (above) R i,obs R i r i CA, [6] r i is the relaxivity defined as the slope of this dependence in units of (s mm) 1 for in vitro experiments (20), and (s nmol/g dry wt.) 1 for ex vivo heart experiments.

3 508 Nordhøy et al. Experimental Groups and Design Five groups of hearts (N 4 6) were included in the ex vivo heart experiments. After 15 min control perfusion, hearts were subjected to a 5-min wash-in period with normal buffer (control) and 30, 100, 300, and 1000 M of MnDPDP. They were finally subjected to a 15-min washout period without MnDPDP. Expression of Results The results are all expressed as the mean and standard deviation (SD). A comparison between groups was performed with the use of a parametric one-way analysis of variance (ANOVA) test for k-independent samples (N 4 6), and the post-tukey test within groups. The regression analysis used is a nonlinear least-square method (Levenberg-Marquardt) (23). The correlation test used was the parametric Pearson correlation test. FIG. 1. In vitro dose-response relaxivity studies with MnCl 2 and MnDPDP dissolved in N 2 -degassed, deionized water with a ph of 7.0. The correlation coefficients ( ) are noted (P 0.05). a: Longitudinal (R 1 ) relaxation rate constants. With a linear curve fit approximation, the relaxivities expressed in (s mm) 1 were 6.9 for MnCl 2 and 1.6 for MnDPDP. The chemical structure of MnDPDP is depicted. b: Transversal (R 2 ) relaxation rate constants. With a linear curve fit approximation, the relaxivities expressed in (s mm) 1 were 37.5 for MnCl 2 and 1.8 for MnDPDP. Two-Site Water Exchange (2SX) Analyses The T 1 data were analyzed according to a 2SX model, as described in detail by Labadie et al. (21) and Landis et al. (22) for use with ec contrast agents, and as recently employed by us (3) for use with ic Mn 2 ions. In the former studies, a stable gadolinium (Gd 3 ) chelate was trapped in the ec space of various cell types. In the latter setting, the 2SX model was used to examine five intrinsic parameters (as defined in Refs. 21 and 22): R 1ec, the ec relaxation rate constant; r 1ic, the ic relaxivity; R 1ic0, the ic relaxation rate constant at [Mn] 0 nmol/g dry wt.; ic, the ic lifetime; and p ic, the ic population fraction. The apparent R 1-1, R 1-2, and p 01 data were accommodated by a simplex minimalization routine (23), which was modified so that we could lock selected parameters and keep them at constant values by varying the five intrinsic 2SX model parameters. To validate the results of ic and p ic, we generated a root mean square (RMS) plot (23) by varying the 2SX variables ic and p ic while holding the other variables constant. The resulting RMS errors were then plotted as a function of ic and p ic. RESULTS In Vitro Relaxography (Fig. 1) With MnDPDP ( M) in N 2 -degassed water at 37 C and 20 MHz, a one-component exponential regression analysis of T 1 and T 2 (Eqs. [1] and [2]) confirmed that the agent was dissolved in a homogenous solution. The corresponding relaxivities were 1.6 (s mm) 1 for r 1 and 1.8 (s mm) 1 for r 2 ( and 0.993, respectively). These values are close to or lower than reported literature values (at the same field and temperature): r (s mm) 1 and r (s mm) 1 (24), r (s mm) 1 and r (s mm) 1 (6), and r (s mm) 1 and r (s mm) 1 (25). In comparison, MnCl 2 presented higher relaxivity values (r (s mm) 1 and r (s mm) 1 ), as would be expected with fully dissociated Mn 2 ions. Cardiac Physiology and Mn Accumulation (Table 1) Concentrations of MnDPDP up to 300 M were well tolerated during exposure, but a minor LVDP depression was noted at Table 1 LVDP in % of Control Values and Tissue Content of Mn After Administration and Washout of MnDPDP for All the Experimental Groups* MnDPDP ( M) N Exposure LVDP (%) Recovery Mn content (nmol/g dry wt.) (14) 102 (11) 45 (9) (12) 110 (11) 46 (19) (8) 98 (4) 72 (16) a,b (8) 93 (11) 106 (23) a c (24) a d 84 (11) a 239 (34) a d *Exposure represents the last min of exposure to MnDPDP, and recovery represents the last min of recovery. The mean values (standard deviation) are expressed in % of absolute values obtained in the last min of control perfusion (n 4 6). The Mn content is expressed in nmol/g dry wt. a Significantly different from 0. b Significantly different from 30. c Significantly different from 100. d Significantly different from 300 M MnDPDP.

4 T 1 Relaxation by Intracellular Manganese Ions 509 FIG. 2. Longitudinal (T 1 ) relaxation recoveries of all ex vivo hearts under study with concentrations of 0, 30, 100, 300, and 1000 M of MnDPDP. The mean experimental data (*) were obtained by (a) one- and (b) two-component exponential regression analysis (solid lines). The results are given in the diagrams for each group (N 4 6). the end of the wash-out period in this group of hearts. Administration of 1000 M of MnDPDP caused a close to 50% reduction in LVDP during the exposure. The Mn content of the control hearts was 45 nmol/g dry wt, which is much in accordance with previous findings (1,3,9,26,27). Treatment with 30 M of MnDPDP did not raise tissue Mn, but 100, 300, and 1000 of M elevated Mn contents to 1.6, 2.4, and 5.3 times the control level, respectively. Relaxography of Excised Ventricular Tissue (Figs. 2 4, Tables 2 4) Longitudinal Relaxation Figure 2a and b present the recovery of magnetization during IR experiments for all groups of hearts. As shown by a detailed analysis of residuals (3), measured values (star symbols) differed from computed values (solid lines) when a monoexponential function was used (Fig. 2a). The mean T 1 values fell gradually from 953 ms in control hearts to about 413 ms in hearts treated with MnDPDP 1000 M. The use of a biexponential equation reduced residuals from about 7% to about 1%, and there was a closer match between measured and computed values for T 1 in both control and Mn 2 -treated hearts (Fig. 2b). As seen in Table 2, the two components T 1-1 and T 1-2 responded closely to the gradual tissue Mn 2 enrichment, with rising concentrations of MnDPDP during the wash-in phase. Accordingly, the ic T 1-1 values fell from about 650 ms in control hearts in a stepwise manner by 11% (30 M), 28% FIG. 3. Dose-response representation of all of the ex vivo hearts (N 24, two duplicates). The longitudinal relaxation rate constant R 1-1 is plotted vs. tissue Mn content. The intrinsic value (dotted line) is shown. The correlation coefficient ( ) is noted. FIG. 4. Dose-response representation of all of the ex vivo hearts (N 24, two duplicates). The transversal relaxation rate constant R 2-1 is plotted vs. tissue Mn content. The correlation coefficient ( ) is noted.

5 510 Nordhøy et al. Table 2 Longitudinal Relaxation Times (T 1 1, T 1 2 ) and Transversal Relaxation Times (T 2 1, T 2 2 ) After Administration and Washout of MnDPDP for All the Experimental Groups* MnDPDP ( M) N T 1 1 (ms) T 1 2 (ms) T 2 1 (ms) T 2 2 (ms) (20) 2712 (134) 62 (3) 1325 (229) (27) a 2536 (85) 59 (2) a 1163 (166) (37) a,b 1842 (523) a,b 56 (1) a,b 1083 (200) (51) a c 2049 (634) a 55 (3) a,b 1011 (328) a (26) a d 1042 (133) a d 46 (1) a d 527 (69) a d *The mean (SD) relaxation values are expressed in ms (N 4 6). a Significantly different from 0. b Significantly different from 30. c Significantly different from 100. d Significantly different from 300 M MnDPDP. (100 M), 36% (300 M), and 61% (1000 M). In parallel, the ec T 1-2 values were reduced below 2712 ms by 6.5% (30 M), 32% (100 M), 24% (300 M), and 62% (1000 M). The relative contributions (p 01 1 p 02 ) to magnetization recovery (Table 3), which on average were 68% (p 01 ) for ic T 1-1, and 32% (p 02 ) for ec T 1-2, indicate that these components represent ic and ec water protons, respectively (28). Figure 3 shows that there was a close correlation between tissue Mn content and the ic longitudinal relaxation rate constant R 1-1 ( 0.934). As shown in Table 2, there was also a clear tendency toward a reduction in T 1-2. When the calculated relaxivity value of ic r 1-1 of (s nmol/g dry wt) 1 was approximated to values per volume of water (assuming ratios for dry-to-wet wt of 1:5 and g-to-ml of 1:1), the apparent value was found to be equal to 56 (s mm) 1. 2SX Analysis The above T 1 and compartmentation data present apparent values and were subjected to a 2SX analysis (21,22) to reveal the intrinsic values. As shown in Table 4, however, there was a close concordance between apparent and intrinsic values for ic relaxivity (identical values for r 1-1 and r 1ic ) and ic population fractions (p 01 68%, p ic 59%). As expected, the excised ventricular tissue was dominated by slow water exchange (3,21,22), with ic and ec water lifetimes of 11.3 s and 7.5 s, respectively. Table 3 Longitudinal and Transversal Intracellular Population Fractions (p 01 ) After Administration and Washout of MnDPDP for All the Experimental Groups* MnDPDP ( M) N Longitudinal p 01 (%) Transversal p 01 (%) a 73 *The values are given in mean expressed in percent of the magnetization vector (M 0 ). a Significantly different from 300 M MnDPDP. Transversal Relaxation (Tables 2 and 3, Fig. 4) T 2 relaxation demonstrated two components: T 2-1 and T 2-2. For control hearts the T 2-1 and T 2-2 values were 62 and 1325 ms, respectively. No reductions were noted for perfusates of 30, 100, or 300 M of MnDPDP. With 1000 M of MnDPDP, T 2-1 and T 2-2 were reduced by 26% and 60%, respectively. The contributions to magnetization (p 01 and p 02 ) were 8.8% higher (ic) and lower (ec), respectively, than observed with the T 1 data. Figure 4 shows the relationship between tissue Mn content and R 2-1 ( 0.932). Transversal relaxation responded more weakly to tissue Mn enhancement compared to longitudinal relaxation. The apparent relaxivity value for r 2-1 was (s nmol/g dry wt) 1 or 130 (s mm) 1. DISCUSSION The present study confirms previous results from studies on cardiac physiology and tissue Mn 2 uptake and retention. It also provides new information relating to relaxography with two-component T 1 analyses and calculations of water exchange. Most importantly, this study documents that ic Mn 2 ions provide a particularly strong T 1 relaxation. Cardiac Physiology and Mn Content As previously shown in isolated hearts from rats (26) and guinea pigs (1), and confirmed by the current results, 300 M of MnDPDP is close to a threshold whereby the released Mn 2 ions might impede the flow through membrane channels of Ca 2 ions needed for cardiac contraction. This also coincides well with a parallel threshold of Table 4 Main Results From the 2SX Analysis* R 1ec (s 1 ) r 1ic (s nmol/g dry wt.) 1 R 1ic0 (s 1 ) ic (s) p ic (%) *The five intrinsic parameters (21, 22) are: R 1ec, the ec relaxation rate constant; r 1ic, the ic relaxivity; R 1ic0, the ic relaxation rate constant at [Mn] 0 nmol/g dry wt.; ic, the ic lifetime; and, p ic, the ic population fraction. ec was calculated from the following formula: ec ic * (1 p ic )/p ic. ec (s)

6 T 1 Relaxation by Intracellular Manganese Ions M during perfusion with free Mn 2 (MnCl 2 ) (3,9), as this latter agent raises tissue Mn content about 8 10 times more than MnDPDP (27). When conversion factors were applied, estimates of the fractional cardiac Mn 2 accumulation (residual Mn 2 after uptake and initial wash-out) vs. the total content of Mn 2 present in MnDPDP could be made. With perfusates of 100, 300, and 1000 M of MnDPDP, the fractional accumulation rates were 1.0, 0.8, and 0.7, respectively. This means that only a very limited transmetallation and Mn 2 release takes place during a single passage of MnDPDP through the coronary circulation. In a previous study (3) that used perfusate concentrations of MnCl 2 25 and 100 M, and the same wash-in and wash-out protocols used in the current study, the fractional accumulation of available Mn 2 was much higher (3.6 and 2.0%, respectively). Together these data amply demonstrate the uptake differences between an immediate Mn 2 ion-releaser like MnCl 2 and a slow Mn 2 ion-releaser like MnD- PDP, and the avidity of cardiomyocytes for Mn 2 uptake. Ex Vivo T 1 Relaxation Major Findings The present study showed that for analyzing T 1 data from excised cardiac tissue, it is more appropriate to use a biexponential than a monoexponential function. The two population fractions contained about 68% (p 01 ) and 32% (p 02 ) of measured data points (Table 3), and the following 2SX analysis revealed an ic population (p ic ) of 59% (Table 4). Since these values are very close to what has been established for ic ( 60%) and ec ( 40%) water compartmentation in excised and blotted rat ventricular myocardium (28), it is justifiable to regard p 01 and p 02 as representing ic and ec water, respectively. The two population fractions were present in all of the hearts, including controls. Both responded to Mn 2 enrichment, but the response was strong for ic R 1-1 and weaker for ec R 1-2. The reduction in ec T 1 (T 1-2 ) was mainly due to restricted exchange across the sarcolemma. This was borne out by the 2SX analysis of the T 1 data, which revealed long ic (11.3 s) and ec (7.5 s) proton lifetimes, signifying slow water exchange. The analysis confirmed that measured and apparent values were identical to intrinsic values for the ic relaxation component. The major finding, however, was the remarkably strong relaxivity of the ic Mn 2 ions. Comparison With Other Studies The above findings are closely similar to those made recently with MnCl 2 in parallel isolated rat heart experiments (3). The one major exception is the much higher tissue Mn metal contents in the latter study. Concerning the use of the 2SX model, we found that our results closely paralleled those of Labadie et al. (21) and Landis et al. (22). In particular, there was a linear relationship between the concentration of applied contrast agent and the intrinsic relaxation rate constants. However, there is a major difference between results obtained with ec Gd 3 -chelates in the original 2SX model (21,22) and those achieved with ic Mn 2 ions in the presently applied 2SX model. Our data did not support the notion of Labadie et al. (21) and Landis et al. (22) that when the concentration of a contrast agent goes toward zero, T 1 relaxation becomes monoexponential. The reason for this may be that in our case there was a large difference in T 1 s between ic and ec water. In the control hearts, ec water had a T 1 of 2700 ms (close to the T 1 of the applied perfusion medium), while ic water had a T 1 of 650 ms possibly due to the high content of relaxation agent (e.g., iron (Fe 2 ) and Mn 2 ) in normal rat hearts (3,9,26,27). An influx of ic Mn 2 results in a reduction of ic T 1 with a subsequent reduction of ec T 1 water due to exchange of water over the cell membrane. Even in a more in vivo-like situation where blood (with its shorter T 1 s of ms) occupies the capillaries, we will not approach the situation where T 1 becomes monoexponential. Our findings differ from those obtained by others. Donahue et al. (29,30) reported proton exchange rates for cellular-to-interstitium and interstitium-to-vasculature of 8 27 Hz and up to 7 Hz, respectively. Judd et al. (31) and Bauer et al. (32) based their myocardial perfusion analysis on a rapid water exchange across the cardiac cell membrane. However, recent papers (33,34) on brain MRI from Charles Springer Jr. s group lend support to our findings of preferably long ic water lifetimes. These papers elaborated on original findings as previously reported by Labadie et al. (21) and Landis et al. (22). The most likely explanation for the discrepancy between our results and those from the myocardial perfusion literature lies in the techniques used to assess tissue T 1. The extraction of multicomponent exponentials from T 1 -based image contrast requires an extremely good signal-to-noise ratio (SNR). Since our results stem from direct relaxography of excised myocardium, SNR is less of a problem. We have confidence in our biexponential T 1 analysis, based on the following facts: the ic and ec water fractions are comparable to water distribution in excised and blotted cardiac tissue (28); going from one to two components reduces residuals from 7% to 1%; and the RMS analysis of errors in p ic and ic, as depicted in Fig. 5, clearly demonstrates that water exchange cannot be fast. Mn Ions and ic Relaxivity Figure 6 incorporates results for ic R 1-1 obtained with MnDPDP in the present study, as shown by closed symbols and a fully drawn line. Also included are results (shown by open symbols and a dotted line) obtained with MnCl 2 in our recently published study (3). [Mn 2 ]in M were obtained by applying correction factors for wet/dry weight and mass vs. volume. MnDPDP and MnCl 2 behave almost identically and present data for the lower and the higher ranges, respectively, of tissue Mn 2. Taken together, these findings strengthen the basic tenet (1,3,10,12,27,35) that MnDPDP and MnCl 2 act through identical mechanisms: first through the initial release of Mn 2 (albeit at different rates), and second through the accompanying rapid cell uptake of these ions. When we calculated ic r 1ic in the ex vivo hearts, we obtained a value of 56 (s mm) 1 with MnDPDP (compared to 60 (s mm) 1 with MnCl 2 as found in Ref. 3). These values are almost one order of magnitude (8.3 times and

7 512 Nordhøy et al. Ex Vivo T 2 Relaxation T 2 relaxation in tissue is multiexponential due to compartmentalized water and susceptibility effects. The trend we found for T 1 was also reflected in T 2, since our T 2 data were dissolved in two components: a short T 2-1 attributed to an average ic water relaxation, and a longer T 2-2 assigned to ec water (Table 2). However, our two-compartment analysis also revealed a good correlation ( 0.932) between R 2-1 and tissue Mn content (Fig. 4). Similar results have been published (38 40). In particular, Zimmerman and Brittin (40) showed that a 2SX analysis is valid also for T 2, as applied in our experimental setting with excised cardiac tissue. FIG. 5. Contour plot of the goodness of fit, RMS, as a function of ic and p ic parameters from the 2SX analysis. The plot shows that the uncertainty of the estimated intrinsic ic value is small below its mean value of 11.3 s, but is very high above that value. The error distribution of the estimated p ic is more symmetric around the minimum value of 0.59, and tailors off rapidly. 8.7 times, respectively) higher than those obtained with MnCl 2 in vitro (Fig. 1a) in the two studies. Also, the present r 1ic value was 36 times r 1 of MnDPDP in water. Altogether, this indicates a remarkably high degree of relaxation enhancement when ic Mn 2 ions are present. Whereas nonspecific protein binding may increase the rotational correlation time ( R ) between proton and electron (Mn 2 ) spins, and enhance relaxivity by a factor of 2 4 in vitro (6), our results indicate that other mechanisms may also have contributed. Inside cells, the Mn 2 binding to many small molecules may decrease, but binding to large molecules increases the relaxivity when compared to Mn 2 aqua ions. This means that our calculated ic r 1-1 represents the mean activity of both highly and less highly responding Mn 2 ligands that with improved relaxography techniques could have been dissolved by the use of a three- or even four-compartment model. Therefore, other preliminary conclusions are that certain specific ic Mn 2 sites may possess even higher relaxivities than (s mm) 1. Obviously, these sites must allow effective water exchange with maintained inner- and outersphere relaxation in the Mn 2 binding sites. This would fit well with both cytoplasmic and mitochondrial proteins (13) or other active large biomolecules. The relaxivity values of 56 and 60 (s mm) 1 for ic Mn 2 are in the range of the highest values yet reported for T 1 -based contrast agents (6,36,37). They are clearly larger than those found for binding of Gd 3 and Mn 2 ions or of ion complexes to human serum albumin (HSA). However, our values are closely similar to those recently published by Aime et al. (36). That study employed in vitro relaxography (20 MHz, 25 C) analyses of different water-soluble Mn 2 EDTA complexes coupled to benzyl-oxymethyl (BOM) moieties, which were again noncovalently bound to HSA. For two Mn 2 -EDTA-BOM-HSA molecules that allowed rapid inner-sphere water exchange, the longitudinal relaxivities were as high as 55.3 and 48.0 (s mm) 1, respectively. Methodological Considerations The experimental model used here is far from in vivo conditions, but it allowed us to perform detailed analyses of dose-response characteristics, ion release-and-uptake mechanisms with MnDPDP, and relationships between tissue Mn 2 content and relaxographic findings. Accordingly, the long ic and ec water lifetimes enabled us to identify two cell compartments in which the intrinsic ic relaxivity could be established with a high degree of accuracy. In describing relaxation phenomena, we have paid attention to the Mn 2 ion only; however, this may not be strictly correct, since our data may have been to a minor extent influenced by Mn 3 ions. Like Mn 2,Mn 3 ions may also bind to ic macromolecules. However, only Mn 2 ions compete with Ca 2 ions and enter cardiac cells. Since Mn 2 has a higher number of unpaired electrons (5 vs. 4), it is also more strongly paramagnetic, and susceptibility effects from Mn 2 are greater than those from Mn 3 (6). In addition, Mn 3 has a much reduced hyperfine relaxivity because of the smaller electron spin relaxation time for Mn 3 (10 10 s vs s) (6). Altogether, Mn 2 -containing compounds are regarded as particularly strong contrast agents for T 1 -weighted imaging (11,25). FIG. 6. Concentration-response graph for Mn 2 ions in ex vivo myocardium. Values for ic R 1 1 obtained with MnDPDP in the present study are compared to those recently obtained with MnCl 2 (3). Observe that tissue Mn contents in nmole/g dry wt. are converted to tissue Mn 2 concentrations in M. The intrinsic values are depicted as fully drawn (MnDPDP) and dotted (MnCl 2 ) lines.

8 T 1 Relaxation by Intracellular Manganese Ions 513 CONCLUSIONS The main conclusions that can be drawn from the present study are that the uptake of Mn 2 ions in cardiac tissue is very effective, and ic Mn 2 ions are strong T 1 relaxation agents when adducts are formed with active ic macromolecules. The free Mn 2 concentration in the interstitial space is low due to a slow release from MnDPDP, and under in vivo conditions an extensive plasma protein binding causes a further lowering of free Mn 2. Nevertheless, cell uptake via physiological Ca 2 channels is surprisingly effective, due to an intermediate channel affinity for Mn 2 (i.e., higher than for Ca 2 ions, but less than required for a total channel blockade). This means that an avid uptake mechanism may compensate for the low ec availability of free Mn 2 ions. The remarkably high ic efficacy of Mn 2 ions can now be added to the list of compensatory mechanisms. As demonstrated in Ref. 3, and confirmed in the present study, the pharmaceutical industry has yet to invent a more powerful T 1 relaxation agent than Mn 2 ions in the ic water compartment. We believe that our results may lead to a search for active ic Mn 2 macromolecular complexes, particularly since their structure may be used for modeling of future blood pool agents. Altogether, the kinetics and dynamics of MnDPDP or other Mn 2 releasers are complex and intriguing, but may be exploited to their full extent for molecular imaging in postgenome research and, most preferably, for future clinical diagnostic applications. A surprising benefit derived from the use of MnDPDP and the metabolite MnPLED is that these Mn 2 -based molecules possess antioxidant properties and may provide cardioprotection against postischemic reperfusion injuries, as shown in a pig myocardial infarction model (41). 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