Manganese and the Heart Intracellular MR relaxation and water exchange across the cardiac cell membrane PhD Thesis in Medical Technology of Wibeke Nordhøy

PhD Thesis of Wibeke Nordhøy2 Content General theory General theory Paper I-III Paper I-III Main conclusions Main conclusions

PhD Thesis of Wibeke Nordhøy3  Hydrogen/Proton: A spinning top

PhD Thesis of Wibeke Nordhøy4 Randomly oriented protons

PhD Thesis of Wibeke Nordhøy5 M0M0M0M0 B0B0B0B0 z Aligned with the external magnetic field M 0 = the netto magnetization vector Resonance Frequency:  0 =  B 0

PhD Thesis of Wibeke Nordhøy6 B X Y Z o time S MzMz M z (t) = M 0 ( 1 - 2e -t/T 1 ) T 1 and T 2 relaxation MoMo B1B1 T 2 (T 2 *) M xy (t) = M 0 e -t/T 2 *

PhD Thesis of Wibeke Nordhøy7 MR and relaxation Magnetic properties of protons in 1 H-MRI Magnetic properties of protons in 1 H-MRI  (H), T 1 and T 2 are essential factors in MRI  (H), T 1 and T 2 are essential factors in MRI Paramagnetic contrast agents (CA) as Mn 2+ act indirectly by decreasing T 1 and T 2 of water protons in near proximity Paramagnetic contrast agents (CA) as Mn 2+ act indirectly by decreasing T 1 and T 2 of water protons in near proximity T 1 –weighted imaging, where T 2 is decreased T 1 –weighted imaging, where T 2 is decreased

PhD Thesis of Wibeke Nordhøy8 Contrast enhancement Shorter and shorter T 1 or stronger and stronger signal in a T 1 weighted image where the CA is present

PhD Thesis of Wibeke Nordhøy9 H 2 O ec H 2 O ic  ec -1 ec:Extracellular ic:Intracellular p ic T 1ic p ec T 1ec  ic -1 T 1 and water transport Symbols  -1 = water exchange p = population fraction of water T 1 = longitudinal relaxation time Intracellular values  ic -1 p ic T 1ic Extracellular values  ec -1 p ec T 1ec

PhD Thesis of Wibeke Nordhøy10 Water exchange rate

PhD Thesis of Wibeke Nordhøy11 Fast exchange M z (t) = M 0 ( 1 - 2e -t/T 1 ) Monoexponential signal

PhD Thesis of Wibeke Nordhøy12 M z (t) = p ic ( 1 - 2e -t/T 1ic ) + p ec ( 1 - 2e -t/T 1ec )) Biexponential signal Slow exchange

PhD Thesis of Wibeke Nordhøy13 Intermediate exchange A more complicated model: Two-site water exchange (2SX) Ref: Springer et al.

PhD Thesis of Wibeke Nordhøy14 Main goals of Paper I-III To establish a model for T 1 measurements in myocardium To establish a model for T 1 measurements in myocardium The examine the influence of water exchange across the cardiac cell membrane The examine the influence of water exchange across the cardiac cell membrane To calculate the T 1 efficacy in each tissue compartment To calculate the T 1 efficacy in each tissue compartment To study the interaction beetween Mn 2+ and Ca 2+ : To study the interaction beetween Mn 2+ and Ca 2+ : uptake and retention of Mn 2+ -ions

PhD Thesis of Wibeke Nordhøy15 Mn 2+ Mn content from freeze-dried hearts LVDP, HR Experimental setup

PhD Thesis of Wibeke Nordhøy16 Mn administration Langendorff-perfused hearts Langendorff-perfused hearts – control perfusion – Mn 2+ ‘wash-in’ – Mn 2+ ‘wash-out’ Single wash-in and wash-out (Paper I and II) Single wash-in and wash-out (Paper I and II) Repeated wash-in and wash-out with Mn- accumulation (Paper III) Repeated wash-in and wash-out with Mn- accumulation (Paper III)

PhD Thesis of Wibeke Nordhøy17 Manganese ions as intracellular contrast agents: proton relaxation and calcium interactions in rat myocardium NMR in biomedicine 16(2): (2003) Paper I

PhD Thesis of Wibeke Nordhøy18 T 1 relaxation in the hearts without Mn (control): which model is most suited?

PhD Thesis of Wibeke Nordhøy19 T 1 relaxation in hearts with 100 µM MnCl 2 hearts:

PhD Thesis of Wibeke Nordhøy20 Strong correlation (r) between ic R 1-1 (R 1ic ) and the Mn content R 1-2 = 1/T 1-2 R 1-1 = 1/T 1-1

PhD Thesis of Wibeke Nordhøy21 T 1 model of rat myocardium Two-components of T 1 : Two-components of T 1 : – T 1-1 rapid, share ~ 60 % (ic component) – T 1-2 slow, share ~ 40 % (ec component) An assumed slow water exchange situation An assumed slow water exchange situation Fast exchange Slow exchange

PhD Thesis of Wibeke Nordhøy22 Multiexponential analyses of T 1 revealed two water compartments (ic and ec) with different chemical environments in the rat myocardiumMultiexponential analyses of T 1 revealed two water compartments (ic and ec) with different chemical environments in the rat myocardium The intracellular R 1 correlated highly with tissue Mn content, which increased R 1 effectivelyThe intracellular R 1 correlated highly with tissue Mn content, which increased R 1 effectively These T 1 components were detectable with a 0.47 T MR spectrometer due to a slow-to-intermediate water exchange across the cardiac cell membraneThese T 1 components were detectable with a 0.47 T MR spectrometer due to a slow-to-intermediate water exchange across the cardiac cell membrane Conclusions of Paper I

PhD Thesis of Wibeke Nordhøy23 Intracellular manganese ions provide strong T 1 relaxation in rat myocardium Magnetic Resonance in Medicine 52: (2004) Paper II

PhD Thesis of Wibeke Nordhøy24 Mn dipyridoxyl-diphosphate (MnDPDP) O-O- Mn NN O O OO O + N H OPO-O- O-O- O + N H OP O OH O

PhD Thesis of Wibeke Nordhøy25 Relaxation rate constants vs. Mn content

PhD Thesis of Wibeke Nordhøy26 2SX water exchange analysis

PhD Thesis of Wibeke Nordhøy27 High intracellular relaxivity with both MnCl 2 and MnDPDP both MnCl 2 and MnDPDP In vitro r 1 of MnCl 2 In vitro r 1 of MnDPDP r 1-1 ~ 60 (s mM) -1

PhD Thesis of Wibeke Nordhøy28 Conclusions of Paper II T 1 relaxography and 2SX analyses revealed two compartments representing ic- and ec- water T 1 relaxography and 2SX analyses revealed two compartments representing ic- and ec- water The ic relaxivity of MnDPDP was as high as for MnCl 2 The ic relaxivity of MnDPDP was as high as for MnCl 2 Protein binding may explain the remarkably high intracellular relaxivity of Mn 2+ ions Protein binding may explain the remarkably high intracellular relaxivity of Mn 2+ ions – Increased correlation time (  c ) between proton- and electron spins of Mn 2+ -ions due to increased rotational correlation time (  R ) of bound protons – Rapid water exchange (  M -1 ) within Mn 2+ sites

PhD Thesis of Wibeke Nordhøy29 Manganese-Calcium interactions with contrast media for cardiac MRI: A study of manganese chloride supplemented with calcium gluconate in isolated guinea pig hearts In Press March 2005: Investigative Radiology Paper III

PhD Thesis of Wibeke Nordhøy30 Background and Goals What will be the optimal formulation of Mn 2+ - releasing contrast media? What will be the optimal formulation of Mn 2+ - releasing contrast media? ‘efficacy’ versus ‘safety’? ‘efficacy’ versus ‘safety’? Authors have suggested different combinations of Ca 2+ - and Mn 2+ -salts (10:1 or 8:1) Authors have suggested different combinations of Ca 2+ - and Mn 2+ -salts (10:1 or 8:1) Three possible Mn 2+ -releasing agents: 1. A slow-release Mn 2+ chelate like MnDPDP 2. Add a ‘cardioprotective’ Ca 2+ salt to a ‘MR effective’ Mn 2+ salt ‘MR effective’ Mn 2+ salt 3. Avoid cardiodepression by controlled infusion of a rapidly dissolving Mn 2+ salt like MnCl 2

PhD Thesis of Wibeke Nordhøy31 LVDP, HR and LVDPxHR Manganese Manganese-Calcium

PhD Thesis of Wibeke Nordhøy32 Normal cell metabolism in all groups (CrP, ATP) Normal cell metabolism in all groups (CrP, ATP) Manganese (660 µM): Manganese (660 µM): – Reduced myocardial contractility (-53 %) – Reduced heart rate (-18 %) – Large Mn metal content (93 times control) Manganese-Calcium (660 µM): Manganese-Calcium (660 µM): – Increased myocardial contractility (+56 %) – Large Mn metal content (41 times control) Slow water exchange and biexponential T 1 Slow water exchange and biexponential T 1 Results

PhD Thesis of Wibeke Nordhøy33 Conclusions of Paper III Alternative 1: High addition of Ca 2+ to Mn 2+ (10:1) increases contractility, but reduces Mn uptake Alternative 1: High addition of Ca 2+ to Mn 2+ (10:1) increases contractility, but reduces Mn uptake MnDPDP is more suited than alternative 1 for clinical MRI studies on the heart MnDPDP is more suited than alternative 1 for clinical MRI studies on the heart Depression of the contractile force can also be avoided by using a slow infusion of MnCl 2 Depression of the contractile force can also be avoided by using a slow infusion of MnCl 2

PhD Thesis of Wibeke Nordhøy34 A biexponential model is best suited for T 1 analyses A biexponential model is best suited for T 1 analyses A slow-intermediate water exchange across the cardiac cell membrane was confirmed for both rat and guinea pig hearts A slow-intermediate water exchange across the cardiac cell membrane was confirmed for both rat and guinea pig hearts Mn 2+ entry dependent on Ca 2+ channel activity Mn 2+ entry dependent on Ca 2+ channel activity The contractile force was not significantly reduced for clinically relevant Mn 2+ concentrations The contractile force was not significantly reduced for clinically relevant Mn 2+ concentrations Close correlation between tissue Mn content and relaxation parameters, especially for T 1ic and high intracellular efficacy Close correlation between tissue Mn content and relaxation parameters, especially for T 1ic and high intracellular efficacy Main conclusions