Manganese and the Heart Intracellular MR relaxation and

Manganese and the Heart Intracellular MR relaxation and water exchange across the cardiac cell membrane Ph. D Thesis in Medical Technology of Wibeke Nordhøy 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy

Content • General theory • Paper I-III • Main conclusions 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 2

Hydrogen/Proton: A spinning top 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 3

Randomly oriented protons 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 4

Aligned with the external magnetic field B 0 z M 0 = the netto magnetization vector 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy Resonance Frequency: 0 = B 0 5

T 1 and T 2 relaxation Mz(t) = M 0 ( 1 - 2 e-t/T 1 ) Mz Z Bo time Mo Y B 1 S X T 2 (T 2*) Mxy(t) = M 0 e-t/T 2* time 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 6

MR and relaxation • Magnetic properties of protons in 1 H-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 • T 1 –weighted imaging, where T 2 is decreased 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 7

Contrast enhancement Shorter and shorter T 1 or stronger and stronger signal in a T 1 weighted image where the CA is present 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 8

T 1 and water transport ec: Extracellular Symbols -1 = water exchange ic: Intracellular H 2 Oic ec-1 ic-1 H 2 Oec p ic T 1 ic p ec T 1 ec p = population fraction of water T 1 = longitudinal relaxation time Intracellular values ic-1 pic T 1 ic Extracellular values ec-1 pec T 1 ec 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 9

Water exchange rate 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 10

Fast exchange Monoexponential signal Mz(t) = M 0 ( 1 - 2 e-t/T 1 ) 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 11

Slow exchange Biexponential signal Mz (t) = pic ( 1 - 2 e-t/T 1 ic ) + pec ( 1 - 2 e-t/T 1 ec )) 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 12

Intermediate exchange A more complicated model: Two-site water exchange (2 SX) Ref: Springer et al. 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 13

Main goals of Paper I-III • To establish a model for T 1 measurements in myocardium • The examine the influence of water exchange across the cardiac cell membrane • To calculate the T 1 efficacy in each tissue compartment • To study the interaction beetween Mn 2+ and Ca 2+: uptake and retention of Mn 2+-ions 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 14

Experimental setup Mn 2+ LVDP, HR Mn content from freeze-dried hearts 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 15

Mn administration • Langendorff-perfused hearts – – – control perfusion Mn 2+ ‘wash-in’ Mn 2+ ‘wash-out’ • Single wash-in and wash-out (Paper I and II) • Repeated wash-in and wash-out with Mnaccumulation (Paper III) 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 16

Paper I Manganese ions as intracellular contrast agents: proton relaxation and calcium interactions in rat myocardium NMR in biomedicine 16(2): 82 -95 (2003) 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 17

T 1 relaxation in the hearts without Mn (control): which model is most suited? 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 18

T 1 relaxation in hearts with 100 µM Mn. Cl 2 hearts: 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 19

Strong correlation (r) between ic R 1 -1 (R 1 ic) and the Mn content R 1 -1 = 1/T 1 -1 R 1 -2 = 1/T 1 -2 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 20

T 1 model of rat myocardium • 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 Fast exchange Slow exchange 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 21

Conclusions of Paper I • Multiexponential 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 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 membrane 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 22

Paper II Intracellular manganese ions provide strong T 1 relaxation in rat myocardium Magnetic Resonance in Medicine 52: 506 - 514 (2004) 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 23

Mn dipyridoxyl-diphosphate (Mn. DPDP) O O O- P OH O O O + N H N Mn N O O + N H P OO- O 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 24

Relaxation rate constants vs. Mn content 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 25

2 SX water exchange analysis 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 26

High intracellular relaxivity with both Mn. Cl 2 and Mn. DPDP r 1 -1 ~ 60 (s m. M)-1 In vitro r 1 of Mn. Cl 2 In vitro r 1 of Mn. DPDP 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 27

Conclusions of Paper II • T 1 relaxography and 2 SX analyses revealed two compartments representing ic- and ec- water • The ic relaxivity of Mn. DPDP was as high as for Mn. Cl 2 • 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 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 28

Paper III 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 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 29

Background and Goals • • • What will be the optimal formulation of Mn 2+releasing contrast media? ‘efficacy’ versus ‘safety’? 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 Mn. DPDP 2. Add a ‘cardioprotective’ Ca 2+ salt to a ‘MR effective’ Mn 2+ salt 3. Avoid cardiodepression by controlled infusion of a rapidly dissolving Mn 2+ salt like Mn. Cl 2 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 30

LVDP, HR and LVDPx. HR Manganese-Calcium 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 31

Results • Normal cell metabolism in all groups (Cr. P, ATP) • Manganese (660 µM): – Reduced myocardial contractility (-53 %) – Reduced heart rate (-18 %) – Large Mn metal content (93 times control) • Manganese-Calcium (660 µM): – Increased myocardial contractility (+56 %) – Large Mn metal content (41 times control) • Slow water exchange and biexponential T 1 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 32

Conclusions of Paper III • Alternative 1: High addition of Ca 2+ to Mn 2+ (10: 1) increases contractility, but reduces Mn uptake • Mn. DPDP 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 Mn. Cl 2 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 33

Main conclusions • 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 • Mn 2+ entry dependent on Ca 2+ channel activity • 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 1 ic and high intracellular efficacy 17. 12. 2004 Ph. D Thesis of Wibeke Nordhøy 34