Introduction

The Purkinje conduction network (composed of Purkinje Fibers (PF)) plays a crucial role in normal cardiac function but it has also been implicated in arrhythmogenesis and sudden cardiac death¹. PF have the particularity of being composed of cardiomyocytes surrounded by a collagen sheath^2,3. Thus, magnetization transfer (MT) techniques are good candidates to increase contrast between cardiac tissue and PF⁴. Beside conventional MT, inhomogeneous MT (ihMT) was developed to explore a different source of contrasts related to dipolar relaxation times T_1D of tissues. Recently Varma et al.⁵ demonstrated successful applications of ihMT on ex vivo tissues with short T_1D (1 ms) components outside the brain, such as in the rat heart. Here, we hypothesize that ihMT can provide images with improved contrast between cardiac muscle and PF based on their different T_1D values. Thus, a rapid sequence incorporating ihMT module was implemented at 9.4T and evaluated on an ex vivo cardiac sample from pig.

Materials & Methods

Sample
A pig’s heart (~40 kg) was surgically collected after a sternal thoracotomy followed by pentobarbital euthanasia in accordance with institutional animal care. The heart was rapidly rinsed with saline solution. A sample (3.5×4×5.5 cm³) was selected and then cut in the longitudinal direction of the heart in the right ventricle including the moderator band (Fig.1), which is a well-known conductive fiber. The sample was fixed and agitated in formalin at 10% for 48 hours. Prior to MRI acquisitions, the sample was removed from formalin and immersed into a perfluoropolyether (3M™ Fluorinert™ FC-770) with a temperature probe (SA Instruments, Stony Brook, NY) positioned in contact with the sample. Hot water was circulated in a tubing system to warm the sample and maintain its temperature at 37°C during MRI acquisition.
Acquisition
MRI acquisitions were performed at 9.4T/30 cm (Bruker BioSpin MRI, Ettlingen Germany) using a Tx/Rx volume antenna (87mm inner diameter). Images were acquired with an in-plane resolution of (250 μm)² and a slice thickness of 2 mm. 2D ihMT-RARE sensitivity-boosted⁷ sequences were implemented (Fig.2) with TR/TE/flip angle/RARE factor/Matrix= 56 ms/6 ms/90°/56/160×100. Frequency alternated pulses (ALT) and four configurations of frequency offsets Δf (7/10/14/20 kHz) from the water peak were tested. For each frequency configuration, the M₀ image was accumulated 4 times and each of the MT⁺, MT^+-, MT^- and MT^-+ images were produced after 60 accumulations. IhMT images with two different T_1D filtering strengths⁸ obtained by tuning the repetition time of saturation pulses to 1.1-ms and 3-ms were evaluated. The saturation period (total duration t=1600 ms) was composed of 12 bursts of N_p=12 pulses repeated every B_TR=130ms, and deposited a root-mean squared saturation power B_1rms=9 μT.
Post-processing
The ihMT datasets were composed of 5 different images: M₀ image (without any MT preparation), MT⁺ image (with a MT preparation pulse at +Δf), MT^- image at -Δf and MT^+- and MT^-+ with dual-frequency MT preparations. The images were post-processed with a homemade program using Matlab (MATLAB 9.5, The Mathworks, Inc). A binary mask was computed based on M₀ image to remove background. The ihMT ratio maps were calculated as $$$\small ihMTR = \left[\left(MT^{+}+MT^{-}\right)-\left(MT^{+-}+MT^{-+}\right)\right]/M_{0}\times100$$$ and conventional MT ratio maps were derived from the same dataset as $$$\small MTR=\left(1-MT^{+}/M_{0}\right)\times100$$$ . Two regions of interest were selected in the cardiac muscle (ROI_mu) and in the moderator band (ROI_fib) on both ihMTR and MTR maps to plot the respective ratio as a function of the frequency offset. Finally, the relative contrast RC defined as $$$\small ROI_{fib}/ROI_{mu}$$$ was calculated.

Results

Cardiac sample exhibits different ihMTR contrast for different frequencies (see ihMTR maps on Fig.3A). The ihMTR graph for fiber and muscle (Fig.3A) shows a maximum signal for frequencies above 15 kHz. With relatively weak filtering of the short T_1D signal (Fig.3B black curve), the ihMTR is 8% for fiber and 11.8% for the tissue with a maximized RC of 0.68. For stronger T_1D filtering (Fig.3B red curve), ihMTR is 3.5% for fiber and 4.5% for the tissue and the RC is 0.78. The MTR graph (Fig.3C) shows an extremum for frequencies below 7 kHz. The MT relative contrast between the tissue and the fiber diminishes to 0.85 with increasing frequency offset at 20 kHz for the 1.1-ms filtering and 0.95 for the 3-ms filtering. For frequencies above 15 kHz, we observe an ihMT relative contrast between the collagen of the PF and the myocardium up to 40% higher compared to the MT relative contrast.

Conclusion

IhMT technique was successfully implemented and tested at 9.4T on heart samples from pig containing conductive fibers. The observed contrast demonstrates that cardiac muscle and PF have different T_1D. Our results show that ihMT provides better contrast between PF and myocardium than conventional MT

Acknowledgements

This work received financial support from the French National Investments for the Future Programs: ANR-10-IAHU-04 (IHU Liryc) and from the Nouvelle-Aquitaine council.

References

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