A technique for in vivo mapping of myocardial creatine kinase metabolism

Abstract

ATP derived from the conversion of phosphocreatine to creatine by creatine kinase provides an essential chemical energy source that governs myocardial contraction. Here, we demonstrate that the exchange of amine protons from creatine with protons in bulk water can be exploited to image creatine through chemical exchange saturation transfer (CrEST) in myocardial tissue. We show that CrEST provides about two orders of magnitude higher sensitivity compared to (1)H magnetic resonance spectroscopy. Results of CrEST studies from ex vivo myocardial tissue strongly correlate with results from (1)H and (31)P magnetic resonance spectroscopy and biochemical analysis. We demonstrate the feasibility of CrEST measurement in healthy and infarcted myocardium in animal models in vivo on a 3-T clinical scanner. As proof of principle, we show the conversion of phosphocreatine to creatine by spatiotemporal mapping of creatine changes in the exercised human calf muscle. We also discuss the potential utility of CrEST in studying myocardial disorders.

Figure 1

CrEST measurement in ex vivo noninfarcted myocardial tissue. (a,b) Non–water-suppressed ¹H nuclear magnetic resonance (NMR) spectra from 100 mM creatine solution (reproduced from ref. 27) (a) and ex vivo myocardial tissue (b) obtained at two different temperatures (25 °C and 37 °C). The broad resonance at ~8.2 p.p.m. (~3.5 p.p.m. downfield of the bulk water) depicts the amide proton resonance from proteins. (c–e) The anatomical CEST-weighted image (2 p.p.m.) (c) and gray (d) and color-coded (e) CrEST maps of ex vivo myocardial tissue are shown. (f) Water spectra obtained from the tissue shown with saturation at ±1.8 p.p.m. using the B_1rms of 155 Hz (3.6 µT) and 250 ms. (g,h) The ratio of the integral of difference water spectrum (g) to the integral of Cr-CH₃ resonance from the same voxel from the water-suppressed spectra (h) is >70. AU, arbitrary units; Cho, choline methyl resonance. (i,j) Saturation pulse amplitude and duration dependence of CrEST are shown. The scale bars shown for anatomical images are MR image intensities in AU and the scale bars for all CrEST maps are CrEST contrasts in % units.

Figure 2

CrEST map of a noninfarcted ex vivo lamb myocardium measured at two separate time points (14 h and 62 h) following excision of the heart. (a–e) Anatomical CEST-weighted image at 2 p.p.m. (a) and gray (b and c) and color-coded CrEST maps (d and e) are shown. The square boxes show the representative ROIs from where the water-suppressed ¹H MRS was measured. (f) ¹H MRS from the ROIs shown in b and c. Clear change in the Cr resonance amplitude measured at two different time points is evident. (g) Plot showing a linear correlation between Cr concentration and CrEST contrast (%). The ex vivo myocardial tissue exhibits 0.8% CrEST contrast per 1 mM Cr. (h,i) Correlations of Cr concentration, measured through PCA extraction, with both CrEST contrast (h) and ¹H MRS–measured Cr concentration (i) are shown. (j) On ³¹P MRS of ex vivo myocardium, only P_i resonance is visible. The scale bars shown for anatomical images are MR image intensities in AU and the scale bars for all CrEST maps are CrEST contrasts in % units.

Figure 3

Ex vivo CrEST of noninfarcted and infarcted myocardial tissue from swine. (a) Anatomical CEST-weighted image (2 p.p.m.) of an ex vivo myocardium tissue. (b,c) The CrEST contrast (%) shown in noninfarcted and infarcted regions in gray (b) and color-coded (c) CEST maps. (d) The trichrome stain of same tissue shows the infarcted region as blue (dotted arrow) and the noninfarcted region as red (solid arrow). The CrEST from the remote region is ~10.4%, whereas from the infarcted region it is ~4.7%. (e) Digital photograph of another myocardial tissue clearly depicts the infarcted (dotted arrow) and noninfarcted regions (solid arrow). (f) Anatomical CEST-weighted image (2 p.p.m.) of the tissue from e with the voxels placed on infarcted (1) and noninfarcted regions (2). (g–i) Grayscale (g) and color-coded (h) CrEST maps show CrEST contrast in noninfarcted and infarcted regions, and ¹HMRS (i) shows Cr resonance in noninfarcted and infarcted regions. The scale bars shown for anatomical images are MR image intensities in AU and the scale bars for all CrEST maps are CrEST contrasts in % units.

Figure 4

In vivo CrEST data from normal and infarcted swine or sheep myocardium. (a–e) CrEST mapping of myocardium of two normal animals (swine, a,b) and three infarcted animals including two sheep (c,d) and one swine (e). The four rows show, from top to bottom, the anatomical CEST-weighted (2 p.p.m.) short-axis images of left ventricle, grayscale CrEST maps, color-coded CrEST maps and overlaid color-coded CrEST maps from all five animals. c and d show a 1-week infarction and e shows an 8-week infarction (arrows show the infarcted regions of the tissue). The process of identifying the wall is described in Supplementary Methods. (f) The fitted Z spectra from normal and infarcted regions are shown (M and M₀ are water signal intensities with and without saturation, respectively). (g) Bar graph showing the CrEST contrast values in normal and infarcted regions of myocardium tissue. Data are expressed as mean ± s.d. (h) Delayed gadolinium-enhanced image of the 8-week infarcted swine myocardium, which shows hyperintensity in the infarcted region. The scale bars shown for anatomical images are MR image intensities in AU and the scale bars for all CrEST maps are CrEST contrasts in % units.

Figure 5

CrEST maps of exercise-induced changes in skeletal muscle. (a) CrEST maps of healthy human calf before and after exercise, at different time points, show an increase in CrEST contrast immediately after exercise that corresponds to an increase in Cr concentration, followed by a return to the basal value CrEST contrast. Anatomic image of calf muscle overlaid with muscle groups highlighted is shown on the bottom right. (b) Kinetics of CrEST changes in different muscle groups depict post-exercise increases in CrEST contrast percentage and recovery to baseline levels. Data are expressed as mean ± s.d. (c) Representative ³¹P MRS before exercise, immediately after exercise and after full recovery from exercise. The decrease in the PCr peak and increase in P_i peak immediately after exercise is shown. (d) PCr kinetics obtained from nonlocalized ³¹P MRS from the same muscle region, showing a decrease in PCr signal immediately following exercise, which recovers back to baseline values. The scale bars shown for anatomical images are MR image intensities in AU and the scale bars for all CrEST maps are CrEST contrasts in % units.