Investigating the effect of trigger delay on cardiac 31P MRS signals

The heart’s geometry and its metabolic activity vary over the cardiac cycle. The effect of these fluctuations on phosphorus (31P) magnetic resonance spectroscopy (MRS) data quality and metabolite ratios was investigated. 12 healthy volunteers were measured using a 7 T MR scanner and a cardiac 31P-1H loop coil. 31P chemical shift imaging data were acquired untriggered and at four different times during the cardiac cycle using acoustic triggering. Signals of adenosine-triphosphate (ATP), phosphocreatine (PCr), inorganic phosphate (Pi) and 2,3-diphosphoglycerate (2,3-DPG) and their fit quality as Cramér-Rao lower bounds (CRLB) were quantified including corrections for contamination by 31P signals from blood, flip angle, saturation and total acquisition time. The myocardial filling factor was estimated from cine short axis views. The corrected signals of PCr and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\gamma$$\end{document}γ-ATP were higher during end-systole and lower during diastasis than in untriggered acquisitions (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$P<0.05$$\end{document}P<0.05). Signal intensities of untriggered scans were between those with triggering to end-systole and diastasis. Fit quality of PCr and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\gamma$$\end{document}γ-ATP peaks was best during end-systole when blood contamination of ATP and Pi signals was lowest. While metabolite ratios and pH remained stable over the cardiac cycle, signal amplitudes correlated strongly with myocardial voxel filling. Triggering of cardiac 31P MRS acquisitions improves signal amplitudes and fit quality if the trigger delay is set to end-systole. We conclude that triggering to end-systole is superior to triggering to diastasis.

A deranged myocardial energy metabolism is associated with many cardiovascular diseases, as heart failure or ischemic heart disease 1,2 . Phosphorus ( 31 P) MRS directly measures the concentrations of the high-energy phosphates adenosine-triphosphate (ATP) and phosphocreatine (PCr) in vivo in the human myocardium. The PCr to ATP ratio has proven to be a strong indicator for cardiac function and a sensitive predictor of mortality 3,4 . 31 P MRS further allows one to study the kinetics of cardiac metabolism in magnetization transfer experiments by measuring rates of the creatine kinase reaction which links ATP and PCr pools 5,6 .
Reproducibility studies of human cardiac 31 P MRS at 1.5 T, 3 T and 7 T 7-9 have shown the difficulty of establishing reliable, predictive and reproducible PCr/ATP. While these studies present various protocol optimizations and emphasize the importance of post-processing steps as saturation and blood correction, surprisingly, a systematic investigation of the effect of cardiac triggering on the quality of 31 P MRS acquisitions has not been reported, yet.
From cardiac 1 H MRS studies on the matter [21][22][23] it is known, that highest spectral quality, SNR, signal stability and repeatability are achievable when triggering to a short period around end-systole. These recommendations, however, are not necessarily directly applicable to 31 P measurements due to several important differences, e.g. voxel size compared to myocardial thickness, acquisition duration or spectral contamination. For the same reasons correction of respiratory motion, important in 1 H MRS, is less relevant in 31 P MRS.
Triggered cardiac 31 P MRS has been used to examine physiological effects of contraction on the complex dynamics of cardiac high-energy phosphates 24  www.nature.com/scientificreports/ contradictory results on physiologic changes over the cardiac cycle [24][25][26] . Similar in vivo studies in humans showed no evidence of such fluctuations 27,28 , and simulations suggested that physiologic variations of cardiac high-energy phosphates are below detectability of current 31 P-MRS techniques 29 . Hence, the case for an optimal triggering strategy for cardiac in vivo 31 P MRS remains. We therefore investigate the influence of cardiac triggering on 31 P MRS data with focus on signal amplitude, spectral quality and blood contamination.

Results
In all 14 data sets, comprising five scans each, the spectra from the four selected voxels were of good quality and fitted successfully (Fig. 1).
For all evaluated voxels, SNRs for PCr were > 7 , and CRLBs were < 19 % . The signal amplitudes and CRLBs of PCr, γ-ATP and 2,3-DPG are shown in Fig. 2 for the voxels individually (colored boxes) and all voxels pooled (gray boxes).
In Fig. 2 the signals are shown after correction for the actual scan times ( S/t acq ) to provide a fair comparison between triggered and untriggered scans. While the numbers differ slightly for signals without this correction, the differences and trends are similar to S/t acq . During end-systole, these corrected metabolite signals showed a significant increase for PCr and γ-ATP, and a decrease for 2,3-DPG, with a generally more pronounced effect in posterior voxels. Diastasis consistently showed the opposite behavior. For PCr and γ-ATP the best fitting accuracy was found during end-systole while CRLBs were highest during diastasis. Selected results are presented in Table 1.
Anterior voxels showed significantly higher PCr signals than posterior voxels, as can be seen from the absolute signals in Fig. 2. CRLBs of PCr and γ-ATP are lower in anterior voxels (Fig. 2g,h) compared to posterior voxels. The P i peak area had a mean CRLB of 29 ± 11 % . No significant variations of P i amplitude, chemical shift or CRLBs over the cardiac cycle were found.
Metabolite ratios PCr/ATP, P i /PCr and pH are presented in Fig. 3. Neither the ratios nor pH values showed significant changes with the trigger delay. The CRLBs of PCr/ATP, however, are significantly lower during endsystole. PCr/ATP is higher in anterior voxels while P i /PCr is higher in posterior voxels. PCr/ATP ratios (mean: 1.75 ± 0.45 ) ranged from 1.47 ± 0.35 in posterior-basal to 2.08 ± 0.37 in anterior-apical voxels (see Fig. 3a,b).

Discussion
In this study, we investigated the effect of triggering on in vivo human cardiac 31 P MR signals. Spectra from untriggered acquisitions were compared to triggered acquisitions at four different time points in the cardiac cycle. Acquiring during end-systole yields the highest signals and lowest CRLBs as compared to other cardiac phases. With our chosen parameters we further present a sound setting for reliably fitting metabolites to evaluate PCr/ ATP, P i /PCr and pH in a clinically feasible measurement time.
We could not confirm significant variations in metabolite ratios PCr/ATP and P i /ATP in the myocardium over the cardiac cycle. This is in agreement with theoretical predictions 29 and implies that results of studies which ignore cardiac motion apparently are not subject to large scale systematic errors. Nonetheless, the sensitivity of myocardial metabolite signals can be significantly improved by acquiring data during end-systole (see Fig. 2) when the heart muscle is the thickest and ventricular filling is the lowest (Fig. 4). www.nature.com/scientificreports/ We conclude that voxel filling is the dominating factor when considering the effect of triggering. Although respiratory motion was not accounted for, ∼ 70 % of variance can be explained by cardiac motion, probably due to large voxel sizes. This is different in 1 H MRS, where voxels are much smaller and typically on the order of respiratory motion. Especially the correlation of CRLBs and filling factor supports the reasonable assumption that the quality of spectral fitting improves with a higher myocardial voxel filling.
We observed a bigger effect of triggering in posterior voxels, which suggests that the anterior ones have some contribution originating from skeletal muscle signals. This is in line with higher PCr/ATP ratios in these voxels. Therefore higher resolution in the anterior-posterior direction or other methods to improve the spatial response function should be used.
In contrast to our results, two prior studies 27,28 reported no significant differences between metabolite concentrations of diastolic and systolic spectra. In our study, however, a smaller nominal voxel size was used. This reduces potential contamination from skeletal muscle but accentuates the susceptibility to partial filling of the voxel, a fact already acknowledged by Grist et al. 27 . Increased SNR at 7 T presumably contributed to our finding.
For cardiac 1 H MRS the optimal trigger delay was found at mid-to end-systole 23 , which agrees well with our findings for 31 P MRS. Results of 1 H MRS are, however, not directly applicable to 31 P MRS as they differ in several important aspects. In 1 H MRS, the predominant water signal is as much in blood as in tissue, small fluctuations in shim and frequencies can lead to severe artifacts. Therefore voxels are much smaller (also due to SNR) which are therefore affected considerably more by motion.
While diastasis offers the longest quiescent period with relatively little cardiac motion, its onset shows a strong dependence on the heart rate which is a liability in long experiments such as 31 P MRS. End-systole, however, provides smaller inter-and intra-subject variability, and is much less affected by the heart-rate 30 . This is particularly relevant if the heart rate is expected to change, e.g., during a long scan or under increased work-load (i.e. stress), hence making end-systole even more favourable for triggering to.
Cardiac motion induced variance can be largely explained by voxel filling, a main limitation of this study lies in the lack of respiratory motion correction. For multi-nuclear sequences currently no robust methods are available by default. Application of respiratory belts can be challenging 31 , as is the implementation of imagebased navigators 32 , and only few successful approaches have been reported on the matter 20 . In our specific case, typical respiratory displacements 33 are smaller than the effective voxel (indicated by the shaded area in Fig. 4a). Even if respiratory motion somewhat obscured the effect of triggering, our results provide conclusive evidence for improvements certainly attributable to triggering. With smaller voxels the correction of breathing motion can substantially improve spectra, as known from 1 H MRS 22 .
Since our signals are normalized to the total acquisition times and the flip angle was lower than the Ernst angle, the potential gain through triggering is underestimated. The SNR of ATP and PCr are especially relevant in kinetic studies, i.e. magnetization transfer measurements of the creatine kinase reaction, and our results are directly applicable there.
Spectral contamination due to signal arising from blood heavily affects the accuracy of cardiac 31 P data. Blood 2,3-DPG is overlapping the small P i peak in 31 P spectra. Blood also contains ATP and P i , adding to their quantification uncertainty proportional to the 2,3-DPG signal. Hence, reducing 2,3-DPG signals directly, as achieved by triggering to end-systole, increases the reliability of the ATP and P i signals.
In most voxels P i was visible at least as a shoulder to 2,3-DPG and quantifiable. P i quantification and hence pH calculation accuracy are limited by the relatively high CRLBs. In particular the chemical shift of P i could be best fitted end-systole, while CRLBs were negatively affected by triggering to diastasis. This demonstrates that even at shorter scan and repetition times than previously used 12 , it can be measured by 7 T 31 P MRS, provided that B 0 shimming is done. www.nature.com/scientificreports/ We conclude, that triggering of cardiac 31 P MRS and the selected cardiac phase have a substantial effect on data quality and signal yield. Cardiac 31 P MRS without triggering is feasible, however, best signals and CRLBs are achieved with triggering to end-systole.

Methods
The study was approved by the ethics committee of the Medical University of Vienna, Austria (EK Nr. 1670/2017) and conducted according to the Declaration of Helsinki in its latest version. Informed consent was obtained from all participants. Twelve healthy subjects (6f / 6m, age 21-39 years, body mass index 20.7-25.2 kg/m 2 ) were studied of which two were scanned twice. The measurements were performed in supine position on a Magnetom 7 T MR scanner (Siemens, Erlangen, Germany) using a cardiac 31 P loop ( d = 14 cm ) and 14 × 22 cm 2 1 H surface coil (Rapid Biomedical, Rimpar, Germany).
Subjects were equipped with an acoustic triggering device (Mri.Tools GmbH, Berlin, Germany) for synchronizing MR acquisitions, and a pulse oximeter as backup. In case of two volunteers with unreliable acoustic trigger www.nature.com/scientificreports/ signal, the pulse oximeter was required for online triggering. In these cases, trigger delays were set accordingly to compensate the temporal delay between pulse and acoustic trigger (see Fig. 1a) 34 . Cardiac localizer views were acquired during exhaled breath-hold state using a retrospectively gated 2D cine FLASH (Fig. 1b-i). Per subject second order B 0 shimming was performed before the first 31 P scan using coronal dual-echo GRE fieldmap scans with the shim volume covering the whole heart. The triggered (no delay) and breath-hold (exhaled) shim scans were repeated several times until shim currents converged. Acquisition weighted (3 averages) pulse-acquire chemical shift imaging (CSI) 35,36 was used to acquire localized cardiac 31 P spectra (Fig. 1j-n) with the following settings: receiver bandwidth 6 kHz and 512 complex samples; sampling duration 85 ms; nominal spatial resolution 8 × 16 × 8 with Hamming filtering; voxel sizes: 27.5 × 13.75 × 25 mm 3 (9.5 ml). The CSI volumes were aligned to the heart's long-axis, the septum and with the highest resolution parallel to the chest wall, to minimize contamination from skeletal muscle. The CSI measurements were repeated in each subject, with one untriggered and four acoustically triggered acquisitions. Trigger delays were identified visually on the cine images reconstructed to 20 cardiac phases and equidistantly distributed between early systole and end-diastole. Cine images were reacquired between CSI scans to confirm trigger delays for the selected cardiac phases. In most volunteers slightly increased heart-rates ( 9 ± 12 % ) were observed during their last compared to the first CSI scan. In most cases the drift was smaller than the temporal resolution of the cine images. This did therefore neither affect the selected trigger delay nor substantially reduce the effective repetition time T R,eff (see Table 1).
Every second trigger signal was used to start 31 P CSI acquisitions, resulting in a T R of approximately 2 s (see also Table 1). In untriggered acquisitions, T R was set individually to two R-R intervals. The seemingly odd choice for the repetition time was selected as a compromise between efficiency and visibility of inorganic phosphate (P i ). Our lower flip angle weighs the signal favorably to long T 1 P i in contrast to 2,3-DPG from flowing blood. The total measurement time was dependent on the individual heart rate, resulting in 12.7 ± 1.5 min for untriggered and 13.9 ± 1.4 min ( P = 0.005 ) for triggered scans, respectively. The whole scan sessions took between 105 min and 115 min.
For analysis, four voxels per subject were selected as shown in Fig. 1b-i, with the positions referred to as: anterior apical, posterior apical, anterior basal and posterior basal. Voxel-wise flip angle estimations were determined using an external reference, as described previously 10 .
In total 280 spectra were analyzed using AMARES as implemented in the OXSA toolbox 37 , fitting the following metabolites as Lorentzian lines: PCr, α -and γ-ATP doublets, two phosphodiester resonances of glycerophosphocholine and glycerophosphoethanolamine, two 2,3-DPG peaks as singlet (3-DPG) and doublet (2-DPG) 38 and inorganic phosphate (P i ). To improve the convergence of the fit for P i , linewidth and phases were locked within two groups: (i) PCr and P i and (ii) phosphodiesters. Additionally, the phases of the 2,3-DPG peaks were locked to the phase of the PCr peak. Correction of ATP and P i signals for contribution from blood was based on the 2,3-DPG signals ( γ-ATP/2,3-DPG = 0.279 and P i /2,3-DPG = 0.070 12,39 ), followed by saturation correction using 7 T 31 P T 1 relaxation times 10,12 . For saturation correction, the effective repetition time T R,eff was estimated as the median of the duration between readouts. To account for potentially longer acquisition time ( t acq ) during triggered scans, peak amplitudes (S) were compared using signal per unit time ( S/t acq ). SNR was determined as the peak height over the baseline standard deviation after application of a matched filter 10 . Changes in signal can be considered equivalent to changes in SNR as long as the noise characteristics are the same between trigger times. When comparing signals acquired with different trigger delay and from different voxels, we used signal amplitudes since the limited number of noise samples introduced additional variance to www.nature.com/scientificreports/ SNR. Cramér-Rao lower bounds (CRLBs) 40 were used to determine uncertainty of metabolite concentrations and ratios. PCr/ATP ratios were calculated using γ-ATP, and P i fits with CRLB > 50 % were excluded. pH was derived from a modified Henderson-Hasselbalch relationship 41 . Two-way analysis of variance (ANOVA) was employed to check for effects of trigger delay and voxel position on the spectral parameters of the metabolites PCr, γ-ATP, 2,3-DPG and P i , and their respective ratios. The trigger delays providing minimal tissue velocities induced by cardiac contraction are the two quiescent phases in the cardiac cycle during end-systole and diastasis, as explored for cardiac MR angiography 42 . Hence, pairwise differences between untriggered, end-systole and diastasis data were examined during post-hoc tests (Bonferroni corrected paired Student's t-tests).
The left ventricle was drawn manually on a stack of short-axis cine images with ten phases per cardiac cycle. The generated mask convolved with the spatial response function delivered the myocardial filling factor for each of the selected CSI voxels. Spearman's rank correlation coefficient r s of the filling factor and the metabolite signals, ratios and CRLBs was evaluated.