mrs studies of creatine kinase metabolism in human heart · mrs studies of creatine kinase...

MRS Studies of Creatine Kinase Metabolism in Human HeartPaul A. BottomleyJohns Hopkins University, Baltimore, MD, USA

The heart is the largest consumer of energy per gram of tissue and the creatine kinase (CK) reaction is its primary cellular energy reserve, providingadenosine triphosphate (ATP) to fuel contraction by shuttling phosphocreatine (PCr) from the mitochondria. For 30 years the measurement of themyocardial PCr/ATP ratio has been a common focus of human cardiac phosphorus (31P) magnetic resonance spectroscopy (MRS). Today, our ability tomeasure the absolute concentrations of PCr and ATP, the CK reaction rate and flux with 31P MRS, and the total creatine pool (CR) using proton (1H)MRS, allows almost complete characterization of CK metabolism in the healthy and diseased human heart. The methods and limitations of humancardiac MRS for measuring CK metabolism are reviewed herein, along with the application and findings in ‘normal’ physiological processes includingaging, diet, sedentary lifestyle, hypoxia, and obesity. The results from studies of patients with myocardial infarction, ischemia, dilated and hypertrophiccardiomyopathies, valve disease, heart failure, diabetes, and other disorders are summarized, with the quantitative findings tabulated. The myocardialPCr/ATP ratio is a sensitive indicator of cardiac energy reserve, existing in a meta-stable state that can be upset in many disorders. Reductions inmetabolite concentrations and the forward flux for delivering ATP are associated with myocardial infarction, congestive heart failure, cardiomyopathy,and disease severity. Moreover, metrics of CK energy reserve and supply can independently predict long-term cardiovascular outcomes, and are nowbeing used to quantify the effect of pharmaceutical and lifestyle intervention on the heart’s energy budget.Keywords: heart, magnetic resonance spectroscopy (MRS), energy metabolism, quantification, heart disease, cardiomyopathy, heart failure, aging,diabetes, creatine kinase reaction

How to cite this article:eMagRes, 2016, Vol 5: 1183–1202. DOI 10.1002/9780470034590.emrstm1488

IntroductionThe heart is the largest consumer of energy per gram of tissue,and disruptions in energy metabolism, including energy supplyand demand, are thought to play a central role in many com-mon diseases of the heart.1,2 It was therefore inevitable that thefirst human in vivo cardiac magnetic resonance spectroscopy(MRS) in 19853,4 focused on those energy metabolites thatwere shown by earlier animal studies5–8 (see also Cardiac MRSStudies in Rodents and Other Animals) to be NMR detectable.Human cardiac MRS required the development of high-field,high-homogeneity magnet systems capable of accommodatingthe torso.9 The performance advantages of such systems forMRI, the sister technology of MRS,10 has so entwined the twothat human cardiac MRS is today inconceivable without MRIguidance and gradient localization.

Human phosphorus (31P) MRS can noninvasively detectendogenous adenosine triphosphate (ATP), the energy thatfuels the cardiac pump via the ATP-ase reaction,1

ATP ⇐⇒ ADP + Pi + ⟨energy⟩. (1)

Inorganic phosphate (Pi) is a by-product whose pH-dependent chemical shift provides a measure of intracellularpH.11,12 It can measure phosphocreatine (PCr),2–7 which servesas the heart’s primary energy reserve. ATP is synthesized fromPCr and adenosine diphosphate (ADP) via the creatine kinase

(CK reaction):

PCr + ADP + H+k−1⇐⇒k1 ATP + Cr (2)

releasing unphosphorylated creatine (Cr). It can even measurethe forward (k1) and reverse (k−1) reaction rate constants(see Measuring Biochemical Reaction Rates In Vivo withMagnetization Transfer).13 Switching to hydrogen (1H) MRSallows documentation of intra- and extra-myocellular lipidfuel deposits in the myocardium,14,15 whose accumulation islinked to cardiac dysfunction, obesity, and diabetes. 1H MRScan also be used to measure the total creatine pool (CR),comprising PCr plus Cr.16–18 Thus, in combination, 31P and1H MRS could almost completely characterize CK metabolismand energy supply in the human heart.16 The depletion of CRin heart failure17 and in myocardial infarction (MI),18 whichis routinely diagnosed by an elevated serum concentration ofmyocardial CK enzyme, suggests its potential as an in situ 1HMRS metabolic marker. Meanwhile, significant reductions inCK energy flux measured by 31P MRS suggest a mechanisticrole for CK metabolism in the failing heart (see MRS in theFailing Heart: From Mice to Humans)13,19 that can be directlytargeted for therapy.20

Access to the myocyte’s metabolic machinery for producingATP energy via oxidative phosphorylation (OXPHOS) usingcarbon (13C) MRS is limited by the low isotopic abundanceof 13C, although it might be useful for seeing endogenouslipids and perhaps glycogen in the human heart.21 However,

Volume 5, 2016 © 2016 John Wiley & Sons, Ltd. 1183

PA Bottomley

Myofibril

Energy sink Energy source

Cytosol Mitochondria

ATP

ADP + Pi ADP + Pi

CK CK

PCr PCr

CK

k1

Cr Cr

OXPHOS

ATP

Figure 1. Illustration of the CK shuttle hypothesis in myocytes.23,24 ATPgenerated by oxidative phosphorylation (OXPHOS) in the mitochondria(right) phosphorylate Cr via the CK reaction, producing PCr. The pro-cess may continue in the cytosol until PCr arriving at the myofibrils (left),combines with ADP via the CK reaction to recreate ATP to fuel an ensu-ing contraction. Unphosphorylated Cr disperses to the mitochondria forrephosphorylation, resulting in a net transfer of biochemical energy frommitochondria to myofibrils. The CK pseudo first-order reaction rate, k1,measured by 31P MRS saturation transfer,13 reflects the tissue average trans-fer rate (center)

recent approaches employing exogenous, hyperpolarized, 13C-enriched metabolic substrates offer new minimally invasivetools for probing OXPHOS ATP synthesis in future humanstudies.22

Human 1H MRS studies of cardiac lipids, 13C MRS, hyperpo-larization, and cardiac MRS in animals are reviewed elsewherein this volume (see Cardiac Lipids by 1H MRS; Integrationof 13C Isotopomer Methods and Hyperpolarization Providesa Comprehensive Picture of Metabolism; HyperpolarizationMethods for MRS; Cardiac MRS Studies in Rodents and OtherAnimals). The present article focuses on CK metabolism, whichinvolves 31P MRS except for the use of 1H MRS to measureCR.16–18 The importance of CK metabolism stems from itsputative role as an intracellular spatial energy shuttle andtemporal buffer to facilitate high-energy phosphate trans-fer – in the form of PCr – from the mitochondria where ATPis produced by OXPHOS, to the myofibrils to fuel muscularcontraction (Figure 1).23,24 The by-product, Cr, ultimatelyreturns to the mitochondria for rephosphorylation. This role issupported by observations that PCr begins to deplete almostimmediately to maintain ATP levels at the onset of criticalischemia or hypoxia,1 and that the resting CK rate is many-foldfaster than OXPHOS,13 as would be required for a temporalbuffer. Accordingly, the ratio of reactant to product, PCr/ATP,has long been considered a sensitive index of energy reserve,and the PCr/ATP ratio is by far the most measured parameterin cardiac MRS.

Since an earlier human cardiac MRS review in 2008,25 thenumber of papers reporting PCr/ATP values in cohorts ofhealthy controls has doubled to over 110 studies of more than1600 volunteers. The NMR field strength (B0) for human car-diac studies has been extended from 1.5 to 7 T (Figure 2).26–28

Protocols for quantifying absolute concentrations of the CKsubstrates, [PCr] and [ATP],29 the CK rate constant k1 and

the flux of the ATP supply, k1⋅[PCr], have also been workedout for human cardiac 31P MRS at 3 T.30–32 The short takein toto is that one or more of these parameters often showprofound differences in acute33 and chronic heart disease andmay independently predict cardiac events and outcomes.34,35

Cardiac MRS methods and results are now reviewed, startingfrom the limitations imposed by the low signal-to-noise ratio(SNR) of CK metabolites. The methods of metabolite detection,localization, and artefacts are then summarized, and the rangesof CK measures in the healthy and diseased human heartpresented.

MethodsSignal-to-noise Ratio (SNR)SNR Relative to 1H MRS. The SNR of a metabolite with an MRSmoiety bearing a number, NX

ml, per unit volume of ‘X-nuclei’with spin IX can be deduced from The Basics (Section 5;Equations 32 and 33 therein). If a set of NX transients aredetected and averaged from a tissue volume VX using a receiverwith bandwidth BWX at an NMR frequency !X, the SNRrelative to a 1H NMR experiment (subscripts H) at the sameB0 is

SNRXSNRH

=!XMXVXNX

ml|B1X|√

NXRHBWH

!HMHVHNHml|B1H|

√NHRXBWX

× 10(NFH−NFX)∕20

(3)Here the M, B1, and R are, respectively, the nuclear magnetiza-tion per Tesla per unit volume, the magnitude of the circularlypolarized transverse RF magnetic field produced at the volumeby the detector excited with unit current, and its RF resistancewhen loaded by the sample. The NFs are the MRI/MRS sys-tem noise figures at the two frequencies. Assuming that thenoise is sample dominant, and that the losses are inductive atboth frequencies36 and derive from the same sample volume,√

RH∕√

RX = !H∕!X. But because

MXMH

= NXml!2

XIX(IX + 1)NH

ml!2HIX(IH + 1) (4)

for spin- 1∕2 nuclei, equation (3) reduces to

SNRXSNRH

=!2

XVXNXml|B1X|

√NXBWH

!2HVHNH

ml|B1H|√

NHBWX(5)

=!2

XVXNXml√

NX

!2HVHNH

ml√

NH(6)

Equation (6) assumes that the detector coil efficiencies (B1 perampere) and receiver bandwidths are the same at the two fre-quencies. These expressions include the intrinsic difference inNMR sensitivity of nucleus X versus 1H (via the !2 terms), butignore differences in the system NF, as well as NMR excitationand relaxation effects.

Cardiac MRS of endogenous metabolism in humans islimited by low metabolite SNR primarily due to low metaboliteconcentrations, as reflected by the NX

ml term above. In thehealthy heart, [ATP], [PCr], and [CR] are present at about 6,10, and 30 μmol g−1 wet weight, respectively.25 This compares

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MRS Studies of Creatine Kinase Metabolism in Human Heart

0

10

(b)

(a)

(c)

2,3-DPG

PDE

PCr

γ-ATP α-ATP

β-ATP

3 T

7 T

5 0 −5 −10 −15 ppm

10 0

10

20

30

40

Figure 2. Comparison of typical cardiac 31P spectra at 3 T (blue) and 7 T (red) from a 5.6 ml voxel in the septum. (a) Spectra are apodized with an expo-nential filter matched to the PCr line width and scaled to illustrate the SNR advantage (y-axes offset for clarity; 2,3-DPG, blood 2,3-diphosphoglycerate; PD,phosphodiesters). (b) Shows short-axis, and (c) four-chamber long-axis cine localizer images acquired at 7 T, and overlaid with the position of the septalvoxel marked in red. The yellow stripe shows the location of a saturation band used to minimize chest contamination. (Reproduced from Ref. 26. © JohnWiley & Sons, Ltd., 2014)

to about 43 mmol g−1 for tissue water (77% by weight), whichhas a 1H concentration of 2× 43= 86 mmol g−1. For PCr andATP, NX

ml∕NHml are thus 1.2× 10−4 and 7× 10−5, respectively.

After accounting for the lower sensitivity of 31P [borne by thequadratic frequency terms in equation (5)], the SNR of PCrin a cardiac 31P MRS experiment is about 1.9× 10−5 timeslower than an equivalent 1H NMR experiment on tissue water(assuming VX/VH =BWH/BWX =B1X/B1H = 1) at the same B0.A 1H MRS CR experiment performed on the 3.0 ppm moietywith its 3 protons will have 45 times higher SNR than the 31PMRS PCr experiment. Nevertheless, CR SNR is still 9× 10−4

times lower than tissue water. These enormous SNR deficitsmust be made up somehow.

Negotiating the Low-SNR Experiment. Since increasingthe B0 by orders of magnitude cannot be contemplated[and would preferentially advantage 1H MRS due to thequadratic frequency dependencies in equation (5)], othersacrifices must be made, and spatial resolution is theprime target. Choosing a 10 ml 31P MRS voxel instead ofa 0.05× 0.05× 0.3= 7.5× 10−4 ml 1H MRI pixel buys a factorof about 13 000, bringing the shortfall to within a factor of4 of tissue water, for example. It is only after this sacrificethat increasing B0 or scan time (or number of averages, NX)by a factor of 2 or 3 can have much practical effect on SNR.Bandwidth is also an easy target for improving SNR, especiallyrelative to 1H MRI whose bandwidths (BWH) are often 16 kHzor higher. For a cardiac MRS experiment, equation (5) showsthat setting BWX ∼ 1 kHz compared to 16 kHz can increaseSNR fourfold. NF should also not be overlooked. Non-1HMRS is often an afterthought in commercial MRI scanners:the author has measured >2-fold losses in cardiac 31P MRSSNR due to insufficient preamplification or inadequate digital

noise suppression in two different leading brands of MRI/MRSsystems operating at 3 and 1.5 T (see The Basics and MagneticResonance Spectroscopy Instrumentation on how to measuresystem NF). Given the huge cost and siting requirements ofMRI/MRS magnets, the option of increasing B0 to obtainhigher SNR is really only justifiable after all the other factors inequation (5) have been optimized.

At the scanner, the total examination time is typically lim-ited by patient tolerance to about an hour. Allowing 20 minfor positioning, performing rudimentary MRI to guide MRS,plus setting up MRI/MRS, means that the total number ofNX transients that can be acquired can consume at most,about 40 min. To detect a 20% change in an MRS metric ofinterest – say, PCr/ATP or [PCr] – requires an SNR> 5 perdata set. If multiple data sets are needed to determine thatmetric within said 40 min acquisition window – say, four fora CK reaction rate13 or a metabolite spin–lattice relaxation(T1) measurement, or for reference scans for quantification29

or studies involving rest and exercise33 or pharmaceuticalintervention20 – then this SNR must often be realized in under10 min. The spatial localization method and study protocolmust be tailored accordingly lest the study fail to achieve itsobjectives.

Nuclear Overhauser Enhancement (nOe). The SNR of car-diac MRS performed on non-1H nuclei such as 13C and 31Pcan be improved by the combined effect of 1H nOe (nuclearoverhauser enhancement) and 1H-decoupling21,37–41 (seeThe Basics). This technique requires the addition of an RFexcitation channel that provides an essentially continuouslow-level 1H irradiation via associated 1H excitation coils, andelectronics to isolate the 1H nOe/decoupling power from the31P (or 13C) receiver.21,38 At 1.5 T, the reported 31P MRS nOe


PA Bottomley

for PCr, "-ATP and #-ATP in the human heart are, respec-tively, $(= the fractional increase in signal)= 0.61± 0.25 SD(standard deviation), 0.6± 0.3, and 0.3± 0.2. Thus, SNR gainsof up to 1.6-fold are possible.38,40 Note that the nOe is likely B0dependent. At 3 T, SNR gains from nOe of 13–43% are reported(mean PCr gain, 1.23-fold, ±0.14; "-ATP, 1.27 ± 0.35; #-ATP,1.14± 0.29).41

Cardiac MRS Detection CoilsSmall ‘surface coils’ implanted directly about the heart inintact animals7 or on isolated perfused hearts8 can be usedto study in vivo high-energy phosphate metabolism duringischemia and monitor response to therapy. Indeed, all humancardiac 31P MRS performed since the first studies in the 1980shave employed surface detection coils placed on the chestclosest to the anterior myocardium.3 Such spectra are typicallydominated by signals originating close to the coil due to itsnonuniform sensitivity profile (see Surface Coil NMR: Detec-tion with Inhomogeneous Radiofrequency Field Antennas;Surface and Other Local Coils for In Vivo Studies; DetectionCoils for MRS; Quantifying Metabolite Ratios and Concen-trations by Non-1H MRS). The nonuniform sensitivity alsoaffords surface coil-based detectors an optimum SNR whenthe noise is sample dominant (which is typical for human torsostudies), by limiting the effective volume of tissue that cancontribute noise (see The Basics). This SNR advantage derivesprimarily from the 1/

√R dependence of equation (3).

The optimum SNR at a region of interest (ROI) lying at depthd on the axis of a tuned loop detector, has a radius a= d/

√5.

Given approximate depths of the heart from the chest of7–15 cm, this translates to an optimum detector comprisedof about 7–15 cm diameter surface coils.42,43 A multichannelphased array comprised of such coils and arranged against thechest adjacent to the heart, can yield, in practice, about 80% ofthe ultimate intrinsic SNR (the maximum SNR that can be hadby any design assuming zero detector noise).42 This translatesto SNR gains of 6- to 10-fold compared to 1H body coils.This has not gone unnoticed in cardiac 1H MRI where torsoarrays employing similar principles are routinely used. CardiacMRS that does not use similar technology may thus be furtherSNR-disadvantaged relative to state-of-the-art cardiac 1H MRI.

Bearing in mind that it is always possible to make a lousydetector of any geometry, in addition to (i) the a= d/

√5 rule,

other ‘rules of thumb’ for cardiac MRS detectors are that:

(ii) Noncircular coils (square, etc.), and ‘figure-8’ or ‘butter-fly’ coils are less-efficient and will generally have lowerSNR than circular loops.

(iii) A ‘quadrature’ coil pair comprised of a figure-8 coilplus circular loop will generally underperform a simplephased array comprised of loop coils.

(iv) A multiturn (distributed capacitance) loop coil will gen-erally outperform a single-turn (distributed capacitance)loop coil, if they are both operating well below the coil’sself resonance frequency.

(v) Finally, because the heart is closer to the surface of thechest when a subject is oriented prone compared to asupine orientation, and due to the nonuniform sensitivity

of the optimized surface coil, a prone orientation can yield2–3 times the SNR achieved by a supine orientation.43,44

Localization in Cardiac MRS31P MRS. By itself, a surface coil placed on the chest is unsuit-able for localizing MRS signals to a heart because the detectedspectrum will be dominated by signals from the interveningsuperficial chest muscle. The first localized 31P MRS of thehuman heart was performed using depth resolved surface coilspectroscopy (DRESS) with slice-selective excitation and MRIgradients positioned using 1H MRI guidance.3 Cardiac 31PMRS studies performed since 2008 have utilized the followingmethods:

1. one-, two-, and three-dimensional (1-D, 2-D, and 3-D)chemical shift imaging (CSI; see CSI and SENSE CSI andQuantifying Metabolite Ratios and Concentrations byNon-1H MRS)33,45,46;

2. 1-D and 3-D image-selected in vivo spectroscopy (ISIS; seeSingle-Voxel MR Spectroscopy)47;

3. 3-D CSI augmented at 3 T with 31P saturation bands48 or‘crusher’ gradients applied using a surface spoiling gradi-ent coil at 7 T,28 to minimize contamination from chestmuscle and surrounding tissue;

4. 3-D CSI using the spatial localization with optimum point-spread function (SLOOP) method (see Accurate and Effi-cient Localized Spectroscopy from Anatomically MatchedRegions: SLOOP and Its Enhancements)40,49; and

5. spectroscopy with linear algebraic modeling (SLAM; seeAccelerated Spatially Encoded Spectroscopy of Arbitrar-ily Shaped Compartments Using Prior Knowledge andLinear Algebraic Modeling).50

The CSI experiments are preferably ‘acquisition weighted’ bycollecting more signals with the lower order phase-encodinggradients than with the higher order gradients in a gradedfashion,51 or by using a Fourier series window (FSW)52 strategy,in order to optimize the point spread function (PSF) whichdefines the spatial resolution of the technique. These methodsand the earlier DRESS (see Single-Voxel MR Spectroscopy)3

and rotating frame zeugmatography (RFZ; Localized MRSEmploying Radiofrequency Field (B1) Gradients)53 meth-ods of cardiac MRS are detailed elsewhere and will not berevisited here.

Often, hybrid methods that combine CSI with DRESS or ISISto encode one or more dimensions are used in cardiac 31P MRSto reduce the dimensionality or number of CSI phase-encodingsteps while retaining full 3-D gradient-mediated localization.Common variants are as follows:

6. 2-D CSI with slice-selective excitation (DRESS) in thethird dimension39,45,54,55; and

7. 1-D CSI combined with 2-D ISIS.12,56,57

In method 6, the slice-selective gradient refocusing lobe is runcoincident with the phase-encoding CSI gradients. In method7, the basic 1-D CSI sequence is preceded by two slice-selectiveinversion pulses applied in the other two non-CSI dimensions,and repeated with all four combinations of inversion pulses



applied and not applied. The resulting four signals are addedand subtracted for each phase encode in the manner of2-D ISIS.

The hybrid 3-D ISIS and 3-D CSI methods yield substan-tially rectangular- or cubic-shaped voxels. SLOOP and SLAMyield arbitrarily shaped voxels that conform to the segmentedportion of the heart.40,49,50 The 1-D methods alone, yield fuzzilydefined voxels whose shape and extent in the two dimensionsnot explicitly defined by the gradients, are determined bythe intersection of the 1-D gradient-localized plane with thedetector coil’s sensitivity profile. This means that the larger thedetector coil or detector array is, the poorer the localizationin those dimensions. It also means that surface coil detectorsshould be positioned with the ROI centered on the coil’s cylin-drical axis and that the 1-D-localizing gradient be directedalong the cylindrical axis to localize a set of ‘sensitive disks’parallel to the surface coil.

Indeed, the sensitivity profile of the detection system mod-ulates the MRS signal acquired by every localization methodanyway. This preferentially weights the signal contributions inevery voxel to favor those that lie closest to, say, an optimum7–15 cm diameter surface coil. The weighting is typically largeenough to warrant sensitivity corrections to measurements ofcardiac tissue volumes when they are used to quantify absolutemetabolite concentrations in voxels with dimensions >1 cm(see Quantifying Metabolite Ratios and Concentrations byNon-1H MRS).29 In the case of the RFZ method which alsogenerates a set of 1-D-resolved spectra, the voxel shapes arefurther modulated by the gradient contours of the B1 field of theexcitation coil (see Localized MRS Employing RadiofrequencyField (B1) Gradients).53

1H MRS. Cardiac 1H MRS could use the same localizationmethods as 31P MRS, and these can work for studies of lipidsand water. However, multiple factors can confound the use ofCSI and ISIS for acquiring uncontaminated 1H spectra of low-concentration cardiac metabolites like CR. First, there is theintense water resonance from tissue and moving blood, and,second, the intense lipid resonance from pericardial fat andthe chest. Third, there is cardiac and breathing motion. Fourth,intense signals can ‘bleed’ into cardiac voxels from adjacentchest voxels due to an imperfect PSF for CSI, or to incompletesignal cancellation in ISIS whose localization depends onspectral subtraction. Fifth, B0 inhomogeneity across the CSIfield of view (FOV) combined with the much smaller chemicalshift dispersion of 1H compared to 31P can exacerbate all theseproblems.

On the other hand, unlike 31P where spin-echo methods areavoided due to signal loss from J-coupling and spin–spin (T2)relaxation, 1H moieties are generally blessed with longer T2s.This renders them amenable to spin-echo localization methodsthat can avoid the excitation of large FOVs containing much ofthe troublesome water and lipid signals. Thus, 1H MRS studiesof human myocardial CR have to date have utilized single-voxel point-resolved surface coil spectroscopy (PRESS)17,58,59

or stimulated-echo acquisition mode (STEAM)18 localiza-tion with water suppression, as detailed in Single-Voxel MRSpectroscopy. These methods yield CR measurements fromabout 3–9 ml voxels in scan times of <10 min at 1.5–3 T.

Known Artefacts of Cardiac MRSSignal ‘Bleed’ and Partial Volume. The primary artefact incardiac 31P MRS is the contamination of cardiac spectra fromextra-voxel signals due to imperfect localization. For CSImethods, the artifact is commonly called ‘bleed’ and arisesfrom the combined effect of the sinc-function-shaped PSFextending into adjacent voxels, and tissue heterogeneity withineach voxel. When the ‘center of mass’ of the signal in eachvoxel coincides with the geometric center of the voxel, its PSFis sampled perfectly at every zero crossing in adjacent voxels,and there is no bleed artifact.60 However, when the signalcenter of mass in the voxel is offset from the voxel’s geometriccenter, its PSF in adjacent voxels is sampled at nonzero pointson the PSF’s side lobes.61 The nonzero signal alternately addsor subtracts to the signal in adjacent voxels because the PSFhas both positive and negative lobes. The likelihood of suchartefacts increases with larger MRS voxel sizes because of theincreased probability of anatomic heterogeneity (it is not due toCSI grid sizes that use eight or more phase-encoding steps60).

When the PCr distribution in a 31P chest voxel is heteroge-neous, the contamination will generally subtract from the PCrin a bordering cardiac voxel, but add to the next deepest car-diac voxel.60 Because [PCr] in chest muscle is over twice that inheart but [ATP] is the same, the effect is to alternately decreaseand increase the apparent PCr/ATP ratio with depth into theheart. The amplitude of the modulation is exacerbated by thehigher sensitivity of a surface detector coil in the chest, whichamplifies the intensity of the muscle signal relative to the car-diac signal. On the distal side, the absence of PCr in ventricularblood which does contain [ATP], albeit at a lower level thanmuscle, reduces the apparent PCr/ATP ratio in voxels that inter-sect the ventricular chamber in proportion to the partial vol-ume present in the voxel. The same is true of the nearby liver,which also contains no PCr.

These problems are alleviated by minimizing the diameterof the detector coil (see section titled ‘Cardiac MRS DetectionCoils’) – since larger coils increase the volume sensitivity toextraneous sources; and/or by applying saturation pulses to thechest28,48; and/or by using acquisition weighting to improve thePSF by reducing the amplitude of the side lobes51,52; or throughthe use of post-acquisition spatial filtering (e.g., applying aFermi filter) which does the same as acquisition weightingbut broadens voxel size; and/or by applying intra-voxel tissueheterogeneity correction methods (see Quantifying MetaboliteRatios and Concentrations by Non-1H MRS).

Motion, Chemical Shift Displacement Artifact and PartialSaturation. The primary artefacts that distinguish the applica-tion of PRESS and STEAM 1H MRS to the heart, from theirapplication to the brain and other organs, are those arisingfrom cardiac motion and breathing, which are more intensethan anywhere else, and from the presence of intense lipidresonances, as noted in section titled ‘1H MRS’. 31P MRS isless susceptible to cardiac motion than 1H MRS because thevoxels are larger, there are no interfering peaks whose intensityis comparable to the fat or water 1H peaks, and because spinechoes are not used. However, protocols employing ISIS havean extended exposure to motion artefacts because localization


PA Bottomley

requires the subtraction of large signal volumes acquired withand without inversion over time periods of twice the pulsesequence repetition period (TR), 4TR, and 8TR for 1-D, 2-D,and 3-D ISIS, respectively. In both cardiac 31P and 1H MRS,motion effects can be ameliorated using a prone orientationand cardiac triggered acquisitions,17,18,33,44 or using a combinedcardiac-respiratory double-triggering strategy for 1H MRS (seePhysiologic Motion: Dealing with Cardiac, Respiratory, andOther Sporadic Motion in MRS).58,59

Both 1H and 31P MRS methods employing slice selectiveexcitation (DRESS, PRESS, STEAM) or selective inversion(ISIS) in one or more dimensions suffer from chemical shiftdisplacement artefact in those dimensions (see MeasuringMetabolite Concentrations I: 1H MRS; Single-Voxel MRSpectroscopy). This problem may be alleviated by usingthe strongest selective gradients and shortest RF pulsesallowed.

ISIS suffers from the added partial saturation effect impartedby its inversion pulses (see Single-Voxel MR Spectroscopy) tothe 31P metabolites, whose T1s are somewhat longer than thoseof 1H metabolites. The use of longer TRs than typical of sim-ple pulse-and-acquire approaches, may be needed to maintainconsistent saturation levels over the full ISIS cycle, albeit at acost to efficiency. A saturation effect may also arise from theecho-producing 180∘ pulses in PRESS since the residual longi-tudinal magnetization is inverted, instead of being refocused, ifthe initial pulse is not set at 90∘.

QuantificationRatios. The PCr/ATP ratio is measured from the ratio of the31P MRS peak areas of PCr and either the "-ATP or the #-ATPresonances. Because the metabolite T1s of PCr and ATP are dif-ferent and long such that 31P acquisitions are usually partiallysaturated, it is standard practice to correct the PCr/ATP ratiosfor partial saturation. This is done using either the known exci-tation flip angle at the heart and prior determined T1 values62,or by directly measuring saturation factors from unlocalizedscans.44 Table 1 lists published relaxation times for CK metabo-lites in the healthy human heart. Metabolite T1 measurementsare unavailable for patients. However, an analysis of the depen-dence of saturation factors on chest muscle content in multi-ple patient populations suggests that saturation corrections formyocardial PCr/ATP ratios do not vary significantly with dis-ease state or chest muscle contamination at 1.5 T.67 Note, how-ever, that the intrinsic T1, T1int, a theoretical measure of the T1in the absence of CK exchange, also does not differ significantlyin patients with heart failure at 3 T,32 while exchange rates dovary.13

It is also standard practice to correct PCr/ATP ratios forthe distortion introduced by ventricular blood, by subtract-ing a signal amount equal to about 0.3 times the integratedblood 2,3-diphosphoglycerate (DPG) peak, from the ATP peakarea.68 Although nOe is often not used in 31P MRS37–39, thedifferences in $ for PCr and ATP are expected to distort thePCr/ATP ratio, necessitating corrections if a physiological ratiois being sought or if interlaboratory comparisons are beingmade.38–41

Table 1. Published relaxation times for CK metabolites in the normalhuman heart

B0 (31P) (T) T1 (PCr) T1 ("-ATP) T1 (#-ATP) T1 (Pi) References

1.5 4.2± 0.2a 2.6± 0.6a 2.24± 0.54a — 621.5 4.3± 0.7 3.0± 0.5 — — 631.5 4.2 1.7 — — 642.0 4.2± 1 2.24± 0.63 2.46± 0.6 4.3± 2.4 623 3.8± 0.7 2.4± 1.1 — — 653 5.8± 0.5 3.1± 0.6 — — 664 5.3± 1.6 2.7± 0.6 — — 567 3.1± 0.4 1.82± 0.09 — — 263 7.4± 1.8b — — — 313 8.2± 1.3c — — — 323 8.4± 1.4d — — — 32

B0 (1H) (T) T1 (CR) T1 (water) T2 (CR) T2 (water) Reference

1.5 1.48 1.21 135 33.1 64

Notes: Values are means± SD; T1 in second, T2 in millisecond. "-ATP and#-ATP are the "- and #-phosphates of ATP.aReported literature average.62bThe ‘intrinsic T1’ of PCr, T1int, calculated assuming no CK chemicalexchange (see Measuring Biochemical Reaction Rates In Vivo with Mag-netization Transfer).cT1int for PCr corrected for spillover irradiation (uncorrected value∼7.5 s).32dThe spillover-corrected T1int for PCr in heart failure patients (P= 0.6 vshealthy controls).32

Total myocardial creatine, CR, in 1H MRS is usuallymeasured from its N-methyl 1H resonance at 3.0 ppm, andquantified relative to water in a nonsuppressed spectrumacquired from the same voxel. The normal CR/water ratiois about 10−3.18 For standardization, the STEAM or PRESSmeasurements of water ratios should be corrected for T2attenuation as well as T1 saturation.16–18 In the absence offormal T2 measurements, this can be done by extrapolatingCR measurements, recorded at long and short echo times,exponentially back to time zero.18

Concentrations and Reaction Rates. Absolute concentrationmeasurements are far fewer, but all that is required after acorrected ratio is obtained is a concentration reference pluscorrections for sensitivity and tissue volume differences whenthe reference and cardiac voxels are at different locations.29,45,69

Tissue water contents are surprisingly stable and fairly wellknown, enabling the water signal from a nonsuppressed 1Hspectrum to serve as a concentration reference for both 1H and31P MRS.16–18,64,69 External phosphate reference solutions areoften used as well.29

The reaction rates for the CK and ATPase reactions can bemeasured by 31P MRS saturation transfer protocols.13 Basi-cally, a first reactant moiety, say PCr, is measured with andwithout the reaction product moiety, "-ATP, saturated, andthe fractional change in amplitude of the first moiety due toits chemical reaction, is recorded. An additional experiment isthen performed to determine the T1 of the first moiety whilesaturation is applied to the second moiety, in order to convertthe fractional change to a rate. The product of the rate with



the concentration, for example, k1⋅[PCr], is the forward fluxthrough the reaction in micromole per gram second.

Protocol Specifics and Derivative Quantities. Detailed proto-cols, equations, and specific examples of quantification of 31Pmetabolite ratios, concentrations, and CK reaction rates inthe human heart are presented in Quantifying MetaboliteRatios and Concentrations by Non-1H MRS and MeasuringBiochemical Reaction Rates In Vivo with MagnetizationTransfer, to which the reader is referred. The formula fordetermining intracellular pH from the chemical shift differ-ence between Pi and PCr is in Cardiac MRS Studies in Rodentsand Other Animals, and the quantification of 1H metabolites iscovered in Measuring Metabolite Concentrations I: 1H MRS.

With measurements of PCr/ATP, PCr, CR, ATP, Pi, pH, k,and CK flux in hand, a complete characterization of cardiac CKmetabolism is but a few short steps away. First, unphosphory-lated creatine, [Cr]= [CR]–[PCr], can be derived from joint 31Pand 1H MRS measurements of PCr and CR.16 [ADP] can thenbe estimated from the CK equilibrium equation:

[ADP] = [ATP][Cr][PCr][H+]Keq

(7)

with [H+] derived from the pH and Keq (= 1.66× 10±9 l mol−1) as the equilibrium constant.13,70 The free energy ofATP hydrolysis available to fuel cardiac contraction, −%G∼ATP,is given by

%G∼ATP = %G0 + RT ln([ADP][Pi]∕[ATP]) kJ mol−1 (8)

with %G0(=−30.5 kJ mol−1) as the standard free energychange, and RT is the universal gas constant times absolutetemperature.13,70

ResultsNormal ValuesTable 2 presents the average (±SD) of the mean values ofhuman cardiac CK metabolic parameters measured by 31P and1H MRS published through 2015. These are unweighted by thenumber of subjects or SDs listed in the source publications. Themean myocardial PCr/ATP has risen slightly from 1.72± 0.26in 200825 to 1.84. The individual study data are visualized in

Figure 3. The B0 at which the studies were conducted does notsignificantly affect the mean PCr/ATP (Figure 2a) – a goodsign – and the scatter appears to decline at 3 and 4 T comparedto 1.5 T, although the 1.5 T data pool includes much olderdata. However, the reported SDs accompanying the individualmean values do not yet make a case that the increase in SNRwith B0,26,65,66 translates to more precise myocardial PCr/ATPmeasurements overall (Figure 2b). Even so, direct comparativemeasurements at 1.5, 3,65 and 7 T26 suggest otherwise, andSNR gains at higher fields are often spent on improving spatialresolution and/or speed [VX and NX in equation (3)] instead ofprecision.56,141

The choice of localization method also does not appearto systematically affect PCr/ATP or its variability overall(Figure 2c). This does not mean that localization is notimportant, but rather suggests that other factors – includingexperimental and physiological variability – are dominantsources of scatter. Regarding the former, about a half of thestudies have SDs ≤15% with a third of them below 10% overall.This demonstrates that such levels of precision are in factcommonly achievable, and suggests that experimental fac-tors unrelated to B0 or localization method per se, representthe primary sources of variability between studies of humanmyocardial PCr/ATP ratios. PCr/ATP does not appear to varywith location in the normal heart.56,106

Despite the added uncertainty in measuring reference con-centrations and other factors needed to convert metaboliteratios to absolute concentrations, cardiac [PCr] and [ATP]measurements appear to be at least as precise as the PCr/ATPmeasurements (Table 2). Although this may reflect bettercontrol of experimental variables in this more quantitativework, a caveat is that these studies remain limited to aboutfour independent research groups. The same is true of the [CR]measurements, which at 28.0± 0.7 μmol g−1 appear tightest ofall. The tabulated values for [CR], the CK rate k1, and the fluxeach reflect the efforts of just two laboratories.

The dearth of measurements of Pi undoubtedly arises fromthe difficulty in resolving it from blood DPG and other phos-phomonoester (PM) resonances in the healthy heart. Although1H decoupling can reduce the line widths of DPG and PM sig-nals and improve the spectral resolution of Pi, it is still unde-tectable in at least half of the normal subjects studied at 1.5 T.12

Moreover, Pi and pH appear ripe for characterization using the

Table 2. Literature average metabolic parameters measured in normal human heart by MRS

Variable Mean± SDa Na Source references

PCr/ATP 1.84± 0.27 1662 3, 4, 12, 13, 19, 26–29, 31, 33–35, 38, 39, 41, 45, 48, 49, 51–57, 63, 68, 69, 71–139 (114 cohorts)[PCr] (μmol g−1 wet) 10.14± 1.36 237 13, 19, 29, 35, 45, 64, 69, 87, 95, 98, 110, 120, 126, 135, 140 (16 cohorts)[ATP] (μmol g−1 wet) 6.02± 1.00 237 13, 19, 29, 35, 45, 64, 69, 87, 95, 98, 110, 120, 126, 135, 140 (16 cohorts)pHb 7.13± 0.04 31 12, 54, 70, 86k1 (s−1) 0.36± 0.06 133 13, 19, 30–32, 35, 126, 140–142CK flux (μmol g−1s−1) 3.35± 0.21 94 13, 19, 31c, 35, 126, 140[CR] (μmol g−1 wet) 28.0± 0.7 66 17, 18, 64, 143, 144

Notes: Values are means± SD of the average of the published mean values, unweighted by individual sample size or uncertainty. Excludes data known to beuncorrected for saturation or nOe.aNumber of subjects – may include same subjects reported in repeat publications.bMeasurements from Pi at 1.5 T, which is undetectable about half the time.12c Value based on a [PCr] determination scaled assuming [ATP]= 5.5 μmol g−1 wet wt.


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Figure 3. Literature measurements of the cardiac PCr/ATP ratio in healthy controls by localized 31P MRS. Each symbol represents a mean published valuefor a study cohort, corrected for saturation and blood ATP contamination. (a) The PCr/ATP ratio as a function of B0 in Tesla. (b) The reported standarddeviation (SD) in the mean PCr/ATP measurements expressed as a percentage of the mean, plotted as a function of B0. (c) PCr/ATP as a function of thelocalization method used to measure it. The data from the three left-most columns utilized 1-D gradient localization (confined in the other two dimensionsonly by a surface detector coil’s sensitivity profile). The 1-D CSI plus DRESS data were acquired with 2-D gradient localization. The right-most three columnsof data were acquired using full 3-D gradient-controlled MRS localization

better spectral resolution and SNR afforded at 7 T. Note that [Pi]in whole human blood is only about 0.08 mM.11 This would beimperceptible to 31P MRS and could not contaminate myocar-dial Pi and pH measurements.84

From Table 2, we see that PCr constitutes 36% of the totalcreatine pool in normal heart, hence [Cr]= 17.9 μmol g−1

wet. Thus, PCr’s fraction of the total creatine pool is a half ofthat available to resting skeletal muscle (i.e., 34/45 mM= 76%from Ref. 145; or 25.6/36.2 μmol g−1 = 71% from Refs 69,146). This is consistent with a more active role of the Cr/PCrsystem in shuttling energy from the mitochondria to fuel thecontracting myofibrils even at rest. ATP synthesis throughOXPHOS has been estimated from myocardial oxygen (O2)consumption, which is about 0.085 ml g−1 wet weight perminute in healthy resting subjects.147,148 Assuming 3 mol ofATP is produced per mole of O2 consumed yields a mean ATPsupply of 0.33 μmol g−1 s−1 via OXPHOS, which is consistentwith invasive measures of ∼0.43 μmol g−1 s−1.149 This meansthat the cardiac CK flux of 3.35 μmol g−1 s−1 (Table 2) would beable to shuttle up to about 10 times the energy provided by aer-obic metabolism at rest to support contraction, consistent witha CK role as a spatiotemporal buffer.13 From equation (7), thevalue of [ADP] is approximately 90 μmol l−1. From equation (8)then, %G∼ATP ≈ 60 kJ mol−1 for the normal heart.13,150

Physiological VariationsCyclic Energy Demand and Cardiac Workload. All of the CKmetabolite values in Table 2 were measured at rest and/orrepresent temporal averages. However, energy demand variesthroughout the cardiac cycle, arguably by at least threefoldbetween peak demand and relaxation.13 If the role of PCr and

the CK reaction is as a rapid energy reserve, then an obviousquestion is whether myocardial PCr/ATP varies cyclicallyin the healthy heart at rest, or during exercise. Measure-ments of PCr/ATP triggered at different points in the cardiaccycle in healthy subjects are few, but any metabolic varia-tions other than those attributable to myocardial volume, areundetectable.71 Indeed, a theoretical analysis of the CK enzy-matic rate and Bloch equations employing empirical parameterranges recently concluded that (i) the maximum intracyclechanges in high-energy phosphate would be ≈0.4 mM, whichare at or below current detection levels and (ii), that satura-tion transfer 31P MRS would be unable to detect intracyclicfluctuations in k1 or CK flux.150

If CK metabolites did vary cyclically, even modestly, thenmuch larger changes would be expected with exercise whenenergy demands would be much higher. PCr/ATP has beenstudied with increased cardiac workloads while subjects layin the scanner using three types of stress test. First, an iso-metric exercise, performed, for example, with a hand-gripdynamometer.33 Second, an aerobic exercise involving thelifting of weights with the legs,75 and, third, stress induced bypharmaceutical agents such as dobutamine.13,80 The increase incardiac workload as indexed by the heart-rate blood-pressureproduct (HR×BP) in these protocols, is limited to: (i) about40% with a hand-grip exercise involving exertion at 30% ofthe subject’s maximum force33; (ii) about twofold with aerobicexercise involving lifting multiple 2.5 kg weights74; and (iii)up to fourfold using dobutamine.80,92,96 Compared to aerobicexercising, the isometric and dobutamine protocols minimizemotion problems during acquisition.

Studies using isometric,33,85 aerobic,48,75,128 and dobutaminestress13,80,138 at up to double the HR×BP did not produce



any significant changes in PCr/ATP. However, at a higherstress of three to four times the resting HR×BP, significantreductions of 14–21% (P< 0.001) in myocardial PCr/ATP wereobserved92,99 even in athletes.96 It is interesting to note thateven though CK starts with a 10-fold buffering capacity forshuttling OXPHOS energy at rest (see section titled ‘NormalValues’), the CK flux might be expected to have difficulty meet-ing ATP demands at three to four times the resting HR×BP,if peak intracycle energy demands are also taken into account.That is of course, unless the CK flux rises to meet the increasedenergy demand at the new stress level.

To test whether CK flux upregulates with cardiac work-load, the CK rate and flux were measured in healthy subjectsduring dobutamine stress using 31P MRS saturation transfermethods.13 No significant changes were observed in PCr/ATP,[PCr], [ATP], k, or CK flux, at least for work-loads at anaverage of double the resting rate.13 If CK flux does not changewith increasing workloads, then the CK ATP supply must belimited. The observed decline in myocardial PCr/ATP at thehighest levels of stress92,96,99 would be a consequence of thislimit, which might even be a determinant of individual limitsto cardiac workload.

Cardiovascular Fitness. A number of studies have exam-ined the effect of cardiovascular fitness on both resting andstressed myocardial PCr/ATP in healthy volunteers. High-leveldobutamine stress at three- to four-fold HR×BP elicitedsimilar 14–17% decreases in myocardial PCr/ATP in bothelite cyclists and age-matched healthy controls, while theirresting PCr/ATP values were also the same (1.4± 0.2).96 Amore recent study, compared resting PCr/ATP in healthymiddle-aged (∼50 years old) volunteers as a function of theirmaximum working capacity, MWC, as gauged by a steppedexercise.55 Unlike the earlier high-level HR×BP study, thisstudy found that resting myocardial PCr/ATP correlatedwith MWC and those with MWC> 230 W had significantlyhigher PCr/ATP than those with MWC <200 W (1.93± 0.36vs 1.59± 0.35; P< 0.001).55 On the other hand, a compar-ison of myocardial PCr/ATP ratios in elite track sprinters,marathon runners, and young sedentary but lean men, pub-lished the same year, found no significant differences amongthe three groups (PCr/ATP= 1.9–2.4), although athletes hadthe highest mean PCr/ATP ratios.123 A follow-up study bythe same authors showed no significant differences betweenyoung sedentary (25± 2 years; PCr/ATP= 1.94± 0.36) andyoung (26± 5 years; PCr/ATP= 2.36± 0.36) and middle-aged(47± 9 years; PCr/ATP= 2.19± 0.34) athletes, but sedentarymiddle-aged (45± 6 years) subjects had significantly lowercardiac PCr/ATP (1.83± 0.27) versus the three other youngand/or fit groups.127

Differences in myocardial PCr/ATP ratios among controlgroups that are supposed to be comparable, combined withobserved changes that often fall within normal ranges pub-lished by others, represent major distractions for these studies.However, if such differences are set aside as being attributableto systematic factors of methodological origin, a picture thatemerges in toto is that most young healthy adults in their 20sare endowed with a high cardiac reserve as indexed by a highPCr/ATP ratio of, say, 1.8–2.0, regardless of their sedentary or

Table 3. Reported age and gender variations in PCr and ATP in normalsubjects

Age/gender n PCr/ATP [PCr](μmol g−1)

[ATP](μmol g−1)

References

32± 3 years 15 1.7± 0.3 13.5± 1.9 8.2± 1.4 9560± 13 years 15 1.6± 0.4 9.7± 2.5* 6.4± 1.8 9530± 6 years 37 2.16± 0.36 — — 3953± 7 years 39 1.83± 0.37† — — 39<40 years 16 1.9± 0.5 9.7± 2.4 5.1± 1.0 120>40 years 14 1.9± 0.4 7.7± 2.5‡ 4.1± 0.8* 120M 18 1.9± 0.4 9.2± 2.4 4.9± 1.0 120F 12 1.9± 0.6 8.0± 2.8 4.2± 0.9 120

Notes: Means± SD as reported or calculated from standard errors; M, male;F, female.*Probability that difference is not significant, P< 0.01 versus youngergroup.†P< 0.001 versus younger group.‡P< 0.05 versus younger group.

athletic lifestyles. However, with aging, a continued sedentarylifestyle is associated with a declining cardiac PCr/ATP by thetime they reach about 50 years old, while maintenance of car-diovascular fitness through exercise can preserve myocardialCK energy reserve at near-youthful levels.

Age and Gender. Indeed, several studies have reported somevariation of PCr/ATP ratios and/or [PCr] and [ATP] con-centrations with age39,95,120 and gender,120 as summarized inTable 3. This work suggests a trend of reduced metaboliteconcentrations,95,120 and probably PCr/ATP,39 with age above40 years, and perhaps in women versus men of similar age aswell.120 Such results are consistent with a decline in cardiacCK energy reserve with age in cohorts of control patients thatinclude more sedentary individuals not selected for cardio-vascular fitness. However, it is prudent to be cautious because(i) there are systematic differences between the results inTables 2 and 3; (ii) there are systematic age- and gender-relatedvariations in chest composition that could affect the results ifchest contamination is present (see section titled ‘Signal ‘Bleed’and Partial Volume’); and (iii) some laboratories that havestudied many subjects have not reported any significant agedependence.

Diet and Obesity. Given that over a third of the adult Americanpopulation are considered obese as of 2012 and that two-thirds are overweight, the inclusion in cardiac MRS studies of‘normal volunteers’ who are overweight but do not otherwisehave exclusion criteria for heart disease, is perhaps inevitable.The effect of diet on myocardial PCr/ATP was tested in acrossover study of young men (age 22± 1 standard error, SE)on whom 31P MRS was performed before and after either a5-day high-fat diet (75% of calories) or a standard 23% fat diet.This was followed by a 2 week ‘washout’ period and crossoverto the alternate diet.134 The high-fat diet increased plasma freefatty acids by 44%, and was associated with a 9% reductionin cardiac PCr/ATP (2.0± 0.1 SE vs 2.3± 0.1 SE vs P< 0.01)along with some cognitive impairment.

Cardiac 31P MRS results from age-matched obese sub-jects (age, 44± 7 years; BMI= 39± SD kg m−2) with no


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history of cardio- or peripheral-vascular disease, smoking,hypertension (HT), or diabetes, were compared with thosefrom ‘normal-weight’ (age, 43± 10 years; body mass index,BMI= 22± 2 SD kg m−2) subjects at rest and during dobu-tamine stress (with a 63± 16% HR increase).138 At rest,diastolic function (peak filling rate) and cardiac PCr/ATPwere 15% lower in obese subjects than in the normal-weightgroup (1.7± 0.4 vs 2.0± 0.3; P< 0.05), which decreased by anadditional 12% (to 1.5± 0.5; P= 0.03 vs rest) with dobutaminestress. As in earlier studies, PCr/ATP was unaltered duringstress in the normal-weight group. A follow-up study by thesame authors compared obese patients (BMI= 34± 4 kg m−2)with ‘normal-weight’ subjects (BMI= 22± 2) before and aftera year in a supervised weight-loss program.151 At the initial31P MRS examination, the PCr/ATP ratio was 1.58± 0.47compared to 2.03± 0.27 in normal-weight subjects (P= 0.002).As a result of the weight-loss program, the obese subjectsshed 9 kg (to BMI= 29± 2), and cardiac PCr/ATP increasedby 24% to 1.96± 0.47 (P< 0.05), which was accompanied byimprovements in diastolic function.

Thus, both dietary fat and obesity are associated withimpaired cardiac CK energy reserve. In obese subjects withdiastolic dysfunction, PCr/ATP may be further reduced duringstress. This transient decrease in PCr/ATP with stress has tobe interpreted as evidence of ischemia, wherein the CK ATPsupply is unable to meet the increased workload. The goodnews is that long-term weight loss may reverse this reductionin myocardial CK energy reserve.

1H MRS studies in obesity have focused not on CR but ontriglyceride elevations in the septum, which are reviewed else-where (see Cardiac Lipids by 1H MRS).

Hypertension (HT). Hypertension, too, may affect myocardialPCr/ATP ratios. Middle-aged hypertensive patients in whomcoronary disease, malignant HT, and diabetes were all ruledout, have been studied at rest and during dobutamine stresswith a 2.2-fold increase in HR×BP. The resting PCr/ATPwas significantly lower than in healthy age-matched con-trols (1.2± 0.18 SD vs 1.39± 0.17, P< 0.05) and decreaseda further 21% with stress (to 0.95± 0.25, P< 0.01 vs rest).99

The metabolic changes were again associated with diastolicdysfunction. However, another study showed no change inresting myocardial PCr/ATP, [PCr], or [ATP] in hypertensivepatients who had a ∼50% increase in left ventricular (LV) masscompared to controls, but otherwise had no symptoms of heartfailure.110

Hypoxia. Chronic exposure to hypobaric hypoxia mayrepresent another ‘normal’ variant for human myocardialPCr/ATP. Six 20- to 30-year-old professional Sherpa trekkerguides who were native to an altitude of ∼3400 m in Nepal,exhibited significant reductions in myocardial PCr/ATPto 1.0± 0.37 SD as compared to control ‘lowlanders’ withPCr/ATP= 1.76± 0.06.52 These results suggest that the heartmay adapt its energy sources and delivery over time in responseto chronic environmental stress.52

Yet even relatively short-term exposure to hypoxia mayreduce myocardial PCr/ATP. A 31P MRS study of 14 trekkers,age 38± 11 SD years, before and immediately after a 17-day

trek to Mt. Everest base camp (5300 m) exemplifies an effect ofhypobaric hypoxia.137 Myocardial PCr/ATP was reduced 18%from 2.05± 0.30 SD before, to 1.68± 0.30 (P= 0.003) after thetrek, accompanied by some alterations in diastolic function.Cardiac metabolism and function returned to pre-trek levelsby 6 months. The effect of shorter normobaric hypoxic expo-sures were investigated by the same authors using a hypoxicchamber with O2 maintained at an end-tidal expiration partialpressure of 50–60 mmHg and 80% peripheral O2 saturation.136

After 20 h of exposure, young (age, 24± 7 SD years) healthyvolunteers exhibited 15% reductions in cardiac PCr/ATP (from2.0± 0.3 to 1.7± 0.3; P< 0.01) and impaired diastolic function.

Thus, cardiac CK energy reserve and high-energy phosphatebalance is depressed in the presence of hypoxia, even at the rel-atively modest O2 reductions that can be tolerated by healthysubjects over periods ranging from hours to lifetimes.

DiseaseMyocardial Infarction (MI). Published 31P MRS studies aremixed on whether resting myocardial PCr/ATP ratios arealtered in MI. The data are summarized in Table 4. Most likely,the mixed message arises from other conditions such as con-gestive heart failure (CHF) or cardiomyopathy (CM) associatedwith post-MI remodeling. These would confound the PCr/ATPmeasurements in chronic MI (see sections titled ‘DilatedCardiomyopathy (DCM) and Heart Failure’ and ‘Hypertrophy,Valve Disease, and Heart Failure’), while playing lesser roles inacute MI in which active ischemia was not present. Meanwhile,in acute anterior MI, Pi may be elevated a week or so post-onset,71 consistent with the post-MI time course of Pi seen incanine studies after an acute decline in PCr and ATP.154

Table 4. Resting myocardial PCr/ATP ratios in patients with MI and/orischemia (ISCH)

MI/ISCH Controls Patients References

MI 1.6± 0.4 1.7± 0.40 71MI + ISCHa 1.72± 0.15 1.45± 0.31 33MI 1.95± 0.45 No change in patients 79MIb 1.85± 0.25 1.24± 0.3* 85ISCHc 1.85± 0.25 1.6± 0.19 85MIb 1.8± 1.03 0.94± 0.41 87ISCHc 1.8± 1.03 1.37± 0.57 87MI 1.61± 0.18 1.51± 0.17 91MI 1.72± 0.31 1.47± 0.38 101MI 1.45± 0.29 0.6± 0.2d,† 108MI 1.87± 0.45 1.74± 0.27 126MI 1.03± 0.39† 152ISCH 1.39± 0.23 153Averagee 1.73± 0.16 1.34± 0.34‡

Notes: Means± SD, as reported or calculated from reported standard errors(SE); ISCH= patients with ischemia.aISCH patients includes 6 with MI (P= 0.052 vs controls).bFixed defect on exercise 201Tl imaging.cReversible defect on exercise 201Tl imaging.dAverage for septal and anterior LV MI.eAverage of the studies listed (omitting duplicate entries).*P< 0.05 in MI versus controls.†P< 0.05 in MI versus uninvolved tissue.‡P< 0.005 versus controls



Indeed, biochemical analyses in animal heart models showdepletion of essentially all ATP and PCr within the first fewhours of an ischemic injury that results in cell death.1 Becausedead cells can contribute no high-energy phosphates, theresting PCr/ATP ratios that are reported in MI must derivefrom ‘metabolically normal’, ‘jeopardized’, and/or ischemicmyocardium adjacent to, or interspersed with the infarction.Note that ‘metabolically normal’ need not mean functionallynormal. A study of 29 reperfused MI patients with dysfunc-tion in the infarct area – ‘myocardial stunning’ – found nosignificant changes in PCr/ATP from normal at 4± 2 dayspost onset, with PCr/ATP remaining unchanged a month latereven though function had improved.91 Regardless, a preservedPCr/ATP has to be interpreted as indicating the presence ofviable surviving myocardium in the MRS voxel.

The main MRS action in MI is the reduction in [PCr],[ATP], and [CR] in the infarction itself, as summarized inTable 5. Reductions of about 50% likely reflect the presence ofboth infarcted and noninfarcted tissue in MRS voxels. There isa significant negative correlation between cardiac ATP levelsand the size of perfusion deficits quantified by thallium 201Tlradionuclide imaging.78 In addition, [PCr] and [ATP] arereduced in patients with fixed 201Tl defects compared to thosewith reversible defects.87 In the tabulated studies, uninvolvedmyocardium without wall motion abnormalities at echocardio-graphy had near-normal PCr, ATP, and CR concentrations.18,64

In addition, CK rate measurements measured by saturationtransfer also showed no change in k1 in MI (Table 5). Becausethe measurements of k1 can only derive from the myocytesthat survive in the infarcted region, this result must reflect apreserved CK metabolism in the surviving tissue, even thoughthe net CK flux given by the product, k1⋅[PCr], is halved due tothe loss of [PCr] in the infarct.126

Myocardial Ischemia. In patients with ischemic heart diseaseinvolving stenosis of the anterior vessels, the resting anteriormyocardial PCr/ATP shows a declining trend (Table 4, shadedentries), with CK metabolite concentrations in reversibledefects reaching statistical significance (Table 5).87 Whilea number of other conditions are associated with reducedmetabolite levels at rest, the transient reduction in PCr/ATPratio that is elicited by increased cardiac workloads, indicatesan excess consumption of PCr that cannot be met by OXPHOS.Thus, stress-induced changes in myocardial PCr/ATP couldserve as a specific metabolic hallmark of ischemia.33

As noted in the section titled ‘Cyclic Energy Demand andCardiac Workload’, in healthy subjects who are free of signif-icant coronary disease, myocardial PCr/ATP is unaltered byup to a ∼2-fold increase in cardiac workload,13,33,75,85,128,138

but a small reduction at three to four times the restingworkload92,96,99 may arise if the CK energy supply cannotcope with demand. However, in patients with severe anteriorcoronary stenosis and ischemia verified by echocardiographyor 201Tl radionuclide imaging, a simple isometric handgripexercise that only elicited a 30–40% increase in HR×BP, wasable to produce a ∼40% reduction in myocardial PCr/ATPcompared to that at rest (Figure 4).33 After exercise, metaboliteratios recovered to near-normal pre-exercise values. Patientswith nonischemic heart disease (CM or valve disease) who

Table 5. CK metabolite levels in patients with MI

[PCr](μmol g−1 wet weight)

[ATP](μmol g−1 wet weight)

References

Control Patients Control Patients

12.1± 4.3 7.6± 3a,* 7.7± 3 6.4± 3.2a 873.9± 2.2b,† 4.4± 1.5b,* 87

11.9± 4.4 11.0± 4.4c 7.0± 1.8 7.9± 4.3c 646.4± 1.8d,* 5.4± 2.2d 645.0± 2.4e ,* 4.9± 2.5e 64

9.6± 1.1 5.4± 1.2‡ 5.5± 1.3 3.4± 1.1‡ 126

[CR](μmol g−1 wet weight)

[CR](μmol g−1 wet weight)

References


28± 6 9.8± 8.6§ 28± 6 26± 11f 1828.8± 3.0 26.5± 4.3c 28.8± 3.0 16.9± 7.1d,* 64

28.8± 3.0 13.2± 5.4e ,* 64

CK rate,k1 (s−1)

CK flux(μmol g−1 wet weight s)

References


0.33± 0.07 0.31± 0.08 3.3± 0.8 1.7± 0.5‡ 126

aPatients with MI and reversible defects on 201Tl scintigraphy.bPatients with MI and fixed defects on 201Tl scintigraphy.cNormo-kinetic by echocardiography.dHypokinetic by echocardiography.eDyskinetic by echocardiography.fMeasurements from uninvolved tissue.*P< 0.05 versus controls.†P< 0.01 versus controls.‡P< 0.001 versus controls.§P< 0.0001 versus controls.

were tested with the same exercise exhibited no PCr/ATPchanges. In addition, patients with ischemia who underwentrepeat stress testing after successful revascularization therapyalso showed no stress-induced PCr/ATP change, consistentwith a resolution of the metabolic abnormality following a suc-cessful clinical outcome.33 A similar 40% decrease in anteriormyocardial PCr/ATP was reported during hand-grip exercisein patients with reversible anterior wall ischemia confirmedby exercise 201Tl radionuclide imaging.85 Those with fixed201Tl defects indicative of MI exhibited no exercise-inducedPCr/ATP changes.

These studies, and observations that dobutamine stress-testing at 1.6-fold resting HR×BP induced no significantreductions in myocardial PCr/ATP in patients with DCM andCHF,80 suggested that stress-induced changes in PCr/ATPmight well be specific to myocardial ischemia. However, asnoted in sections titled ‘Diet and Obesity’ and ‘Hypertension(HT)’, 12% and 21% reductions in myocardial PCr/ATP havealso been observed with dobutamine stress in obese patients138

and those with HT,99 respectively, in the absence of inde-pendent evidence of ischemia. More recently, a 9% transientreduction in cardiac PCr/ATP was reported in patients who


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Figure 4. Hand-grip exercise stress testing of a patient with myocardial ischemia performed at 1.5 T.33 (a) Axial 1H surface coil image of the chest andanterior LV of a patient with an occluded right coronary and 70–90% stenosis of the left anterior coronary artery. The image is annotated with the locationsof three 1 cm thick coronal slices localized in the 31P MRS exam (SE, septum; RV, right ventricle; ENDO, endocardial; EPI, epicardial; ST, sternum; REF,reference vial in the 31P coil). (b–d) Cardiac-gated 1-D CSI surface coil 31P spectra from the three slices (b) at rest, and during (c), an 8 min isometrichand-grip exercise, and (d) a recovery acquisition commencing 2 min post exercise. The PCr/ATP decreases from 2.0 to 0.9, and 1.6 to 1.3 in the endo- andepicardial slices respectively, consistent with ischemia. The PCr/ATP ratio is unchanged in the chest (slice #14)

had HCM of a genetic etiology but no evidence of coronaryartery disease, during aerobic exercise testing at a 1.7-foldincrease in HR×BP workload (see also section titled ‘Hyper-trophy, Valve Disease, and Heart Failure’).48 This work suggeststhat factors other than frank vessel disease may also lead totransient reductions in energy reserve – that is, an ischemicresponse – in obesity, hypertensive, and hypertrophic disease.

Two additional studies have used isometric hand-gripexercise to measure myocardial ischemia. One studied‘women’s ischemic syndrome’ involving chest pain – alsoin the absence of significant coronary vessel disease –reportingthat some women had cardiac PCr/ATP ratios during stressthat were two SDs below the mean of control subjects.155

However, the overall variations in PCr/ATP were higher thanin controls and exhibited both increases and decreases duringstress. The second study was a double-blinded application ofstress 31P MRS to test the efficacy of an experimental anti-ischemic pharmaceutical that reduces the binding affinity ofoxygen to hemoglobin, thereby potentially increasing oxygenavailability.153 While the stress produced a 31% PCr/ATPreduction during control studies, the lesser 20% decline seenwith anti-ischemic therapy in the same subjects was notsignificant and the outcome was inconclusive.

Because LV dysfunction and heart failure are commonprognoses for ischemic events, another study examined theeffect of trimetazidine (TMZ) on resting cardiac PCr/ATP postischemia. TMZ is an anti-ischemic therapy that shifts energyutilization from free fatty acids to glucose.156 A double-blinded90-day crossover protocol with placebo or TMZ was imple-mented in patients with CHF but not active ischemia, acuteMI, or anterior lesions at locations where the 31P MRS wasperformed. It was found that TMZ reduced the symptom-basedNew York Heart Association (NYHA) CHF class, improvedLV ejection fraction (EF), and increased myocardial PCr/ATPfrom an abnormally low 1.35± 0.33 to 1.8± 0.5 (P= 0.03).

Dilated Cardiomyopathy (DCM) and Heart Failure. Initial 31PMRS studies of DCM in the early 1990s revealed significant68

and nonsignificant46,77 reductions in myocardial PCr/ATP. Anelevated phosphodiester (PD) peak,46,77 and an elevated Pi/PCr

Table 6. Myocardial PCr/ATP in patients with dilated cardiomyopathy(DCM)

Controls DCM NYHA References

1.54± 0.11 1.51± 0.29 I–III 771.8± 0.21 1.46± 0.31* (CHF) 681.65± 0.26 1.52± 0.58 II–III 122.09± 0.44 1.88± 0.4 n.s. 811.95± 0.45 1.78± 0.51 II–IV 79

1.44± 0.52† ≥III1.94± 0.43 <III

1.86± 0.17 1.63± 0.24a I–III 802.02± 0.41 1.54± 0.48† II–III 891.94± 0.60 1.63± 0.43b I–III 341.75± 0.25 1.26± 0.29 n.s. 1072.07± 0.17 1.31± 0.38* III–IV 122

1.63± 0.33* I–IIn.s. 1.54± 0.34 n.s. 40n.s. 1.58± 0.41 II–III 201.81± 0.49 1.59± 0.48c I–III 35n.s. 1.5± 0.4 n.s. 271.86± 0.17c 1.56± 0.16d,* Average

Notes: Values are means± SD, as reported or calculated from reported dataor standard errors (SEs) excluding repeat entries or data uncorrected forsaturation when known; NYHA, New York Heart Association classificationfor congestive heart failure (CHF); n.s., not specified.aNo change with dobutamine stress at a 1.6-fold increase in HR×BP.80bPCr/ATP< 1.6 predicted mortality at 2.5 years (P< 0.02).34cPatients had DCM or left ventricular hypertrophy (LVH).35dAverage of the studies listed.*P< 0.001 versus controls.†P< 0.05 versus controls.



Table 7. CK metabolite concentrations and flux rates in patients with cardiomyopathy

[PCr] (μmol g−1 wet weight) [ATP] (μmol g−1 wet weight) References


DCM+CHFa 10.1± 1.3 8.3± 2.6* 5.7± 1.3 5.2± 1.3 13DCM — 6.2± 1.5b — 4.1± 0.8b 40DCM+CHFc 8.8± 1.3 4.3± 1.2† 5.7± 1.0 3.7± 0.5* 110LVH+CHFc ,d — 6.3± 1.5* — 4.9± 0.9 110LVH+CHF 9.4± 1.1 7.2± 3.7 5.5± 1.3 5.0± 1.1 19LVH no CHF — 6.1± 2.0† — 4.7± 1.3 19CM+CHFc , f — 7.6± 1.8 — 4.9± 1.3 20CM+CHFa 9.75± 1.33 7.39± 2.5‡ 5.7± 1.3 4.8± 1.4* 35LVH 9.7± 2.5 6.1± 2.2† 6.4± 1.8 4.1± 1.3* 95HCMg 9.4± 1.2 7.1± 2.3† 5.8± 1.2 5.0± 0.8 140HCMh 8.8± 2.6 6.1± 1.9e ,† 4.6± 1.0 3.9± 1.5* 135

[CR] (μmol g−1 wet weight) [CR] (μmol g−1 wet weight) References


DCM+CHFc 27.6± 4.1 15.0± 5.4‡ — — 143DCM+CHFa 27.6± 4.1 16.1± 4.5* — — 17HCM+CHFa — 22.6± 8.1* — — 17CM+CHFa 27.1± 3.2 16.5± 6‡ — — 144

CK rate, k1 (s−1) CK flux (μmol g−1 wt s) References


DCM+CHFa 0.32± 0.07 0.21± 0.07‡ 3.2± 0.9 1.6± 0.6‡ 13CM+CHFc , f 0.28± 0.13 2.07± 1.27 20LVH no CHF 0.32± 0.06 0.36± 0.04 3.1± 0.8 2.2± 0.7* 19LVH+CHF 0.17± 0.06‡ 1.1± 0.4‡ 19HCMg 0.38± 0.07 0.28± 0.15* 3.6± 0.9 2.0± 1.4‡ 140CM+CHFa 0.38± 0.08 0.24± 0.12‡ 3.6± 0.8 1.75± 0.96‡ 35

Notes: LVH, left ventricular hypertrophy; DCM, nonischemic dilated cardiomyopathy; CM, nonischemic cardiomyopathy; CHF, congestive heart failure;HCM, hypertrophic cardiomyopathy. Control values are listed only once per reference.aPatients with NYHA class I–IV CHF.bControl study for patients undergoing exercise training: training improved LV function but [PCr] and [ATP] were unchanged.cPatients with NYHA class II–III CHF.dPatients with LVH due to valve disease.eEnzyme replacement therapy increased [PCr] to 7.1± 1.5 μmol g−1 (P= 0.012).fControl study for double-blind evaluation of acute allopurinol treatment: allopurinol increased [PCr] by ∼11% and CK flux by 39%.20gHCM due to a familial myosin heavy chain Arg403Gln mutation.hHCM due to Fabry disease.*P< 0.05 versus controls.†P< 0.01 versus controls.‡P< 0.001 versus controls.

in several patients12 were also reported. PD was subsequentlyfound to correlate with blood DPG signal, and attributed toblood contamination, exacerbated by the thin ventricular wallin these patients (see section titled ‘Ratios’).12 The publishedPCr/ATP ratios for DCM are summarized in Table 6. Thepreponderance of studies suggests that myocardial PCr/ATPis reduced on average by nearly 20% in DCM, an amount thatdoes not always reach statistical significance. The PCr/ATPratios generally correlate only weakly with functional and mor-phological indices of disease severity such as EF and/or frac-tional shortening. However, significant correlations between anincreasing severity of NYHA class and a decreasing PCr/ATPhave been reported.79,110,122 In addition, PCr/ATP has beenobserved to recover in patients whose NYHA class improves

due to drug therapy.79 Conversely, a significantly higher riskof cardiovascular death within 2.5 years was associated withthose who had myocardial PCr/ATP< 1.6, compared to thosewith PCr/ATP> 1.6 (mortality 40% vs 11%, P< 0.02).34

Because the relative reduction in PCr represents a dropin myocardial high-energy phosphate reserve, these findingssupport the old hypothesis that the failing heart is energystarved.2 The picture is reinforced by quantitative 31P and 1HMRS measurements of myocardial [PCr], [ATP], and [CR] inpatients with CM and CHF, summarized in Table 7. Reductionsin both [ATP] and [PCr] mean that measurements of PCr/ATPratios often underestimate the loss in high-energy phosphatereserves.110 Importantly, [CR] also declines with increasingNYHA severity.144 Whether the fraction of the total creatine


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pool that is phosphorylated is preserved – which is criticalfor CK reaction kinetics in equations (2) and (7) – is as yetunknown, but is at least within the grasp of MRS.16,64 As isevident from Table 7, for all of these CM studies, the reductionsin the creatine pool are striking. Mindlessly averaging the tab-ulated results as a whole reveals a 30% reduction in [PCr] from9.4 to 6.6 μmol g−1 (P< 0.001) and a 40% reduction in [CR]from 27 to 18 μmol g−1, which presumably all has somethingto do with a 20% loss in [ATP] (P= 0.001).

Measurements of CK reaction kinetics in these patientsreveal that even more fuel has been taken from the fire. Ontop of the reductions in [PCr] as substrate, the CK reactionrate is also reduced by about a third (Table 7, lower panel).Consequently, the CK ATP supply at rest in those exhibitingsymptoms of CHF is approximately half that in healthy subjects(Figure 5).13 Given variable intracyclic energy demands, thisloss in CK ATP supply could be enough to compromise energysupply, leading to contractile dysfunction and symptoms dur-ing periods of moderate exercise, by limiting the heart’s abilityto work (see section titled ‘Cyclic Energy Demand and CardiacWorkload’).13

So what mechanism could slow the CK chemical reactionrate in CHF patients? Focus shifts to the possibility that CKenzyme is somehow being damaged, for example, by reactiveoxygen species (ROS) generated from increased xanthine oxi-dase (XO) activity, a terminal reaction in purine degradation. Ifso, an XO inhibitor could reduce ROS, thereby increasing CKactivity and flux. A double-blinded saturation transfer study ofallopurinol, a ROS inhibitor used for treating gout, was recently

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Figure 5. The forward CK flux (micromole per gram wet weight per sec-ond) in healthy controls and patients with heart disease as measured bysaturation transfer 31P MRS at 1.5 T. Healthy subjects at rest and with dobu-tamine stress at a 200% in workload (HR×BP) have the highest flux (lefttwo columns).13 Patients with pressure-overload LVH19 and anterior MI126

(third and fourth columns) were not in failure and had a normal CK rateconstant, k1, but a reduced [PCr], which resulted in a significantly lowerCK flux – although only locally in the case of the MI patients. Patients withfamilial HCM140 had both significantly reduced CK rates and a lower [PCr],as well as symptoms of CHF (NYHA class I–III, but mostly I). DCM13

and LVH19 patients in the two right-most columns had CHF. These datalink a decline in CK ATP supply with heart failure (n, number of subjects;*P< 0.01 and **P< 0.001 vs healthy controls)

reported in patients with CHF. Acute administration of allop-urinol increased cardiac PCr/ATP and [PCr] by about 10%(P< 0.02), but the mean CK flux – the CK ATP supply – went upby nearly 40% (P< 0.007).20 This provides direct evidence thatcardiac energy delivery can be pharmaceutically augmented inthe failing heart. The CK reaction may thus be a viable targetfor heart failure therapy, and 31P MRS is a suitable – if notunique – noninvasive tool for evaluating such approaches (seeMRS in the Failing Heart: From Mice to Humans).

Hypertrophy, Valve Disease, and Heart Failure. The first local-ized 31P MRS study of HCM appeared in the late 1980s.72 Study-ing cardiac hypertrophy with MRS has the practical advantagethat the MRS signal from the thickened ventricle is less likelyto be contaminated by chamber blood whose lack of PCr couldsystematically reduce PCr/ATP and [PCr] measurements (seesection titled ‘Ratios’). Common underlying causes of HCMinclude familial and genetic factors, while HT and valve dis-ease are common causes of left ventricular hypertrophy (LVH).As in DCM, CHF is a common outcome, and from an MRSstandpoint, the CK metabolism of HCM and LVH are indistin-guishable from DCM. Published myocardial PCr/ATP valuesfor nonischemic hypertrophic disease to date are summarizedin Table 8: the concentrations and fluxes are included in Table 7.

Overall, the 31P MRS data suggest that myocardial PCr/ATPis reduced by about 23% in hypertrophic disease, includingHCM of various genetic origins, LVH, and underlying HTand valve disease. Myocardial PCr/ATP tends to decrease withthe presence and severity of symptoms of CHF,46,53,93,97,110

and/or the degree of severity of underlying valve disease,97,121

or dysfunction in HT (Table 8).121 Yet reduced PCr/ATP isalso seen in hypertrophy that is not explicitly linked to CHFor severity.19,46,94,140 Indeed, a study of LVH patients whohad comparable cardiac function and morphology reportedthat PCr/ATP was the same in those with and those withoutsymptoms of CHF: in fact, mean [PCr] was lowest in thoseLVH patients who were not in heart failure, though the differ-ence vs. CHF was not significant19 (Table 7). So high-energyphosphate concentrations – mostly PCr – are reduced inhypertrophy.19,95,110,135,140 As if to preserve the relative frac-tions of phosphorylated and unphosphorylated creatine, [CR]is down by a similar fraction as [PCr] in the only reportedstudy (Table 7).17 While [CR] reductions were less in HCMthan in DCM in that study,17 this may reflect the more severedisease present in the DCM cohort.

A couple of studies have reported elevated Pi in hypertrophicdisease.12,94As in healthy subjects (see section titled ‘NormalValues’) given the potential for blood DPG and PM contam-ination, the dearth of Pi data likely reflects quantificationdifficulties rather than these findings being anomalous, a situa-tion that may be resolved at higher B0. As also noted in sectiontitled ‘Myocardial Ischemia’, a recent study of 40-year-olds withgenetic HCM found not only a lower resting cardiac PCr/ATPratio (1.71± 0.35 vs 2.14± 0.35 in controls, P< 0.0001), butthat PCr/ATP declined further (to 1.56± 0.29; P= 0.02) whencardiac workload was increased 1.7-fold.48 These results, whichimply a CK response in HCM analogous to that seen withtransient ischemia due to coronary artery disease, differ fromprevious, more-limited studies of isometric handgrip exercise



Table 8. Literature myocardial PCr/ATP ratios for patients with hypertrophy (LVH, HCM), including associated valve disease (VAL) and heart failure(CHF)

Cause/CHF Controls Patients References

1.55± 0.2 0.9± 0.2 72LVH-mild 0.89± 0.30 0.78± 0.16 46LVH-severe 0.8± 0.29 46VAL-CHF 1.5± 0.2 1.1± 0.32* 53VAL-non-CHF — 1.56± 0.15 53VAL 1.76± 0.22 1.34± 0.25 67HCM — 1.73± 0.23 67HCM 2.09± 0.44 1.43± 0.36* 81LVH-HT, VAL — 1.89± 0.2 81HCM 1.65± 0.26 1.32± 0.29† 12HCM 1.71± 0.32 1.07± 0.44‡ 83VAL, NYHA III 2.02± 0.41 1.51± 0.09* SE 93VAL, NYHA I-II — 1.86± 0.18 SE 93AS, CHF — 1.55± 0.43‡ 93AI, CHF — 1.77± 0.36 93VAL, NYHA I-III — 1.64± 0.42‡ 93HCM 2.15± 0.51 1.93± 0.57 94HCM 2.46± 0.53 1.98± 0.37† 54HCM-familial 2.46± 0.53 1.81± 0.28† 103HCM 1.6± 0.4 1.6± 0.6 95MR-severe, NYHA I-III 1.61± 0.3 1.29± 0.3† 97MR-dyspnea — 1.21± 0.24‡ 97MR, NYHA I-III — 1.29± 0.29‡ 97MR-mild, NYHA I — 1.73± 0.17§ 97MR-moderate, NYHA I-II — 1.49± 0.18|| 97HT 1.39± 0.17 1.2± 0.18† 99LVH, VAL 1.65± 0.21 0.8± 0.25* 102AS, LVH 1.43± 0.14 1.24± 0.17‡ ,b 57HCM, FRD 2.39± 0.13 1.47± 0.55* 109HT, non-CHF 1.59± 0.33 No change 110AS, NYHA II-III — 1.3± 0.2 110NYHA II, CM, AS — 1.36± 0.22 110NYHA III, CM, AS — 1.21± 0.3† 110HCM-genetic origin 2.44± 0.3 1.7± 0.43* 115LVH, HT, CHF 1.9± 0.3 1.3± 0.5‡ 19LVH, HT, non-CHF — 1.3± 0.3‡ 19HT 2.07± 0.17 1.65± 0.25*,c 121HT — 1.43± 0.21*,d 121LVH, NYHA II-IIIe 2.14± 0.63 1.57± 0.53‡ 125HCM 2.41± 0.3 2.18± 0.41† 130HCMf, NYHA I-IV 1.92± 0.5 1.68± 0.43 135HCM-genetic origin 1.7± 0.3 1.4± 0.4 140HCM 2.14± 0.35 1.71± 0.35*,g 48Averagea 1.91± 0.35 1.47± 0.31*

Notes: Means± SD as reported or calculated from SE where possible or specified SE where not; HT, hypertension; AS, aortic valve stenosis; AI, aorticincompetence; MR, mitral regurgitation; FRD, Friedreich ataxia. Control values are listed only once per reference.aUnweighted average of means, excluding Ref. 46 (not saturation corrected).bPCr/ATP improved to 1.47± 0.14 (P< 0.05) after aortic valve replacement.cPatients with diastolic dysfunction only.dPatients with both diastolic and systolic dysfunction.ePatients with preserved ejection fraction.fDue to Fabry disease.gExercise that increased HR×BP by 70% reduced PCr/ATP further to 1.56± 0.29 (P= 0.02 vs rest).*P< 0.001 versus controls.†P< 0.05 versus controls.‡P< 0.01 versus controls.§P< 0.01 versus severe.||P< 0.05 versus mild MR.


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in nonischemic patients with valve disease, HCM, or DCM,33

and with DCM patients undergoing dobutamine stress,80

although the diagnoses and levels of stress differ.All told, the work in hypertrophic disease suggests a com-

promised CK energy reserve regardless of the underlyingcause. Disease severity, including but not limited to heartfailure, appears to exacerbate a deterioration in CK energymetabolism. Measurements of CK reaction kinetics, k1 andCK flux, might offer better specificity for CHF. LVH patientswith CHF have lower k1, while the k1 in LVH patients with noCHF but comparable cardiac function and morphology wasshown to be the same as in healthy controls (Table 7).19 Thecombined effect of the reduction in k and reduced [PCr] meansthat the CK ATP supply or flux, k1⋅[PCr], can be reducedto as little as a third of that in healthy controls (1.1± 0.4 vs3.1± 0.8 μmol g−1 s−1; P< 0.001).19 It is difficult to see howa threefold hit to cardiac ATP supply would not negativelyimpact cardiac energetics and function.

Indeed, a 31P MRS study of 58 patients with a mix of nonis-chemic LVH or DCM, and with NYHA class I–IV heart failuresymptoms, found that myocardial CK flux was an independentpredictor of all-cause and cardiac death, CHF hospitalization,cardiac transplantation, and ventricular-assist device place-ment at 5 years.35 The other independent predictors wereNYHA class, EF, and African-American race.

Other Specific Disorders. A number of other specific diseaseshave been studied by cardiac 31P MRS including congenital car-diomegaly in an infant;4 muscular dystrophy, cardiac beri–beriand amyloidosis;81 and diseases associated with progressivesystemic sclerosis.86 All these showed reductions in myocar-dial PCr/ATP ratios compared to healthy subjects. Similarly,patients with untreated familiar hypercholesterolemia arereported as having reduced myocardial PCr/ATP compared tostatin-treated patients (1.78± 0.34 vs 2.15± 0.26, P< 0.001)and healthy controls (2.04± 0.26, P< 0.01).116 PCr/ATP islower in patients with hereditary hemochromatosis as well(1.6± 0.41 vs 1.93± 0.36 in healthy controls; P= 0.004).117

31P MRS studies of type I diabetes mellitus (T1DB) showa modest decrease in myocardial PCr/ATP (to 1.9± 0.4 vs2.15± 0.3, P< 0.05) in patients with no other symptoms ofcardiac disease.111 More significant PCr/ATP reductions thatwere not correlated with perfusion reserve as measured bystress MRI, were reported in another study of asymptomaticT1DB patients (1.5–1.6 vs 2.1 in controls; P< 0.0001). InT1DB patients with uremia and diastolic dysfunction, an evenlower PCr/ATP has been reported (1.36± 0.4 vs 1.91± 0.18;P< 0.01).119

Two studies of patients who had type II diabetes (T2DB)but no coronary disease or systolic dysfunction, again showeda reduced PCr/ATP (1.47± 0.28 vs 1.88± 0.34, P< 0.01;113

and 1.5± 0.11 vs 2.3± 0.12; P< 0.001).114 On the other hand,another study found no change in PCr/ATP in T2DB patientswho had no cardiovascular disease or impaired perfusionCHF.129

Heart Transplant Patients. The management of patients withtransplanted hearts requires the timely detection of graft rejec-tion to assess whether augmented immunosuppressive therapy

Table 9. Summary of MRS findings in common heart diseases through2015

Disorder PCr/ATP Pi [PCr] [ATP] k CK flux [CR]

MI acute – ↑ ↓ ↓MI chronic ↓ ↓ ↓ – ↓ ↓ISCH rest –ISCH exercise ↓ ↑LVH, HCM ↓ ↑ ↓ – or ↓ – or ↓ ↓ ↓DCM ↓ – or ↑ ↓ – or ↓ ↓ ↓CHF+CM ↓ ↓ – or ↓ ↓ ↓ ↓Valve disease ↓ ↓ – or ↓XPLANT ↓T1DB ↓T2DB ↓

Notes: XPLANT, transplanted heart (post operation); ↓, decrease versusnormal; ↑, increase versus normal; –, unchanged versus normal; – or ↓ (↑),unchanged or reduced (increased) versus normal. Blank entries are not yetdetermined.

is warranted. The standard of practice requires that endomy-ocardial biopsies be acquired at regularly scheduled cardiaccatheterization procedures, from which histological evidenceof myocyte necrosis is read. Clearly, a noninvasive methodof assessing rejection would be most welcome. The idea thatchanges in myocardial metabolite ratios might predict histo-logical rejection in human heart transplants arose from animal31P MRS studies of nonimmunosuppressed allografts, in whichmetabolic changes and histological evidence for acute rejectionin the first week or so post-transplantation were seen.157–159

A first paper applying 31P MRS to patients with hearttransplants studied up to 5.5 years post-transplantation didfind significantly lower resting anterior myocardial PCr/ATPratios compared to normal controls (1.57± 0.5 vs 1.93± 0.21;P< 0.01).76 However, 31P MRS agreed with histological evi-dence of necrosis that distinguished mild from moderaterejection (which would trigger augmented therapy), in onlyabout 60–70% of examinations. Consequently, it was concludedthat 31P MRS did not precisely predict significant histologicalrejection in many transplant patients.76 This finding was con-firmed in two other studies.104,160 One of these followed up13 patients serially for 13–294 days post-transplantation andfound a significant correlation between improving PCr/ATPand time post-transplantation, independent of rejection.160

That PCr/ATP ratios are imprecise predictors of histologicalrejection likely reflects fundamental differences between histo-logical and metabolic indices. Thus, myocyte necrosis, which iskey to the histological evaluation, cannot directly cause alteredPCr/ATP ratios because dead cells can contribute no high-energy phosphates. The correlation between PCr/ATP and timepost-transplantation160 suggests that the low PCr/ATP acutelyfollowing transplantation is associated with the transplant pro-cedure itself, with factors that include graft harvesting, storage,duration of hypoxia, and so on. Metabolic recovery requires anextended period of months, which are typically punctuated byepisodes of acute rejection. The use of 31P MRS to assess the via-bility of excised donor hearts at transplantation is reviewed else-where (see Assessing Cardiac Transplant Viability with MRS).



Conclusions31P and 1H MRS can almost completely characterize CKmetabolism in the healthy and diseased human heart withmeasurements of [PCr], [ATP], [CR], k1, and CK flux. The‘almost’ is because not all measures have been obtained from allmajor diseases; [PCr] and [CR] have not both been measuredon the same subjects to quantify [Cr] unambiguously; [Pi] andpH are not reliably quantified; nor is [ADP]. The measure-ments are made possible in patient examinations primarily bysacrificing spatial resolution. Accordingly, focal disease – MIand ischemia – have taken a back seat to studies of diseasesthat affect the heart globally – CMs, CHF, obesity, diabetes, andso on.

The vast majority of published work has focused on just asingle variable, the myocardial PCr/ATP ratio. It is clear fromTables 2–8 that quantification is an ongoing challenge: thenormal control values are listed because the inter-laboratoryvariations (e.g., 1.4–2.5 in Table 8) are often comparable to theintralaboratory variations being attributed to various diseases.Establishing reproducibility for measurements performed atthe same site, and between different sites, is essential for anymulticenter trial and for establishing normal ranges of valuesthat can be relevant to individual patients. As noted in sectiontitled ‘Normal Values’, about one-third of studies are able toachieve SDs that are about 10% of the mean, so the rest ofthe range of variation between 1.4 and 2.5 in control subjectsmust relate to extraneous factors. A larger variation in thevalues of k1 and flux is expected due to the accumulation offactors required for those measurements: for example, when k1is measured from four different spectra, plus a concentrationmeasurement for flux.13

Nevertheless, it is now evident that there are physiologicalvariations as well. Before in vivo MRS came along, someassumed that the CK reaction in healthy hearts would behavejust like the CK reaction in skeletal muscle: as energy demandincreased, PCr would go down to supply more ATP, andPi would rise. After MRS showed that this did not happen,the assumption then was that PCr/ATP was fixed: regulatedsomehow to a single value whose scatter primarily reflected aninability to measure it accurately. While the latter is true, intral-aboratory comparisons of different cohorts of healthy subjectswith various conditions that are not normally designatedas heart disease (as reviewed in section titled ‘PhysiologicalVariations’) now indicate that there are variations in restingmyocardial PCr/ATP with age, cardiovascular fitness, hyper-tension, exposure to hypoxia, obesity, and diet that might beconsidered ‘preclinical’. In patients, the presence of ischemicdisease, heart failure, CM, diabetes, or having had an MI ora transplanted heart appear as even stronger risk factors fora reduced CK energy reserve as indexed by a lower myocar-dial PCr/ATP ratio (Table 9). All these point not to a fixedmyocardial PCr/ATP ratio but to a meta-stable operating valuethat may be eroded and compounded by one or more of theaforementioned factors. The erosion probably commences witha decline in the creatine pool, which noninvasive 1H MRS isideally suited to document.

When CK ATP flux was measured in the human heartwith 31P MRS, two of the possible outcomes could well have

rendered the CK reaction irrelevant to cardiac energetics. First,if the rate was either much smaller than that of OXPHOSor, secondly, if it far exceeded OXPHOS, then the changesin creatine substrate levels, flux, or PCr/ATP being observedcould not have a relevant effect on energy supply in the contextof ongoing energy demands. Instead, the measured CK fluxfell right in a range of about 7–10 times OXPHOS, and doesnot appear to vary much with cardiac workload in the healthyheart. This means that the reductions in flux and/or ATPsupply seen in patients with ischemia, CM, or heart failurecould be enough during stress to consume some of the PCrreserve and ultimately limit cardiac work.13,33 While we do notknow yet whether limited CK reserve limits physical activity,we do know that compromised CK metabolism is a long-termpredictor of poor cardiovascular outcomes.34,35 Given thatpredicting bad things is less rewarding than fixing them, it ismost encouraging to see cardiac 31P MRS metrics being usedto quantify the status of the cardiovascular energy reserve inorder to evaluate the benefits of both pharmaceutical20,153,156

and lifestyle interventions.40,127,134,137,151

AcknowledgmentsI thank RG Weiss for years of fruitful collaboration and discus-sions and the National Institutes of Health for past support.

Biographical SketchPaul A. Bottomley. BSc (Hon), 1975, PhD, 1978, Physics, Univer-sity of Nottingham, United Kingdom. Research Associate, JohnsHopkins University, Baltimore, 1978–1980. Physicist, G. E. Researchand Development Center, 1980–1994. Currently Russell H MorganProfessor and Director of the Division of MR Research of the RussellH Morgan Department of Radiology and Radiological Sciences, JohnsHopkins University. Fellow and Gold Medal recipient of the Societyof Magnetic Resonance in Medicine, 1989; Coolidge Fellowship andmedal, G.E. Company, 1990; Gold, Silver, and Bronze patent medals,G.E. Company; Gold Medal, American Roentgen Ray Society 2015;Member National Academy of Inventors; over 40 issued patents, about180 peer-reviewed papers, 24 book chapters, 13 editorials, and over225 published abstracts. Research specialties: in vivo NMR, MRI,tissue relaxation times, localized NMR spectroscopy, human cardiacNMR spectroscopy, interventional MRI, MRI safety.

Related ArticlesCardiac MRS Studies in Rodents and Other Animals; MRS inthe Failing Heart: From Mice to Humans; Cardiac Lipids by 1HMRS; Assessing Cardiac Transplant Viability with MRS; Mea-suring Biochemical Reaction Rates In Vivo with MagnetizationTransfer; Quantifying Metabolite Ratios and Concentrations byNon-1H MRS

References1. R. B. Jennings and K. A. Reimer, Am. J. Pathol., 1981, 102, 241.

2. S. Neubauer, N. Engl. J. Med., 2007, 356, 1140.

3. P. A. Bottomley, Science, 1985, 229, 769.

4. J. R. Whitman, B. Chance, H. Bode, J. Maris, J. Haselgrove, R. Kelley, B. J.Clark, and A. H. Harken, J. Am. Coll. Cardiol., 1985, 5, 745.


PA Bottomley

5. W. E. Jacobus, G. J. Taylor, D. P. Hollis, and R. L. Nunnally, Nature, 1977, 263,756.

6. P. B. Garlick, G. K. Radda, P. J. Seeley, and B. Chance, Biochem. Biophys. Res.Commun., 1977, 74, 1256.

7. T. H. Grove, J. J. H. Ackerman, G. K. Radda, and P. J. Bore, Proc. Natl. Acad.Sci. U. S. A., 1980, 77, 299.

8. R. L. Nunnally and P. A. Bottomley, Science, 1981, 211, 177.

9. P. A. Bottomley, H. R. Hart, W. A. Edelstein, J. F. Schenck, L. S. Smith, W. M.Leue, O. M. Mueller, and R. W. Redington, Lancet , 1983, 322, 273.

10. P. A. Bottomley, H. R. Hart, W. A. Edelstein, J. F. Schenck, L. S. Smith, W. M.Leue, O. M. Mueller, and R. W. Redington, Radiology , 1984, 150, 441.

11. D. G. Gadian, Nuclear Magnetic Resonance and its Applications to LivingSystems, Oxford University Press: Oxford, 1982, 30.

12. A.de Roos, J. Doornbos, P. R. Luyten, L. J. M. P. Oosterwaal, E. E.van der Wall,and J. A.den Hollander, J. Magn. Reson. Imaging, 1992, 2, 711.

13. R. G. Weiss, G. Gerstenblith, and P. A. Bottomley, Proc. Natl. Acad Sci. U. S.A., 2005, 102, 808.

14. J. A.den Hollander, W. T. Evanochko, and G. M. Pohost, Magn. Reson. Med.,1994, 32, 175.

15. L. S. Szczepaniak, R. L. Dobbins, G. J. Metzger, G. Sartoni-D’Ambrosia,D. Arbique, W. Vongpatanasin, R. Unger, and R. G. Victor, Magn. Reson.Med., 2003, 49, 417.

16. P. A. Bottomley and R. G. Weiss, Radiology , 2001, 219, 411.

17. I. Nakae, K. Mitsunami, T. Omura, T. Yabe, T. Tsutamoto, S. Matsuo, M. Taka-hashi, S. Morikawa, T. Inubushi, Y. Nakamura, M. Kinoshita, and M. Horie, J.Am. Coll. Cardiol., 2003, 42, 1587.

18. P. A. Bottomley and R. G. Weiss, Lancet , 1998, 351, 714.

19. C. S. Smith, P. A. Bottomley, S. P. Schulman, G. G. Gerstenblith, and R. G.Weiss, Circulation, 2006, 114, 1151.

20. G. A. Hirsch, P. A. Bottomley, G. Gerstenblith, and R. G. Weiss, J. Am. Coll.Cardiol., 2012, 59, 802.

21. P. A. Bottomley, C. J. Hardy, P. B. Roemer, and O. M. Mueller, Magn. Reson.Med., 1989, 12, 348.

22. M. A. Schroeder, K. Clarke, S. Neubauer, and D. J. Tyler, Circulation, 2011,124, 1580.

23. T. Wallimann, Curr. Biol., 1994, 4, 42.

24. P. P. Dzeja and A. Terzic, J. Exp. Biol., 2003, 206, 2039.

25. P. A. Bottomley, in Encyclopedia of Magnetic Resonance, eds R. K. Har-ris and R. E. Wasylishen, John Wiley & Sons, Ltd: Chichester, 2009. DOI:10.1002/9780470034590.emrstm0345.pub2.

26. C. T. Rodgers, W. T. Clarke, C. Snyder, J. T. Vaughan, S. Neubauer, and M. D.Robson, Magn. Reson. Med., 2014, 72, 304.

27. V. Stoll, W. T. Clarke, E. Levelt, S. G. Myerson, M. D. Robson, S. Neubauer,and C. Rodgers, J. Cardiovasc. Magn. Reson., 2015, 17, 249.

28. B. Schaller, W. T. Clarke, S. Neubauer, M. D. Robson, and C. T. Rodgers, Magn.Reson. Med., 2015. DOI: 10.1002/mrm.25755.

29. A. E. M. El-Sharkawy, R. E. Gabr, M. Schar, R. G. Weiss, and P. A. Bottomley,NMR Biomed., 2013, 26, 1363.

30. M. Schar, A. E. M. El-Sharkawy, R. G. Weiss, and P. A. Bottomley, Magn.Reson. Med., 2010, 63, 1493.

31. A. Bashir and R. Gropler, NMR Biomed., 2014, 27, 663.

32. M. Schär, R. E. Gabr, A. M. El-Sharkawy, A. Steinberg, P. A. Bottomley, and R.G. Weiss, J. Cardiovasc. Magn. Reson., 2015, 17, 70. DOI: 10.1186/s12968-015-0175-4.

33. R. G. Weiss, P. A. Bottomley, C. J. Hardy, and G. Gerstenblith, N. Engl. J. Med.,1990, 323, 1593.

34. S. Neubauer, M. Horn, M. Cramer, K. Harre, J. B. Newell, W. Peters, T. Pabst,G. Ertl, D. Hahn, J. S. Ingwall, and K. Kochsiek, Circulation, 1997, 96, 2190.

35. P. A. Bottomley, G. S. Panjrath, S. Lai, G. A. Hirsch, K. Wu, S. S. Najjar, A.Steinberg, G. Gerstenblith, and R. G. Weiss, Sci. Trans. Med., 2013, 5, 215re3.

36. D. I. Hoult and P. C. Lauterbur, J. Magn. Reson., 1979, 34, 425.

37. P. R. Luyten, G. Bruntink, F. M. Sloff, J. I. Vermeulen, J. I.van der Heijden, J.A.den Hollander, and A. Heerschap, NMR Biomed., 1989, 1, 177.

38. P. A. Bottomley and C. J. Hardy, Magn. Reson. Med., 1992, 24, 384.

39. M. F. H. Schocke, B. Metzler, C. Wolf, P. Steinboeck, C. Kremser, O. Pachinger,W. Jaschke, and P. Lukas, Magn. Reson. Imaging, 2003, 21, 553.

40. M. Beer, D. Wagner, J. Myers, J. Sandstede, H. Köstler, D. Hahn, S. Neubauer,and P. Dubach, J. Am. Coll. Cardiol., 2008, 51, 1883.

41. D. J. Tyler, Y. Emmanuel, L. E. Cochlin, L. E. Hudsmith, C. J. Holloway,S. Neubauer, K. Clarke, and M. D. Robson, NMR Biomed., 2009, 22, 405.

42. P. A. Bottomley, C. H. Lugo-Olivieri, and R. Giaquinto, Magn. Reson. Med.,1997, 37, 591.

43. C. J. Hardy, P. A. Bottomley, K. W. Rohling, and P. B. Roemer, Magn. Reson.Med., 1992, 28, 54.

44. P. A. Bottomley and C. J. Hardy, Phil. Trans. R. Soc. Lond. A, 1990, 333, 531.

45. P. A. Bottomley, C. J. Hardy, and P. B. Roemer, Magn. Reson. Med., 1990, 14,425.

46. S. Schaefer, J. R. Gober, G. G. Schwartz, D. B. Twieg, M. W. Weiner, andB. Massie, Am. J. Cardiol., 1990, 65, 1154.

47. S. Schaefer, J. Gober, M. Valenza, G. S. Karczmar, G. B. Matson, S. A. Camacho,E. H. Botvinick, B. Massie, and M. W. Weiner, J. Am. Coll. Cardiol., 1988, 12,1149.

48. S. Dass, L. E. Cochlin, J. J. Suttie, C. J. Holloway, O. J. Rider, L. Carden, D. J.Tyler, T. D. Karamitsos, K. Clarke, S. Neubauer, and H. Watkins, Eur. Heart. J.,2015, 36, 1547.

49. R. Löffler, R. Sauter, H. Kolem, A. Haase, and M.von Kienlin, J. Magn. Reson.,1998, 134, 287.

50. Y. Zhang, R. E. Gabr, M. Schär, R. G. Weiss, and P. A. Bottomley, J. Magn.Reson., 2012, 218, 66. ol., 1988, 12, 1449.

51. R. Pohmann and M.von Kienlin, Magn. Reson. Med., 2001, 45, 817.

52. P. W. Hochachka, C. M. Clark, J. E. Holden, C. Stanley, K. Ugurbil, and R. S.Menon, Proc. Natl. Acad. Sci. U. S. A., 1996, 93, 1215.

53. M. A. Conway, J. Allis, R. Ouwerkerk, T. Niioka, B. Rajagopalan, and G. K.Radda, Lancet , 1991, 338, 973.

54. W. I. Jung, L. Sieverding, J. Breuer, T. Hoess, S. Widmaier, O. Schmidt,M. Bunse, F.van Erckelens, J. Apitz, O. Lutz, and G. J. Dietze, Circulation,1998, 97, 2536.

55. G. Klug, R. H. Zwick, M. Frick, C. Wolf, M. F. H. Schocke, E. Conci, W. Jaschke,O. Pachinger, and B. Metzler, Int. J. Sports Med., 2007, 28, 667.

56. R. S. Menon, K. Hendrich, X. Hu, and K. Ugurbil, Magn. Reson. Med., 1992,26, 368.

57. H. P. Beyerbacht, H. J. Lamb, A.van der Laarse, H. W. Vliegen, F. Leujes, M. G.Hazekamp, A.de Roos, and E. E.van der Wall, Radiology , 2001, 219, 637.

58. J. Felblinger, B. Jung, J. Slotboom, C. Boesch, and R. Kreis, Magn. Reson.Med., 1999, 42, 903.

59. M. Schär, S. Kozerke, and P. Boesiger, Magn. Reson. Med., 2004, 51, 1091.

60. P. A. Bottomley, C. J. Hardy, P. B. Roemer, and R. G. Weiss, NMR Biomed.,1989, 2, 284.

61. Y. Zhang, J. Zhou, and P. A. Bottomley, Magn. Reson. Med., 2015, 74, 320.

62. P. A. Bottomley and R. Ouwerkerk, Magn. Reson. Med., 1994, 32, 137.

63. J. O. Van Dobbenburgh, C. Lekkerkerk, R.de Beer, and C. J. A. Van Echteld,NMR Biomed., 1994, 7, 218.



64. I. Nakae, K. Mitsunami, T. Yabe, T. Inubushi, S. Morikawa, S. Matsuo, T. Koh,and M. Horie, J. Cardiovasc. Magn. Reson., 2004, 6, 685.

65. D. J. Tyler, L. E. Hudsmith, K. Clarke, S. Neubauer, and M. D. Robson, NMRBiomed., 2008, 21, 793.

66. A. E. M. El-Sharkawy, M. Schär, R. Ouwerkerk, R. G. Weiss, and P. A. Bottom-ley, Magn. Reson. Med., 2009, 61, 785.

67. P. A. Bottomley, C. J. Hardy, and R. G. Weiss, J. Magn. Reson., 1991, 95, 341.

68. C. J. Hardy, R. G. Weiss, P. A. Bottomley, and G. Gerstenblith, Am. Heart J.,1991, 122, 795.

69. P. A. Bottomley, E. A. Atalar, and R. G. Weiss, Magn. Reson. Med., 1996, 35,664.

70. C. Gibbs, J. Mol. Cell. Cardiol., 1985, 17, 727.

71. P. A. Bottomley, R. J. Herfkens, L. S. Smith, and T. M. Bashore, Radiology ,1987, 165, 703.

72. B. Rajagopalan, M. J. Blackledge, W. J. McKenna, N. Bolas, and G. K. Radda,Ann. N. Y. Acad. Sci., 1987, 508, 321.

73. M. A. Conway, J. D. Bristow, M. J. Blackledge, B. Rajagopalan, and G. K.Radda, Lancet , 1988, 332, 692.

74. T. M. Grist, J. B. Kneeland, W. R. Rilling, A. Jesmanowicz, W. Froncisz, andJ. S. Hyde, Radiology , 1989, 170, 357.

75. M. A. Conway, J. D. Bristow, M. J. Blackledge, B. Rajagopalan, and G. K.Radda, Br. Heart J., 1991, 65, 25.

76. P. A. Bottomley, R. G. Weiss, C. J. Hardy, and W. A. Baumgartner, Radiology ,1991, 181, 67.

77. W. Auffermann, W. M. Chew, C. L. Wolfe, N. J. Tavares, W. W. Parmley, R. C.Semelka, T. Donnelly, K. Chatterjee, and C. B. Higgins, Radiology , 1991, 179,253.

78. K. Mitsunami, M. Okada, T. Inoue, M. Hachisuka, M. Kinoshita, and T.Inubushi, Jpn. Circ. J., 1992, 56, 614.

79. S. Neubauer, T. Krahe, R. Schindler, M. Horn, H. Hillenbrand, C. Entzeroth,H. Mader, E. P. Kromer, G. A. J. Riegger, K. Lackner, and G. Ertl, Circulation,1992, 86, 1810.

80. S. Schaefer, G. G. Schwartz, S. K. Steinman, D. J. Meyerhoff, B. M. Massie,and W. M. Weiner, Magn. Reson. Med., 1992, 25, 260.

81. Y. Masuda, Y. Tateno, H. Ikehira, T. Hashimoto, F. Shishido, M. Sekiya, Y.Imazeki, H. Imai, S. Watanabe, and Y. Inagaki, Jpn. Circ. J., 1992, 56, 620.

82. S. Neubauer, T. Krahe, R. Schindler, H. Hillenbrand, C. Entzeroth, M. Horn, W.R. Bauer, T. Stephan, K. Lackner, A. Haase, and G. Ertl, Magn. Reson. Med.,1992, 26, 300.

83. H. Sakuma, K. Takeda, T. Tagami, T. Nakagawa, S. Okamoto, T. Konishi, andT. Nakano, Am. Heart J., 1993, 125, 1323.

84. H. Sakuma, S. J. Nelson, D. B. Vigneron, J. Hartiala, and C. B. Higgins, Magn.Reson. Med., 1993, 29, 688.

85. T. Yabe, K. Mitsunami, M. Okada, S. Morikawa, T. Inubushi, and M. Kinoshita,Circulation, 1994, 89, 1709.

86. J. Doornbos, P. R. Luyten, M. Janssen, M. Wasser, and A. De Roos, J. Magn.Reson. Imaging, 1994, 4, 165.

87. T. Yabe, K. Mitsunami, T. Inubushi, and M. Kinoshita, Circulation, 1995, 92,15.

88. H. P. Hetherington, D. J. E. Luney, J. T. Vaughan, J. W. Pan, S. L. Ponder, O.Tschendel, D. B. Twieg, and G. M. Pohost, Magn. Reson. Med., 1995, 33, 427.

89. S. Neubauer, M. Horn, T. Pabst, M. Godde, D. Lubke, B. Jilling, D. Hahn, andG. Ertl, Eur. Heart J., 1995, 16, 115.

90. H. J. Lamb, J. Doornbos, J. A.den Hollander, P. R. Luyten, H. P. Beyerbacht,E. E.van der Wall, and A.de Roos, NMR Biomed., 1996, 9, 217.

91. R. Kalil-Filho, C. P.de Albuquerque, R. G. Weiss, A. Mocelim, G. Bellotti,G. Cerri, and F. Pileggi, J. Am. Coll. Cardiol., 1997, 30, 1228.

92. H. J. Lamb, H. P. Beyerbacht, R. Ouwerkerk, J. Doornbos, B. M. Pluim, E. E.vander Wall, A.van der Laarse, and A.de Roos, Circulation, 1997, 96, 2969.

93. S. Neubauer, M. Horn, T. Pabst, K. Harre, H. Stromer, G. Bertsch, J. Sandstede,G. Ertl, D. Hahn, and K. Kochsiek, J. Invest. Med., 1997, 45, 453.

94. L. Sieverding, W. I. Jung, J. Breuer, S. Widmaier, A. Staubert, F.van Erckelens,O. Schmidt, M. Bunse, T. Hoess, O. Lutz, G. J. Dietze, and J. Apitz, Am. J.Cardiol., 1997, 80, 34A.

95. M. Okada, K. Mitsunami, T. Inubushi, and M. Kinoshita, Magn. Reson. Med.,1998, 39, 772.

96. B. M. Pluim, H. J. Lamb, H. W. M. Kayser, F. Leujes, H. P. Beyerbacht, A. H.Zwinderman, A.van der Laarse, H. W. Vliegen, A.de Roos, and E. E.van derWall, Circulation, 1998, 97, 666.

97. M. A. Conway, P. A. Bottomley, R. Ouwerkerk, G. K. Radda, and B.Rajagopalan, Circulation, 1998, 97, 1716.

98. M. Meininger, W. Landschutz, M. Beer, T. Seyfarth, M. Horn, T. Pabst, A.Haase, D. Hahn, S. Neubauer, and M.von Kienlin, Magn. Reson. Med., 1999,41, 657.

99. H. J. Lamb, H. P. Beyerbacht, A.van der Laarse, B. C. Stoel, J. Doornbos,E. E.van der Wall, and A.de Roos, Circulation, 1999, 99, 2261.

100. J. O. Van Dobbenburgh, M. C. H. De Groot, N. De Jonge, C. Klopping, J. R.Lahpor, S. R. Woolley, E. O. R.de Medina, and C. J. A. Van Echteld, NMRBiomed., 1999, 12, 515.

101. M. Beer, J. Sandstede, W. Landschutz, M. Viehrig, K. Harre, M. Horn, M.Meininger, T. Pabst, W. Kenn, A. Haase, M.von Kienlin, S. Neubauer, andD. Hahn, Eur. Radiol., 2000, 10, 1323.

102. M. Beer, M. Viehrig, T. Seyfarth, J. Sandstede, C. Lipke, T. Pabst, W. Kenn, K.Harre, M. Horn, W. Landschutz, M.von Kienlin, S. Neubauer, and D. Hahn,Radiologie, 2000, 40, 162.

103. W. I. Jung, T. Hoess, M. Bunse, S. Widmaier, L. Sieverding, J. Breuer, J. Apitz,O. Schmidt, F.van Erckelens, G. J. Dietze, and O. Lutz, Circulation, 2000, 101,e121.

104. S. D. Buchthal, T. O. Noureuil, J. A.den Hollander, R. C. Bourge, J. K. Kirklin, C.R. Katholi, J. B. Caulfield, G. M. Pohost, and W. T. Evanochko, J. Cardiovasc.Magn. Reson., 2000, 2, 51.

105. J. G. Crilley, E. A. Boehm, B. Rajagopalan, A. M. Blamire, P. Styles, F. Muntoni,D. Hilton-Jones, and K. Clarke, J. Am. Coll. Cardiol., 2000, 36, 1953.

106. H. Kostler, M. Beer, W. Landschutz, S. Buchner, J. Sandstede, T. Pabst, W.Kenn, S. Neubauer, M.von Kienlin, and D. Hahn, ROFO, 2001, 173, 1093.

107. M.von Kienlin, M. Beer, A. Greiser, D. Hahn, K. Harre, H. Kostler, W. Land-schutz, T. Pabst, J. Sandstede, and S. Neubauer, J. Magn. Reson. Imaging,2001, 13, 521.

108. M. Beer, S. Buchner, J. Sandstede, M. Viehrig, C. Lipke, A. Krug, H. Kostler,T. Pabst, W. Kenn, W. Landschutz, M.von Kienlin, K. Harre, S. Neubauer, andD. Hahn, Magn. Reson. Mater. Phys., 2001, 13, 70.

109. R. Lodi, B. Rajagopalana, A. M. Blamire, J. M. Cooper, C. H. Davies, J. L.Bradley, P. Styles, and A. H. V. Schapira, Cardiovasc. Res., 2001, 52, 111.

110. M. Beer, T. Seyfarth, J. Sandstede, W. Landschutz, C. Lipke, H. Kostler, M.vonKienlin, K. Harre, D. Hahn, and S. Neubauer, J. Am. Coll. Cardiol., 2002, 40,1267.

111. B. Metzler, M. F. H. Schocke, P. Steinboeck, C. Wolf, W. Judmaier, M. Lech-leitner, P. Lukas, and O. Pachinger, J. Cardiovasc. Magn. Reson., 2002, 4, 493.

112. W. T. Evanochko, S. D. Buchthal, J. A.den Hollander, C. R. Katholi, R. C. Bourge,R. L. Benza, J. K. Kirklin, and G. M. Pohost, J. Heart Lung Transplant., 2002,21, 522.

113. M. Diamant, H. J. Lamb, Y. Groeneveld, E. L. Endert, J. W. A. Smit, J. J. Bax, J.A. Romijn, A.de Roos, and J. K. Radder, J. Am. Coll. Cardiol., 2003, 42, 328.

114. M. Scheuermann-Freestone, P. L. Madsen, D. Manners, A. M. Blamire, R. E.Buckingham, P. Styles, G. K. Radda, S. Neubauer, and K. Clarke, Circulation,2003, 107, 3040.


PA Bottomley

115. J. G. Crilley, E. A. Boehm, E. Blair, B. Rajagopalan, A. M. Blamire, P. Styles, W. J.McKenna, I. Ostman-Smith, K. Clarke, and H. Watkins, J. Am. Coll. Cardiol.,2003, 41, 1776.

116. M. F. H. Schocke, M. Martinek, C. Kremser, C. Wolf, P. Steinboeck, M. Lech-leitner, W. Jaschke, O. Pachinger, and B. Metzler, J. Cardiovasc. Magn. Reson.,2003, 5, 595.

117. M. F. H. Schocke, H. Zoller, W. Vogel, C. Wolf, C. Kremser, P. Steinboeck, G.Poelzl, O. Pachinger, W. R. Jaschke, and B. Metzler, Magn. Reson. Imaging,2004, 22, 515.

118. T. Caus, F. Kober, A. Mouly-Bandini, A. Riberi, D. R. Metras, P. J. Cozzone, andM. Bernard, Eur. J. Cardiothorac. Surg., 2005, 28, 576.

119. G. Perseghin, P. Fiorina, F. De Cobelli, P. Scifo, A. Esposito, T. Canu, M. Danna,C. Gremizzi, A. Secchi, L. Luzi, and A. Del Maschio, J. Am. Coll. Cardiol., 2005,46, 1085.

120. H. Kostler, W. Landschutz, S. Koeppe, T. Seyfarth, C. Lipke, J. Sandstede, M.Spindler, M.von Kienlin, D. Hahn, and M. Beer, Magn. Reson. Med., 2006, 56,907.

121. J. P. Heyne, R. Rzanny, A. Hansch, U. Leder, J. R. Reichenbach, and W. A.Kaiser, Eur. Radiol., 2006, 16, 1796.

122. A. Hansch, R. Rzanny, J. P. Heyne, U. Leder, J. R. Reichenbach, and W. A.Kaiser, Eur. Radiol., 2005, 15, 319.

123. G. Perseghin, F. De Cobelli, A. Esposito, G. Lattuada, I. Terruzzi, A. La Torre,E. Belloni, T. Canu, P. Scifo, A. Del Maschio, L. Luzi, and G. Alberti, Am. Heart.J., 2007, 154, 937.

124. G. Perseghin, G. Lattuada, F. De Cobelli, A. Esposito, E. Belloni, G. Ntali,F. Ragogna, T. Canu, P. Scifo, A. Del Maschio, and L. Luzi, Hepatology , 2008,47, 51.

125. T. T. Phan, K. Abozguia, G. N. Shivu, G. Mahadevan, I. Ahmed, L. Williams,G. Dwivedi, K. Patel, P. Steendijk, H. Ashrafian, A. Henning, and M. Frenneaux,J. Am. Coll. Cardiol., 2009, 54, 402.

126. P. A. Bottomley, K. C. Wu, G. Gerstenblith, S. P. Schulman, A. Steinberg, andR. G. Weiss, Circulation, 2009, 119, 1918.

127. G. Perseghin, F. De Cobelli, A. Esposito, E. Belloni, G. Lattuada, T. Canu, P. L.Invernizzi, F. Ragogna, A. La Torre, P. Scifo, G. Alberti, A. Del Maschio, and L.Luzi, Heart , 2009, 95, 630.

128. L. E. Hudsmith, D. J. Tyler, Y. Emmanuel, S. E. Petersen, J. M. Francis, H.Watkins, K. Clarke, M. D. Robson, and S. Neubauer, Int. J. Cardiovasc. Imag-ing, 2009, 25, 819.

129. L. J. Rijzewijk, R. W.van der Meer, H. J. Lamb, H. W. A. M.de Jong, M. Lub-berink, J. A. Romijn, J. J. Bax, A.de Roos, J. W. Twisk, R. J. Heine, A. A.Lammertsma, J. W. A. Smit, and M. Diamant, J. Am. Coll. Cardiol., 2009, 54,1524.

130. A. Esposito, F. De Cobelli, G. Perseghin, M. Pieroni, E. Belloni, R. Mellone, T.Canu, F. Gentinetta, P. Scifo, C. Chimenti, A. Frustaci, L. Luzi, A. Maseri, andA. Del Maschio, Heart , 2009, 95, 228.

131. G. N. Shivu, T. T. Phan, K. Abozguia, I. Ahmed, A. Wagenmakers, A. Henning,P. Narendran, M. Stevens, and M. Frenneaux, Circulation, 2010, 121, 1209.

132. G. N. Shivu, K. Abozguia, T. T. Phan, I. Ahmed, A. Henning, and M. Frenneaux,Eur. J. Radiol., 2010, 73, 255.

133. D. E. J. Jones, K. Hollingsworth, G. Fattakhova, G. MacGowan, R. Taylor, A.Blamire, and J. L. Newton, Am. J. Physiol. Gastrointest. Liver Physiol., 2010,298, G764.

134. C. J. Holloway, L. E. Cochlin, Y. Emmanuel, A. Murray, I. Codreanu, L. M.Edwards, C. Szmigielski, D. J. Tyler, N. S. Knight, B. K. Saxby, B. Lambert, C.Thompson, S. Neubauer, and K. Clarke, Am. J. Clin. Nutr., 2011, 93, 748.

135. W. Machann, F. Breunig, F. Weidemann, J. Sandstede, D. Hahn, H. Kostler, S.Neubauer, C. Wanner, and M. Beer, Eur. J. Heart Fail., 2011, 13, 278.

136. C. J. Holloway, L. E. Cochlin, I. Codreanu, E. Bloch, M. Fatemian, C. Szmigiel-ski, H. Atherton, L. Heather, J. Francis, S. Neubauer, P. Robbins, H. E. Mont-gomery, and K. Clarke, FASEB J., 2011, 25, 3130.

137. C. J. Holloway, H. E. Montgomery, A. J. Murray, L. E. Cochlin, I. Codreanu, N.Hopwood, A. W. Johnson, O. J. Rider, D. Z. H. Levett, D. J. Tyler, J. M. Francis,S. Neubauer, M. P. W. Grocott, and K. Clarke, FASEB J., 2011, 25, 792.

138. O. J. Rider, J. M. Francis, M. K. Ali, C. Holloway, T. Pegg, M. D. Robson, D.Tyler, J. Byrne, K. Clarke, and S. Neubauer, Circulation, 2012, 125, 1511.

139. O. Geier, A. M. Weng, A. Toepell, D. Hahn, M. Spindler, M. Beer, and H. Köstler,Z. Med. Phys., 2014, 24, 49.

140. M. R. Abraham, P. A. Bottomley, V. L. Dimaano, A. Pinheiro, A. Steinberg, T.A. Traill, T. P. Abraham, and R. G. Weiss, Am. J. Cardiol., 2013, 112, 861.

141. P. A. Bottomley and C. J. Hardy, J. Magn. Reson., 1992, 99, 443.

142. R. E. Gabr, R. G. Weiss, and P. A. Bottomley, J. Magn. Reson., 2008, 191, 248.

143. I. Nakae, K. Mitsunami, S. Matsuo, T. Matsumoto, S. Morikawa, T. Inubushi,T. Koh, and M. Horie, Magn. Reson. Med. Sci., 2004, 3, 19.

144. I. Nakae, K. Mitsunami, S. Matsuo, T. Inubushi, S. Morikawa, T. Tsutamoto, T.Koh, and M. Horie, Circ. J., 2005, 69, 711.

145. G. J. Kemp, M. Meyerspeer, and E. Moser, NMR Biomed., 2007, 20, 555.

146. P. A. Bottomley, Y. H. Lee, and R. G. Weiss, Radiology , 1997, 204, 403.

147. K. T. Sun, L. A. Yeatman, D. B. Buxton, K. Chen, J. A. Johnson, S.-C. Huang,K. F. Kofoed, S. Weismueller, J. Czernin, M. E. Phelps, et al., J. Nucl. Med.,1998, 39, 272.

148. B. L. Gerber, W. Wijns, J. J. Vanoverschelde, G. R. Heyndrickx, B. DeBruyne,J. Bartunek, and J. A. Melin, J. Am. Coll. Cardiol., 1999, 34, 1939.

149. A. C. Cave, J. S. Ingwall, J. Friedrich, R. Liao, K. W. Saupe, C. S. Apstein, andF. R. Eberli, Circulation, 2000, 101, 2090.

150. K. Weiss, P. A. Bottomley, and R. G. Weiss, NMR Biomed., 2015, 28, 694.

151. O. J. Rider, J. M. Francis, D. Tyler, J. Byrne, K. Clarke, and S. Neubauer, Int. J.Cardiovasc. Imaging, 2013, 29, 1043.

152. M. Beer, M. Spindler, J. J. W. Sandstede, H. Remmert, S. Beer, H. Kostler, andD. Hahn, J. Magn. Reson. Imaging, 2004, 20, 798.

153. S. S. Najjar, P. A. Bottomley, S. P. Schulman, M. M. Waldron, R. P. Steffen,G. Gerstenblith, and R. G. Weiss, J. Cardiovasc. Magn. Reson., 2005, 7, 1.

154. P. A. Bottomley, L. S. Smith, S. Brazzamano, L. W. Hedlund, R. W. Redington,and R. J. Herfkens, Magn. Reson. Med., 1987, 5, 129.

155. S. D. Buchthal, J. A. Den Hollander, C. N. B. Merz, W. J. Rogers, C. J. Pepine,N. Reichek, B. L. Sharaf, S. Reis, S. F. Kelsey, and G. M. Pohost, N. Engl. J.Med., 2000, 342, 829.

156. G. Fragasso, G. Perseghin, F.de Cobelli, A. Esposito, A. Palloshi, G. Lattuada,P. Scifo, G. Calori, A.del Maschio, and A. Margonato, Eur. Heart J., 2006, 27,942.

157. R. C. Canby, W. T. Evanochko, L. V. Barrett, J. K. Kirklin, D. C. McGiffen, T. T.Sakai, M. E. Brown, R. E. Foster, R. C. Reeves, and G. M. Pohost, J. Am. Coll.Cardiol., 1987, 9, 1067.

158. C. E. Haug, J. L. Shapiro, L. Chan, and R. Weil, Transplantation, 1987, 44,175.

159. C. D. Fraser, V. P. Chacko, W. E. Jacobus, G. M. Hutchins, J. Glickson, B. A.Reitz, and W. A. Baumgartner, Transplantation, 1989, 48, 1068.

160. J. O. Van Dobbenburgh, J. R. Lahpor, S. R. Woolley, N. de Jonge, C. Klopping,and C. J. Van Echteld, Circulation, 1996, 94, 2831.


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