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MRS Studies of Creatine Kinase Metabolism in Human Heart Paul A. Bottomley Johns 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, providing adenosine triphosphate (ATP) to fuel contraction by shuttling phosphocreatine (PCr) from the mitochondria. For 30 years the measurement of the myocardial PCr/ATP ratio has been a common focus of human cardiac phosphorus (31 P) magnetic resonance spectroscopy (MRS). Today, our ability to measure the absolute concentrations of PCr and ATP, the CK reaction rate and flux with 31 P MRS, and the total creatine pool (CR) using proton (1 H) MRS, allows almost complete characterization of CK metabolism in the healthy and diseased human heart. The methods and limitations of human cardiac MRS for measuring CK metabolism are reviewed herein, along with the application and findings in ‘normal’ physiological processes including aging, diet, sedentary lifestyle, hypoxia, and obesity. The results from studies of patients with myocardial infarction, ischemia, dilated and hypertrophic cardiomyopathies, valve disease, heart failure, diabetes, and other disorders are summarized, with the quantitative findings tabulated. The myocardial PCr/ATP ratio is a sensitive indicator of cardiac energy reserve, existing in a meta-stable state that can be upset in many disorders. Reductions in metabolite 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 now being 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 Introduction (CK reaction): The heart is the largest consumer of energy per gram of tissue, and disruptions in energy metabolism, including energy supply and demand, are thought to play a central role in many common diseases of the heart.1,2 It was therefore inevitable that the first human in vivo cardiac magnetic resonance spectroscopy (MRS) in 19853,4 focused on those energy metabolites that were shown by earlier animal studies5–8 (see also Cardiac MRS Studies in Rodents and Other Animals) to be NMR detectable. Human cardiac MRS required the development of high-field, high-homogeneity magnet systems capable of accommodating the torso.9 The performance advantages of such systems for MRI, the sister technology of MRS,10 has so entwined the two that human cardiac MRS is today inconceivable without MRI guidance and gradient localization. Human phosphorus (31 P) MRS can noninvasively detect endogenous adenosine triphosphate (ATP), the energy that fuels the cardiac pump via the ATP-ase reaction,1 ATP ⇐⇒ ADP + Pi + ⟨energy⟩. (1) Inorganic phosphate (Pi) is a by-product whose pHdependent chemical shift provides a measure of intracellular pH.11,12 It can measure phosphocreatine (PCr),2–7 which serves as the heart’s primary energy reserve. ATP is synthesized from PCr and adenosine diphosphate (ADP) via the creatine kinase Volume 5, 2016 PCr + ADP + H+k−1 ⇐⇒k1 ATP + Cr (2) releasing unphosphorylated creatine (Cr). It can even measure the forward (k1 ) and reverse (k−1 ) reaction rate constants (see Measuring Biochemical Reaction Rates In Vivo with Magnetization Transfer).13 Switching to hydrogen (1 H) MRS allows documentation of intra- and extra-myocellular lipid fuel deposits in the myocardium,14,15 whose accumulation is linked to cardiac dysfunction, obesity, and diabetes. 1 H MRS can also be used to measure the total creatine pool (CR), comprising PCr plus Cr.16–18 Thus, in combination, 31 P and 1 H MRS could almost completely characterize CK metabolism and energy supply in the human heart.16 The depletion of CR in heart failure17 and in myocardial infarction (MI),18 which is routinely diagnosed by an elevated serum concentration of myocardial CK enzyme, suggests its potential as an in situ 1 H MRS metabolic marker. Meanwhile, significant reductions in CK energy flux measured by 31 P MRS suggest a mechanistic role for CK metabolism in the failing heart (see MRS in the Failing Heart: From Mice to Humans)13,19 that can be directly targeted for therapy.20 Access to the myocyte’s metabolic machinery for producing ATP energy via oxidative phosphorylation (OXPHOS) using carbon (13 C) MRS is limited by the low isotopic abundance of 13 C, although it might be useful for seeing endogenous lipids and perhaps glycogen in the human heart.21 However, © 2016 John Wiley & Sons, Ltd. 1183 PA Bottomley Myofibril Cytosol Mitochondria OXPHOS ATP Cr CK ATP Cr CK CK k1 ADP + Pi Energy sink PCr PCr ADP + Pi Energy source myocytes.23,24 Figure 1. Illustration of the CK shuttle hypothesis in ATP generated by oxidative phosphorylation (OXPHOS) in the mitochondria (right) phosphorylate Cr via the CK reaction, producing PCr. The process 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 ensuing contraction. Unphosphorylated Cr disperses to the mitochondria for rephosphorylation, resulting in a net transfer of biochemical energy from mitochondria to myofibrils. The CK pseudo first-order reaction rate, k1 , measured by 31 P MRS saturation transfer,13 reflects the tissue average transfer rate (center) recent approaches employing exogenous, hyperpolarized, 13 Cenriched metabolic substrates offer new minimally invasive tools for probing OXPHOS ATP synthesis in future human studies.22 Human 1 H MRS studies of cardiac lipids, 13 C MRS, hyperpolarization, and cardiac MRS in animals are reviewed elsewhere in this volume (see Cardiac Lipids by 1 H MRS; Integration of 13 C Isotopomer Methods and Hyperpolarization Provides a Comprehensive Picture of Metabolism; Hyperpolarization Methods for MRS; Cardiac MRS Studies in Rodents and Other Animals). The present article focuses on CK metabolism, which involves 31 P MRS except for the use of 1 H MRS to measure CR.16–18 The importance of CK metabolism stems from its putative role as an intracellular spatial energy shuttle and temporal buffer to facilitate high-energy phosphate transfer – in the form of PCr – from the mitochondria where ATP is produced by OXPHOS, to the myofibrils to fuel muscular contraction (Figure 1).23,24 The by-product, Cr, ultimately returns to the mitochondria for rephosphorylation. This role is supported by observations that PCr begins to deplete almost immediately to maintain ATP levels at the onset of critical ischemia or hypoxia,1 and that the resting CK rate is many-fold faster than OXPHOS,13 as would be required for a temporal buffer. 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 parameter in cardiac MRS. Since an earlier human cardiac MRS review in 2008,25 the number of papers reporting PCr/ATP values in cohorts of healthy controls has doubled to over 110 studies of more than 1600 volunteers. The NMR field strength (B0 ) for human cardiac studies has been extended from 1.5 to 7 T (Figure 2).26–28 Protocols for quantifying absolute concentrations of the CK substrates, [PCr] and [ATP],29 the CK rate constant k1 and 1184 the flux of the ATP supply, k1 ⋅[PCr], have also been worked out for human cardiac 31 P MRS at 3 T.30–32 The short take in toto is that one or more of these parameters often show profound differences in acute33 and chronic heart disease and may independently predict cardiac events and outcomes.34,35 Cardiac MRS methods and results are now reviewed, starting from 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 ranges of CK measures in the healthy and diseased human heart presented. Methods Signal-to-noise Ratio (SNR) SNR Relative to 1 H MRS. The SNR of a metabolite with an MRS X , per unit volume of ‘X-nuclei’ moiety bearing a number, Nml with spin IX can be deduced from The Basics (Section 5; Equations 32 and 33 therein). If a set of NX transients are detected and averaged from a tissue volume VX using a receiver with bandwidth BWX at an NMR frequency 𝜈 X , the SNR relative to a 1 H NMR experiment (subscripts H) at the same B0 is √ X 𝜈X MX VX Nml |B1X | NX RH BWH SNRX √ = × 10(NFH −NFX )∕20 H SNRH 𝜈H MH VH Nml |B1H | NH RX BWX (3) Here the M, B1 , and R are, respectively, the nuclear magnetization per Tesla per unit volume, the magnitude of the circularly polarized transverse RF magnetic field produced at the volume by the detector excited with unit current, and its RF resistance when loaded by the sample. The NFs are the MRI/MRS system noise figures at the two frequencies. Assuming that the noise is sample dominant, and that the losses are inductive at 36 and derive from the same sample volume, both √ frequencies √ RH ∕ RX = 𝜈H ∕𝜈X . But because X 2 MX Nml 𝜈X IX (IX + 1) = H MH Nml 𝜈H2 IX (IH + 1) (4) for spin- 1∕2 nuclei, equation (3) reduces to √ X |B1X | NX BWH 𝜈X2 VX Nml SNRX √ = 2 H SNRH 𝜈H VH Nml |B1H | NH BWX √ X N 𝜈X2 VX Nml √ X = 2 H 𝜈H VH Nml NH (5) (6) Equation (6) assumes that the detector coil efficiencies (B1 per ampere) and receiver bandwidths are the same at the two frequencies. These expressions include the intrinsic difference in NMR sensitivity of nucleus X versus 1 H (via the 𝜈 2 terms), but ignore differences in the system NF, as well as NMR excitation and relaxation effects. Cardiac MRS of endogenous metabolism in humans is limited by low metabolite SNR primarily due to low metabolite X concentrations, as reflected by the Nml term above. In the healthy heart, [ATP], [PCr], and [CR] are present at about 6, 10, and 30 μmol g−1 wet weight, respectively.25 This compares © 2016 John Wiley & Sons, Ltd. Volume 5, 2016 MRS Studies of Creatine Kinase Metabolism in Human Heart 3T PCr 40 7T γ-ATP 30 α-ATP 20 2,3-DPG (b) 10 β-ATP PDE 10 0 0 10 (a) 5 0 −5 −10 −15 ppm (c) Figure 2. Comparison of typical cardiac 31 P spectra at 3 T (blue) and 7 T (red) from a 5.6 ml voxel in the septum. (a) Spectra are apodized with an exponential 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 septal voxel marked in red. The yellow stripe shows the location of a saturation band used to minimize chest contamination. (Reproduced from Ref. 26. © John Wiley & Sons, Ltd., 2014) to about 43 mmol g−1 for tissue water (77% by weight), which has a 1 H concentration of 2 × 43 = 86 mmol g−1 . For PCr and X H ATP, Nml ∕Nml are thus 1.2 × 10−4 and 7 × 10−5 , respectively. After accounting for the lower sensitivity of 31 P [borne by the quadratic frequency terms in equation (5)], the SNR of PCr in a cardiac 31 P MRS experiment is about 1.9 × 10−5 times lower than an equivalent 1 H NMR experiment on tissue water (assuming VX /VH = BWH /BWX = B1X /B1H = 1) at the same B0 . A 1 H MRS CR experiment performed on the 3.0 ppm moiety with its 3 protons will have 45 times higher SNR than the 31 P MRS PCr experiment. Nevertheless, CR SNR is still 9 × 10−4 times lower than tissue water. These enormous SNR deficits must be made up somehow. Negotiating the Low-SNR Experiment. Since increasing the B0 by orders of magnitude cannot be contemplated [and would preferentially advantage 1 H MRS due to the quadratic frequency dependencies in equation (5)], other sacrifices must be made, and spatial resolution is the prime target. Choosing a 10 ml 31 P MRS voxel instead of a 0.05 × 0.05 × 0.3 = 7.5 × 10−4 ml 1 H MRI pixel buys a factor of about 13 000, bringing the shortfall to within a factor of 4 of tissue water, for example. It is only after this sacrifice that 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, especially relative to 1 H MRI whose bandwidths (BWH ) are often 16 kHz or higher. For a cardiac MRS experiment, equation (5) shows that setting BWX ∼ 1 kHz compared to 16 kHz can increase SNR fourfold. NF should also not be overlooked. Non-1 H MRS is often an afterthought in commercial MRI scanners: the author has measured >2-fold losses in cardiac 31 P MRS SNR due to insufficient preamplification or inadequate digital Volume 5, 2016 noise suppression in two different leading brands of MRI/MRS systems operating at 3 and 1.5 T (see The Basics and Magnetic Resonance Spectroscopy Instrumentation on how to measure system NF). Given the huge cost and siting requirements of MRI/MRS magnets, the option of increasing B0 to obtain higher SNR is really only justifiable after all the other factors in equation (5) have been optimized. At the scanner, the total examination time is typically limited by patient tolerance to about an hour. Allowing 20 min for positioning, performing rudimentary MRI to guide MRS, plus setting up MRI/MRS, means that the total number of NX transients that can be acquired can consume at most, about 40 min. To detect a 20% change in an MRS metric of interest – say, PCr/ATP or [PCr] – requires an SNR > 5 per data set. If multiple data sets are needed to determine that metric within said 40 min acquisition window – say, four for a CK reaction rate13 or a metabolite spin–lattice relaxation (T1 ) measurement, or for reference scans for quantification29 or studies involving rest and exercise33 or pharmaceutical intervention20 – then this SNR must often be realized in under 10 min. The spatial localization method and study protocol must be tailored accordingly lest the study fail to achieve its objectives. Nuclear Overhauser Enhancement (nOe). The SNR of cardiac MRS performed on non-1 H nuclei such as 13 C and 31 P can be improved by the combined effect of 1 H nOe (nuclear overhauser enhancement) and 1 H-decoupling21,37–41 (see The Basics). This technique requires the addition of an RF excitation channel that provides an essentially continuous low-level 1 H irradiation via associated 1 H excitation coils, and electronics to isolate the 1 H nOe/decoupling power from the 31 P (or 13 C) receiver.21,38 At 1.5 T, the reported 31 P MRS nOe © 2016 John Wiley & Sons, Ltd. 1185 PA Bottomley for PCr, 𝛾-ATP and 𝛽-ATP in the human heart are, respectively, 𝜂(= the fractional increase in signal) = 0.61 ± 0.25 SD (standard deviation), 0.6 ± 0.3, and 0.3 ± 0.2. Thus, SNR gains of up to 1.6-fold are possible.38,40 Note that the nOe is likely B0 dependent. At 3 T, SNR gains from nOe of 13–43% are reported (mean PCr gain, 1.23-fold, ±0.14; 𝛾-ATP, 1.27 ± 0.35; 𝛽-ATP, 1.14 ± 0.29).41 Cardiac MRS Detection Coils Small ‘surface coils’ implanted directly about the heart in intact animals7 or on isolated perfused hearts8 can be used to study in vivo high-energy phosphate metabolism during ischemia and monitor response to therapy. Indeed, all human cardiac 31 P MRS performed since the first studies in the 1980s have employed surface detection coils placed on the chest closest to the anterior myocardium.3 Such spectra are typically dominated by signals originating close to the coil due to its nonuniform sensitivity profile (see Surface Coil NMR: Detection with Inhomogeneous Radiofrequency Field Antennas; Surface and Other Local Coils for In Vivo Studies; Detection Coils for MRS; Quantifying Metabolite Ratios and Concentrations by Non-1 H MRS). The nonuniform sensitivity also affords surface coil-based detectors an optimum SNR when the noise is sample dominant (which is typical for human torso studies), by limiting the effective volume of tissue that can contribute noise (see √ The Basics). This SNR advantage derives primarily from the 1/ R dependence of equation (3). The optimum SNR at a region of interest (ROI) lying at depth √ d on the axis of a tuned loop detector, has a radius a = d/ 5. Given approximate depths of the heart from the chest of 7–15 cm, this translates to an optimum detector comprised of about 7–15 cm diameter surface coils.42,43 A multichannel phased array comprised of such coils and arranged against the chest adjacent to the heart, can yield, in practice, about 80% of the ultimate intrinsic SNR (the maximum SNR that can be had by any design assuming zero detector noise).42 This translates to SNR gains of 6- to 10-fold compared to 1 H body coils. This has not gone unnoticed in cardiac 1 H MRI where torso arrays employing similar principles are routinely used. Cardiac MRS that does not use similar technology may thus be further SNR-disadvantaged relative to state-of-the-art cardiac 1 H MRI. Bearing in mind that it is always possible to make√a lousy detector of any geometry, in addition to (i) the a = d/ 5 rule, other ‘rules of thumb’ for cardiac MRS detectors are that: (ii) (iii) (iv) (v) 1186 Noncircular coils (square, etc.), and ‘figure-8’ or ‘butterfly’ coils are less-efficient and will generally have lower SNR than circular loops. A ‘quadrature’ coil pair comprised of a figure-8 coil plus circular loop will generally underperform a simple phased array comprised of loop coils. A multiturn (distributed capacitance) loop coil will generally outperform a single-turn (distributed capacitance) loop coil, if they are both operating well below the coil’s self resonance frequency. Finally, because the heart is closer to the surface of the chest when a subject is oriented prone compared to a supine orientation, and due to the nonuniform sensitivity of the optimized surface coil, a prone orientation can yield 2–3 times the SNR achieved by a supine orientation.43,44 Localization in Cardiac MRS 31 P MRS. By itself, a surface coil placed on the chest is unsuitable for localizing MRS signals to a heart because the detected spectrum will be dominated by signals from the intervening superficial chest muscle. The first localized 31 P MRS of the human heart was performed using depth resolved surface coil spectroscopy (DRESS) with slice-selective excitation and MRI gradients positioned using 1 H MRI guidance.3 Cardiac 31 P MRS studies performed since 2008 have utilized the following methods: 1. one-, two-, and three-dimensional (1-D, 2-D, and 3-D) chemical shift imaging (CSI; see CSI and SENSE CSI and Quantifying Metabolite Ratios and Concentrations by Non-1 H MRS)33,45,46 ; 2. 1-D and 3-D image-selected in vivo spectroscopy (ISIS; see Single-Voxel MR Spectroscopy)47 ; 3. 3-D CSI augmented at 3 T with 31 P saturation bands48 or ‘crusher’ gradients applied using a surface spoiling gradient coil at 7 T,28 to minimize contamination from chest muscle and surrounding tissue; 4. 3-D CSI using the spatial localization with optimum pointspread function (SLOOP) method (see Accurate and Efficient Localized Spectroscopy from Anatomically Matched Regions: SLOOP and Its Enhancements)40,49 ; and 5. spectroscopy with linear algebraic modeling (SLAM; see Accelerated Spatially Encoded Spectroscopy of Arbitrarily Shaped Compartments Using Prior Knowledge and Linear Algebraic Modeling).50 The CSI experiments are preferably ‘acquisition weighted’ by collecting more signals with the lower order phase-encoding gradients than with the higher order gradients in a graded fashion,51 or by using a Fourier series window (FSW)52 strategy, in order to optimize the point spread function (PSF) which defines the spatial resolution of the technique. These methods and the earlier DRESS (see Single-Voxel MR Spectroscopy)3 and rotating frame zeugmatography (RFZ; Localized MRS Employing Radiofrequency Field (B1 ) Gradients)53 methods of cardiac MRS are detailed elsewhere and will not be revisited here. Often, hybrid methods that combine CSI with DRESS or ISIS to encode one or more dimensions are used in cardiac 31 P MRS to reduce the dimensionality or number of CSI phase-encoding steps while retaining full 3-D gradient-mediated localization. Common variants are as follows: 6. 2-D CSI with slice-selective excitation (DRESS) in the third 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 run coincident with the phase-encoding CSI gradients. In method 7, the basic 1-D CSI sequence is preceded by two slice-selective inversion pulses applied in the other two non-CSI dimensions, and repeated with all four combinations of inversion pulses © 2016 John Wiley & Sons, Ltd. Volume 5, 2016 MRS Studies of Creatine Kinase Metabolism in Human Heart applied and not applied. The resulting four signals are added and subtracted for each phase encode in the manner of 2-D ISIS. The hybrid 3-D ISIS and 3-D CSI methods yield substantially rectangular- or cubic-shaped voxels. SLOOP and SLAM yield arbitrarily shaped voxels that conform to the segmented portion of the heart.40,49,50 The 1-D methods alone, yield fuzzily defined voxels whose shape and extent in the two dimensions not explicitly defined by the gradients, are determined by the intersection of the 1-D gradient-localized plane with the detector coil’s sensitivity profile. This means that the larger the detector coil or detector array is, the poorer the localization in those dimensions. It also means that surface coil detectors should be positioned with the ROI centered on the coil’s cylindrical axis and that the 1-D-localizing gradient be directed along the cylindrical axis to localize a set of ‘sensitive disks’ parallel to the surface coil. Indeed, the sensitivity profile of the detection system modulates the MRS signal acquired by every localization method anyway. This preferentially weights the signal contributions in every voxel to favor those that lie closest to, say, an optimum 7–15 cm diameter surface coil. The weighting is typically large enough to warrant sensitivity corrections to measurements of cardiac tissue volumes when they are used to quantify absolute metabolite concentrations in voxels with dimensions >1 cm (see Quantifying Metabolite Ratios and Concentrations by Non-1 H MRS).29 In the case of the RFZ method which also generates a set of 1-D-resolved spectra, the voxel shapes are further modulated by the gradient contours of the B1 field of the excitation coil (see Localized MRS Employing Radiofrequency Field (B1 ) Gradients).53 1H MRS. Cardiac 1 H MRS could use the same localization methods as 31 P MRS, and these can work for studies of lipids and water. However, multiple factors can confound the use of CSI and ISIS for acquiring uncontaminated 1 H spectra of lowconcentration cardiac metabolites like CR. First, there is the intense water resonance from tissue and moving blood, and, second, the intense lipid resonance from pericardial fat and the chest. Third, there is cardiac and breathing motion. Fourth, intense signals can ‘bleed’ into cardiac voxels from adjacent chest voxels due to an imperfect PSF for CSI, or to incomplete signal cancellation in ISIS whose localization depends on spectral subtraction. Fifth, B0 inhomogeneity across the CSI field of view (FOV) combined with the much smaller chemical shift dispersion of 1 H compared to 31 P can exacerbate all these problems. On the other hand, unlike 31 P where spin-echo methods are avoided due to signal loss from J-coupling and spin–spin (T2 ) relaxation, 1 H moieties are generally blessed with longer T2 s. This renders them amenable to spin-echo localization methods that can avoid the excitation of large FOVs containing much of the troublesome water and lipid signals. Thus, 1 H MRS studies of human myocardial CR have to date have utilized singlevoxel point-resolved surface coil spectroscopy (PRESS)17,58,59 or stimulated-echo acquisition mode (STEAM)18 localization with water suppression, as detailed in Single-Voxel MR Spectroscopy. These methods yield CR measurements from about 3–9 ml voxels in scan times of <10 min at 1.5–3 T. Volume 5, 2016 Known Artefacts of Cardiac MRS Signal ‘Bleed’ and Partial Volume. The primary artefact in cardiac 31 P MRS is the contamination of cardiac spectra from extra-voxel signals due to imperfect localization. For CSI methods, the artifact is commonly called ‘bleed’ and arises from the combined effect of the sinc-function-shaped PSF extending into adjacent voxels, and tissue heterogeneity within each voxel. When the ‘center of mass’ of the signal in each voxel coincides with the geometric center of the voxel, its PSF is sampled perfectly at every zero crossing in adjacent voxels, and there is no bleed artifact.60 However, when the signal center of mass in the voxel is offset from the voxel’s geometric center, its PSF in adjacent voxels is sampled at nonzero points on the PSF’s side lobes.61 The nonzero signal alternately adds or subtracts to the signal in adjacent voxels because the PSF has both positive and negative lobes. The likelihood of such artefacts increases with larger MRS voxel sizes because of the increased probability of anatomic heterogeneity (it is not due to CSI grid sizes that use eight or more phase-encoding steps60 ). When the PCr distribution in a 31 P chest voxel is heterogeneous, the contamination will generally subtract from the PCr in a bordering cardiac voxel, but add to the next deepest cardiac voxel.60 Because [PCr] in chest muscle is over twice that in heart but [ATP] is the same, the effect is to alternately decrease and increase the apparent PCr/ATP ratio with depth into the heart. The amplitude of the modulation is exacerbated by the higher sensitivity of a surface detector coil in the chest, which amplifies the intensity of the muscle signal relative to the cardiac signal. On the distal side, the absence of PCr in ventricular blood which does contain [ATP], albeit at a lower level than muscle, reduces the apparent PCr/ATP ratio in voxels that intersect the ventricular chamber in proportion to the partial volume present in the voxel. The same is true of the nearby liver, which also contains no PCr. These problems are alleviated by minimizing the diameter of the detector coil (see section titled ‘Cardiac MRS Detection Coils’) – since larger coils increase the volume sensitivity to extraneous sources; and/or by applying saturation pulses to the chest28,48 ; and/or by using acquisition weighting to improve the PSF by reducing the amplitude of the side lobes51,52 ; or through the use of post-acquisition spatial filtering (e.g., applying a Fermi filter) which does the same as acquisition weighting but broadens voxel size; and/or by applying intra-voxel tissue heterogeneity correction methods (see Quantifying Metabolite Ratios and Concentrations by Non-1 H MRS). Motion, Chemical Shift Displacement Artifact and Partial Saturation. The primary artefacts that distinguish the application of PRESS and STEAM 1 H MRS to the heart, from their application to the brain and other organs, are those arising from cardiac motion and breathing, which are more intense than anywhere else, and from the presence of intense lipid resonances, as noted in section titled ‘1 H MRS’. 31 P MRS is less susceptible to cardiac motion than 1 H MRS because the voxels are larger, there are no interfering peaks whose intensity is comparable to the fat or water 1 H peaks, and because spin echoes are not used. However, protocols employing ISIS have an extended exposure to motion artefacts because localization © 2016 John Wiley & Sons, Ltd. 1187 PA Bottomley requires the subtraction of large signal volumes acquired with and without inversion over time periods of twice the pulse sequence repetition period (TR), 4TR, and 8TR for 1-D, 2-D, and 3-D ISIS, respectively. In both cardiac 31 P and 1 H MRS, motion effects can be ameliorated using a prone orientation and cardiac triggered acquisitions,17,18,33,44 or using a combined cardiac-respiratory double-triggering strategy for 1 H MRS (see Physiologic Motion: Dealing with Cardiac, Respiratory, and Other Sporadic Motion in MRS).58,59 Both 1 H and 31 P MRS methods employing slice selective excitation (DRESS, PRESS, STEAM) or selective inversion (ISIS) in one or more dimensions suffer from chemical shift displacement artefact in those dimensions (see Measuring Metabolite Concentrations I: 1 H MRS; Single-Voxel MR Spectroscopy). This problem may be alleviated by using the strongest selective gradients and shortest RF pulses allowed. ISIS suffers from the added partial saturation effect imparted by its inversion pulses (see Single-Voxel MR Spectroscopy) to the 31 P metabolites, whose T1 s are somewhat longer than those of 1 H metabolites. The use of longer TRs than typical of simple pulse-and-acquire approaches, may be needed to maintain consistent saturation levels over the full ISIS cycle, albeit at a cost to efficiency. A saturation effect may also arise from the echo-producing 180∘ pulses in PRESS since the residual longitudinal magnetization is inverted, instead of being refocused, if the initial pulse is not set at 90∘ . Quantification Ratios. The PCr/ATP ratio is measured from the ratio of the 31 P MRS peak areas of PCr and either the 𝛾-ATP or the 𝛽-ATP resonances. Because the metabolite T1 s of PCr and ATP are different and long such that 31 P acquisitions are usually partially saturated, it is standard practice to correct the PCr/ATP ratios for partial saturation. This is done using either the known excitation flip angle at the heart and prior determined T1 values62 , or by directly measuring saturation factors from unlocalized scans.44 Table 1 lists published relaxation times for CK metabolites in the healthy human heart. Metabolite T1 measurements are unavailable for patients. However, an analysis of the dependence of saturation factors on chest muscle content in multiple patient populations suggests that saturation corrections for myocardial PCr/ATP ratios do not vary significantly with disease state or chest muscle contamination at 1.5 T.67 Note, however, that the intrinsic T1 , T1int , a theoretical measure of the T1 in the absence of CK exchange, also does not differ significantly in patients with heart failure at 3 T,32 while exchange rates do vary.13 It is also standard practice to correct PCr/ATP ratios for the distortion introduced by ventricular blood, by subtracting a signal amount equal to about 0.3 times the integrated blood 2,3-diphosphoglycerate (DPG) peak, from the ATP peak area.68 Although nOe is often not used in 31 P MRS37–39 , the differences in 𝜂 for PCr and ATP are expected to distort the PCr/ATP ratio, necessitating corrections if a physiological ratio is being sought or if interlaboratory comparisons are being made.38–41 1188 Table 1. Published relaxation times for CK metabolites in the normal human heart B0 (31 P) (T) T1 (PCr) T1 (𝛾-ATP) T1 (𝛽-ATP) T1 (Pi) References 1.5 1.5 1.5 2.0 3 3 4 7 3 3 3 4.2 ± 0.2a 2.6 ± 0.6a 2.24 ± 0.54a — 4.3 ± 0.7 3.0 ± 0.5 — — 4.2 1.7 — — 4.2 ± 1 2.24 ± 0.63 2.46 ± 0.6 4.3 ± 2.4 3.8 ± 0.7 2.4 ± 1.1 — — 5.8 ± 0.5 3.1 ± 0.6 — — 5.3 ± 1.6 2.7 ± 0.6 — — 3.1 ± 0.4 1.82 ± 0.09 — — — — — 7.4 ± 1.8b — — — 8.2 ± 1.3c — — — 8.4 ± 1.4d B0 (1 H) (T) T1 (CR) T1 (water) T2 (CR) T2 (water) Reference 1.48 1.21 135 33.1 64 1.5 62 63 64 62 65 66 56 26 31 32 32 Notes: Values are means ± SD; T1 in second, T2 in millisecond. 𝛾-ATP and 𝛽-ATP are the 𝛾- and 𝛽-phosphates of ATP. a Reported literature average.62 b The ‘intrinsic T ’ of PCr, T 1 1int , calculated assuming no CK chemical exchange (see Measuring Biochemical Reaction Rates In Vivo with Magnetization Transfer). cT 1int for PCr corrected for spillover irradiation (uncorrected value ∼7.5 s).32 d The spillover-corrected T 1int for PCr in heart failure patients (P = 0.6 vs healthy controls).32 Total myocardial creatine, CR, in 1 H MRS is usually measured from its N-methyl 1 H resonance at 3.0 ppm, and quantified relative to water in a nonsuppressed spectrum acquired from the same voxel. The normal CR/water ratio is about 10−3 .18 For standardization, the STEAM or PRESS measurements of water ratios should be corrected for T2 attenuation as well as T1 saturation.16–18 In the absence of formal T2 measurements, this can be done by extrapolating CR measurements, recorded at long and short echo times, exponentially back to time zero.18 Concentrations and Reaction Rates. Absolute concentration measurements are far fewer, but all that is required after a corrected ratio is obtained is a concentration reference plus corrections for sensitivity and tissue volume differences when the reference and cardiac voxels are at different locations.29,45,69 Tissue water contents are surprisingly stable and fairly well known, enabling the water signal from a nonsuppressed 1 H spectrum to serve as a concentration reference for both 1 H and 31 P MRS.16–18,64,69 External phosphate reference solutions are often used as well.29 The reaction rates for the CK and ATPase reactions can be measured by 31 P MRS saturation transfer protocols.13 Basically, a first reactant moiety, say PCr, is measured with and without the reaction product moiety, 𝛾-ATP, saturated, and the fractional change in amplitude of the first moiety due to its chemical reaction, is recorded. An additional experiment is then performed to determine the T1 of the first moiety while saturation is applied to the second moiety, in order to convert the fractional change to a rate. The product of the rate with © 2016 John Wiley & Sons, Ltd. Volume 5, 2016 MRS Studies of Creatine Kinase Metabolism in Human Heart the concentration, for example, k1 ⋅[PCr], is the forward flux through the reaction in micromole per gram second. Protocol Specifics and Derivative Quantities. Detailed protocols, equations, and specific examples of quantification of 31 P metabolite ratios, concentrations, and CK reaction rates in the human heart are presented in Quantifying Metabolite Ratios and Concentrations by Non-1 H MRS and Measuring Biochemical Reaction Rates In Vivo with Magnetization Transfer, to which the reader is referred. The formula for determining intracellular pH from the chemical shift difference between Pi and PCr is in Cardiac MRS Studies in Rodents and Other Animals, and the quantification of 1 H metabolites is covered in Measuring Metabolite Concentrations I: 1 H MRS. With measurements of PCr/ATP, PCr, CR, ATP, Pi, pH, k, and CK flux in hand, a complete characterization of cardiac CK metabolism is but a few short steps away. First, unphosphorylated creatine, [Cr] = [CR]–[PCr], can be derived from joint 31 P and 1 H MRS measurements of PCr and CR.16 [ADP] can then be 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 of ATP hydrolysis available to fuel cardiac contraction, −𝛥G∼ATP , is given by 𝛥G∼ATP = 𝛥G0 + RT ln([ADP][Pi]∕[ATP]) kJ mol−1 (8) with 𝛥G0 (=−30.5 kJ mol−1 ) as the standard free energy change, and RT is the universal gas constant times absolute temperature.13,70 Results Normal Values Table 2 presents the average (±SD) of the mean values of human cardiac CK metabolic parameters measured by 31 P and 1 H MRS published through 2015. These are unweighted by the number of subjects or SDs listed in the source publications. The mean myocardial PCr/ATP has risen slightly from 1.72 ± 0.26 in 200825 to 1.84. The individual study data are visualized in Figure 3. The B0 at which the studies were conducted does not significantly affect the mean PCr/ATP (Figure 2a) – a good sign – and the scatter appears to decline at 3 and 4 T compared to 1.5 T, although the 1.5 T data pool includes much older data. However, the reported SDs accompanying the individual mean values do not yet make a case that the increase in SNR with B0 ,26,65,66 translates to more precise myocardial PCr/ATP measurements overall (Figure 2b). Even so, direct comparative measurements at 1.5, 3,65 and 7 T26 suggest otherwise, and SNR gains at higher fields are often spent on improving spatial resolution and/or speed [VX and NX in equation (3)] instead of precision.56,141 The choice of localization method also does not appear to systematically affect PCr/ATP or its variability overall (Figure 2c). This does not mean that localization is not important, but rather suggests that other factors – including experimental and physiological variability – are dominant sources of scatter. Regarding the former, about a half of the studies have SDs ≤15% with a third of them below 10% overall. This demonstrates that such levels of precision are in fact commonly achievable, and suggests that experimental factors unrelated to B0 or localization method per se, represent the primary sources of variability between studies of human myocardial PCr/ATP ratios. PCr/ATP does not appear to vary with location in the normal heart.56,106 Despite the added uncertainty in measuring reference concentrations and other factors needed to convert metabolite ratios to absolute concentrations, cardiac [PCr] and [ATP] measurements appear to be at least as precise as the PCr/ATP measurements (Table 2). Although this may reflect better control of experimental variables in this more quantitative work, a caveat is that these studies remain limited to about four independent research groups. The same is true of the [CR] measurements, which at 28.0 ± 0.7 μmol g−1 appear tightest of all. The tabulated values for [CR], the CK rate k1 , and the flux each reflect the efforts of just two laboratories. The dearth of measurements of Pi undoubtedly arises from the difficulty in resolving it from blood DPG and other phosphomonoester (PM) resonances in the healthy heart. Although 1 H decoupling can reduce the line widths of DPG and PM signals and improve the spectral resolution of Pi, it is still undetectable 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 [PCr] (μmol g−1 wet) [ATP] (μmol g−1 wet) pHb k1 (s−1 ) CK flux (μmol g−1 s−1 ) [CR] (μmol g−1 wet) 1.84 ± 0.27 10.14 ± 1.36 6.02 ± 1.00 7.13 ± 0.04 0.36 ± 0.06 3.35 ± 0.21 28.0 ± 0.7 1662 237 237 31 133 94 66 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) 13, 19, 29, 35, 45, 64, 69, 87, 95, 98, 110, 120, 126, 135, 140 (16 cohorts) 13, 19, 29, 35, 45, 64, 69, 87, 95, 98, 110, 120, 126, 135, 140 (16 cohorts) 12, 54, 70, 86 13, 19, 30–32, 35, 126, 140–142 13, 19, 31c, 35, 126, 140 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 be uncorrected for saturation or nOe. a Number of subjects – may include same subjects reported in repeat publications. b Measurements from Pi at 1.5 T, which is undetectable about half the time.12 c Value based on a [PCr] determination scaled assuming [ATP] = 5.5 μmol g−1 wet wt. Volume 5, 2016 © 2016 John Wiley & Sons, Ltd. 1189 PA Bottomley PCr/ATP 60 2.5 SD (%) 2.3 2.3 50 2.1 1.9 2.1 1.9 40 1.7 1.7 1.5 30 1.5 0.9 10 0.7 0.7 B0 (T) 1 2 (a) 3 4 5 6 7 8 0 B0 (T) 0.5 1 2 3 4 5 6 7 8 (b) (c) 3-DCSI, 2-DCSI+DRESS 0.9 1.1 1-D CSI+ 2-D ISIS 1.1 DRESS 20 Surface crusher 1-DCSI, RFZ 1-DCSI+DRESS ISIS, SLOOP, FSW 1.3 1.3 0.5 PCr/ATP 2.5 Figure 3. Literature measurements of the cardiac PCr/ATP ratio in healthy controls by localized 31 P MRS. Each symbol represents a mean published value for 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 standard deviation (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 the localization method used to measure it. The data from the three left-most columns utilized 1-D gradient localization (confined in the other two dimensions only 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 columns of 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 be imperceptible to 31 P MRS and could not contaminate myocardial Pi and pH measurements.84 From Table 2, we see that PCr constitutes 36% of the total creatine pool in normal heart, hence [Cr] = 17.9 μmol g−1 wet. Thus, PCr’s fraction of the total creatine pool is a half of that 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/PCr system in shuttling energy from the mitochondria to fuel the contracting myofibrils even at rest. ATP synthesis through OXPHOS has been estimated from myocardial oxygen (O2 ) consumption, which is about 0.085 ml g−1 wet weight per minute in healthy resting subjects.147,148 Assuming 3 mol of ATP is produced per mole of O2 consumed yields a mean ATP supply of 0.33 μmol g−1 s−1 via OXPHOS, which is consistent with invasive measures of ∼0.43 μmol g−1 s−1 .149 This means that the cardiac CK flux of 3.35 μmol g−1 s−1 (Table 2) would be able to shuttle up to about 10 times the energy provided by aerobic metabolism at rest to support contraction, consistent with a CK role as a spatiotemporal buffer.13 From equation (7), the value of [ADP] is approximately 90 μmol l−1 . From equation (8) then, 𝛥G∼ATP ≈ 60 kJ mol−1 for the normal heart.13,150 Physiological Variations Cyclic Energy Demand and Cardiac Workload. All of the CK metabolite values in Table 2 were measured at rest and/or represent temporal averages. However, energy demand varies throughout the cardiac cycle, arguably by at least threefold between peak demand and relaxation.13 If the role of PCr and 1190 the CK reaction is as a rapid energy reserve, then an obvious question is whether myocardial PCr/ATP varies cyclically in the healthy heart at rest, or during exercise. Measurements of PCr/ATP triggered at different points in the cardiac cycle in healthy subjects are few, but any metabolic variations other than those attributable to myocardial volume, are undetectable.71 Indeed, a theoretical analysis of the CK enzymatic rate and Bloch equations employing empirical parameter ranges recently concluded that (i) the maximum intracycle changes in high-energy phosphate would be ≈0.4 mM, which are at or below current detection levels and (ii), that saturation transfer 31 P MRS would be unable to detect intracyclic fluctuations in k1 or CK flux.150 If CK metabolites did vary cyclically, even modestly, then much larger changes would be expected with exercise when energy demands would be much higher. PCr/ATP has been studied with increased cardiac workloads while subjects lay in the scanner using three types of stress test. First, an isometric exercise, performed, for example, with a hand-grip dynamometer.33 Second, an aerobic exercise involving the lifting of weights with the legs,75 and, third, stress induced by pharmaceutical agents such as dobutamine.13,80 The increase in cardiac workload as indexed by the heart-rate blood-pressure product (HR × BP) in these protocols, is limited to: (i) about 40% with a hand-grip exercise involving exertion at 30% of the subject’s maximum force33 ; (ii) about twofold with aerobic exercise involving lifting multiple 2.5 kg weights74 ; and (iii) up to fourfold using dobutamine.80,92,96 Compared to aerobic exercising, the isometric and dobutamine protocols minimize motion problems during acquisition. Studies using isometric,33,85 aerobic,48,75,128 and dobutamine stress13,80,138 at up to double the HR × BP did not produce © 2016 John Wiley & Sons, Ltd. Volume 5, 2016 MRS Studies of Creatine Kinase Metabolism in Human Heart any significant changes in PCr/ATP. However, at a higher stress of three to four times the resting HR × BP, significant reductions of 14–21% (P < 0.001) in myocardial PCr/ATP were observed92,99 even in athletes.96 It is interesting to note that even though CK starts with a 10-fold buffering capacity for shuttling OXPHOS energy at rest (see section titled ‘Normal Values’), the CK flux might be expected to have difficulty meeting 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 increased energy demand at the new stress level. To test whether CK flux upregulates with cardiac workload, the CK rate and flux were measured in healthy subjects during dobutamine stress using 31 P MRS saturation transfer methods.13 No significant changes were observed in PCr/ATP, [PCr], [ATP], k, or CK flux, at least for work-loads at an average of double the resting rate.13 If CK flux does not change with increasing workloads, then the CK ATP supply must be limited. The observed decline in myocardial PCr/ATP at the highest levels of stress92,96,99 would be a consequence of this limit, which might even be a determinant of individual limits to cardiac workload. Cardiovascular Fitness. A number of studies have examined the effect of cardiovascular fitness on both resting and stressed myocardial PCr/ATP in healthy volunteers. High-level dobutamine stress at three- to four-fold HR × BP elicited similar 14–17% decreases in myocardial PCr/ATP in both elite cyclists and age-matched healthy controls, while their resting PCr/ATP values were also the same (1.4 ± 0.2).96 A more recent study, compared resting PCr/ATP in healthy middle-aged (∼50 years old) volunteers as a function of their maximum working capacity, MWC, as gauged by a stepped exercise.55 Unlike the earlier high-level HR × BP study, this study found that resting myocardial PCr/ATP correlated with MWC and those with MWC > 230 W had significantly higher PCr/ATP than those with MWC <200 W (1.93 ± 0.36 vs 1.59 ± 0.35; P < 0.001).55 On the other hand, a comparison of myocardial PCr/ATP ratios in elite track sprinters, marathon runners, and young sedentary but lean men, published the same year, found no significant differences among the three groups (PCr/ATP = 1.9–2.4), although athletes had the highest mean PCr/ATP ratios.123 A follow-up study by the same authors showed no significant differences between young sedentary (25 ± 2 years; PCr/ATP = 1.94 ± 0.36) and young (26 ± 5 years; PCr/ATP = 2.36 ± 0.36) and middle-aged (47 ± 9 years; PCr/ATP = 2.19 ± 0.34) athletes, but sedentary middle-aged (45 ± 6 years) subjects had significantly lower cardiac PCr/ATP (1.83 ± 0.27) versus the three other young and/or fit groups.127 Differences in myocardial PCr/ATP ratios among control groups that are supposed to be comparable, combined with observed changes that often fall within normal ranges published by others, represent major distractions for these studies. However, if such differences are set aside as being attributable to systematic factors of methodological origin, a picture that emerges in toto is that most young healthy adults in their 20s are endowed with a high cardiac reserve as indexed by a high PCr/ATP ratio of, say, 1.8–2.0, regardless of their sedentary or Volume 5, 2016 Table 3. Reported age and gender variations in PCr and ATP in normal subjects Age/gender n 32 ± 3 years 60 ± 13 years 30 ± 6 years 53 ± 7 years <40 years >40 years M F 15 15 37 39 16 14 18 12 PCr/ATP 1.7 ± 0.3 1.6 ± 0.4 2.16 ± 0.36 1.83 ± 0.37† 1.9 ± 0.5 1.9 ± 0.4 1.9 ± 0.4 1.9 ± 0.6 [PCr] (μmol g−1 ) 13.5 ± 1.9 9.7 ± 2.5* — — 9.7 ± 2.4 7.7 ± 2.5‡ 9.2 ± 2.4 8.0 ± 2.8 [ATP] References (μmol g−1 ) 8.2 ± 1.4 6.4 ± 1.8 — — 5.1 ± 1.0 4.1 ± 0.8* 4.9 ± 1.0 4.2 ± 0.9 95 95 39 39 120 120 120 120 Notes: Means ± SD as reported or calculated from standard errors; M, male; F, female. * Probability that difference is not significant, P < 0.01 versus younger group. † P < 0.001 versus younger group. ‡ P < 0.05 versus younger group. athletic lifestyles. However, with aging, a continued sedentary lifestyle is associated with a declining cardiac PCr/ATP by the time they reach about 50 years old, while maintenance of cardiovascular fitness through exercise can preserve myocardial CK energy reserve at near-youthful levels. Age and Gender. Indeed, several studies have reported some variation of PCr/ATP ratios and/or [PCr] and [ATP] concentrations with age39,95,120 and gender,120 as summarized in Table 3. This work suggests a trend of reduced metabolite concentrations,95,120 and probably PCr/ATP,39 with age above 40 years, and perhaps in women versus men of similar age as well.120 Such results are consistent with a decline in cardiac CK energy reserve with age in cohorts of control patients that include more sedentary individuals not selected for cardiovascular fitness. However, it is prudent to be cautious because (i) there are systematic differences between the results in Tables 2 and 3; (ii) there are systematic age- and gender-related variations in chest composition that could affect the results if chest contamination is present (see section titled ‘Signal ‘Bleed’ and Partial Volume’); and (iii) some laboratories that have studied many subjects have not reported any significant age dependence. Diet and Obesity. Given that over a third of the adult American population are considered obese as of 2012 and that twothirds are overweight, the inclusion in cardiac MRS studies of ‘normal volunteers’ who are overweight but do not otherwise have exclusion criteria for heart disease, is perhaps inevitable. The effect of diet on myocardial PCr/ATP was tested in a crossover study of young men (age 22 ± 1 standard error, SE) on whom 31 P MRS was performed before and after either a 5-day high-fat diet (75% of calories) or a standard 23% fat diet. This was followed by a 2 week ‘washout’ period and crossover to the alternate diet.134 The high-fat diet increased plasma free fatty acids by 44%, and was associated with a 9% reduction in cardiac PCr/ATP (2.0 ± 0.1 SE vs 2.3 ± 0.1 SE vs P < 0.01) along with some cognitive impairment. Cardiac 31 P MRS results from age-matched obese subjects (age, 44 ± 7 years; BMI = 39 ± SD kg m−2 ) with no © 2016 John Wiley & Sons, Ltd. 1191 PA Bottomley history of cardio- or peripheral-vascular disease, smoking, hypertension (HT), or diabetes, were compared with those from ‘normal-weight’ (age, 43 ± 10 years; body mass index, BMI = 22 ± 2 SD kg m−2 ) subjects at rest and during dobutamine stress (with a 63 ± 16% HR increase).138 At rest, diastolic function (peak filling rate) and cardiac PCr/ATP were 15% lower in obese subjects than in the normal-weight group (1.7 ± 0.4 vs 2.0 ± 0.3; P < 0.05), which decreased by an additional 12% (to 1.5 ± 0.5; P = 0.03 vs rest) with dobutamine stress. As in earlier studies, PCr/ATP was unaltered during stress in the normal-weight group. A follow-up study by the same authors compared obese patients (BMI = 34 ± 4 kg m−2 ) with ‘normal-weight’ subjects (BMI = 22 ± 2) before and after a year in a supervised weight-loss program.151 At the initial 31 P MRS examination, the PCr/ATP ratio was 1.58 ± 0.47 compared to 2.03 ± 0.27 in normal-weight subjects (P = 0.002). As a result of the weight-loss program, the obese subjects shed 9 kg (to BMI = 29 ± 2), and cardiac PCr/ATP increased by 24% to 1.96 ± 0.47 (P < 0.05), which was accompanied by improvements in diastolic function. Thus, both dietary fat and obesity are associated with impaired cardiac CK energy reserve. In obese subjects with diastolic dysfunction, PCr/ATP may be further reduced during stress. This transient decrease in PCr/ATP with stress has to be interpreted as evidence of ischemia, wherein the CK ATP supply is unable to meet the increased workload. The good news is that long-term weight loss may reverse this reduction in myocardial CK energy reserve. 1 H MRS studies in obesity have focused not on CR but on triglyceride elevations in the septum, which are reviewed elsewhere (see Cardiac Lipids by 1 H MRS). Hypertension (HT). Hypertension, too, may affect myocardial PCr/ATP ratios. Middle-aged hypertensive patients in whom coronary disease, malignant HT, and diabetes were all ruled out, have been studied at rest and during dobutamine stress with a 2.2-fold increase in HR × BP. The resting PCr/ATP was significantly lower than in healthy age-matched controls (1.2 ± 0.18 SD vs 1.39 ± 0.17, P < 0.05) and decreased a further 21% with stress (to 0.95 ± 0.25, P < 0.01 vs rest).99 The metabolic changes were again associated with diastolic dysfunction. However, another study showed no change in resting myocardial PCr/ATP, [PCr], or [ATP] in hypertensive patients who had a ∼50% increase in left ventricular (LV) mass compared to controls, but otherwise had no symptoms of heart failure.110 Hypoxia. Chronic exposure to hypobaric hypoxia may represent another ‘normal’ variant for human myocardial PCr/ATP. Six 20- to 30-year-old professional Sherpa trekker guides who were native to an altitude of ∼3400 m in Nepal, exhibited significant reductions in myocardial PCr/ATP to 1.0 ± 0.37 SD as compared to control ‘lowlanders’ with PCr/ATP = 1.76 ± 0.06.52 These results suggest that the heart may adapt its energy sources and delivery over time in response to chronic environmental stress.52 Yet even relatively short-term exposure to hypoxia may reduce myocardial PCr/ATP. A 31 P MRS study of 14 trekkers, age 38 ± 11 SD years, before and immediately after a 17-day 1192 trek to Mt. Everest base camp (5300 m) exemplifies an effect of hypobaric hypoxia.137 Myocardial PCr/ATP was reduced 18% from 2.05 ± 0.30 SD before, to 1.68 ± 0.30 (P = 0.003) after the trek, accompanied by some alterations in diastolic function. Cardiac metabolism and function returned to pre-trek levels by 6 months. The effect of shorter normobaric hypoxic exposures were investigated by the same authors using a hypoxic chamber with O2 maintained at an end-tidal expiration partial pressure of 50–60 mmHg and 80% peripheral O2 saturation.136 After 20 h of exposure, young (age, 24 ± 7 SD years) healthy volunteers exhibited 15% reductions in cardiac PCr/ATP (from 2.0 ± 0.3 to 1.7 ± 0.3; P < 0.01) and impaired diastolic function. Thus, cardiac CK energy reserve and high-energy phosphate balance is depressed in the presence of hypoxia, even at the relatively modest O2 reductions that can be tolerated by healthy subjects over periods ranging from hours to lifetimes. Disease Myocardial Infarction (MI). Published 31 P MRS studies are mixed on whether resting myocardial PCr/ATP ratios are altered in MI. The data are summarized in Table 4. Most likely, the mixed message arises from other conditions such as congestive heart failure (CHF) or cardiomyopathy (CM) associated with post-MI remodeling. These would confound the PCr/ATP measurements in chronic MI (see sections titled ‘Dilated Cardiomyopathy (DCM) and Heart Failure’ and ‘Hypertrophy, Valve Disease, and Heart Failure’), while playing lesser roles in acute MI in which active ischemia was not present. Meanwhile, in acute anterior MI, Pi may be elevated a week or so postonset,71 consistent with the post-MI time course of Pi seen in canine studies after an acute decline in PCr and ATP.154 Table 4. Resting myocardial PCr/ATP ratios in patients with MI and/or ischemia (ISCH) MI/ISCH MI MI + ISCHa MI MIb ISCHc MIb ISCHc MI MI MI MI MI ISCH Averagee Controls Patients References 1.6 ± 0.4 1.72 ± 0.15 1.95 ± 0.45 1.85 ± 0.25 1.85 ± 0.25 1.8 ± 1.03 1.8 ± 1.03 1.61 ± 0.18 1.72 ± 0.31 1.45 ± 0.29 1.87 ± 0.45 1.7 ± 0.40 1.45 ± 0.31 No change in patients 1.24 ± 0.3* 1.6 ± 0.19 0.94 ± 0.41 1.37 ± 0.57 1.51 ± 0.17 1.47 ± 0.38 0.6 ± 0.2d,† 1.74 ± 0.27 1.03 ± 0.39† 1.39 ± 0.23 1.34 ± 0.34‡ 71 33 79 85 85 87 87 91 101 108 126 152 153 1.73 ± 0.16 Notes: Means ± SD, as reported or calculated from reported standard errors (SE); ISCH = patients with ischemia. a ISCH patients includes 6 with MI (P = 0.052 vs controls). b Fixed defect on exercise 201 Tl imaging. c Reversible defect on exercise 201 Tl imaging. d Average for septal and anterior LV MI. e Average 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 © 2016 John Wiley & Sons, Ltd. Volume 5, 2016 MRS Studies of Creatine Kinase Metabolism in Human Heart Indeed, biochemical analyses in animal heart models show depletion of essentially all ATP and PCr within the first few hours of an ischemic injury that results in cell death.1 Because dead cells can contribute no high-energy phosphates, the resting PCr/ATP ratios that are reported in MI must derive from ‘metabolically normal’, ‘jeopardized’, and/or ischemic myocardium adjacent to, or interspersed with the infarction. Note that ‘metabolically normal’ need not mean functionally normal. A study of 29 reperfused MI patients with dysfunction in the infarct area – ‘myocardial stunning’ – found no significant changes in PCr/ATP from normal at 4 ± 2 days post onset, with PCr/ATP remaining unchanged a month later even though function had improved.91 Regardless, a preserved PCr/ATP has to be interpreted as indicating the presence of viable 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 in Table 5. Reductions of about 50% likely reflect the presence of both infarcted and noninfarcted tissue in MRS voxels. There is a significant negative correlation between cardiac ATP levels and the size of perfusion deficits quantified by thallium 201 Tl radionuclide imaging.78 In addition, [PCr] and [ATP] are reduced in patients with fixed 201 Tl defects compared to those with reversible defects.87 In the tabulated studies, uninvolved myocardium without wall motion abnormalities at echocardiography had near-normal PCr, ATP, and CR concentrations.18,64 In addition, CK rate measurements measured by saturation transfer also showed no change in k1 in MI (Table 5). Because the measurements of k1 can only derive from the myocytes that survive in the infarcted region, this result must reflect a preserved CK metabolism in the surviving tissue, even though the net CK flux given by the product, k1 ⋅[PCr], is halved due to the loss of [PCr] in the infarct.126 Myocardial Ischemia. In patients with ischemic heart disease involving stenosis of the anterior vessels, the resting anterior myocardial PCr/ATP shows a declining trend (Table 4, shaded entries), with CK metabolite concentrations in reversible defects reaching statistical significance (Table 5).87 While a number of other conditions are associated with reduced metabolite levels at rest, the transient reduction in PCr/ATP ratio that is elicited by increased cardiac workloads, indicates an excess consumption of PCr that cannot be met by OXPHOS. Thus, stress-induced changes in myocardial PCr/ATP could serve as a specific metabolic hallmark of ischemia.33 As noted in the section titled ‘Cyclic Energy Demand and Cardiac Workload’, in healthy subjects who are free of significant coronary disease, myocardial PCr/ATP is unaltered by up to a ∼2-fold increase in cardiac workload,13,33,75,85,128,138 but a small reduction at three to four times the resting workload92,96,99 may arise if the CK energy supply cannot cope with demand. However, in patients with severe anterior coronary stenosis and ischemia verified by echocardiography or 201 Tl radionuclide imaging, a simple isometric handgrip exercise that only elicited a 30–40% increase in HR × BP, was able to produce a ∼40% reduction in myocardial PCr/ATP compared to that at rest (Figure 4).33 After exercise, metabolite ratios recovered to near-normal pre-exercise values. Patients with nonischemic heart disease (CM or valve disease) who Volume 5, 2016 Table 5. CK metabolite levels in patients with MI [PCr] (μmol g−1 wet weight) Control 12.1 ± 4.3 11.9 ± 4.4 9.6 ± 1.1 [ATP] (μmol g−1 wet weight) Patients Control Patients 7.6 ± 3a, * 3.9 ± 2.2b,† 11.0 ± 4.4c 6.4 ± 1.8d, * 5.0 ± 2.4e, * 5.4 ± 1.2‡ 7.7 ± 3 6.4 ± 3.2a 4.4 ± 1.5b, * 7.9 ± 4.3c 5.4 ± 2.2d 4.9 ± 2.5e 3.4 ± 1.1‡ [CR] (μmol g−1 wet weight) 7.0 ± 1.8 5.5 ± 1.3 [CR] (μmol g−1 wet weight) Control Patients Control Patients 28 ± 6 28.8 ± 3.0 9.8 ± 8.6§ 28 ± 6 28.8 ± 3.0 28.8 ± 3.0 26 ± 11f 16.9 ± 7.1d, * 13.2 ± 5.4e, * 26.5 ± 4.3c CK rate, k1 (s−1 ) Control 0.33 ± 0.07 CK flux (μmol g−1 wet weight s) Patients Control Patients 0.31 ± 0.08 3.3 ± 0.8 1.7 ± 0.5‡ References 87 87 64 64 64 126 References 18 64 64 References 126 a Patients with MI and reversible defects on 201 Tl scintigraphy. with MI and fixed defects on 201 Tl scintigraphy. c Normo-kinetic by echocardiography. d Hypokinetic by echocardiography. e Dyskinetic by echocardiography. f Measurements from uninvolved tissue. * P < 0.05 versus controls. † P < 0.01 versus controls. ‡ P < 0.001 versus controls. § P < 0.0001 versus controls. b Patients were tested with the same exercise exhibited no PCr/ATP changes. In addition, patients with ischemia who underwent repeat stress testing after successful revascularization therapy also showed no stress-induced PCr/ATP change, consistent with a resolution of the metabolic abnormality following a successful clinical outcome.33 A similar 40% decrease in anterior myocardial PCr/ATP was reported during hand-grip exercise in patients with reversible anterior wall ischemia confirmed by exercise 201 Tl radionuclide imaging.85 Those with fixed 201 Tl defects indicative of MI exhibited no exercise-induced PCr/ATP changes. These studies, and observations that dobutamine stresstesting at 1.6-fold resting HR × BP induced no significant reductions in myocardial PCr/ATP in patients with DCM and CHF,80 suggested that stress-induced changes in PCr/ATP might well be specific to myocardial ischemia. However, as noted in sections titled ‘Diet and Obesity’ and ‘Hypertension (HT)’, 12% and 21% reductions in myocardial PCr/ATP have also been observed with dobutamine stress in obese patients138 and those with HT,99 respectively, in the absence of independent evidence of ischemia. More recently, a 9% transient reduction in cardiac PCr/ATP was reported in patients who © 2016 John Wiley & Sons, Ltd. 1193 PA Bottomley Exercise Rest Recovery PCr PCr 0.9 2.0 #16 endo. Pi 1.6 #15 PCr 1.3 3.2 epi. Chest muscle 3.2 #14 ATP (a) (b) 0 20 (c) 0 20 (d) 0 20 ppm Figure 4. Hand-grip exercise stress testing of a patient with myocardial ischemia performed at 1.5 T.33 (a) Axial 1 H surface coil image of the chest and anterior 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 locations of three 1 cm thick coronal slices localized in the 31 P MRS exam (SE, septum; RV, right ventricle; ENDO, endocardial; EPI, epicardial; ST, sternum; REF, reference vial in the 31 P coil). (b–d) Cardiac-gated 1-D CSI surface coil 31 P spectra from the three slices (b) at rest, and during (c), an 8 min isometric hand-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- and epicardial 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 coronary artery disease, during aerobic exercise testing at a 1.7-fold increase in HR × BP workload (see also section titled ‘Hypertrophy, Valve Disease, and Heart Failure’).48 This work suggests that factors other than frank vessel disease may also lead to transient reductions in energy reserve – that is, an ischemic response – in obesity, hypertensive, and hypertrophic disease. Two additional studies have used isometric hand-grip exercise to measure myocardial ischemia. One studied ‘women’s ischemic syndrome’ involving chest pain – also in the absence of significant coronary vessel disease –reporting that some women had cardiac PCr/ATP ratios during stress that were two SDs below the mean of control subjects.155 However, the overall variations in PCr/ATP were higher than in controls and exhibited both increases and decreases during stress. The second study was a double-blinded application of stress 31 P MRS to test the efficacy of an experimental antiischemic pharmaceutical that reduces the binding affinity of oxygen to hemoglobin, thereby potentially increasing oxygen availability.153 While the stress produced a 31% PCr/ATP reduction during control studies, the lesser 20% decline seen with anti-ischemic therapy in the same subjects was not significant and the outcome was inconclusive. Because LV dysfunction and heart failure are common prognoses for ischemic events, another study examined the effect of trimetazidine (TMZ) on resting cardiac PCr/ATP post ischemia. TMZ is an anti-ischemic therapy that shifts energy utilization from free fatty acids to glucose.156 A double-blinded 90-day crossover protocol with placebo or TMZ was implemented in patients with CHF but not active ischemia, acute MI, or anterior lesions at locations where the 31 P MRS was performed. It was found that TMZ reduced the symptom-based New York Heart Association (NYHA) CHF class, improved LV ejection fraction (EF), and increased myocardial PCr/ATP from an abnormally low 1.35 ± 0.33 to 1.8 ± 0.5 (P = 0.03). 1194 Dilated Cardiomyopathy (DCM) and Heart Failure. Initial 31 P MRS studies of DCM in the early 1990s revealed significant68 and nonsignificant46,77 reductions in myocardial PCr/ATP. An elevated phosphodiester (PD) peak,46,77 and an elevated Pi/PCr Table 6. Myocardial PCr/ATP in patients with dilated cardiomyopathy (DCM) Controls 1.54 ± 0.11 1.8 ± 0.21 1.65 ± 0.26 2.09 ± 0.44 1.95 ± 0.45 1.86 ± 0.17 2.02 ± 0.41 1.94 ± 0.60 1.75 ± 0.25 2.07 ± 0.17 n.s. n.s. 1.81 ± 0.49 n.s. 1.86 ± 0.17c DCM NYHA References 1.51 ± 0.29 1.46 ± 0.31* 1.52 ± 0.58 1.88 ± 0.4 1.78 ± 0.51 1.44 ± 0.52† 1.94 ± 0.43 1.63 ± 0.24a 1.54 ± 0.48† 1.63 ± 0.43b 1.26 ± 0.29 1.31 ± 0.38* 1.63 ± 0.33* 1.54 ± 0.34 1.58 ± 0.41 1.59 ± 0.48c 1.5 ± 0.4 1.56 ± 0.16d, * I–III (CHF) II–III n.s. II–IV ≥III <III I–III II–III I–III n.s. III–IV I–II n.s. II–III I–III n.s. 77 68 12 81 79 80 89 34 107 122 40 20 35 27 Average Notes: Values are means ± SD, as reported or calculated from reported data or standard errors (SEs) excluding repeat entries or data uncorrected for saturation when known; NYHA, New York Heart Association classification for congestive heart failure (CHF); n.s., not specified. a No change with dobutamine stress at a 1.6-fold increase in HR × BP.80 b PCr/ATP < 1.6 predicted mortality at 2.5 years (P < 0.02).34 c Patients had DCM or left ventricular hypertrophy (LVH).35 d Average of the studies listed. * P < 0.001 versus controls. † P < 0.05 versus controls. © 2016 John Wiley & Sons, Ltd. Volume 5, 2016 MRS Studies of Creatine Kinase Metabolism in Human Heart Table 7. CK metabolite concentrations and flux rates in patients with cardiomyopathy [PCr] (μmol g−1 wet weight) DCM + CHFa DCM DCM + CHFc LVH + CHFc,d LVH + CHF LVH no CHF CM + CHFc,f CM + CHFa LVH HCMg HCMh [ATP] (μmol g−1 wet weight) Control Patients Control Patients 10.1 ± 1.3 — 8.8 ± 1.3 — 9.4 ± 1.1 — — 9.75 ± 1.33 9.7 ± 2.5 9.4 ± 1.2 8.8 ± 2.6 8.3 ± 2.6* 6.2 ± 1.5b 4.3 ± 1.2† 6.3 ± 1.5* 7.2 ± 3.7 6.1 ± 2.0† 7.6 ± 1.8 7.39 ± 2.5‡ 6.1 ± 2.2† 7.1 ± 2.3† 6.1 ± 1.9e,† 5.7 ± 1.3 — 5.7 ± 1.0 — 5.5 ± 1.3 — — 5.7 ± 1.3 6.4 ± 1.8 5.8 ± 1.2 4.6 ± 1.0 5.2 ± 1.3 4.1 ± 0.8b 3.7 ± 0.5* 4.9 ± 0.9 5.0 ± 1.1 4.7 ± 1.3 4.9 ± 1.3 4.8 ± 1.4* 4.1 ± 1.3* 5.0 ± 0.8 3.9 ± 1.5* [CR] (μmol g−1 wet weight) DCM + CHFc DCM + CHFa HCM + CHFa CM + CHFa [CR] (μmol g−1 wet weight) Control Patients Control Patients 27.6 ± 4.1 27.6 ± 4.1 — 27.1 ± 3.2 15.0 ± 5.4‡ — — — — — — — — 16.1 ± 4.5* 22.6 ± 8.1* 16.5 ± 6‡ CK rate, k1 (s−1 ) DCM + CHFa CM + CHFc,f LVH no CHF LVH + CHF HCMg CM + CHFa CK flux (μmol g−1 wt s) Control Patients Control Patients 0.32 ± 0.07 0.21 ± 0.07‡ 3.2 ± 0.9 1.6 ± 0.6‡ 2.07 ± 1.27 2.2 ± 0.7* 1.1 ± 0.4‡ 2.0 ± 1.4‡ 1.75 ± 0.96‡ 0.32 ± 0.06 0.38 ± 0.07 0.38 ± 0.08 0.28 ± 0.13 0.36 ± 0.04 0.17 ± 0.06‡ 0.28 ± 0.15* 0.24 ± 0.12‡ 3.1 ± 0.8 3.6 ± 0.9 3.6 ± 0.8 References 13 40 110 110 19 19 20 35 95 140 135 References 143 17 17 144 References 13 20 19 19 140 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. a Patients with NYHA class I–IV CHF. b Control study for patients undergoing exercise training: training improved LV function but [PCr] and [ATP] were unchanged. c Patients with NYHA class II–III CHF. d Patients with LVH due to valve disease. e Enzyme replacement therapy increased [PCr] to 7.1 ± 1.5 μmol g−1 (P = 0.012). f Control study for double-blind evaluation of acute allopurinol treatment: allopurinol increased [PCr] by ∼11% and CK flux by 39%.20 g HCM due to a familial myosin heavy chain Arg403Gln mutation. h HCM 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 subsequently found to correlate with blood DPG signal, and attributed to blood contamination, exacerbated by the thin ventricular wall in these patients (see section titled ‘Ratios’).12 The published PCr/ATP ratios for DCM are summarized in Table 6. The preponderance of studies suggests that myocardial PCr/ATP is reduced on average by nearly 20% in DCM, an amount that does not always reach statistical significance. The PCr/ATP ratios generally correlate only weakly with functional and morphological indices of disease severity such as EF and/or fractional shortening. However, significant correlations between an increasing severity of NYHA class and a decreasing PCr/ATP have been reported.79,110,122 In addition, PCr/ATP has been observed to recover in patients whose NYHA class improves Volume 5, 2016 due to drug therapy.79 Conversely, a significantly higher risk of cardiovascular death within 2.5 years was associated with those who had myocardial PCr/ATP < 1.6, compared to those with PCr/ATP > 1.6 (mortality 40% vs 11%, P < 0.02).34 Because the relative reduction in PCr represents a drop in myocardial high-energy phosphate reserve, these findings support the old hypothesis that the failing heart is energy starved.2 The picture is reinforced by quantitative 31 P and 1 H MRS measurements of myocardial [PCr], [ATP], and [CR] in patients with CM and CHF, summarized in Table 7. Reductions in both [ATP] and [PCr] mean that measurements of PCr/ATP ratios often underestimate the loss in high-energy phosphate reserves.110 Importantly, [CR] also declines with increasing NYHA severity.144 Whether the fraction of the total creatine © 2016 John Wiley & Sons, Ltd. 1195 PA Bottomley pool that is phosphorylated is preserved – which is critical for CK reaction kinetics in equations (2) and (7) – is as yet unknown, but is at least within the grasp of MRS.16,64 As is evident from Table 7, for all of these CM studies, the reductions in the creatine pool are striking. Mindlessly averaging the tabulated results as a whole reveals a 30% reduction in [PCr] from 9.4 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 something to do with a 20% loss in [ATP] (P = 0.001). Measurements of CK reaction kinetics in these patients reveal that even more fuel has been taken from the fire. On top of the reductions in [PCr] as substrate, the CK reaction rate is also reduced by about a third (Table 7, lower panel). Consequently, the CK ATP supply at rest in those exhibiting symptoms of CHF is approximately half that in healthy subjects (Figure 5).13 Given variable intracyclic energy demands, this loss in CK ATP supply could be enough to compromise energy supply, leading to contractile dysfunction and symptoms during periods of moderate exercise, by limiting the heart’s ability to work (see section titled ‘Cyclic Energy Demand and Cardiac Workload’).13 So what mechanism could slow the CK chemical reaction rate in CHF patients? Focus shifts to the possibility that CK enzyme is somehow being damaged, for example, by reactive oxygen species (ROS) generated from increased xanthine oxidase (XO) activity, a terminal reaction in purine degradation. If so, an XO inhibitor could reduce ROS, thereby increasing CK activity and flux. A double-blinded saturation transfer study of allopurinol, a ROS inhibitor used for treating gout, was recently CK flux (μmol−1 (g. wt. s) ) 5 4 Rest 200% Stress CHF LVH HCM MI DCM LVH 3 * 2 † † 1 0 n = 16 n=6 n = 11 n=9 n =15 † † n = 17 n = 10 Normal Figure 5. The forward CK flux (micromole per gram wet weight per second) in healthy controls and patients with heart disease as measured by saturation transfer 31 P MRS at 1.5 T. Healthy subjects at rest and with dobutamine stress at a 200% in workload (HR × BP) have the highest flux (left two columns).13 Patients with pressure-overload LVH19 and anterior MI126 (third and fourth columns) were not in failure and had a normal CK rate constant, k1 , but a reduced [PCr], which resulted in a significantly lower CK flux – although only locally in the case of the MI patients. Patients with familial 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 data link a decline in CK ATP supply with heart failure (n, number of subjects; *P < 0.01 and ** P < 0.001 vs healthy controls) 1196 reported in patients with CHF. Acute administration of allopurinol increased cardiac PCr/ATP and [PCr] by about 10% (P < 0.02), but the mean CK flux – the CK ATP supply – went up by nearly 40% (P < 0.007).20 This provides direct evidence that cardiac energy delivery can be pharmaceutically augmented in the failing heart. The CK reaction may thus be a viable target for heart failure therapy, and 31 P MRS is a suitable – if not unique – noninvasive tool for evaluating such approaches (see MRS in the Failing Heart: From Mice to Humans). Hypertrophy, Valve Disease, and Heart Failure. The first localized 31 P MRS study of HCM appeared in the late 1980s.72 Studying cardiac hypertrophy with MRS has the practical advantage that the MRS signal from the thickened ventricle is less likely to be contaminated by chamber blood whose lack of PCr could systematically reduce PCr/ATP and [PCr] measurements (see section titled ‘Ratios’). Common underlying causes of HCM include familial and genetic factors, while HT and valve disease are common causes of left ventricular hypertrophy (LVH). As in DCM, CHF is a common outcome, and from an MRS standpoint, the CK metabolism of HCM and LVH are indistinguishable from DCM. Published myocardial PCr/ATP values for nonischemic hypertrophic disease to date are summarized in Table 8: the concentrations and fluxes are included in Table 7. Overall, the 31 P MRS data suggest that myocardial PCr/ATP is reduced by about 23% in hypertrophic disease, including HCM of various genetic origins, LVH, and underlying HT and valve disease. Myocardial PCr/ATP tends to decrease with the 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 is also seen in hypertrophy that is not explicitly linked to CHF or severity.19,46,94,140 Indeed, a study of LVH patients who had comparable cardiac function and morphology reported that PCr/ATP was the same in those with and those without symptoms of CHF: in fact, mean [PCr] was lowest in those LVH patients who were not in heart failure, though the difference vs. CHF was not significant19 (Table 7). So high-energy phosphate concentrations – mostly PCr – are reduced in hypertrophy.19,95,110,135,140 As if to preserve the relative fractions of phosphorylated and unphosphorylated creatine, [CR] is down by a similar fraction as [PCr] in the only reported study (Table 7).17 While [CR] reductions were less in HCM than in DCM in that study,17 this may reflect the more severe disease present in the DCM cohort. A couple of studies have reported elevated Pi in hypertrophic disease.12,94 As in healthy subjects (see section titled ‘Normal Values’) given the potential for blood DPG and PM contamination, the dearth of Pi data likely reflects quantification difficulties rather than these findings being anomalous, a situation that may be resolved at higher B0 . As also noted in section titled ‘Myocardial Ischemia’, a recent study of 40-year-olds with genetic HCM found not only a lower resting cardiac PCr/ATP ratio (1.71 ± 0.35 vs 2.14 ± 0.35 in controls, P < 0.0001), but that PCr/ATP declined further (to 1.56 ± 0.29; P = 0.02) when cardiac workload was increased 1.7-fold.48 These results, which imply a CK response in HCM analogous to that seen with transient ischemia due to coronary artery disease, differ from previous, more-limited studies of isometric handgrip exercise © 2016 John Wiley & Sons, Ltd. Volume 5, 2016 MRS Studies of Creatine Kinase Metabolism in Human Heart Table 8. Literature myocardial PCr/ATP ratios for patients with hypertrophy (LVH, HCM), including associated valve disease (VAL) and heart failure (CHF) Cause/CHF LVH-mild LVH-severe VAL-CHF VAL-non-CHF VAL HCM HCM LVH-HT, VAL HCM HCM VAL, NYHA III VAL, NYHA I-II AS, CHF AI, CHF VAL, NYHA I-III HCM HCM HCM-familial HCM MR-severe, NYHA I-III MR-dyspnea MR, NYHA I-III MR-mild, NYHA I MR-moderate, NYHA I-II HT LVH, VAL AS, LVH HCM, FRD HT, non-CHF AS, NYHA II-III NYHA II, CM, AS NYHA III, CM, AS HCM-genetic origin LVH, HT, CHF LVH, HT, non-CHF HT HT LVH, NYHA II-IIIe HCM HCMf, NYHA I-IV HCM-genetic origin HCM Averagea Controls 1.55 ± 0.2 0.89 ± 0.30 1.5 ± 0.2 — 1.76 ± 0.22 — 2.09 ± 0.44 — 1.65 ± 0.26 1.71 ± 0.32 2.02 ± 0.41 — — — — 2.15 ± 0.51 2.46 ± 0.53 2.46 ± 0.53 1.6 ± 0.4 1.61 ± 0.3 — — — — 1.39 ± 0.17 1.65 ± 0.21 1.43 ± 0.14 2.39 ± 0.13 1.59 ± 0.33 — — — 2.44 ± 0.3 1.9 ± 0.3 — 2.07 ± 0.17 — 2.14 ± 0.63 2.41 ± 0.3 1.92 ± 0.5 1.7 ± 0.3 2.14 ± 0.35 1.91 ± 0.35 Patients 0.9 ± 0.2 0.78 ± 0.16 0.8 ± 0.29 1.1 ± 0.32* 1.56 ± 0.15 1.34 ± 0.25 1.73 ± 0.23 1.43 ± 0.36* 1.89 ± 0.2 1.32 ± 0.29† 1.07 ± 0.44‡ 1.51 ± 0.09* SE 1.86 ± 0.18 SE 1.55 ± 0.43‡ 1.77 ± 0.36 1.64 ± 0.42‡ 1.93 ± 0.57 1.98 ± 0.37† 1.81 ± 0.28† 1.6 ± 0.6 1.29 ± 0.3† 1.21 ± 0.24‡ 1.29 ± 0.29‡ 1.73 ± 0.17§ 1.49 ± 0.18|| 1.2 ± 0.18† 0.8 ± 0.25* 1.24 ± 0.17‡,b 1.47 ± 0.55* No change 1.3 ± 0.2 1.36 ± 0.22 1.21 ± 0.3† 1.7 ± 0.43* 1.3 ± 0.5‡ 1.3 ± 0.3‡ 1.65 ± 0.25*,c 1.43 ± 0.21*,d 1.57 ± 0.53‡ 2.18 ± 0.41† 1.68 ± 0.43 1.4 ± 0.4 1.71 ± 0.35*,g 1.47 ± 0.31* References 72 46 46 53 53 67 67 81 81 12 83 93 93 93 93 93 94 54 103 95 97 97 97 97 97 99 102 57 109 110 110 110 110 115 19 19 121 121 125 130 135 140 48 Notes: Means ± SD as reported or calculated from SE where possible or specified SE where not; HT, hypertension; AS, aortic valve stenosis; AI, aortic incompetence; MR, mitral regurgitation; FRD, Friedreich ataxia. Control values are listed only once per reference. a Unweighted average of means, excluding Ref. 46 (not saturation corrected). b PCr/ATP improved to 1.47 ± 0.14 (P < 0.05) after aortic valve replacement. c Patients with diastolic dysfunction only. d Patients with both diastolic and systolic dysfunction. e Patients with preserved ejection fraction. f Due to Fabry disease. g Exercise 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. Volume 5, 2016 © 2016 John Wiley & Sons, Ltd. 1197 PA Bottomley 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 compromised CK energy reserve regardless of the underlying cause. Disease severity, including but not limited to heart failure, appears to exacerbate a deterioration in CK energy metabolism. Measurements of CK reaction kinetics, k1 and CK flux, might offer better specificity for CHF. LVH patients with CHF have lower k1 , while the k1 in LVH patients with no CHF but comparable cardiac function and morphology was shown to be the same as in healthy controls (Table 7).19 The combined effect of the reduction in k and reduced [PCr] means that the CK ATP supply or flux, k1 ⋅[PCr], can be reduced to as little as a third of that in healthy controls (1.1 ± 0.4 vs 3.1 ± 0.8 μmol g−1 s−1 ; P < 0.001).19 It is difficult to see how a threefold hit to cardiac ATP supply would not negatively impact cardiac energetics and function. Indeed, a 31 P MRS study of 58 patients with a mix of nonischemic LVH or DCM, and with NYHA class I–IV heart failure symptoms, found that myocardial CK flux was an independent predictor of all-cause and cardiac death, CHF hospitalization, cardiac transplantation, and ventricular-assist device placement at 5 years.35 The other independent predictors were NYHA class, EF, and African-American race. Other Specific Disorders. A number of other specific diseases have been studied by cardiac 31 P MRS including congenital cardiomegaly in an infant;4 muscular dystrophy, cardiac beri–beri and amyloidosis;81 and diseases associated with progressive systemic sclerosis.86 All these showed reductions in myocardial PCr/ATP ratios compared to healthy subjects. Similarly, patients with untreated familiar hypercholesterolemia are reported as having reduced myocardial PCr/ATP compared to statin-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 is lower in patients with hereditary hemochromatosis as well (1.6 ± 0.41 vs 1.93 ± 0.36 in healthy controls; P = 0.004).117 31 P MRS studies of type I diabetes mellitus (T1DB) show a modest decrease in myocardial PCr/ATP (to 1.9 ± 0.4 vs 2.15 ± 0.3, P < 0.05) in patients with no other symptoms of cardiac disease.111 More significant PCr/ATP reductions that were not correlated with perfusion reserve as measured by stress MRI, were reported in another study of asymptomatic T1DB patients (1.5–1.6 vs 2.1 in controls; P < 0.0001). In T1DB patients with uremia and diastolic dysfunction, an even lower 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 showed a 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 patients who had no cardiovascular disease or impaired perfusion CHF.129 Heart Transplant Patients. The management of patients with transplanted hearts requires the timely detection of graft rejection to assess whether augmented immunosuppressive therapy 1198 Table 9. Summary of MRS findings in common heart diseases through 2015 Disorder MI acute MI chronic ISCH rest ISCH exercise LVH, HCM DCM CHF + CM Valve disease XPLANT T1DB T2DB PCr/ATP Pi – ↓ – ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↑ ↑ ↑ – or ↑ [PCr] [ATP] ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ k CK flux [CR] – – or ↓ – or ↓ – or ↓ ↓ – or ↓ ↓ – or ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ Notes: XPLANT, transplanted heart (post operation); ↓, decrease versus normal; ↑, increase versus normal; –, unchanged versus normal; – or ↓ (↑), unchanged or reduced (increased) versus normal. Blank entries are not yet determined. is warranted. The standard of practice requires that endomyocardial biopsies be acquired at regularly scheduled cardiac catheterization procedures, from which histological evidence of myocyte necrosis is read. Clearly, a noninvasive method of assessing rejection would be most welcome. The idea that changes in myocardial metabolite ratios might predict histological rejection in human heart transplants arose from animal 31 P MRS studies of nonimmunosuppressed allografts, in which metabolic changes and histological evidence for acute rejection in the first week or so post-transplantation were seen.157–159 A first paper applying 31 P MRS to patients with heart transplants studied up to 5.5 years post-transplantation did find significantly lower resting anterior myocardial PCr/ATP ratios compared to normal controls (1.57 ± 0.5 vs 1.93 ± 0.21; P < 0.01).76 However, 31 P MRS agreed with histological evidence of necrosis that distinguished mild from moderate rejection (which would trigger augmented therapy), in only about 60–70% of examinations. Consequently, it was concluded that 31 P MRS did not precisely predict significant histological rejection in many transplant patients.76 This finding was confirmed in two other studies.104,160 One of these followed up 13 patients serially for 13–294 days post-transplantation and found a significant correlation between improving PCr/ATP and time post-transplantation, independent of rejection.160 That PCr/ATP ratios are imprecise predictors of histological rejection likely reflects fundamental differences between histological and metabolic indices. Thus, myocyte necrosis, which is key to the histological evaluation, cannot directly cause altered PCr/ATP ratios because dead cells can contribute no highenergy phosphates. The correlation between PCr/ATP and time post-transplantation160 suggests that the low PCr/ATP acutely following transplantation is associated with the transplant procedure itself, with factors that include graft harvesting, storage, duration of hypoxia, and so on. Metabolic recovery requires an extended period of months, which are typically punctuated by episodes of acute rejection. The use of 31 P MRS to assess the viability of excised donor hearts at transplantation is reviewed elsewhere (see Assessing Cardiac Transplant Viability with MRS). © 2016 John Wiley & Sons, Ltd. Volume 5, 2016 MRS Studies of Creatine Kinase Metabolism in Human Heart Conclusions 31 P 1H and MRS can almost completely characterize CK metabolism in the healthy and diseased human heart with measurements of [PCr], [ATP], [CR], k1 , and CK flux. The ‘almost’ is because not all measures have been obtained from all major diseases; [PCr] and [CR] have not both been measured on the same subjects to quantify [Cr] unambiguously; [Pi] and pH are not reliably quantified; nor is [ADP]. The measurements are made possible in patient examinations primarily by sacrificing spatial resolution. Accordingly, focal disease – MI and ischemia – have taken a back seat to studies of diseases that affect the heart globally – CMs, CHF, obesity, diabetes, and so on. The vast majority of published work has focused on just a single variable, the myocardial PCr/ATP ratio. It is clear from Tables 2–8 that quantification is an ongoing challenge: the normal control values are listed because the inter-laboratory variations (e.g., 1.4–2.5 in Table 8) are often comparable to the intralaboratory variations being attributed to various diseases. Establishing reproducibility for measurements performed at the same site, and between different sites, is essential for any multicenter trial and for establishing normal ranges of values that can be relevant to individual patients. As noted in section titled ‘Normal Values’, about one-third of studies are able to achieve SDs that are about 10% of the mean, so the rest of the range of variation between 1.4 and 2.5 in control subjects must relate to extraneous factors. A larger variation in the values of k1 and flux is expected due to the accumulation of factors required for those measurements: for example, when k1 is measured from four different spectra, plus a concentration measurement for flux.13 Nevertheless, it is now evident that there are physiological variations as well. Before in vivo MRS came along, some assumed that the CK reaction in healthy hearts would behave just like the CK reaction in skeletal muscle: as energy demand increased, PCr would go down to supply more ATP, and Pi would rise. After MRS showed that this did not happen, the assumption then was that PCr/ATP was fixed: regulated somehow to a single value whose scatter primarily reflected an inability to measure it accurately. While the latter is true, intralaboratory comparisons of different cohorts of healthy subjects with various conditions that are not normally designated as heart disease (as reviewed in section titled ‘Physiological Variations’) now indicate that there are variations in resting myocardial PCr/ATP with age, cardiovascular fitness, hypertension, exposure to hypoxia, obesity, and diet that might be considered ‘preclinical’. In patients, the presence of ischemic disease, heart failure, CM, diabetes, or having had an MI or a transplanted heart appear as even stronger risk factors for a reduced CK energy reserve as indexed by a lower myocardial PCr/ATP ratio (Table 9). All these point not to a fixed myocardial PCr/ATP ratio but to a meta-stable operating value that may be eroded and compounded by one or more of the aforementioned factors. The erosion probably commences with a decline in the creatine pool, which noninvasive 1 H MRS is ideally suited to document. When CK ATP flux was measured in the human heart with 31 P MRS, two of the possible outcomes could well have Volume 5, 2016 rendered the CK reaction irrelevant to cardiac energetics. First, if the rate was either much smaller than that of OXPHOS or, secondly, if it far exceeded OXPHOS, then the changes in creatine substrate levels, flux, or PCr/ATP being observed could not have a relevant effect on energy supply in the context of ongoing energy demands. Instead, the measured CK flux fell right in a range of about 7–10 times OXPHOS, and does not appear to vary much with cardiac workload in the healthy heart. This means that the reductions in flux and/or ATP supply seen in patients with ischemia, CM, or heart failure could be enough during stress to consume some of the PCr reserve and ultimately limit cardiac work.13,33 While we do not know yet whether limited CK reserve limits physical activity, we do know that compromised CK metabolism is a long-term predictor of poor cardiovascular outcomes.34,35 Given that predicting bad things is less rewarding than fixing them, it is most encouraging to see cardiac 31 P MRS metrics being used to quantify the status of the cardiovascular energy reserve in order to evaluate the benefits of both pharmaceutical20,153,156 and lifestyle interventions.40,127,134,137,151 Acknowledgments I thank RG Weiss for years of fruitful collaboration and discussions and the National Institutes of Health for past support. Biographical Sketch Paul A. Bottomley. BSc (Hon), 1975, PhD, 1978, Physics, University of Nottingham, United Kingdom. Research Associate, Johns Hopkins University, Baltimore, 1978–1980. Physicist, G. E. Research and Development Center, 1980–1994. Currently Russell H Morgan Professor and Director of the Division of MR Research of the Russell H Morgan Department of Radiology and Radiological Sciences, Johns Hopkins University. Fellow and Gold Medal recipient of the Society of Magnetic Resonance in Medicine, 1989; Coolidge Fellowship and medal, 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, about 180 peer-reviewed papers, 24 book chapters, 13 editorials, and over 225 published abstracts. Research specialties: in vivo NMR, MRI, tissue relaxation times, localized NMR spectroscopy, human cardiac NMR spectroscopy, interventional MRI, MRI safety. 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