Human Subjects All studies were approved by The Johns Hopkins Institutional Review Board on human investigation and all subjects gave informed consent following explanation of the study and protocol. Fourteen healthy subjects (mean age 41±7years; two women) without a history of
hypertension, coronary artery disease, or diabetes were studied contemporaneous with the patients and served as controls. Some healthy subjects were included in a prior report on dilated cardiomyopathy 1. All subjects with LVH (n = 20; age 56±12; 11 women) had stage II
hypertension of at least one-year duration with echocardiographic evidence of concentric LVH (septal and posterior wall thickness >1.2cm). The left ventricular mass index (LVMI) and ejection fraction (EF) of those subjects with LVH were calculated from echocardiography data as previously reported2;3. All patients had no evidence of ischemic disease determined by either a cardiac catheterization within the prior 12 months demonstrating no coronary stenosis greater than 50% or a negative stress test.
Patients were excluded from the study if they had a history of myocardial infarction or unexplained wall motion abnormalities on echocardiography. Other exclusion criteria included a history of pulmonary disease, cor pulmonale, obstructive sleep apnea or any other pulmonary condition requiring baseline oxygen administration, pregnancy, or renal failure as defined by baseline serum creatinine of >2.0mg/dl. Lastly, patients with a history of recent cerebrovascular disease, uncontrolled thyroid disease, or active use of allopurinol were also ineligible. 1
CHF was defined using a modified Framingham scoring system 4. CHF was diagnosed at the time of the patient’s hospitalization with two major criteria (paroxysmal nocturnal dyspnea or orthopnea, jugular venous distention, rales, cardiomegaly, radiographic pulmonary edema, S3 gallop, hepatojugular reflux, or >4.5 lb. weight loss in response to diuresis); or one major and two minor criteria (ankle edema, night cough, dyspnea on exertion, hepatomegaly, or pleural effusion). If possible, the echocardiogram used to define ejection fraction and hypertrophy was obtained within 72 hours of hospitalization for CHF. No patients underwent MRI/MRS studies during acute episodes of decompensated CHF.
Cardiac MRS Subjects were positioned prone, rotated on their left side, in a clinical broadband 1.5T GE Rev 5.8 Signa MRI scanner on a 31P MRS 6-cm receive, 20-cm transmit surface coil probe. Cardiacgated scout trans-axial fast-spin-echo 1H MRI was performed to aid in positioning the coil immediately anterior to the myocardium. Next, the four-angle saturation transfer (FAST)
method was applied with one-dimensional chemical shift imaging (slice thickness, 1 cm) via the
P MRS coils to measure ATP flux through the CK reaction of the human heart in vivo1;5. The
FAST method consisted of two pairs of measurements of the PCr signal, M, with adiabatic “BIRP” 6 pulse flip-angles of 15° and 60°. One pair were acquired with the -ATP resonance at – 2.7 ppm saturated, to obtain PCr signals denoted by primes, M’15 and M’60. The other pair were acquired with the same irradiation applied at +2.7 ppm as a control, to yield PCr (unprimed) signals M15 and M60. A fifth
P MRS data set was acquired with a 60° pulse and without
selective saturation in order to measure the spillover of the saturation and to provide a basis for
phosphate metabolite concentrations. A cardiac 1H MRS data set was acquired with a 60° pulse using the
P MRS receiver coil to provide a water reference signal for the concentration
measurements7. After the exam, fully-relaxed 1H and
P MRS were acquired from a reference phantom of
phosphate with the same coils to calibrate the ratio of 31P to 1H signals for the water-referenced concentration measurement7;8. The availability of
P MRS data sets from both the heart and
phantom permitted, in addition, semi-independent phosphate-referenced concentration estimates9;10. [PCr] and [ATP] were calculated by two previously validated techniques that
used water 7 and phosphate
as internal and external references, respectively, and the results
averaged for the myocardial slices in each subject7;9;10. Concentration measurements were determined from MRS peak areas that were Gaussian-fitted in the frequency-domain, and corrected for blood and saturation11;12. The water-reference concentration measures (umol/g tissue) assume substantially equivalent tissue water content among the groups10.
The forward CK rate constant, Kfor, was calculated from the FAST method using Equations (5), (6) and (9) of Ref 5. The fully-relaxed PCr signal in the presence of control, M0, and saturating irradiation, M0', are first derived from M15, M60, M’15, and M’60 quantified as peak heights via5:
M 60 [cos15 cos 60] sin 60[cos15 1] R.sin15[cos 60 1]
where R= M60/M15,
M0' obtains from the same expression with M60 and R replaced by M’60
and R’= M’60/M’15, respectively The spin-lattice relaxation time in the presence of -ATP saturation was calculated from the dual angle formula 13;14:
sin 60 R'sin15 T '1 TR / ln cos15sin 60 R'cos 60sin15
where TR is the pulse sequence repetition period. The forward rate constant was then obtained from 5 Kf T1' = Q(1-M0'/M0) , 
where Q is the ratio of M0 measured with control irradiation to that measured with no irradiation. The CK forward flux rate was calculated from the product Kfor[PCr] for each subject. Statistics Continuous variables are presented as mean ± standard deviation (SD), while categorical variables are presented as either absolute counts or percentages. Statistical analysis of demographic variables was performed with an unpaired, 2-tailed Student’s t test. The ShapiroWilk test was employed for testing the normality of the key metabolic variables and it could not reject the hypothesis that all these variables are normally distributed. Therefore parametric testing was performed and differences in means among groups were assessed by ANOVA followed by groupwise comparisions with the Tukey-Kramer Adjustment to control the overall error rate. In all instances, statistical significance was assumed at p<0.05.
References 1. Weiss RG, Gerstenblith G, Bottomley PA. ATP flux through creatine kinase in the normal, stressed, and failing human heart. Proc Natl Acad Sci. 2005;102:808-813. 2. Devereux RB, Alonso DR, Lutas EM, Gottlieb GJ, Campo E, Sachs I, Reichek N. Echocardiographic assessment of left ventricular hypertrophy: comparison to necropsy findings. Am J Cardiol. 1986;15:450-458. 3. American Society of Echocardiography Committee on Standards SoQoT-DE. Recommendations for quantitation of the left ventricle by two-dimensional echocardiography. J AM Soc Echocardiogr. 1989;2:367.
4. Kannel WB, Castelli WP, McKee PA, Feinleib M. Role of blood pressure in the development of congestive heart failure. The Framingham Study. NEJM. 1972;287:781787. 5. Bottomley PA, Ouwerkerk R, Lee RF, Weiss RG. The four-angle saturation transfer (FAST) method for measuring creatine kinase reaction rates in vivo. Magnetic Resonance in Medicine. 2002;47:850-863. 6. Bottomley PA, Ouwerkerk R. BIRP, an improved implementation of low-angle adiabatic (BIR-4) excitation pulses. J Mag Res. 1993;103A:242-244. 7. Bottomley PA, Atalar E, Weiss RG. Human cardiac high-energy phosphate metabolite concentrations by 1D-resolved NMR spectroscopy. Magnetic Resonance in Medicine. 1996;35:664-670. 8. Bottomley PA, Weiss RG. Noninvasive localized MR quantification of creatine kinase metabolites in normal and infarcted canine myocardium. Radiology. 2001;219:411-418. 9. Bottomley PA, Hardy CJ, Roemer PB. Phosphate metabolite imaging and concentration measurements in human heart by nuclear magnetic resonance. Magnetic Resonance in Medicine. 1990;14:425-434. 10. Yabe T, Mitsunami K, Inubushi T, Kinoshita M. Quantitative measurements of cardiac phosphorus metabolites in coronary artery disease by 31P magnetic resonance spectroscopy. Circulation. 1995;92:15-23. 11. Hardy CJ, Weiss RG, Bottomley PA, Gerstenblith G. Altered myocardial high-energy phosphate metabolites in patients with dilated cardiomyopathy. Am Heart J. 1991;122:795801. 12. Bottomley PA, Hardy CJ, Weiss RG. Correcting human heart 31P NMR spectra for partial saturation. Evidence that saturation factors for PCr/ATP are homogeneous in normal and diseased states. J Mag Res. 1991;95:341-355. 13. Bottomley PA, Ouwerkerk R. Optimum flip-angles for exciting NMR with uncertain T1 values. Magnetic Resonance in Medicine. 1994;32:137-141. 14. Bottomley PA, Ouwerkerk R. Fast sensitive T1 measurement in vivo with low angle adiabatic pulses: the dual-angle method. J Mag Res. 1994;104:159-167.