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Nicotinic acid receptor agonists have previously been shown to cause acute reductions in cardiac contractility. We sought to uncover the changes in cardiac metabolism underlying these alterations in function. In nine humans, we recorded cardiac energetics and function before and after a single oral dose of nicotinic acid using cardiac MRI to demonstrate contractile function and Phosphorus-31 ( 31 P) magnetic resonance spectroscopy to demonstrate myocardial energetics. Nicotinic Acid 400 mg lowered ejection fraction by 4% (64 ± 8% to 60 ± 7%, P = .03), and was accompanied by a fall in phosphocreatine/ATP ratio by 0.4 (2.2 ± 0.4 to 1.8 ± 0.1, P = .04). In four groups of eight Wistar rats, we used pyruvate dehydrogenase (PDH) flux studies to demonstrate changes in carbohydrate metabolism induced by the nicotinic acid receptor agonist, Acipimox, using hyperpolarized Carbon-13 ( 13 C) magnetic resonance spectroscopy. In rats which had been starved overnight, Acipimox caused a fall in ejection fraction by 7.8% (67.5 ± 8.9 to 60 ± 3.1, P = .03) and a nearly threefold rise in flux through PDH (from 0.182 ± 0.114 to 0.486 ± 0.139, P = .002), though this rise did not match pyruvate dehydrogenase flux observed in rats fed carbohydrate rich chow (0.726 ± 0.201). In fed rats, Acipimox decreased pyruvate dehydrogenase flux (to 0.512 ± 0.13, P = .04). Concentration of plasma insulin fell by two-thirds in fed rats administered Acipimox (from 1695 ± 891 ng/L to 550 ± 222 ng/L, P = .005) in spite of glucose concentrations remaining the same. In conclusion, we demonstrate that nicotinic acid receptor agonists impair cardiac contractility associated with a decline in cardiac energetics and show that the mechanism is likely a combination of reduced fatty acid availability and a failure to upregulate carbohydrate metabolism, essentially starving the heart of fuel., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Background: Why some but not all patients with severe aortic stenosis (SevAS) develop otherwise unexplained reduced systolic function is unclear. We investigate the hypothesis that reduced creatine kinase (CK) capacity and flux is associated with this transition., Methods: We recruited 102 participants to 5 groups: moderate aortic stenosis (ModAS) (n=13), SevAS, left ventricular (LV) ejection fraction ≥55% (SevAS-preserved ejection fraction, n=37), SevAS, LV ejection fraction <55% (SevAS-reduced ejection fraction, n=15), healthy volunteers with nonhypertrophied hearts with normal systolic function (normal healthy volunteer, n=30), and patients with nonhypertrophied, non-pressure-loaded hearts with normal systolic function undergoing cardiac surgery and donating LV biopsy (non-pressure-loaded heart biopsy, n=7). All underwent cardiac magnetic resonance imaging and 31 P magnetic resonance spectroscopy for myocardial energetics. LV biopsies (AS and non-pressure-loaded heart biopsy) were analyzed for CK total activity, CK isoforms, citrate synthase activity, and total creatine. Mitochondria-sarcomere diffusion distances were calculated by using serial block-face scanning electron microscopy., Results: In the absence of failure, CK flux was lower in the presence of AS (by 32%, P =0.04), driven primarily by reduction in phosphocreatine/ATP (by 17%, P <0.001), with CK k f unchanged ( P =0.46). Although lowest in the SevAS-reduced ejection fraction group, CK flux was not different from the SevAS-preserved ejection fraction group ( P >0.99). Accompanying the fall in CK flux, total CK and citrate synthase activities and the absolute activities of mitochondrial-type CK and CK-MM isoforms were also lower ( P <0.02, all analyses). Median mitochondria-sarcomere diffusion distances correlated well with CK total activity ( r =0.86, P =0.003)., Conclusions: Total CK capacity is reduced in SevAS, with median values lowest in those with systolic failure, consistent with reduced energy supply reserve. Despite this, in vivo magnetic resonance spectroscopy measures of resting CK flux suggest that ATP delivery is reduced earlier, at the moderate AS stage, where LV function remains preserved. These findings show that significant energetic impairment is already established in moderate AS and suggest that a fall in CK flux is not by itself a necessary cause of transition to systolic failure. However, because ATP demands increase with AS severity, this could increase susceptibility to systolic failure. As such, targeting CK capacity and flux may be a therapeutic strategy to prevent and treat systolic failure in AS.

Background: Obesity is strongly associated with exercise intolerance and the development of heart failure. Whereas myocardial energetics and diastolic function are impaired in obesity, systolic function is usually preserved. This suggests that the rate of ATP delivery is maintained, but this has never been explored in human obesity. We hypothesized that ATP transfer rate through creatine kinase (CK) ( k f CKrest ) would be increased, compensating for depleted energy stores (phosphocreatine/ATP), but potentially limiting greater ATP delivery during increased workload. We hypothesized that these changes would normalize with weight loss., Methods: We recruited 80 volunteers (35 controls [body mass index 24±3 kg/m 2 ], 45 obese [body mass index 35±5 kg/m 2 ]) without coexisting cardiovascular disease. Participants underwent body composition analysis, magnetic resonance imaging of abdominal, liver, and myocardial fat content, left ventricular function, and 31 P magnetic resonance spectroscopy to assess phosphocreatine/ATP and CK kinetics, at rest and during dobutamine stress. Obese volunteers were assigned to a dietary weight loss intervention, before reexamination., Results: At rest, although myocardial phosphocreatine/ATP was 14% lower in obesity (1.9±0.3 versus 2.2±0.2, P <0.001), k f Ckrest was 33% higher (0.23±0.07 s -1 versus 0.16±0.08 s -1 , P =0.002), yielding no difference in overall resting ATP delivery (obese 2.5±0.9 µmol·g -1 ·s -1 versus control 2.2±1.1 µmol·g -1 ·s -1 , P =0.232). In controls, increasing cardiac workload led to an increase in both k f CK (+86%, P <0.001) and ATP delivery (+80%, P <0.001). However, in obesity, similar stress led to no significant increase in either k f CK ( P =0.117) or ATP delivery ( P =0.608). This was accompanied by reduced systolic augmentation (absolute increase in left ventricular ejection fraction, obese +16±7% versus control +21±4%, P =0.031). Successful weight loss (-11±5% body weight) was associated with improvement of these energetic changes such that there was no significant difference in comparison with controls., Conclusions: In the obese resting heart, the myocardial CK reaction rate is increased, maintaining ATP delivery despite reduced phosphocreatine/ATP. During increased workload, although the nonobese heart increases ATP delivery through CK, the obese heart does not; this is associated with reduced systolic augmentation and exercise tolerance. Weight loss reverses these energetic changes. This highlights myocardial energy delivery through CK as a potential therapeutic target to improve symptoms in obesity-related heart disease, and a fascinating modifiable pathway involved in the progression to heart failure, as well.

Mitochondrial creatine kinase (Mt-CK) is a major determinant of cardiac energetic status and is down-regulated in chronic heart failure, which may contribute to disease progression. We hypothesised that cardiomyocyte-specific overexpression of Mt-CK would mitigate against these changes and thereby preserve cardiac function. Male Mt-CK overexpressing mice (OE) and WT littermates were subjected to transverse aortic constriction (TAC) or sham surgery and assessed by echocardiography at 0, 3 and 6 weeks alongside a final LV haemodynamic assessment. Regardless of genotype, TAC mice developed progressive LV hypertrophy, dilatation and contractile dysfunction commensurate with pressure overload-induced chronic heart failure. There was a trend for improved survival in OE-TAC mice (90% vs 73%, P = 0.08), however, OE-TAC mice exhibited greater LV dilatation compared to WT and no functional parameters were significantly different under baseline conditions or during dobutamine stress test. CK activity was 37% higher in OE-sham versus WT-sham hearts and reduced in both TAC groups, but was maintained above normal values in the OE-TAC hearts. A separate cohort of mice received in vivo cardiac 31 P-MRS to measure high-energy phosphates. There was no difference in the ratio of phosphocreatine-to-ATP in the sham mice, however, PCr/ATP was reduced in WT-TAC but preserved in OE-TAC (1.04 ± 0.10 vs 2.04 ± 0.22; P = 0.007). In conclusion, overexpression of Mt-CK activity prevented the changes in cardiac energetics that are considered hallmarks of a failing heart. This had a positive effect on early survival but was not associated with improved LV remodelling or function during the development of chronic heart failure.

Background: Cardiovascular phosphorus MR spectroscopy ( 31 P-CMRS) is a powerful tool for probing energetics in the human heart, through quantification of phosphocreatine (PCr) to adenosine triphosphate (ATP) ratio. In principle, 31 P-CMRS can also measure cardiac intracellular pH (pH i ) and the free energy of ATP hydrolysis (ΔG ATP ). However, these require determination of the inorganic phosphate (Pi) signal frequency and amplitude that are currently not robustly accessible because blood signals often obscure the Pi resonance. Typical cardiac 31 P-CMRS protocols use low (e.g. 30°) flip-angles and short repetition time (TR) to maximise signal-to-noise ratio (SNR) within hardware limits. Unfortunately, this causes saturation of Pi with negligible saturation of the flowing blood pool. We aimed to show that an adiabatic 90° excitation, long-TR, 7T 31 P-CMRS protocol will reverse this balance, allowing robust cardiac pH i measurements in healthy subjects and patients with hypertrophic cardiomyopathy (HCM)., Methods: The cardiac Pi T 1 was first measured by the dual TR technique in seven healthy subjects. Next, ten healthy subjects and three HCM patients were scanned with 7T 31 P-MRS using long (6 s) TR protocol and adiabatic excitation. Spectra were fitted for cardiac metabolites including Pi., Results: The measured Pi T 1 was 5.0 ± 0.3 s in myocardium and 6.4 ± 0.6 s in skeletal muscle. Myocardial pH was 7.12 ± 0.04 and Pi/PCr ratio was 0.11 ± 0.02. The coefficients of repeatability were 0.052 for pH and 0.027 for Pi/PCr quantification. The pH in HCM patients did not differ (p = 0.508) from volunteers. However, Pi/PCr was higher (0.24 ± 0.09 vs. 0.11 ± 0.02; p = 0.001); Pi/ATP was higher (0.44 ± 0.14 vs. 0.24 ± 0.05; p = 0.002); and PCr/ATP was lower (1.78 ± 0.07 vs. 2.10 ± 0.20; p = 0.020), in HCM patients, which is in agreement with previous reports., Conclusion: A 7T 31 P-CMRS protocol with adiabatic 90° excitation and long (6 s) TR gives sufficient SNR for Pi and low enough blood signal to permit robust quantification of cardiac Pi and hence pH i . Pi was detectable in every subject scanned for this study, both in healthy subjects and HCM patients. Cardiac pH i was unchanged in HCM patients, but both Pi/PCr and Pi/ATP increased that indicate an energetic impairment in HCM. This work provides a robust technique to quantify cardiac Pi and pH i .

Background: Heart failure (HF) is characterized by altered myocardial substrate metabolism which can lead to myocardial triglyceride accumulation (steatosis) and lipotoxicity. However its role in mild HF with preserved ejection fraction (HFpEF) is uncertain. We measured myocardial triglyceride content (MTG) in HFpEF and assessed its relationships with diastolic function and exercise capacity., Methods: Twenty seven HFpEF (clinical features of HF, left ventricular EF >50%, evidence of mild diastolic dysfunction and evidence of exercise limitation as assessed by cardiopulmonary exercise test) and 14 controls underwent 1 H-cardiovascular magnetic resonance spectroscopy ( 1 H-CMRS) to measure MTG (lipid/water, %), 31 P-CMRS to measure myocardial energetics (phosphocreatine-to-adenosine triphosphate - PCr/ATP) and feature-tracking cardiovascular magnetic resonance (CMR) imaging for diastolic strain rate., Results: When compared to controls, HFpEF had 2.3 fold higher in MTG (1.45 ± 0.25% vs. 0.64 ± 0.16%, p = 0.009) and reduced PCr/ATP (1.60 ± 0.09 vs. 2.00 ± 0.10, p = 0.005). HFpEF had significantly reduced diastolic strain rate and maximal oxygen consumption (VO 2 max), which both correlated significantly with elevated MTG and reduced PCr/ATP. On multivariate analyses, MTG was independently associated with diastolic strain rate while diastolic strain rate was independently associated with VO 2 max., Conclusions: Myocardial steatosis is pronounced in mild HFpEF, and is independently associated with impaired diastolic strain rate which is itself related to exercise capacity. Steatosis may adversely affect exercise capacity by indirect effect occurring via impairment in diastolic function. As such, myocardial triglyceride may become a potential therapeutic target to treat the increasing number of patients with HFpEF.

Heart failure is a major cause of morbidity and mortality in type 2 diabetes. Type 2 diabetes contributes to the development of heart failure through a variety of mechanisms, including disease-specific myocardial structural, functional and metabolic changes. This review will focus on the contemporary contributions of state of the art non-invasive technologies to our understanding of diabetic cardiomyopathy, including data on cardiac disease phenotype, cardiac energy metabolism and energetic deficiency, ectopic and visceral adiposity, diabetic liver disease, metabolic modulation strategies and cardiovascular outcomes with new classes of glucose-lowering therapies., (© 2018 The authors.)

The energy starvation hypothesis proposes that maladaptive metabolic remodeling antedates, initiates, and maintains adverse contractile dysfunction in heart failure (HF). Better understanding of the cardiac metabolic phenotype and metabolic signaling could help identify the role metabolic remodeling plays within HF and the conditions known to transition toward HF, including "pathological" hypertrophy. In this review, we discuss metabolic phenotype and metabolic signaling in the contexts of pathological hypertrophy and HF. We discuss the significance of alterations in energy supply (substrate utilization, oxidative capacity, and phosphotransfer) and energy sensing using observations from human and animal disease models and models of manipulated energy supply/sensing. We aim to provide ways of thinking about metabolic remodeling that center around metabolic flexibility, capacity (reserve), and efficiency rather than around particular substrate preferences or transcriptomic profiles. We show that maladaptive metabolic remodeling takes multiple forms across multiple energy-handling domains. We suggest that lack of metabolic flexibility and reserve (substrate, oxidative, and phosphotransfer) represents a final common denominator ultimately compromising efficiency and contractile reserve in stressful contexts., (Copyright © 2017 the American Physiological Society.)

Ketosis, the metabolic response to energy crisis, is a mechanism to sustain life by altering oxidative fuel selection. Often overlooked for its metabolic potential, ketosis is poorly understood outside of starvation or diabetic crisis. Thus, we studied the biochemical advantages of ketosis in humans using a ketone ester-based form of nutrition without the unwanted milieu of endogenous ketone body production by caloric or carbohydrate restriction. In five separate studies of 39 high-performance athletes, we show how this unique metabolic state improves physical endurance by altering fuel competition for oxidative respiration. Ketosis decreased muscle glycolysis and plasma lactate concentrations, while providing an alternative substrate for oxidative phosphorylation. Ketosis increased intramuscular triacylglycerol oxidation during exercise, even in the presence of normal muscle glycogen, co-ingested carbohydrate and elevated insulin. These findings may hold clues to greater human potential and a better understanding of fuel metabolism in health and disease., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Mice over-expressing the creatine transporter have elevated myocardial creatine levels [Cr] and are protected against ischaemia/reperfusion injury via improved energy reserve. However, mice with very high [Cr] develop cardiac hypertrophy and dysfunction. To investigate these contrasting effects, we applied a non-biased hypothesis-generating approach to quantify global protein and metabolite changes in the LV of mice stratified for [Cr] levels: wildtype, moderately elevated, and high [Cr] (65-85; 100-135; 160-250 nmol/mg protein, respectively). Male mice received an echocardiogram at 7 weeks of age with tissue harvested at 8 weeks. RV was used for [Cr] quantification by HPLC to select LV tissue for subsequent analysis. Two-dimensional difference in-gel electrophoresis identified differentially expressed proteins, which were manually picked and trypsin digested for nano-LC-MS/MS. Principal component analysis (PCA) showed efficient group separation (ANOVA P ≤ 0.05) and peptide sequences were identified by mouse database (UniProt 201203) using Mascot. A total of 27 unique proteins were found to be differentially expressed between normal and high [Cr], with proteins showing [Cr]-dependent differential expression, chosen for confirmation, e.g. α-crystallin B, a heat shock protein implicated in cardio-protection and myozenin-2, which could contribute to the hypertrophic phenotype. Nuclear magnetic resonance (¹H-NMR at 700 MHz) identified multiple strong correlations between [Cr] and key cardiac metabolites. For example, positive correlations with α-glucose (r² = 0.45; P = 0.002), acetyl-carnitine (r² = 0.50; P = 0.001), glutamine (r² = 0.59; P = 0.0002); and negative correlations with taurine (r² = 0.74; P < 0.0001), fumarate (r² = 0.45; P = 0.003), aspartate (r² = 0.59; P = 0.0002), alanine (r² = 0.66; P < 0.0001) and phosphocholine (r² = 0.60; P = 0.0002). These findings suggest wide-ranging and hitherto unexpected adaptations in substrate utilisation and energy metabolism with a general pattern of impaired energy generating pathways in mice with very high creatine levels.

Background: Left ventricular (LV) hypertrophy in aortic stenosis (AS) is characterized by reduced myocardial perfusion reserve due to coronary microvascular dysfunction. However, whether this hypoperfusion leads to tissue deoxygenation is unknown. We aimed to assess myocardial oxygenation in severe AS without obstructive coronary artery disease, and to investigate its association with myocardial energetics and function., Methods: Twenty-eight patients with isolated severe AS and 15 controls underwent cardiovascular magnetic resonance (CMR) for assessment of perfusion (myocardial perfusion reserve index-MPRI) and oxygenation (blood-oxygen level dependent-BOLD signal intensity-SI change) during adenosine stress. LV circumferential strain and phosphocreatine/adenosine triphosphate (PCr/ATP) ratios were assessed using tagging CMR and 31P MR spectroscopy, respectively., Results: AS patients had reduced MPRI (1.1 ± 0.3 vs. controls 1.7 ± 0.3, p < 0.001) and BOLD SI change during stress (5.1 ± 8.9% vs. controls 18.2 ± 10.1%, p = 0.001), as well as reduced PCr/ATP (1.45 ± 0.21 vs. 2.00 ± 0.25, p < 0.001) and LV strain (-16.4 ± 2.7% vs. controls -21.3 ± 1.9%, p < 0.001). Both perfusion reserve and oxygenation showed positive correlations with energetics and LV strain. Furthermore, impaired energetics correlated with reduced strain. Eight months post aortic valve replacement (AVR) (n = 14), perfusion (MPRI 1.6 ± 0.5), oxygenation (BOLD SI change 15.6 ± 7.0%), energetics (PCr/ATP 1.86 ± 0.48) and circumferential strain (-19.4 ± 2.5%) improved significantly., Conclusions: Severe AS is characterized by impaired perfusion reserve and oxygenation which are related to the degree of derangement in energetics and associated LV dysfunction. These changes are reversible on relief of pressure overload and hypertrophy regression. Strategies aimed at improving oxygen demand-supply balance to preserve myocardial energetics and LV function are promising future therapies.

Aims: To determine whether post-infarction LV dysfunction is due to low energy availability or inefficient energy utilization, we compared energy metabolism in normal and failing hearts. We also studied whether improved coupling of glycolysis and glucose oxidation by knockout of malonyl CoA decarboxylase (MCD-KO) would have beneficial effects on LV function and efficiency., Methods and Results: Male C57BL/6 mice were subjected to coronary artery ligation (CAL) or sham operation (SHAM) procedure. After 4 weeks and echocardiographic evaluation, hearts were perfused (working mode) to measure LV function and rates of energy metabolism. Similar protocols using MCD-KO mice and wild-type (WT) littermates were used to assess consequences of MCD deficiency. Relative to SHAM, CAL hearts had impaired LV function [lower % ejection fraction (%EF, 49%) and LV work (46%)]. CAL hearts had higher rates (expressed per LV work) of glycolysis, glucose oxidation, and proton production. LV work per ATP production from exogenous sources was lower in CAL hearts, indicative of inefficient exogenous energy substrate utilization. Fatty acid oxidation rates, ATP, creatine, and creatine phosphate contents were unaffected. Utilization of endogenous substrates, triacylglycerol and glycogen, was similar in CAL and SHAM hearts. MCD-KO CAL hearts had 31% higher %EF compared with that of WT-CAL, and lower rates of glycolysis, glucose oxidation, proton production, and ATP production, indicative of improved efficiency., Conclusion: CAL hearts are inefficient in utilizing energy for mechanical function, possibly due to higher proton production arising from mismatched glycolysis and glucose oxidation. MCD deficiency lessens proton production, LV dysfunction, and inefficiency of exogenous energy substrate utilization.

Disruption of the creatine kinase (CK) system in hearts of CK-deficient mice leads to changes in the ultrastructure and regulation of mitochondrial respiration. We expected to see similar changes in creatine-deficient mice, which lack the enzyme guanidinoacetate methyltransferase (GAMT) to produce creatine. The aim of this study was to characterize the changes in cardiomyocyte mitochondrial organization, regulation of respiration, and intracellular compartmentation associated with GAMT deficiency. Three-dimensional mitochondrial organization was assessed by confocal microscopy. On populations of permeabilized cardiomyocytes, we recorded ADP and ATP kinetics of respiration, competition between mitochondria and pyruvate kinase for ADP produced by ATPases, ADP kinetics of endogenous pyruvate kinase, and ATP kinetics of ATPases. These data were analyzed by mathematical models to estimate intracellular compartmentation. Quantitative analysis of morphological and kinetic data as well as derived model fits showed no difference between GAMT-deficient and wild-type mice. We conclude that inactivation of the CK system by GAMT deficiency does not alter mitochondrial organization and intracellular compartmentation in relaxed cardiomyocytes. Thus, our results suggest that the healthy heart is able to preserve cardiac function at a basal level in the absence of CK-facilitated energy transfer without compromising intracellular organization and the regulation of mitochondrial energy homeostasis. This raises questions on the importance of the CK system as a spatial energy buffer in unstressed cardiomyocytes.

The energetic requirements of the heart are, weight for weight, higher than for any other organ. The heart provides non-stop function for a lifetime, while maintaining energy in reserve in order to respond to increased demand. This demand is met by continuously recycling a relatively small pool of ATP, with the creatine kinase (CK) system acting as a spatial and temporal buffer. In the failing heart, key components of this system are downregulated, but whether these energetic changes are biomarkers or drivers of dysfunction and whether they represent therapeutic targets are the subjects of ongoing research. Key methodologies are now becoming available in vivo to help address these questions in mouse models, such as (31)P magnetic resonance spectroscopy to detect high-energy phosphates and (1)H magnetic resonance spectroscopy to detect total creatine. This report briefly discusses the challenges involved in using these technologies, the application and pitfalls of murine surgical models of heart failure, and how this has contributed to our understanding of pathophysiology in recent years.

Objective: To determine the effects of short-term exercise training on cardiac function and metabolism during rest and physical exercise in patients with heart failure from dilated cardiomyopathy (DCM)., Design: Patients with DCM (n=15, age 58±2 years, NYHA class I-III) were studied before and after 8 weeks of cycle exercise for 20 min, five times per week., Main Outcome Measures: Cardiac volumes, function and high energy phosphate metabolism were measured using cardiac magnetic resonance during rest and 7 min of acute physical exercise (leg-raising)., Results: At baseline, average left ventricular ejection fraction (LVEF) was 38±3%, which did not alter during 7 min of exercise. After 8 weeks of home exercise training, there was a 16% improvement in resting LVEF to 44±3% (p<0.01). Training caused a further 20% improvement in LVEF (p<0.05) during acute physical exercise. There was a negative correlation between subjects' baseline level of exercise and change in LVEF (r=-0.67, p<0.05), with sedentary patients having the greatest improvement. Cardiac phosphocreatine (PCr) to ATP ratio did not change during acute physical exercise or after exercise training., Conclusions: Short-term exercise training improves resting LVEF and LVEF with acute physical exercise with sedentary patients having the greatest improvement. There were no changes in cardiac PCr to ATP, before or after exercise training, suggesting that the improved cardiac function was not caused by improved energetics. Therefore, peripheral factors likely underlie the improved cardiac function in patients with heart failure after short-term exercise.

Cardiac magnetic resonance spectroscopy (MRS) is a noninvasive tool for the assessment of myocardial metabolism, without the use of radiation or intravenous contrast agents. Using the intrinsic magnetic resonance signals from nuclei, including (31)Phosphorus, (1)Hydrogen, (23)Sodium, and (13)Carbon and, more recently, hyperpolarization techniques, MRS provides a comprehensive metabolic assessment of cardiac muscle. This highly versatile technique has provided insights into the pathophysiology of cardiac metabolism in a wide range of conditions, including ischemic heart disease, heart failure, genetic cardiomyopathies, heart transplantation, hypertensive heart disease, valvular heart disease, and diabetes. In addition, MRS has value in the assessment of prognosis and for monitoring therapeutic strategies in heart failure. However, because of the low temporal and spatial resolution of the technique, MRS has so far been limited to research applications. With higher field strength magnets and novel hyperpolarization techniques, the promise of using MRS for clinical applications may eventually be fulfilled., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Hypoxia causes left ventricular dysfunction in the human heart, but the biochemical mechanism is poorly understood. Here, we tested whether short-term normobaric hypoxia leads to changes in cardiac energetics and early cardiac dysfunction. Healthy male volunteers (n=12, age 24 ± 2 yr) were exposed to normobaric hypoxia in a purpose-built hypoxic chamber. The partial pressure of oxygen during end-tidal expiration (P(ET)o₂) was kept between 50 and 60 mmHg, and peripheral oxygen saturation (Sao₂) was kept above 80%. Cardiac morphology and function were assessed using magnetic resonance imaging and echocardiography, both before and after 20 h of hypoxic exposure, and high-energy phosphate metabolism [measured as the phosphocreatine (PCr)/ATP ratio] was measured using ³¹P magnetic resonance spectroscopy. During hypoxia, P(ET)o₂ and Sao₂ averaged 55 ± 1 mmHg and 83.6 ± 0.4%, respectively. Hypoxia caused a 15% reduction in cardiac PCr/ATP (from 2.0 ± 0.1 to 1.7 ± 0.1, P<0.01) and reduced diastolic function (measured as E/E', rising from 6.1 ± 0.4 to 7.5 ± 0.7, P<0.01). Normobaric hypoxia causes a rapid decrease in high-energy phosphate metabolism in the human cardiac left ventricle, which may lead to a decline in diastolic function. These findings are important in understanding the response of normal individuals to environmental hypoxia, and to situations in which disease reduces cardiac oxygen delivery.

We recently showed that a week-long, high-fat diet reduced whole body exercise efficiency in sedentary men by >10% (Edwards LM, Murray AJ, Holloway CJ, Carter EE, Kemp GJ, Codreanu I, Brooker H, Tyler DJ, Robbins PA, Clarke K. FASEB J 25: 1088-1096, 2011). To test if a similar dietary regime would blunt whole body efficiency in endurance-trained men and, as a consequence, hinder aerobic exercise performance, 16 endurance-trained men were given a short-term, high-fat (70% kcal from fat) and a moderate carbohydrate (50% kcal from carbohydrate) diet, in random order. Efficiency was assessed during a standardized exercise task on a cycle ergometer, with aerobic performance assessed during a 1-h time trial and mitochondrial function later measured using (31)P-magnetic resonance spectroscopy. The subjects then underwent a 2-wk wash-out period, before the study was repeated with the diets crossed over. Muscle biopsies, for mitochondrial protein analysis, were taken at the start of the study and on the 5th day of each diet. Plasma fatty acids were 60% higher on the high-fat diet compared with moderate carbohydrate diet (P < 0.05). However, there was no change in whole body efficiency and no change in mitochondrial function. Endurance exercise performance was significantly reduced (P < 0.01), most probably due to glycogen depletion. Neither diet led to changes in citrate synthase, ATP synthase, or mitochondrial uncoupling protein 3. We conclude that prior exercise training blunts the deleterious effect of short-term, high-fat feeding on whole body efficiency.

Background: High-fat, low-carbohydrate diets are widely used for weight reduction, but they may also have detrimental effects via increased circulating free fatty acid concentrations., Objective: We tested whether raising plasma free fatty acids by using a high-fat, low-carbohydrate diet results in alterations in heart and brain in healthy subjects., Design: Men (n = 16) aged 22 ± 1 y (mean ± SE) were randomly assigned to 5 d of a high-fat, low-carbohydrate diet containing 75 ± 1% of calorie intake through fat consumption or to an isocaloric standard diet providing 23 ± 1% of calorie intake as fat. In a crossover design, subjects undertook the alternate diet after a 2-wk washout period, with results compared after the diet periods. Cardiac (31)P magnetic resonance (MR) spectroscopy and MR imaging, echocardiography, and computerized cognitive tests were used to assess cardiac phosphocreatine (PCr)/ATP, cardiac function, and cognitive function, respectively., Results: Compared with the standard diet, subjects who consumed the high-fat, low-carbohydrate diet had 44% higher plasma free fatty acids (P < 0.05), 9% lower cardiac PCr/ATP (P < 0.01), and no change in cardiac function. Cognitive tests showed impaired attention (P < 0.01), speed (P < 0.001), and mood (P < 0.01) after the high-fat, low-carbohydrate diet., Conclusion: Raising plasma free fatty acids decreased myocardial PCr/ATP and reduced cognition, which suggests that a high-fat diet is detrimental to heart and brain in healthy subjects.

Aims: In vitro studies have shown impairment of energy metabolism in cardiac fibroblasts from Fabry patients. A recent in vivo study reported an association between cardiac energy metabolism and increased myocardial mass in Fabry patients. We therefore assessed possible disturbances of cardiac energy metabolism in Fabry patients by in vivo (31)P-MR-spectroscopy. Additionally, the effect of enzyme replacement therapy (ERT) on cardiac energetics was tested., Methods and Results: Twenty-three patients (41 ± 9 years; 10 females) with genetically proven Fabry disease were examined with a 1.5 T Scanner, and compared with an age-matched healthy control group. Eight patients underwent ERT and had follow-up examinations after 3 and 14 months. The high-energy phosphate molecules phosphocreatine (PCr) and adenosine triphosphate (ATP) were quantified in localized 31P-spectra by SLOOP (spectral localization with optimum point spread function). Cine- and late gadolinium enhancement (LGE) studies were also performed. When compared with healthy controls, Fabry patients demonstrated reduced PCr- (6.1 ± 1.9 vs. 8.8 ± 2.6 mmol/kg; P = 0.003) and ATP concentrations (3.9 ± 1.5 vs. 4.6 ± 1.0 mmol/kg; P = 0.048). During ERT, PCr concentrations increased (7.1 ± 1.5 mmol/kg vs. 6.1 ± 1.9; P < 0.05) and left ventricular mass decreased (215 ± 55 vs. 185 ± 45 g; P = 0.012). Disturbances in cardiac energetics were not correlated to the presence or absence of cardiac fibrosis on LGE., Conclusion: Cardiac energy metabolism is disturbed in Fabry disease; this may play an important role in the pathogenesis of Fabry cardiomyopathy. Enzyme replacement therapy ameliorates energetic depression.

We postulated that changes in cardiac high-energy phosphate metabolism may underlie the myocardial dysfunction caused by hypobaric hypoxia. Healthy volunteers (n=14) were studied immediately before, and within 4 d of return from, a 17-d trek to Mt. Everest Base Camp (5300 m). (31)P magnetic resonance (MR) spectroscopy was used to measure cardiac phosphocreatine (PCr)/ATP, and MR imaging and echocardiography were used to assess cardiac volumes, mass, and function. Immediately after returning from Mt. Everest, total body weight had fallen by 3% (P<0.05), but left ventricular mass, adjusted for changes in body surface area, had disproportionately decreased by 11% (P<0.05). Alterations in diastolic function were also observed, with a reduction in peak left ventricular filling rates and mitral inflow E/A, by 17% (P<0.05) and 24% (P<0.01), respectively, with no change in hydration status. Compared with pretrek, cardiac PCr/ATP ratio had decreased by 18% (P<0.01). Whether the abnormalities were even greater at altitude is unknown, but all had returned to pretrek levels after 6 mo. The alterations in cardiac morphology, function, and energetics are similar to findings in patients with chronic hypoxia. Thus, a decrease in cardiac PCr/ATP may be a universal response to periods of sustained low oxygen availability, underlying hypoxia-induced cardiac dysfunction in healthy human heart and in patients with cardiopulmonary diseases.

Magnetic resonance spectroscopy (MRS) is an established technique for the non-invasive assessment of myocardial metabolism. MRS is ideal for the evaluation of heart failure, as it allows quantification of the primary energy source for all myocardial cellular functions (ATP), the energy reserve phosphocreatine (PCr), and the creatine kinase reaction, which maintains cellular energy equilibrium. PCr forms the primary ATP buffer in the cell via the creatine kinase (CK) reaction and is involved in transporting the chemical energy from the ATP-producing mitochondria to the ATP-consuming contractile proteins. Using 31phosphorus (31P) MRS, a low cardiac PCr/ATP has consistently been found in patients with heart failure, supporting the hypothesis that the failing heart is energy starved. The use of 1H MRS has allowed the detection of total creatine, which when combined with 31P MRS, provides an in depth examination of the creatine kinase reaction. MRS signals from 31P, 1H, 23Na and 13C, including novel hyperpolarization techniques, have provided considerable insight into the understanding of energy metabolism in the healthy and diseased heart.

Many disease states are associated with regional or systemic hypoxia. The study of healthy individuals exposed to high-altitude hypoxia offers a way to explore hypoxic adaptation without the confounding effects of disease and therapeutic interventions. Using (31)P magnetic resonance spectroscopy and imaging, we investigated skeletal muscle energetics and morphology after exposure to hypobaric hypoxia in seven altitude-naïve subjects (trekkers) and seven experienced climbers. The trekkers ascended to 5300 m while the climbers ascended above 7950 m. Before the study, climbers had better mitochondrial function (evidenced by shorter phosphocreatine recovery halftime) than trekkers: 16+/-1 vs. 22+/-2 s (mean +/- SE, p<0.01). Climbers had higher resting [Pi] than trekkers before the expedition and resting [Pi] was raised across both groups on their return (PRE: 2.6+/-0.2 vs. POST: 3.0+/-0.2 mM, p<0.05). There was significant muscle atrophy post-CXE (PRE: 4.7+/-0.2 vs. POST: 4.5+/-0.2 cm(2), p<0.05), yet exercising metabolites were unchanged. These results suggest that, in response to high altitude hypoxia, skeletal muscle function is maintained in humans, despite significant atrophy.

Investigation of phosphorus ((31)P) magnetic resonance spectroscopy under stress conditions provides a non-invasive tool to examine alterations in cardiac high-energy phosphate metabolism that may not be evident at rest. Our aim was to establish cardiac (31)P MR spectroscopy during leg exercise at 3T. The increased field strength should provide a higher signal to noise ratio than at lower field strengths. Furthermore, relatively high temporal resolution at a sufficiently fine spatial resolution should be feasible. (31)P MR spectra were obtained with a 3D acquisition weighted chemical shift imaging sequence in 20 healthy volunteers at rest, during dynamic physiological leg exercise and after recovery at 3T. Haemodynamic measurements were made throughout and the rate pressure product calculated. With exercise, the mean heart rate increased by 73%, achieving a mean increase in rate pressure product of 115%. The corrected PCr/ATP ratio for subjects at rest was 2.02 +/- 0.43, exercise 2.14 +/- 0.67 (P = 0.54 vs. rest) and at recovery 2.03 +/- 0.52 (P = 0.91 vs. rest, P = 0.62 vs. exercise). A cardiac (31)P MR spectroscopy physiological exercise-recovery protocol is feasible at 3T. There was no significant change in high-energy cardiac phosphate metabolite concentrations in healthy volunteers at rest, during physiological leg exercise or during recovery. When applied to patients with heart disease, this protocol should provide insights into physiological and pathological cardiac metabolism.

Magnetic resonance spectroscopy (MRS) is the only noninvasive, nonradiation exposure technique for the investigation of cardiac metabolism in vivo. MRS uses magnetic resonance signals from nuclei, such as (31)phosphorus, (1)hydrogen, and (23)sodium, to provide comprehensive metabolic and biochemical information about cardiac muscle. This method is highly versatile and can provide metabolic insights into the role of cardiac metabolism, in particular, cardiac energetics, in a wide number of conditions, including hypertensive, valvular, and ischemic heart disease, heart failure, and cardiac transplantation, as well as cardiomyopathies. This method can also be used to monitor patient responses to therapeutic interventions: pharmacologic, surgical, or interventional. When combined with cardiovascular magnetic resonance imaging, MRS enables detailed pathophysiologic insights into the inter-relations among cardiac structure, function, perfusion, and metabolism. However, MRS is currently used primarily as a research tool because of low temporal and spatial resolution and low reproducibility. It is hoped that future technical developments and use of higher magnetic field strengths (such as 7-T) may enable application of cardiac MRS in clinical practice.

Patients with muscular dystrophy have abnormal cardiac function and decreased high-energy phosphate metabolism. Here, we have determined whether the 8 month old mdx mouse, an animal model of muscular dystrophy, also has abnormal cardiac function and energetics. In vivo cardiac MRI revealed 33% and 104% larger right ventricular end-diastolic and end-systolic volumes, respectively, and 17% lower right ventricular ejection fractions in mdx mice compared with controls. Evidence of left ventricular diastolic dysfunction included 18% lower peak filling rates in mdx mouse hearts. Abnormal cardiac function was accompanied by necrosis and lower citrate synthase activity in the mdx mouse heart, suggesting decreased mitochondrial content. Decreased mitochondrial numbers were associated with 38% lower phosphocreatine concentration, 22% lower total creatine, 36% higher cytosolic free ADP concentration and 1.3 kJ/mol lower free-energy available from ATP hydrolysis in whole isolated, perfused mdx mouse hearts than in controls. Transsarcolemmal creatine uptake was 12% lower in mdx mouse hearts. We conclude that the absence of dystrophin in adult mdx mouse heart, as in the heart of human patient, is associated with right ventricular dilatation, left ventricular diastolic dysfunction and abnormal energy metabolism.

Objectives: The present study investigated changes in cardiac energy metabolism and function in patients with dilated cardiomyopathy (DCM) before and after exercise training (ET) with phosphorus-31 magnetic resonance spectroscopy (MRS) in combination with magnetic resonance imaging (MRI)., Background: Exercise training might have a beneficial role on myocardial function and oxidative metabolism in DCM, but it is unclear whether the additional load on the failing heart leads to deterioration of cardiac energy metabolism., Methods: Twenty-four patients were randomized to an exercise (age 53 +/- 12 years) or a control (age 56 +/- 6 years) group. Supervised ET was performed for 2 months, followed by 6 months of self-regulated training. At baseline and 2 and 8 months, maximal exercise testing along with quantitative MRS and MRI studies were performed., Results: The effectiveness of ET was demonstrated by a 17% increase in peak oxygen uptake (p < 0.05). Exercise training improved left ventricular (LV) end-systolic volume (p < 0.05) and LV ejection fraction (30 +/- 15% vs. 37 +/- 15%; p < 0.01) but not right ventricular parameters. The improvement in cardiac function was not accompanied by changes in cardiac high-energy phosphate concentrations; phosphocreatine, adenosine triphosphate, and the phosphocreatine/adenosine triphosphate ratio were all unchanged after training., Conclusions: The observation that LV function improved and LV energy metabolism remained unchanged suggests that the beneficial effect of ET on LV function is achieved without adversely affecting metabolism. These findings lend further support for the use of ET as an adjunct therapy in DCM.

Characteristic alterations of the creatine kinase (CK) system occur in heart failure and may contribute to contractile dysfunction. We examined two mouse models of chronic cardiac stress, transverse aortic constriction (TAC) and coronary artery ligation (CAL), and examined the relationship of CK system changes with hypertrophy and heart failure development. C57Bl/6 mice were subjected to TAC or sham surgery and sacrificed after 2-10 weeks according to echocardiographic criteria of myocardial hypertrophy and function to create four groups representing progressive dysfunction from normal, through compensated hypertrophy, to heart failure. Only mice with congestive heart failure had LV total creatine concentration and total CK activity significantly lower than sham values (11% and 30% lower, respectively). However for all aortic banded mice, a linear relationship was observed between ejection fraction and estimated maximal CK reaction velocity. Mice with heart failure also had corresponding decreases in the activities of the Mito-, MM-, and MB-CK isoenzymes, while the BB isoform remained unchanged. To determine whether these changes were model specific, mice were subjected to CAL or sham operation and followed for 7 weeks. Quantitative changes in total creatine, total CK activity, Mito-CK and MM-CK activities were similar for CAL and TAC mice. We conclude that alterations in the creatine kinase system occur during heart failure in mice qualitatively similar to those occurring in larger animals and humans, suggesting that mice are a suitable model for studying the role of such changes in the pathogenesis of heart failure.

Objective: Many patients with heart failure have whole-body insulin resistance and reduced cardiac fluorodeoxyglucose uptake, but whether these metabolic changes have detrimental effects on the heart is unknown. Here, we tested whether there is a link between insulin resistance and ischemic damage in the chronically infarcted Wistar rat heart, postulating that the heart would have decreased insulin sensitivity, with lower GLUT4 glucose transporter protein levels due to high circulating free fatty acid (FFA) concentrations. A decreased capacity for glucose uptake would lower glycolytic adenosine triphosphate (ATP) production and thereby increase ischemic injury in the infarcted heart., Methods and Results: In vivo left ventricular ejection fractions, measured using echocardiography, were 40% lower in rats 10 weeks after coronary artery ligation than in sham-operated control rats. Insulin-stimulated D[2-3H]glucose uptake was 42% lower in isolated, perfused, infarcted hearts. Myocardial GLUT4 glucose transporter protein levels were 28% lower in the infarcted hearts and correlated negatively with ejection fractions and with fasting plasma FFA concentrations. Compared with controls, chronically infarcted hearts had 46% lower total glucose uptake and three-fold faster ATP hydrolysis rates, measured using phosphorus-31 nuclear magnetic resonance spectroscopy, during 32-min ischemia at 0.4 ml/min/gww. During reperfusion, recovery of left ventricular developed pressure in infarcted hearts was 42% lower than in control hearts., Conclusions: Glucose uptake, in response to insulin or ischemia, was lower in the chronically infarcted rat heart and associated with increased circulating FFA concentrations and decreased GLUT4 levels. Thus, infarcted hearts had greater ATP depletion, and consequently incurred greater damage, during ischemia.

Background: The role of the creatine kinase (CK)/phosphocreatine (PCr) energy buffer and transport system in heart remains unclear. Guanidinoacetate-N-methyltransferase-knockout (GAMT-/-) mice represent a new model of profoundly altered cardiac energetics, showing undetectable levels of PCr and creatine and accumulation of the precursor (phospho-)guanidinoacetate (P-GA). To characterize the role of a substantially impaired CK/PCr system in heart, we studied the cardiac phenotype of wild-type (WT) and GAMT-/- mice., Methods and Results: GAMT-/- mice did not show cardiac hypertrophy (myocyte cross-sectional areas, hypertrophy markers atrial natriuretic factor and beta-myosin heavy chain). Systolic and diastolic function, measured invasively (left ventricular conductance catheter) and noninvasively (MRI), were similar for WT and GAMT-/- mice. However, during inotropic stimulation with dobutamine, preload-recruitable stroke work failed to reach maximal levels of performance in GAMT-/- hearts (101+/-8 mm Hg in WT versus 59+/-7 mm Hg in GAMT-/-; P<0.05). (31)P-MR spectroscopy experiments showed that during inotropic stimulation, isolated WT hearts utilized PCr, whereas isolated GAMT-/- hearts utilized P-GA. During ischemia/reperfusion, GAMT-/- hearts showed markedly impaired recovery of systolic (24% versus 53% rate pressure product recovery; P<0.05) and diastolic function (eg, left ventricular end-diastolic pressure 23+/-9 in WT and 51+/-5 mm Hg in GAMT-/- during reperfusion; P<0.05) and incomplete resynthesis of P-GA., Conclusions: GAMT-/- mice do not develop hypertrophy and show normal cardiac function at low workload, suggesting that a fully functional CK/PCr system is not essential under resting conditions. However, when acutely stressed by inotropic stimulation or ischemia/reperfusion, GAMT-/- mice exhibit a markedly abnormal phenotype, demonstrating that an intact, high-capacity CK/PCr system is required for situations of increased cardiac work or acute stress.

Background: It is well known that patients with type 2 diabetes have increased risk of cardiovascular disease, but it is not known whether they have underlying abnormalities in cardiac or skeletal muscle high-energy phosphate metabolism., Methods and Results: We studied 21 patients with type 2 diabetes with no evidence of coronary artery disease or impaired cardiac function, as determined by echocardiography, and 15 age-, sex-, and body mass index-matched control subjects. Cardiac high-energy phosphate metabolites were measured at rest using 31P nuclear magnetic resonance spectroscopy (MRS). Skeletal muscle high-energy phosphate metabolites, intracellular pH, and oxygenation were measured using 31P MRS and near infrared spectrophotometry, respectively, before, during, and after exercise. Although their cardiac morphology, mass, and function appeared to be normal, the patients with diabetes had significantly lower phosphocreatine (PCr)/ATP ratios, at 1.50+/-0.11, than the healthy volunteers, at 2.30+/-0.12. The cardiac PCr/ATP ratios correlated negatively with the fasting plasma free fatty acid concentrations. Although skeletal muscle energetics and pH were normal at rest, PCr loss and pH decrease were significantly faster during exercise in the patients with diabetes, who had lower exercise tolerance. After exercise, PCr recovery was slower in the patients with diabetes and correlated with tissue reoxygenation times. The exercise times correlated negatively with the deoxygenation rates and the hemoglobin (Hb)A1c levels and the reoxygenation times correlated positively with the HbA1c levels., Conclusions: Type 2 diabetic patients with apparently normal cardiac function have impaired myocardial and skeletal muscle energy metabolism related to changes in circulating metabolic substrates.

The individual functional significance of the various creatine kinase (CK) isoenzymes for myocardial energy homeostasis is poorly understood. Whereas transgenic hearts lacking the M subunit of CK (M-CK) show unaltered cardiac energetics and left ventricular (LV) performance, deletion of M-CK in combination with loss of sarcomeric mitochondrial CK (ScCKmit) leads to significant alterations in myocardial high-energy phosphate metabolites. To address the question as to whether this alteration is due to a decrease in total CK activity below a critical threshold or due to the specific loss of ScCKmit, we studied isolated perfused hearts with selective loss of ScCKmit (ScCKmit(-/-), remaining total CK activity approximately 70%) using (31)P NMR spectroscopy at two different workloads. LV performance in ScCKmit(-/-) hearts (n = 11) was similar compared with wild-type hearts (n = 9). Phosphocreatine/ATP, however, was significantly reduced in ScCKmit(-/-) compared with wild-type hearts (1.02 +/- 0.05 vs. 1.54 +/- 0.07, P < 0.05). In parallel, free [ADP] was higher (144 +/- 11 vs. 67 +/- 7 microM, P < 0.01) and free energy release for ATP hydrolysis (DeltaG(ATP)) was lower (-55.8 +/- 0.5 vs. -58.5 +/- 0.5 kJ/mol, P < 0.01) in ScCKmit(-/-) compared with wild-type hearts. These results demonstrate that M- and B-CK containing isoenzymes are unable to fully substitute for the loss of ScCKmit. We conclude that ScCKmit, in contrast to M-CK, is critically necessary to maintain normal high-energy phosphate metabolite levels in the heart.

Aims: Despite substantial improvements over the last three decades, heart failure (HF) remains associated with a poor prognosis. The sodium‐glucose co‐transporter‐2 inhibitor empagliflozin demonstrated significant reductions of HF hospitalization in patients with HF independent of the presence or absence of type 2 diabetes mellitus in the EMPEROR‐Reduced trial and cardiovascular mortality in the EMPA‐REG OUTCOME trial. To further elucidate the mechanisms behind these positive outcomes, this study aims to determine the effects of empagliflozin treatment on cardiac energy metabolism and physiology using magnetic resonance spectroscopy (MRS) and cardiovascular magnetic resonance (CMR). Methods and results: The EMPA‐VISION trial is a double‐blind, randomized, placebo‐controlled, mechanistic study. A maximum of 86 patients with HF with reduced ejection fraction (n = 43, Cohort A) or preserved ejection fraction (n = 43, Cohort B), with or without type 2 diabetes mellitus, will be enrolled. Participants will be randomized 1:1 to receive either 10 mg of empagliflozin or placebo for 12 weeks. Eligible patients will undergo cardiovascular magnetic resonance, resting and dobutamine stress MRS, echocardiograms, cardiopulmonary exercise tests, serum metabolomics, and quality of life questionnaires at baseline and after 12 weeks. The primary endpoint will be the change in resting phosphocreatine‐to‐adenosine triphosphate ratio, as measured by 31Phosphorus‐MRS. Conclusions: EMPA‐VISION is the first clinical trial assessing the effects of empagliflozin treatment on cardiac energy metabolism in human subjects in vivo. The results will shed light on the mechanistic action of empagliflozin in patients with HF and help to explain the results of the safety and efficacy outcome trials (EMPEROR‐Reduced and EMPEROR‐Preserved). [ABSTRACT FROM AUTHOR]

Aims: Adenylate kinase 1 (AK1) catalyses the reaction 2ADP ↔ ATP + AMP, extracting extra energy under metabolic stress and promoting energetic homeostasis. We hypothesised that increased AK1 activity would have negligible effects at rest, but protect against ischaemia/reperfusion (I/R) injury. Methods and Results: Cardiac-specific AK1 overexpressing mice (AK1-OE) had 31% higher AK1 activity (P = 0.009), with unchanged total creatine kinase and citrate synthase activities. Male AK1-OE exhibited mild in vivo dysfunction at baseline with lower LV pressure, impaired relaxation, and contractile reserve. LV weight was 19% higher in AK1-OE males due to higher tissue water content in the absence of hypertrophy or fibrosis. AK1-OE hearts had significantly raised creatine, unaltered total adenine nucleotides, and 20% higher AMP levels (P = 0.05), but AMP-activated protein kinase was not activated (P = 0.85). 1H-NMR revealed significant differences in LV metabolite levels compared to wild-type, with aspartate, tyrosine, sphingomyelin, cholesterol all elevated, whereas taurine and triglycerides were significantly lower. Ex vivo global no-flow I/R, caused four-of-seven AK1-OE hearts to develop terminal arrhythmia (cf. zero WT), yet surviving AK1-OE hearts had improved functional recovery. However, AK1-OE did not influence infarct size in vivo and arrhythmias were only observed ex vivo , probably as an artefact of adenine nucleotide loss during cannulation. Conclusion: Modest elevation of AK1 may improve functional recovery following I/R, but has unexpected impact on LV weight, function and metabolite levels under basal resting conditions, suggesting a more nuanced role for AK1 underpinning myocardial energy homeostasis and not just as a response to stress. [ABSTRACT FROM AUTHOR]

Aim: Guanidinoacetate N -methyltransferase (GAMT) is the second essential enzyme in creatine (Cr) biosynthesis. Short-term Cr deficiency is metabolically well tolerated as GAMT–/– mice exhibit normal exercise capacity and response to ischemic heart failure. However, we hypothesized long-term consequences of Cr deficiency and/or accumulation of the Cr precursor guanidinoacetate (GA). Methods: Cardiac function and metabolic profile were studied in GAMT–/– mice >1 year. Results: In vivo LV catheterization revealed lower heart rate and developed pressure in aging GAMT–/– but normal lung weight and survival versus age-matched controls. Electron microscopy indicated reduced mitochondrial volume density in GAMT–/– hearts (P < 0.001), corroborated by lower mtDNA copy number (P < 0.004), and citrate synthase activity (P < 0.05), however, without impaired mitochondrial respiration. Furthermore, myocardial energy stores and key ATP homeostatic enzymes were barely altered, while pathology was unrelated to oxidative stress since superoxide production and protein carbonylation were unaffected. Gene expression of PGC-1α was 2.5-fold higher in GAMT–/– hearts while downstream genes were not activated, implicating a dysfunction in mitochondrial biogenesis signaling. This was normalized by 10 days of dietary Cr supplementation, as were all in vivo functional parameters, however, it was not possible to differentiate whether relief from Cr deficiency or GA toxicity was causative. Conclusion: Long-term Cr deficiency in GAMT–/– mice reduces mitochondrial volume without affecting respiratory function, most likely due to impaired biogenesis. This is associated with hemodynamic changes without evidence of heart failure, which may represent an acceptable functional compromise in return for reduced energy demand in aging mice. [ABSTRACT FROM AUTHOR]

Purpose We present a new Bloch-Siegert four Angle Saturation Transfer (BOAST) method for measuring the creatine kinase (CK) first-order effective rate constant kf in human myocardium at 7 tesla (T). BOAST combines a variant of the four-angle saturation transfer (FAST) method using amplitude-modulated radiofrequency pulses, phosphorus Bloch-Siegert [ABSTRACT FROM AUTHOR]

Background: Multiple studies suggest creatine mediates anti-oxidant activity in addition to its established role in cellular energy metabolism. The functional significance for the heart has yet to be established, but antioxidant activity could contribute to the cardioprotective effect of creatine in ischaemia/reperfusion injury. Objectives: To determine whether intracellular creatine levels influence responses to acute reactive oxygen species (ROS) exposure in the intact beating heart. We hypothesised that mice with elevated creatine due to over-expression of the creatine transporter (CrT-OE) would be relatively protected, while mice with creatine-deficiency (GAMT KO) would fare worse. Methods and Results: CrT-OE mice were pre-selected for creatine levels 20–100% above wild-type using in vivo 1H–MRS. Hearts were perfused in isovolumic Langendorff mode and cardiac function monitored throughout. After 20 min equilibration, hearts were perfused with either H2O2 0.5 µM (30 min), or the anti-neoplastic drug doxorubicin 15 µM (100 min). Protein carbonylation, creatine kinase isoenzyme activities and phospho-PKCδ expression were quantified in perfused hearts as markers of oxidative damage and apoptotic signalling. Wild-type hearts responded to ROS challenge with a profound decline in contractile function that was ameliorated by co-administration of catalase or dexrazoxane as positive controls. In contrast, the functional deterioration in CrT-OE and GAMT KO hearts was indistinguishable from wild-type controls, as was the extent of oxidative damage and apoptosis. Exogenous creatine supplementation also failed to protect hearts from doxorubicin-induced dysfunction. Conclusions: Intracellular creatine levels do not influence the response to acute ROS challenge in the intact beating heart, arguing against creatine exerting (patho-)physiologically relevant anti-oxidant activity. [ABSTRACT FROM AUTHOR]

Background: Reduced levels of creatine and total adenine nucleotides (sum of ATP, ADP and AMP) are hallmarks of chronic heart failure and restoring these pools is predicted to be beneficial by maintaining the diseased heart in a more favourable energy state. Ribose supplementation is thought to support both salvage and re-synthesis of adenine nucleotides by bypassing the rate-limiting step. We therefore tested whether ribose would be beneficial in chronic heart failure in control mice and in mice with elevated myocardial creatine due to overexpression of the creatine transporter (CrT-OE). Methods and Results: Four groups were studied: sham; myocardial infarction (MI); MI+ribose; MI+CrT-OE+ribose. In a pilot study, ribose given in drinking water was bioavailable, resulting in a two-fold increase in myocardial ribose-5-phosphate levels. However, 8 weeks post-surgery, total adenine nucleotide (TAN) pool was decreased to a similar amount (8–14%) in all infarcted groups irrespective of the treatment received. All infarcted groups also presented with a similar and substantial degree of left ventricular (LV) dysfunction (3-fold reduction in ejection fraction) and LV hypertrophy (32–47% increased mass). Ejection fraction closely correlated with infarct size independently of treatment (r2 = 0.63, p<0.0001), but did not correlate with myocardial creatine or TAN levels. Conclusion: Elevating myocardial ribose and creatine levels failed to maintain TAN pool or improve post-infarction LV remodeling and function. This suggests that ribose is not rate-limiting for purine nucleotide biosynthesis in the chronically failing mouse heart and that alternative strategies to preserve TAN pool should be investigated. [ABSTRACT FROM AUTHOR]

Aims To measure the activity of the key phosphotransfer enzymes creatine kinase (CK), adenylate kinase (AK), and glycolytic enzymes in two common mouse models of chronic heart failure. Methods and results C57BL/6 mice were subjected to transverse aortic constriction (TAC), myocardial infarction induced by coronary artery ligation (CAL), or sham operation. Activities of phosphotransfer enzymes CK, AK, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 3-phosphoglycerate kinase (PGK), and pyruvate kinase were assessed spectrophotometrically. Mice were characterized by echocardiography or magnetic resonance imaging 5- to 8-week post-surgery and selected for the presence of congestive heart failure. All mice had severe left ventricular hypertrophy, impaired systolic function and pulmonary congestion compared with sham controls. A significant decrease in myocardial CK and maximal CK reaction velocity was observed in both experimental models of heart failure. However, the activity of AK and its isoforms remained unchanged, despite a reduction in its protein expression. In contrast, the activities of glycolytic phosphotransfer mediators GAPDH and PGK were 19 and 12% higher in TAC, and 31 and 23% higher in CAL models, respectively. Conclusion Chronic heart failure in the mouse is characterized by impaired CK function, unaltered AK, and increased activity of glycolytic phosphotransfer enzymes. This pattern of altered phosphotransfer activity was observed independent of the heart failure aetiology. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: Cardiac fatty acid uptake occurs predominantly via sarcolemmal transporter proteins; fatty acid translocase (FAT/CD36), plasma membrane fatty acid binding protein (FABPpm) and fatty acid transporter proteins (FATP) 1 and 6. We hypothesised that levels of the fatty acid transporters would be reduced in the chronically infarcted rat heart, in parallel with reduced dependence on fatty acid utilisation. Methods and results: In vivo left ventricular ejection fractions, measured using echocardiography, were 36% lower in rats six months after coronary artery ligation than in sham-operated control rats. In isolated, perfused, infarcted hearts, 3H-palmitate oxidation was 30% lower, and correlated with in vivo ejection fractions. As myocardial lipid incorporation was also reduced by 25%, total palmitate utilisation was 29% lower in the infarcted rat heart. The protein levels of the cardiac fatty acid transporters were reduced in the infarcted rat heart; FAT/CD36 by 36%, FABPpm by 12%, FATP6 by 21% and FATP1 by 26%, and the cytosolic fatty acid binding protein (cFABP) was 47% lower than in sham-operated rat hearts. Fatty acid transporter levels correlated with both palmitate oxidation rates and cardiac ejection fractions. Conclusions: Reductions in fatty acid oxidation and lipid incorporation rates were accompanied by downregulation of the cardiac fatty acid transporters. The metabolic shift away from fatty acid utilisation was proportional to the degree of functional impairment in the chronically infarcted rat heart. [Copyright &y& Elsevier]

Changes in creatine kinase (CK) and lactate dehydrogenase (LDH) isoform expression occur in residual tissue after myocardial infarction. It is unknown how these changes correlate with cardiac remodeling, contractile performance and efficiency. Rats were subjected to left coronary artery ligation (MI) or sham operation (sham). Left ventricular end-diastolic pressure (EDP) was measuredin vivo8 weeks later. Hearts were isolated, buffer-perfused (Langendorff) at constant pressure and isovolumetric left ventricular (LV) pressure-volume (PV) curves were recorded. LV PV areas (PVA) were calculated and related to oxygen consumption. Biopsies of intact left ventricular tissue were taken for biochemical measurements. Correlations betweenin vivoEDP and biochemical parameters were found: Total CK activity (r = -.47, p = .022), CK isoenzyme percentage for BB (r = +.57, p = .004), MB (r = +.54, p = .006) and CK-mito (r = -.51, p = .012), total creatine content (r = -.61, p = .002) and the ratio of LDH5/LDH1 (r = .49, p = .016). Correlations were also detected for left ventricular volume and PVAs atin vivoEDP demonstrating that the extent of CK and LDH system alterations correlate with the extent of LV dilatation and mechanical energy requirements. The slope of the MVO2-PVA relation decreased significantly with increasing values ofin vivoEDP (r = -.68, p = 0.0003) indicating increased contractile ef.ciency. Improved efficiency correlated with the increase in fetal CK isoenzyme expression. Thus, contractile efficiency increases parallel to the extent of left ventricular dilatation and dysfunction. CK and LDH system changes in residual intact myocardium also occur proportional to LV dysfunction. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Creatine kinase (CK) is responsible for the transport of high-energy phosphates in excitable tissue and is of central importance in myocardial energy homeostasis. Significant changes in myocardial energetics have been reported in mice lacking the various CK isoenzymes. Our hypothesis was that ablation of CK isoenzymes leads to cardiac hypertrophy, impaired function, and aggravation of left ventricular remodeling post-myocardial infarction. Methods: CK-deficient mice (CK KO) were examined by cardiac magnetic resonance imaging (MRI) to determine left ventricular volumes, ejection fraction, and mass: ten wild-type (WT), 6 mitochondrial CK KO (Mito-CK−/−), 10 cytosolic CK KO (M-CK−/−), and 10 mice with combined KO (M/Mito-CK−/−). Results: While ejection fraction was similar in all groups, there was significant LV dilatation with a ∼30% increase in LV end-diastolic volumes in Mito-CK−/− and in M/Mito-CK−/−. Compared to WT, there was a striking 73% and 64% increase of LV mass in Mito-CK−/− and in M/Mito-CK−/− mice, respectively, but no significant increase of LV mass (+33%; p=n.s.) in M-CK−/−. Furthermore, significant re-expression of β-MHC, a marker of myocardial hypertrophy, was found in all CK-deficient hearts. LV remodeling was investigated by MRI in hearts of 7 WT and 10 M/Mito-CK−/− mice 4 weeks postmyocardial infarction (MI). Four weeks post-LAD ligation (MI size ∼32%), WT and M/Mito-CK−/− showed a similar degree of cardiac dysfunction, dilatation, and hypertrophy. Conclusion: Mito-CK−/− and M/Mito-CK−/− mice show significant LV dilatation and marked LV hypertrophy, but LV remodeling post-MI is not aggravated. CK ablation leads to substantial adaptational changes in heart. [Copyright &y& Elsevier]

The importance of heart rate for left ventricular remodeling and prognosis after myocardial infarction is not known. We examined the contribution of heart rate reduction by zatebradine, a direct sinus node inhibitor without negative inotropic effects on left ventricular function and dilatation, on mortality, energy metabolism, and neurohormonal changes in rats with experimental myocardial infarction (MI). Thirty minutes after left coronary artery ligation or sham operation, the rats were randomized to receive either placebo or zatebradine (100 mg·kg-1·day-1 per gavage) continued for 8 wk. Mortality during 8 wk was 33.3% in the placebo and 23.0% in the zatebradine group (P < 0.05); MI size was 36 ± 2% and 30 ± 1% (means ± SE, P < 0.05), respectively. Zatebradine improved stroke volume index in all treated rats but increased left ventricular v01ume in rats with small MI (2.43 ± 0.10 vs. 1.81 ± 0.10 ml/kg, P < 0.05) but not in rats with large MI (2.34 ± 0.09 vs. 2.35 ± 0.11 ml/kg, not significant). Zatebradine reduced left and right ventricular norepinephrine and increased left and right ventricular 3,4-dihydroxyphenyl ethylene glycol-to-norepinephrine ratio suggesting aggravation of cardiac sympathetic activation by zatebradine after MI. Creatine kinase and lactate dehydrogenase isoenzymes in rats with MI remained unchanged by zatebradine. Lowering heart rate per se reduces mortality and MI size in this model but induces adverse effects on left ventricular remodeling in rats with small MI. [ABSTRACT FROM AUTHOR]

This review describes the temporal changes of high-energy phosphate metabolites during the cardiac cycle. Under baseline condition, the extent of cyclical changes (“cycling”) of ATP, phosphocreatine and inorganic phosphate varies between 10–15 %. Such energy oscillations are not augmented under various forms of metabolic stress including 1) inotropic stimulation, 2) acute hypoxia and 3) failing, chronically infarcted hearts. Thus, the concentrations of high-energy phosphates over the cardiac cycle are a tightly regulated entity, even when energetic needs are substantially augmented. [ABSTRACT FROM AUTHOR]

Changes in the kinetics of the creatine kinase (CK) shuttle are sensitive markers of cardiac energetics but are typically measured at rest and in the prone position. This study aims to measure CK kinetics during pharmacological stress at 3 T, with measurement in the supine position. A shorter "stressed saturation transfer" (StreST) extension to the triple repetition time saturation transfer (TRiST) method is proposed. We assess scanning in a supine position and validate the MR measurement against biopsy assay of CK activity. We report normal ranges of stress CK forward rate (kfCK) for healthy volunteers and obese patients. TRiST measures kfCK in 40 min at 3 T. StreST extends the previously developed TRiST to also make a further kfCK measurement during <20 min of dobutamine stress. We test our TRiST implementation in skeletal muscle and myocardium in both prone and supine positions. We evaluate StreST in the myocardium of six healthy volunteers and 34 obese subjects. We validated MR‐measured kfCK against biopsy assays of CK activity. TRiST kfCK values matched literature values in skeletal muscle (kfCK = 0.25 ± 0.03 s−1 vs 0.27 ± 0.03 s−1) and myocardium when measured in the prone position (0.32 ± 0.15 s−1), but a significant difference was found for TRiST kfCK measured supine (0.24 ± 0.12 s−1). This difference was because of different respiratory‐ and cardiac‐motion‐induced B0 changes in the two positions. Using supine TRiST, cardiac kfCK values for normal‐weight subjects were 0.15 ± 0.09 s−1 at rest and 0.17 ± 0.15 s−1 during stress. For obese subjects, kfCK was 0.16 ± 0.07 s−1 at rest and 0.17 ± 0.10 s−1 during stress. Rest myocardial kfCK and CK activity from LV biopsies of the same subjects correlated (R = 0.43, p = 0.03). We present an independent implementation of TRiST on the Siemens platform using a commercially available coil. Our extended StreST protocol enables cardiac kfCK to be measured during dobutamine‐induced stress in the supine position. [ABSTRACT FROM AUTHOR]