Creatine Kinase Flux Research Articles

Vasoplegia induces high energy demand and depletes cardiac energetics: a new insight into physiological concepts of cardiac work and a model for acute heart failure?

Abstract Funding Acknowledgements Type of funding sources: Other. Main funding source(s): British Heart Foundation Background / Introduction Hearts subjected to constant high workload will, over time, fail. We hypothesized vasoplegia would create a high cardiac energy demand and deplete cardiac energy reserves, a mechanism causing failure. With 31Phosphorus Magnetic Resonance Spectroscopy, we can measure energy: ATP usage (via creatine kinase flux) and reserves of phosphocreatine (PCr/ATP ratio). Methods: Ten healthy volunteers were recruited mean age 37, mean BMI 22.4, 5 male, 5 female. A nitrate vasodilator (Glyceryl trinitrate, GTN) was used to induce vasoplegia, with MRI and MRS measurements taken before and during. Results There was reduced preload (falls in end diastolic volume and right atrial area) and afterload (mean blood pressure fall 14mmHg, p <0.0001). Subjects mounted reflex tachycardia (mean heart rate rise 9bpm, p < 0.0001) and inotropy (mean baseline ejection fraction increase 5%, p = 0.0002) hence cardiac output and rate pressure product were maintained (baseline CO 6.24 ± 1.45 L/min vs GTN 6.49 ± 1.43 L/min, p = 0.37; baseline RPP = 6929 ± 976 mmHg.bpm vs GTN 7214 ± 1051 mmHg.bpm, p = 0.06). There was a 58% increase in Creatine Kinase activity (kf) (baseline 0.158 s-1 vs GTN 0.249 s-1, p = 0.006) and CK flux (baseline 1.79 ± 0.78 umol/g/s vs GTN 2.59 ± 1.07 umol/g/s, p = 0.03), demonstrating increased ATP transfer. PCr/ATP fell (from 2.17 ± 0.2 to 1.99 ± 0.22, p = 0.027). Baseline PCr/ATP positively correlated with the change in stroke volume (R2 = 0.66, p = 0.005) during administration of GTN. In addition, whilst at rest, CK kf correlated with rate pressure product (R2 = 0.56, p = 0.03), there was no correlation seen during GTN. Conclusions We have shown vasoplegia increases energy demand upon the heart, despite unchanged cardiac output and rate pressure product. Further, we have shown a fall in Phosphocreatine/ATP ratio, suggesting this ATP transfer outstripped oxidative phosphorylation and the phosphocreatine pool was depleted in order to maintain ATP delivery. We speculate that progressive depletion of energetics in the context of high energy demand drives hearts under constant stress into failure. In addition, we demonstrate that the magnitude of the resting phosphocreatine pool relates to ability to augment stroke volume during stress and suggest that this may be protective against a decline into failure. Further work is needed, but PCr/ATP may be an important biomarker for treatments that target myocardial energetics to protect against failure. Abstract Figure 1

Bioenergetics and abnormal functional connectivity in psychotic disorders.

Psychotic Disorders such as schizophrenia (SZ) and bipolar disorder (BD) are characterized by abnormal functional connectivity (FC) within neural networks such as the default mode network (DMN), as well as attenuated anticorrelation between DMN and task-positive networks (TPN). Bioenergetic processes are critical for synaptic connectivity and are also abnormal in psychotic disorders. We therefore examined the association between brain energy metabolism and FC in psychotic disorders. 31P magnetization transfer spectroscopy from medial prefrontal cortex (MPFC) and whole-brain fMRI data were collected from demographically matched groups of SZ, BD, and healthy control (HC) subjects. The creatine kinase (CK) reaction flux calculated from spectroscopy was used as an index of regional energy production rate. FC maps were generated with MPFC as the seed region. Compared to HC, SZ showed significantly lower CK flux, while both BD and SZ patients showed decreased anticorrelation between MPFC and TPN. CK flux was significantly correlated with FC between MPFC and other DMN nodes in HC. This positive correlation was reduced modestly in BD and strongly in SZ. CK flux was negatively correlated with the anticorrelation between MPFC and TPN in HC, but this relationship was not observed in BD or SZ. These results indicate that MPFC energy metabolism rates are associated with stronger FC within networks and stronger anticorrelation between networks in HC. However, this association is decreased in SZ and BD, where bioenergetic and FC abnormalities are evident. This pattern may suggest that impairment in energy production in psychotic disorders underlies the impaired neural connectivity.

Nitrate induced vasoplegia depletes cardiac energy reserves: a model for acute cardiomyopathy in septic shock?

Abstract Introduction The healthy heart is at its most efficient when contractile filaments are stretched with preload. To produce the same cardiac output, oxygen requirement rises are proportionally greater with increased inotropy or heart rate than with increased preload. We hypothesized that in vasoplegia, loss of preload (owing to dilation of capacitance veins) and compensatory rises in heart rate and contractility would compromise the efficiency of the heart. We speculated that this may be one factor impairing cardiac function in conditions of distributive shock such as sepsis and looked to model the effects. We used cardiac magnetic resonance imaging to capture changes in cardiac volumes and contractility and magnetic resonance spectroscopy to investigate changes in ATP metabolism within the myocardium in healthy volunteers given GTN as a vasodilator. Methods We recruited 7 healthy volunteers (mean age 40 years, range 28–62 years; mean BMI 21.9, range 18.5–24.5) and measured their baseline cardiac volumes and function, PCr/ATP ratio and Creatine Kinase first order rate constant (CKkf), using cardiac magnetic resonance imaging and magnetic resonance spectroscopy and saturation transfer at 3 Tesla. At the same visit, they received a glyceryl trinitrate (GTN) infusion to induce vasoplegia and the measurements were repeated. We targeted GTN infusion rate to a fall in mean arterial pressure of 15mmHg. Results The GTN infusion brought about a fall in mean arterial pressure (from a baseline of 79±7mmHg to 64±7 mmHg, p<0.0001) and a fall in LV end diastolic volume (169±57 ml vs 148±58 ml, p=0.003) indicating a reduction in preload. As expected, there was a compensatory rise in heart rate (61±7 bpm vs 69±10bpm, p=0.0005) and ejection fraction (62±3% vs 67±3%, p=0.001), however cardiac output remained unchanged (6.72±1.49 L/min vs 6.68±1.48 L/min, p=0.87). Cardiac work (calculated as stroke volume x MAP x heart rate) fell (477±123 vs 424±119 L.mmHg/min, p=0.03). There was a fall in PCr/ATP ratio on GTN (2.18±0.25 vs 1.91±0.2, p 0.03) while CKkf more than doubled (0.14±0.06 s-1 vs 0.23±0.08 s-1, p=0.02) and creatine kinase flux also showed a significant increase (1.65±0.78 μmol/g/s vs 2.28±0.71 μmol/g/s, p=0.05). Conclusions What is novel here is that we show a fall in PCr/ATP ratio: as ATP concentrations in the cell are strictly maintained, this suggests phosphocreatine pool depletion occurs when preload is lost and cardiac output is maintained by an increase in inotropy and chronotropy. The rise in CKkf and CK flux confirm the increased energy demand. Progressive energetic depletion during high demand may give rise to contractile dysfunction over time as the heart is unable to keep up with increased requirements for ATP, and progressively becomes more starved of energy. This could be a mechanism of cardiac dysfunction in septic shock and other vasoplegic states. Figure 1 Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): British Heart Foundation

Increased myocardial fat supply improves cardiac energetics and function in heart failure

Abstract Introduction The failing heart is starved of energy, in part accounting for its contractile dysfunction. Reduced uptake of fat and sugar required for energy production has frequently been demonstrated in heart failure, therefore altering metabolism of glucose and/or fat is therefore attractive as a therapy. We hypothesized increasing glucose supply would be beneficial over increasing fat supply so measured ATP usage (via PCr/ATP ratio and flux through creatine kinase) and cardiac function during fat emulsion infusion or euglycaemic hyperinsulinaemic clamp. Methods 11 patients with a diagnosis of heart failure and nonischaemic cardiomyopathy were recruited, mean age 66 (range 49–80), mean BMI 27.7 (range 21.3–37.5), F:M 3:8, 3 diabetic and 8 non-diabetic. On the first visit they had a baseline cardiac magnetic resonance (CMR), collecting cardiac volumes and function, then were randomised to receive either fat infusion or euglycaemic clamp. Following an hour of infusion, CMR was repeated followed by 31P cardiac magnetic resonance spectroscopy, then a dobutamine stress sequence at 65% maximum heart rate. They received the alternate infusion at the next visit. Results Data was normally distributed. Baseline ejection fraction was 37±9%. PCr/ATP ratio was greater with the fat infusion compared to euglycaemic clamp (1.82±0.26 vs 1.68±0.24, p=0.04). Fat emulsion infusion also brought about a greater ejection fraction increase over the baseline, compared to the euglycaemic clamp in which there was little difference (+5.3±5.3% vs −0.6±3.1%, p=0.004). Calculated cardiac work was greater in the fat infusion group than the Insulin/glucose group (682±156 L.mmHg/min vs 581±85 L.mmHg/min, p=0.009). There was no significant difference in creatine kinase first order rate constant (fat infusion 0.2±0.09/s vs euglycaemic clamp 0.16±0.07/s, p=0.32) nor creatine kinase flux (fat infusion 1.85±0.92 μmol/g/s vs euglycaemic clamp 1.46±0.58 μmol/g/s, p=0.22). The increment in cardiac output on stress over baseline was not significantly different between arms (fat infusion +3.39±3.07 L/min vs euglycaemic clamp +3.08±2.57 L/min, p=0.42). The PCr/ATP ratio showed positive correlation with the stress ejection fraction (R2=0.656, p=0.001), but not with resting ejection fraction. Conclusions Increased supply of fat to the myocardium brought about improved contractility and cardiac energetics compared to an increased glucose supply. The increase in PCr/ATP ratio would imply (given ATP concentrations are kept constant in the myocardium) there is a greater availability of phosphocreatine, suggesting increased mitochondrial ATP synthesis. These results were unexpected as it has traditionally been thought that increased glucose metabolism would yield greater cardiac function in the failing heart. These data suggest targeting myocardial fat metabolism may provide novel treatments for cardiac dysfunction. Figure 1 Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): British Heart Foundation

Cardiac Energetics in Patients With Aortic Stenosis and Preserved Versus Reduced Ejection Fraction

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. 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 31P 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. 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 kf 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). 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.

P2272Determinants of left ventricular ATP availability measured in vivo and ex vivo in patients with severe aortic stenosis: correlation of creatine kinase activity with LVEF and ATP diffusion distance

Abstract Introduction The transition to systolic failure in severe aortic stenosis (AS) increases mortality. There are currently no reliable markers of transition, and the guideline LVEF &lt;50% threshold for intervention in asymptomatic severe AS does not capture all subjects at increased risk. In animal models, reduced ATP delivery capacity through creatine kinase (CK) is important, with modest increases in CK capacity conferring cardioprotection. ATP may also diffuse (independent of CK) from mitochondria to the contractile site. We have performed the first human study to test whether ATP diffusion distance relates to CK activity and whether CK activity is reduced in low LVEF severe AS. Methods 19 patients with severe AS, LVEF ≥55% (AS-pEF, mean±SD LVEF 63±5%, mean gradient 48±14 mmHg) and 10 with severe AS, LVEF &lt;55% (AS-rEF, LVEF 42±8%, mean gradient 32±11) underwent 31P-MRS for CK rate constant (kf) and phosphocreatine/ATP (PCr/ATP) ratio, and MRI for LV volumes. LV biopsies were taken during AVR and analysed for CK total activity, CK isoforms, total creatine, and citrate synthase (CS) activity. 9 biopsies also underwent serial block face scanning electron microscopy and mitochondria-sarcomere 3D distance distributions were plotted. Results were compared to 24 controls (LVEF 61±4%), of which 4 had LV biopsy (3 severe MS, 1 LA myxoma, MS-pEF). Surgical patients had flow-limiting atheroma excluded with invasive angiography and prior myocardial infarction excluded with late gadolinium enhancement MRI. Results When compared to controls, both CK total activity and CS activity were lower in AS-pEF (by 27% and 23% respectively, both p&lt;0.05, Panels A-B). Although PCr/ATP reduced in AS-pEF (by 20%, p&lt;0.001, panel C), kf (panel D) and CK flux estimated by kf × total creatine were not different. CK-MB expression reduced in AS-pEF (19 vs 27% of total CK, p=0.003), reflecting compensatory increases in CK-MM (p=0.26) and CK-BB (p=0.18) in the face of reduced CK activity. AS-rEF was associated with further reduction in both CK and CS activities (by 32% and 22% respectively, both p&lt;0.05, Panels A-B), but no differences in PCr/ATP, CK kf or relative CK isozyme expression were seen. There were no significant between-group differences in total creatine (Panel E). Overall this suggests that CK reserve and oxidative capacity potentially reduce in pressure overload, with further falls commensurate with systolic dysfunction. When median mitochondria-sarcomere ATP diffusion distances were plotted against CK total activity a strong positive correlation was observed (r=0.86, p=0.003, Panel F). This suggests a compensatory reduction in diffusion distance develops when CK activity falls. Conclusions Transition to failure in severe AS is associated with lower oxidative capacity and maximal ATP delivery capacity through CK. Despite compensatory falls in ATP diffusion distance and altered CK isozyme expression, these changes may underlie susceptibility to EF decline in AS. Acknowledgement/Funding British Heart Foundation Clinical Research Training Fellowship (FS/15/80/31803) and Programme Grant (RG/18/12/34040).

P1611Non-invasive predictors of LV biopsy-obtained creatine kinase activity in patients with non-failing and failing myocardial hypertrophy

Abstract Introduction Myocardial creatine kinase (CK) activity represents an important metabolic reserve: it correlates closely with contractile reserve and post-ischaemic function, keeps cytosolic [ADP] low, and optimises the free energy for ATP hydrolysis. It may also contribute to the transition to failure in the heart hypertrophied by chronic haemodynamic overload; modest up-regulation has been shown to be cardio-protective. Total CK activity measurement requires chemical freeze-and-extract methods, which destroy tissue, precluding repeated measures. To date, non-invasive assessments of human myocardial CK flux (calculated as kf × [PCr], where kf is the pseudo-first-order forward rate constant measured by 31P-magnetic resonance spectroscopy (MRS) have not assessed creatine content or total CK activity. Thus we aimed to validate kf measurement against total CK activity and investigate predictors of CK activity. Methods 39 subjects (median age 71, range 43–84) undergoing clinically indicated cardiac surgery had CK total activity measured from LV biopsy. 31 had severe AS (10 with impaired LVEF); 2 had severe primary MR; 5 had severe mitral stenosis (2 with impaired LVEF) and 1 had an LA mass. 35 of 39 contributed triplicate datasets: CK total activity, kf (31P-MRS TRiST sequence at 3T) and LV volumes (cine-MRI, 3T Siemens). 27 had severe AS (8 with impaired LVEF); other groups were the same. Exclusion criteria were prior myocardial infarction and flow-limiting coronary disease. Flash-frozen LV biopsies obtained within 15 min of cardiopulmonary bypass were analysed for CK total activity, total creatine, and citrate synthase (CS) activity (a marker of oxidative phosphorylation capacity). Results Multiple, novel correlations were observed between CK total activity (IU/mg protein) and CS activity (r=0.87, p=9e-13), total creatine (r=0.59, p=8e-5), kf (r=0.42, p=0.013), total creatine × kf (r=0.64, p=4e-5), LVESVi (r=−0.52, p=8e-4), LVEF (r=0.48, p=0.002) and LVMi (r=−0.42, p=0.009) (Panels A-E) (LVEDVi and non-indexed counterparts were also significant correlates.) The most predictive linear regression model incorporating elements of the CK rate equation included total creatine (nmol/mg protein), kf (/s), and kf × LVESVi (ml/m2) (adjusted R2=0.56, beta=0.426, 0.618, −1.968 and p=0.001, 5e-5, 0.003 respectively, Panel F). Figure 1 Conclusions These results are the first evidence of agreement between non-invasive estimates of human cardiac CK activity (kf) and freeze-extracted chemical methods. The key original finding is that it is feasible to attempt to predict CK capacity in vivo by using a combination of techniques (creatine by 1H MRS, CK kf by 31P-MRS and LVESVi by cine imaging). The key insight is that CK capacity is best estimated not simply from what the rate equation would predict (creatine and kf), but that other factors relating to failing contractility reduce CK activity independently from creatine content and enzyme kf. Acknowledgement/Funding British Heart Foundation Clinical Research Training Fellowship (FS/15/80/31803) and Programme Grant (RG/18/12/34040).

Neural-network classification of cardiac disease from 31P cardiovascular magnetic resonance spectroscopy measures of creatine kinase energy metabolism

BackgroundThe heart’s energy demand per gram of tissue is the body’s highest and creatine kinase (CK) metabolism, its primary energy reserve, is compromised in common heart diseases. Here, neural-network analysis is used to test whether noninvasive phosphorus (31P) cardiovascular magnetic resonance spectroscopy (CMRS) measurements of cardiac adenosine triphosphate (ATP) energy, phosphocreatine (PCr), the first-order CK reaction rate kf, and the rate of ATP synthesis through CK (CK flux), can predict specific human heart disease and clinical severity.MethodsThe data comprised the extant 178 complete sets of PCr and ATP concentrations, kf, and CK flux data from human CMRS studies performed on clinical 1.5 and 3 Tesla scanners. Healthy subjects and patients with nonischemic cardiomyopathy, dilated (DCM) or hypertrophic disease, New York Heart Association (NYHA) class I-IV heart failure (HF), or with anterior myocardial infarction are included. Three-layer neural-networks were created to classify disease and to differentiate DCM, hypertrophy and clinical NYHA class in HF patients using leave-one-out training. Network performance was assessed using ‘confusion matrices’ and ‘area-under-the-curve’ (AUC) analyses of ‘receiver operating curves’. Possible methodological bias and network imbalance were tested by segregating 1.5 and 3 Tesla data, and by data augmentation by random interpolation of nearest neighbors, respectively.ResultsThe network differentiated healthy, HF and non-HF cardiac disease with an overall accuracy of 84% and AUC > 90% for each category using the four CK metabolic parameters, alone. HF patients with DCM, hypertrophy, and different NYHA severity were differentiated with ~ 80% overall accuracy independent of CMRS methodology.ConclusionsWhile sample-size was limited in some sub-classes, a neural network classifier applied to noninvasive cardiac 31P CMRS data, could serve as a metabolic biomarker for common disease types and HF severity with clinically-relevant accuracy. Moreover, the network’s ability to individually classify disease and HF severity using CK metabolism alone, implies an intimate relationship between CK metabolism and disease, with subtle underlying phenotypic differences that enable their differentiation.Trial registrationClinicalTrials.gov Identifier: NCT00181259.

Cardiac work is related to creatine kinase energy supply in human heart failure: a cardiovascular magnetic resonance spectroscopy study

BackgroundIt has been hypothesized that the supply of chemical energy may be insufficient to fuel normal mechanical pump function in heart failure (HF). The creatine kinase (CK) reaction serves as the heart’s primary energy reserve, and the supply of adenosine triphosphate (ATP flux) it provides is reduced in human HF. However, the relationship between the CK energy supply and the mechanical energy expended has never been quantified in the human heart. This study tests whether reduced CK energy supply is associated with reduced mechanical work in HF patients.MethodsCardiac mechanical work and CK flux in W/kg, and mechanical efficiency were measured noninvasively at rest using cardiac pressure-volume loops, magnetic resonance imaging and phosphorus spectroscopy in 14 healthy subjects and 27 patients with mild-to-moderate HF.ResultsIn HF, the resting CK flux (126 ± 46 vs. 179 ± 50 W/kg, p < 0.002), the average (6.8 ± 3.1 vs. 10.1 ± 1.5 W/kg, p <0.001) and the peak (32 ± 14 vs. 48 ± 8 W/kg, p < 0.001) cardiac mechanical work-rates, as well as the cardiac mechanical efficiency (53% ± 16 vs. 79% ± 3, p < 0.001), were all reduced by a third compared to healthy subjects. In addition, cardiac CK flux correlated with the resting peak and average mechanical power (p < 0.01), and with mechanical efficiency (p = 0.002).ConclusionThese first noninvasive findings showing that cardiac mechanical work and efficiency in mild-to-moderate human HF decrease proportionately with CK ATP energy supply, are consistent with the energy deprivation hypothesis of HF. CK energy supply exceeds mechanical work at rest but lies within a range that may be limiting with moderate activity, and thus presents a promising target for HF treatment.Trial registrationClinicalTrials.gov Identifier: NCT00181259.

Decreased ATP production and myocardial contractile reserve in metabolic heart disease

Metabolic syndrome is a cluster of obesity-related metabolic abnormalities that lead to metabolic heart disease (MHD) with left ventricular pump dysfunction. Although MHD is thought to be associated with myocardial energetic deficiency, two key questions have not been answered. First, it is not known whether there is a sufficient energy deficit to contribute to pump dysfunction. Second, the basis for the energy deficit is not clear. To address these questions, mice were fed a high fat, high sucrose (HFHS) ‘Western’ diet to recapitulate the MHD phenotype. In isolated beating hearts, we used 31P NMR spectroscopy with magnetization transfer to determine a) the concentrations of high energy phosphates ([ATP], [ADP], [PCr]), b) the free energy of ATP hydrolysis (∆G~ATP), c) the rate of ATP production and d) flux through the creatine kinase (CK) reaction. At the lowest workload, the diastolic pressure-volume relationship was shifted upward in HFHS hearts, indicative of diastolic dysfunction, whereas systolic function was preserved. At this workload, the rate of ATP synthesis was decreased in HFHS hearts, and was associated with decreases in both [PCr] and ∆G~ATP. Higher work demands unmasked the inability of HFHS hearts to increase systolic function and led to a further decrease in ∆G~ATP to a level that is not sufficient to maintain normal function of sarcoplasmic Ca2+-ATPase (SERCA). While [ATP] was preserved at all work demands in HFHS hearts, the progressive increase in [ADP] led to a decrease in ∆G~ATP with increased work demands. Surprisingly, CK flux, CK activity and total creatine were normal in HFHS hearts. These findings differ from dilated cardiomyopathy, in which the energetic deficiency is associated with decreases in CK flux, CK activity and total creatine. Thus, in HFHS-fed mice with MHD there is a distinct metabolic phenotype of the heart characterized by a decrease in ATP production that leads to a functionally-important energetic deficiency and an elevation of [ADP], with preservation of CK flux.

Creatine kinase rate constant in the human heart measured with 3D-localization at 7 tesla.

PurposeWe 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 B1+‐mapping to determine the per‐voxel flip angles, and nonlinear fitting to Bloch simulations for postprocessing.MethodsOptimal flip angles and repetition time parameters were determined from Monte Carlo simulations. BOAST was validated in the calf muscle of two volunteers at 3T and 7T. The myocardial CK forward rate constant was then measured in 10 volunteers at 7T in 82 min (after 1H localization).ResultsBOAST kfCK values were 0.281 ± 0.002 s−1 in the calf and 0.35 ± 0.05 s−1 in myocardium. These are consistent with literature values from lower fields. Using a literature values for adenosine triphosphate concentration, we computed CK flux values of 4.55 ± 1.52 mmol kg−1 s−1. The sensitive volume for BOAST depends on the B1 inhomogeneity of the transmit coil.ConclusionBOAST enables measurement of the CK rate constant in the human heart at 7T, with spatial localization in three dimensions to 5.6 mL voxels, using a 10‐cm loop coil. Magn Reson Med 78:20–32, 2017. © 2016 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Decline of Phosphotransfer and Substrate Supply Metabolic Circuits Hinders ATP Cycling in Aging Myocardium.

Integration of mitochondria with cytosolic ATP-consuming/ATP-sensing and substrate supply processes is critical for muscle bioenergetics and electrical activity. Whether age-dependent muscle weakness and increased electrical instability depends on perturbations in cellular energetic circuits is unknown. To define energetic remodeling of aged atrial myocardium we tracked dynamics of ATP synthesis-utilization, substrate supply, and phosphotransfer circuits through adenylate kinase (AK), creatine kinase (CK), and glycolytic/glycogenolytic pathways using 18O stable isotope-based phosphometabolomic technology. Samples of intact atrial myocardium from adult and aged rats were subjected to 18O-labeling procedure at resting basal state, and analyzed using the 18O-assisted HPLC-GC/MS technique. Characteristics for aging atria were lower inorganic phosphate Pi[18O], γ-ATP[18O], β-ADP[18O], and creatine phosphate CrP[18O] 18O-labeling rates indicating diminished ATP utilization-synthesis and AK and CK phosphotransfer fluxes. Shift in dynamics of glycolytic phosphotransfer was reflected in the diminished G6P[18O] turnover with relatively constant glycogenolytic flux or G1P[18O] 18O-labeling. Labeling of G3P[18O], an indicator of G3P-shuttle activity and substrate supply to mitochondria, was depressed in aged myocardium. Aged atrial myocardium displayed reduced incorporation of 18O into second (18O2), third (18O3), and fourth (18O4) positions of Pi[18O] and a lower Pi[18O]/γ-ATP[18 O]-labeling ratio, indicating delayed energetic communication and ATP cycling between mitochondria and cellular ATPases. Adrenergic stress alleviated diminished CK flux, AK catalyzed β-ATP turnover and energetic communication in aging atria. Thus, 18O-assisted phosphometabolomics uncovered simultaneous phosphotransfer through AK, CK, and glycolytic pathways and G3P substrate shuttle deficits hindering energetic communication and ATP cycling, which may underlie energetic vulnerability of aging atrial myocardium.

Creatine Kinase-Overexpression Improves Myocardial Energetics, Contractile Dysfunction and Survival in Murine Doxorubicin Cardiotoxicity

Doxorubicin (DOX) is a commonly used life-saving antineoplastic agent that also causes dose-dependent cardiotoxicity. Because ATP is absolutely required to sustain normal cardiac contractile function and because impaired ATP synthesis through creatine kinase (CK), the primary myocardial energy reserve reaction, may contribute to contractile dysfunction in heart failure, we hypothesized that impaired CK energy metabolism contributes to DOX-induced cardiotoxicity. We therefore overexpressed the myofibrillar isoform of CK (CK-M) in the heart and determined the energetic, contractile and survival effects of CK-M following weekly DOX (5mg/kg) administration using in vivo 31P MRS and 1H MRI. In control animals, in vivo cardiac energetics were reduced at 7 weeks of DOX protocol and this was followed by a mild but significant reduction in left ventricular ejection fraction (EF) at 8 weeks of DOX, as compared to baseline. At baseline, CK-M overexpression (CK-M-OE) increased rates of ATP synthesis through cardiac CK (CK flux) but did not affect contractile function. Following DOX however, CK-M-OE hearts had better preservation of creatine phosphate and higher CK flux and higher EF as compared to control DOX hearts. Survival after DOX administration was significantly better in CK-M-OE than in control animals (p<0.02). Thus CK-M-OE attenuates the early decline in myocardial high-energy phosphates and contractile function caused by chronic DOX administration and increases survival. These findings suggest that CK impairment plays an energetic and functional role in this DOX-cardiotoxicity model and suggests that metabolic strategies, particularly those targeting CK, offer an appealing new strategy for limiting DOX-associated cardiotoxicity.

Creatine Kinase Adenosine Triphosphate and Phosphocreatine Energy Supply in a Single Kindred of Patients With Hypertrophic Cardiomyopathy

A lethal and extensively characterized familial form of hypertrophic cardiomyopathy (HC) is due to a point mutation (Arg403Gln) in the cardiac β-myosin heavy chain gene. Although this is associated with abnormal energy metabolism and progression to heart failure in an animal model, in vivo cardiac energetics have not been characterized in patients with this mutation. Noninvasive phosphorus saturation transfer magnetic resonance spectroscopy was used to measure the adenosine triphosphate supplied by the creatine kinase (CK) reaction and phosphocreatine, the heart's primary energy reserve, in 9 of 10 patients from a single kindred with HC caused by the Arg403GIn mutation and 17 age-matched healthy controls. Systolic and diastolic function was assessed by echocardiography in all 10 patients with HC. The patients with HC had impairment of diastolic function and mild systolic dysfunction, when assessed using global systolic longitudinal strain. Myocardial phosphocreatine was significantly decreased by 24% in patients (7.1 ± 2.3 μmol/g) compared with the controls (9.4 ± 1.2 μmol/g; p = 0.003). The pseudo-first-order CK rate-constant was 26% lower (0.28 ± 0.15 vs 0.38 ± 0.07 s⁻¹, p = 0.035) and the forward CK flux was 44% lower (2.0 ± 1.4 vs 3.6 ± 0.9 μmol/g/s, p = 0.001) than in the controls. The contractile abnormalities did not correlate with the metabolic indexes. In conclusion, myocardial phosphocreatine and CK-ATP delivery are significantly reduced in patients with HC caused by the Arg403Gln mutation, akin to previous results from mice with the same mutation. A lack of a relation between energetic and contractile abnormalities suggests the former result from the sarcomeric mutation and not a late consequence of mechanical dysfunction.

Skeletal muscle ATP kinetics during exercise in patients with systolic heart failure

Background Creatine kinase (CK) reaction is the primary energy reserve reaction in skeletal muscle (SM), providing additional ATP during exercise. Although exercise intolerance is a major limitation in systolic heart failure (SHF), the rate of ATP delivery by CK (CK flux) has not been studied during exercise in healthy subjects (HS) or SHF patients. We hypothesized that SM high-energy phosphates and CK flux are reduced at rest and during low intensity plantar flexion exercise (PFE) in mild-moderate SHF patients. Methods 14 non-ischemic SHF patients (EF<40) and 11 HS underwent 31P magnetic resonance spectroscopy with saturation transfer (MRST) measures of gastrocnemius creatine phosphate-to-ATP (PCr/ATP) and CK pseudo-first-order rate constant, k, at rest and during PFE in a Philips 3T MRI/ MRS system. Single-leg PFE involved lifting 0.9 kg/sec on a custom built non-magnetic device for 8.5 minutes. [ATP] (umol/g) was taken as 5.2umol/g for both groups and [PCr](umol/g) was calculated as (PCr/ATP)*(5.2umol/g) and CK flux (umol/g/s) as ([PCr]*k). Comparisons were made with paired and non-paired t-testing using XLSTAT package.

Sensitivity Analysis of Flux Determination in Heart by H218O -provided Labeling Using a Dynamic Isotopologue Model of Energy Transfer Pathways

To characterize intracellular energy transfer in the heart, two organ-level methods have frequently been employed: inversion and saturation transfer, and dynamic labeling. Creatine kinase (CK) fluxes obtained by following oxygen labeling have been considerably smaller than the fluxes determined by saturation transfer. It has been proposed that dynamic labeling determines net flux through CK shuttle, whereas saturation transfer measures total unidirectional flux. However, to our knowledge, no sensitivity analysis of flux determination by oxygen labeling has been performed, limiting our ability to compare flux distributions predicted by different methods. Here we analyze oxygen labeling in a physiological heart phosphotransfer network with active CK and adenylate kinase (AdK) shuttles and establish which fluxes determine the labeling state. A mathematical model consisting of a system of ordinary differential equations was composed describing enrichment in each phosphoryl group and inorganic phosphate. By varying flux distributions in the model and calculating the labeling, we analyzed labeling sensitivity to different fluxes in the heart. We observed that the labeling state is predominantly sensitive to total unidirectional CK and AdK fluxes and not to net fluxes. We conclude that measuring dynamic incorporation of into the high-energy phosphotransfer network in heart does not permit unambiguous determination of energetic fluxes with a higher magnitude than the ATP synthase rate when the bidirectionality of fluxes is taken into account. Our analysis suggests that the flux distributions obtained using dynamic labeling, after removing the net flux assumption, are comparable with those from inversion and saturation transfer.

Creatine kinase overexpression improves ATP kinetics and contractile function in postischemic myocardium

Reduced myofibrillar ATP availability during prolonged myocardial ischemia may limit post-ischemic mechanical function. Because creatine kinase (CK) is the prime energy reserve reaction of the heart and because it has been difficult to augment ATP synthesis during and after ischemia, we used mice that overexpress the myofibrillar isoform of creatine kinase (CKM) in cardiac-specific, conditional fashion to test the hypothesis that CKM overexpression increases ATP delivery in ischemic-reperfused hearts and improves functional recovery. Isolated, retrograde-perfused hearts from control and CKM mice were subjected to 25 min of global, no-flow ischemia and 40 min of reperfusion while cardiac function [rate pressure product (RPP)] was monitored. A combination of (31)P-nuclear magnetic resonance experiments at 11.7T and biochemical assays was used to measure the myocardial rate of ATP synthesis via CK (CK flux) and intracellular pH (pH(i)). Baseline CK flux was severalfold higher in CKM hearts (8.1 ± 1.0 vs. 32.9 ± 3.8, mM/s, control vs. CKM; P < 0.001) with no differences in phosphocreatine concentration [PCr] and RPP. End-ischemic pH(i) was higher in CKM hearts than in control hearts (6.04 ± 0.12 vs. 6.37 ± 0.04, control vs. CKM; P < 0.05) with no differences in [PCr] and [ATP] between the two groups. Post-ischemic PCr (66.2 ± 1.3 vs. 99.1 ± 8.0, %preischemic levels; P < 0.01), CK flux (3.2 ± 0.4 vs. 14.0 ± 1.2 mM/s; P < 0.001) and functional recovery (13.7 ± 3.4 vs. 64.9 ± 13.2%preischemic RPP; P < 0.01) were significantly higher and lactate dehydrogenase release was lower in CKM than in control hearts. Thus augmenting cardiac CKM expression attenuates ischemic acidosis, reduces injury, and improves not only high-energy phosphate content and the rate of CK ATP synthesis in postischemic myocardium but also recovery of contractile function.

Allopurinol Acutely Increases Adenosine Triphospate Energy Delivery in Failing Human Hearts

This study tested the hypothesis that acute administration of the xanthine oxidase (XO) inhibitor allopurinol improves cardiac high-energy phosphate concentrations in human heart failure (HF) and increases the rate of adenosine triphosphate (ATP) synthesis through creatine kinase (CK), the primary myocardial energy reserve. Studies of patients and animal models implicate impaired myocardial high-energy phosphate availability in HF. The XO reaction is a critical terminal step in ATP and purine degradation and an important source of reactive oxygen species. Thus, XO inhibition is a potentially attractive means to improve energy metabolism in the failing human heart. We randomized 16 patients with nonischemic cardiomyopathy in a double-blind fashion to allopurinol (300 mg intravenously) or placebo infusion, 4-to-1, the latter for purposes of blinding only. The myocardial concentrations of ATP and creatine phosphate (PCr) and the rate of ATP synthesis through CK (CK flux) were determined by (31)P magnetic resonance spectroscopy. Allopurinol infusion increased mean cardiac PCr/ATP and PCr concentration by ∼11% (p < 0.02), and mean CK flux by 39% (2.07 ± 1.27 μmol/g/s to 2.87 ± 1.82 μmol/g/s, p < 0.007). Calculated cytosolic adenosine diphosphate concentration decreased, whereas the free energy of ATP hydrolysis (ΔG(∼ATP)) increased with allopurinol. The increased CK flux was disproportionate to substrate changes, indicating increased CK enzyme activity. Intravenous administration of the XO inhibitor allopurinol acutely improves the relative and absolute concentrations of myocardial high-energy phosphates and ATP flux through CK in the failing human heart, offering direct evidence that myofibrillar CK energy delivery can be pharmaceutically augmented in the failing human heart. (Intravenous Allopurinol in Heart Failure; NCT00181155).

Abstract 9004: Creatine Kinase Over-Expression Improves ATP Kinetics and Contractile Function in the Post-Ischemic Myocardium

ATP is the biochemical fuel for myocardial contraction but it is depleted during prolonged ischemia and its depletion may limit post-ischemic mechanical function. Because it has been difficult to augment ATP synthesis during and after ischemia, we generated mice that overexpress the myofibrillar isoform of Creatine Kinase (CKM), the primary energy reserve reaction of the heart, in cardiac-specific, conditional fashion to test the hypothesis that enhanced CKM expression increases ATP delivery via Creatine Kinase (CK) in ischemic-reperfused hearts and improves functional recovery. CKM mice were generated using a tet-off system. Gene induction was achieved by switching from a doxycycline diet (650mg/kg diet) to regular diet 4 weeks prior to the study. Isolated, retrograde-perfused hearts from control and CKM mice were subjected to 25 minutes of global ischemia and 40 minutes of reperfusion while cardiac function (rate pressure product, RPP) was monitored using a left ventricular balloon connected to a pressure transducer. A combination of 31P nuclear magnetic resonance magnetization transfer experiments at 11.7T and biochemical assays were used to measure the CK pseudo first-order rate constant (kf), the rate of ATP synthesis via CK (CK flux), intracellular pH and in vitro CK activity. Pre-ischemic kf and CK flux were three-fold higher in CKM than control hearts with no difference in RPP (Table). During ischemia, intracellular pH fell more in control than CKM hearts (6.10±0.29 vs. 6.46±0.08, p&lt;0.05) with no difference in PCr and ATP levels between the two groups. Post-ischemic kf, PCr, CK flux and functional recovery were significantly higher in CKM vs. control hearts as was CK activity (21.6±7.23 vs 7.59±1.42 IU/mg protein, p&lt;0.01). These observations demonstrate that augmenting CKM expression attenuates ischemic acidosis and improves not only [PCr] and rate of ATP synthesis in the post-ischemic myocardium but also recovery of contractile function.

Abstract 16924: Multimodality Imaging Reveals Decreased Creatine-Kinase Flux and Systolic Strain in Patients with Hypertrophic Cardiomyopathy and Preserved LVEF

Background/Objectives: We investigated in vivo cardiac creatine kinase (CK) ATP supply and function in a family with HCM who carry an extensively characterized point mutation (R403Q) in the cardiac β-myosin heavy chain gene. Methods: Quantitative phosphorus ( 31 P) saturation transfer MRS was used to noninvasively measure cardiac CK energy transfer-the forward CK flux and the CK rate constant, k f -and CK metabolite concentrations (phosphocreatine, [PCr], and [ATP]) in patients with HCM (n=10), and in age-matched healthy controls (n=15). Systolic strain, LV ejection fraction (EF), diastolic function and cardiac morphology were assessed by echocardiography in HCM patients (n=11) and a second group of healthy controls (n=10). Results: LV mass was higher in HCM patients than controls (307.21 ± 12.37 vs 167.63 ± 42.53; p=0.0037). [PCr] was significantly decreased by 22% in HCM patients (7.36 ± 2.29µmol/g wet weight) as compared to controls (9.41 ± 1.2µmol/g; p=0.005), but PCr/ATP ratio was not significantly altered (1.54 ± 0.4 in HCM vs. 1.69 ± 0.29, p=0.26). The pseudo-first-order rate-constant for the CK reaction, k f was reduced by 26% (0.266 ± 0.14 vs. 0.36 ± 0.08, p&lt;0.04) and the forward CK flux was reduced by 43% (1.94 ± 1.27 vs 3.39 ± 0.96, p&lt;0.003) in HCM patients when compared to controls. Diastolic function (E/A) was impaired (0.90 ± 0.31 in HCM vs 1.48 ± 0.27 controls; p=0.0003) and peak systolic strain (longitudinal; εL) was significantly decreased in HCM patients (-13.40 ± 5.32 vs -18.93 ± 2.46; p=0.0076). Peak longitudinal strain and diastolic function (E/A) correlated with NYHA class (R²=0.50; p =0.0007); E/A &lt;1 correlated with NYHA class progression over 5 years of follow up (R 2 =0.6, p=0.024). Conclusions: PCr, k for and CK flux are significantly reduced in patients compared to age-matched healthy control subjects, indicating impaired CK ATP energy supply even at rest, consistent with prior work on a HCM (R403Q) mouse model. However, only systolic strain and diastolic function were correlated with NYHA class and progression respectively, suggesting that while bioenergetic alterations are associated with the underlying mutation, other downstream molecular effects contribute to the severity of dysfunction and clinical status of these patients.