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.

Myosin-driven rescue of contractile reserve and energetics in mouse hearts bearing familial hypertrophic cardiomyopathy-associated mutant troponin T is mutation-specific.

The thin filament protein troponin T (TnT) is a regulator of sarcomere function. Whole heart energetics and contractile reserve are compromised in transgenic mice bearing missense mutations at R92 within the tropomyosin-binding domain of cTnT, despite being distal to the ATP hydrolysis domain of myosin. These mutations are associated with familial hypertrophic cardiomyopathy (FHC). Here we test the hypothesis that genetically replacing murine αα-MyHC with murine ßß-MyHC in hearts bearing the R92Q cTnT mutation, a particularly lethal FHC-associated mutation, leads to sufficiently large perturbations in sarcomere function to rescue whole heart energetics and decrease the cost of contraction. By comparing R92Q cTnT and R92L cTnT mutant hearts, we also test whether any rescue is mutation-specific. We defined the energetic state of the isolated perfused heart using (31)P-NMR spectroscopy while simultaneously measuring contractile performance at four work states. We found that the cost of increasing contraction in intact mouse hearts with R92Q cTnT depends on the type of myosin present in the thick filament. We also found that the salutary effect of this manoeuvre is mutation-specific, demonstrating the major regulatory role of cTnT on sarcomere function at the whole heart level.

Study design for the Immediate Myocardial Metabolic Enhancement During Initial Assessment and Treatment in Emergency Care (IMMEDIATE) Trial: A double-blind randomized controlled trial of intravenous glucose, insulin, and potassium for acute coronary syndromes in emergency medical services.

BACKGROUND: Experimental studies suggest that metabolic myocardial support by intravenous (IV) glucose, insulin, and potassium (GIK) reduces ischemia-induced arrhythmias, cardiac arrest, mortality, progression from unstable angina pectoris to acute myocardial infarction (AMI), and myocardial infarction size. However, trials of hospital administration of IV GIK to patients with ST-elevation myocardial infarction (STEMI) have generally not shown favorable effects possibly because of the GIK intervention taking place many hours after ischemic symptom onset. A trial of GIK used in the very first hours of ischemia has been needed, consistent with the timing of benefit seen in experimental studies. OBJECTIVE: The IMMEDIATE Trial tested whether, if given very early, GIK could have the impact seen in experimental studies. Accordingly, distinct from prior trials, IMMEDIATE tested the impact of GIK (1) in patients with acute coronary syndromes (ACS), rather than only AMI or STEMI, and (2) administered in prehospital emergency medical service settings, rather than later, in hospitals, after emergency department evaluation. DESIGN: The IMMEDIATE Trial was an emergency medical service-based randomized placebo-controlled clinical effectiveness trial conducted in 13 cities across the United States that enrolled 911 participants. Eligible were patients 30 years or older for whom a paramedic performed a 12-lead electrocardiogram to evaluate chest pain or other symptoms suggestive of ACS for whom electrocardiograph-based acute cardiac ischemia time-insensitive predictive instrument indicated a ≥75% probability of ACS, and/or the thrombolytic predictive instrument indicated the presence of a STEMI, or if local criteria for STEMI notification of receiving hospitals were met. Prehospital IV GIK or placebo was started immediately. Prespecified were the primary end point of progression of ACS to infarction and, as major secondary end points, the composite of cardiac arrest or in-hospital mortality, 30-day mortality, and the composite of cardiac arrest, 30-day mortality, or hospitalization for heart failure. Analyses were planned on an intent-to-treat basis, on a modified intent-to-treat group who were confirmed in emergency departments to have ACS, and for participants presenting with STEMI. CONCLUSION: The IMMEDIATE Trial tested whether GIK, when administered as early as possible in the course of ACS by paramedics using acute cardiac ischemia time-insensitive predictive instrument and thrombolytic predictive instrument decision support, would reduce progression to AMI, mortality, cardiac arrest, and heart failure. It also tested whether it would provide clinical and pathophysiologic information on GIK's biological mechanisms.

Rearrangement of energetic and substrate utilization networks compensate for chronic myocardial creatine kinase deficiency.

Plasticity of the cellular bioenergetic system is fundamental to every organ function, stress adaptation and disease tolerance. Here, remodelling of phosphotransfer and substrate utilization networks in response to chronic creatine kinase (CK) deficiency, a hallmark of cardiovascular disease, has been revealed in transgenic mouse models lacking either cytosolic M-CK (M-CK(-/-)) or both M-CK and sarcomeric mitochondrial CK (M-CK/ScCKmit(-/-)) isoforms. The dynamic metabolomic signatures of these adaptations have also been defined. Tracking perturbations in metabolic dynamics with (18)O and (13)C isotopes and (31)P NMR and mass spectrometry demonstrate that hearts lacking M-CK have lower phosphocreatine (PCr) turnover but increased glucose-6-phosphate (G-6-P) turnover, glucose utilization and inorganic phosphate compartmentation with normal ATP γ-phosphoryl dynamics. Hearts lacking both M-CK and sarcomeric mitochondrial CK have diminished PCr turnover, total phosphotransfer capacity and intracellular energetic communication but increased dynamics of ß-phosphoryls of ADP/ATP, G-6-P and γ-/ß-phosphoryls of GTP, indicating redistribution of flux through adenylate kinase (AK), glycolytic and guanine nucleotide phosphotransfer circuits. Higher glycolytic and mitochondrial capacities and increased glucose tolerance contributed to metabolic resilience of M-CK/ScCKmit(-/-) mice. Multivariate analysis revealed unique metabolomic signatures for M-CK(-/-) and M-CK/ScCKmit(-/-) hearts suggesting that rearrangements in phosphotransfer and substrate utilization networks provide compensation for genetic CK deficiency. This new information highlights the significance of integrated CK-, AK-, guanine nucleotide- and glycolytic enzyme-catalysed phosphotransfer networks in supporting the adaptivity and robustness of the cellular energetic system.

Reducing the late sodium current improves cardiac function during sodium pump inhibition by ouabain.

Inhibition by cardiac glycosides of Na(+), K(+)-ATPase reduces sodium efflux from myocytes and may lead to Na(+) and Ca(2+) overload and detrimental effects on mechanical function, energy metabolism, and electrical activity. We hypothesized that inhibition of sodium persistent inward current (late I(Na)) would reduce ouabain's effect to cause cellular Na(+) loading and its detrimental metabolic (decrease of ATP) and functional (arrhythmias, contracture) effects. Therefore, we determined effects of ouabain on concentrations of intracellular sodium (Na(+)(i)) and high-energy phosphates using (23)Na and (31)P NMR, the amplitude of late I(Na) using the whole-cell patch-clamp technique, and contractility and electrical activity of guinea pig isolated hearts, papillary muscles, and ventricular myocytes in the absence and presence of inhibitors of late I(Na). Ouabain (1-1.3 µM) increased Na(+)(i) and late I(Na) of guinea pig isolated hearts and myocytes by 3.7- and 4.2-fold, respectively. The late I(Na) inhibitors ranolazine and tetrodotoxin significantly reduced ouabain-stimulated increases in Na(+)(i) and late I(Na). Reductions of ATP and phosphocreatine contents and increased diastolic tension in ouabain-treated hearts were also markedly attenuated by ranolazine. Furthermore, the ouabain-induced increase of late I(Na) was also attenuated by the Ca(2+)-calmodulin-dependent kinase I inhibitors KN-93 [N-[2-[[[3-(4-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulphonamide] and autocamide-2 related inhibitory peptide, but not by KN-92 [2-[N-(4'-methoxybenzenesulfonyl)]amino-N-(4'-chlorophenyl)-2-propenyl-N-methylbenzylamine phosphate]. We conclude that ouabain-induced Na(+) and Ca(2+) overload is ameliorated by the inhibition of late I(Na).

An improved isolation procedure for adult mouse cardiomyocytes.

Isolated adult mouse cardiomyocytes are an important tool in cardiovascular research, but are challenging to prepare. Because the energy supply determines cell function and viability, we compared total creatine ([Cr]) and [ATP] in isolated cardiomyocytes with the intact mouse heart. Isolated myocytes suffered severe losses of Cr (-70%) and ATP (-53%). Myocytes were not able to replete [Cr] during a 5 h incubation period in medium supplemented with 1 mM Cr. In contrast, adding 20 mM Cr to the digestion buffers was sufficient to maintain normal [Cr]. Supplementing buffers with 5 mM of inosine (Ino) and adenosine (Ado) to prevent loss of cellular nucleosides partially protected against loss of ATP. To test whether maintaining [ATP] and [Cr] improves contractile function, myocytes were challenged by varying pacing rate from 0.5 to 10 Hz and by adding isoproterenol (Iso) at 5 and 10 Hz. All groups performed well up to 5 Hz, showing a positive cell shortening-frequency relationship; however, only 16% of myocytes isolated under standard conditions were able to sustain pacing with Iso challenge at 10 Hz. In contrast, 30-50% of the myocytes with normal Cr levels were able to contract and maintain low diastolic [Ca(2+)]. Cell yield also improved in Cr and the Cr/Ino/Ado-treated groups (85-90% vs. 70-75% rod shaped in untreated myocytes). These data suggest that viability and performance of isolated myocytes are improved when they are protected from the severe loss of Cr and ATP during the isolation, making them an even better research tool.

Compromised myocardial energetics in hypertrophied mouse hearts diminish the beneficial effect of overexpressing SERCA2a.

The sarcoplasmic reticulum calcium ATPase (SERCA) plays a central role in regulating intracellular Ca(2+) homeostasis and myocardial contractility. Several studies show that improving Ca(2+) handling in hypertrophied rodent hearts by increasing SERCA activity results in enhanced contractile function. This suggests that SERCA is a potential target for gene therapy in cardiac hypertrophy and failure. However, it raises the issue of increased energy cost resulting from a higher ATPase activity. In this study, we determined whether SERCA overexpression alters the energy cost of increasing myocardial contraction in mouse hearts with pressure-overload hypertrophy using (31)P NMR spectroscopy. We isolated and perfused mouse hearts from wild-type (WT) and transgenic (TG) mice overexpressing the cardiac isoform of SERCA (SERCA2a) 8 weeks after ascending aortic constriction (left ventricular hypertrophy (LVH)) or sham operation. We found that overexpressing SERCA2a enhances myocardial contraction and relaxation in normal mouse hearts during inotropic stimulation with isoproterenol. Energy consumption was proportionate to the increase in contractile function. Thus, increasing SERCA2a expression in the normal heart allows an enhanced inotropic response with no compromise in energy supply and demand. However, this advantage was not sustained in LVH hearts in which the energetic status was compromised. Although the overexpression of SERCA2a prevented the down-regulation of SERCA protein in LVH hearts, TG-LVH hearts showed no increase in inotropic response when compared with WT-LVH hearts. Our results suggest that energy supply may be a limiting factor for the benefit of SERCA overexpression in hypertrophied hearts. Thus, strategies combining energetic support with increasing SERCA activity may improve the therapeutic effectiveness for heart failure.

Cardiac myosin heavy chain isoform exchange alters the phenotype of cTnT-related cardiomyopathies in mouse hearts.

Familial hypertrophic cardiomyopathy, FHC, is a clinically heterogeneous, autosomal-dominant disease of the cardiac sarcomere leading to extensive remodeling at both the whole heart and molecular levels. The remodeling patterns are mutation-specific, a finding that extends to the level of single amino acid substitutions at the same peptide residue. Here we utilize two well-characterized transgenic FHC mouse models carrying independent amino acid substitutions in the TM-binding region of cardiac troponin T (cTnT) at residue 92. R92Q and R92L cTnT domains have mutation-specific average peptide conformation and dynamics sufficient to alter thin filament flexibility and cross-bridge formation and R92 mutant myocytes demonstrate mutation-specific temporal molecular remodeling of Ca(2+) kinetics and impaired cardiac contractility and relaxation. To determine if a greater economy of contraction at the crossbridge level would rescue the mechanical defects caused by the R92 cTnT mutations, we replaced the endogenous murine alpha-myosin heavy chain (MyHC) with the beta-MyHC isoform. While beta-MyHC replacement rescued the systolic dysfunction in R92Q mice, it failed to rescue the defects in diastolic function common to FHC-associated R92 mutations. Surprisingly, a significant component of the whole heart and molecular contractile improvement in the R92Q mice was due to improvements in Ca(2+) homeostasis including SR uptake, [Ca2+](i) amplitude and phospholamban phosphorylation. Our data demonstrate that while genetically altering the myosin composition of the heart bearing a thin filament FHC mutation is sufficient to improve contractility, diastolic performance is refractory despite improved Ca(2+) kinetics. These data reveal a previously unrecognized role for MyHC isoforms with respect to Ca(2+) homeostasis in the setting of cardiomyopathic remodeling and demonstrate the overall dominance of the thin filament mutation in determining the degree of diastolic impairment at the myofilament level.

Progressive loss of creatine maintains a near normal DeltaG approximately (ATP) in transgenic mouse hearts with cardiomyopathy caused by overexpressing Gsalpha.

Myocardial [ATP] falls in the failing heart. One potential compensatory mechanism for maintaining a near normal free energy of ATP hydrolysis (DeltaG approximately (ATP)), despite a fall in [ATP], may be the reduction of myocardial creatine (Cr). To test this, we conducted a longitudinal study using transgenic mice overexpressing cardiac Gsalpha, which slowly developed cardiomyopathy. Myocardial energetics measured using (31)P NMR spectroscopy and isovolumic contractile performance were determined in perfused hearts isolated from 5-, 10-, 17-month-old Gsalpha and age-matched littermate wild type (WT) mice. In young Gsalpha hearts, contractile performance was enhanced with near normal cardiac energetics. With age, as contractile performance progressively decreased in Gsalpha hearts, [ATP] and [PCr] progressively decreased while [Pi] increased only modestly; no changes were observed in WT hearts. Myocardial (but not skeletal) [Cr] in Gsalpha mice decreased, beginning at an early age (1.5 months). Consequently, cytosolic [ADP] and the free energy available from ATP hydrolysis were maintained at normal levels in Gsalpha hearts, despite decreased [ATP]. During increased cardiac work caused by supplying isoproterenol, the relationship between the rate pressure product (RPP) and DeltaG approximately (ATP) in Gsalpha mouse hearts demonstrated an increased cost of contraction in failing hearts. Thus, our results suggest that the decrease of myocardial [Cr] and net Pi efflux play compensatory roles by maintaining a nearly normal free energy of ATP hydrolysis in the dysfunctional heart; however, it also increased the cost of contraction, which may contribute to the lower contractile reserve in the failing heart.

Early beneficial effects of bone marrow-derived mesenchymal stem cells overexpressing Akt on cardiac metabolism after myocardial infarction.

Administration of mesenchymal stem cells (MSCs) is an effective therapy to repair cardiac damage after myocardial infarction (MI) in experimental models. However, the mechanisms of action still need to be elucidated. Our group has recently suggested that MSCs mediate their therapeutic effects primarily via paracrine cytoprotective action. Furthermore, we have shown that MSCs overexpressing Akt1 (Akt-MSCs) exert even greater cytoprotection than unmodified MSCs. So far, little has been reported on the metabolic characteristics of infarcted hearts treated with stem cells. Here, we hypothesize that Akt-MSC administration may influence the metabolic processes involved in cardiac adaptation and repair after MI. MI was performed in rats randomized in four groups: sham group and animals treated with control MSCs, Akt-MSCs, or phosphate-buffered saline (PBS). High energy metabolism and basal 2-deoxy-glucose (2-DG) uptake were evaluated on isolated hearts using phosphorus-31 nuclear magnetic resonance spectroscopy at 72 hours and 2 weeks after MI. Treatment with Akt-MSCs spared phosphocreatine stores and significantly limited the increase in 2-DG uptake in the residual intact myocardium compared with the PBS- or the MSC-treated animals. Furthermore, Akt-MSC-treated hearts had normal pH, whereas low pH was measured in the PBS and MSC groups. Correlative analysis indicated that functional recovery after MI was inversely related to the rate of 2-DG uptake. We conclude that administration of MSCs overexpressing Akt at the time of infarction results in preservation of normal metabolism and pH in the surviving myocardium.

Energy metabolism in heart failure and remodelling.

Myocytes of the failing heart undergo impressive metabolic remodelling. The time line for changes in the pathways for ATP synthesis in compensated hypertrophy is: flux through the creatine kinase (CK) reaction falls as both creatine concentration ([Cr]) and CK activity fall; increases in [ADP] and [AMP] lead to increases in glucose uptake and utilization; fatty acid oxidation either remains the same or decreases. In uncompensated hypertrophy and in other forms of heart failure, CK flux and fatty acid oxidation are both lower; any increases in glucose uptake and utilization are not sufficient to compensate for overall decreases in the capacity for ATP supply and [ATP] falls. Metabolic remodelling is under transcriptional and post-transcriptional control. The lower metabolic reserve of the failing heart contributes to impaired contractile reserve.

Creatine kinase inhibitor iodoacetamide antagonizes calcium-stimulated inotropy in cardiomyocytes.

1. Inhibition of creatine kinase is known to suppress cardiac contractile reserve in intact hearts, although the underlying mechanism has not been elucidated. 2. The present study was designed to examine whether cardiac depression induced by creatine kinase inhibition was due to action at the level of the essential contractile element, namely cardiomyocytes. Adult rat cardiomyocytes were perfused with the creatine kinase inhibitor iodoacetamide (90 micromol/L) for 90 min. Mechanical and intracellular Ca(2+) properties were evaluated using edge-detection and fluorescence microscopy, respectively. Myocytes were superfused with normal (1.3 mmol/L) or high (3.3 mmol/L) extracellular Ca(2+) contractile buffer. Mechanical function was examined, including peak shortening (PS), maximal velocity of shortening/relengthening (+/-dL/dt), time to 90% PS (TPS(90)), time to 90% relengthening (TR(90)) and integration of shortening/relengthening (normalized to PS). Intracellular Ca(2+) transients were evaluated using the following indices: resting and rise of fura-2 fluorescence intensity (Delta FFI) and intracellular Ca(2+) decay time constant. 3. The results indicate that elevated extracellular Ca(2+) stimulated cardiomyocyte positive inotrope, manifested as increased PS, +/-dL/dt, area of shortening, resting FFI and Delta FFI associated with a shortened TR(90) and intracellular Ca(2+) decay time constant. High extracellular Ca(2+) did not affect TPS(90) and area of relengthening. Iodoacetamide ablated high Ca(2+)-induced increases in PS, +/-dL/dt, area of shortening, resting FFI, Delta FFI and shortened TR(90) and intracellular Ca(2+) decay time constant. Iodoacetamide itself significantly enhanced the area of relengthening and TR(90) without affecting other indices. 4. Collectively, these data demonstrate that inhibition of creatine kinase blunts high extracellular Ca(2+)-induced increases in cardiomyocyte contractile response (i.e. cardiac contractile reserve).

Low over-expression of TNFalpha in the mouse heart increases contractile performance via TNFR1.

TNFalpha is a cytokine wit pleiotropic functions in many organs. In the heart increased TNFalpha levels are not only associated with heart failure, but also, paradoxically, with protection from ischemic damage. To test whether the protective role of TNFalpha in the heart is concentration-dependent, we studied two mouse heart models with low (two- to threefold) over-expression of endogenous TNFalpha: mice deficient in a translational repressor of TNFalpha mRNA, TIA-1(-/-), and mice over-expressing human TNFalpha. Hearts lacking TIA-1 were characterized for their endogenous TNFalpha over-expression during normal Langendorff perfusion. To define which TNFalpha receptor mediates cardiac protection, we also used mice lacking the TNFR1 receptor. Contractile function was assessed in isolated hearts perfused in the isovolumic Langendorff mode during and following global no-flow ischemic stress and in response to varying extracellular [Ca(2+)] to determine their contractile response and Ca(2+) sensitivity. All hearts with low over-expression of TNFalpha, independent of human or murine origin, have improved contractile performance and increased Ca(2+) sensitivity (by 0.2-0.26 pCa). Hearts lacking TNFR1 have contractile performance equal to wild type hearts. Recovery from ischemia was greater in TIA-1(-/-) and was diminished in TNFR1(-/-). Better contractile function in TNFalpha over-expressing hearts is not due to improved cardiac energetics assessed as [ATP] and glucose uptake or to differences in expression of SERCA2a or calmodulin. We suggest that low levels of TNFalpha increase the Ca(2+) sensitivity of the heart via a TNFR1-mediated mechanism.

Calcineurin-induced energy wasting in a transgenic mouse model of heart failure.

Overexpression of calcineurin (CLN) in the mouse heart induces severe hypertrophy that progresses to heart failure, providing an opportunity to define the relationship between energetics and contractile performance in the severely failing mouse heart. Contractile performance was studied in isolated hearts at different pacing frequencies and during dobutamine challenge. Energetics were assessed by 31P-NMR spectroscopy as ATP and phosphocreatine concentrations ([ATP] and [PCr]) and free energy of ATP hydrolysis (|Delta G( approximately ATP)|). Mitochondrial and glycolytic enzyme activities, myocardial O2 consumption, and myocyte ultrastructure were determined. In transgenic (TG) hearts at all levels of work, indexes of systolic performance were reduced and [ATP] and capacity for ATP synthesis were lower than in non-TG hearts. This is the first report showing that myocardial [ATP] is lower in a TG mouse model of heart failure. [PCr] was also lower, despite an unexpected increase in the total creatine pool. Because Pi concentration remained low, despite lower [ATP] and [PCr], |Delta G( approximately ATP)| was normal; however, chemical energy did not translate to systolic performance. This was most apparent with beta-adrenergic stimulation of TG hearts, during which, for similar changes in |Delta G( approximately ATP)|, systolic pressure decreased, rather than increased. Structural abnormalities observed for sarcomeres and mitochondria likely contribute to decreased contractile performance. On the basis of the increases in enzyme activities of proteins important for ATP supply observed after treatment with the CLN inhibitor cyclosporin A, we also conclude that CLN directed inhibition of ATP-producing pathways in non-TG and TG hearts.