Increasing mitochondrial ATP synthesis with butyrate normalizes ADP and contractile function in metabolic heart disease.

Metabolic heart disease (MHD), which is strongly associated with heart failure with preserved ejection fraction, is characterized by reduced mitochondrial energy production and contractile performance. In this study, we tested the hypothesis that an acute increase in ATP synthesis, via short chain fatty acid (butyrate) perfusion, restores contractile function in MHD. Isolated hearts of mice with MHD due to consumption of a high fat high sucrose (HFHS) diet or on a control diet (CD) for 4 months were studied using 31 P NMR spectroscopy to measure high energy phosphates and ATP synthesis rates during increased work demand. At baseline, HFHS hearts had increased ADP and decreased free energy of ATP hydrolysis (ΔG~ATP ), although contractile function was similar between the two groups. At high work demand, the ATP synthesis rate in HFHS hearts was reduced by over 50%. Unlike CD hearts, HFHS hearts did not increase contractile function at high work demand, indicating a lack of contractile reserve. However, acutely supplementing HFHS hearts with 4mM butyrate normalized ATP synthesis, ADP, ΔG~ATP and contractile reserve. Thus, acute reversal of depressed mitochondrial ATP production improves contractile dysfunction in MHD. These findings suggest that energy starvation may be a reversible cause of myocardial dysfunction in MHD, and opens new therapeutic opportunities.

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.

Spectral improvement by fourier thresholding of in vivo dynamic spectroscopy data.

PURPOSE: MR spectroscopy (MRS) typically requires averaging of multiple acquisitions to achieve adequate signal-to-noise ratio (SNR). In systems undergoing dynamic changes this can compromise the temporal resolution of the measurement. One such example is (31) P MRS of exercising skeletal muscle. Spectral improvement by Fourier thresholding (SIFT) offers a way of suppressing noise without averaging. In this study, we evaluate the performance of SIFT in healthy subjects and clinical cases. METHODS: (31) P MRS of the calf or thigh muscle of subjects (n = 12) was measured continuously before, during, and after exercise. The data were processed conventionally and with the addition of SIFT before quantifying peak amplitudes and frequencies. The postexercise increase in the amplitude of phosphocreatine was also characterized by fitting with an exponential function to obtain the recovery time constant. RESULTS: Substantial reductions in the uncertainty of peak fitting for phosphocreatine (73%) and inorganic phosphate (60%) were observed when using SIFT relative to conventional processing alone. SIFT also reduced the phosphocreatine recovery time constant uncertainty by 38%. CONCLUSION: SIFT considerably improves SNR, which improved quantification and parameter estimation. It is suitable for any type of time varying MRS and is both straightforward and fast to apply. Magn Reson Med 76:978-985, 2016. © 2015 Wiley Periodicals, Inc.

Cardiac-specific VLCAD deficiency induces dilated cardiomyopathy and cold intolerance.

The very long-chain acyl-CoA dehydrogenase (VLCAD) enzyme catalyzes the first step of mitochondrial ß-oxidation. Patients with VLCAD deficiency present with hypoketotic hypoglycemia and cardiomyopathy, which can be exacerbated by fasting and/or cold stress. Global VLCAD knockout mice recapitulate these phenotypes: mice develop cardiomyopathy, and cold exposure leads to rapid hypothermia and death. However, the contribution of different tissues to development of these phenotypes has not been studied. We generated cardiac-specific VLCAD-deficient (cVLCAD(-/-)) mice by Cre-mediated ablation of the VLCAD in cardiomyocytes. By 6 mo of age, cVLCAD(-/-) mice demonstrated increased end-diastolic and end-systolic left ventricular dimensions and decreased fractional shortening. Surprisingly, selective VLCAD gene ablation in cardiomyocytes was sufficient to evoke severe cold intolerance in mice who rapidly developed severe hypothermia, bradycardia, and markedly depressed cardiac function in response to fasting and cold exposure (+5°C). We conclude that cardiac-specific VLCAD deficiency is sufficient to induce cold intolerance and cardiomyopathy and is associated with reduced ATP production. These results provide strong evidence that fatty acid oxidation in myocardium is essential for maintaining normal cardiac function under these stress conditions.

SGLT2 inhibitor ertugliflozin decreases elevated intracellular sodium, and improves energetics and contractile function in diabetic cardiomyopathy.

BACKGROUND: Elevated myocardial intracellular sodium ([Na+]i) was shown to decrease mitochondrial calcium ([Ca2+]MITO) via mitochondrial sodium/calcium exchanger (NCXMITO), resulting in decreased mitochondrial ATP synthesis. The sodium-glucose co-transporter 2 inhibitor (SGLT2i) ertugliflozin (ERTU) improved energetic deficit and contractile dysfunction in a mouse model of high fat, high sucrose (HFHS) diet-induced diabetic cardiomyopathy (DCMP). As SGLT2is were shown to lower [Na+]i in isolated cardiomyocytes, we hypothesized that energetic improvement in DCMP is at least partially mediated by a decrease in abnormally elevated myocardial [Na+]i. METHODS: Forty-two eight-week-old male C57BL/6J mice were fed a control or HFHS diet for six months. In the last month, a subgroup of HFHS-fed mice was treated with ERTU. At the end of the study, left ventricular contractile function and energetics were measured simultaneously in isolated beating hearts by 31P NMR (Nuclear Magnetic Resonance) spectroscopy. A subset of untreated HFHS hearts was perfused with vehicle vs. CGP 37157, an NCXMITO inhibitor. Myocardial [Na+]i was measured by 23Na NMR spectroscopy. RESULTS: HFHS hearts showed diastolic dysfunction, decreased contractile reserve, and impaired energetics as reflected by decreased phosphocreatine (PCr) and PCr/ATP ratio. Myocardial [Na+]i was elevated > 2-fold in HFHS (vs. control diet). ERTU reversed the impairments in HFHS hearts to levels similar to or better than control diet and decreased myocardial [Na+]i to control levels. CGP 37157 normalized the PCr/ATP ratio in HFHS hearts. CONCLUSIONS: Elevated myocardial [Na+]i contributes to mitochondrial and contractile dysfunction in DCMP. Targeting myocardial [Na+]i and/or NCXMITO may be an effective strategy in DCMP and other forms of heart disease associated with elevated myocardial [Na+]i.

Role of peroxisome proliferator-activated receptor {delta}/{beta} in hepatic metabolic regulation.

Pharmacological activation of peroxisome proliferator-activated receptor δ/ß (PPARδ/ß) improves glucose handling and insulin sensitivity. The target tissues of drug actions remain unclear. We demonstrate here that adenovirus-mediated liver-restricted PPARδ activation reduces fasting glucose levels in chow- and high fat-fed mice. This effect is accompanied by hepatic glycogen and lipid deposition as well as up-regulation of glucose utilization and de novo lipogenesis pathways. Promoter analyses indicate that PPARδ regulates hepatic metabolic programs through both direct and indirect transcriptional mechanisms partly mediated by its co-activator, PPARγ co-activator-1ß. Assessment of the lipid composition reveals that PPARδ increases the production of monounsaturated fatty acids, which are PPAR activators, and reduces that of saturated FAs. Despite the increased lipid accumulation, adeno-PPARδ-infected livers exhibit less damage and show a reduction in JNK stress signaling, suggesting that PPARδ-regulated lipogenic program may protect against lipotoxicity. The altered substrate utilization by PPARδ also results in a secondary effect on AMP-activated protein kinase activation, which likely contributes to the glucose-lowering activity. Collectively, our data suggest that PPARδ controls hepatic energy substrate homeostasis by coordinated regulation of glucose and fatty acid metabolism, which provide a molecular basis for developing PPARδ agonists to manage hyperglycemia and insulin resistance.

Novel Small-Molecule Troponin Activator Increases Cardiac Contractile Function Without Negative Impact on Energetics.

BACKGROUND: Current heart failure therapies unload the failing heart without targeting the underlying problem of reduced cardiac contractility. Traditional inotropes (ie, calcitropes) stimulate contractility via energetically costly augmentation of calcium cycling and worsen patient survival. A new class of agents-myotropes-activates the sarcomere directly, independent of calcium. We hypothesize that a novel myotrope TA1 increases contractility without the deleterious myocardial energetic impact of a calcitrope dobutamine. METHODS: We determined the effect of TA1 in bovine cardiac myofibrils and human cardiac microtissues, ex vivo in mouse cardiac fibers and in vivo in anesthetized normal rats. Effects of increasing concentrations of TA1 or dobutamine on contractile function, phosphocreatine and ATP concentrations, and ATP production were assessed by 31P nuclear magnetic resonance spectroscopy on isolated perfused rat hearts. RESULTS: TA1 increased the rate of myosin ATPase activity in isolated bovine myofibrils and calcium sensitivity in intact mouse papillary fibers. Contractility increased dose dependently in human cardiac microtissues and in vivo in rats as assessed by echocardiography. In isolated rat hearts, TA1 and dobutamine similarly increased the rate-pressure product. Dobutamine increased both developed pressure and heart rate accompanied by decreased phosphocreatine-to-ATP ratio and decreased free energy of ATP hydrolysis (ΔG~ATP) and elevated left ventricular end diastolic pressure. In contrast, the TA1 increased developed pressure without any effect on heart rate, left ventricular end diastolic pressure, phosphocreatine/ATP ratio, or ΔG~ATP. CONCLUSIONS: Novel myotrope TA1 increased myocardial contractility by sensitizing the sarcomere to calcium without impairing diastolic function or depleting the cardiac energy reserve. Since energetic depletion negatively correlates with long-term survival, myotropes may represent a superior alternative to traditional inotropes in heart failure management.

Active trans-plasma membrane water cycling in yeast is revealed by NMR.

Plasma membrane water transport is a crucial cellular phenomenon. Net water movement in response to an osmotic gradient changes cell volume. Steady-state exchange of water molecules, with no net flux or volume change, occurs by passive diffusion through the phospholipid bilayer and passage through membrane proteins. The hypothesis is tested that plasma membrane water exchange also correlates with ATP-driven membrane transport activity in yeast (Saccharomyces cerevisiae). Longitudinal (1)H(2)O NMR relaxation time constant (T(1)) values were measured in yeast suspensions containing extracellular relaxation reagent. Two-site-exchange analysis quantified the reversible exchange kinetics as the mean intracellular water lifetime (τ(i)), where τ(i)(-1) is the pseudo-first-order rate constant for water efflux. To modulate cellular ATP, yeast suspensions were bubbled with 95%O(2)/5%CO(2) (O(2)) or 95%N(2)/5%CO(2) (N(2)). ATP was high during O(2), and τ(i)(-1) was 3.1 s(-1) at 25°C. After changing to N(2), ATP decreased and τ(i)(-1) was 1.8 s(-1). The principal active yeast ion transport protein is the plasma membrane H(+)-ATPase. Studies using the H(+)-ATPase inhibitor ebselen or a yeast genetic strain with reduced H(+)-ATPase found reduced τ(i)(-1), notwithstanding high ATP. Steady-state water exchange correlates with H(+)-ATPase activity. At volume steady state, water is cycling across the plasma membrane in response to metabolic transport activity.

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).

Aberrant activation of AMP-activated protein kinase remodels metabolic network in favor of cardiac glycogen storage.

AMP-activated protein kinase (AMPK) responds to impaired cellular energy status by stimulating substrate metabolism for ATP generation. Mutation of the gamma2 regulatory subunit of AMPK in humans renders the kinase insensitive to energy status and causes glycogen storage cardiomyopathy via unknown mechanisms. Using transgenic mice expressing one of the mutant gamma2 subunits (N488I) in the heart, we found that aberrant high activity of AMPK in the absence of energy deficit caused extensive remodeling of the substrate metabolism pathways to accommodate increases in both glucose uptake and fatty acid oxidation in the hearts of gamma2 mutant mice via distinct, yet synergistic mechanisms resulting in selective fuel storage as glycogen. Increased glucose entry in the gamma2 mutant mouse hearts was directed through the remodeled metabolic network toward glycogen synthesis and, at a substantially higher glycogen level, recycled through the glycogen pool to enter glycolysis. Thus, the metabolic consequences of chronic activation of AMPK in the absence of energy deficiency is distinct from those previously reported during stress conditions. These findings are of particular importance in considering AMPK as a target for the treatment of metabolic diseases.

Ets-1 is a critical transcriptional regulator of reactive oxygen species and p47(phox) gene expression in response to angiotensin II.

Angiotensin (Ang) II is a potent mediator of vascular inflammation. A central mechanism by which Ang II promotes inflammation is through the generation of reactive oxygen species (ROS). In the current study, we investigated the role of the transcription factor Ets-1 in regulating Ang II-induced ROS generation. ROS generation was measured in the thoracic aorta of Ets-1(-/-) mice compared with littermate controls after continuous infusion of Ang II. H2O2 and superoxide anion (O2(-)) production were significantly blunted in the Ets-1(-/-) mice. Inhibition of Ets-1 expression by small interfering RNA in primary human aortic smooth muscle cells also potently inhibited ROS production and the induction of the NAD(P)H oxidase subunit p47(phox) in response to Ang II. To evaluate the therapeutic potential of inhibiting Ets-1 in wild-type mice, dominant negative Ets-1 membrane-permeable peptides were administered systemically. Ang II-induced ROS production and medial hypertrophy in the thoracic aorta were markedly diminished as a result of blocking Ets-1. In summary, Ets-1 functions as a critical downstream transcriptional mediator of Ang II ROS generation by regulating the expression of NAD(P)H oxidase subunits such as p47(phox).

Energetic Dysfunction Is Mediated by Mitochondrial Reactive Oxygen Species and Precedes Structural Remodeling in Metabolic Heart Disease.

Aims: Metabolic syndrome is associated with metabolic heart disease (MHD) that is characterized by left ventricular (LV) hypertrophy, interstitial fibrosis, contractile dysfunction, and mitochondrial dysfunction. Overexpression of catalase in mitochondria (transgenic expression of catalase targeted to the mitochondria [mCAT]) prevents the structural and functional features of MHD caused by a high-fat, high-sucrose (HFHS) diet for ≥4 months. However, it is unclear whether the effect of mCAT is due to prevention of reactive oxygen species (ROS)-mediated cardiac remodeling, a direct effect on mitochondrial function, or both. To address this question, we measured myocardial function and energetics in mice, with or without mCAT, after 1 month of HFHS, before the development of cardiac structural remodeling. Results: HFHS diet for 1 month had no effect on body weight, heart weight, LV structure, myocyte size, or interstitial fibrosis. Isolated cardiac mitochondria from HFHS-fed mice produced 2.2- to 3.8-fold more H2O2, and 16%-29% less adenosine triphosphate (ATP). In isolated beating hearts from HFHS-fed mice, [phosphocreatine (PCr)] and the free energy available for ATP hydrolysis (ΔG∼ATP) were decreased, and they failed to increase with work demands. Overexpression of mCAT normalized ROS and ATP production in isolated mitochondria, and it corrected myocardial [PCr] and ΔG∼ATP in the beating heart. Innovation: This is the first demonstration that in MHD, mitochondrial ROS mediate energetic dysfunction that is sufficient to impair contractile function. Conclusion: ROS produced and acting in the mitochondria impair myocardial energetics, leading to slowed relaxation and decreased contractile reserve. These effects precede structural remodeling and are corrected by mCAT, indicating that ROS-mediated energetic impairment, per se, is sufficient to cause contractile dysfunction in MHD.

Adrenaline is a critical mediator of acute exercise-induced AMP-activated protein kinase activation in adipocytes.

Exercise increases AMPK (AMP-activated protein kinase) activity in human and rat adipocytes, but the underlying molecular mechanisms and functional consequences of this activation are not known. Since adrenaline (epinephrine) concentrations increase with exercise, in the present study we hypothesized that adrenaline activates AMPK in adipocytes. We show that a single bout of exercise increases AMPKalpha1 and alpha2 activities and ACC (acetyl-CoA carboxylase) Ser79 phosphorylation in rat adipocytes. Similarly to exercise, adrenaline treatment in vivo increased AMPK activities and ACC phosphorylation. Pre-treatment of rats with the beta-blocker propranolol fully blocked exercise-induced AMPK activation. Increased AMPK activity with exercise and adrenaline treatment in vivo was accompanied by an increased AMP/ATP ratio. Adrenaline incubation of isolated adipocytes also increased the AMP/ATP ratio and AMPK activities, an effect blocked by propranolol. Adrenaline incubation increased lipolysis in isolated adipocytes, and Compound C, an AMPK inhibitor, attenuated this effect. Finally, a potential role for AMPK in the decreased adiposity associated with chronic exercise was suggested by marked increases in AMPKalpha1 and alpha2 activities in adipocytes from rats trained for 6 weeks. In conclusion, both acute and chronic exercise are significant regulators of AMPK activity in rat adipocytes. Our findings suggest that adrenaline plays a critical role in exercise-stimulated AMPKalpha1 and alpha2 activities in adipocytes, and that AMPK can function in the regulation of lipolysis.

Tai Chi Improves Brain Metabolism and Muscle Energetics in Older Adults.

BACKGROUND AND PURPOSE: Tai Chi is a mind-body exercise that has been shown to improve both mental and physical health. As a result, recent literature suggests the use of Tai Chi to treat both physical and psychological disorders. However, the underlying physiological changes have not been characterized. The aim of this pilot study is to assess the changes in brain metabolites and muscle energetics after Tai Chi training in an aging population using a combined brain-muscle magnetic resonance spectroscopy (MRS) examination. METHODS: Six healthy older adults were prospectively recruited and enrolled into a 12-week Tai Chi program. A brain 1 H MRS and a muscle 31 P MRS were scanned before and after the training, and postprocessed to measure N-acetylaspartate to creatine (NAA/Cr) ratios and phosphocreatine (PCr) recovery time. Wilcoxon-signed rank tests were utilized to assess the differences between pre- and post-Tai Chi training. RESULTS: A significant within-subject increase in both the NAA/Cr ratios (P = .046) and the PCr recovery time (P = .046) was observed between the baseline and the posttraining scans. The median percentage changes were 5.38% and 16.51% for NAA/Cr and PCr recovery time, respectively. CONCLUSIONS: Our pilot study demonstrates significant increase of NAA/Cr ratios in posterior cingulate gyrus and significantly improved PCr recovery time in leg muscles in older adults following short-term Tai Chi training, and thus provides insight into the beneficial mechanisms.

Decreased contractile and metabolic reserve in peroxisome proliferator-activated receptor-alpha-null hearts can be rescued by increasing glucose transport and utilization.

BACKGROUND: Downregulation of peroxisome proliferator-activated receptor-alpha (PPARalpha) in hypertrophied and failing hearts leads to the reappearance of the fetal metabolic pattern, ie, decreased fatty acid oxidation and increased reliance on carbohydrates. Here, we sought to elucidate the functional significance of this shift in substrate preference. METHODS AND RESULTS: We assessed contractile function and substrate utilization using 13C nuclear magnetic resonance spectroscopy and high-energy phosphate metabolism using 31P nuclear magnetic resonance spectroscopy in perfused hearts isolated from genetically modified mice (PPARalpha(-/-)) that mimic the metabolic profile in myocardial hypertrophy. We found that the substrate switch from fatty acid to glucose (3-fold down) and lactate (3-fold up) in PPARalpha(-/-) hearts was sufficient for sustaining normal energy metabolism and contractile function at baseline but depleted the metabolic reserve for supporting high workload. Decreased ATP synthesis (measured by 31P magnetization transfer) during high workload challenge resulted in progressive depletion of high-energy phosphate content and failure to sustain high contractile performance. Interestingly, the metabolic and functional defects in PPARalpha(-/-) hearts could be corrected by overexpressing the insulin-independent glucose transporter GLUT1, which increased the capacity for glucose utilization beyond the intrinsic response to PPARalpha deficiency. CONCLUSIONS: These findings demonstrate that metabolic remodeling in hearts deficient in PPARalpha increases the susceptibility to functional deterioration during hemodynamic overload. Moreover, our results suggest that normalization of myocardial energetics by further enhancing myocardial glucose utilization is an effective strategy for preventing the progression of cardiac dysfunction in hearts with impaired PPARalpha activity such as hearts with pathological hypertrophy.

On-resonance low B1 pulses for imaging of the effects of PARACEST agents.

Application of the exchange-sensitive, low-power RF pulses positioned on the bulk water resonance for imaging of the effects of PARACEST agents is proposed as an alternative to the standard CW off-resonance irradiation. Specifically, we applied a low-power WALTZ-16 RF train, with the 90 degrees pulse unit replaced by a pulse of the fixed length (WALTZ-16*). Using this sequence, the bulk water signal was found to be sensitive to exchange lifetimes with PARACEST complex bound protons, the transverse relaxation time of bulk water, and longitudinal relaxation time of bound protons. In this report, the concept of using WALTZ-16* to "activate" a PARACEST effect is introduced and some of the salient features of this technique with respect to experimental conditions and performance levels are discussed. Computational predictions are verified and explored by comparison with experimental spectroscopic and imaging data. It is shown that WALTZ-16* can be used to detect PARACEST agents with an RF intensity as low as 200 Hz for concentrations as low as a few tens of microM for lanthanide chelates having appropriate water-exchange rates (Tm,Dy).

Rosiglitazone causes cardiotoxicity via peroxisome proliferator-activated receptor γ-independent mitochondrial oxidative stress in mouse hearts.

This study aims to test the hypothesis that thiazolidinedione rosiglitazone (RSG), a selective peroxisome proliferator-activated receptor γ (PPARγ) agonist, causes cardiotoxicity independently of PPARγ. Energy metabolism and mitochondrial function were measured in perfused hearts isolated from C57BL/6, cardiomyocyte-specific PPARγ-deficient mice, and their littermates. Cardiac function and mitochondrial oxidative stress were measured in both in vitro and in vivo settings. Treatment of isolated hearts with RSG at the supratherapeutic concentrations of 10 and 30 µM caused myocardial energy deficiency as evidenced by the decreases in [PCr], [ATP], ATP/ADP ratio, energy charge with a concomitant cardiac dysfunction as indicated by the decreases in left ventricular systolic pressure, rates of tension development and relaxation, and by an increase in end-diastolic pressure. When incubated with tissue homogenate or isolated mitochondria at these same concentrations, RSG caused mitochondrial dysfunction as evidenced by the decreases in respiration rate, substrate oxidation rates, and activities of complexes I and IV. RSG also increased complexes I- and III-dependent O2⁻ production, decreased glutathione content, inhibited superoxide dismutase, and increased the levels of malondialdehyde, protein carbonyl, and 8-hydroxy-2-deoxyguanosine in mitochondria, consistent with oxidative stress. N-acetyl-L-cysteine (NAC) 20 mM prevented RSG-induced above toxicity at those in vitro settings. Cardiomyocyte-specific PPARγ deletion and PPARγ antagonist GW9662 did not prevent the observed cardiotoxicity. Intravenous injection of 10 mg/kg RSG also caused cardiac dysfunction and oxidative stress, 600 mg/kg NAC antagonized these adverse effects. In conclusion, this study demonstrates that RSG at supratherapeutic concentrations causes cardiotoxicity via a PPARγ-independent mechanism involving oxidative stress-induced mitochondrial dysfunction in mouse hearts.

pCEST: Positive contrast using Chemical Exchange Saturation Transfer.

Chemical Exchange Saturation Transfer (CEST) contrast utilizes selective pre-saturation of a small pool of exchanging protons and subsequent detection of the decrease in bulk water signal. The CEST contrast is negative and requires detection of small signal change in the presence of a strong background signal. Here we develop a Positive CEST (pCEST) detection scheme utilizing the analogous nature of the CEST and off-resonance T(1)(ρ) experiments and exploring increased apparent relaxation rates in the presence of the selective pre-saturation. pCEST leads to the positive contrast, i.e., increased signal intensity as the result of the presence of the agent and RF pre-saturation. Simultaneously substantial background suppression is achieved. The contrast can be switched "ON" and "OFF", similar to the original CEST.

Proteomic analyses of transgenic LQT1 and LQT2 rabbit hearts elucidate an increase in expression and activity of energy producing enzymes.

Various biochemical and genomic mechanisms are considered to be a hallmark of metabolic remodeling in the stressed heart, including the hypertrophied and failing heart. In this study, we used quantitative proteomic 2-D Fluorescence Difference In-Gel Electrophoresis (2-D DIGE) in conjunction with mass spectrometry to demonstrate differential protein expression in the hearts of transgenic rabbit models of Long QT Syndrome 1 (LQT1) and Long QT Syndrome 2 (LQT2) as compared to littermate controls (LMC). The results of our proteomic analysis revealed upregulation of key metabolic enzymes involved in all pathways associated with ATP generation, including creatine kinase in both LQT1 and LQT2 rabbit hearts. Additionally, the expression of lamin-A protein was increased in both LQT1 and LQT2 rabbit hearts as was the expression of mitochondrial aldehyde dehydrogenase and desmoplakin in LQT1 and LQT 2 rabbit hearts, respectively. Results of the proteomic analysis also demonstrated down regulation in the expression of protein disulfide-isomerase A3 precuorsor and dynamin-like 120 kDa protein (mitochondrial) in LQT1, and of alpha-actinin 2 in LQT2 rabbit hearts. Up regulation of the expression of the enzymes associated with ATP generation was substantiated by the results of selective enzyme assays in LQT1 and LQT2 hearts, as compared to LMC, which revealed increases in the activities of glycogen phosphorylase (+50%, +65%, respectively), lactate dehydrogenase (+25%, +25%) pyruvate dehydrogenase (+31%, +22%), and succinate dehydrogenase (+32%, +60%). The activity of cytochrome c-oxidase, a marker for the mitochondrial function was also found to be significantly elevated (+80%) in LQT1 rabbit hearts as compared with LMC. Western blot analysis in LQT1 and LQT2 hearts compared to LMC revealed an increase in the expression of very-long chain-specific acyl-CoA dehydrogenase (+35%, +33%), a rate-limiting enzymes in ß-oxidation of fatty acids. Collectively, our results demonstrate similar increases in the expression and activities of key ATP-generating enzymes in LQT1 and LQT2 rabbit hearts, suggesting an increased demand, and in turn, increased energy supply across the entire metabolic pathway by virtue of the upregulation of enzymes involved in energy generation.