Biochemical factors limiting myocardial energy in a chicken genotype selected for rapid growth

Broiler chickens (Gallus gallus) genetically selected for rapid growth are inherently predisposed to heart failure. In order to understand the biochemical mechanisms associated with the deterioration of heart function and development of congestive heart failure (CHF) in fast-growing chickens, this study examined several factors critical for myocardial energy metabolism. Measured variables included cardiac energy substrates [creatine phosphate (CrP), adenosine triphosphate (ATP), l-carnitine], activity of selected cytosolic enzymes [creatine kinase (CK; EC 2.7.3.2), lactate dehydrogenase (LDH; EC 1.1.1.27)] and mitochondrial enzymes [pyruvate dehydrogenase (PDH; EC 1.2.4.1), alpha-ketoglutarate dehydrogenase (alpha-KGDH; EC 1.2.4.2)]. The CK activities were higher in fast-growing and CHF broilers as compared to slow-growing broilers (p<0.05). Cardiac LDH and alpha-KGDH activities were not changed (p>0.05), whereas PDH activity was highest (p<0.05) in broilers with CHF. Deterioration of heart function is correlated with lowered cardiac ATP, CrP, and l-carnitine levels (all p<0.05). Depletion of high energy phosphate substrates, ATP and CrP, is evident in fast-growing chickens and those that developed CHF. Increased activity of CK suggests that cardiac energy management in fast-growing broilers and those with CHF largely depends on contribution of this pathway to regeneration of ATP from CrP. In this scenario, inadequate level of CrP is a direct cause of ATP insufficiency, whereas low cardiac l-carnitine, because of its role in fatty acid transport, is most likely an important factor contributing to shortage of key substrate required for synthesis of cardiac ATP. The insufficiencies in cardiac energy substrate synthesis provide metabolic basis of myocardial dysfunction in chickens predisposed to heart failure.     

Comparative study of myocardial high energy phosphate substrate content in slow and fast growing chicken and in chickens with heart failure and ascites

In order to explain the biochemical mechanisms associated with deteriorating heart function in broiler chickens, this study compared myocardial high energy phosphate substrates in leghorns, feed restricted (Broilers-Res) broilers, ad libitum fed broilers (Broilers-AL), and in broilers that developed heart failure and ascites. The profile of adenine nucleotide content in the heart tissue did not differ between leghorns and Broilers-Res, but there were significant differences among Broilers-Res, Broilers-AL, and broilers with ascites. During intensive growth periods, leghorns and Broilers-Res showed increasing trends in heart ATP levels, whereas in fast growing broilers the heart ATP declined (p<0.021). ATP:ADP and ATP:CrP ratios increased with age in both leghorn and Broilers-Res, declined in fast growing broilers, and were the lowest in broilers that developed heart failure. The changes in heart high energy phosphate profile in broilers suggest that the energy demand of the heart during a rapid growth phase may exceed the bird's metabolic capacity to supply adequate levels of high energy phosphate substrate. The insufficiency of energy substrate likely contributes to the declining heart rate. In some individuals this may lead to impaired heart pump function, and in more severe cases may progress to heart pump failure.     

The aetiology of hypoxaemia in chickens selected for rapid growth

In comparison to other classes of chickens, broilers selected for rapid growth tend to be hypoxaemic, and many develop congestive heart failure (CHF). In order to explain the physiological mechanisms associated with hypoxaemia in fast-growing broiler chickens (Gallus gallus), this study examined several basic physiological parameters including the blood gas profile in arterial [left atrial (LA)] and mixed venous [right atrial (RA)] blood, systemic oxygen extraction ratio, and intrapulmonary shunt fraction. These parameters were further studied in the context of blood flow in the pulmonary circulation, structural characteristics of the lungs, and cardiac function [measured as cardiac index (CI)]. Overall, broilers had lower arterial and mixed venous blood pO(2) levels and higher pCO(2) levels compared to leghorns. The cardiac index was lower in fast-growing and CHF broilers compared to leghorn chickens or feed-restricted broilers. Systemic oxygen extraction ratio (ER) and intrapulmonary shunt fraction were significantly higher in fast-growing broilers and birds with CHF (all P<0.01). Lungs of all broilers, but not leghorns, contained ectopic, irregular nodular formations located within air spaces. Broilers with clinical signs of hypoxaemia revealed the highest number of these formations in their lung. Taken together, the present findings indicate that key factors associated with the development of hypoxaemia in fast-growing broilers include: (1) high demand for oxygen as evidenced by high oxygen ER; (2) inadequate cardiac output (CO) to fulfill the higher oxygen demands, leading to severe depletion of O(2) in mixed venous blood; and (3) elevated intrapulmonary shunt fraction and possibly dead space associated with specific pathological and anatomical characteristics within the lung.     

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.     

Energy status of the rapidly paced canine myocardium in congestive heart failure.

Rapid ventricular pacing (RVP) is used as an experimental model of congestive heart failure (CHF). The purpose of this study was to determine the energy status of the dog myocardium after the development of CHF via chronic RVP. The myocardium had a significantly lower (P < 0.05) energy charge (EC) during CHF (0.63 +/- 0.01) than in sham-operated controls (0.82 +/- 0.02). This was due to significant differences in concentrations in ATP (-48%), ADP (29%), and AMP (275%) in the RVP group. However, the total adenine nucleotide pool was not different between groups. Myocardial lactate concentration was also similar. Glycogen was significantly lower (P < 0.05) by 20% at peak CHF. The adenine nucleotides were similar among the different myocardial layers (endo-, mid-, and epicardium). The administration of enalapril (an inhibitor of angiotension-converting enzyme) to decrease vascular resistance had no effect on the myocardial energy status of CHF dogs. These findings suggest that the lower EC in CHF animals is not the result of subendocardial ischemia. Also, lower EC is not associated with endogenous glycogen depletion or increased lactate concentration. The energy status of the myocardium in RVP-induced CHF is unlike that seen in ischemia-induced heart failure. This suggests that CHF in RVP is not vascular in origin.     

Failing atrial myocardium: energetic deficits accompany structural remodeling and electrical instability

The failing ventricular myocardium is characterized by reduction of high-energy phosphates and reduced activity of the phosphotransfer enzymes creatine kinase (CK) and adenylate kinase (AK), which are responsible for transfer of high-energy phosphoryls from sites of production to sites of utilization, thereby compromising excitation-contraction coupling. In humans with chronic atrial fibrillation (AF) unassociated with congestive heart failure (CHF), impairment of atrial myofibrillar energetics linked to oxidative modification of myofibrillar CK has been observed. However, the bioenergetic status of the failing atrial myocardium and its potential contribution to atrial electrical instability in CHF have not been determined. Dogs with (n = 6) and without (n = 6) rapid pacing-induced CHF underwent echocardiography (conscious) and electrophysiological (under anesthesia) studies. CHF dogs had more pronounced mitral regurgitation, higher atrial pressure, larger atrial area, and increased atrial fibrosis. An enhanced propensity to sustain AF was observed in CHF, despite significant increases in atrial effective refractory period and wavelength. Profound deficits in atrial bioenergetics were present with reduced activities of the phosphotransfer enzymes CK and AK, depletion of high-energy phosphates (ATP and creatine phosphate), and reduction of cellular energetic potential (ATP-to-ADP and creatine phosphate-to-Cr ratios). AF duration correlated with left atrial area (r = 0.73, P = 0.01) and inversely with atrial ATP concentration (r = -0.75, P = 0.005), CK activity (r = -0.57, P = 0.054), and AK activity (r = -0.64, P = 0.02). Atrial levels of malondialdehyde, a marker of oxidative stress, were significantly increased in CHF. Myocardial bioenergetic deficits are a conserved feature of dysfunctional atrial and ventricular myocardium in CHF and may constitute a component of the substrate for AF in CHF.     

Thyroid hormone regulation of cardiac bioenergetics: role of intracellular creatine

The effect of thyroid hormone (T(3)) on the content of myocardial creatine (Cr), Cr phosphate (CrP), and high-energy adenine nucleotides and on cardiac function was examined. In the hearts of control and T(3)-treated rats perfused in vitro, while "low" and "high" contractile work was performed, T(3) treatment resulted in a approximately 50% reduction in CrP, Cr, total Cr content (Cr + CrP), and in the CrP-to-Cr ratio. In addition, there was a slight fall in myocardial content of ATP and a large rise in calculated free ADP (fADP), resulting in a significant decrease in the ATP-to-fADP ratio in the hearts of hyperthyroid compared with euthyroid rats. Moreover, there was a substantial decrease in the level of ATP in hearts of T(3)-treated rats under high work conditions. Importantly, the ratio of cardiac work to oxygen consumption was not altered by thyroid status. Treatment with T(3) also resulted in an almost threefold reduction in the content of Na(+)/Cr transporter mRNA in the ventricular myocardium and skeletal muscle but not in the brain. We conclude with the following: 1) changes in the expression of the Na(+)/Cr transporter mRNA correlate with Cr + CrP in the myocardium; 2) hearts of hyperthyroid rats contain lower levels of ATP and higher levels of fADP under both low and high work conditions but no reduction in efficiency of work output; and 3) the reduction in Cr and ATP in hearts of hyperthyroid rats may be the basis for the reduced maximal work capacity of the hyperthyroid heart.     

Mapping hypoxia-induced bioenergetic rearrangements and metabolic signaling by 18O-assisted 31P NMR and 1H NMR spectroscopy

Brief hypoxia or ischemia perturbs energy metabolism inducing paradoxically a stress-tolerant state, yet metabolic signals that trigger cytoprotection remain poorly understood. To evaluate bioenergetic rearrangements, control and hypoxic hearts were analyzed with 18O-assisted 31P NMR and 1H NMR spectroscopy. The 18O-induced isotope shift in the 31P NMR spectrum of CrP, betaADP and betaATP was used to quantify phosphotransfer fluxes through creatine kinase and adenylate kinase. This analysis was supplemented with determination of energetically relevant metabolites in the phosphomonoester (PME) region of 31P NMR spectra, and in both aromatic and aliphatic regions of 1H NMR spectra. In control conditions, creatine kinase was the major phosphotransfer pathway processing high-energy phosphoryls between sites of ATP consumption and ATP production. In hypoxia, creatine kinase flux was dramatically reduced with a compensatory increase in adenylate kinase flux, which supported heart energetics by regenerating and transferring beta- and gamma-phosphoryls of ATP. Activation of adenylate kinase led to a build-up of AMP, IMP and adenosine, molecules involved in cardioprotective signaling. 31P and 1H NMR spectral analysis further revealed NADH and H+ scavenging by alpha-glycerophosphate dehydrogenase (alphaGPDH) and lactate dehydrogenase contributing to maintained glycolysis under hypoxia. Hypoxia-induced accumulation of alpha-glycerophosphate and nucleoside 5'-monophosphates, through alphaGPDH and adenylate kinase reactions, respectively, was mapped within the increased PME signal in the 31P NMR spectrum. Thus, 18O-assisted 31P NMR combined with 1H NMR provide a powerful approach in capturing rearrangements in cardiac bioenergetics, and associated metabolic signaling that underlie the cardiac adaptive response to stress.     

Cytosolic adenylates and adenosine release in perfused working heart. Comparison of whole tissue with cytosolic non-aqueous fractionation analyses

Free cytosolic adenylates were examined in relation to adenosine plus inosine released from perfused working guinea-pig hearts. Whole-tissue adenylate data from freeze-clamped hearts were quantitatively compared with corresponding values obtained by subcellular fractionation of homogenized myocardium in non-aqueous media. Adenosine and inosine in venous cardiac effluents were measured by high-performance liquid chromatography. Hearts, perfused at their natural flows, were subjected to various workloads, substrates and catecholamines to alter myocardial energy metabolism and respiration over a wide physiological range. Non-aqueous cytosolic ATP and creatine phosphate (CrP) accounted for more than 80% of the respective total myocardium content. The cytosolic CrP/Pi ratio was in near-quantitative agreement with the overall tissue CrP/Pi ratio when the latter parameter was corrected for extracellular Pi. This was conclusive evidence that ATP, CrP and Pi were predominantly located in the cytosol of the well-oxygenated cardiomyocyte. Measured myocardial oxygen uptake (MVO2) was reciprocally related to the phosphorylation state of CrP [( CrP]/[Cr] X [Pi]) and hence that of ATP [( ATP]/[ADP] X [Pi]) assuming the creatine kinase at near-equilibrium at a near-constant pH of 7.2. On the other hand, calculated mean free cytosolic ADP concentrations increased essentially linearly up to threefold with increasing MVO2 in the presence of virtually unchanged or only slightly decreased ATP levels; this was found both according to the whole tissue and the special subcellular fractionation data. Employing the myokinase mass-action ratio and substituting total cardiac ADP by the mean free cytosolic ADP concentrations, the mean free cytosolic AMP concentrations proved to be in the nanomolar range, i.e. up to three orders of magnitude lower than the overall tissue AMP content. We propose, therefore, that in the normoxic heart, AMP is located predominantly in the mitochondrial compartment. Nevertheless, both free cytosolic AMP concentration and release of adenosine plus inosine were apparently square or even higher-power functions of the rate of cardiac respiration. On the other hand, the mean purine nucleoside release seemed linearly correlated (r = 0.920) with the calculated free cytosolic AMP concentration. Our observations seem to suggest that the concentrations of free ADP and AMP in the cytosol are major determinants of the production of inosine and coronary vasodilator adenosine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Restricted feeding improves postischemic recovery of Langendorff-perfused rat hearts

The goal of the present study was to assess the effects of a restricted feeding schedule (RFS) on postischemic contractile recovery in relation to triacylglycerol (TAG), glycogen, and ATP content. Glucose-6-phosphate dehydrogenase (G6PDH) activity, reduced/oxidized glutathione ratio (GSH/GSSG), and thiobarbituric acid reactive substances (TBARS) levels were also determined. Isolated rat hearts entrained to daily RFS (2 h food access starting at 1200) or fed ad libitum (FED) for 3 weeks were Langendorff-perfused (25 min ischemia, 30 min reperfusion) with Krebs-Ringer bicarbonate solution (10?mmol/L glucose). RFS improved the recovery of contractility and reduced creatine kinase (CK) release upon reperfusion. Further, at the end of reperfusion, RFS hearts exhibited increased G6PDH activity and repletion of tissue glycogen, TAG, and ATP that was not observed in the FED hearts. GSH/GSSG at the end of reperfusion fell to the same value in both nutritional states, and TBARS levels were higher in the RFS hearts. In conclusion, RFS improved postischemic functional recovery, which was accompanied by a reduction in CK release and a striking energy recovery. Although enhanced G6PDH activity was displayed, RFS was unable to reduce lipid peroxidation, supporting a clear dissociation between protection against mechanical dysfunction and CK release on the one hand and oxidative damage on the other.     

Analyzing the functional properties of the creatine kinase system with multiscale 'sloppy' modeling

In this study the function of the two isoforms of creatine kinase (CK; EC 2.7.3.2) in myocardium is investigated. The 'phosphocreatine shuttle' hypothesis states that mitochondrial and cytosolic CK plays a pivotal role in the transport of high-energy phosphate (HEP) groups from mitochondria to myofibrils in contracting muscle. Temporal buffering of changes in ATP and ADP is another potential role of CK. With a mathematical model, we analyzed energy transport and damping of high peaks of ATP hydrolysis during the cardiac cycle. The analysis was based on multiscale data measured at the level of isolated enzymes, isolated mitochondria and on dynamic response times of oxidative phosphorylation measured at the whole heart level. Using 'sloppy modeling' ensemble simulations, we derived confidence intervals for predictions of the contributions by phosphocreatine (PCr) and ATP to the transfer of HEP from mitochondria to sites of ATP hydrolysis. Our calculations indicate that only 15±8% (mean±SD) of transcytosolic energy transport is carried by PCr, contradicting the PCr shuttle hypothesis. We also predicted temporal buffering capabilities of the CK isoforms protecting against high peaks of ATP hydrolysis (3750 μM*s(-1)) in myofibrils. CK inhibition by 98% in silico leads to an increase in amplitude of mitochondrial ATP synthesis pulsation from 215±23 to 566±31 μM*s(-1), while amplitudes of oscillations in cytosolic ADP concentration double from 77±11 to 146±1 μM. Our findings indicate that CK acts as a large bandwidth high-capacity temporal energy buffer maintaining cellular ATP homeostasis and reducing oscillations in mitochondrial metabolism. However, the contribution of CK to the transport of high-energy phosphate groups appears limited. Mitochondrial CK activity lowers cytosolic inorganic phosphate levels while cytosolic CK has the opposite effect.     

心元胶囊对慢性心力衰竭心血瘀阻证患者心脏超声参数、脂质代谢及血液流变学的影响

摘要 目的：探讨心元胶囊对慢性心力衰竭（CHF）心血瘀阻证患者心脏超声参数、脂质代谢及血液流变学的影响。方法：根据随机数字表法,将我院2020年3月~2022年3月期间收治的的CHF心血瘀阻证患者120例分为对照组（常规西药治疗,n=60）和观察组（心元胶囊联合常规西药治疗,n=60）。对比两组中医疗效、心功能指标、血脂指标、肌酸激酶（CK）、肌酸激酶同工酶（CK-MB）、N-末端脑钠肽前体（NT-proBNP）、血液流变学指标,并观察两组不良反应情况。结果：观察组的中医总有效率明显高于对照组（P<0.05）。两组治疗后左室射血分数升高,左室舒张末期容量、左室收缩末期容量缩小,且观察组的改善效果优于对照组（P<0.05）。两组治疗后三酰甘油（TG）、总胆固醇（TC）、低密度脂蛋白胆固醇（LDL-C）下降,高密度脂蛋白胆固醇（HDL-C）升高,且观察组的改善效果优于对照组（P<0.05）。两组治疗后CK、CK-MB、NT-proBNP下降,且观察组的改善效果优于对照组（P<0.05）。两组治疗后全血高切黏度、全血低切黏度、血浆黏度、纤维蛋白原下降,且观察组的改善效果优于对照组（P<0.05）。两组不良反应发生率对比,差异无统计学意义（P>0.05）。结论：心元胶囊治疗CHF心血瘀阻证可提高临床疗效,改善心功能,调节脂质代谢、血液流变学水平,安全有效。     

Effects of nitroglycerin on energy metabolism of rat reticulocytes.

Nitric oxide (NO) in many cells inactivates aconitase and mitochondrial respiratory chain, and influenced glyceraldehyde 3-phosphate dehydrogenase activity. The aim of this study was to evaluate role of nitroglycerin (NTG), a widely used NO donor, on energy metabolism of rat reticulocytes. Rat reticulocyte rich red blood cell suspensions containing 70-100% of reticulocytes, were aerobically incubated without (control) or in the presence of different concentrations of (a) NTG (0.1, 0.25, 0.5, 1.0, 1.5 mmol/l), (b) 8-Br-cGMP (0.1, 0.5, 1.0 mmol/l) and (c) NaNO2 and NaNO3 (1 mmol/l). NTG in dose- and time-dependent manner decreased total (p>0.05; EC50 = 0.78+/-0.05 mmol/l) and coupled (p<0.05; EC50 = 0.50+/-0.04 mmol/l) and increased uncoupled oxygen consumption (p<0.05: EC50 = 0.36+/-0.01 mmol/l). They were accompanied by stimulation of glycolysis, as measured by increased glucose consumption and lactate accumulation (p<0.001 EC50 = 0.53 and 0.53 mmol/l, respectively). Levels of all glycolytic intermediates in the presence of NTG indicate stimulation of HK-PFK, GA3PDH and PK activity. NTG significantly decreased ATP level, which accompanied by increased ADP and AMP levels. However, level of total adenine nucleotides (TAN) was significantly lower, which was consequence of increased catabolism of adenine nucleotides (increased hypoxanthine level; p<0.05). Stimulation of glycolysis accompanied with inhibition of the OxP, activation of HK-PFK, decrease of ATP and simultaneous rise of ADP and AMP levels, all together represent an example of Pasteur effect occurring in NTG-treated reticulocytes. In rat reticulocytes under steady state conditions 93% of overall energy was produced by OxP, but only 7% by glycolysis. Due to decrease of coupled oxygen consumption in the presence of NTG, ATP production via OxP was significantly diminished. Simultaneous increase of glycolytic ATP production is not enough to provide constant either ATP production or concentration. Calculated mean ATP-turnover time was prolonged even for 45% in the presence of 1.5 mmol/l NTG. Metabolic effects of NTG were not mimic by exogenous 8-Br-cGMP, NaNO2 or NaNO3, which indicate that NTG induced a) inhibition of coupled respiration and b) stimulation of glycolysis in rat reticulocytes are mediated by NO as an effector molecule.     

Excessive dietary vitamin D supplementation as a risk factor for sudden death syndrome in fast growing commercial broilers.

Broiler diets are frequently fortified with vitamin D (D3) above the recommended levels in an attempt to prevent commonly occurring leg problems. Since the basal levels of dietary D3 are rarely known, there is a risk of over-supplementation. Over-supplementation of D3 has been shown to have detrimental effects on the heart. Sudden death syndrome (SDS) is a condition commonly observed in broiler flocks and is associated with acute heart failure. The present study examines the effects of excessive levels of vitamin D3 on cardiac health in fast growing broiler chickens. Commercial male broilers (Gallus gallus) were exposed to either a commercial diet or a commercial diet supplemented with D3. Throughout the trial all birds were monitored several times daily for overt signs of heart disease, and periodically electrocardiographic measurements were obtained. Morbidity and mortality data were collected daily. On day 32 a simulated stress challenge consisting of a single injection of epinephrine (100 microg/kg BW) was administered under continuous ECG monitoring. Broilers fed the high D3 diet were 2.5 fold more likely to succumb to acute heart failure and die of SDS (p<0.05). Electrocardiographic examination showed a higher rate of cardiac arrhythmia in birds fed the high D3 diet (22.6%), in comparison to those fed the control diet (11.8%). The stress challenge test revealed that broilers exposed to high dietary D3 were more susceptible to ventricular arrhythmia. Our findings indicate that over-supplementation of vitamin D increases the risk of SDS in broilers, and that the most likely mechanism is associated with increased susceptibility of the ventricular myocardium to arrhythmia.     

Diisopropylamine Dichloroacetate,a Novel Pyruvate Dehydrogenase Kinase 4 Inhibitor,as a Potential Therapeutic Agent for Metabolic Disorders and Multiorgan Failure in Severe Influenza

Severe influenza is characterized by cytokine storm and multiorgan failure with metabolic energy disorders and vascular hyperpermeability. In the regulation of energy homeostasis, the pyruvate dehydrogenase (PDH) complex plays an important role by catalyzing oxidative decarboxylation of pyruvate, linking glycolysis to the tricarboxylic acid cycle and fatty acid synthesis, and thus its activity is linked to energy homeostasis. The present study tested the effects of diisopropylamine dichloroacetate (DADA), a new PDH kinase 4 (PDK4) inhibitor, in mice with severe influenza. Infection of mice with influenza A PR/8/34(H1N1) virus resulted in marked down-regulation of PDH activity and ATP level, with selective up-regulation of PDK4 in the skeletal muscles, heart, liver and lungs. Oral administration of DADA at 12-h intervals for 14 days starting immediately after infection significantly restored PDH activity and ATP level in various organs, and ameliorated disorders of glucose and lipid metabolism in the blood, together with marked improvement of survival and suppression of cytokine storm, trypsin up-regulation and viral replication. These results indicate that through PDK4 inhibition, DADA effectively suppresses the host metabolic disorder-cytokine cycle, which is closely linked to the influenza virus-cytokine-trypsin cycle, resulting in prevention of multiorgan failure in severe influenza.     

Modeling of oxygen transport and cellular energetics explains observations on in vivo cardiac energy metabolism

Observations on the relationship between cardiac work rate and the levels of energy metabolites adenosine triphosphate (ATP), adenosine diphosphate (ADP), and phosphocreatine (CrP) have not been satisfactorily explained by theoretical models of cardiac energy metabolism. Specifically, the in vivo stability of ATP, ADP, and CrP levels in response to changes in work and respiratory rate has eluded explanation. Here a previously developed model of mitochondrial oxidative phosphorylation, which was developed based on data obtained from isolated cardiac mitochondria, is integrated with a spatially distributed model of oxygen transport in the myocardium to analyze data obtained from several laboratories over the past two decades. The model includes the components of the respiratory chain, the F₀F₁-ATPase, adenine nucleotide translocase, and the mitochondrial phosphate transporter at the mitochondrial level; adenylate kinase, creatine kinase, and ATP consumption in the cytoplasm; and oxygen transport between capillaries, interstitial fluid, and cardiomyocytes. The integrated model is able to reproduce experimental observations on ATP, ADP, CrP, and inorganic phosphate levels in canine hearts over a range of workload and during coronary hypoperfusion and predicts that cytoplasmic inorganic phosphate level is a key regulator of the rate of mitochondrial respiration at workloads for which the rate of cardiac oxygen consumption is less than or equal to approximately 12 μmol per minute per gram of tissue. At work rates corresponding to oxygen consumption higher than 12 μmol min⁻¹ g⁻¹, model predictions deviate from the experimental data, indicating that at high work rates, additional regulatory mechanisms that are not currently incorporated into the model may be important. Nevertheless, the integrated model explains metabolite levels observed at low to moderate workloads and the changes in metabolite levels and tissue oxygenation observed during graded hypoperfusion. These findings suggest that the observed stability of energy metabolites emerges as a property of a properly constructed model of cardiac substrate transport and mitochondrial metabolism. In addition, the validated model provides quantitative predictions of changes in phosphate metabolites during cardiac ischemia.     

Regulation of pyruvate dehydrogenase in the common killifish, Fundulus heteroclitus, during hypoxia exposure

We examined the metabolic responses of the hypoxia-tolerant killifish (Fundulus heteroclitus) to 15 h of severe hypoxia and recovery with emphasis on muscle substrate usage and the regulation of the mitochondrial protein pyruvate dehydrogenase (PDH), which controls carbohydrate oxidation. Hypoxia survival involved a transient activation of substrate-level phosphorylation in muscle (decreases in [creatine phospate] and increases in [lactate]) during which time mechanisms to reduce overall ATP consumption were initiated. This metabolic transition did not affect total cellular [ATP], but had an impact on cellular energy status as indicated by large decreases in [ATP]/[ADP(free)] and [ATP]/[AMP(free)] and a significant loss of phosphorylation potential and Gibbs free energy of ATP hydrolysis (DeltafG'). The activity of PDH was rapidly (within 3 h) decreased by approximately 50% upon hypoxia exposure and remained depressed relative to normoxic samples throughout. Inactivation of PDH was primarily mediated via posttranslational modification following the accumulation of acetyl-CoA and subsequent activation of pyruvate dehydrogenase kinase (PDK). Estimated changes in cytoplasmic and mitochondrial [NAD(+)]/[NADH] did not parallel one another, suggesting the mitochondrial NADH shuttles do not function during hypoxia exposure. Large increases in the expression of PDK (PDK isoform 2) were consistent with decreased PDH activity; however, these changes in mRNA were not associated with changes in total PDK-2 protein content assessed using mammalian antibodies. No other changes in the expression of other known hypoxia-responsive genes (e.g., lactate dehydrogenase-A or -B) were observed in either muscle or liver.     

31P NMR detection of subcellular creatine kinase fluxes in the perfused rat heart: contractility modifies energy transfer pathways

The subcellular fluxes of exchange of ATP and phosphocreatine (PCr) between mitochondria, cytosol, and ATPases were assessed by (31)P NMR spectroscopy to investigate the pathways of energy transfer in a steady state beating heart. Using a combined analysis of four protocols of inversion magnetization transfer associated with biochemical data, three different creatine kinase (CK) activities were resolved in the rat heart perfused in isovolumic control conditions: (i) a cytosolic CK functioning at equilibrium (forward, F(f) = PCr --> ATP, and reverse flux, F(r) = ATP --> PCr = 3.3 mm.s(-1)), (ii) a CK localized in the vicinity of ATPases (MM-CK bound isoform) favoring ATP synthesis (F(f) = 1.7 x F(r)), and (iii) a mitochondrial CK displaced toward PCr synthesis (F(f) = 0.3 and F(r) = 2.6 mm.s(-1)). This study thus provides the first experimental evidence that the energy is carried from mitochondria to ATPases by PCr (i.e. CK shuttle) in the whole heart. In contrast, a single CK functioning at equilibrium was sufficient to describe the data when ATP synthesis was partly inhibited by cyanide (0.15 mm). In this case, ATP was directly transferred from mitochondria to cytosol suggesting that cardiac activity modified energy transfer pathways. Bioenergetic implications of the localization and activity of enzymes within myocardial cells are discussed.     

PRE- AND POSTSYNAPTIC UTILIZATION OF ATP AND CREATINE PHOSPHATE AT THE NERVE-ELECTROPLAQUE JUNCTION

Abstract— When the electric organ of the Torpedo fish was stimulated in vivo and in vitro , there was a large rapid breakdown of creatine phosphate (CrP), and a slower decrease in the level of ATP. The acetylcholine content showed a parallel fall to that of ATP. Curare was used to interrupt synaptic transmission, allowing the presynaptic nerve endings to remain sensitive to stimulation, but simultaneously keeping electroplaques inactive. In stimulated curarized tissue, the level of ATP was not changed, while the breakdown of CrP was still 25% of that observed in non-curarized controls. These observations suggest that presynaptic nerve endings, despite their small size, utilize a very high amount of energy.     

Geldanamycin prevents hemorrhage-induced ATP loss by overexpressing inducible HSP70 and activating pyruvate dehydrogenase

Hemorrhage in mice results in decreased ATP levels in the jejunum, lung, kidney, heart, and brain but not in liver tissue lysates, albeit at variable levels and time kinetics. The decreased protein expression and activity of pyruvate dehydrogenase (PDH) accounted for the hemorrhage-induced ATP loss. Treatment with geldanamycin (GA; 1 microg/g body wt), a known inducer of heat shock protein (HSP)70, inhibited the hemorrhage-induced ATP loss in the jejunum, lung, heart, kidney, and brain. GA was found to increase PDH protein, preserve PDH enzymatic activity, and inhibit mucosal injury in jejunum tissues. GA-induced HSP70i was found to form complexes with PDH protein. HSP70 gene transfer into intestinal epithelial cells promoted PDH and ATP levels, whereas HSP70 short interfering RNA limited them. We conclude that agents able to increase the expression of HSP70 and PDH may be of value in reducing pathology resulting from hemorrhage-associated ATP loss.