Improvement in survival and cardiac metabolism after gene transfer of sarcoplasmic reticulum Ca(2+)-ATPase in a rat model of heart failure.

BACKGROUND: In heart failure, sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a) activity is decreased, resulting in abnormal calcium handling and contractile dysfunction. We have previously shown that increasing SERCA2a expression by gene transfer improves ventricular function in a rat model of heart failure created by ascending aortic constriction. METHODS AND RESULTS: In this study, we tested the effects of gene transfer of SERCA2a on survival, left ventricular (LV) volumes, and metabolism. By 26 to 27 weeks after aortic banding, all animals developed heart failure (as documented by >25% decrease in fractional shortening) and were randomized to receive either an adenovirus carrying the SERCA2a gene (Ad.SERCA2a) or control virus (Ad.betagal-GFP) by use of a catheter-based technique. Sham-operated rats, uninfected or infected with either Ad.betagal-GFP or Ad.SERCA2a, served as controls. Four weeks after gene transfer, survival in rats with heart failure treated with Ad.betagal-GFP was 9%, compared with 63% in rats receiving Ad.SERCA2a. LV volumes were significantly increased in heart failure (0.64+/-0.05 versus 0.35+/-0.03 mL, P<0.02). Overexpression of SERCA2a normalized LV volumes (0.46+/-0.07 mL) in the failing hearts. (31)P NMR analysis showed a reduced ratio of phosphocreatine to ATP content in failing+Ad.betagal-GFP compared with sham+Ad.betagal-GFP (0.82+/-0.13 versus 1.38+/-0.14, P<0.01). Overexpression of SERCA2a in failing hearts improved the phosphocreatine/ATP ratio (1.23+/-0.28). CONCLUSIONS: In this study, we show that unlike inotropic agents that improve contractile function at the expense of increased mortality and worsening metabolism, gene transfer of SERCA2a improves survival and the energy potential in failing hearts.

Nuclear magnetic resonance evaluation of metabolic and respiratory support of work load in intact rabbit hearts.

Pre-steady-state 13C nuclear magnetic resonance (NMR) spectra can provide a nondestructive probe of metabolic events associated with the physiology of intact organs. Therefore, the relation between phosphorylation state and intermediary metabolism in rabbit hearts, oxidizing [2-13C]acetate, was examined with a combination of 31P and 13C NMR. Multiple enrichment of the tissue glutamate pool with 13C as an index of metabolic turnover within the tricarboxylic acid cycle was readily observed as a function of work load. Dynamic changes in pre-steady-state 13C spectra evolved according to work load and correlated closely to respiratory rate in rabbit hearts perfused 1) under normal conditions (n = 7), 2) at basal metabolic rates (20 mM KCl arrest, n = 5), 3) and at heightened contractile state (10(-7) M isoproterenol, n = 7). The ratio of signal intensity arising from the secondary labeling sites within glutamate (C-2 and C-3) to that of the initial labeling site (C-4) reached steady state within 8.5 minutes in isoproterenol-treated hearts versus 18.5 minutes in control hearts. Work load did not affect glutamate concentration or fractional enrichment at the C-4 position, although an unlabeled fraction of glutamate persisted. Arrested hearts displayed slowed evolution of steady-state 13C enrichment with increased contributions from anaplerotic sources for tricarboxylic acid intermediate formation (32%) as compared with control (9%). Thus, the response of mitochondrial dehydrogenase activity to the demands of cardiac performance is likely to influence the recruitment of anabolic sources supplying the tricarboxylic acid cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic heterogeneity of carbon substrate utilization in mammalian heart: NMR determinations of mitochondrial versus cytosolic compartmentation.

Carbon-13 (13C) nuclear magnetic resonance (NMR) spectroscopy can be used to target specific pathways of intermediary metabolism within intact tissues and was employed in this study to evaluate the compartmentation of pyruvate metabolism between the cytosol and mitochondrial matrix. The distribution of 13C into the tissue alanine, lactate, and glutamate pools was evaluated during metabolism of [3-13C]-pyruvate in intact, isolated perfused rabbit hearts with and without activation of pyruvate dehydrogenase activity by dichloroacetate (5 mM). Equilibrium between the intracellular alanine and pyruvate pools was in evidence from the rapid evolution of the steady-state 13C signal arising from the 3-carbon of alanine in intact hearts perfused with 2.5 mM 99.4% [3-13C]pyruvate. Augmented pyruvate oxidation, in response to perfusion with dichloroacetate, was evident within 13C NMR spectra of intact hearts as a relative increase in signal intensity of 53-62% (p less than 0.05) from the 4-carbon resonance of 13C-enriched glutamate when compared to the unaffected alanine signal. The increased bulk flow of [3-13C]pyruvate into the tricarboxylic acid cycle in response to dichloroacetate resulted in elevated fractional enrichment of glutamate from 68% in controls to 83% in the treated group (p less than 0.04), via interconversion with alpha-ketoglutarate, without changes in the actual tissue content of glutamate. Evidence of metabolic heterogeneity of cytosolic and mitochondrial pyruvate pools was also obtained from analysis of tissue extracts with in vitro NMR spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

Inosine preserves ATP during ischemia and enhances recovery during reperfusion.

The effects of exogenous inosine (IN) on high-energy phosphate metabolism and function in isolated, working rabbit hearts were monitored with 31P-nuclear magnetic resonance spectroscopy. Dynamic measurements of ATP and phosphocreatine (PCr) were made along with concomitant functional recordings during normal perfusion, global ischemia (IS), and reperfusion (RE). We found that 0.1 mM IN enhanced the rate of pressure development (dP/dt) within the left ventricle by 10 +/- 5% (n = 7). Although IN levels in treated hearts were elevated during normal perfusion, no effect was observed on ATP or PCr levels. However during IS, pretreatment with IN minimized ATP loss for the first 20 min relative to untreated controls (UNT, P less than 0.05). Both IN and UNT hearts that were ischemic for only 13.5 min regained function during a 60-min RE period. However, at the end of IS, IN hearts (n = 8) displayed 88 +/- 10% of the pre-IS ATP levels, whereas UNT hearts (n = 7) retained only 60 +/- 10%. With RE, ATP in IN hearts remained elevated over that of UNT hearts for the entire 60 min. IN treatment also increased the rate of recovery of dP/dt and maintained improved function over 60 min of RE. No correlation was found between post-IS ATP levels and dP/dt values during RE in either IN or UNT hearts. These data indicate that IN was protective against ATP loss during IS and improved functional recovery on RE.

Reduced substrate oxidation in postischemic myocardium: 13C and 31P NMR analyses.

13C and 31P nuclear magnetic resonance (NMR) spectra were used to assess substrate oxidation and high-energy phosphates in postischemic (PI) isolated rabbit hearts. Phosphocreatine (PCr) increased in nonischemic controls on switching from glucose perfusion to either 2.5 mM [3-13C]pyruvate (120%, n = 7) or [2-13C]acetate (114%, n = 8, P less than 0.05). ATP content, oxygen consumption (MVO2), and hemodynamics (dP/dt) were not affected by substrate availability in control or PI hearts. dP/dt was 40-60% lower in PI hearts during reperfusion after 10 min ischemia. Hearts reperfused with either pyruvate (n = 11) or acetate (n = 8) regained preischemic PCr levels within 45 s. Steady-state ATP levels were 55-70% of preischemia with pyruvate and 52-60% with acetate. Percent maximum [4-13C]glutamate signal showed reduced conversion of pyruvate to glutamate via the tricarboxylic acid (TCA) cycle at 4-min reperfusion (PI = 24 +/- 4%, means +/- SE; Control = 48 +/- 4%). The increase in 13C signal from the C-4 position of glutamate was similar to control hearts within 10.5 min. The increase in [4-13C]glutamate signal from acetate was not different between PI and control hearts. The ratio of [2-13C]Glu:[4-13C]Glu, reflecting TCA cycle activity, was reduced in PI hearts with acetate for at least 10 min (Control = 0.76 +/- 0.03; PI = 0.51 +/- 0.09) until steady state was reached. Despite rapid recovery of oxidative phosphorylation, contractility remained impaired and substrate oxidation was significantly slowed in postischemic hearts.

Fatty acid metabolism and contractile function in the reperfused myocardium. Multinuclear NMR studies of isolated rabbit hearts.

The hypothesis that substrate availability can alter contractile function in reperfused myocardium after global ischemia was investigated in this study. Isolated rabbit hearts were placed in a dual tuned (31P/13C) NMR probe with a 9.4-T magnet and perfused with the following substrates given individually or in combination: 10 mM glucose, 2 mM palmitate, and 2.5 mM [3-13C]pyruvate. Glucose was the sole substrate present for all groups of hearts before the onset of 10 or 20 minutes of zero-flow ischemia. Contractility (dP/dt) was significantly higher in hearts reperfused with glucose compared with hearts reperfused with palmitate or the combination. In addition, myocardial oxygen consumption/unit of work at reperfusion was more efficient with glucose than with palmitate. ATP content during reperfusion was similar with glucose and palmitate and did not account for improved function with glucose. To determine if inhibition of pyruvate metabolism by palmitate might result in altered postischemic function, additional hearts were reperfused with 2.5 mM [3-13C]pyruvate provided alone or in combination with palmitate. Using 13C NMR spectroscopy, it was shown that with the addition of palmitate, pyruvate oxidation was decreased in control and 10-minute ischemic hearts as is consistent with inhibition of pyruvate dehydrogenase by fatty acids. However, palmitate/pyruvate did not worsen postischemic function as compared with palmitate or pyruvate alone. Tricarboxylic acid cycle activity was slowed in reperfused pyruvate hearts, but no further reduction was observed when palmitate was present. In conclusion, palmitate reduces the mechanical function of the reperfused isolated rabbit heart as compared with glucose. This effect of palmitate does not appear to be caused by suppression of pyruvate oxidation or by a change in high energy phosphate content.

Dynamic changes in 13C NMR spectra of intact hearts under conditions of varied metabolite enrichment.

Dynamic changes in 13C NMR signal from enriched glutamate pools within hearts have been examined under varied conditions of metabolite pool size and fractional enrichment. Relative signal intensities of 13C-enriched glutamate isotope isomers were similar within spectra from both intact hearts and corresponding in vitro samples. The parameters used to assess metabolic activity with 13C NMR proved independent of fractional enrichment and pool size. The data show the importance of acknowledging unlabeled, 13C NMR invisible metabolites.

Pyruvate dehydrogenase influences postischemic heart function.

BACKGROUND: The pyruvate dehydrogenase (PDH) enzyme complex determines the extent of carbohydrate oxidation in the myocardium. PDH is in a largely inactive state during early reperfusion of postischemic myocardium. The resultant decrease in pyruvate oxidation in postischemic hearts has been documented with 13C nuclear magnetic resonance (NMR) spectroscopy. This study demonstrates that counteracting depressed pyruvate oxidation can enhance contractile recovery in the absence of increases in either glycolytic activity or glucose oxidation. The findings indicate that increased incorporation of carbon units from pyruvate into the intermediates of the oxidative pathways by PDH influences the metabolic efficiency and mechanical work of postischemic hearts. METHODS AND RESULTS: Isolated rabbit hearts were situated in an NMR magnet and perfused or reperfused (10 minutes of ischemia) with 2.5 mmol/L [3-13C]pyruvate as sole substrate to target PDH directly and bypass the glycolytic pathway. Hearts were observed with or without activation of PDH with dichloroacetate. Mechanical function and oxygen consumption (MVO2) were monitored. 13C and 31P NMR spectroscopy allowed observations of pyruvate oxidation and bioenergetic state in intact, functioning hearts. Metabolite content and 13C enrichment levels were then determined with in vitro NMR spectroscopy and biochemical assay. PDH activation did not affect performance of normal hearts. Postischemic hearts with augmented pyruvate oxidation (dichloroacetate-treated) sustained improved mechanical function throughout 40 minutes of reperfusion. Rate-pressure-product (RPP) increased from 8300 +/- 1800 (mean +/- SEM) in untreated postischemic hearts to 21,300 +/- 2400 in hearts treated with dichloroacetate (P < .05). Oxygen use per unit work [MVO2 multiplied by 10(4) divided by RPP] was improved from 1.50 +/- 0.13 to 1.14 +/- 0.11 (P < .05) without differences in high-energy phosphate content between treated and untreated hearts. Values of dP/dt were also consistently higher, by as much as 185%, during reperfusion with dichloroacetate. Postischemic hearts displayed reduced pyruvate oxidation from the incorporation of 13C into the tissue glutamate pool. With the tissue alanine level as a marker of 13C-enriched pyruvate availability in the cell, the ratio of labeled glutamate to alanine was only 58% of the control value during early reperfusion. With dichloroacetate, that ratio was 167% greater than that of untreated hearts (P < .05). By the end of the reperfusion period, the 13C enrichment of the tissue glutamate pool by pyruvate oxidation was elevated from dichloroacetate treatment (untreated, 62 +/- 7%; DCA-treated, 81 +/- 6%; P < .05), but glycogen content was similar in both groups and 13C enrichment of tissue alanine remained unchanged, near 60%, indicating no increases in glycolytic end-product formation. CONCLUSIONS: Metabolic reversal of contractile dysfunction was achieved in isolated hearts by counteracting depressed PDH activity in the postischemic myocardium. Improved cardiac performance did not result from, nor require, increased glycolysis secondary to the activation of PDH. Rather, restoring carbon flux through PDH alone was sufficient to improve mechanical work by postischemic hearts.

High-energy phosphates and function in isolated, working rabbit hearts.

An isolated, working, rabbit heart has been developed for use with nuclear magnetic resonance (NMR) spectroscopy. This model is functionally stable over a 4-h period and displays classic hemodynamic responses to work-load changes. Control 31P spectra of this preparation (n = 5) were obtained with simultaneous recordings of left ventricular pressure (LVP), LVP differentiated with respect to time (dP/dt), heart rate (HR), and cardiac output (CO). ATP, phosphocreatine (PCr), and hemodynamics remained stable over a 90-min perfusion. Hearts were also subjected to 13.5 min of global ischemia (IS) at 37 degrees C followed by 60 min of reperfusion (RE, n = 7) or 45 min of chronic IS (n = 6). Contraction ceased within 60 s of IS. PCr loss was rapid, reaching undetectable limits by 11 min. ATP loss was gradual and bore no relationship to functional loss. ATP fell to 60 +/- 4% (means +/- SE) of pre-IS levels after 13.5 min of IS. With RE, PCr returned to control levels, whereas ATP values remained depressed for the entire 60 min. Functional activity resumed with RE, but dP/dt did not rise above 85 +/- 7% of preischemic values. No correlation between residual ATP at the end of IS and functional recovery during RE was evident.

Effects of inosine on glycolysis and contracture during myocardial ischemia.

The effects of inosine (INO) on substrate metabolism and rigor formation in ischemic myocardium were examined in isolated rabbit hearts. Metabolite content was assessed in tissue extracts by chemical analysis and in the whole heart by 13C and 31P nuclear magnetic resonance spectroscopy. In ischemic hearts metabolizing either [3-13C]pyruvate or [1-13C]glucose, 1 mM INO increased both total and 13C-labeled alanine content; lactate content was unaffected. At 3 minutes of ischemia, tissue alanine was 1.81 +/- 0.11 microM/g wet wt (mean +/- SEM) in hearts perfused with pyruvate+INO versus 1.23 +/- 0.15 microM/g wet wt in hearts perfused with pyruvate alone (p less than 0.05). INO reduced tissue glycogen during ischemia in pyruvate-perfused hearts. Tissue alanine content in ischemic hearts that were supplied glucose+INO (1.29 +/- 0.13 microM/g wet wt) was greater than in ischemic hearts supplied glucose alone (0.65 +/- 0.14 microM/g wet wt). Alanine was found to originate from pyruvate and was a glycolytic end product in glucose-perfused hearts. INO raised the [3-13C]alanine/[3-13C]lactate ratio in ischemic, intact hearts (glucose = 0.24 +/- 0.07 versus glucose+INO = 0.60 +/- 0.09; pyruvate = 0.49 +/- 0.08 versus pyruvate+INO = 0.89 +/- 0.08). At 7 minutes of ischemia, ATP content fell to 70 +/- 3% with glucose+INO versus 58 +/- 5% with glucose alone. Rigor (stone heart) was delayed from 14.7 +/- 1.3 to 23.2 +/- 1.6 minutes with INO. INO did not change ATP content in ischemic hearts that were supplied pyruvate but delayed rigor (pyruvate = 9.9 +/- 1.2 minutes; pyruvate+INO = 15.6 +/- 1.0 minutes), possibly at the expense of glycogen. Supplemental glucose improved the effectiveness of INO with pyruvate to preserve ATP (pyruvate+glucose = 42 +/- 6%; pyruvate+glucose+INO = 72 +/- 6%) and further delayed rigor (pyruvate+glucose = 13.3 +/- 1.5 minutes; pyruvate+glucose+INO = 20.3 +/- 1.8 minutes). Glucose metabolism supported improved energetic and contractile states in ischemic hearts treated with INO. Thus, cardioprotection of the ischemic heart by INO was associated with preservation of functional integrity and improved energy production due to increased glycolytic activity. Activation of glycolysis in the presence of INO was accommodated by augmented alanine production without the additional accumulation of lactate.

Modeling enrichment kinetics from dynamic 13C-NMR spectra: theoretical analysis and practical considerations.

Measurements of oxidative metabolism in the heart from dynamic 13C nuclear magnetic resonance (NMR) spectroscopy rely on 13C turnover in the NMR-detectable glutamate pool. A kinetic model was developed for the analysis of isotope turnover to determine tricarboxylic acid cycle flux (VTCA) and the interconversion rate between alpha-ketoglutarate and glutamate (F1) by fitting the model to NMR data of glutamate enrichment. The results of data fitting are highly reproducible when the noise level is within 10%, making this model applicable to single or grouped experiments. The values for VTCA and F1 were unchanged whether obtained from least-squares fitting of the model to mean experimental enrichment data with standard deviations in the cost function (VTCA = 10.52 mumol.min-1.g dry wt-1, F1 = 10.67 mumol.min-1.g dry wt-1) or to the individual enrichment values for each heart with the NMR noise level in the cost function (VTCA = 10.67 mumol.min-1.g dry wt-1, F1 = 10.18 mumol.min-1.g dry wt-1). Computer simulation and theoretical analysis indicate that glutamate enrichment kinetics are insensitive to the fractional enrichment of acetyl-CoA and changes in small intermediate pools (< 1 mumol/g dry wt). Therefore, high-resolution NMR analysis of tissue extracts and biochemical assays for intermediates at low concentrations are unnecessary. However, a high correlation between VTCA and F1 exists, as anticipated from competition for alpha-ketoglutarate, which indicates the utility of introducing independent experimental constraints into the data fitting for accurate quantification.

Substrate-dependent proton load and recovery of stunned hearts during pyruvate dehydrogenase stimulation.

Stimulation of pyruvate dehydrogenase (PDH) improves functional recovery of postischemic hearts. This study examined the potential for a mechanism mediated by substrate-dependent proton production and intracellular pH. After 20 min of ischemia, isolated rabbit hearts were reperfused with or without 5 mM dichloroacetate (DCA) in the presence of either 5 mM glucose, 5 mM glucose + 2.5 mM lactate, or 5 mM glucose + 2.5 mM pyruvate. DCA inhibits PDH kinase, increasing the proportion of dephosphorylated, active PDH. Unlike pyruvate or glucose alone, lactate + glucose did not support the effects of DCA on the recovery of rate-pressure product (RPP) (without DCA, RPP = 14,000 +/- 1,200, n = 6; with DCA, RPP = 13,700 +/- 1,800, n = 9). Intracellular pH, from (31)P nuclear magnetic resonance spectra, returned to normal within 2.1 min of reperfusion with all substrates except for lactate + glucose + DCA or lactate + DCA, which delayed pH recovery for up to 12 min (at 2.1 min pH = 6. 00 +/- 0.08, lactate + glucose + DCA; pH = 6.27 +/- 0.34, for lactate + DCA). Hearts were also reperfused after 10 min of ischemia with 0.5 mM palmitate + 5 mM DCA and either 2.5 mM pyruvate or 2.5 mM lactate. Again, intracellular pH recovery was delayed in the presence of lactate. PDH activation in the presence of lactate also decreased coupling of oxidative metabolism to mechanical work. These findings have implications for therapeutic use of stimulated carbohydrate oxidation in stunned hearts.

Chemical versus isotopic equilibrium and the metabolic fate of glycolytic end products in the heart.

Recent studies of isotope exchange across lactate dehydrogenase (LDH) and alanine aminotransferase (AAT) in hearts call into question whether both reactions are in equilibrium. To compare the oxidative and non-oxidative fates of glycolytic end products, isolated rabbit hearts were perfused with 5 mM [2-13C] glucose and 2.5 mM [3-13C] pyruvate: with (n = 6) and without (n = 7) stimulation of pyruvate oxidation using dichloroacetate (DCA), and during normal perfusion or hypoxia (n = 7/n = 6, +/- DCA). 13C NMR spectroscopy of intact hearts confirmed a steady-state enrichment level in both alanine and lactate. 1H- and 13C-NMR spectroscopy of tissue extracts identified the fractions of lactate, alanine and glutamate pools formed from each exogenous substrate. Glycolysis from glucose accounted for 22 +/- 7% of lactate formed and 10 +/- 2% of alanine formed in control hearts, and 16 +/- 2% lactate and 15 +/- 2% alanine in hypoxic hearts (mean +/- S.E.M.). In contrast, exogenous pyruvate formed 36 +/- 5% of the lactate pool, and 86 +/- 3% of the alanine pool in controls and 47 +/- 3% of lactate and of 67 +/- 3% alanine during hypoxia. [2(-13)C] glucose did not contribute to oxidative energy production via the TCA cycle as determined from low 13C enrichment of glutamate C5 from glucose (< 2%), while [3-13C] pyruvate accounted for 84 +/- 7% of labeled glutamate C4. Thus, exogenous pyruvate out-competed the metabolism of glucose, indicating low glycolytic activity. At 40 min, 96 +/- 2% of the total alanine was labeled from either glucose or pyruvate, confirming equilibrium at AAT. However, only 55 +/- 10% of total lactate was labeled, suggesting that the LDH reaction is not in rapid equilibrium within the myocardium.

NMR studies of beta-oxidation and short-chain fatty acid metabolism during recovery of reperfused hearts.

The effects of beta-oxidation on the contractile recovery and metabolic activity of postischemic (10 min) rabbit hearts were examined during reperfusion with the short-chain fatty acid butyrate. Hearts received either 13C-enriched butyrate or acetate to evaluate metabolic targeting with 13C nuclear magnetic resonance (NMR) spectroscopy. Acetate and butyrate supported similar contractility (rate of pressure development, dP/dt) and 31P-NMR-detected, high-energy phosphate (HEP) levels during normal perfusion. In postischemic hearts, butyrate sustained a greater percentage of preischemic dP/dt (83 +/- 4%) than did acetate reperfusion (44 +/- 6%, P less than 0.05) with no differences in HEP. The efficiency of oxygen consumption per unit of work was greater in hearts reperfused with butyrate (2.8 +/- 0.2 microM.g-1.mmHg-1) vs. acetate (3.4 +/- 0.1). Inhibition of butyrate oxidation with 4-bromocrotonic acid (4-BCA) during normal perfusion severely reduced dP/dt and HEP. Acetate supported normal dP/dt and HEP levels during perfusion with 4-BCA and butyrate, but contractile recovery during reperfusion with acetate, 4-BCA, and butyrate (46 +/- 6%) was similar to that with acetate alone. With acetate and butyrate combined at reperfusion, acetate accounted for 56% of substrate entering oxidative metabolism at acetyl CoA and delayed contractile recovery (57 +/- 5% at midpoint and 80 +/- 6% at end). Thus improved respiratory efficiency of contraction in reperfused hearts was related to the activity of beta-oxidation.

Assessment of experimental pericardial effusion using nuclear magnetic resonance imaging techniques.

An experimental canine model of pericardial effusion was designed to validate previous clinical nuclear magnetic resonance imaging (NMR) studies. Saline (n = 7), serum (n = 4), blood (10% hematocrit [n = 5]; 20% hematocrit [n = 5]), and lipid (n = 4) effusions were chosen to resemble: (1) transudative/exudative, (2) nonhemorrhagic/hemorrhagic, and (3) chylous effusions, respectively. There was a linear correlation between the infused volume and the pericardial/epicardial distance measurements on the nuclear magnetic resonance images. Hemorrhagic and nonhemorrhagic exudative effusions were distinguished from transudative effusions by the low signal intensity of transudative effusions images obtained at a TR (repetition time) of 400 and 800 msec. Nonhemorrhagic effusions had significantly lower effusion-to-myocardial signal intensity ratio at TR of 400 msec than did hemorrhagic effusions. Differences in hematocrit were not appreciated qualitatively or quantitatively. Compared with other effusion types, only chylous effusions were hyperintense to myocardium at a TR of 400 msec. Chylous effusions were further uniquely characterized by a decreasing effusion-to-myocardial signal intensity ratio with increasing TR. These experimental findings corroborate the findings of earlier clinical reports and suggest that NMR can provide important assistance in the evaluation of pericardial effusions.

Cytosolic redox state mediates postischemic response to pyruvate dehydrogenase stimulation.

Augmented pyruvate oxidation via pharmacological stimulation of pyruvate dehydrogenase (PDH) during reperfusion has been related to improved recovery of postischemic hearts independent of glycolytic activity. This study examined recovery of postischemic rabbit hearts during activation of PDH with dichloroacetate (DCA) in the presence of lactate, as a source of pyruvate, to determine the response to substrate-dependent changes in cytosolic redox state. After 10 min of ischemia, isolated hearts were reperfused with either 2.5 mM or 0. 5 mM pyruvate (Pyr) or 2.5 mM lactate (Lac), with or without 5 mM DCA. (13)C-enriched substrates allowed NMR assessment of metabolic perturbations. During normal perfusion, Pyr and Lac supported similar mechanical work. Increasing Pyr oxidation restored postischemic rate-pressure product to 82 +/- 4 and 88 +/- 6% of preischemic values during reperfusion with 2.5 and 0.5 mM Pyr, respectively, vs. 61 +/- 6 and 45 +/- 14% for untreated 2.5 and 0.5 mM Pyr, respectively (P < 0.05). In contrast, increasing Lac oxidation did not benefit recovery of RPP in untreated (44 +/- 7%) vs. DCA-treated 36 +/- 4% hearts. Thus the benefit of PDH activation for contractile recovery of postischemic hearts is mediated by the source of pyruvate, which also influences cytosolic redox state.

Cardiac responses to induced lactate oxidation: NMR analysis of metabolic equilibria.

The role of lactate as a source of pyruvate oxidation in supporting cardiac work, energetics, and formation of oxidative metabolites was examined in normal myocardium. 13C- and 31P-nuclear magnetic resonance (NMR) spectra were acquired from isolated rabbit hearts supplied 2.5 mM [3-13C]lactate or [3-13C]pyruvate with or without stimulation of pyruvate dehydrogenase (PDH) by dichloroacetate (DCA). Similar workloads determined by rate-pressure products were noted with pyruvate (21,700 +/- 2,400; mean +/- SE) and lactate (18,970 +/- 1,510). Oxygen consumption was similar in all four groups with means between 19.0 and 22.2 mumol.min-1.g dry weight-1 (SE = 1.6-2.0) as was the ratio of phosphocreatine to ATP with means between 1.8 and 2.1 (SE = 0.1-0.6). Intracellular pH, determined from 31P-NMR spectra, was essentially the same with pyruvate (7.06 +/- 0.02) and lactate (7.05 +/- 0.04). 13C enrichment of glutamate was higher with lactate (92%) than with pyruvate (70%). Pyruvate plus DCA induced no change in glutamate content at 9-10 mumol/g, but 13C enrichment increased to 83%, while lactate plus DCA maintained enrichment at 90%. Levels of alpha-ketoglutarate were lower with lactate (1.81 mumol/g) than with pyruvate (2.36 mumol/g). Lactate plus DCA elevated glutamate by 60% with a proportional increase in alpha-ketoglutarate. Thus the balance between glutamate and alpha-ketoglutarate was affected by substrate supply only and not by PDH activation. The results suggest that the equilibrium between alpha-ketoglutarate and glutamate is sensitive to cytosolic redox state, an important consideration for 13C-NMR analyses that rely on glutamate.(ABSTRACT TRUNCATED AT 250 WORDS)

Subcellular metabolite transport and carbon isotope kinetics in the intramyocardial glutamate pool.

The pathophysiological state of the cell must be translated into the mitochondria to meet the demands for oxidative energy production. Metabolite exchange across the mitochondrial membrane provides this communication and was observed with 13C NMR spectroscopy of hearts oxidizing [2-13C]-butyrate at normal or high cytosolic redox state. Previous NMR observations of 13C turnover within the glutamate pool of intact tissues have indicated its relationship with metabolic flux through the tricarboxylic acid (TCA) cycle, but the direct influence of isotope exchange between the TCA cycle intermediates in the mitochondria and the cytosolic glutamate pool has been much less considered. This current study was designed to determine whether the physical transport of metabolites across the mitochondrial membrane of intact heart tissues could be discerned as a rate determinant for isotope turnover in the NMR-detectable glutamate pool. 13C entry into glutamate provided measures of TCA cycle flux and the interconversion between mitochondrial intermediates and cytosolic glutamate. The influence of the malate-aspartate shuttle activity was examined by comparing two groups of hearts: one group oxidizing 2.5 mM [2-13C]-butyrate (n = 5) and the other oxidizing 2.5 mM [2-13C]butyrate in the presence of a lactate (2.5 mM)-induced elevation in the cytosolic redox to stimulate shuttle activity (n = 5). High redox state did not affect TCA cycle flux but increased the rate of interconversion between alpha-ketoglutarate and glutamate from 3.1 +/- 0.2 mumol min-1 (g dry)-1 to 14.3 +/- 2.0. High resolution 13C NMR spectra of tissue extracts confirmed that the exogenous lactate did not contribute as a carbon source for the formation of either the TCA cycle intermediates or glutamate. In both groups, over 95% of the acetyl-CoA was derived from the short-chain fatty acid butyrate, irrespective of the presence of lactate. Additional hearts perfused with unlabeled butyrate and [3-13C]lactate showed no label entry into glutamate, but rather the formation of [3-13C]alanine, indicating the net reverse flux through lactate dehydrogenase to increase NADH production. Thus, the addition of lactate served only to augment cytosolic redox state to drive the malate-aspartate shuttle. The dynamic-mode acquisition of 13C NMR data from intact hearts, oxidizing [2-13C]-butyrate with or without additional lactate, demonstrated the influence of malate-aspartate shuttle activity on the 13C enrichment rates within glutamate. These data indicate metabolic communication between the mitochondria and cytosol in response to the physiological state of intact tissues.

Mitochondrial transporter responsiveness and metabolic flux homeostasis in postischemic hearts.

The transport of metabolites between mitochondria and cytosol via the alpha-ketoglutarate-malate carrier serves to balance flux between the two spans of the tricarboxylic acid (TCA) cycle but is reduced in stunned myocardium. To examine the mechanism for reduced transporter activity, we followed the postischemic response of metabolite influx/efflux from mitochondria to stimulation of the malate-aspartate (MA) shuttle. Isolated rabbit hearts were either perfused with 2.5 mM [2-13C]acetate (n = 7) or similarly reperfused (n = 5) after 10-min ischemia. In other hearts, the MA shuttle was stimulated with a high cytosolic redox state (NADH) induced by 2.5 mM lactate in normal (n = 6) or reperfused hearts (n = 7). In normal hearts, the MA shuttle response accelerated transport from 8.3 +/- 3.4 to 16.2 +/- 5.0 micromol. min(-1). g dry wt(-1). Although transport was reduced in stunned hearts, the MA shuttle was responsive to cytosolic NADH load, increasing transport from 3.4 +/- 1.0 to 9.8 +/- 3.7 micromol. min(-1). g dry wt(-1). Therefore, metabolite exchange remains intact in stunned myocardium but responds to changes in TCA cycle flux regulation.