J. Michael O’Donnell

Altered metabolite exchange between subcellular compartments in intact postischemic rabbit hearts.

To examine metabolic regulation in postischemic hearts, we examined oxidative recycling of 13C within the glutamate pool (GLU) of intact rabbit hearts. Isolated hearts oxidized 2.5 mmol/L [2-13C]acetate during normal conditions (n = 6) or during reperfusion after 10 minutes of ischemia (n = 5). 13C-Nuclear magnetic resonance spectra were acquired every 1 minute. Kinetic analysis of 13C incorporation into GLU provided both tricarboxylic acid (TCA) cycle flux and the interconversion rate (F1) between the TCA cycle intermediate, alpha-ketoglutarate (alpha-KG), and the largely cytosolic GLU. The rate-pressure product in postischemic hearts was 46% of normal (P < .05). No difference in substrate utilization occurred between groups, with acetate accounting for 92% of the carbon units entering the TCA cycle at the citrate synthase step. TCA cycle flux in postischemic hearts was normal (normal hearts, 10.7 mumol.min-1.g-1; postischemic hearts, 9.4 mumol.min-1.g-1), whereas F1 was 72% lower at 2.9 +/- 0.4 versus 10.2 +/- 2.5 mumol.min-1.g-1 (mean +/- SE) in normal hearts (P < .05). From additional hearts perfused with 2.5 mmol/L [2-13C]acetate plus supplemental 5 mmol/L glucose, any potential differences in endogenous carbohydrate availability were proved not to account for the reduced rate alpha-KG and GLU exchange, which remained depressed in postischemic hearts. However, specific activities of the transaminase enzyme, catalyzing chemical exchange of alpha-KG and GLU, were the same, and transaminase flux was 100 mumol.min-1.g-1 in postischemic hearts versus 68 mumol.min-1.g-1 in normal hearts. Normal transaminase activity and the increased flux in postischemic hearts are contrary to the reduced F1. The findings indicate reduced metabolite transport rates across the mitochondrial membranes of stunned myocardium, particularly through the reversible alpha-KG-malate carrier.

Coupling of Mitochondrial Fatty Acid Uptake to Oxidative Flux in the Intact Heart

The coordination of long chain fatty acid (LCFA) transport across the mitochondrial membrane (V(PAL)) with subsequent oxidation rate through beta-oxidation and the tricarboxylic acid (TCA) cycle (V(tca)) has been difficult to characterize in the intact heart. Kinetic analysis of dynamic (13)C-NMR distinguished these flux rates in isolated rabbit hearts. Hearts were perfused in a 9.4 T magnet with either 0.5 mM [2,4,6,8,10,12,14,16-(13)C(8)] palmitate (n = 4), or 0.5 mM (13)C-labeled palmitate plus 0.08 mM unlabeled butyrate (n = 4). Butyrate is a short chain fatty acid (SCFA) that bypasses the LCFA transporters of mitochondria. In hearts oxidizing palmitate alone, the ratio of V(TCA) to V(PAL) was 8:1. This is consistent with one molecule of palmitate yielding eight molecules of acetyl-CoA for the subsequent oxidation through the TCA cycle. Addition of butyrate elevated this ratio; V(TCA)/V(PAL) = 12:1 due to an SCFA-induced increase in V(TCA) of 43% (p < 0.05). However, SCFA oxidation did not significantly reduce palmitate transport into the mitochondria: V(PAL) = 1.0 +/- 0.2 micromol/min/g dw with palmitate alone versus 0.9 +/- 0.1 with palmitate plus butyrate. Thus, the products of beta-oxidation are preferentially channeled to the TCA cycle, away from mitochondrial efflux via carnitine acetyltransferase.

Tight Control of Exogenous SERCA Expression Is Required to Obtain Acceleration of Calcium Transients With Minimal Cytotoxic Effects in Cardiac Myocytes

Abstract— Collateral effects of exogenous sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) expression were characterized in neonatal rat and chicken embryo cardiac myocytes, and the conditions required to produce acceleration of Ca2+ transients with minimal toxicity were established. Cultured myocytes were infected with adenovirus vector carrying the cDNA of wild-type SERCA1, an inactive SERCA1 mutant, or enhanced green fluorescence protein under control of the cytomegalovirus promoter. Controls were exposed to empty virus vector. Each group was tested with and without phenylephrine (PHE) treatment. Under conditions of limited calf-serum exposure, the infected rat myocytes manifested a more rapid increase in size, protein content, and rate of protein synthesis relative to noninfected controls. These changes were not accompanied by reversal to fetal transcriptional pattern (as observed in hypertrophy triggered by PHE) and may be attributable to facilitated exchange with serum factors. SERCA virus titers >5 to 6 plaque-forming units per cell produced overcrowding of ATPase molecules on intracellular membranes, followed by apoptotic death of a significant number of rat but not chicken myocytes. Enhanced green fluorescence protein virus and empty virus also produced cytotoxic effects but at higher titers than SERCA. Expression of exogenous SERCA and enhancement of Ca2+ transient kinetics could be obtained with minimal cell damage in rat myocytes if the SERCA virus titer were maintained within 1 to 4 plaque-forming units per cell. Expression of endogenous SERCA was unchanged, but expression of exogenous SERCA was higher in myocytes rendered hypertrophic by treatment with PHE than in nontreated controls.

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.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. 13C-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.

Mitochondrial transporter responsiveness and metabolic flux homeostasis in postischemic hearts

The transport of metabolites between mitochondria and cytosol via the α-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 μmol ⋅ 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 μmol ⋅ min-1 ⋅ g dry wt-1. Therefore, metabolite exchange remains intact in stunned myocardium but responds to changes in TCA cycle flux regulation.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.

Acute liver carnitine palmitoyltransferase I overexpression recapitulates reduced palmitate oxidation of cardiac hypertrophy.

Rationale: Muscle carnitine palmitoyltransferase I is predominant in the heart, but the liver isoform (liver carnitine palmitoyltransferase I [L-CPT1]) is elevated in hearts with low long chain fatty acid oxidation, such as fetal and hypertrophied hearts. Objective: This work examined the effect of acute L-CPT1 expression on the regulation of palmitate oxidation and energy metabolism in intact functioning rat hearts for comparison with findings in hypertrophied hearts. Methods and Results: L-CPT1 was expressed in vivo in rat hearts by coronary perfusion of Adv.cmv.L-CPT1 (L-CPT1, n=15) vs phosphate-buffered saline (PBS) infusion (PBS, n=7) or empty virus (empty, n=5). L-CPT1 was elevated 5-fold at 72 hours after Adv.cmv.L-CPT1 infusion (P<0.05), but muscle carnitine palmitoyltransferase I was unaffected. Despite similar tricarboxylic acid cycle rates, palmitate oxidation rates were reduced with L-CPT1 (1.12 ± 0.29 &mgr;mol/min per gram of dry weight, mean±SE) vs PBS (1.6 ± 0.34). Acetyl CoA production from palmitate was reduced with L-CPT1 (69 ± 0.02%; P<0.05; PBS=79 ± 0.01%; empty=81 ± 0.02%), similar to what occurs in hypertrophied hearts, and with no difference in malonyl CoA content. Glucose oxidation was elevated with L-CPT1 (by 60%). Surprisingly, L-CPT1 hearts contained elevated atrial natriuretic peptide, indicating induction of hypertrophic signaling. Conclusions: The results link L-CPT1 expression to reduced palmitate oxidation in a nondiseased adult heart, recapitulating the phenotype of reduced long chain fatty acid oxidation in cardiac hypertrophy. The implications are that L-CPT1 expression induces metabolic remodeling hypertrophic signaling and that regulatory factors beyond malonyl CoA in the heart regulate long chain fatty acid oxidation via L-CPT1.

In Vivo, Cardiac-Specific Knockdown of a Target Protein, Malic Enzyme- 1, in Rat via Adenoviral Delivery of DNA for Non-Native miRNA

This study examines the feasibility of using the adenoviral delivery of DNA for a non-native microRNA to suppress expression of a target protein (cytosolic NADP(+)-dependent malic-enzyme 1, ME1) in whole heart in vivo, via an isolated-heart coronary perfusion approach. Complementary DNA constructs for ME1 microRNA were inserted into adenoviral vectors. Viral gene transfer to neonatal rat cardiomyocytes yielded 65% suppression of ME1 protein. This viral package was delivered to rat hearts in vivo (Adv.miR_ME1, 10(13) vp/ml PBS) via coronary perfusion, using a cardiac-specific isolation technique. ME1 mRNA was reduced by 73% at 2-6 days post-surgery in heart receiving the Adv.miR_ME1. Importantly, ME1 protein was reduced by 66% (p < 0.0002) at 5-6 days relative to sham-operated control hearts. Non-target protein expression for GAPDH, calsequestrin, and mitochondrial malic enzyme, ME3, were all unchanged. The non-target isoform, ME2, was unchanged at 2-5 days and reduced at day 6. This new approach demonstrates for the first time significant and acute silencing of target RNA translation and protein content in whole heart, in vivo, via non-native microRNA expression.

Enhanced Redox State and Efficiency of Glucose Oxidation With miR Based Suppression of Maladaptive NADPH-Dependent Malic Enzyme 1 Expression in Hypertrophied HeartsNovelty and Significance

Rationale: Metabolic remodeling in hypertrophic hearts includes inefficient glucose oxidation via increased anaplerosis fueled by pyruvate carboxylation. Pyruvate carboxylation to malate through elevated ME1 (malic enzyme 1) consumes NADPH necessary for reduction of glutathione and maintenance of intracellular redox state. Objective: To elucidate upregulated ME1 as a potential maladaptive mechanism for inefficient glucose oxidation and compromised redox state in hypertrophied hearts. Methods and Results: ME1 expression was selectively inhibited, in vivo, via non-native miR-ME1 (miRNA specific to ME1) in pressure-overloaded rat hearts. Rats subjected to transverse aortic constriction (TAC) or Sham surgery received either miR-ME1 or PBS. Effects of ME1 suppression on anaplerosis and reduced glutathione (GSH) content were studied in isolated hearts supplied 13C-enriched substrate: palmitate, glucose, and lactate. Human myocardium collected from failing and nonfailing hearts during surgery enabled RT-qPCR confirmation of elevated ME1 gene expression in clinical heart failure versus nonfailing human hearts (P<0.04). TAC induced elevated ME1 content, but ME1 was lowered in hearts infused with miR-ME1 versus PBS. Although Sham miR-ME1 hearts showed no further reduction of inherently low anaplerosis in normal heart, miR-ME1 reduced anaplerosis in TAC to baseline: TAC miR-ME1=0.034±0.004; TAC PBS=0.081±0.005 (P<0.01). Countering elevated anaplerosis in TAC shifted pyruvate toward oxidation in the tricarboxylic acid cycle. Importantly, via the link to NADPH consumption by pyruvate carboxylation, ME1 suppression in TAC restored GSH content, reduced lactate production, and ultimately improved contractility. Conclusions: A maladaptive increase in anaplerosis via ME1 in TAC is associated with reduced GSH content. Suppressing increased ME1 expression in hypertrophied rat hearts, which is also elevated in failing human hearts, reduced pyruvate carboxylation thereby normalizing anaplerosis, restoring GSH content, and reducing lactate accumulation. Reducing ME1 induced favorable metabolic shifts for carbohydrate oxidation, improving intracellular redox state and enhanced cardiac performance in pathological hypertrophy.

Acute Liver Carnitine Palmitoyltransferase I Overexpression Recapitulates Reduced Palmitate Oxidation of Cardiac HypertrophyNovelty and Significance

Rationale: Muscle carnitine palmitoyltransferase I is predominant in the heart, but the liver isoform (liver carnitine palmitoyltransferase I [L-CPT1]) is elevated in hearts with low long chain fatty acid oxidation, such as fetal and hypertrophied hearts. Objective: This work examined the effect of acute L-CPT1 expression on the regulation of palmitate oxidation and energy metabolism in intact functioning rat hearts for comparison with findings in hypertrophied hearts. Methods and Results: L-CPT1 was expressed in vivo in rat hearts by coronary perfusion of Adv.cmv.L-CPT1 (L-CPT1, n=15) vs phosphate-buffered saline (PBS) infusion (PBS, n=7) or empty virus (empty, n=5). L-CPT1 was elevated 5-fold at 72 hours after Adv.cmv.L-CPT1 infusion (P<0.05), but muscle carnitine palmitoyltransferase I was unaffected. Despite similar tricarboxylic acid cycle rates, palmitate oxidation rates were reduced with L-CPT1 (1.12 ± 0.29 &mgr;mol/min per gram of dry weight, mean±SE) vs PBS (1.6 ± 0.34). Acetyl CoA production from palmitate was reduced with L-CPT1 (69 ± 0.02%; P<0.05; PBS=79 ± 0.01%; empty=81 ± 0.02%), similar to what occurs in hypertrophied hearts, and with no difference in malonyl CoA content. Glucose oxidation was elevated with L-CPT1 (by 60%). Surprisingly, L-CPT1 hearts contained elevated atrial natriuretic peptide, indicating induction of hypertrophic signaling. Conclusions: The results link L-CPT1 expression to reduced palmitate oxidation in a nondiseased adult heart, recapitulating the phenotype of reduced long chain fatty acid oxidation in cardiac hypertrophy. The implications are that L-CPT1 expression induces metabolic remodeling hypertrophic signaling and that regulatory factors beyond malonyl CoA in the heart regulate long chain fatty acid oxidation via L-CPT1.